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WO1997033173A1 - Muscular dystrophy, stroke, and neurodegenerative disease diagnosis and treatment - Google Patents

Muscular dystrophy, stroke, and neurodegenerative disease diagnosis and treatment Download PDF

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Publication number
WO1997033173A1
WO1997033173A1 PCT/US1997/003897 US9703897W WO9733173A1 WO 1997033173 A1 WO1997033173 A1 WO 1997033173A1 US 9703897 W US9703897 W US 9703897W WO 9733173 A1 WO9733173 A1 WO 9733173A1
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nnos
psd
gly
leu
dystrophin
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PCT/US1997/003897
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French (fr)
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David S. Bredt
Jay E. Brenman
Daniel S. Chao
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The Regents Of The University Of California
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Publication of WO1997033173A1 publication Critical patent/WO1997033173A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • C12N9/0075—Nitric-oxide synthase (1.14.13.39)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2878—Muscular dystrophy

Definitions

  • This invention concerns nitric oxide, neuronal nitric oxide synthase, neuronal nitric oxide synthase binding proteins, their inhibitors and a method of use of neuronal
  • nitric oxide synthase 10 nitric oxide synthase, its binding proteins and their inhibitors for diagnosis and treatment of muscular dystrophy, stroke and other neurodegenerative diseases.
  • one aspect of the invention concerns involvement of neuronal nitric oxide synthase and its
  • the invention also concerns diagnostic assay for detection of absence of dystrophin or its mutated forms, neuronal nitric oxide synthase or its
  • binding proteins as well as a method for treatment of muscular dystrophies by restoration of a functional dystrophin molecule in dystrophic muscles using gene therapy.
  • the second aspect of the invention concerns
  • the invention also concerns diagnosis as well as prevention and treatment of stroke and the other neurodegenerative diseases.
  • invention concerns the cloning and expression of the neuronal nitric oxide synthase binding proteins. Finally, the invention concerns a binding assay for monitoring of binding of nitric oxide binding proteins with neuronal nitric oxide synthase and appropriate synaptic
  • Muscular dystrophy i ⁇ a debilitating disease caused by a motor dysfunction due to a genetic abnormality resulting in the absence or mutation of the protein dystrophin.
  • Muscular dystrophies consist of a group of inherited diseases characterized by progressive weakness and degeneration of muscle fibers, without evidence of neural degeneration. The group includes dystrophies such as pseudohypertrophic Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy (Leyden- Mobius pelvifemoral type) , and facioscapulohumoral (Landouzy-Dejerine) muscular dystrophy.
  • Duchenne dystrophy is an X-linked recessive disorder caused by a mutation at the Xp21 locus, which results in the absence of the gene product dystrophin, normally localized in the sarcolemma of muscle cells.
  • Becker muscular dystrophy also a X-linked disorder, is a milder clinical variant of Duchenne dystrophy with the same genomic mutation at Xp21 where patients do not lack dystrophin completely but their dystrophin has an abnormal molecular weight and is somehow dysfunctional.
  • Duchenne muscular dystrophy is a severe, and ultimately fatal, disease.
  • Duchenne dystrophy patients typically boys 3-7 years old, experience muscle weakness, waddling gait, toe-walking, lordosis, frequent falls, and difficulty in standing up and in climbing stairs. Progression of the disease is steady and most patients are confined to a wheelchair at age 10 to 12. Few patients survive age of 20 years. Clinical symptoms of Becker muscular dystrophy are less severe; very few patient are confined to a wheelchair and more than 90% of these patients survive.
  • Dystrophin is a large intracellular protein containing several defined sequence motifs (Cell. 80:675-679 (1995)).
  • An amino terminal ⁇ -actinin-like domain binds to F-actin and is followed by a large rod domain that shares sequence homology with the structural repeats in spectrin.
  • the carboxyl terminus is unique to dystrophin and dystrophin related proteins as this region directly binds to a glycoprotein complex in skeletal muscle.
  • the structural dystrophin glycoprotein complex includes intracellular proteins syntrophins as well as integral membranes proteins, the dystroglycans and sarcoglycans.
  • Dystrophin was originally identified by positional cloning as the gene product mutated in Duchenne muscular dystrophy (Nature, 323: 646-650 (1986)). Subsequent studies have identified a family of intracellular and transmembrane glycoproteins in a dystrophin-associated complex that links the extracellular matrix with the actin- ba ⁇ ed cytoskeleton. Recent ⁇ tudies indicate a major role for this complex in neuromuscular development and disease.
  • ⁇ -Dystroglycan an extracellular glycoprotein linked to dystrophin, serves as a physiologic receptor for agrin, which mediates clustering of acetylcholine receptors (Cell. 77: 663-674 (1994), ibid. 77: 675-686 (1994)).
  • disruption of dystrophin or other proteins in this complex results in muscular dystrophy in both humans and animals (Cell. 80: 675-679 (1995)).
  • the dystrophin complex is involved in signalling function in muscle, including regulation of a stretch-activated calcium channel. Signal transduction by the dystrophin complex must be somehow mediated. It has now been discovered that nitric oxide may be that mediator.
  • Nitric oxide is a major endogenous mediator involved in diverse developmental and physiological processes (Annu. Rev. Biochem. , 63: 175-195 (1994)). In addition to controlling diverse cellular processes, NO also participates in certain pathophysiological conditions. In skeletal muscle NO has been shown to depress the muscle contractile function (Nature. 372: 546-548 (1994)). In the brain, nitric oxide plays important physiological role in neurotransmission and synaptic modulation. In primary cortical cultures, NO mediates glutamate neurotoxicity (PNAS. 88: 6368-6371 (1991)). Neuronal NO production contributes to the development of ischemic brain necrosis (Science. 265:1883-1885 (1994)).
  • NMDA N-methyl-D-aspartate
  • NO nitric oxide synthase
  • these three NOSs are endothelial (eNOS) , neuronal NOS (nNOS) and inducible NOS (iNOS) .
  • eNOS endothelial
  • nNOS neuronal NOS
  • iNOS inducible NOS
  • the nNOS and eNOS enzymes are discretely expressed in specific tissues and rapidly transduce signaling events in a calciu - dependent manner.
  • eNOS activity accounts for endothelium- dependent blood vessel relaxation, while nNOS occurs discretely in a variety of cell types, including neurons, epithelial cells, esangial cells, and skeletal muscle cells.
  • Inducible iNOS is a calcium-independent form of NOS expressed at highest levels in immunologically activated cells.
  • nNOS n-nitrosarcomasergic-cholinergic transmitter in numerous pathways, including the gastrointestinal and urogenital tracts.
  • NOS proteins are all regulated by calmodulin (PNAS.. USA f 87: 682-685 (1990)), which links NO formation to increases in cellular calcium.
  • calmodulin PNAS.. USA f 87: 682-685 (1990)
  • Activation of nNOS in neurons is regulated by the steep gradients of calcium that exist in the vicinity of open calcium channels.
  • calcium influx through the N-methyl-glutamic acid receptor is selectively coupled to nNOS activity.
  • nNOS is enriched in fast-twitch muscle fibers, where NO opposes contractile force (Nature. 372:546-548 (1994)). It has now been found that the physiological actions of NO in muscle are facilitated by restriction of the nNOS protein to the sarcolemmal membrane.
  • the sarcolemma of skeletal muscle is a complex structure reinforced by an actin-containing cytoskeleton.
  • actin-containing cytoskeleton In addition to ubiquitous structural elements such as spectrin, skeletal muscle sarcolemma contains a unique network formed around dystrophin and related proteins (Curr. Qpin. Cell Biol.. 5: 82-84 (1993)).
  • nNOS The N-terminal domain of nNOS is unique to the neuronal isoform and contains a PDZ motif of approximately 100 amino acids that is found in a diverse group of cytoskeletal proteins and enzymes (Neuron. 9: 929-942 (1992)). This domain has now been found to mediate association of nNOS with the dystrophin complex. Therefore it would seem that nNOS must play a distinct role in the muscular dystrophy development and control, and could be advantageously used for early detection of the dystrophic disease.
  • the invention discloses a role of NO, nNOS and its binding proteins in development or controlling of muscular dystrophy in one aspect, and in prophylaxis, treatment and diagnosis of stroke and other neurodegenerative diseases in another aspect.
  • One aspect of the current invention concerns a function of nitric oxide, neuronal nitric oxide synthase, and neuronal nitric oxide synthase binding proteins in muscular dystrophies.
  • Another aspect of the current invention concerns a function of nitric oxide, neuronal nitric oxide synthase, and neuronal nitric oxide synthase binding proteins in stroke and other neurodegenerative diseases.
  • Another aspect of the current invention concerns nitric oxide synthase binding proteins, their cloning and expression.
  • Another aspect of the current invention concerns identification of two brain proteins, namely, postsynaptic density PSD-95 and postsynaptic density PSD-93 proteins that bind to neuronal nitric oxide synthase. Still yet another aspect of the current invention concerns a discovery that neuronal nitric oxide synthase is functionally connected to calcium influx through a N- methyl-D-aspartate receptor where, at a receptor synaptic junction, neuronal nitric oxide synthase is enriched with post-synaptic density proteins.
  • Still another aspect of the current invention concerns identification of inhibitors of nitric oxide synthase binding proteins.
  • Another aspect of the current invention concerns identification of a small 9-mer peptide that potently blocks binding of neuronal nitric oxide synthase with post- synaptic density proteins.
  • Still another aspect of the current invention is a method of use of neuronal nitric oxide synthase, its binding proteins, and their inhibitors, for diagnosis and treatment of muscular dystrophy.
  • Yet another aspect of the current invention concerns diagnostic assay for detection of absence of dystrophin or its mutated form, as well as a method for treatment of muscular dystrophy by restoration of a functional dystrophin molecule, or a functional fragment thereof in dystrophic muscles using gene therapy.
  • Still yet another aspect of the current invention concerns a binding assay for monitoring of binding of nitric oxide binding proteins with neuronal nitric oxide synthase and with appropriate synaptic receptors, useful for development of compounds for treatment of stroke and other neurodegenerative diseases.
  • Still another aspect of the current invention is a method of use of neuronal nitric oxide synthase, its binding proteins and their inhibitors for diagnosis and treatment of stroke or other neurodegenerative diseases.
  • Still another aspect of the current invention concerns a method for prevention of brain damage due to nitric oxide, by blocking the binding between neuronal nitric oxide synthase and postsynaptic density proteins resulting in uncoupling neuronal nitric oxide synthase from neurotransmitter receptors.
  • Figure 1 illustrates differential extractability of nNOS and eNOS in skeletal muscle homogenates.
  • Figure 2 is a schematic alignment of eNOS and nNOS domains showing the extended N-terminus of nNOS containing a PDZ domain.
  • Figure 3 shows association of nNOS and dystrophin in skeletal muscle in wild-type, mdx and NOS knockout skeletal muscle.
  • Figure 4 shows extraction of skeletal muscle membrane in build-type and mdx mice and nNOS displacement from particulate fractions of the mdx skeletal muscles.
  • Figure 5 show ⁇ immunofluorescent staining for nNOS of cryostat muscle section from quadriceps of wild-type, mdx, homozygous dystrophic and nNOS knockout mice showing that nNOS is selectively absent from sarcolemma of the mdx skeletal muscle.
  • Figure 6 are skeletal muscle cryosections of normal and DMD patients showing nNOS to be absent from sarcolemma of DMD muscle fibers.
  • Figure 7 is a SDS-PAGE of human skeletal muscle tissue homogenates from three cases of Duchenne muscular dystrophy and from three normal muscle biopsies.
  • Figure 8 shows localization of nNOS, dystrophin and other dystrophin associated proteins during postnatal development.
  • Figure 9 are immunofluorescent stained cryosections from mouse quadriceps labeled for nNOS, ⁇ l-syntrophin and ⁇ -BGT showing localization of nNOS and ⁇ l-syntrophin in wild type, mdx and transgenic mdx mice.
  • Figure 10 are Western blots of mouse skeletal muscle homogenates showing subcellular distribution of nNOS in transgenic mdx mice.
  • Figure 11 are Western blots of solubilized membranes from mouse quadriceps showing selective interaction of nNOS and ⁇ l-syntrophin.
  • Figure 12 are immunostained cryosections of skeletal muscle sarcolemma in Becker muscular dystrophy patients showing absence of nNOS from skeletal muscle sarcolemma in patients with Becker muscular dystrophy.
  • Figure 13 is a molecular model of nNOS and NMDA receptor binding PSD-95.
  • Figure 14 shows alignment of PSD-93 and PSD-95 three PDZ repeats, a SH3 domain and a region homologous to guanylate kinase.
  • Figure 15 shows expression of PDS-93, PDS-95 and nNOS in a rat brain and E15 embryos.
  • Figure 16 illustrates PSD-95 colocalization with nNOS in developing neurons.
  • Figure 17 illustrates nNOS binding to PSD-95 through PDZ motif interaction.
  • Figure 18 shows alternative splicing of exons 1 and 2 of nNOS.
  • Figure 19 shows that catalytically active nNOS isoform lacking exon 2 are expressed in the brain in nNOS ⁇ / ⁇ .
  • Figure 20 shows that nNOS isoforms lacking the PDZ motif do not bind to PSD-95 or to brain membranes.
  • Figure 21 illustrates binding of ⁇ l syntrophin to the N-terminal PDZ containing domain of nNOS.
  • Figure 22 shows direct binding of nNOS to ⁇ l- syntrophin PDZ domain.
  • NOS means nitric oxide synthase, an enzyme that regulates production of nitric oxide.
  • NO means nitric oxide.
  • nNOS means neuronal nitric oxide synthase.
  • NMDA N-methyl-D-aspartate receptor, which is a glutamate type receptor.
  • PSD-95 means postsynaptic density-95 protein, which is present at the brain synaptic junction.
  • PSD-93 means po ⁇ t-synaptic den ⁇ ity-93 protein, which is present at the brain synaptic junction.
  • PZ means a N-terminal domain of nNOS, containing a 66-amino acid motif, bearing homology to a heterogeneous family of signaling enzymes localized at cell-cell junctions.
  • CAM means calmodulin
  • FMN flavin mononucleotide
  • FAD flavin adenine dinucleotide
  • T-SYN or "SYN-1” means syntrophins.
  • DMD Duchenne muscular dystrophy
  • BMD Becker muscular dystrophy.
  • mdx or “mdx mice” means mice that specifically lack dystrophin due to a nonsense mutation, but express nNOS at near normal levels.
  • “dy” mouse means a mouse which has severe muscular dystrophy due to an absence of an extracellular matrix protein, merosin, but has a normal distribution of dystrophin at the sarcolemma.
  • Sptrophins means a family of dystrophin-binding proteins which colocalize with nNOS beneath the sarcolemmal membrane.
  • the current invention involves a discovery that nitric oxide, neuronal nitric oxide synthase and neuronal nitric oxide synthase binding proteins are involved in the development and management of a group of muscular dystrophic and neurodegenerative diseases such as stroke. Muscular dystrophic diseases are characterized by the complete absence, or by diminished level of a fully functional dystrophin. Stroke and other neurodegenerative diseases are characterized by overactive N-methyl-D- aspartate receptors linked to nitric oxide formation in neurons.
  • the invention comprises two parts.
  • the first part is directed to the diagnosis and treatment of muscular dystrophies.
  • the second part is directed to the diagnosis, prophylaxis and treatment of stroke and other neurodegenerative diseases.
  • Neuronal nitric oxide synthase is localized in sarcolemma of fast-twitch fibers and it has now been shown that nNOS partitions with skeletal muscle membranes on account of its association with dystrophin.
  • the dystrophin is associated with intracellular and transmembrane glycoproteins forming a dystrophin-associated complex.
  • the dystrophin complex interacts with a N-terminal domain of nNOS that contains a PDZ motif. Muscles of muscular dystrophy patients show selective loss of nNOS protein and loss of catalytic activity from muscle membrane.
  • nNOS is concentrated at synaptic junctions at motor endplates of the skeletal muscle where the N-terminus domain of nNOS, which contains a PDZ protein motif, binds to the PDZ motif present in ⁇ l-syntrophin.
  • the PDZ domain thus mediates binding of nNOS to skeletal muscle syntrophin, a dystrophin associated protein.
  • the invention also describes a method of use of nitric oxide, neuronal nitric oxide synthase, its binding proteins and their inhibitors for diagnosis and treatment of muscular dystrophy.
  • a diagnostic assay for detection of absence of dystrophin or its mutated form and a method for treatment of muscular dystrophy by restoration of a functional dystrophin molecule in dystrophic muscles using gene therapy are also disclosed. Absence or Deficiency in Formation of Dvstrophin-nNOS Complex in Muscular Dystrophy
  • Muscular dystrophies have been characterized by the complete absence of dystrophin in Duchenne dystrophy or by truncated dystrophin in Becker muscular dystrophy. It has now been discovered that additionally, these diseases also lack the normal level of neuronal nitric oxide synthase.
  • Nitric oxide (NO) is synthesized in skeletal muscle by neuronal-type nitric oxide synthase (nNOS) , which is localized to sarcolemma of fast-twitch fibers. Synthesis of NO in active muscle opposes contractile force. It has now been shown and described in studies below that nNOS partitions with skeletal muscle membranes owing to association of nNOS with dystrophin, the protein missing in Duchenne muscular dystrophy (DMD) . In healthy muscle, the dystrophin complex interacts with the N-terminal domain of nNOS that contains a PDZ motif. In the muscular dystrophy
  • mice model and in human DMD skeletal muscle samples, a selective loss of nNOS protein as well as loss of catalytic activity from muscle membranes was found, demonstrating a novel role for dystrophin in localizing a signaling enzyme to the myocyte sarcolemma. Aberrant regulation of nNOS production is therefore suspected to contribute to preferential degeneration of fast-twitch muscle fibers in DMD.
  • Neuronal NOS is present in cytoskeletal extracts from a healthy skeletal muscle and have been found to be associated with membrane.
  • Figure 1 illustrates differential extractability of nNOS and eNOS in mice skeletal muscle homogenates.
  • tissue was prepared, extracted and submitted to Western blot analysis as described in Example 1. After subcellular fractionation and 7.5% SDS-PAGE (100 ⁇ g protein per lane) , nNOS and eNOS were sequentially detected by protein immunoblot. Positions of molecular size markers are indicated in kilodaltons.
  • Figure IA is a Western blot showing an association on nNOS of an insoluble pellet (P) , as well as mouse quadriceps homogenates sequentially extracted as indicated.
  • Western blotting indicates that significant nNOS remains in an insoluble pellet (P) following sequential extraction of mouse quadriceps homogenates with 100 mM NaCl (S,) , 500 mM NaCl (S 2 ) , and 0.5% Triton X-100 (S 3 ) , lower levels of nNOS are present in each of these fractions.
  • Figure IB is a Western blot showing association of eNOS with particulate fractions. As seen in Figure IB, eNOS is found only in membrane-associated fractions but is not present in cytosol and in the insoluble pellet. Probing the same blot with an eNOS monoclonal antibody indicates the eNOS is completely extracted by 500 mM NaCl (S 2 ) and 0.5% Triton X-100 (S 3 ) .
  • the amino acid sequence of nNOS contains a 230 amino acid N-terminal domain that is not present in eNOS. Beyond this extended N-terminus of nNOS, the two proteins share >60% sequence identity. Similar enzymatic activities of eNOS and nNOS suggest that the unique N-terminus of nNOS is not required for catalytic activity and has another function.
  • FIG. 2B shows a schematic alignment of the PDZ domain of nNOS with syntrophins and a family of other cytoskeletal-associated proteins.
  • T-SYN and SYN-1 indicate syntrophins
  • DLG indicates disks large
  • DSH indicates disheveled
  • PTP indicates protein-tyrosine phosphatase.
  • nNOS nNOS nNOS -terminal domain
  • nNOS contains a 66 amino acid motif that bears homology to a heterogeneous family of signaling enzymes that share the property of being localized to specialized cell-cell junctions as seen in Figure 2B. Proteins containing this motif, which is named PDZ for a con ⁇ erved tetrapeptide (Neuron.
  • dlg-l the product of the lethal discs large tumor suppressor gene that localizes to the undercoat of the septate junction in Drosophila; disheveled, a gene required for planar cell polarity in Drosophila; PSD-95, a brain-specific protein; ZO-1, a protein that localizes to tight junctions (zona occludens) of epithelial and endothelial cells; and certain protein- tyrosine phosphatases such as PTPIE, which are localized at the junction between the plasma membrane and the cytoskeleton. Homology to syntrophins, a family of recently cloned dystrophin-binding proteins, which colocalize with nNOS beneath the sarcolemmal membrane of skeletal muscle, was observed.
  • nNOS ⁇ l-226, lacking the first 226 amino acids was constructed.
  • Expre ⁇ ion vector ⁇ containing full-length nNOS and nNOS ⁇ l-226 were transiently transfected into COS cells.
  • COS cells were transfected with 10 ⁇ g of the expression vector using a cytomegalovirus promoter to drive expression of either full-length nNOS (Fig. 2C) or the truncation mutant nNOS ⁇ l-226 (Fig. 2D) .
  • NOS activity was mea ⁇ ured in cell homogenates 3 days following transfection in the presence of either 200 ⁇ M free calcium (full squares) or 2 mM EDTA (full circles) .
  • Kinetic constants V ⁇ and K. were calculated by Scatchard plot analysi ⁇ . Data are means of triplicate determinations that varied by ⁇ 10%. This experiment was replicated twice with similar result ⁇ .
  • a dystrophin-nNOS a ⁇ sociation complex was investigated by means of succinylated wheat germ agglutinin ( ⁇ WGA) using ⁇ WGA-Sepharo ⁇ e affinity chromatography. Thi ⁇ technique allow ⁇ distinction between nNOS which, due to its lack of glycosylation sites does not bind to wheat germ column, while dystrophin binds to a glycoprotein complex.
  • Figure 3A shows dystrophin-as ⁇ ociated glycoprotein complex and nNOS purified by ⁇ WGA chromatography from wild- type (WT) , and mdx skeletal muscle.
  • Dystrophin has no nucleotide-binding ⁇ ite, and would not be expected to adhere to a 2',5'-ADP column.
  • parallel purifications from skeletal muscle of nNOS knockout mice, which are devoid of full length nNOS protein yet express dy ⁇ trophin at normal levels were conducted.
  • Salt-washed heavy microsomes from wild-type and full length nNOS knockout mouse quadriceps were solubilized in 1% digitonin and allowed to adhere to 2' ,5'-ADP-agarose columns. The columns were extensively washed with buffers containing 0.5 M NaCl and 0.5% Triton X-100. Tightly bound proteins were eluted with buffers containing 20 mM NADPH. Results are seen in Figure 3B.
  • FIG. 3B shows dystrophin-associated glycoprotein complex and nNOS purified by 2' ,5'-ADP-agaro ⁇ e chromatography from wild-type (WT) , mdx, and NOS knockout (NOS 1 ) skeletal muscle.
  • WT wild-type
  • NOS 1 NOS knockout
  • Western blotting for dystrophin reveals that dystrophin level ⁇ are equivalent in crude ⁇ ample ⁇ from wild-type (WT) and nNOS knockout (NOS 1 ) skeletal muscle.
  • Dystrophin coelutes with NOS on a 2',5'-ADP affinity column in muscle homogenate ⁇ from wild-type but not for nNOS knockout mice.
  • nNOS N-terminal domain of nNOS
  • GST-nNOS(1-299) beads and control GST beads were incubated with ⁇ olubilized homogenate ⁇ of mouse skeletal muscle. After extensive washing of the beads, bound proteins were eluted with sample buffer. Results are seen in Figure 3C.
  • GST-nNOS(1-299) beads bound to GST or GST-nNOS(1-299) were incubated with solubilized skeletal muscle membranes.
  • Figure 3D depicts Western blotting for nNOS from equally loaded fractions (5 ⁇ g per lane) from sWGA chromatography ⁇ how ⁇ large enrichment of nNOS in NAG eluate fractions.
  • Figure 3F shows an immunoprecipitation of NAG eluate fractions with a monoclonal antibody to dystrophin (2.0 / xg/ml; 12 nM) which precipitates nNOS (lane 1) .
  • Western blot analysi ⁇ seen in Figure 3F revealed potent immunoprecipitation of nNOS with ⁇ -dystrophin antibody. Control immunoprecipitations lacking the primary dystrophin antibody or containing an alternate monoclonal antibody anti-Myc did not precipitate detectable nNOS, demon ⁇ trating ⁇ pecificity of the interaction.
  • nNOS coelutes with dystrophin on a ⁇ WGA or a 2',5'-ADP affinity column in healthy muscle homogenates but not in extracts from mdx or knockout mice and that dystrophin binds to the N-terminal domain of nNOS.
  • Neuronal NOS from equally loaded fractions from sWGA chromatography shows large enrichment of nNOS in NAG eluate fractions and nNOS and dystrophin, but not eNOS, were purified by sWGA. This show ⁇ that a large fraction of nNOS actually binds to the dystrophin complex.
  • Figure 4 illustrates nNOS displacement from particulate fractions of the mdx skeletal muscle.
  • Figure 4B shows that dystrophin is enriched in detergent extract (S 3 ) and cytoskeletal pellet (P) fractions in wild-type mice (WT) and is completely absent from the mdx muscle.
  • nNOS levels and enzyme activity were modestly decreased (-80% of control levels) in skeletal muscle from mdx mice.
  • Subcellular analysis revealed that nNOS distributed with dystrophin in membrane-associated and cytoskeletal fractions from wild- type skeletal muscle.
  • nNOS was quantitatively solubilized from microsomal membranes washed with 0.5 M NaCl. No nNOS protein was detected in detergent extract or cytoskeletal fractions ( Figure 4A) .
  • Data are means of triplicate determinations that varied by ⁇ 10%. "In counts per minute per milligram of protein.
  • NOS catalytic activity wa ⁇ ob ⁇ erved in ⁇ oluble and particulate fraction ⁇ in ⁇ keletal u ⁇ cle and in brain in WT mouse tissue.
  • nNOS was found in the soluble but not in the particulate fraction.
  • NOS activity in the soluble fraction of mdx skeletal muscle occurred at levels 75% greater than wild- type, but NOS activity was not detectable in the particulate fraction from mdx muscle.
  • NOS- specific activity was nearly equivalent in soluble and particulate fractions.
  • Thi ⁇ distribution was unchanged in the mdx brain, suggesting that proteins other than dystrophin anchor nNOS to neuronal membranes.
  • Figure 5 shows immunofluorescent ⁇ taining for nNOS in quadricep ⁇ of wild-type, mdx, dy, and nNOS knockout mice performed u ⁇ ing an affinity-purified polyclonal antibody.
  • Cryostat mice sections from wild-type, mdx, dy, and nNOS knockout mouse were stained under identical conditions using an affinity-purified nNOS antiserum and a FITC-linked ⁇ econdary antibody.
  • Figures 5A and 5B show that nNOS immunostaining is present at the surface membranes of skeletal muscle fibers from wild-type (WT) mouse (A) , but is absent from mdx mouse (B) skeletal muscle sarcolemma.
  • WT wild-type
  • B mdx mouse
  • nNOS immunostaining is present at the surface membranes of skeletal muscle fibers from wild-type (WT) mouse (A) , but is absent from mdx mouse (B) skeletal muscle sarcolemma.
  • Figure 5C shows nNOS distribution in homozygous dystrophic dy mice displaying normal sarcolemmal nNOS labeling of intact fibers. Results show that nNOS is present normally at the sarcolemma of dy dystrophic mice ( Figure 5C) .
  • Figure 5D shows that skeletal muscle from NOS knockout (NOS" 1 ”) mice is entirely devoid of immunostaining.
  • NOS NOS knockout
  • nNOS immunofluorescence found in Figure 5 was restricted to the sarcolemma of a subset of WT skeletal muscle fibers. These fibers were previously noted to be fast twitch fibers (Nature. 372: 546-548 (1994)). However, nNOS immunoreactivity was absent from the sarcolemma of mdx muscle.
  • Figure 6 are skeletal muscle cryosection ⁇ of normal (NI) or DMD (DI) skeletal muscles immunostained with antibodies to dystrophin, nNOS, and spectrin.
  • NI normal
  • DI DMD
  • Figure 6B show ⁇ repre ⁇ entative mu ⁇ cle ⁇ ection ⁇ , labeled with antibodies, from two normal patients (N2 and
  • Figure 6C which represents control experiments using two independently generated nNOS anti ⁇ era ⁇ hows similar staining of human tissue ⁇ . No immunofluorescence was detected in the absence of primary (1°) antibody.
  • Results seen in Figure 6 shows that nNOS is absent from sarcolemma of human DMD muscle fibers. Absence of dystrophin in DMD results in disruption of the dystrophin-associated glycoprotein complex and in a dramatic reduction of overall levels of certain dy ⁇ trophin- associated proteins in mu ⁇ cle.
  • Western blot analysis was conducted. Results are seen in Figure 7.
  • skeletal muscle tis ⁇ ue homogenates from three cases of DMD and three normal muscle biopsie ⁇ were resolved by SDS-PAGE as described in Example 1.
  • Immunoblot analysis seen in Figure 7A confirms that dystrophin is present in normal human muscle but is essentially absent from the human DMD muscle. Densitometric scanning of nNOS immunoreactive bands in equally loaded Western blots revealed -75% decrease of nNOS in DMD tissues when compared to the normal human muscle. Immunoblotting for spectrin seen in Figure 7C confirmed that similar amounts of protein were loaded in all cases and that the structural cytoskeleton of these samples remained intact.
  • nNOS nNOS-containing human and mouse skeletal muscle sarcolemmal nNOS
  • the dystrophin complex interacts with the N-terminus of nNOS, which contains a PDZ motif.
  • nNOS is pre ⁇ ent in sarcolemma.
  • nNOS i ⁇ ab ⁇ ent from the sarcolemma and accumulate ⁇ in the cyto ⁇ ol. This derangement of nNOS i ⁇ specific for dystrophin abnormalities, as nNOS disposition is unaffected in other muscular diseases.
  • the obtained results provide molecular evidence for a specific intracellular signaling molecule linked to the dy ⁇ trophin-a ⁇ sociated complex and suggest roles for NO in processes of neuro-muscular development and disea ⁇ e associated with thi ⁇ complex.
  • the PDZ domain is a protein motif that is present in a heterogeneous family of enzymes.
  • the current invention investigated and discovered that deletion of the PDZ domain of nNOS does not alter NOS catalytic activity in transfected cell ⁇ .
  • a 299 amino acid fu ⁇ ion protein containing the PDZ domain in nNOS selectively retained dy ⁇ trophin from skeletal muscle extracts, indicating that this domain is capable of interacting with the dystrophin- associated complex.
  • nNOS neuromuscular signaling and disease associated with dystrophin.
  • As ⁇ ociation of nNOS with dy ⁇ trophin completes the link between the extracellular matrix and intracellular signal-transducing enzymes.
  • nNOS has been unque ⁇ tionably implicated in the DMD where the dy ⁇ trophin i ⁇ ab ⁇ ent, and nNOS was ⁇ hown to be di ⁇ placed, the ⁇ econd ⁇ eries of studies was designed to investigate whether these findings would also be valid for other, not so severe, types of muscular dystrophies where dystrophin is not completely absent but is mutated and to a certain degree dysfunctional.
