WO2010088729A1 - Compositions and uses therefor - Google Patents

Abstract

The invention is directed inter alia to the use of receptor-associated protein (RAP) polypeptide or a functional analog thereof to reduce amyloid oligomerisation and deposition and to reduce Aβ associated neuropathologic features. Thus, the invention provides a composition comprising RAP or an analog thereof which binds β-amyloid peptide (Aβ) for use in the treatment or prophylaxis of a symptom of Alzheimer's disease (AD) or a related condition in a subject. One illustrative symptom is memory loss or consolidation. In another aspect, the invention provides methods of screening for RAP polypeptide functional variants or analogs by testing the ability of an agent to compete successfully with RAP for binding to Aβ.

Description

COMPOSITIONS AND USES THEREFOR

FIELD

The subject specification relates generally to compositions and methods for treating or preventing symptoms of amyloidoses, Alzheimer's disease or related conditions characterised by amyloid deposits in the brain, memory loss and dementia. In particular, the specification considers compositions that reduce the activity or pathogenesis of Aβ comprising agents capable of binding to β-amyloid peptide (Aβ).

BACKGROUND

Bibliographic details of references in the subject specification are also listed at the end of the specification.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

The proteopathies comprise a group of clinically diverse disorders characterised by the damaging accumulation of aggregated proteins in cells and tissues of the body. Proteopathies include, inter alia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, prion diseases, inclusion body myopathy, and the systemic amyloidoses.

Proteins normally fold into preferred, 'native' conformations in which they can carry out their customary functions in the cell. However, in proteopathies a protein assumes an atypical, three-dimensional conformation, which often is enriched in β-sheet structure. 13

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Proteins in this non-native conformation are highly stable, resistant to degradation, and have an enhanced tendency to aggregate with similar protein molecules.

Each proteopathy has a characteristic signature that includes the accumulation of a particular protein as extracellular deposits and/or intracellular inclusions or aggregations in certain organs. Such deposits and inclusions are considered central to the pathology of proteopathies and attempts to develop effective therapies for the proteopathies have been directed inter alia toward reducing the production of the proteins, blocking their aggregation, or augmenting their removal.

Alzheimer's disease is a common and debilitating neurodegenerative proteopathy resulting in progressive loss of memory and cognitive ability that eventually lead to dementia and death. Related conditions include conditions that are characterised by amyloid deposits in the brain and memory loss, such as found in Lewy body dementia, in muscles such as in inclusion body mycositis, or in cerebral blood vessels such as in cerebral amyloid angiopathy.

Alzheimer's disease is characterized by accumulation of β-amyloid protein (Aβ) in the brain, extracellularly as amyloid plaques and cerebral amyloid angiopathy, and intracellularly as neurofibrillary tangles (NFTs). It is generally accepted that Aβ is neurotoxic and that oligomeric forms of Aβ are the most potent neurotoxin (Lambert et al, Proc Natl Acad Sci USA 25:6448-6453, 1998; Small et al, Nat Rev Neurosci 2:595-598, 2001; Walsh et al, Nature 416:535-539, 2002; Gong et al, Proc Natl Acad Sci U S A 700:10417-10422, 2003; Cleary et al, Nat Neurosci 5:79-84, 2005; Lacor et al, J Neurosci 27:796-807, 2007).

The mechanism responsible for Aβ's neurotoxic effects remains unclear. Several studies have shown that the binding of Aβ to the plasma membrane correlates with Aβ-induced neurotoxicity (Small et al, 2001 (supra); Subasinghe et al, J. Neurochem. 84: 471-479, 2003 ; Verdier and Penke, Curr Protein Pept Sci 5:19-31 , 2004; Small et al, J. Neurochem. 101: 1527-1538, 2007). A number of Aβ receptors have been proposed including LRPl (Deane et ah, Neuron 43:333-344, 2004), the α7-nicotinic acetylcholine receptor (ct7nAChR) (Wang et ah, J. Biol. Chern. 275: 5626-5632, 2000), the ρ75 neurotrophin receptor (Yaar et ah, J. Clin. Invest. 100: 2333-2340, 1997) and the receptor for advanced glycation endproducts (RAGE) (Yan et ah, Proc. Natl. Acad. Set USA 94: 5296-5301, 1997). Studies by Yamada et ah, J. Biol. Chem. 283: 34554-34562, 2008 have shown that Aβ does not bind directly to LRPl in endothelial cells and our own studies (Small et ah, 2007 (supra)) have shown that Aβ does not bind directly to the α7nAChR. Instead, our studies (Subasinghe et ah, 2003 (supra); Small et ah, 2007 (supra)) and the work of others (Simakova and Arispe, J. Neurosci. 27: 13719-13729, 2007; Davis and Berkowitz, Biophys. J. 96: 785-797, 2009; Ciccotosto et ah, Neurobioh Aging, [Epub ahead of print, March 24] doi:10.1016/j.neurobiolaging.2009.02.018., 2009) have shown that Aβ binds to lipids in the plasma membrane.

The 39 kDa receptor-associated protein (RAP) is a major ligand of many low-density lipoprotein receptor family members (Bu, Int. Rev. Cytol. 209: 79-116, 2001). RAP is of interest, as a carrier, for the therapy of brain diseases as the protein is actively transported across the blood-brain barrier (Pan et ah, J Cell Sci. 117: 5071-5078, 2004) and has been proposed as a vehicle for drug delivery of active agents to the brain (Prince et ah, J Biol

Chem. 279: 35037-35046, 2004).

There is a need for agents that are effective in preventing or reducing the symptoms of amyloidoses, AD and related conditions in a subject.

SUMMARY

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are now described.

As used herein the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a single cell, as well as two or more cells; reference to "an agent" includes one agent, as well as two or more agents; and so forth.

Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

The present invention is based upon the finding by the inventors that receptor associated protein (RAP) binds to β-amyloid protein (Aβ) and directly modulates various activities of Aβ in vitro or in vivo. Previously, RAP has been known for its ability within a cell to bind to and antagonise lipoprotein receptors such as LRPl. These receptors have been genetically linked to AD and studies of reduced receptor function have been conducted. As knockout mice for the lipoprotein receptor related protein (LRPl) do not survive, scientists have down regulated RAP in order to down regulate LRPl and investigate LRPl function. Surprisingly, in some embodiments, RAP is found herein to bind to extracellular Aβ in a lipoprotein receptor independent manner and to enhance binding of Aβ to neuronal cells. As exemplified in the Examples, when RAP and Aβ are added to neuronal cells in vitro, RAP polypeptide binds strongly to Aβ and enhances binding and/or uptake of Aβ to/by neuronal cells. Further, RAP down modulates Aβ aggregation. Furthermore, RAP reduced the neurotoxic ability of Aβ to induce memory loss or cognitive impairment in an animal model of AD.

These results are of relevance to AD as the direct action of aggregated Aβ on neurons underlies the neurological dysfunction seen in AD (Lambert et ah, 1998 (supra); Walsh et ah, 2002 {supra); Gong et at., 2003 (supra)). It is proposed herein -without limitation that the mechanism by which RAP influences Aβ deposition is via a direct interaction between Aβ and RAP.

The RAP-Aβ interaction is clearly demonstrated by the co-immunoprecipitation of both Aβ and RAP using an anti-RAP antibody. This procedure also revealed that some RAP and Aβ remained associated during SDS-PAGE suggesting that the interaction is particularly stable. It is likely that the 46 kDa RAP-Aβ complex contains one molecule of Aβ and one molecule of RAP, based on the apparent molecular mass of the complex during SDS-PAGE. However, the possibility that higher or lower molecular weight forms of Aβ may bind to RAP cannot be excluded at this stage and will be determined experimentally as described herein. The co-localization of RAP and Aβ at the cell membrane suggests that the two proteins can remain associated while bound to the cell surface.

The results of experiments on Aβ-induced Ca²⁺ dysregulation and chick memory show that RAP protects against Aβ toxicity in vivo. Co-injection of RAP blocked Aβ-induced amnesia in day-old chicks. As further shown herein, levels of neuronal RAP are lowered in the hippocampus of subjects with AD.

Accordingly, in one embodiment the present invention provides a composition comprising a molecule having the activity of RAP including the herein disclosed ability to bind Aβ and reduce Aβ oligomerisation. In some embodiments, the molecule is a RAP polypeptide or a variant thereof including a functional part (fragment or portion of RAP), or an analog or agonist thereof which is capable of binding to Aβ or a precursor thereof. The subject molecule or agent is for use in the treatment or prophylaxis of symptoms of AD or a related condition in a subject. In a preferred embodiment, the symptoms of AD include memory loss associated with AD in a subject.

In some embodiments, the RAP polypeptide is capable of binding to a lipoprotein receptor under physiological conditions. In other embodiments, the RAP polypeptide is incapable of binding to a lipoprotein receptor under physiological conditions.

Accordingly, a composition comprising a RAP polypeptide or variant thereof as defined herein as the active ingredient, active in preventing the pathogenesis associated with Aβ is provided.

In some embodiments, the RAP polypeptide is RAP or a fragment of RAP.

In other embodiments, the RAP polypeptide is a variant of RAP.

In further embodiments, the RAP polypeptide is a RAP peptidomimetic or other peptide such as a stapled peptide.

In still further embodiments, the RAP polypeptide is a small molecule analog or agonist of RAP.

In a preferred embodiment, the RAP polypeptide is human or mammalian.

In another preferred embodiment, the analog or agonist is a small molecule, an antibody, a nucleic acid or a peptide.

In one embodiment, the molecules of the present invention are conveniently provided in a medicament form such as a pharmaceutical composition. In another aspect, the present invention provides a method of identifying a candidate agent that modulates Aβ activity. In some embodiments, the methods are suitable for identifying agents that modulate amyloid activity in related conditions other than AD. In some embodiments, the activity of Aβ is selected from the group consisting of Aβ aggregation or oligomerisation, Aβ fibril formation, Aβ induced increase in intracellular calcium (Ca²⁺),

Aβ induced neuropathy and memory loss or dementia. In some embodiments, the candidate agent (also a RAP polypeptide or agonist) reduces Aβ aggregation or oligomerisation, Aβ fibril formation, Aβ induced neuropathy and/or enhances or preserves memory performance. In some embodiments said method comprises:

i) contacting the candidate agent with a system comprising Aβ; and ii) determining the presence of a complex between the agent and Aβ or a change in the level of an indicator of the activity of the complex or of a component thereof.

In another embodiment of this aspect, the method comprises:

i) contacting Aβ with a system comprising the candidate agent; and ii) determining the presence of a complex between the agent and Aβ or a change in the level of an indicator of the activity of the complex or of a component thereof.

In some embodiments, the system comprises an in vitro cell, such as a brain cell or a neural cell. Conveniently, the cell is a neuroblastoma cell.

In other embodiments, the system comprises an animal model of AD.

In some embodiments of the method, the system further comprises a RAP polypeptide and step ii) comprises instead determining the ability of the agent to reduce the binding between the RAP polypeptide and Aβ. In particular, the present invention contemplates a method of generating a RAP polypeptide (RAP fragment, mutant, agonist, mimetic) capable of agonising RAP activity by binding to Aβ or other amyloid peptide and down-regulating the activity of Aβ. In some embodiments, the method comprises the steps of:

i) mutating one or more residues of an Aβ binding domain of RAP; ii) contacting the mutated Aβ-binding domain with Aβ; iii) detecting the presence of binding between the mutated RAP and Aβ thereby identifying amino acid residues associated with a binding interaction between the Aβ-binding domain of RAP and Aβ or other amyloid peptide; and iv) generating a RAP polypeptide agonist which mimics the wild-type RAP polypeptide at the residues essential for binding to occur between RAP polypeptide and Aβ and which inhibits the activity of Aβ.

In particular, the present invention contemplates a method of generating a RAP polypeptide (RAP fragment, mutant (variant), agonist, mimetic) capable of agonising or mimicking the functional activity of RAP by binding to an Aβ peptide and down- regulating the activity of Aβ. In some embodiments, the method comprises the steps of:

i) mutating one or more residues of an Aβ binding domain of RAP; ii) contacting the mutated Aβ-binding domain with Aβ; iii) detecting the presence of binding between the mutated RAP and Aβ thereby identifying amino acid residues essential for a binding interaction between the Aβ-binding domain of RAP and Aβ; and iv) generating a RAP polypeptide or a RAP polypeptide analog which mimics a RAP polypeptide at the residues essential for binding to occur between RAP polypeptide and Aβ. In another particular, the present invention contemplates a method of generating a RAP polypeptide (a RAP fragment, mutant or variant, agonist, mimetic or analog) capable of agonising or mimicking RAP activity by binding to Aβ and down-regulating the activity of Aβ. In some embodiments, the method comprises the steps of:

i) mutating one or more residues of a RAP polypeptide or a fragment thereof; ii) contacting the mutated RAP with Aβ; iii) detecting the presence of binding between the mutated RAP and Aβ thereby identifying amino acid residues associated with a binding interaction between RAP and Aβ; and iv) generating a RAP polypeptide or analogs which mimics the wild-type RAP polypeptide at the residues essential for binding to occur between RAP polypeptide and Aβ and which inhibits the activity of Aβ.

