WO2009143556A1

WO2009143556A1 - TREATMENTS FOR EXCITOTOXICITY AND Aß-MEDIATED TOXICITY AND AGE-ASSOCIATED NEURONAL DYSFUNCTION

Info

Publication number: WO2009143556A1
Application number: PCT/AU2009/000608
Authority: WO
Inventors: Jurgen Gotz; Lars Ittner
Original assignee: The University Of Sydney
Priority date: 2008-05-30
Filing date: 2009-05-15
Publication date: 2009-12-03

Abstract

The invention relates to compounds for preventing, inhibiting or antagonising fyn from phosphorylating an amino acid residue in a molecule having an amino acid sequence shown in SEQ ID NO: 1. Typically the compound is capable of binding to fyn, to prevent fyn from migrating to a region of a nerve cell cytosol adjacent a post synaptic membrane and in one embodiment the compound holds or otherwise sequesters fyn within the nerve cell soma. The invention also relates to the treatment or prevention of conditions, diseases or syndromes that are particularly, but not exclusively, a consequence of excitotoxicity or associated with excitotoxicity.

Description

Treatments for excitotoxicity and A/?-mediated toxicity and age- associated neuronal dysfunction

Field of the invention

The invention relates to conditions involving neuronal dysfunction including those associated with excitotoxicity, Aβ-mediated toxicity and age associated conditions, to the treatment of these and other conditions and to models for screening for compounds having therapeutic application.

Background of the invention

Nerve cells and tissues may be damaged and killed by glutamate and similar substances when receptors for excitatory transmitters, examples being the NMDA receptor and AMPA receptor are over -activated. According to the process, NMDA, kainic acid and other molecules that bind to these types of receptors, as well as pathologically high levels of glutamate facilitate the ingress of calcium ions into a cell, leading to activation of enzymes such as phospholipases, endonucleases, proteases and concomitant damage to cytoskeleton, membrane and DNA.

The pathology is known as "excitotoxicity" and it is believed to be involved in many diseases, conditions and syndromes of the nervous system including spinal cord injury, stroke, traumatic brain injury, MS, Alzheimer's disease, ALS, Parkinson's disease, Huntington's disease, and other neurodegenerative diseases.

At a molecular level, the phosphorylation of residues on NMDA receptors by the tyrosine kinase fyn is understood to be an important event in the transmission of certain excitotoxic signals. This event is understood to be important for the interaction of proteins such as PSD-93, PSD-95 and nNOS with NMDA.

One approach to blocking the transmission of an excitotoxic signal has been to introduce a peptide into a cell, the peptide having a sequence that is more or less identical to a region of a NMDA receptor (a NR2B receptor) containing a residue for phosphorylation by fyn such as a tyrosine residue. It is believed that saturation of a compartment of a neuron at a post synaptic cleft where NR2B receptor is located blocks the phosphorylation of a subject tyrosine on the NR2B receptor by fyn, probably by creating competition for phosphorylation by fyn that is skewed in favour of phosphorylation of the peptide. It is not clear whether this approach would be useful for blocking transmission of excitotoxic signals in the treatment of a condition or disease because without adequate delivery and saturation at a majority of post synaptic clefts, it would be possible for fyn to phosphorylate a NMDA receptor tyrosine residue leading to interaction with proteins such as PSD-93, PSD-95 and nNOS and generation of the signal.

Another approach is to introduce a peptide that has a sequence for binding to molecular domains on either side of the PSD-95/NMDA receptor interaction complex. Again this approach does not stop fyn from phosphorylating NR2B so signal transmission remains possible without adequate delivery and saturation at a majority of post synaptic clefts.

There is a need for new approaches to blocking an excitotoxic signal, to compounds having this application and to screening methods for identifying said compounds.

Summary of the invention

The invention seeks to at least minimise the above identified need and in one embodiment provides a compound for preventing, inhibiting or antagonising fyn from phosphorylating an amino acid residue in a molecule having an amino acid sequence shown in SEQ ID NO: 1. Typically the compound is capable of binding to fyn, to prevent fyn from migrating to a region of a nerve cell cytosol adjacent a post synaptic membrane and in one embodiment the compound holds or otherwise sequesters fyn within the nerve cell soma.

In further embodiments there is provided a peptide which has an amino acid sequence of a projection domain of a Tau protein. In certain embodiments the peptide is useful for preventing, inhibiting or antagonising src kinases such as fyn and related enzymes from phosphorylating a tyrosine residue in a molecule having an amino acid sequence shown in SEQ ID NO: 1. In certain embodiments of the invention the compound is a peptide or a peptidomimetic.

In further embodiments, there is provided a nucleic acid encoding a peptide of the invention.

In certain embodiments, there is provided a cell including a peptide of the invention.

In other embodiments there is provided a non human transgenic animal including a peptide or protein of the invention.

In further embodiments there is provided a molecular complex including a compound, peptide or peptidomimetic of the invention bound to fyn.

In other embodiments, there is provided an antibody for binding to a compound or peptide of the invention.

In certain embodiments, there is provided a pharmaceutical composition including a compound, peptide, peptiodomimetic or an antibody of the invention and a pharmaceutically effective carrier, diluent or excipient.

In further embodiments there is provided a method for antagonising, preventing or inhibiting fyn from phosphorylating a tyrosine residue in a molecule having an amino acid sequence shown in SEQ ID No: 1 including the step of contacting fyn with a compound or peptide according to the invention.

In yet further embodiments there is provided a method for treating or preventing a condition, disease or syndrome that is a consequence of excitotoxicity or associated with excitotoxicity in an individual including providing an individual with a compound or peptide according to the invention.

In further embodiments there is provided a method for maintaining neuron function or preventing the loss of neuron function or preventing abnormal neuron function in an individual including administering to the individual a Tau projection domain or fragment thereof. The abnormal neuron function may or may not be a consequence of excitotoxicity or associated with excitotoxicity.

In further embodiments there is provided a method for preventing or reducing decline in neuron function associated with age in an individual including administering to the individual a Tau projection domain or fragment thereof.

In one embodiment, the individual is an individual at risk of developing abnormal neuron function or losing neuron function.

In still further embodiments there is provided a method for monitoring the treatment of an individual having a condition, disease or syndrome including:

- selecting an individual who has been provided with a compound, peptide or peptidomimetic according to the invention for treatment of a condition, disease or syndrome;

- determining whether the individual contains a compound, peptide, peptidomimetic or molecular complex according to the invention;

thereby monitoring the treatment of an individual having a condition, disease or syndrome.

In one embodiment the condition, disease or syndrome is a consequence of excitotoxicity or associated with excitotoxicity.

In another embodiment there is provided a method for identifying a compound for preventing, inhibiting or antagonising fyn from phosphorylating a tyrosine residue in a molecule having an amino acid sequence shown in SEQ ID No: 1 including:

- providing a compound for which capacity to prevent, inhibit or antagonise fyn function is to be determined;

- contacting the compound with fyn; - determining whether fyn is capable of phosphorylating a molecule having an amino acid sequence shown in SEQ ID No: 1.

In a further embodiment there is provided a use of a compound or peptide capable of preventing, inhibiting or antagonising fyn from phosphorylating an amino acid residue in a molecule having an amino acid sequence shown in SEQ ID NO: 1 in the preparation of a medicament for treating or preventing a condition, disease or syndrome in an individual.

In another embodiment the invention provides a composition for the treatment or prevention of a condition, disease or syndrome in an individual comprising as an active ingredient a compound or peptide capable of preventing, inhibiting or antagonising fyn from phosphorylating an amino acid residue in a molecule having an amino acid sequence shown in SEQ ID NO: 1.

In another embodiment the invention provides a pharmaceutical composition comprising an effective amount of a compound or peptide capable of preventing, inhibiting or antagonising fyn from phosphorylating an amino acid residue in a molecule comprising an amino acid sequence shown in SEQ ID NO: 1 as a main ingredient.

In another embodiment the invention provides a composition comprising a compound or peptide capable of preventing, inhibiting or antagonising fyn from phosphorylating an amino acid residue in a molecule having an amino acid sequence shown in SEQ ID NO: 1 for use in the treatment or prevention of a condition, disease or syndrome.

In another embodiment the invention provides a composition comprising a compound or peptide capable of preventing, inhibiting or antagonising fyn from phosphorylating an amino acid residue in a molecule having an amino acid sequence shown in SEQ ID NO: 1 for use as a medicament.

In certain embodiments, the condition, disease or syndrome may be a consequence of excitotoxicity or associated with excitotoxicity. In a further embodiment there is provided a use of a Tau projection domain or fragment thereof in the preparation of a medicament for maintaining neuron function or preventing the loss of neuron function in an individual.

In a further embodiment there is provided a use of a Tau projection domain or fragment thereof in the preparation of a medicament for preventing or reducing decline in neuron function associated with age in an individual.

In another embodiment the invention provides a composition for maintaining neuron function or preventing the loss of neuron function in an individual comprising as an active ingredient a Tau projection domain or fragment thereof.

In another embodiment the invention provides a pharmaceutical composition comprising an effective amount of a Tau projection domain or fragment thereof as a main ingredient.

In another embodiment the invention provides a composition comprising a Tau projection domain or fragment thereof for use in maintaining neuron function or preventing the loss of neuron function.

In another embodiment the invention provides a composition comprising a Tau projection domain or fragment thereof for use in preventing or reducing decline in neuron function associated with age.

In another embodiment the invention provides a composition comprising a Tau projection domain or fragment thereof for use as a medicament.

In certain embodiments, there is provided a peptidomimetic based on a Tau projection domain or fragment thereof.

Brief description of the drawings

Figure 1. Expression of truncated tau (Δtau) prevents premature mortality of APP23 mice, (a) Δtau mice express truncated tau comprising only the amino-terminal projection domain (Δtau) under the control of an mThyl.2 promoter in neurons of hippocampus, cortex and amygdala, as revealed by immunohistochemistry with a human tau-specific antibody (HT7), (b) Both, Δtau and APP^swe (APP23) transgenic mice were crossed of a fau-deficient (tau-/-) background, to obtain APP23.tau^+/' and Δtau.tau^+/" founder mice (F), respectively. Their offspring included all genotype combinations studied subsequently, (c) APP23 mice present a pronounced premature mortality that is ameliorated by reducing tau levels in APP23.tau^+/' and even more in APP23.tau^"/" mice. Expression of Δtau improved the survival of APP23 mice similar to tau^+/'. Combining Δtau expression with heterozygous tau-deficiency rescued APP23.Δtau.tau^+/" mice completely.

