WO2005103707A1

WO2005103707A1 - Novel compounds for the treatment of alzheimer’s disease and methods for identifying same

Info

Publication number: WO2005103707A1
Application number: PCT/EP2004/005625
Authority: WO
Inventors: Koenraad Frederik Florentina Spittaels; Marcel Hoffmann; Pascal Gerard Merchiers
Original assignee: Galapagos N.V.
Priority date: 2004-04-27
Filing date: 2004-05-21
Publication date: 2005-11-03

Abstract

The present invention relates to methods of identifying compounds that influence the amyloid-beta precursor protein processing in a cell, and/or that influence the activity or levels of proteins involved in the processing of the amyloid beta precursor protein. It furthermore relates to the compounds that may be identified using the methods of the invention and to use of said compounds in the treatment of neurological disorders such as Alzheimer's disease. More specifically, it relates to compounds that target the AVPR1a, AVPR1b and/or AVPR2 receptors.

Description

TITLE Novel compounds for the treatment of Alzheimer' s disease and methods for identifying same. FIELD OF THE INVENTION The invention relates to novel compounds and to methods for identifying such compounds. The compounds typically target the proteins AVPRla, AVPRlb and/or AVPR2 and are useful in the treatment of neurological disorders such as Alzheimer's disease. More specifically, the invention relates to compounds that influence the processing of the amyloid-beta precursor protein.

BACKGROUND OF THE INVENTION Alzheimer's disease (AD) is a neurological disorder that is clinically characterized by the progressive loss of intellectual capacities: initial loss of memory, and later on by disorientation, impairment of judgment and reasoning, commonly referred to as cognitive impairment, progressing to full dementia. The patients finally fall into a severely debilitated, immobile state between 4 and 12 years after onset of the disease. Worldwide, about 20 million people suffer from AD. The pathological hallmarks of the disease are the presence of extracellular amyloid plaques and intracellular tau tangles in the brain, which are associated with neuronal degeneration (Ritchie and Lovestone, 2002). A small fraction of AD cases are caused by autosomal dominant mutations in the genes encoding presenilin 1 and 2 (PSl; PS2) and the amyloid-beta precursor protein (APP) . It has been shown that mutations in APP, PSl and/or PS2 alter the amyloid-beta precursor protein metabolism (processing) such that more of the insoluble, pathogenic amyloid beta 1- 42 is produced in the brain. Following secretion, these amyloid beta 1-42 peptides form amyloid fibrils more readily than the amyloid beta 1-40 peptides, the latter being predominantly produced in healthy people. The insoluble amyloid fibrils resulting from the amyloid beta 1-42 peptides are subsequently deposited in the amyloid plaques, resulting in the onset and/or the progression of the disease. The amyloid beta peptides are generated from the membrane anchored APP after cleavage by beta secretase and gamma secretase at position 1 and 42, respectively (Figure 1, Annaert and De Strooper, 2002) . The gamma secretase can also cleave at position 40. In addition, it was found that an increased activity of beta secretase might result in a shift of the cleavage at position 1 to position 11. Cleavage of amyloid-beta precursor protein by alpha secretase activity and gamma secretase activity at position 17 and 40 or 42 generates the non-pathological p3 peptide. The beta secretase protein was identified as the membrane anchored aspartyl protease BACE, while gamma secretase is a protein complex comprising presenilin 1 (PSl) or presenilin 2 (PS2), nicastrin, Anterior Pharynx Defective 1 (APH1) and Presenilin Enhancer 2 (PEN2) . Of these proteins, the presenilins are thought to constitute the catalytic activity of the gamma secretase, while the other components play a role in the maturation and localization of the complex. The identity of the alpha secretase is still illustrious, although some results point towards the proteases ADAM 10 and TACE, which could have redundant functions. It has been shown that injection of amyloid beta fibrils in the brains of P301L tau transgenic mice enhances the formation of neurofibrillary tangles, placing the amyloid beta peptide upstream of the neurotoxic cascade (Gotz et al. 2001). Although no mutations in PSl, PS2 and amyloid-beta precursor protein have been identified in late onset AD patients, the pathological hallmarks are highly similar to the early onset AD patients. Therefore, it is generally accepted that aberrant increased amyloid peptide levels in the brains of late onset AD patients are also the cause of the disease. These increased levels of amyloid beta peptide could originate progressively with age from disturbed amyloid-beta precursor protein processing (e.g. high cholesterol levels enhance amyloid beta peptide production) or from decreased catabolism of the peptide. Because the cholinergic neurons are the first neurons to degenerate during AD, levels of the neurotransmitter acetylcholine decrease, which results in the progressive loss of memory. Therefore, the major current AD therapies are focused on the inhibition of the acetylcholinesterase enzyme, leading to an increased concentration of the acetylcholine. However, this therapy does not halt the progression of the disease. Due to the fact that the socio- economical impact of AD is worldwide considered very significant, the need for an effective therapy is extremely high. Therapies aimed at decreasing the levels of amyloid beta peptides in the brain, are investigated in the field. Most of these therapies are focused on the perturbed amyloid-beta precursor protein processing (metabolism) and target directly beta- or gamma secretase activity. However, targeting these proteins has not yielded any new drugs to date, because of the difficulty to find specific drugs and of suspected serious side effects. Thus, the identification of alternative drug targets within the amyloid-beta precursor protein-processing pathway is of great importance. This would allow a direct interference with the production of the pathological amyloid beta 1-42 peptide, which preferably should block the neurotoxic cascade induced by the latter.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. APP processing: The membrane anchored amyloid precursor protein (APP) is processed by two pathways: the amyloidogenic and non amyloidogenic pathway. In the latter pathway, APP is cleaved first by alpha secretase and then by gamma secretase, yielding the p3 peptides (17-40 or 17-42) . The amyloidogenic pathway generates the pathogenic amyloid beta peptides (A beta) after cleavage by beta- and gamma- secretase respectively. The numbers depicted are the positions of the amino acids comprising the A beta sequences.

Figure 2. Evaluation of the APP processing assay: Positive (PS1G384L; PS1L392V and BACE1) and negative (eGFP, LacZ and empty) control viruses are infected in Hek293APPwt at random MOI, mimicking a screening. A and B: Transduction is performed respectively with 1 and 0.2 ?1 of virus and amyloid beta 1-42 levels are performed. Data are represented as relative light units and correlate to pM of amyloid beta 1-42.

Figure 3. Involvement of the Arginine Vasopressin Receptor 2 (AVPR2) in APP processing: differentiated SH-SY5Y APPwt cells are transduced with Ad5/AVPR2 or with negative control virus Ad5/empty at two different multiplicities of infection (MOI 50 and 250) . Resulting amyloid beta 1-42 peptides were measured with the appropriate ELISA. Data are represented in pM.

Figure 4. Involvement of AVPR2 in APP processing: HEK293 APPwt cells are transduced with Ad5/CRE reporter (MOI 400) plus Ad5/AVPR2 (MOI 50) or negative control virus Ad5/empty (MOI 50) . Infected cells are challenged with different concentrations (10^~13-10^~6) of arginine vasopressin (DDAVP) for 12 hours. Resulting amyloid beta 1-42, peptides were measured with the appropriate ELISA. Data are represented as Relative light units (URL) as measure for the reporter luciferase activity that represents receptor activity (A) and as % of maximum levels of amyloid beta 1-42 (B) .

Figure 5. Knock-down screen results for AVPR2. Ad5/AVPR2 knock-down virus at MOI 1800, 600 and 200 was used to infect SH-SY5Y cells that are co-infected with hAPP695 (Swedish clinical mutant) . Amyloid beta 1-42 levels (expressed as relative light units) were significantly lower for Ad5/AVPR2 compared to the negative controls (knock-down Ad5 viruses for eGFP, Luciferase and alkaline phosphatase liver/bone/kidney) . The cut-off is set at the mean of the negative controls minus 2.5 times the standard deviation on the mean of these negative controls.

Figure 6. ClustalW protein sequence alignment of AVPR2, AVPRla and AVPRlb.

Figure 7. Structure of AVP-receptor antagonist Tolvaptan. SUMMARY OF THE INVENTION The present invention relates to the association between vasopressin receptors and the production of amyloid beta. More specifically, the invention provides novel compounds that interfere with the processing of the amyloid beta precursor protein processing through the targeting of certain vasopressin receptors. The present invention provides novel methods to identify compounds that change the amyloid-beta precursor protein processing in cells. It also relates to methods to influence the processing of the amyloid-beta precursor protein by using said compounds. Methods are directed to decrease the expression of the G-protein coupled receptor Arginine Vasopressin Receptor 2 (AVPR2) . Decrease of expression of AVPR2 results in reduction of amyloid-beta peptide 1-42. Different compounds that can be identified according to the methods provided by the present invention and used in methods for the treatment of diseases such as Alzheimer's disease are antisense RNA, Ribozymes, antisense oligodeoxynucleotides, small molecules and small interfering RNA. These compounds typically cause the decrease of expression of AVPR2. Polypeptides and polynucleotides, and vectors comprising same, are also provided. The compounds can also be polyclonal or monoclonal antibodies that interact with the polypeptides and inhibit their activity. The compounds may also be ^Λnanobodies' , the smallest functional fragment of naturally occurring single-domain antibodies (Cortez-Retamozo et al. 2004). The compounds according to the present invention are useful as a medicament, preferably in the treatment of Alzheimer's disease. The present invention furthermore provides means and methods to change the processing, the activity and the levels of the amyloid-beta precursor protein, for instance by inhibiting the activity of the protein AVPR2. Thus, compounds that inhibit the activity of AVPR2 are provided. Also provided are methods for diagnosis of pathological conditions involving cognitive impairment, such as AD. This invention also provides methods as outlined above for AVPRla and AVPRlb, which proteins are close relatives of AVPR2.

