WO2009121176A1 - Insulin-induced gene (insig) peptide compositions and methods for cytoprotection - Google Patents

Abstract

Insulin-induced gene (INSIG) peptide compositions and methods for cytoprotection are provided. Peptides are derived from an INSIG region that is ubiquitinated at two lysine residues and may be coupled to a cell transduction or other delivery or targeting or localization moiety. Methods are for cytoprotection may be directed to stroke or amyotrophic lateral sclerosis.

Description

INSULIN-INDUCED GENE (INSIG) PEPTIDE COMPOSITIONS AND METHODS FOR CYTOPROTECTION

FIELD OF THE INVENTION

The preseNt invention relates to lipid metabolism pathways, and more specifically to relationship between these pathways and cytotoxic cell death. Furthermore, therapeutic compositions and methods are presented.

BACKGROUND

Sterol regulatory element binding proteins (SREBPs) are endoplasmic reticulum (ER) membrane bound transcription factors (TFs) involved in activating genes required for lipid biosynthesis. There are two different SREBPs genes in most mammals (SREBFl and SREBF2). SREBP-I, is involved in the regulation of genes involved in fatty acids, phospholipids and triglyceride (TG) synthesis and SREBP-2 is responsible in regulating genes in cholesterol biosynthesis pathway (Goldstein et al. 2006; and Espenshade PJ. & Hughes AL. 2007). SREBP-I expression produces two different isoforms, SREBP-1a and -1c, due to alternative transcriptional start sites present in the SREBP-I gene. Immature SREBPs exist within the ER membrane in a tight association with SREBP cleavage activating protein (SCAP) (Nohturfft et al. 1998). SCAP responds to cellular cholesterol levels or cellular stress to regulate SREBP (Brown and Goldstein 1999). Sterol depletion or cellular stress, results in SCAP interaction with a member of COPII coat proteins to escort immature SREBPs (full-length) to Golgi where they are proteolytically processed and N-terminal is released as an active (or mature) TF, which can translocate to the nucleus (Sun et al 2005). Active TFs are then able to bind to sterol regulatory element DNA sequences to upregulate production of enzymes associated with sterol biosynthesis.

When cellular cholesterol levels are high (or there is no cellular stress), SCAP binds to another ER membrane protein called Insulin-Induced Gene (INSIG) protein, which has two isoforms INSIG-I and -2 (Flury et al. 2005), and which retain SREBP- SCAP complex in the ER membrane (Yang et al. 2002; Sun et al. 2005). In cholesterol- depleted cells, INSIG-I protein is rapidly ubiquitinated at lysine- 156 and lysine-158 and degraded by proteasomes (Gong et al. 2006a; Gong et al. 2006b; Lee et al.2006a; Lee et al. 2006b). Furthermore, it has been shown that INSIG-I, but not INSIG-2, is ubiquitinated and rapidly degraded and that ubiquitin ligase gp78 binds with much higher affinity to INSIG-I than INSIG-2 (Lee et al. 2006b). Cellular stress such as hypotonic shock and ER stress also activates SREBP via rapid turnover of INSIG-I (Lee and Ye 2004). The degradation of INSIG-I in the ER is a key determining step in the release of immature SREBPl and its subsequent proteolytic activation in Golgi.

A large number of in vitro and in vivo experimental studies have suggested that neuronal excitotoxicity caused by overactivation of the N-methyl-D-aspartate subtype of glutamate receptors (NMDARs) may be one of the primary neuropathological processes contributing to neuronal injury following insults of stroke and brain trauma (Choi 1988; and Lipton 2006). However, several clinical trials have failed to find the expected efficacy of NMDAR antagonists in reducing the brain injuries of stroke patients (Ikonomidou & Turski 2002; Kemp & McKernan 2002; and Lee et al. 1999).

SUMMARY OF INVENTION

Embodiments of this invention are based in part on the surprising discovery that in primary cortical neuronal cultures, excitotoxic neuronal death produced by a brief NMDA stimulation or ischemic insult simulated with oxygen-glucose deprivation (OGD) is associated with a dose- and time-dependent activation and nuclear translocation of sterol regulatory element binding protein- 1 (SREBPl).

Further embodiments are also based in part on the surprising discovery that inhibition of SREBPl activation and nuclear translocation by inhibiting INSIG-I degradation with a membrane-permeant INSIG-I derived interference peptide (Indip) significantly reduces either NMDA or OGD-induced neuronal death and that agents capable of reducing SREBPl activation such as Indip may represent a new class of NMDAR-based neuroprotective therapeutics against stroke and other neurologic diseases and conditions.

Further embodiments are also based in part on the surprising discovery that SREBPl activation is significant in mediating excitotoxic neuronal injuries.

Further embodiments are also based in part on the surprising discovery that the rat transient medial cerebral artery occlusion (MCAO) focal ischemic model of stroke revealed that a 90 min of ischemic insult produces a dramatic and infarct area-specific activation and nuclear translocation of SREBPl .

Further embodiments are also based in part on the surprising discovery that systemic application of Indip peptide not only prevents the SREBPl activation, but also significantly reduces the infarct volume and improves neurobehavioural outcomes in stroke model animals.

Further embodiments are also based in pan on the surprising discovery that activation of SREBP1 as a novel step in cascades leading to ejtcitotoxic neuronal death, suggesting an important role for lipid homeostasis in ischemic brain damage.

Further embodiments are also based in part on the surprising discovery that activation of SREBP1 in human spinal cord of both sporadic and familial amyotrophic lateral sclerosis (ALS).

Further embodiments are also based in part on the surprising discovery that Indip peptide protects against glutamate induced excitotoxicity cell death in both wild type and AI-S (G93A) transgenic mouse embryonic spinal cord neurons.

Further embodiments are also based in part on the surprising discovery that Indip peptide protects against glutamate induced DNA fragmentation (apoptosis) in G93 A transgenic embryonic spinal cord neurons.

In a first embodiment there is provided an isolated bioactive polypeptide, wherein the amino acid sequence of the polypeptide comprises a polypeptide selected from the group consisting of: a) PHKFKX₁ (SEQ ID NO: 21), where X₁ = R or K; b) X₁X₂PHKFKX₃X₄X₅ (SEQ ID NO: 22), wherein X₁ = is a linker of 1 -5 G or is absent, X₂ « E or D or is absent, X₃ = R Or K₁ X₄ = E Or D oTiS absent, and Xs = W or F or is absent; c) X₁X₂PHKFKX₃X₄X₅ (SEQ ID NO: 23), wherein X_t = is a linker of 1-5 G or is absent, Xi = E or D or N or Q or is absent, X₃ = R or K or H, X4 = E or D or N or Q or is absent, and X₅ = W or F or Y or is absent; and d) XIX₂PHKFKX₃X₄XS (SEQ ID NO: 24), wherein X₁ = G or A or V or L or I or is a linker of 1-5 G or is absent, X₂ = E or D or N or Q, X₃ = R or Kor H, X₄ - E or D or N or Q, and X₅ = W or F or Y.

In a further embodiment there is provided a pharmaceutical composition including a polypeptide and a carrier suitable for facilitating delivery of the polypeptide to a cell, wherein the polypeptide comprises one or more of the amino acid sequences set forth in SEQ ID NOS: 1 - 24 and 26-45. The pharmaceutical composition may be for the treatment of stroke or amyotrophic lateral sclerosis (ALS). In a further embodiment there is provided a method of inhibiting cytotoxic cell death or reducing cytoxic stress, including, treating a cell, a mammal including the cell, or a tissue including the cell, with an effective amount a polypeptide described herein. In a further embodiment there is provided a method of treating stroke or ALS including, treating a cell, a mammal including the cell, or a tissue including the cell, with an effective amount a polypeptide described herein.

In a further embodiment there i$ provided a use of a polypeptide for treating stroke or ALS in a subject, wherein the polypeptide is selected from any one or more of SEQ ID NOS: 1-24 and 26-45.

In a further embodiment there is provided a use of a polypeptide to formulate a ^' medicament treating stroke or ALS in a subject, wherein the polypeptide is selected from any one or more of SEQ ID NOS: 1-24 and 26-45.

In a further embodiment mere is provided a commercial package, including a polypeptide, wherein the polypeptide is selected from any one or more of SEQ ID NOS : 1 - 24 and 26-45 or a pharmaceutical composition thereof. There may also be provide a kit including a polypeptide, wherein the polypeptide is selected from any one or more of SEQ ID NOS: 1-24 and 26-45.

The polypeptide may be selected from one or mote of the following: GEPHKFOJEW (SEQ ID NO: 1); GGEPHKFKREW (SEQ ID NO; 3); GGGEPHKFKREW (SEQ ID NO: 4); GGGGEPHKFKREW (SEQ ID NO: 5); GGGGGEFHKFKREW (SEQ ID NO: 6); EPHKFKREW (SEQ ID NO: 7); GEPHKFKRE (SEQ ID NO: 8); EPHKFKRE (SEQ ID NO: 9); GEPHKFKRE (SEQ ID NO: 10); EPHKFKRE (SEQ ID NO: 11); PHKFKRE (SEQ ID NO: 12); PHKFKR (SEQ ID NO: 13); PHKFKK (SEQ ID NO: 14); DPHKFKREW (SEQ ID NO: 15); EPHKFKREF (SEQ ID NO: 16); DPHKFKREF (SEQ ID NO: 17); EPHKFKRDW (SEQ ID NO: 18); EPHKFKRD (SEQ ID NO: 19); and PHKFKRD (SEQ ID NO: 20). The peptide may also have one or more conserved amino acid changes. The polypeptide may further include a delivery moiety. The delivery moiety may allow for localization to the central nervous system. The delivery moiety may be a TAT peptide. The polypeptide may be selected from one or more of the following: YGRKKRRQRRRGEPHKFKREW (SEQ ID NO: 26); YGRKXRRQRRRGGEPHKFKREW (SEQ ID NO: 27); YGRKKRRQRRP-GGGEPHKFKREW (SEQ ID NO: 28); YGRKKRRQRRRGGGGEPHKFKREW (SEQ ID NO: 29); YGRKK RRQRRRGGGGGEPrIKFK-REW (SEQ ID NO: 30); YGRKKRRQRRREPHKFKREW (SEQ ID NO: 31); YGRKKRRQRRRGEPHKFKRE (SEQ ID NO: 32); YGRKKRRQRRREPHKFKRE (SEQ ID NO: 33); YGRKKRRQRRRGEPHKFKRE (SEQ ID NO: 34); YGRKKRRRQRRREPHKFKRE (SEQ ID NO: 35); YGRKKRRQRRRPHKFKRE (SEQ ID NO:-36); YGRKKKKKQRKR (SEQ ID NO: 37); YGRKKKKQ RRRPHKEKK (SEQ ID NO: 38); YGRKKRRQRRRDPHKFKREW (SEQ ID NO: 39); YGRXKRRQRRREPHKFKREF (SEQ ID NO: 40); YGRKKRRQRRRDPHKFKREF (SEQ ID NO: 41); YGRKKRRQRRREPHKFKRDW (SEQ ID NO: 42); YGRKKRRQRRREPHKFKRD (SEQ ID NO: 43); YGRKKRRQRRRPHKFKRD (SEQ ID NO: 44); and GEPHKFKREWYGRKKRRQRRR (SEQ ID NO: 45). The polypeptide may be in the form of a pharMaceUtically acceptable salt thereof.

In a further embodiment there is provided a nucleic acid including a non-naturally occurring nucleic acid capable of encoding a polypeptide selected from any one or more of SEQ ID NOS: 1-24 and 26-45. The nucleic acid maybe encoded by SEQ ID NO: 25, The nucleic acid may be contained in an expression vector. The nucleic acid may be contained in a cell.

Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE IA is a bar graph of protein/DNA microarray for transcription factors with characterized response elements revealed altered activity of a large number of transcription factors. Nuclear extracts from cortical cultured neurons following NMDA- induced excitotoxicity (50μM NMDA for Ih followed by 4h incubation) were compared to control cells, and up or down regulated transcription factors were identified. Note an about two-fold increase in SREBP1 -binding activity in response to NMDA stimulation (Black Bar).

FIGURE IB is a bar graph showing the appearance of cleaved n-terminal (active) SREBP1 (nt-SREBP1) at 6 hours following NMDA insult as compared to β-tubulin.

FIGURE 1C is a bar graph showing that NMDA-induced activation (NMDA) is prevented by treatment of cortical cultures with NMDAR antagonist AP5 (50μM) or NR2B-subunit specific antagonist Ro25-6981 (Ro 0.5μM), but not NR2A-preferential antagonist NVP-AAM077 (NVP 0.4μM). Group data from 4 individual experiments were summarized in the bar graph below the blots. FIGURE ID is a western blot showing that chelating of intracellular Ca2+ by prior incubation of neurons with BAPTA-AM (10μM) effectively inhibits NMDA-induced nt-SREBP1 production, and suggests a calcium dependency in SREBP1 activation.