  • nNOS occurs normally at the sarcolemma in human neurogenic muscle atrophy, central core disease, and severe childhood autosomal recessive muscular dystrophy but that it is, however, displaced from ⁇ arcolemma of Becker mu ⁇ cular dystrophy ⁇ ugge ⁇ ting ⁇ pecificity of the defect of nNOS in DMD and BMD.
  • Becker muscular dystrophy is a clinical variant of Duchenne muscular dystrophy. It differs from the more severe Duchenne dy ⁇ trophy in that Becker mu ⁇ cular dy ⁇ trophy patients do not lack dystrophin completely but their dystrophin is of abnormal molecular weight due to chronic mutation at Xp21. Becker muscular dystrophy patients, therefore, have reduced amounts of normal-sized dystrophin protein.
  • Becker muscular dy ⁇ trophy is an X-linked disease due to mutations of the dystrophin gene. Mutations causing Becker's dystrophy are often in-frame deletions in the central rod-like domain of dystrophin that do not generally affect formation of the structural glycoprotein complex formed around dy ⁇ trophin in the mu ⁇ cle.
  • nNOS neuronal-type nitric oxide synthase
  • nNOS does not as ⁇ ociate with ⁇ l- ⁇ yntrophin on the sarcolemma in certain human Becker's dystrophy patients and in transgenic mdx mice expressing truncated dystrophin proteins. This suggests a macromolecular interaction of nNOS, ⁇ l- syntrophin and dystrophin in vivo , a conclusion supported by developmental ⁇ tudie ⁇ in mu ⁇ cle.
  • the data below indicate that proper a ⁇ embly of the dy ⁇ trophin complex i ⁇ dependent upon the structure of the central rod-like domain and have implications for the design of dystrophin- containing vectors for gene therapy.
  • nNOS occurred only at extrajunctional sarcolemma and enrichment of nNOS at neuromuscular endplates did not become apparent until P12, which coincided with as ⁇ embly of dystrophin complexes at endplates.
  • mice models carrying either the full length dystrophin or truncated dystrophin were investigated.
  • nNOS dystrophin as ⁇ ociated proteins
  • ⁇ -BGT dystrophin as ⁇ ociated proteins
  • ⁇ l-syntrophin expression were compared in skeletal muscle of wild type, mdx and various transgenic mice that express mutant forms of dystrophin, such as full-dys, ⁇ 330, mdx ⁇ EXON 17-48, ini-dys or Dp71 mutant lines.
  • nNOS in wild type mice was expressed at extrajunctional sarcolemma of a subset of fibers and was enriched at all neuromuscular endplates.
  • nNOS was absent from junctional and extrajunctional sarcolemma in mdx mice.
  • nNOS ⁇ taining in mdx transgenic mice expres ⁇ ing full length dy ⁇ trophin (full-dys) or truncated dystrophin lacking the C-terminal 330 nucleotides ( ⁇ 330) , resembled that of wild type mice.
  • mdx mice expressing dystrophin lacking exons 17-48 (mini-dys) or lacking the C-terminal 71 kDa of dystrophin did not show nNOS staining at sarcolemma. These results corresponded to results observed in non-transgenic mdx mice. ⁇ l-Syntrophin staining was observed at extra- junctional sarcolemma and was concentrated at neuromuscular endplates in wild type mice but was restricted to the endplates in mdx mouse. ⁇ l-Syntrophin expression was restored to sarcolemma in the four transgenic mdx mouse lines expressing different portions of the dystrophin gene.
  • Figure 10 shows subcellular distribution of nNOS in WT, mdx, and transgenic mdx mice.
  • Mouse quadriceps skeletal muscle homogenates were sequentially extracted with buffers containing 100 mM NaCl (SI) , 500 mM NaCl (S2) , and 0.5% Triton X-100 (S3), leaving an insoluble cytoskeletal pellet (P) .
  • SI 100 mM NaCl
  • S2 500 mM NaCl
  • S3 0.5% Triton X-100
  • Figure 10A is Western blotting indicating that nNOS was enriched in membrane as ⁇ ociated and in pellet fractions in wild type mice (lanes 1) and in transgenic mdx mice expressing full length dystrophin (lanes 4) .
  • nNOS was fully extracted by 500 mM NaCl and was absent from the membrane associated and cytoskeletal fractions.
  • nNOS did not as ⁇ ociate with ⁇ arcolemma in mdx mice or tran ⁇ genic mdx mice expre ⁇ ing either Dp71 or ⁇ E17-48.
  • nNOS was enriched in membrane as ⁇ ociated and cyto ⁇ keletal fraction ⁇ , while in mdx, Dp71 and ⁇ E17-48 lines, nNOS was present only in soluble fractions of muscle, ⁇ l- ⁇ yntrophin occurred in sarcolemmal fractions of all four lines of transgenic mdx mice evaluated.
  • nNOS connections between nNOS and the presence of normal length or truncated dystrophin have been clearly establi ⁇ hed by these ⁇ tudies.
  • dystrophin was absent from the transgenic mouse phenotype, nNOS presence was only observed in soluble but not in sarcolemmal fractions.
  • Figure 11A illu ⁇ trate ⁇ ⁇ elective interaction of nNOS and ⁇ l-syntrophin using Western blotting.
  • Crude solubilized membranes from mouse quadriceps were titrated with NaOH to pH 11, to dissociate the dystrophin complex, and were neutralized to pH 7.4 with 1 M TrisHCl.
  • Native (native) and dissociated (dissoc) preparation ⁇ were incubated with agarose beads linked to either GST or GST fused to the first 299 amino acids of nNOS (G-NOS) . After extensive washing, beads were eluted with loading buffer and proteins resolved by SDS/PAGE.
  • FIG 11A shows that ⁇ l-syntrophin was selectively retained by G-NOS beads in both native and dis ⁇ ociated preparations.
  • neither dystrophin nor ⁇ - ⁇ arcoglycan bound to G-NOS neither dystrophin nor ⁇ - ⁇ arcoglycan bound to G-NOS.
  • the 55 kD band ob ⁇ erved in input lanes from ⁇ - ⁇ arcoglycan blot appear ⁇ to be mouse IgG and was reactive with the secondary antibody used for western blotting.
  • Yeast HF7c and Y187 cells were cotransfor ed with expression vectors encoding various GAL4-binding domain and GAL4 activation domain fusion proteins.
  • Each transformation mixture was plated on two synthetic dextrose plates, one lacking tryptophan and leucine and the other lacking tryptophan, leucine and histidine. Growth was , measured on histidine-deficient plates and color was measured by a 0-galactosidase colorimetric filter assay according to Nature. 340: 245-6 (1989) .
  • Figure 21 illustrates that ⁇ l-syntrophin binds to the N-terminal PDZ containing domain of nNOS.
  • Figure 21A shows result ⁇ of "pull down" assays of solubilized mu ⁇ cle extract ⁇ from wild type or mdx mice using an nNOS (amino acids 1-299)-GST fu ⁇ ion protein which were done a ⁇ described in Figure 19.
  • Western blotting shows that ⁇ l-syntrophin from both wild type and mdx mice is selectively retained by the G-NOS column. Input was 20% protein.
  • Figures 2IB and 21C show immunoprecipitations of solubilized muscle extracts with a polyclonal antibody to ⁇ l-syntrophin which show co-precipitation of (Figure 2IB, lane 2) nNOS but not eNOS ( Figure 21C, lane 2) .
  • Control experiments with non-immune serum show no precipitation of nNOS or eNOS (lanes 1) .
  • Bands at 55 kD represent immunoglobulin heavy chains.
  • Figures 2ID and 2IE are subcellular fractionation of nNOS which is altered in nNOS*'' mouse mu ⁇ cle.
  • Dystrophin is retained by a G-NOS column.
  • the absence of dystrophin in mdx mice results in disruption of the dystrophin glycoprotein complex. Therefore, association of skeletal muscle syntrophin from mdx mouse with G-NOS was evaluated. Total ⁇ l-syntrophin levels were decreased -50% in mdx muscle. Binding of ⁇ l-syntrophin to G-NOS was unaffected by the dystrophin deficiency (Figure 21A; lanes 2,5,6).
  • Skeletal muscle homogenates were sequentially extracted with physiologic saline, the 500 nM NaCl, and finally 0.5% triton X-100. Following 2'5' ADP agarose purification of the muscle extracts, We ⁇ tern blotting indicated that only the nNOS ⁇ form is expre ⁇ sed in muscle of nNOS ⁇ / ⁇ . nNOS in skeletal muscle runs as a doublet due to a 102 bp (34 amino acid) alternative splice near the middle of the gene.
  • Figure 22 shows the interaction between the nNOS-GST (1-299) fusion protein and the four ⁇ l-syntrophin domain fusion proteins, PHI, PDZ, PH2 and SU. Notably, only PDZ associates with nNOS. Moreover, no prominent bands were detected when the domains were overlayed with GST alone. Syntrophin domain fusion proteins (containing the T7 «Tag epitope) were also used to overlay GST and nNOS-GST ( Figure 22) . Only the PDZ domain of ⁇ l-syntrophin bound to nNOS and no binding was observed to GST alone.
  • Figure 22A is purified ⁇ l-syntrophin PHI (25kDa, lanes 1), PDZ domain (15kDa; lanes 2), PH2 (18 kDa; lanes 3) and SU domain (16 kDa; lanes 4) fusion protein ⁇ were resolved and overlayed with GST or nNOS (1-299)-GST.
  • the position and relative amounts of the ⁇ l-syntrophin domain fu ⁇ ion proteins are indicated by immunoreactivity with a monoclonal antibody against the T7*Tag. Bound GST fusion proteins were detected by blotting with a monoclonal antibody to GST. Only nNOS-GST bound specifically to the PDZ domain of ⁇ l-syntrophin.
  • FIG 22B GST (lanes l) and nNOS-GST (lanes 2) were separated and overlayed with ⁇ l-syntrophin domain fusion proteins (PHI, PDZ, or PH2) or blotted with a monoclonal antibody to GST. Bound syntrophin fusion proteins were detected with monoclonal antibody to T7*Tag. Of the syntrophin fu ⁇ ion protein ⁇ te ⁇ ted, only PDZ bound to nNOS; no binding to GST wa ⁇ detected.
  • PKI ⁇ l-syntrophin domain fusion proteins
  • Figure 22C ⁇ how ⁇ co-localization of nNOS and ⁇ l- syntrophin immunofluorescence at skeletal muscle sarcolemma and neuromuscular junctions which was labeled by rhoda ine ⁇ -bungarotoxin (BGT) .
  • Figure 22D is a schematic model showing interaction of nNOS via with skeletal muscle ⁇ l-syntrophin (59K syn) connected to dystrophin dimer. The interaction of nNOS with syntrophin is via their respective PDZ domains. DG indicates dystroglycan.
  • Figure 22 shows the interaction between the nNOS-GST (1-299) fusion protein and the four ⁇ l-syntrophin domain fu ⁇ ion proteins, PHI, PDZ, PH2 and SU. Notably, only PDZ associates with nNOS. Moreover, no prominent bands were detected when the domains were overlayed with GST alone. Syntrophin domain fusion proteins (containing the T7*Tag epitope) were also used to overlay GST and nNOS-GST ( Figure 22) . Again, only the PDZ domain of ⁇ l-syntrophin bound to nNOS and no binding was observed to GST alone. (1989) . Sarcolemmal nNOS Expres ⁇ ion in Becker' ⁇ Dv ⁇ trophv
  • nNOS and ⁇ l-syntrophin expression in 12 BMD patients with molecularly defined deletions in the dystrophin gene were immunohi ⁇ tochemically evaluated. Immunohi ⁇ tochemical expression of nNOS, dystrophin and syntrophin was asses ⁇ ed blindly. Results are seen in Figure 12.
  • Figure 12 shows skeletal muscle cryosections from human biopsie ⁇ from normal patients or from patients having DMD
  • BMD ⁇ EXON 45-47, BMD ⁇ EXON 10-42, and ⁇ -sarcoglycan disturbances were immunostained with antibodies to dystrophin, syntrophin, ⁇ -sarcoglycan, or nNOS. All four antibodies showed sarcolemmal staining in normal patients and essentially no sarcolemmal labeling in patient ⁇ with
  • DMD Duchenne muscular dystrophy
  • nNOS is absent from skeletal mu ⁇ cle ⁇ arcolemma in certain patients with Becker muscular dy ⁇ trophy.
  • Mild BMD (S90-14162) 45-47 0 ++++ ++++ Mild BMD (1987) 52 +++ ++++ ++++ Mild BMD (KF22) 45-48 + ++++ ++++++
  • Sev. BMD (CS9004625) 8 0 + +++ Sev. BMD (S88-2698) 3-7 0 + +++ Sev. BMD (S90-14163) 45-47 0 +++ ++++ Sev. BMD (S90-107002) 51-52 0 + +++ ⁇ -sarcoglycanopathy 1 ++++ ++++ ++++ ⁇ -sarcoglycanopathy 2 - +++ +++ ++++++++
  • Table 3 shows that loss of sarcolemmal nNOS, but not ⁇ l- syntrophin expression was highly correlated with disease phenotype. Some of the patients, with mild to intermediate disease, showed reduced but detectable nNOS staining of sarcolemma. In several patients, los ⁇ of sarcolemmal nNOS occurred despite apparently normal as ⁇ embly of other components of the dystrophin-a ⁇ sociated glycoprotein complex seen in Figure 12. By contrast, nNOS expression was intact in two patients with ⁇ -sarcoglycan deficiency, ⁇ ugge ⁇ ting that abnormalities of nNOS are not a consequence of muscular dystrophy, but are specific for dystrophin-linked disea ⁇ e.
  • nNOS expression in BMD patients demonstrate that non-overlapping deletions in the N-terminal or central domain of dystrophin disrupt recruitment of nNOS to the sarcolemma. These results indicate that a unique nNOS interaction domain may not be present in dystrophin, but that proper conformation is required for assembly of nNOS into the dystrophin complex.
  • utrophin complexes at neuromuscular endplates of mdx mice specifically lack nNOS.
  • biochemical studie ⁇ showing a selective and direct interaction of nNOS with ⁇ l-syntrophin in vitro
  • sarcolemmal localization of nNOS seems to require a presence of both syntrophin and dystrophin.
  • nNOS expression is specific for dy ⁇ trophin-related di ⁇ eases. Immunohistochemical analysis for nNOS, therefore, is able to provide a reliable diagnostic test for detection of these disea ⁇ e ⁇ .
  • nNOS nNOS-a ⁇ ociated protein absent from the sarcolemma.
  • loss of sarcolemmal nNOS expression broadly correlates with the severity of the disease.
  • a primary goal of muscular dy ⁇ trophy therapy is restoration of fully functional dystrophin.
  • the therapy therefore, involves either the replacement of full length dystrophin or replacement of a fragment of dystrophin which assures binding of dystrophin with nNOS through syntrophin.
  • the muscular dystrophy therapy thus involves replacement of dysfunctional or missing dystrophin with functional dystrophin or a functional fragment thereof.
  • dystrophin is missing in DMD and is dysfunctional in BMD, so far such therapy has not been successive ⁇ ful.
  • di ⁇ covery according to the invention that in the normal nondy ⁇ trophic skeletal muscle dystrophin is colocalized with nNOS which binds to a PdZ motif of syntrophin, a dystrophin associated protein, and that in dystrophic muscle ⁇ not only dystrophin but al ⁇ o nNOS is missing from sarcolemma of the skeletal muscle, it is clear that it is not necessary to replace the whole dystrophin but only the dystrophin fragments which are involved in formation of nNOS/sarcolemma/dy ⁇ trophin complex. Con ⁇ equently, the dy ⁇ trophin fragments binding to syntrophin which in turn binds to nNOS, as seen in Figure 22D, in sarcolemma suffice for treatment of muscular dys
  • vectors used for production of proteins useful for treatment of muscular dystrophy to be u ⁇ ed for gene therapy need to encode truncation mutants of dystrophin, that provide mutated gene replacement with dystrophin constructs that properly assemble nNOS which complete rescue of muscle function requires.
  • dystrophin constructs Prior to their use in gene therapy, dystrophin constructs are analyzed to ensure that the y recruit nNOS to sarcolemma.
  • Diagnosis of muscular dystrophy is based on detection of nNOS using immunohistochemical detection of nNOS, histologic analysis of nNOS or a combination of both.
  • NADPH diaphorase staining method which is fast, easy and practical for routine use, is most preferred.
  • PSD-93 proteins bind to the PDZ domain of nNOS in the brain is also novel and was never before disclosed or described.
  • Neuronal NOS is concentrated at synaptic junctions in the brain where the N-terminus domain of nNOS, which contains a PDZ protein motif, interacts both in vivo and in vitro with the second PDZ motif present in postsynaptic density-95 or -93 proteins.
  • the second PDZ domain mediates binding of nNOS to the N-methyl-D-asparagine (NMDA) receptor located at the synapse through the first and/or the third PDZ domains of the PSD-95 or PSD-93.
  • NMDA N-methyl-D-asparagine
  • NMDA N- methyl-D-aspartate
  • the invention also describes a method of use of nitric oxide, neuronal nitric oxide syntha ⁇ e, it ⁇ binding protein ⁇ and their inhibitor ⁇ , for diagnosi ⁇ , prophylaxis and treatment of stroke and other neurodegenerative diseases, such as Huntington disease, amyotropic lateral sclerosis, Alzheimer disease, etc. as well as a diagnostic as ⁇ ay for detection of ab ⁇ ence of binding protein ⁇ or nNOS.
  • the invention concerns a binding assay for monitoring of binding of nNOS binding proteins with nNOS and appropriate synaptic receptors useful for development of compounds for treatment of neurodegenerative diseases.
  • Nitric oxide plays important physiological role in neurotransmission and synaptic modulation in central nervous tissue. Endogenous neuronal NO participates in development of some forms of neurotoxic injury, including stroke and other neurodegenerative processes. Functionally, NO mediates certain aspects of synaptic plasticity and neurotoxicity associated with NMDA receptors, but it does not play a major role in other pathways.
  • nNOS activity is selectively activated by a calcium influx through the NMDA receptor. Both nNOS and NMDA receptors are concentrated at synaptic junctions in the brain. Consequently, understanding of NO neurotoxicity requires identification of the functional connection of nitric oxide synthetic enzyme nNOS with NMDA receptors. For interacting connection of nNOS with NMDA receptor, a linker able to bind these two entities together is necessary. Two proteins, PSD-95 and PSD-93, have been identified as pos ⁇ ible binding linkers.
  • nNOS postsynaptic density-95 protein
  • PSD-95 postsynaptic density-95 protein
  • Figure 13 is a molecular model of nNOS/N-methyl-D-aspartate receptor (NMDAR) bindingmediated by PSD-95 or PSD-93 proteins. These proteins are known to associate with the glutamate type receptors to which NMDA receptor belongs.
  • nNOS neuronal NO synthase
  • PSD-95 and PSD-93 are physically able to associate with nNOS through their respective PDZ domains. This shows that NMDA and nNOS are able interact with nearby binding sites in the second PDZ domain of PSD-95.
  • Nitric Oxide Synthase Binding Proteins In the brain nNOS is thus functionally coupled to N- methyl-D-a ⁇ partate receptors.
  • the N-terminal domain of nNOS is unique to the neuronal isoform and contains a PDZ motif of about 100 amino acids that is found in a diverse group of cytoskeletal protein ⁇ and enzymes. Because this domain was shown to mediate as ⁇ ociation of nNOS with the dystrophin complex, a ⁇ described in section I, attempts were made to identify interacting proteins in the brain to perform the same function.
  • nNOS is enriched at synaptic junctions in the brain owing to association of nNOS with the postsynaptic density proteins, specifically with the two proteins identified as PSD-95 and PSD-93.
  • PSD-95 protein clusters NMDA receptors at central nervous system synapses.
  • PSD-95 and PSD-93 proteins therefore act as interacting proteins between nNOS and NMDA receptors.
  • PSD-95 Postsynaptic den ⁇ ity protein PSD-95 was originally identified as an abundant detergent-insoluble component of brain postsynaptic density. Subcellular and electron micrographic studie ⁇ have determined that PSD-95 is localized at both pre- and post-synaptic membrane and has a similar distribution to nNOS. PSD-95 contains three PDZ repeat' , a SH3 domain and a region homologous to guanylate ki .se (Neuron. 9: 929-942 (1992)). As seen in Figure 13, the second domain of PSD-95 provides the connecting link between nNOS and NMDA receptor.
  • PSD-95 and PSD-93 proteins Schematic representation of PSD-95 and PSD-93 proteins is seen in Figure 14.
  • Figure 14 cloning and sequencing of the PSD-95 related gene derived a protein PSD-93 of 93 kD, that has the same domain structure as PSD-95 and shares with PSD-95 about 60% amino acid identity.
  • the nucleotide sequence of PSD-93 (SEQ ID NO: 1:) has been deposited in GenBank.
  • the PSD-93 nucleic acid sequence contains 2963 base pairs.
  • the amino acid ⁇ equence of PSD-93 are depicted by SEQ ID NO ⁇ : 2-6 containing cumulatively 987 amino acid ⁇ .
  • the amino acid sequence of PSD-95 is seen in Figure 14 and is identified as SEQ ID NOs: 15-19.
  • This invention thus identifies for the first time two brain proteins that physically associate with the enzyme neuronal nitric oxide synthase (nNOS) .
  • nNOS neuronal nitric oxide synthase
  • PSD-95 postsynaptic density-95
  • PSD-93 nNOS binding protein
  • the yeast two-hybrid system was used to identify interacting proteins. Screening a brain library demonstrated that the PDZ containing domain of nNOS binds to PDZ repeats present in PSD-95 and in a novel related protein, PSD-93. PSD-95 was found to be co-expressed with nNOS in several neuronal populations in the developing and mature nervous system, and a specific PSD-95/nNOS interaction was detected in transfected cell lines and solubilized cerebellar membranes. On the other hand, residual catalytically active nNOS isoforms identified in nNOS ⁇ / ⁇ mice, that specifically lack a PDZ motif, did not interact with PSD-95.
  • Figure 15 shows expression of PSD-93, PSD-95 and nNOS in rat brain and in E15 embryo. In situ hybridization was used to localize transcripts for PSD-93 (in ⁇ et A), PSD-95 (in ⁇ et B) , nNOS (inset C) or sense control (inset D) in adjacent cryosection ⁇ prepared according to Example 5.
  • Figure 15A shows ⁇ that in an adult rat brain, PSD-95 was observed only in neurons and was co-expressed with nNOS in certain neurons in hypothalamus, hippocampus and cerebellum.
  • PSD-93 also appeared to be neuron specific, but had a more restricted distribution than did PSD-95.
  • Figure 15B shows that in the cerebellum, PSD-95 and nNOS were co-expressed in cerebellar granule cells in the granular layer (G) and basket cells (B) in the molecular layer.
  • PSD-93 was restricted to Purkinje neurons (P) of the cerebellum, which lack nNOS or PSD-95.
  • Double labeling with NADPH diaphorase and in situ hybridization identified the PSD- 95 and nNOS expressing cells in molecular layer as basket cells.
  • FIG. 15C show ⁇ that in E15 embryo, PSD-95 wa ⁇ found ubiquitously expressed in differentiated central neurons, but not in neuronal precursors.
  • PSD-95 was co-expressed with nNOS in the cerebral cortical plate (CP) , dor ⁇ al root ganglia (DRG) and neurons of the olfactory epithelium (OE) .
  • PSD-93 was specifically co-expressed in neurons of the spinal cord (SC) , DRG and trigeminal nerve (V) .
  • PSD-93 was specifically co- expressed with nNOS in secretory cells of the submandibular gland (SG) and in (Figure 15D) chromaffin cells of the developing adrenal gland, which lack PSD-95.
  • K identifies the kidney.
  • A identifies the adrenal gland.
  • nNOS-containing cells in embryonic day 15 were differentially co-expressed with either PSD-95 or PSD-93 ( Figures 15C and 15D) .
  • transient NOS neurons were detected in developing cerebral cortical plate, olfactory epithelium, and sensory ganglia.
  • PSD-95 mRNA was found.
  • PSD-93 mRNA and nNOS mRNA were co- expressed in these glands, while PSD-95, which is neuron specific, was absent. Co-localization of nNOS and the PSD-95 protein was additionally evaluated by immunohistochemical staining of adjacent section ⁇ from an E19 rat. Results are seen in Figure 16.
  • Figure 16 shows that PSD-95 co-localizes with nNOS in developing neurons.
  • Immunohistochemical staining of adjacent sagittal sections of an E19 rat fetus indicates that PSD-95 (Figure 16A and 16C) and nNOS ( Figures 16B and 16D) are co- localized in primary olfactory epithelium (OE) and in nerve processes projecting to the olfactory bulb (OB) (Magnification in Figures 16A and 16B is 50X; in Figures 16C and 16D is 400X) .
  • OE primary olfactory epithelium
  • OB olfactory bulb
  • PSD- 95 and nNOS are also co-localized. Both proteins are most concentrated in neuronal processes of the intermediate zone (IZ) and cell bodies of the cortical plate (CP) , while the ventricular zone (VZ) is devoid of staining (Magnification 100X) .
  • both PSD-95 and nNOS were enriched in dendritic specialization ⁇ in olfactory cilia and in axonal processes projecting to the olfactory bulb, which itself does not contain either nNOS or PSD-95, as seen in
  • FIG 16A-D nNOS also occurs in fetal myenteric neurons and its absence is associated with hypertrophic pyloric stenosis. Immunohistochemical analysis revealed a co-localization of nNOS with PSD-95 in myenteric neurons ( Figures 16 E-H) . nNOS and PSD-95 were similarly co-localized in embryonic cerebral cortex. Staining for both proteins was enriched in the intermediate zone and in developing cortical plate, while lesser staining was found in the subplate region. The ventricular zone wa ⁇ devoid of staining ( Figures 161 and 16) .
  • yeast constructs encoding appropriate fragments of PSD- 95 were fused to the GAL4 activation domain. Constructs encoding the second PDZ motif of PSD-95 interacted with nNOS while those lacking this region were inactive. Results are seen in Table 4.
  • nNOS amino acids 1-195
  • SV 40 amino acids 84-708
  • PSD-95 and PSD-93 are related, both structurally, as seen from Figure 14, and also functionally, as their respective clones interacted with nNOS.
  • the second PDZ domain of PSD-95 provides a binding link between the nNOS and NMDA receptor.
  • confirmation of nNOS interaction with the second PDZ domain of PSD-95 was investigated.
  • immunoprecipitation studies were conducted. Results are seen in Figure 17 .
  • Figure 17 shows co-immunoprecipitation of nNOS and PSD- 95.
  • COS cells were transfected with an expression construct PSD-myc, encoding amino acids 1-386 of PSD-95 with a 10 amino acid c-myc epitope tag alone (lanes 1) or were co-transfected with PSD-myc and nNOS (lanes 2) .
  • Cell homogenates were immunoprecipitated with nNOS and probed with a monoclonal antibody to c-myc. Input was 5% protein loaded onto columns.
  • solubilized cerebellar membranes were immunoprecipitated with antibody to PSD-95 (lanes 2) or a non- immune serum (lanes 1) .
  • Western blotting ⁇ how ⁇ ⁇ pecific co- immunoprecipitation of nNOS but not eNOS with PSD-95.
  • Figure 17C shows identical immunoprecipitations from cerebellar cytosol, which lack ⁇ PSD-95 but contain ⁇ high concentrations of nNOS.
  • Figure 17C shows that the PSD-95 antibody (lane 2) does not directly interact with nNOS.
  • the eNOS blot and the nNOS blot from cerebellar cytosol were intentionally overexposed, but failed to show specific immunoprecipitated bands.
  • Figure 17D shows affinity chromatography which demonstrates that nNOS is selectively retained by an immobilized PSD-95 protein fragment (amino acids 1-386) fused to GST. eNOS is not retained by the PSD column. Solubilized brain extracts were incubated with G-PSD or control GST beads, columns were washed with a buffer containing 0.5 M NaCl and 1% triton X-100, and eluted with SDS. Bound proteins were detected by Western blotting. Inpu was 10% protein.
  • Figure 17E shows that NMDA rece or 2B carboxy terminal peptide displaces nNOS and 1 ⁇ 1.4 fr PSD-95.
  • "Pull-down" assays from brain were conducted as a ve containing 0 (lanes 6, 7), 10 ⁇ M (lanes 4,5) or 30 ⁇ M (1-nes 2,3) NMDA receptor peptide or 200 ⁇ M control peptide (lanes 8,9). Input was 10% protein.
  • Figure 17 shows and confirms that nNOS binds to PSD-95 through PDZ motif interactions.
  • nNOS-PSD-95 complex was immunoprecipitated from COS cells co-transfected with expression vectors for nNOS and the PDZ repeats of PSD-95, indicating that this interaction occurs in a cellular environment ( Figure 17A) .
  • Figure 17A shows that only a small fraction of PSD-95 can be solubilized from brain densities with non- denaturing detergents
  • a nNOS/PDS-95 complex was specifically immunoprecipitated from cerebellum ( Figures 17B and 17C) , where both proteins are co-expre ⁇ ed at high levels.
  • mice carrying a targeted disruption of exon 2 of nNOS express residual nNOS isoforms specifically lacking the PDZ domain. Thus these mice are extremely suitable for investigation whether nNOS isoform lacking the PDZ motif will bind to PSD-95 or PSD-93.
  • Neuronal NOS ⁇ mice were generated by deleting the first translated exon, which is exon 2, of nNOS in both mice and humans which encodes the PDZ motif. Results are shown in Figure 18.
  • Figure 18 illustrates that exons 1 and 2 of nNOS are alternatively spliced.
  • Figure 18A is Northern blot analysi ⁇ of brain mRNA from wild type (WT) and nNOS * mice hybridized with a full length nNOS cDNA probe. A broad band of 10.5 kb i ⁇ recognized in the wild type mouse brain and weaker bands of 11 and 9.5 kb are recognized in nNOS ⁇ / ⁇ mice.
  • Figures 18B and 18C show RT-PCR analysis of 5' splicing of nNOS gene.
  • cDNA was amplified wir primers 1 and 2 (lanes 1,2).
  • Figure 18B is ethidium bromide staining which shows a band of 1 kb amplified from nNOS 7 and a band of 2.2 kb from wild type.
  • Figure 18C is Southern hybridization with a full length nNOS probe showing hybridization to the ethidium stained bands. A weaker band of 1 kb in amplifications of wild type cDNA is also detected by hybridization (lane 2) . A similar analysis using primers 3 and 4 confirms that exon 2 sequences are only detected in wild type cDNA (lanes 3,4).
  • Figure 19 shows that catalytically active nNOS isoforms lacking exon 2 are expressed in the brain of nNOS ⁇ 4 mice.
  • Figure 19A is Western blotting of crude (lanes 1,2) and 2' 5'- ADP affinity purified (lanes 3,4) brain extracts, indicating that the major nNOS band in wild type brain migrates at 160 kD (lanes 1,3) while in nNOS ⁇ , co-purifying bands of 125 and 136 kD (lanes 2,4) are observed. Partial tryptic digestion of 2'5-ADP agarose-purified proteins reveals a similar proteolytic "fingerprint" from wild type (lane 5) and nN0S ⁇ (lane 6) .