DETAILED DESCRIPTION The primary physiological roles of the arginine vasopressin receptor 2 (AVPR2) involve its regulation of cardiovascular activities by acting on vascular smooth muscle cells and its antidiuretic actions on the kidney.

Vasopressin is synthesized in the perikarya of magnocellular neurons in the supraoptic nucleus and paraventricular nucleus of the hypothalamus . Vasopressin-containing granules are transported through the axon not only to classical storage sites in the neurohypophysis but also to the adenohypophyse to play a role as a corticotropin-releasing factor. Next to the hypothalamus, vasopressin is also synthesized in the heart. An increase in plasma osmolarity is the principal physiological stimulus for vasopressin secretion, but severe hypovolumia/hypotension is also a powerful trigger for vasopressin release. The cellular effects of vasopressin are mediated by interactions of the hormone with two principal types of receptors, VI and V2, which show significant structural homology to one another (Figure 6) . VI receptors have been subclassified further as Via and Vlb. The Via receptor is the most widespread subtype; it is expressed in a lot of different tissues, including many central nervous system structures. The adenohypophysis and the adrenal medulla are known to contain Vlb receptors, whereas V2 receptors are predominantly located in principal cells of the renal collecting duct system. These vasopressin receptors are typical G protein-coupled receptors containing seven transmembrane-spanning domains (Hardman and Limbird, 2001) . The biological effects mediated by the VI receptor include vasoconstriction, glycogenolysis, platelet aggregation, ACTH release and growth of vascular smooth muscle cells. The function of the V2 receptor is to increase the permeability of the renal collecting duct for water (Laycock and Hanoune, 1998). Several human diseases affecting the vasopressin system are known. Central Diabetes Insipidus (DI) is a disease of impaired renal conservation of water due to an inadequate secretion of vasopressin from the neurohypophysis because of head injury, hypothalamic or pituitary tumors, CNS ischemia, brain infection or mutations in the arginine- vasopressin preprohormone . Nephrogenic Diabetes Insipidus may be acquired or genetic. A number of mutations in the gene encoding the V2 receptor have been identified in patients with familial, X-linked nephrogenic DI . These mutations result predominantly in abnormal synthesis, processing or intracellular transport of V2 receptors (Morello and Bichet, 2001) . The Syndrome of Inappropriate Secretion of Antidiuretic hormone (SIADH) is a disease of impaired water excretion with accompanying hyponatremia and hypoosmolality caused by inappropriate (too high) secretion of vasopressin. In patients with congestive heart failure and liver cirrhosis, effective blood volume is reduced, which stimulates vasopressin release. As many human diseases are associated with aberrant functionalities of the vasopressin system, pharma' s objective is to discover the most effective drug to cope with these disorders. Antidiuretic drugs (antidiuretic peptides such as vasopressin, lypressin and desmopressin) are already on the market and new compounds are being tested in clinical trials. The therapeutic uses of vasopressin and its congeners depend on the type of vasopressin receptor involved. In view of the high cost of the drug (therapeutic peptides), the search for nonpeptide small molecule compounds, both agonists and antagonists, continues intrepidly. The impetus for the development of specific vasopressin receptor antagonists is the believe that such drugs may be useful in a number of clinical settings: selective Via antagonists may be beneficial in congestive heart failure (e.g. OPC-21268, SR 49059); selective V2 antagonists can be useful whenever reabsorption of solute- free water is excessive (e.g. SR 121463A, VPA-985, VPA-343, OPC-41061, see Tolvaptan, Gheorghiade et al. 2003) and Vla/V2 antagonists may be applicable for a combined indication of dilutional hyponatremia and hypertension (e.g. OPC-31260, YM 087, see Serradeil-Le Gal et al . 2002) . Thus, the invention relates to the use of a compound that inhibits a polypeptide according to SEQ ID NO: 4, 5 and/or 6 in the manufacture of a medicament for the treatment of Alzheimer's Disease. In one embodiment, the compound for such use is selected from the group consisting of OPC-21268, SR 49059, SR 121463A, VPA-985, VPA-343, OPC- 41061, OPC-31260 and YM-087 and any of the compounds as described in US 6,653,478 and US 5,512,563. In another embodiment, the compound for such use is selected from the group of compounds that may be identified according to the methods of the present invention, including those compounds that have been described intra. It has become clear that vasopressin is also critically involved in numerous central processes including higher cognitive functions such as memory and learning (Barberis and Tribollet, 1996) . Vasopressin content in the brain declines with age. Consequently, vasopressin has been used to treat memory deficits due to aging, senile dementia and amnesia. Vasopressin improves attention, retention and recall (both short-term and long-term) , and it is well known to enhance short-term memory in normal young adults, as well as in those with age-associated memory impairment. In addition, it has been shown to improve both mood and memory in the elderly and those with Alzheimer's disease. The effect of vasopressin receptor modulation by agonists on the processing of APP has been studied (Nitsch et al . 1995 and 1998) . These publications report that receptor stimulation rapidly increases the rate of release of the alpha-secretase processing product APPs, which is cleaved within the amyloid beta domain. This invention relates to the targeting of the vasopressin receptor V2 (AVPR2) and to the knock-down of AVPR2, which typically results in a reduction of amyloid beta 1-42 levels in the conditioned medium of transduced cells. The presence of AVPR2 (and its related proteins AVPRla and AVPRlb) in the central nervous system renders these GPCRs as putative drug targets for the (prophylactic) treatment and/or the diagnosis of Alzheimer's disease. Inhibition or reduction of AVPR2, AVPRla and/or AVPRlb activity has been associated with possible treatment of a variety of diseases such as Diabetes Insipidus (AVPR2) and congestive heart failure (AVPRla) but compounds that reduce or inhibit the activity of AVPR2, AVPRla and/or AVPRlb have never been associated with treatment or prevention or amelioration of a pathological condition involving cognitive impairment, such as AD. The present invention clearly indicates that reduction of expression or reduction of activity of AVPR2 is related to the reduction of the amyloid-beta peptide level, which reduction is desired.

