WO2013034805A1

WO2013034805A1 - Neuroprotective cell-penetrating peptides

Info

Publication number: WO2013034805A1
Application number: PCT/FI2012/050859
Authority: WO
Inventors: Urmas Arumäe; Mart Saarma; Pia Runeberg-Roos
Original assignee: Arumaee Urmas; Mart Saarma; Pia Runeberg-Roos
Priority date: 2011-09-05
Filing date: 2012-09-05
Publication date: 2013-03-14
Also published as: EP2753641A4; FI20115870A0; EP2753641A1

Abstract

The present invention relates to the fields of bioactive peptides and cell-penetrating peptides as well as to the field of neurotrophic factors, i.e. growth factors for neural cells. The present invention provides peptides with length of 4 to 40 amino acids comprising the sequence CKGC (SEQ ID NO:1 ) or CRAC (SEQ ID NO:2). Pharmaceutical compositions comprising said peptides are also provided as well as the peptides for use in the treatment of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, stroke, peripheral neuropathy, epilepsy, diabetes or drug addiction. The invention is also directed to a method for promoting survival of dopaminergic neurons. In addition, peptides with length of 4 to 40 amino acids comprising the sequence CXXC (SEQ ID NO: 5) for use in the above mentioned diseases are also provided.

Description

NEUROPROTECTIVE CELL-PENETRATING PEPTIDES

The present invention relates to the fields of bioactive peptides and cell-penetrating peptides as well as to the field of neurotrophic factors, i.e. growth factors for neural cells. The present invention provides CXXC peptides with length of 4 to 40 amino acids having the sequence CKGC (SEQ ID NO: l) or CRAC (SEQ ID NO:2). The invention is also directed to a method for promoting survival of dopaminergic neurons.

BACKGROUND OF THE INVENTION

Neurotrophic factors cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) (Lindholm and Saarma, 2010) are currently the most efficient proteins for the treatment of rats in the 6-OHDA model of Parkinson's disease. Both factors potently prevent the 6-OHDA-induced behavioral and histological symptoms of Parkinson's disease when applied before the toxin (Lindholm et al., 2007), (Voutilainen et al., 2009). More importantly, post-treatment with either factor efficiently restored the normal motor behavior and dopaminergic innervations of the striatum when applied at the stage when the 6-OHDA-induced symptoms of the Parkinson's disease are already far-reaching

(Lindholm et al., 2007), (Voutilainen et al., 2011). CDNF protects and repairs dopamine neurons also in mouse MPTP model of Parkinson's disease and in a severe 6-OHDA model it is more efficient than glial cell line-derived neurotrophic factor (GDNF) (Voutilainen et al., 2011). The mechanism of action of CDNF and MANF is, however, not elucidated. We found that in vitro, MANF and CDNF protect the apoptotic neurons only intracellularly but not in the culture medium (Hellman et al., 2011).

In the studies of the mechanism of neuroprotection of MANF/CDNF we paid attention to the evolutionarily conserved CXXC motif in the sequence of both factors: CKGC in MANF and CRAC in CDNF. Structural studies of CDNF and MANF showed that these proteins consist of two domains: the saposin-like N-terminal domain (Parkash et al., 2009) and a SAP- domain-like C-terminal domain (Hellman et al., 2011). The CXXC motif (residues 149-152 of human MANF, NCBI Reference Sequence: NP_006001.3) is located in the C-terminal domain (C-MANF) in the loop region outside the helical core of the domain, and the cysteines are connected with the disulfide bond (Hellman et al., 2011). Corresponding motif of CDNF is located at the same position (NCBI Reference Sequence: NP_001025125.2). We have shown that C-MANF is potently anti-apoptotic in vitro, when expressed inside the

sympathetic neurons (Hellman et al., 2011). Importantly, mutation of cysteine 152 of the CXXC motif into alanine inactivated C-MANF in this assay (unpublished). Further, an antioxidant compound comprising an amidated CGPC structure is disclosed in US20030109457 (Atlas, Daphne). This antioxidant compound is disclosed to be useful in the treatment of a disease associated with formation of oxidative stress such as a central nervous system disease. However, the publication seems to contain examples of in vitro inhibition of oxidative stress and ROS production in a mouse fibroblast cell line only.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows MANF-derived peptides used in this study.