  • Figure 19B shows cDNA clones encoding nNOS, nNOS/3 (5'a spliced to exon 3) or nNOS (5'b spliced to exon 3) were transfected (trx) into COS cells and protein extracts were resolved by SDS/PAGE.
  • Full length NOS (nNOS-trx) comigrates at 160 kD with the major product from the wild type brain (lanes 1,2).
  • Transfection of nNOS/3 and nNOS yields proteins of 136 and 125 kD respectively that comigrate with immunoreactive bands from nNOS ⁇ 4 (lanes 3,4,5).
  • Figure 19C shows NOS catalytic activity of nNOS isoforms.
  • COS cells were transfected with 10 ⁇ g of expres ⁇ ion vector encoding full length nNOS, nNOS/3, or nN0S .
  • NOS activity was measured in cell homogenates three days following transfection in the presence of 200 ⁇ M free calcium. This experiment was replicated twice with similar results.
  • Figure 19D shows that nNOS isoforms are discretely expressed in the nNOS ⁇ brain.
  • Highest densities of nNOS in wild type (10 ⁇ g/lane) are found in the cerebellum (Cb) .
  • Cb cerebellum
  • highest levels of nNOS isoforms are found in striatum (St) and hippocampus (Hi)
  • Bs brainstem
  • Cx cerebral cortex
  • Figure 19E eNOS is homogeneously distributed in forebrain (Fb) , cerebellum (Cb) as well as the peripheral tissues liver (Li) , lung (Lu) and kidney (Ki) . All lanes in Figure 19C were loaded with 100 ⁇ g of solubilized membrane extract. Transfection of the 5'a containing construct generated a prominent immunoreactive protein band of 136 kD that comigrated with nNOS3 from nNOS ⁇ 4 brain ( Figure 19B; lanes 3,4). Transfection of the 5'b containing construct yielded a nNOS band of 125 kD ( Figure 19B, lane 5) .
  • NOS activity in the wild type brain i ⁇ highest in the cerebellum.
  • NOS activity in nNOS ⁇ is highest in the striatum and lowest in the cerebellum.
  • the regional distribution of residual nNOS isoform ⁇ in nNOS' 1 ' 4 brain extracts paralleled the pattern of residual nNOS activity previously reported in Cell. 75: 1273-86 (1993) . Absence of a PDZ Motif Prevents A ⁇ sociation of nNOS
  • nNOS isoform ⁇ lacking a PDZ motif was investigated.
  • nNOS 4 ' 4 mice express nNOS isoforms specifically lacking the PDZ motif were used as an important tool to determine the functions for this domain in vivo .
  • Association of residual nNOS isoforms with PSD-95 was investigated.
  • nNOS proteins purified from wild type and nNOS ⁇ / ⁇ mouse forebrain were subjected to pull-down as ⁇ ay ⁇ a ⁇ de ⁇ cribed above. Re ⁇ ults are seen in Figure 20.
  • Figure 20 shows that nNOS isoforms lacking the PDZ motif do not bind to PSD-95 or to brain membranes.
  • FIG 20A partially purified nNOS protein from wild type (WT) or nNOS 4 ⁇ brains were analyzed by PS "pull-down" assay. Full length nNOS hi "is to PSD-95 while the residual isoforms lacking the PDZ mot- do not. Input was 20% protein.
  • Figure B show ⁇ that residual nNOS isoforms are restricted to cytosol of nNOS 4 .
  • Brain homogenates extracted with 100 mM NaCl (lanes l) , l M KCl + 1% triton X-100 (lanes 2) or insoluble pellet (lanes 3), from wild type (20 ⁇ g/lane) or nNOS 474 (200 ⁇ g/lane) were probed by Western blotting. Only full length nNOS protein containing the PDZ motif was retained by G-PSD bead ⁇ ; the alternatively ⁇ pliced forms in nNOS 474 did not adhere to G-PSD ( Figure 20A) .
  • nNOS in wild type and nNOS 4 ' 4 mice were compared. Brain homogenates were first extracted with physiological saline, then with buffer containing 1M KCl and 1% triton X-100, leaving a cytoskeletal pellet. nNOS in wild type brain was present in all fractions while residual nNOS isoforms in nNOS 474 occurred only in the first soluble fraction (Figure 2OB) .
  • nNOS is functionally coupled to N-methyl-D-a ⁇ partate receptor ⁇ through the interaction with binding protein ⁇ .
  • the N-terminal domain of nNOS is unique to the neuronal isoform and contains a PDZ motif of about 100 amino acids.
  • nNOS is enriched at synaptic junctions in the brain owing to association of nNOS with the postsynaptic density proteins PSD-95 and PSD-93 which act as interacting proteins between nNOS and NMDA receptors.
  • PSD-95 and PSD-93 which act as interacting proteins between nNOS and NMDA receptors.
  • the interaction of nNOS and PSD-95 or PSD-93 via their respective PDZ domains mediates synaptic- association of nNOS with the NMDA receptor.
  • nNOS PDZ domain interacts with the second PDZ domain of the PSD-95 or PSD- 3 and the PDZ domain of nNOS is important for its interaction with the NMDA receptor. When this domain is missing, the interaction between nNOS and PSD-95 is also missing. Absence of a PDZ domain thus prevents the binding of nNOS/PSD-95 or PSD-93 proteins and nNOS interaction with the NMDA receptor. Neuronal NOS and its specific binding proteins are therefore physiologically very important for neuronal functionality.
  • NMDA receptor stimulates influx of calcium ions into the cell and through the effect of calmodulin it activates nNOS, which in turn increases production of NO in the neuron.
  • Neuronal NO either causes or at least participates in development of neurotoxic injury, including stroke.
  • the increased activity of the NMDA receptor is to a certain extent dependent on binding of NMDA to nNOS through PDZ domains of nNOS binding proteins PSD-95 or PSD-93. This can be advantageously utilized for early detection of impending stroke or development of other neurodegenerative diseases by detecting a level of nNOS, PSD-95 or PSD-93 proteins.
  • nNOS When the level of nNOS is high, the probability of impending stroke or other neurodisturbance is high.
  • nNOS The detection of nNOS is according to Example 22 and as described above for diagnostic test for detection of muscular dystrophy. Instead of mu ⁇ cle tissue, brain or central nervous tissue biopsy is used. Alternatively, in situ imaging method is used using labeled PSD-95 protein inhibitors.
  • nNOS neuron-derived NO
  • NMDA receptor activity is responsible for and mediates brain injury following cerebral ischemia. Therefore, by blocking nNOS activity by disruption of its binding with binding proteins, the action of NO can be controlled and further damage to neurons is prevented.
  • Screening for drugs that block interaction of NMDA receptors with PSD-95 or PSD-93 could be done by an analogous procedure to that described in Example 23.
  • a 9-mer peptide identified as a SEQ.ID No. 3, corresponding to the final 9 amino acids of NMDA receptor 2B potently interact ⁇ with GST-fu ⁇ ion protein ⁇ encoding the fir ⁇ t 2 PDZ domain ⁇ of PSD-95 and PSD-93.
  • a C-terminal 9-mer peptide of NMDA 2B receptor can be advantageously used for a large scale screening assay for compounds which block its binding to PSD-95 or PSD-93. Those compounds would then be able to inhibit, or block by competition, the binding of PSD-95 or PSD-93 to nNOS.
  • PSD-95 or PSD-93 GST-fusion proteins One way how to produce these inhibitors is to label the peptide and to incubate it with PSD-95 or PSD-93 GST-fusion proteins to reach a binding equilibrium and immobilize the fusion proteins on glutathione resin.
  • PSD-95 or PSD-93 fragments are retained by immobilized glutathione resin, and the resin is washed to elute unbound peptide.
  • this assay one could perform large scale screening of compounds for drug di ⁇ covery.
  • Potent inhibitor ⁇ of this binding are therefore useful in treatment and in prevention of stroke and neurodegenerative disease. They would be administered in any suitable pharmaceutically acceptable route either before impending stroke or after the stroke developed, to prevent further neuronal damage.
  • Inhibitors of nNOS and Postsynaptic PSD-93 and PSD-95 Proteins are therefore useful in treatment and in prevention of stroke and neurodegenerative disease. They would be administered in any suitable pharmaceutically acceptable route either before impending stroke or after the stroke developed, to prevent further neuronal damage.
  • This invention also identifie ⁇ a small 9-mer peptide that potently (Kd-1 ⁇ M) blocks association of nNOS with PSD-95 and PSD-93.
  • This invention demonstrates a novel mode to block brain damage due to nitric oxide, that is, the identification of small molecules that disrupt interaction of nNOS with PSD-95 and PSD-93.
  • This invention demonstrates that such drugs would uncouple nNOS from neurotransmitter receptors and would prevent NO mediated brain damage.
  • Binding Assay for Screening Inhibitors of PSD-95 and PSD-93 Binding with nNOS GST-fusion proteins linked to the first two or three PDZ motifs of PSD-95 or PSD-93 are expressed in E. coli as described in Example 6. Binding interactions to this fragment are monitored by a variety of assays known in the art. To detect binding of endogenous nNOS or NMDA receptor subunits to PSD-95 or PSD-93, pull down assays are done as described. Screening for drugs that block interaction of NMDA receptors with PSD-95 or PSD-93 could be done by an analogous procedure as described above, and in Example 23.
  • UTILITY Current invention is useful for diagnosis and treatment of muscular diseases, primarily for diagnosis and treatment of Duchenne dystrophy, Becker muscular dystrophy and other types of muscular dystrophies. Detection of presence or absence of nNOS in human biopsies, for example, immunohistochemically, detects the disease and its severity. Treatment of muscular dystrophie ⁇ utilize ⁇ the restoration of fully functional dystrophin able to bind to nNOS, using, for example, gene therapy. Restoration of a functional dystrophin molecule to muscle represents a primary goal for therapy. The invention is also useful for management of neurodegenerative diseases.
  • EXAMPLE 1 Tissue Extraction and Western Blot Analysis This example describes methods used for skeletal muscle tissue extraction.
  • Mouse quadriceps skeletal muscle was homogenized in 10 vol (w/v) of buffer A (25 mM Tris-HCl, pH 7.4, 100 mM NaCl, ImM EDTA, 1 mM EGTA, 1 mM PMSF) , and heavy micro ⁇ omes were prepared by a standard protocol with minor modifications according to (J. Cell Biol.. 96: 1008-16 (1983)). Nuclei were pelleted by centrifugation at 1000 x g. The supernatant was then centrifuged at 20,000 x g, yielding supernatant S,.
  • buffer A 25 mM Tris-HCl, pH 7.4, 100 mM NaCl, ImM EDTA, 1 mM EGTA, 1 mM PMSF
  • the re ⁇ ulting heavy micro ⁇ omal pellet was resu ⁇ pended in buffer A containing 500 mM NaCl, incubated for 30 min at 4°C with agitation, and centrifuged at 15,000 x g, yielding supernatant S 2 .
  • the resulting pellet was resuspended in buffer A containing 500 mM NaCl plus 0.5% Triton X-100, incubated for 30 min at 4°C with agitation, and centrifuged at 15,000 x g, yielding supernatant S 3 and a final pellet, P.
  • Tissue extracts were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (7.5% polyacrylamide) , and proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore) .
  • PVDF polyvinylidene difluoride
  • Membranes were incubated overnight with primary antisera bNOS, (1:250) and eNOS, (1:250), obtained from Transduction Laboratories; dystrophin (1:100), and spectrin (1:100), obtained from Novacastra Laboratorie ⁇ diluted in Tris-HCl-buffered saline containing 3% bovine serum albumin. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL) according to the specifications of the manufacturer (Amersham) .
  • ECL enhanced chemiluminescence
  • mice Mouse quadriceps from wild-type and mdx mice were homogenized and solubilized in 10 vol of buffer B (50 mM Tris- HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, 1 mM PMSF) containing 1% digitonin. Solubilized membranes (4 mg) from wild-type and mdx mice were circulated for 1 hour with 250 ⁇ l of sWGA- agarose obtained from Vector Labs at 4°. Columns were washed sequentially with 5 ml of buffer B containing 0.1% digitonin, buffer B containing 0.1% digitonin and 500 mM NaCl, and buffer B containing 500 mM NaCl and 0.5% Triton X-100.
  • buffer B 50 mM Tris- HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, 1 mM PMSF
  • Solubilized membranes (4 mg) from wild-type and mdx mice
  • mice quadriceps from wild-type and nNOS knockout mice were homogenized in 10 vol (w/v) of buffer B, and heavy microsomes were prepared and solubilized in buffer B containing 1% digitonin. Solubilized membranes (4 mg) from wild-type and nNOS knockout mice were applied to 150 ⁇ l columns of 2' ,5'-ADP-agarose (Sigma).
  • Glutathione-S-Transfera ⁇ e-Fu ⁇ ion Proteins This example describes preparation of glutathione-S- transferase (GST) fusion proteins.
  • a GST-nNOS(1-299) construct was generated by cloning sequences encoding the first 299 amino acids of rat brain NOS into the EcoRI site of the pGEX-2T vector.
  • GST-fusion proteins were expressed in Escherichia coli and purified on glutathione-Sepharose beads according to Gene. 67:31-40 (1988) and according to the specifications of the manufacturer Pharmacia. Solubilized skeletal muscle membranes (2 mg) were incubated with control (GST) or GST-nNOS (1-299) beads for 1 hr. Beads were washed with buffer containing 0.5% Triton X- 100 plus 300 mM NaCl, and proteins were eluted with 150 ⁇ l of loading buffer.
  • Dystrophin Immunoprecipitation This example describes method used for dystrophin immunoprecipitation.
  • IgG (lO ⁇ g) obtained from Cappel was then added, and after 30 min, 50 ⁇ l of protein A-Sepharo ⁇ e was used to precipitate antibodies. Protein A pellets were washed three times with buffer containing 200 mM NaCl and 0.1% Triton X-100. Immunoprecipitated proteins were denatured with loading buffer and resolved by SDS-PAGE.
  • EXAMPLE 5 Immunohistochemical and Immunoblotting Procedures This example describes methods used for preparation of, skeletal muscle samples for immunohistochemical and immunoblotting procedures.
  • nNOS antibody 1:250 prepared according to Nature. 372: 546-548 (1994) were applied to sections overnight at 4°
  • FITC secondary goat anti-rabbit fluorescence isothiocyanate
  • donkey anti-mouse Cy-3 conjugated antibodies were used according to the specifications of the manufacturer (1:200), Jackson Laboratories.
  • EXAMPLE 6 Mammalian Cell Transfections This example illustrates the method used for mammalian cell transfection.
  • nNOS cDNAs were cloned into the mammalian expression vector pcDNA-3 obtained from Invitrogen.
  • Monkey COS cells were grown in culture medium consisting of DMEM (GIBCO BRL) supplemented with 10% fetal bovine serum.
  • Cell ⁇ were plated in 10 cm dishes at a density of 2 x 10 4 per square centimeter and transfected the following day using calcium phosphate as previously described in Nature, 351:714-718.
  • Cells were washed with PBS 3 days following transfection, harvested in 2 ml of buffer containing 25 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, and disrupted using a polytron.
  • Tris-HCl pH 7.4
  • This example describe ⁇ procedure used for assessment of for NOS catalytic activity.
  • Quadriceps skeletal muscle from wild-type and mdx mouse were homogenized in 10 vol of buffer containing 25 mM Tris-HCl (pH 7.4) , 1 mM EDTA, ImM EGTA, and 0.1 M NaCl. The homogenate was centrifuged at 20,000 x g, yielding the soluble fraction. The pellet was extracted in the same buffer containing 0.5 M NaCl and centrifuged at 20,000 x g, yielding the particulate fraction.
  • This example describes methods used for evaluation of the nNOS localization in human tissue.
  • Tissues were snap frozen in liquid nitrogen-cooled isopentane.
  • cryostat sections were collected into plastic tubes and sonicated in buffer containing 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, l mM EGTA, and
  • nNOS staining was performed using two independently raised antisera. The first antiserum ( ⁇ -nNOSl) reacts only with determinants in the N- terminal domain of nNOS (Neuron. 13:301-313, 1994b) , while the second ( ⁇ -nN0S2) reacts only with the C-terminal region, ⁇ - nNOS was obtained from Transduction Laboratories. Unless otherwise noted, all histologic section ⁇ were labeled with ⁇ - nNOSl.
  • nNOS polyclonal antibody raised against homogenou ⁇ nNOS protein purified from rat cerebellum prepared according to Nature. 347: 768-770 (1990).
  • nNOS monoclonal antibody obtained from Tran ⁇ duction Lbas.
  • ⁇ l-syntrophin polyclonal antibody prepared according to Neuroreoort. 5: 1577-1580 (1994) .
  • Dystrophin monoclonal antibody wa ⁇ obtained from Sigma.
  • 3-dystroglycan monoclonal, utrophin monoclonal, and ⁇ arcoglycan monoclonal antibodie ⁇ were obtained from Novacastra.
  • This example describe ⁇ the assay used for immunofluore ⁇ cent studies on skeletal muscle samples from Becker muscular dystrophy patients and transgenic mice.
  • Unfixed skeletal muscle samples obtained from human patients or from mdx or transgenic mice were flash frozen in liquid nitrogen cooled isopentane, sectioned on a cryostat (10 ⁇ m) and melted directly onto glass slides. Sections were then post-fixed in 2% parafor aldehyde in phosphate buffered saline (PBS) or cold acetone. Tissues were "blocked" in PBS containing 1% normal goat serum. Primary antibodies were diluted in blocking reagent and were applied to sections overnight at 4 ⁇ C. For indirect immunofluorescence secondary goat anti-rabbit FITC (1:200), or donkey anti-mouse Cy-3 (1:200) conjugated antibodies were used according to the manufacturer's specifications (Jackson Laboratories) . Cy-3 conjugated ⁇ -BGT was diluted together with the secondary antibody for double labeling motor endplates.
  • EXAMPLE 11 Antibodies Immunoprecipitation This example describe ⁇ procedure used for precipitation of polyclonal antibodies to ⁇ l-syntrophin. Polyclonal antibodies (1 ⁇ g) to ⁇ l-syntrophin or non- immune serum were added to 0.5 ml aliquots of solubilized skeletal muscle membranes (2 mg/ml) or muscle cytosol (1 mg/ml) , and ⁇ araple ⁇ were incubated on ice for 1 hour. Protein A sepharose (50 ⁇ l) was used to precipitate antibodies. Protein A pellets were washed 3 times with buffer containing 100 mM NaCl and 1% tritox X-100. Immunoprecipitated proteins were denatured with a loading buffer and resolved by SDS-PAGE.
  • EXAMPLE 12 Fusion Protein Affinity Chromatography This example describes fusion protein affinity chromatography procedure used for GST-nNOS (1-299) fusion protein.
  • a fusion protein of GST fused to the first 299 amino acids of nNOS was expressed in Escherichia coli and purified on glutathione sepharose bead ⁇ as described Cell. 82: 743-752 (1995) . Solubilized skeletal muscle membranes were incubated with control (GST) or GST-nNOS (1-299) beads. Samples were loaded into disposable columns, which were washed with 50 volumes of buffer containing 0.5% triton X-100 + 300 mM NaCl, and proteins eluted with 150 ⁇ l of SDS/PAGE loading buffer.
  • EXAMPLE 13 Antibodies and Western Blotting Used for Studie ⁇ of Interaction of NOS with PSD-95 and ⁇ l-Syntropin This example list ⁇ specific antibodie ⁇ used for ⁇ tudies of interaction of NOS with the ⁇ ynaptic density protein PSD-95 and ⁇ l-syntropin.
  • nNOS monoclonal and eNOS monoclonal antibodies were obtained from Transduction Labs.
  • ⁇ l- ⁇ yntrophin polyclonal as described in Example 9 PSD- 95 polyclonal were prepared according to Neuron. 9: 929-942 (1992).
  • Kyl.4 polyclonal antibodies were prepared according to Nature, 378: 85-88 (1995) .
  • c-myc monoclonal antibody 9E10 was obtained from BABCO.
  • T7-Tag monoclonal and GST 12 monoclonal antibodies were obtained for Santa Cruz Biotechnology, Inc.
  • EXAMPLE 14 In situ hybridization This example describe ⁇ the procedure used for in situ hybridization used for coexpression of nNOS and PSD-95 transcripts.
  • Rats were perfused with 4% paraformaldehyde, tissues were harvested, post-fixed at 4°C for 3 hours, and cryoprotected in 20% sucrose overnight. Twenty micron sections were cut on a cryostat and melted onto glass slides (Plus) , obtained from Fisher. Sections were then blocked for one hour in a buffer containing 2% goat serum, 0.1% triton X-100 in PBS. Primary antibodies to nNOS (polyclonal nNOS) , PSD 95, or ⁇ l-syntrophin were diluted into a blocking reagent and incubated with sections overnight. Immunoperoxidase histochemistry wa ⁇ performed using the ABC method according to a kit obtained from Vector. Immunofluorescent ⁇ taining of rat extensor digitorum longus muscle was done as de ⁇ cribed in Cell. 82:
  • Neuronal NOS cDNA were cloned into the EcoRV and Xba I sites of the mammalian expression vector pcDNA 3 (Invitrogen) .
  • 5'a and 5'b containing constructs were amplified by PCR, sequenced, and cloned into the unique Nar I re ⁇ triction site of nNOS.
  • PSD-95-myc construct containing amino acids 1-386 with a C-terminal myc-epitope tag was amplified by PCR and cloned into the BamHI and EcoRI sites of pcDNAIII.
  • Monkey COS cells were grown and transfected using calcium phosphate as previously described in Nature. 351: 714-718 (1991) .
  • Cerebellar membrane ⁇ were ⁇ olubilized with 1% digitonin and 100 mM NaCl and centrifuged to remove the in ⁇ oluble cyto ⁇ keleton.
  • Three ⁇ l of PSD-95 polyclonal anti ⁇ erum to PSD-95 or 3 ⁇ l non-immune serum were added to l ml (500 ⁇ g) of cerebellar cytosol or solubilized membranes. After a 60 minute incubation on ice, 50 ⁇ l of protein A sepharose was added to precipitate antibodies. Protein A pellets were washed 3 times with a buffer containing 200 mM NaCl and 1% triton X-100.
  • Immunoprecipitated proteins were denatured with a loading buffer and resolved by SDS-PAGE. Heavy microsomes of rat gastrocnemius were prepared and solubilized with 1% triton X-100 as described in Cell. 82: 743-752 (1995) . Five ⁇ g polyclonal antiserum to ⁇ l-syntrophin or non-immune serum were added to 1 ml (500 ⁇ g) solubilized muscle samples. Immunoprecipitations from transfected COS cells used polyclonal antibody to nNOS.
  • EXAMPLE 18 GST Fusion Protein Affinity Chromatography This example describes methods used for construction of GST fusion constructs.
  • GST fusion construct ⁇ were con ⁇ tructed by PCR and fusion proteins purified as described in Example 3.
  • solubilized tissue samples were incubated with control or GST-fusion protein beads for 1 hour. Beads were wa ⁇ hed with a buffer containing 0.5% triton X-100 and 350 mM NaCl, and protein ⁇ were eluted with SDS loading buffer.
  • NMDA receptor peptide (SEQ ID NO: 7:) (lys leu ser ser ile glu ser asp val) or control peptide (SEQ ID NO: 8:) (lys pro lys his ala lys his pro asp gly his ser gly asn leu cys) were added where indicated during tissue incubation with the fusion protein.
  • SEQ ID NO: 7: lys leu ser ser ser ile glu ser asp val
  • control peptide SEQ ID NO: 8:
  • cDNAs encoding mouse ⁇ l-syntrophin domains (PHla domain, amino acids 1-77; PHlb, 162-271; PDZ, 75-170; PH2, 281-402; SU domain 401-499) were amplified by PCR and cloned into pET28a vector (Novagen, Inc.) with the exception of PHla and PHlb that were ligated together to produce the intact PHI domain.
  • Blots were washed 3 x 10 minute ⁇ in TBS-Tween, incubated with primary antibody T7.Tag or GST for 30 minutes and bands visualized by ECL.
  • EXAMPLE 20 mRNA Isolation and cDNA Analvsi ⁇ This example describes procedures used for isolation of mRNA and cDNA analysis.
  • RNA was isolated using the guanidine isothiocyanate/CsCl method and mRNA was selected using oligo dT sepharose. For Northern blotting, mRNA was separated on a formaldehyde agarose gel and transferred to a Nylon membrane. A random primed probe 32 P probe wa ⁇ generated using the full-length (5057 bp) nNOS cDNA as described in Nature. 351: 714-718 (1991) a ⁇ a template. The filter was washed at high stringency, 68 ⁇ C, 0.1% SSC, 0.1% SDS and exposed to X-ray film overnight at -70°C.
  • mRNA was reverse transcribed with RTth polymerase using random hexamer primers.
  • the sequence of the PCR primers used were: PI: SEQ ID NO: 11: P2: SEQ ID NO: 12: P3: SEQ ID NO: 13: P4: SEQ ID NO: 14: Clones encoding PSD-93 were isolated from a rat brain cDNA library (Stratagene) by plaque hybridization.
  • EXAMPLE 21 nNOS Protein Purification and Catalytic Assays This example describes purification procedure used for solubilized tissue homogenated and nNOS protein catalytic assays.
  • Solubilized tis ⁇ ue homogenates were incubated with 100 ⁇ l of 2'5'-ADP agarose (Sigma), columns were washed with 5 ml of buffer containing 0.35 M NaCl, and were eluted with 10 mM NADPH. Catalytic NOS activity was quantitated by monitoring the conversion of [ 3 H]arginine to [ 3 H]citrulline as described in PNAS USA. 87:682-685 (1990) .
  • This example describe ⁇ a diagno ⁇ tic te ⁇ t useful for detecting muscle disease.
  • nNOS immunofluorescence is performed as described in Example 10. Briefly, nNOS antibodies are applied to cryostat sections of muscle samples overnight at 4°C. Secondary goat anti-rabbit Cy-3 conjugated antibodies (1:200) are obtained from Jackson Laboratories and are used according to the manufacturer's specifications.
  • NADPH diaphorase staining is performed as described in
  • cryosections are incubated with ImM NADPH, 0.2 mM nitroblue tetrazoliu in a 0.1 M Tris-HCl buffer (pH 7.4) containing 0.2% triton X-100 for 90 minutes at room temperature.
  • nNOS Presence of nNOS is detected by the presence of blue staining.
  • the presence of sarcolemmal nNOS staining is consistent with presence of a functional dystrophin molecule.
  • the absence of sarcolemmal nNOS is a sensitive and specific indicator of abnormal dystrophins.
  • EXAMPLE 23 Binding Assay to Screen for compounds that Disrupt Interaction of nNOS. NMDA Receptors or Other Ion Channels with PSD-95 or PSD-93 This example describes a binding as ⁇ ay u ⁇ eful for screening compounds which prevent, inhibit or disrupt binding of nNOS, NMDA receptors or other ion channels with PSD-95 and PSD-93 proteins.
  • nNOS 1-299 is expressed in E-coli with a N-terminal hexahistidine tag and a heart muscle protein kinase site. This fragment is radiolabeled with 32 P using [ 32 P] ATP and heart muscle kinase.
  • PSD-95 or PSD-93 GST-fu ⁇ ion proteins are then incubated with the labeled nNOS fragment.
  • PSD-95 or PSD-93 fragments are retained by immobilized glutathione resin, and the resin is washed to elute unbound nNOS fragments. Bound nNOS fragments on the resin are quantitated by scintillation counting or by an ELISA. Using this as ⁇ ay, large scale screening of compounds for drug discovery is possible.
  • GENERAL INFORMATION (i) APPLICANT: BREDT, DAVID S. BRENMAN, JAY E. CHAO, DANIEL S.
  • MOLECULE TYPE protein
  • MOLECULE TYPE genomic DNA
  • SEQUENCE DESCRIPTION SEQ ID NO: 9: CCACAGATCA TTGAAGACTC G 21
  • MOLECULE TYPE genomic DNA
  • SEQUENCE DESCRIPTION SEQ ID NO: 10: GGGGAATTCC CCGCCCCAGG GGCGGGGAGC TTT 33
  • MOLECULE TYPE genomic DNA
  • SEQUENCE DESCRIPTION SEQ ID NO: 11: GTCCCTGCGT ATTGATGCA 19
  • MOLECULE TYPE genomic DNA
  • MOLECULE TYPE genomic DNA
  • SEQUENCE DESCRIPTION SEQ ID NO: 13:

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Abstract

Nitric oxide, neuronal nitric oxide synthase, neuronal nitric oxide synthase binding proteins, their inhibitors and a method of use of neuronal nitric oxide synthase, its binding proteins and their inhibitors for diagnosis and treatment of muscular dystrophy, stroke and other neurodegenerative diseases. A diagnostic assay for detection of absence of dystrophin or its mutated forms, neuronal nitric oxide synthase or its binding proteins. A method for treatment of muscular dystrophies by restoration of a functional dystrophin molecule in dystrophic muscles using gene therapy. Neuronal nitric oxide binding proteins PSD-95 and PSD-93 involved in management of stroke and other neurodegenerative diseases. The cloning and expression of the neuronal nitric oxide synthase binding proteins.

Description

MUSCULAR DYSTROPHY, STROKE, AND NEURODEGENERATIVE DISEAS DIAGNOSIS AND TREATMENT
5 BACKGROUND OF THE INVENTION
Field of Invention
This invention concerns nitric oxide, neuronal nitric oxide synthase, neuronal nitric oxide synthase binding proteins, their inhibitors and a method of use of neuronal
10 nitric oxide synthase, its binding proteins and their inhibitors for diagnosis and treatment of muscular dystrophy, stroke and other neurodegenerative diseases.
In particular, one aspect of the invention concerns involvement of neuronal nitric oxide synthase and its
15 binding protein αl-syntrophin in muscular dystrophic diseases, and their use in diagnosis and therapy of muscular dystrophies. The invention also concerns diagnostic assay for detection of absence of dystrophin or its mutated forms, neuronal nitric oxide synthase or its
20 binding proteins as well as a method for treatment of muscular dystrophies by restoration of a functional dystrophin molecule in dystrophic muscles using gene therapy.
The second aspect of the invention concerns
25 involvement of neuronal nitric oxide, neuronal nitric oxide synthase and its binding proteins PSD-95 and PSD-93 in stroke and other neurodegenerative diseases. The invention also concerns diagnosis as well as prevention and treatment of stroke and the other neurodegenerative diseases.
30 Additionally, invention concerns the cloning and expression of the neuronal nitric oxide synthase binding proteins. Finally, the invention concerns a binding assay for monitoring of binding of nitric oxide binding proteins with neuronal nitric oxide synthase and appropriate synaptic
35 receptors useful for development of compounds for treatment of neurodegenerative diseases. BACKGROUND ART AND RELATED ART DISCLOSURES Muscular dystrophy iε a debilitating disease caused by a motor dysfunction due to a genetic abnormality resulting in the absence or mutation of the protein dystrophin. Muscular dystrophies consist of a group of inherited diseases characterized by progressive weakness and degeneration of muscle fibers, without evidence of neural degeneration. The group includes dystrophies such as pseudohypertrophic Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy (Leyden- Mobius pelvifemoral type) , and facioscapulohumoral (Landouzy-Dejerine) muscular dystrophy.