The present invention relates to a method of identifying a compound that modifies the amyloid-beta precursor protein processing (metabolism) in a cell, which method comprises providing a population of cells expressing a polypeptide having an amino acid sequences selected from the group consisting of SEQ ID NO: 4-6, or a functional fragment or derivative thereof; determining a first amyloid- beta precursor processing level in the cell; exposing the cell to a compound; determining a second amyloid-beta precursor processing level in the cell; and identifying the compound that modifies the amyloid-beta precursor processing in said cell. Preferably, the compound identified according to the methods of the present invention is selected from the group consisting of an antisense RNA molecule, a ribozyme (that cleaves the polyribonucleotide) , an antisense oligodeoxynucleotide (ODN) , a small interfering RNA (siRNA, that is sufficiently homologous to a portion of the polyribonucleotide such that the siRNA is capable of inhibiting the polyribonucleotide that would otherwise cause the production of the polypeptide) , a nucleic acid expressing the antisense RNA, an antibody, a polypeptide, a peptide, a lipid, a polynucleotide, or a vector carrying a polynucleotide or peptide according to the invention. Preferably, the invention relates to a method wherein the modification in the processing of the amyloid-beta precursor protein in a cell is a reduction of the level of amyloid- beta peptide 1-42, 1-40, 11-42 or 11-40, most preferably a reduction of the amyloid beta peptide 1-42. The present invention further relates to a method of identifying a compound that modifies the amyloid-beta precursor protein processing (metabolism) in a cell, which method comprises expressing a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6, or a functional fragment or derivative thereof; determining a first activity level of said polypeptide; exposing the cell to a compound; determining a second activity level of said polypeptide; and identifying the compound that influences the activity level. Preferably, the activity is measured through measuring the level of one or more second messengers of the polypeptide. Typically, the invention relates to a method wherein the second activity level is lower than the first activity level. More preferably, the second activity level is close to zero, meaning that the activity of the target protein has been lowered to levels that would be beneficial in the treatment and/or prevention of the disease that needs to be treated and/or prevented. Although a reduction of the levels may differ and may be multifold, it is held here that a reduction of 30% in a patient (in vivo) is a preferred level. Thus the influence on the processing on the amyloid-beta precursor protein (APP) as used herein refers to a preferred reduction of said expression and/or activity which is more or less comparable to a reduction of 30% (or more) in vivo. It can however not be excluded that levels found in vitro do not perfectly correlate with levels found in vivo, such that a slightly reduced level in vitro may still result in a higher reduction in vivo when the compound is applied in a therapeutic setting. It is therefore preferred to have reduced in vitro levels of at least 10%, more preferably more than 30%, even more preferably more than 50% and most preferably a reduction between 50% and 100%, which would mean an almost complete disappearance of the amyloid-beta peptides 1-42, 1-40, 11-42 and/or 11-40. The invention relates to a method of identifying a compound that decreases the expression and/or activity of a polypeptide selected from SEQ ID NO: 4, 5 or 6 in a cell, said method comprising the steps of incubating a cell; determining a first level of expression and/or activity of said polypeptide in said cell; incubating said cell with a compound; determining a second level of expression and/or activity in said cell of said polypeptide following or during the previous step; and identifying a compound that decreases the expression and/or activity of said polypeptide. Amongst other amyloid beta peptides, the amyloid beta peptide 1-42 was found in amyloid plaques. Thus, reducing the level of this amyloid beta peptide would be beneficial for patients with cognitive impairment. The pharmacological inhibition of these targets results in a decrease of amyloid beta levels. A preferred polypeptide that is targeted by the compounds of the present invention is a G-protein coupled receptor (GPCR) and can typically be inhibited by small molecules. GPCRs, their general structure and the signal transduction pathways that involve GPCRs are well known in the art. All GPCRs share a common architecture of 7 transmembrane domains, an extracellular N-terminus and an intracellular C-terminus . The major signal transduction cascades activated by GPCRs are initiated by the activation of heterotrimeric G-proteins, built from three different proteins; the G_? , G_? and G_? subunits. The signal transduction cascade starts with the activation of the receptor by an agonist. Transformational changes in the receptor are then translated down to the G-protein. The G-protein dissociates into the G? subunit and the G?? subunit. Both subunits dissociate from the receptor and are both capable of initiating different cellular responses. Best known are the cellular effects that are initiated by the G? subunit. It is for this reason that G-proteins are categorized by their G_? subunit. The G-proteins are divided into four groups: G_s ,Gi_/0, G_q and Gι₂ι₃. All G-proteins are capable of activating an effector protein, which in turn results in changes in second messenger levels in the cell. The changes in second messenger level are the triggers that make the cell respond to the extracellular signal in a specific manner. The inventors of the present invention have made use of this: The activity of a GPCR can be measured by measuring the activity level of the second messenger. The two most important second messengers in the cell are cAMP and Ca²⁺, and are the preferred second messengers used in the methods according to the present invention for determining the activity level of the targeted polypeptide. The ? -subunit of the G_s class of G-proteins is able to activate adenylyl cyclase, resulting in an increased turnover from ATP to cAMP. The ? -subunit of Gι₀ G-proteins does exactly the opposite and inhibits adenylyl cyclase activity resulting in a decrease of cellular cAMP levels. Together, these two classes of G-proteins regulate the second messenger cAMP. Ca²⁺ is regulated by the ? -subunit of the G_q class of G-proteins. Through the activation of phospholipase C phosphatidylinositol 4, 5-bisphosphate (PIP2) from the cell membrane are hydrolyzed to inositol 1,4,5- trisphosphate and 1, 2-diacylglycerol, both these molecules act as second messengers. Inositol 1, 4, 5-trisphosphate binds specific receptors in the endoplasmatic reticulum, resulting in the opening of Ca²⁺ channels and release of Ca²⁺ in the cytoplasm. Determining the level of the second messenger is hence found useful in determining the biological activity of the GPCR. The second messenger levels can be measured by several techniques known in the art, either directly by ELISA or radioactive technologies or indirectly by reporter gene analysis. A host cell expressing a polypeptide of the present invention can be a cell with endogenous expression of the polypeptide or a cell overexpressing the polypeptide e.g. by transduction. When the endogenous expression of the polypeptide of the present invention is not sufficient for a first activity level of the second measure that can easily be measured, overexpression of the polypeptide can be applied, or an agonist of the polypeptide can be added. Overexpression has the advantage that the first activity level of the second messenger is significantly higher than the activity level by endogenous expression and differences can be measured more easily. In one embodiment of the present invention, the method according to the present invention comprises the step of contacting the host cell with an agonist for the polypeptide before determining the first activity level. The addition of an agonist further stimulates the polypeptide of the present invention, thereby further increasing the activity level of the second messenger if the endogenous levels do not allow proper measurements. According to one embodiment, the AVPR2, AVPRla and AVPR2b proteins are activated with a ligand, here also referred to as an agonist. Several such agonists (ligands) are known in the art, and are useful in the methods of the present invention: arginine vasopressin, desmopressin (DDAVP) or lypressin (porcine vasopressin), amongst others.

The amyloid-beta precursor protein is processed into several different amyloid beta peptides species. According to the methods provided by the present invention, compounds are identified that change the APP processing and reduce the level of secreted pathological amyloid beta peptides. Levels of amyloid beta peptides can be measured with specific ELISA' s using an antibody specifically recognizing the amyloid beta peptide species 1-42 (see e.g. Example 1). Levels of amyloid beta peptides can also be measured by Mass spectrometry analysis. A particularly preferred embodiment of the present invention relates to a method wherein the polypeptide is AVPR2 (SEQ ID NO: 6) . A further embodiment of the present invention relates to methods for identifying a compound that influences the amyloid-beta precursor protein processing in a cell, wherein the activity level of the polypeptide is measured by determining the level of one or more second messengers of the polypeptide, and wherein the level of the one or second messenger is determined with a reporter comprising a promoter that is responsive to the second messenger. The reporter typically comprises a gene under the control of a promoter that responds to the cellular level of a second messenger. Preferred second messengers are Cyclic AMP or Ca2⁺. The reporter gene encodes a gene product that can easily be measured. The reporter gene can either be stably transfected (integrated into the host cell genome) or be present in the cell outside the genome. The reporter gene is preferably selected from the group consisting of alkaline phosphatase, Green fluorescent protein (GFP) , enhanced green fluorescent protein (eGFP) , destabilized green fluorescent protein (dGFP) , luciferase, and ?-galactosidase. Other reporter genes may be applied. Preferably the promoter controlling the reporter construct is a cyclic AMP-responsive promoter, an NF-KB responsive promoter, or a NF-AT responsive promoter. The cyclic-AMP responsive promoter is responsive to the cyclic- AMP levels in the cell. The NF-AT responsive promoter is sensitive to cytoplasmic Ca²⁺-levels in the cell. The NF-KB responsive promoter is sensitive for activated NF-?B levels in the cell. Preferably the reporter is luciferase or ?- galactosidase. Luciferase and ?-galactosidase are easily available and have a large dynamic range for measuring. In addition, luciferase and ?-galactosidase are less expensive which is favorable especially when performing the method of the present invention in a high throughput format.

The present invention further relates to methods for identifying compounds that influences the amyloid-beta precursor protein processing (metabolism) in a cell, which ^• methods comprise the steps of contacting a compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4 to 6, or a functional derivative or fragment thereof; determining the binding affinity of the compound to the polypeptide; contacting a population of cells expressing the polypeptide with the compound that exhibits a binding affinity of at most 10 micromolar; and identifying the compound that influences the amyloid-beta precursor protein processing in the cells. Preferably, the levels of amyloid-beta are decreased. The decrease of amyloid-beta precursor protein is typically measured by determining the levels of one or more second messenger molecules. More in particular, the method relates to a method identifying a compound that influences the amyloid-beta precursor protein processing in a cell, wherein said modification of amyloid-beta precursor protein processing in a cell results in a reduction of the level of amyloid-beta peptide 1-42, 1-40, 11-42 or 11-40, most preferably the 1-42 peptide. The binding affinity of the compound with the polypeptide or polynucleotide can be measured by methods generally known in the art, such as using surface plasmon resonance biosensors (Biacore) , by saturation binding analysis with a labeled compound (e.g. Scatchard and Lindmo analysis) , by differential UV spectrophotometer, fluorescence polarisation assay, Fluorometric Imaging Plate Reader (FLIPR^®) system, Fluorescence resonance energy transfer, and Bioluminescence resonance energy transfer. The binding affinity of compounds can also be expressed in dissociation constant (Kd) or as IC50 or EC50. The IC50 represents the concentration of a compound that is required for 50% inhibition of binding of another ligand to the polypetide. The EC50 represents the concentration required for obtaining 50% of the maximum effect in any assay that measures receptor function. The dissociation constant, Kd, is a measure of how well a ligand binds to the polypeptide, it is equivalent to the ligand concentration required to saturate exactly half of the binding-sites on the polypeptide. Compounds with a high affinity binding have low Kd, IC50 and EC50 values, i.e. in the range of 100 nM to 1 pM; a moderate to low affinity binding relates to a high Kd, IC50 and EC50 values, i.e. in the micromolar range. For high-throughput purposes, libraries of compounds can be used such as peptide libraries (e.g. LOPAP™, Sigma Aldrich) , lipid libraries (BioMol) , synthetic compound libraries (e.g. LOPAC™, Sigma Aldrich) or natural compound libraries (Specs, TimTec) . One preferable type of compound that can be identified by the methods of the present invention is a low molecular weight compound. Low molecular weight compounds, i.e. with a molecular weight of approximately 500 Dalton or less, are likely to have good absorption and permeation in biological systems and are consequently likely to be successful drug candidates (Lipinski et al. 1997) . According to another embodiment the compounds are peptides. Many GPCRs have a peptide as an antagonist. Peptides can be excellent drug candidates and there are multiple examples of commercially valuable peptides such as fertility hormones and platelet aggregation inhibitors. According to another embodiment the compounds are natural compounds . Natural compounds are compounds that have been extracted from e.g. plants, soil or tissues, or compounds or that may be synthesized on the basis of a natural occurring molecule. Using natural compounds in screens may have the advantage that one is able to screen more diverse kinds of molecules. According to another embodiment the compounds are lipids. GPCRs listed in table 1 (SEQ ID NO: 1-3, 4-6) can have lipids as antagonists. Using lipids as candidate compounds can increase the chance of finding a specific antagonist or inverse agonist for the polypeptides of the present invention. According to another embodiment, the compound is an antibody. The generation of specific antibodies against target proteins and/or other cellular factors is well known in the art . According to another embodiment, the compounds are low molecular weight compounds selected from the group of OPC- 31260, OPC-41061 (Tolvaptan, Otsuka Pharma) , YM-087 (Conivaptan, Yamanouchi Pharma) , VP-343 (Wakamoto Pharma) , SR121463A, VPA-985, OPC-21268, SR49059, or any of the compounds as described in US patents 6,653,478 and 5,512,563 (see Figure 7) . It is preferred that the compounds are able to pass the blood-brain barrier. Small molecule compounds that may be identified by using the present invention preferably act as antagonists of AVPR2. In another aspect of the present invention, the compound is an expression inhibitory agent inhibiting the expression and/or translation of a nucleotide sequence encoding a polypeptide selected from SEQ ID No: 4-6. Expression levels and/or translation levels of the proteins may be determined using general methods known in the art. Such methods include mRNA analysis by Northern blots,