In some embodiments, the RAP polypeptide is a fragment of RAP comprising all or part of Domain 1 (amino acids 1 to 112), Domain 2 (amino acids 113 to 215), or Domain 3 (amino acids 216 to 323) of the mature protein. The amino acid sequence of these fragments are set out in SEQ ID NOs: 2, 3 and 4. The amino acid sequence of the RAP precursor protein is set out in SEQ ID NO: 1. In some embodiments, the RAP polypeptide has the amino acid sequence set out in SEQ ID NO: 5, lacking the first 34 amino acid residues set out in SEQ ID NO: 1. In some embodiments, the RAP polypeptide has the ER retention signal C-terminal (HDEL) removed. Analogs and variants of all fragments are contemplated as a matter of routine.

In some embodiments, the RAP polypeptide is also tested for its ability to cross biological membranes.

In some embodiments, an indicator of the activit y of the complex between the RAP polypeptide or RAP polypeptide analog and Aβ is the memory performance of an animal model of AD. In one embodiment, the candidate agent (RAP polypeptide) is generated by methods such as, but not limited to, in silico screening, high throughput chemical screening, function based assay or structure-activity relationships. The agents may be a proteinaceous or non- proteinaceous molecule derived from natural, synthetic or recombinant sources. Useful sources include screening libraries such as natural product libraries, chemical molecule libraries, peptide libraries, pharmaceutical product libraries, combinatorial libraries, phage display libraries and in vitro translation libraries, as known in the art.

In some embodiments of the method, RAP and/or Aβ are endogenously produced within a cell. In other instances, these agents are supplied exogenously. In some embodiments, the cell is a brain cell or a neural cell as referred to hereinabove as a component of the system.

In another aspect, the invention provides a composition comprising a complex comprising an isolated RAP polypeptide and Aβ. In some non-essential embodiments, RAP is covalently bound to Aβ .

In another aspect, kits comprising the complex or a specific-binding agent thereto for the diagnosis or prognosis of AD and related conditions are contemplated.

In yet another aspect, the present invention provides for the use of RAP or a complex comprising RAP and Aβ in the manufacture of a medicament in the treatment of AD or a related condition in a subject. Reference herein to manufacture, includes selection or design of a medicament.

In some embodiments, the invention provides a method for the treatment or prophylaxis of a subject with AD or a related condition or who is at risk of developing same said method comprising administering to the subject an amount of a RAP polypeptide or an agent capable of producing same or a RAP analog or agonist capable of binding to Aβ (or an Aβ precursor for a time and under conditions effective to reduce Aβ aggregation, Aβ oligomerisation, Aβ fibril formation, amyloid neuropathy and/or enhance memory performance. In a preferred embodiment, the subject is a human.

In a related embodiment, the invention contemplates a combination therapeutic protocol for the treatment or prophylaxis of symptoms of a condition characterised by aggregation of Aβ in brain tissue, said protocol comprising the administration of a composition as defined above or herein and one or more other treatments, In an illustrative embodiment, the other treatment is administration of a neurotrophin or other agent to enhance nerve cell regeneration, growth or development.

Reference to a "RAP polypeptide" includes all biologically active naturally occurring forms of RAP as well as biologically active portions or fragments thereof. In addition, variants (including mutants, analogs and mimetics) or derivatives of a RAP polypeptide that bind Aβ as disclosed herein. Biological activity as used herein refers to the ability of RAP in accordance with the present invention to bind to Aβ, or modify the activity of Aβ and/or reduce one or more symptoms of AD, such as memory loss. Mutants, analogs and mimetics are therefore selected for their ability to target Aβ at the structural and/or functional level.

The invention provides for and includes methods of screening for functional variants of RAP wherein the functional variant inter alia retains the ability to bind to Aβ and/or prevent or ameliorate the development of symptoms of proteopathies including neurodegenerative changes in a subject. Without being bound by any particular theory, as RAP binding to Aβ and internalisation by neural cells is independent of binding to lipoprotein receptors such as LRPl, RAP polypeptides are tested for their ability to bind Aβ. In some embodiments, an ability to bind LRPl is not required.

Accordingly, in some embodiments, the invention provides a method for modulating aggregation of Aβ. Oligomeric forms of Aβ rather than the monomeric or fibrillar forms are the most potent neurotoxins, and accordingly, reducing the formation of oligomers

(aggregates) of Aβ will be useful in treating or preventing symptoms of AD. In other embodiments, the methods are useful in vitro. In some embodiments, the method comprises contacting Aβ or cells producing Aβ with a RAP polypeptide. Reference herein to "Aβ" includes without limitation Aβ_1-40 and Aβ_1-42 peptides.

In another embodiment, the invention provides a method for modulating cellular uptake of Aβ. In an illustrative embodiment, full length RAP polypeptide enhances the cell surface association and uptake of Aβ by cells. In another illustrative embodiment, the cell is a neuronal call. In some embodiments, the method comprises contacting Aβ with a RAP polypeptide

Accordingly, in another embodiment the present invention provides a method for preventing or reducing symptoms of AD, said method comprising administering to a subject an effective amount of a RAP polypeptide. The subject may be administered in vivo or ex vivo. In some embodiments, the method comprises administering RAP polypeptide or an agent from which RAP polypeptide is producible. In further embodiments, the subject is tested for AD, cognitive impairment and/or loss of memory before or after administration of RAP polypeptide.

In some embodiments, the RAP polypeptide is a portion or fragment of a full length RAP. In other embodiments, the RAP polypeptide is a variant or derivative of RAP comprising conservative amino acid changes as described herein and known in the art. In some embodiments, basic residues are conserved. In some embodiments, the RAP polypeptide comprises Domain 1 (Dl)₅ Domain 2 (D2) or Domain 3 (D3), of any combination of one or more of these domains of RAP. In other embodiments, the RAP polypeptide variant has at least 80%, 90% or 95% amino acid sequence identity to a naturally occurring RAP polypeptide over a reference region of at least 40, 50, 100, 150, 200 or 250 contiguous amino acids. In other embodiments, the variant RAP is encoded by a sequence of nucleotides that hybridise in conditions of medium or high stringency to the complement of the nucleotide sequences encoding a RAP polypeptide, such as described herein and including a fragment thereof. In other embodiments described in greater detail herein RAP polypeptide is a variant, analog or mimetic of RAP that may comprise, for example, a protein, peptide, nucleic acid, small or large molecule or aptamer or antibody.

In still further embodiments, the invention provides a genetically modified cell genetically modified to express or over express a RAP polypeptide.

In other embodiments, the invention provides an antibody that recognises (binds to) RAP or Aβ when RAP and Aβ are bound to each other but not when unbound.

In other embodiments the invention provides methods of diagnosis and diagnostic markers for subjects at risk for or exhibiting one or more symptoms of AD. In an illustrative embodiment, labelled RAP is provided as a marker for Aβ; alternatively labelled Aβ is provided as a marker for RAP. In another embodiment, antibodies to RAP or a RAP binding agent is provided suitable for detection of a RAP molecule in biological samples.

In some embodiments, extracellular RAP is to be detected. In other embodiments, intracellular RAP is detected.

The above summary is not and should not be seen in any way as an exhaustive recitation of all embodiments of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Some figures contain colour representations or entities. Coloured versions of the figures are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

Figure 1 is a photographic representation depicting binding and internalisation of FluoAβ!- ₄₂ by SH-SY5Y cells. Cells were incubated with FluoAβ_1-42 (1 μM) for 1 hours (h) (upper panel), 4 h (middle panel) or 24 h (lower panel), then the cell membrane was labeled by incubation with ice-cold Alexa-555-CTX B subunit (Alexa555-CTX) (250 ng/ml). Fluorescence images were captured by confocal microscopy. Scale bar, 20 μm.

Figure 2 is a representation of data illustrating that cell-bound Aβ colocalizes with exogenous RAP but not endogenous LRPl. Panel A. Effect of RAP and anti-LRPl antibody on association of FluoAβi_-42 with SH-S Y5 Y cells. Cells were treated with freshly prepared FluoAβ_1-42 (1 μM) in the presence of RAP (5 μg/ml) (grey bars) or in the absence of RAP (black bars). Incubations were also performed in the absence (Control) or presence of anti-LRP antibody R2629 (10 μg/ml). AU incubations were for 4 h. Cells were analyzed for fluorescence by flow cytometry. Data represent means of three independent experiments ± SEM. Two-way ANOVA revealed a significant effect of RAP on FluoAβ_1-42 binding (Fl,35 = 11.34, p < 0.01), but no effect of R2629 treatment (F1.28 = 0.01, p = 0.94). Panel B. Anti-LRPl R2629 antibody inhibits binding and uptake of RAP. Cells were incubated with RAP (5 μg/ml) in the presence or absence of anti-LRP R2629 (10 μg/ml) for 4 h, then the lysates were measured for RAP levels by slot-blot analysis. Total 7Fl (anti-RAP monoclonal antibody) irnmunoreactivity, above endogenous levels, was detected and quantitated. Band density was quantitated using ImageQuant. *p < 0.05, as compared to controls (Student's Mest). Panels C and D. Localization of FluoAβi.₄₂ RAP and LRPl on SH-SY5Y cells. Cells were incubated with freshly prepared FluoAβ_1-42 (1 μM) in the presence of RAP (5 μg/ml) for 6 h, then rinsed, fixed and permeabilized. In panel C₅ LRPl was detected using antibody R2629 (1 μg/ml). Arrows indicate cell surface-bound FluoAβ_1-42 and arrow heads show LRPl immunoreactivity. Scale bar, 10 μm. Panel D shows co-localization of Aβ and RAP at the cell surface. The cells were fixed, permeabilized, and stained for RAP with anti-RAP monoclonal antibody 7Fl. Fluorescence was visualized by confocal microscopy. Asterisks show regions of co- localization. Blue channel shows AlexaFluor-555-CTX fluorescence. Scale bar, 20 μm.

Figure 3 is a photographic representation depicting Aβ and RAP co-immunoprecipitation. Aβ_1-40 or Aβ_1-42 (1 μM) and/or RAP (5 μg/ml) were incubated in NaCl/P_i5 pH 7.4 at 37°C for 6 h. Incubation mixtures were then further incubated with monoclonal antibody 7Fl- or an unrelated transferrin receptor antibody H68.4-bound protein G agarose. Beads were then washed thoroughly and then bound proteins denatured by heating in SDS sample buffer. Proteins were separated on a 12% Tris-glycine gel, transferred to nitrocellulose membrane, and immunoblotted using anti-Aβ monoclonal antibody 6E10 (left panel). The membrane was then stripped and re-probed using anti-RAP monoclonal antibody 7Fl (right panel), f Under these conditions of electrophoresis, Aβ migrated with the solvent front at -4-6 kDa.

Figure 4 is a photographic representation of SDS-PAGE analysis showing that RAP alters Aβ_1-4o oligomerization and induces the formation of an SDS-stable RAP-Aβ complex. Aβ_1-4o (upper panel) or Aβ_1-42 (lower panel) (1 μM) and/or RAP (5 μg/ml) were incubated in NaCl/Pj, pH 7.4 at 37°C for the times indicated (A) or for 96 h (B). At the end of incubation, proteins (20 ng A$ per lane) were separated by 16.5% Tris-tricine (A) or 15% Tris-glycine (JS) SDS-PAGE, transferred to nitrocellulose and immunoblotted with 6E10. B, The membrane was then stripped and immunoblotted with 7Fl (right panel).

Figure 5 is a photographic representation illustrating that RAP inhibits Aβ_1-42 aggregation. Aβ_1-42 (1 μM) and/or RAP (5 μg/ml) were incubated in NaCl/Pj, pH 7.4 at 37°C for the times indicated. Proteins were then deposited onto a surface of freshly cleaved highly oriented pyrolytic graphite, washed, dried and visualized by AFM. Left panels show images of Aβ_1-42 incubated alone. Middle panels show incubations with Aβ_1-42 incubated in the presence of RAP. Right panel shows RAP alone. Scale bar, 100 nm. Figure 6 are a graphical representations showing the effect of RAP on the Aβ_1-42-induced increase in intracellular Ca²⁺. Freshly prepared Aβ_1-42 was first dissolved in HFIP prior to dilution into calcium imaging buffer. Cells were loaded with fluo-4 and treated with Ap_{1- 42} in the absence or presence of RAP, with or without R2629 pre-treatment. Changes in intracellular Ca²⁺ were detected as a change in fluorescence over time. A , Graph shows the change in fluorescence from baseline (AF/F) with time. Values are means of AF/F calculated for 4 wells + SEM. B. Effect of RAP and an anti-LRPl antibody (R2629) on Aβ-induced Ca2+ increase. Maximum AF/F was defined as the average AF/F over the period between 208-256 s following commencement of Aβm₂ treatment. A two-way ANOVA found a significant main effect of RAP (F_hn = 59.85, p < 0.0001), but no effect of R2629 (F\_ti2 ⁼ 0.73, p = 0.4107). Similar results were obtained in two independent experiments. ***p < 0.0001 (Bonferroni post-test). Similar results were also obtained with Aβ_M0.

Figure 7 is a graphical representation of data showing that RAP co-injection blocks the amnestic effect of Aβ_1-42 in day-old chicks. Physiological saline containing Aβ_1-42 (10 pmo I/hemisphere) with or without RAP (50 pg/hemisphere) was injected into the avian cortical region intermediate medial mesopallium of each hemisphere 45 min before bead discrimination training. As a measure of memory retention, bead discrimination was tested at 120 min after training, providing a discrimination ratio (DR) score for each chick. For complete memory retention a DR of 1.0 is obtained, whereas for complete loss of memory, a DR of 0.5 is obtained. Data are presented as Means + SEM (n = 11-15) **p < 0.001 (One-way ANOVA, F_3t49 = 7A0,p < 0.001, Dunnett's t-test) compared to vehicle control.

