CA2273622C - Inhibition of nmda receptor signalling in reducing neuronal damage - Google Patents

Abstract

Ischemic or traumatic injuries to the brain or spinal cord often produce irreversible damage to central nervous system (CNS) neurons and to their processes. These injuries are major problems to society as they occur frequently, the damage is often severe. The invention provides a tSXV motif-containing peptide in the manufacture of a medicament for reducing the damaging effect of excitotoxicity in a mammal as a solution to these problems. The tSXV motif-containing peptide is particularly useful for treatment of stroke. The invention further provides a tSXV motif-containing peptide linked to an internalization peptide and pharmaceutical compositions containing the same. Such peptides inhibit the binding between N-methyl-D-aspartate receptors and neuronal proteins, such as PSD95.

Description

INHIBITION OF NMDA RECEPTOR SIGNALLING IN REDUCING
NEURONAL DAMAGE

FIELD OF THE INVENTION

This invention relates to methods of reducing the damaging effect of an injury to mammalian cells by treatment with compounds which reduce the binding between N-methyl-D-aspartate receptors and neuronal proteins; pharmaceutical compositions comprising said compounds and methods for the preparation of said pharmaceutical compositions.

BACKGROUND TO THE INVENTION

Ischemic or traumatic injuries to the brain or spinal cord often produce irreversible damage to central nervous system (CNS) neurons and to their processes.
These injuries are major problems to society as they occur frequently, the damage is often severe, and at present there are still no effective treatments for acute CNS
injuries. Clinically, ischemic cerebral stroke or spinal cord injuries manifest themselves as acute deteriorations in neurological capacity ranging from small focal defects, to catastrophic global dysfunction, to death. It is currently felt that the final magnitude of the deficit is dictated by the nature and extent of the primary physical insult, and by a time-dependent sequence of evolving secondary phenomena which cause further neuronal death. Thus, there exists a theoretical time-window, of uncertain duration, in which a timely intervention might interrupt the events causing delayed neurotoxicity. However, little is known about the cellular mechanisms triggering and maintaining the processes of ischemic or traumatic neuronal death, making it difficult to devise practical preventative strategies. Consequently, there are currently no clinically useful treatments for cerebral stroke or spinal cord injury.
In vivo, a local reduction in CNS tissue perfusion mediates neuronal death in both hypoxic and traumatic CNS injuries. Local hypoperfusion is usually caused

2 by a physical disruption of the local vasculature, vessel thrombosis, vasospasm, or luminal occlusion by an embolic mass. Regardless of its etiology, the resulting ischemia is believed to damage susceptible neurons by impacting adversely on a variety of cellular homeostatic mechanisms. Although the nature of the exact disturbances is poorly understood, a feature common to many experimental models of neuronal injury is a rise in free intracellular calcium concentration ([Ca2+]i).
Neurons possess multiple mechanisms to confine [Ca2+]; to the low levels, about 100nM necessary for the physiological function. It is widely believed that a prolonged rise in [Ca2+]; deregulates tightly-controlled Ca 2+-dependent processes, causing them to yield excessive reaction products, to activate normally quiescent enzymatic pathways, or to inactivate regulatory cytoprotective mechanisms.
This, in-turn, results in the creation of experimentally observable measures of cell destruction, such as lipolysis, proteolysis, cytoskeletal breakdown, pH
alterations and free radical formation.
The classical approach to preventing Ca 2+ neurotoxicity has been through pharmacological blockade of Ca2+ entry through Ca 2+ channels and/or of excitatory amino acid (EAA) - gated channels. Variations on this strategy often lessen EAA -induced or anoxic cell death in vitro, lending credence to the Ca2+-neurotoxicity hypothesis. However, a variety of Ca 2+ channel- and EAA- antagonists fail to protect against neuronal injury in vivo, particularly in experimental Spinal Cord Injury (SCI), head injury and global cerebral ischemia. It is unknown whether this is due to insufficient drug concentrations, inappropriate Ca 2+ influx blockade, or to a contribution from non-Ca2+ dependent neurotoxic processes. It is likely that Ca 2+
neurotoxicity is triggered through different pathways in different CNS neuron types.
Hence, successful Ca2+-blockade would require a polypharmaceutical approach.
As a result of investigations, I have discovered methods of reducing the damaging effect of an injury to mammalian cells by treatment with compounds to reduce the binding between N-methyl-D-aspartate (NMDA) receptors and neuronal proteins.

3 PUBLICATIONS
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7. H. Dong, et al, Nature 386, 279 (1997).; P.R. Brakeman, et al, Nature 386, (1997).

8. S.E. Craven, D.S. Bredt, Cell 93, 495 (1998).; M. Niethammer, et al, Neuron 20, 693 (1998).; J.H. Kim, D. Liao, L.F. Lau, R.L. Huganir, Neuron 20, 683 (1998).; T.
Tezuka, H. Umemori, T. Akiyama, Nakanishi, T. Yamamoto, Proc Natl Acad Sci USA 96,435 (1999).

