WO2005003288A2

WO2005003288A2 - Diagnostic methods , prognostic methods and pharmaceutical compositions for neurodegenerative diseases like alzheimer’s disease by modulating and investigating genes rgs4 and itpkb, their gene products or fragments and derivatives thereof

Info

Publication number: WO2005003288A2
Application number: PCT/SE2004/001079
Authority: WO
Inventors: Elena Jazin; Lina Emilsson
Original assignee: Elena Jazin; Lina Emilsson
Priority date: 2003-07-02
Filing date: 2004-07-02
Publication date: 2005-01-13
Also published as: WO2005003288A3

Abstract

The present invention relates to diagnostic and prognostic methods for Alzheimer's Disease. The methods comprise measuring altered expression levels of the genes RGS4 and ITPKB in samples from a subject. The inventions also relate to kits for performing the method, and methods for screening for agents modulating expression of RGS4 and/or ITPKB, as well as the use of such agents for treating Alzheimer's Disease and manufacturing pharmaceutical compositions.

Description

DIAGNOSTIC MARKERS

Background of the invention

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive memory deterioration and disordered cognitive function. Most cases of AD arise during advanced age and this syndrome is called late-onset Alzheimer's disease (LOAD). A small percentage of AD cases are inherited in a mendelian autosomal dominant fashion, and are named early-onset Alzheimer's disease (EOAD). All known EOAD mutations, including the genes encoding B- amyloid precursor protein (APP), presenilin 1 (PSEN1), a d presenilin 2

(PSEN2), are associated with the metabolism of APP. Altered APP processing is involved in the formation of senile plaques (SP), one of the hallmarks of the disease. Other hallmarks are intracellular neurofibrillary tangles (NFT) and neuronal degeneration. All of these pathological changes are seen in the brains of subjects suffering from both EOAD and LOAD (LaFerla, Nat Rev Neurosci V^" 2002,3: 862-72). --

AD related molecular changes are most probably initiated several years before cognitive impairment, memory loss and large pathological changes become apparent. Memory loss and impairment of cognitive ability are one of the earliest clinical manifestations. In terms of improving the quality of life of the subjects, the success of new therapeutic treatments for this illness will be judged upon its ability to prevent memory loss or restore memory ability. However, the main targets responsible for alterations of memory function in the subjects are not known. On the other hand, mounting evidence suggests that IP3-mediated calcium levels (LaFerla, Nat Rev Neurosci 2002,3: 862-72), synaptic failure, and glutamate deregulation may be among the earliest alterations in AD (Selkoe, Science 2002,298: 789-91).

RGS4. Regulator ofG-protein signalling 4. RGS4 (accession number NM_005613) is a member of a family of GTPase activating proteins that is only expressed in brain (Hepler, Trends Pharmacol Sci 1999,20: 376-82). RGS4 regulates Gq and Gi protein signalling, by accelerating the rate of GTPase reactions, and thereby driving G proteins into their inactive GDP-bound forms. RGS proteins are involved in the signal cascade that, via glycogen synthase kinase 3 (GSK3), can regulate the formation of both amyloid plaques and neurofibrillary tangles, two pathological hallmarks of Alzheimer's disease (Fhiel, Wilson et al., Nature 2003,423: 435-9). In the inositol triphospate (IP₃) -mediated calcium signalling cascade, RGS4 acts by regulating Gq and Gi protein signaling by interacting with PLCB1, Ca²⁺/calmodulin (CaM) and dipalmitoylphosphatidylinositol-3,4,5- trisphosphate (PIP3), driving G-proteins into their inactive GDP-bound forms. Therefore, RGS4 regulates indirectly intracellular calcium release. The family of RGS proteins have shown good potential as targets for drug development (Zhong and Neubig, J Pharmacol Exp Ther 2001,297: 837-45). The possibility that RGS4 levels are also modified at the protein level has recently been suggested (Muma, Mariyappa et al., Synapse 2003,47: 58-65).

ITPKB. Inositol- 1 ,4, 5-trisphospkate (IP3) 3-kinase B ITPKB (accession number NM_002221) is one out of three isoforms (A, B and C) that catalyse the phosphorylation of IP3 to inositol 1,3,4,5- tetrakisphosphate (IP4). IP3 is involved in the biological response of a large number of hormones and neurotransmitters, by regulating calcium release, with functional implications for synaptic plasticity. By phosphorylating IP3 to IP4, ITPKB protects IP3 against hydrolysis, enhancing IP3's ability to activate store-operated Ca²⁺ entry. The protective effect of IP3 is achieved by the fact that IP4 has a 10-fold higher affinity for IP3 than inositol phosphatase 5- phosphatase. Furthermore, IP₄ can act as an antagonist at the IP3 receptor, helping Ca²⁺ pools to re-load, thus sharpening up Ca²⁺ oscillations (Irvine, Curr Biol 2001, 11: R172-4). IP3K1, a Drosophila gene homologous to mammalian IP3Ks is involved in oxidative damage in the flies (Monnier, Girardot et al., Free Radic Biol Med 2002,33: 1250-9).