Figure 8 is a photographic and graphical representation illustrating decreased expression of RAP in AD brain. A&B. Representative micrographs of anti-RAP immunhistochemistry in the CAl of a control (A) and sporadic AD (B) case. Scale bar in A is equivalent for B. The majority of neurons in control CAl are RAP immunoreactive (A) whereas many neurons in AD cases were RAP-negative (B). C. Quantitative analysis revealed that a significantly greater percentage of CAl neurons were immunoreactive for RAP in controls compared with AD (p=0.003). Graph shows means ± SEM. BRIEF DESCRIPTION OF THE TABLES

Table 1 provides a description of the SEQ ID NOs provided herein.

Table 2 provides a list of abbreviations

Table 3 provides an amino acid sub-classification.

Table 4 provides exemplary amino acid substitutions.

Table 5 provides a list of non-natural amino acids contemplated in the present invention.

Table 6 provides control and AD cases used for RAP immunohistochemical analysis.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

As used herein, the term "about" refers to a quantity, level, value, percentage, dimension, size, or amount that varies by as much as 30%, 20% or 10% to a reference quantity, level, value, percentage, dimension, size, or amount.

The phrase "RAP polypeptide" includes compounds that induce the desired pharmacological and/or physiological effect of RAP as disclosed herein. The term also encompasses pharmaceutically acceptable and pharmacologically active ingredients of those compounds specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, derivatives, analogs and the like. When the above term is used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc. The phrase is not to be construed narrowly but extends to proteinaceous molecules including all forms of peptide, polypeptide and protein as well as mimetics and chemical analogs thereof as well as cellular agents.

The term "agent" in the phrase "agent from which RAP polypeptide is producible" includes a cell which is capable of producing and secreting RAP polypeptide as well as a polynucleotide comprising a nucleotide sequence that encodes a RAP polypeptide. In illustrative examples of this type, the RAP-encoding nucleotide sequence is operably connected to a regulatory element in a nucleic acid construct. Thus, the term "agent" extends to nucleic acid constructs including vectors such as viral or non-viral vectors, expression vectors and plasmids for expression in and secretion in a range of cells. "Analogs" contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs.

By "biologically active portion" is meant a portion part or fragment of a RAP polypeptide such as for example a RAP polypeptide whose amino acid sequence is set out in SEQ ID NO: 1 or SEQ ID NO: 5. In accordance with the present invention, the portion retains at least one of the herein described activities of a RAP polypeptide including binding to Aβ. As used herein, the term "biologically active portion" includes peptides, for example, of at least about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, 350 contiguous amino acids (and every integer in between) which comprise an activity of a reference RAP polypeptide including binding to Aβ. Another example of a biologically active portion is a RAP polypeptide without a signal sequence or ER retention signal. Another example of a fragment is a contiguous sequence of amino acids that comprises or consists of one or more of domain 1 (SEQ ID NO: 2), domain 2 (SEQ ID NO: 3), or domain 3 (SEQ ID NO: 4) of RAP. Portions of this type may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled "Peptide Synthesis" by Atherton and Shephard which is included in a publication entitled "Synthetic Vaccines" edited by Nicholson and published by Blackwell Scientific Publications. Alternatively, peptides can be produced by digestion of a peptide or polypeptide of the invention with proteinases such as endoLys-C, endoArg- C, endoGlu-C and staphylococcus V8-protease. The digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques. Recombinant nucleic acid or synthetic techniques can also be used to produce such portions. The biological activities of portions are tested in vivo and/or in vitro. In some embodiments, furin cleavage is preferred. By "cell" is meant any prokaryotic or eukaryotic cell. In particular non-limiting embodiments, the cell is a neural cell. A syngeneic cell is preferred which is genetically identical to the subject or is genetically compatible to minimize any immune response.

By "co-administered" is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. For example, the subject composition may be administered together with another agent in order to enhance its activity. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules. These molecules may be administered in any order.

"Complementary" as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA₅ RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other.

By "derivative" is meant a polypeptide that has been derived from the basic sequence by modification, for example by conjugation or complexing with other chemical moieties or by post-translational modification techniques as would be understood in the art. The term "derivative" also includes within its scope alterations that have been made to a RAP polypeptide including additions, or deletions that provide for functionally equivalent molecules.

A functional derivative of a polynucleotide encoding a RAP polypeptide comprises a sequence of nucleotides having at least 80% or 90% or 95% similarity identity to the polynucleotide over a reference window of comparison. A "part" or "portion" of a polynucleotide is defined as having a minimal size of at least about 10 nucleotides or preferably about 13 nucleotides or more preferably at least about 20 nucleotides and may have a minimal size of at least about 35 nucleotides. This definition includes all sizes in the range of 10-35 nucleotides including 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides as well as greater than 35 nucleotides including 50, 100, 300, 500, 600 nucleotides or nucleic acid molecules having any number of nucleotides within these values.

By "effective amount," in the context of treating AD is meant the administration of that amount of active to a subject, either in a single dose or as part of a series or slow release system, that is effective for treatment typically in a statistically significant number of subjects. The effective amount will vary depending upon the health and physical condition of the subject and the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

The terms "expression" or "gene expression" refer to either production of RNA message or translation of RNA message into proteins or polypeptides. Detection of either types of gene expression in use of any of the methods described herein are part of the invention.

By "expression vector" is meant any autonomous genetic element capable of directing the transcription of a polynucleotide contained within the vector and suitably the synthesis of a peptide or polypeptide encoded by the polynucleotide. Such expression vectors are known to practitioners in the art.

The term "gene" as used herein refers to any and all discrete coding regions of the cell's genome, as well as associated non-coding and regulatory regions. The gene is also intended to mean the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression. In this regard, the gene may further comprise control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals. The DNA sequences may be cDNA or genomic DNA or a fragment thereof. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host.

"Hybridization" is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid. Complementary base sequences are those sequences that are related by the base-pairing rules. In DNA, A pairs with T and C pairs with G. In RNA U pairs with A and C pairs with G. In this regard, the terms "match" and "mismatch" as used herein refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances as known to those of skill in the art.

The phrase "hybridizing specifically to" and the like refer to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g. total cellular) DNA or RNA.

By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated polynucleotide", as used herein, refers to a polynucleotide, isolated from the sequences which flank it in a naturally-occurring state, e.g. a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment. Alternatively, an "isolated peptide" or an "isolated polypeptide" and the like, as used herein, refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell. Without limitation, an isolated composition, complex, polynucleotide, peptide, or polypeptide can refer to a native sequence that is isolated by purification or to a sequence that is produced by recombinant or synthetic means.

By "modulation" or "modulator" in relation to a particular target is meant directly or indirectly up-regulating or down-regulating the level, effects or activity of the target. For example, the effects of Aβ may be down-regulated by binding to RAP polypeptide.

Reference to "mutants" or "mutations" includes the substitution or deletion of one or more amino acids within one or more domains of RAP. Insertional amino acids sequence mutants are those in which one or more amino acid residues are introduced into a predetermined site in a protein although random insertion is also possible with suitable screening of the resulting product. Deletional mutants include the removal of one or more amino acids. Substitutional mutants contain at least one residue that have been inserted in place of the wild-type (parent or naturally) occurring residue. Substitutions are either conservative or non-conservative.

Neurotrophins (nerve growth factors) include without limitation nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin 4/5 and neurotrophin 6. Neurotrophins are a group of small structurally and chemically related proteins which support the survival and development of neurones and maintain neuronal phenotypes.

The term "operably connected" or "operably linked" as used herein means placing a structural gene under the regulatory control of a promoter, which then controls the transcription and optionally translation of the gene. The terms "polynucleotide," "genetic material," "genetic forms," "nucleic acids" and "nucleotide sequence" include RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog (such as the morpholine ring), internucleotide modifications such as uncharged linkages (e.g. methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g. phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g. polypeptides), intercalators (e.g. acridine, psoralen, etc.), chelators, alkylators and modified linkages (e.g. α-anomeric nucleic acids, etc.).

Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen binding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. RNA forms of the genetic molecules of the present invention are generally niRNA or iRNA including siRNAs. The genetic form may be in isolated form or integrated with other genetic molecules such as vector molecules and particularly expression vector molecules. The terms "nucleotide sequence", "polynucleotide" and "nucleic acid molecule" used herein interchangeably and encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference nucleotide sequence whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide. The terms "polynucleotide variant" and "variant" refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains a biological function or activity of the reference polynucleotide. The terms "polynucleotide variant" and "variant" also include naturally-occurring allelic variants.

The terms "polypeptide," "proteinaceous molecule," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers. Reference to peptides includes a foldamer, peptido- including cyclic peptidomimetic, constrained or stapled peptides. These terms do not exclude modifications, for example, glycosylations, aceylations, phosphorylations and the like. Soluble forms of the subject proteinaceous molecules are particularly useful. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids or polypeptides with substituted linkages. The term "polypeptide variant" refers to polypeptides which are distinguished from a reference polypeptide by the addition, deletion or substitution of at least one amino acid residue. In certain embodiments, one or more amino acid residues of a reference polypeptide are replaced by different amino acids. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide (conservative substitutions) as described hereinafter.

By "obtained from" means derived from, either directly or indirectly.

The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, GIy₅ VaI, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, GIu₅ Asn, GIn₅ Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e. the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by an appropriate method. For example, sequence identity analysis may be carried out using the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.

Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP₅ BESTFIT, FASTA₅ and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al, Nucl. Acids Res. 25:3389, 1997. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al, "Current Protocols in Molecular Biology", John Wiley & Sons Inc, Chapter 15, 1994-1998.

The term "small molecule" refers to a non-peptide molecule that has a molecular mass of up to about 1500 Daltons, such as from about 200 or 400 to 1000 Daltons, or 600 to about 1200 Daltons, or from about 500 to 1500 Daltons.

"Stringency" as used herein refers to the temperature and ionic strength conditions, and presence or absence of certain organic solvents, during hybridization. The higher the stringency, the higher will be the observed degree of complementarity between sequences.

"Stringent conditions" as used herein refers to temperature and ionic conditions under which only polynucleotides having a high proportion of complementary bases, preferably having exact complementarity, will hybridize. The stringency required is nucleotide sequence dependent and depends upon the various components present during hybridization, and is greatly changed when nucleotide analogues are used. Generally, stringent conditions are selected to be about 10°C to 2O⁰C less than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a target sequence hybridizes to a complementary probe.

It will be understood that a preferred polynucleotide will hybridize to a RAP sequence or its complement under at least low stringency conditions, preferably under at least medium stringency conditions and more preferably under high stringency conditions. Reference herein to low stringency conditions include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42°C, and at least about 1 M to at least about 2 M salt for washing at 42°C. Low stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 niM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 2xSSC, 0.1% SDS; or (ii) 0.5% BSA₃ 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS for washing at room temperature. Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42⁰C, and at least about 0.5 M to at least about 0.9 M salt for washing at 42°C. Medium stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA₅ 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS for washing at 42⁰C. High stringency conditions include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization at 42°C, and at least about 0.01 M to at least about 0.15 M salt for washing at 42°C. High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 0.2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, ImM EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS for washing at a temperature in excess of 65°C. Other stringent conditions are well known in the art. A skilled addressee will recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization. For detailed examples, see Current Protocols in Molecular Biology (supra) at pages 2.10.1 to 2.10.16 and Sambrook et at, eds. Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, 1989 at sections 1.101 to 1.104.

The term "subject" as used herein refers to an animal, in particular a mammal and more particularly a primate including a lower primate and even more particularly, a human who can benefit from the medical protocols of the present invention. A subject regardless of whether a human or non-human animal or embryo may be referred to as an individual, subject, animal, patient, host or recipient. The present invention has both human and veterinary applications. For convenience, an "animal" specifically includes livestock animals such as cattle, horses, sheep, pigs, camelids, goats and donkeys, as well as companion animals. With respect to horses, these include horses used in the racing industry as well as those used recreationally or in the livestock industry. The terms "treatment" or "therapy" are used interchangeably in their broadest context and include any measurable or statistically significant change in one or more symptoms or frequency of one or more assessable indications of AD.

By "vector" is meant a polynucleotide molecule, suitably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast, virus, mammal, avian, reptile or fish into which a polynucleotide can be inserted or cloned. A vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector can be an autonomously replicating vector, i.e. a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain any means for assuring self-replication. Alternatively, the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are known to those of skill in the art.

The present invention provides, inter alia methods for treating symptoms of AD in a subject, as disclosed in the Summary, including the administration to a subject of an effective amount of RAP polypeptide or an agent from which a RAP polypeptide is producible.

RAP species homologs sharing more that about 60% amino acid sequence similarity have been identified in man, mice, rat, chicken, zebrafish, pig, invertebrates such as worms, mosquitoes and fruit flies. In accordance with the present invention, a RAP polypeptide encompasses any naturally-occurring RAP polypeptide from any animal species as well as their biologically active portions and variants or derivatives of these, as defined herein.