Figure 2. Δtau expression and fau-deficiency does not affect APP mRNA expression, Aβ levels or plaque burden, (a) Levels of APP mRNA are not altered in APP23 mice in the presence of Δtau or faw-deficiency. (b) Aβ_1-40 and Aβ ₁.₄₂ levels are comparable in APP23, APP23.Δtau and APP23.tau^~/' mice. (c,d) Thioflavin S staining reveals Aβ- plaques (arrows; insets) at similar numbers (c) and morphology (d) in APP23 mice independent of Δtau expression and tau reduction.

Figure 3. Δtau localizes to axons and cell bodies, but not dendrites, (a) Immunohistochemistry with a human tau-specific antibody (HT7) reveals a membrane- like pattern of Δtau in cortical neurons (A₁B). (b) Whereas expression of full-length P301L mutant tau in pR5 mice results in somatodendritic localization of transgenic tau, as shown by HT7 staining , in Δtau mice, transgenic truncated tau is excluded from dendrites (open arrowhead) despite abundant staining of cell bodies, (c) Western blot of micro-dissected hippocampal CA1 cell body layer (S) and dendrites (D) as indicated by the boxes in (b), confirms Δtau expression in cell bodies and its virtual absence from dendrites, whereas P301 L mutant tau distributes to soma and dendrites. As a control for the two compartments, we included the post-synaptic density protein PSD-95 that is enriched in dendritic extracts (D), whereas the nuclear TAR-binding protein TDP43 is more abundant in soma extracts (S). (d) Axons of CA1 neurons project to neurons within the subiculum (arrows; scheme). Immunohistochemistry with the human tau specific antibody HT7 (red) of the subiculum shows transgenic Δtau in axonal boutons (arrows), suggesting accumulation of Δtau in axons of CA1 neurons. Δtau localizes to axon terminals even in the absence of endogenous tau in Δtau. tau^"'^" mice, excluding hitchhiking by full-length tau as a transport mechanism, (e) Similarly, in Δtau and Δtau.tau^"'^" brains, Δtau is found in the posterior limb (pi) of the anterior commissure that contains axons of amygdala and cortex. The anterior limb (al), build by axons of the olfactory system, does not contain Δtau consistent with the expression pattern.

Figure 4. Δtau expression reduces susceptibility to excitotoxic seizures, (a) The mean seizure severity induced by i.p. injection of PTZ (50mg/kg) is significantly reduced in both Δtau and tau^'1' mice compared to wild-type (wt) controls (**p<0.01). (b) Δtau expression and tau reduction in tau^"'^" mice increases the latency for more severe seizures, (c) In APP23 mice, PTZ-induced seizures are mostly lethal (10 of 11), whereas expression of Δtau or crossing on a tau^"'^" background reduces the seizure severity markedly (*p<0.05; **p<0.01). (D) Δtau expression and tau reduction in tau^"'^" mice increases the latency for more severe seizures in and APP23 mice.

Figure 5. Reduced synaptic Fyn is associated with decreased NRI/PSD-95-interaction. (a) Levels of NR subunits are not changed in Δtau and tau^'1' brains, compared to wt controls (see Figure 7). Accordingly, intracellular staining for NR1 (top row) are similar in wt and tau^'1' neurons. Staining of drebrin suggests normal morphology of dendrites in wt and tau^'1' neurons. Cell surface staining shows presentation of NMDA receptor (NR) 1 (bottom row) on dendrites of primary hippocampal wt neurons, whereas NR1 is hardly detectable on dendrites of tau^'1' neurons, suggesting decreased surface expression rather than reduced levels of NR in tau^'1' neurons, (b) Quantification of wt and tau^'1' hippocampal neurons (a) reveals significantly reduced NR1 surface expression in tau^'1' compared to wt dendrites (*p<0.0001). (c) NR surface presentation depends on interaction with PSD-95. lmmunoprecipitation with PSD-95 antibodies shows co- immunoprecipitation of NR1 from wt hippocampi, but hardly any NR1 from Δtau and tau^' '^" hippocampi (*p<0.005). (d) NR2b phosphorylation by Src-kinases such as Fyn is known to stabilize the NR/PSD-95 interaction. Levels of the NR subunit NR2b were similar in wt, Δtau and tau^'1' hippocampi (for quantification see Figure 6 & 7). Phosphorylation of NR2b at the Fyn epitope Y1472 was, however, significantly reduced in Δtau and tau^"7" mice compared to wt controls (*p<0.005). (e) Expression of Δtau results in reduced interaction of Fyn with endogenous tau, as revealed by co- immunoprecipitation with antibodies to full-length tau that does not recognize Δtau (^*p<0.05). (f) Western blots of micro-dissected CA1 cell bodies (S) and their dendrites (D) show accumulation of Fyn in cell bodies of Δtau and tau^'1' CA1 neurons, different from wt neurons (*p<0.005). (g) Accordingly, levels of Fyn were reduced in synaptosomal preparations from Δtau and tau^'1' hippocampi compared to wt controls (^*p<0.005).

Figure 6. Neuronal expression of truncated tau (Δtau) in Δtau transgenic mice, (a) Scheme of the longest human tau isoform, htau40 (441 aa), comprising an amino- terminal projection domain (PD), followed by the microtubule binding domain (MTB) with four repeats (grey boxes), and a carboxy-terminal tail (C). Truncated Δtau comprises only the PD. Transgenic expression of Δtau is under the control of the neuronal mThyl.2 promoter (bottom), (b) Western blot of wt and Δtau (A) hippocampal extract reveals endogenous murine tau (55 kD) in all extracts and Δtau protein (37 kD) in transgenic samples, (c) Endogenous and Δtau levels add to 2.6-fold increased total tau levels in Δtau transgenic mice compared to wt controls, (d) lmmunohistochemistry with the human tau-specific antibody HT7 (red) shows expression of Δtau in neurons of the hippocampus, amygdala and cortex. Scale bar; 250 μm.

Figure 7. Unaltered expression of NMDA receptor (NR) subunits, Fyn-kinase and PSD- 95 in Δtau and tau^'1' mice. Western blots of hippocampal extracts from wt, Δtau and tau^' '^' brains show comparable expression levels of NR1 , NR2a, NR2b (blot included in Figure 5 main text), PSD-95 and Fyn. The quantification is normalized to Gapdh expression.

Figure 8. Aβ plaque-forming mice were icv (internal cerebral vein) implanted with an osmotic pump releasing either the Tat-NR2B9c peptide in artificial CSF (cerebral spinal fluid) (aCSF) or mock (only aCSF) for 28 days. Then the pump was replaced by a second pump using the same regimen. After another 28 days the pump was removed. This treatment within a therapeutic window was sufficient to rescue the lethality of the Aβ-producing mice. Detailed description of the embodiments

The inventors have surprisingly found that it is possible to sequester src kinases such as fyn from cell compartments adjacent a post synaptic cleft of a neuron where it is normally expressed so as to retain the enzyme in a soma or body of a nerve cell. This sequestration effectively stops the phosphorylation of NMDA receptor and like receptors by fyn and like enzymes and hence prevents the transmission of an excitotoxic signal.

This finding is distinct from another approach for interfering with transmission of an excitotoxic signal which requires the establishment of competition for fyn-mediated phosphorylation of residues on an NMDA receptor. As mentioned above, one problem with such an approach is that fyn -mediated phosphorylation and hence signal transmission remains a possibility where effective saturation of a cell compartments adjacent a post synaptic cleft is not achieved.

One key advantage of the present invention is that it is possible to target src kinase function at a specific cell compartment while not effecting src kinase function at other cell compartments.

Thus in certain embodiments, there is provided a compound for preventing, inhibiting or antagonising a src kinase such as fyn from phosphorylating a tyrosine residue, preferably tyrosine 1472, in a molecule having an amino acid sequence shown in SEQ ID No: 1.

By "preventing, inhibiting or antagonising a src kinase such as fyn" it is meant that the relevant NMDA receptor cannot be phosphorylated by fyn. The underlying mechanism is understood to be based on the capacity of a compound of the invention to fix fyn at a cellular compartment that is spatially apart from the location of the relevant NMDA receptor so that fyn is unable to contact the receptor. This is distinguished from where fyn is blocked from contacting the receptor but fyn is not fixed at a cellular compartment that is spatially apart from the location of the relevant NMDA receptor.

The phrase "preventing, inhibiting or antagonising a src kinase such as fyn from phosphorylating" will generally be understood as being that the compound prevents phosphorylation of a peptide having the sequence of SEQ ID No: 1 whether the peptide is comprised in a monomeric, dimeric or trimeric form or in a form as generally observed that is capable of tranducing an excitotoxic signal.

Neuron function relates to any cellular process that a neuron is involved in. Non-limiting examples of neuron function are the production, release, binding, and uptake of certain neurotransmitters or transmission of action potentials. Neuron function effects central control of metabolism in humans including, but not limited to, regulation of vegetative processes, such as regulation of blood glucose levels, circadian rhythm, body temperature, gluconeogenesis and motor functions.

Abnormal neuron function relates to a change in any cellular process of a neuron that can contribute to the initiation, development or progression of a condition, disease or syndrome described herein. For example, any change in the ability of a neuron to communicate with another neuron may be an abnormal neuron function. A change in the ability of a neuron to communicate with another neuron may be the reduced or elevated capacity to transmit or receive signals from other neurons, for example a change in the ability to release or detect neurotransmitters or generate or propagate action potentials.

SEQ ID No: 1 is the amino acid sequence of the human NR2B receptor.

In a further embodiment the compound is capable of binding to fyn to prevent fyn from migrating to a region of a nerve cell compartment adjacent a post synaptic or dendritic membrane where otherwise located in this compartment fyn is capable of phosphorylating an NMDA receptor, including an NR2B receptor.

In a further embodiment the compound is capable of binding to fyn to substantially inhibit fyn from migrating to a region of a nerve cell compartment adjacent a post synaptic membrane. In certain circumstances not all fyn molecules present in a cell will be inhibited from migrating to a region of a nerve cell compartment adjacent a post synaptic membrane, however any inhibition of fyn migration to a region of a nerve cell compartment adjacent a post synaptic membrane is sufficient to have an effect on excitotoxicity or neuron function.

In a further embodiment the compound is capable of binding to fyn to hold fyn within a nerve cell soma or preventing it from migrating to a compartment adjacent a post synaptic or dendritic membrane.

In certain embodiments of the invention the compound is a peptide or a peptidomimetic.

