+

WO1997033173A1 - Traitement et diagnostic de la dystrophie musculaire, de l'ictus et d'autres maladies neurodegeneratives - Google Patents

Traitement et diagnostic de la dystrophie musculaire, de l'ictus et d'autres maladies neurodegeneratives Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
nnos
psd
gly
leu
dystrophin
Prior art date
Application number
PCT/US1997/003897
Other languages
English (en)
Inventor
David S. Bredt
Jay E. Brenman
Daniel S. Chao
Original Assignee
The Regents Of The University Of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Priority to AU22080/97A priority Critical patent/AU2208097A/en
Publication of WO1997033173A1 publication Critical patent/WO1997033173A1/fr

Links

Classifications

    • 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:

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Monoxyde d'azote, monoxyde d'azote synthétase neuronal, protéines liantes du monoxyde d'azote synthétase neuronal, leurs protéines liantes et leurs inhibiteurs, ainsi que leur utilisation pour le traitement et le diagnostic de la dystrophie musculaire, de l'ictus et d'autres maladies neurodégénératives. Dosage diagnostique permettant de déceler l'absence de la dystrophine ou de ses mutations, du monoxyde d'azote synthétase neuronal ou de ses protéines liantes. Méthode de traitement des dystrophies musculaires par la restauration de la molécule fonctionnelles de la dystrophine dans les muscles dystrophes au moyen de la thérapie génique. Protéines liantes du monoxyde d'azote neuronal PSD-95 et PSD-93 utilisés dans la gestion de l'ictus et d'autres maladies neurodégénératives. Clonage et expression des protéines liantes du monoxyde d'azote synthétase neuronal.
PCT/US1997/003897 1996-03-08 1997-03-06 Traitement et diagnostic de la dystrophie musculaire, de l'ictus et d'autres maladies neurodegeneratives WO1997033173A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU22080/97A AU2208097A (en) 1996-03-08 1997-03-06 Muscular dystrophy, stroke, and neurodegenerative disease diagnosis and treatment

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US61311496A 1996-03-08 1996-03-08
US08/613,114 1996-03-08

Publications (1)

Publication Number Publication Date
WO1997033173A1 true WO1997033173A1 (fr) 1997-09-12

Family

ID=24455919

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/003897 WO1997033173A1 (fr) 1996-03-08 1997-03-06 Traitement et diagnostic de la dystrophie musculaire, de l'ictus et d'autres maladies neurodegeneratives

Country Status (2)

Country Link
AU (1) AU2208097A (fr)
WO (1) WO1997033173A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999040112A1 (fr) * 1998-02-10 1999-08-12 Protarga, Inc. Peptides neuroprotecteurs et leur utilisation
WO2000053191A2 (fr) * 1999-03-11 2000-09-14 The University Of Manitoba Modulation de l'activation du precurseur du muscle squelettique
FR2794647A1 (fr) * 1999-06-11 2000-12-15 Centre Nat Rech Scient Compositions pharmaceutique comprenant du no ou au moins un compose capable de liberer ou d'induire la formation de no dans les cellules
WO2001077170A2 (fr) * 2000-04-06 2001-10-18 The University Court Of The University Of Edinburgh Incorporated Under The Universities (Scotland) Acts Matiere biologique et utilisations de celle-ci
WO2001084159A2 (fr) * 2000-05-01 2001-11-08 Board Of Regents, The University Of Texas System Chemins de signalisation de recepteur de lipoproteines basse densite
WO2001087285A2 (fr) * 2000-05-12 2001-11-22 The Johns Hopkins University Inhibition de l'interaction de l'antisens psd93 et psd95 avec des recepteurs de nnos et nmda
US20050059597A1 (en) * 1999-06-02 2005-03-17 Michael Tymianski Method of reducing injury to mammalian cells
US6967102B1 (en) 1999-03-11 2005-11-22 University Of Manitoba Nitric oxide manipulation of muscle satellite cell activation
US20060252677A1 (en) * 2001-11-20 2006-11-09 Osamu Ohara Postsynaptic proteins
US7494981B2 (en) 2000-05-12 2009-02-24 The Johns Hopkins University Inhibition of interaction of PSD93 and PSD95 with nNOS and NMDA receptors
US7595297B2 (en) * 1999-06-02 2009-09-29 Michael Tymianski Method of reducing injury to mammalian cells
WO2010125358A1 (fr) * 2009-04-30 2010-11-04 The University Of Birmingham Méthode de diagnostic et de traitement de la dystrophie musculaire

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5260209A (en) * 1990-05-23 1993-11-09 University Of Iowa Research Foundation Nucleic acids encoding dystrophin-associated proteins
US5266594A (en) * 1992-05-12 1993-11-30 Dawson Valina L Inhibitors of nitric oxide synthase and use thereof to prevent glutamate neurotoxicity
US5466676A (en) * 1990-02-12 1995-11-14 Board Of Regents, The University Of Texas At Austin Satellite cell proliferation in adult skeletal muscle