9. Hertz, E., Yu, A.C.H., Hertz, L., Juurlink, B.H.J. & Schousboe, A. in A
dissection and tissue culture manual of the nervous system (eds Shahar, A., de Vellis, J., Vernadakis, A. & Haber, B.) Vol.1, 183-186 (Alan R. Liss Inc., New York, 1989).

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Uhl, S.H. Snyder, J Neurosci 13, 2651 (1993).; T.M. Dawson, D.S. Bredt, M.
Fotuhi, P.M. Hwang, S.H. Snyder, Proc Natl Acad Sci USA 88, 7797 (1991).

13. Xiong, Z., Lu, W. & MacDonald, J.F. proc Natl Acad Sci USA 94, 7012-7017 (1997).

14. R. Sattler, M.P. Charlton, M. Hafner, M. Tymianski, J Cereb Blood Flow Metab 17, 455 (1997).

15. N. Bumashev, Z. Zhou, E. Neher, B. Sakmann, JPhysiol 485, 403 (1995).

16. M. Migaud, et al, Nature 396, 433 (1998).

17. J.R. Brorson, P.T. Schumacker, H. Zhang, JNeurosci 19, 147 (1999).

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19. S.R. Jaffrey, S.H. Snyder, Annual Review of Cell & Developmental Biology 11, 417 (1995).

SUMMARY OF THE INVENTION

It is a preferred object of the present invention to provide in its broadest aspect a method of reducing the damaging effect of an injury to mammalian cells.
In a further preferred object, the invention provides pharmaceutical compositions for use in treating mammals to reduce the damaging effect of an injury to mammalian tissue.
The present invention is based on the discovery of a neuroprotective effect against excitotoxic and ischemic injury by inhibiting the binding between N-methyl-D-aspartate (NMDA) receptors and neuronal proteins in a neuron.
Accordingly, in one aspect the invention provides a method of inhibiting the binding between N-methyl-D-aspartate receptors and neuronal proteins in a neuron said method comprising administering to said neuron an effective inhibiting amount of a peptide replacement agent for the NMDA receptor neuronal protein interaction domineer or precursor therefor to effect said inhibition.

In a further aspect, the invention provides a method of reducing the damaging effect of ischemia or traumatic injury to the brain or spinal chord in a mammal, said method comprising treating said mammal with a non-toxic, damage-reducing, effective amount of a peptide replacement agent for the NMDA receptor neuronal 5 protein interaction domain or precursor therefor.
The NMDA agent is, preferably, bindable with membrane associated guanylate kinases, and most preferably, is selected from postsynaptic density-95 proteins, PSD-95, PSD-93 and SAP102.
I have found that the replacement agent is a tSXV-containing peptide or precursor therefor, preferably KLSSLESDV.
In a yet further aspect the invention provides a pharmaceutical composition comprising a peptide replacement agent for the NMDA receptor neuronal protein interaction domain or a precursor therefor in a mixture with a pharmaceutically acceptable carrier when used for reducing the damaging effect of an ischemic or traumatic injury to the brain or spinal chord of a mammal; preferably further comprising antessapedia internalisation peptide.
In a further aspect, the invention provides a method of inhibiting the binding between NMDA receptors and neuronal proteins in a neuron, said method comprising administering to said neuron an effective inhibiting amount of an antisense DNA to prevent expression of said neuronal proteins to effect inhibition of said binding. Preferably, this aspect provides a method wherein said antisense DNA
reduces the expression of a membrane associated guanylate kinase bindable to said NMDA receptor. More preferably, the guanylate kinase is selected from PSD-95, PSD-93 and SAP 102.

In the mammalian nervous system, the efficiency by which N-methyl-D-aspartate receptor (NMDAR) activity triggers intracellular signaling pathways governs neuronal plasticity, development, senescence and disease. I have studied excitotoxic NMDAR signaling by suppressing the expression of the NMDAR
scaffolding protein PSD-95. In cultured cortical neurons, this selectively attenuated NMDAR excitotoxicity, but not excitotoxicity by other glutamate or Ca 2+
channels.
NMDAR function was unaffected, as receptor expression, while NMDA-currents and 45Ca loading via NMDARs were unchanged. Suppressing PSD-95 selectively blocked Ca2+ -activated nitric oxide production by NMDARs, but not by other pathways, without affecting neuronal nitric oxide synthase (nNOS) expression or function. Thus, PSD-95 is required for the efficient coupling of NMDAR
activity to nitric oxide toxicity and imparts specificity to excitotoxic Ca2+ signaling.

It is known that calcium influx through NMDARs plays key roles in mediating synaptic transmission, neuronal development, and plasticity (1). In excess, Ca influx triggers excitotoxicity (2), a process that damages neurons in neurological disorders that include stroke, epilepsy, and chronic neurodegenerative conditions (3).
Rapid Ca2+-dependent neurotoxicity is triggered most efficiently when Ca 2+ influx occurs through NMDARs, and cannot be reproduced by loading neurons with equivalent quantities of Ca 2+ through non-NMDARs or voltage-sensitive Ca 2+ channels (VSCCs) (4). This observation suggests that Ca 2+ influx through NMDAR
channels is functionally coupled to neurotoxic signaling pathways.