A 'peptidomimetic' is a synthetic chemical compound that has substantially the same structure and/or functional characteristics of a peptide of the invention, the latter being described further herein. Typically, a peptidomimetic has the same or similar structure as a peptide of the invention, for example the same of similar sequence of a Tau projection domain or fragment thereof. A peptidomimetic generally contains at least one residue that is not naturally synthesised. Non-natural components of peptidomimetic compounds may be according to one or more of: a) residue linkage groups other than the natural amide bond ('peptide bond') linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.

In an embodiment the peptide or peptidomimetic has an amino acid sequence that is identical to or that has homology or identity with a sequence of a Tau protein projection domain or fragment thereof.

In a further embodiment the peptide or peptidomimetic has an amino acid sequence that is identical to or that has homology or identity with a sequence of a human Tau protein projection domain or fragment thereof.

The human Tau protein can occur in the brain in six alternatively spliced isoforms. The longest human tau isoform, htau40 (441 aa) (NCBI sequence reference NP_005901), comprises an amino-terminal projection domain (PD; also known as Tau projection domain or projection domain of Tau), followed by a microtubule binding domain (MTB) with four repeats and a carboxy-terminal tail. The amino-terminal projection domain of Tau protrudes from the microtubule surface when the Tau protein is bound to microtubules.

htau40 is can also be referred to as 2N4R as it contains 2 amino-terminal inserts (2N) and 4 microtubule-binding repeats (4R). The two amino-terminal inserts are encoded by two alternatively spliced exons, E2 and E3, and encode 29 amino acids each. The various isoforms of the Tau protein arise from alternative splicing of exon 2, 3 and 10. The isoforms differ in either 0, 1 or 2 inserts of the 29 amino acid amino-terminal part and three or four microtubule-binding repeats. The isoforms of human Tau are summarised below:

The 0N3R isoform is 352 amino acids in length (NCBI sequence reference NP_058525.1), with the amino-terminal projection domain being 197 amino acids. The amino acid sequence of the amino-terminal projection domain of the 0N3R isoform is shown in SEQ ID NO: 2.

The 0N4R isoform is 383 amino acids in length (NCBI sequence reference NP 058518.1), with the amino-terminal projection domain being 197 amino acids. The amino acid sequence of the amino-terminal projection domain of the 0N4R isoform is shown in SEQ ID NO: 2.

The 1 N3R isoform is 383 amino acids in length, with the amino-terminal projection domain being 226 amino acids. The amino acid sequence of the amino-terminal projection domain of the 1 N3R isoform is shown in SEQ ID NO: 3.

The 1 N4R isoform is 412 amino acids in length, with the amino-terminal projection domain being 226 amino acids. The amino acid sequence of the amino-terminal projection domain of the 1 N4R isoform is shown in SEQ ID NO: 3.

The 2N3R isoform is 410 amino acids in length, with the amino-terminal projection domain being 255 amino acids. The amino acid sequence of the amino-terminal projection domain of the 2N3R isoform is shown in SEQ ID NO: 4. The 2N4R isoform is 441 amino acids in length, with the amino-terminal projection domain being 255 amino acids. The amino acid sequence of the amino-terminal projection domain of the 2N4R isoform is shown in SEQ ID NO:4.

The amino terminal projection domain of Tau is 255 amino acids in length or shorter.

The amino acid sequence of human Tau isoforms can be found in publicly available databases, for example those supported by NCBI (National Center for Biotechnology Information), including GenBank^®. The projection domain of any Tau isoform found in a database may be used in any embodiment or aspect of the invention.

In a further embodiment the peptide or peptidomimetic has an amino acid sequence of a fragment of a projection domain of a Tau protein, whether it is human or not, the fragment having a length of from about 7 residues to about 70 residues.

Typically, the fragment of a projection domain of a Tau protein binds src kinases such as fyn.

In a further embodiment the peptide or peptidomimetic has a length of from about 20 to about 60 residues.

In a further embodiment the peptide or peptidomimetic has a length of from about 30 to 50 residues.

In a further embodiment the peptide or peptidomimetic has a length of about 40 residues.

Smaller peptides or peptidomimetics may have a length of from 3 to 25 residues, preferably 5 to 10 residues.

In a further embodiment the peptide or peptidomimetic has a sequence that is identical to or that has homology or identity with a sequence shown in one of:

SEQ ID NO: 2 (197 amino acid projection domain); MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSL EDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRI PAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAV VRTPPKSPSSAKSRLQTAPVPMPDLKN

SEQ ID NO: 3 (226 amino acid projection domain);

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKDVTAPLVDEGA PGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKK AKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYS SPGSPGTPGSRSRTPSLPTPPTREPKKVAWRTPPKSPSSAKSRLQTAPVPMPDLKN

SEQ ID NO:4. (255 amino acid projection domain);

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDG SEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPS LEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANAT RIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVA WRTPPKSPSSAKSRLQTAPVPMPDLKN

SEQ ID NO:5 (exon 1 of tau, part of exon 2 of tau);

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTE

SEQ ID NO: 6 (exon 4,5);

AEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAK

SEQ ID NO:7 (exon 7);

GADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSS

SEQ ID NO:8 (part of exon 7, exon 8); IPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREP

SEQ ID NO:9 (exon 8, part of exon 9 to position 255);

GEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAWRTPPKSPSSAKSR LQTAPVPMPDLKN

and SEQ ID NO: 10 (part of exon 8 and exon 9; amino acids 199-238 of 2N4R);

SPGSPGTPGSRSRTPSLPTPPTREPKKVAWRTPPKSPSS

In a further embodiment the peptide has a sequence that is at least 90% identical to the sequence shown in any one of SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO: 4; SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO:10.

In a further embodiment the peptide has a sequence that is at least 95% identical to the sequence shown in any one of SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO: 4; SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.

In a further embodiment the peptide has a sequence that is at least 90% identical to the sequence of SEQ ID NO:9 or 10 and includes one or more of the following sequences:

SEQ ID NO:11 (PGSPGTP)

SEQ ID NO:12 (PSLPTPP)

SEQ ID NO:13 (PKSP)

In one embodiment, a peptide or peptidomimetic of the invention consists of a sequence of a Tau projection domain. In other words, it does not include any other domains of the full length Tau protein such as a microtubule binding domain. Examples of these types of peptides or peptidomimetics include SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO: 4; SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. In certain embodiments the peptide or peptidomimetics has a sequence that is at least 90, 95, 96, 97, 98 or 99% identical to the sequence shown in any one of SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO: 4; SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO:10.

In certain embodiments, a peptide or peptidomimetic of the invention includes a sequence of a Tau projection domain and a fragment of a microtubule binding domain of a Tau protein or a fragment of a c-terminal domain of a Tau protein. The microtubule binding domain of a Tau protein and the carboxy-terminal tail domain of a Tau protein is typically located c-terminal to the Tau projection domain. In certain embodiments, the c- terminal domain of a Tau protein is a microtubule binding domain or a carboxy-terminal tail domain. In certain embodiments the part of the peptide or peptidomimetic contributed by the microtubule binding domain or c-terminal domain of a Tau protein is less than 155 amino acids.

In certain embodiments, a peptide or peptidomimetic of the invention includes a sequence of a Tau projection domain and another domain or domains of the Tau protein, wherein the peptide or peptidomimetic is characterised in that it does not substantially aggregate with another peptide or peptidomimetic of the invention.

In one embodiment, there is provided a fusion protein comprising a fusion or carrier domain and a Tau projection domain. In one example the fusion or carrier has a sequence of a c-terminal domain of a Tau protein including a microtubule binding domain or a carboxy-terminal tail domain.

In one embodiment, the compound, peptide or peptidomimetic of the invention may be capable of reducing, inhibiting or preventing a post-translational modification of fyn.

In certain embodiments, the compound, peptide or peptidomimetic of the invention may be capable of reducing, inhibiting or preventing a modification of fyn that would otherwise result in an attachment of a fatty acid molecule to fyn. In one example the modification may be palmitoylation.

In one embodiment, the compound, peptide or peptidomimetic of the invention may be capable of modulating cellular fatty acid levels and/or composition, such as modulating omega 3 unsaturated fatty acids. By reducing, inhibiting or preventing modification of fyn with a fatty acid molecule or modulating fatty acid levels and/or composition, the amount of synaptic fyn may be reduced, for example, by preventing fyn from anchoring in the post-synaptic membrane.

It will be understood to a person skilled in the relevant art that conservative substitutions may be made to the peptides or peptidomimetics of the invention while still maintaining the desired function. Whilst the concept of conservative substitution is well understood by the person skilled in the art, for the sake of clarity conservative substitutions are those set out below.

GIy, Ala, VaI, lie, Leu, Met;

Asp, GIu, Ser;

Asn, GIn ;

Ser, Thr;

Lys, Arg, His;

Phe, Tyr, Trp, His; and

Pro, Nα-alkalamino acids.

"Percent (%) amino acid sequence identity" or " percent (%) identical" with respect to a peptide or polypeptide sequence, i.e. a peptide of the invention defined herein, is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, i.e. a peptide of the invention, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms (non-limiting examples described below) needed to achieve maximal alignment over the full-length of the sequences being compared. When amino acid sequences are aligned, the percent amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain percent amino acid sequence identity to, with, or against a given amino acid sequence B) can be calculated as: percent amino acid sequence identity = X/Y100, where X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not equal the percent amino acid sequence identity of B to A.

In calculating percent identity, typically exact matches are counted. The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J. MoI. Biol. 215:403. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. Alignment may also be performed manually by inspection. Another non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22:4673- 4680). ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence, and thus can provide data about the sequence conservation of the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, CA). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed. A non- limiting example of a software program useful for analysis of ClustalW alignments is GENEDOC™. GENEDOC™ allows assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA). When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

Any of the peptides or peptidomimetics of the invention can be linked to an internalization peptide that facilities translocation through the plasma membrane of a cell. For example, the HIV TAT internalization peptide YGRKKRRQRRR can be used. Any suitable internalization peptide or compound is one that facilitates translocation of the peptide or peptidomimetic of the invention into a cell and does not prevent the peptide or peptidomimetic of the invention from holding fyn within a nerve cell soma or preventing fyn from migrating to a compartment adjacent a post synaptic or dendritic membrane. Further, any suitable internalization peptide or compound is one that facilitates translocation of the Tau projection domain or fragment thereof into a cell and does not prevent the Tau projection domain or fragment thereof from maintaining neuron function or preventing the loss of neuron function or preventing abnormal neuron function.