RAP polypeptides may be prepared by any suitable procedure known to those of skill in the art. For example, the polypeptides may be prepared by a procedure including the steps of: (a) preparing a construct comprising a polynucleotide sequence that encodes RAP polypeptide and that is operably linked to a regulatory element; (b) introducing the construct into a host cell; (c) culturing the host cell to express the RAP polypeptide; and (d) isolating the RAP polypeptide from the host cell. In illustrative examples, the nucleotide sequence encodes at least a biologically active portion of the sequence set forth in SEQ ID NO: 1, or a variant thereof. Recombinant RAP polypeptides can be conveniently prepared using standard protocols as described for example in Sambrook et al, 1989 (supra), in particular Sections 16 and 17; Ausubel et al, 1994 (supra), in particular Chapters 10 and 16; and Coligan et al, Current Protocols in Protein Science John Wiley & Sons, Inc. 1995-1997, in particular Chapters 1, 5 and 6. Alternatively, the RAP polypeptides may be synthesized by chemical synthesis, e.g. using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 of Atherton and Shephard (supra) and in Roberge et al, Science 269:202, 1995. The RAP polypeptide may be produced by any convenient method such as by purifying the polypeptide from naturally- occurring reservoirs including blood or serum. Methods of purification include lectin (e.g. wheat genu agglutinin) affinity chromatography or separation. The identity and purity of derived RAP is determined for example by SDS-polyacrylamide electrophoresis or chromatographically such as by high performance liquid chromatography (HPLC).

The RAP polypeptide of the present invention includes all biologically active naturally occurring forms of RAP as well as biologically active portions (fragments) thereof, and variants or derivatives of these. Biological activity as used herein refers to the ability of RAP polypeptide to modulate an activity of Aβ or reduce or otherwise ameliorate a symptom of AD or a related proteopathy. Biologically active portions of RAP polypeptide include parts of the amino acid sequence set out in SEQ ID NO: 1 or SEQ ID NO: 5. A biologically active portion of a full-length RAP polypeptide may comprise, for example, at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120 or 150, or even at least about 200, 220, 240, 260, 280, 300, 310, 320, or 330 contiguous amino acid residues, or almost up to the total number of amino acids present in a full-length RAP polypeptide. Suitably, the portion is a "biologically- active portion" having no less than about 10%, 20%, 30%, 40% 50%, 60%, 70%, 80%, 90%, 99% of the activity of the full-length RAP polypeptide from which it is derived. Suitable biologically active portions include soluble forms of the polypeptide without a leader or signal peptide.

RAP polypeptides include "variant" polypeptides that are distinguished from a naturally- occurring RAP polypeptide or from a biologically active portion thereof by the addition, deletion and/or substitution of at least one amino acid residue. Thus, variants include proteins derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.

Variant proteins encompassed by the present invention are biologically active, that is, they continue to possess the desired biological activity of the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native RAP polypeptide will have at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, preferably about 90% to 95% or more, and more preferably about 98% or more or 99% or more sequence similarity or identity with the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters. A biologically active variant of a

RAP polypeptide may differ from that polypeptide generally by as much 100, 50 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. A naturally occurring isolated RAP polypeptide or its encoding sequences may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a RAP polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, Proc. Natl. Acad. ScL USA 52:488-492, 1985, Kunkel et al, Methods in Enzymol. 254:367-382, 1987, U.S. Pat. No. 4,873,192, Watson et al, "Molecular Biology of the Gene", Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987 and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al,. Atlas of protein sequence and structure, National Biomedical Research Foundation, Washington DC, Vol. 5, pp. 345-358, 1978. Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of RAP polypeptides. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify RAP polypeptide variants (Arkin and Yourvan, Proc. Natl. Acad. ScL USA 59:7811-7815, 1992; Delgrave et al., Protein Engineering 6:321-331, 1993). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be desirable as discussed in more detail below.

Variant RAP polypeptides containing conservative amino acid substitutions at one or various locations along their sequence, as compared to the parent RAP amino acid sequence are encompassed. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.

Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows: Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having an acidic side chain include glutamic acid and aspartic acid.

Basic: The residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g. histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having a basic side chain include arginine, lysine and histidine.

Charged: The residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e. glutamic acid, aspartic acid, arginine, lysine and histidine).

Hydrophobic: The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan.

Neutral/polar: The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.

This description also characterizes certain amino acids as "small" since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity. With the exception of proline, "small" amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not. Amino acids having a small side chain include glycine, serine, alanine and threonine. The gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains. The structure of proline differs from all the other naturally-occurring amino acids in that its side chain is bonded to the nitrogen of the α- amino group, as well as the α-carbon. Several amino acid similarity matrices (e.g. PAM120 matrix and PAM250 matrix as disclosed for example by Dayhoff et al, 1978 {supra), A model of evolutionary change in proteins. Matrices for determining distance relationships in Dayhoff et ah, 1978 {supra); and by Gonnet et al., Science 256(5062): 11443-11445, 1992), however, include proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes of the present invention, proline is classified as a "small" amino acid.

The degree of attraction or repulsion required for classification as polar or nonpolar is arbitrary and, therefore, amino acids specifically contemplated by the invention have been classified as one or the other. Most amino acids not specifically named can be classified on the basis of known behavior.

Amino acid residues can be further sub-classified as cyclic or noncyclic, and aromatic or nonaromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always nonaromatic. Dependent on their structural properties, amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, sub-classification according to this scheme is presented in the Table 3.

Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional RAP polypeptide can readily be determined by assaying its activity. Conservative substitutions are shown in Table 3 below under the heading of exemplary substitutions. More preferred substitutions are shown under the heading of preferred substitutions. Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity.

Alternatively, similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains. The first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains; the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine; and the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G., Biochemistry, third edition, Wm.C. Brown Publishers, 1993.

Thus, a predicted non-essential amino acid residue in a RAP polypeptide is typically replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of a RAP polynucleotide coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity of the parent polypeptide to identify mutants which retain that activity. Following mutagenesis of the coding sequences, the encoded peptide can be expressed recombinantly and the activity of the peptide can be determined.

Accordingly, the present invention also contemplates variants of the naturally-occurring RAP polypeptide sequences or their biologically-active fragments, wherein the variants are distinguished from the naturally-occurring sequence by the addition, deletion, or substitution of one or more amino acid residues. In general, variants will display at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % similarity to a parent RAP polypeptide sequence as, for example, set forth in SEQ ID NO: 1. Desirably, variants will have at least 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity to a parent RAP polypeptide sequence as, for example, set forth in SEQ ID NO: 1 or the mature polypeptides lacking residues 1 to 34 of SES ID NO: 1. Moreover, sequences differing from the native or parent sequences by the addition, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60 ,70, 80, 90, 100 or more amino acids but which retain the properties of the parent RAP polypeptide are contemplated. RAP polypeptides also include polypeptides that are encoded by polynucleotides that hybridize under stringency conditions as defined herein, especially high stringency conditions, to RAP polynucleotide sequences, or the non-coding strand thereof. Illustrative RAP polynucleotide sequences encode the polypeptide described in SEQ ID NO: 1 , 2, 3 or 4.

In some embodiments, variant polypeptides differ from a RAP sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In another, variant polypeptides differ from the corresponding sequence in any one of SEQ ID NO: 1, 2, 3 or 4 by at least 1% but less than 20%, 15%, 10% or 5% of the residues. (If this comparison requires alignment the sequences should be aligned for maximum similarity. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, suitably, differences or changes at a non-essential residue or a conservative substitution. Naturally-occurring RAP polypeptides contain a significant number of structural characteristics in common with each other. An alignment shows positions that are amenable to conservative substitution and others that accommodate non- conservative substitutions.

A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of an embodiment polypeptide without abolishing or substantially altering one or more of its activities. Suitably, the alteration does not substantially alter one of these activities, for example, the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type.

An "essential" amino acid residue is a residue that, when altered from the wild-type sequence of a RAP polypeptide, results in abolition of an activity of the parent molecule such that less than 20% of the wild-type activity is present. For example, amino acid residues that are absolutely conserved between the RAP polypeptides of human, mice, and zebrafish may be unamenable to alteration.

In other embodiments, a variant polypeptide includes an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or more similarity to a corresponding sequence of a RAP polypeptide as, for example, set forth in any one of SEQ ID NO: 1, 2 or 4 and has the activity of a RAP polypeptide as described herein.

Another useful group of compounds that function as RAP polypeptides are functional derivatives, analogs and mimics (mimetics) of RAP. In accordance with the present invention, these molecules retain the ability to ameliorate symptoms of AD or related proteopathies, or to bind to Aβ and ameliorate its effects, enhance its uptake for degradation or transport away from neurons. Analogs and may also possess additional characteristics which improve their efficacy, such as exhibiting a longer half life in vivo or alternatively which are, for example, readily synthesized or readily taken up across the blood-brain barrier or by neurons or other cells. A peptide mimetic or mimic has some chemical similarity to the parent molecule, e.g. RAP, but agonizes its activity. A peptide mimic may be a peptide-containing molecule which mimics elements of protein secondary structure (as described for example in Johnson et at, "Peptide Turn Mimetics" in Biotechnology and Pharmacy, Pezzuto et a!., Eds., Chapman and Hall, New York, 1993). Non-peptide "small molecules" are often preferred for many in vivo pharmaceutical applications and accordingly mimetics may be designed for pharmaceutical use. Mimetic design, synthesis and testing is available to avoid randomly screening large numbers of molecules for a particular property, particularly where a lead compound has already been identified. As a first step, residues critical for binding are identified and this framework used as a pharmacophore. The structure may then be modeled using computational and other analyses. Alternatively, the three dimensional structure of RAP and Aβ are know and RAP polypeptides may be designed in silico along the same lines.

The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g. agonists, antagonists, inhibitors or enhancers) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g. enhance or interfere with the function of a polypeptide in vivo. See, e.g. Hodgson, Bio/Technology, P:19-21, 1991, Henchy et al, Curr Opin Chem Biol 12(6):692-7 ^', 2008 and reference referred to therein). In one approach, one first determines the three-dimensional structure of a protein of interest by x-ray crystallography, by computer modeling or most typically, by a combination of approaches. Useful information regarding the structure of a polypeptide may also be gained by modeling based on the structure of homologous proteins. An example of rational drug design is the development of HIV protease inhibitors (Erickson et al, Science, 249:527-533, 1990). In addition, target molecules may be analyzed by an alanine scan (Wells, Methods Enzymol, 202:2699-2705, 1991). In this technique, an amino acid residue is replaced by Ala and its effect on the peptide's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.

It is also possible to isolate a target-specific antibody, selected by a functional assay and then to solve its crystal structure. In principle, this approach yields a pharmacophore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides. Selected peptides would then act as the pharmacophore.

Analogs contemplated herein include but are not limited to modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs. This term also does not exclude modifications of the polypeptide, for example, glycosylations, aceylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid or polypeptides with substituted linkages. Such polypeptides may need to be able to enter the cell.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH₄; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide. Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino- 3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenyl glycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.

Suitable β-amino acids include, but are not limited to, L-β-homoalanine, L-β- homoarginine, L-β-homoasparagine, L-β-homoaspartic acid, L-β-homoglutamic acid, L-β- homoglutamine, L-β-homoisoleucine, L-β-homoleucine, L-β-homolysine, L-β- homomethionine, L-β-homophenylalanine, L-β-homoproline, L-β-homoserine, L-β- homothreonine, L-β-homotryptophan, L-β-homotyrosine, L-β-homovaline, 3-amino- phenylpropionic acid, 3-amino-chlorophenylbutyric acid, 3-amino-fluorophenylbutyric acid, 3-amino-bromopheynyl butyric acid, 3-amino-nitrophenylbutyric acid, 3-amino- methylphenylbutyric acid, 3-amino-pentanoic acid, 2-amino-tetrahydroisoquinoline acetic acid, 3 -amino-naphthyl -butyric acid, 3-amino-pentafluorophenyl-butyric acid, 3-amino- benzothienyl-butyric acid, 3-amino-dichIorophenyl-butyric acid, 3-amino-difluorophenyl- butyric acid, 3-amino-iodophenyl-butyric acid, 3-amino-trifluoromethylphenyl-butyric acid, 3-amino-cyanophenyl-butyric acid, 3-amino-thienyl-butyric acid, 3-amino-5- hexanoic acid, 3-arnino-furyl-butyric acid, 3-amino-diphenyl-butyric acid, 3-amino-6- phenyl-5-hexanoic acid and 3-amino-hexynoic acid.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid contemplated herein is shown in Table 5.