FIGURE IE is a western blot showing NMDA-induced SREBP1 requires calpain activity, whereby neurons treated with calpain inhibitors calpeptin (Calpeptin; 25 μM) or MDL (MDL; 50μM) prior to NMDA-insult show reduced NMDA-induced SREBP1 activation.

FIGURE 2A is a western blot showing purified nuclear fractions from cortical cultured neurons probed for the active form of SREBP1 (Nuclear SREBP1 or nt-SREBP1) and the membranes were then stripped and re-probed for TATA-binding protein (TBP) as nuclear fraction confirmation and loading control (bottom blots). Mature SREBP1 is detected in the nucleus in response to NMDA stimulation in a time-dependent fashion and the SREBP1 nuclear translocation is blocked by AP5, demonstrating its dependence on specific activation of NR2B-contaiπing NMDARs.

FIGURE 2B is a bar graph showing purified nuclear fractions from cortical cultured neurons probed for the active form of SREBP1 (Nuclear SREBP1 or nt-SREBP1) as compared to Lamin A/C as nuclear fraction confirmation and loading control. Mature SREBP1 is detected in the nucleus in response to NMDA stimulation and the SREBP1 nuclear translocation is blocked by AP5 and Ro, but not NVP (at least 4 independent experiments in each groups; **p<0.01), demonstrating its dependence on specific activation of NR2B-containing NMDARs.

FIGURE 2C is a series of immuno fluorescent micrographs showing nuclear translocation of SREBP1 is dependent on NMDAR activation and associated with NMDA-induced apoptosis. Neurons were treated without (Control) or with NMDA alone (NMDA; 20μM for 1 hour) or in the presence 50 μM AP5 (NMDA+AP5) and were stained for SREBP1 four hours later. SREBP1 translocates into the nucleus following NMDA stimulation (NMDA) as demonstrated by the increased colocalization with Hoechst 33358 and this translocation is prevented by co-application of AP5. scale bar 40μm.

FIGURE 2D is a scatter plot for individual neurons correlating nuclear SREBP 1 signal with Hoechst 33358 fluorescence and shows a positive correlation (R²=0.43) of apoptosis with increased SREBP1 in the nucleus, suggesting a contribution of SREBP1 activation to NMDA-induced excitotoxicity. **ρ<0.01 FIGURE 3A is a western blot of total lysate 6 hours after NMDA insult probed for nt- SREBP1 (and with β-tubulin as a control) and shows reduced SREBP1 activation at higher cholesterol concentrations, whereby cholesterol was supplemented to cortical cultures 12 hours prior to NMDA insult at the indicated concentrations.

FIGURE 3B is a bar graph showing cortical cultures were assayed for LDH release after excitotoxicNMDA treatment at various cholesterol concentrations. The addition of lOOμM cholesterol significantly reduced excitoxicity-induced cell death. *p<0.05 from 3 independent experiments.

FIGURE 3 C is a western blot showing endogenous SREBP1 expression is reduced by between 40 and 50% after 48 hour expression of a shRNA knockdown construct in HEK293 cells compared to empty vector or scrambled control shRNA.

FIGURE 3D is a series of immunofluorescent micrographs showing flag- SREBP1 expression is dramatically reduced in bippocampal cultures upon co-transfection with SREBP1 shRNA construct as compared to control shRNA and GFP alone neurons. 4 to 5 days after co-transfection, anti-flag immunofluoresence was used to detect flag- SREBP1 expression in green cells, nuclear concentrated flag-SREBP1 was absent in SREBP1 shRNA co-transfected neurons.

FIGURE 3 E is a series of immunofluorescent micrographs showing hippocampal neurons transfected with SREBP1 shRNA construct are resistant to NMDA induced excitotoxicity. 4 to 5 days after transfection, neurons were exposed to NMDA challenge (50μM) and allowed 2 hours recovery before fixation. Apoptotic nuclear condensations were assessed by Hoechst 33358 (5μg/ml) fluorescent stain (transfected nuclei indicated by arrow).

FIGURE 3F is a bar graph showing results of coverslips that were scored for the percentage of green apoptotic neurons (bar chart for >10 coverslips per condition from 3 independent experiments). **p<0.01, scale bar lOμm.

FIGURE 4A is a proposed mechanism underlying SREBP1 activation and its inhibition by high level cholesterol or TAT-Indip peptide, showing that under basal conditions, immature SREBP1 forms a SREBP1-SCAP complex and is retained in the ER by the interaction between SCAP and ER anchoring protein Insig-1, with low cholesterol or excitotoxicity leads to the sequential ubiquitination of and degradation of the anchoring protein Insig-1, causing the release SREBP1 -SCAP from ER for SREBP1 cleavage/activation in the Golgi, and with high levels of cholesterol or lnsig-1 degradation interference peptide (Indip, that carries ubiquitination sites of Insig-1) is predicted to inhibit Insig-1 degradation and hence stabilize the Insig-1 SCAP interaction, thereby preventing the release of and activation of SREBP1. A control peptide (TAT-Indip_K-R) lacking the Insig-1 ubiquitination lysine residues is predicted not to affect Insig-1 degradation.

FIGURE 4B is an immunoblot of HEK 293 cell lysates, where the cells were treated with 2 μM peptide followed by 24 h stimulation with 10 μM simvastatin. The simvastatin-induced degradation of Insig-1 was inhibited by TAT-Indip, but not TAT-Indip_K-R.

FIGURE 4C is a bar graph showing Insig-1 protein as compared to β-tubulin in cortical culture lysates pretreated with TAT-Indip or TAT-Indip_K-R peptide and probed for Insig- 1. Insig-1 relative to β-tubulin control is unaffected by NMDA treatment in the presence of TAT-Indip, but not TAT-Indip_K-R peptide, showing that TAT-Indip peptide can reduce NMDA-induced Insig-1 degradation. 4 experiments ρ<0.01

FIGURE 4D is a bar graph showing that TAT-Indip prevents NMDA-induced SREBP1 activation in neuronal cultures, where western blots of cortical cultures 6 hours after NMDA insult and the bar graph represents data from 4 independent experiments, p<0.01.

FIGURE 5A is a bar graph that shows cholesterol treatment reduces NMDA- induced LDH release. Cholesterol was incubated with cortical cultures 12 hours prior to NMDA insult and LDH assay was then performed 24 hours after NMDA insult (n=4).

FIGURE 5B is a bar graph that shows TAT-Indip reduces NMDA-induced LDH release. Cortical cultures were pretreated with 1 μM TAT-Indip peptide for 20 minutes before NMDA induced excitotoxicity assessed by LDH assay.

FIGURE 5C is a bar graph that shows TAT-Indip prevents NMDA-induced apoptosis. Cortical cultures were treated without (Control) or with NMDA in the presence or absence of TAT-Indip (1 μM 20 min prior to NMDA application). Apoptosis was measured by detecting genomic DNA cleavage using ELISA assay 24 hours after treatment of NMDA (C; n=6).

FIGURE 5D is a scatter plot quantification of Hoechst 33358 condensations showing that TAT-Indip peptide reduces apoptosis progression (data points for >60 neurons from 2 independent experiments). **p<0.01

FIGURE 6A is a bar graph showing TAT-Indip reduces NMDA-induced cell death in cultured neurons. Cortical cultures were pretreated with 2 μM TAT-Indip or control peptide (Indip_K-R) for 20 minutes before NMDA induced excitotoxicity. Cell death assessed by LDH release 24 h later.

FIGURE 6B is a series of immunofluorescent micrographs that show TAT-Indip effectively reduces NMDA-induced neuronal death. Individual neurons assessed for propidum iodide uptake 24 h after NMDA induced apoptosis in presence of TAT-Indip or TAT-Indip_K-R- Neurons were identified by NeuN counterstain and nuclear propidiura iodide localized with Hoechst 33358. TAT-Indip peptide (but not TAT-Indip_K-R) effectively blocks NMDA induced neuronal death (quantification for 6 coverslips from 2 independent experiments).

FIGURE 6C is a bar graph showing that Oxygen Glucose Deprivation (OGD) induces activation (nt-SREBP1) of SREBP1 as compared to β-Tubulin in cortical cultures exposed to 1 h OGD and immunobloted for nt-SREBP1 at indicated times post-OGD.

FIGURE 6D is a bar graph showing OGD-induced activation of SREBP 1 is NMDA-receptor dependent OGD treated cortical cultures in the presence of NMDA- receptor antagonists were immunoblotted. The pan NMDA-receptor antagonist (AP5, 50 μM) and NR2B specific antagonist (Ro25-698l, 0.5 μM) showed inhibition of OGD- dependent SREBP1 activation, but NR2A antagonist (NVP-AAM077, 0.4 μM) showed no effect.

FIGURE 6E is a bar graph showing TAT-Indip prevents QGD-raduced cell death. Cortical cultures pretreated with TAT-Indip or control peptide (2 μM for 20 minutes) exposed to 1 h OGD. Cell death was assayed by LDH release 24 h later. TAT- Indip peptide (but not TAT-Indip_K-R) reduces OGD-induced cell death. **p<0.01

FIGURE 7A is a schematic of pre-TAT-Indip peptide treatment.

FIGURE 7B is an immunoblot showing MCAO-induced Insig-1 degradation is reduced in i.v. injected TAT-Indip (3 nmol/g) treated animals. Cortical tissue collected from indicated areas and probed for Insig-1 on an immunoblot. MCAO causes Insig-1 loss in the occluded hemisphere (saline, occ), but TAT-Indip treated animals show no loss of Insig-1.

FIGURE 7C is an immunoblot showing SREBP1 activation is reduced in TAT- Indip treated animals. The immunoblot is of cortical tissue collected at indicated time point in 7A. nt-SREBP1 is increased with MCAO (saline, occ), but not in the presence of TAT-Indip. FIGURE 7D is a bar graph showing TAT-Indip peptide protects from ischemia induced neuronal damage. TTC stain was used to show healthy tissue (pink). MCAO induces dramatic neuronal death 24 h after occlusion (saline). TAT-Indip effectively reduces. MCAO induced damage. ρ<0.05, n=9.

FIGURE 7E is a bar graph showing that cognitive loss of function due to ischemia is reduced with TAT-hidip treatment. Combined scores for behavioral deficit 24 h after MCAO, wher the treatment of animals with TAT-Indip significantly improves behavioral function. p<0.05, n=9.

FIGURE 7F is a Schematic of post-TAT-Indip peptide treatment, where TAT- Indip and TAT-Indip_K__R injected i.v, 0.5 h after MCAO.

FIGURE 7G is a bar graph showing that post-MCAO TAT-Indip peptide treatment reduces ischemic cell death. FluoroJade immunohistochemisty for dead cells 7 days post-MCAO. Quantification of FuoroJade signal shows reduction of MCAO damage in the occluded hemisphere with TAT-Indip treatment, however saline injected or TAT-Indip_K-R injected animais showed significantly more cell death. ρ<0.01, n>6.

FIGURE 8 . is a plot showing that TAT-Indip peptide reduces tactile response deficit post-ischaemia. Animals were treated with TAT-Indip or TAT-Indip_K-R peptide 30 minutes after MCAO were assessed for limb response due to tactile stimuli on the indicated days post-ischaemia (O - no deficit, 1 - mild deficit, 2 - severe deficit). Immediately after MCAO all animals showed severe deficits in tactile response, but 7 days post-ischaemia TAT-Indip treated animals show significantly improved responses. **p<0.01.

FIGURE 9 is a an immunoblot from spinal cord lysates of wild type and ALS (G93A) transgenic mice that were subjected to SDS-PAGE and immunoblotting for Insig- 1 and mature SREBP1. ALS tg mice showed increased SREBP1 activation and Insig-1 degradation. Data from 4 individual experiments were summarized in the Bar graph.

FIGURE 10 is a bar graph showing TAT-Indip reduces glutamate-induced cell death in cultured mouse spinal coxd neurons. Mouse embryonic spinal cord cultured neurons were pretreated with 2 μM TAT-Indip or TAT-Indip_K-R peptide for 45 minutes before glutamate (100 μM for 30 min) induced excitotoxicity. Cell death assessed by LDH release after 6 hours.

FIGURE 11 is a bar graph showing G93A transgenic mouse embryonic spinal cord cultured neurons were pretreated with.2 μM TAT-Indip or TAT-Indip_K-R peptide for 45 minutes before glutamate (100 μM for 30 rain) induced excitotoxicity. Cells were washed and then transferred to the regular culture media for additional 6 h. Neurons were then fixed and subjected to the Tunel assay to measure percentage of DNA fragmented cells (apoptotic cells). TAT-Indip effectively blocked glutamate induced apoptosis in G93A spinal cord neurons while TAT-Indip_K-R had some neuronal toxic effect on its own and failed to block glutamate-induced toxicity. Data from 4 independent experiments.