In certain embodiments, peptides and peptidomimetics of the invention prevent, inhibit or otherwise antagonise phosphorylation of NMDA receptor (NMDAR) residues by fyn, leading to preventing or decoupling of the NMDAR -PSD95 (or PSD-93 or nNOS) interaction. Hence, pharmacological activity of peptides or peptidomimetics can be confirmed by screening for inhibition of src related activity, especially fyn activity or for preventing or decoupling of these interactions. This can be achieved using standard techniques in the art. Useful peptides and peptidomimetics typically have IC50 values of less than 50μM, 25 μM, 10 μM, 0.1 μM or 0.01 μM in such an assay. Preferred peptides typically have an IC50 value of between 0.001-1 μM, and more preferably 0.05-0.5 or 0.05 to 0.1 μM.

Peptides and peptidomimetics of the invention can be derivatized (e.g., acetylated, phosphorylated and/or glycosylated) to improve the binding affinity with fyn or a src related kinase, to improve ability to be transported across a cell membrane or to improve stability.

Peptides of the invention and heterologous proteins containing said peptides such as those with a fusion domain, for example for controlling half life and those including an intemalisation domain as described above can be synthesized by solid phase synthesis or recombinant methods.

Peptidomimetics can be synthesized using a variety of procedures and methodologies described in the scientific and patent literatures, e.g., Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley & Sons, Inc., NY, al-Obeidi (1998) MoI. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1 :114-119; Ostergaard (1997) MoI. Divers. 3:17-27; Ostresh (1996) Methods Enzymol. 267:220-234.

The compounds, peptides or peptidomimetics of the invention can be administered in the form of a pharmaceutical composition. These compositions may be manufactured under GMP conditions or in some embodiments by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The ingredients may facilitate processing peptides or peptidomimetics into preparations which can be used pharmaceutically.

Administration for treatment can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Pharmaceutical compositions for parenteral administration are generally sterile and substantially isotonic. Physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer may be used. The solution may also contain suspending, stabilizing and/or dispersing agents. The peptides or peptidomimetics may be provided in powder form to be dissolved in solvent such as sterile pyrogen-free water, before use.

Compositions for transmucosal administration, including nasal cavity or sublingual administration include penetrants appropriate to the barrier to be permeated in the formulation.

Compounds, peptides or peptidomimetics may be formulated for oral administration by combining the compounds, peptides or peptiodmimetics with pharmaceutically acceptable carriers to form formulations for oral ingestion such as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. Suitable excipients for oral solid formulations such as powders, capsules and tablets include fillers such as sugars (lactose, sucrose, mannitol and sorbitol); cellulose (maize starch, wheat starch, rice starch, potato starch, gelatine, gum tragacanth, methyl cellulose, hydroxyproplylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP)); granulating agents; and binding agents. Disintegrating agents (cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate) may also be used. Sugar or enteric coatings can be applied to solid dosage forms using standard techniques. Carriers, excipients or diluents including water, glycols, oils alcohols can be used for oral liquid preparations. Flavoring agents, preservatives, coloring agents and the like may be added.

The compounds, peptides or peptidomimetics can also be formulated for sustained release for example by subcutaneous or intramuscular implantation or by intramuscular injection. Polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivates, or salt may be used or a semipermeable matrix of solid polymers containing the therapeutic agent. The compounds, peptides or peptidomimetics of the invention may be released for a few weeks up to 3 months.

Alternatively, other pharmaceutical delivery systems can be employed. Liposomes and emulsions can be used to deliver compounds of the invention.

The compounds, peptides or peptidomimetics of the invention can be included in any of the above-described formulations as the free acids or bases or as pharmaceutically acceptable salts.

Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration) containing any of the dosages indicated above.

The compounds, peptides or peptidomimetics of the invention are used in an amount effective to achieve the intended purpose. An amount is generally considered to be therapeutically effective if an individual treated patient achieves an outcome more favourable than the mean outcome in a control population of comparable patients not treated by methods of the invention.

Where the compound of the invention is a peptide or peptidomimetic, preferred dosage ranges include 0.001 to 20 μmol peptide or peptidomimetic per kg patient body weight

The amount of peptide or peptidomimetic administered depends on the subject being treated, on the subject's weight, the severity and nature of the condition, the manner of administration and the judgment of the prescribing physician. The therapy can be repeated intermittently while symptoms are detectable or even when they are not detectable. The therapy can be provided alone or in combination with other drugs.

Thus certain embodiments, there is provided a compound of the invention and a pharmaceutically effective carrier, diluent or excipient.

In certain embodiments, there is provided a method for antagonising, preventing or inhibiting fyn or a src related kinase from phosphorylating a tyrosine residue in a molecule having an amino acid sequence shown in SEQ ID No: 1 including the step of contacting the enzyme with a compound described above or composition including same.

In certain embodiments, there is provided a method for treating or preventing a condition, disease or syndrome that is a consequence of excitotoxicity or associated with excitotoxicity in an individual including providing an individual with a compound, peptide or peptidomimetic, said compound, peptide or peptidomimetic being characterised in that it is capable of binding to fyn, to prevent fyn from migrating to a region of a nerve cell cytosol adjacent a post synaptic membrane and in one embodiment the compound holds or otherwise sequesters fyn within the nerve cell soma.

Example of conditions, diseases or syndromes that are a consequence of excitotoxicity or associated with excitotoxicity include epilepsy, hypoxia, traumatic injury to the CNS, stroke, Alzheimer's disease and Parkinson's disease. By preventing or treating these dieases, conditions or syndromes the lifespan of the individual may be increased compared to the lifespan of an individual without treatment or prevention.

In a preferable embodiment the condition, disease or syndrome is Alzheimer's disease. In these embodiments the individual to be treated may display one or more of the following symptoms:

• Age-associated cognitive impairment

• Age-associated neuronal dysfunction not restricted to cognitive impairment

• short term memory loss

• inability to acquire new information • semantic memory impairments

• apathy

• mild cognitive impairment

• language, executive or visuoconstructional problems or apraxia

• long term memory impairment • irritability and aggression

• exhaustion

In another embodiment the individual may also have a tauopathy such as:

• Amyotrophic lateral sclerosis/parkinsonism-dementia complex

• Argyrophilic grain dementia (AgD) • Corticobasal degeneration (CBD)

• Creutzfeldt-Jakob disease (CJD)

• Dementia lacking distinct histopathology (DLDH)

• Dementia pugilistica • Diffuse neurofibrillary tangles with calcification

• Down's syndrome

• Frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17)

• Frontotemporal lobar degeneration with ubiquitin-positive lesions (FTLD-U)

• Gerstmann-Straussler-Scheinker disease

• Hallervorden-Spatz disease

• Myotonic dystrophy

• Niemann-Pick disease, type C (NPC)

• Non-Guamanian motor neuron disease with neurofibrillary tangles

• Pick's disease (PD)

• Postencephalitic parkinsonism

• Prion protein cerebral amyloid angiopathy

• Progressive subcortical gliosis

• Progressive supranuclear palsy (PSP)

• Subacute sclerosing panencephalitis

• Tangle only dementia

As shown in the examples the mice having the Tau projection domain are rescued from a disease phenotype. The inventors understand from these data that in certain embodiments the Tau projection domain, fragment or peptidomimetic thereof may be useful for treatment of a wide range of diseases irrespective of interaction with fyn and whether those diseases are a consequence of excitotoxicity or associated with excitotoxicity.

It is understood that while the Tau projection domain, fragment or peptidomimetic thereof is capable of binding to fyn, to prevent fyn from migrating to a region of a nerve cell cytosol adjacent a post synaptic membrane and in turn having effects on certain downstream molecular events, other molecular events or pathways are affected by the projection domain, fragment or peptidomimetic thereof in the soma. It therefore follows that conditions, diseases or syndromes other than those that are a consequence of excitotoxicity or associated with excitotoxicity can be treated or prevented by a compound or peptide of the invention. By treating or preventing these conditions, diseases or syndromes other than those that are a consequence of excitotoxicity or associated with excitotoxicity, the lifespan of an individual may be increased compared to an individual without treatment or prevention.

In certain embodiments, a method of treatment of the invention may improve cognitive function, wherein the cognitive function of the treated individual is defined by consciousness (alertness and orientation), memory, and/or attention span

In certain embodiments, a method of treatment of the invention may improve quality of life (QOL) in a treated individual. QOL can be assessed using, for example, the known SF-12^® or SF-36^® health survey scoring procedures. SF-36^® assesses a patient's physical and mental health in the eight domains of physical functioning, role limitations due to physical problems, social functioning, bodily pain, general mental health, role limitations due to emotional problems, vitality, and general health perceptions. Scores obtained can be compared to published values available for various general and patient populations. QOL of life may be considered to be improved if there is a positive change in at least one of the eight domains of physical functioning.

Effective dosages and routes of administration of compounds, peptides or pharmaceutical compositions of the invention are conventional. The exact amount (effective dose) of the compound, peptide or pharmaceutical composition will vary from subject to subject, depending on, for example, the species, age, weight and general or clinical condition of the subject, the severity or mechanism of any disorder being treated, the particular agent or vehicle used, the method and scheduling of administration, and the like. A therapeutically effective dose can be determined empirically, by conventional procedures known to those of skill in the art. See, e.g., The Pharmacological Basis of Therapeutics, Goodman and Gilman, eds., Macmillan Publishing Co., New York. For example, an effective dose can be estimated initially either in cell culture assays or in suitable animal models. The animal model may also be used to determine the appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes for administration in humans.

Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, intramuscular, subcutaneous, inhalation, intranasal, transdermal, topical, transmucosal, intra-tumoral, intra-synovial and rectal administration. In a specific embodiment, a pharmaceutical composition of the invention is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, intranasal or topical administration to human beings. In one embodiment, a pharmaceutical composition is formulated in accordance with routine procedures for subcutaneous administration to human beings. Typically, pharmaceutical compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Examples of dosage forms include, but are not limited to: liquid dosage forms suitable for parenteral administration to a subject; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a subject.

The particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (e.g., the subject, the condition, disease or syndrome, the condition, disease or syndrome state involved, and whether the treatment is prophylactic). Treatment may involve daily or multi-daily doses of compound(s), peptide(s) or pharmaceutical compositions(s) over a period of a few days to months, or even years.

In general, however, a suitable dose will be in the range of from about 0.001 to about 100 mg/kg, e.g., from about 0.01 to about 100 mg/kg of body weight per day, such as above about 0.1 mg per kilogram, or in a range of from about 1 to about 10 mg per kilogram body weight of the recipient per day. For example, a suitable dose may be about 0.1 mg/kg, 1 mg/kg, 10 mg/kg, or 50 mg/kg of body weight per day.

The compounds, peptides, peptidomimetics or pharmaceutical compositions of the invention are conveniently administered in unit dosage form; for example, containing 0.05 to 10000 mg, 0.5 to 10000 mg, 5 to 1000 mg, or about 100 mg of active ingredient per unit dosage form.