Duchenne dystrophy is an X-linked recessive disorder caused by a mutation at the Xp21 locus, which results in the absence of the gene product dystrophin, normally localized in the sarcolemma of muscle cells. Becker muscular dystrophy, also a X-linked disorder, is a milder clinical variant of Duchenne dystrophy with the same genomic mutation at Xp21 where patients do not lack dystrophin completely but their dystrophin has an abnormal molecular weight and is somehow dysfunctional.
Duchenne muscular dystrophy is a severe, and ultimately fatal, disease. Duchenne dystrophy patients, typically boys 3-7 years old, experience muscle weakness, waddling gait, toe-walking, lordosis, frequent falls, and difficulty in standing up and in climbing stairs. Progression of the disease is steady and most patients are confined to a wheelchair at age 10 to 12. Few patients survive age of 20 years. Clinical symptoms of Becker muscular dystrophy are less severe; very few patient are confined to a wheelchair and more than 90% of these patients survive.
There is no known diagnostic procedure available for diagnosis, particularly early diagnosis of the disease and its severity, other than muscle biopsy or electromyography. Currently, the diagnosis is based chiefly on clinical signs and on the patient's genetic pedigree. Similarly, there is no specific therapy for muscular dystrophy other than supervised exercise programs and weight management (The Merck Manual. 16th Ed., 1526 (1992)).
It would be, therefore, advantageous to provide and have available a method for early diagnosis of the muscular dystrophy by using, for example, immunoblotting for detection of dystrophin, syntrophin or nNOS presence or absence in the patient's skeletal muscles, aε well as a method for treatment of muscular dystrophy by providing a functional dystrophin molecule or functional fragment thereof or generating means for synthesis of normal nonmutated dystrophin in dystrophic muscles using the method of the invention, including gene therapy.
Dystrophin which has been shown to be misεing or mutated in muscular dystrophy physically links the extracellular matrix to the muscle skeleton. Dystrophin is a large intracellular protein containing several defined sequence motifs (Cell. 80:675-679 (1995)). An amino terminal α-actinin-like domain binds to F-actin and is followed by a large rod domain that shares sequence homology with the structural repeats in spectrin. The carboxyl terminus is unique to dystrophin and dystrophin related proteins as this region directly binds to a glycoprotein complex in skeletal muscle. The structural dystrophin glycoprotein complex includes intracellular proteins syntrophins as well as integral membranes proteins, the dystroglycans and sarcoglycans. The absence of dystrophin in Duchenne dystrophy causes a disruption of this complex (Nature. 345: 315-319 (1990)). Dystrophin was originally identified by positional cloning as the gene product mutated in Duchenne muscular dystrophy (Nature, 323: 646-650 (1986)). Subsequent studies have identified a family of intracellular and transmembrane glycoproteins in a dystrophin-associated complex that links the extracellular matrix with the actin- baεed cytoskeleton. Recent εtudies indicate a major role for this complex in neuromuscular development and disease. α-Dystroglycan, an extracellular glycoprotein linked to dystrophin, serves as a physiologic receptor for agrin, which mediates clustering of acetylcholine receptors (Cell. 77: 663-674 (1994), ibid. 77: 675-686 (1994)). On the other hand, disruption of dystrophin or other proteins in this complex results in muscular dystrophy in both humans and animals (Cell. 80: 675-679 (1995)). Despite these data, it is not known how the dystrophin complex mediates signal transduction, nor is it clear why disruption leads to muscle disease. Indeed, none of the previously identified dystrophin-aεsociated proteins have known catalytic activities.
In addition to this structural role, the dystrophin complex is involved in signalling function in muscle, including regulation of a stretch-activated calcium channel. Signal transduction by the dystrophin complex must be somehow mediated. It has now been discovered that nitric oxide may be that mediator.
Nitric oxide (NO) is a major endogenous mediator involved in diverse developmental and physiological processes (Annu. Rev. Biochem. , 63: 175-195 (1994)). In addition to controlling diverse cellular processes, NO also participates in certain pathophysiological conditions. In skeletal muscle NO has been shown to depress the muscle contractile function (Nature. 372: 546-548 (1994)). In the brain, nitric oxide plays important physiological role in neurotransmission and synaptic modulation. In primary cortical cultures, NO mediates glutamate neurotoxicity (PNAS. 88: 6368-6371 (1991)). Neuronal NO production contributes to the development of ischemic brain necrosis (Science. 265:1883-1885 (1994)). A fundamental understanding of NO actions in the brain requires identification of the functional connections of NO synthetic enzyme NNOS with N-methyl-D-aspartate (NMDA) receptors. Deregulation of nNOS in the brain is asεociated with glutamate type receptor overactivity, of which the NMDA receptor is a member, and contributes to neuronal damage in animal stroke models (Ann. Neurol.. 32: 297-311 (1992)) and, conceivably, nNOS is therefore also involved in stroke and other neurodegenerative diseases in humans. Thus, it would be important to have a means to prevent development of and/or to treat stroke and the other neurodegenerative diseases by providing inhibitors of neuronal nitric oxide, neuronal nitric oxide synthase or inhibitors of its binding to the NMDA receptors.
Because NO is a short-lived free radical, regulation of signaling occurs largely at the level of NO biosynthesis. Three mammalian nitric oxide synthase (NOS) genes have been identified, and each forms NO from the guanidine nitrogen of L-arginine in a unique cytochrome P- 450-type reaction that consumes reduced nicotinamide adenine dinucleotide phosphate.
Specifically, these three NOSs are endothelial (eNOS) , neuronal NOS (nNOS) and inducible NOS (iNOS) . The nNOS and eNOS enzymes are discretely expressed in specific tissues and rapidly transduce signaling events in a calciu - dependent manner. eNOS activity accounts for endothelium- dependent blood vessel relaxation, while nNOS occurs discretely in a variety of cell types, including neurons, epithelial cells, esangial cells, and skeletal muscle cells. Inducible iNOS is a calcium-independent form of NOS expressed at highest levels in immunologically activated cells.
Physiological actions for nNOS have been characterized in the peripheral nervous system, where NO functions as a noradrenergic-cholinergic transmitter in numerous pathways, including the gastrointestinal and urogenital tracts.
In addition to transcriptional control, NOS proteins are all regulated by calmodulin (PNAS.. USAf 87: 682-685 (1990)), which links NO formation to increases in cellular calcium. Activation of nNOS in neurons is regulated by the steep gradients of calcium that exist in the vicinity of open calcium channels. In many central neurons, calcium influx through the N-methyl-glutamic acid receptor is selectively coupled to nNOS activity.
A more complex level of regulation is reflected by targeting of NOS proteins to intracellular membranes. This subcellular targeting restricts NO signaling to specific targets. Membrane association of eNOS is mediated by two fatty acid modifications (J. Biol. Chem.. 270: 995-998 (1995)). Neuronal NOS lacks consensus sequences for fatty acid modification (J. Neurochem.. 62: 1524-1529 (1994)). The majority of nNOS immunoreactivity in neurons is associated with rough endoplasmic reticulum and specialized electron-dense synaptic membrane structures. In skeletal muscle, nNOS is associated with the sarcolemma (Nature. 372: 546-548 (1994)).
Recent studies have identified nNOS expression at higher levels in human skeletal muscle than in human brain and thus would point toward a role of NO in skeletal muscle (FEBS Lett..316:175-180 (1993)). In mature skeletal muscle, nNOS is enriched in fast-twitch muscle fibers, where NO opposes contractile force (Nature. 372:546-548 (1994)). It has now been found that the physiological actions of NO in muscle are facilitated by restriction of the nNOS protein to the sarcolemmal membrane.
The sarcolemma of skeletal muscle is a complex structure reinforced by an actin-containing cytoskeleton. In addition to ubiquitous structural elements such as spectrin, skeletal muscle sarcolemma contains a unique network formed around dystrophin and related proteins (Curr. Qpin. Cell Biol.. 5: 82-84 (1993)).
The N-terminal domain of nNOS is unique to the neuronal isoform and contains a PDZ motif of approximately 100 amino acids that is found in a diverse group of cytoskeletal proteins and enzymes (Neuron. 9: 929-942 (1992)). This domain has now been found to mediate association of nNOS with the dystrophin complex. Therefore it would seem that nNOS must play a distinct role in the muscular dystrophy development and control, and could be advantageously used for early detection of the dystrophic disease.
It is therefore a primary objective of this invention to provide means for early diagnosis of muscular dystrophies as well as means for developing and identifying means and compounds suitable for treatment of muscular dystrophies, stroke and other neurodegenerative diseases. To this end the invention discloses a role of NO, nNOS and its binding proteins in development or controlling of muscular dystrophy in one aspect, and in prophylaxis, treatment and diagnosis of stroke and other neurodegenerative diseases in another aspect.
All cited patents, patent applications or publications are hereby incorporated by reference in their entirety.
SUMMARY One aspect of the current invention concerns a function of nitric oxide, neuronal nitric oxide synthase, and neuronal nitric oxide synthase binding proteins in muscular dystrophies.
Another aspect of the current invention concerns a function of nitric oxide, neuronal nitric oxide synthase, and neuronal nitric oxide synthase binding proteins in stroke and other neurodegenerative diseases.
Another aspect of the current invention concerns nitric oxide synthase binding proteins, their cloning and expression.
Another aspect of the current invention concerns identification of two brain proteins, namely, postsynaptic density PSD-95 and postsynaptic density PSD-93 proteins that bind to neuronal nitric oxide synthase. Still yet another aspect of the current invention concerns a discovery that neuronal nitric oxide synthase is functionally connected to calcium influx through a N- methyl-D-aspartate receptor where, at a receptor synaptic junction, neuronal nitric oxide synthase is enriched with post-synaptic density proteins.
Still another aspect of the current invention concerns identification of inhibitors of nitric oxide synthase binding proteins.
Another aspect of the current invention concerns identification of a small 9-mer peptide that potently blocks binding of neuronal nitric oxide synthase with post- synaptic density proteins.
Still another aspect of the current invention is a method of use of neuronal nitric oxide synthase, its binding proteins, and their inhibitors, for diagnosis and treatment of muscular dystrophy. Yet another aspect of the current invention concerns diagnostic assay for detection of absence of dystrophin or its mutated form, as well as a method for treatment of muscular dystrophy by restoration of a functional dystrophin molecule, or a functional fragment thereof in dystrophic muscles using gene therapy.
Still yet another aspect of the current invention concerns a binding assay for monitoring of binding of nitric oxide binding proteins with neuronal nitric oxide synthase and with appropriate synaptic receptors, useful for development of compounds for treatment of stroke and other neurodegenerative diseases.
Still another aspect of the current invention is a method of use of neuronal nitric oxide synthase, its binding proteins and their inhibitors for diagnosis and treatment of stroke or other neurodegenerative diseases.
Still another aspect of the current invention concerns a method for prevention of brain damage due to nitric oxide, by blocking the binding between neuronal nitric oxide synthase and postsynaptic density proteins resulting in uncoupling neuronal nitric oxide synthase from neurotransmitter receptors.
BRIEF DESCRIPTION OF FIGURES Figure 1 illustrates differential extractability of nNOS and eNOS in skeletal muscle homogenates. Figure 2 is a schematic alignment of eNOS and nNOS domains showing the extended N-terminus of nNOS containing a PDZ domain. Figure 3 shows association of nNOS and dystrophin in skeletal muscle in wild-type, mdx and NOS knockout skeletal muscle.
Figure 4 shows extraction of skeletal muscle membrane in build-type and mdx mice and nNOS displacement from particulate fractions of the mdx skeletal muscles.
Figure 5 showε immunofluorescent staining for nNOS of cryostat muscle section from quadriceps of wild-type, mdx, homozygous dystrophic and nNOS knockout mice showing that nNOS is selectively absent from sarcolemma of the mdx skeletal muscle.
Figure 6 are skeletal muscle cryosections of normal and DMD patients showing nNOS to be absent from sarcolemma of DMD muscle fibers. Figure 7 is a SDS-PAGE of human skeletal muscle tissue homogenates from three cases of Duchenne muscular dystrophy and from three normal muscle biopsies.
Figure 8 shows localization of nNOS, dystrophin and other dystrophin associated proteins during postnatal development.
Figure 9 are immunofluorescent stained cryosections from mouse quadriceps labeled for nNOS, αl-syntrophin and α-BGT showing localization of nNOS and αl-syntrophin in wild type, mdx and transgenic mdx mice. Figure 10 are Western blots of mouse skeletal muscle homogenates showing subcellular distribution of nNOS in transgenic mdx mice.
Figure 11 are Western blots of solubilized membranes from mouse quadriceps showing selective interaction of nNOS and αl-syntrophin.
Figure 12 are immunostained cryosections of skeletal muscle sarcolemma in Becker muscular dystrophy patients showing absence of nNOS from skeletal muscle sarcolemma in patients with Becker muscular dystrophy. Figure 13 is a molecular model of nNOS and NMDA receptor binding PSD-95. Figure 14 shows alignment of PSD-93 and PSD-95 three PDZ repeats, a SH3 domain and a region homologous to guanylate kinase.
Figure 15 shows expression of PDS-93, PDS-95 and nNOS in a rat brain and E15 embryos.
Figure 16 illustrates PSD-95 colocalization with nNOS in developing neurons.
Figure 17 illustrates nNOS binding to PSD-95 through PDZ motif interaction. Figure 18 shows alternative splicing of exons 1 and 2 of nNOS.
Figure 19 shows that catalytically active nNOS isoform lacking exon 2 are expressed in the brain in nNOS Δ/Δ.
Figure 20 shows that nNOS isoforms lacking the PDZ motif do not bind to PSD-95 or to brain membranes.
Figure 21 illustrates binding of αl syntrophin to the N-terminal PDZ containing domain of nNOS.
Figure 22 shows direct binding of nNOS to αl- syntrophin PDZ domain. DEFINITIONS
As used herein:
"NOS" means nitric oxide synthase, an enzyme that regulates production of nitric oxide.
"NO" means nitric oxide. "nNOS" means neuronal nitric oxide synthase.
"NMDA" means N-methyl-D-aspartate receptor, which is a glutamate type receptor.
"PSD-95" means postsynaptic density-95 protein, which is present at the brain synaptic junction. "PSD-93" means poεt-synaptic denεity-93 protein, which is present at the brain synaptic junction.
"PDZ" means a N-terminal domain of nNOS, containing a 66-amino acid motif, bearing homology to a heterogeneous family of signaling enzymes localized at cell-cell junctions.
"CAM" means calmodulin.
"FMN" means flavin mononucleotide. "FAD" means flavin adenine dinucleotide.
"T-SYN" or "SYN-1" means syntrophins.
"DMD" or "DD" means Duchenne muscular dystrophy.
"BMD" means Becker muscular dystrophy. "mdx" or "mdx mice" means mice that specifically lack dystrophin due to a nonsense mutation, but express nNOS at near normal levels.
"dy" mouse means a mouse which has severe muscular dystrophy due to an absence of an extracellular matrix protein, merosin, but has a normal distribution of dystrophin at the sarcolemma.
"Syntrophins" means a family of dystrophin-binding proteins which colocalize with nNOS beneath the sarcolemmal membrane. DETAILED DESCRIPTION OF THE INVENTION
The current invention involves a discovery that nitric oxide, neuronal nitric oxide synthase and neuronal nitric oxide synthase binding proteins are involved in the development and management of a group of muscular dystrophic and neurodegenerative diseases such as stroke. Muscular dystrophic diseases are characterized by the complete absence, or by diminished level of a fully functional dystrophin. Stroke and other neurodegenerative diseases are characterized by overactive N-methyl-D- aspartate receptors linked to nitric oxide formation in neurons.
The invention comprises two parts. The first part is directed to the diagnosis and treatment of muscular dystrophies. The second part is directed to the diagnosis, prophylaxis and treatment of stroke and other neurodegenerative diseases.
I. Diagnosis and Treatment of Muscular Dystrophies
Patients suffering from muscular dystrophy have been known to either lack the protein dystrophin completely or to possess dystrophin which is somehow dysfunctional. It has now been discovered that not only dystrophin but also nitric oxide synthase is absent or its level is lower in the skeletal muscle of the muscular dystrophy patients.
Neuronal nitric oxide synthase is localized in sarcolemma of fast-twitch fibers and it has now been shown that nNOS partitions with skeletal muscle membranes on account of its association with dystrophin. The dystrophin is associated with intracellular and transmembrane glycoproteins forming a dystrophin-associated complex. The dystrophin complex interacts with a N-terminal domain of nNOS that contains a PDZ motif. Muscles of muscular dystrophy patients show selective loss of nNOS protein and loss of catalytic activity from muscle membrane.
While patients with Duchenne dystrophy lack dystrophin completely and dystrophin of patients with Becker muscular dystrophy is mutated, it has now been found that in both cases there is a loss of nNOS in the skeletal muscle. In healthy individuals, nNOS is concentrated at synaptic junctions at motor endplates of the skeletal muscle where the N-terminus domain of nNOS, which contains a PDZ protein motif, binds to the PDZ motif present in αl-syntrophin. The PDZ domain thus mediates binding of nNOS to skeletal muscle syntrophin, a dystrophin associated protein.
The invention also describes a method of use of nitric oxide, neuronal nitric oxide synthase, its binding proteins and their inhibitors for diagnosis and treatment of muscular dystrophy. A diagnostic assay for detection of absence of dystrophin or its mutated form and a method for treatment of muscular dystrophy by restoration of a functional dystrophin molecule in dystrophic muscles using gene therapy are also disclosed. Absence or Deficiency in Formation of Dvstrophin-nNOS Complex in Muscular Dystrophy
Muscular dystrophies have been characterized by the complete absence of dystrophin in Duchenne dystrophy or by truncated dystrophin in Becker muscular dystrophy. It has now been discovered that additionally, these diseases also lack the normal level of neuronal nitric oxide synthase. Duchenne Dystrophy and a Function of Neuronal
Nitric Oxide Svnthase in Skeletal Muscle
Nitric oxide (NO) is synthesized in skeletal muscle by neuronal-type nitric oxide synthase (nNOS) , which is localized to sarcolemma of fast-twitch fibers. Synthesis of NO in active muscle opposes contractile force. It has now been shown and described in studies below that nNOS partitions with skeletal muscle membranes owing to association of nNOS with dystrophin, the protein missing in Duchenne muscular dystrophy (DMD) . In healthy muscle, the dystrophin complex interacts with the N-terminal domain of nNOS that contains a PDZ motif. In the muscular dystrophy
(mdx) mice model and in human DMD skeletal muscle samples, a selective loss of nNOS protein as well as loss of catalytic activity from muscle membranes was found, demonstrating a novel role for dystrophin in localizing a signaling enzyme to the myocyte sarcolemma. Aberrant regulation of nNOS production is therefore suspected to contribute to preferential degeneration of fast-twitch muscle fibers in DMD.
Presence and Absence of nNOS in Skeletal Muscle
Sarcolemma
Neuronal NOS is present in cytoskeletal extracts from a healthy skeletal muscle and have been found to be associated with membrane.
In order to elucidate a function of nNOS in muscular dystrophies and to understand the mechanism of membrane association of nNOS, the extractability of nNOS from mouse quadriceps was investigated and compared to extractability of eNOS. Results are seen in Figure 1.
Figure 1 illustrates differential extractability of nNOS and eNOS in mice skeletal muscle homogenates. For these studies, tissue was prepared, extracted and submitted to Western blot analysis as described in Example 1. After subcellular fractionation and 7.5% SDS-PAGE (100 μg protein per lane) , nNOS and eNOS were sequentially detected by protein immunoblot. Positions of molecular size markers are indicated in kilodaltons.
Obtained results, shown in Figures IA and IB, show that nNOS is anchored both to microsomal membranes (S2 and S3) and to cytoskeleton (P) whereas eNOS is only found in microsomal membranes (S2 and S3) .
Figure IA is a Western blot showing an association on nNOS of an insoluble pellet (P) , as well as mouse quadriceps homogenates sequentially extracted as indicated. Western blotting indicates that significant nNOS remains in an insoluble pellet (P) following sequential extraction of mouse quadriceps homogenates with 100 mM NaCl (S,) , 500 mM NaCl (S2) , and 0.5% Triton X-100 (S3) , lower levels of nNOS are present in each of these fractions.
Figure IB is a Western blot showing association of eNOS with particulate fractions. As seen in Figure IB, eNOS is found only in membrane-associated fractions but is not present in cytosol and in the insoluble pellet. Probing the same blot with an eNOS monoclonal antibody indicates the eNOS is completely extracted by 500 mM NaCl (S2) and 0.5% Triton X-100 (S3) .
Results of this study show that nNOS is anchored both to microsomal membranes and to cytoskeleton. Thus, the majority of nNOS protein remained membrane-associated following extensive washing of skeletal muscle heavy microsomes with 0.5 M NaCl. Solubilization of washed membranes with 0.5% Triton X-100 released about half of this particulate nNOS, with the remainder found to be in an insoluble cytoskeletal pellet (Figure IA) . By contrast, eNOS, which is membrane-associated owing to N-terminal myristoylation, was quantitatively solubilized from these same preparations by Triton X-100 (Figure IB) .
The differential fractionation of nNOS and eNOS seen in Figure 1 suggests that unique determinants present in nNOS anchor this isoform to the skeletal muscle cytoskeleton.
To further investigate these findings, cofactor- binding domains of eNOS and nNOS were compared using a schematic alignment. Results are seen in Figure 2 which shows that the extended N-terminus of nNOS contains a PDZ domain that iε not present in eNOS and not required for enzyme normal activity. Figure 2A shows schematic alignment of cofactor- binding domains of eNOS and nNOS, indicating N-terminal myristoylation (Myr) of eNOS and extended N-terminus of nNOS. The jagged line indicates the region deleted for the nNOSΔl-226 mutant. CaM indicates calmodulin, FMN indicates flavin mononucleotide and FAD indicates flavin adenine dinucleotide regions.
As seen in Figure 2A, the amino acid sequence of nNOS contains a 230 amino acid N-terminal domain that is not present in eNOS. Beyond this extended N-terminus of nNOS, the two proteins share >60% sequence identity. Similar enzymatic activities of eNOS and nNOS suggest that the unique N-terminus of nNOS is not required for catalytic activity and has another function.
Figure 2B shows a schematic alignment of the PDZ domain of nNOS with syntrophins and a family of other cytoskeletal-associated proteins. T-SYN and SYN-1 indicate syntrophins, DLG indicates disks large, DSH indicates disheveled and PTP indicates protein-tyrosine phosphatase.
Rather than regulating catalytic activity, the N- terminal domain of nNOS instead εeemε to target nNOS to skeletal muscle sarcolemma. Within this domain, nNOS contains a 66 amino acid motif that bears homology to a heterogeneous family of signaling enzymes that share the property of being localized to specialized cell-cell junctions as seen in Figure 2B. Proteins containing this motif, which is named PDZ for a conεerved tetrapeptide (Neuron. 9: 929-942 (1992)), include dlg-l, the product of the lethal discs large tumor suppressor gene that localizes to the undercoat of the septate junction in Drosophila; disheveled, a gene required for planar cell polarity in Drosophila; PSD-95, a brain-specific protein; ZO-1, a protein that localizes to tight junctions (zona occludens) of epithelial and endothelial cells; and certain protein- tyrosine phosphatases such as PTPIE, which are localized at the junction between the plasma membrane and the cytoskeleton. Homology to syntrophins, a family of recently cloned dystrophin-binding proteins, which colocalize with nNOS beneath the sarcolemmal membrane of skeletal muscle, was observed.
In fact, aε seen in Figure 2B, syntrophins are more closely related to nNOS in this domain than any other known gene, which suggests a dystrophin role in regulating the sarcolemmal localization of nNOS.
To analyze the role of the extended N-terminal region of nNOS, a deletion mutant nNOSΔl-226, lacking the first 226 amino acids was constructed. Expreεεion vectorε containing full-length nNOS and nNOSΔl-226 were transiently transfected into COS cells.
In Figures 2C and 2D, COS cells were transfected with 10 μg of the expression vector using a cytomegalovirus promoter to drive expression of either full-length nNOS (Fig. 2C) or the truncation mutant nNOSΔl-226 (Fig. 2D) . NOS activity was meaεured in cell homogenates 3 days following transfection in the presence of either 200 μM free calcium (full squares) or 2 mM EDTA (full circles) . Kinetic constants (V^ and K. were calculated by Scatchard plot analysiε. Data are means of triplicate determinations that varied by <10%. This experiment was replicated twice with similar resultε.
As seen in Figures 2C and 2D, kinetic characteriεticε of NOS activity for the nNOSΔl-226 mutant waε eεsentially indistinguishable from the full-length isoform. Both constructs displayed similar Vωι and K,,, for arginine as well as regulation by calcium/calmodulin.
Results of these experimentε εhow that nNOS and eNOS aεεociate with different cellular membrane fractions and that nNOS but not eNOS is present in cytoskeletal extractε from εkeletal muεcle. Association of nNOS with Dystrophin
Based on the above findings of nNOS homology to syntrophins, dystrophin binding proteins, association of nNOS and dystrophin in skeletal muscle extracts waε investigated.
Results of several studies of association of nNOS and dystrophin using affinity chromatography in skeletal muscle are seen in Figure 3. Affinity chromatography waε performed according to Example 2. For Figures 3A-3C, protein homogenates (200 μg per lane) and aliquots of affinity eluates (150 μl per lane) were reεolved on 7.5%
(α-nNOS) or 6% (α-dyεtrophin) SDS gelε and tranεferred to
PVDF membranes, and immunoreactive bands were visualized by chemiluminescence. A dystrophin-nNOS aεsociation complex was investigated by means of succinylated wheat germ agglutinin (εWGA) using εWGA-Sepharoεe affinity chromatography. Thiε technique allowε distinction between nNOS which, due to its lack of glycosylation sites does not bind to wheat germ column, while dystrophin binds to a glycoprotein complex.
To ensure specificity of this asεociation, parallel experimentε were conducted with mdx mice that εpecifically lack dyεtrophin owing to a nonsense mutation, but expresε nNOS at near-normal levels. Quadriceps from wild-type and mdx mice were homogenized and solubilized in a buffer containing 0.2 M NaCl and 1% digitonin. Solubilized homogenates were applied to sWGA-Sepharose columns that were extensively washed with a buffer containing 0.5 M NaCl and 0.5% Triton X-100. Tightly bound proteins were affinity eluted with 0.3 M N-acetyl-D-glucosamine (NAG). Results are seen in Figure 3A.
Figure 3A shows dystrophin-asεociated glycoprotein complex and nNOS purified by εWGA chromatography from wild- type (WT) , and mdx skeletal muscle.
As seen in Figure 3A, Western blotting indicates that total nNOS levels in crude extracts are similar in wild- type (WT) and mdx muscle and revealε the presence of nNOS in NAG eluates from wild-type but not mdx tisεue. nNOS coelutes with dystrophin on a sWGA affinity column in muscle homogenates from wild-type mice. nNOS does not adhere to an sWGA column in extracts from mdx mice.
Additionally, analogous experiments were conducted and association of dystrophin with nNOS was evaluated using a 2 ' ,5'-ADP-agaroεe column, which tightly binds nNOS at its C-terminal NADPH-binding motif. Dystrophin has no nucleotide-binding εite, and would not be expected to adhere to a 2',5'-ADP column. To enεure εpecificity of a potential nNOS-dystrophin interaction, parallel purifications from skeletal muscle of nNOS knockout mice, which are devoid of full length nNOS protein yet express dyεtrophin at normal levels, were conducted. Salt-washed heavy microsomes from wild-type and full length nNOS knockout mouse quadriceps were solubilized in 1% digitonin and allowed to adhere to 2' ,5'-ADP-agarose columns. The columns were extensively washed with buffers containing 0.5 M NaCl and 0.5% Triton X-100. Tightly bound proteins were eluted with buffers containing 20 mM NADPH. Results are seen in Figure 3B.
Figure 3B shows dystrophin-associated glycoprotein complex and nNOS purified by 2' ,5'-ADP-agaroεe chromatography from wild-type (WT) , mdx, and NOS knockout (NOS1) skeletal muscle. As seen in Figure 3B, Western blotting for dystrophin reveals that dystrophin levelε are equivalent in crude εampleε from wild-type (WT) and nNOS knockout (NOS1) skeletal muscle. Dystrophin coelutes with NOS on a 2',5'-ADP affinity column in muscle homogenateε from wild-type but not for nNOS knockout mice.
To evaluate directly the binding of dystrophin- associated complexes to the N-terminal domain of nNOS, a protein containing glutathione S-transferase (GST) fused to the first 299 amino acids of nNOS was coupled to glutathione beads according to the method described in Example 3. The GST-nNOS(1-299) beads and control GST beads were incubated with εolubilized homogenateε of mouse skeletal muscle. After extensive washing of the beads, bound proteins were eluted with sample buffer. Results are seen in Figure 3C. In Figure 3C, glutathione-Sepharose beads bound to GST or GST-nNOS(1-299) were incubated with solubilized skeletal muscle membranes. After extensive washing, the beads were eluted with 0.2% SDS and proteins were separated by SDS- PAGE, and retention of dystrophin protein was analyzed by Western blotting. Western blotting indicated that GST- nNOS(1-299) beads but not control beadε retained the dyεtrophin protein.
Other proteins which were concurrently evaluated, such aε myoεin, were retained in very εmall but equivalent amounts by both GST and GST-nNOS(1-299) beads indicating specificity of the association with dystrophin.
To quantitate enrichment of nNOS by sWGA chromatography, scale purifications from rat skeletal muscle tissue were conducted as seen in Figure 3D where L indicates load, F indicates flowthrough, Wl indicates 500 mM/NaCl wash, W2 indicates 500 mM NaCl and 0.5% Triton X- 100 wash, and E indicates 0.3 M NAG eluate. Results are seen in Figure 3D.
Figure 3D depicts Western blotting for nNOS from equally loaded fractions (5 μg per lane) from sWGA chromatography εhowε large enrichment of nNOS in NAG eluate fractions.
To evaluate the retention of nNOS by sWGA, fractions from sWGA chromatography were reloaded onto SDS gelε with 6-fold more total protein in load (L) and flowthrough (F) laneε (21 μg) than in waεh (Wl and W2) and eluate (E) lanes (3.5 μg) . Results are seen in Figure 3E.
In Figure 3E reloaded samples from the sWGA column εhow purification of nNOS and dyεtrophin, but not eNOS, by εWGA. Clearly, the majority of particulate nNOS adhered to the εWGA column as evidenced by the minimal amount of nNOS present in the flowthrough. When the blot shown in Figure 3E was probed with antisera to dystrophin, it was found that essentially all the solubilized dystrophin adhered to the sWGA column and that dystrophin was enriched to a somewhat greater degree than nNOS by this procedure (- 150-fold purification) . The somewhat higher recovery of dyεtrophin appears to be due to slow disεociation of nNOS from the εWGA column due to itε binding within the dyεtrophin complex under stringent conditions, as evidenced by some "bleeding" of nNOS with the 500 mM NaCl and 0.5% Triton X-100 wash steps (Figure 3E) .