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5466676A (en) * 1990-02-12 1995-11-14 Board Of Regents, The University Of Texas At Austin Satellite cell proliferation in adult skeletal muscle
US5260209A (en) * 1990-05-23 1993-11-09 University Of Iowa Research Foundation Nucleic acids encoding dystrophin-associated proteins
US5266594A (en) * 1992-05-12 1993-11-30 Dawson Valina L Inhibitors of nitric oxide synthase and use thereof to prevent glutamate neurotoxicity

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ANNU. REV. BIOCHEM., 1994, Vol. 63, BREDT et al., "Nitric Oxide: A Physiologic Messenger Molecule", pages 175-195. *
CELL, 08 September 1995, Vol. 82, BRENMAN et al., "Nitric Oxide Synthase Complexed with Dystrophin and Absent from Skeletal Muscle Sarcolemma in Duchenne Muscular Dystrophy", pages 743-752. *
CELL, 10 March 1995, Vol. 80, CAMPBELL, "Three Muscular Dystrophies: Loss of Cytoskeleton-Extracellular Matrix Linkage", pages 675-679. *
JOURNAL OF BIOLOGICAL CHEMISTRY, 27 October 1995, Vol. 270, No. 43, ADAMS et al., "Mouse alpha1- and beta2-Syntrophin Gene Structure, Chromosome Localization and Homology with a Discs Large Domain", pages 25859-25865. *
NEURON, August 1994, Vol. 13, BREDT et al., "Transient Nitric Oxide Synthase Neurons in Embryonic Cerebral Cortical Plate, Sensory Ganglia and Olfactory Epithelium", pages 301-313. *
NEURON, November 1992, Vol. 9, CHO et al., "The Rat Brain Postsynaptic Density Fraction Contains a Homolog of the Drosophila Discs-Large Tumor Suppressor Protein", pages 929-942. *
NEUROREPORT, 15 August 1994, Vol. 5, No. 13, PETERS et al., "Beta2-Syntrophin: Localization at the Neuromuscular Junction in Skeletal Muscle", pages 1577-1580. *
PROC. NATL. ACAD. SCI. U.S.A., July 1991, Vol. 88, DAWSON et al., "Nitric Oxide Mediates Glutamate Neurotoxicity in Primary Cortical Cultures", pages 6368-6371. *

Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999040112A1 (fr) * 1998-02-10 1999-08-12 Protarga, Inc. Peptides neuroprotecteurs et leur utilisation
US6627601B2 (en) 1998-02-10 2003-09-30 Protarga, Inc. Neuroprotective peptides and uses thereof
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
WO2000053191A2 (fr) * 1999-03-11 2000-09-14 The University Of Manitoba Modulation de l'activation du precurseur du muscle squelettique
WO2000053191A3 (fr) * 1999-03-11 2001-09-13 Univ Manitoba Modulation de l'activation du precurseur du muscle squelettique
US7846897B2 (en) 1999-06-02 2010-12-07 Michael Tymianski Agents for reducing injury to mammalian cells
US7595297B2 (en) * 1999-06-02 2009-09-29 Michael Tymianski Method of reducing injury to mammalian cells
US20150190459A1 (en) * 1999-06-02 2015-07-09 Nono Inc. Method Of Reducing Injury To Mammalian Cells
US20050059597A1 (en) * 1999-06-02 2005-03-17 Michael Tymianski Method of reducing injury to mammalian cells
US7510824B2 (en) 1999-06-02 2009-03-31 Nono Inc. Method of screening peptides useful in treating traumatic injury to the brain or spinal cord
US8648043B2 (en) 1999-06-02 2014-02-11 Nono Inc. Method of reducing injury to mammalian cells
US8071548B2 (en) 1999-06-02 2011-12-06 Nono, Inc. Method of reducing injury to mammalian cells
WO2000076451A3 (fr) * 1999-06-11 2002-06-20 Centre Nat Rech Scient Composition pharmaceutique comprenant du no un compose donneur de no, ou inducteur de la formation de no et son utilisation en therapie
JP2003506320A (ja) * 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 Ｎｏもしくは少なくとも１つのｎｏ供与化合物または細胞においてｎｏを放出するかもしくはｎｏの形成を誘導することが可能な他の化合物を含む薬学的組成物
AU776692B2 (en) * 1999-06-11 2004-09-16 Centre National De La Recherche Scientifique - Cnrs Pharmaceutical composition comprising NO or at least a NO donor compound or another compound capable of releasing or inducing no formation in cells
US6872751B2 (en) 1999-06-11 2005-03-29 Centre National De La Recherche Scientifique - Cnrs Composition and method for augmenting or restoring the production of fetal protein in patient in need thereof
WO2000076451A2 (fr) * 1999-06-11 2000-12-21 Centre National De La Recherche Scientifique - Cnrs Composition pharmaceutique comprenant du no un compose donneur de no, ou inducteur de la formation de no et son utilisation en therapie
FR2794647A1 (fr) * 1999-06-11 2000-12-15 Centre Nat Rech Scient Compositions pharmaceutique comprenant du no ou au moins un compose capable de liberer ou d'induire la formation de no dans les cellules
WO2001077170A3 (fr) * 2000-04-06 2002-03-28 Univ Edinburgh Inc Under The U Matiere biologique et utilisations de celle-ci
WO2001077170A2 (fr) * 2000-04-06 2001-10-18 The University Court Of The University Of Edinburgh Incorporated Under The Universities (Scotland) Acts Matiere biologique et utilisations de celle-ci
WO2001084159A2 (fr) * 2000-05-01 2001-11-08 Board Of Regents, The University Of Texas System Chemins de signalisation de recepteur de lipoproteines basse densite
US7056688B2 (en) 2000-05-01 2006-06-06 Board Of Regents, The University Of Texas System LDL receptor signaling assays
WO2001084159A3 (fr) * 2000-05-01 2003-01-16 Univ Texas Chemins de signalisation de recepteur de lipoproteines basse densite
US7494981B2 (en) 2000-05-12 2009-02-24 The Johns Hopkins University Inhibition of interaction of PSD93 and PSD95 with nNOS and NMDA receptors
US8148347B2 (en) 2000-05-12 2012-04-03 The Johns Hopkins University Inhibition of interaction of PSD93 and PSDS95 with nNOS and NMDA receptors
WO2001087285A3 (fr) * 2000-05-12 2002-08-15 Univ Johns Hopkins Inhibition de l'interaction de l'antisens psd93 et psd95 avec des recepteurs de nnos et nmda
WO2001087285A2 (fr) * 2000-05-12 2001-11-22 The Johns Hopkins University Inhibition de l'interaction de l'antisens psd93 et psd95 avec des recepteurs de nnos et nmda
US20060252677A1 (en) * 2001-11-20 2006-11-09 Osamu Ohara Postsynaptic proteins
WO2010125358A1 (fr) * 2009-04-30 2010-11-04 The University Of Birmingham Méthode de diagnostic et de traitement de la dystrophie musculaire