FIGURE 12 is an immunoblot showing twenty micrograms of homogenate prepared from post-mortem spinal cord samples as well as sex with age matched normal samples run on SDS-PAGE and immunobloted for SREBP-I and β-Tubulin (as loading control). SREBP1 was significantly activated in spinal cord of both sporadic and familial ALS patients compared to the control subjects. A representative blot is shown (n=2).

DETAILED DESCRIPTION

Definitions

Any terms not directly defined herein shall be understood to have the meanings commonly associated with them as understood within the art of the invention. As employed throughout the specification, the following terms, unless otherwise indicated, shall be understood to have the following meanings.

The term 'antibody' as used herein refers to immune system proteins, also called immunoglobulins, produced in response to antigens. Antibodies typically contain two heavy chains and two light chains, which are joined. Variability in the structure of these chains provides antigen specificity, thereby allowing individual antibodies to recognize specific antigens. The term antibody may include polyclonal and monoclonal antibodies, chimeric, single chain, or humanized antibodies, as well as Fab or F(ab)2 fragments, including the products of an Fab or other immunoglobulin expression library. Methods of making such antibodies or fragments are known in the art and may be found in, for example Harlow E. and Lane D. Antibodies: A Laboratory Manual. (1988). Cold Spring Harbor Laboratory Press. Antibodies may also include intracellular antibodies, sometimes referred to as intrabodies. Methods for designing, making and/or using such antibodies has been described in the art, for instance Lecerf et al. 2001 ; Hudson and Sourjau 2003. Selection or identification of specific peptides for use as epitopes for production of antibodies that differentiate between proteins, or isoforms of proteins may be made using sequence comparisons - one of skill in the art will be able to identify suitable peptide or protein sequences that may be useful for producing antibodies with the desired selectivities. Polyclonal antibodies axe antibodies that are derived from different B-cell lines. They are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognising a different epitope. These antibodies are typically produced by immunization of a suitable mammal, .such as a mouse, rabbit or goat. Larger mammals are often preferred as the amount of serum that can be collected is greater. An antigen is injected into the mammal, which induces the B-lymphocytes to produce IgG immunoglobulins specific for the antigen. This IgG is purified from the mammal's serum. By contrast, monoclonal antibodies may be derived from a single cell line. Adjuvants may be used to improve or enhance an immune response to antigens.

As used herein 'bioactive' refers to one or more of the following: protection against glutamate induced DNA fragmentation; protection against glutamate induced excitotoxicity cell death; reduction of INSIG-I degradation; reduction of NMDA and/or OGD-induced neuronal death; neuroprotection; reduction of SREBP1 activation; prevention the SREBPI activation; mediation of excitotoxic neuronal injuries; reduction of nuclear translocation of SREBP1 ; reduction of infarct volume following stroke; reduction of SREBP1 activation in sporadic and familial amyotrophic lateral sclerosis; and reduction of ischemic injury.

As used herein a 'subject' refers to an animal, for example a bird or a mammal. Animals may include a zebra fish, a rat, a mouse, a dog, a cat, a cow, a sheep, a horse, a pig or a primate (for example, a monkey or an ape). A subject may further be a human, alternatively referred to as a patient. Alternatively, a subject may be a transgenic animal.

The term 'NMDA' refers to N-methyl-D-asparate, and 'NMDAR' refers to NMDA receptor(s). NMDAR is an ionotropic receptor for glutamate. NMDA is a selective specific agonist of NMDAR, and the activation of NMDAR leads to ion channel opening, allowing for Na⁺ and Ca²⁺ ions into the cell and K⁺ out of the cell. Although the amount of Ca²⁺ flux is small, it is thought that the movement of Ca²⁺ plays a critical role in synaptic plasticity, which is implicated in learning and memory. There are 8 NRl variants formed by alternative splicing of the GRINl gene or NMDARl (i,e. NRl-Ia, NRl-Ib, NRl -2a, NRl -2b, NRl -3 a, NRl -3b, NRl -4a, NRl -4b) and 4 isoforms of NR2 (i.e. NR2A- D) are generated by 4 genes (GRIN2A-D or NMDAR2A-D). Multiple receptor isoforms with distinct brain distributions and functional properties arise by selective splicing of the NRl transcripts and differential expression of the NR2 subunits. NMDAR forms a heterodimer between NRl and NR2 subunits.

The term 'SCAP' refers to SREBP cleavage activating protein (EntrezGene ID: 22937), which is required for proteolytic cleavage of SRBBP. SCAP is an integral membrane protein located in the endoplasmic reticulum (ER), with a cytosolic region that contains a hexapeptide amino acid sequence (MELADL), which detects cellular cholesterol. When cholesterol is present, SCAP undergoes a conformational change, which prevents activation of SREBP and thereby inhibiting cholesterol synthesis. .

The term 'SREBP-I ' refers to sterol regulatory element binding protein 1, which may alternately be referred to as SREBF-I or SREBP1 (EntrezGene ID: 6720). SREBPs belong to the basic-helix-loop-helix leucine zipper class of transcription factors and are attached to the endoplasmic reticulum membrane in an inactive form. In cells with low levels of sterols (i.e. cholesterol) SREBPs are cleaved to form a soluble N-terminal domain that translocates to the nucleus. These activated SREBPs then bind to specific sterol regulatory elements (SREs) on the DNA, which in turn upregulates the synthesis of enzymes involved in sterol biosynthesis. Sterols in turn inhibit the cleavage of SREBPs and therefore synthesis of additional sterols is reduced through a negative feedback loop.

The term 'ϊnsig-1 ' (or INSIG-1) refers to the protein product of insulin-induced gene 1 (EntrezGene H): 3638), which produces a 277 amino acid polypeptide. This protein binds to the sterol-sensing domains of SREBP cleavage-activating protein (SCAP) and HMG CoA reductase, and is involved in sterol-mediated trafficking of SCAP and HMG CoA reductase. Alternatively spliced transcript variants encoding distinct isoforms have been observed and degradation of Insϊg-1 by ubiquitin-mediated pathways is. important for SCAP/SREBP regulation, as described herein. Provided herein are various compositions and methods for cytoprotection. Provided herein are various compositions and methods for modulating ubiquitination of Insig-1.

The term "Insig degradation peptide inhibitor" (Indip) referes to the lysines 156 and 158 and flanking sequences as set out herein and represented by human Ihsig-1 (i.e. GenBank Accession no. AYl 12745) as set out in Gong et al. (2006b). Based on a proposed membrane topology of human Insig-1, the region containing lysines 156 and 158 and flanking sequence is shown as loop outside of the ER membrane on the cytosolic surface (Gong et al. 2006b). Indip peptides or polypeptides may be selected from one or more of the following: GEPHKPKREW (amino acids 152 to 162 of AYl 12745); EPHKFKREW (amino acids 153 to 162 of AYl 12745); HLGEPHKFKREW (amino acids 148 to 162 OfAYl 12745); GEPHKFKREW (amino acids 96 to 105 of AY527632). Alternatively, the Indip peptide or polypeptide may be represented by one or more of the following formulas: PHKFKX₁ where X₁ = R or K; X₁X₂PHKFKX₃X₄X₅, wherein X₁ = is a linker of 1-5 G or is absent, X₂ - E or D or is absent, X₃ = R or K, X₄ = E or D or is absent, and X₅ = W or F or is absent; X₁X₂PHKFKX₃X₄X₅, wherein X₁ = is a linker of 1 - 5 G or is absent, X₂ = E or D or N or Q oris absent, X₃ = R or K or H, X₄ - E or D or N or Q or is absent, and X₅ = W or F or Y or is absent; and X₁X₂PHKFKX₃X4X₅, wherein X₁ = G or A or V or L or I or is a linker of 1-5 G or is absent, X₂ = E or D or N or Q, X₃ = R orK orH, X₄ = E orD orN or Q, and X₅ = W orF or Y.

Indip peptides may be found in TABLE 1 below. TABLE 1

The terms 'peptide' or 'polypeptide' are used interchangeably herein, and refer to a compound comprised of at least two amino acid residues, but not a full length protein, covalently linked by peptide bonds or modified peptide bonds (for example peptide isosteres), whereby the modified peptide bonds may provide additional desired properties to the peptide or polypeptide, such as increased half-life. Alternatively, a peptide or a polypeptide may comprise portions of one or more different proteins. For example, SEQ ID NO: 2 shows a TAT, derived HTV TAT protein transduction domain, linked to an Indip peptide, derived from human insig protein. The amino acids comprising a peptide or polypeptide as described herein may also be modified either by other processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in a peptide or polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It is understood that the same type of modification may be present in the same or varying degrees at several sites in a given peptide. Furthermore, it is well known in the art how to select an appropriate expression system depending on the type of modification desired.

As used herein, the term 'conserved amino acid substitution' or substitutions refers to the substitution of one or more amino acids for another at a given location or locations in the peptide or polypeptide, where the substitution can be made without substantial loss of the relevant function, hi making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like (described in more detail below), and such substitutions may be assayed for their effect on the function of the peptide or polypeptide by routine testing.

The term 'recombinant' means that something has been recombined, so that when made in reference to a nucleic acid construct the term refers to a molecule that is comprised of nucleic acid sequences that are joined together or produced by means of molecular biological techniques. The term "recombinant" when made in reference to a protein or a polypeptide or a peptide refers to a protein or polypeptide or peptide molecule, which is expressed using a recombinant nucleic acid construct created by means of molecular biological techniques. The term 'recombinant' when made in reference to genetic composition refers to a gamete or progeny with new combinations of alleles that did not occur in the parental genomes. Recombinant nucleic acid constructs may include a nucleotide sequence, which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Recombinant nucleic acid constructs, therefore, indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e. by human intervention. Recombinant nucleic acid constructs may for example be introduced into a host cell by transformation. Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species, which have been isolated and reintroduced into cells of the host species. Recombinant nucleic acid construct sequences may become integrated into a host cell genome, either as a result of transformation of the host cells, or as the result of subsequent recombination events. . .

In alternative embodiments, there are provided, isolated compounds such as nucleic acids and amino acids (i.e. peptides or polypeptides). 'Isolated' as used herein, is meant to convey that the isolated substance has been substantially separated or purified away from other components, such as biological components, with which it would otherwise be associated, for example in vivo, so that the isolated substance may be itself be manipulated or processed. The term 'isolated' therefore includes substances purified by purification methods known in the art, as well as substances prepared by recombinant expression in a host, as well as chemically synthesized substances. In some embodiments, a compound is 'isolated' when it is separated from the components that naturally accompany it so that it is at least 60%, more generally 75% or over 90%, by weight, of the total relevant material in a sample. Thus, for example, a polypeptide that is chemically synthesised or produced by recombinant technology maybe generally substantially free from its naturally associated components. A nucleic acid molecule is substantially pure when it is not immediately contiguous with (i.e., covalently linked to) the coding sequences with which it is normally contiguous in the naturally occurring genome of the organism from which the DNA of the invention is derived. An isolated compound can be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid molecule encoding a polypeptide compound; or by chemical synthesis. Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis or HPLC.

The term 'medicament' as used herein, refers to a composition that may be administered to a patient or test subject and is capable of producing an effect in the patient or test subject.. The medicament may be comprised of the effective chemical entity (i.e. one or more of the peptides or polypeptides described herein) alone or in combination with a pharmaceutically acceptable excipient.

The term 'pharmaceutically acceptable excipient' may include any and all solvents, dispersion media, coatings, antibacterial, antimicrobial or antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. An excipient may be suitable for intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, topical or oral administration. An excipient may include sterile aqueous solutions or dispersions for extemporaneous preparation of sterile injectable solutions or dispersion- Use of such media for preparation of medicaments is known in the art.

As used herein, the term 'polynucleotide' includes RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both, sense and antisense strands, and may be chemically or biochemically modified or may contain non- natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.

As used herein, the term 'vector' refers to a polynucleotide compound used for introducing exogenous or endogenous polynucleotide into host cells. A vector comprises a nucleotide sequence which may encode one or more polypeptide molecules. Plasmids, cosmids, viruses and bacteriophages, in a natural state or which have undergone recombinant engineering, are examples of commonly used vectors to provide an isolated polynucleotide molecule to a cell.

Peptide or Polypeptide Modifications

Examples of modifications to peptides or polypeptides may include, but are not limited to acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodjnation, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer- RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, Proteins-Structure and Molecular Properties, 2nd ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold F, Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Posttranslational Covalent Modification of Proteins, B. C. Johnson, ed., Academic Press, New York, 1983; Seifter et a!., Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. (1990) 182: 626-646 and Rattan et al: (1992), Protein Synthesis: Posttranslational Modifications and Aging," Ann NY Acad Sci 663: 48-62. Furthermore, such modifications, if made, would preferably change the circulating half-life of the peptide or polypeptide or the bioavailability, of the peptide or polypeptide QΓ other characteristics of the peptide or polypeptide, without adversely affecting their biological activity.