Figure 2. MANF-derived peptides protect sympathetic neurons against nerve growth factor (NGF) deprivation-induced death when microinjected into the cytoplasm. Neurons from the neonatal mouse superior cervical ganglion were grown with NGF for 6 days. The peptides or control substances were microinjected directly into the cytoplasm and NGF was then deprived. The living neurons were counted after three days (72 h) and expressed as percentage of initial number of injected neurons, counted 3-4 hours after injection. The mean + SEM of three independent experiments is shown. p<0.01 (**); p<0.05 (*) compared to buffer (PBS)- injected controls (ANOVA, Dunnett's post hoc test, p<0.05 is significant). C152S: one cysteine mutated into serine; C149/152S: both cysteines mutated into serine; w/o S-S: peptide without S-S bond.

Figure 3. MANF-derived peptides protect sympathetic neurons against nerve growth factor (NGF) deprivation-induced death when added to the culture medium. Neurons from the neonatal mouse superior cervical ganglion were grown with NGF for 6 days. Then the cultures were deprived of NGF and the peptides added to the culture medium. Living neurons were counted 72 h later and expressed as percentage of initial number of injected neurons, counted 3-4 hours after injection. The mean + SEM of three independent experiments is shown. p<0.01 (**) compared to no peptide-control (ANOVA, Dunnett's post hoc test, p<0.05 is significant). C149/152S: both cysteines mutated into serine. The NGF+ neurons were maintained in the presence of NGF to show that the neurons did not deteriorate due to poor culture conditions.

Figure 4. FITC-conjugated peptide of MANF (FITC- ETCKGCAE) spontaneously enters the sympathetic neurons. Three typical confocal microscopic images of the sympathetic neurons cultured in the presence of fluorescent MANF-derived peptide (FITC-conjugate) for two days. No efforts was made to facilitate the entry or uptake of the peptide.

Figure 5. MANF-derived peptides protect cultured dopaminergic neurons against trophic factor deprivation-induced death when added to the culture medium. Dissociated cultures of the El 3 mouse midbrain floors were cultured with the indicated peptides at 20 mM, with GDNF (100 micro grams/ml), or without factor, for 5 days. The cultures were then fixed and immunostained for tyrosine hydroxylase, a marker of the dopaminergic neurons. The immunopositive neurons were counted and expressed as percent of GDNF-maintained control neurons. The mean + SEM of four independent experiments is shown. p<0.05 (*) compared to no factor-control (ANOVA, Dunnett's post hoc test, p<0.05 is significant). C149/152S: both cysteines mutated into serine; w/o S-S: peptide without S-S bond.

Figure 6. MANF-derived peptide and inactive peptide with two mutated cysteines (MANF 2C-2S) did not significantly change the number of rat pheochromocytoma PC6 cells. The cells were plated at equal density, treated with the peptides at indicated concentrations for two days, and the number of cells counted by Cell-IQ machine. Shown are the averages of three parallels + standard deviations of one experiment.

Figure 7. Survival of cultured dopaminergic neurons with increasing doses of the

tetrapeptides CKGC and SKGS. Dissociated cultures of E13 NMRI mouse midbrain floors were grown on the microislands for 5 days with the peptides at increasing concentrations, with GDNF (100 ng/ml), or without added factors. The cultures were immunostained for tyrosine hydroxylase, all immunopositive neurons were counted manually under the microscope in the "blind" manner and expressed as % of GDNF-maintained neurons. The peptides were ordered from Casio Laboratory (Lyngby, Denmark) and were end- modified (N-terminal acetylation and C-terminal amidation). The experiments were repeated seven times on the independent cultures. Peptides from two different batches were used that gave well comparable results. Means +/- S.E.M. (n=7). One-way ANOVA / Dunnett post hoc test (versus no-factor control), n.s., not significant

Figure 8. Combined cumulative rotational behavior measured 2 and 4 weeks after lesion and treatment with MANF peptide. When the cumulative rotations at 2 and 4 weeks after the lesion were summed, there were significant differences between MANF4 peptide (i.e. Ac- CKGC-Am) 10 μg and vehicle treated groups (p<0,01) and between MANF4 peptide 30 μg and vehicle treated groups (p<0,05). The maximum protective effect was observed with MANF4 peptide dose of 10 μg. Levene: p < 0,001 ; Kruskal-Wallis: p = 0,009; Mann-Whitney U with Bonferroni correction: PBS vs. 10 μg: p < 0,01 (**); PBS vs. 30 μg: p < 0,05 (*); PBS vs. GDNF: p = 0,081.