Without being bound by theory, I believe that lethal Ca 2+ signaling by NMDARs is determined by the molecules with which they physically interact. The NR2 NMDAR subunits, through their intracellular C-terminal domains, bind to PSD-95/SAP90 (5), chapsyn-110/PSD-93, and other members of the membrane-associated guanylate kinase (MAGUK) family (6). NMDAR-bound MAGUKs are generally distinct from those associated with non-NMDARs (7). I have found that the preferential activation of neurotoxic Ca2+ signals by NMDARs is determined by the distinctiveness of NIVIDAR-bound MAGUKs, or of the intracellular proteins that they bind. PSD-95 is a submembrane scaffolding molecule that binds and clusters NMDARs preferentially and, through additional protein-protein interactions, may link them to intracellular signaling molecules (8). Perturbing PSD-95 would impact on neurotoxic Ca 2+ signaling through NMDARs.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be better understood preferred embodiments will now be described by way of example only with reference to the accompanying drawings wherein:
Fig.1 a is an immunublot;
Fig. lb is a bar chart providing densitometric analysis of PSD-95 expression;
Fig. I c represents representative phase contrast and propidium fluorescence images;
Fig.1 d is a bar chart of NMDA concentration against fraction of dead cells;
Fig. 1 e is a bar chart of NMDA concentration against Calcium accumulation.
Fig.2a1-b2 represent bar charts of selective activations of AMPA/Kainate receptors with Kainate (2a1 and 2-a2); and loadings with Vscc's (2-bl) and calcium loading (2-b2).

Fig.3a-d represent: immunoblots (3a); NMSa dose-response curves (3b); NMDA
current density measurements (3c); and current/time graph (3d) dialyzed with hucifer yellow; and Fig.4 bar charts (4a; 4c-4f) and immublot of effect on nNOS expression in cultures;
hereinafter better described and explained.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
METHODS:

Cultured cortical neurons were prepared by standard techniques (4,9) and switched to serum-free media at 24h [Neurobasal with B27 supplement (Gibco)].
The AS ODN corresponded to nucleotides 435-449 (5'-GAATGGGTCACCTCC-3') of mouse PSD-95/SAP90 mRNA (GeneBank Acc. No. D50621). Filter-sterilized phosphodiester AS (5'-GAATGGGTCACCTCC-3'), SE, and MS (5'-CCGCTCTATCGAGGA-3') ODNs (5 M) were added in culture medium during feedings at 4,6,8 and 10 days after plating. Cultures were used for all experiments (figs. 1-4) on day 12. ODN sequences exhibited no similarity to any other known mammalian genes (BLAST search (10) ).

Immunoblotting was done as described in ref "26". Tissue was harvested and pooled from 2 cultures/lane. The blotted proteins were probed using a monoclonal anti-PSD-95 mouse IgGI (Transduction Labs, 1:250 dilution), polyclonal anti PSD-93 (1:1000 dilution) and anti SAP-102 (1:2000 dilution) rabbit serum antibodies (Synaptic Systems GmbH), a monoclonal anti NR1 mouse IgG2a (PharMingen Canada, 1:1000 dilution) or a monoclonal anti nNOS (NOS type I) mouse IgG2a (Transduction Labs, 1:2500 dilution). Secondary antibodies were sheep anti-mouse, or donkey anti-rabbit Ig conjugated to horseradish peroxidase (Amersham).
Immunoblots for PSD-95 were obtained for all experiments (Figs 1-4) from sister cultures, and all gels quantified using an imaging densitometer (Bio-Rad GS-670).

cGMP determinations were performed 10 min after challenging the cultures with NMDA, kainate, or high-K (Figs. 4c-e) with the Biotrak* cGMP
enzymeimmunoassay system according to the kit manufacturer's instructions (Amersham). Staining for NADPH diaphorase (Fig 4b) was done as described in ref.
12.

Electrophysiology. Whole cell patch-clamp recordings in the cultured neurons were performed and analyzed as described in ref. 13. During each experiment a voltage step of -10 mV was applied from holding potential and the cell capacitance was calculated by integrating the capacitative transient. The extracellular solution contained (in mM): 140 NaCl, 5.4 KCI, 1.3 CaCI2, 25 HEPES, 33 glucose.. 0.01 glycine, and 0.001 tetrodotoxin (pH =7.3-7.4, 320 - 335 mOsm). A multi-barrel perfusion system was employed to rapidly exchange NNIDA containing solutions.