It is well known in the art that some modifications and changes can be made in the structure of a peptide or polypeptide without substantially altering the biological function, to obtain a biologically equivalent peptide or polypeptide. In one aspect of the invention, proteins that differ from the native polypeptide sequence by conservative amino acid substitutions are provided. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the protein by routine testing.

Alternatively, in another aspect of the invention, peptides that differ from the 10 amino acid Indip sequence by removal of one or more N-terminal or C-terminal amino acids are provided. For example, the 6 AA sequence of PHKFKXl (where Xl = R or K) is a possible alternative peptide. Furthermore, such polypeptides with N-terminal or C- terminal amino acids removed may be assayed for their effect on the function of the protein by routine testing.

Amino acids may be described as, for example, polar, non-polar, acidic, basic, aromatic or neutral, A polar amino acid is an amino acid that may interact with water by hydrogen bonding at biological or near-neutral pH. The polarity of an amino acid is an indicator of the degree of hydrogen bonding at biological or near-neutral pH. Examples of polar amino adds include serine, proline, threonine, cysteine, asparagine, glutarnine, lysine, histidine, arginine, aspartate, tyrosine and glutamate. Examples of non-polar amino acids include glycine, alanine, valine leucine, isoleucrae, methionine, phenylalanine, and tryptophan. Acidic amino acids have a net negative charge at a neutral pH. Examples of acidic amino acids include aspartate and glutamate. Basic amino acids have a net positive charge at a neutral pH. Examples of basic amino acids include arginine, lysine and histidine. Aromatic amino acids are generally nonpolar, and may participate in hydrophobic interactions. Examples of aromatic amino acids include phenylalanine, tyrosine and tryptophan. Tyrosine may also participate in hydrogen bonding through the hydroxyl group on the aromatic side chain. Neutral, aliphatic amino acids are generally nonpolar and hydrophobic. Examples of neutral amino acids include alanine, valine, leucine, isoleucine and methionine, An amino acid may be described by more than one descriptive category. Amino acids sharing a common descriptive category may be substitutable for each other in a peptide.

In some embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0), where the following may be an amino acid having a hydropathic index of about -1.6 such as Tyr (-1.3) or Pro (-1.6)s are assigned to ammo acid residues (as detailed in United States Patent No. 4,554,101) Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); GLa (+0.2); Gly (0); Pro (-0,5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); VaI (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); and Trp (-3.4).

Alternatively, the hydropathy index of an ammo add may be represented as a scale indicating the tendency of an amino acid to seek out an aqueous environment (negative value) or a hydrophobic environment (positive value). Hydropathy indices of the standard amino acids include alanine (+1.8), arginine (-4.5), asparagine (-3.5), aspartic acid (-3.5), cysteine (+2.5), glutamine (-3.5), glutamic acid (-3.5), glycine (-0.4), histidine (-3.2), isoleucine (+4.5), leucine (+3.8), lysine (-3.9), methionine (+1.9), phenylalanine (+2.8), proline (-1.6), serine (-0.8), threonine (-0.7), tryptophan (-0.9), tyrosine (-1.3), and valine (+4.2). Amino acids with similar hydropathy indices may be substitutable for each other in a peptide (Kyte & Doolittle 1982). In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e.g., within a value of plus or minus 2.0). In such embodiments, each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: Ile (+4.5); VaI (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gln (-3.5); Asp (-3,5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, VaI, Leu, He, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn, Gln, Tyr. . .

In alternative embodiments, conservative amino acid changes include changes based on considerations, of hydrophilicity or hydrophobicity, size or volume, or charge. Amino acids can be generally characterized as hydrophobic or hydropbilic, depending primarily on the properties of the amino acid side chain. A hydrophobic amino acid exhibits a hydrophobicity of greater than zero, and a hydrophilic amino acid exhibits a hydrophilicity of less than zero, based on the normalized consensus hydrophobicity scale of Eiseriberg et al. (1984). Genetically encoded hydrophobic amino adds include Gly, Ala, Phe, VaI, Leu, lie, Pro, Met and Trp, and genetically encoded hydrophilic Eanrao acids include Thr, His, Glu, Gln, Asp, Arg, Ser, and Lys. Non-genetically encoded hydrophobic amino acids include t-butylalanine, while non-genetically encoded hydxophilic amino acids include citrulhne and homocysteine.

Hydrophobic or hydrophilic amino acids can be further subdivided based on the characteristics of their side chains. For example, an aromatic amin,o acid is a hydrophobic amino acid with a side chain containing at least one aromatic or heteroaromatic ring, which may contain one or more substiruents such as -OH, -SH, -CN, -F, -Cl, -Br, -I, - NO2, -NO, -NH2, -NHR, -NRR, -C(O)R, -C(O)OH₅ -C(O)OR₁ -C(O)NH2, -C(O)NHR, - C(O)NRR, etc., where R is independently (C1-C6) alkyl, substituted (C1-C6) alkyl, (Cl- C6) allcenyl, substituted (C1-C6) allcenyl, (C1-C6) alkynyl, substituted (C1-C6) alkynyl, (C5-C20) aryl, substituted (C5-C20) aryl, (C6-C26) alkaryl, substituted (C6-C26) alkaryl, 5-20 membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 membered alkhetqroaryl or substituted 6-26 membered alkheteroaryl. Genetically encoded aromatic amino acids include Phe, Tyr, and Trp.

An apolax amino acid is a hydrophobic amino acid "with a side chain that is uncharged at physiological pH and which has bonds in which a pair of electrons shared in common by two atoms is generally held equally by each of the two atoms (i.e., the side chain is not polar). Genetically encoded apolar amino acids include Gly, Leu, VaI, Be, Ala, and Met. Apolar amino acids can be further subdivided to include aliphatic amino acids, which is a hydrophobic amino acid having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include Ala, Leu, VaI, and lie.

A polar amino acid is a hydrophilic amino acid with a side chain that is uncharged at physiological pH, hut which has one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include Ser, Thr, Asn, and Gln.

An acidic amino acid is a hydrophilic amino acid with a side chain pKa value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include Asp and Glu. A basic amino acid is a hydrophilic amino acid with a side chain pKa value of greater than 7. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Genetically encoded basic amino acids include Arg, Lys, and His.

Alternatively, conserved amino acid substitutions may be made by substituting an amino acid for another amino acid within the group containing similar amino acids (if there are others) and provided the amino acid is not known to be responsible for the desired activity, as per the following groupings: GAVLI; FYW; CM; ST; KRH; DENQ; P (Stothard 2000). For example, the lysines (K) at positions 5 and 7 of the 10 amino acid sequence represented by SEQ ID NO: 1 are needed for ubiquitination and are preferably not both substituted with any other amino acids.

It will be appreciated, by one skilled in the art, that the above classifications are not absolute and that an amino acid may be classified in more than one category. In addition, amino acids can be classified based on known behaviour and or characteristic chemical, physical, or biological properties based on specified assays or as compared with previously identified amino acids. Amino acids contained within the peptides described herein may be understood to be in the L- or D- configuration. In peptides and peptidomimetics, D-amino acids may be substitutable for L-amino acids.

Nonstandard amino acids may occur in nature, and may or may not be genetically encoded. Examples of genetically encoded nonstandard amino acids may include selenocysteine, sometimes incorporated into some peptides at a UGA codon, which may normally be a stop codon, or pyrrolysine, sometimes incorporated into some proteins at a UAG codon, which may normally be a stop codon. Some nonstandard amino acids that are not genetically encoded may result from modification of standard amino acids already incorporated in a peptide, or may be for example metabolic intermediates or precursors. Examples of nonstandard amino acids may include, but are not limited to 4- hydroxyproline, 5-hydroxylysine, 6-N-methyllysine, gamma-carboxyglutamate, desmosine, selenocysteine, ornithine, citrulline, lanthionine, 1-aminocyclopropane-1- carboxylic acid, gamma-aminobutyric add, carnitine, sarcosine, or N-formylmethionine. Synthetic variants of $tandaχd and non-standard amino acids are also known and may include chemically derivatized amino acids, amino acids labeled for identification or tracking, or amino acids with a variety of side groups on the alpha carbon. Example of such side groups are known in the art end may include aliphatic, single aromatic, polycyclic aromatic, heterocyclic, heteronuclear, amino, alkylamino, carboxyl, carboxamide,.carboxyl ester, guanidine, amidine, hydroxyl, alkoxy, mercapto-, alkylmercapto-, or other heteroatom-containing side chains. Other synthetic amino adds may include alpha-imino adds, non-alpha amino acids such as beta-amino adds, des- carboxy or des-amino acids. Synthetic variants of amino acids may be synthesized using general methods known in the art, or may be. purchased from commercial suppliers, for example RSP Amino Adds LLC™ (Shirley, MA)..

Alternatively, the peptide or polypeptide may be represented by one or more of the . sequences represented in TABLE 1 or elsewhere herein.

A linker sequence may be used to join an Indip peptide to a targeting, delivery or localization moiety, referred to herein collectively as a 'delivery moiety', and may be advantageous. Such a linker may be used to link, for example, a transduction domain like TAT to an tidip peptide. For example 1-5 G residues may be used as a linker (see for example SEQ ID NOs: 1, and 3-6). Alternatively, linker sequences maybe at the C- terminus of an Indip peptide. Alternative linkers are known in the art. For example, a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) or glutaraldehyde are well known..

Synthesis and Expression

Peptides or peptide analogues can be synthesised by chemical techniques known in the art, for example, by automated synthesis using solution or solid phase synthesis methodology. Automated peptide synthesisers are commercially available and use techniques well known in the art. Peptides and peptide analogues can also be prepared using recombinant DNA technology using methods such as those described in, for example, Sambrook J. and Russell D. (2000) Molecular Cloning: A Laboratory Manual (Third Edition) Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) or Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Suns, 1994).

Targeting, Delivery, and Localization with 'Delivery Moieties'

The peptides or polypeptides may be used in isolation, or may be linked to or in combination with another moiety, such as a targeting, delivery or localization moiety, referred to herein collectively as a 'delivery moiety', for example, tracer compounds, protein transduction domains or sequences, antibodies, liposomes, carbohydrate carriers, polymeric carriers or other agents or excipients as will be apparent to one of skill in the art.

Delivery of bioactive molecules such as peptides, to a cell or cells in a reasonably efficient manner may require more than just the 'dumping' of the naked peptide onto the cell, or administering the naked peptide into the patient or test subject. Agents that enable delivery of bioactive molecules into cells (or 'delivery moieties') in a suitable manner so as to provide an effective amount, such as a pharmacologically effective amount are known in the art, and are described in, for example, Dietz et al 2004. MoI Cell. Neurosci 27:85-131. Examples of such agents include liposomes, antibodies or receptor ligands that may be coupled to the bioactive molecule, viral vectors, and protein transduction domains (PTD). Examples of PTDs include Antennapedia homeodomain (Perez et al 1992 J. Cell Sci 102:717-722), transportan (Pooga et al 1998 FASEB J 12: 67-77), the translocation domains of diphtheria toxin (Stenmark et al 1991 J Cell Biol 113:1025-1032; Wiedlocha et al 1994 Cell 76:1039-1051), anthrax toxin (Ballard et al 1998 Infect. Immun 66:615-619; Blanke et al 1996 Proc Natl Acad Sci 93 : 8437-8442) and Pseudomonas exotoxin A (Prior et al 1992 Biochemistry 31:3555-3559), protegrin derivatives such as deπnaseptin S4 (Hariton-Gazal et a] 2002 Biochemistry 41:9208-9214), HSV-I VP22 (Dilber et al 1999 Gene Ther. 6:12-21), PEP-I (Morris et al 2001 Nature Biotechnol 19:1173-1176), basic peptides such as poly-L and poly-D-lysine (Wolfert et al 1996 Gene Ther. 3 :269-273 ; Rysex et al 1980 Cancer 45:1207-1211; Shen et al 1978 ProcNatl Acad Sci 75:1872- 1876), HSP70.(Fujihara et al 1999 EMBO J 18:411-419) and HIV-TAT (Demarchi et al 1996 J Virol 70:4427-4437). Other examples and related details of such protein transduction domains are described in Dietz 2004 above.

Furthermore, delivery across the blood brain barrier (BBB) maybe facilitated by a number of methods known in the art. For example, the TAT peptide has been used to transduce ghal-derived neurotrophic factor (GDNF) into the central nervous system (CNS) ofmice (Kilic E. et aL 2005). Similarly, the Schwarze et al. (1999) introduced TAT tagged proteins itito the brains of mice. Alternatively, intravenous administration of brain- . derived neurotrophic factor conjugated to the OX26 monoclonal antibody (MAb) specific for the transferring recptor to cross the blood-brain barrier (Pardridge (2002); and Zhang & Partridge (2001)). Alternatively, charged liposomes (Haas et al; (2009)), • rhenacarborane (Hawkins et al. (2009)), or hyaluronan (Horvat et al. (2008)) could be used.