In certain embodiments, there is provided a molecular complex including a compound, peptide or peptidomimetic of the invention bound to a src related kinase such as fyn. The molecular complex is useful as a biomarker for monitoring the efficacy of treatments as described herein. Specifically, the detection of a complex including a compound of the invention bound to fyn indicates an at least progression towards treatment of a condition, disease or syndrome described herein. It is also useful in the drug screening methods described further herein wherein detection of the complex may indicate a drug candidate for prevention or treatment of a condition, disease or syndrome described herein.

Typically the compound is a Tau projection domain or fragment or peptidomimetic thereof.

The complex may be contained in a cell such as a cell transfectant such as a stable or transient transfectant, or an explant of a transgenic animal described further herein. It may also be contained in a nerve cell, especially a nerve cell of a patient undergoing treatment.

Thus in certain embodiments, there is provided a cell including a compound of the invention. In other embodiments, there is provided a cell including a molecular complex of the invention. Typically the cell is a nerve cell. A nerve cell includes unipolar, pseudounipolar, bipolar and multipolar neurons, Basket cells, Betz cells, medium spiny neurons, Purkinje cells, pyramidal cells, Renshaw cells and granule cells. The cell may also be other cells found in the brain including glial cells, such as microglia, astrocytes, oligodendrocytes. The cell may be macrophages have the capacity to or have entered the brain. The cells may be other cells of the central nervous system. The cell may be a cell line including neuroblastoma cells of human or non-human origin or any nerve cell lines available from the ATCC (American Type Culture Collection).

In other embodiments the complex is located in a region of the nerve cell cytosol adjacent a post synaptic membrane.

In certain embodiments, there is provided a method for monitoring the treatment of an individual having a condition, disease or syndrome that is a consequence of excitotoxicity or associated with excitotoxicity including:

- selecting an individual who has been provided with a compound, peptide or peptidomimetic according to the invention for treatment of a condition, disease or syndrome that is a consequence of excitotoxicity or associated with excitotoxicity;

- determining whether the individual contains a compound, peptide or peptidomimetic or molecular complex according to the invention;

thereby monitoring the treatment of an individual having a condition, disease or syndrome that is a consequence of excitotoxicity or associated with excitotoxicity.

Biopsy of relevant tissue and subsequent histology with or without the antibodies discussed further herein can be used to determine the presence of the molecular complex. As described herein, when the complex is found in a cell, fyn tends to be held in a cell soma.

In certain embodiments, there is provided an antibody for binding to a compound of the invention. In certain embodiments, there is provided an immune complex formed from the binding of an antibody of the invention with a compound of the invention.

In certain embodiments, there is provided a method including the steps of:

-providing a sample of cells or tissue obtained from the individual;

- contacting the sample with an antibody as described herein;

thereby determining whether the individual contains a compound, peptide, peptiodmimetic or molecular complex according to any one of the preceding claims.

In certain embodiments, there is provided a nucleic acid encoding a peptide of the invention.

Nucleic acids or nucleic acid sequences according to the invention can be an oligonucleotide, nucleotide, polynucleotide or a fragment of any of these, DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may be a sense or antisense strand, peptide nucleic acid (PNA) or any DNA- like or RNA-like material, natural or synthetic in origin.

In certain embodiments, there is provided a vector including a nucleic acid of the invention. Examples of these vectors include expression vectors, especially vectors capable of constitutive or inducible expression of a peptide of the invention described herein in a mammalian cell.

Other vectors are those useful for cloning and sub cloning of nucleic acid molecules, such as those used in bacterial cloning systems.

Expression vectors commonly include one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers can be provided on separate vectors, and replication of the exogenous DNA can be provided by integration into the host cell genome. The design of expression vectors depends on such factors as the choice of the host cells or the desired expression levels. Selection of promoters, enhancers, selectable markers, and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.

Expression vectors can be derived from a variety of sources, such as plasmids, viruses, or any combination thereof. Suitable viral vectors include, but are not limited to, retroviral, lentiviral, adenoviral, adeno-associated viral (AAV), herpes viral, alphavirus, astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picomavirus, poxvirus, or togavirus vectors.

In one embodiment, the expression vector is an E. coli vector which has a constitutive or inducible promoter. Sequences encoding additional peptides can be fused to the the relevant coding sequence in order to serve desirable purposes, such as increasing the expression or solubility of the recombinant protein or aiding its purification, in one example, the fused peptide(s) is cleavable from the recombinant protein. Expression vectors suitable for this purpose include, but are not limited to, pGEX (Pharmacia Piscataway, NJ), pMAL (New England Biolabs, Beverly, MA), and pRITS (Pharmacia, Piscataway, NJ).

Various methods can be used to maximize the expression of the recombinant protein in E. coli. One strategy is to use a host bacterium that has an impaired capacity to proteolytically cleave the recombinant protein. Another strategy is to alter the coding sequence such that the individual codon for each amino acid is preferentially utilized by E. coli.

In another embodiment, the expression vector is a yeast expression vector. Exemplary yeast expression vectors include, but are not limited to, pYepSecl, pMFa, pJRY88, pYES2 (Invitrogen Corporation, San Diego, CA), and picZ (Invitrogen Corp, San Diego, CA). In yet another embodiment, the expression vector is an insect cell expression vector. Commonly used insect cell expression vectors include baculovirus expression vectors, such as the pAc and pVL series.

In still another embodiment, the expression vector is a mammalian expression vector. Suitable mammalian expression vectors include, but are not limited to, pCDMδ, pMT2PC, pJL3, pJL4, pMT2 CXM, and pEMC2βl . When used in mammalian cells, the expression control sequences are often provided by viral regulatory elements. For example, promoters derived from polyoma, adenovirus 2, cytomegalovirus, or Simian virus 40 are commonly employed in mammalian expression vectors.

As those skilled in the art will recognize based upon the present invention, a wide variety of cloning vectors may be used as vector backbones in the construction of a gene targeting vector of the present invention, including pBluescript-related plasmids (e.g., Bluescript KS+11 ), pQE70, pQE60, pQE-9, pBS, pD10, phagescript, phiX174, pBK Phagemid, pNHδA, pNH16a, pNH18Z, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 PWLNEO, pSV2CAT, pXTI, pSG (Stratagene), pSVK3, PBPV, PMSG, and pSVL, pBR322 and pBR322-based vectors, pMB9, pBR325, pKH47, pBR328, pHC79, phage Charon 28, pKB11 , pKSV-10, pK19 related plasmids, pUC plasmids, and the pGEM series of plasmids. These vectors are available from a variety of commercial sources (e.g., Boehringer Mannheim Biochemicals, Indianapolis, IN; Qiagen, Valencia, CA; Stratagene, La JoIIa, CA; Promega, Madison, Wl; and New England Biolabs, Beverly, MA). However, any other vectors, e.g. plasmids, viruses, or parts thereof, may be used as long as they are replicable and viable in the desired host. The vector may also comprise sequences which enable it to replicate in the host whose genome is to be modified. The use of such a vector can expand the interaction period during which recombination can occur, increasing the efficiency of targeting (see Molecular Biology, ed. Ausubel et al, Unit 9.16, Fig. 9.16.1).

Vectors contemplated include gene therapy vectors. Such gene therapy vectors include, for example, viral vectors (such as adenoviruses ("Ad"), adeno-associated viruses

(AAV), and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a polynucleotide to a host cell. Vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells. Such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell- type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide. Such components also might include markers, such as detectable and/or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector. Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities. A large variety of such vectors are known in the art and are generally available. Additional references describing adenovirus vectors and other viral vectors which could be used in the methods of the present invention include the following: Horwitz, M.S., Adenoviridae and Their Replication, in Fields, B., et al (eds.) Virology, Vol. 2, Raven Press New York, pp. 1679-1721 , 1990); Graham, F., et al, pp. 109-128 in Methods in Molecular Biology, Vol. 7: Gene Transfer and Expression Protocols, Murray, E. (ed.), Humana Press, Clifton, NJ. (1991); Miller, N., et al, FASEB Journal 9: 190-199, 1995; Schreier, H, Pharmaceutica Acta Helvetiae 68: 145-159, 1994; Schneider and French, Circulation 88:1937-1942, 1993; Curiel D.T., et al, Human Gene Therapy 3: 147-154, 1992; Graham, F.L., et al, WO 95/00655 (5 January 1995); Falck-Pedersen, E.S., WO 95/16772 (22 June 1995); Denefle, P. et al, WO 95/23867 (8 September 1995); Haddada, H. et al, WO 94/26914 (24 November 1994); Perricaudet, M. et al, WO 95/02697 (26 January 1995); Zhang, W., et al, WO 95/25071 (12 October 1995).

In certain embodiments, there is provided a cell including a nucleic acid or a vector of the invention. The cell may be any cell capable of containing or expressing a peptide of the invention. Examples include prokaryotic and eukaryotic cells. Nerve cells and cells of the CNS are particular examples. The cell may be a cell line including neuroblastoma cells of human or non-human origin or any nerve cell lines available from the ATCC (American Type Culture Collection).

The specific host cells that may be employed for propagating the vectors of the present invention include E. coli K12 RR1 (Bolivar et al., Gene 2: 95, 1977), E. coli K12 HB101 (ATCC No. 33694), E. coli MM21 (ATCC No. 336780), E. coli DH1 (ATCC No. 33849), E. coli strain DH5σ, and E. coli STBL2. Alternatively, hosts such as S. cerevisiae or B. subtilis can be used. The above-mentioned hosts are available commercially (e.g., Stratagene, La JoIIa, CA; and Life Technologies, Rockville, MD).

In certain embodiments, there is provided a non human mammal including a nucleic acid or a vector of the invention. Typically the nucleic acid or vector encodes a peptide or protein as described herein. In one embodiment, the peptide is a Tau projection domain or fragment thereof.

In a further embodiment, the mammal further includes a nucleic acid encoding a β amyloid protein or a fragment derived thereof any other amyloidogenic proteins or peptides, including but not limited to the British peptide, the Prion peptide and amylin. Furthermore, in one embodiment the mammal includes a nucleic acid that encodes a protein may form a cross-beta sheet quarternary structure.

In a further embodiment, the β amyloid protein is a β amyloid precursor protein.

The transgenic animals of the invention can be generated by the standard techniques as exemplified herein.