DETAILED DESCRIPTION OF THE INVENTION

To study the role of the CXXC motif of MANF in more detail we turned to MANF-derived peptides containing the CXXC. As a first approach, we chose an octapeptide ETCKGCAE (residues 149-156 of human MANF, SEQ ID NO:3). This sequence is evolutionarily conserved and the Protein-Blast search found it only on MANF and CDNF protein sequences. We ordered (CASLO Laboratory ApS, Lyngby, Denmark) this peptide with and without the disulfide bond. The control peptides having one (Cys 154) or both cysteines (Cys 151 and Cys 154) mutated into serines were also ordered. Finally, to study the intracellular localization of the peptides we ordered the S-S-bond-containing octapeptide with N-terminally conjugated fluorochrome FITC. The design of the peptides is shown on Figure 1.

As our data had shown intracellular but not extracellular action of MANF (and also CDNF) in vitro (Hellman et al. 2011) we first applied the peptides also intracellularly. We first turned to the cultured neonatal mouse sympathetic neurons, our common well-elaborated model of neurotrophic factors. These neurons survive in vitro in the presence of nerve growth factor (NGF) and die apoptotically when deprived of NGF. We directly microinjected the plasmids into the cytoplasm of NGF-maintained neurons and then deprived the cultures of NGF. As shown on Figure 2, the octapeptide efficiently protected the neurons, whereas the peptide with both cysteines mutated was without effect. The peptides with one mutated cysteine or without the S-S bond were less effective than the peptide with the S-S bond.

In the next series of experiments we applied the peptides to the culture medium of NGF- deprived sympathetic neurons. Surprisingly, the octapeptide with S-S bond protected the neurons also on this paradigm, whereas the control peptide with two mutated cysteines was without effect (Figure 3). Thus, differently from the full MANF protein, the MANF-derived octapeptide protected the neurons also "from the outside", i.e. when added to the culture medium.

To study the localization of the octapeptide in the protected neurons we turned to FITC- conjugated peptide. The sympathetic neurons were cultured in the presence of this peptide for two days and then examined by confocal microscope. Surprisingly, we found strong FITC fluorescence inside the neurons (Figure 4). We concluded that the MANF-derived octapeptide is able to enter the cells and thus belongs to the class of cell-penetrating peptides (for recent review, see Madani et al., 2011). Importantly, the N-terminally added FITC moiety did not hamper the neuroprotective activity of the peptide (Figure 2).

As MANF can potently rescue and repair the 6-OHDA-damaged dopaminergic neurons in the animal experiments, we also studied the effect of the MANF-derived octapeptide on the cultured dopaminergic neurons, a model well established in our lab (Yu et al., 2008) (Yu and Arumae, 2008). We cultured the embryonic mouse midbrain cultures, containing the dopaminergic neurons, in the presence of the MANF-derived peptides or control GDNF. In this assay, a great portion of the dopaminergic neurons die apoptotically unless the appropriate trophic factor is provided. As shown on Figure 5, the octapeptide with S-S bond potently protected the dopaminergic neurons from death, whereas the mutant peptide with two cysteines mutated was without effect. Absence of the S-S bond somewhat reduced the protective potency of the peptide, whereas the FITC-conjugated peptide was active again.

To study the effect of the MANF-derived octapeptide on the non-neuronal cells, we have started the experiments with several cell lines and primary glial cells. In the pilot experiments with the rat pheochromocytoma PC6 cells we did not observe any significant effect of the peptides on the number of the cell (Figure 6).

Accordingly, the present invention provides a peptide with the length of 4 - 40 amino acids comprising the sequence CKGC (SEQ ID NO: 1) or CRAC (SEQ ID NO:2). In one

embodiment of the invention, the peptide comprises or consists of the sequence ETCKGCAE (SEQ ID NO:3).

In the embodiments of the invention, the length of the peptide is in the range of 4 - 40, 4 - 35, 4 - 30, 4 - 25, 4 - 20, 4 - 15, or 4 - 10 amino acids. Preferably, the length of the peptide is in the range of 5 - 40, 6 - 40, 7 -40, or 8 - 40 amino acids. More preferably, the length of the peptide is in the range of 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 6-35, 6-30, 6-25, 6-20, 6-15, 6-10, 7-35, 7-30, 7-25, 7-20, 7-15, 7-10, 8-35, 8-30, 8-25, 8-20, or 8-15 amino acids. For example, the preferred peptides can consist of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids. The peptides may comprise any of the naturally occurring amino acids such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine as well as non-conventional or modified amino acids. Preferably, the peptide has 100% homology with the sequence of human CDNF or MANF protein.

