WO2004019044A1

WO2004019044A1 - Diagnostic and therapeutic use of proteolipid protein for alzheimer's disease

Info

Publication number: WO2004019044A1
Application number: PCT/EP2003/009130
Authority: WO
Inventors: Heinz Von Der Kammer; Johannes Pohlner
Original assignee: Evotec Neurosciences Gmbh
Priority date: 2002-08-19
Filing date: 2003-08-18
Publication date: 2004-03-04
Also published as: AU2003251709A1

Abstract

The present invention discloses the differential expression of the PLP gene coding for PLP/DM20 protein in specific brain regions of Alzheimer's disease patients. Based on this finding, this invention provides a method for diagnosing or prognosticating a neurodegenerative disease, in particular Alzheimer's disease, in a subject, or for determining whether a subject is at increased risk of developing such a disease. Furthermore, this invention provides therapeutic and prophylactic methods for treating or preventing Alzheimer's disease and related neurodegenerative disorders using a PLP gene and its corresponding gene products. A method of screening for modulating agents of neurodegenerative diseases is also disclosed.

Description

DIAGNOSTIC AND THERAPEUTIC USE OF PROTEOLIPID PROTEIN FOR

ALZHEIMER'S DISEASE

The present invention relates to methods of diagnosing, prognosticating and monitoring the progression of neurodegenerative diseases in a subject. Furthermore, methods of therapy control and screening for modulating agents of neurodegenerative diseases are provided. The invention also discloses pharmaceutical compositions, kits, and recombinant animal models.

Neurodegenerative diseases, in particular Alzheimer's disease (AD), have a strongly debilitating impact on a patient's life. Furthermore, these diseases constitute an enormous health, social, and economic burden. AD is the most common neurodegenerative disease, accounting for about 70% of all dementia cases, and it is probably the most devastating age-related neurodegenerative condition affecting about 10% of the population over 65 years of age and up to 45% over age 85 (for a recent review see Vickers et al. , Progress in Neurobioiogy 2000, 60: 139-165). Presently, this amounts to an estimated 12 million cases in the US, Europe, and Japan. This situation will inevitably worsen with the demographic increase in the number of old people ("aging of the baby boomers") in developed countries. The neuropathological hallmarks that occur in the brains of individuals with AD are senile plaques, composed of amyloid-β protein, and profound cytoskeletal changes coinciding with the appearance of abnormal filamentous structures and the formation of neurofibrillary tangles.

The amyloid-β (Aβ) protein evolves from the cleavage of the amyloid precursor protein (APP) by different kinds of proteases. The cleavage by the β/γ-secretase leads to the formation of Aβ peptides of different lengths, typically a short more soluble and slow aggregating peptide consisting of 40 amino acids and a longer 42 amino acid peptide, which rapidly aggregates outside the cells, forming the characteristic amyloid plaques (Selkoe, Physiological Rev 2001 , 81 : 741-66; Greenfield et al., Frontiers Bioscience 2000, 5: D72-83). Two types of plaques, diffuse plaques and neuritic plaques, can be detected in the brain of AD patients, the latter ones being the classical, most prevalent type. They are primarily found in the cerebral cortex and hippocampus. The neuritic plaques have a diameter of 50μm to 200μm and are composed of insoluble fibrillar amyloids, fragments of dead neurons, of microglia and astrocytes, and other components such as neurotransmitters, apolipoprotein E, glycosaminoglycans, 1 -antichymotrypsin and others. The generation of toxic Aβ deposits in the brain starts very early in the course of AD, and it is discussed to be a key player for the subsequent destructive processes leading to AD pathology. The other pathological hallmarks of AD are neurofibrillary tangles (NFTs) and abnormal neurites, described as neuropil threads (Braak and Braak, Ada Neuropathol 1991 , 82: 239-259). NFTs emerge inside neurons and consist of chemically altered tau, which forms paired helical filaments twisted around each other. Along the formation of NFTs, a loss of neurons can be observed. It is discussed that said neuron loss may be due to a damaged microtubule-associated transport system (Johnson and Jenkins, J Alzheimers Dis 1996, 1 : 38-58; Johnson and Hartigan, J Alzheimers Dis 1999, 1 : 329-351). The appearance of neurofibrillary tangles and their increasing number correlates well with the clinical severity of AD (Schmitt et al., Neurology 2000, 55: 370-376). AD is a progressive disease that is associated with early deficits in memory formation and ultimately leads to the complete erosion of higher cognitive function. The cognitive disturbances include among other things memory impairment, aphasia, agnosia and the loss of executive functioning. A characteristic feature of the pathogenesis of AD is the selective vulnerability of particular brain regions and subpopulations of nerve cells to the degenerative process. Specifically, the temporal lobe region and the hippocampus are affected early and more severely during the progression of the disease. On the other hand, neurons within the frontal cortex, occipital cortex, and the cerebellum remain largely intact and are protected from neurodegeneration (Terry et al., Annals of Neurology 1981 , 10: 184-92). The age of onset of AD may vary within a range of 50 years, with early-onset AD occurring in people younger than 65 years of age, and late-onset of AD occurring in those older than 65 years. About 10% of all AD cases suffer from early-onset AD, with only 1 -2% being familial, inherited cases.

