CA2203084A1 - The inositol phosphate pathway and its implication in alzheimer's disease - Google Patents

Abstract

The present invention discloses nucleic acid molecules encoding genes involved in CNS disorders such as Alzheimer's disease and epilepsy. Also disclosed are gene products, vectors and host cells suitable for expression of such gene products. Methods are providing for detecting the presence of a genes involved in Alzheimer's disease in human subjects and for treating humans suffering from Alzheimer's disease. Furthermore, markers which are associated with Alzheimer's disease are disclosed. Methods in kits for the detection of Alzheimer disease in a subject are additionally provided.

Description

TITLE OF THE INVENTION
THE INOSITOL PHOSPHATE PATHWAY AND ITS
IMPLICATION IN ALZHEIMER'S DISEASE

This invention relates generally to central nervous system (CNS) disorders. More particularly, this invention relates to Alzheimer's disease. In addition the invention relates to the diagnosis and treatment of Alzheimer's disease.
BACKGROUND OF THE INVENTION
Alzheimer's disease (AD) is the most common cause of progressive cognitive decline in the aged population. It causes 100 000 deaths each year in the United States where it is the fourth leading cause 15 of death. Alzheimer described amyloid plaques, neurofibrillary tangles and dementia that characterize AD in 1907. The usual presenting symptoms are deficits of recent memory often in association with with language and visuosp~ti~l and attention problems.
To date, three genes have been identified that, when 20 mutated, can lead to early onset forms of AD and variation in a fourth one has been implicated as a risk or susceptibility factor for AD.
~-amyloid precursorprotein The major protein of the senile plaques is ~amyloid (A~), a 39 to 43 amino acid peptide (Glenner and Wong, 1984; Masters 25 et al., 1985; ) derived from the ~-amyloid precursor protein (APP).
Plaques are found mainly in the hippocampus and in the temporal lobe cortex. APP was the first gene in which mutations were found to cause familial Alzheimer's disease (FAD). The APP gene, located on chromosome 21, has 19 exons and A~ is encoded by parts of exons 16 and 17 (Lemaire et al., 1989). Four mutations in the APP gene have been described (Chartier-Harlin etal., 1991; Fidani etal., 1992; Goate et al., 1991; Karlinsky etal., 1992; Mullan etal., 1992; Murrell etal., 1991;
Naruse et al., 1991; but they account for only 5% of published early-onset FAD .
Presenilins In 1992, Schellenberg etal (Schellenberg etal., 1992) reported a second locus causing early-onset AD on chromosome 14q24.3. A positional cloning strategy permitted the identification of a candidate gene, the S182 gene (Sherrington et al., 1995) later renamed presenilin-1 or PS1, that carried coding region mutations in families multiply affected by early-onset AD. The PS1 gene, composed of 10 exons, encodes a 467 amino acids protein with 7 to 10 transmembrane domains. More than 35 different mutations have been found in the PS1 gene in over 50 families of different ethnic origins (see van Broeckhoven, 1995 for review). The proportion of early-onset familial AD cases due to mutations in the PS1 gene is around 50%.
A genome -wide search conducted on another polulation with familial early-onset AD indicated another locus on chromosome 1 (Levy-Lahad et al,1995a). The chromosome 1 FAD gene was cloned by virtue of its homology to PS1. The PS2 gene is composed of 12 exons and encodes a 448 amino acids protein (Levy-Lahad et al., 1995b). It shows 67% identity with the PS1 protein. Only two mutations have been identified in the PS2 gene suggesting that mutations in this gene are a rare cause of FAD protein (Levy-Lahad et a/., 1995b; Rogaev et al., 1 995).
APO e4 The apolipoprotein E (APOE) gene, located on 5 chromosome 1 9q13.2 has been identified as a susceptibilty factor for AD
by genetic analysis of late-onset FAD pedigrees (Pericak-Vance et al, 1991). APO E is a major serum lipoprotein involved in cholesterol metabolism. Three common isoforms of APOE are encoded by alleles e2, e3, and e4 as a result of amino acids changes at codons 112 and 10 158. The APO e4 allele shows a dose dependent increase in risk for AD, apparently mediated through a decrease in the age of onset of disease (Corderetal., 1993).
Not everyone having the susceptibility e4 allele will develop illness and many who lackcthe allele will also develop AD. APOE
15 testing is therefore not useful for predicting whether someone will develop AD.
Research on the molecular ethiology of the a complex disease such as Alzheimer disease has been confounded by the large number of hereditary and environmental factors involved and by the 20 paucity of neuropathological and neurochemical studies on brains for affected individual. The finding of a linked marker involved in one hereditary form of Alzheimer disease will help to resolve the number of different genes underlying this complex disease. This markers can be used eventually to provide genetic counselling in some affected families.
25 Most importantly, the delineation of the genomic region containing Alzheimer disease gene will provide a mean to eventually discover and characterize this gene(s) in its encoded protein(s). The finding of link-markers will also make it possible to evaluate the role of gene(s) in this chromosomal region in the different levels of severity and onset of Alzheimer's disease.