A transgenic animal of the invention can be created by introducing a nucleic acid encoding a polypeptide into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal, lntronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the polypeptide to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly, animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866, 4,870,009, and 4,873,191 and in Hogan, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of mRNA encoding the transgene in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying the transgene can further be bred to other transgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a gene encoding a polypeptide into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the gene. The vector may be designed such that, upon homologous recombination, the endogenous gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" or "knock-in" vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous protein). In the homologous recombination vector, the altered portion of the gene is flanked at its 5¹ and 3¹ ends by additional nucleic acid of the gene to allow for homologous recombination to occur between the exogenous gene contained in the vector and an endogenous gene in an embryonic stem cell. The additional flanking nucleic acid sequences are of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see, e.g., Thomas and Capecchi (1987) Cell 51 :503 for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced gene has homologously recombined with the endogenous gene are selected (see, e.g., Li et al. (1992) Cell 69:915). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see, e.g., Bradley in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley (1991 ) Current Opinion in Bio/Technology 2:823-829 and in PCT Publication NOS. WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169.

In certain embodiments of the present invention, a functional disruption of an endogenous gene or expression of a transgene occurs at specific developmental or cell cycle stages (temporal disruption or expression) or in specific cell types (spatial disruption or expression). In other embodiments, the gene disruption is inducible when certain conditions are present. A recombinase excision system, such as a Cre-Lox system, may be used to activate or inactivate a gene at a specific developmental stage, in a particular tissue or cell type, or under particular environmental conditions. Generally, methods utilizing Cre-Lox technology are carried out as described by Torres and Kuhn, Laboratory Protocols for Conditional Gene Targeting, Oxford University Press, 1997. Methodology similar to that described for the Cre-Lox system can also be employed utilizing the FLP-FRT system. Further guidance regarding the use of recombinase excision systems for conditionally disrupting genes by homologous recombination or viral insertion is provided, for example, in U.S. Pat. No. 5,626,159, U.S. Pat. No. 5,527,695, U.S. Pat. No. 5,434,066, WO 98/29533, U.S. Pat. No. 6,228,639, Orban et al., Proc. Nat. Acad. Sci. USA 89: 6861-65, 1992; O'Gorman et al., Science 251 : 1351-55, 1991 ; Sauer et al., Nucleic Acids Research 17: 147-61 , 1989; Barinaga, Science 265: 26-28, 1994; and Akagi et al., Nucleic Acids Res. 25: 1766-73, 1997. More than one recombinase system can be used to genetically modify a non- human mammal or animal cell of the present invention. When using homologous recombination to disrupt a gene in a temporal, spatial, or inducible fashion, using a recombinase system such as the Cre-Lox system, a portion of the gene coding region is replaced by a targeting construct comprising the gene coding region flanked by loxP sites. Non-human mammals and animal cells carrying this genetic modification contain a functional, loxP-flanked gene. The temporal, spatial, or inducible aspect of the gene disruption is caused by the expression pattern of an additional transgene, a Cre recombinase transgene, that is expressed in the non-human mammal or animal cell under the control of the desired spatially-regulated, temporally-regulated, or inducible promoter, respectively. A Cre recombinase targets the loxP sites for recombination. Therefore, when Cre expression is activated, the LoxP sites undergo recombination to excise the sandwiched gene coding sequence, resulting in a functional disruption of the gene (Rajewski et al., J. Clin. Invest. 98: 600-03, 1996; St.-Onge et al., Nucleic Acids Res. 24: 3875-77, 1996; Agah et al., J. Clin. Invest. 100: 169-79, 1997; Brocard et al., Proc. Natl. Acad. Sci. USA 94: 14559-63, 1997; Feil et al., Proc. Natl. Acad. Sci. USA 93: 10887-90, 1996; and Kϋhn et al., Science 269: 1427-29, 1995). A cell containing both a Cre recombinase transgene and loxP-flanked gene can be generated through standard transgenic techniques or, in the case of genetically-modified, non-human mammals, by crossing genetically-modified, non-human mammals wherein one parent contains a loxP flanked gene and the other contains a Cre recombinase transgene under the control of the desired promoter. Further guidance regarding the use of recombinase systems and specific promoters to temporally, spatially, or conditionally disrupt a gene is found, for example, in Sauer, Meth. Enz. 225: 890-900, 1993, Gu et al., Science 265: 103-06, 1994, Araki et al., J. Biochem. 122: 977-82, 1997, Dymecki, Proc. Natl. Acad. Sci. 93: 6191-96, 1996, and Meyers et al., Nature Genetics 18: 136- 41 , 1998. An inducible disruption of a gene can also be achieved by using a tetracycline responsive binary system (Gossen and Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-51 , 1992). This system involves genetically modifying a cell to introduce a Tet promoter into the endogenous gene regulatory element and a transgene expressing a tetracycline- controllable repressor (TetR). In such a cell, the administration of tetracycline activates the TetR which, in turn, inhibits gene expression and, therefore, disrupts the gene (St.- Onge et al., Nucleic Acids Res. 24: 3875-77, 1996, U.S. Patent No. 5,922,927). The above-described systems for temporal, spatial, and inducible disruptions of a gene can also be adopted when using gene trapping as the method of genetic modification, for example, as described, in WO 98/29533 and U.S. Pat. No. 6,288,639, for creating the genetically modified non-human mammals and animal cells of the invention. Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nature 385:810-813 and PCT Publication Nos. WO 97/07668 and WO 97/07669.

To insert a foreign sequence into a genome of a cell to create the genetically modified non-human mammals and animal cells of the invention based upon the present description, the foreign DNA sequence is introduced into the cell according to a standard method known in the art such as electroporation, calciunrphosphate precipitation, retroviral infection, microinjection, biolistics, liposome transfection, DEAE- dextran transfection, or transferr infection (see, e.g., Neumann et al., EMBO J. 1 : 841- 845, 1982; Potter et al., Proc. Natl. Acad. Sci USA 81 : 7161-65, 1984; Chu et al., Nucleic Acids Res. 15: 1311-26, 1987; Thomas and Capecchi, Cell 51: 503-12, 1987; Baum et al., Biotechniques 17: 1058-62, 1994; Biewenga et al., J. Neuroscience Methods 71 : 67-75, 1997; Zhang et al., Biotechniques 15: 868-72, 1993; Ray and Gage, Biotechniques 13: 598-603, 1992; Lo, MoI. Cell. Biol. 3: 1803-14, 1983; Nickoloff et al., MoI. Biotech. 10: 93-101 , 1998; Linney et al., Dev. Biol. (Orlando) 213: 207-16, 1999; Zimmer and Grass, Nature 338: 150-153, 1989; and Robertson et al., Nature 323: 445- 48, 1986). One preferred method for introducing foreign DNA into a cell is electroporation.

In one embodiment the transgenic animal is a rodent, such as a mouse or rat.

In certain embodiments, there is provided a method for identifying a compound, peptide or peptidomimetic for preventing, inhibiting or antagonising fyn from phosphorylating a tyrosine residue in a molecule having an amino acid sequence shown in SEQ ID No: 1. including:

-providing a compound, peptide or peptidomimetic

-contacting the compound, peptide or peptidomimetic with fyn

-determining whether fyn is capable of phosphorylating a molecule having an amino acid sequence shown in SEQ ID No: 1. In a further embodiment of the method, the compound is contacted with a cell as described herein.

In a further embodiment of the method, the cell is contained in a non human mammal as described herein.

The examples set forth below demonstrate the following aspects and/or principles of the invention:

1. that excitotoxic signal transmission can be prevented, inhibited or antagonised by sequestering fyn in a nerve cell soma so as to substantially prevent expression of fyn at a post synaptic cleft;

2. that this can be achieved by compounds that are capable of binding to fyn; these compounds include but are not limited to peptides or peptidomimetics having sequence related to a Tau projection domain or fragment thereof;

3. that this sequestration of fyn has particular application for the treatment of conditions, diseases or syndromes that are a consequence of excitoxicity or associated with excitoxicity including but not limited to AD;

4. that a peptide having an amino acid sequence related to the Tau projection domain or fragment thereof can be used in a cell or animal model for screening for compounds capable of modulating excitotoxic signal transmission; and

5. that the Tau projection domain or fragments or peptidomimetics thereof may be used to treat or prevent other conditions, diseases or syndromes that are not a consequence of consequences of excitoxicity or associated with excitoxicity.

It will be understood that these examples are intended to demonstrate these and other aspects of the invention and although the examples describe certain embodiments of the invention with reference to use of a Tau projection domain for the treatment of Alzhiemer's disease, it will be understood that the examples do not limit these embodiments to these things. Various changes can be made and equivalents can be substituted and modifications made without departing from the aspects and/or principles of the invention mentioned above. All such changes, equivalents and modifications are intended to be within the scope of the claims set forth herein.

Examples

EXAMPLE 1 Materials and methods

Animals. APP23 and tau^1' mice have been described previously (Sturchler-Pierrat, C, et al. Two amyloid precursor protein transgenic mouse models with Alzheimer disease- like pathology. Proc Natl Acad Sci U S A 94, 13287-13292 (1997) and Tucker, K.L., Meyer, M. & Barde, Y.A. Neurotrophins are required for nerve growth during development. Nat Neurosci 4, 29-37 (2001)). To generate Δtau mice, we used site- directed mutagenesis to truncate amino acids 256-441 of human tau40 (Ittner, L.M., Koller, D., Muff, R., Fischer, J.A. & Born, W. The N-terminal extracellular domain 23-60 of the calcitonin receptor-like receptor in chimeras with the parathyroid hormone receptor mediates association with receptor activity-modifying protein 1. Biochemistry 44, 5749-5754 (2005) and Probst, A., et al. Axonopathy and amyotrophy in mice transgenic for human four-repeat tau protein. Acta Neuropathol (Berl) 99, 469-481 (2000)). The resulting cDNA was equipped with a human Kozak sequence and sub- cloned into the Xhol site of a mThy1.2 promotor vector for neuronal expression (Gotz, J., Chen, F., Barmettler, R. & Nitsch, R.M. Tau filament formation in transgenic mice expressing P301 L tau. J Biol Chem 276, 529-534 (2001 )). The construct was linearized and purified prior to injection. Pronuclear injection was carried out as described (Ittner, L.M. & Gotz, J. Pronuclear injection for the production of transgenic mice. Nat Protoc 2, 1206-1215 (2007 and Gotz, J., et al. Somatodendritic localization and hyperphosphorylation of tau protein in transgenic mice expressing the longest human brain tau isoform. Embo J 14, 1304-1313 (1995)). Transgenic mice were identified by PCR with the primers 5'-aagtcacccagcagggaggtgctcag-3 and 5'- gggtgtctccaatgcctgcttcttcag-3'. Four Δtau strains were established from independent founder animal by backcrossing onto the C57BI/6 background. All animal experiments were approved by the Animal Ethics Committee of the University of Sydney. Histology, lmmunohistochemistry on paraffin sections was performed as described (Ittner, L.M., et al. Compound developmental eye disorders following inactivation of TGFbeta signaling in neural-crest stem cells. J Biol 4, 11 (2005)). Primary antibodies was to human tau (HT7; Pierce), visualised by Alexa-coupled secondary antibodies or DAB (Pierce). Nuclei were stained with 2-(4-amidinophenyl)-6-indolecarbamidine (Molecular Probes, USA) or hematoxylin. Thioflavine S (Sigma, USA) stainings were done following standard protocols.