Currently, there is no cure for AD, nor is there an effective treatment to halt the progression of AD or even to diagnose AD ante-mortem with high probability. Several risk factors have been identified that predispose an individual to develop AD, among them most prominently the epsilon 4 allele of the three different existing alleles (epsilon 2, 3, and 4) of the apolipoprotein E gene (ApoE) (Strittmatter et al., Proc Natl Acad Sci USA 1993, 90: 1977-81 ; Roses, Ann NY Acad Sci 1998, 855: 738-43). The polymorphic plasmaprotein ApoE plays a role in the intercellular cholesterol and phospholipid transport by binding low-density lipoprotein receptors, and it seems to play a role in neurite growth and regeneration. Efforts to detect further susceptibility genes and disease-linked polymorphisms, lead to the assumption that specific regions and genes on human chromosomes 10 and 12 may be associated with late-onset AD (Myers et al., Science 2000, 290: 2304-5; Bertram et al., Science 2000, 290: 2303; Scott et al., Am J Hum Genet 2000, 66: 922-32). Although there are rare examples of early-onset AD which have been attributed to genetic defects in the genes for amyloid precursor protein (APP) on chromosome 21 , presenilin-1 on chromosome 14, and presenilin-2 on chromosome 1 , the prevalent form of late-onset sporadic AD is of hitherto unknown etiologic origin. The mutations found to date account for only half of the familial AD cases, which is less than 2% of all AD patients. The late onset and complex pathogenesis of neurodegenerative disorders pose a formidable challenge to the development of therapeutic and diagnostic agents. It is crucial to expand the pool of potential drug targets and diagnostic markers. It is therefore an object of the present invention to provide insight into the pathogenesis of neurological diseases and to provide methods, materials, agents, compositions, and animal models which are suited inter alia for the diagnosis and development of a treatment of these diseases. This object has been solved by the features of the independent claims. The subclaims define preferred embodiments of the present invention.

Neurons of the vertebrate nervous system conduct signals in the form of electrical impulses, which are transmitted at very high speed and over long distances. To make this rapid impulse conduction possible, the neuronal axons are insulated with segments of a highly specialized membrane, which consists of a unique composition and a unique segmental structure, the so called myelin sheaths. These multiiayered myeiin sheaths are formed by oligodendrocytes in the central nervous system (CNS) and by Schwann cells in the peripheral nervous system (PNS). Oligodendrocytes and astrocytes belong to glia cells, which constitute 90% of all cells in the human brain, and besides functioning in myelin formation and maintenance, are necessary for correct neuronal development and functioning of mature neurons.

The process of myelination includes neuron-glia cell recognition, molecular assembly of myelin components, and compaction of membranes to form lamellar structures. In humans, myelin formation occurs within the first year after birth. Oligodendrocytes myelinate nearby axons by sending out extensions of their plasma membrane, spirally wrapping around the axons, and thereby creating the multiiayered structure of the myelin sheath. Removal of the cytoplasmic content brings the plasma membranes close together. This process of compaction results in a characteristic ultrastructure of compact myelin lamellae with two different appositions, the cytoplasmic "major dense line" (MDL) and the extracellular "intraperiod line" (IPL). Between one segment of sheath and the next, a small region remains unsheathed. These so called "nodes of Ranvier" are focal points of electrical activity that mediate saltatory conduction, i.e. the rapid jumping of the action potential from node to node along a single nerve fibre (Baumann and Pham- Dinh, Physiological Reviews 2001 , 81 : 871-927). A single oligodendrocytic cell is able to myelinate several axons, but adjacent myelin segments on the same axon belong to different oligodendrocytes. In contrast, Schwann cells of the PNS myelinate only one segment of an axon.