The pipette solution contained (in mM): 140 CsF, 35 CsOH, 10 HEPES, 11 EGTA, 2 tetraethylammonium chloride (TEA), 1 CaCl2, 4 MgATP, pH 7.3 at 300 mOsm.
Lucifer yellow (LY; 0.5% w/v) was included in the pipette for experiments in figure 3d.

* Trade-mark Excitotoxicity and Ca2+ accumulation measurements were performed identically to the methods described and validated in refs. 4 and 14. We used measurements of propidium iodide fluorescence as an index of cell death, and of radiolabelled 45Ca2+
accumulation for Ca2+ load determinations in sister cultures on the same day.

Experimental solutions were as previously described (4). Ca 2+ influx was pharmacologically channeled through distinct pathways as follows: To NMDARs by applying NMDA (x60 min) in the presence of both CNQX (Research Biochemicals Inc) and nimodipine (Miles Pharmaceuticals), to non-NMDARs by applying kainic acid (x60 min or 24h) in the presence of both MK-801 (RBI) and nimodipine, and to VSCCs using 50 mM K+ solution (x60 min) containing 10mM Ca2+ and S(-)-Bay K
8644, an L-type channel agonist (300-500nM; RBI), MK-801 and CNQX.
Antagonist concentrations were (in M): MK-801 10, CNQX 10, nimodipine 2. All three antagonists were added after the 60 min agonist applications for the remainder of all experiments (24 h). A validation of this approach in isolating Ca 2+
influx to the desired pathway in our cortical cultures has been published (4).

Whole cell patch-clamp recordings in the cultured neurons were performed and analyzed as described in Z. Xiong, W. Lu, J.F. MacDonald, Proc Natl Acad Sci USA 94, 7012 (1997). During each experiment a voltage step of -10 mV was applied from holding potential and the cell capacitance was calculated by integrating the capacitative transient. The extracellular solution contained (in mM): 140 NaCl, 5.4 KCI, 1.3 CaC12, 25 HEPES, 33 glucose, 0.01 glycine, and 0.001 tetrodotoxin (pH
=7.3-7.4, 320 - 335 mOsm). A multi-barrel perfusion system was employed to rapidly exchange NMDA containing solutions. The pipette solution contained (in mM): 140 CsF, 35 CsOH, 10 HEPES, 11 EGTA, 2 tetraethylammonium chloride (TEA), 1 CaCl2, 4 MgATP, pH 7.3 at 300 mOsm. Lucifer yellow (LY; 0.5% w/v) was included in the pipette for experiments in figure 3D.

Data analysis: data in all figures were analyzed by ANOVA, with a post-hoc Student's t-test using the Bonferroni correction for multiple comparisons. All means are presented with their standard errors.

In greater detail:
5 Figure 1, shows increased resilience of PSD-95 deficient neurons to NMDA
toxicity in spite of Ca 2+ loading. A. Immunoblot showing representative effects of sham (SH) washes, and PSD-95 AS, SE and MS ODNs, on PSD-95 expression. PC:
positive control tissue from purified rat brain cell membranes. Asterisk: non-specific band produced by the secondary antibody, useful to control for protein loading and 10 blot exposure times. B. Densitometric analysis of PSD-95 expression pooled from N
experiments. Asterisk: different from other groups, one-way ANOVA, F = 102, p<0.0001. ODNs were used at 5 M except where indicated (AS 2 M). C.
Representative phase contrast and propidium iodide fluorescence images of PSD-deficient (AS) and control (SE) cultures 24 h after a 60 min challenge with 30 M

NMDA. Scale bar: 100 m. D. Decreased NMDA toxicity at 24h in PSD-95 deficient neurons following selective NMDAR activation x 60 min (n=16 cultures/bar pooled from N=4 separate experiments). Asterisk: differences from SE, MS and SH (Bonferroni t-test, p<0.005). Death is expressed as the fraction of dead cells produced by 100 M NMDA in sham-ODN-treated controls (validated in 4,14).

E. No effect of PSD-95 deficiency on NMDAR-mediated Ca 2+ loading (n = 12/bar, N = 3; reported as the fraction of 45Ca2+ accumulation achievable over 60 min in the sham controls by 100 M NMDA, which maximally loads the cells with calcium (4).

Figure 2, shows thatPSD-95 deficiency does not affect toxicity and Ca 2+
loading produced by activating non-NMDARs and Ca 2+ channels. Cultures were treated with SH washes or AS or SE ODNs as in Fig. 1. A. Selective activation of AMPA/kainate receptors with kainate in MK-801 (10 M) and nimodipine (NIM;
2 M) produces toxicity over 24h (Al) irrespective of PSD-95 deficiency, with minimal 45Ca2+ loading (A2). B. Selective activation of VSCCs produces little toxicity (B 1), but significant 45Ca2+ loading (B2) that is also insensitive to PSD-95 deficiency. n = 4 cultures/bar in all experiments.