Additionally, this same blot was probed with an antibody to eNOS, which is similar to nNOS but lacks a PDZ domain. eNOS did not specifically adhere to the sWGA column, and only residual amounts of eNOS were found in NAG eluate fractions.
To determine whether the nNOS associates with dystrophin complexes eluting from sWGA, immunoprecipitation experiments were conducted. NAG eluate samples were incubated for 1 hour with a monoclonal antibody to α- dystrophin, and immunocomplexes were pelleted with anti- ouεe immunoglobulin G (IgG) linked to protein A-Sepharoεe as described in Example 4. Results are seen in Figure 3F.
Figure 3F shows an immunoprecipitation of NAG eluate fractions with a monoclonal antibody to dystrophin (2.0 /xg/ml; 12 nM) which precipitates nNOS (lane 1) . Control experiments containing a monoclonal anti-Myc antibody (12 nM) obtained from BABCO, or lacking any primary antibody (lanes 2 and 3) fail to precipitate nNOS. Western blot analysiε seen in Figure 3F revealed potent immunoprecipitation of nNOS with α-dystrophin antibody. Control immunoprecipitations lacking the primary dystrophin antibody or containing an alternate monoclonal antibody anti-Myc did not precipitate detectable nNOS, demonεtrating εpecificity of the interaction.
These studies show that nNOS coelutes with dystrophin on a εWGA or a 2',5'-ADP affinity column in healthy muscle homogenates but not in extracts from mdx or knockout mice and that dystrophin binds to the N-terminal domain of nNOS. Neuronal NOS from equally loaded fractions from sWGA chromatography shows large enrichment of nNOS in NAG eluate fractions and nNOS and dystrophin, but not eNOS, were purified by sWGA. This showε that a large fraction of nNOS actually binds to the dystrophin complex.
Immunoprecipitation of nNOS was found upon incubation with a monoclonal antibody to α-dystrophin, but not with other control antibodies.
These results clearly indicate association of nNOS with dystrophin or dystrophin-aεεociated complex. Displacement of nNOS from Sarcolemma The absence of dystrophin was shown (J. Cell. Biol.. 112: 135-148 (1991)) to lead to a dramatic reduction of other dystrophin-aεεociated proteins, including α- and β- dystroglycan and syntrophins, in the sarcolemma of mdx mice and in patientε with DMD (J. Biol. Chem.. 266: 9161-9165 (1991)). In order to determine whether the same is valid for nNOS, the distribution of nNOS in the mdx mice brain and skeletal muscle tissue was evaluated. Reεultε are seen in Figure 4.
Figure 4 illustrates nNOS displacement from particulate fractions of the mdx skeletal muscle.
In Figure 4, subcellular fractions (S,, S2, S3, and P) of mouse quadriceps from healthy wild-type (WT) and mdx mice (age matched at 7-8 weeks) were prepared as described in Figure 1 and nNOS and dystrophin were detected by Western blotting. Results are shown in Figures 4A and 4B and in Table 1.
Western blotting seen in Figure 4A shows that nNOS in the skeletal muscle of mdx mice is largely extracted from membranes with 100 mM NaCl (S,) and is completely removed with 500 mM NaCl (S2) . In wild-type mice, the majority of nNOS remains membrane-associated following the 500 mM NaCl waεh. Of this remaining nNOS, approximately half is removed by a 0.5% Triton X-100 extraction (S3) and half is present in an insoluble pellet (P) .
Figure 4B shows that dystrophin is enriched in detergent extract (S3) and cytoskeletal pellet (P) fractions in wild-type mice (WT) and is completely absent from the mdx muscle.
As seen in Figures 4A and 4B, overall nNOS levels and enzyme activity were modestly decreased (-80% of control levels) in skeletal muscle from mdx mice. Subcellular analysis revealed that nNOS distributed with dystrophin in membrane-associated and cytoskeletal fractions from wild- type skeletal muscle. However, in preparations from mdx mice, nNOS was quantitatively solubilized from microsomal membranes washed with 0.5 M NaCl. No nNOS protein was detected in detergent extract or cytoskeletal fractions (Figure 4A) .
Displacement of nNOS from skeletal muscle and brain tisεue of WT and mdx mouse is seen in Table 1.
TABLE 1
NOS Activity in Extracts from Wild-Type and mdx Mouβe-TiβBuee
NOS Activity Wild Type" ϊnδP
S Skkeelleettaall mmuussccllee
Soluble fraction 33. 3 58.3
Particulate fraction 252 <0.5
Brain
Soluble fraction 1020 1060
Particulate fraction 1080 1210
Particulate and soluble fractions of quadriceps εkeletal muscle from wild-type and mdx mice were prepared and assayed for NOS activity aε deεcribed in
Example 1.
Data are means of triplicate determinations that varied by <10%. "In counts per minute per milligram of protein.
Aε εeen in Table 1, NOS catalytic activity waε obεerved in εoluble and particulate fractionε in εkeletal uεcle and in brain in WT mouse tissue. In mdx tisεue, nNOS was found in the soluble but not in the particulate fraction. NOS activity in the soluble fraction of mdx skeletal muscle occurred at levels 75% greater than wild- type, but NOS activity was not detectable in the particulate fraction from mdx muscle. In the brain, NOS- specific activity was nearly equivalent in soluble and particulate fractions. Thiε distribution was unchanged in the mdx brain, suggesting that proteins other than dystrophin anchor nNOS to neuronal membranes.
The above findings were confirmed with immunohisto- chemical findings of an absence of sarcolemmal nNOS in mdx skeletal muscle. Cryosections were prepared and immunostaining was performed according to Example 5. Results are seen in Figure 5.
Figure 5 shows immunofluorescent εtaining for nNOS in quadricepε of wild-type, mdx, dy, and nNOS knockout mice performed uεing an affinity-purified polyclonal antibody. Cryostat mice sections from wild-type, mdx, dy, and nNOS knockout mouse were stained under identical conditions using an affinity-purified nNOS antiserum and a FITC-linked εecondary antibody.
Figures 5A and 5B show that nNOS immunostaining is present at the surface membranes of skeletal muscle fibers from wild-type (WT) mouse (A) , but is absent from mdx mouse (B) skeletal muscle sarcolemma. To determine whether this derangement of nNOS was specific for dystrophin abnormalities, the distribution of nNOS in dy mice, which have severe muscular dystrophy due to absence of an extracellular matrix protein, merosin, but have a normal diεtribution of dyεtrophin at the sarcolemma, was evaluated.
Figure 5C shows nNOS distribution in homozygous dystrophic dy mice displaying normal sarcolemmal nNOS labeling of intact fibers. Results show that nNOS is present normally at the sarcolemma of dy dystrophic mice (Figure 5C) .
Figure 5D shows that skeletal muscle from NOS knockout (NOS"1") mice is entirely devoid of immunostaining. The above results show that nNOS is selectively absent from sarcolemma of mdx skeletal muscle.
Using a polyclonal antiserum, nNOS immunofluorescence found in Figure 5 was restricted to the sarcolemma of a subset of WT skeletal muscle fibers. These fibers were previously noted to be fast twitch fibers (Nature. 372: 546-548 (1994)). However, nNOS immunoreactivity was absent from the sarcolemma of mdx muscle.
Several control experiments were conducted to ensure a specificity of the immunofluorescence. No specific labeling was present in muscle tiεεues incubated with preimmune serum or without primary antiserum. Most importantly, labeling waε not preεent in tranεgenic knockout mice (NOS1) containing a targeted mutation of the nNOSε gene (Figure 5D) .
Thiε εtudy clearly shows that in dystrophic muscle tisεue nNOS iε displaced from εarcolemma but accumulateε in cytosol.
Absence of nNOS from Skeletal Muscle Sarcolemma in Human Duchenne Muscular Dvstrophv Patients
In follow-up studieε described above, and to show that the same results are observed in animal models and controls, the localization of nNOS in healthy and DMD human muscle tissues was evaluated. A total of 20 human tissues were prepared according to Example 8 and evaluated. These tissues included thirteen normal specimens and seven specimens from patients with DMD. Skeletal muscle crosε εections were processed for immunofluorescence for nNOS, dystrophin, and spectrin. Results are seen in Figure 6.
Figure 6 are skeletal muscle cryosectionε of normal (NI) or DMD (DI) skeletal muscles immunostained with antibodies to dystrophin, nNOS, and spectrin.
Figure 6B showε repreεentative muεcle εectionε, labeled with antibodies, from two normal patients (N2 and
N3) and two DMD cases (D2 and D3) , to dystrophin and nNOS.
Figure 6C which represents control experiments using two independently generated nNOS antiεera εhows similar staining of human tissueε. No immunofluorescence was detected in the absence of primary (1°) antibody.
All normal specimens showed colocalization of nNOS, dystrophin, and spectrin beneath the sarcolemma of muscle fibers (Figures 6A and 6B) , indicating that nNOS shares a similar distribution in both human and rodent skeletal muscle. In all seven biopsies from patients with DMD, the disruption of dystrophin resulted in absence of nNOS staining of sarcolemma (Figures 6A and 6B) . Normal sarcolemma labeling for spectrin confirmed that this structural cytoskeleton was not disrupted in the DMD tiεsues (Figure 6A, spectrin) . Two independently raised nNOS antibodieε according to Example 3 yielded εimilar immunofluorescent staining patterns. No specific labeling was found in muscle εectionε incubated without primary antibody (Figure 6C) .
Results seen in Figure 6 shows that nNOS is absent from sarcolemma of human DMD muscle fibers. Absence of dystrophin in DMD results in disruption of the dystrophin-associated glycoprotein complex and in a dramatic reduction of overall levels of certain dyεtrophin- associated proteins in muεcle. To evaluate total nNOS levels in skeletal muscle tissues from human DMD, Western blot analysis was conducted. Results are seen in Figure 7. In Figure 7, skeletal muscle tisεue homogenates from three cases of DMD and three normal muscle biopsieε were resolved by SDS-PAGE as described in Example 1.
Immunoblot analysis seen in Figure 7A confirms that dystrophin is present in normal human muscle but is essentially absent from the human DMD muscle. Densitometric scanning of nNOS immunoreactive bands in equally loaded Western blots revealed -75% decrease of nNOS in DMD tissues when compared to the normal human muscle. Immunoblotting for spectrin seen in Figure 7C confirmed that similar amounts of protein were loaded in all cases and that the structural cytoskeleton of these samples remained intact.
The above series of studies demonstrates that both human and mouse skeletal muscle sarcolemmal nNOS is complexed with dystrophin. The dystrophin complex interacts with the N-terminus of nNOS, which contains a PDZ motif. In normal healthy muscle, nNOS is preεent in sarcolemma. In human DMD muscle and mdx mice, which lack dyεtrophin, nNOS iε abεent from the sarcolemma and accumulateε in the cytoεol. This derangement of nNOS iε specific for dystrophin abnormalities, as nNOS disposition is unaffected in other muscular diseases.
The obtained results provide molecular evidence for a specific intracellular signaling molecule linked to the dyεtrophin-aεsociated complex and suggest roles for NO in processes of neuro-muscular development and diseaεe associated with thiε complex.
The PDZ domain is a protein motif that is present in a heterogeneous family of enzymes. The current invention investigated and discovered that deletion of the PDZ domain of nNOS does not alter NOS catalytic activity in transfected cellε. A 299 amino acid fuεion protein containing the PDZ domain in nNOS selectively retained dyεtrophin from skeletal muscle extracts, indicating that this domain is capable of interacting with the dystrophin- associated complex.
These results suggest potential roles for nNOS in neuromuscular signaling and disease associated with dystrophin. Asεociation of nNOS with dyεtrophin completes the link between the extracellular matrix and intracellular signal-transducing enzymes. Aberrant translocation of nNOS from sarcolemma to cytosol in DMD and mdx muεcle haε implicationε for the pathogeneεis of muscular dystrophy.
As nNOS has been unqueεtionably implicated in the DMD where the dyεtrophin iε abεent, and nNOS was εhown to be diεplaced, the εecond εeries of studies was designed to investigate whether these findings would also be valid for other, not so severe, types of muscular dystrophies where dystrophin is not completely absent but is mutated and to a certain degree dysfunctional.
Consequently, evaluation of nNOS distribution in a variety of muscle diseases and dystrophies was undertaken. In these follow-up studies it was found that nNOS occurs normally at the sarcolemma in human neurogenic muscle atrophy, central core disease, and severe childhood autosomal recessive muscular dystrophy but that it is, however, displaced from εarcolemma of Becker muεcular dystrophy εuggeεting εpecificity of the defect of nNOS in DMD and BMD.
Selective Loss of Sarcolemmal NOS in Becker Muscular
Dystrophy
Becker muscular dystrophy is a clinical variant of Duchenne muscular dystrophy. It differs from the more severe Duchenne dyεtrophy in that Becker muεcular dyεtrophy patients do not lack dystrophin completely but their dystrophin is of abnormal molecular weight due to chronic mutation at Xp21. Becker muscular dystrophy patients, therefore, have reduced amounts of normal-sized dystrophin protein.
Becker muscular dyεtrophy is an X-linked disease due to mutations of the dystrophin gene. Mutations causing Becker's dystrophy are often in-frame deletions in the central rod-like domain of dystrophin that do not generally affect formation of the structural glycoprotein complex formed around dyεtrophin in the muεcle.
In the studies described below, it was discovered that neuronal-type nitric oxide synthase (nNOS) , an identified signaling component of the dystrophin complex, is uniquely absent from skeletal muscle sarcolemma in many human Becker's patients and in mouse models of Becker's dystrophy. This is in agreement with finding the same displacement of nNOS from sarcolemma in DMD patientε. A N-terminal PDZ domain of nNOS directly interactε with αl-syntrophin but not with other proteins in the dystrophin complex analyzed. However, nNOS does not asεociate with αl-εyntrophin on the sarcolemma in certain human Becker's dystrophy patients and in transgenic mdx mice expressing truncated dystrophin proteins. This suggests a macromolecular interaction of nNOS, αl- syntrophin and dystrophin in vivo , a conclusion supported by developmental εtudieε in muεcle. The data below indicate that proper aεεembly of the dyεtrophin complex iε dependent upon the structure of the central rod-like domain and have implications for the design of dystrophin- containing vectors for gene therapy.
Sarcolemma nNOS Expression During Postnatal Muscle Development
This section deεcribes studies performed to investigate sarcolemmal nNOS expresεion during poεtnatal muεcle development.
The studies deεcribed above εhow that association of nNOS with the dystrophin complex is mediated by direct binding of the N-terminus of nNOS to the PDZ domain of αl- syntrophin. In the current study, illustrated in Figure 8, localization of nNOS and other dystrophin asεociated proteins during postnatal development was investigated. In the postnatal day 3 (P3) , day 7 (P7) , day 12 (P12) and day 60 (P60) samples of rat quadriceps were obtained and investigated. Adjacent sections of postnatal rat quadriceps muscle were stained for dystrophin, nNOS, α-BGT, and utrophin and nearby sections were stained for αl- syntrophin and α-BGT. Staining was performed according to Nature Genetics. (1996) . Aε seen in Figure 8, dystrophin and nNOS stained extrajunctional sarcolemma at days P3 and P7 and both became concentrated at neuromuscular endplates at days P12 and P60. αl-syntrophin was present at extrajunctional sarcolemma and was enriched at neuromuscular endplates at all ages evaluated. Utrophin staining was restricted to neuromuscular endplates and was specifically enriched at neuromuscular endplates. By contrast at postnatal day 3 (P3) and P7, nNOS occurred only at extrajunctional sarcolemma and enrichment of nNOS at neuromuscular endplates did not become apparent until P12, which coincided with asεembly of dystrophin complexes at endplates. Enrichment of αl-syntrophin at endplates of P3 and P7 muscle likely occurs by association with the dystrophin related protein, utrophin, which was enriched at endplates in all stages evaluated.
These studies indicate an apparent requirement of dystrophin for nNOS/αl-syntrophin colocalization at neuromuεcular endplates during development and suggest that nNOS doeε not aεεociate with utrophin-containing protein complexes.
Association of nNOS with Mutant Dvεtrophin Complexeε in Transgenic Mice
In order to investigate whether nNOS association with the dystrophin complex correlates with the dystrophic disease phenotype, several tranεgenic mice models carrying either the full length dystrophin or truncated dystrophin were investigated.
In studieε to inveεtigate association of nNOS with mutant dystrophin complexes in transgenic mice, nNOS, dystrophin asεociated proteins (α-BGT) and αl-syntrophin expression were compared in skeletal muscle of wild type, mdx and various transgenic mice that express mutant forms of dystrophin, such as full-dys, Δ330, mdx ΔEXON 17-48, ini-dys or Dp71 mutant lines.
Results are seen in Figure 9 which shows localization of nNOS and αl-syntrophin in transgenic mdx mice. In Figure 9, cryosections from mouse quadriceps were immunofluorescently double labeled for either nNOS, -αl- syntrophin and α-BGT.
Aε seen in Figure 9, immunofluorescent εtaining showed that nNOS in wild type mice was expressed at extrajunctional sarcolemma of a subset of fibers and was enriched at all neuromuscular endplates. nNOS was absent from junctional and extrajunctional sarcolemma in mdx mice. nNOS εtaining in mdx transgenic mice expresεing full length dyεtrophin (full-dys) or truncated dystrophin lacking the C-terminal 330 nucleotides (Δ330) , resembled that of wild type mice. mdx mice expressing dystrophin lacking exons 17-48 (mini-dys) or lacking the C-terminal 71 kDa of dystrophin did not show nNOS staining at sarcolemma. These results corresponded to results observed in non-transgenic mdx mice. αl-Syntrophin staining was observed at extra- junctional sarcolemma and was concentrated at neuromuscular endplates in wild type mice but was restricted to the endplates in mdx mouse. αl-Syntrophin expression was restored to sarcolemma in the four transgenic mdx mouse lines expressing different portions of the dystrophin gene. These studies confirmed that αl-syntrophin was abεent from extrajunctional εarcolemma of mdx mice, but remained at its neuromuscular endplates. nNOS was absent from both junctional and extrajunctional sarcolemma of mdx mice. By contrast nNOS was restored to the sarcolemma only by full length dystrophin and the Δ330 mutant. The absence of sarcolemmal nNOS was thus closely correlated with diseaεe phenotype in theεe tranεgenics.
Biochemical confirmation of the above studies is illustrated in Figure 10. Figure 10 shows subcellular distribution of nNOS in WT, mdx, and transgenic mdx mice. Mouse quadriceps skeletal muscle homogenates were sequentially extracted with buffers containing 100 mM NaCl (SI) , 500 mM NaCl (S2) , and 0.5% Triton X-100 (S3), leaving an insoluble cytoskeletal pellet (P) .
Figure 10A is Western blotting indicating that nNOS was enriched in membrane asεociated and in pellet fractions in wild type mice (lanes 1) and in transgenic mdx mice expressing full length dystrophin (lanes 4) . In mdx mice (lanes 2) and Dp71 transgenic mdx mice (lanes 3) , nNOS was fully extracted by 500 mM NaCl and was absent from the membrane associated and cytoskeletal fractions. In Figures 10B and IOC a similar fractionation was performed on muscle homogenates from wild type mouse (lanes 1) , mdx mice expreεsing dystrophin lacking exons 17-48 (lanes 2) , or mdx mice expresεing full length dyεtrophin (lanes 3) . In Figure 10B, nNOS was absent from membrane associated (S3) and cytoskeletal pellet (P) in mdx mice expressing the truncated dystrophin. In Figure IOC, reprobing the blot for αl-syntrophin had a generally similar fractionation in muscle from all three mice lines.
Biochemical studies illustrated in Figure 10 confirmed that nNOS did not asεociate with εarcolemma in mdx mice or tranεgenic mdx mice expreεεing either Dp71 or ΔE17-48. In wild type mice and mdx tranεgenic mice expressing full length dystrophin or Δ330, nNOS was enriched in membrane asεociated and cytoεkeletal fractionε, while in mdx, Dp71 and ΔE17-48 lines, nNOS was present only in soluble fractions of muscle, αl-εyntrophin occurred in sarcolemmal fractions of all four lines of transgenic mdx mice evaluated.
Connections between nNOS and the presence of normal length or truncated dystrophin have been clearly establiεhed by these εtudies. When dystrophin was absent from the transgenic mouse phenotype, nNOS presence was only observed in soluble but not in sarcolemmal fractions.
Specific Binding of PDZ Domain of nNOS to αl-Syntrophin Further studies were directed to answering the question whether the PDZ domain of nNOS binds to dyεtrophin or dyεtrophin-associated proteins. As previously demonstrated, a sepharoεe column linked to the fusion protein εelectively retained several components of the dystrophin complex from crude skeletal muscle extracts. To determine which components directly interact with nNOS in vitro the dystrophin complex was disεociated by briefly adjuεting the pH of muscle extracts to 11 and then repeating the binding asεays immediately after neutralizing the extracts. Previous studies (J. Biol. Chem.. 266: 9161-9165 (1991)) have demonstrated that this procedure reversibly dissociates dystrophin from asεociated proteins.
Consequently, the current studies were directed to investigation whether the PDZ domain of nNOS specifically binds to αl-syntrophin. In these studies, a consideration was given to reεultε obtained in DMD εtudieε εhowing that the N-terminal domain of nNOS is necessary and sufficient for interaction with the dystrophin complex. Therefore, interaction of dystrophin-aεsociated proteins with a purified fusion protein containing the first 299 amino acids of nNOS was evaluated. Reεults are seen in Figure 11.
Figure 11A illuεtrateε εelective interaction of nNOS and αl-syntrophin using Western blotting. Crude solubilized membranes from mouse quadriceps were titrated with NaOH to pH 11, to dissociate the dystrophin complex, and were neutralized to pH 7.4 with 1 M TrisHCl. Native (native) and dissociated (dissoc) preparationε were incubated with agarose beads linked to either GST or GST fused to the first 299 amino acids of nNOS (G-NOS) . After extensive washing, beads were eluted with loading buffer and proteins resolved by SDS/PAGE.
Figure 11A shows that αl-syntrophin was selectively retained by G-NOS beads in both native and disεociated preparations. Reprobing the same blot with dystrophin as seen in Figure 11B, or α-sarcoglycan, aε seen in Figure 11C, revealed that G-NOS beads retained these proteins from native protein preparations. However, following dissociation of the complex, neither dystrophin nor α- εarcoglycan bound to G-NOS. The 55 kD band obεerved in input lanes from α-εarcoglycan blot appearε to be mouse IgG and was reactive with the secondary antibody used for western blotting. On the other hand, as seen in Figures 11A - 11C, αl-syntrophin continued to interact with the nNOS column but dystrophin and other components of the dystrophin complex were not retained. Binding of nNOS to Skeletal Muscle Svntroohin through
PDZ Interactions
In this study, nNOS binding to skeletal muscle syntrophin through PDZ interactions was determined.
Interaction of PDZ motifs found to be present in nNOS and PSD-95 protein in the brain, aε described in Section II raised the possibility that the PDZ domain of syntrophin represents the binding εite for association of nNOS with the dystrophin complex. Interaction between the PDZ containing domains of nNOS (amino acids 1-195) and αl- syntrophin (amino acids 59-166) was first evaluated by the yeast two hybrid system.
Results are seen in Table 2. As seen in Table 2 when co-transferred, these constructs reconstituted GAL4 transcriptional activity.
TABLE 2 Interaction Between nNOS and αl-Syntrophin
Ga DNA Gal4 Activation Colony Color Growth Binding Hybrid Hybrid
Lamin C ( 66-230) αl-βyntrophin (59-166) White p53 ( 72-390) αl-syntrophin (59-166) White nNOS ( 1-195 ) αl-syntrophin (59-166) t. Blue
Yeast HF7c and Y187 cells were cotransfor ed with expression vectors encoding various GAL4-binding domain and GAL4 activation domain fusion proteins. Each transformation mixture was plated on two synthetic dextrose plates, one lacking tryptophan and leucine and the other lacking tryptophan, leucine and histidine. Growth was, measured on histidine-deficient plates and color was measured by a 0-galactosidase colorimetric filter assay according to Nature. 340: 245-6 (1989) .
To biochemically evaluate this association, "pull-down" assays from skeletal muscle extracts using GST fused to the PDZ domain of nNOS (G-NOS) were conducted according to Example 12. Results are seen in Figure 21.
Figure 21 illustrates that αl-syntrophin binds to the N-terminal PDZ containing domain of nNOS. Figure 21A shows resultε of "pull down" assays of solubilized muεcle extractε from wild type or mdx mice using an nNOS (amino acids 1-299)-GST fuεion protein which were done aε described in Figure 19. Western blotting shows that αl-syntrophin from both wild type and mdx mice is selectively retained by the G-NOS column. Input was 20% protein.
Figures 2IB and 21C show immunoprecipitations of solubilized muscle extracts with a polyclonal antibody to αl-syntrophin which show co-precipitation of (Figure 2IB, lane 2) nNOS but not eNOS (Figure 21C, lane 2) . Control experiments with non-immune serum show no precipitation of nNOS or eNOS (lanes 1) . Bands at 55 kD represent immunoglobulin heavy chains. Figures 2ID and 2IE are subcellular fractionation of nNOS which is altered in nNOS*'' mouse muεcle. Homogenized muεcle was sequentially extracted in buffer containing 100 mM NaCl (lanes 1) , 500 mM NaCl (lanes 2) and 500 mM NaCl + 1% triton X-100 (lanes 3) . These extracts were purified by 2'5'-ADP agarose chromatography and were resolved by SDS- PAGE. Figure 2ID nNOS is preεent in both soluble and membrane associated fractions of wild type mice, while nNOS isoforms in nNOS^ are reεtricted to the cytoεol (Figure 21E) . αl-Syntrophin waε εelectively retained by the G-NOS column but did not aεεociate with a control GST protein column (Figure 21A; lanes 1,3,4). Dystrophin is retained by a G-NOS column. The absence of dystrophin in mdx mice results in disruption of the dystrophin glycoprotein complex. Therefore, association of skeletal muscle syntrophin from mdx mouse with G-NOS was evaluated. Total αl-syntrophin levels were decreased -50% in mdx muscle. Binding of αl-syntrophin to G-NOS was unaffected by the dystrophin deficiency (Figure 21A; lanes 2,5,6). To demonstrate asεociation of nNOS with εyntrophin in muεcle extractε, we conducted immunoprecipitation experimentε. A polyclonal antibody specific for αl-syntrophin selectively precipitated nNOS from solubilized skeletal muεcle microsomes (Figure 2IB) . No precipitation of nNOS occurred with non-immune antibody, and eNOS, was not specifically precipitated by the αl-syntrophin antibody (Figure 21C) . To determine the role of the PDZ domain for association of nNOS with sarcolemmal dystrophin complexeε in vivo we evaluated the subcellular distribution of nNOS isoformε in nNOS Δ/Δ mouse. Skeletal muscle homogenates were sequentially extracted with physiologic saline, the 500 nM NaCl, and finally 0.5% triton X-100. Following 2'5' ADP agarose purification of the muscle extracts, Weεtern blotting indicated that only the nNOSΦ form is expreεsed in muscle of nNOS Δ/Δ. nNOS in skeletal muscle runs as a doublet due to a 102 bp (34 amino acid) alternative splice near the middle of the gene. Subcellular fractionation indicated that nN0S waε reεtricted to soluble fractionε of skeletal muεcle (Figure 2IE) , contraεting with the soluble/particulate distribution of full length nNOS in muscle (Figure 2ID) . Protein overlay assays were performed to evaluate association of nNOS with individual domains of αl- syntrophin. The three known syntrophinε (αl, βl , and β2 ) have a common domain structure consisting of two pleckstrin homology (PH) domains, a PDZ domain and a carboxy terminal domain unique to syntrophins (SU, syntrophin unique) . Resultε are εeen in Figure 22.
Figure 22 shows the interaction between the nNOS-GST (1-299) fusion protein and the four αl-syntrophin domain fusion proteins, PHI, PDZ, PH2 and SU. Notably, only PDZ associates with nNOS. Moreover, no prominent bands were detected when the domains were overlayed with GST alone. Syntrophin domain fusion proteins (containing the T7«Tag epitope) were also used to overlay GST and nNOS-GST (Figure 22) . Only the PDZ domain of αl-syntrophin bound to nNOS and no binding was observed to GST alone.
Figure 22A is purified αl-syntrophin PHI (25kDa, lanes 1), PDZ domain (15kDa; lanes 2), PH2 (18 kDa; lanes 3) and SU domain (16 kDa; lanes 4) fusion proteinε were resolved and overlayed with GST or nNOS (1-299)-GST. The position and relative amounts of the αl-syntrophin domain fuεion proteins are indicated by immunoreactivity with a monoclonal antibody against the T7*Tag. Bound GST fusion proteins were detected by blotting with a monoclonal antibody to GST. Only nNOS-GST bound specifically to the PDZ domain of αl-syntrophin.
Figure 22B GST (lanes l) and nNOS-GST (lanes 2) were separated and overlayed with αl-syntrophin domain fusion proteins (PHI, PDZ, or PH2) or blotted with a monoclonal antibody to GST. Bound syntrophin fusion proteins were detected with monoclonal antibody to T7*Tag. Of the syntrophin fuεion proteinε teεted, only PDZ bound to nNOS; no binding to GST waε detected.
Figure 22C εhowε co-localization of nNOS and αl- syntrophin immunofluorescence at skeletal muscle sarcolemma and neuromuscular junctions which was labeled by rhoda ine α-bungarotoxin (BGT) . Figure 22D is a schematic model showing interaction of nNOS via with skeletal muscle αl-syntrophin (59K syn) connected to dystrophin dimer. The interaction of nNOS with syntrophin is via their respective PDZ domains. DG indicates dystroglycan. Figure 22 shows the interaction between the nNOS-GST (1-299) fusion protein and the four αl-syntrophin domain fuεion proteins, PHI, PDZ, PH2 and SU. Notably, only PDZ associates with nNOS. Moreover, no prominent bands were detected when the domains were overlayed with GST alone. Syntrophin domain fusion proteins (containing the T7*Tag epitope) were also used to overlay GST and nNOS-GST (Figure 22) . Again, only the PDZ domain of αl-syntrophin bound to nNOS and no binding was observed to GST alone. (1989) . Sarcolemmal nNOS Expresεion in Becker'ε Dvεtrophv
In thiε section, sarcolemmal expresεion of nNOS in Becker'ε dystrophy was investigated in order to clarify whether mutationε in the N-terminal or rod-like domains of dystrophin that cause BMD in humans are associated with altered localizations of nNOS. For this purpose, nNOS and αl-syntrophin expression in 12 BMD patients with molecularly defined deletions in the dystrophin gene were immunohiεtochemically evaluated. Immunohiεtochemical expression of nNOS, dystrophin and syntrophin was assesεed blindly. Results are seen in Figure 12.