Sugar amino acids are sugar moieties containing at least one amino group as well as at least one carboxyl group. Sugar amino acids may be based on pyranose sugars or furanose sugars. Suitable sugar amino acids may have the amino and carboxylic acid groups attached to the same carbon atom, α-sugar amino acids, or attached to adjacent carbon atoms, β-sugar amino acids. Suitable sugar amino acids include but are not limited to

Sugar amino acids may be synthesized starting from commercially available monosaccharides, for example, glucose, glucosamine and galactose. The amino group may be introduced as an azide, cyanide or nitromethane group with subsequent reduction. The carboxylic acid group may be introduced directly as CO₂, by Wittig reaction with subsequent oxidation or by selective oxidation of a primary alcohol. In some embodiments, the RAP analog or agonist is a small chemical molecule. New chemical entities, natural products, combinatorial synthetic organic or inorganic compounds, peptide/polypeptide/protein, nucleic acid molecules and libraries or phage or other display technology comprising these are all available to screen or test for suitable agents. Natural products include those from coral, soil, plant, or the ocean or Antarctic environments. Libraries of small organic molecules can be generated and screened using high-throughput technologies known to those of skill in this art. See for example United States Patent No. 5,763,623 and United States Application No. 20060167237. Combinatorial synthesis provides a very useful approach wherein a great many related compounds are synthesized having different substitutions of a common or subset of parent structures. Such compounds are usually non-oligomeric and may be similar in terms of their basic structure and function, for example, varying in chain length, ring size or number or substitutions. Virtual libraries are also contemplated and these may be constructed and compounds tested in silico (see for example, US Publication No. 20060040322) or by in vitro or in vivo assays known in the art. Libraries of small molecules suitable for testing are available in the art (see for example, Amezcua et al, Structure (London) 70:1349-1361, 2002 and Rapisarda et al, Cancer Res 62:4316-24, 2002). Yeast SPLINT antibody libraries are available for testing for intrabodies which are able to disrupt protein-protein interactions (see Visintin et al, J. Biotechnol, 735:1-15, 2008; Visintin et al, J. Immunol. Methods, 290(l-2):135-53, 2008). Examples of suitable methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et ah, Proc. Natl. Acad. ScI USA 90:6909, 1993; Erb et al., Proc. Natl Acad. ScI USA 91:11422, 1994; Zuckermann et al., J. Med. Chem. 37:2678, 1994; Cho et al, Science 261:1303, 1993; Carrell et al, Angew. Chem. Int. Ed. Engl 33:2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl. 33:2061, 1994; and Gallop et al.J. Med. Chem. 37:1233, 1994.

Thus, agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is suited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145, 1997; United States Patent No. 5,738,996; and United States Patent No. 5,807,683). Libraries of compounds may be presented, for example, in solution (e.g. Houghten, Bio/Techniques 75:412-421, 1992), or on beads (Lam, Nature 354:82-84, 1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (United States Patent No. 5,223,409), spores (United States Patent Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. ScL USA S9:1865- 1869, 1992) or phage (Scott and Smith, Science 249:386-390, 1990; Devlin, Science 249:404-406, 1990; CwMa et al., Proc. Natl. Acad. Sci. USA 57:6378-6382, 1990; and Felici, J. MoI Biol. 222:301-310, 1991).

The proteins necessary for high capacity assays may be produced in bacteria. One useful assay suitable for high throughput is Amplified Luminescent Proximity Homogenous Assay (ALPHA) technology described in Glickman et al, J Biomol Screen. 7(l):3-10, 2002. By revealing changes in fluorescent output as two partner proteins interact, it can monitor protein interactions with exquisite sensitivity. Compounds that pass the initial tests can be checked for purity and identity such as by liquid chromatography, mass spectrometry and them tested for target specificity. Active compounds may be analysed by NMR spectrometry which can greatly facilitate drug discovery but informing regarding target protein binding and low affinity interactions. Active compounds are also tested in assays designed to predict inactive absorption, hepatocytotoxicity. In silico methods can be used to predict compound biodistribution properties, and to exclude pharmacophores.

The three-dimensional structures of RAP and Aβ have been determined and this facilitates the design of binding agents that modulate Aβ activity. Peptide or non-peptide mimetics are anticipated to mimic elements of protein secondary structure and permit molecular interactions similar to the natural molecule. Leads selected may require some or considerable modification to enhance their biological, biochemical and pharmacological properties. Lead compounds identified in screening process can be optimised by molecular modelling in silico.

Three-dimensional representations of the structure of one or more binding sites of Aβ and/or RAP or a variant, derivative or analog of either of these molecules are used to identify interacting molecules that, as a result of their shape, reactivity, charge potential etc. favourably interacts or associate. In a preferred aspect, the skilled person can screen three-dimensional structure databases of compounds to identify those compounds having functional groups that will fit into one or more of the binding sites. Combinational chemical libraries can be generated around such structures to identify those with high affinity binding to appropriate binding sites. Agents identified from screening compound databases or libraries are then fitted to three-dimensional representations of RAP or Aβ binding sites in fitting operations using, for example docking software programs.

A potential modulator may be evaluated "in silico" for its ability to bind to a Aβ binding site prior to its actual synthesis and testing. The quality of the fit of such entities to binding sites may be assessed by, for example, shape complementarity by estimating the energy of the interaction (Meng et al, J. Comp. Chem., i3:505-524, 1992).

The design of chemical entities that associate with Aβ or other amyloid proteins will involve consideration of two factors. The compound must be capable of physically and structurally associating with Aβ. Non-covalent molecular interactions important in the association of a compound with its interacting partners include hydrogen bonding, van der Waal's and hydrophobic interactions. Second, the compound must be able to assume a conformation that allows it to associate with Aβ. Although certain portions of the compound will not directly participate in this association with Aβ, those portions may still influence the overall conformation of the molecule. Such conformation requirements include the overall three-dimensional structure and orientation of the chemical entity or compound in relation to all or a portion of the active site, or the spacing between functional groups of a compound comprising several chemical entities that directly interact with amyloid peptides.

Once a binding compound has been optimally selected or designed, as described above, substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties. Generally, initial substitutions are conservative, i.e. the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should of course be understood that components known in the art to alter conformation should be avoided.

Putative binding agents may be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with the one or more binding sites. Selected fragments or chemical entities may then be positioned in a variety of orientations, or "docked," to target binding sites. Docking may be accomplished using software, such as QUANTA and SYBYL, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM or AMBER. Specialised computer programs may be of use for selecting interesting fragments or chemical entities. These programs include, e.g. GRID (Oxford University, Oxford, UK), 5 MCSS (Molecular Simulations, USA), AUTODOCK (Scripps Research Institute, USA), DOCK (University of California, USA), XSITE (University College of London, UK) and CATALYST (Accelrys).

Useful programs to aid the skilled addressee in connecting chemical entities or fragments include CAVEAT (University of California, USA), 3D database systems and HOOK (Molecular Simulations, USA) De-novo ligand design methods include those described in LUDI (Molecular Simulations, USA), LEGEND (Molecular Simulations, USA), LeapFrog (Tripos Inc.,) SPROUT (University of Leeds, UK) and the like.

Structure based ligand design is well known in the art and various strategies are available which can build on structural information provided herein to determine ligands which effectively modulate the activity of amyloid proteins. Molecular modelling techniques 0 000113

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include those described by Cohen et al, J. Med. Chem., 33:883-894, 1990, and Navia et al, Current Opinions in Structural Biology, 2:202-210, 1992.

In another aspect, the medicament is an autologous cell derived from the subject to be treated or a syngeneic cell. In some embodiments, the cell is genetically modified in order to secrete a RAP polypeptide. Other cells, such as neurones secrete RAP polypeptide naturally. In other embodiments, the cell is a genetically modified neuronal cell capable of producing RAP polypeptide. In still further embodiments, the cell is a stem cell or progenitor cell for a neuronal cell. In an illustrative example of this type, the stem cell is an neuronal cell progenitor cell. Genetically modified neuronal stem cells are conveniently used in order to treat proteopathies.

Recombinant methods for producing genetically modified cells from which RAP polypeptide is producible are routine in the art. Essentially, a polynucleotide encoding a RAP polypeptide is engineered within an expression construct or shuttle vector and operably linked to a regulatory element (e.g. a promoter) that is operable in the cell in which it is desired to express the polynucleotide. In other embodiments, the nucleic acid construct that is delivered remains episomal and induces an endogenous and otherwise silent gene. Generally, a selective marker gene such as an antibiotic resistance marker gene is employed to facilitate selection of appropriately modified cells. In some embodiments, the polynucleotide (cDNA) is selected (amplified) or modified by removal of sequences encoding signal sequences to facilitate secretion of a soluble or mature RAP polypeptide. Mammalian expression vectors capable of expression in mammalian epidermal cells are, for example, routinely available. Construction of recombinant DNAs comprising RAP polynucleotides and a mammalian vector capable of expressing inserted DNAs in cultured human or animal cells, can be carried out by standard gene expression technology using methods well known in the art for expression of such a relatively simple polypeptide. Promoters for selective expression in a range of cells have also been identified and documented. A number of viruses have been used as gene transfer vectors or as the basis for preparing gene transfer vectors, including papovaviruses, adenovirus, vaccinia virus, adeno- associated virus, herpesviruses including HSV and EBV, Antiviruses, Sindbis and Semliki Forest virus and retroviruses of avian and murine origin. Viral and non-viral methods of polynucleotide delivery are available. Various viral vectors are routinely used to transform a range of different cell types with adequate efficiency.

In some embodiments, the RAP polynucleotide comprises a nucleotide sequence that encodes wholly or partially the amino acid sequence set forth in SEQ ID NO: 1, or a sequence having at least 80% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 1. In some embodiments, the RAP polynucleotide comprises all or part of the nucleotide sequence encoding the polypeptide described in SEQ ID NO: 1, or a sequence having at least 80% sequence identity to SEQ ID NO: 1, or a sequence that hybridizes to the nucleotide sequence encoding the sequence set out in SEQ ID NO: 1 or to a complementary form thereof under at least medium stringency conditions.

In another embodiment, the present invention provides contemplates an antibody or antigen-binding fragment thereof which mimics the ability of RAP to bind to Aβ and modulate its functional activity.

Hence, compounds which have the potential to act as modulators include small chemical molecules which can penetrate a cell membrane or via an ion channel or other pore and an antigen binding agent which has the capacity for intracellular transmission such as cartilage fish-derived antibodies (e.g. shark antibodies, see for example, Lui et al., BMC Biotechnol. 7:78, 2007). The latter are described in International Patent Publication No. WO 2005/118629. An antigen binding agent, or functionally active fragment thereof, which has the capacity for intracellular transmission also includes antibodies such as camelids and llama antibodies, scFv antibodies, intrabodies or nanobodies, e.g. scFv intrabodies and VH_H intrabodies. Such antigen binding agents can be made as described by Harmsen & De Haard, Appl. Microbiol. Biotechnol. 77(l):l3-22, 2007; Tibary et al, Soc. Reprod. Fertil. Suppl. 64:291-313, 2007; Muyldermans, J Biotechnol. 74:211-302, 2001; and references cited therein. For use in the methods of the invention, such agents may comprise a cell-penetrating peptide sequence or nuclear-localizing peptide sequence such as those disclosed in Constantini et al., Cancer Biotherm. Radiopharm. 25(7^:3-24, 2008. Also useful for in vivo delivery are Vectocell or Diato peptide vectors such as those disclosed in De Coupade et al, Biochem J. 390(ρt2):407-418, 2005 and Meyer-Losic et al., J Med Chem. 49(23^:6908-6916, 2006. Thus, the invention provides the therapeutic use of fusion proteins of the agents (or functionally active fragments thereof), for example but without limitation, where the antibody or fragment thereof is fused via a covalent bond (e.g. a peptide bond), at optionally the N-terminus or the C-terminus, to a cell-penetrating peptide or nuclear-localizing peptide sequence.

In another aspect, the present invention provides a composition for use in therapy. In some embodiments the composition comprises a pharmaceutically acceptable carrier, diluent and/or excipient.

Diseases associated with the accumulation of insoluble protein deposits (amyloid) are known as "proteopathies" or "proteinopathies amyloidoses" or "conformational diseases" and comprise clinically and pathologically diverse disorders. Examples of proteopathies are AA amyloidosis, AH (heavy chain) amyloidosis, AL (light chain) amyloidosis, Alexander disease, Alzheimer's disease, amyotrophic lateral sclerosis, aortic medial amyloidosis, apoAl amyloidosis, apoA2 amyloidosis, apoA4 amyloidosis, CADASIL, cardiac atrial amyloidosis, cataract, cerebral amyloid angiopathy, corneal lactoferrin amyloidosis, Creutzfeld-Jacob disease, mad cow disease, critical illness myopathy, cutaneous lichen amyloidosis, dialysis amyloidosis, Familial amyloidotic neuropathy, familial British dementia, familial Danish dementia, familial visceral amyloidosis, fibrinogen amyloidosis, Finnish hereditary amyloidosis, frontotemporal lobar dementia, hereditary cerebral hemorrhage with amyloidosis - Dutch type, hereditary cerebral hemorrhage with amyloidosis - Icelandic type, Huntington's disease, hereditary lattice corneal dystrophy, inclusion body myositis/myopathy, lysozyme amyloidosis, Creutzfeld- Jacob disease, mad cow disease, medullary thyroid carcinoma, Odontogenic (Pindborg) tumor amyloid, Parkinson's disease, pituitary prolactinoma, primary systemic amyloidosis, primary cutaneous amyloidosis, prion disease, pulmonary alveolar proteinosis, seminal vesicle amyloid, seipinopathy, senile systemic amyloidosis, serpinopathy, sickle cell disease, synucleinopathy, tauopathy, type 2 diabetes. Reference herein to the phrase "related conditions" in the context of AD in some embodiments includes conditions such as those listed supra which are characterised by amyloid deposits in the brain, memory loss and dementia. In other embodiments, the term extends to amyloidoses generally, including those listed supra.