Figure 3, shows that there is no effect of perturbing PSD-95 on receptor function. A. Immunoblots of PSD-95 ODN-treated cultures probed for PSD-95, NR1, PSD-93, and SAP-102 using specific antibodies. PC: positive control tissue from purified rat brain cell membranes. B. NMDA dose-response curves and representative NMDA currents (inset) obtained with 3-300 M NMDA. C. NMDA
current density measurements elicited with 300 M NMDA (AS: n = 18; SE: n =19;
SH: n = 17; one-way ANOVA F=1.10, p=0.34), and analysis of NMDA current desensitization. Iss = steady-state current; Ipeak = peak current. AS: n=15;
SE: n = 16;
SH: n = 16 (ANOVA,, F=0.14, p=0.87). Time constants for current decay were AS:
1310 158 ms; SE, 1530 185 ms; SH: 1190 124 ms (ANOVA, F= 1.22, p=
0.31). D. Currents elicited with 300 M NMDA in neurons dialyzed with LY
(insert) and 1 mM tSXV or control peptide.

Figure 4, shows the effect of coupling of NMDAR activation to nitric oxide signaling by PSD-95. A. L-NAME protects against NMDA toxicity (n = 4, N = 2).
Asterisk: difference from 0 M L-NAME (Bonferroni t-test, p<0.05). B. No effect of SH and of PSD-95 AS and MS ODNs on nNOS expression in cultures (immunoblot) and on NADPH diaphorase staining in PSD-95 AS and SE-treated neurons. PC:
positive control tissue from purified rat brain cell membranes. C. Effect of isolated NMDAR activation on cGMP formation (n=12 cultures/bar pooled from N=3 separate experiments) D,E. Effects of VSCC activation (n = 8/bar, N = 2), and AMPA/kainate receptor activation (n = 4/bar, N= 1) on cGMP
formation. Data in C-E are expressed as the fraction of cGMP produced in SE-treated cultures by 100 M NMDA. Asterisk: differences from both SH and SE
controls (Bonferroni t-test, p<0.0001). F. Sodium nitroprusside toxicity is similar in PSD-95 AS, SE and SH treated cultures.

PSD-95 expression was suppressed in cultured cortical neurons to < 10% of control levels, using a 15-mer phosphodiester antisense (AS) oligodeoxynucleotide (ODN) (Fig. IA,B) Sham (SH) washes, sense (SE) and missense (MS) ODNs (9) had no effect. The ODNs had no effect on neuronal survivability and morphology as gauged by viability assays, herein below, and phase-contrast microscopy (not shown).

To examine the impact of PSD-95 on NMDAR-triggered excitotoxicity, ODN-treated cultures were exposed to NMDA (10-100 .tM) for 60 min, washed, and either used for 45Ca2+ accumulation measurements, or observed for a further 23 h.
Ca 2+ influx was isolated to NMDARs by adding antagonists of non-NMDARs and Ca2+ channels (4). NMDA toxicity was significantly reduced in neurons deficient in PSD-95 across a range of insult severities (Figs. 1C,D; EC50: AS: 43.2 4.3;
SE:

26.3 3.4, Bonferroni t-test, p <0.005). Concomitantly however, PSD-95 deficiency had no effect on Ca2+ loading into identically treated sister cultures (Fig.
1E).
Therefore, PSD-95 deficiency induces resilience to NMDA toxicity despite maintained Ca 2+ loading.

I next examined whether the increased resilience to Ca2+ loading in PSD-95 deficient neurons was specific to NMDARs. Non-NMDAR toxicity was produced using kainic acid (30-300 M), a non-desensitizing AMPA/kainate receptor agonist (15), in the presence of NMDAR and Ca 2+ channel antagonists (4). Kainate toxicity was unaffected in PSD-95 deficient in neurons challenged for either 60 min (not shown) or 24 h (Fig. 2A1). Non-NMDAR toxicity occurred without significant 45Ca2+ loading (Fig. 2A2), as >92% of neurons in these cultures express impermeable AMPA receptors (4). However, Ca 2+ loading through VSCCs, which is non-toxic (4) (Fig. 2B1), was also unaffected by PSD-95 deficiency (Fig. 2B2).
Thus, suppressing PSD-95 expression affects neither toxicity nor Ca2+ fluxes triggered through pathways other than NMDARs.

Immunoblot analysis (11) of PSD-95 deficient cultures revealed no alterations in the expression of the essential NMDAR subunit NR1, nor of two other NMDAR-associated MAGUKs, PSD-93 and SAP-102 (Fig. 3A). This indicated that altered expression of NN DARs and their associated proteins was unlikely to explain reduced NMDA toxicity in PSD-95 deficiency (Fig. 1 C,D). Therefore, I examined the possibility that PSD-95 modulates NMDAR function. NMDA currents were recorded using the whole-cell patch technique (16) (Fig. 3B). PSD-95 deficiency had no effect on passive membrane properties, including input resistance and membrane capacitance [Capacitance: AS 55.0 2.6 pF (n =18 ); SE 52.7 3.2 pF (n=19);
SH
48.1 3.4 pF (n = 17; ANOVA, F=1.29, p=0.28)]. Whole-cell currents elicited with 3-300 M NMDA were also unaffected. Peak currents were AS: 2340 255 pA
(n=18); SE: 2630 276 (n=19); SH: 2370 223 (n=17) (Fig. 3B, inset; one-way ANOVA, F = 0.43, p = 0.65). NMDA dose-response relationships also remained unchanged (Fig. 3B; EC50 AS: 16.1 0.8 M (n=7); SE: 15.5 2.1 (n=6); SH:
15.9 2.9; one-way ANOVA, F= 0.02, p = 0.98), as were NMDA current density and desensitization (Figs. 3C).