As used herein in the specification and in the claims section below, the term "peptide" includes native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and modified peptides, which may have, for example, modifications rendering the peptides more stable or less immunogenic. Such modifications include, but are not limited to, cyclization, N-terminus modification, C-terminus modification, peptide bond modification, backbone modification and residue modification.

The present invention also shows that the peptide may be conjugated to a detectable chemical or biochemical moiety such as a FITC-label. As used herein, a "detectable chemical or biochemical moiety" means a tag that exhibits an amino acid sequence or a detectable chemical or biochemical moiety for the purpose of facilitating detection of the peptide; such as a detectable molecule selected from among: a visible, fluorescent, chemiluminescent, or other detectable dye; an enzyme that is detectable in the presence of a substrate, e.g., an alkaline phosphatase with NBT plus BCIP or a peroxidase with a suitable substrate; a detectable protein, e.g., a green fluorescent protein. Preferably, the tag does not prevent or hinder the penetration of the peptide into the target cell.

Acetylation - amidation of the termini of the peptide (i.e., N-terminal acetylation and C- terminal amidation) increases the stability and cell permeability of the peptides. Examples of acetylated-amidated peptides are:

Ac-ETCKGCAE-Am

Ac-TCKGCA-Am

Ac-CKGC-Am Since the peptides potently protected the dopaminergic neurons from death (see Fig. 5), the prior art such as WO/2009/133247 and EP 1 969 003 shows that the peptides can also be used in the treatment of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, stroke, peripheral neuropathy, epilepsy, or drug addiction. Our recent results also predict that the peptides of the present invention can be effective in the treatment of type I and type II diabetes. Accordingly, the present invention is directed to a method for treatment of

Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, stroke, peripheral neuropathy, epilepsy, diabetes or drug addiction, wherein a pharmaceutically effective amount of the peptide with the length of 4 - 40 amino acids comprising the sequence CKGC (SEQ ID NO:l) or CRAC (SEQ ID NO:2) is administered to a patient. In other words, the peptide according to the present invention is for use in the treatment of Alzheimer's disease,

Parkinson's disease, amyotrophic lateral sclerosis, stroke, peripheral neuropathy, epilepsy, diabetes or drug addiction (e.g., abuse of cocaine, morphine, amphetamine, or alcohol). More preferably, the peptide is for use in the treatment of Parkinson's disease or diabetes such as type I and type II diabetes. In one embodiment of the present invention, the peptide with the length of 4 - 40 amino acids comprising the sequence CKGC (SEQ ID NO: l) or CRAC (SEQ ID NO:2) can be

incorporated into pharmaceutical compositions. Such compositions of the invention are prepared for storage by mixing the peptide having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A., Ed., (1980)), in the form of lyophilized cake or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or

immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter-ions such as sodium; and/or non- ionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).

The peptides may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano- particles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in

Remington's Pharmaceutical Sciences, supra.

The route of peptide administration is in accord with known methods as well as the general routes of injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, or intralesional means, or sustained release systems as noted below. The peptide is administered continuously by infusion or by bolus injection. Generally, where the disorder permits, one should formulate and dose the peptide for site-specific delivery. Administration can be continuous or periodic. Administration can be accomplished by a constant- or programmable-flow implantable pump or by periodic injections.

Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the peptide, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels as described by Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982) or polyvinylalcohol, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), or non-degradable ethylene-vinyl acetate (Langer et al., supra). Preferably, a pharmaceutical composition comprising the peptide of the invention as defined above is for use in the treatment of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, stroke, peripheral neuropathy, epilepsy, diabetes or drug addiction (e.g., abuse of cocaine, morphine, amphetamine, or alcohol). In another embodiment, the present invention provides a method for promoting survival of dopaminergic neurons comprising the step of contacting dopaminergic neurons with the peptide of 4 - 40 amino acids comprising the sequence CKGC (SEQ ID NO: l) or CRAC (SEQ ID NO:2) . Preferably, the method is performed in vitro as shown below in the

Experimental Section. Said dopaminergic neurons are preferably cultured non-human neurons, such as mouse or rat sympathetic neurons.

Based on the results provided by the present invention, the invention is also directed to a peptide with the length of 4 - 40 amino acids comprising the sequence CXXC (SEQ ID NO:5) for use in the treatment of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, stroke, peripheral neuropathy, epilepsy, diabetes, such as type I and type II diabetes, or drug addiction (e.g., abuse of cocaine, morphine, amphetamine, or alcohol).