The unique composition of the myelin membrane favors its insulating properties. It is poorly hydrated, very rich in lipids, and harbors specific myelin-constituting proteins. The lipid content of myelin is more than twice as much as the protein content. This is the reverse ratio as observed for other cellular membranes. Myelin contains cholesterol, phospholipids, and glycolipids in molar ratios of 4:3:2 to 4:4:2. The architecture of compacted myelin is determined by abundant membrane proteins that are specific to oligodendrocytes and/or Schwann cells. The cytosolic membrane-associated myelin basic proteins (MBP) and the integral membrane proteolipid proteins (PLP, also referred to as proteolipid protein, myelin proteolipid protein, lipophilin or SPG2, and its isoform DM20, also referred to as PLP splice variant or myelin PLP splice variant) are the major CNS myelin proteins. They constitute 80% of total proteins, whereby PLP alone accounts for up to 50 %. These proteins feature hydrophobic properties, mainly due to acylation by covalently attached palmitic, oleic, and stearic acids. PLP/DM20 are integral membrane proteins with four hydrophobic transmembrane α-helices, two extracytoplasmic and three cytoplasmic domains, and molecular mass weights of 25 kDa for PLP, and of 20 kDa for the DM20 isoform, respectively (Popot et al., Journal Membrane Biology 1991 , 120: 233-246). Interaction of PLP with cholesterol and with lipid rafts was described (Simons et al., Journal Cell Biology 2000, 151 : 143-153). Lipid rafts are cholesterol- and sphingolipid-rich membrane domains, which are implicated in intracellular transport and cell signaling processes.

The isoforms PLP and DM20 are encoded by the same gene (further referred to herein as the PLP gene; GenBank accession number M27110), which spans approximately 17 kb and is organized in 7 exons and 6 introns (Diehl et al., Proc Natl Acad Sci USA 1986, 83: 9807-9811 ; Kronquist et al., J Neurosci Res 1987, 18: 395-401 ). A family of PLP mRNAs with sizes of about 1 .6 kb, 2.4 kb and 3.2 kb, and two corresponding amino acid sequences, containing 276 and 241 residues, were detected. Isolation of the alternatively spliced PLP gene product DM20 from a mouse brain cDNA library revealed the 241 amino acid protein (Nave et al., Proc Natl Acad Sci USA 1987, 84: 5665-5669). In DM20, a portion of exon 3 (exon 3B) of the PLP gene is spliced out, leading to the deletion of amino acid residues 1 16 to 150 of the PLP protein. Willard and his colleagues assigned the human PLP gene to the long arm of the X-chromosome, at position Xq13-Xq22, and the murine PLP gene to the mouse X-chromosome (Willard et al., Science 1985, 230:940-942). The primary structure of PLP and DM20 is completely homologous between man and mouse, and only four amino acids are exchanged in the bovine PLP sequence (Stoffel et al., Biol Chem Hoppe Seyler 1985, 366:627-635; Diehl et al., Proc Natl Acad Sci USA 1986, 83: 9807-981 1 ). Because of this unusually high degree of conservation, it is speculated that PLP is the prototype of a new protein family, which also includes the two neuronal DM20-like proteins M6A and M6B, showing 56% and 46% sequence identity with DM20 (Yan et al., Neuron 1993, 11 : 423-431 ). Recently, two more isoforms of the mouse PLP gene were identified, which, respective to their soma-restricted appearence within the cells, are designated as srPLP and srDM20 (Bongarzone at el., J Neurosci 1999, 19: 8349-8357; Bongarzone et al., J Neurosci Res 2001 , 65: 485-492). These isoforms contain a new exon, which is spliced into PLP and DM20 mRNAs between exon 1 and exon 2. As a result, the srPLP and srDM20 proteins have a 12 amino acid leader sequence at their N-terminus, targeting them away from myelin membranes to different cellular compartments.