To further examine the effect of PSD-95 binding on NMDAR function, a 9 as peptide (KLSSIESDV) corresponding to the C-terminal domain of the NR2B
subunit characterized by the tSXV motif (6) was injected into the neurons. At 0.5mM, this peptide competitively inhibited the binding of PSD-95 to GST-NR2B
fusion proteins (6), and was therefore predicted to uncouple NMDARs from PSD-95. Intracellular dialysis of 1mM tSXV or control peptide (CSKDTMEKSESL) (6) was achieved through patch pipettes (3-5 MCI) also containing the fluorescent tracer Lucifer Yellow (LY). This had no effect on NMDA currents over 30 min despite extensive dialysis of LY into the cell soma and dendrites (Fig. 3D). Peak current amplitudes were tSXV: 2660 257 pA (n=
9), control: 2540 281 pA (n= 10; t(17) = 0.31, p = 0.76).

The data is consistent with that obtained from recently generated mutant mice expressing a truncated 40K PSD-95 protein that exhibited enhanced LTP and impaired learning (17). Hippocampal CAI neurons in PSD-95 mutants exhibited no changes in NMDAR subunit expression and stoichiometry, cell density, dendritic cytoarchitecture, synaptic morphology, or NMDAR localization using NR1 immunogold labeling of asymmetric synapses. NMDA currents, including synaptic currents, were also unchanged (16). I also found no effects of PSD-95 deficiency on NMDAR expression, on other NMDAR associated MAGUKs, nor on NMDA-evoked currents. In addition, NMDAR function gauged by measuring NMDA-evoked 45Ca2+-accumulation was unaffected. Thus, the neuroprotective consequences of PSD-95 deficiency must be due to events downstream from NMDAR activation, rather than to altered NMDAR function.

The second PDZ domain of PSD-95 binds to the C-terminus of NR2 subunits and to other intracellular proteins (8). Among these is nNOS (18), an enzyme that catalyzes the production of nitric oxide (NO), a short-lived signaling molecule that also mediates Cat+-dependent NMDA toxicity in cortical neurons (12). Although never demonstrated experimentally, the NMDAR/PSD-95/nNOS complex was postulated to account for the preferential production of NO by NMDARs over other pathways (8). To determine whether NO signaling plays a role in NMDA toxicity in the present cultures, we treated the cells with 1VG-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor (12). L-NAME protected the neurons against NMDA
toxicity (Fig. 4A), indicating the possibility that suppressing PSD-95 might perturb this toxic signaling pathway.

The effect of suppressing PSD-95 expression on NO signaling and toxicity was examined using cGMP formation as a surrogate measure of NO production by Cat+-activated nNOS (20,21). PSD-95 deficiency had no impact on nNOS expression (Fig. 4B), nor on the morphology (Fig. 4B) or counts of NADPH diaphorase-staining (12) neurons (SH: 361 60, SE: 354 54, AS: 332 42 staining neurons /10mm coverslip, 3 coverslips/group). However, in neurons lacking PSD-95 challenged with NMDA under conditions that isolated Ca2+ influx to NMDARs (4), cGMP
production was markedly attenuated (>60%; Fig. 4C, one-way ANOVA, p<0.0001).
Like inhibited toxicity (Figs. 1,2), inhibited cGMP formation in neurons lacking PSD-95 was only observed in response to NMDA. It was unaffected in neurons loaded with Ca2+ through VSCCs (Fig. 4D), even under high neuronal Ca 2+ loads matching those attained by activating NMDARs (compare Figs. 1E and 2B2) (4).
nNOS function therefore, was unaffected by PSD-95 deficiency. AMPA/kainate receptor activation failed to load the cells with Ca2+ (Fig. 2A2), and thus failed to 5 increase cGMP levels (Fig. 4E). Our findings indicate that suppressing PSD-selectively reduces NO production efficiency by NMDAR-mediated Ca 2+ influx, but preserves NO production by Ca 2+ influx through other pathways.

Bypassing nNOS activation with NO donors restored toxicity in neurons lacking PSD-95. The NO donors sodium nitroprosside (12) (Fig. 4F; EC50 300 M) and S-10 nitrosocysteine (17) (not shown) were highly toxic, irrespective of PSD-95 deficiency. Thus, reduced NMDA toxicity in PSD-95 deficient cells was unlikely to be caused by altered signaling events downstream from NO formation.