Reverse transcriptase PCR and Real-time PCR, amongst others. Other useful methods are protein analysis by Western blots or Elisa. One type of expression-inhibitory agent concerns a nucleic acid that is antisense to a nucleic acid comprising SEQ ID NO: 1-3. For example, an antisense nucleic acid (e.g. DNA) may be introduced into cells in vitro, or administered to a subject in vivo, as gene therapy to inhibit cellular expression of nucleic acids comprising SEQ ID NO: 1-3. Antisense oligonucleotides preferably comprise a sequence containing from about 17 to about 100 nucleotides and more preferably the antisense oligonucleotides comprise from about 18 to about 30 nucleotides. Antisense nucleic acids may be prepared by expression of all or part of a sequence selected from the group consisting of SEQ ID NO: 1- 3, in the opposite orientation. Antisense oligonucleotides can also contain a variety of modifications that confer resistance to nucleolytic degradation such as, for example, modified internucleoside linkages, modified nucleic acid bases and/or modified sugars and the like. The antisense oligonucleotides can also be modified by chemically linking the oligonucleotide to one or more moieties or conjugates to enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide. Such moieties or conjugates include lipids such as cholesterol, cholic acid, thioether, aliphatic chains, phospholipids, polyamines, polyethylene glycol (PEG), or pal ityl moieties. Another type of expression-inhibitory agent as a compound relates to a nucleic acid that is able to catalyze cleavage of RNA molecules. The expression "ribozymes" relates to catalytic RNA molecules capable of cleaving other RNA molecules at phosphodiester bonds in a manner specific to the sequence. The hydrolysis of the target sequence to be cleaved is initiated by the formation of a catalytically active complex consisting of ribozyme and substrate RNA. All ribozymes capable of cleaving phosphodiester bonds in trans, that is to say intra olecularly, are suitable for the purposes of the invention. Apart from ribonuclease P the known naturally occurring ribozymes (hammerhead ribozyme, hairpin ribozyme, hepatitis delta virus ribozyme, Neurospora mitochondrial VS ribozyme, group I and group II introns) are catalysts, which cleave or splice themselves and which act in cis (intramolecularly) . A preferred method of identifying compounds according to the present invention relates to the identification of small interfering RNAs (siRNAs). siRNAs mediate the post- transcriptional process of gene silencing by double stranded RNA (dsRNA) that is homologous in sequence to the silenced RNA. siRNAs can also contain a variety of modifications that confer resistance to nucleolytic degradation such as, for example, modified internucleoside linkages, modified nucleic acid bases and/or modified sugars and the like. siRNAs can also be modified by chemically linking the oligonucleotide to one or more moieties or conjugates to enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide. Such moieties or conjugates include lipids such as cholesterol, cholic acid, thioether, aliphatic chains, phospholipids, polyamines, polyethylene glycol (PEG), or pal ityl moieties. One embodiment of the present invention relates to a method wherein the siRNA comprises a sense strand of 17-23 nucleotides homologous to a 17-23 nucleotide long nucleotide sequence selected from the group consisting of SEQ ID NO: 1- 3 and an antisense strand of 17-23 nucleotides complementary to the sense strand. All nucleotides in the sense and antisense strand base pair, or alternatively there may be mismatches between the sense and antisense strand. Preferably the siRNA further comprises a loop region connecting the sense and the antisense strand. A self-complementing single stranded siRNA molecule polynucleotide according to the present invention comprises a sense portion and an antisense portion connected by a loop region. Preferably, the second sequence is 4-30 nucleotides long, more preferably 5-15 nucleotides long and most preferably 8 nucleotides long. In a most preferred embodiment the linker sequence is GTTTGCTATAAC (SEQ ID NO: 231) . Self-complementary single stranded siRNAs form hairpin loops and are more stable than ordinary dsRNA. In addition, they are more easily produced from vectors. It is therefore preferred that the nucleotide comprising the siRNA present within a vector, which is preferably an adenoviral, retroviral, adeno-associated viral (AAV) , lentiviral, herpes simplex viral (HSV) , an alphaviral or a sendai viral vector. Thus, the expression inhibitory agent may be an antisense RNA, ribozyme, antisense oligodeoxynucleotide, or siRNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 7 to 230. Nucleotide sequences of the siRNAs are generally selected according to siRNA designing rules known in the art that give an improved reduction of the target sequences compared to nucleotide sequences that do not comply with these siRNA designing rules. A further aspect of the invention relates to a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-3, 7-230, or a nucleotide sequence complementary thereto, or a functional derivative or fragment thereof, or a pharmaceutical acceptable salt thereof for use as a medicament for the treatment of a pathological condition involving cognitive impairment or a susceptibility to the condition. Such pathological condition is preferably Alzheimer's disease. In another embodiment, a polynucleotide according to the invention is modified to confirm resistance to nucleolytic degradation or to enhance the activity, cellular distribution, or cellular uptake. Such modification may consist of modified internucleoside linkages, modified nucleic acid bases, modified sugars, and/or chemically linking the oligonucleotide to one or more moieties or conjugates . Vectors that may be applied according to the present invention and that comprise nucleic acids according to any one of SEQ ID NO: 1-3 or 7-230 may also be used as a medicament, as discussed intra. The nucleotide sequence in the vector may be an siRNA, an antisense RNA, a ribozyme, or an antisense oligodeoxynucleotide according to the invention. A further aspect of the invention relates to the use of a compound or a vector comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-3, 7-230 or a derivative, or a fragment thereof, or complementary thereto (separate or enclosed in a vector) for the manufacture of a medicament for the treatment of a pathological condition involving cognitive impairment or a susceptibility to the condition. Another aspect of the invention relates to a method for treatment, prevention or amelioration of a pathological condition involving cognitive impairment or a susceptibility to the condition, which comprises administration to a subject a compound or pharmaceutical acceptable salt or derivative thereof, or a vector encoding said compound, which compound inhibits the activity of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6 or a derivative or a fragment thereof.

Yet another aspect of the invention relates to a method of reducing or inhibiting the level of amyloid-beta peptide 11-42 or 11-40, 1-42 or 1-40 in a subject, comprising contacting said subject with a compound according to the invention or a pharmaceutical acceptable salt or derivative thereof, which compound inhibits the activity of polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6 or a functional derivative or fragment thereof. A further aspect of the invention relates to a pharmaceutical composition comprising a compound according to the present invention further comprising a pharmaceutical acceptable carrier and/or diluent and/or excipient.

A "pharmaceutically acceptable carrier and/or diluent and/or excipient" refers to any useful solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic and absorption delaying agent, and the like, compatible with pharmaceutical administration. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non- aqueous vehicles such as fixed oils may also be used. Supplementary active compounds can also be incorporated into the compositions. A pharmaceutical composition of the compound is formulated to be compatible with its intended route of administration, including intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA) ; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Such compositions are preferably stable during manufacture and storage and must generally be preserved against contamination from microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol) , and suitable mixtures. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. Various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal, can contain microorganism contamination. Isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride can be included in the composition. Compositions that can delay absorption include agents such as aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients as required, followed by sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium, and the other required ingredients. Sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying that yield a powder containing the active ingredient and any desired ingredient from a sterile solution. Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included. Tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch; a lubricant such as magnesium stearate or STEROTES; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the compounds are generally delivered as an aerosol spray from a nebulizer or a pressurized container that contains a suitable propellant, e.g., a gas such as carbon dioxide. Systemic administration can also be transmucosal or transdermal. For transmucosal or transdermal administration, penetrants that can permeate the target barrier (s) are generally selected. Transmucosal penetrants include detergents, bile salts, and fusidic acid derivatives. Nasal sprays or suppositories can be used for transmucosal administration. For transdermal administration, the active compounds are generally formulated into ointments, salves, gels, or creams. The compounds can also be prepared in the form of suppositories (e.g., with bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. In one embodiment, the active compounds are prepared with carriers that protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable or biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Polyethylene glycols, e.g. PEG, are also good carriers. Such materials can be obtained commercially from ALZA Corporation (Mountain View, Calif.) and NOVA Pharmaceuticals, Inc. (Lake Elsinore, Calif.), or prepared by one of skill in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as in US 4,522,811. Oral formulations or parenteral compositions in unit dosage form can be created to facilitate administration and dosage uniformity. Unit dosage form refers to physically discrete units suited as single dosages for the subject to be treated, containing¹ a therapeutically effective quantity of active compound in association with the required pharmaceutical carrier. The specification for the unit dosage forms of the invention are dictated by, and directly dependent on, the unique characteristics of the active compound and the particular desired therapeutic effect, and the inherent limitations of compounding the active compound.