The expression of the PLP gene starts preceding myelination in the human spinal cord, as early as 11 weeks after conception and persists during development (Kronquist et al., J Neurosci Res 1987, 18: 395-401 ). The appearance of the DM20 isoform precedes that of the PLP isoform and predominates during early stages of myelination. Later in development, the PLP transcript increases until it surmounts the DM20 isoform (Campagnoni and Skoff, Brain Pathol 2001 , 1 1 : 74-91 ). In addition to the expression in oligodendrocytes, the PLP/DM20 transcripts have been found in neurons of the CNS, in embryonic CNS, in the PNS, in the heart, and in cells of the human immune system, i.e. in peripheral blood lymphocytes and in macrophages of the human thymus (Pribyl et al., J Neurosci Res 1996, 45: 812-819). The srPLP and srDM20 proteins have been identified in neuronal cells of postnatal brain, in the cerebellum, hippocampus, and the olfactory system (Bongarzone at el., J Neurosci 1999, 19: 8349-8357). It has been proposed that the expression of the PLP gene is spatially and temporally tightly regulated, which in part, is due to a cis-acting DNA antisilencer region in intron 1 of the gene (Dobretsova et al., J Neurochem 2000, 75: 1368-1376). The tight regulation of PLP gene expression is necessary for correct differentiation and survival of oligodendrocytes. (Ikenaka and Kagawa, Dev Neurosci 1995, 17: 127-136).

To date, a number of mutations, including point mutations, deletions and duplications in the PLP gene of human and animals have been described, which result in an abnormal structure of myelin, different types of dysmyelination, and reduction in the number and survival of oligodendrocytes. But, despite the high degree of conservation, the same mutation may have different pathological consequences for mouse and man.

Mutations in the PLP gene are associated with heterogeneous dysmyelinating syndromes such as Pelizaeus-Merzbacher disease and spastic paraplegia (NGBI database Online Inheritance in Man; OMIM:312080) in humans. PMD is a rare myelination disorder, affecting the white matter of the CNS. The disorder is defined according to genetic and clinical criteria, including X-linked recessive inheritance, leukodystrophy with preservation of axons and persistence of perivascular islands of myelin, nystagmus within the first months of life, psychomotor deterioration and progressive dystonic and cerebellar signs. The majority of PMD cases are caused by duplications of the PLP gene. Patients with absent PLP expression present an associated peripheral neuropathy and the mild form of PMD/SPG-2, whereas in mouse, complete loss of PLP function causes a very severe phenotype. The severity of symptoms varies according to the type of mutation, from early onset of the disease (already perinatal symptoms) with severe and lethal dysmyelination, to very late onset with mild demyelination and axonal degeneration, and from cases with lack of myelin to only mild myelin abnormalities.

The presence of misfolded PLP proteins or of overexpressed PLP protein, e.g. an overdosage of PLP proteins, leads to mistrafficking of myelin components within the cells. Transportation of these abnormal PLP proteins to the cell surface is blocked. As a consequence, PLP, and in addition cholesterol, are accumulated in the endoplasmatic reticulum and lysosomes. It is speculated that these depositions disturb myelination processes and later cause oligodendrocyte cell death. A direct correlation between the severity of phenotypes and transport of abnormal PLP proteins was observed (Thomson et al., Glia 1997, 20: 322-332; Simons et al., J Cell Biol 2002, 157: 327-336).

Similar dysmyelinating disorders, with the same genetic defects, are described for a number of animal species. They are very useful models for studying diseases associated with PLP gene mutations (Yool et al., Hum Mol Genet 2000, 9: 987-992). In the so called jimpy (jp) mouse, the mutation specifically affects the PLP gene and produces a shortened protein with 242 residues only (Duncan et al., Glia 1989, 2: 148-154). The jimpy mouse has a lethal phenotype with an extremely shortened life span (death occuring 3-4 weeks postnatally) and shows dysmyelination of most of the axons in the CNS, abnormal myelin sheaths, reduced number of mature oligodendrocytes, and premature oligodendroglial cell death. The rumpshaker mouse (rsh) is characterized by a mild phenotype and carries an amino acid substitution, which has also been identified in patients with SPG-2 (Schneider et al., Nature 1992, 358: 758-761 ). The myelin deficient (md) rat (Koeppen et al., J Neurol Sci 1988, 84: 315-327), the canine shaking pub (Griffiths et al., J Neurol Sci 1981 , 50: 423-433), and the paralytic tremor (pt) rabbit (Tosic et al., Brain Res 1993, 625: 307-312) all harbor a missense mutation in the PLP gene. They display a less severe phenotype with a normal number of oligodendrocytes and comparatively more myelin than observed in the other animal models, and they survive into adulthood. In animals lacking PLP/DM20 gene expression (null allele or knock-outs), the oligodendrocytes were still able to myelinate axons, although an abnormal condensation of the IPL, correlating with reduced physical stability, was observed. Very early in development, axonal swelling and axonal degeneration occurs (Klugmann et al., Neuron 1997, 18: 59-70).