The invention seeks to provide diagnosis and therapeutic tools for CNS disorders. Particularly the inventio seeks to provide diagnosis and therapeutic tools for Alzheimer's disease (AD). Herein, the term AD-related nucleic acid is not meant to be restrictiv eto AD only, 10 since other CNS disorders are herein shown to share common genes and products thereof.
The present invention seeks to provide a nucleic acid segment isolated from human comprising at least a portion of a gene responsible for CNS disorders and particularly to AD. The AD-related 15 nucleic acid segment can be isolated using conventional methods which include for example YAC and BAC cloning, exon trapping and the like.
Such nucleic acids could also be synthesized chemically. Having the AD-related nucleic acid segments of the present invention, parts thereof or oligos derived therefrom, other AD-related sequences using methods 20 described herein or other well known methods.
The invention also seeks to provide prokaryotic and eukaryotic expression vectors harboring the AD-related nucleic acid segment of the invention in an expressible from, and cells transformed with same. Such cells can serve a variety of purposes such as in vitro 25 models for the function of AD-related gene as well as for screening pharmaceutical compounds that could regulate the expression of the gene or the activity of the protein encoded therefrom. For example, such . CA 02203084 1997-04-18 a cell, expressing a DNA sequence encoding a protein involved in proper neural function through the inositol phosphate pathway could serve to screen for pharmaceutical compounds that regulate neural function or inositol phosphate pathway.
An expression vector harboring AD-related nucleic acid segment or part thereof, can be used to obtain substantially pure protein.
Well-known vectors can be used to obtain large amounts of the protein which can then be purified by standard biochemical methods based on charge, molecular weight, solubility or affinity of the protein or alternatively, the protein can be purified by using gene fusion techniques such as GST fusion, which permits the purification of the protein of interest on a gluthathion column. Other types of purification methods or fusion proteins could also be used.
Antibodies both polyclonal and monoclonal can be prepared from the protein encoded by the Ad-related nucleic acid segment of the invention. Such antibodies can be used for a variety of purposes including affinity purification of the AD-related protein and diagnosis of a predisposition to AD or othre CNS disorders.
The AD-related nucleic acid segment, parts thereof or oligonucleotides derived therefrom, can further be used to identify differences between AD affected individuals and non AD-affected individuals. Similarly such segments can be used to identify a predisposition to AD in individulas. The AD-related sequences can further be used to obtain animal models for the study of CNS disorders.
Transgenic animals can be obtained. The functional activity of the AD
protein encoded by these nucleic acids, whether native or mutated, can be tested in in vitro or in vivo models.