Figure 12 shows skeletal muscle cryosections from human biopsieε from normal patients or from patients having DMD,
BMD Δ EXON 45-47, BMD Δ EXON 10-42, and α-sarcoglycan disturbances were immunostained with antibodies to dystrophin, syntrophin, α-sarcoglycan, or nNOS. All four antibodies showed sarcolemmal staining in normal patients and essentially no sarcolemmal labeling in patientε with
Duchenne muscular dystrophy (DMD) . In two patients with
BMD, due to loss of exons 45-47 or exons 10-42 of dyεtrophin, immunofluorescent labeling for dystrophin, syntrophin, and α-sarcoglycan was detected at the membrane. By contrast, nNOS sarcolemmal staining for nNOS was undetectable in these two BMD patients. nNOS labeling was present in a patient with α-sarcoglycan deficiency.
As seen in Figure 12, nNOS is absent from skeletal muεcle εarcolemma in certain patients with Becker muscular dyεtrophy.
Sarcolemmal expression of nNOS, dystrophin and εyntrophin in Becker muεcular dystrophy is illustrated in Table 3.
TABLE 3
Sarcolemmal Expression of nNOS. Dystrophin and Syntrophin in BMD
Immunofluorescence at Sarcolemma
Diagnosis Exons Deleted nNOS Dystrophin Syntrophin Normal (S92-10906) ++++ ++++ ++++ Normal (94x-243) ++++ ++++ ++++
Mild BMD (S90-14162) 45-47 0 ++++ ++++ Mild BMD (1987) 52 +++ ++++ ++++ Mild BMD (KF22) 45-48 + ++++ ++++
Int. BMD (91-1670) 3-6 + +++ ++++ Int. BMD (78-113) 1042 0 ++++ ++++ Int. BMD (S84-2812) 13-41 ++ +++ ++++ Int. BMD (590-536) 45 0 + ++
Sev. BMD (CS9004625) 8 0 + +++ Sev. BMD (S88-2698) 3-7 0 + +++ Sev. BMD (S90-14163) 45-47 0 +++ ++++ Sev. BMD (S90-107002) 51-52 0 + +++ α-sarcoglycanopathy 1 ++++ ++++ ++++ α-sarcoglycanopathy 2 - +++ +++ ++++
Human muscle biopsies were labeled by immunofluorescence. Sarcolemmal labeling was blindly evaluated by three observers from 0 to ++++. Variation between observers never varied by more than one +, and in those cases, the majority score is reported here.
Table 3 shows that loss of sarcolemmal nNOS, but not αl- syntrophin expression was highly correlated with disease phenotype. Some of the patients, with mild to intermediate disease, showed reduced but detectable nNOS staining of sarcolemma. In several patients, losε of sarcolemmal nNOS occurred despite apparently normal asεembly of other components of the dystrophin-aεsociated glycoprotein complex seen in Figure 12. By contrast, nNOS expression was intact in two patients with α-sarcoglycan deficiency, εuggeεting that abnormalities of nNOS are not a consequence of muscular dystrophy, but are specific for dystrophin-linked diseaεe. A principal finding of this study is that assembly of nNOS into the dystrophin complex is dependent upon the structure of the N-terminal and rod-like domains of dystrophin. Understanding the mechanism for nNOS association with the dystrophin complex is particularly important because disruption of this interaction broadly correlateε with diεease phenotype in certain animal models of muscular dystrophy and in patients with BMD. Absence of sarcolemmal nNOS in mdx mice expressing a dystrophin mini-gene indicates a role for the rod-like domain of dystrophin.
Studies of nNOS expression in BMD patients demonstrate that non-overlapping deletions in the N-terminal or central domain of dystrophin disrupt recruitment of nNOS to the sarcolemma. These results indicate that a unique nNOS interaction domain may not be present in dystrophin, but that proper conformation is required for assembly of nNOS into the dystrophin complex.
Studies described above further indicate that direct interaction of nNOS with αl-syntrophin accounts for association of nNOS with the dystrophin complex. Three syntrophin genes have been identified and each contains two pleckstrin homology (PH) domains. The first PH domain is split by a PDZ motif, and the second PH domain is followed by a C-terminal region unique to the syntrophins (J. Biol. Chem.. 270: 25859-25865, (1995)). Interaction of nNOS with αl- syntrophin is mediated by direct association of PDZ protein- binding interfaces near the N-terminus of nNOS and αl- syntrophin.
Studies described in this section are consistent with findings of the nNOS function in DMD and further demonstrate that αl-syntrophin, but not dystrophin, β-dystroglycan or α- sarcoglycan, directly binds to the PDZ domain of nNOS following dissociation of the dystrophin complex. NOS isoforms lacking a PDZ motif do not associate with the dystrophin complex further confirming that the PDZ domain of nNOS represents the relevant domain for interaction. Neuronal NOS does not to interact with utrophin containing complexes. During muscle development, nNOS occurs only at sites co-localized for both αl-syntrophin and dystrophin at developing neuromuscular endplates. Similarly, utrophin complexes at neuromuscular endplates of mdx mice specifically lack nNOS. Taken together with biochemical studieε showing a selective and direct interaction of nNOS with αl-syntrophin in vitro , sarcolemmal localization of nNOS seems to require a presence of both syntrophin and dystrophin. Based on the cumulative evaluation of above described studieε, it is evident that abnormality of nNOS expression is specific for dyεtrophin-related diεeases. Immunohistochemical analysis for nNOS, therefore, is able to provide a reliable diagnostic test for detection of these diseaεeε. Additionally, abnormal expression of nNOS seemε to play a role in the pathophysiology of BMD. Endogenous NO is involved in regulation of skeletal muscle development and its contractility. Disruption of these signaling pathways may contribute to abnormal muscle function and incomplete myofiber regeneration seen in muscular dystrophy. In mdx mice expressing Dp71 or dystrophin mini-gene, nNOS is the only known dystrophin-aεεociated protein absent from the sarcolemma. In human BMD, loss of sarcolemmal nNOS expression broadly correlates with the severity of the disease. Muscular Dvstrophv Therapy
The above findings allow a design of genetic therapies for DMD, BMD and other muscular dyεtrophies.
A primary goal of muscular dyεtrophy therapy is restoration of fully functional dystrophin. The therapy, therefore, involves either the replacement of full length dystrophin or replacement of a fragment of dystrophin which assures binding of dystrophin with nNOS through syntrophin. The muscular dystrophy therapy thus involves replacement of dysfunctional or missing dystrophin with functional dystrophin or a functional fragment thereof.
All fragments of dystrophin which meet the requirement of binding with nNOS in sarcolemma through syntrophin are intended to be within the scope of this invention.
Because of the large size of the dystrophin protein, the replacement of the whole dystrophin is difficult and therefore, although it is known that dystrophin is missing in DMD and is dysfunctional in BMD, so far such therapy has not been succesεful. With the diεcovery according to the invention that in the normal nondyεtrophic skeletal muscle dystrophin is colocalized with nNOS which binds to a PdZ motif of syntrophin, a dystrophin associated protein, and that in dystrophic muscleε not only dystrophin but alεo nNOS is missing from sarcolemma of the skeletal muscle, it is clear that it is not necessary to replace the whole dystrophin but only the dystrophin fragments which are involved in formation of nNOS/sarcolemma/dyεtrophin complex. Conεequently, the dyεtrophin fragments binding to syntrophin which in turn binds to nNOS, as seen in Figure 22D, in sarcolemma suffice for treatment of muscular dystrophic disorders.
Therefore, vectors used for production of proteins useful for treatment of muscular dystrophy to be uεed for gene therapy need to encode truncation mutants of dystrophin, that provide mutated gene replacement with dystrophin constructs that properly assemble nNOS which complete rescue of muscle function requires. Prior to their use in gene therapy, dystrophin constructs are analyzed to ensure that the y recruit nNOS to sarcolemma.
Any and all such constructs are intended to be included in a method for treatment of muscular dystrophies.
Gene therapy is performed according to methods known in the art for these purposeε. Muscular Dystrophy Diagnosis
Diagnosis of muscular dystrophy is based on detection of nNOS using immunohistochemical detection of nNOS, histologic analysis of nNOS or a combination of both.
NADPH diaphorase staining method, which is fast, easy and practical for routine use, is most preferred.
A diagnostic test is described in Example 22. II. Diagnosis. Prophylaxis and Treatment of Stroke and Other
Neurodegenerative Diseases In addition to findings that nNOS plays a role in development and control of muscular dystrophies, it has now been discovered that in the brain certain proteins, particularly postsynaptic density proteins PSD-95 and PSD-93 localized at synaptic junctions, also bind to PDZ domain of nNOS. In this way they may become important for management, therapy and diagnosis of stroke and other neurodegenerative diseases. Binding protein PSD-93 is novel and has never before been disclosed or described. Finding that PSD-95 and
PSD-93 proteins bind to the PDZ domain of nNOS in the brain is also novel and was never before disclosed or described.
Use of nNOS Binding Proteins in Diagnosis. Prophylaxis and Therapy of Stroke and Other Neurodegenerative Diseases
Neuronal NOS is concentrated at synaptic junctions in the brain where the N-terminus domain of nNOS, which contains a PDZ protein motif, interacts both in vivo and in vitro with the second PDZ motif present in postsynaptic density-95 or -93 proteins. The second PDZ domain mediates binding of nNOS to the N-methyl-D-asparagine (NMDA) receptor located at the synapse through the first and/or the third PDZ domains of the PSD-95 or PSD-93.
Two binding proteins were found and identified to be involved in the binding of the nNOS to the NMDA receptor. They were cloned and expressed, and fusion proteins were prepared. Additionally, both these proteins were found to colocalize with nNOS. Additionally, small peptides corresponding to the carboxy terminal nine amino acids of N- methyl-D-aspartate (NMDA) type glutamate receptor were identified as inhibitors of binding of the nNOS binding proteins with the NMDA.
The invention also describes a method of use of nitric oxide, neuronal nitric oxide synthaεe, itε binding proteinε and their inhibitorε, for diagnosiε, prophylaxis and treatment of stroke and other neurodegenerative diseases, such as Huntington disease, amyotropic lateral sclerosis, Alzheimer disease, etc. as well as a diagnostic asεay for detection of abεence of binding proteinε or nNOS.
Finally, the invention concerns a binding assay for monitoring of binding of nNOS binding proteins with nNOS and appropriate synaptic receptors useful for development of compounds for treatment of neurodegenerative diseases.
Interaction of NOS with the Svnaptic Density Proteins
PSD-95 and PSD-93
Nitric oxide plays important physiological role in neurotransmission and synaptic modulation in central nervous tissue. Endogenous neuronal NO participates in development of some forms of neurotoxic injury, including stroke and other neurodegenerative processes. Functionally, NO mediates certain aspects of synaptic plasticity and neurotoxicity associated with NMDA receptors, but it does not play a major role in other pathways. In the brain, nNOS activity is selectively activated by a calcium influx through the NMDA receptor. Both nNOS and NMDA receptors are concentrated at synaptic junctions in the brain. Consequently, understanding of NO neurotoxicity requires identification of the functional connection of nitric oxide synthetic enzyme nNOS with NMDA receptors. For interacting connection of nNOS with NMDA receptor, a linker able to bind these two entities together is necessary. Two proteins, PSD-95 and PSD-93, have been identified as posεible binding linkers.
The N-terminuε domain of nNOS, which contains a PDZ protein motif was shown to interact with the second PDZ motif in postsynaptic density-95 protein (PSD-95) and a related novel PSD-93 protein. Synaptic organization of nNOS at a synaptic junction is schematically shown in Figure 13 which is a molecular model of nNOS/N-methyl-D-aspartate receptor (NMDAR) bindingmediated by PSD-95 or PSD-93 proteins. These proteins are known to associate with the glutamate type receptors to which NMDA receptor belongs.
As seen in Figure 13, endogenous NO is derived from L- arginine by nNOS. nNOS is concentrated at synaptic junction in close vicinity of NMDAR. In the brain, neuronal NO synthase (nNOS) activity is selectively regulated through calcium influx controlled by NMDA receptors. Two post¬ synaptic density proteins PSD-95 and PSD-93, also located in the vicinity of the synaptic junction, are physically able to associate with nNOS through their respective PDZ domains. This shows that NMDA and nNOS are able interact with nearby binding sites in the second PDZ domain of PSD-95. Nitric Oxide Synthase Binding Proteins In the brain nNOS is thus functionally coupled to N- methyl-D-aεpartate receptors. The N-terminal domain of nNOS is unique to the neuronal isoform and contains a PDZ motif of about 100 amino acids that is found in a diverse group of cytoskeletal proteinε and enzymes. Because this domain was shown to mediate asεociation of nNOS with the dystrophin complex, aε described in section I, attempts were made to identify interacting proteins in the brain to perform the same function.
This invention demonstrates that nNOS is enriched at synaptic junctions in the brain owing to association of nNOS with the postsynaptic density proteins, specifically with the two proteins identified as PSD-95 and PSD-93. The PSD-95 protein clusters NMDA receptors at central nervous system synapses. PSD-95 and PSD-93 proteins therefore act as interacting proteins between nNOS and NMDA receptors.
Postsynaptic denεity protein PSD-95 was originally identified as an abundant detergent-insoluble component of brain postsynaptic density. Subcellular and electron micrographic studieε have determined that PSD-95 is localized at both pre- and post-synaptic membrane and has a similar distribution to nNOS. PSD-95 contains three PDZ repeat' , a SH3 domain and a region homologous to guanylate ki .se (Neuron. 9: 929-942 (1992)). As seen in Figure 13, the second domain of PSD-95 provides the connecting link between nNOS and NMDA receptor.
Schematic representation of PSD-95 and PSD-93 proteins is seen in Figure 14. As seen in Figure 14, cloning and sequencing of the PSD-95 related gene derived a protein PSD-93 of 93 kD, that has the same domain structure as PSD-95 and shares with PSD-95 about 60% amino acid identity. The nucleotide sequence of PSD-93 (SEQ ID NO: 1:) has been deposited in GenBank. The PSD-93 nucleic acid sequence contains 2963 base pairs. The amino acid εequence of PSD-93 are depicted by SEQ ID NOε: 2-6 containing cumulatively 987 amino acidε. The amino acid sequence of PSD-95 is seen in Figure 14 and is identified as SEQ ID NOs: 15-19. This invention thus identifies for the first time two brain proteins that physically associate with the enzyme neuronal nitric oxide synthase (nNOS) . One of these nNOS binding proteins, postsynaptic density-95 (PSD-95) waε previously cloned. The other, PSD-93 nNOS binding protein, is a novel protein whose cloning, sequencing and functional expression has never before been disclosed.
These proteins have now been shown to be involved in development and progresεion of stroke and other neurodegenerative diseases. Their inhibition, inhibition of their binding domain PDZ (second domain) or the inhibition of their binding with nNOS are all important for treatment, prophylaxis, detection and diagnosis of stroke and neurodegenerative diseaεeε.
Colocalization of nNOS and PDS-95 in Adult and Developing Neurons
In order to determine whether the association of nNOS with PSD-95 and PSD-93 is physiologically relevant, colocalization of nNOS and PSD-95 or PSD-93 in adult and developing neurons was investigated. Their co-expression in neurons was studied using in situ hybridization.
The yeast two-hybrid system was used to identify interacting proteins. Screening a brain library demonstrated that the PDZ containing domain of nNOS binds to PDZ repeats present in PSD-95 and in a novel related protein, PSD-93. PSD-95 was found to be co-expressed with nNOS in several neuronal populations in the developing and mature nervous system, and a specific PSD-95/nNOS interaction was detected in transfected cell lines and solubilized cerebellar membranes. On the other hand, residual catalytically active nNOS isoforms identified in nNOSΔ/Δ mice, that specifically lack a PDZ motif, did not interact with PSD-95. These data demonstrate a physiological role for PDZ domain interactions in organizing proteins at synaptic membranes. Interaction of nNOS and PSD-95 or PSD-93 via their PDZ domains therefore mediates synaptic-asεociation of nNOS, with the NMDA receptor and may play a more general role in formation of macromolecular signaling complexes.
The study was based on the premise that if the association of nNOS with PSD-95 identified in yeast is physiologically relevant, the two proteins must be co-expressed in neurons. Results are seen in Figure 15. Figure 15 shows expression of PSD-93, PSD-95 and nNOS in rat brain and in E15 embryo. In situ hybridization was used to localize transcripts for PSD-93 (inεet A), PSD-95 (inεet B) , nNOS (inset C) or sense control (inset D) in adjacent cryosectionε prepared according to Example 5. Figure 15A εhowε that in an adult rat brain, PSD-95 was observed only in neurons and was co-expressed with nNOS in certain neurons in hypothalamus, hippocampus and cerebellum. PSD-93 also appeared to be neuron specific, but had a more restricted distribution than did PSD-95. Figure 15B shows that in the cerebellum, PSD-95 and nNOS were co-expressed in cerebellar granule cells in the granular layer (G) and basket cells (B) in the molecular layer. By contrast, PSD-93 was restricted to Purkinje neurons (P) of the cerebellum, which lack nNOS or PSD-95. Double labeling with NADPH diaphorase and in situ hybridization identified the PSD- 95 and nNOS expressing cells in molecular layer as basket cells.
Figure 15C showε that in E15 embryo, PSD-95 waε found ubiquitously expressed in differentiated central neurons, but not in neuronal precursors. PSD-95 was co-expressed with nNOS in the cerebral cortical plate (CP) , dorεal root ganglia (DRG) and neurons of the olfactory epithelium (OE) . PSD-93 was specifically co-expressed in neurons of the spinal cord (SC) , DRG and trigeminal nerve (V) . PSD-93 was specifically co- expressed with nNOS in secretory cells of the submandibular gland (SG) and in (Figure 15D) chromaffin cells of the developing adrenal gland, which lack PSD-95. The signal in liver (L) seen in all εampleε including control repreεentε non-εpecific hybridization to an aberrant fold in the tiεεue. K identifies the kidney. A identifies the adrenal gland.
In situ hybridization in a rat brain (Figures 15A and 15B) demonstrated co-expression of nNOS and PSD-95 transcripts in several neuronal populations, particularly in cerebellar granule and basket cells, which have previously been shown to express high denεitieε of nNOS and PSD-95 proteins. On the other hand, PSD-93 occurred at highest densitieε in cerebellar Purkinje neuronε, complimentary to the distribution to nNOS and PSD-95.
When expression of nNOS with PSD-95/PSD-93 during embryonic development was compared, it was found that nNOS- containing cells in embryonic day 15 (E15) were differentially co-expressed with either PSD-95 or PSD-93 (Figures 15C and 15D) . As previously reported, transient NOS neurons were detected in developing cerebral cortical plate, olfactory epithelium, and sensory ganglia. In all of these neuronal groups PSD-95 mRNA was found. nNOS waε alεo developmentally expressed in certain non-neuronal cells including chromaffin cellε of the adrenal gland and secretory cells of the submandibular gland. PSD-93 mRNA and nNOS mRNA were co- expressed in these glands, while PSD-95, which is neuron specific, was absent. Co-localization of nNOS and the PSD-95 protein was additionally evaluated by immunohistochemical staining of adjacent sectionε from an E19 rat. Results are seen in Figure 16.
Figure 16 shows that PSD-95 co-localizes with nNOS in developing neurons. Immunohistochemical staining of adjacent sagittal sections of an E19 rat fetus indicates that PSD-95 (Figure 16A and 16C) and nNOS (Figures 16B and 16D) are co- localized in primary olfactory epithelium (OE) and in nerve processes projecting to the olfactory bulb (OB) (Magnification in Figures 16A and 16B is 50X; in Figures 16C and 16D is 400X) . In the intestine (Int) of an E19 rat, as seen in Figures 16E and 16G, PSD-95 and, as seen in Figures 16F and 16H, nNOS are co-localized in myenteric neurons (MN) . (Magnification 50X, and 200X, respectively.)
In an E19 rat cerebral cortex, as seen in Figure 161, PSD- 95 and nNOS (Figure 16J) are also co-localized. Both proteins are most concentrated in neuronal processes of the intermediate zone (IZ) and cell bodies of the cortical plate (CP) , while the ventricular zone (VZ) is devoid of staining (Magnification 100X) .
Within the olfactory system, both PSD-95 and nNOS were enriched in dendritic specializationε in olfactory cilia and in axonal processes projecting to the olfactory bulb, which itself does not contain either nNOS or PSD-95, as seen in
Figure 16A-D. nNOS also occurs in fetal myenteric neurons and its absence is associated with hypertrophic pyloric stenosis. Immunohistochemical analysis revealed a co-localization of nNOS with PSD-95 in myenteric neurons (Figures 16 E-H) . nNOS and PSD-95 were similarly co-localized in embryonic cerebral cortex. Staining for both proteins was enriched in the intermediate zone and in developing cortical plate, while lesser staining was found in the subplate region. The ventricular zone waε devoid of staining (Figures 161 and 16) .
These results show that both PSD-95 and PSD-93 are co- localized with nNOS in neuronal tissue and therefore must have a physiological importance connected with this tissue in conjunction with nNOS.
The Binding of nNOS Containing PDZ Domain to Similar
Motifs of PSD-95 and PSD-93
In this study, binding of the PDZ containing domain of nNOS to similar motifs in PSD-95 and to a related protein, PSD-93 was investigated.
To determine which domain or domains of PSD-95 bind to nNOS, yeast constructs encoding appropriate fragments of PSD- 95 were fused to the GAL4 activation domain. Constructs encoding the second PDZ motif of PSD-95 interacted with nNOS while those lacking this region were inactive. Results are seen in Table 4.
Table 4 Interactions Between nNOS and PSD-95 or PSD-93
Gal4 ONA Gal4 Activation Colony Color Growth Binding Hybrid Hybrid
nNOS (amino acids 1-195) SV 40 (amino acids 84-708) White - p53 *72-390) PSD-95, PDZ1-3 (20-364) White -
Lamin C (66-230) PSD-95, PDZ1-3 (20-364) White - nNOS (1-195) PSD-95, PDZ1-3 (20-364) Blue + nNOS (1-195) PSD-95, PDZ1-2 (20-294) Blue + nNOS (1-195) PSD-95, PDZ2-3 (138-364) Blue + nNOS (1-195) PSD-95, PDZ1 (20-144) White - nNOS (1-195) PSD-95, PDZ2 (138-294) Blue + nNOS (1-195) PSD-95, PDZ3 (291-364) White - p53 (72-390) PSD-93 (116-421) White -
Lamin C (66-230) PSD-93 (116-421) White - nNOS (1-195) PSD-93 (116-421) Blue + p53 (72-390) SV 40 (84-708) Blue +
When the PDZ domain of nNOS (amino acids 1-195) was fused to the DNA binding domain of GAL4 and screened by a human brain library for interacting proteins using the yeast two- hybrid system according to Nature. 340: 245-246 (1989) (Clonetech) , two families of interacting clones were identified from a screen of 106 plasmids as seen in Table 4.
One family represented isolates encoding the PDZ motifs of PSD-95, and the other family encoded PDZ repeats of a related novel gene product, protein PSD-93. These findings show that PSD-95 and PSD-93 are related, both structurally, as seen from Figure 14, and also functionally, as their respective clones interacted with nNOS.
Interaction of nNOS with the PDZ Motif of PSD-95
As seen in Figure 13, the second PDZ domain of PSD-95 provides a binding link between the nNOS and NMDA receptor. In this study, confirmation of nNOS interaction with the second PDZ domain of PSD-95 was investigated. To evaluate formation of a nNOS/PDS-95 complex in the brain, immunoprecipitation studies were conducted. Results are seen in Figure 17 .
Figure 17 shows co-immunoprecipitation of nNOS and PSD- 95. In Figure 17A, COS cells were transfected with an expression construct PSD-myc, encoding amino acids 1-386 of PSD-95 with a 10 amino acid c-myc epitope tag alone (lanes 1) or were co-transfected with PSD-myc and nNOS (lanes 2) . Cell homogenates were immunoprecipitated with nNOS and probed with a monoclonal antibody to c-myc. Input was 5% protein loaded onto columns. In Figure 17B solubilized cerebellar membranes were immunoprecipitated with antibody to PSD-95 (lanes 2) or a non- immune serum (lanes 1) . Western blotting εhowε εpecific co- immunoprecipitation of nNOS but not eNOS with PSD-95.
Figure 17C shows identical immunoprecipitations from cerebellar cytosol, which lackε PSD-95 but containε high concentrations of nNOS. Figure 17C shows that the PSD-95 antibody (lane 2) does not directly interact with nNOS. The eNOS blot and the nNOS blot from cerebellar cytosol were intentionally overexposed, but failed to show specific immunoprecipitated bands.
Figure 17D shows affinity chromatography which demonstrates that nNOS is selectively retained by an immobilized PSD-95 protein fragment (amino acids 1-386) fused to GST. eNOS is not retained by the PSD column. Solubilized brain extracts were incubated with G-PSD or control GST beads, columns were washed with a buffer containing 0.5 M NaCl and 1% triton X-100, and eluted with SDS. Bound proteins were detected by Western blotting. Inpu was 10% protein.
Figure 17E shows that NMDA rece or 2B carboxy terminal peptide displaces nNOS and 1^1.4 fr PSD-95. "Pull-down" assays from brain were conducted as a ve containing 0 (lanes 6, 7), 10 μM (lanes 4,5) or 30 μM (1-nes 2,3) NMDA receptor peptide or 200 μM control peptide (lanes 8,9). Input was 10% protein. Figure 17 shows and confirms that nNOS binds to PSD-95 through PDZ motif interactions. A nNOS-PSD-95 complex was immunoprecipitated from COS cells co-transfected with expression vectors for nNOS and the PDZ repeats of PSD-95, indicating that this interaction occurs in a cellular environment (Figure 17A) . Though only a small fraction of PSD-95 can be solubilized from brain densities with non- denaturing detergents, a nNOS/PDS-95 complex was specifically immunoprecipitated from cerebellum (Figures 17B and 17C) , where both proteins are co-expreεεed at high levels.
To biochemically evaluate the interactions of nNOS and PSD-95, a fusion protein linking glutathione-S-transferase (GST) to the first 386 amino acids of PSD-95 (G-PSD) was generated. Solubilized brain extracts were incubated with glutathione beads linked to GST or G-PSD. Following extensive washing with a buffer containing 350mM NaCl and 0.5% triton X-100, bound proteins were eluted with a loading buffer. Western blotting indicated selective retention of nNOS to G- PSD beads but not of eNOS, which is 60% identical to nNOS but lacks a PDZ motif. These results indicate specificity of the interaction with nNOS (Figure 17D) .
Because the second PDZ motif of PSD-95 binds to both nNOS and to tSXV, a protein motif of the NMDA receptor, containing ion channels, the investigation waε undertaken to determine whether theεe binding εiteε are independent or overlapping. Studies described above demonstrated that a peptide corresponding to the carboxy terminal nine amino acids of NMDA receptor type 2B blocks interaction of NMDA receptor with PSD-95 (Science. 269: 1737-1740 (1995)). As seen in Figure 17E, this NMDA receptor peptide potently blocks association of nNOS with PSD-95. Half maximal inhibition of binding was achieved at <10 μM NMDA receptor peptide, while control peptides were inactive at 200 μM. As a control K„ 1.4, a voltage-dependent K+ channel containing a tSXV sequence, was found to be displaced from PSD-95 by similar concentrations of NMDA receptor peptide.
The importance of the second PDZ domain of PSD-95 or PSD- 93 for nNOS/PSD-95 binding was confirmed when the second PDZ domain of PSD-93 expressed as a GST-fusion protein was found to bind to nNOS in a manner competitive with the NMDA receptor peptide.
Presence of Catalytically Active nNOS Isoforms Lacking a PDZ Motif in nNOS Δ/Δ Mice
To confirm that a PDZ motif is responsible for interaction of nNOS to PSD-95 or PSD-93, catalytically active nNOS isoforms lacking a PDZ motif observed in nNOSΔA mice were investigated.
Mice carrying a targeted disruption of exon 2 of nNOS express residual nNOS isoforms specifically lacking the PDZ domain. Thus these mice are extremely suitable for investigation whether nNOS isoform lacking the PDZ motif will bind to PSD-95 or PSD-93.
Neuronal NOSΔΔ mice were generated by deleting the first translated exon, which is exon 2, of nNOS in both mice and humans which encodes the PDZ motif. Results are shown in Figure 18.
Figure 18 illustrates that exons 1 and 2 of nNOS are alternatively spliced.
Figure 18A is Northern blot analysiε of brain mRNA from wild type (WT) and nNOS * mice hybridized with a full length nNOS cDNA probe. A broad band of 10.5 kb iε recognized in the wild type mouse brain and weaker bands of 11 and 9.5 kb are recognized in nNOSΔ/Δ mice.
Figures 18B and 18C show RT-PCR analysis of 5' splicing of nNOS gene. For this study, cDNA was amplified wir primers 1 and 2 (lanes 1,2).
Figure 18B is ethidium bromide staining which shows a band of 1 kb amplified from nNOS7 and a band of 2.2 kb from wild type. Figure 18C is Southern hybridization with a full length nNOS probe showing hybridization to the ethidium stained bands. A weaker band of 1 kb in amplifications of wild type cDNA is also detected by hybridization (lane 2) . A similar analysis using primers 3 and 4 confirms that exon 2 sequences are only detected in wild type cDNA (lanes 3,4).
Figure 19 shows that catalytically active nNOS isoforms lacking exon 2 are expressed in the brain of nNOS^4 mice. Figure 19A is Western blotting of crude (lanes 1,2) and 2' 5'- ADP affinity purified (lanes 3,4) brain extracts, indicating that the major nNOS band in wild type brain migrates at 160 kD (lanes 1,3) while in nNOS^, co-purifying bands of 125 and 136 kD (lanes 2,4) are observed. Partial tryptic digestion of 2'5-ADP agarose-purified proteins reveals a similar proteolytic "fingerprint" from wild type (lane 5) and nN0SΔΔ (lane 6) . 20 fold more protein was loaded from nNOS4'4 (lanes 2,4,6) than from wild type samples (lanes 1,3,5). Figure 19B shows cDNA clones encoding nNOS, nNOS/3 (5'a spliced to exon 3) or nNOS (5'b spliced to exon 3) were transfected (trx) into COS cells and protein extracts were resolved by SDS/PAGE. Full length NOS (nNOS-trx) comigrates at 160 kD with the major product from the wild type brain (lanes 1,2). Transfection of nNOS/3 and nNOS yields proteins of 136 and 125 kD respectively that comigrate with immunoreactive bands from nNOS^4 (lanes 3,4,5).
Figure 19C shows NOS catalytic activity of nNOS isoforms. COS cells were transfected with 10 μg of expresεion vector encoding full length nNOS, nNOS/3, or nN0S . NOS activity was measured in cell homogenates three days following transfection in the presence of 200 μM free calcium. This experiment was replicated twice with similar results.