The precise function of the PLP proteins remains to be elucidated. To date, most assumptions are based on the findings as described above. It is speculated that the PLP/DM20 gene products play a fundamental role in oligodendrocyte maturation and in the formation of stabilizing membrane junctions after myelin compaction (Ikenaka and Kagawa, Developmental Neuroscience 1995, 17: 127-136). In addition to their function as myelin structural proteins, there is evidence that the PLP/DM20 proteins are involved in neuronal development, neuronal cell death (Boucher et al., J Neurosci 2002, 1 : 1772-1783), autocrine and paracrine regulation processes, intracellular transport mechanisms, and migration of oligodendrocytes. The soma- restricted proteins srPLP and srDM20, also encoded by the PLP gene, appear to be components of intracellular transport vesicles and of transcriptional complexes (Campagnoni et al., Brain Pathol 2001 , 1 1 : 74-91 ). The soma-restricted proteins srPLP and srDM20 have not been detected in myelin structures.

In the present invention, using suppressive subtractive hybridization and screening of DNA biochips, transcription products of the gene coding for PLP/DM20 are analyzed in human brain samples. Importantly, the present invention discloses a dysregulation of PLP gene expression on the transcriptional level in the inferior temporal lobe or in the hippocampus relative to the frontal cortex of brain samples taken from AD patients and healthy control persons, respectively. No such dysregulation is observed in samples derived from age-matched, healthy controls. In contrast to its well-documented role in so called myelin diseases, to date, no experiments have been described that demonstrate a relationship between the dysregulation of PLP gene expression and the pathology of neurodegenerative disorders, in particular AD. Such a link, as disclosed in the present invention, offers new ways, inter alia, for the diagnosis and treatment of said neurodegenerative diseases, in particular AD.

The singular forms "a", "an", and "the" as used herein and in the claims include plural reference unless the context dictates otherwise. For example, "a cell" means as well a plurality of cells, and so forth. The term "and/or" as used in the present specification and in the claims implies that the phrases before and after this term are to be considered either as alternatives or in combination. For instance, the wording "determination of a level and/or an activity" means that either only a level, or only an activity, or both a level and an activity are determined. The term "level" as used herein is meant to comprise a gage of, or a measure of the amount of, or a concentration 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 for 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. 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. "Dysregulation" shall mean an upregulation or downregulation of gene expression. A gene product comprises either RNA or protein and is the result of expression of a gene. The amount of a gene product can be used to measure how active a gene is. 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 "ORF" is an acronym for "open reading frame" and refers to a nucleic acid sequence that does not possess a stop codon in at least one reading frame and therefore can potentially be translated into a sequence of amino acids. "Regulatory elements" shall comprise inducible and non-inducible promoters, enhancers, operators, and other elements that drive and regulate gene expression. 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 acetylation, 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, for instance, may 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 terms "agent", "reagent", or "compound" refer to any substance, chemical, composition or extract that have a positive or negative biological effect on a cell, tissue, body fluid, or within the context of any biological system, or any assay system examined. They can be agonists, antagonists, partial agonists or inverse agonists of a target. Such agents, reagents, or compounds may be nucleic acids, natural or synthetic peptides or protein complexes, or fusion proteins. They may also be antibodies, organic or anorganic molecules or compositions, small molecules, drugs and any combinations of any of said agents above. They may be used for testing, for diagnostic or for therapeutic purposes. The terms "oligonucleotide primer" or "primer" refer to short nucleic acid sequences which can anneal to a given target polynucleotide by hybridization of the complementary base pairs and can be extended by a polymerase. They may be chosen to be specific to a particular sequence or they may be randomly selected, e.g. they will prime all possible sequences in a mix. The length of primers used herein may vary from 10 nucleotides to 80 nucleotides. "Probes" are short nucleic acid sequences of the nucleic acid sequences described and disclosed herein or sequences complementary therewith. They may comprise full length sequences, or fragments, derivatives, isoforms, or variants of a given sequence. The identification of hybridization complexes between a "probe" and an assayed sample allows the detection of the presence of other similar sequences within that sample. As used herein, "homolog or homology" is a term used in the art to describe the relatedness of a nucleotide or peptide sequence to another nucleotide or peptide sequence, which is determined by the degree of identity and/or similarity between said sequences compared. The term "variant" as used herein refers to any polypeptide or protein, in reference to polypeptides and proteins disclosed in the present invention, in which one or more amino acids are added and/or substituted and/or deleted and/or inserted at the N-terminus, and/or the C-terminus, and/or within the native amino acid sequences of the native polypeptides or proteins of the present invention. Furthermore, the term "variant" shall include any shorter or longer version of a polypeptide or protein. "Variants" shall also comprise a sequence that has at least about 80% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity with the amino acid sequences of SEQ ID NO.1 and/or SEQ ID NO. 2. "Variants" of a protein molecule shown in SEQ ID NO. 1 and/or SEQ ID NO. 2 include, for example, proteins with conservative amino acid substitutions in highly conservative regions. "Proteins and polypeptides" of the present invention include variants, fragments and chemical derivatives of the protein comprising SEQ ID NO. 1 and/or SEQ ID NO.2. They can include proteins and polypeptides which can be isolated from nature or be produced by recombinant and/or synthetic means. Native proteins or polypeptides refer to naturally-occurring truncated or secreted forms, naturally occurring variant forms (e.g. splice-variants) and naturally occurring allelic variants. The term "isolated" as used herein is considered to refer to molecules that are removed from their natural environment, i.e. isolated from a cell or from a living organism in which they normally occur, and that are separated or essentially purified from the coexisting components with which they are found to be associated in nature. This notion further means that the sequences encoding such molecules can be linked by the hand of man to polynucleotides, to which they are not linked in their natural state, and that such molecules can be produced by recombinant and/or synthetic means. Even if for said purposes those sequences may be introduced into living or non-living organisms by methods known to those skilled in the art, and even if those sequences are still present in said organisms, they are still considered to be isolated. In the present invention, the terms "risk", "susceptibility", and "predisposition" are tantamount and are used with respect to the probability of developing a neurodegenerative disease, preferably Alzheimer's disease.