As discussed in relation the methods of the present invention, the composition of the present invention, in some embodiments, comprises a cellular agent. In an illustrative example of this type the cell is a genetically modified syngeneic cell that produces a RAP polypeptide. In other embodiments, the cell is a viral cell capable of transforming cells and causing them to produce RAP polypeptide. In other embodiments, the cell naturally produces RAP polypeptide such as a cell of the neural lineage.

As described above, in another aspect the present invention provides a use of a RAP polypeptide or an agent from which a RAP polypeptide is producible or a RAP analog or mimetic in the manufacture of a medicament for the treatment of AD.

In some embodiments, the medicament is suitable for local or systemic administration by any route, such as without limitation by patch, cellular transfer, implant, orally, intravenously, intravesicaly, intracerebrally, intradermally, intramuscularly, intraperitoneally, intrathecally, subcutaneously, sublingually, rectally, vaginally, iiitraocularly, nasally, respiratorialy, nasopharyngeal, subcutaneously, cutaneously, topically and transdermally.

Pharmaceutical compositions are conveniently prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing, Company, Easton, PA, U.S.A., 1990). The composition may contain the active agent or pharmaceutically acceptable salts of the active agent. These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. intravenous, oral or parenteral.

For oral administration, the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques. The active agent can be encapsulated to make it stable to passage through the gastrointestinal tract. See for example, International Patent Publication No. WO 96/11698. For parenteral administration, the compound may dissolved in a pharmaceutical carrier and administered as either a solution or a suspension. Illustrative of suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin. The carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.

The actual amount of active agent administered and the rate and time-course of administration will depend on the nature and severity of the burn injury. Prescription of treatment, e.g. decisions on dosage, timing, etc. is within the responsibility of general practitioners or specialists and typically takes into account the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences {supra). The pharmaceutical composition is contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal and the agent chosen. A broad range of doses may be applicable. Considering a patient, for example, from about 0.1 ng, 0.2 ng, 0.3 ng, 0.4 ng, 0.5 ng, 0.6 ng, 0.7 ng, 0.8 ng. 0.9 ng, or 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg. 0.9 mg to about 1 to 10 mg or from 5 to 50 mg of RAP polypeptide oragent may be administered per kilogram of body weight per day. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.

The agents may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intraperitoneal, intramuscular, subcutaneous, intradermal or suppository routes or implanting (e.g. using slow release molecules). The agent or composition comprising the agent may be administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g. with zinc, iron or the like (which are considered as salts for purposes of this application). Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is to be administered in tablet form, the tablet may contain a binder such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate.

The present invention is further described by the following non-limiting Examples. EXAMPLE 1

Preparation and Testing of RAP polypeptide

Materials. Human recombinant RAP was expressed and purified as described (Medved et al, J Biol Chem 274:111-121, 1999). Rabbit polyclonal IgG against LRP, R2629, was produced and affinity-purified as described (Kounnas et al, J Biol Chem 2(57:12420-12423, 1992). Mouse monoclonal antibodies were purchased as follows: anti- RAP (clone 7Fl) from Merck-Calbiochem (Kilsyth, VIC₅ Australia), anti-Aβ (clone 6E10) from Sigma- Aldrich (Castle Hill, NSW, Australia), anti-transferrin receptor (clone H68.4) from Invitrogen-Zymed (Mt Waverley, VIC, Australia). Heparin from porcine intestinal mucosa and methyl anthranilate were purchased from Sigma-Aldrich. Hoechst 33342, fiuo-4 acetoxymethyl (AM) ester, and all AlexaFluor-conjugated secondary antibodies and choleratoxin B subunits (CTX) were purchased from Invitrogen-Molecular Probes (Mt Waverley, VIC, Australia). Protein G agarose was purchased from Roche (Castle Hill, NSW, Australia). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG was purchased from GE Lifesciences-Amersham (Rydalmere, NSW, Australia).

Aβ peptide preparation and aggregation. Synthetic Aβ_1-42 N-terminally labeled with fluorescein (FluoAβ_1-42) and recombinant Aβ_1-40 were purchased from rPeptides (Athens, GA, USA). Aβ_1-42 was purchased from Keck Laboratories (New Haven, CT, USA). All peptides were of >95% purity as assessed by high performance liquid chromatography and mass spectrometry. Aβ peptides were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/ml and stored at -8O⁰C. For intracellular Ca²⁺ measurement, Aβ_1-42 (2.5 mg/ml) was dissolved in hexafluoroisopropanol, diluted in distilled water at 250 μg/ml, then centrifuged at 14,000 x g at room temperature for 20 min prior to addition to cells. "Aging" of Aβ peptides was performed by incubating the peptide (1 μM) in the absence or presence of RAP (5 μg/ml) in 50 mM NaH₂PO₄ containing 100 mM NaCl (NaCl/Pi), pH 7.4 at 37⁰C.

SH-SY5Y neuroblastoma cell culture. SH-SY5Y cells were obtained from the American Type Culture Collection (CRL-2266; Manassas, VA, USA) and were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 (Invitrogen-Gibco, Mt Waverley, VIC, Australia) containing 100 U/ml penicillin, 100 U/ml streptomycin, 2 mM L-glutamine and 10% (v/v) fetal bovine serum (FBS)₅ in an atmosphere of 5% CO₂. Cells were passaged by rinsing with warm phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 6.5 mM Na₂HPO₄, 1.76 mM KH₂PO₄), pH 7.4, then dissociating with trypsin. Serum-free medium was DMEM/F12 containing penicillin, streptomycin and 10 mM HEPES, pH 7.4 at 37°C. For confocal microscopy, cells were plated on 13 mm autoclaved coverslips in 24-well plates and grown to 50% confluence before treatment. For flow cytometry and for analysis of RAP uptake, cells were plated in 48-well plates and 96-well plates, respectively, and grown to 80-90% confluence before treatment. For intracellular Ca²⁺ measurement, cells were plated in black, clear-bottomed 96-well plates (Coming, Lindfield, NSW, Australia) and grown to 50% confluence before treatment. Cells were rinsed once in serum-free medium, then treated with FluoAβ_1-42 (1 μM), or Aβ_1-42 (1 μM), with or without RAP (5 μg/ml), in serum-free medium at 37⁰C.

Immunocytochemistry and confocal microscopy. Following Aβ treatment, SH-S Y5Y cells were incubated on ice with AlexaFluor-555 or 647 conjugated CTX (Alexa555-CTX or CTX-647; 250 ng/ml) in serum-free medium for 15 min. The cells were then rinsed once with ice-cold PBS containing 0.5 mM MgCl₂, 0.5 mM MgSO₄ and 1 mM CaCl₂ (PBS/Mg²⁺/Ca²⁺) then fixed with 4% paraformaldehyde in PBS/Mg²⁺/Ca²⁺. Cells were then permeabilized in 1% (v/v) Triton X-100 for 5 min before rinsing and blocking in PBS containing 0.05% Tween-20 (PBS-T) with 2% (w/v) bovine serum albumin for 1 h. Primary (7Fl, 1:4000; R2629, 1 μg/ml) and secondary (anti-mouse conjugated to AlexaFluor 647 [anti-mouse 647], 1:1000; anti-rabbit conjugated to AlexaFluor 546 [anti- rabbit 546], 1:1000) antibody incubations were for 1 h in PBS-T containing 1% (w/v) bovine serum albumin. Immunolabeled cells on coverslips were rinsed, incubated with Hoechst 33342 (1 μg/ml) in PBS for 5 min, then rinsed and mounted on microscopy slides in Fluoromount G (Southern Biotech, Birmingham, AL, USA). Images were captured on an Olympus FVlOOO confocal microscope, using Kalman integration of two scans and sequential scanning of channels to minimize bleed-through. Images were processed using the ImageJ software (Abramoff et at, Biophotlnt 11:36-42, 2004). Flow cytometry. SH-S Y5 Y cells were incubated with FluoAβ_1-42 for 4 h then placed on ice, rinsed with ice-cold PBS and allowed to lift in PBS containing 1 mM EDTA and 1% (v/v) FBS. After 30 min, cells were transferred to polystyrene tubes and triturated. Twenty min prior to analysis, propidium iodide (PI; 0.1 μg/ml) was added to each tube. Ten thousand cells were read from each well at a rate of 100-300 cells per second in a FC500 cytometer (Beckman Coulter, Gladesville, NSW, Australia). Each incubation was performed in triplicate and experiments were performed at least twice. Pi-negative single cells were selected for inclusion in data analysis using WinMDI software (v2.9).

Analysis of RAP uptake. SH-SY5Y cells were treated with RAP (5 μg/ml) in the absence or presence of R2629 (10 μg/ml) for 4 h, then rinsed and lysed in lysis buffer (50 mM Tris- HCl, pH 7.5, 150 mM NaCl, 1% [v/v] Nonidet P40, 0.5% [v/v] sodium deoxycholate). Lysates were applied to an UltraBind polyvinylsulfone membrane using a Bio-Rad slot blot apparatus, then the membrane was probed for RAP using 7Fl and anti-mouse AlexaFluor 647. Fluorescence on the membrane was detected using a Typhoon gel scanner (GE Lifesciences-Amersham), and band density was quantitated using ImageQuant software.

Immunoprecipitation and immunoblotting. Protein G agarose beads were incubated in lysis buffer containing 1 μg of either 7Fl or H68.4 for 2 h at 4⁰C. Solutions containing Aβ and/or RAP in NaCl/Pj were diluted 1:1 in lysis buffer, and then 500 μl of this solution was incubated with 20 μl of antibody-coated beads for 3 h at 4⁰C. The beads were then rinsed thrice in lysis buffer and twice in 50 mM Tris-HCl, pH 7.5 containing 0.1% (v/v) Nonidet P40 and 0.05% (v/v) sodium deoxycholate, then boiled in an equal volume of 2* SDS sample buffer (125 mM Tris-HCl, pH 6.8, 4% [v/v] SDS, 20% [v/v] glycerol, 0.01% [w/v] bromophenol blue) and β-mercaptoethanol (β-ME) to 5% (v/v). For non- immunoprecipitated samples, three parts of Aβ solution were combined with one part of 4x SDS sample buffer (250 mM Tris-HCl, pH 6.8, 8% [v/v] SDS, 40% [v/v] glycerol, 0.02% [w/v] bromophenol blue) and β-ME to 10% (v/v) and heated at 90⁰C for 5 min. Equal volumes of samples were loaded onto 16.5% Tris-tricine, 12% Tris-glycine, or 15% Tris- glycine gels and separated electrophoretically. Proteins were transferred onto 0.22 μm nitrocellulose membranes then the membranes were air dried, boiled in PBS for 5 min, and immunoblotted. Incubations with primary (6E10, 1:1000; 7Fl, 1:4000) and secondary (anti-mouse HRP, 1:4000) antibodies were carried out in blocking buffer for 1.5 h and 1 h, respectively, and detection was achieved via enhanced chemiluminescence (GE Lifesciences-Amersham). When required, antibodies were stripped from the membrane by incubating in 62.5 mM Tris-HCl, pH 6.7, containing 2% (v/v) SDS and 100 mM β-ME at 50°C for 30 min before reprobing.

Atomic force microscopy (AFM). Aβ peptide samples were stored at -2O⁰C before analysis. AFM was performed essentially as previously described (Hou et al, J Neurochem 100:446-457, 2007). Samples were applied to a substrate of highly oriented pyrolytic graphite, which was then briefly rinsed with distilled deionized water and dried under a constant flow of nitrogen. Imaging was performed by tapping mode in air using NSCl 5 silicon probes (Mikromasch, Tallinn, Estonia) on a Nanoscope IV Multimode scanning probe microscope (Veeco Corp., Santa Barbara, CA, USA). AFM images were analysed using the WSxM 4.0 software (Horcas et al, Rev Sci lustrum 75:013705, 2007).

Measurement of intracellular Ca²⁺. Intracellular Ca²⁺ measurements were performed in DMEM/F12 without phenol red (Invitrogen). Cells were loaded with fluo-4 AM (2 μM) for 7-10 min at 37⁰C. Following removal of fluo-4 AM, cells were rinsed and incubated for a further 30 min in 100 μl medium at 37⁰C to allow complete de-esterification. Fluorescence was measured in a microplate reader (FluoStar Optima, BMG Labtechnologies, Offenburg, Germany) equipped with fluorescence optics (excitation 485 nm, emission 520 nm). Fluorescence measurements were made every 8 s. Baseline was determined for 56 s before addition of Aβ_1-42 (10 μM; final concentration of 1 μM) with or without RAP (50 μg/ml; final concentration of 5 μg/ml), and the response was monitored for a further 200 s. Quantitation of intracellular Ca²⁺ response was achieved by subtracting the average background response to vehicle treatments from Aβ treatments, then calculating AF/F values where AF represents the fluorescence change of cells compared to 13

- 57 -

baseline fluorescence (F). For R2629 pre-treatment of cells, fluo-4 AM loading was performed in the presence of R2629 (10 μg/ml).