Currently, there is no cure for AD, nor is there an effective treatment to halt the progression of AD or even an effective method to prognose and/ or diagnose AD prior to death with high probability. The late onset and complex pathology of neurodegenerative diseases pose a very large challenge to the development of therapeutic and diagnostic agents. It is crucial to expand the pool of potential drug targets and diagnostic markers.

Summary of the invention

This invention is based on the discovery of the down-regulation of the gene coding for RGS4 and the up-regulation of the ITPKB gene in the brain samples of AD subjects. No such down-regulation of RGS4 and up-regulation of ITPKB is observed in brain samples from age-matched healthy controls. These discoveries offer new ways for the diagnosis, prognosis and treatment of AD as described below.

In a first aspect this invention concerns a method for diagnosing a neurodegenerative disease, preferably Alzheimer's disease, comprising analysing the expression of the genes RGS4 and/or ITPKB, or measuring the level of the products of those genes in a sample from a subject suspected to suffer from AD. The levels and activities of fragments or derivatives of the gene products may also be measured. These levels may be compared to the corresponding levels in healthy individuals or known AD-patients. This aspect also includes a method for prognosticating AD in a subject diagnosed with AD, and assessing the risk of developing AD for a person not suffering from AD.

In a second aspect, the invention relates to a method for evaluating a treatment of AD. In this aspect of the invention, several samples are taken from the subject and analysed for the expression of RGS4 and or ITPKB. Preferably, the samples are taken both before treatment and during treatment. Additional samples may be taken after the treatment.

In a third aspect, the invention relates to a kit for performing the method according the first two aspects.

In a fourth aspect, the invention relates to a method of treating neurodegenerative diseases, preferably Alzheimer's disease, by restoring either the expression levels of RGS4 and/ or ITPKB or the activity or level of their gene products to levels found in subjects not suffering from said neurodegenerative disease. This aspect also includes pharmaceutical compositions comprising compounds capable of restoring said levels and administration of the compositions to subjects I need thereof. These compounds could be, for example, modulators of RGS4- or ITPKB-expression, agonists and antagonists of the gene products of RGS4 and ITPKB, siRNA, etc.

In a fifth aspect, the invention relates to a method for screening for compounds useful in the fourth aspect. Definitions

The term "level" is meant to comprise a measure of the amount of a transcription product, for instance an mRNA, or a translation product, for instance a protein or polypeptide. The term "activity" as used herein shall be understood as a measure for the ability of a transcription product or a translation product to produce a biological effect or a measure of a level of biologically active molecules. The term "activity" also refers to enzymatic activity. The terms "level" and/ or "activity" as used herein further refer to gene expression levels or gene activity.

The term "gene expression" can be defined as the utilization of the information contained in a gene by transcription and translation leading to the production of a gene product.

A "gene product" is a molecule which is the result of the expression of a gene. The level of a gene product can be used to measure how active a gene is. The term gene product should be construed as comprising fragments and derivatives of the original gene product. For example, mRNA transcribed from a gene, a protein translated from that mRNA and the post-translationally modified protein and are all included in the term "gene product".

The term "gene" as used in the present specification and in the claims comprises both coding regions (exons) as well as non-coding regions (e.g. non- coding regulatory elements such as promoters or enhancers, introns, leader and trailer sequences). The term "fragment" as used herein is meant to comprise e.g. an alternatively spliced, or truncated, or otherwise cleaved transcription product or translation product.

The term "derivative" as used herein refers to a mutant, or an RNA-edited, or a chemically modified, or otherwise altered transcription product, or to a mutant, or chemically modified, or otherwise altered translation product. For instance, a "derivative" may be generated by processes such as altered phosphorylation, or glycosylation, or lipidation, or by altered signal peptide cleavage or other types of maturation cleavage. These processes may occur post-translationally.

The term "modulator" as used in the present invention and in the claims refers to a molecule capable of changing or altering the level and/ or the activity of a gene, or a transcription product of a gene, or a translation product of a gene. Preferably, a "modulator" is capable of changing or altering the biological activity of a transcription product or a translation product of a gene. Said modulation may, for instance, be an increase or a decrease in enzyme activity, a change in binding characteristics, or any other change or alteration in the biological, functional, or immunological properties of said translation product of a gene.

