WO2008135790A1 - Biologically active c-terminal fragment of acetylcholinesterase - Google Patents

Abstract

The C-terminal (30) amino acid fragment of the T-isoform of acetylcholinesterase, exhibits biological activity consistent with a role in the aetiology of neurodegenerative disease, particularly Alzheimer's Disease, Parkinson's Disease and Motor Neurone Disease. Useof this polypeptide (T30 peptide) is proposed both as a screening tool in assays for therapeutics and as adiagnostic marker for neurodegenerative disease.

Description

Biologically Active C-terminal Fragment Of Acetylcholinesterase

Field of the invention

The present invention relates to the isolated polypeptide consisting of the C-terminal 30 amino acid residues of the T-isoform of the enzyme acetylcholinesterase (T-AChE).

This polypeptide fragment, designated T30 peptide, exhibits non-enzymatic biological activities consistent with a role in causation of neurodegenerative disease. Hence, it is of interest in relation to both diagnosing, and providing new treatments for, such disease, especially Alzheimer's Disease, Parkinson's Disease and Motor Neuron Disease.

Background to the invention

Alzheimer's disease (AD), Parkinson's disease (PD), and Motor Neuron disease (MND) are the three most common neurodegenerative disorders, but it is still not known why some neurons, and not others in the brain, are particularly vulnerable to neurodegeneration. One familiar idea is that, for AD at least, the key pathological process is the formation of amyloid plaques and fibrils, derived from abnormal cleavage of amyloid precursor protein (APP). However, the 'Amyloid Hypothesis' (Hardy & Higgns, Science (1992) 256, 84-85, 'Alzheimer's disease: the amyloid cascade hypothesis') does not account for the regional neuronal selectivity which characterizes neurodegeneration in AD. In contrast, the 'Cholinergic Hypothesis' (Vickers et al. Prog. Neurobiol. (2000) 60, 139-165, The cause of neuronal degeneration in Alzheimer's disease') suggested the primary cause of AD is a deficiency in the transmitter acetylcholine (ACh). However, there is a mismatch between the neurons that are especially vulnerable and the cholinergic systems in the brain.

An alternative proposal centres on neurodegeneration being attributable to the slow death of cells extending from the spinal cord to the midbrain. This phylogenetically old group of cells, referred to as 'global' neurons are fundamentally different from their counterparts elsewhere in the CNS, in diverse characteristics ranging from fetal provenance to biochemical properties (Woolf, Neurosci. (1996) 74, 625-651. 'Global and serial neurons form a hierarchically arranged interface proposed to underlie memory and cognition'). Of particular interest is that global neurons, irrespective of the transmitter they express, all contain acetylcholinesterase (AChE). Moreover, although Alzheimer's Disease and Parkinson's Disease have different clinical profiles, it has long been acknowledged that the underlying pathologies can overlap and both diseases can be attributed to different degrees of disruption of global neuronal groups.

In line with this proposal, it has for some years been further proposed that AChE can give rise to associated non-enzymatic action in the brain and that this could provide an underlying causation for neurodegenerative disease (Greenfield SA (1984) Trends Neurosci. 1_, 364-368, 'Acetylcholinesterase may have novel functions in the brain'; Greenfield SA (1996) Neurochem. Int. 28, 485-490, 'Non-classical actions of cholinesterases: role in cellular differentiation, tumorigenesis and Alzheimer's disease'; Greenfield and Vaux (2002) Neuroscience 113, 485-490, 'Parkinson's disease, Alzheimer's disease and motor neurone disease: identifying a common mechanism.'). Consistent with this hypothesis a 14 mer fragment of T-AChE (Synaptica Peptide or T14 peptide) was previously identified and shown to be able to modulate calcium flux through alpha 7 nicotinic receptors (see published International Applications WO 97/35962 and WO 01/73446; see also Greenfield et al., J. Neurochem. (2004) 90, 325-331 , ' A novel peptide modulates alpha 7 nicotinic responses: implications for a possible trophic-toxic mechanism within the brain.'). A key tenet of this hypothesis has been that vulnerability of global neurons to degeneration might be associated with aberrant activation of a developmental mechanism mediated by Ca²⁺ entry into neurons. However, a query has remained over whether Synaptica Peptide is operative in vivo.

Multiple C-terminal isoforms of AChE adds increasing complexity to its functional capability (Massoulie et al. et al. (1998) J. Physiol. Paris 92, 183-190, 'Acetylcholinesterase: C-terminal domains, molecular forms and functional localization'). Whereas lower weight monomeric (G1 ) and dimeric (G2) forms are most abundant in the developing CNS, in adult neurons, the AChE protein predominantly exists in tetrameric (G4) membrane-bound T-AChE form on cell surface membranes, where it hydrolyses ACh in the synaptic cleft. R-AChE always remains monomeric, soluble and secretable (see Figures 1 and 3). As shown in Figure 3, all isoforms of AChE are derived from a single gene transcript and contain the invariable exons 2, 3 and 4. The T-AChE isoform arises through alternative imRNA splicing of exon 6 to the invariable exons.

Whereas R-AChE upregulation correlates with early developmental events and stress responses, evidence suggests that the T-AChE isoform is associated with neurodegenerative processes (Sternfeld et al. (2000) Proc. Natl Acad, Sci. USA 97, 8647-8652; Zhang et al. (2002) Cell Death Differen. 9, 790-800). In the AD brain, T- AChE reverts to a more immature developmental form (G1 ) (Arendt et al. (1992) Neurochem.lnt. 21_, 381-396) and all sub-cortical nuclei showing pathology, irrespective of transmitter, express AChE (Smith & Cuello (1984) Lancet 1_, 513; Shortridge et al. (1985) Clin. Neuropathol. 4,227-237; Greenfield 1996 ibid). AChE and amyloid beta- peptide (AβP) have been co-localized to senile plaques in AD (Inestrosa et al. (2005) Subcell. Biochem. 38, 299-317) and AChE increases the neurotoxicity of amyloid fibrils (Inestrosa et al. (1996) Neuron 16 , 881-891 ; Alvarez et al. (1998) J. Neurosci 18, 3213-3223). AChE and the amyloid precursor protein (APP) exhibit functional similarities and co-dependence. Both proteins are transiently expressed during specific developmental stages, display properties of cell adhesion molecules and are secreted by neurons and glia. AChE and APP are decreased in cerebrospinal fluid (CSF) of AD patients (Appleyard et al. (1983) Lancet U_, 452; Arendt et al. (1984) Lancet 1_, 173; Farlow et al. (1992) Lancet 340, 453-454) and both proteins can enhance calcium entry into cells (Webb et al. (1996) Eur. J. Neurosci.8, 837-841 ; Ueda et al. (1997) J. Neurochem. 68, 265-271 ). Moreover, AChE can induce the expression of APP and accumulation of AβP (von Bernhardi et al. (2003) Neurobiol. Dis. J_4, 447-457) and, conversely, AβP induces AChE production (Saez-Valero et al. (2003) Biochem. 43, 15292-15299) in both neurons and glia, suggesting that the synthesis and metabolism of these molecules are linked physiologically. Thus there has grown a large body of evidence that AChE plays an integral role in neurodegenerative pathology.