Chick discriminative avoidance memory task. The ability of RAP to influence the functional effects of Aβ was tested in a recently established in vivo model of Aβ-induced inhibition of memory consolidation using a discriminative avoidance task (Gibbs et ah, Neurobiol Aging, [Available online, accessed July 15, 2008] doi:10.1016/j.neurobiolaging.2008.05.018₅ 2008). Aβ_1-42 was diluted from DMSO in cold physiological saline (0.9% [w/v] NaCl) to yield a final peptide concentration of 2 μM. RAP (10 μg/ml) was immediately added and samples were stored at 4⁰C for no more than 1 h prior to injecting into day-old chicks. At 45 min prior to training, Aβ with or without RAP (5 μl) was injected directly into the intermediate medial mesopallium of each hemisphere of each chick, as described previously (Gibbs et ah, 2008 (supra)). During the training trial, chicks were presented for 10 s with a red bead that had been dipped in 100% methyl anthranilate, which tastes bitter and induces an avoidance response. At 120 min after training, chicks were tested for memory retention by presentation of both red and blue beads for 10 s. The number of pecks at the blue and red beads, respectively, were counted and used to calculate a discrimination ratio (DR; no. of pecks at blue bead / total no. of pecks) for each chick (Gibbs and Ng, Biobehav Rev 1:113-136, 1977; Ng et at, Neurosci Biobehav Rev 21:45-54, 1997). Each condition was tested on a group of 16 chicks, according to Gibbs et al, 2008 (supra).

Brain tissue. Brain tissue from 7 AD and 8 clinical and neuropathological controls were available for immunohistochemical analysis (Table 6). Diagnosis of AD (and no other neurodegenerative condition) or control (without neurological or neuropathological disease) was based on longitudinal clinical and systematic neuropathological assessments, as previously described (Shepherd et al, Neurobiol. Dis. 9: 249-257, 2002). AU cases were matched for sex, age and post-mortem delay. The post-mortem delay was 24 + 6 h for control tissue and 22 + 8 h for AD tissue (mean + SEM). Immunohisto chemistry of brain tissue. For quantitative analysis of RAP in brain, 10 μm thick sections were cut from paraffin-embedded formalin-fixed tissue blocks of the hippocampus taken from 7 AD and 8 control cases. Peroxidase immunohistochemistry was carried out using anti-RAP mouse monoclonal antibody (7Fl). Briefly, antigen retrieval was carried out by microwaving the tissue sections on high for 12 min in Tris-buffered saline followed by incubation at room temperature for 1 hr. Endogenous peroxidase activity was then inhibited by treatment with 1% H₂O₂ in ethanol for 30 min. The tissue was then incubated in 10% serum to eliminate non-specific binding. The primary antibody was incubated overnight at 4⁰C at a concentration of lμg/ml. Sections were then sequentially incubated with biotinylated secondary antibodies (Vector biotinylated secondary immunoglobulin G antibodies) for 2 hr at room temperature, streptavidin- conjugated horseradish peroxidase (Vector Elite ABC) for 30 minutes at 25°C, and with 3,3'-diaminobenzidine in H₂O₂ until the reaction products were visualised (5-10 min). All sections were lightly counterstained with cresyl violet (0.5% aqueous solution) then rapidly dehydrated through alcohol (70%, 95%, and twice with 100%), cleared in xylene and coverslipped with p-xylene-όzs-pyridinium bromide (DPX). The specificity of the immunohistochemical reactions was tested by omitting the primary antiserum. No reaction was observed in the negative control sections.

Quantification of RAP -positive neurons. Quantification was carried out using an 11x11 eye piece grid at 20Ox magnification on a Zeiss microscope in the CAl of the hippocampus. Neurons were identified by the presence of a clear nucleolus and defined cytoplasm. Both total (cresyl violet positive) and RAP-imrnunoreactive neurons were counted in the eye piece grid and the percentage of RAP-immunoreactive neurons calculated. Ten repeated measurements on all slides from one case at different time intervals did not vary by more than 5%. Counts on the same slides from four cases by different investigators varied on average by 7 %.

Statistical analysis. For RAP immunohistochemical studies, statistical analysis was performed using Statview (Abacus, Berkeley, CA, USA). Statistical tests were performed using GraphPad Prism software (v5.0). ANOVA followed by post-hoc comparisons or Student's t-tests were used to compare groups. Values of p < 0.05 were considered statistically significant.

EXAMPLE 2

Extracellular RAP polypeptide binds strongly to Aβ and increases Aβ binding to neuronal cells independent of presence of LDL receptor

As extracellular Aβ binds to neuronal cell membranes (Lambert et al, 1998 {supra)), it has been hypothesized that this interaction may underlie the neurological dysfunction seen in AD (Small et al, 2001 {supra)). Human SH-SY5Y neuroblastoma cells were tested because of their well documented responses to Aβ treatment (Demuro et al., J Biol Chem 250:17294-17300, 2005; Petratos et al, Brain 737:90-108, 2008). Binding of labelled Aβ to SH-SY5Y cells was examined.

Fluorescein-labeled Aβ_1-42 was incubated with SH-SY5Y cells and its cell association was evaluated by confocal microscopy (Figure 1). A brief incubation on ice with Alexa 555- conjugated CTX allowed visualization of the plasma membrane. Following 1 hour (h) of incubation, little, if any, FluoAβ_1-42 was observed in association with the cells. However, after 4 h of incubation, cell-associated FluoAβi-₄₂ was observed. Most of the FluoAβ_1-42 was intracellular, distributed in a punctate fashion. However, some of the FluoAβi.₄₂ bound to the cell surface. After a longer (24 h) incubation, almost all of the FluoAβ_1-42 fluorescence was intracellular, with little fluorescence detected at the plasma membrane.

Previous studies have reported that Aβ can bind LRPl both directly (Deane et al, 2004 {supra)) although this has been disputed (Yamada et al., 2008 {supra)), and indirectly (Narita et al, J Neurochem (59:1904-1911, 1997; Zerbinatti et al, 2006 {supra)), and that this interaction can mediate Aβ uptake into cells. Since SH-SY5Y cells are known to express LRPl, LRPl -mediated Aβ uptake was assessed by analyzing the effect of the endogenous LRPl ligand, RAP on the binding and uptake of FluoAβi-^. However, contrary to previous studies which have reported that RAP inhibits Aβ binding to cells (Deane et al, 2004 {supra); Nazer et al, Neurobiol Dis 30:94-102, 2008), it is shown here that RAP increases FluoAβ_1-42 binding to SH-SY5Y cells (Figure 2). At higher magnification, it was apparent that in the presence of RAP₅ FluoAβ_1-42 was accumulating both at the cell surface and intracellular^. This suggested that RAP acts to increase Aβ binding and uptake extracellularly, prior to internalization.

There was a small, marginally significant increase (30%) in FluoAβ_1-42 associated with cells treated with RAP. Furthermore, a neutralising anti-LRPl antibody (R2629), which is known to block LRPl -mediated uptake (Yamada et ah, 2008 (supra)), did not block Aβ uptake into SH-SY5Y cells (Figure 2A). A parallel experiment confirmed that the antibody inhibited association of RAP when it was incubated with the cells in the absence of Aβ (Figure 2B), indicating that the lack of effect on Ab uptake was not due to a lack of inhibition of LRPl by the antibody. FluoAβ_1-42 did not bind specifically to regions of LRPl immunoreactivity (Figure 2C), indicating that most of the peptide was associated with sites distinct from LRP 1.

Since RAP appeared to be altering the binding of FluoAβ_1-42 to the cell surface, and because mature LRPl is localized at the cell surface, experiments were undertaken to determine whether LRPl is involved in Aβ-cell binding. To test this hypothesis, an affinity-purified LRPl polyclonal antibody, R2629, was employed which binds to the extracellular portion of LRPl, including the ligand-binding domains and can block extracellular ligand binding to LRPl (Mikhailenko et al, J Biol Chem 27(5:39484-39491, 2001). First, the effectiveness of the antibody was evaluated by measuring the amount of cell-associated RAP following an incubation of RAP with SH-SY5Y cells. Endogenous RAP was beyond the detection limit of this assay (data not shown). A co-incubation of R2629 with RAP resulted in an inhibition of RAP uptake by the cells. Therefore, this demonstrated that R2629 can inhibit LRPl -mediated binding and uptake.

To determine whether LRPl might mediate the binding of Aβ to cells, immunofluorescent labeling of LRPl was performed and its localization compared to that of cell-bound FluoAβi-₄2. High levels of LRPl were detected at the cell surface by immunofluorescent detection using R2629. In the absence of RAP, FluoAβ_1-42 fluorescence did not co-localize to R2629 immunoreactivity, suggesting that the binding site for Aβ_1-42 on these cells was not LRPl . Also, the enhancement of cell association by RAP co-incubation did not induce the localization of FlUoAp_1-42 to LRPl. Thus, FlUoAp_1-42 did not co-localize to LRPl in the absence or presence of RAP.

The potential involvement of LRPl in Ap binding to SH-S Y5 Y cells was further tested by measuring the cell-association of FluoAβi_-42 following incubation in the presence of R2629. Cell-associated fluorescence was quantitated using flow cytometry. Neither the Aβ-cell binding, nor the RAP -induced enhancement of Aβ-cell binding was affected by R2629. From these data, it was concluded that the cell binding of FluoAβ_1-42, in the absence or presence of RAP, was not mediated by LRPl.

It has previously been reported that the binding of Aβ to cells is sensitive to heparin (Yang et ah, Neuroscience 90: 1217-1226, 1999), possibly due to heparin's ability to inhibit an interaction between Ap and heparan sulfate proteoglycans (Buee et ah, Brain Res 601 :154- 163, 1993). Here, heparin inhibited binding of FluoAβ_1-42 to SH-S Y5 Y cells. An additional co-incubation with RAP enhanced FluoAβ_1-42 cell binding in the presence of heparin. However, the inhibitory effect of heparin was independent of RAP as the amount of heparin inhibition was similar in the presence or absence of RAP. Together, these data confirmed that LRPl was not involved in the observed binding of Ap to SH-S Y5 Y cells. These data are consistent with a mechanism whereby RAP co-incubation enhances the ability of Ap to bind the cell membrane.

The fact that most of the Ap did not co-localise with LRPl was not surprising, as we had previously shown that Ap binds principally to lipids on the cell membrane (Subasinghe et al, 2003 (supra); Small et at, 2007 (supra)). While most of the FluoAβ_1-42 did not co- localize with LRPl, the peptide did co-localize with RAP at the cell surface (Figure 2D). This result suggested to us the possibility that the RAP and Aβ may bind to each other. To examine this possibility, purified Aβ (1 μM) and RAP (5 μg/ml; 130 nM) were incubated together, then RAP was immunoprecipitated using an anti-RAP antibody (7Fl), and the immunoprecipitate was analyzed by immunoblotting with an anti-Aβ antibody (6El 0). Following coincubation, both Aβ and RAP were recovered in the immunoprecipitate (Figure 3). When an irrelevant monoclonal antibody (H68.4, anti-transferrin receptor) was used instead of the anti-RAP antibody very little Ap_1-40 or Ap_1-42 was recovered in the immunoprecipitate. The anti-RAP antibody did not immunoprecipitate Aβl-40 or Aβl-42 in the absence of RAP, thus confirming the specificity of the immunoprecipitation.

The binding of Aβ to RAP was sufficiently stable to allow the Aβ-RAP complex to be seen on SDS-PAGE. A -46 kDa Aβ-immunoreactive band was observed on western blots after 7Fl immunoprecipitation (Figure 3). This band was not clearly visible on blots stained with the 7Fl antibody; probably because the heavily stained RAP band that migrated with an apparent relative molecular mass of 42 kDa, was too close to allow easy visualization of a 46 kDa complex. However, a 46 kDa band was seen when samples that had not been immunoprecipitated were analyzed by SDS-PAGE and stained with 6E10 (Figure 4A). Under these conditions, the intensity of the 46 kDa band increased with increasing incubation time (Figure 4A). The apparent molecular weight of the band was similar to the expected molecular weight of a RAP-Aβ complex. In order to confirm that the 46 kDa band was a complex of Aβ and RAP, Aβ and RAP were incubated together for 96 h to maximise the amount of complex formation, and then samples were analyzed electrophoretically under conditions designed to clearly separate the 46 kDa band from the 42 kDa RAP band. This experiment confirmed that the 46 kDa band was immunoreactive for both Aβ (6E10) and RAP (7Fl) and it was possible to see that in the presence of Aβ_1-40 or Aβμ₄₂, an additional band of 46 kDa was seen on the immunoblot stained with 7Fl antibody (Figure 4B). This result demonstrated that the 46 kDa band was an SDS-stable complex of RAP and Aβ. EXAMPLE 3 RAP polypeptide reduces oligomerization ofAβ

A time course study showed that with co-incubation of RAP and Aβ, the amount of the 46 IcDa RAP-Aβ complex increased for up to 7 d (Figure 4A, arrows). Over the 7 d of incubation, in the absence of RAP, Aβ_1-40 oligomerized to form species with apparent molecular masses ranging from those of dimers (8 kDa) to octamers (-30 kDa) (Figure 4A). However, in the presence of RAP, Aβl-40 aggregation was inhibited and the total pattern of oligomerization was similar to that seen after only 1 d in the absence of RAP.

In contrast to Aβ_1-40, Aβ_1-42 aggregated to form higher molecular weight structures that failed to enter the polyacrylamide gel (data not shown). Some oligomeric species were observed (Figure 4B), but overall, the pattern of aggregation was not easily analyzed using gel electrophoresis. Therefore, we examined Aβi_-42 aggregation by AFM, a technique that gives a qualitative assessment of the morphology of Aβ aggregation. Aβ_1-42 was diluted in the absence or presence of RAP then either examined immediately or aged for 6 h prior to analysis (Figure 5).