The invention also relates to a method for diagnosing a pathological condition in a subject comprising comparing the nucleic acid sequence of the subject's mRNA or genomic DNA with a nucleic acid selected from the group consisting of SEQ ID NO: 1-3; and identifying any difference (s) between the nucleic acid sequence of the subject's mRNA or genomic DNA and the nucleic acid selected from the group consisting of SEQ ID NO: 1-3. The invention also relates to a method for diagnosing a pathological condition in a subject, comprising determining the amount of polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6 in a sample from said subject, and comparing the amount with the amount of the polypeptide in a sample of a healthy subject. In such a method, an increase of the amount of polypeptide compared to the healthy subject is indicative of the presence of a pathological condition. It is well understood in the art that databases such as GenBank, can be searched to identify genomic sequences that contain regions of identity (exons) to a nucleic acid. Such genomic sequences encode for the nucleic acid. The term "amyloid beta peptide" refers to amyloid beta peptides with different composition that are processed from the amyloid beta precursor protein (APP) . Examples of the species comprise 1-40, 1-42, y-42, whereby y ranges from 1- 17, and 1-x whereby x ranges from 24-42, and 11-40 and 11- 42. The term "compound", besides relating to the entities outlined above, also relates to organic and inorganic compounds, such as synthetic molecules, peptides, lipids, antibodies and natural compounds . The term "agonist" predominantly refers to a ligand that activates the receptor the ligand binds to. However, other types of agonists (such as for instance ^triggering' antibodies) may be applicable as long as they stimulate the activation of the receptor. The term "functional derivatives of a polypeptide" relate to those peptides, oligopeptides, polypeptides, proteins and enzymes that retain the biological activity (functionality) of the protein, e.g. polypeptides that have amino acid mutations compared to the amino acid sequence of a naturally-occurring form of the polypeptide. A derivative may further comprise additional naturally occurring, altered, glycosylated, acylated or non-naturally occurring amino acid residues compared to the amino acid sequence of a naturally occurring form of the polypeptide. It may also contain one or more non-amino acid substituents compared to the amino acid sequence of a naturally occurring form of the polypeptide, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence. The term "functional fragment of a polypeptide" relates to peptides, oligopeptides, polypeptides, proteins and enzymes that exhibit substantially a similar, but not necessarily identical, activity as the complete sequence. The term "polynucleotide" refers also to nucleic acids with modified backbones such as peptide nucleic acid, polysiloxane, and 2 ' -0- (2-methoxy) ethylphosphorothioate. The term "derivatives of a polynucleotide" relates to DNA- and RNA- molecules, nucleic acids, and oligonucleotides that may have nucleic acid mutations compared to the nucleic acid sequence of a naturally occurring form of the polynucleotide. A derivative may further comprise nucleic acids with modified backbones such as peptide nucleic acid (PNA) , polysiloxane, and 2 ' -0- (2-methoxy) ethylphosphorothioate, non-naturally occurring nucleic acid residues, or one or more nuclei acid substituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-, amino-, propyl- , chloro-, and methanocarbanucleosides, or a reporter molecule to facilitate its detection. The term "fragment of a polynucleotide" relates to oligonucleotides that exhibit substantially a similar, but not necessarily identical, activity as the complete sequence. TABLE 1: GPCRs involved in APP processing (SEQ ID 1-3; 4-6), Sequences for expression inhibitory agent (SEQ ID 7 to 118, 119 to 178, 179-230) and the hairpin loop sequence of the RNAi (SEQ ID 231) :

TABLE2 : buffers and solutions used for ELISA

TABLE 3: Primers used in the quantitative real time PCR analysis for AVPR2 expression levels

TABLE 4 : Ct values obtained during quantitative real time PCR: Human cerebral cortex or human hippocampus RNA is tested for the presence of AVPR2 RNA via quantitative real time PCR. GAPDH RNA is used to normalize all samples (?Ct) ,

TABLE 5: Effective knock-down sequence for AVPR2.

EXAMPLES

EXAMPLE 1: Overexpression of AVPR2 plus incubation with arginine vasopressin increases amyloid beta 1-42 levels in SH-SY5Y APPwt cell cultures. To identify novel drug targets that change the APP processing, a stable cell line overexpressing APP, SH-SY5Y APPwt, was transduced with adenoviral cDNA libraries and the resulting amyloid beta 1-42 levels were detected via ELISA. This stable cell line was created^' after transfection of SH- SY5Y cells with the APP695wt cDNA cloned in pcDNA3.1 and selection with G418 during 3 weeks. At this time point colonies were picked and stable clones were expanded and tested for their secreted amyloid beta peptide levels. The assay was performed as follows. Cells were seeded in collagen-coated plates at a cell density of 15,000 cells/well (384 well plate) in DMEM glutamax 15%FBS. Cells were differentiated towards a neuronal phenotype with retinoic acid for the next 8 days. Next, cells were infected with 1 ?1 of adenovirus (? MOI 200). The following day, the virus was washed away. Cultures infected with Ad5/AVPR2 were incubated with 10 nM of [Arg8] vasopressin in DMEM glutamax with 25 mM Hepes and 15%FBS. Amyloid beta peptides were allowed to accumulate during 24h. The ELISA plate was prepared by coating the capture antibody (JRF/cAbeta42/26) (obtained from M Mercken, Johnson and Johnson Pharmaceutical Research and Development, B-2340 Beerse, Belgium) overnight in buffer 42 (table 2) at a concentration of 2,5 ?g/ml. The excess capture antibody was washed away the next morning with PBS and the ELISA plate was then blocked overnight with casein buffer (table 2) at 4°C. Upon removal of the blocking buffer, 30 ?1 of the sample was transferred to the ELISA plate and incubated overnight at 4°C. After extensive washing with PBS-Tween20 and PBS, 30 ?1 of the horse reddish peroxidase (HRP) labeled detection antibody (Peroxidase Labeling Kit, Roche), JRF/AbetaN/25-HRP (obtained from M Mercken, Johnson and Johnson Pharmaceutical Research and Development, B-2340 Beerse, Belgium) was diluted 1/5000 in buffer C (table 2) and added to the wells for another 2h. Following the removal of excess detection antibody by a wash with PBS-Tween20 and PBS, HRP activity was detected via addition of luminol substrate (Roche) , which was converted into a chemiluminescent signal by the HRP enzyme. In order to validate the assay, the effect of adenoviral overexpression with random titre of two clinical PSl mutants and BACE on amyloid beta 1-42 production was evaluated in the Hek293 APPwt cells. As is shown in Figure 2, all constructs induce amyloid beta 1-42 levels as expected. An adenoviral cDNA library containing almost all GPCRs was constructed as follows. DNA fragments covering the full coding region of the GPCRs, were amplified by PCR from a pooled placental and fetal liver cDNA library (InvitroGen) . All fragments were cloned into an adenoviral vector as disclosed in US 6,340,595 and subsequently adenoviruses were made harboring the corresponding cDNAs. During the screening of the adenoviral GPCR library in the SH-SY5Y APPwt cells, AVPR2 was identified as a modulator of APP processing. These results indicated that over-expression of AVPR2 {+ additional stimulation with its ligand) leads to increased levels of amyloid beta 1-42 peptides in the conditioned medium of SH-SY5Y APPwt cells, showing that this GPCR modulates APP processing. The stimulatory effect of AVPR2 + arginine vasopressin was confirmed upon re-analysis using the viruses with a known titer (viral particles/ml) , as determined by quantitative real time PCR. AVPR2 virus was infected at MOI 50 and 250 and the experiment was performed as described above. Amyloid beta 1-42 levels were moderately but significantly higher compared to the negative controls for Ad5/AVPR2 (Figure 3). In addition, other ELISA' s were performed as described above, with following antibodies: for the amyloid beta 1-40 ELISA, the capture and detection antibody were respectively JRF/cAbeta40/10 and JRF/AbetaN/25-HRP (obtained from M Mercken, Johnson and Johnson Pharmaceutical Research and Development, B-2340 Beerse, Belgium) , for the amyloid beta 11-42 ELISA, the capture and detection antibody were respectively JRF/cAbeta42/26 and JRF/hAbll/1 (obtained from M Mercken, Johnson and Johnson Pharmaceutical Research and Development, B-2340 Beerse, Belgium) and for amyloid beta 11-40 ELISA, the capture and detection antibody were respectively JRF/cAbeta40/10 and JRF/hAbll/1 (obtained from M Mercken, Johnson and Johnson Pharmaceutical Research and Development, B-2340 Beerse, Belgium) . The same procedure is used for the analysis of APP processing by AVPRla and AVPRlb.

EXAMPLE 2 : Identification of close relatives of AVPR2. The amino acid sequence of the human AVPR2 receptor was used as query in a BLAST search against all the human GPCRs in order to find its closest homologues. Table 1 (SEQ ID 4- 5) shows the 2 closest homologues of the AVPR2 receptor. Using Clustal an alignment was constructed showing the degree of homology between the AVPR2 and its closest homologues, the AVPRla and AVPRlb (figure 6) .