The term "AD" shall mean Alzheimer's disease. The term "Neurodegenerative disease" comprise Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Pick's disease, fronto-temporal dementia, progressive nuclear palsy, corticobasal degeneration, cerebrovascular dementia, multiple system atrophy, and mild-cognitive impairment. Further conditions involving neurodegenerative processes are, for instance, ischemic stroke, age- related macular degeneration and narcolepsy.

Brief description of the figures

Figure 1. Gene Ontology classification of the top ranked results from the microarray experiments. From each set of microarray experiments the 30 genes top ranked after the statistical analysis of the results were selected. These genes were classified into categories according to gene ontology. Several genes with unknown functions could not be classified as described in the results. The figure shows the number of genes that belong to the categories containing at least 3 members. Figure 2. Real-Time PCR experiments for the reference genes GAPD and actin, and for the candidate genes ITPKB and RGS4. The figure shows the normalized and covariate adjusted mRNA expression patterns in AD and control individuals. Each part of the figure represents the correlation between the levels of expression of the particular gene (Y axys) and the levels of expression of a reference gene (ACTB). All expression values are presented in a logarithmic scale. The solid line represents the regression line for all the control samples while the broken line represents the regression line for the subjects. The difference in intercept- values for the two lines represent the approximate difference in expression values for subjects and controls. For example, a 0,4 difference in the intercept value represents a 2, 5-fold difference between subjects and controls. Fig.2A GAPD vs. ACTB, Fig.2B = APP vs ACTB, Fig.2C = ITPKB vs ACTB, Fig.2D = RGS4 vs ACTB. Figure 2B shows a small and non- significant difference in intercepts of the regression lines for AD and control samples indicating that APP mRNA expression is not modified in the AD subjects. On the other hand, Figure 2C and 2D illustrate an enriched (Fig.2C) or a restricted (Fig.2D) normalized mRNA expression in AD samples visualized by the larger or smaller (respectively) value for the intercept of the regression lines.

Detailed description of the invention

It is an object of the present invention to provide methods that are suited for the diagnosis and development of a treatment of AD and/ or related neurodegenerative disorders. The features of the independent claims solve this objective. The first step towards the invention is based on our discoveries described below.

The present invention is based on the finding of the down-regulation of the mRNA for the gene RGS4 and the up-regulation of the gene ITPKB in the brain samples from Alzheimer's disease subjects compared to controls. These genes may be involved in a deterioration of cognitive capacity and at the same time may contribute to the formation of amyloid plaques and/ or neurofibrillary tangles in the AD subjects.

Recent advances in cDNA microarray technology make it possible to investigate thousands of genes simultaneously and allowed screening for altered molecular pathways in different diseases. We used two different sets of cDNA microarrays and we identified several genes differentially expressed in AD subjects after the analysis of a large sample collection, including 114 human brain autopsies. The success of the strategy presented to identify, with high resolution and precision, genes that are differentially expressed in AD subjects, is based in several factors: 1. We have one of the largest collections of autopsy samples from subjects and controls ever investigated by microarray analysis. A large collection of individuals reduces the "noise" caused by large levels of inter- individual variation in expression levels and allows the identification of targets with altered expression levels in multiple subjects. 2. We have used two sets of microarrays . This allowed independent replication of the results. 3. All candidate genes identified in steps 1 and 2 are re-analyzed using real-time RT PCR: This step is crucial to confirm the results of the arrays by an independent method. Also, it is important to unequivocally identify the gene target among multiple members of gene families that are not possible to distinguish by microarray analysis. We used a high resolution strategy well established by our group previously (Castensson, Emilsson et al., Genome Res 2000, 10: 1219-29; Emilsson, Saetre et al., Neurosci Lett 2002,326: 56-60; Castensson, Emilsson et al., Biol Psychiatry 2003,54: 1212-21). 4. We have developed a statistical model to distinguish the effect of the disease on expression levels from many other factors that could modify expression levels including age, time post-mortem, sex, pH of the brain and mRNA quality and quantity of the samples investigated.

All the genes confirmed as differentially expressed are involved in IP3-mediated calcium signaling, vesicle regulation and/ or glutamate signaling In particular, the most significant results were obtained for Regulator of G-protein signalling 4 (RGS4) and Type B inositol 1,4,5-triphosphate 3 kinase (ITPKB).

The present invention discloses the differential expression of the genes coding for RGS4 and ITPKB in the brain of AD subjects. The decrease in mRNA for RGS4 that we observed in the subjects with the disease can have two effects: (i). Disturb signal transduction that leads to memory formation (ii). Affect the processing of APP leading to an increased deposition of amyloid plaques. Drug targeting directed to an increase in RGS4 in the Alzheimer's subjects could be used to modify memory impairment, amyloid plaque formation and /or tangle formation.