The C-terminal region of the T-AChE protein has many structural and functional similarities to the N-terminus of the predominant amyloid beta protein (Aβ42) found in

AD. The 14 amino acid residue portion corresponding to Synaptica Peptide, within the

T30 peptide, has high homology to the N-terminal region of Aβ42 (Greenfield & Vaux

(2002) ibid). Figure 2 shows the sequence alignment of the amyloid precursor protein and T-AChE with the relevant peptides (T30, Synaptica Peptide (T14) and Aβ42) highlighted for comparison.

T-AChE and R-AChE have identical sequences except for their C-terminal exons (see Figure 3). Moreover, it has previously been reported that a natural C-terminal truncated form of T-AChE is present in fetal bovine serum relying on sequencing of tryptic peptides (Saxena et al. (2003) Biochem. 43, 15292-15299, 'Natural Monomeric Form of Fetal Bovine Acetylcholinesterase Lacks the C-terminal Tetramerization Domain'). However, the truncated monomer size given is 543-547 compared to 583 for mature T- AChE and no information was provided by Saxena et al. on any C-terminal fragment, or more especially reason to isolate and investigate properties of the C terminal 30 amino acid residues of T-AChE.

Published International Application no. WO 00/73427 suggests use of the C-terminal region of R-AChE or T-AChE as a source of therapeutic peptides, although the data provided, if anything, makes a case for focussing on the C-terminal 26 mer of R-AChE (ARP) and only the C-terminal 40 mer of T-AChE was tested. There is no suggestion of any isoform of AChE being a source of any peptide having physiological relevance to disease causation; WO 00/73427 provides no indication that unusual properties beyond those investigated might be found by selecting the C-terminal 30 amino acid residues of T-AChE. Thus, it is to be noted that only the much shorter T14 peptide was previously recognised as exhibiting properties consistent with a mechanism for neurodegeneration

Evidence is now presented that in a number of test systems in which the T14 peptide exhibits biological activity, isolated T30 peptide also exhibits biological activity but is more potent or more efficacious. Significantly, from the point of view of linking T-AChE with harmful calcium flux into neurons, T30 peptide has been shown in live cell binding experiments to bind like Synaptica Peptide to alpha 7 nicotinic acetylcholine receptors, but to bind with higher affinity to the allosteric modulation site targeted by T14 and to have a second binding site on the receptor.

The α7-nicotinic acetylcholine receptor (α7-nAChR) is a potent calcium ionophore, which has been suggested to be pivotal in neural development, neural responses to injury and neurodegeneration. Consistent with its role in degenerative processes, α7- nAChR mRNA is upregulated in affected areas of the AD brain (Nagele et al. (2002) Neurosci. 110, 199-211 ) and in transgenic mice overexpressing APP. Aβ42 and T14 peptide exhibit modulatory activity at the α7-nAChR and potentiate calcium influx through L-type voltage gated calcium channels (L-VGCC) (Dineley et al. (2001 ) J Neurosci. 21 , 4125-4133; Bon & Greenfield (2003) Eur. J. Neurosci. 17, 1991-1995). AβP binds α7-nAChR with picomolar affinity (Wang et al. (2000) J. Neurochem.75, 1 155-1161 ), promoting rapid Ca²⁺ influx into hippocampal neurons and activating kinase signalling cascades. Moreover, during critical developmental periods, the transient expression of this receptor correlates spatially and temporally with the transient appearance of AChE (Taylor et al (1994) Biochem. Soc. Trans. 22, 740-745; Broide et al. (1996) J. Neurosci. 16, 2956-2971 ).

The newly presented evidence herein that T30 peptide binds to the α7-nAChR, coupled with the fact that it can be generated by a single protease cleavage in vivo (see in Figure 4 the indicated beta-secretase cleavage site at the N-terminus of the T30 sequence), means that it is now proposed as a physiologically relevant peptide of particular interest in relation to, for example, screening for agents of potential use in treating neurodegenerative disease, especially for example AD, PD and MND. It is postulated that such agents also include agents which inhibit proteolytic release of the T30 peptide from T-AChE. Importantly, the findings now presented also open new possibility for diagnosis of neurodegenerative disease, for example AD, PD and MND, especially AD.

Summary of the invention

Many forms and fragments of AChE have previously been suggested to be of physiological interest, but as indicated above results now presented herein lead to selection of the T30 peptide as an isolated peptide having particularly significant properties for use as a tool in advancing diagnosis and treatment of neurodegenerative disease.

In a first aspect, the present invention therefore provides an isolated polypeptide (T30 peptide) consisting of the C-terminal 30 amino acid residues of the T isoform of acetylcholinesterase (T-AChE), said polypeptide consisting of the sequence

KAEFHRWSSYMVHWKNQFDHYSKQDRCSDL (SEQ. ID. NO.1 )

and functional analogues thereof, including same length analogues. Such functional analogues will be understood to retain substantially the same biological activity as T30 peptide in any of the tests for biological activity set out in the examples and like T30 peptide itself to be distinguishable from T14 peptide (AEFH RWSSYMVHWK; SEQ. ID no.2) in such tests. Thus, for example such analogues may be selected on the basis of binding ability to the α7-nAChR and will show higher affinity binding to the modulatory site targeted by T14 than T14 itself and binding to the same second site as T30. Like Aβ42, the T30 peptide binds the α7-nAChR with picomolar affinity whereas T14 is less potent at this receptor. The data presented in Example 1 below is consistent with T30 interacting with both the active ligand binding site and the allosteric modulatory site on the α7-nAChR.

For the purpose of use in assays for biological activity, it will be understood that "isolated" will equate with isolation in soluble form, although a polypeptide of the invention may for some purposes be immobilized on a solid support. A polypeptide of the invention may be provided as part of a fusion construct. It may also be labelled.

In a further aspect, the invention provides use of a polypeptide of the invention as an antigen and/or screening agent to provide an antibody or antibody fragment which binds T30 peptide. Antibodies or antibody fragments thus obtained may be of value, for example, for use in detecting the T30 peptide for diagnostic purpose.

Thus, in another aspect, the invention provides a method of diagnosing in an individual a neurodgenerative disorder, especially AD, PD or MND, most especially AD, which comprises detecting an abnormal T30 peptide level in a cerebrospinal fluid (CSF) sample.

As indicated above, also provided is use of such a polypeptide as a screening tool to screen for agents capable of antagonising biological activity of the polypeptide in one or more in vitro tests for such activity. Such agents may be of interest as therapeutic agents for treatment of neurodegenerative disorders, possibly after modification or packaging for delivery across the blood-brain barrier.

In a still further aspect, there is provided an agent which prevents proteolytic cleavage of T30 peptide from T-AChE in the brain for use in treating a neurodegenerative disorder, for example AD, PD or MND, especially, for example, AD. Such agents may include antisense oligonucleotides, antisense PNAs and siRNAs, which target expression of, for example, the brain β-secretase implicated in production of T30 peptide in vivo (see again Figure 4). Again such agents may be modified or packaged for delivery across the blood-brain barrier. The invention also extends to pharmaceutical compositions containing such an agent together with a pharmaceutical carrier or diluent. Identification of such agents may also be by means of simple in vitro screening assays in which T-AChE is exposed to physiological proteolytic conditions whereby it is cleaved to release T30 peptide and such assays form a still further aspect of the invention. The invention will be further described below with reference to the figures already noted above and further figures which are now detailed.