Compositions or compounds according to some embodiments may be administered in any of a variety of known routes. Examples of methods that may be suitable for the administration of a compound include orally, intravenous, inhalation, intramuscular, subcutaneous, topical, intraperitoneal, intra-rectal or intra-vaginai suppository, sublingual, and the like. The compounds of the present invention may be administered as a sterile aqueous solution, or may be administered in a fat-soluble excipient, or in another solution, suspension, patch, tablet or paste format as is appropriate. A composition comprising the compounds of the invention may be formulated for administration by inhalation. For instance, a compound may be combined with an excipient to allow dispersion in an aerosol. Examples of inhalation formulations will be known to those skilled in the art. Other agents may be included in combination with the compounds of the present invention to aid "uptake or metabolism, or delay dispersion within the host, such as in a controlled- release formulation. Examples of controlled release formulations will be known to those of skill in the art, and may include microencapsulation, embolism within a carbohydrate or polymer matrix, and the like. Other methods known in the art for making formulations are found in, for example, "Remington's Pharmaceutical Sciences", (19th edition), ed. A. Gennaro, 1995, Mack Publishing Company, Easton, Pa.

The dosage of the compositions or compounds of some embodiments of the invention may vary depending on the route of administration (oral, intravenous, inhalation, or the like) and the form in which the composition or compound is administered (solution, controlled release or the like). Determination of appropriate dosages is within the ability of one of skill in the art. As used herein, an 'effective amount', a 'therapeutically effective amount', or a 'pharmacologically effective amount' of a medicament refers to an amount of a medicament present in such a concentration to result in a therapeutic level of drug delivered over the term that the drug is used. This may be dependent on mode of delivery, time period of the dosage, age, weight, general health, sex and diet of the subject receiving the medicament. Methods of determining effective amounts are known in the art.

Nucleic Acid Manipulation

The nucleic acid molecules can be inserted into any suitable vector. Suitable vectors may include, viral or bacterial vectors. Suitable viral vectors may include, retroviral vectors, alphaviral, vaccinial, adenoviral, adenoassociated viral, herpes viral, and fowl pox viral vectors. The vectors preferably have a native or engineered capacity to transform eukaryotic cells, e.g., CHO-Kl cells. Additionally, the vectors useful in the context of the invention can be 'naked' nucleic acid vectors (i.e., vectors having Httle or no proteins, sugars, and/oϊ lipids encapsulating them) such as plasmids or episomes, or the vectors can be complexed with other molecules. Other molecules that may be combined with the present nucleic acids are viral coats, cationic lipids, liposomes, polyamines, gold particles, and targeting moieties such as ligands, receptors, or antibodies that target cellular molecules.

The nucleic acid molecules described herein may comprise a region coding for one or more of the ϊndip peptides described herein, may be operatively linked to a suitable promoter, which promoter is preferably functional in eukaryotic cells. Viral promoters, such as, the RSV promoter and the adenovirus major late promoter. Suitable non-viral promoters may include, the phosphoglycerokinase (PGK) promoter and the elongation factor 1α promoter. Non- viral promoters may be human promoters. Additional suitable genetic elements, known in the art, may also be ligated to, attached to, or inserted into the nucleic acid and constructs to provide additional functions, such as level of expression, or pattern of expression. The native promoters for expression of the Insig-1 nucleotides may also be used, in which event they are preferably not -used in the chromosome naturally encoding them unless modified by a process that substantially changes that chromosome. Such substantially changed chromosomes can include chromosomes transfected and altered by a retroviral vector or similar process. Alternatively, such substantially changed chromosomes can comprise an artificial chromosome such as a HAC, YAC, or BAC.

In addition, the nucleic acid molecules described herein maybe operatively linked to enhancers to facilitate transcription. Enhancers are cis-acting elements of DNA that stimulate the transcription of adjacent genes. Examples of enhancers which confer a high level of transcription on linked genes in a number of different cell types from many species include, without liinitation, the enhancers from SV40 and the RSV-LTR. Such enhancers can be combined with other enhancers which have cell type-specific effects, or any enhancer may be used alone. .

To optimize protein production the inventive nucleic acid molecule can further comprise a polyadenylation site following the coding region of the nucleic acid molecule. Also, preferably all the proper transcription signals (and translation signals, where appropriate) will be correctly arranged such that the exogenous nucleic acid will be properly expressed in the cells into which it is introduced. If desired, the exogenous nucleic acid also can incorporate, splice sites (i.e., splice acceptor and splice donor sites) to facilitate mRNA production while maintaining an inframe, full length transcript Moreover, the inventive nucleic acid molecules can further comprise the appropriate sequences for processing, . secretion, intracellular localization, and the like.

The 9 amino acid sequence "EPHKFKREW", maybe reverse translated to a 27 base sequence based on the most likely codons as follows: gaaccgcataaatttaaacgcgaatgg (SEQ ID NO : 25) .

Methods

General Antibodies, Peptides, and Reagents

Rabbit anti-SREBP1 was obtained from Santa Cruz Biotechnology™ (Santa Cruz, CA). Mouse mAb MAP2 was obtained from BD Pharmagen™ (San Diego, CA). ProLong Gold™ mounting medium and secondary fluorophore-bound IgGs (Alexa Fluor™ series) for immunocytochemistry were obtained from Invitrogen-Molecular Probes™ (Portland, OR). Rabbit anti-Lamin A/C was obtained from Cell Signaling Technology™ (Danvers, MA). Mouse anti β-tubulin was from Sigma Aldrich™ (St. Louis, MO). Mouse anti- neuronal nuclei (NeuN) was purchased from Chemicon, Inc.™ (Temecula, CA). Mouse anti-Myc tag was from. Upstate™ (Temecula, CA). Mouse anti TATA binding protein (TBP) was purchased from Abeam Inc.™ (Cambridge, MA). Leupeptin protease inhibitor was obtained from Peptides International™ (Louisville, Kentucky) and all other lysate buffer components, including phosphatase and protease inhibitors, were obtained from Sigma Aldrich™ (St Louis, MO). BAPTA-AM was obtained from Invitrogen-Molecular Probes™ (Portland, OR). Calpeptin and MDL were obtained from Calbiochera™. (San Diego, CA). NMDA was purchased from Ascent Scientific™ (Weston, UK). Mouse anti Insig-1 antibody was a generous gift from Prof. Stephen A. Johnston, Arizona State University (Tempe, AZ). TAT-Indip (YGRKKRRQRRRGEPHKFKREW) was custom synthesized by Peptide Synthesizing Facility at UBC (Vancouver, BC, Canada), Nuclear extraction kit was purchased from Panomics™ (Redwood City,. CA), NR2A-specific antagonist NVP-AAM077 was a generous gift from YP Auberson, Novartis Phanna AG™ (Basel, Switzerland). NR2B-specific antagonist Ro 25-6981 was from Sigma Aldrich™ (St. Louis, MO). AP5 was purchased from Tocris™ (Cookson Bristol, UK).

Western Blotting

For western blotting, 10 μg of the nuclear extract or 40 μg of total cell lysate (solubilized by 1% SDS;. ImM EGTA, ImM EDTA, ImM DTT, 2mM sodium orthovanadate, and protease inhibitors cocktail in PBS) from each treatment condition was separated with SDS-PAGE, transferred onto a polyvinylidene difluoride (PVDF) membrane, and probed with the relevant antibodies. For sequential reprobing of the same blots, the membranes were stripped of the initial primary and secondary antibodies and subjected to immunoblotting with another target antibody. Blots were developed using enhanced chemiluminescence detection (Amersham™). Band intensities were quantified using N1H ImageJ™ software and normalized to the quantity of a nuclear marker or β-tubulin in each sample lane.

Primary culture of cortical neurons and spinal cord neurons

Dissociated cultures of rat cortical neurons were prepared from 18-d-old Sprague Dawley rat embryos as described previously (Meilke & Wang 2005). To obtain mixed cortical cultures enriched with neurons, uridine (10 μM) and 5-Fluor-2'-deoxyuridine (10 μM) were added to the culture medium at 3 d in vitro (DIV) and maintained. for 48 h to inhibit non- neuronal cell proliferation before the cultures were returned to the normal culture medium. Mature neurons (12-14 DIV) were used for experiments. Dissociated spinal cord neurons were prepared from 16-d-old mice embryos hSOD-G93A or wt controls. Cultures were maintained for 10 d in vitro before excitotoxic challenge.

Experimental in vitro excitotoxicity insults

Oxygen-glucose deprivation (OGD) was achieved by transferring cortical cultures to an anaerobic chamber (Thermo EC™) containing a 5% CO₂, 10% H₂, and 85% N₂ (<0.01% O₂) atmosphere (Aarts et al. 2002; Goldberg & Choi 1.993; and Meilke & Wang 2005). Cultured neurons were then washed three times with glucose-free bicarbonate-buffered solution (deoxygenated in the anaerobic chamber for 30 min before use) and maintained anoxic for 1 h at 37°C. OGD was terminated by removing the cultures from the chamber, washing them twice with normal ECS, and returning them to the original growth conditions until additional assay. Alternatively, in order to induce excitotoxicity, 12-14 DW. cultured neurons were washed and transferred to Mg²⁺-free extracellular solution (ECS) containing the following (in mM); 25 HEPES. acid, 140 NaCl, 33 glucose, 5.4.KCl,. and 1.3 CaCl₂, with pH 7.35 and osmolality 320-330 mOsm. ECS and subjected to NMDA-induced excitotoxicity (50μM NMDA, 10μM glycine, 20 min at room temperature) or any other experimental conditions as indicated in the texts. Cells were then washed twice with normal ECS, and returned to the original growth conditions until additional assay. .

Nuclear extract isolation and protein/DNA array assay

Nuclear extracts were isolated from control and NMDA-treated cell cultures (10⁸ cells) using the Panomics™ nuclear extraction kit (Panomics™; catalog number AY2002) as recommended by the manufacturer. Biorin-labeled DNA binding oligonucleotides (TranSignal™ probe mix; Panomics™) were incubated with 10 μg of nuclear extract for 30 min. at 15°C to allow the formation of protein/DNA (or transcription factor/DNA) complexes. The protein/DNA complexes were then separated from the free probes by column purification as described by the manufacture's recommendations (Panomics™). Probes were then hybridized to the TranSignal Protein/DNA Combo Arrays™ overnight at 42°C followed by post-hybridization washes. Each TranSignal Protein/DNA Combo Array™ contained 345 putative transcription factor nucleotide sequences. In order to quantify the presence of the transcriptional factors in the experimental nuclear extracts, hybridized arrays were incubated with horseradish peroxidase-labeled streptavidin and bound signals were detected using chemiluminescence imaging system

Assessment of neuronal death

Necrotic neuronal death was quantified by measuring lactate dehydrogenase (LDH) release 20 h after treatments using a Cyto Tox 96™ assay kit (Promega™, Madison, WI); Apoptotic neuronal death was determined either by visualizing neurons stained with Hoechst-33342 or using a cell ELISA assay. For visualizing apoptotic neurons, Hαechest- 33342™ (ϊ μg/ml) was added to the culture medium after treatments and incubated for 30 min. Images were taken with a Leica DMIRE2™ fluorescence microscope. Cells with condensed or fragmented chromatin were considered apoptotic. These observations were quantified by double-blind counting of apoptotic and total neurons in each, visual field and expressed as percentage apoptosis. Cell ELISA quantitative assessment of neuronal apoptosis was performed 20 h after treatments using a Cell Death Detection ELISA Kit™ (Roche Products™, Welwyn Garden City, UK).. Absorbance readings were measured using a spectrophotometric microplate reader. Data analyses were performed according to the instructions of the manufacturer. Data are expressed as the difference in apoptosis relative to control and are expressed as a percentage. Propidium Iodide assessment of cell death was performed by adding 10 μg/ml Propidium Iodide for 30 minutes prior to fixation. Neurons identified by NeuN counterstain were scored blind for Propidium Iodide positive nuclei. TUNEL stain (Roche ProductsTM) determination of apoptotic cells was performed according to the manufacturer's direction.

Middle cerebral arterial occlusion

Adult male Sprague-Dawley rats (330 - 360 g, Charles River) were used in this study.

They had free access to rat pellet chow and water throughout the entire study. Each rat was anaesthetized with 4% isofluorane in a nitrous oxide/oxygen (70:30%) mixture and thereafter maintained with 1.5-2.0% isoflurane in the same gas mixture. Reversible middle cerebral arterial occlusion (MCAo) was induced using the suture-insertion method described previously (Liu et al. 2007; and Aarts et al. 2002). Briefly, a nylon suture with a blunted tip was inserted through the right external carotid artery, and advanced slowly down the right internal carotid artery until the right MCA was occluded. The operation wound was sutured, and the animal was allowed to recover from anaesthesia. After 90 minutes of occlusion, the animal was re-anaesthetized to facilitate removal of the occlusion. Sham-operated animals received the same surgical operation, except without the final occlusion. Body temperature was monitored and maintained between 36.5 and 37.5 °C throughout the surgical procedure with a heating pad. _.