An up-regulation of ITPKB, as we observed in the AD subjects, could lead to increased hydrolysis of IP3 to IP4 affecting the cellular balance of Ca²⁺-release, disturbing synaptic plasticity and thereby affecting the cognitive abilities of AD suffering individuals. In particular, the B isoform is the only one that can be localized in the plasma membrane. This suggest the possibility that the up- regulation of this gene in the AD subjects may specifically modify Ca ²⁺ regulation of vesicle formation and release at synaptic terminals.

Consequently, the RGS4 and/ or ITPKB genes and their corresponding translation products may have a causative role in the symptoms, progression and/or pathology of AD. Based on these disclosures, the present invention has utility for the diagnostic evaluation, prognosis, identification of a predisposition to AD, diagnostic monitoring of subjects undergoing treatment and /or for the development of treatment methods for AD.

According to the first aspect, the invention features a method of diagnosing or prognosticating AD, determining whether a subject is at increased risk of developing the disease, and or monitoring the progression of AD in subjects. The method comprises: determining a level, or an activity, or both said levels and said activity of (i) a transcription product of a gene coding for

RGS4 and/or ITPKB, and/or of (ii) a translation product of any of those two genes, and/ or of (iii) a fragment or derivative of the transcription or translation product of the mentioned two genes. The samples to be analyzed could be brain tissue or other tissue, organs, or body cells. The sample can preferably consist of cerebrospinal fluid or other body fluids such as saliva, urine, blood, serum plasma, or nasal mucosa. An increase of transcription /translation product for ITPKB and/or a decrease of RGS4 in a sample from an AD subject relative to a reference value obtained from healthy individuals indicates a diagnosis, or prognosis, or increased risk for AD. The measurement of levels of transcription products of a gene coding for RGS4 and/ or ITPKB is performed in a sample from a subject using a quantitative PCR-analysis and/ or hybridization methods such as microarray technologies. Levels of translation can be detected using an immunoassay, an activity assay, and/ or binding assay. These assays can measure the amount of binding between said protein molecule and an anti-protein antibody by the use of enzymatic, chromodynamic, radioactive, magnetic, or luminescent labels which are attached to either the anti-protein antibody or a secondary antibody.

According to the second aspect, the method above could be used to monitor subjects undergoing treatment for AD. A series of samples are then taken over a period of time before, during and after treatment and the results of the samples are compared to each other to evaluate the treatment.

According to a third aspect, a diagnostic kit using any of the mentioned strategies may serve as a means for targeting identified individuals for early preventive measures, therapeutic intervention and/ or monitoring success or failure of therapeutic treatment for AD. Preferably the kit comprises: (a) at least one reagent that selectively detects the level and /or activity a gene product of the RGS 4 and/ or ITPKB genes, and (b) means for correlating the level and/or activity of said gene product to a reference value indicating a known health status or disease state, and (c) optionally additional instructions and means for carrying out the method.

In a fourth aspect, the invention features a method of treating or preventing AD, comprising the administration of a therapeutically or prophylactically effective amount of an agent or agents which directly or indirectly affect a level, or an activity, or both said level and said activity, of (i) a transcription product of a gene coding for RGS4 and/or ITPKB, and/ or of (ii) a translation product of any of those two genes, and/or of (iii) a fragment or derivative of the transcription or translation product of the mentioned two genes. Said agent may be a small molecule, a peptide, and/or an oligonucleotide. Alternatives also include the application of gene therapy with nucleic acids encoding all or part(s) of RGS4 and/ or ITPKB with suitable control elements known in the art, grafting of donor cells into the nervous system, therapeutic cloning, transplantation, stem cell therapy and/or antisense nucleic acid technology to administer said agent.

In a fifth aspect, the invention features a method for screening for a modulator for AD, using as an assay the measurement of (i) a transcription product of a gene coding for RGS4 and/ or ITPKB, and/ or of (ii) a translation product of any of those two genes, and /or of (iii) a fragment or derivative of the transcription or translation product of the mentioned two genes. This screening method comprises (a) contacting a cell or organism with a test compound, and (b) measuring the activity, or the level, or both the activity and the level of one or more substances indicated in (i) to (iii), and (c) measuring the activity, or the level, or both the activity and the level of said substances in a control cell or organism not contacted with said test compound, and (d) comparing the levels of the substance in the cells /organisms of step (b) and (c), wherein an alteration in the activity and/or level of said substances in the contacted cells/organisms indicates that the test compound is a modulator of said diseases and disorders.