Brief description of the figures

Figure 1 : Summary of the major protein isoforms of derived form the AChE gene. E- AChE is specifically found in blood cells, whereas R-AChE and T-AChe, as already noted above, are the predominant isoforms in brain tissue. T-AChE is the adult CNS isoform that can be post-translationally modified to allow formation of membrane-bound tetramers, whereas R-AChE always remains monomeric, soluble and secretable. Globular forms represent the monomeric and multimeric assemblies of individual subunits.

Figure 2: Sequence alignment of the amyloid precursor protein (APP) and the C- terminal region of T-AChE with the T30 peptide of AChE and Aβ42 fragment of APP light shaded. Also shown by dark shading are the T14 (Synaptica Peptide) portion of the T30 peptide and the homologous portion of Aβ42.

Figure 3: Schematic diagram showing the C-terminal exons of R-AChE and T-AChE and the AChE and control polypeptides used in the examples. As indicated above, all isoforms of AChE are derived from a single gene transcript and contain the invariable exons 2, 3 and 4. The T-AChE isoform arises through alternative mRNA splicing of exon 6 to the invariable exons. Truncated AChE (T548) is a recombinant protein, translated from cDNA containing exons 2, 3, and 4, but lacking a C-terminal exon, which was produced from the glycophospholipid-linked form of mouse AChE (Marchot et al. (1996) 'Soluble monomeric acetylcholiesterase from mouse; expression, purification and crystallization in complex with fasciculin' Protein Sci. 5, 672-679). The underlined amino acid sequence highlights the unique C-terminus of the T-AChE isoform derived from exon 6 of the AChE gene with the location and sequence of AChE peptides indicated. Control peptides which were used in experimentation discussed in the examples include: S14, which is a scrambled version of AChE T14 peptide; B14, which comprises the 14 amino acid region in butyrylcholinesterase (BuChE) that is homologous to AChE T14; and SB14, which is the scrambled version of the same region of BuChE. Figure 4: Diagram showing protease cleavage sites in APP and the C-terminal region of T-AChE. Cleavage sites for α- and β-secretase are indicated. Small arrows indicate sites cleaved in APP by other proteases. Lightly shaded box highlights Aβ42. Dark shaded boxes indicate area of homology between APP and the Synaptica Peptide (T14 region) of T-AChE.

Figure 5: Results of binding of T30 and T14 peptides to the α7-nAChR in live cell binding assays. (A) Comparison of T30 and T14 peptide binding with known α7-nAChR ligands at concentrations indicated (n=6). MLA=methylyllcaconitine, αBTX=α- bungarotoxin, Ach= acetylcholine, IVM = invermectin (B) Displacement of [¹²⁵I]-O- bungarotoxin with T14 and T30 peptides at a various concentrations.

Figure 6: Western blot analysis showing upregulation of α7nAChR expression in cultured GH4-hα7 cells expressing the receptor following treatment with T30 and T14 peptides. Representative western blots of α7-nAChR protein levels in control (C) and peptide (T14, T30) treated GH4-hα7 cells. All experiments were performed a minimum of 2-3 times. A. Protein levels as assessed in total cell homogenate and in membrane compartments after 6 or 24 hr peptide exposure. The filled and open arrow heads indicate α7-nAChR and actin at 55 and 42 kDa MW, respectively. Actin was used as an internal standard. B. After 24 hour peptide treatment, cells were treated with the membrane-impermeant cross-linking reagent bis(sulfosuccinimidyl)suberate sodium salt (BS³). The filled and open arrow heads indicate α7-nAChR and high molecular weight aggregated species of the α7-nAChR receptor, respectively. C. Representative western blot of α7-nAChR protein levels in control and T15 (control peptide) treated cells.

Figure 7: Results showing increased astroglia cell proliferation/metabolism in the presence of low concentrations of T30 and T14 peptides by assay determining bioreduction by metabolically active cells, of a tetrazolium compound (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) to form a coloured formazan product.

Figure 8: Determination of secreted AChE, secreted BuCHe and intracellular AChE in astroglia cell culture when such cells are exposed to T30 and T14 peptides for 24 hrs.

Figure 9: Effect of T30 and T14 peptides on substrate inhibition of AChE. Figure 10: RT-PCR analysis of α7-nAChR mRNA showing upregulation of α7-nAChR mRNA in GH4-hα7 cells after 24 hrs exposure to T30 and T14 peptides. GAPDH expression was used as an internal standard. Data shown are representative results of experiments performed a minimum of 3 times. A. Effect of varying concentrations of AChE peptides T14 and T30 on α7-nAChR expression. B. Effects of 100 nM control peptides, 10 nM full-length T-AChE, or 10 nM truncated T-AChE on α7-nAChR expression. Lane 1 = Control, 2 = T15, 3 = S14, 4 = B14, 5 = SB14, 6 = full-length T- AChE, 7 = truncated T-AChE (T548). C. Effect of T-AChE peptides T14 (100 nM) and T30 (100 nM) on α4-nAChR expression. D. Effects of MLA (10 μM), T30 (100 nM), and MLA (10 μM) + T30 (100 nM) on α7-nAChR expression.

Figure 1 1 : Schematic diagram showing envisaged role of APP and AChE in neurodgeneration.

Figure 12: Acute live cell binding in GH4-hα7 cells. Cells were pre-treated with peptides or drugs for 30 min, then [¹²⁵I] α-bungarotoxin (α-BTX) was added and cells were incubated for 1.5 hr at 37⁰C. Data shown is the average ± SEM of 3 separate experiments. A. Raw saturation binding data shows total, nonspecific, and specific binding for [¹²⁵I] α-BTX to α7-nAChR in this cell line. B. Specific binding of [¹²⁵I] α-BTX to α7-nAChR in fmol/mg protein with Scatchard analysis. C. Comparison of maximal specific [¹²⁵I] α-BTX binding displacement by AChE peptides T14 and T30 as compared with known α7-nAChR antagonists and agonists. MLA = methyllycaconitine; ACh = acetylcholine. D. Maximal specific [¹²⁵I] α-BTX binding displacement by control peptides, full length T-AChE, and truncated AChE (T548). E. Displacement binding profiles for AChE C-terminal peptides T14 and T30.

Figure13: Acute membrane binding in GH4-hα7 cells. Purified membranes (50-100 μg) from GH4-hα7 cells were incubated with peptides and/or drugs, 2 nM [¹²⁵I] α-BTX, and binding buffer overnight at 4⁰C. Data shown are the combined results of a minimum of 2 experiments each performed in triplicate and expressed as percent control specific binding. A. Competition binding with T30 concentrations varying from 1 pM to 10 μM. B. Effect of varying concentrations of T30 on α-BTX competition binding with ACh, MLA, and choline. C. Competition binding curve for ACh vs. ACh + T30 (100 nM). D. Competition binding curve for MLA vs. MLA + T30 (100 nM). E. Competition binding curve for choline vs. choline + T30 at various concentrations of T30. Figure 14: Specific saturation binding on cell membranes after chronic treatment of GH4-hα7 cells with 100 nM T-AChE peptides for A. 24 hr and B. 72 hr. Data shown are the average ± SEM of 2 separate experiments each performed in triplicate.