Figure 19D shows that nNOS isoforms are discretely expressed in the nNOSΔΔ brain. Highest densities of nNOS in wild type (10 μg/lane) are found in the cerebellum (Cb) . In nNOSΔΔ (100 μg/lane) highest levels of nNOS isoforms are found in striatum (St) and hippocampus (Hi) , lower amounts are found in brainstem (Bs) and cerebral cortex (Cx) while the cerebellum is devoid of nNOS isoforms in nNOS^A. Figure 19E eNOS is homogeneously distributed in forebrain (Fb) , cerebellum (Cb) as well as the peripheral tissues liver (Li) , lung (Lu) and kidney (Ki) . All lanes in Figure 19C were loaded with 100 μg of solubilized membrane extract. Transfection of the 5'a containing construct generated a prominent immunoreactive protein band of 136 kD that comigrated with nNOS3 from nNOS^4 brain (Figure 19B; lanes 3,4). Transfection of the 5'b containing construct yielded a nNOS band of 125 kD (Figure 19B, lane 5) . Catalytic assays indicated that the 136 kD nNOSβ form had activity -80% that of full length nNOS under these transfection conditions (Figure 19C) . Enzyme activity was fully dependent on calciu /calmodulin and the K,,, for arginine was similar to that of full-length nNOS from the brain. By contrast, the activity of the 125 kD nNOS was -3% that of nNOS.
NOS activity in the wild type brain iε highest in the cerebellum. By contrast, NOS activity in nNOSΔΔ is highest in the striatum and lowest in the cerebellum. The regional distribution of residual nNOS isoformε in nNOS'1'4 brain extracts paralleled the pattern of residual nNOS activity previously reported in Cell. 75: 1273-86 (1993) . Absence of a PDZ Motif Prevents Aεsociation of nNOS
Isoforms with PSD-95 or with Brain Membranes
In this study, association of nNOS isoformε lacking a PDZ motif with PSD-95 or brain membranes was investigated. nNOS4'4 mice express nNOS isoforms specifically lacking the PDZ motif were used as an important tool to determine the functions for this domain in vivo . Association of residual nNOS isoforms with PSD-95 was investigated. nNOS proteins purified from wild type and nNOSΔ/Δ mouse forebrain were subjected to pull-down asεayε aε deεcribed above. Reεults are seen in Figure 20.
Figure 20 shows that nNOS isoforms lacking the PDZ motif do not bind to PSD-95 or to brain membranes.
In Figure 20A partially purified nNOS protein from wild type (WT) or nNOS4^ brains were analyzed by PS "pull-down" assay. Full length nNOS hi "is to PSD-95 while the residual isoforms lacking the PDZ mot- do not. Input was 20% protein.
Figure B showε that residual nNOS isoforms are restricted to cytosol of nNOS 4. Brain homogenates, extracted with 100 mM NaCl (lanes l) , l M KCl + 1% triton X-100 (lanes 2) or insoluble pellet (lanes 3), from wild type (20 μg/lane) or nNOS474 (200 μg/lane) were probed by Western blotting. Only full length nNOS protein containing the PDZ motif was retained by G-PSD beadε; the alternatively εpliced forms in nNOS474 did not adhere to G-PSD (Figure 20A) . The distribution of nNOS in wild type and nNOS4'4 mice by subcellular fractionation was compared. Brain homogenates were first extracted with physiological saline, then with buffer containing 1M KCl and 1% triton X-100, leaving a cytoskeletal pellet. nNOS in wild type brain was present in all fractions while residual nNOS isoforms in nNOS474 occurred only in the first soluble fraction (Figure 2OB) .
The studies represented in Section II show that in the brain nNOS is functionally coupled to N-methyl-D-aεpartate receptorε through the interaction with binding proteinε. The N-terminal domain of nNOS is unique to the neuronal isoform and contains a PDZ motif of about 100 amino acids. nNOS is enriched at synaptic junctions in the brain owing to association of nNOS with the postsynaptic density proteins PSD-95 and PSD-93 which act as interacting proteins between nNOS and NMDA receptors. The interaction of nNOS and PSD-95 or PSD-93 via their respective PDZ domains mediates synaptic- association of nNOS with the NMDA receptor. Association of nNOS with PSD-95 is physiologically relevant as the two proteins are co-expresεed in neurons. nNOS PDZ domain interacts with the second PDZ domain of the PSD-95 or PSD- 3 and the PDZ domain of nNOS is important for its interaction with the NMDA receptor. When this domain is missing, the interaction between nNOS and PSD-95 is also missing. Absence of a PDZ domain thus prevents the binding of nNOS/PSD-95 or PSD-93 proteins and nNOS interaction with the NMDA receptor. Neuronal NOS and its specific binding proteins are therefore physiologically very important for neuronal functionality.
Diagnosis of Stroke and Neurodegenerative Diεeaεes Stroke or other neurodegenerative diseaseε develop and are a consequence of an overly active NMDA receptor at the neuronal εynaptic junction. The active NMDA receptor stimulates influx of calcium ions into the cell and through the effect of calmodulin it activates nNOS, which in turn increases production of NO in the neuron. Neuronal NO either causes or at least participates in development of neurotoxic injury, including stroke. According to the findings of this invention, the increased activity of the NMDA receptor is to a certain extent dependent on binding of NMDA to nNOS through PDZ domains of nNOS binding proteins PSD-95 or PSD-93. This can be advantageously utilized for early detection of impending stroke or development of other neurodegenerative diseases by detecting a level of nNOS, PSD-95 or PSD-93 proteins.
When the level of nNOS is high, the probability of impending stroke or other neurodisturbance is high.
The detection of nNOS is according to Example 22 and as described above for diagnostic test for detection of muscular dystrophy. Instead of muεcle tissue, brain or central nervous tissue biopsy is used. Alternatively, in situ imaging method is used using labeled PSD-95 protein inhibitors.
Treatment and Prophylaxis of Stroke and Other Neurodegenerative Diseases
Compounds that block association of nNOS with PSD-95 or PSD-93 are candidates for novel therapeutic agents useful for treatment or prophylaxis of stroke or other degenerative diseases. As shown, neuron-derived NO, associated with NMDA receptor activity, is responsible for and mediates brain injury following cerebral ischemia. Therefore, by blocking nNOS activity by disruption of its binding with binding proteins, the action of NO can be controlled and further damage to neurons is prevented. Screening for drugs that block interaction of NMDA receptors with PSD-95 or PSD-93 could be done by an analogous procedure to that described in Example 23.
For this purpose, a 9-mer peptide identified as a SEQ.ID No. 3, corresponding to the final 9 amino acids of NMDA receptor 2B potently interactε with GST-fuεion proteinε encoding the firεt 2 PDZ domainε of PSD-95 and PSD-93.
Therefore, a C-terminal 9-mer peptide of NMDA 2B receptor, either radiolabeled or epitope tagged, can be advantageously used for a large scale screening assay for compounds which block its binding to PSD-95 or PSD-93. Those compounds would then be able to inhibit, or block by competition, the binding of PSD-95 or PSD-93 to nNOS.
One way how to produce these inhibitors is to label the peptide and to incubate it with PSD-95 or PSD-93 GST-fusion proteins to reach a binding equilibrium and immobilize the fusion proteins on glutathione resin. PSD-95 or PSD-93 fragments are retained by immobilized glutathione resin, and the resin is washed to elute unbound peptide. Using this assay one could perform large scale screening of compounds for drug diεcovery.
Potent inhibitorε of this binding are therefore useful in treatment and in prevention of stroke and neurodegenerative disease. They would be administered in any suitable pharmaceutically acceptable route either before impending stroke or after the stroke developed, to prevent further neuronal damage. Inhibitors of nNOS and Postsynaptic PSD-93 and PSD-95 Proteins
This invention also identifieε a small 9-mer peptide that potently (Kd-1 μM) blocks association of nNOS with PSD-95 and PSD-93. This invention demonstrates a novel mode to block brain damage due to nitric oxide, that is, the identification of small molecules that disrupt interaction of nNOS with PSD-95 and PSD-93. This invention demonstrates that such drugs would uncouple nNOS from neurotransmitter receptors and would prevent NO mediated brain damage.
Previous efforts to identify enzyme inhibitors of nNOS have been unsuccesεful becauεe there are two other iεoforms of NOS (eNOS and iNOS) , and nNOS selective drugs have not yet been identified. However, this invention clearly shows that only nNOS associates with PSD-95 and PSD-93, so that drugs which block these interactions would be specific for nNOS. To that effect, a binding assay suitable for screening for suitable inhibitors of nNOS/PSD-95 or PSD-93 protein has been developed and is described in Example 23. Binding Assay for Screening Inhibitors of PSD-95 and PSD-93 Binding with nNOS GST-fusion proteins linked to the first two or three PDZ motifs of PSD-95 or PSD-93 are expressed in E. coli as described in Example 6. Binding interactions to this fragment are monitored by a variety of assays known in the art. To detect binding of endogenous nNOS or NMDA receptor subunits to PSD-95 or PSD-93, pull down assays are done as described. Screening for drugs that block interaction of NMDA receptors with PSD-95 or PSD-93 could be done by an analogous procedure as described above, and in Example 23.
UTILITY Current invention is useful for diagnosis and treatment of muscular diseases, primarily for diagnosis and treatment of Duchenne dystrophy, Becker muscular dystrophy and other types of muscular dystrophies. Detection of presence or absence of nNOS in human biopsies, for example, immunohistochemically, detects the disease and its severity. Treatment of muscular dystrophieε utilizeε the restoration of fully functional dystrophin able to bind to nNOS, using, for example, gene therapy. Restoration of a functional dystrophin molecule to muscle represents a primary goal for therapy. The invention is also useful for management of neurodegenerative diseases.
EXAMPLE 1 Tissue Extraction and Western Blot Analysis This example describes methods used for skeletal muscle tissue extraction.
Mouse quadriceps skeletal muscle was homogenized in 10 vol (w/v) of buffer A (25 mM Tris-HCl, pH 7.4, 100 mM NaCl, ImM EDTA, 1 mM EGTA, 1 mM PMSF) , and heavy microεomes were prepared by a standard protocol with minor modifications according to (J. Cell Biol.. 96: 1008-16 (1983)). Nuclei were pelleted by centrifugation at 1000 x g. The supernatant was then centrifuged at 20,000 x g, yielding supernatant S,. The reεulting heavy microεomal pellet was resuεpended in buffer A containing 500 mM NaCl, incubated for 30 min at 4°C with agitation, and centrifuged at 15,000 x g, yielding supernatant S2. The resulting pellet was resuspended in buffer A containing 500 mM NaCl plus 0.5% Triton X-100, incubated for 30 min at 4°C with agitation, and centrifuged at 15,000 x g, yielding supernatant S3 and a final pellet, P.
Tissue extracts were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (7.5% polyacrylamide) , and proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore) . Membranes were incubated overnight with primary antisera bNOS, (1:250) and eNOS, (1:250), obtained from Transduction Laboratories; dystrophin (1:100), and spectrin (1:100), obtained from Novacastra Laboratorieε diluted in Tris-HCl-buffered saline containing 3% bovine serum albumin. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL) according to the specifications of the manufacturer (Amersham) .
EXAMPLE 2 Affinity Chromatography
This example describes affinity chromatography used for sWGA affinity chromatography. sWGA Sepharose Affinity Chromatography
Mouse quadriceps from wild-type and mdx mice were homogenized and solubilized in 10 vol of buffer B (50 mM Tris- HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, 1 mM PMSF) containing 1% digitonin. Solubilized membranes (4 mg) from wild-type and mdx mice were circulated for 1 hour with 250 μl of sWGA- agarose obtained from Vector Labs at 4°. Columns were washed sequentially with 5 ml of buffer B containing 0.1% digitonin, buffer B containing 0.1% digitonin and 500 mM NaCl, and buffer B containing 500 mM NaCl and 0.5% Triton X-100. Columns were affinity eluted with 1 ml of buffer B containing 0.3 M NAG with 0.1% digitonin. Chromatography of rat skeletal muscle followed a similar procedure, except 20 mg of solubilized membranes was loaded onto 1 ml of sWGA that was washed with 10 ml of buffers containing 500 mM NaCl and Triton X-100 and eluted with 3 ml of 0.3 M N-methyl-D-glucosamine. 2'.5'-ADP Affinity Chromatography
For 2',5'-ADP affinity chromatography, mouse quadriceps from wild-type and nNOS knockout mice were homogenized in 10 vol (w/v) of buffer B, and heavy microsomes were prepared and solubilized in buffer B containing 1% digitonin. Solubilized membranes (4 mg) from wild-type and nNOS knockout mice were applied to 150 μl columns of 2' ,5'-ADP-agarose (Sigma).
Columns were sequentially washed with 5 ml of buffer B containing 0.1% digitonin, 1 ml of buffer B containing 500 mM
NaCl and 0.1% digitonin, and 1 ml of buffer B containing 500 mM NaCl and 0.5% Triton X-100. Columns were affinity eluted with 1 ml of buffer B containing 20 mM NADPH and 0.1% digitonin. EXAMPLE 3
Glutathione-S-Transferaεe-Fuεion Proteins This example describes preparation of glutathione-S- transferase (GST) fusion proteins.
A GST-nNOS(1-299) construct was generated by cloning sequences encoding the first 299 amino acids of rat brain NOS into the EcoRI site of the pGEX-2T vector. GST-fusion proteins were expressed in Escherichia coli and purified on glutathione-Sepharose beads according to Gene. 67:31-40 (1988) and according to the specifications of the manufacturer Pharmacia. Solubilized skeletal muscle membranes (2 mg) were incubated with control (GST) or GST-nNOS (1-299) beads for 1 hr. Beads were washed with buffer containing 0.5% Triton X- 100 plus 300 mM NaCl, and proteins were eluted with 150 μl of loading buffer. EXAMPLE 4
Dystrophin Immunoprecipitation This example describes method used for dystrophin immunoprecipitation.
Monoclonal antibodies (2 μg) to dystrophin or Myc epitope (BABCO) were added to l ml aliquots of NAG eluate (15 μg) , and samples were incubated on ice for 1 hour. Rabbit anti-mouse
IgG (lOμg) obtained from Cappel was then added, and after 30 min, 50 μl of protein A-Sepharoεe was used to precipitate antibodies. Protein A pellets were washed three times with buffer containing 200 mM NaCl and 0.1% Triton X-100. Immunoprecipitated proteins were denatured with loading buffer and resolved by SDS-PAGE.
EXAMPLE 5 Immunohistochemical and Immunoblotting Procedures This example describes methods used for preparation of, skeletal muscle samples for immunohistochemical and immunoblotting procedures.
Unfixed skeletal muscle samples were flash frozen in liquid nitrogen cooled isopentane, sectioned on a cryostat (10 μm) , and melted directly onto glass slides. Sections were then postfixed in 2% paraformaldehyde-phosphate-buffered saline (PBS) . Tisεues were blocked in PBS containing 1% normal goat serum. Monoclonal antibodies to dystrophin
(1:200) obtained from Sigma, nNOS (1:100) obtained from
Transduction Laboratories, spectrin (1:50) obtained from
Novacastra Laboratories, or a polyclonal nNOS antibody (1:250) prepared according to Nature. 372: 546-548 (1994) were applied to sections overnight at 4° For indirect immunofluorescence, secondary goat anti-rabbit fluorescence isothiocyanate (FITC) or donkey anti-mouse Cy-3 conjugated antibodies were used according to the specifications of the manufacturer (1:200), Jackson Laboratories.
EXAMPLE 6 Mammalian Cell Transfections This example illustrates the method used for mammalian cell transfection. nNOS cDNAs were cloned into the mammalian expression vector pcDNA-3 obtained from Invitrogen. Monkey COS cells were grown in culture medium consisting of DMEM (GIBCO BRL) supplemented with 10% fetal bovine serum. Cellε were plated in 10 cm dishes at a density of 2 x 104 per square centimeter and transfected the following day using calcium phosphate as previously described in Nature, 351:714-718. Cells were washed with PBS 3 days following transfection, harvested in 2 ml of buffer containing 25 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, and disrupted using a polytron.
EXAMPLE 7 Nitric Oxide Synthase Catalytic Assays
This example describeε procedure used for assessment of for NOS catalytic activity.
Quadriceps skeletal muscle from wild-type and mdx mouse were homogenized in 10 vol of buffer containing 25 mM Tris-HCl (pH 7.4) , 1 mM EDTA, ImM EGTA, and 0.1 M NaCl. The homogenate was centrifuged at 20,000 x g, yielding the soluble fraction. The pellet was extracted in the same buffer containing 0.5 M NaCl and centrifuged at 20,000 x g, yielding the particulate fraction. Aliquots from these fractions were assayed in 125 μl reactions containing 100,000 cpm, of [3H]arginine (60 Ci/mmol) , 1 mM NADPH, 400 μM free calcium, 1 μM calmodulin,
3 μM each of tetrahydrobiopterin, FAD, and FMN. After incubation for 25 min at 22°C, asεayε were terminated with 4 ml of H20. Samples were applied to 0.5 ml Dowex AG50WX-8 (Na+ form) columnε. [3H] citruline waε quantified by liquid scintillation spectroscopy of the 4 ml flowthrough. Crude homogenates of transfected COS cells were asεayed using an identical procedure.
EXAMPLE 8 Characterization of Human Tissues
This example describes methods used for evaluation of the nNOS localization in human tissue.
Human tissues were obtained from the pathology department at University of California, San Francisco. Clinical diagnosis of DMD was made on the basis of onset and progresεion of disease, the presence of creatinine kinase in serum, and histologic study of the biopsied muscles.
Tissues were snap frozen in liquid nitrogen-cooled isopentane. For Western blotting, cryostat sections were collected into plastic tubes and sonicated in buffer containing 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, l mM EGTA, and
1 mM PMSF. Crude protein sampleε (200 μg per lane) were resolved by SDS-PAGE and analyzed by Western blotting as described in Example 1. Immunohistochemiεtry was performed on 10 μm human tissue samples as described in Example 5. To ensure specificity, immuno-fluorescent nNOS staining was performed using two independently raised antisera. The first antiserum (α-nNOSl) reacts only with determinants in the N- terminal domain of nNOS (Neuron. 13:301-313, 1994b) , while the second (α-nN0S2) reacts only with the C-terminal region, α- nNOS was obtained from Transduction Laboratories. Unless otherwise noted, all histologic sectionε were labeled with α- nNOSl.
EXAMPLE 9 Preparation of Polyclonal and Monoclonal Antibodies This example lists a primary antibodies used for sarcolemmal studies.
In the studies directed to selective losε of εarcolemmal nitric oxide synthase in Becker muscular dystrophy, the following primary antibodies were used: nNOS polyclonal antibody raised against homogenouε nNOS protein purified from rat cerebellum, prepared according to Nature. 347: 768-770 (1990). nNOS monoclonal antibody obtained from Tranεduction Lbas. αl-syntrophin polyclonal antibody prepared according to Neuroreoort. 5: 1577-1580 (1994) . Dystrophin monoclonal antibody waε obtained from Sigma. 3-dystroglycan monoclonal, utrophin monoclonal, and αεarcoglycan monoclonal antibodieε were obtained from Novacastra.
EXAMPLE 10 Immunofluorescence Assay
This example describeε the assay used for immunofluoreεcent studies on skeletal muscle samples from Becker muscular dystrophy patients and transgenic mice.
Unfixed skeletal muscle samples obtained from human patients or from mdx or transgenic mice were flash frozen in liquid nitrogen cooled isopentane, sectioned on a cryostat (10 μm) and melted directly onto glass slides. Sections were then post-fixed in 2% parafor aldehyde in phosphate buffered saline (PBS) or cold acetone. Tissues were "blocked" in PBS containing 1% normal goat serum. Primary antibodies were diluted in blocking reagent and were applied to sections overnight at 4βC. For indirect immunofluorescence secondary goat anti-rabbit FITC (1:200), or donkey anti-mouse Cy-3 (1:200) conjugated antibodies were used according to the manufacturer's specifications (Jackson Laboratories) . Cy-3 conjugated α-BGT was diluted together with the secondary antibody for double labeling motor endplates.
EXAMPLE 11 Antibodies Immunoprecipitation This example describeε procedure used for precipitation of polyclonal antibodies to αl-syntrophin. Polyclonal antibodies (1 μg) to αl-syntrophin or non- immune serum were added to 0.5 ml aliquots of solubilized skeletal muscle membranes (2 mg/ml) or muscle cytosol (1 mg/ml) , and εarapleε were incubated on ice for 1 hour. Protein A sepharose (50 μl) was used to precipitate antibodies. Protein A pellets were washed 3 times with buffer containing 100 mM NaCl and 1% tritox X-100. Immunoprecipitated proteins were denatured with a loading buffer and resolved by SDS-PAGE.
EXAMPLE 12 Fusion Protein Affinity Chromatography This example describes fusion protein affinity chromatography procedure used for GST-nNOS (1-299) fusion protein.
A fusion protein of GST fused to the first 299 amino acids of nNOS was expressed in Escherichia coli and purified on glutathione sepharose beadε as described Cell. 82: 743-752 (1995) . Solubilized skeletal muscle membranes were incubated with control (GST) or GST-nNOS (1-299) beads. Samples were loaded into disposable columns, which were washed with 50 volumes of buffer containing 0.5% triton X-100 + 300 mM NaCl, and proteins eluted with 150 μl of SDS/PAGE loading buffer. EXAMPLE 13 Antibodies and Western Blotting Used for Studieε of Interaction of NOS with PSD-95 and αl-Syntropin This example listε specific antibodieε used for εtudies of interaction of NOS with the εynaptic density protein PSD-95 and αl-syntropin.
The following primary antibodies were used: nNOSl polyclonal antibody raised against homogenous nNOS protein purified from rat cerebellum as described in Example 9. nNOS monoclonal and eNOS monoclonal antibodies were obtained from Transduction Labs. αl-εyntrophin polyclonal as described in Example 9, PSD- 95 polyclonal were prepared according to Neuron. 9: 929-942 (1992).
Kyl.4 polyclonal antibodies were prepared according to Nature, 378: 85-88 (1995) . c-myc monoclonal antibody 9E10 was obtained from BABCO. T7-Tag monoclonal and GST 12 monoclonal antibodies were obtained for Santa Cruz Biotechnology, Inc.
For Western blotting, protein extracts were resolved by SDS/PAGE and transferred to PVDF membranes. Primary antibodies were diluted in block solution containing 3% BSA, 0.1% Tween-20 in TBS and incubated with membranes overnight at 4° Labeled bands were visualized using ECL. (Amersham) . All nNOS Western blots used in nNOS monoclonal antibodies.
EXAMPLE 14 In situ hybridization This example describeε the procedure used for in situ hybridization used for coexpression of nNOS and PSD-95 transcripts.
In situ hybridization used 35S-labeled RNA probes as described in Methods Enzvmol.. 225: 384-404 (1993). Antisense probes to nNOS (nucleotides 4119-5057, PSD-95 (1-1155), PSD 93 (237-927) or sense control PSD-93 (237-927) were synthesized from bluescript vectorε. EXAMPLE 15 PSD-95 and nNOS Colocalization-Immunochemistrv Assay This example describeε immunohiεtochemistry assay used in studies of the association of nNOS with PSD-95 in neuronal populations and during embryonic development.
Rats were perfused with 4% paraformaldehyde, tissues were harvested, post-fixed at 4°C for 3 hours, and cryoprotected in 20% sucrose overnight. Twenty micron sections were cut on a cryostat and melted onto glass slides (Plus) , obtained from Fisher. Sections were then blocked for one hour in a buffer containing 2% goat serum, 0.1% triton X-100 in PBS. Primary antibodies to nNOS (polyclonal nNOS) , PSD 95, or αl-syntrophin were diluted into a blocking reagent and incubated with sections overnight. Immunoperoxidase histochemistry waε performed using the ABC method according to a kit obtained from Vector. Immunofluorescent εtaining of rat extensor digitorum longus muscle was done as deεcribed in Cell. 82:
743-752 (1995) . Control sectionε lacking primary antisera were stained in parallel. EXAMPLE 16
Cell Culture and Transfection This example describes assays used for cell culture and cloning of nNOS and transfection assays.
Neuronal NOS cDNA were cloned into the EcoRV and Xba I sites of the mammalian expression vector pcDNA 3 (Invitrogen) . 5'a and 5'b containing constructs were amplified by PCR, sequenced, and cloned into the unique Nar I reεtriction site of nNOS. PSD-95-myc construct containing amino acids 1-386 with a C-terminal myc-epitope tag was amplified by PCR and cloned into the BamHI and EcoRI sites of pcDNAIII. Monkey COS cells were grown and transfected using calcium phosphate as previously described in Nature. 351: 714-718 (1991) . Two days following transfection cells were washed with PBS, harvested in 2 ml of buffer containing 25 mM TrisHCl pH 7.4, 100 mM NaCl, ImM EDTA, ImM EGTA, ImM PMSF and disrupted using a polytron. EXAMPLE 17 Immunoprecipitations of PSD-95 Proteins Thiε example deεcribeε procedure used for immunoprecipitation of PSD-95 proteins. Rat cerebellum was homogenized in 20 volumeε of a buffer containing 25 mM Triε (pH 7.5), 150 mM NaCl and centrifuged at l00,000x g to yield cytoεol. Cerebellar membraneε were εolubilized with 1% digitonin and 100 mM NaCl and centrifuged to remove the inεoluble cytoεkeleton. Three μl of PSD-95 polyclonal antiεerum to PSD-95 or 3 μl non-immune serum were added to l ml (500 μg) of cerebellar cytosol or solubilized membranes. After a 60 minute incubation on ice, 50 μl of protein A sepharose was added to precipitate antibodies. Protein A pellets were washed 3 times with a buffer containing 200 mM NaCl and 1% triton X-100. Immunoprecipitated proteins were denatured with a loading buffer and resolved by SDS-PAGE. Heavy microsomes of rat gastrocnemius were prepared and solubilized with 1% triton X-100 as described in Cell. 82: 743-752 (1995) . Five μg polyclonal antiserum to αl-syntrophin or non-immune serum were added to 1 ml (500 μg) solubilized muscle samples. Immunoprecipitations from transfected COS cells used polyclonal antibody to nNOS.
EXAMPLE 18 GST Fusion Protein Affinity Chromatography This example describes methods used for construction of GST fusion constructs.
GST fusion constructε were conεtructed by PCR and fusion proteins purified as described in Example 3. For "pull down" assays, solubilized tissue samples were incubated with control or GST-fusion protein beads for 1 hour. Beads were waεhed with a buffer containing 0.5% triton X-100 and 350 mM NaCl, and proteinε were eluted with SDS loading buffer. NMDA receptor peptide (SEQ ID NO: 7:) (lys leu ser ser ile glu ser asp val) or control peptide (SEQ ID NO: 8:) (lys pro lys his ala lys his pro asp gly his ser gly asn leu cys) were added where indicated during tissue incubation with the fusion protein. EXAMPLE 19 Generation of αl-Svntrophin Fusion Proteinε and Protein Overlay Assay This example describes production of αl-syntrophic fusion proteins and procedures used therefore. cDNAs encoding mouse αl-syntrophin domains (PHla domain, amino acids 1-77; PHlb, 162-271; PDZ, 75-170; PH2, 281-402; SU domain 401-499) were amplified by PCR and cloned into pET28a vector (Novagen, Inc.) with the exception of PHla and PHlb that were ligated together to produce the intact PHI domain.
Clones were sequenced and electroporated into BL21
(λDE3)pLysS cells. Overnight cultures were diluted 1:10, incubated 2 hours and induced for 3 hours with IPTG. Expressed proteins, which contain a T7.Tag epitope encoded in the vector, were purified on Nickel columns obtained from
Novagen, Inc. The PHI and PDZ domains were purified from the soluble friction; PH2 and SU were purified from urea solubilized inclusions. Fusion proteins were separated on 15% SDS/PAGE gelε, transferred to nitrocellulose membranes, blocked with 5% skim milk in 25 mM Tris (pH 7.5) , 150 mM NaCl and 0.1% tween-20 (TBS-Tween) and incubated with purified fusion proteins (20 μg/ml) in this buffer for 1 hour at 25°C.
Blots were washed 3 x 10 minuteε in TBS-Tween, incubated with primary antibody T7.Tag or GST for 30 minutes and bands visualized by ECL.
EXAMPLE 20 mRNA Isolation and cDNA Analvsiε This example describes procedures used for isolation of mRNA and cDNA analysis.
RNA was isolated using the guanidine isothiocyanate/CsCl method and mRNA was selected using oligo dT sepharose. For Northern blotting, mRNA was separated on a formaldehyde agarose gel and transferred to a Nylon membrane. A random primed probe 32P probe waε generated using the full-length (5057 bp) nNOS cDNA as described in Nature. 351: 714-718 (1991) aε a template. The filter was washed at high stringency, 68βC, 0.1% SSC, 0.1% SDS and exposed to X-ray film overnight at -70°C.
Thermal RACE-PCR was performed as described in Method Enzymol.. 218: 340-356 (1993). The sequence of the nNOS specific primers in exon 3 were: Race 1: SEQ ID NO: 9: Race 2: SEQ ID NO: 10:
For RT-PCR, mRNA was reverse transcribed with RTth polymerase using random hexamer primers. The sequence of the PCR primers used were: PI: SEQ ID NO: 11: P2: SEQ ID NO: 12: P3: SEQ ID NO: 13: P4: SEQ ID NO: 14: Clones encoding PSD-93 were isolated from a rat brain cDNA library (Stratagene) by plaque hybridization.
EXAMPLE 21 nNOS Protein Purification and Catalytic Assays This example describes purification procedure used for solubilized tissue homogenated and nNOS protein catalytic assays.
Solubilized tisεue homogenates were incubated with 100 μl of 2'5'-ADP agarose (Sigma), columns were washed with 5 ml of buffer containing 0.35 M NaCl, and were eluted with 10 mM NADPH. Catalytic NOS activity was quantitated by monitoring the conversion of [3H]arginine to [3H]citrulline as described in PNAS USA. 87:682-685 (1990) .
EXAMPLE 22 Histologic Analysis of Nitric Oxide Synthase as a Diagnostic Test for Muscle Disease
This example describeε a diagnoεtic teεt useful for detecting muscle disease.
Human muscle biopsies are harvested and span frozen, and cryosectioned according to a standard protocol. Sarcolemmal localization of nNOS is detected by either immunofluorescence analysis or by histochemical εtaining for NADPH diaphorase, which reflects nNOS activity according to Nature: 347:768-770 ( 1991 ) . nNOS immunofluorescence is performed as described in Example 10. Briefly, nNOS antibodies are applied to cryostat sections of muscle samples overnight at 4°C. Secondary goat anti-rabbit Cy-3 conjugated antibodies (1:200) are obtained from Jackson Laboratories and are used according to the manufacturer's specifications.
NADPH diaphorase staining is performed as described in
Nature, ibid. Briefly, the cryosections are incubated with ImM NADPH, 0.2 mM nitroblue tetrazoliu in a 0.1 M Tris-HCl buffer (pH 7.4) containing 0.2% triton X-100 for 90 minutes at room temperature.
Presence of nNOS is detected by the presence of blue staining. The presence of sarcolemmal nNOS staining is consistent with presence of a functional dystrophin molecule. The absence of sarcolemmal nNOS is a sensitive and specific indicator of abnormal dystrophins.
EXAMPLE 23 Binding Assay to Screen for compounds that Disrupt Interaction of nNOS. NMDA Receptors or Other Ion Channels with PSD-95 or PSD-93 This example describes a binding asεay uεeful for screening compounds which prevent, inhibit or disrupt binding of nNOS, NMDA receptors or other ion channels with PSD-95 and PSD-93 proteins.