In a sixth aspect, the present invention provides a method for producing a medicament comprising the separate steps of (i) identifying a modulator of AD and (ii) admixing the thus identified modulator with a pharmaceutical carrier. This aspects also includes pharmaceutical compositions comprising a modulator according to the fifth aspect, optionally mixed with pharmaceutically acceptable carriers and excipients.

Other features and advantages of the invention will be apparent from the following description of the experimental part that illustrates but does not limit the invention.

Experimental part

Methods

Methods Tissue samples Post-mortem human brains samples were obtained from 114 individuals. They were provided by the Maudsley Brain Bank (Department of Neuropathology Institute of Psychiatry, King's College London, SES 8AF, United Kingdom and corresponded to the frontal cortex, Brodmann areas 8 and 9. Sixty-one samples were obtained from patients diagnosed with AD (31 females and 30 males) and 53 from individuals without any psychiatric disorder (27 females and 26 males). The mean age of the AD patients was 78 years at death (spanning from 49 to 97 years), and for the controls was 73 (range 41 to 72 years). Mean brain pH at death was for AD patients 6.4 (spanning from 5.8 to 7.2), and for the controls 6.6 (spanning from 6.1 to 7.0). Mean postmortem time was 33 h for AD (range from 4 to 103 h) and the control mean was 45 (spanning from 8 to 96 hours). All brains were examined by a senior neuropathologist, and the neuropathological diagnosis of was made according to the CERAD criteria. All patients were CERAD-positive (Mirra, Heyman et al., Neurology 1991,41 : 479-86.).

RNA and mRNA purification Each autopsy sample was divided into pieces of 50-100 mg of tissue, and homogenized in 2 ml of Trizol reagent (Life Technologies, Sweden) using an Ultra-Turrax T25 basic homogenizer (Ika Labortecnhik, Germany) and stored at -70C prior to use. Total RNA and mRNA purification from tissue homogenates were performed as described previously (Castensson, Emilsson et al., Genome Res 2000, 10: 1219-29). Total RNA and mRNA quantity was measured with RiboGreen, RNA Quantification Reagent kit (Molecular Probe, Sweden) following the manufacturer's protocol. The samples were concentrated using Microcon, Centrifugal Filter YM 30 (Millipore Corporation, USA. cDNA microarrays. Two different manufactured cDNA microarrays were used. One set of cDNA Microarrays was produced by the WCN Expression Platform at Rudbeck Laboratory, Uppsala University and each slide contains 7762 human cDNA clones in duplicates ( http: / / www.genpat.uu.se/wcn/uppsala.html, 2004-06-30 ). This slides are called "Uppsala array". The other set of arrays were at the KTH microarray resource centre in Stockholm, and contains 20000 human cDNA clones ( http:/ /www.biotech.kth.se/molbio/cores/arrav core.html , 2004-06-30), and are called "KTH arrays". Messenger RNA from all individuals was combined into two sample pools, one AD pool (61 individuals) and one Control pool (53 individuals, each individual contributing with the same amount of mRNA. 1 OOng of each mRNA pool was added to each cDNA microarray. Two different experimental designs were used. For the Uppsala arrays, the mRNA pools of control and AD samples were hybridized independently with a new pool that included 110 reference mRNA samples (common reference design). Four Uppsala arrays were hybridized, with dye swap, resulting in signals obtained eight times per cDNA clone. For the KTH arrays, the AD pool was hybridized five times with the control pool, including dye swap, and resulting in five replications per cDNA clone. cDNA synthesis, labelling and hybridisation The MICROMAX TSA ™

Labelling and Detection kit (NEN ® Life Science Products, Inc.) was used to label the sample pools with Cyanine-3 (Cy3) and Cyanine_5 (Cy5) respectively. The TSA ™ procedure was performed according to manufacturer's protocols, with an extended RT incubation time of three hours. The cDNA microarray were scanned at lOμm resolution using a GenePix 4000B scanner(Axon Instruments, Inc.).

Image processing and Statistical Analysis of microarray data. GenePix Pro 3.0 Microarray Analysis Software (Axon Instruments, Inc.) was used to process the tiff-image files developed from the Uppsala arrays while the tiff- image files from the KTH were processed using Spot (Jung and Cho, Bioinformatics 2002,18 Suppl 2: S 141-51). The mean intensity of the Cy5- labelled sample (R) and the Cy3 -labelled reference (G) were used to calculate the log transformed ratio between the sample and the reference for each spot: M = log2 (R/G). The robust scatter plot smoother owess' (Proc loess, SAS v.8.2) was used to perform a sub-array intensity normalization of M with the smoothing parameter set to 40% (Yang, Dudoit et al., Nucleic Acids Res 2002,30: el 5). All spots with a mean spot intensity below the local median background were excluded from the analysis. To identify genes that were differentially expressed with respect to diagnosis we ranked the genes with respect to their penalized t-statistic (Smyth, Methods in Molecular Biology series, Humana PRess, Totowa, NJ 2002). We choose ao to be the 90^th percentile of s² values (Efron, Journal of the American Statistical Association 2001,96: 1151-1160) identify genes with large average differences between tAD and Controls, with no respect to the variation between individuals within the two groups. Reverse transcription and replica plate design for Real-time RT PCR.