The human AD-related sequences can be used in a DNA-based diagnostic assay to identify these individuals in the population who are at risk for the above mentioned types of diseases.
Further, the present invention seeks to provide the use of 5 the AD-related protein as a pharmacological target for modulating neuronal function and the like.
As used herein in the specifications and appended claims, the term "oligonucleotide" includes both oligomers of ribonucleotides and oligomers of deoxyribonucleotides.
The term high stringency hybridization conditions, as used herein and well known in the art, includes, for example: 5 X SSPE (1 X
SSPE is 10 mM Na-phosphate, pH 7.0; 0.18 M NaCI; 1 mM Na2 EDTA), 5 x Denhardt's solution (from a 100 X solution containing 2% BSA, 2%
Ficoll, 2% polyvinyl pyrollidone), 0.1% SDS, and 0,5 mg/ml denatured 15 salmon sperm DNA, at 65~C. Other conditions considered stringent include the use of formamide. An example of washing conditions for the blot includes, as a final stringency wash, an incubation of the blot at 65~C
in 0.1 X SSPE, 0.1% SDS for 1 hour.
In the specifications and appended claims, it is to be 20 understood that absolute complementarity between the primers and the template is not required. Any oligonucleotide having a sufficient complementarity with the template, so that a stable duplex is formed, is suitable. Since the formation of a stable duplex depends on the sequence and length of the oligonucleotide and its complementarity to the 25 template it hybridizes to, as well as the hybridization conditions, one skilled in the art may readily determine the degree of mismatching that can be tolerated between the oligonucleotide and its target sequence for any given hybridization condition.
The invention features the means to identify factors that modulate the transcriptional activity of AD-related genes. Such factors 5 include, without being limited thereto, other kinases, phosphatases, nuclear receptors and transcriptionally regulatory proteins.
The present invention is also related to the use of AD-realted sequences of the present invention and functional derivatives thereof to screen for agents that modulate gene expression or the actity 10 of the products of these segments. Such modulators can be used as lead compounds to design or search drugs that can modulate the level of expression of these genes or the activity of their products.
Further, the present invention concerns a method for measuring the ability of a compound to act as an agonist or antagonist of 15 AD-related gene products comprising (a) contacting the compound with a transfected host cell expressing an AD-related sequence or mutant threof, and (b) comparing the level of activity of the product thereof or the level of expression of the AD related sequence. It is herein co"ler"plated to use the control regions of AD-related nucleic acids hooked to 20 heterologous genes such as any appropriate reporter gene (i.e.
Iuciferase, chloramphenicol acetyl transferase, green fluorescent protein or ~-galactosidase).
The invention is based on the results of an association study in recently founded populations in which a linkage disequilibrium 25 mapping of Alzheimer's disease was carried out. This analysis permitted the construction of haplotypes and enabled the identification of additional markers in the vicinity of the most significant markers identified by the association analysis.
From these data, it was inferred that the Alzheimer's disease loci comprise D10S212, D6S273, D1S228, D1S232, Gata89a1, D2S126, and D8S552.
Now that the location of Alzheimer's disease markers have been identified, other markers can be found using methods known in the art. Generally, primers are utilized which will identify markers associated with Alzheimer disease, for example (GD)n and RFLP markers.
The invention also extents to products useful for carrying out the assay, such as DNA probes (labelled or unlabelled), kits, and the like.
As broadest, the invention comprises detecting: the presence of genes involved in Alzheimer's disease by analysing human chromosomes, particularly chromosome 10, 6, 1, 9, 2 and 8 for further markers or DNA polymorphisms or the like linked to Alzheimer's disease.
The use RFLP's is only one preferred embodiment of detecting the polymorphisms. The most common methodology for detecting the presence of RLFP is to carry out restriction analysis using a given enzyme, perfomm a Southem procedure with a desired probe and identify a given RFLP or RFLPs. The use RLFPs in linkage analysis and genetic testing is well known in the art (for example, see Gusella, US 4, 666,828 incorporated herein by reference in Donnus-Keller et al., 1987, Cell. 51:319-337). It should be clear that other methods to identify differences at the DNA level, or RNA level which are not related to RFLPs can also be used. These methods are well known in the art of human genetics. Any method capable of directing the polymorphisms can also be used. Techniques such as amplification of the desired regional chromosome coupled with direct sequencing, a location of polymorphisms and the chromosome by radio-labelling, fluorescent-labelling and enzyme-labelling can also be utilized.
DNA and/or RNA can be amplified using an amplificable 5 RNA sequence as a probe and q~-replicas.
The polynucleotide probes may be RNA or DNA and preferably DNA, and can be labelled by standard labelling techniques such as with the radio-label, enzyme-label, fluorescent-label, biotin-avidin label and the like, which allow for the detection after hybridization as 10 commonly known in the art.
Comparison of the RLFP or RLFPs for affected and unaffected individuals in the family line of the subject, with the RLFP or RLFPs (or other methods) for the subject under investigation will quickly reveal the presence or absence of the Alzheimer disease gene(s) in the 15 subject. Results of this expresses in terms of probability of presence of the Alzheimer disease gene(s) in the subject.
A number of methods are available to the person of ordinary skill to obtain other genetic sequences useful for probes in accordance with the present invention. Non limiting examples of such methods 20 include random DNA sequences which can be tested for their specificity, construction of DNA libraries and isolation of clones therefrom. The results of such methods is to identify a probe which can detect a polymorphism useful for testing for Alzheimer disease. The polymorphism must be found to be linked to Alzheimer disease or the other useful 25 markers in families studies, all to be adjacent to preexisting markers.
A particular probe can have any desired sequence as long as its is capable of identifying the polymorphism in the involved DNA