Detailed description

The T30 peptide of the invention and functional analogues thereof may be produced synthetically or by recombinant methods well known in the peptide synthesis field. In identifying such functional analogues, or antagonists of T30 peptide biological activity, one or more of the following identified biological effects of T30 peptide may be relied upon:

1. ability to bind the α7-nAChR;

2. ability to increase expression of α7-nAChR in the membrane of cells which express the receptor, e.g. GH4-hα7 cells as commercially available (Merck, USA)

3. ability to increase cultured astroglial cell proliferation/metabolism, e.g. as measured by MTS assay;

4. ability to increase secretion of AChE from cultured astroglial cells;

5. ability to increase intracellular AChE activity in cultured astroglial cells; 6. ability to inhibit substrate inhibition of AChE activity;

7. ability to change gene expression in cells, e.g. upregulate expression of α7- nAChR mRNA in cells expressing that receptor, e.g. GH4-hα7 cells.

As indicated above, tests for such biological effects may, for example, conform with the assay systems described in the examples. It is notable that in the case of all the biological effects so determined using the T30 and T14 peptides, T30 peptide was more potent or more efficacious than the T14 peptide.

Functional analogues of T30 peptide thus identified may include variants having one or more additions and /or deletions and /or substitutions, e.g. one or more conservative substitutions, compared to SEQ. ID No. 1 and which exhibit substantially the same activity or activities as T30 peptide tested. As indicated above, in all instances distinction will be maintained compared to activity of T14 peptide in the same test.

In identifying antagonists of T30 peptide as potential therapeutics for use in treating neurodegenerative disease, it may be particularly preferred to look at the ability of test agents to inhibit binding of T30 peptide to the α7-nAChR. For this purpose, membranes or cultured cells may be employed presenting functional α7-nAChR or a functional analogue thereof. Such a receptor will exhibit in the presence of Ca²⁺ ions and acetylcholine induced Ca²⁺ ion flux which can be blocked by α-bungarotoxin and modulated by T30 peptide binding at the allosteric modulation site, also targeted by T14. Such a receptor may be a native homomeric α7-nAChR, preferably a human α7- nAChR, in its normal membrane environment. However, preferably synthetic membranes, or more preferably cultured cells transformed to express a functional recombinant α7-nAChR, may be employed, e.g. Xenpous oocytes or other cells engineered to express human α7-nAChR as described in Published International Applications nos WO 94/20617 and WO 01/73446 and by Seguela et al. (J. Neurosci. (1993) 13, 596-604). SH-EP1-hα7 cells or GH4-hα7 cells may, for example, be preferred, both of which express high levels of AChE and the human α7-nAChR. (SH- EP1-hα7 cells are human epithelial— like clonal cells derived from the human SK-N-SK human cell line that have been stably transfected to express human α7-nAChR. GH4- hα7 cells are a well-known rat pituitary tumour-derived cell line, again transfected to express the human α7-nAChR.) In this case, binding of T30 peptide alone or in the presence of the test agent may be determined as in Example 1.

A potential therapeutic identified as an antagonist as above may be formulated into a pharmaceutical composition together with pharmaceutical carrier or diluent. Where delivery is desired across the blood-brain barrier, as indicated above it may be desirable to modify or package the antagonist to facilitate such delivery.

In addition to use as a screening tool, an isolated polypeptide of the invention may be used to as antigen and/or a screening agent to obtain antibodies or antibody fragments which bind T30 peptide, including such monoclonal antibodies and fragments thereof. Production of such antibodies may be followed by labelling with detectable label for use in assays. Such assays may include diagnostic assays as noted above.

It will be appreciated that envisaged diagnostic assays for neurodegenerative disease relying on identification of abnormal T30 peptide level in CSF fluid samples may take many formats, the critical requirement being to distinguish T30 peptide from AChE and other peptides/proteins sharing homology with T30 peptide in the sample. Thus, in carrying out such diagnostic assays anti-T30 antibodies may be employed together with other peptide/protein separation techniques well known in the diagnostic assay field. Such a diagnostic assay may be combined with other clinical observations in making diagnosis of a particular neurodegenerative disorder such as Alzheimer's Disease or Parkinson's Disease.

While it is envisaged that therapeutically useful agents for treatment of neurodegenerative disease may directly interfere with T30 peptide action in the brain, e.g. by inhibiting binding of T30 peptide to α7-nicotinic receptors, another immediately evident approach to treating neurodegenerative disease is to intervene with processing of T-AChE to produce T30 peptide. As indicated above, such processing is consistent with action of β-secretase on AChE and hence, agents which target β-secretase including for example, antisense oligonucleotides or PNAs or siRNAs which target expression of that protease are of immediate interest in this connection. Such an agent may be an antibody or antibody fragment. However, other agents which will inhibit processing of AChE to give the T30 peptide may be screened for. Such a screen may take the form of a specific assay for β-secretase inhibition.

In a still further aspect, the invention moreover provides a method of identifying a potential therapeutic agent for use in treating a neurodegenerative disease which comprises contacting a test agent with T-AChE exposed in vitro to physiological proteolytic conditions whereby in the absence of the test agent it is cleaved to release T30 peptide and determining whether such cleavage is prevented.

Means for packaging or modifying nucleic acids and polypeptides for delivery across the blood-brain barrier are known and may possibly be employed for delivering therapeutic agents as discussed above into the brain, but direct delivery into the brain may also be considered. It will be understood that treatment may include prophylactic treatment or treatment at an early stage before symptoms of neurodegeneration are observable.

The following examples illustrate the invention. Examples

Materials and Methods

All reagents were purchased from Sigma-Aldrich Co. Ltd., Poole, UK, unless otherwise noted. Disposables and cell culture plasticware were from Fisher Scientific, Loughborough, UK.

Preparation of Astroglia and Cell Culture Methods

Astroglia were prepared as previously described (Whyte & Greenfield (2003) Exp. Neurol. 184, 496-509: ' Effects of acetylcholinesterase and butrylcholinesterase on cell survival, neurite outgrowth and voltage-dependent calcium currents of embryonic ventral mesencephalic neurons'). Briefly, P1-P3 Wistar rats were treated with an overdose of isofluorane anaesthetic (Schedule 1 , Animal Scientific Procedures Act, UK, 1986), and then decapitated in a sterile environment. The cerebrum was removed by blunt dissection, rolled on sterile filter paper to remove meninges, cut into ~1 mm³ pieces, and dissociated with gentle trituration in Dulbecco's modified Eagle's medium with 4500 mg/l glucose and GlutaMAX (DMEM; Life Technologies Ltd., Paisley, UK) containing 10% fetal calf serum, 1 % penicillin/streptomycin, and 2.5 μg/ml amphotericin B. The dissociated tissue was plated into 75 cm² flasks precoated with poly-D-lysine (PDL), then incubated at 37 ^°C in a humidified atmosphere (95% air: 5% CO₂) for 7 days. Before passaging, confluent flasks were agitated on a shaking platform to dislodge any contaminating microglia adhering to the astroglial monolayer. Astroglia were washed with 1x Hanks balanced salt solution (HBSS), lifted with 1x trypsin, placed into clean PDL-coated 75 cm² flasks and allowed to reach confluency. Astroglia were again passaged into clean PDL-coated flasks or 6-well plates and allowed to recover for 1-3 days before experimentation. Identification of the cultures as Type 1 reactive astrocytes was confirmed by microscopic examination of morphology and immunocytochemical detection with a glial fibrillary acidic protein antibody (>99% positive). To reduce inter-animal variability, each experiment using astroglia was performed on a pool of astrocytes derived from the cortices of approximately 20 pups from two litters.