EXAMPLE 3 : Reduction of the amyloid beta production via siRNA-mediated knock down of the expression levels of AVPR2. The effect of an antagonist can be mimicked through the use of siRNA-based strategies, which result in decreased expression levels of the targeted protein, in our case AVPR2. HEK293 APPwt, SH-SY5Y APPwt or SK-N-MC APPwt cells are transfected with a smart pool of siRNAs or single siRNAs of AVPR2 (Dharmacon, USA), eGFP, Luciferase and BACE with Oligofectamine. 24 hours after transfection, medium is refreshed and the cells are allowed to accumulate amyloid beta peptides in the conditioned medium for 24 hours prior analysis with the ELISAs described above. The same procedure is used for the analysis of APP processing by AVPRla and AVPRlb.

EXAMPLE 4: Infection of SH-SY5Y cells with Ad5-AVPR2 knock- down virus reduces amyloid beta 1-42 levels . An adenoviral-siRNA technology was developed to knockdown the expression of selected genes in human cells. A collection of _. adenoviruses containing knock-down sequences targeting over 4,000 human genes belonging to the drugable gene families was built, generally following the principles as disclosed in WO 99/64582, WO 03/020931 and described in PCT/EP03/04362. The drugable collection includes splice variants of the proteins and consists of genes belonging to Kinases, Phosphatases, GPCRs, Transporters, Proteases, Receptors, Nuclear Hormone Receptors, Ion Channels and Phosphodiesterases . The assay was performed as follows. SH-SY5Y cells were seeded in collagen-coated plates at a cell density of 15,000 cells/well (384 well plate) in DMEM glutamax 10%FBS. Two days after seeding, the cultures were incubated with 1 μM retinoic acid to differentiate the cells towards a neuronal phenotype. Next day, cells were co-infected with an adenovirus holding the cDNA of human APP695 that exhibit the Swedish mutation and with the knock-down adenovirus library. The following day, viruses were washed away and cells were further differentiated with 1 μM retinoic acid for the following 4 days. Then, the medium was refreshed again and amyloid beta peptides were allowed to accumulate during 48h. The ELISA plate was prepared by coating the capture antibody (JRF/cAbeta42/26) (obtained from M Mercken, Johnson and Johnson Pharmaceutical Research and Development, B-2340 Beerse, Belgium) overnight i buffer 42 (table 2) at a concentration of 2,5 ?g/ml. The excess capture antibody was washed away the next morning with PBS and the ELISA plate was then blocked overnight with casein buffer (table 2) at 4°C. Upon removal of the blocking buffer, 50 ?1 of the sample was transferred to the ELISA plate and incubated overnight at 4°C. The ELISA was continued according to the procedure outlined in example 1. During the screen of the adenoviral knock-down library in the SH-SY5Y cells, AVPR2 was identified as a modulator of APP processing. These results indicate that reduction in AVPR2 expression leads to decreased levels of amyloid beta 1-42 peptides in the conditioned medium of these cells, showing that this GPCR modulates APP processing. The inhibitory effect on amyloid beta 1-42 levels by AVPR2 elimination was confirmed upon a three-MOI screen of the viruses with a known titer (viral particles/ml), as determined by quantitative real time PCR. Ad5/AVPR2 knockdown virus was used to infect the cells at MOI 1800, 600 and 200, and the experiment was performed as described above. Amyloid beta 1-42 levels were significantly lower for

Ad5/AVPR2 compared to the negative controls (Figure 5) . The same procedure is used for the analysis of APP processing by AVPRla and AVPRlb.

EXAMPLE 5: Expression of AVPR2 in the human brain. Upon identification of a modulator of APP processing, it is important to evaluate whether the modulator is expressed in the tissue and in the cells of interest. This can be achieved by measuring the RNA and/or protein levels. In recent years, RNA levels were being quantified through real time PCR technologies, whereby the RNA is first transcribed to cDNA and then the amplification of the cDNA of interest is monitored during a PCR reaction. The amplification plot and the resulting Ct value are indicators for the amount of RNA present in the sample. Determination of the levels of household keeping genes allows the normalization of RNA levels of the target gene between different RNA samples, represented as delta Ct values. To assess whether the GPCR of the invention is expressed in the human brain, real time PCR with GAPDH specific primers and specific primers for GPCR of the invention was performed on human cerebral cortex and human hippocampal total RNA (BD Biosciences) . GAPDH was detected with a Taqman probe, while for the GPCR SybrGreen was used. In short, 40 ng of RNA was transcribed to DNA using the

MultiScribe Reverse Transcriptase (50 U/μl) enzyme (Applied BioSystems) . The resulting cDNA was amplified with AmpliTaq Gold DNA polymerase (Applied BioSystems) during 40 cycles using an ABI PRISM® 7000 Sequence Detection System. Cerebral cortex and hippocampal total RNA was analyzed for the presence of the GPCR transcripts via quantitative real time PCR. For AVPR2, the obtained Ct values indicate that it is detected in all RNA samples (table 4) . To gain more insight into the specific cellular expression, immunohistochemistry (protein level) and/or in situ hybridization (RNA level) were carried out on sections from human normal and Alzheimer' s brain hippocampal, cortical and subcortical structures. These results indicate whether expression occurs in neurons, microglia cells or astrocytes. The comparison of diseased tissue with healthy tissue, teaches us whether AVPR2 is expressed in the diseased tissue and whether its expression level was changed compared to the non-pathological situation. The same procedure is used for expression profiling of AVPRla and AVPRlb.

EXAMPLE 6: Amyloid beta production in rat primary neuronal cells . In order to investigate whether AVPR2 of the invention affects amyloid beta production in a real neuron, human or rat primary hippocampal or cortical neurons are used. In first instance, these neurons are transduced with adenovirus containing the AVPR2 and amyloid beta levels are determined by ELISA (see EXAMPLE 1) after incubation of the cells with arginine vasopressin. In second instance, as hippocampal and cortical neurons endogenously express the AVPR2 receptor, neuron cultures are treated with the agonist arginine vasopressin. Since rodent APP genes carry a number of mutations in APP compared to the human sequence, they produce less amyloid beta 1-40 and 1-42. In order to achieve higher amyloid beta levels, co-transduction with human wild type APP or human Swedish mutant APP (which enhances Abeta production) cDNA is performed. In third instance, hippocampal and cortical neurons are treated with an agonist (e.g. arginine vasopressin) and known AVPR2 antagonists (e.g. Tolvaptan) are added to these cultures to determine the levels of extracellular amyloid beta 1-42 peptides. This procedure proves that modulation of the AVPR2 can regulate the levels of amyloid beta peptides. Human primary neurons are purchased from Cellial Technologies, France. Rat primary neuron cultures are prepared from brain of E18-E19-day-old fetal Sprague Dawley rats according to Goslin and Banker (Culturing Nerve cells, second edition, 1998 ISBN 0-262-02438-1) . Briefly, single cell suspensions obtained from the hippocampus or cortices are prepared. The number of cells is determined (only taking into account the living cells) and cells are plated on poly- L-lysine-coated plastic 96-well plates in minimal essential medium (MEM) supplemented with 10% horse serum. The cells are seeded at a density between 30,000 and 60,000 cells per well (i.e. about 100,000 - 200,000 cells/cm², respectively). After 3-4 h, culture medium is replaced by 150 μl serum-free neurobasal medium with B27 supplement (GIBCO BRL) . Cytosine arabinoside (5 μM) is added 24 h after plating to prevent nonneuronal (glial) cell proliferation. Neurons are used at day 5-7 after plating. Before adenoviral transduction, 150 μl conditioned medium of these cultures is transferred to the corresponding wells in an empty 96-well plate and 50 μl of the conditioned medium returns to the cells. The remaining 100 μl/well is stored at 37°C and 5% C0₂. Both hippocampal and cortical primary neuron cultures are co-infected with the crude or purified lysate of virus containing the cDNA of AVPR2, and human wild type APP or human Swedish mutant APP, at different MOIs, ranging from 100 to 3000. Sixteen to twenty-four hours after transduction, virus is removed and cultures are washed with 100 μl pre-warmed fresh neurobasal medium. After removal of the wash solution, the remaining 100 μl of the stored conditioned medium is transferred to the corresponding cells. From now on, cells accumulate amyloid beta in the conditioned medium and its concentration is determined by myloid beta 1-42 ELISA (see EXAMPLE 1) . In addition, other amyloid beta species are determined by use of specific ELISAs. The conditioned media are collected 24, 48 and 96 hours after exchanging virus-containing medium by stored conditioned medium. In addition, the following ELISA' s are performed as described above: amyloid beta 1-40, amyloid beta 11-42 and amyloid beta 11-40. In addition, the procedure as described above is used to analyse rodent amyloid beta by means of an ELISA protocol described in EXAMPLE 1. The ELISA plate is prepared by coating the capture antibody (JRF/cAbeta42/26) (obtained from M Mercken, Johnson and Johnson Pharmaceutical Research and Development, B-2340 Beerse, Belgium) and rodent amyloid beta levels are determined by the horse reddish peroxidase (HRP) labeled detection antibody (Peroxidase Labeling Kit, Roche), JRF/rAb/2-HRP (obtained from M Mercken, Johnson and Johnson Pharmaceutical Research and Development, B-2340 Beerse, Belgium) . The same procedure is used for the analysis of AVPRla and AVPRlb.

EXAMPLE 7: Ligand screens for GPCRs.