The publications and other materials used herein to illuminate the background of the invention, and in particular, to provide additional details with respect to its practice, are incorporated herein by reference. The present invention is further described in the following examples, which are not intended to limit the scope of the invention.

EXAMPLES

EXAMPLE 1

The peptides (CASLO Laboratory ApS, Lyngby, Denmark) were dissolved in the water according to the manufacturer's instructions, divided into aliquots and stored at -80

centigrades. The working concentration of the peptides was 20 μΜ. For the cultures of sympathetic neurons (Hellman et al., 2011; Hamner et al., 2001; Lindholm et al., 2002; Sun et al., 2001; Aalto et al., 2007) the superior cervical ganglia from the postnatal day (P) 0-3 were digested with collagenase (2.5 mg/ml; Worthington), dispase (5 mg/ml; Roche Molecular Biochemicals), and trypsin (10 mg/ml; Worthington) for 45 min at 37 °C and dissociated mechanically with a siliconized glass Pasteur pipette. Non-neuronal cells were removed by extensive preplating. Almost pure neurons were cultured in

polyornithine/laminin (Sigma)-coated 35-mm plastic dishes in the small-size standard microislands in the Neurobasal medium and B27 supplement (Invitrogen/Gibco) in the presence of 30 ng/ml mouse 2.5 S NGF (Promega) for 5-6 days. NGF was deprived by extensive washing and addition of the function-blocking anti-NGF antibodies (Roche). The neurons were pressure-microinjected with our special equipment (Hellman et al., 2011;

Hamner et al, 2001; Lindholm et al, 2002; Sun et al, 2001; Yu et al, 2003)(Sun et al, 2001; Sun et al., 2003). For the survival assay, all neurons on the microislands were counted in the beginning (initial number) and the end (three days) of the experiment and expressed as % of initial. The experiments were repeated 3-5 times on the independent cultures.

To study the dopaminergic neurons (Yu et al., 2008; Yu and Arumae, 2008), the midbrain floors were dissected from the ventral mesencephali of 13-d-old NMRI strain mouse embryos. Tissues were incubated with 0.5% trypsin (ICN Biomedical), then mechanically dissociated using a large fire-polished Pasteur pipette. The neurons were grown on the poly-L-ornithine- coated (Sigma) culture dishes on the microislands of the standard size in DMEM/F12 medium (Invitrogen) containing N2 supplement (Invitrogen) in the presence or absence of GDNF (100 ng/ml) or the peptides for three days. The cultures without added neurotrophic factors served as the negative control. As the midbrain cultures contain several neuronal types, the cultures were fixed and immunostained with the antibodies to tyrosine hydroxylase (Millipore), a specific marker for the dopaminergic neurons. All immunopositive neurons were counted from each microisland and expressed as percent of GDNF-maintained neurons. The experiments were repeated 3-5 times on the independent cultures.

Rat pheochromocytoma PC6 cells were grown in the DMEM (Invitrogen) containing 10% of horse serum (PAA Laboratories, Pasching, Austria), 5% of fetal bovine serum (HyClone, Thermo Scientific, UK). The number of the neurons was counted in real-time using Cell-IQ machine (Chipman).

The 6-OHDA model of Parkinson's disease will be applied in two paradigms: neuroprotective and neurorestorative. In the model of neuroprotection, the factors MANF, CDNF or the respective peptides are stereotaxically injected into the left striata of the rats 6 h before injection of neurotoxin 6-OHDA. The concentrations of the peptides must be determined empirically, taking into account the results of the in vitro titration experiments. Two and four weeks later, amphetamine is injected to the same site and the rotational behavior induced by the amphetamine is measured. The more the neurons are damaged by 6-OHDA, the more the rats rotate. At the end of the experiment, the brains of the rats are fixed, immunostained for the tyrosine hydroxylase, and the dopaminergic neurons and their fibers quantified stereologically. In the neurorestorative experiments, the neurotoxin 6-OHDA is applied first, and the growth factors or the peptides four weeks later. The behavioural tests are carried out 1 week before (that is, 3 weeks after 6-OHDA injection) and 2, 4, 6 and 8 weeks after the growth factor injections. In the MPTP model of Parkinson's disease, the neurotoxin MPTP is applied systemically to the peritoneum of 2the mice either 20 h before the factors or peptides (neuroprotective model) or one week before the factors or peptides (neurorestorative model) are performed essentially as in the case of 6-OHDA. The behavioural tests will be performed two weeks later and the histological analysis at the end of the experiment. In the model of stroke, the right middle cerebral artery and bilateral common carotids of the rats are ligated with specific suture for 60 min to cause infarction in one cortical hemisphere. The factors (MANF, CDNF) or the corresponding CXXC-peptides are applied about 20 min before (neuroprotective model). Several behavioural tests will be performed 2, 7 and 14 days after the treatment and the histological analysis will be performed at the end of the

experiment, where the infarction volume is measured and other histochemical parameters (e.g. number of apoptotically dying cells) are counted.