Suppressing PSD-95 expression uncoupled NO formation from NMDAR
activation (Fig. 4C), and protected neurons against NMDAR toxicity (Fig. 1 C,D) 15 without affecting receptor function (Figs I E, 3A-D), by mechanisms downstream from NMDAR activation, and upstream from NO-mediated toxic events (Fig. 4F).
Therefore, PSD-95 imparts NMDARs with signaling and neurotoxic specificity through the coupling of receptor activity to critical second messenger pathways. Our results have broader consequences, as NMDAR activation and NO signaling are also critical to neuronal plasticity, learning, memory, and behavior (1,18,19).
Thus, our report provides experimental evidence for a mechanism by which PSD-95 protein may govern important physiological and pathological aspects of neuronal functioning.

Although this disclosure has described and illustrated certain preferred embodiments of the invention, it is to be understood that the invention is not restricted to those particular embodiments. Rather, the invention includes all embodiments which are functional or mechanical equivalence of the specific embodiments and features that have been described and illustrated.

SEQUENCE LISTING
1. GENERAL INFORMATION

(i) APPLICANT:
(A) : TYMIANSKI, Michael (ii) TITLE OF INVENTION: METHOD OF REDUCING INJURY TO MN-1MALIAN CELLS
(iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS:
(A) NAME: DEETH WILLIAMS WALL LLP
(B) STREET ADDRESS: 150 York Street, Suite 400 (C) CITY: Toronto (D) PROVINCE: Ontario (E) POSTAL CODE: M5H 3S5 (v) COMPUTER-READABLE FORM
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM Compatible/Dell (C) OPERATING SYSTEM: Windows XP
(D) SOFTWARE: Word 2003 (vi) CURRENT APPLICATION DATA
(A) APPLICATION NO.: 2273622 (B) FILING DATE: June 2, 1999 (vii) PATENT AGENT INFORMATION
(A) NAME: DEETH WILLIAMS WALL LLP
(B) REFERENCE NUMBER: 4438 0005 2. INFORMATION FOR SEQ ID NO.: 1 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 (B) TYPE: PRT
(C) ORGANISM: Unknown (ii) FEATURE:
(A) OTHER INFORMATION: Description of Unknown Organism: peptide (iii) SEQUENCE DESCRIPTION: SEQ ID NO: 1 Lys Leu Ser Ser Ile Glu Ser Asp Val 3. INFORMATION FOR SEQ ID NO.: 2 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 (B) TYPE: PRT
(C) ORGANISM: Unknown (ii) FEATURE:
(A) OTHER INFORMATION: Description of Unknown Organism: peptide (iii) SEQUENCE DESCRIPTION: SEQ ID NO: 2 Cys Ser Lys Asp Thr Met Glu Lys Ser Glu Ser Leu 4. INFORMATION FOR SEQ ID NO.: 3 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 (B) TYPE: PRT
(C) ORGANISM: Unknown (ii) FEATURE:
(A) OTHER INFORMATION: Description of Unknown Organism: peptide (iii) SEQUENCE DESCRIPTION: SEQ ID NO: 3 Lys Leu Ser Ser Ile Glu Thr Asp Val

Claims (45)

Claims:

1. The use of a tSXV motif-containing peptide in the manufacture of a medicament for reducing the damaging effect of excitotoxicity in a mammal.

2. The use of a tSXV motif-containing peptide for reducing the damaging effect of excitotoxicity in a mammal.

3. The use of a tSXV motif-containing peptide in the manufacture of a medicament to protect against the damaging effect of excitotoxicity to the brain or spinal cord in a mammal.

4. The use of a tSXV motif-containing peptide to protect against the damaging effect of excitotoxicity to the brain or spinal cord in a mammal.

5. The use of a tSXV motif-containing peptide in the manufacture of a medicament for reducing the damaging effect of stroke in a mammal.

6. The use of a tSXV motif-containing peptide for reducing the damaging effect of stroke in a mammal.

7. A tSXV motif-containing peptide comprising the amino acid sequence KLSSIESDV [SEQ ID NO: 1] or KLSSIETDV (SEQ ID NO:3) linked to an internalization peptide that facilitates uptake of the tSXV-motif containing peptide into cells.

8. The use according to any one of claims 1 to 6, wherein the tSXV motif-containing peptide comprises the amino acid sequence KLSSIESDV [SEQ ID NO: 1].

9. The use according to any one of claims 1 to 6 or 8, wherein the tSXV motif-containing peptide consists of the amino acid sequence KLSSIESDV [SEQ ID NO:
1].

10. The use according to any one of claims 1 to 6, wherein the tSXV motif-containing peptide comprises the amino acid sequence KLSSIETDV [SEQ ID NO: 3].

11. The use according to any one of claims 1 to 6, wherein the tSXV-motif containing peptide consists of the amino acid sequence KLSSIETDV [SEQ ID NO:
3].