Western blotting and immunoprecipitation. Western blotting and immunoprecipitation has been described before (3). Primary antibodies were to human tau (HT7 (Pierce); tau-13 (Chemicon)), total tau (tau-5 (Bioscource)); Tau (Dako) immunoreactive with the MTB domain), NR1 , NR2a, NR2b, PSD-95, Gapdh (Chemicon), TDP-43 (Proteintec), Fyn (Santa Cruz) and Y1472 phospho NR2b (Affinity Bioreagents) and alkaline phosphatase-coupled secondary antibodies (Sigma, USA) were used.

Synaptosom preparation. Synaptosomes (also referred to as synaptoneurosomes) were prepared from hippocampi using a volume-adjusted protocol established for whole brains before. Briefly, the hippocampal of one brain were homogenized in 1 ml Sucrose Buffer (SB; 0.32M sucrose, 1 mM NaHCO₃, 1mM MgCI₂, 0.5mM CaCI₂) with a RSR2020 homogenizer (Heidorf) by 12 strokes at 700 rpm on ice. Cell debris and nuclei were pelleted twice by centrifugation at 1 ,400xg for 10 minutes (min) at 4⁰C. The supernatants (S) were combined, cleared by centrifugation at 720xg for 10 min at 4⁰C and then centrifuged at 13,800xg for 10 min at 4⁰C to obtain the crude synaptosomes in the pellet (P). The pellet was resuspended in 300 μl_ SB, layered over 1 ml_ pre-cooled 5%Ficoll and centrifuged at 45,000xg for 45 min at 4⁰C. The supernatant was removed carefully, and the pellet was resuspended in 100//L pre-cooled 5% Ficoll, which was then layered over 1 ml_ pre-cooled 13% Ficoll and centrigured at 45,000 for 45 min at 4⁰C. A high interface containing synaptosomes was recovered, the remaining supernatant removed and the pellet containing purified mitochondria was kept. The interface was diluted with SB and centrifuged at 45,000 for 45 min at 4⁰C to pellet the synaptosomes. Micro-dissection. Brains were harvested from WT, Δtau and tau^1' mice, and hippocampi prepared using Dumont #5 forceps (FST, Canada). Then, the hippocampi were sliced transversely under a SZX7 stereo-microscope (Olympus, Japan) using micro-scissors (FST, Canada). Superficial cells were visualized with hematoxylin (HDS, Australia), and subsequently, the cell body layer (CA1 ) was micro-dissected from their dendrites. The resulting material was lysed in RIPA buffer and analysed by Western blotting as described above.

Experimental seizures. Seizures were induced by intraperitoneal injection of (50mg/kg body weight) pentylenetetrazole (PTZ; Sigma, USA) at as described (Roberson, E.D., et a/. Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model. Science 316, 750-754 (2007)). Mice were video- monitored and the seizure severity was rated by an independent, blinded person, as 0: no seizures, 1 : immobility, 2: tail extension, 3: forelimb clonus, 4: generalized clonus, 5: bouncing seizures, 6: full extension and 7: death.

Quantitative PCR. Total mRNA was isolated from forebrains using Trizol® (Invitrogen, USA) following the manufacturer's recommended procedure. Second strand DNA was generated from DNase (Promega, USA)-treated mRNA using Superscript III Reverse Transcriptase (Invitrogen, USA). Quantitative PCR with SYBR green (ABI, USA) was performed in an Mx4000 real-time PCR cycler (Stratagene, USA). Primers were for human APP 5'-atttgaaggacttggggagg-3'and 5'-atcatggtgtggtggaggtt-3' and for actin 5'- ggctgtattcccctccatcg-3' and 5'-ccagttggtaacaatgccatgt-3'.

A/? levels. Aβ1-40 and Aβ1-42 levels were measured in brain lysates by ELISA (Genetics Company, Switzerland) following the manufacturers instructions.

Primary cultures and immunocytochemistry. For hippocampal low-density cultures, brains were harvested in HBSS (pH 7.3; Sigma) and meninges removed. The midbrain and hippocampus was removed from both hemispheres, followed by incubation of the cortices for 30 min in trypsin at 37^°C. The tissue was titrated with fire-polished Pasteur pipettes after adding DNase, resulting in a homogeneous cell suspension. The cell suspension was adjusted to a cell density of 5 x 10³ hippocampal and 1 x 10⁶ cortical cells/ml respectively. 200//L hippocampal cells were plated on poly-D-lysine-coated cover slips which were mounted in the centre of a 12-well culture plate well. Subsequently, 200μl_ cortical cells were plated in a circle around the coverslips to support hippocampal cell growth. The plating medium (DMEM/FBS) was replaced after 2 hours by 1 mL Neurobasal medium containing B27 supplement and Glutamax (Gibco). Cortical neurons were cultured from E16.5 embryonic brains at a density of 22,000 cells/cm² as described.

Cells were used for experiments after 20 days in culture, lmmunohistochemistry was performed as described. Briefly cells were fixed in 4% paraformaldehyde and subsequently washed with PBS. For surface staining of NR1 , cells were blocked with blocking buffer (PBS containing 3% heat inactivated goat serum and 2% bovine serum albumin), followed by incubation with mouse-antiNR1 (Millipore) at room temperature for 2 hours. Cells were then washed, fixed, permeabilized with 0.2% NP-40 (Sigma) in PBS and stained with rabbit anti-drebrin (Sigma). Primary antibodies were visualized using Alexa-coupled secondary antibodies (Molecular Probes). For cell toxicity, H2O2, staurosporine, NMDA (all Sigma) and A/?i_-42 (Bachem) were used at indicated concentrations diluted in culture medium. A/?-^ was pre-aggregated at a concentration of 100/yM as described. The Tat-NR2B9c peptide was purchased from Auspep (Australia) at the highest possible purity (>95%).

Statistics. Statistics was done with the Prizm 4 software (GraphPad Software, USA). Values are given as mean ± standard error.

EXAMPLE 2 Truncated tau (Δtau) attenuates premature mortality of APP23 mice

This example demonstrates that compounds including those having a conformation comparable to Δtau are useful for treatment of disease characterized by excitotoxicity.

Neuronal expression of A/?-forming human mutant APP in transgenic mice is associated with a marked early lethality. Compared to other APP expressing models, the premature mortality is even more pronounced in APP23 mice expressing K670N/M671L mutant human APP (APP^swe). Tau is both a target of Aβ as well as essential for mediating its toxicity, also with regards to memory impairment. By crossing the APP23 on a tau- deficient background, we found that heterozygous and to a higher extenttau-deficiency is sufficient to improve the survival of APP23 transgenic mice (Fig. 1c).

Tau has two principal domains, a microtubule (MT)-binding domain (MTB) that stabilizes MTs, and the amino-terminal projection domain involved in yet ill-defined MT- independent functions. We hypothesized that expression of the projection domain alone (Δtau) would uncouple MT-dependent and -independent functions of tau by competing for specific interaction partners, and thereby allow an assignment of specific signaling functions to these domains. To test this in vivo, we generated transgenic mice that express Δtau under control of the neuronal mThy1.2 promoter (Figure 6). Four strains that expressed Δtau at comparable levels (data not shown), with line 74 expressing Δtau at 1.6-fold higher levels than endogenous tau. The transgene was expressed in brain areas including the hippocampus, cortex and amygdala (Fig. 1a and Figure 6). Whereas tau was strongly phosphorylated in transgenic mice expressing full-length P301 L mutant tau, hardly any phosphorylation of transgenic Δtau was observed (data not shown).

When we crossed APP23 mice with Δtau transgenic or tau-deficient mice (Fig. 1b), this resulted both in a significantly delayed onset of mortality and an improved the overall survival (Fig. 1c). Whereas any rescue (either on a tau'^' background or by expressing Δtau) was partial, expression of Δtau on a heterozygous teu-deficient (tau^+/~) background rescued the lethality of APP23 completely. This suggests additive beneficial effects of tau-deficiency and Δtau expression on APP23 survival.

We next determined whether Aβ levels would be reduced in the crosses because of the decreased expression of APP₁ due to titration of transcription factors used by the respective promoters, or due to altered processing and aggregation of APP, protecting the APP23.Δtau or APP23.tau^'/~ mice from premature mortality. This, however, is unlikely as neither human APP mRNA levels (Fig 2a), nor Aβ levels (Fig. 2b) nor plaque burden (Fig. 2c,d) were changed upon Δtau expression or taw-deficiency. Taken together, expression of truncated tau in APP23 mice attenuated A/?-associated premature mortality, without changing Aβ levels. EXAMPLE 3 Somato-axonal distribution of Δtau is independent of endogenous tau

This Example demonstrates interference with an excitotoxic signal can be implemented by administration of a compound that prevents fyn from localizing to dendrites and post synaptic cleft.

We next determined the subcellular distribution of Δtau, as this may be related to the rescue of A/?-mediated toxicity. In the transgenic mice Δtau showed a distribution pattern consistent with a localization to the cell membrane as shown for cortical neurons (Fig. 3a). However, whereas full-length P301L tau transgenic mice showed a somatodendritic distribution of transgenic tau in addition to the physiological axonal localization, Δtau was found in the soma, but mainly excluded from the dendrites as shown for hippocampal CA1 neurons (Fig. 3b,c).

When we further examined axonal structures in different brain regions, we found that Δtau, despite the lack of an MTB, was localized to axons and their terminals of CA1 neurons projecting to subiculum and fornix (Fig. 3d and data not shown), and of amygdaloid and cortical neurons in the anterior commissure (Fig. 3e). Similarly, in transfected primary hippocampal neurons, Δtau localized to axons showing an increasing proximo-distal gradient similar to endogenous tau (data not shown).

We next determined whether in the presence of endogenous full-length tau, Δtau may be hitchhiked to the axonal compartment. However, when we examined the distribution of Δtau on a tau^1' background, it was still localised to the axon, suggesting that axonal localization is independent from MT association (Fig. 3d,e). Taken together, Δtau associates with or close to the cell membrane, localizes to the soma and axon, and is excluded from dendrites.