GH4-hα7 cells (Merck & Co, Inc, Rahway, USA) were maintained in DMEM (4500 mg/l glucose with GlutaMAX containing 10% fetal bovine serum, 1 % penicillin/streptomycin, 2.5 μg/ml amphotericin B and 500 μg/ml active G418 (geneticin). Ca²⁺-free, serum-free media was prepared with calcium-free DMEM (4500 mg/ml glucose; Invitrogen, Paisley, UK), 25 mM HEPES, 2 mM GlutaMAX, 1 % penicillin/streptomycin, 2.5 μg/ml amphotericin B, and 1.8 mM BaCI₂. Medium was sterilized by filtration through a 0.22 μm polyethersulfone low protein binding membrane and stored at 4 ^°C.

Measurement of Cholinesterase Activity

The method of Ellman et al (Biochem. Pharamcol. (1961 ) 7, 88-95) was used to determine the levels of cholinesterase released into media samples. Briefly, media was removed from the cells at various time intervals, centrifuged, and 25 μl aliquots were taken from the supernatant for analysis. To ensure that observed esterase activity was exclusively attributable to AChE, rather than the alternative esterase butyrylcholinesterase (BuChE), samples were assayed in the presence of the specific BuChE antagonist tetra-isopropyl pyrophosphoramide (Iso-OMPA, 100 μM). BuChE activity was determined by measuring total cholinesterase activity of samples in the presence of the AChE specific antagonist BW284c51 (1.5-bis(4- allydimethylammoniumphenyl)-pentan-3-one dibromide).Absorbance was read at 405 nm over 20 min using a Molecular Devices plate reader (Alpha Laboratories Ltd, Hampshire, UK). Baseline control values from cell media were subtracted from appropriate experimental values. AChE activity was calculated (V_max x enzyme efficiency factor x dilution factor = mU/ml/min) and statistical analyses were performed by ANOVA, followed by multiple comparison post-tests, using GraphPad Prism 4 data analysis program (GraphPad Software, San Diego, USA).

Live Cell Radioligand Binding Assay

Confluent cells were seeded into 12-well plates at a density of 5 x 10⁴ cells/well and allowed to recover for 24-48 hours before experimentation. Live cell binding was performed by treating cells with indicated peptides or α7-nAChR inhibitors for 1 hr at 37^°C in cell media with 1 % FBS. Then [125l]-α-bungarotoxin (150 Ci/mmol, GE Healthcare Bio-Sciences, Amersham, UK) was added and cells were incubated at 37^°C for a further 1.5 hr. Cell layers were washed 3x with 2 ml cell media, then 0.5 ml 1 M NaOH was added to each well to lyse cells. Cell lysates were transferred to 5 ml scintillation fluid and radioactivity was determined using a Beckman LS6000IC scintillation counter.

Peptide binding to the α7-nAChR in purified membrane preparations For membrane binding experiments, confluent cells were scraped off culture plates into ice-cold lysis buffer (20 mM Tris-HCI, pH 7.0, 5 mM EDTA, and 1X protease inhibitor cocktail (Roche Diagnostics, Ltd., West Sussex, UK). After pelleting by centrifugation for 10 min at 13,000 rpm, cells were resuspended in 7 ml ice-cold lysis buffer, lysed by Dounce homogenization, and then centrifuged at 1000 X g for 10 min. Supernatant was removed and the extraction process repeated. The supernatants were combined and centrifuged at 50,000 rpm (70 Ti rotor) for 30 min (Beckman Ultracentrifuge). The membrane pellet was washed with 5 ml lysis buffer and centrifuged as above. All centrifugations were carried out at 4^°C. The pelleted membranes were resuspended in binding buffer (50 mM Tris-HCI, 120 mM NaCI, 5 mM KCI, 1 mM MgCI₂, 2.5 mM CaCI₂, pH 7.0) and protein concentration determined using the DC Protein Assay kit (Bio-Rad Laboratories, Ltd., Hemel Hempstead, UK). Binding assays were assembled on ice in borosilicate glass test tubes with 50-100 μg membrane protein in binding buffer in a final volume of 250 μl. Binding reactions were incubated at 4^°C overnight, and then terminated by rapid vacuum filtration using a Brandel Cell Harvester onto Whatman GF/B glass fibre filters pre-soaked in 0.4% polyethylenimine.

Cell Proliferation Assay

Confluent cells were seeded into 96-well plates at a density of 1x10⁴ cells/well and allowed to recover for 24 hr before experimentation. Cells were treated with peptides at indicated concentrations for 48 hours, then cell proliferation was measured using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit (Promega, Southampton, UK) as per the manufacturer's instructions. This assay is based on the bioreduction, by metabolically active cells, of a tetrazolium compound (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) to form a colored formazan product that is soluble in tissue culture medium. Briefly, 20 μl Cell Titer Aqueous One Solution Reagent was added to each well of the 96-well assay plate containing the cells in 100 μl serum-free culture medium. Plates were incubated for 3 hr at 37^°C in a humidified 5% CO₂ atmosphere. Absorbance was read at 490 nm using a Molecular Devices plate reader (Alpha Laboratories Ltd, Hampshire, UK).

Total RNA Isolation, cDNA Preparation and PCR Amplification

Total RNA was isolated from astroglial and GH4 cells using the Sigma GenElute™ Mammalian Total RNA kit. RNA was reverse transcribed into cDNA using Superscript First-Strand Synthesis System (Invitrogen, Paisley, UK) as per the manufacturer's instructions. Briefly, 2 μg total RNA was annealed with 50 ng random hexamers and dNTP mix, then remaining reaction components were added to a final volume of 20 μl. Reactions were incubated at 42 ^°C for 50 min, terminated at 70 ^°C, and then RNA was removed by incubation with RNase H for 20 min at 37 ^°C. 100 ng cDNA was amplified by PCR with 50 pmol gene-specific primers, 1.5 mM MgCb, 200 μM dNTPs, and 1.25 U Taq DNA polymerase (Promega, Southampton, UK) in a 50 μl final reaction volume. After an initial denaturation of 95 ^°C for 2 min, reactions were amplified for 35 cycles: 95 ^°C for 30 sec, 55 ^°C for 30 sec, 72 ^°C for 1 min, followed by a final extension of 72 ^°C for 10 min. Reaction products were separated by electrophoresis on 1.5-2.0% agarose TAE gels and visualized by UV illumination. Images were captured and band density determined using a Bio-Rad Gel Doc 2000 and QuantityOne software (Bio-Rad, Hempstead, UK).