Reporter gene screen. Mammalian cells such as HEK293 or CHO-K1 cells are either stably transfected with a plasmid harboring the luciferase gene under the control of a cAMP dependent promoter (CRE elements) or transduced with an adenovirus harboring a luciferase gene under the control of a cAMP dependent promoter. In addition reporter constructs can be used with the luciferase gene under the control of a Ca²⁺ dependent promoter (NF-AT elements) or a promoter that is controlled by activated NF-?B. These cells, expressing the reporter construct, are then transduced with an adenovirus harboring the cDNA of the GPCR of the present invention. 40 h after transduction the cells are treated with a) an agonist for the receptor (e.g. arginine vasopressin, lypressin or desmopressin) and screened against a large collection of reference compounds comprising peptides (LOPAP, Sigma Aldrich), lipids (Bio ol, TimTech) , carbohydrates (Specs), natural compounds (Specs, TimTech), and small chemical compounds (Tocris), or b) compounds, which decrease the agonist-induced increase in luciferase activity, and which are considered to be antagonists or inverse agonists for AVPR2. These compounds are screened again for verification and screened against their effect on secreted amyloid beta peptide levels. In addition, cells expressing the NF-AT reporter gene can be transduced with an adenovirus harboring the cDNA encoding the ? -subunit of G₁₅ or chimerical G? subunits. G₁₅ is a promiscuous G protein of the G_q class that couples to many different GPCRs and as such re-directs their signaling towards the release of intracellular Ca²⁺ stores. The chimerical G alpha subunits are members of the G_s and Gι₀ family by which the last 5 C-terminal residues are replaced by those of G?q, these chimerical G-proteins also redirect cAMP signaling to Ca²⁺ signaling. FLIPR screen. Mammalian cells such as HEK293 or CHO-K1 cells are stably transfected with an expression plasmid construct harboring the cDNA of a GPCR according to the present invention. Cells are seeded and grown until sufficient stable cells can be obtained. Cells are loaded with a Ca²⁺ dependent fluorophore such as Fura3 or Fura4. After washing away the excess of fluorophore the cells are screened against a large collection of reference compounds comprising peptides (LOPAP, Sigma Aldrich), lipids (Biomol, TimTech), carbohydrates (Specs), natural compounds (Specs, TimTech), and small chemical compounds (Tocris) by simultaneously adding an agonist and a compound to the cells. As a reference just the agonist is added. Activation of the receptor is measured as an almost instantaneously increase in fluorescence due to the interaction of the fluorophore and the Ca⁺ that is released. Compounds that reduce or inhibit the agonist induced increase in fluorescence are considered to be antagonists or inverse agonists for the receptor they are screened against. These compounds will be screened again to measure the secreted amyloid beta peptide.

AequoScreen. CHO cells, stably expressing Apoaequorin are stably transfected with a plasmid construct harboring the cDNA of a GPCR. Cells are seeded and grown until sufficient stable cells can be obtained. The cells are loaded with coelenterazine, a cofactor for apoaequorin. Upon receptor activation intracellular Ca²⁺ stores will be emptied and the aequorin will react with the coelenterazine in a light emitting process. The emitted light is a measure for receptor activation. The CHO, stable expressing both the apoaequorin and the receptor are screened against a large collection of reference compounds comprising peptides (LOPAP, Sigma Aldrich), lipids (Biomol, TimTech), carbohydrates (Specs), natural compounds (Specs, TimTech), and small chemical compounds (Tocris) by simultaneously adding an agonist and a compound to the cells or by only adding a compound. As a reference just the agonist is added. Activation of the receptor is measured as an almost instantaneously light flash due to the interaction of the apoaequorin, coelenterazine and the Ca²⁺ that is released. Compounds that reduce or inhibit the agonist induced increase in light or the constitutive activity are considered to be antagonists or inverse agonists for the receptor they are screened against. These compounds will be screened again for verification and secreted amyloid beta levels. In addition, CHO cells stable expressing the apoaequorin gene are stably transfected with a plasmid construct harboring the cDNA encoding the ? -subunit of Gι₅ or chimerical G_? subunits. G₁₅ is a promiscuous G protein of the G_q class that couples to many different GPCRs and as such redirects their signaling towards the release of intracellular Ca2+ stores. The chimerical G alpha subunits are members of the G_s and Gι_/0 family by which the last 5 C- terminal residues are replaced by those of G_?q, these chimerical G-proteins also redirect cAMP signaling to Ca2+ signaling.

Screening for compounds that bind to AVPR2, AVPRla and/or AVPRlb (displacement experiment) Compounds are screened for binding. The affinity of the compounds to the polypeptides is determined in a displacement experiment. In brief, the polypeptides of the present invention are incubated with a radiolabeled (e.g. [3HJ-AVP and [3H] -desglycinamide-d(CH2) [D-Ile2, Ile4]AVP) ligand that is known to bind to the polypeptide and with an unlabeled compound. The displacement of the labeled ligand from the polypeptide is determined by measuring the amount of labeled ligand that is still associated with the polypeptide. The amount associated with the polypeptide is plotted against the concentration of the compound to calculate IC₅₀ values. This value reflects the binding affinity of the compound to its target, i.e. the polypeptides of the present invention. Strong binders have an IC50 in the nanomolar and even picomolar range. Compounds that have an IC₅₀ of at least 10 micromol or better (nmol to pmol) are applied in beta amyloid secretion assay to check for their effect on the beta amyloid secretion and processing. The polypeptides of the present invention can be prepared in a number of ways depending on whether the assay will be run on cells, cell fractions or biochemically, on purified proteins.

Screening for compounds that bind to AVPR2, AVPRla and/or AVPRlb (generic GPCR screening assay) When a G protein receptor becomes active, it binds to a G protein (Gq, Gs, Gi, Go) and stimulates the binding of GTP to the G protein. The G protein then acts as a GTPase and slowly hydrolyses the GTP to GDP, whereby the receptor, under normal conditions, becomes deactivated. A non- hydrolyzable analog of GTP, [³⁵S]GTP?S, can be used to monitor enhanced binding to membranes which express activated receptors. It is reported that [³⁵S]GTP?S can be used to monitor G protein coupling to membranes in the absence and presence of ligand. A pin-tool is used to transfer a candidate compound in each well plus [³⁵S]GTP?S, followed by an incubation on a shaker for 60 minutes at room temperature. The assay is stopped by spinning of the plates at 4000 RPM for 15 minutes at 22 °C. The plates are then aspirated and radioactivity is then read. This procedure is used for analysis of AVPR2, AVPRla and AVPRlb.

Receptor-ligand binding study on cell surface The receptor is expressed in mammalian cells (HEK293, CHO, COS7) by adenoviral transduction of the cells (see US 6,340,595). The cells are incubated with both labeled ligand (iodinated, tritiated, or fluorescent) and the unlabeled compound at various concentrations, ranging from 10 pM to

10?M (3 hours at 4°C: 25 mM HEPES, 140 mM NaCl, 1 mM CaCl₂, 5 mM MgCl₂ and 0.2% BSA, adjusted to pH 7.4). Reaction mixtures are aspirated onto PEI-treated GF/B glass filters using a cell harvester (Packard) . The filters are washed twice with ice cold wash buffer (25 mM HEPES, 500 mM NaCl, 1 mM CaCl₂, 5 mM MgCl₂, adjusted to pH 7.4). Scintillant (MicroScint-10; 35 μl) is added to dried filters and the filters counted in a (Packard Topcount) scintillation counter. Data are analyzed and plotted using Prism software (GraphPad Software, San Diego, Calif.). Competition curves are analyzed and IC50 values calculated. If 1 or more datapoints do not fall within the sigmoidal range of the competition curve or close to the sigmoidal range the assay is repeated and concentrations of labeled ligand and unlabeled compound adapted to have more data points close to or in the sigmoidal range of the curve.

Receptor-ligand binding studies on membrane preparations Membrane preparations are isolated from mammalian cells (HEK293, CHO, C0S7) overexpressing the receptor and the membrane generation is done as follows: Medium is aspirated from the transduced cells and cells are harvested in 1 x PBS by gentle scraping. Cells are pelleted (2500 rpm 5 min) and resuspended in 50 mM Tris pH 7.4 (10 x 10E6 cells/ml). The cell pellet is homogenized by sonicating 3 x 5 sec (UP50H; sonotrode MSI; max amplitude: 140 μm; max Sonic Power Density: 125W/cm²) . Membrane fractions are prepared by centrifuging 20 min at maximal speed (13000 rpm -15 000 to 20 OOOg or ref) . The resulting pellet is resuspended in 500 μl 50 mM Tris pH 7.4 and sonicated again for 3 x 5 sec. The membrane fraction is isolated by centrifugation and finally resuspended in PBS. Binding competition and derivation of IC₅₀ values are determined as described above.

Internalisation screen (1) Activation of a GPCR-associated signal transduction pathway commonly leads to translocation of specific signal transduction molecules from the cytoplasma to the plasma membrane or from the cytoplasma to the nucleus . Norak has developed a transfluor assay based on agonist-induced translocation of receptor-β-arrestin-GFP complex from the cytosol to the plasma membrane and subsequent internalization of this complex, which occurs during receptor desensitization. A similar assay uses GFP tagged receptor instead of β-arrestin. HEK293 cells are transduced with a AVPR2-eGFP vector that translates for an AVPR2-eGFP fusion protein. 48 hours after transduction, the cells are set to fresh serum-free medium for 60 minutes and treated with a ligand (e.g. arginine vasopressin) for 15, 30, 60 or 120 minutes at 37 °C and 5% C0₂. After indicated exposure times, cells are washed with PBS and fixed with 5% paraformaldehyde for 20 minutes at RT. GFP fluorescence is visualized with a Zeiss microscope with a digital camera. This method aims for the identification of compounds that inhibit a ligand-mediated translocation of the fusion protein to intracellular compartments. The same procedure is used for analysis of AVPRla and AVPRlb.