EXAMPLE 4 Δtau mediates resistance to fatal excitotoxicitv

This example demonstrates that compounds including those having a conformation comparable to Δtau are useful for treatment of symptoms of diseases characterized by excitotoxicity. The full extension posture observed in APP23 mice that died premature is consistent with fatal status epilepticus. Moreover, we observed repeatedly that APP23 mice presented with generalized seizures. For comparison, experimentally induced excitotoxicity characteristically presents as generalized seizures in mice. Surprisingly, tau-deficiency were reported to be protected from experimentally induced excitotoxicity. Thus, we speculated that Δtau expression may similarly reduce excitotoxic damage in APP23 mice. Therefore, we induced convulsions with pentylenetetrazole (PTZ) to examine the susceptibility to excitotoxicity of APP23, Δtau, tau^"7" and wild-type mice and crosses derived thereof. We found that seizure severity was significantly reduced in Δtau and tau^"Λ mice compared to WT controls (Fig.4a). Similarly, the latency for severe convulsion was increased in Δtau and tau^'7' mice compared to WT controls (Fig. 4b). In contrast, APP23 mice showed the most severe seizure response with all mice reaching status epilepticus (n=11), together with a reduced convulsion latency, and hardly any mouse surviving the experiment (1/11 ) (Fig. 4c). However, both the expression of Δtau and tøu-deficiency decreased seizure severity, reduced fatality and increased convulsion latency in APP23 mice significantly (Fig. 4c,d). Taken together, similarly to tau-deficiency, expression of Δtau decreases susceptibility of APP23/Δtau mice to experimentally induced seizures.

EXAMPLE 5 Fvn is aberrantly localised in Δtau and tau-deficient mice

This Example demonstrates interference with an excitotoxic signal can be implemented by administration of a compound that prevents fyn from localizing to dendrites and post synaptic cleft

Excitotoxicity is mediated by NRs. Hence, alterations in the NR and its signaling may underlie the decreased susceptibility of Δtau and tau^"7" mice to excitotoxicity. However, levels of the NR subunits NR1 , NR2a and NR2b were not altered in Δtau and tau^"7" mice compared to wild-type controls (Figure 7). Accordingly, NR1 staining of permeabilized primary hippocampal neurons obtained from wild-type and tau^'Λ mice appeared similar, as was the dendritic morphology (Fig. 5a). In contrast, when we stained for NR1 present on the dendritic surface, NR1 staining intensity of dendrites was markedly reduced in tau^'Λ neurons compared to wild-type cells, reflecting reduced surface expression of NR. (Fig. 5a, b). Stabilization of NR at the cell membrane is known to be promoted by its interaction with PSD-95, which also couples NRs to intracellular signaling pathways including those mediating excitotoxicity. Therefore, we next addressed the interaction of the NR with the post-synaptic density protein PSD-95 by co-immunoprecipitation. We found that hardly any NR1 co-precipitated with PSD-95 from Δtau and tau^'7' compared to wild-type extracts, which is consistent with a decreased interaction of NR and PSD- 95 (Fig. 5c). Phosphorylation of the NR subunit NR2b by Src-kinases such as Fyn strengthens the interaction of NR and PSD-95, and increases NR activity. Whereas levels of NR2b were unaltered in hippocampal extracts from Δtau and tau^"7" mice compared to wild-type controls (Fig. 5d and Fig. 7), levels of NR2b phosphorylated at the Fyn epitope Y1472 were significantly reduced (Fig. 5d). Therefore, we next addressed Fyn in Δtau mice. Total levels of Fyn were unchanged (Figure 7). However, immunoprecipitation with antibodies specific to endogenous tau co-precipitated less Fyn from Δtau hippocampi than from wild-type controls (Fig 5e), suggesting that Δtau competes with endogenous tau for interaction with Fyn. To determine the subcellular distribution of Fyn in the absence of tau or in the presence of Δtau, we performed a micro-dissection of the CA1 region of wild-type, Δtau and tau^"7" hippocampi. In wild-type preparations, Fyn was mainly found in the dendrite extract, consistent with a postsynaptic distribution (Fig. 5f). In contrast, Fyn accumulated in the cell body fraction of both tau-/- and Δtau hippocampi. We next obtained synaptosomal hippocampal preparations from wild-type,Δtau and tau^"7" hippocampi. Western blotting revealed that levels of Fyn in the synaptic preparation were markedly reduced in Δtau and tau^'7' mice compared to wild-type controls (Fig. 5g), suggesting that the synaptic localization of Fyn is tau-dependent. Taken together, we found in both tau^"7" and Δtau brains a decreased post-synaptic activity of the Src kinase Fyn, a reduced phosphorylation of its substrate NR2b, a reduced interaction of NR and PSD-95 and a reduced surface expression of NR.

EXAMPLE 6 Animal ΔTau model for screening for compounds that interfere with excitotoxicitv

Excitotoxicity in mice can be induced by intraperitoneal injection of pentylenetetrazol (PTZ, Sigma, USA) at 50mg/kg body weight as described (Roberson, E.D., et al. Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model. Science 316, 750-754 (2007)). Mice are video-monitored and the seizure severity is rated by an independent, blinded person, ranging from 0: no seizures, 1 : immobility, 2: increased exploratory activity, 3: automatisms, 4: mild limbic convulsions, 5: major limbic convulsions, 6: full extension, and 7: death.

In addition to the rating of the seizure the latency for more severe seizures is recorded.

Wild-type and APP FAD mutant mice are highly susceptible to the experimental induction of seizures, whereas ΔTau transgenic are highly resistant.

A compound having properties similar to ΔTau expression or in preventing the formation of an NR-PSD-95 complex confer both wild-type and APP FAD mutant mice with an increased resistance to excitotoxicity as determined by the degree of seizure and the latency.

The ΔTau strain would be included in the above excitotoxicity study as a negative control. Furthermore, any drug working on the above principle should not improve (or should not considerably improve) the resistance of mice to excitotoxicity when the ΔTau transgene is present.

EXAMPLE 7. Cell based model for screening for compounds that interfere with excitotoxicitv

Uncoupling the NR/PSD-95 interaction reduces Aβ toxicity. This is achieved either by transfection of ΔTau or fragments derived thereof (in the following labelled ΔTau for simplicity) or by using the Tat-NR2B9c peptide. This peptide is a fusion peptide composed of a fragment of the NR2 receptor and the Tat peptide that has been shown to disrupt the NR/PSD-95 interaction (Aarts, M. et al. (2002). Treatment of ischemic brain damage by perturbing NMDA receptor- PSD-95 protein interactions. Science 298, 846-50), similar to what we see in ΔTau mice and in ΔTau transfected cell lines and cortical primary cultures. Mature primary cortical neurons (20 DIV) are pre-incubated with either ΔTau or Tat- NR2B9c before treatment with either NMDA or A/? peptide. 24 h later, cell death is analysed by propidium iodide (Pl) uptake. Compared to cultures treated without preincubation with ΔTau or Tat-NR2B9c (vehicle only) this results in less Pl-positive cells. Numbers of Pl-positive cells are not changes by Tat-NR2B9c treatment of controls that have not been treated with NMDA or A/?.

All publications and patents cited in this specification are incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Further, any polypeptide sequence, polynucleotide sequences or annotation thereof, are incorporated by reference herein. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

Although the present invention has been described with reference to the specific embodiments thereof various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. IN addition, many modifications can be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, sprit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Claims

I . A compound for preventing, inhibiting or antagonising fyn from phosphorylating an amino acid residue in a molecule having an amino acid sequence shown in SEQ ID NO: 1.

2. The compound is capable of binding to fyn, to prevent fyn from migrating to a region of a nerve cell cytosol adjacent a post synaptic membrane.

3. The compound holds or otherwise sequesters fyn within the nerve cell soma.

4. A peptide which has an amino acid sequence of a projection domain of a Tau protein or fragment thereof capable of binding fyn or other src related kinases.

5. A peptidomimetic which has an amino acid sequence of a projection domain of a Tau protein or fragment thereof capable of binding fyn or other src related kinases.

6. A nucleic acid encoding a peptide according to claim 4.

7. A cell including a peptide according to claim 4.

8. A non human transgenic animal including a peptide according to claim 4.

9. A molecular complex including a compound, peptide or peptidomimetic according to one of the preceding claims bound to fyn.

10. An antibody for binding to a compound, peptide or peptidomimetic according to one of the preceding claims.

I I . A pharmaceutical composition including a compound, peptide, peptidomimetic or an antibody of according to one of the preceding claims and a pharmaceutically effective carrier, diluent or excipient.

12. A method for maintaining neuron function or preventing the loss of neuron function in an individual including administering to the individual a Tau projection domain or fragment thereof.

13. A method for antagonising, preventing or inhibiting fyn from phosphorylating a tyrosine residue in a molecule having an amino acid sequence shown in SEQ ID No: 1 including the step of contacting fyn with a compound, peptide or peptidomimetic according to one of the preceding claims.

14. A method for treating or preventing a condition, disease or syndrome that is a consequence of excitotoxicity or associated with excitotoxicity in an individual including providing an individual with a compound, peptide or peptidomimetic according to one of the preceding claims.

15. A method according to claim 14, wherein the compound is a Tau projection domain, fragment or peptidomimetic thereof.

16. A method according to claim 14 or 15, wherein the condition, disease or syndrome is selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease or other neurodegenerative diseases.

17. A method according to any one of claims 14 to 16, wherein the condition, disease or syndrome is a consequence of excitotoxicity or associated with excitotoxicity.

18. A method for monitoring the treatment of an individual having a condition, disease or syndrome that is a consequence of excitotoxicity or associated with excitotoxicity including:

- selecting an individual who has been provided with a compound, peptide or peptidomimetic according to one of the preceding claims for treatment of a condition, disease or syndrome that is a consequence of excitotoxicity or associated with excitotoxicity; - determining whether the individual contains a compound, peptide, peptidomimetic or molecular complex according to compound, peptide or peptidomimetic according to any one of the preceding claims;

19. A method for identifying a compound for preventing, inhibiting or antagonising fyn from phosphorylating a tyrosine residue in a molecule having an amino acid sequence shown in SEQ ID No: 1 including:

- providing a compound, peptide or peptidomimetic for which capacity to prevent, inhibit or antagonise fyn function is to be determined;

- contacting the compound, peptide or peptidomimetic with fyn;

- determining whether fyn is prevented from localising to a post synaptic cleft thereby preventing phosphorylation of a tyrosine residue in a molecule having an amino acid sequence shown in SEQ ID No: 1.