In the model of Alzheimer' s disease the APdE9 mice carrying mutated human APPswe and PSldE genes (Jankowsky et al., 2004) will be used. The MANF/CDNF proteins or the corresponding CXXC-peptides will be stereotaxically injected into the hippocampi of these mice. A battery of behavioural tests will be performed 3 weeks after the injection. In the histological analysis, the number of amyloid plaques and the neuroinflammation (microglia, reactive astroglia) around the plaques, as well as the adult neurogenesis (doublecortin immunostaining) will be analysed at the end of the experiment.

Discussion

We have found an octapeptide sequence ETCKGCAE from the C-terminal domain of MANF that is potently anti-apoptotic in the neurons and can spontaneously enter the cells. This peptide could be potentially very powerful in the protection of the degenerating neurons, but potentially also other cells.

The mechanism of action of the peptide is currently being studied. The neuroprotective activities seem to depend on the cysteines of the CXXC motif. This motif is known to be critical in the active center of the enzymes of thioredoxin superfamily. The reactive cysteines can also be modified (S-glutathionation, S-sulfenation, S-nitrosation) and participate in the redox-regulation of proteins, in both physiological and pathological (against oxidative or nitrosative stress) situations. In addition, the CXXC motif can bind and coordinate metal ions (Zn²⁺, Cd²⁺, Cu²⁺), thereby controlling metal detoxifixation, metal ion transport etc. For review, see Ying et al., 2007, or Fomenko et al., 2008.

Penetration of the cell membrane seems to be critical for the neuroprotective properties of the peptide. Importantly, our data show that the peptide is able to carry a cargo (FITC moiety) through the membrane. Thus, it could be used as a vehicle to transport the associated molecules inside the cells. It is of note that the conjugated FITC did not change the activity of the peptide itself.

CDNF has a similar evolutionarily conserved CXXC-containing sequence EECRACAE in the corresponding position of the C-terminal domain. We thus disclose that MANF/CDNF- derived peptides can also protect and repair the degenerating neurons in vivo, just as the full- length proteins (Lindholm et al., 2007; Voutilainen et al., 2009; Voutilainen et al., 2011; Airavaara et al., 2010; Airavaara et al., 2009). Importantly, however, such short peptides will certainly diffuse better in the brain parenchyma than the large protein factors. More importantly, such cell-penetrating peptides could also penetrate the brain-blood barrier, such that these could potentially applied systemically via the bloodstream.

Finally, as MANF and CDNF proteins can protect a wide range of cells when expressed intracellularly by transfection of the plasmids (unpublished), we predict that the peptides could be applied also to the non-neuronal pathological conditions. In particular, MANF has been related to the alleviation of the endoplasmic reticulum stress (Mizobuchi et al., 2007; Apostolou et al., 2008). Thus, MANF and its CXXC-peptide (but also CDNF and its CXXC- peptide) has potential to be effective on the disease models of the endoplasmic stress-related conditions, in particular the endocrinological disease, and in particular, the type I and type II diabetes. Indeed, a major cause for the development of diabetes is the endoplasmic reticulum stress in the beta-cells of the Langerhans islands and according to our preliminary results, MANF is related to the development of the conditions for type I diabetes.

EXAMPLE 2 MANF-peptide CKGC studied with PD neuroprotection model in rats Materials and methods

Rats were operated stereotaxically in two sessions using isoflurane anesthesia. In the first surgery the rats were injected PBS-vehicle, GDNF 10 μg, MANF peptide CKGC 1, 10 or 30 μg in 4 μL· unilaterally into the left striatum (coordinates relative to bregma and dura: A/P +1,0; L/M +2,7; D/V -4,0). In the second surgery 6 hours after the first injection the rats received 6-OHDA-injection (10 μg in 4 μί) into the left striatum (the same coordinates as previously). Inside neurons 6-OHDA has two ways of action that act synergistically: 1) it accumulates in the cytosol and forms free radicals causing oxidative stress; 2) it is a potent inhibitor of the mitochondrial respiratory chain complexes I and IV. Noradrenergic neurons were protected by using a NAT-inhibitor desipramine (15 mg/kg, i.p., 30 mins before 6- OHDA -injection).