Two replicate mRNA samples from each of the 114 individuals were reverse transcribed with reagents for reverse transcription (Life Technologies, Sweden), the protocol used is earlier described by Castensson et al (Castensson, Emilsson et al., Genome Res 2000, 10: 1219-29). After the RT reaction, the cDNA in each well was diluted 1:20 andlOO replica plates were produced, dried and stored at 4 °C for future use. To analyze each gene, a set of three 96 well plates was used, covering the 61 patients and 53 controls in duplicate.

Real-time RT PCR assay. To each cDNA well in the 96 well optical plates 16 ul of TaqMan Universal master mix (APPlied Biosystems) was added, and the reaction was carried out as described earlier (Castensson, Emilsson et al., Genome Res 2000,10: 1219-29). The fluorescence data produced was analyzed and converted into threshold cycle values (Ct-values) by the Sequence detector 1.6.3 software system. The Ct-value for each sample was then translated into number of copies using a standard curve prepared from a reference sample pool of cDNAs. Primers and probe were ordered from BAPPlied Biosystems "assay on demand" facility, with the exception of ACTB, and RGS4 . In these cases the sequences of primers and probes were designed in-house: ACTB primers: 5 - GAGCTACGAGCTGCCTGACG-3' (SEQ ID NO: 1) and 5'- GTAGTTTCGTGGATGCCACAGGACT-3' (SEQ ID NO: 2); ACTB probe: 5'- ATCACCATTGGCAATGAGCGGTTCC-3' (SEQ ID NO: 3); RGS4 primers: 5'- CGCTTCCTCAAGTCTCGATTCTAT-3' (SEQ ID NO: 4) and 5'- CTCCTTTCTGCTTTTCTGCCC-3' (SEQ ID NO: 5); RGS4 probe: 5'- TTGATTTGGTCAACCCGTCCAGCTG-3' (SEQ ID NO: 6).

Statistical analysis of real-time RT PCR.

To test whether the mRNA expression levels of a target gene differed between Alzheimer's subjects and control individuals, we used an Ancova type of model, with disease and plate as main factors and the amount of reference gene mRNA as a covariate. The mRNA levels of the reference genes (ACTB and GAPD) were highly correlated (r=0.95). To improve normalization accuracy, the geometric mean of the two genes was used as reference gene expression. To account for variation in mRNA that was due to pH, age and time post-mortem, we also included these factors as covariates in the analysis. We used a logarithmic transformed expression data and we averaged the results obtained from the duplicate samples from each individual before the statistical analysis. To control for multiple testing, p-values were adjusted so that the rate of false discovery was controlled to be less than one in twenty for differentially expressed genes (adjusted p-values < 0.05). The GLM procedure was used for statistical modelling, and the correction for multiple testing was done with the MULTTEST procedure in SAS © (Statistical Analysis System: SAS Institute Inc). RESULTS High throughput microarray screening of pooled AD and control samples- Two sets of microarray experiments were performed as described in the Methods. In the first set of microarrays, mRNA from each pool (AD or controls) was hybridized together with a common reference sample to four separate arrays with duplicate spots from 7762 human cDNA clones (Uppsala arrays). In the second set of experiments, mRNA from both pools was hybridized with each other to five separate arrays with single spots from 20,000 human cDNA clones (KTH arrays). Genes were ranked after their likelihood of having an average expression difference between the AD and the control group (penalized t- statistic), and the top thirty genes from each set of microarray experiments were selected. This resulted in a list of 57 genes since 3 genes, including RGS4, ITPKB and RAB3A, overlapped in the top ranks of each array set. These 57 genes were classified into functional categories provided by the Gene Ontology Consortium. Thirty-three of them corresponded to one or more of the G04 category classes. Twenty-four genes were not included in any GO class and were excluded of this analysis. The proportion of the remaining genes corresponding to each functional group is shown in Figure 1. We found that the categories best represented were intracellular signalling cascade/ signal transduction, and transport (in particular vesicle-mediated transport). Each of these groups contained eight members. In general, the results of the two sets of array experiments correlated well, but the magnitude of the differential expression was different, probably due to the different clones on each set of arrays and the different hybridization conditions for different segments of each gene (Table 1).