regional or locus, it can be a DNA or RNA fragment, maybe synthesized chemical, enzymatically or isolated from a plasmid as well known to the person of ordinary skill. If a polymorphism is found in a gene product, such as a mRNA, the presence of that polymorphic mRNA may be 5 assayed directly with the probe, especially with antisense RNA probe.
Now that chromosomal location of the Alzheimer disease genes have been identified and defined to a small region, the region can be cloned and characterized by general methods known in the art.
The method lends itself readily to the formulation of kits 10 which can utilized in diagnosis.
Having now generally described the invention, the same will be understood by reference to certain specific examples that are provided here in exemplary form only and are not intended to be limiting unless otherwise specified.
DESCRIPTION OF THE PREFERRED EMBODIMENT
Data analysis from a genome wide screenil lg of Alzheimer's palients (23), using 700 microsatellites (positioned at an average of 4 to 7 cM), reveals seven (excluding ApoE) different regions in the genome 20 which seem to be implicated in the physiopathology of AD. Genetic markers representing these regions have been sorted with relative P
values, and are ordered from greatest importance as follows: D10S212 D6S273 ~ D1S228 > D1S232 ~ Gata89a1 > D2S126 > ApoE >
D8S552. Other potential sites of interest have also been detected in the 25 genomic regions containing the Presenilin gene which have previously been shown to be implicated in AD pathology. The P values for these regions, however, were found to be weaker than those observed for the microsatellites listed above.
The microsatellite D10S212 coincides with the region of principal interest as revealed by fine mapping, and is found to be adjacent 5 to an intron of the inositol polyphosphate-5-phosphatase gene (IPP1).
This gene encodes a 43-Kda protein involved in the inositol phosphate pathway, its role being that of a downregulator within the cascade by inactivating inositol phosphate signalling molecules.
Biochemical messengers within most cells effect diverse 10 and complex responses that often depend on the mobilization of Ca2 from intracellular stores within the sarcoplasmic (in muscle) or endoplasmic reticulum (S-ER). Two types of S-ER Ca2+ stores have been functionally characterized and identified by immunocyto-chemical localization of receptors (reviewed by Golovina and Blaustein, 1997), and 15 release of Ca2+ from one of the stores requires myo-inositol 1,4,5-trisphosphate (IP3).
Two distinct human genes coding for 5-phosphatase (Types I and ll) have been cloned, and encode for 43-kDa and 75-kDa proteins respectively. The Type I protein is phosphorylated and activated by 20 protein kinase C, while Type ll is not phosphorylated by this kinase. 5-phosphatase enzymes hydrolyze three substrates involved in calcium mobilization: inositol 1,4,5-triphosphate (IP3), inositol cyclic 1:2,4,5-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate (IP4).
Several studies suggest that alterations in the receptor-25 mediated phosphoinositide cascade and cytosolic free calciumconcentration [Ca2+]j are involved in the pathophysiology of aging, and in Alzheimer's disease. Cellular calcium ion signalling is induced by inositol phosphates formed directly or indirectly by the action of phosphatidylinositol-specific phospholipase C on phosphatidylinositol 4,5-bisphosphate in response to extracellular agonists (Berridge and Irvine, 1989; Bansal and Majerus, 1990; Rana and Hokin, 1990). These inositol phosphate signaling molecules are inactivated by inositol polyphosphate-5-phosphatase enzymes (5-phosphatase). Thus, by analogy with the adenylate cyclase/cyclic nucleotide phosphodiesterase system (Ross and Gilman, 1990), phospholipase C forms the active signalling molecules, while the 5-phosphatase acts to degrade them. Changes in the activity of either of these enzymes may alter cellular responses to agonists.
The second-most promising region, represented by the microsatellite D6S273, is in the vicinity (at a distance of about 2.7 cM) of a gene functionaly related to the IPP1, namely: the inositol 1,4,5-trisphosphate receptor type 3 gene (IP3R3). There is also on chromosome 12 another member of the inositol 1,4,5-trisphosphate receptor gene family, namely the type 2 receptor gene (IP3R2).
Three inositol 1,4,5-trisphosphate receptors have so far been cloned in humans. They mapped to three different chromosomal regions: the Types 2 and 3 respectively in chromosome 12p11 and 6p21, respectively, and the Type 1 in chromosome 1p. The inositol 1,4,5-triphosphate receptors (IP3R) act as IP3-gated Ca2+ release channels in a variety of cell types. The Type 1 receptor (IP3R1) is the major neuronal member of the IP3R family in the central nervous system. It is predominantly enriched in cerebellar Purkinje cells, but is also concentrated in neurons of the hippocampal CA1 region, caudate-putamen, and cerebral cortex. We have shown recently (unpublished results), that Type 2 and Type 3 receptors are also expressed in specific regions of the brain. Matsumoto et al.(1996) have shown that IP3R1-dericienl mice exhibit severe ataxia and tonic or tonic-clonic seizures, and die by the weaning period. Electroencephalograms demonstrate that such mice suffer from epilepsy, indicating that IP3R1 is essential for 5 proper brain function. Liu et al. (1995), in studies on juvenile myoclonic epilepsy (JME) in human families with classical JME, shown that in a region of about 7cM on chromosome 6p21.2-p11 an epilepsy locus exists whose mutated phenotype consists of classic JME with convulsions and/or electroencephalographic (EEG) rapid multispike wave complexes.
10 Again our marker D6S273 is within this interval.
IP3R binding sites were studied in autopsied brains from subjects with dementia of the Alzheimer type (DAT) and, in the parietal cortex and hippocampus, there was a 50-70% loss of (3H( IP3 binding, whereas no significant changes were observed in frontal, occipital and 15 temporal cortices, caudate or amygdala (L.Trevor Young et al., 1988).
Another candidate gene maps to a region of approximately 6 cM surrounding the microsatellite D1S228. This gene, the FK506 binding protein ripamycin ~ssoci~tcd protein, (FRAP) whose precise function is unknown, shows a ho m ~ layy with the C-terminal regions (21 %
20 identity on average) of several phosphatidylinositol kinases (Brown et al., 1994; Moore et al.,1996).
Another region of interest derived from our investigations highlights the region a~jacenl to the microsatellite D2S126, located within the chromosomal region 2q37, where the SH2-containing inositol-5-25 phosphatase gene (SHIP) has already been mapped.