GST fusion proteins linked to the first two or three PDZ motifs of PSD-95 or PSD-93 are expressed in E. coli as described in Example 6. Binding interactions to this fragment are monitored by a variety of asεays known and used in teh art. To detect binding of endogenouε nNOS or NMDA receptor subunits to PSD-05 or PSD-93 pull down assays are done as described below.
Larger scale screening assays are facilitated by expressing an appropriate N-terminal fragment of nNOS in a bacterial expression system. This fragment is radiolabeled or epitope tagged and binding of these fragment to expressed PSD-95 or PSD-93 GST-fusion protein is monitored by a filtration binding asεay. nNOS 1-299 is expressed in E-coli with a N-terminal hexahistidine tag and a heart muscle protein kinase site. This fragment is radiolabeled with 32P using [32P] ATP and heart muscle kinase. PSD-95 or PSD-93 GST-fuεion proteins are then incubated with the labeled nNOS fragment. After incubation to reach binding equilibrium, PSD-95 or PSD-93 fragments are retained by immobilized glutathione resin, and the resin is washed to elute unbound nNOS fragments. Bound nNOS fragments on the resin are quantitated by scintillation counting or by an ELISA. Using this asεay, large scale screening of compounds for drug discovery is possible.
SEQUENCE LISTING
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(ii) TITLE OF INVENTION: NITRIC OXIDE SYNTHASE BINDING PROTEINS AND A METHOD FOR THEIR USE IN TREATMENT AND DIAGNOSIS OF MUSCULAR DYSTROPHY
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
CTAATTGAAA TTACTGGGCA TAATGCTATA TATAGCCAAT GAAGAGATTC TGAGCTCTCA 60
CTCAGTGCCT TCAAGACATG TCGTTTTGTA GTCAGAGGAA ACAGGGATCA ATGCATTTTC 120
AAACTGACAG AGGGAACGGA TGCTCCTTAG CAGCACATGC CCAGGATCGT GTGTGTGGGG 180
CTTGCGCTGT GCTGAGAAGC TGATCACCGG CCCATATGCC CCTTATT AC TGCAATGTTC 240
TTTGCATGTT ATTGTGCACT CCGGACTAAC GTGAAGAAGT ATCGATACCA AGATGAGGAC 300
GGTCCACATG ATCATTCCTT ACCTCGGCTA ACTCATGAAG TAAGAGGTCC AGAACTGGTG 360
CATGTGTCGG AAAAGAACCT CTCTCAAATA GAAAATGTCC ACCGATATGT CTTACAGTCT 420
CACATTTCTC CTCTGAAGGC TAGCCCTGCT CCTATAATTG TCAACACAGA CACTTTGGAC 480
ACTATTCCTT ATGTCAATGG AACAGAAATT GAATATGAAT TTGAAGAAAT TACATTGGAG 540
AGGGGAAATT CAGGTCTGGG ATTCAGTATT GCTGGAGGGA CAGATAATCC TCACATTGGA 600 GATGACCCTG GCATATTTAT TACGAAGATT ATTCCAGGAG GTGCTGCAGC AGAGGATGGC 660 AGACTCAGGG TCAACGATTG TATCTTGCGG GTGAATGAAG TTGATGTGTC GGAGGTTTCC 720 CACAGTAAAG CAGTGGAGGC CCTCAAGGAA GCAGGCTCTA TTGTTCGACT GTATATACTT 780 AGAAGACGAC CCATCCTGGA GACTGTTGTG GAAATCAAAC TTTTCAAAGG GCCAAAAGGT 840 TTAGGCTTCA GTATTGCTGG AGGGGTGGGG AACCAGCACA TACCCGGAGA CAACAGCATT 900 TATGTAACGA AAATTATGGA TGGTGGAGCT GCACAGAAAG ATGGGAGGTT GCAAGTAGGA 960 GACAGACTGC TAATGGTAAA TAACTATAGT TTAGAAGAAG TTACACATGA AGAGGCTGTA 1020 GCGATATTGA AAAATACATC TGATGTTGTT TATCTAAAAG TTGGCAAACC CACAACCATT 1080 TATATGACTG ATCCTTATGG GCCACCGGAT ATCACTCACT CTTATTCTCC ACCAATGGAA 1140 AATCATCTAC TGTCTGGTAA CAATGGCACG TTAGAATACA AAACATCCCT GCCGCCCATC 1200 TCTCCAGGGA AGTACTCACC AATTCCAAAG CACATGCTGG TTGAAGATGA CTACACAAGG 1260 CCTCCGGAAC CTGTTTACAG CACTGTGAAT AAACTGTGTG ATAAACCTGC TTCTCCCAGG 1320 CACTATTCCC CTGTTGAGTG TGACAAAAGC TTCCTTCTCT CAACTCCTTA CCCCCACTAC 1380 CACCTAGGCC TGCTCCCTGA CTCTGACATG ACCAGTCATT CTCAGCACAG TACTGCAACT 1440 CGTCAGCCCT CAGTGACTCT CCAACGGGCC ATCTCCCTGG AAGGGGAGCC CCGAAAGGTG 1500 GTCCTTCACA AAGGCTCCAC TGGCCTGGGC TTCAACATTG TGGGTGGAGA AGACGGAGAA 1560 GGTATTTTTG TATCCTTCAT TCTGGCCGGT GGACCAGCAG ACCTGAGTGG GGAGCTCCAG 1620 AGAAGAAAAC AGATTTTATC GGTGAATGGT ATCCATCTCC CAGGAGACTC TCATGAACAG 1680 GCACTTCCCC TGAAGGGGGC GGGGCAGACA GTGACAATCA TAGCACAATA TCAACCTGAA 1740 GATTACTCTC GATTCGAGGC CAAAATCCAT GACCTACGAG AGCAGATGAT GAACCACAGC 1800 ATGAGTTCCG GGTCCGGGTC CCTTCGAACC AATCAGAAAC GCTCCCTGTA TGTCAGAGCC 1860 ATGTTTGACT ATGACAAGAG CAAGGACAGT GGACTGCCTA GCCAAGGACT TAGTTTTAAA 1920 TATGGAGACA TCCTTAATGT CATCAATGCC TCTGATGATG AGTGGTGGCA AGCCAGAAGG 1980 GTCATACAAG ATGGGGACAG CGAGGAGATG GGAGTCATTC CCAGCAAACG GAGGGTGGAλ 2040 AGAAAGGAGC GTGCCCGATT GAAGACAGTG AAGTTCAATG CAAAACCTGG TGTGATTGAT 2100 TCCAAAGGGT CATTCAATGA CAAGCGTAAA AAGAGCTCCA TCTTTTCACG AAAATTCCCA 2160 TTCTACAAGA ACAAGGAGCA GAGTGAGCAG GAAACCAGTG ATCCTGAACG AGGACAAGAA 2220 GATCTCATTC TTTCCTATGA ACCTGTCACG AGGCAGGAAA TAAACTACAC CCGACCAGTG 2280 ATTATCCTGG GCCCCATGAA GGATCGAATC AATGATGACT TGATATCTGA ATTTCCTGAT 2340 AAATTTGGCT CCTGTGTGCC TCATACTACG AGGCCAAAGC GTGACTACGA AGTCGACGGC 2400 AGAGACTATC ACTTTGTCAT TTCTAGAGAA CAAATGGAGA AAGATATCCA AGAGCACAAA 2460 TTTATAGAAG CCGGCCAGTA CTATGACAAT TTATATGGAA CCAGTGTGCA GTCTGTGAGA 2520 TTTGTAGCAG AAAGGGGCAA ACACTGTATA CATGATGTAT CGGGAAATGC TATTAAGCGG 2580 TTACAAGTTG CCCAGCTCTA TCCCATTGCT ATCTTCATAA AGCCCAAGTC TCTGGAACCT 2640 CTGATGGAGA TGAATAACGG TCTAATGGAG GAACAAGCCA AGAAAACCTA TGACCGGGCA 2700 ATTAAGCTAG AACAAGAATT TGGAGAATAT TTTACAGCTA TTGTCCAAGG AGATACCTTA 2760 GAAGATATTT ACAACCAATG CAAGCTTGTT ATTGAAGAGC AGTCTGGACC TTTCATCTGG 2820 ATTCCCTCAA AGGAGAAGTT ATAAATTAGC TACTGCACCT CTGACAACGA CGAAGAGCAT 2880 ATAGAAGAAC AAATATATAT AATATACACT GAGGCTTTAT GTTTTGTTGC ATTATGTTTT 2940 GCAGTCAATG TGAATCTTAT GAA 2963
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: leu ile glu ile thr gly his asn ala ile tyr ser gin 1 5 10 13
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: arg asp ser glu leu ser leu ser ala phe lys thr cys arg phe 1 5 10 15 val val arg gly asn arg asp gin cys ile phe lys leu thr glu
20 25 30 gly thr asp ala pro
35
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: gin his met pro arg ile val cys val gly leu ala leu cys 1 5 10 14
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 882 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: glu ala asp his arg pro ile cys pro leu phe thr ala met phe
1 5 10 15 phe ala cys tyr cys ala leu arg thr asn val lys lys tyr arg
20 25 30 tyr gin asp glu asp gly pro his asp his ser leu pro arg leu
35 40 45 thr his glu val arg gly pro glu leu val his val ser glu lys
50 55 60 asn leu ser gin ile glu asn val his gly tyr val leu gin ser
65 70 75 his ile ser pro leu lys ala ser pro ala pro ile ile val asn
80 85 90 thr asp thr leu asp thr ile pro tyr val asn gly thr glu ile
95 100 105 glu tyr glu phe glu glu ile thr leu glu arg gly asn ser gly
110 115 120 leu gly phe ser ile ala gly gly thr asp asn pro his ile gly
125 130 135 asp asp pro gly ile phe ile thr lys ile ile pro gly gly ala
140 145 150 ala ala glu asp gly arg leu arg val ar asp cys ile leu arg
155 : 165 val asn glu val asp val ser glu val ε -ιis ser lys ala val
170 1 180 glu ala leu lys glu ala gly ser ile va. arg leu tyr ile leu
185 190 195 arg arg arg pro ile leu glu thr val val glu ile lys leu phe
200 205 210 lys gly pro lys gly leu gly phe ser ile ala gly gly val gly
215 220 225 asn gin his ile pro gly asp asn ser ile tyr val thr lys ile
230 235 240 met asp gly gly ala ala gin lys asp gly arg leu gin val gly
245 250 255 asp arg leu leu met val asn asn tyr ser leu glu glu val thr 260 265 270 his glu glu ala val ala ile leu lys asn thr ser asp val val
275 280 285 tyr leu lys val gly lye pro thr thr ile tyr met thr asp pro
290 295 300 tyr gly pro pro asp ile thr his ser tyr ser pro pro met glu
305 310 315 asn his leu leu ser gly asn asn gly thr leu glu tyr lys thr
320 325 330 ser leu pro pro ile ser pro gly lys tyr ser pro ile pro lys
335 340 345 his met leu val glu asp asp tyr thr arg pro pro glu pro val
350 355 360 tyr ser thr val asn lys leu cys asp lys pro ala ser pro arg
365 370 375 his tyr ser pro val glu cys asp lys ser phe leu leu ser thr
380 385 390 pro tyr pro his tyr his leu gly leu leu pro asp ser asp met
395 400 405 thr βer his ser gin his ser thr ala thr arg gin pro ser val
410 415 420 thr leu gin arg ala ile βer leu glu gly glu pro arg lys val
425 430 435 val leu his lys gly ser thr gly leu gly phe asn ile val gly
440 445 450 gly glu asp gly glu gly ile phe val ser phe ile leu ala gly
455 460 465 gly pro ala asp leu ser gly glu leu gin arg arg lys gin ile
470 475 480 leu ser val asn gly ile his leu pro gly asp ser his glu gin
485 490 495 ala leu pro leu lys gly ala gly gin thr val thr ile ile ala
500 505 510 gin tyr gin pro glu asp tyr ser arg phe glu ala lys ile his
515 520 525 aβp leu arg glu gin met met asn his ser met ser ser gly ser
530 535 540 gly ser leu arg thr asn gin lys arg ser leu tyr val arg ala
545 550 555 met phe asp tyr asp lys ser lys asp ser gly leu pro ser gin
560 565 570 gly leu ser phe lys tyr gly asp ile leu asn val ile asn ala
575 580 585 βer asp asp glu trp trp gin ala arg arg val ile gin asp gly
590 595 600 asp ser glu glu met gly val ile pro ser lys arg arg val glu
605 610 615 arg lys glu arg ala arg leu lys thr val lys phe asn ala lys
620 625 630 pro gly val ile asp ser lys gly ser phe asn asp lys arg lys
635 640 645 lys ser phe ile phe ser arg lys phe pro phe tyr lys asn lys
650 655 660 glu gin ser glu gin glu thr ser asp pro glu arg gly gin glu
665 670 675 asp leu ile leu ser tyr glu pro val thr arg gin glu ile asn
680 685 690 tyr thr arg pro val ile ile leu gly pro met lys aβp arg ile
695 700 705 asn aβp asp leu ile ser glu phe pro asp lys phe gly ser cys
710 715 720 val pro his thr thr arg pro lys arg asp tyr glu val asp gly
725 730 735 arg asp tyr his phe val ile ser arg glu gin met glu lys asp
740 745 750 ile gin glu his lys phe ile glu ala gly gin tyr tyr asp asn
755 760 765 leu tyr gly thr ser val gin ser val arg phe val ala glu arg
770 775 780 gly lys his cys ile his asp val ser gly asn ala ile lys arg
785 790 795 leu gin val ala gin leu tyr pro ile ala ile phe ile lys pro
800 805 810 lys ser leu glu pro leu met glu met asn asn gly leu met glu
815 820 825 glu gin ala lys lys thr tyr asp arg ala ile lys leu glu gin
830 835 840 glu phe gly glu tyr phe thr ala ile val gin gly asp thr leu
845 850 855 glu asp ile tyr asn gin cys lys leu val ile glu glu gin βer
860 865 870 gly pro phe ile trp ile pro ser lys glu lys leu
875 880 882
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ile ser tyr cys thr ser asp asn asp glu glu his ile glu glu
1 5 10 15 gin ile tyr ile ile tyr thr glu ala leu cys phe val ala leu
20 25 30 cys phe ala val asn val asn leu met
35 39
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: lys leu ser ser ile glu βer asp val 1 5 9
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acid
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: lye pro lys his ala lys his pro asp gly his ser gly asn leu cys 1 5 10 15 16
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CCACAGATCA TTGAAGACTC G 21
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GGGGAATTCC CCGCCCCAGG GGCGGGGAGC TTT 33
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GTCCCTGCGT ATTGATGCA 19
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GGCCGACCTG AGATTCCC 18
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
CTCTGCATCT GTCAAGCTGG 20
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
CCTTCACCAG GAAGCCCAGA 20
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE: (A) ORGANISM:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: met phe phe ala eye tyr cys ala leu arg thr asn val lys lys 1 5 10 15 tyr arg tyr gin asp glu asp gly pro his 20 25
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: asp his ser leu pro arg leu thr his glu val arg gly pro glu
1 5 10 15 leu val his val ser glu lys asn leu ser gin ile glu asn val
20 25 30 his gly tyr val leu gin ser his ile ser pro leu lys ala ser
35 40 45 pro ala pro ile ile val asn thr asp thr leu asp thr ile pro
50 55 60 tyr 61
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 396 amino acids
(B) TYPE: amino acid (C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: val asn gly thr glu ile glu tyr glu phe glu glu ile thr leu
1 5 10 15 glu arg gly asn βer gly leu gly phe ser ile ala gly gly thr
20 25 30 aβp asn pro his ile gly asp asp pro gly ile phe ile thr lys
35 40 45 ile ile pro gly gly ala ala ala glu asp gly arg leu arg val
50 55 60 asn asp cys ile leu arg val asn glu val aβp val ser glu val
65 70 75 βer his βer lye ala val glu ala leu lys glu ala gly ser ile
80 85 90 val arg leu tyr ile leu arg arg arg pro ile leu glu thr val
95 100 105 val glu ile lys leu phe lye gly pro lys gly leu gly phe βer
110 115 120 ile ala gly gly val gly asn gin his ile pro gly asp asn ser
125 130 135 ile tyr val thr lys ile met asp gly gly ala ala gin lys asp
140 145 150 gly arg leu gin val gly aβp arg leu leu met val asn asn tyr
155 160 165 βer leu glu glu val thr his glu glu ala val ala ile leu lys
170 175 180 asn thr ser asp val val tyr leu lys val gly lys pro thr thr
185 190 195 ile tyr met thr asp pro tyr gly pro pro asp ile thr his ser
200 205 210 tyr βer pro pro met glu asn his leu leu βer gly asn asn gly
215 220 225 thr leu glu tyr lys thr ser leu pro pro ile βer pro gly lys
230 235 240 tyr βer pro ile pro lys his met leu val glu asp asp tyr thr
245 250 255 arg pro pro glu pro val tyr ser thr val asn lys leu cys asp
260 265 270 lys pro ala βer pro arg his tyr βer pro val glu cys asp lys
275 280 285 βer phe leu leu βer thr pro tyr pro his tyr his leu gly leu
290 295 300 leu pro aβp βer aβp met thr βer his βer gin his ser thr ala
305 310 315 thr arg gin pro ser val thr leu gin arg ala ile ser leu glu
320 325 330 gly glu pro arg lys val val leu his lys gly ser thr gly leu
335 340 345 gly phe asn ile val gly gly glu asp gly glu gly ile phe val
350 355 360 βer phe ile leu ala gly gly pro ala aβp leu ser gly glu leu
365 370 375 gin arg arg lys gin ile leu βer val asn gly ile his leu pro
380 385 390 gly asp βer hie glu gin
Figure imgf000081_0001
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: ala leu pro leu lye gly ala gly gin thr val thr ile ile ala
1 5 10 15 gin tyr gin pro glu aβp tyr βer arg phe glu ala lys ile hie
20 25 30 aβp leu arg glu gin met met asn his βer met ser ser gly βer
35 40 45 gly βer leu arg thr aβn gin lys arg ser leu tyr val arg ala
50 55 60 met phe aβp tyr asp lye βer lys asp ser gly leu pro βer gin
65 70 75 gly leu ser phe lye tyr gly asp ile leu asn val ile asn ala
80 85 90 βer aβp aβp glu trp trp gin ala arg arg val ile gin aβp gly
95 100 105 aβp βer glu glu met gly val ile pro βer lys arg arg val glu
110 115 120 arg lys glu arg ala arg leu lys thr val lys phe asn ala lys
125 130 135 pro gly val ile asp βer lys arg gly gin glu asp leu ile leu
140 145 150 βer tyr glu pro val thr arg gin glu ile asn tyr thr arg pro
155 160 165 val ile ile leu gly pro met lys asp arg ile asn asp asp leu
170 175 180 ile ser glu phe pro aβp lys phe gly ser cys val pro his thr
185 190 195 thr arg pro lye arg aβp tyr glu val asp gly arg aβp tyr hie
200 205 210 phe val ile ser arg glu gin met glu lys asp ile gin glu his
215 220 225 lye phe ile glu ala gly gin tyr tyr asp asn leu tyr gly thr
230 235 240 βer val gin βer val arg phe val ala glu arg gly lys his cys
245 250 255 ile his asp val βer gly aβn ala ile lys arg leu gin
260 265 268
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 85 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: val ala gin leu tyr pro ile ala ile phe ile lys pro lys ser
1 5 10 15 leu glu pro leu met glu met asn asn gly leu met glu g u gin
20 25 30 ala lys lys thr tyr asp arg ala ile lys leu glu gin giu phe
35 40 45 gly glu tyr phe thr ala ile val gin gly asp thr leu glu asp
50 55 60 ile tyr asn gin eye lye leu val ile glu glu gin ser gly pro
65 70 75 phe ile trp ile pro ser lys glu lys leu
80 85
(2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 20: met asp eye leu cys ile val thr thr 1 5 9
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: lye lye tyr arg tyr gin asp glu aβp thr pro pro leu glu hie 1 5 10 15 βer pro ala hie leu
Figure imgf000083_0001
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 280 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22 pro aβn gin ala aβn βer pro pro val ile val aβn thr aβp thr
1 5 10 15 leu glu ala pro gly tyr glu leu gin val aβn gly thr glu gly
20 25 30 glu met glu tyr glu glu ile thr leu glu arg gly asn ser gly
35 40 45 leu gly phe βer ile ala gly gly thr aβp asn pro his ile gly
50 55 60 aβp asp pro ser ile phe ile thr lys ile ile pro gly gly ala
65 70 75 ala ala gin asp gly arg leu arg val asn asp ser ile leu phe
80 85 90 val aβn glu val aβp val arg glu val thr his ser ala ala val
95 100 105 glu ala leu lys glu ala gly ser ile val arg leu tyr val met
110 115 120 arg arg lys pro pro ala glu lys val met glu ile lys leu ile
125 130 135 lys gly pro lys gly leu gly phe ser ile ala gly gly val gly
140 145 150 aβn gin hie ile pro gly aβp asn ser ile tyr val thr lys ile
155 160 165 ile glu gly gly ala ala his lys asp gly arg leu gin ile gly
170 175 180 asp lys ile leu ala val asn ser val gly leu glu asp val met
185 190 195 his glu asp ala val ala ala leu lys asn thr tyr asp val val 200 205 210 tyr leu lys val ala lys pro ser aβn ala tyr leu ser asp ser
215 220 225 tyr ala pro pro asp ile thr thr ser tyr βer gin hie leu aβp
230 235 240 aβn glu ile βer his βer βer tyr leu gly thr asp tyr pro thr
245 250 255 ala met thr pro thr βer pro arg arg tyr βer pro val ala lys
260 265 270 aβp leu leu gly glu glu aβp ile pro arg
275 280
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 203 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: glu pro arg arg ile val ile his arg gly ser thr gly leu gly
1 5 10 15 phe asn ile val gly gly glu asp gly glu gly ile phe ile ser
20 25 30 phe ile leu ala gly gly pro ala asp leu ser gly glu leu arg
35 40 45 lys gly aβp gin ile leu ser val asn gly val asp leu arg asn
50 55 60 ala ser his glu gin ala ala ile ala leu lys aβn ala gly gin
65 70 75 thr val thr ile ile ala gin tyr lye pro glu glu tyr βer arg
80 85 90 phe glu ala lye ile his asp leu arg glu gin leu met aβn βer
95 100 105 βer leu gly ser gly thr ala ser leu arg ser asn pro lys arg
110 115 120 gly phe tyr ile arg ala leu phe asp tyr asp lys thr lys aβp
125 130 135 eye gly phe leu βer gin ala leu ser phe arg phe gly aβp val
140 145 150 leu hie val ile aβp ala gly asp glu glu trp trp gin ala arg
155 160 165 arg val his βer aβp βer glu thr asp asp ile gly phe ile pro
170 175 180 βer lys arg arg val glu arg arg glu trp ser arg leu lys ala
185 190 195 lye aβp trp gly βer βer ser gly
200 203
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 127 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: βer gin gly arg glu asp ser val leu ser tyr glu thr val thr
1 5 10 15 gin met glu val his tyr ala arg pro ile ile ile leu gly pro
20 25 30 thr lys asp arg ala asn asp asp leu leu ser glu phe pro asp 35 40 45 lys phe gly ser cys val pro his thr thr arg pro lys arg glu
50 55 60 tyr glu ile aβp gly arg aβp tyr hie phe val βer βer arg glu
65 70 75 lye met glu lye aβp ile gin ala his lye phe ile glu ala gly 80 85 90 gin tyr aβn βer his leu tyr gly thr ser val gin βer val arg 95 100 105 glu val ala glu gin gly lys his cys ile leu asp val βer ala 110 115 120 aβn ala val arg arg leu gin 125 127
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 85 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: ala ala his leu his pro ile ala ile phe ile arg pro arg ser
1 5 10 15 leu glu asn val leu glu ile asn lys arg ile thr glu glu gin
20 25 30 ala arg lys ala phe asp arg ala thr lys leu glu gin glu phe
35 40 45 thr glu cys phe ser ala ile val glu gly aβp ser phe glu glu
50 55 60 ile tyr his lys val lys arg val ile glu asp leu ser gly pro
65 70 75 tyr ile trp val pro ala arg glu arg leu
80 85
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: val arg leu phe lys arg lys val gly gly leu gly phe leu val 1 5 10 15 lye 16
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: glu arg val βer lys pro pro val ile ile βer asp leu ile arg
1 5 10 15 gly gly ala ala glu gin βer gly leu ile gin ala gly aβp ile
20 25 30 ile leu ala val aβn aβp arg pro leu val asp leu βer tyr aβp
35 40 45 βer ala leu glu val leu
Figure imgf000086_0001
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acidβ
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: val arg ile val lye gin glu ala gly gly leu gly ile βer ile 1 5 10 15 lys gly gly 18
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: arg glu asn hie met pro ile leu ile ser lye ile phe arg gly leu
1 5 10 15 ala ala glu gin βer arg leu leu phe val gly asp ala ile leu βer
20 25 30 val aβn gly thr aβp leu arg asp ala thr his asp gin ala val gin
35 40 45 ala leu
47
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: val arg val val lys gin glu ala gly gly leu gly ile ser ile 1 5 10 15 lys gly gly 18
(2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: arg glu asn arg met pro ile leu ile βer lye ile phe pro gly
1 5 10 15 leu ala ala asp gin βer arg ala leu arg leu gly asp ala ile
20 25 30 leu βer val aβn gly thr asp leu arg gin ala thr his aβp gin
35 40 45 ala val gin ala leu
50
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: arg ile val ile his arg gly ser thr gly leu gly phe asn ile 1 5 10 15 val gly gly 18
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: glu asp gly glu gly ile phe ile ser phe ile leu ala gly gly
1 5 10 15 pro ala aβp leu βer gly glu leu arg lys gly asp gin ile leu
20 25 30 βer val aβn gly val aβp leu arg asn ala ser his glu gin ala
35 40 45 ala ile ala leu
49
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 34: thr ile thr ile gin lye gly pro gin gly leu gly phe asn ile 1 5 10 15 val gly gly 18
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: glu aβp gly gin gly ile tyr val ser phe ile leu ala gly gly
1 5 10 15 pro ala asp leu gly ser glu leu lys arg gly asp gin ile leu
20 25 30 βer val asn asn val asn leu thr his ala thr his glu glu ala
35 40 45 ala gin ala leu
49
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: βer ile aβn met glu ala val asn phe gly leu gly ile βer ile 1 5 10 15 val gly gin 18
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 52 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: βer aβn arg gly gly asp gly gly ile tyr val gly ser ile met
1 5 10 15 lys gly gly ala ala val leu asp gly arg ile glu pro gly asp
20 25 30 met ile leu gin val aβn aβp val asn phe glu asn met thr asn
35 40 45 aβp glu ala val arg val leu
50 52
(2) INFORMATION FOR SEQ ID NO: 38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 74 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: glu val lys leu phe lys asn ser ser gly leu gly phe ser phe
1 5 10 15 ser arg glu asp asn leu ile pro glu gin ile asn ala βer ile
20 25 30 val arg val lye lye leu phe pro gly gin pro ala ala glu βer
35 40 45 gly lye ile aβp val gly aβp val ile leu lye val aβn gly ala
50 55 60 βer leu lye gly leu ser gin gin glu ala ile βer ala leu
65 70 74
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: val arg phe lys lys gly asp ser val gly leu arg leu ala 1 5 10 14
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: gly gly aβn asp
Figure imgf000089_0001
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: val gly ile phe val ala gly ile gin glu gly thr βer ala glu
1 5 10 15 gin glu gly leu leu gin glu gly asp gin ile leu lys val asn
20 25 30 thr gin asp phe arg gly leu val arg glu asp ala val leu val
35 40 45 leu val arg phe lys lys gly asp ser val gly leu arg leu ala
50 55 60

Claims

WHAT IS CLAIMED IS:
1. A method for diagnosing muscular dystrophy in a mammal comprising determination of absence or decreaεe of neuronal-type nitric oxide synthaεe protein in a mammal skeletal muscle sample.
2. The method of Claim 1 comprising steps:
(a) obtaining the skeletal muscle biopsy sample; and (b preparing the muscle sample cryosections; and
(c) detecting the presence or absence of nitric oxide synthase protein by immunofluorescence assay, by histochemical staining or biochemically.
3. The method of Claim 2 wherein the immunofluorescent assay compriseε a detection with a primary antibody.
4. The method of Claim 2 wherein the detection of nitric oxide synthase iε by histochemical staining for NADPH diaphorase.
5. The method of Claim 4 wherein the cryosections are incubated with NADPH and stained with nitroblue tetrazolium.
6. The method of Claim 5 wherein the presence of nitric oxide synthaεe iε detected by a presence of blue color.
7. The method of Claim 1 wherein nitric oxide synthase is detected biochemically and the detection comprises analysiε of the nitric oxide synthase protein by Western blotting.
8. A method for treatment of muscular dystrophy by restoration to a patient in need of such treatment of functional dystrophin or a functional fragment thereof able to bind neuronal nitric oxide to syntrophin in muscle sarcolemma.
9. The method of Claim 8 wherein neuronal nitric oxide is bound to syntrophin through its PDZ domain.
10. The method of Claim 9 wherein the method for treatment of muscular dyεtrophy involveε a production of vectorε encoding conεtructε or fragments thereof that assemble funct i on a l neur on a l n i tr i c ox i d e synthaεe/syntrophin/dystrophin complex.
11. A method for detection, prevention and treatment of neurodegenerative diseases by administering to a patient in need of such treatment an inhibitor of binding of neuronal nitric oxide synthaεe and a binding protein.
12. The method of Claim 11 wherein the binding protein iε a protein identified as PSD-95 or PSD-93.
13. The method of Claim 12 wherein neuronal nitric oxide and the binding protein are bound through their respective PDZ domains.
14. An inhibitor of binding of neuronal nitric oxide synthase and a binding protein.
15. A diagnostic kit for detection of muscular dystrophy in a mammal muscle biopsy sample.
16. A binding protein identified as PSD-93 having sequences identified as SEQ ID NOε: 2-6.
17. A nucleic acid εequence identified aε SEQ ID NO: 1 encoding a protein having sequences SEQ ID NOs: 2-6.
18. A binding assay to monitor interaction of N-methyl-D- aspartate receptors and neuronal nitric oxide synthase through binding interaction with binding proteins PDS-95 and PDS-93.
PCT/US1997/003897 1996-03-08 1997-03-06 Muscular dystrophy, stroke, and neurodegenerative disease diagnosis and treatment WO1997033173A1 (en)

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US6225444B1 (en) 1998-02-10 2001-05-01 Protarga, Inc. Neuroprotective peptides and uses thereof
US6967102B1 (en) 1999-03-11 2005-11-22 University Of Manitoba Nitric oxide manipulation of muscle satellite cell activation
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US8648043B2 (en) 1999-06-02 2014-02-11 Nono Inc. Method of reducing injury to mammalian cells
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JP2003506320A (en) * 1999-06-11 2003-02-18 センター ナショナル デ ラ レシェルルシェ サイエンティフィック−シーエヌアールエス Pharmaceutical compositions comprising NO or at least one NO donating compound or other compound capable of releasing NO or inducing NO formation in cells
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