12. The use according to any one of claims 1 to 6, or 8 to 11, wherein the tSXV
motif-containing peptide further comprises an internalization peptide that facilitates uptake of the tSXV-motif containing peptide into cells.

13. The use according to any one of claims 1 to 4, or 8 to 12, wherein the excitotoxicity is due to ischemia.

14. The use according to claim 13, wherein the ischemia is selected from the group consisting of cerebral ischemia, global cerebral ischemia and cerebral stroke.

15. The use according to claim 13 or 14, wherein said ischemia results from a decrease in central nervous system tissue perfusion with blood.

16. The use according to claim 15, wherein said decrease in central nervous system tissue perfusion is associated with a pathological condition selected from the group consisting of vessel thrombosis, vasospasm, and luminal occlusion by an embolic mass.

17. The use according to any one of claims 1 to 4 or 8 to 12, wherein the excitotoxicity is due to traumatic injury.

18. The tSXV motif-containing peptide of claim 7, wherein the peptide comprises the amino acid sequence KLSSIESDV [SEQ ID NO:1].

19. The tSXV motif-containing peptide of claim 7, wherein the peptide consists of the amino acid sequence KLSSIESDV [SEQ ID NO:1].

20. The tSXV motif-containing peptide of claim 7, wherein the peptide comprises the amino acid sequence KLSSIETDV [SEQ ID NO:3].

21. The tSXV motif-containing peptide of claim 7, wherein the peptide consists of the amino acid sequence KLSSIETDV [SEQ ID NO:3].

22. A pharmaceutical composition comprising the tSXV motif-containing peptide of claim 7; and, a pharmaceutical carrier.

23. The pharmaceutical composition of claim 22, for use in reducing the damaging effect of excitotoxicity in a mammal.

24. The pharmaceutical composition of claim 22, for use in protecting against the damaging effect of excitotoxicity to the brain or spinal cord in a mammal.

25. The pharmaceutical composition of claim 22, for use in reducing the damaging effect of stroke in a mammal.

26. The pharmaceutical composition of claim 23 or 24, wherein the excitotoxicity is due to ischemia.

27. The pharmaceutical composition of claim 26, wherein the ischemia is selected from the group consisting of cerebral ischemia, global cerebral ischemia and cerebral stroke.

28. The pharmaceutical composition of claim 26 or 27, wherein said ischemia results from a decrease in central nervous system tissue perfusion with blood.

29. The pharmaceutical composition of claim 28, wherein said decrease in central nervous system tissue perfusion is associated with a pathological condition selected from the group consisting of vessel thrombosis, vasospasm, and luminal occlusion by an embolic mass.

30. A tSXV motif-containing peptide for use in the manufacture of a medicament for reducing the damaging effect of excitotoxicity in a mammal.

31. A tSXV motif-containing peptide for use in reducing the damaging effect of excitotoxicity in a mammal.

32. A tSXV motif-containing peptide for use in the manufacture of a medicament to protect against the damaging effect of excitotoxicity to the brain or spinal cord in a mammal.

33. A tSXV motif-containing peptide for use to protect against the damaging effect of excitotoxicity to the brain or spinal cord in a mammal.

34. A tSXV motif-containing peptide for use in the manufacture of a medicament for reducing the damaging effect of stroke in a mammal.

35. A tSXV motif-containing peptide for use in reducing the damaging effect of stroke in a mammal.

36. The peptide according to any one of claims 30 to 35, wherein the tSXV
motif-containing peptide comprises the amino acid sequence KLSSIESDV [SEQ ID NO:1].

37. The peptide according to any one of claims 30 to 36, wherein the tSXV
motif-containing peptide consists of the amino acid sequence KLSSIESDV [SEQ ID
NO:1].

38. The peptide according to any one of claims 30 to 35, wherein the tSXV
motif-containing peptide comprises the amino acid sequence KLSSIETDV [SEQ ID NO: 3].

39. The peptide according to any one of claims 30 to 35, wherein the tSXV
motif-containing peptide consists of the amino acid sequence KLSSIETDV [SEQ ID NO:
3].

40. The peptide according to any one of claims 30 to 39, wherein the tSXV
motif-containing peptide further comprises an internalization peptide that facilitates uptake of the tSXV-motif containing peptide into cells.

41. The peptide according to any one of claims 30 to 33, wherein the excitotoxicity is due to ischemia.

42. The peptide according to claim 41, wherein the ischemia is selected from the group consisting of cerebral ischemia, global cerebral ischemia and cerebral stroke.

43. The peptide according to claim 41 or 42, wherein said ischemia results from a decrease in central nervous system tissue perfusion with blood.

44. The peptide according to claim 43, wherein said decrease in central nervous system tissue perfusion is associated with a pathological condition selected from the group consisting of vessel thrombosis, vasospasm, and luminal occlusion by an embolic mass.

45. The peptide according to any one of claims 30 to 33, wherein the excitotoxicity is due to traumatic injury.