Table 1

Table 1. Differentially expressed genes in AD found by cDNA microarray experiments The table shows the relative expression levels of selected genes in the two sets of microarray experiments performed. The first set, called "Uppsala Array" in table 1, consisted on four microarrays, each of them including 7762 clones spotted on duplicates on the slides. The second set, named "KTH array", consisted on five arrays including 20,000 human clones each. A pool of 61 AD frontal cortex cDNA samples were hybridysed to a cDNA pool prepared from 53 frontal cortex samples from controls. The two pools of samples were balanced regarding sex, post-mortem time and different ages at death. Four different hybridisations were performed on the Uppsala array and a summary of the results is presented on the left part of the table. The right segment of the table shows the summary of the analysis of five hybridisations for the KTH arrays. Both sets of experiments gave in total 130800 signals. The results were analysed as described in the methods and results section and the top ranked 30 genes were selected from each set of arrays. These genes were classified using Gene Ontology and the genes with known classification belonged to the categories shown in Figure 1. Among them, the two categories with the largest number of members were intracellular signalling cascade/ signal transduction, and vesicle-mediated transport.. Some genes were down-regulated (D) while others were up-regulated (U) in the AD subjects. The fold-change in expression as well as the ranking of the genes are indicated. Other symbols (1) =classification outside Gene Ontology, obtained from other publications.

Real-time RT-PCR (Taqman) analysis of selected genes

We used a high resolution real time RT-PCR strategy (Castensson, Emilsson et al., Genome Res 2000, 10: 1219-29; Emilsson, Saetre et al., Neurosci Lett 2002,326: 56-60; Castensson, Emilsson et al., Biol Psychiatry 2003,54: 1212- 21) to examine whether the mRNA expression levels of selected target genes differed between Alzheimer's subjects and control individuals. In total 25 target genes were selected for the analysis. We used an Ancova based model to test if observed expression differences were significant, and adjusted the p-values so that the rate of false discovery was controlled to be less than one in twenty for differentially expressed genes (adjusted p-values ≤ 0.05). The statistical model is discussed in more detail in the Methods.

RGS4 and ITPKB correspond to the category intracellular signalling, and showed clearly altered normalized expression levels in AD. That is, the expression levels of these genes had a disease effect added on top of the mRNA concentration differences between AD and controls. ITPKB showed 2.2 fold higher normalized mRNA levels in AD than in controls, whereas the mRNA levels of RGS4 were 2.3 fold lower in AD than in controls. In Figure 2 C-D the expression differences of ITPKB and RGS4, as well as the lack of differences for APP are shown in detail for each of the individual subjects. The expression levels of ACTB and GAPD were highly correlated (r=0.95, Fig la) indicating that the genes were proper control genes. However, mean reference gene expression was lower in AD thanin control (p=0.004, Figla-d) indicating lower mRNA concentrations in AD. This means that when we use the expression of the reference genes to remove differences in mRNA concentrations between AD and control samples, the remaining mRNA difference in a target gene can be interpreted as the disease effect on top of the mRNA concentration differences. Figure 1B-D illustrates how we used reference gene expression to remove the effect of a lower average mRNA concentration in the AD samples. The absolute mRNA level of APP is clearly lower in AD than in control samples. However, the difference in mRNA levels of APP is similar to that of the reference genes. Thus when APP mRNA levels are normalized with reference gene expression there is no significant disease effect on top of the mRNA concentration difference. On the other hand, RGS4 is clearly down-regulated while ITPKB is significantly up- regulated. The difference can be best visualized by the difference in the intercept of the regression lines for AD and control samples.

Three genes (RAB3A, STX1A, and SYNGR3) involved in transport showed lower normalized expression levels in AD than in controls (Table 2). For RAB3A and STX1A mRNA levels were 2J and 1.9 fold lower in AD than in controls, and the difference was highly significant. For SYNGR3 the decreases in mRNA levels were less pronounced (1.6 fold) and the p-value was one order of magnitude lower.

In the signal transduction group, GRN1, a gene involved in glutamate signaling showed lower normalized expression levels in AD than in controls (Table 2). The expression of GRIN1 was clearly affected in AD and the mRNA levels were approximately 2 fold lower in AD than in the controls. Of all the investigated genes that are involved in APP metabolism or the formation of NFT only the expression of PSENl was significantly higher in AD samples.