The size of the unilateral lesion and the effect of the treatments were measured with amphetamine induced rotational behavior 2 and 4 weeks after the lesion. The number of amphetamine-induced (2,5 mg/kg, i.p.) full (360°) ipsi- and contralateral turns were recorded for 120 mins after a 30 mins habituation period. The results are expressed as net ipsilateral turns to the lesion side (see Figure 8). Exclusion criterion was Mean (net rotations) + 2 x STDEV.

REFERENCES

Aalto, A.P., L.P. Sarin, A.A. van Dijk, M. Saarma, M.M. Poranen, U. Arumae, and D.H. Bamford. 2007. Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage phi6 RNA-dependent RNA polymerase. RNA 13:422-429. Airavaara, M., M.J. Chiocco, D.B. Howard, K.L. Zuchowski, J. Peranen, C. Liu, S. Fang, B.J. Hoffer, Y. Wang, and B.K. Harvey. 2010. Widespread cortical expression of MANF by AAV serotype 7: localization and protection against ischemic brain injury. Exp. Neurol. 225: 104- 113.

Airavaara, M. Harvey, B.K. Voutilainen, M.H. Shen, H. Chou, J Lindholm, P. Lindahl, M. Tuominen. R.K. Saarma, M. Wang, Y., and B. Hoffer. 2011. CDNF protects the nigrostriatal dopamine system and promotes recovery after MPTP treatment in mice. /. Neurosci. in press .

Airavaara, M., H. Shen, C.C. Kuo, J. Peranen, M. Saarma, B. Hoffer, and Y. Wang. 2009. Mesencephalic astrocyte-derived neurotrophic factor reduces ischemic brain injury and promotes behavioral recovery in rats. J. Comp. Neurol. Apostolou, A., Y. Shen, Y. Liang, J. Luo, and S. Fang. 2008. Armet, a UPR-upregulated protein, inhibits cell proliferation and ER stress-induced cell death. Exp. Cell Res. 314:2454- 2467.

Fomenko, D.E., S.M. Marino, and V.N. Gladyshev. 2008. Functional diversity of cysteine residues in proteins and unique features of catalytic redox-active cysteines in thiol

oxidoreductases. Mol.Cells. 26:228-235.

Hamner, S., U. Arumae, Y. Li-Ying, Y.F. Sun, M. Saarma, and D. Lindholm. 2001.

Functional characterization of two splice variants of rat bad and their interaction with Bcl-w in sympathetic neurons. Mol.Cell.Neurosci. 17:97-106.

Hellman, M., U. Arumae, L.Y. Yu, P. Lindholm, J. Peranen, M. Saarma, and P. Permi. 2011. Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a unique mechanism to rescue apoptotic neurons. J.Biol.Chem. 286:2675-2680.

Jankowsky, J.L., D.J. Fadale, J. Anderson, G.M. Xu, V. Gonzales, N.A. Jenkins, N.G.

Copeland, M.K. Lee, L.H. Younkin, S.L. Wagner, S.G. Younkin, and D.R. Borchelt. 2004. Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific gamma secretase. Hum.Mol.Genet. 13: 159- 170.

Lindholm, D., E.A. Mercer, L.Y. Yu, Y. Chen, J. Kukkonen, L. Korhonen, and U. Arumae. 2002. Neuronal apoptosis inhibitory protein: Structural requirements for hippocalcin binding and effects on survival of NGF-dependent sympathetic neurons. Biochim.Biophys.Acta.

1600: 138-147.

Lindholm, P., and M. Saarma. 2010. Novel CDNF/MANF family of neurotrophic factors. Dev.Neurobiol. 70:360-371.

Lindholm, P., M.H. Voutilainen, J. Lauren, J. Peranen, V.M. Leppanen, J.O. Andressoo, M. Lindahl, S. Janhunen, N. Kalkkinen, T. Timmusk, R.K. Tuominen, and M. Saarma. 2007. Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo. Nature. 448:73-77.

Madani, F., S. Lindberg, U. Langel, S. Futaki, and A. Graslund. 2011. Mechanisms of cellular uptake of cell-penetrating peptides. J.Biophys. 2011:414729. doi: 10.1155/2011/414729. Mizobuchi, N., J. Hoseki, H. Kubota, S. Toyokuni, J. Nozaki, M. Naitoh, A. Koizumi, and K. Nagata. 2007. ARMET is a soluble ER protein induced by the unfolded protein response via ERSE-II element. Cell Struct.Funct. 32:41-50.