Primers for cDNA amplification. GAPDH:

5' = GAACATCATCCCTGCATCCA (SEQ. ID no. 3) 3' = CCAGTGAGCTTCCCGTTCA (SEQ. ID no. 4)

α7-nAChR:

5' = GGAGGCTGTACAAGGAGCTG (SEQ ID no. 5)

3' = GCCATCTGGAAAACGAACA (SEQ. ID no. 6)

Lvsate Preparation, Cellular Fractionation, Protein Evaluation, SDS-PAGE and Western Blot Analysis

Cells were harvested in Tris HCI 25 mM containing EDTA 2 mM and a complete set of protease inhibitors (Complete; Roche, Mannheim, Germany) and pelleted at 4 ⁰C and 10,000 x g for 10 minutes. For preparation of cell lysates, lysis buffer containing Tris HCI 25 mM pH 7.4, NaCI 150 mM, EDTA 2 mM, phenylmethylsulfonyl fluoride PMSF 0.1 mM, Nonidet 0.1 % and a complete set of protease inhibitors was added. Cells were vortexed vigorously for 5 minutes placing on ice intermittently. After separation of nuclei, % of cell lysate was kept as total homogenate for further analysis, while the remaining ³A were used to separate out the membrane fraction as described in earlier work (Zimmermann et al. (2004) J. Neurochem. 90, 1489-1499). Briefly, cell lysates were pelleted by centrifugation for 50 minutes at 4 ⁰C and 100,000 x g. The resulting pellets (membranes) were resuspended in lysis buffer and protein levels (alpha7 nAChR and actin) were determined by Western blot analysis. To this end, protein amount in both total homogenates and membrane lysates was determined using the method established by Bradford (Bradford (1976) Anal. Biochem. 72, 248-254). Samples were prepared in Laemmli buffer, separated in 7% SDS-PAGE and blotted onto nitrocellulose membrane. For immuno-detection, the following antibodies were used: anti-alpha7 nAChR (Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution 1 :200), anti-actin (Sigma-Aldrich, Poole, Dorset, UK; dilution 1 :5,000), peroxidase conjugated donkey anti-goat (Sigma-Aldrich, Poole, Dorset, UK; dilution 1 :10,000), and peroxidase conjugated goat anti-rabbit (Pierce, Rockford, IL, USA, dilution 1 :10,000). Blots were developed with enhanced chemiluminescence (ECL; GE Healthcare, Little Chalfont, Buckinghamshire, UK).

Example 1 : Binding of the T30 and T14 peptides to the α7-nicotinic acetylcholine receptor (α7nAChR).

Binding of the T30 and T14 peptides to the α7nAChR was investigated employing live cells expressing the receptor (GH4-hα7 cells) in culture as described above.

Preliminary results.

T30 binds the α7-nicotinic acetylcholine receptor (α7nAChR). In live cell binding experiments, T14 has a K = 1.0 nM for the α7nAChR, whereas T30 binding fits a two- site competition model with a high affinity K₁ = 50 pM and a low affinity K₁ = 500 nM. Thus T30 shows higher affinity than T14 for their common binding site and has an additional effect at a second binding site on the receptor (see Figure 5).

Further results

Further experiments were performed to characterize the binding parameters of the human α7-nAChR heterologously expressed in the rat GH4-hα7 cell line using a live- cell binding method. Saturation binding was carried out with [¹²⁵I] alpha-bungarotoxin ([¹²⁵I] α-BTX) concentrations ranging from 0.03 nM to 100 nM. Non-specific binding was determined in the presence of 20 μM methyllycaconitine (MLA). Total radioligand bound was consistently <10% of free radioligand in the assay. High levels of concentration-dependent and saturable specific [¹²⁵I] α-BTX binding in this cell line were observed (Fig. 12A, 12B). The high correlation coefficient of hyperbolic curve fitting (R² = 0.9775) is consistent with binding to a single class of receptors. Binding parameters were established as B_max = 965.8 ± 28.9 fmol/mg protein; K_d = 4.68 ± 0.61 nM. Saturation binding repeated at intervals throughout the experimental period verified that the levels observed initially were maintained through multiple passages (up to 30), demonstrating the stable nature of α7-nAChR expression by this cell line. Preliminary screening of the binding of both peptides to the α7-nAChR, when compared with that of acknowledged receptor agonists and antagonists, revealed significant, but incomplete, competition with α-BTX for receptor binding sites (Fig. 12C). T14 exhibited approximately 40% efficacy at 10 μM concentration, whereas the same concentration of T30 was 70% efficacious. Higher concentrations of these peptides did not further displace radioligand binding. In contrast, none of the control peptides were able to compete with α-BTX for binding to the α7-nAChR (Fig. 12D). Similarly, neither full-length T-AChE, nor truncated T548, had an effect on α-BTX binding to the receptor (Fig. 12D).

To elucidate further T14 and T30 binding parameters, full displacement binding profiles were performed (Fig. 12E). Interestingly, a two-phased activity was observed. First, a high affinity binding was apparent that fit the classic one-site competition model expected for displacement from a single class of receptors. T14 and T30 were equally efficacious at the high affinity site, with maximum displacement at about 45% of total specific binding, however T30 (K₁ = 16.8±1.8 pM) displayed much greater potency than T14 (K, = 653.3±12.6 pM). A second, lower affinity site was identified for T30 that accounts for a further 25% binding displacement by the peptide (K₁ = 47.1 ±2.8 nM). Comparative analysis of the data for T30, using Akaike's Information Criteria method, confirmed that the two-site competition model fits the data better than a one-site competition model with a >99.99% probability that it is correct. In contrast, for T14, although a one-site competition model provided an acceptable fit to the data in the 1 pM to 10 nM range, with increasing concentrations of peptide >10 nM, a reversal of displacement efficacy was observed.

To eliminate possible interference from cellular physiological processes or interaction of the peptides with intracellular molecules, we examined peptide binding to α7-nAChR in purified membrane preparations. Both T14 and T30 displacement of α-BTX binding to the α7-nAChR were markedly reduced as compared with that seen in live cell preparations (Fig. 13A). To explore the possibility that the peptide might act through an allosteric site to affect binding of other α7-nAChR ligands to the receptor, varying concentrations of T30 were incubated with cell membranes in the presence of MLA, acetylcholine (ACh), or choline at constant concentrations equivalent to their measured EC50 values (Fig. 13B). Specific binding displacement was altered by T30 in a concentration-dependent manner, with significant decreases in specific binding efficacy of 18% for ACh (p = 0.0426), 24% for MLA (p = 0.0032), and 36% for choline (p = 0.0064) as compared with the individual ligands alone. To determine if this shift in efficacy was due to an alteration in receptor affinity for the ligands, displacement binding was performed for each ligand in the presence and absence of a constant concentration of T30. Global fitting analysis was performed to compare whole binding curve differences. A small but statistically significant (p = 0.032) rightward shift was observed for ACh + T30 (IC50 = 9.5 ± 0.5 μM) as compared with ACh alone (IC50 = 7.2 ± 0.3 μM; Fig. 13C). In the presence of T30, the binding profiles for MLA and choline were similarly shifted to the right, though to a greater degree than that seen for ACh. Comparison of binding parameters revealed a highly significant (p = 0.0006) decrease in competitive potency for MLA + T30 (IC50 = 15.5 ± 0.7 nM) as compared with MLA alone (IC50 = 9.5 ± 0.4 nM; Fig. 13D). Since the effect of T30 on choline binding was greater than that seen for either ACh or MLA, we expanded the experimental method to examine the effect of a range of T30 concentrations on choline binding profiles. As shown in Fig. 13E, we observed a highly significant (p < 0.0001 ) concentration-dependent decrease in choline competitive potency in the presence of T30. Comparative IC50 and K₁ values are shown in Table 1. K₁ was calculated from the IC50 using the equation of Cheng and Prusoff ( Biochem. Pharmacol. (1973) 22, 3099- 3108) based on a constant radioligand concentration of 2 nM with a K_d = 4.68 nM.

Ligand IC50 : t SEM Ki ■ ± SEM

(μM) (μM)

Choline alone 122 .9 ± 7 .4 85 .9 ± 5. 1

+ T30 1 nM 126 .0 ± 12 .8 88 .9 ± 8. 9

+ T30 1O nM 225 .0 ± 21 .2 157 .3 ± 16. 9

+ T30 10O nM 357 .4 ± 33 .0 249 .9 ± 24. 5

+ T30 1 μM 736 .6 ± 68 .2 515 .1 ± 44. 6

Table 1 : Comparison of EC50 and K₁ values showing the effect of increasing concentrations of T30 on choline binding to the α7-nAChR.