AFM studies showed that RAP inhibited formation of Aβ_1-42 protofibrils. The freshly prepared Aβ_1-42 formed globular structures with an apparent diameter of 20 nm that displayed a strong tendency to align along the graphite surface, as previously reported (Losic et al._% 2006). In contrast, fresh Aβ_1-42 in the presence of RAP generally did not form these aligned patterns, but instead consisted of smaller structures with an apparent diameter ranging from 15 nm through to 35 nm. RAP alone formed discrete structures of approximately 25 or 40 nm in diameter; it was not clear which structures in the sample containing both RAP and Aβ_1-42 represented Aβi.42, RAP, or a complex of the two proteins.

After 6 h of aging, in the absence of RAP, Aβ_1-42 formed smooth elongated protofibrils with an apparent width of 20-25 nm (Figure 5). However, in the presence of RAP, elongated structures were not observed. Instead, amorphous globular structures with appareiit diameters ranging from 30-50 nm were seen. Thus, together with the SDS-PAGE experiments, these results demonstrated that RAP can decrease the amount of Aβ aggregation in vitro.

EXAMPLE 4

RAP polypeptide blocks Aβ-induced neurotoxic effects in vitro and in vivo

The ability of Aβ to induce neurotoxicity is highly dependent on its aggregation state (Walsh et al, 2002 (supra)). Therefore, it was hypothesized that RAP, which inhibited Aβ aggregation, would influence Aβ neurotoxicity. One response of cells to extracellular Aβ is an increase in intracellular Ca²⁺ (Small et al, J Alzheimer s Dis 16: 225-233, 2008). In particular, neurons and neuron-like cells display immediate intracellular Ca²⁺ changes in response to oligomeric, but not monomeric or fibrillar, Aβ (Demuro et al, 2005 (supra); Kelly and Ferreira, J Biol Chem 281:28079-28089, 2006). To examine the response of intracellular Ca²⁺ to Aβ_1-42 treatment, SH-SY5Y cells were loaded with fluro-4 and the change in fluorescence over time was measured. Following addition of Aβ_1-42 there was an immediate and steady rise in intracellular Ca²⁺ (Figure 6A). A maximal response of AF/ F ~ 0.051 was achieved in response to Aβ_1-42 following 180 s of treatment. In the presence of RAP, Aβ_1-42 induced a rise in intracellular Ca²⁺, but the maximal response was only about 65% of the response in the absence of RAP. To exclude the possibility that LRPl was involved in the RAP effect, the experiment was performed in cells pre-treated with R2629. The Ca²⁺ response of SH-SY5Y cells to Aβ_1-42, with or without RAP, was unaffected by R2629 pre-treatment (Figure 6B). These findings support the hypothesis that the inhibitory effect of RAP on Aβ-induced intracellular Ca²⁺ levels was not mediated via an LRPl -dependent mechanism. Similar results were also obtained with Aβ_1-40.

The effect of RAP on Aβ neurotoxicity was further examined in vivo, using a well established discriminative avoidance learning task (Gibbs and Ng, 1977 (supra); Ng et al, 1997 (supra)). It has been previously shown (Gibbs et al, 2008 (supra)) that injection of Aβ into the brains of day-old chicks inhibited consolidation of long-term memory. Importantly, this effect was quite specific as it was highly dependent on the aggregation state of Aβ. Indeed, oligomeric Aβ was found to be the most potent amnestic species (Gibbs et al, 2008 (supra)).

Freshly prepared Aβ_1-42 was used, which was previously shown to contain toxic oligomeric species (Gibbs et al, 2008 (supra)) and to potently inhibit memory consolidation in the chick (Gibbs et al, 2008 (supra)). Aβ_1-42 was freshly diluted from stocks in the absence or presence of RAP, and then injected into the brains of chicks 45 min prior to their training for discriminative avoidance of red versus blue beads. Consistent with our previous report (Gibbs et al, 2008 (supra)), chicks injected with Aβ_1-42 alone did not avoid the red beads upon testing 120 min after training, thus their discrimination ratio (DR) score was close to that of chance alone (0.5) (Figure 9). In contrast, chicks injected with an Aβ_1-42 solution that also contained RAP exhibited less amnesia as they avoided the red beads, giving a DR of close to 1.0. The behavior of chicks injected with Aβ_1-42 and RAP together was indistinguishable from that of chicks injected with vehicle alone. Thus, the co-injection of RAP prevented the amnestic effect of Aβ_1-42 in chicks.

EXAMPLE 5 Characterisation of RAP in AD subjects

Tissue from a transgenic mouse model of AD (such as Tg2576) taken at different ages is analysed for RAP by western blotting and immunohistochemical staining. The distribution of RAP is compared with that of age-matched background strain control mice. The tissue distribution of staining is compared with that of various markers of Aβ deposition and toxicity such as Aβ, tau and ubiquitin. Tissue from AD brain is analysed for RAP by western blotting and immunohistochemical staining. The distribution of RAP is compared with that of age-matched controls or other neurological diseases. Wherever possible, tissue are matched for sex, age and post-mortem interval. The tissue distribution of staining are compared with that of various markers of Aβ deposition and toxicity such as Aβ, tau and ubiquitin. EXAMPLE 6

Therapeutic effects of RAP polypeptide in AD subjects

The role of peripheral administration of a RAP polypeptide in reducing Aβ levels, amyloid deposition and associated neuropathologic features are investigated in a transgenic mouse model of AD. RAP₅ as it occurs naturally in the body or as administered in its naturally occurring form, is actively taken up across the blood-brain barrier via a receptor-mediated mechanism (Pan et al., 2004 (supra)) and is relatively stable in blood. Therefore, peripheral intravenous administration of RAP results in rapid incorporation of the brain into the brain parenchyma. RAP polypeptide is administered intravenously to Tg2576 mice. The effects of a RAP polypeptide on amyloid load (as measured with Thioflavin S and Aβ immunohistochemistry), Aβ levels (ELISA and western blotting) and various markers of amyloid neuropathology (such as tau and ubiquitin) are determined. Treated Tg2576 mice are also be tested for memory using a Morris Water Maze and the effects of RAP polypeptide examined.

EXAMPLE 7 Level of RAP in hippocampus of AD brain

The distribution of RAP immunoreactivity in sections of AD and age-matched control (non-AD) hippocampus. Sections of CAl region of hippocampus from 7 AD and 8 control cases were stained for RAP using monoclonal antibody 7Fl (Figure 8). Immunohistochemical staining showed that most of the RAP immunoreactivity in non-AD was neuronal and intracellular (Figure 8A, arrowheads). In contrast to the control tissue, there was less RAP immunoreactivity in the AD tissue. Quantitative analysis revealed that 89 ± 3% (± SEM) of control CAl neurons were RAP-immunoreactive whereas only 38 ± 11% of AD CAl neurons showed RAP positivity (t-test ρ= 0.003).

Many modifications will be apparent to those skilled in the art without departing from the scope of the present invention.

The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

TABLE 1 Summary of sequence identifiers

TABLE 2 Abbreviations

The following abbreviations are used throughout the application:

nts = nucleotides g = gram aa = amino acid(s) mg = milligram kb = kilobase(s) or kilobase ρair(s) μg = microgram

IcDa = kilodalton(s) ng = nanogram h = hour μl = microlitre

°C ⁼ degrees Celcius cm = cenitmetre

HlM = millimolar % = percent μm = micrometer min = minute

TABLE 3 Amino acid sub-classification

TABLE 4

Exemplary and Preferred Amino Acid Substitutions

TABLE 5 Codes for non-conventional amino acids

Non-conventional Code Non-conventional Code amino acid amino acid α-aminobutyric acid Abu L-N-methylalamne Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine DgIn L-N-methylnorvaline Nmnva

D-glutamic acid DgIu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine DiIe L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nnithr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine NIe

D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval α-methyl-γ-aminobutyrate Mgabu

D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen

D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap

D-α-methylaspartate Dmasp α-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine NgIu

D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-α-methylhistidine Dmhis N-(3 -aminopropyl)glycine Norn

D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-α-methylleucine Dmleu α-napthylalanine Anap

D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine NgIn

D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine NgIu

D-α-methylproline Dmpro N-(carboxymethyl) glycine Nasp

D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-α-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3 ,3 -diphenylpropyl)glycine Nbhe

D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-(l-hydroxyethyl)glycine Nthr

D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl)) glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycme Nhtrp

D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-niethylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine NaIa D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro N-(l-methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtrp N-(l-methylethyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr

L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-α-methylalanine Mala

L-α-methylarginine Marg L-α-methylasparagine Masn

L-α-methylaspartate Masp L-α-methyR-butylglycine Mtbug

L-α-methylcysteine Mcys L-methylethylglycine Metg

L-α-methylglutamine MgIn L-α-methylglutamate MgIu

L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe

L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

L-α-methylleucine Mleu L-α-methyllysine Mlys

L-α-methylmethionine Mmet L-α-methylnorleucine MnIe

L-α-methylnorvaline Mnva L-α-methylornithine Morn

L-α-methylphenylalanine Mphe L-α-methylproline Mpro

L-α-methylserine Mser L-cc-methylthreonine Mthr

L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe

N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylproρyl) Nnbhe carbaniylmethyl)glycine carbamylmethyl)glycine

1 -carboxy- 1 -(2,2-diphenyl- Nmbc ethylamino)cyclopropane

Table 6 Control and AD cases used for RAP immunohistochemical analysis.

Case Control or AD Gender Age at death PMl

(years)

1 Control M 92 43

2 Control F 85 23

3 Control F 88 45.5

4 Control M 79 8

5 Control M 68 11

6 Control F 98 20

7 Control F 64 5

8 Control M 41 35

9 AD F 78 24

10 AD F 86 15

11 AD F 84 6

12 AD F 87 19

13 AD M 69 18

14 AD M 67 6

15 AD M 79 67

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Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A composition comprising a Receptor Associated Protein (RAP) polypeptide or an analog thereof which binds to β-amyloid peptide (Aβ) for use in the treatment or prophylaxis of a symptom, of Alzheimer's disease (AD) or a related condition in a subject.

2. The composition of claim 1 wherein the RAP polypeptide is RAP or a fragment of RAP.

3. The composition of claim 1 wherein the RAP polypeptide is a variant of RAP.

4. The composition of claim 3 wherein the RAP polypeptide is a variant of a fragment of RAP.

5. The composition of claim 1 wherein the RAP polypeptide is a RAP peptidomimetic or stapled peptide.

6. The composition of claim 1 wherein the RAP polypeptide is a small molecule analog of RAP.

7. The composition of any one of claims 1 to 5 wherein the RAP polypeptide is human or mammalian.

8. The composition of claim 1 wherein the analog is selected from the group comprising a small molecule, an antibody, a nucleic acid or a peptide.

9. The composition of any one of claims 1 to 8 wherein the symptom of AD is memory loss.

10. An isolated composition comprising a complex comprising RAP bound to Aβ.

11. A pharmaceutical composition comprising a RAP polypeptide or an analog or agonist thereof capable of binding to Aβ.

12. A method of identifying a candidate agent that reduces Aβ aggregation or oligomerisation, Aβ fibril formation, Aβ neuropathy and/or enhances memory performance, said method comprising:

i. contacting the candidate agent with a system comprising Aβ and a RAP polypeptide; and ii. determining the presence of a complex between the agent and Aβ or a change in the level of an indicator of the activity of the complex or a component thereof.

13. A method of identifying a candidate agent that reduces Aβ aggregation or oligomerisation, Aβ fibril formation, Aβ neuropathy and/or enhances memory performance, said method comprising:

i. contacting Aβ with a system comprising the agent and a RAP polypeptide; and ii. determining the presence of a complex between the agent and Aβ or a change in the level of an indicator of the activity of the complex or a component thereof.

14. The method of claim 12 or 13 wherein the system comprises an in vitro cell.

15. The method of claim 12 or 13 wherein the cell is a brain cell or neuronal cell.

16. The method of claim 12 or 13 wherein the cell is a neuroblastoma cell.

17. The method of claim 12 or 13 wherein the system is an animal model of AD or related condition.

18. The method of claim 12 or 13 wherein step ii) comprises determining the ability of the agent to reduce the binding between the RAP polypeptide and Aβ.

19. The method of any one of claims 12 to 18 wherein the indicator of the activity of the complex is memory performance in an animal model of AD or a related condition.

20. The method of any one of claims 12 to 19 wherein RAP polypeptide and/or Aβ are produced endogenously by a cell within the system.

21. The method of any one of claims 12 to 19 wherein RAP polypeptide and/or Aβ are supplied exogenously.

22. Use of RAP or a complex comprising RAP and Aβ in the manufacture of a medicament for use in the treatment of symptoms of AD in a subject.

23. A method for the treatment or prophylaxis of a subject with AD or who is at risk of developing same said method comprising administering to the subject an amount of a RAP polypeptide or a RAP analog capable of binding to Aβ for a time and under conditions effective to reduce Aβ aggregation, Aβ oligomerisation, Aβ fibril formation, Aβ induced neuropathy and/or enhance memory performance.

24. The method of claim 23 wherein the subject is a human.