Parkash, V., P. Lindholm, J. Peranen, N. Kalkkinen, E. Oksanen, M. Saarma, V.M. Leppanen, and A. Goldman. 2009. The structure of the conserved neurotrophic factors MANF and CDNF explains why they are bifunctional. Protein Eng.Des.Sel. 22:233-241.

Sun, Y.F., L.Y. Yu, M. Saarma, and U. Arumae. 2003. Mutational analysis of N-Bak reveals different structural requirements for antiapoptotic activity in neurons and proapoptotic activity in nonneuronal cells. Mol.Cell.Neurosci. 23: 134-143.

Sun, Y.F., L.Y. Yu, M. Saarma, T. Timmusk, and U. Arumae. 2001. Neuron-specific Bcl-2 homology 3 domain-only splice variant of Bak is anti-apoptotic in neurons, but pro-apoptotic in non-neuronal cells. J.Biol.Chem. 276: 16240-16247 ^'.

Voutilainen, M.H., S. Back, J. Peranen, P. Lindholm, A. Raasmaja, P.T. Mannisto, M. Saarma, and R.K. Tuominen. 2011. Chronic infusion of CDNF prevents 6-OHDA-induced deficits in a rat model of Parkinson's disease. Exp. Neurol. 228:99-108. Voutilainen, M.H., S. Back, E. Porsti, L. Toppinen, L. Lindgren, P. Lindholm, J. Peranen, M. Saarma, and R.K. Tuominen. 2009. Mesencephalic astrocyte-derived neurotrophic factor is neurorestorative in rat model of Parkinson's disease. J.Neurosci. 29:9651-9659. doi:

10.1523/JNEUROSCI.0833-09.2009.

Ying, J., N. Clavreul, M. Sethuraman, T. Adachi, and R.A. Cohen. 2007. Thiol oxidation in signaling and response to stress: detection and quantification of physiological and

pathophysiological thiol modifications. Free Radic.Biol.Med. 43: 1099-1108.

Yu, L.Y., and U. Arumae. 2008. Survival assay of transiently transfected dopaminergic neurons. J.Neurosci.Methods. 169:8-15.

Yu, L.Y., E. Jokitalo, Y.F. Sun, P. Mehlen, D. Lindholm, M. Saarma, and U. Arumae. 2003. GDNF-deprived sympathetic neurons die via a novel nonmitochondrial pathway. J. Cell Biol. 163:987-997.

Yu, L.Y., M. Saarma, and U. Arumae. 2008. Death receptors and caspases but not

mitochondria are activated in the GDNF- or BDNF-deprived dopaminergic neurons.

J.Neurosci. 28:7467-7475.

Claims

1. Peptide with the length of 4 - 40 amino acids comprising the sequence CKGC (SEQ ID NO: l) or CRAC (SEQ ID NO:2).

2. The peptide according to claim 1 comprising the sequence ETCKGCAE (SEQ ID NO:3).

3. The peptide according to claim 1 or 2 conjugated to a detectable chemical or biochemical moiety.

4. The peptide according to any one of claims 1 - 3 for use in the treatment of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, stroke, peripheral neuropathy, epilepsy, diabetes or drug addiction.

5. Method for promoting survival of dopaminergic neurons comprising the step of contacting dopaminergic neurons with the peptide according to any of claims 1-3.

6. The method according to claim 5, wherein the method is performed in vitro.

7. Method for treatment of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, stroke, peripheral neuropathy, epilepsy, diabetes or drug addiction, wherein a pharmaceutically effective amount of the peptide according to any one of claims 1-3 is administered to a patient.

8. The method according to claim 7, wherein said patient suffers a peripherial neuropathy.

9. The method according to claim 7, wherein said patient suffers from Alzheimer's disease.

10. The method according to claim 7, wherein said patient suffers from Parkinson's disease.

11. Use of the peptide according to any one of claims 1-3 for the manufacture of a medicament for the treatment of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, stroke, peripheral neuropathy, epilepsy, diabetes or drug addiction.

12. Pharmaceutical composition comprising the peptide according to any one of claims 1-3.

13. The pharmaceutical composition according to claim 12 for use in the treatment of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, stroke, peripheral neuropathy, epilepsy, diabetes or drug addiction.

14. Peptide with the length of 4 - 40 amino acids comprising the sequence CXXC (SEQ ID NO:5) for use in the treatment of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, stroke, peripheral neuropathy, epilepsy, diabetes or drug addiction.