Behavioural tests

Each rat received behaviour tests one hour following occlusion to confirm successful stroke induction. . The behaviour tests used were described previously, and included a postural reflex test and five forelimb placement tests (Liu et al. 2007; and Aarts et al. 2002).. Each test gave a score between 0 (no deficit) and 2 (complete deficit) for a total score between 0 and 12. Only rats with a neurological score of 11 one hour following stroke were included in this study. The rats were scored again the next day to determine functional neurological outcome.

Data analysis .

Data are expressed as mean ± SEM. ANOVA was used for comparison among multiple groups. Statistical significance was defined asp < 0.05.

The following examples are provided for illustrative purposes and are not intended to be limiting as such.

Results Activation of NMDARs has been linked to the modulation of a number of transcription factors (TF), with either pro-neuronal survival or pro-death activity (Camandola & Mattson 2007; Hardingham & Bading 2001; Hetman & Kharebava 2006; Rao & Finkbeiner 2007; West et al. 2002; Zhang et al. 2007; and zou & Crews 2006), suggesting that' alteration of TF activity may critically contribute to excitotoxic neuronal injuries following stroke. To systemically study changes in transcriptional regulation following exitotoxic insults, we isolated nuclear extracts from cortical neuronal cultures 4 hours after a 60 minute challenge with 50 μM NMDA and screened 345 transcription factors (TFs) against their characterized transcriptional binding sites (TBS) (FIGURE IA). From the total population of TFs analyzed, we identified 16 that showed a 2 fold or greater increase in activity (NMDA/Control) and 6 with a 2 fold or more decrease (<0.5) (confirmed by electrophoretic mobility shift assay (EMSA), results not shown). Among those TFs elevated more than two fold upon NMDA-induced excitotoxicity we were particularly interested in further characterizirig SREBP1, a member of membrane bound SREBP TFs that are. best known for their role in regulating genes required for cholesterol, fatty acids, triglycerides and phospholipids biosynthesis (Espenshade & Hughes 2007; and Goldstein et al. 2006). The present disclosure is focused on SREBP1 for the following reasons: 1) evidence accumulated in recent years suggests there is a role for SREBP1- regulated lipid products in mediating cell damage in ischemia and other neiirodegerative conditions (Adibhatla et al. 2006; and Siesjo and Katsura 1992); 2) SREBP1 activation has recently been linked to glucolipotbxic cell death in pancreatic β-cells (Sandberg et al; 2005; Takahashi et al. 2005; Wang et al. 2005; Wang et al. 2003; and Yamashita et al, 2004); and 3) activation of SREBPl has also been demonstrated in response to anaerobic and hypoxic stress in fission yeast and Cryptococcus neoformans (Chang et al 2007; and Todd et al. 2006). The present data and l)-3) above suggests that activation of SREBP1 may also pjay a role in mediating excitotoxic neuronal damage following ischemic insult.

Activation of SREBP1 requires the cleavage of the inactive membrane integral precursor protein of 130 kDa by twp dedicated proteases in the Golgi, leading to the release and translocation of the soluble mature N-terminal SREBP1 (nt-SREBP1) of 68kDa for transcriptional activity. To further confirm ability of NMDA stimulation to activate SREBP1, changes in the amount of activated nt-SREBP1 in cortical neuronal cultures treated with and without NMDA (50 μM; 20 min) were determined, As shown in FIGURE IB, NMDA stimulation produced an about 4.5 fold increase in expression of mature nt-SREBP1 after 6 hours (FIGURE IB). Activation is specifically mediated by NMDARs as it was fully prevented when NMDA stimulation was performed in the presence of NMDAR. antagonist AP5 (50μM) and. NR2B-subunit specific antagonist RO25-6981 (Ro 0.5μM), but not NR2A-ρreferential agonist NVP-AAM077 (NVP 0.4μM) as shown in FIGURE 1C. Emerging evidence has suggested that activation of NMDAR could lead to pro-survival or pro-death of neuronal cells depending on the subunit composition and/or subcellular location of the receptors. Thus, activation of synaptic, predominantly NR2A-containing NMDARs could activate cell survival signaling, whereas stimulating the extrasvnaptic, predominantly NR2B-containing NMDARs could mediate excitotoxic/ischemic neuronal injuries (Hardingham et al. 2002; Liu et al. 2007; Zhou & Baudry 2006; Liu et al. 2004; Tigaret et al. 2006; and Mutel et al. 1998). As shown in FIGURE 1C, it was found that the NMDA-induced SREBP1 activation is resistant to NVP-AAMQ77, a NR2A-containing NMDAR preferable antagonist, but blocked by Ro25- 6981, a NR2B subunit specific antagonist. These results suggest that NMDA-induced SREBP1. activation is primarily mediated by NR2B-subuπit-containing NMDARs, and hence may specifically contribute to the mediation of excitotoxic neuronal injuries.

Rising intracellular Ca²⁺ concentration and subsequent activation of some of Ca²⁺'depeήdent cell death signaling steps such as activation of calpain has been considered a downstream step leading to NMDAR-mediated excitotoxicity (Choi 1988; Lipton 2006; and Mattson 2007). Consistent with the involvement of intracellular Ca²⁺ and calpain activation in mediating NMDA-induced SREBP1 activation, pre-incubation of the neurons with either Ca²⁺ chelator BAPTA-AM (lOμM) (FIGURE ID), or . calpain rnhibitor calpeptin (25μM) or MDL (50μM) (FIGURE IE) inhibited NMDAR-mediated SREBP1 activation, further supporting a potential role of SREBP1 activation in NMDAR-mediated excitotoxicity. .

Pollowing activation, the mature nt-SREBP.l translocates into the nucleus to . regulate the expression of genes containing sterol regulatory elements (SRE). . Nuclear translocation of SREBP1 . (nt-SREBP1) following NMDA-induced excitotoxicity was examined. As shown in FIGURE 2A, western blotting of nuclear extract fractions, of control or NMDA-treated cortical neuronal cultures revealed a time dependent and NMDAR-dependent appearance of active form of SREBP1 in the nucleus (nt-SREBP1). Similar to SREBP1 activation, SREBP1 nuclear translocation was also primarily mediated by NR2B-containing NMDARs as it was resistant to NR2A antagonist NVP, but prevented by NR2B antagonist Ro (FIGURE 2B). The NMDAR-dependent activation and nuclear translocation of SREBP1 was also further confirmed with co-immunolocalization of endogenous SREBP1 with nuclei stained using Hoechst 33342. An increase nuclear SREBP1 was detected after NMDA-induced excitotoxicity and which was sensitive to AP5 (FIGURE 2C).

The requirement for NR2B activation and calpain activity by both SREBP1 activation and excitotoxic/ischemic neuronal injuries suggests that SREBP1 may causatjvely relate to NMDAR-mediated excitotoxic neuronal injuries. By measuring the intensity of nuclear SREBP1 signal and Hoechst 33342 stain (as an index of nucleus condensation and thus progression of apoptosis), it was found that there was a strong correlation between the two factors, implying that SREBP1 may have a causative role in mediating NMD AR-mediated excitotoxicity (FIGURE 2D).

To directly investigate a causative role of SREBP1 activation in mediating NMDAR-tnediated cell death, inhibition of NMDAR-mediated SREBP1 activation and nuclear translocation with two mechanistically and structurally distinct inhibitors: . extracellular cholesterol and Insig degradation peptide inhibitor (Indip) was performed as follows. . . .

As illustrated in FIGURE 4A, under basal unstimulated conditions, immature SREBPs form a stable complex with SREBP cleavage activating protein (SCAP) (Nohturftt et al. 1998). This complex, is retained in the ER by the interaction between SCAP and the ER membrane resident protein insulin-induced gene-1 (Insig- 1) (Sun et al 2005; and Yang et al. 2002). When cholesterol is low, Insig- 1 protein is rapidly ubiquitinated on lysine-156 and lysine-158 and degraded by the proteasome (Gong et al. . 2006a; Gong et al. 2006b; Lee et al. 2006a; and Lee et al. 2006b),- SCAP is then released from ER membrane and to chaperone SREBP to the Golgi for proteolytic processing, thereby producing the active mature nt-SREBPs. Since cellular stress suoh as hypotonic shock and ER stress also activate SREBPs via rapid turnover of Insig-1 (Lee & Ye 2004), NMDAR activation may produce the mature nt-SREBP1 via a similar Insig-1 dependent mechanism. Accordingly, increasing extracellular concentrations of cholesterol by stabilizing Insig-1 and SCAP interaction to prevent Insig-1 degradationj should also inhibit NMDA-induced SREBP1 activation.- Cortical neuronal cultures were treated with cholesterol added into the extracellular solution for 12 hours prior to NMDA-induced exitotoxicity and then SREBP1 activation was assessed by Western blot. The addition of cholesterol showed no observable effect on SREBPi activation on its own, but reduced NMDA-induced activation of SREBP1 in a dose-dependent fashion (FIGURES 3A). Using these same cholesterol concentrations we then looked for NMDA-induced excitotoxicity after 12 h cholesterol treatment (FIGURE 3B). Similar to the inhibition of SREBP1 activation we found a dose dependent reduction of NMDA-induced excitoxiciτy in our cultured neurons due to cholesterol treatment as determined by LDH release 24 h later. Lower doses of cholesterol which were ineffective at reducing NMDA-induced SREBP1 activation were also ineffective at reducing NMDA-induced excitotoxicity; The results are consistent with a role of SREBP1 activation in mediating NMDA-induced cell death,. .

To directly modify SREBP1 a knockdown approach was investigated. Endogenous SREBPl expression was reduced by between 40 and 50% after 48 hour expression of a shRNA knockdown construct in HEK293 cells as compared to empty vector or scrambled control. shRNA, as assessed by SREBP1 Western blot (FIGURE 3C). Expression of flag-SREBP1 was dramatically reduced in hippocampal cultures upon co- trahsfection with SREBP1 sbRNA construct as compared to control shRNA and GFP alone neurons (FIGURE 3D). Hippocampal neurons transfected with SREBP1 shRNA construct were resistant to NMDA induced excitotoxicity (FIGURE 3E). Four to five days after transfection, neurons were exposed to NMDA challenge (50μM) and allowed 2 hours recovery before fixation and apoptotic nuclear condensations were assessed by Hoechst 33358 (5 μg/ml) fluorescent stain (transfected nuclei indicated by arrow) and coverslips were scored for the percentage of apoptotic neurons (FIGURE 3F). .

The ability of cholesterol to inhibit NMDAR-mediated activation of SREBP1 suggests that the degradation of ER membrane resident protein Insig-1 may be involved in NMDAR-mediated activation of SREBP1.

To test the requirement of Insig-1 degradation in NMDAR-mediated SREBP1 activation during excitotoxicity, an Insig-1 degradation interference peptide (Indip) was designed (GEPHKFKREW (SEQ ID NO: I)) and which contains the lysine-156 and lysine-158 ubiquitination sites (positions 5 and 7 in bold of the 10 AA peptide) based on the prediction that the peptide would competitively block Insig-1 ubiquitination and hence reduce Insig- 1 degradation in the proteasσme (FIGURE 4A) . The peptide was rendered membrane permeable by fusing the cell-membrane transduction domain of the human immunodeficiency virus-type 1 Tat protein (YGRXKRRQKRR) as per Schwarze et al. 1999 to the N-terminal of the Indip to generate TAT-Indip peptide (FIGURE 4A). However, it would be appreciated that the delivery moiety, such as TAT, could be joined to the C-termihus or otherwise associated with the Indip peptide as described herein, _. Simvastatin-induced degradation of Insig-1 was inhibited by TAT-Indip in HEK 293 cells treated with 2 μM of Insig-1 peptide followed by 24 h stimulation with 10 μM simvastatin. Cell lysates irαmunoblotted for. Insig-1 showed reduced Insig-1 in control cells as compared to TAT-Indip treated cells, but not TAT-Indipκ_-R treated cells (FIGURE 4B). Furthermore, rapid tn$ig-l ubiquitination in response to NMDA-induced excitotoxicity was inhibited by TAT-ϋαdip, but not control peptide. Cortical cell cultures were pretreated with TAT-Indrp or TAT-Indip_K._R peptide (30 minutes, 2 μM peptide) prior to NMDA insult and ubiqutrnated proteins were precipitated from total lysates for 30 minutes after NMDA treatment with ρ62 ubiqitin binding domain and immunoblotted for Insig-1. There was an increase in ubiquitinated ϋisig-l detected with NMDA insult, but not in the presence of TAT-Indip (results not shown).