Internalisation screen (2) Various variations on translocation assays exists using β-arrestin and β-galactosidase enzyme complementation and BRET based assays with receptor as energy donor and β- arrestin as energy acceptor. Also the use of specific receptor antibodies labeled with pH sensitive dyes are used to detect agonist induced receptor translocation to acidic lysosomes. All of he translocation assays are used for screening for both agonistic and antagonistic acting ligands .

Melanophore assay (Arena Pharmaceutical) The melanophore assay is based on the ability of GPCRs to alter the distribution of melanin cotaining melanosomes in Xenopus melanophores. The distribution of the melanosomes depends on the exogenous receptor that is either Gi/o or Gs/q coupled. The distribution of the melanosomes (dispersed or aggregated) is easily detected by measuring light absorption. This type of assay is used for both agonist as well as antagonist compound screens.

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Claims

1. Method for identifying a compound that influences the processing of an amyloid-beta precursor protein, said method comprising the steps of: (a) expressing a polypeptide of SEQ ID NO: 4, 5 or 6, or a functional fragment or derivative thereof, in a cell; (b) determining a first level of processing of an amyloid- beta precursor protein in said cell; (c) exposing said cell to a compound; (d) determining a second level of processing of said amyloid-beta precursor protein in said ^"cell; and (e) identifying the compound, that influences the processing of said amyloid-beta precursor protein.

2. Method for identifying a compound that influences the processing of an amyloid-beta precursor protein, said method comprising the steps of: (a) expressing a polypeptide selected from the group consisting of SEQ ID NO: 4, 5 or 6, or a functional fragment or derivative thereof, in a cell; (b) determining a first activity level of an amyloid-beta precursor protein; (c) exposing said cell to a compound; (d) determining a second activity level of said precursor protein; and (e) identifying the compound that influences the activity level .

3. Method for identifying a compound that decreases the expression or activity of a polypeptide selected from SEQ ID NO: 4, 5 or 6 in a cell, said method comprising the steps of: (a) incubating a cell; (b) determining a first level of expression or activity of said polypeptide in said cell; (c) incubating said cell with a compound; (d) determining a second level of expression or activity in said cell of said polypeptide following or during step (c) ; and (e) identifying a compound that decreases the expression or activity of said polypeptide.

4. Method according to any one of claims 1-3, wherein the polypeptide is AVPR2 (SEQ ID NO: 6).

5. Method according to any one of claims 1-4, wherein the influence on the processing of said precursor protein or the decrease in expression of the polypeptide is such that the level of amyloid-beta peptide 1-42, 1-40, 11-42 or 11-40 is reduced.

6. Method according to claim 2, wherein said second activity level is lower than the first activity level, preferably wherein the second activity level is close to zero.

7. Method according to any one of claims 1-6, further comprising the step of exposing said cell to an agonist of the polypeptide.

8. Method according to claim 7, wherein the agonist is arginine vasopressin, lypressin or desmopressin

9. Method according to claim 2, wherein the activity is measured by determining the level of the second messengers cyclic AMP or Ca²⁺.

10. Method according to claim 9, wherein the activity level of at least one second messenger is determined with a reporter gene under the control of a promoter that is responsive to the second messenger.

11. Method according to claim 10, wherein the promoter is a cyclic AMP-responsive promoter, an NF-KB responsive promoter, or a NF-AT responsive promoter.

12. Method according to claim 10 or 11, wherein the reporter gene is selected from the group consisting of: alkaline phosphatase, GFP, eGFP, dGFP, luciferase and ??galactosidase .

13. Method according to any one of claims 1-12, wherein the compound exhibits a binding affinity to the polypeptide of at most 10 micromolar.

14. Method according to any of the claims 1-13, wherein the compound is selected from the group consisting of: a small molecule (low molecular weight) compound, an antisense RNA, an antisense oligodeoxynucleotide (ODN) , an siRNA, a ribozyme, an RNAi, an antibody, a nanobody, a peptide, a polypeptide, a nucleic acid, a lipid and a natural compound.

15. Method according to any one of claims 1-14, wherein said compound is an expression inhibitory agent that inhibits the expression and/or the translation of the nucleic acid encoding the polypeptide.

16. Method according to any one of claims 1-15, wherein the compound is provided by a vector.

17. Method according to claim 16, wherein the vector is an adenovirus, a retrovirus, an alphavirus, an adeno-associated virus (AAV) , a lentivirus, a herpes simplex virus (HSV) or a sendai virus.

18. Method according to any one of claims 14-17, wherein the compound is an siRNA comprising a sense strand of 17-23 nucleotides homologous to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-3 and an antisense strand of 17-23 nucleotides complementary to the sense strand.

19. Method according to claim 18, wherein the siRNA further comprises a loop region connecting the sense and the antisense strand.

20. Method according to claim 19, wherein the loop region comprises the nucleic acid of SEQ ID NO: 231.

21. Method according to any one of claims 1-20, wherein the compound is modified to confirm resistance to nucleolytic degradation or to enhance the activity, cellular distribution, or cellular uptake.

22. Method according to claim 21, wherein the modification comprises a modified internucleoside linkage, a modified nucleic acid base, a modified sugar, and/or a chemical linkage of the oligonucleotide to one or more moieties or conjugates.

23. An isolated nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-3 and 7-230, or a functional derivative or fragment thereof, or an isolated nucleic acid sequence complementary thereto.

24. An isolated nucleic acid sequence according to claim

23, wherein the nucleic acid sequence is further modified to confirm resistance to nucleolytic degradation or to enhance the activity, cellular distribution, or cellular uptake.

25. An isolated nucleic acid sequence according to claim

24, wherein the modification comprises a modified internucleoside linkage, a modified nucleic acid base, a modified sugar and/or a chemical linkage of the oligonucleotide to one or more moieties or conjugates.

26. An isolated nucleic acid sequence according to any one of claims 23-25 for use as a medicament.

27. An isolated nucleic acid according to claim 26, wherein said use is in the treatment of a pathological condition involving cognitive impairment or a susceptibility to the condition, such as Alzheimer's Disease.

28. An siRNA molecule comprising a sense strand of 17-23 nucleotides homologous to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-3 and an antisense strand of 17-23 nucleotides complementary to the sense strand.

29. An siRNA according to claim 28, further comprising a loop region connecting the sense and the antisense strand.

30. An siRNA molecule according to claim 29, wherein the loop region comprises the nucleic acid sequence of SEQ ID NO: 231.

31. A vector comprising a nucleic acid sequence according to any one of claims 23-25.

32. A vector according to claim 31, wherein said vector is selected from the group consisting of an adenovirus, a retrovirus, an alphavirus, a lentivirus, an adeno-associated virus, a herpes simplex virus and a sendai virus.

33. A pharmaceutical composition comprising a nucleic acid sequence according to any one of claims 23-25, and a ^• pharmaceutically acceptable solvent, diluent, excipient and/or carrier.

34. Method for treatment, prevention or amelioration of a pathological condition involving cognitive impairment or a susceptibility to the condition in a mammalian subject, said method comprising the step of administering a pharmaceutical composition according to claim 33.

35. Method of reducing the level of amyloid-beta peptide 11-42 or 11-40, 1-42 or 1-40 in a subject, said method comprising the step of administering a pharmaceutical composition according to claim 33.

36. Method for diagnosis of a pathological condition involving cognitive impairment or a susceptibility to the condition in a subject, said method comprising the steps of: (a) determining the nucleic acid sequence of at least one of the genes of SEQ ID NO: 1, 2 or 3 within the genomic DNA of said subject; (b) comparing the sequence from step (a) with the nucleic acid sequence obtained from a database and/or a healthy subject; and (c) identifying any difference (s) related to the onset of the pathological condition.

37. Method for diagnosis of a pathological condition involving cognitive impairment or a susceptibility to the condition in a subject, said method comprising the steps of: (a) determining the amount of polypeptide of SEQ ID NO: 4, 5 or 6 in a sample from said subject; and (b) comparing the amount determined in step (a) with the amount of said polypeptide is a sample from a. healthy individual; wherein the increase in the sample of said subject as compared to the healthy individual is indicative for the onset or presence of said pathological condition.

38. Method according to claim 36 or 37, wherein the pathological condition is Alzheimer's disease.

39. Use of a compound that inhibits the activity and/or the expression of a polypeptide according to SEQ ID NO: 4, 5 and/or 6 in the manufacture of a medicament for the treatment of Alzheimer's Disease.

40. Use according to claim 39, wherein said compound is selected from the group consisting of OPC-21268, SR 49059, SR 121463A, VPA-985, VPA-343, OPC-41061, OPC-31260, YM-087 and any of the compounds as described in US 6,653,478 and US 5,512,563.

41. Use according to claim 39, wherein said compound is identified according to any one of the methods of claims 1- 22.

42. Use according to claim 39, wherein said compound is any one of the isolated nucleic acid sequences of claims 23-25.

43. Use according to claim 39, wherein said compound is any one of the siRNA' s of claims 28-30.