Example 2: Effect of the T30 and T14 peptides on expression of the α7-nAChR in GH4- hα7 cells

GH4-hα7 cells were treated for 6 hours or 24 or more hours with 100 nM of T14 or T30 peptide. Preliminary Results. α7-nAChR protein expression increases after chronic treatment (24-48 hr) with T14 or T30 peptide. This increased expression has been specifically localized to the plasma membrane indicating that the peptides stimulate increased production or trafficking of receptors to the cell surface. T30 induces significantly more protein expression than than does T14 (see Figure 6). These results are further expanded upon below in Example 6 in relation to investigation of the effects of the same peptides on α7-nAChR mRNA expression.

Further results

Further investigation was undertaken of the effects of chronic peptide exposure on the number of α7-nAChR receptor binding sites and receptor affinity for α-BTX binding (Fig. 14). Cells in culture were exposed to AChE peptides for 24 hr, then saturation binding assays were performed on purified cell membranes using [¹²⁵I] α-BTX in concentrations ranging from 0.033 to 33.0 nM. After 24 hr treatment with T14 or T30, a significant increase (p = 0.0004 and p < 0.0001 respectively) in the number of α7- nAChR binding sites, as determined by maximal binding values, was observed (Fig. 14). Additionally, the affinity of receptors for [¹²⁵I] α-BTX was significantly decreased (T14, p = 0.0035; T30, p = 0.0018) as compared with controls. In contrast to that seen for T14 and T30 peptides, T15 treatment for 24 hr had no effect on specific binding affinity of [¹²⁵I] α-BTX to the α7-nAChR or on the number of available receptor binding sites (Fig. 14). Average B_max and K_d values for α7-nAChR binding after chronic peptide exposure are summarized in Table 2.

24 hr

Treatment Bmax (fmol/mg) K_d (nM)

Control 1908 ± 66.9 4.66 ± 0.54

T14 2626 ± 101.7 9.06 ± 0.93

T30 3147 ± 136.8 10.57 ± 1.17

T15 1938 ± 65.9 4.91 ± 0.53

72 hr

Treatment Bmax (fmol/mg) K_d (nM)

Control 1975 ± 54.1 4.90 ± 0.43

T14 1960 ± 104.3 11.60 ± 1.52

T30 2024 ± 157.8 16.43 ± 2.83

T15 2007 ± 139.0 5.65 ± 1.13

Table 2: Summary of saturation binding parameters showing the effects of chronic T- AChE peptide treatment on the number of α7-nAChR binding sites (B_max) and receptor affinity (K_d) for α-BTX. Example 3: Effect of T30 and T14 peptides on proliferation/metabolism of astrogial cells as measured by MTS assay

Although both T14 and T30 exhibit equal cytotoxic effects at high concentrations (-20% inhibition of cell proliferation at 10 μM), the proliferative effect of low concentrations on astroglia is greater with T30 (120±4% at 1 pM and 135±9% at 10 pM) than with T14 (96±5% at 1 pM and 121±8% at 10 pM); see Figure 7.

Example 4: Effect of T30 and T14 peptides on astroglial cell AChE intracellular activity and secretion of cholinesterases

Exposure of astroglia to peptides for 24 hr induced increased AChE and butyrylcholinesterase (BuChE) release and increased intracellular AChE activity. The effect of T30 on astroglial cell cholinesterase expression and secretion is greater than that induced by T14 (see Figure 8).

Example 5: Effect of T30 and T14 peptides on substrate inhibition of AChE

Substrate inhibition is a characteristic of AChE activity whereby excessive concentrations of substrate cause a feedback inhibition of the enzymatic activity. T30 and T14 decrease substrate inhibition thus enhancing AChE activity at higher concentrations of substrate. T30 demonstrates a significantly greater effect than does T14 (see Figure 9).

Example 6: Effect of T30 and T14 peptides on gene expression in GH4-hα7 cells

Exposure of cultured GH4-hα7 cells for 24 hrs to the AChE peptides in concentrations ranging from 1 nM to 1 μM results in changes in gene expression similar to that seen when cells are exposed to oxidative stress-inducing agents such as peroxides. Increased expression of R-AChE mRNA and decreased expression of L-type voltage- gated calcium channel mRNA was observed in GH4-hα7 cells after 24 hr peptide treatment. Consistent with the observed increase in α7-nAChR protein expression, α7- nAChR mRNA was also upregulated in response to peptide exposure. T30 elicited greater changes in gene expression at low concentrations than did T14 (see Figure 10). Further investigation of peptide-induced changes in α7-nAChR mRNA expression. Specifics of primer design are described above, while sequences used in RT-PCR experiments are detailed in Table 3.

Gene Accession 5' Primer 3' Primer Product

GAPDH NM_017008 1455-1474 1514-1532 78 bp α7-nAChR L31619 102-121 319-337 236 bp α4-nAChR L31620 392-41 1 587-606 215 bp Table 3: Primers used in RT-PCR for mRNA expression analysis.

RT minus controls were negative and gene expression in control cells did not change noticeably throughout the series of experiments. RT-PCR analysis was performed in control GH4-hα7 cells and those exposed to AChE peptides at concentrations ranging from 1 nM to 1 μM for 24 hr (Fig. 10A). After T14 or T30 peptide treatment, α7-nAChR mRNA expression was markedly upregulated for all concentrations of the peptides tested. Levels of α7-nAChR mRNA displayed a concentration-dependent increase with T14 treatment, with maximal expression at 100 nM. A similar high level of α7-nAChR expression was achieved after treatment with only 1 nM T30. As peptide concentrations increased further, α7-nAChR expression levels declined, however they remained significantly enhanced as compared with controls at all peptide concentrations tested.

To test whether the increased expression observed was directly attributable to specific T-AChE peptide interaction with the receptor, rather than random non-specific peptide effects, cells were similarly treated with control peptides, followed by analysis of α7- nAChR mRNA expression (Fig. 10B). No significant change in α7-nAChR mRNA levels was observed after exposure to T15, S14, B14 or SB14 peptides. In addition, neither the full-length T-AChE molecule, nor the truncated T548, effected a change in α7- nAChR mRNA expression. Similarly, to ascertain the receptor specificity of AChE peptide-induced α7-nAChR enhancement, cells treated with T14 and T30 were analysed by RT-PCR for changes in mRNA expression of α4-nAChR (Fig. 10C). No significant change in α4-nAChR mRNA levels was observed.

We next attempted to block peptide-induced α7-nAChR mRNA enhancement using the α7-nAChR specific inhibitor MLA. Exposure of GH4-hα7 cells to 10 μM MLA for 24 hr induced upregulation of α7-nAChR expression, although to a lesser degree than did

100 nM T30 (Fig. 10D). When MLA and T30 were co-applied, however, the greater enhancement of T30-induced α7-nAChR expression was suppressed to levels observed after MLA treatment alone.