Pretreatment of neurons with the TAT-Indip peptide (2μM), but not TAT- Indip_{K-R j} was able to block the NMDA-induced reduction of Insig-1 in cortical cultures (FIGURE 4C). Similarly, myc-Insig-1 fluorescent signal was also protected in cultures pretreated with TAT-Indip peptide (results not shown), demonstrating its effectiveness as an Insig-1 degradation interference peptide in blocking NMDA-induced Insig-1 degradation. Consistent with the role of Insig-1 degradation in NMDAR-mediated SREBP1 activation and nuclear translocation, western blotting and immunocytochemical studies revealed that TAT-Indip pretreatment was able to reduce NMDA-induced SREBPl activation (FIGURE 4D). These results support the role of Insig-1 degradation as a significant step in NMDAR-mediated activation of SREBP1, but also suggests that this Indip peptide may be used as a specific inhibitor for SREBP 1 activation.

An investigation of whether NMDAR-induced excitotoxic neuronal injuries could be reduced by inhibition of SREBP1 activation using ϋidip was made by measuring cell death dependent lactate dehydrogenase (LDH) release. Cortical cultures were pretreated for 12 hours with high and low cholesterol concentrations before NMDA treatment and LDH assessment 24 hours later. At higher concentrations of cholesterol (100 μM) NMDA induced excitotoxicity was reduced, but lower concentrations resulted in. poor blocking of SREBP1 activation and did not offer protection (FIGURE 5A). Similarly, LDH signal in cortical cultures pretreated with TAT-Indip peptide (2μM; given 30 minutes prior to NMDA treatment), but not TAT-ϊndipκ-_R, showed a dramatic decrease in media levels of LDH 24 hours after NMDA treatment almost to control levels (FIGURE 5B). The potential neuroprotective effect of Indip against NMDA-induced apoptotic cell death was examined using both DNA fragmentation and nuclear condensation imaging. Using an ELISA-based DNA fragmentation assay as an unbiased quantitative measure of apoptosis, we found that treatment of neurons with TAT-Indip also reduced NMDA- induced apoptosis to the control levels (FIGURE 5C), suggesting that TAT-Indip is also able to attenuate apoptotic neuronal injuries caused by excitotoxicity. This was further corroborated by nuclear condensation imaging with Hoechst 33358 fluorescence in response to NMDA stimulation. Neurons (identified with doubled staining with NeuN in green) treated with TAT-Indip (NMDA+TAT-Indip) showed fewer condensations than neurons treated with NMDA alone (data not shown). The neuroprotective effect of TAT- Indip was time-dependent as quantifying Hoechst signal revealed that although the protection could be noticeable as early as 30 min, it only became statistically significant 4 hours after NMDA treatments (FIGURE 5D). This delayed protective action of TAT- Indip corresponds well to the time course required for SREBP1 activation, consistent with TAT-Indip acting via the suppression of SREBP1.

Together the above results support an important role for SREBPl activation in mediating excitotoxic neuronal injuries. Since NMDA-mediated excitotoxicity is thought to be a primary event leading to neuronal injuries following ischemic brain insults and SREBP1 activation has recently been demonstrated following hypoxic stimulation, it wa$ predicted that SREBP1 activation may also play a causative role in mediating neuronal injury following ischemic insults such as stroke and brain trauma. To examine this prediction, a well-characterized in vitro stroke model, oxygen and glucose deprivation (OGD) was used (Aarts et al. 2002; and Goldberg and Choi 1993). Treatment of cortical neuronal cultures with OGD, similar to NMDA stimulation, resulted in time-dependent activation of SREBP1 and the OGD-induced activation could be blocked by either AP5 or NR2B antagonist, but not NR2A preferable antagonist (results not shown). Consistent with, a critical role of SREBPl activation in OGD-induced neuronal injuries, pretieatcaent of the neurons with TAT-Indip (1 μM; 1 hour prior to OGD challenge and throughout the entire observation period) prevented OGD-induced increase m LDH release assayed 24 hour following OGD challenge (results not shown). TAT-Indip peptide was shown to protect against NMDA induced excitotoxicity and Oxygen Glucose Deprivation neuronal death in cultured neurons. Cortical cultures were pretreated with TAT-Indip or TAT- Indip_K-R before NMDA induced excitotoxicity and cell death was assessed by LDH release or propidum iodide uptake after NMDA induced apoptosis and showed that TAT- Indip effectively reduces NMDA-induced neuronal death (FIGURES 6A & 6B). Cortical cultures exposed oxygen glucose deprivation (OGD) were immunobloted for nt-SREBP1 at various times post-OGD and showed increased nt-SREBP1 over tune following OGD (FIGURE 6C). OGD-induced activation of SREBP1 is NMDA-receptor dependent. OGD treated cortical cultures in the presence of NMDA-receptor antagonists AP5 and Ro25- 69Sl inhibit OGD-dependent SREBP1 activation, but NVP-AAM077 has no effect (FIGURE 6D). TAT-Indip is also shown to prevent OGD-induced cell death in cortical cultures pretreated with TAT-Indip or TAT-mdipκ-_R before 1 h OGD. Cell death was assayed by LDH release and TAT-Indip, but not TAT-Indipκ__R reduces OGD-induced cell death (FIGURE 6E). .

The role of SREBP1 activation in neuronal injuries following stroke and whether Indip might be used as a potential neuroprotective stroke therapeutic was investigated . using the rat focal ischemic stroke model, middle cerebral artery occlusion (MCAo) as described by Liu et al. (2007) and Bederson et al. (1986). TAT-Indip (8.4 mg/kg animal weight) or vehicle (saline) was infused intravenously in the rats 45 minutes prior to stroke onset. Neurological scores were determined 1 and 24 hours^' and cerebral infarction were examined 24 h after the MCAo onset. All experiments and measurements were done in a double blinded manner. Similar to the results observed with. OGD in vitro, it was found that MCAo challenge produced a significant activation of SKEBPl only m tissue from the ischemic hemisphere (R: Right) of stroked animals, but not in tissues from either the contralateral hemisphere (L: Left) of stroked animals or either hemispheres from animals subjected sham surgery. Intravenous infusion of TAT-Indip peptide, while having no . ^•. effect on basal SREBP1 activation, prevented MCAo-induced activation of SREBP1 in the stroke hemisphere. Suggesting that following systemic application, the TAT-ϊndip peptide was successful in reaching inside of the stroke affected area and effectively interfering with the degradation of Instg-1 and subsequently blocking SREBP1 activation. Most importantly, compared with saline^treated controls, the TAT-Indip peptide infusion significantly reduced the infarct areas. In non-treated animals MCAo produced an averaged infarct area represented 45 ± 7.6 % of the total hemisphere volume. In contrast, in TAT-Indip treated animals, MCAo-induced infarct was significantly reduced to 22 ± 4.2 % of the hemisphere (FIGURE 7D). Neurological behavioral tests further showed that the TAT-Indip peptide treatment also reduced the impact of MCAo on. animals' behavioural performance on forelimb placing and postural reflex tests. Thus, when scored on a scale of 12 for failure, TAT-Indip treated animals (7±1.2) performed significantly better than control animals (ll±0.3) 24 hours after MCAo surgery (FIGURE 7E). Thus the in vivo results suggest that SREBP1 is activated by MCAo challenge and that . ; inhibiting this activation with TAT-Indip not only reduces ischemic neuronal damage, but also improves neurological performance. ^' Furthermore, FIGURE 8 shows TAT-Indip peptide reduces tactile response deficit following an ischaemic insult Animals that were treated with TAT-Indip or TATMndipκ-_R peptide after MCAo were assessed for limb response due to tactile stimuli for several days post-ischaemia and although all animals showed severe deficits in tactile response initially, TAT-Indip treated animals show significantly improved responses after 7 days.

The results demonstrate NMD AR-mediated. SREBP1 activation m signaling pathways leading to excitotoxic neuronal injuries following ischemic insults. The NMD AR-mediated SREBP1 activation appears to be dependent on elevated intracellular Ca²⁺ concentration and to share a similar mechanism with lipid homeostasis regulation, involving rapid degradation of lhsig-1. SREBP1 activation following NMDA-induced excitotoxicity could potentially function as either a neuroprotective event as part of self- defense mechanism or a critical event that leads to cell death. However, the present results provide evidence supporting a causative role of SREBP1 in mediating neuronal injuries.. Specifically,. there is a positive relationship between SREBP1 nuclear translocation and neuronal injuries and most importantly, inhibiting SREBP1 with two structurally distinct inhibitors, cholesterol and Tat-Indip, seems to provide protection against excitoxic neuronal injuries. Accordingly, the present disclosure identifies activation of SREBP1 as a potential target in the cascade leading to ischemic neuronal damage, upon which new compounds, such as ϊndip, TAT-Indip and conserved peptides thereof may be developed as potentially a new_: class of neuroprotective thereapeutics to reduce, neuronal damage following ischemic insults such as stroke and brain trauma! lnsig-1 degradation and SREBP1 activation are shown in spinal cord lysate of ALS (G93A) transgenic mice-immunoblots as compared to wild type mouse spinal cord lysates. SREBP1 activation is increased in ALS tg mice, while Insig-1 shows degradation (FIGURE 9).. Also, TAT-Indip peptide appears to protect against glutamate induced excitotoxicity cell death in both wild type and G93A transgenic embryonic spinal cord neurons. Mice embryonic spinal cord cultured neurons were pretreated with TAT-Indip or TAT-Indip_K__R peptide before glutamate induced excitotoxicity, then assayed for cell death by LDH release and showed significant inhibition of cell death with TAT-Indip (FIGURE 10). Similarly, TAT-Indip peptide appears to protect against glutamate induced DNA fragmentation (apoptosis) in G93A transgenic embryonic spinal cord neurons. G93A transgenic mouse embryonic spinal cord cultured neurons were pretreated with TAT-Indip or TAT-Indipκ-R peptide before glutamate induced excitotoxicity and assayed by Tunel assay to measure percentage of DNA fragmented cells (apoptotic cells), showing that TAT-Indip effectively blocked glutamate induced apoptosis in G93A spinal cord neurons while TAT-Indip_K-R appeared to have some neuronal toxic effect on its own and failed to block glutam^'ate-induced toxicity (FIGURE 11). Furthermore, FIGURE 12 shows that SREBP1 is activated in post-mortem spinal cord samples from both sporadic and familial ALS patients, but not normal controls as compared to the β-Tubulin.

Although SREBPs are the major transcription factors that regulate the expression of a large number genes involved in cellular cholesterol and lipid biogenesis and metabolic alterations in some of these lipid products may be implicated in mediating neuronal damage following ischaemic injury and activation of SREBP1 may exert its pro- neuronal death action by altering one or more of these lipid products, there are also reports that SREBPl may regulate the expression of SRE-containing genes that are not directly involved in lipid metabolism, including genes encoding G proteins (Park et al. 2002) and voltage-gated ion channels (Park et al. 2008). Thus, it may equally plausible that SREBP1 contributes to neuronal damage by a mechanism independent of lipid metabolism. Given to the fact that excitotoxicity is thought to be a common neuropathology associated with a large number of neurological disorders ranging from acute brain insults such as stroke to chronic neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), the present disclosure may have broad implications for new therapeutics for^'clinical treatment of these neurological disorders. . . .

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Claims

WHAT IS CLAIMED IS:

1. An isolated bioactive polypeptide, wherein the amino acid sequence of the polypeptide comprises a polypeptide selected from the group consisting of: a) PHKFKX₁ (SEQ ID NO: 21), where X₁ = R or K; b) XtX₂PHKFKX₃X₄X₅ (SEQ ID NO: 22), wherein X₁ = is a linker of 1-5 G or is absent, X₂ = E or D or is absent, Xj = R or K, X₄ = E or D or is absent, and Xj = W or F or is absent; . . c) X₁X₂PHKFKX₃X₄X₅ (SEQ ID NO: 23), wherein X₁ = is a linker of 1-5 G or is absent, X₂ = E or D or N or Q or is absent, X₃ - R or K or H..X₄ = E or D or N or Q or is ab$ent, and X₅ = W or F or Y or is absent; and d) X_IX₂PHKFKX₃X₄XS (SEQ ID NO: 24), wherein X₁ = G or A or V or L or I or is a linker of 1-5 G or is absent, X₂ = E or D or N or Q, X₃ = R or K or H, X₄. = E or D orN or Q, and X₅ = W or F or Y.