Table 2

Table 2. Real-time RT-PCR analysis of selected candidate genes. The genes described in table 1, together with additional members of the same gene families and additional candidates selected for their potential involvement in the same metabolic pathways constituted the list genes tested by real-time RT-PCR and some of them are presented in the table. In this case, the expression levels of each individual were tested separately, in duplicated experiments. In total, 246 autopsy samples were analysed.. Two of these genes were up regulated (ITPKB, and PSENl) while the others had a lower expression in the subjects. The Twelve candidates can be classified in three groups, according the level of significance of the results. RGS4, RAB3A, and ITPKB are strongly significant results, with p<0.0005. The second group consisted of STXIA and GRINl, with p<0.005 and the third group, with p<0.05 consisted of SYNGR3, and PSENl. Five genes did not show significant differential expression between subjects and controls.

Functional Studies of RGS4 and ITPKB Mammalian cell line studies

Functional studies of RGS4 and ITPKB include down-regulation, using RNA silencing technology, and up-regulation of gene expression in cell lines (SH- SY5Y and HEK 293). In a first stage the most effective small interfering RNA's (siRNA) are identified by transient transfections. RNAi experiment are verified with real-time RT PCR (RNA level) and Western blot (protein level). Stable cell lines with silenced RGS4 expression are then to be produced, by RGS4 adenovirus constructs and/ or plasmid constructs that we have obtained, which can be used in both cell lines and in animal models. Modified cell lines are used to test relevant phenotypes that may be associated with the diseases, such as intracellular calcium levels and APP cleavage. These cell lines can provide new assays to screen for compounds that modulate the expression of the selected genes. In-vivo functional studies in Drosophila melanogaster

RNA interference is also used to perform functional studies in APP mutated Drosophila. This allows the tissue specific silencing of RGS4 and ITPKB, and also the evaluation of RGS4 effect during different developmental stages in AD. By performing different time scale measurements of silenced RGS4 and/or ITPKB in Drosophila early changes in AD can be identified, as well as possible connections between RGS4, neuron degeneration, senile plaque and tangles. An in-vivo reporter system has been developed in Drosophila melanogaster to analyse the processing of human amyloid precursor protein (APP) in the living organism (Loewer, Soba et al., EMBO Rep 2004,5: 405-11). A similar system can be used to study in-vivo the effect of changes in the expression of RGS4 and ITPKB on APP processing and other phenotypes.

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Claims

1. A method of diagnosing or prognosticating a neurodegenerative disease in a subject, or determining an elevated risk for a subject to develop a neurodegenerative disease, comprising determining an expression level of RGS4 and/or ITPKB, or a level and/or an activity of a gene product of RGS4 and/or ITPKB, or a fragment or derivative of said gene product, in a sample from said subject.

2. The method according to claim 1 wherein said neurodegenerative disease is Alzheimer's disease.

3. A method according to any of claims 1 or 2, wherein, compared to a reference value indicating a known health or disease status, a decreased activity and/ or level of a RGS4 gene product indicates that the subject suffers from said neurodegenerative disease.

4. A method according to any of claims 1 to 3, wherein, compared to a reference value indicating a known health or disease status, an increased activity and/ or level of an ITPKB gene product indicates that the subject suffers from said neurodegenerative disease.

5. The method according to any of claims 1 to 4 wherein said sample comprises a cell, a tissue, an organ or a body fluid, preferably cerebrospinal fluid or blood.

6. A method for evaluating the treatment of a neurodegenerative disease, comprising performing the method according to any of claims 1-5 and comparing said level and/or activity in a series of samples taken from said subject over a period of time.

7. A kit for performing the method according to any of claims 1-6 said kit comprising: (a) at least one reagent that selectively detects the level and/ or activity a gene product of the RGS4 and/or ITPKB genes, and (b) means for correlating the level and/or activity of said gene product to a reference value indicating a known health status or disease state, and (c) optionally additional instructions and means for carrying out the method.

8. A method of treating or preventing a neurodegenerative disease, in particular Alzheimer's disease, in a subject comprising administering to said subject in a therapeutically or prophylactically effective amount an agent or agents which directly or indirectly affect an activity and/ or a level of (i) a gene coding for RGS4 and/ or ITPKB, and/ or (ii) a gene product of the RGS4 and/or ITPKB genes, and/or (iii) a fragment or derivative of (i) or (ii) .

9. Use of a modulator of the expression of the genes RGS4 and/or ITPKB for the production of a pharmaceutical composition for treatment of Alzheimer's disease.

10. A method for screening for a modulator of neurodegenerative diseases, in particular Alzheimer's disease, or related diseases or disorders, said method comprising the steps: (i) contacting a cell with a test compound, and (ii) measuring the activity and/ or level of a gene product of RGS4 and/or ITPKB in the cell, and

(iii) measuring said activity /level in a control cell not contacted with said test compound; and

(iv) comparing the levels and/ or activities of the gene product in the cells of steps (ii) and (iii) wherein a difference in the activity and/ or level of the substances in the contacted cells indicates that the test compound is a modulator of said diseases or disorders.