Also Published As

Publication number Publication date
AU2208097A (en) 1997-09-22

Similar Documents

Publication Publication Date Title
Brenman et al. Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy
WANG et al. Structural requirement of the calcium-channel subunit α2δ for gabapentin binding
Wang et al. Characterization of NMDA receptor subunit‐specific antibodies: distribution of NR2A and NR2B receptor subunits in rat brain and ontogenic profile in the cerebellum
US8394929B2 (en) Nogo receptor-mediated blockade of axonal growth
US20020012965A1 (en) Nogo receptor-mediated blockade of axonal growth
US7465549B2 (en) Methods for identifying agents that bind a levetiracetam binding site (LBS) or compete with LEV binding to a LBS of a synaptic vesicle protein 2 (SV2)
JP2008500266A (ja) Ａｍｐａ受容体エンドサイトーシスの阻害剤を用いた神経細胞反応のモジュレート方法
Schraw et al. A role for Sec1/Munc18 proteins in platelet exocytosis
JP4580618B2 (ja) 神経変性疾患におけるタンパク質凝集に関する材料および方法
JP2003521247A (ja) Ｎｍｄａ受容体のプロテインチロシンホスファターゼとの相互作用
WO1997033173A1 (fr) Traitement et diagnostic de la dystrophie musculaire, de l'ictus et d'autres maladies neurodegeneratives
Bredt Targeting nitric oxide to its targets
WO1997018230A1 (fr) CLONAGE ET EXPRESSION DU RECEPTEUR DE βAPP-C100 (C100-R)
WO1997018230A9 (fr) CLONAGE ET EXPRESSION DU RECEPTEUR DE βAPP-C100 (C100-R)
US5723301A (en) Method to screen compounds that affect GAPDH binding to polyglutamine
JP2002507892A (ja) ヒト・アデニル酸シクラーゼのクローニングおよび特性評価
US20030219739A1 (en) Novel nucleic acid and polypeptide molecules
WO2005050170A2 (fr) Procedes et compositions permettant d'inhiber la formation d'agregats intracellulaires
JP2002330769A (ja) ヒトバニロイド受容体様タンパク質
US6365337B1 (en) Genes encoding neuronal voltage-gated calcium channel gamma subunits
WO1995022609A2 (fr) Proteines des recepteurs humains du glutamate
US5854392A (en) β APP-C100 receptor
JP2003530125A (ja) 生物学的物質およびその使用方法
US6465210B1 (en) Nucleic acid molecules encoding CASPR/p190
US20040241165A1 (en) Novel rfrp-3 and dna thereof

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 97532017

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载