Peptide-induced changes in α7-nAChR protein expression. Given that changes in RNA expression are not necessarily reflected in equivalent alterations in protein levels, we also analysed AChE peptide effects on protein expression by SDS-PAGE and Western blot. As already noted above (see Example 2), a slight, but discernable, upregulation of α7-nAChR protein expression was detected after only 6 hr exposure to 100 nM T14 or T30 in both total cell homogenates and purified membrane fractions (see Fig. 6A, filled arrowheads). After 24 hr treatment with peptides, a profound increase in α7-nAChR protein levels was observed. This increase was particularly pronounced in the membrane fractions of cells treated with the AChE peptides for 24 hr. Furthermore, T30 treatment induced a greater increase in receptor protein levels at 24 hr than did T14.

To see whether the induced increase in α7-nAChR protein levels in the membrane compartment reflects an upregulation of receptor protein at the plasma membrane, peptide-treated GH4-hα7 cells were exposed to the cross-linking agent BS³ prior to harvesting for analysis. As can be seen in Fig. 16B, both T14 and T30 increase α7- nAChR levels in the membrane compartment. In addition, higher amounts of high molecular weight aggregated species of the α7-nAChR were observed for T30 incubated cells as compared with controls (Fig. 6B, empty arrowheads). In order to verify that BS³ does not permeate cell membranes and, thus, exclude that the observations described are confounded by cross-linked intracellular α7-nAChR protein, actin levels were assessed in BS³-treated cells. Importantly, the immunoreactivity for this intracellular protein was unaffected by the BS³ treatment (data not shown). In contrast to that seen for T14 and T30, treatment of the cells for 24 hours with the control peptide T15 had no effect on α7-nAChR levels in cell membranes (Fig. 6C).

In addition, we examined changes in α7-nAChR protein further using immunofluorescent staining. High levels of α7-nAChR protein were detected in cellular membranes, but not in cytoplasmic or perinuclear regions. Background control cells incubated with secondary antibodies, but lacking primary antibodies, did not produce a discernable signal. After treatment for 24 hr with T30, enhanced signal intensity was evident in cell membranes as compared with controls. In summary, like Aβ42 peptide, T30 peptide binds the α7-nAChR with picomolar affinity, whereas T14 is less potent at this receptor. The data indicate that T30 peptide interacts with both the active ligand-binding site and the allosteric modulatory site on the receptor, whereas T14 peptide only interacts with one receptor binding site. In addition, T30 peptide induces upregulation of α7-nAChR mRNA and protein expression to a greater degree than does T14 peptide. In astroglia, T30 peptide stimulates both AChE and BuChE upregulation and secretion, whereas T14 peptide exposure has no effect on BuChE release and a less potent effect on AChE upregulation. Furthermore, T30 peptide has a greater effect on astroglial cell proliferation at low doses than does T14 peptide. These results are consistent with T30 peptide being a more physiologically active and relevant peptide than T14 peptide, just as Aβ42 is more potent and physiologically relevant than the shorter APP peptides (Aβ40, Aβ16, etc) in the pathology of AD.

The evidence now presented supports a model for neurodegeneration (summarised schematically in Figure 1 1 ) in which in affected brain areas containing global neurons, neurons and glia revert to a more immature phenotype, expressing increased levels of APP and monomeric AChE. Hence, accumulation of AChE in extracellular spaces is an early event in neurodegeneration. It is envisaged that cleavage of T30 peptide from monomeric T-AChE exacerbates neurodegenerative processes, particularly through interaction with APP, AβP and the α7-nAChR. Hence, T30 peptide in CSF samples is anticipated to provide an early marker of progression of neurodegenerative disease. Moreover, blocking action of T30 peptide in such circumstances can be anticipated to provide a therapeutic intervention that addresses the underlying causative factor in neurodgeneration, while circumventing the toxic side effects of blocking the catalytic function of whole AChE.

Claims

Claims:

1. An isolated polypeptide (T30 peptide) consisting of the C-terminal 30 amino acid residues of the T isoform of acetylcholinesterase (T-AChE), said polypeptide consisting of the sequence

KAEFHRWSSYMVHWKNQFDHYSKQDRCSDL (SEQ. ID. NO.1 )

and functional analogues thereof.

2. A method of identifying an antagonist of the T30 peptide as defined in claim 1 which comprises screening a test agent for ability to inhibit one or more of the following biological effects of said peptide in vitro;

(i) ability to bind the α7-nicotinic acetylcholine receptor (α7-nAChR);

(ii) ability to increase expression of α7-nAChR in the membrane of cells which express the receptor;

(ii) ability to increase cultured astroglial cell proliferation/metabolism;

(iii) ability to increase secretion of AChE from cultured astroglial cells;

(iv) ability to increase intracellular AChE activity in cultured astroglial cells;

(v) ability to inhibit substrate inhibition of AChE activity;

(vi) ability to change gene expression in cells.

3. A method as claimed in claim 2 which comprises determining whether the test agent inhibits binding of T30 peptide or a functional analogue thereof to α7-nicotinic acetylcholine receptors.

4. A method as claimed in claimed in claim 3 wherein cultured cells are employed engineered to express recombinant α7-nicotinic acetylcholine receptor or a functional analogue thereof.

5. A method as claimed in any one of claims 2 to 4 which further comprises formulating a test agent identified as an antagonist into a pharmaceutical composition.

6. A method as claimed in claim 5 wherein said agent is modified or packaged to facilitate delivery across the blood brain barrier.

7. Use of a polypeptide as claimed in claim 1 as an antigen and/or screening agent to provide an antibody or antibody fragment capable of binding T30 peptide.

8. A use as claimed in claim 7 for providing a monoclonal antibody or fragment thereof capable of binding T30 peptide.

9. A use as claimed in claim 8, which further comprises labelling of said antibody or antibody fragment with a detectable label.

10. A method of diagnosing in an individual a neurodegenerative disorder which comprises detecting an abnormal T30 peptide level in a cerebrospinal fluid (CSF) sample.

1 1. A method as claimed in claim 10 wherein said disorder is Alzheimer's Disease, Parkinson's Disease or Motor Neurone Disease.

12. A method as claimed in claim 11 wherein the disorder diagnosed is Alzheimer's Disease.

13. An agent which prevents proteolytic cleavage of T30 peptide from T-AChE in the brain for use in treating a neurodegenerative disorder.

14. An agent as claimed in claim 13 which targets beta-secretase.

15. An agent as claimed in claim 13 or claim 14 for use in treating a neurodegenerative disorder selected from Alzheimer's Disease, Parkinson's Disease and Motor Neurone Disease.

16. An agent as claimed in claim 15 for use in treating Alzheimer's Disease.

17. An agent as claimed in any one of claims 13 to 116 which is an antisense oligonucleotide, antisense PNA, siRNA, antibody or antibody fragment.

18. An agent as claimed in any one of claims 13 to 17 which is modified or packaged to facilitate delivery across the blood-brain barrier.

19. A pharmaceutical composition comprising an agent as claimed in any one of claims 13 to 18 together with a pharmaceutical carrier or diluent.

20. A method of identifying a potential therapeutic agent for use in treating a neurodegenerative disease which comprises contacting a test agent with T-AChE exposed in vitro to physiological proteolytic conditions whereby in the absence of the test agent it is cleaved to release T30 peptide and determining whether such cleavage is prevented.

21. A method as claimed in claim 20 which further comprises modifying or packaging of an agent thus identified to facilitate delivery across the blood-brain barrier.

22. A method as claimed in claim 20 or claim 21 which further comprises formulating said agent into a pharmaceutical composition.