The published cDNA of the inositol polyphosphate-5-phosphatase is not complete, but we have cloned the full length cDNA
from a humain brain cDNA library as well as part of the promoter region, and have determined the intron/exon junction sequences. The genomic 5 DNA corresponding to the exons and intron/exon junctions of the gene have been amplified using PCR and screened for mutations by the method of single strand conformation polymorphism (SSCP), from which some nucleotide changes have been observed. All the polymorphisms except one are found within non-coding sequences, or in the third codon 10 position in exons, which do not alter the amino acid residue. The only non- conservative mutation found is within the third exon of the gene, which changes the amino acid Iysine (MG) to arginine (AGG). However, this transition is conservative if we consider the presence of a putative phosphorylation consensus sequence serine/threonine-X-lysine/arginine 15 at that site. Experiments employing RT-PCR to analyze this polymorphism on the basis of differential expression levels within a set of patient samples did not show any differences.
All of this data strongly suggests that one or more components of the inositol pathway should be considered as excellent 20 candidates for the development of a physiopathological model of Alzheimer disease. In light of the fact that the IP3R1-/- (from human chromosome 1) transgenic mice develop epilepsy, and that studies on human families affected by the JME reveal that the affected loci cosegregate with chromosome 6p21 where the homolog gene (IP3R3) is 25 located, it appears highly probable that alterations in this pathway could be shared by different forms of genetic neurodisorders. If this proposed scenario is correct, we would expect to find in our population of AD some , CA 02203084 1997-04-18 incidence of epilepsy, and this is indeed the case; the incidence of epilepsy in our examined population is significantly higher than that normally expected. These observations point tantilizingly towards the hypothesis that various alterations within the inositol biochemical pathway 5 may result in vastly differing phenotypic manifestations, including epilepsy and Alzheimer's disease.
Having now identified the inositol phosphate pathway as a key player in CNS disorders and especially in AD, the present invention now permits a biochemical dissection of these diseases. Further, genetic 10 analysis can now be more focussed, and should enable the identification of other genes or products thereof which are part of the pathway or which affect it indirectly. Such analyses should also enable the identification of the critical role of the inositol pathway in other CNS disorders.
The present description refers to a number of documents, 15 the contents of which are incorporated by reference.

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Claims (2)

1. Use of the Inositol phosphate pathway as a target for diagnosis and/or therapy of CNS disorders.

2. Use accordaing to claim 1, wherein said disorder is AD.