2. The polypeptide of claim 1, wherein the polypeptide is selected from one or more of the following: GEPHKFKREW (SEQ ID NO: 1); GGEPHKFKREW (SEQ ID NO: 3); GGGEPHKFKREW (SEQ ID NO: 4); GGGGEPHKFKREW (SEQ_. ID NO: 5); GGGGGEPHKFKREW (SEQ ID NO:- <5); EPHKFKREW (SEQ ID NO: 7); GEPHKFKRE (SEQ ID NO: 8); EPHKFKRE (SEQ ID NO: 9); GEPHKFKRE (SEQ ID NO: 10); EPHKFKRE (SEQ ID NO: 11); PHKFKRE (SEQ ID NO: 12); PHKFKR (SEQ ID NO: 13); PHKFKK (SEQ ID NO: 14); DPHKFKREW (SEQ ID NO: 15); EPHKFKBJEF (SEQ ID NO: 16); DPHKJFKREF (SEQ ID NO: 17); EPHKFKRDW (SEQ ID NO: 18); EPHKFKKJD (SEQ ID NO: 19); and PHKFKRD (SEQ ID NO: 20).

3. The polypeptide. of claim 1 or 2, wherein the polypeptide ftuther comprises a delivery moiety. .

4. The polypeptide of claim 3, wherein the delivery moiety, allows for localization to the central nervous system.

5. The polypeptide of claim 3, wherein the delivery moiety is a TAT peptide.

6. . The polypeptide of any one of claims 3-5, wherein the polypeptide is selected from one or more of the following: YGRKKRRQRRRGEPHKFKREW (SEQ ID NO: 26); YGRKKRRQRRRGGEPHKFKREW (SEQ ID NO: 27); YGRKKRRQRRRGGGEPHKFKREW (SEQ ID NO: 28); YGRKKRRQRRRGGGGEPHKFKREW (SEQ ID NO: 29); YGRKKRRQRRRGGGGGEPHKFKREW (SEQ ID NO: 30);

YGRKKRRQRJRREPHKFKREW (SEQ ID NO: 31); YGRKKRRQRRRGEPHKFKRE (SEQ ID NO: 32); YGRKΕ-RRQRJRREPHKFKJRE (SEQ ED NO: 33); YGRJKKRRQRRRGEPHKFKRE (SEQ DD NO: 34); YGRKKRJR.QRRREPHKFKRE (SEQ ID NO: 35); YGRJKKItRQRRRPHKFKRE (SEQ ID NO: 36); YGRKKRRQRRRPHKFKR (SEQ BD NO: 37); YGRKKRRQRRRPHKJFKK (SEQ ID . NO: 38); YGRKKRRQRJRRDPHKFKREW (SEQ ID NO: 39); ^■

YGRKKRRQRRREPHKFKREF (SEQ ID NO: 40); YGRKKRRQRRRDPHKFKREF (SEQ ID NO: 41); YGRKKRJRQRRREPHKFKJRDW (SEQ ID NO: 42); YGRKKRRQRRJREPHKFKRD (SEQ ID NO: 43); YGJRKKJRRQRRRPHKFKRD (SEQ ΪD NO: 44); and GEPHKFKREWYGRKKRRQRRR (SEQ ID NO: 45).

7. The polypeptide of any one of claims 1 -6, wherein the polypeptide is in the form of a pharmaceutically acceptable salt.

8. A pharmaceutical composition comprising a polypeptide and a carrier

^■ suitable for facilitating delivery of the polypeptide to a cell, wherein said polypeptide comprises.one or more of "the amino acid sequences set forth in SEQ ID NOS: 1- 24 and 26^5. ^{' ■ ■ ■} • ^' :

9: The pharmaceutical composition of claim 8, for the treatment of stroke or amyotrophic lateral sclerosis (ALS). . .. ^'

10. A method of inhibiting cytotoxic cell death or reducing cytoxic stress, comprising, treating a cell, a mammal comprising the cell, or a tissue comprising the cell, with an effective amount an polypeptide, wherein the polypeptide is selected from the group consisting of: ^• a) PHKFKX₁ (SEQ ID NO: 21), where X₁ =. R or K; b) X₁X₂PHKFKX₃X₄X₅ (SEQ ID NO: 22), wherein X₁ - is a linker of 1-5 G or is absent, X₂ = E or D ox is absent, X₃ = R or K, X» = E or D or is absent, and X5 = W or F or is absent; c) X₁X₂PHKFKX₃X₄X₅ (SEQ ID NO: 23), wherein X₁ = is a linker of 1-5 G or is absent, X2 = E or D or N or Q or is absent, X₃ = R or. K or H, X₄ = E or D or N or Q or. is absent, and X₅ = W or F or Y or is absent; and d) X₁X₂PHKFKX₃X₄X₃ (SEQ ID NO: 24), wherein X₁ = G or A or V or L or I or is a linker of 1-5 G or is absent, Xj = E or D or N or Q, X₃ = R or K or H, X₄ - E or D or N or Q, and X₅ = W or F or Y.

11. The method of claim 10, wherein the polypeptide is selected from one or more of the following: GBPHKFKREW (SEQ ID NO: 1); GGBPHKFKREW (SEQ DD NO: 3); GGGEPHKFKREW (SEQ ID NO: 4); GGGGEPHKFKREW (SEQ ID NO: 5); GGGGGEPHKFKREW (SEQ ID NO: 6); EPHKFKREW (SEQ ID NO: 7); GEPHKFKRE (SEQ ID NO: 8); EPHKFKRE (SEQ ID NO: 9); GEPHKFKRE (SEQ ID NO: 10); EPHKPKRE (SEQ ID NO: 11); PHKFKRE (SEQ ED NO: 12);.PHKFKR (SEQ ID NO: _.13); PHKFKK (SEQ ID NO: 14); DPHKFKRJBW (SEQ ID NO: 15); EPHKFKREF (SEQ ID NO: 16); DPHKFKREF (SEQ ID NO: 17); EPHKFKRDW (SEQ ID NO: 1 S); EPHKFKRD (SEQ ID NO: 19) ; and PHKFKRD (SEQ ID NO: 20).

_.12. The method of claim 10 or 11, wherein the polypeptide further comprises a delivery moiety.

13. The method of claim 12, wherein the delivery moiety, allows for localization to the central nervous system. .

14. The method of claim 12, wherein the delivery moiety is a TAT peptide.

15. The method of any one of claims 10-14, wherein the polypeptide is selected from one or more of the following: YGRKKRRQRRRGEPHKFKREW (SEQ ID NO: 26); YGRKKRRQRRRGGEPHKFKREW (SEQ ID NO: 27); YGRKKRRQRRRGGGEPHKFKREW (SEQ ID NO: 28); YGRKKRRQRRRGGGGEPHKFKREW (SEQ ID NO: 29); YGRKKRRQRRRGGGGGEPHKFKREW (SEQ ID NO: 30);

YGRKKRRQRRREPHKFKREW (SEQ ID NO: 31); YGRKKRRQRRRGEPHKFKRE (SEQ JD NO: 32); YGRKKRRQRRREPHKFKRJE (SEQ ID NO: 33); YGRKKRRQRRRGEPHKFKRE (SEQ ED NO: 34); YGRKKRRQRRREPHKFKRE (SEQ ID NO: 35); YGRKKRRQRRRPHKFKRE (SEQ ID NO: 36); YGRKKRRQRRRPHKFKR (SEQ ID NO: 37); YGRKKRRQRRRPHKFKK (SEQ ID NO: 38); YGRKKRRQRRRDPHKFKREW (SEQ ID NO: 39); YGPVKKRRQRRREPHKFKRΈF (SEQ ID NO: 40); YGRKKRRQRRRDPHKFK.REF (SEQ ID NO: 41); YGRKKRRQRRREPHKFKRDW (SEQ ID NO: 42); YGRKKRRQRRREPHKFKRD (SEQ ID NO: 43); YGRKK.RRQRRRPHKFKRD (SEQ ID NO: 44); and GEPHKFKREWYGRKKRRQRRR (SEQ ID NO: 45).

16. A method of treating stroke or ALS comprising, treating a cell, a mammal comprising the cell, or a tissue comprising the cell, with an effective amount a polypeptide, wherein the polypeptide is selected from the group consisting of: a) PHKFKX₁ (SEQ ID NO: 21), where X₁ - R or K; b) X₁X₂PHKFKX₃X₄X₅ (SEQ ID NO: 22), wherein X₁ = is a linker of 1-5 G or is absent, X₂ = E or D or is absent, X3 = R or K, X₄ - E or D or is absent, and X₅ = W or F or is absent; ^• . c) X₁X₂PHKFKX₃X₄X₅ (SEQ ID NO: 23), wherein X₁ = is a linker of 1-5 G or is absent, X₂ = E or D_. or N or Q or is absent, X₃ - R or K or H, X₄ = E or D or N or Q or is absent, and Xs = W or F or Y or is absent; and d).X₁X₂PHKFKX3X4X5 (SEQ ID NO: 24), wherein X₁ = G or A or V or L or I or is a linker of 1-5 G or is absent, X₂ = E or D or N or Q, X₃ = R or K or H, X₄ = E or D or N or Q, and X₅ = W or F or Yl

17. The method of claim 16, wherein the polypeptide is selected from one or more of the following: GEPHKFKREW (SEQ ID NO: 1); GGEPHKFKREW (SEQ ID NO: 3); GGGEPHKFKREW (SEQ ID NO: 4); GGGGEPHKFKREW (SEQ ID NO: 5); GGGGGEPHKFKREW (SEQ ID NO: 6); EPHKFKREW (SEQ ID NO: 7); GEPHKFKRE (SEQ ID NO: 8); EPHKFKRE (SEQ ID NO: 9); GEPHKFKRE (SBQ ID NO: 10); EPHKFKKE (SEQ ID NO: 11); PHKFKRE (SEQ ID NO: 12); PHKFKR (SEQ ID NO: 13); PHKFKK (SEQ ID NO: 14); DPHKFKREW (SEQ ID NO: 15); EPHKFKREF (SEQ ID NO: 16); DPHKFKREF (SEQ ID NO: 17); EPHKFKRDW (SEQ ID NO: 18); EPHKFKRD (SEQ ID NO: 19); and PHKFKRD (SEQ ID NO: 20).

18. The method of clatrsk, 16 or 17, wherein the polypeptide further comprises a delivery moiety.

19. The method of claim 1 S, wherein the delivery moiety, allows for localization to the central nervous system:

.

20. The method of claim 18, wherein the delivery moiety is a TAT peptide.

21. The method of any one of claims 16-20, wherein the polypeptide is selected from one or more of the following: YGRKKRRQRRRGEPHKFKREW (SEQ ID NO: 26); YGRKKRRQRRRGGEPHKFKREW (SEQ ID NO: 27); YGRKKRRQRRRGGGEPHKFKKJΞW (SEQ JD NO: 28); YGRKK^RQRRRGGGGEPHKFKREW (SEQ ID NO: 29); YGRJfCKRRQRRRGGGGGEPHKFKREW (SEQ ID NO: 30);

YGRKK-RRQRRREPHKFKREW (SEQ JD NO: 31); YGRKKRRQRRRGEPHKFKRE (SEQ ID NO: 32); YGRKKRRQRRREPHKFKRE (SEQ ID NO: 33); YGRKKRRQRRRGEPHKFKRE (SEQ ID NO: 34); YGRKKRRQRRREPHKFKRE \ . (SEQ JD NO: 35); YGRKK.RRQRRRPHKFKRE (SEQ ID_. NO: 36); YGRKKRRQRRRPHKFKR (SEQ ID NO: 37); YGRKKRRQRRRPHKFKK (SEQ ID NO: 38); YGRKKRRQRRRDPHKFKREW (SEQ ID NO: 39);

YGROaOlQRRREPHKFKR-EF (SEQ ID NO^O)J YGRKKRRQRRRDPHKFKREF (SEQ ID NO: 41); YGRKKRRQRRREPHKFKRDW (SEQ ID NO: 42); YGRKKRRQRRREPHKFKRD (SEQ ID NO: 43); YGRKKRRQRRRPHKFKRD (SEQ ω NO: 44); and GEPHKPKJREWYGRKKRRQRRR (SEQ ID NO: 45).

22- A nucleic acid comprising a npn-naturally occurring nucleic acid capable of encoding a polypeptide selected from any one or more of SEQ ID NOS: 1-24 and 26-45.

23. The nucleic acid of claim 22, wherein the nucleic acid is contained in an expression vector. _. ;

24. The nucleic acid of claim 22 or 23, wherein the nucleic acid is contained in a cell. . .

25. Use of a polypeptide for treating stroke or ALS in a subject, wherein the polypeptide is selected from any one or more of SEQ ID NOS: 1-24 and 26-45.

26. . Use of a polypeptide to formulate a medicament treating stroke or ALS in a subject, wherein the polypeptide is selected from any one or more of SEQ ID NOS : 1 -24 and 2ό-45. . ■ ■

27- The use according of claim 25 or 26, wherein the subject is a human.

28. The use of any one of claims 25-27, wherein the polypeptide is formulated for intravenous, subcutaneous, intramuscular, intraperitoneal, intratumoral, oral, or ϊnhalational use. . .

29. A commercial package, comprising a polypeptide, wherein the polypeptide is selected from any one or more of SEQ ID NOS: 1-24 and 26-45 or a pharmaceutical composition thereof. .