The term 'AD' shall mean Alzheimer's disease. "AD-type neuropathology" as used herein refers to neuropathological, neurophysiological, histopathological and clinical hallmarks as described in the instant invention and as commonly known from state- of-the-art literature (see: Iqbal, Swaab, Winblad and Wisniewski, Alzheimer^'s Disease and Related Disorders (Etiology, Pathogenesis and Therapeutics), Wiley & Sons, New York, Weinheim, Toronto, 1999; Scinto and Daffner, Early Diagnosis of Alzheimer^'s Disease, Humana Press, Totowa, New Jersey, 2000; Mayeux and Christen, Epidemiology of Alzheimer^'s Disease: From Gene to Prevention, Springer Press, Berlin, Heidelberg, New York, 1999; Younkin, Tanzi and Christen, Presenilins and Alzheimer^'s Disease, Springer Press, Berlin, Heidelberg, New York, 1998). Neurodegenerative diseases or disorders according to the present invention comprise Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Pick's disease, fronto-temporal dementia, progressive nuclear palsy, corticobasal degeneration, cerebro-vascular dementia, multiple system atrophy, argyrophilic grain dementia and other tauopathies, and mild- cognitive impairment. Further conditions involving neurodegenerative processes are, for instance, age-related macular degeneration, narcolepsy, motor neuron diseases and prion diseases.

In one aspect, the invention features a method of diagnosing or prognosticating a neurodegenerative disease in a subject, or determining whether a subject is at increased risk of developing said disease. The method comprises: determining a level, or an activity, or both said level and said activity of (i) a transcription product of a gene coding for PLP, and/or of (ii) a translation product of a gene coding for PLP, and/or of (iii) a fragment, or derivative, or variant of said transcription or translation product in a sample from said subject and comparing said level, and/or said activity to a reference value representing a known disease or health status, thereby diagnosing or prognosticating said neurodegenerative disease in said subject, or determining whether said subject is at increased risk of developing said neurodegenerative disease. The invention also relates to the construction and the use of primers and probes which are unique to the nucleic acid sequences, or fragments, or variants thereof, as disclosed in the present invention. The oligonucleotide primers and/or probes can be labeled specifically with fluorescent, bioluminescent, magnetic, or radioactive substances. The invention further relates to the detection and the production of said nucleic acid sequences, or fragments and variants thereof, using said specific oligonucleotide primers in appropriate combinations. PCR-analysis, a method well known to those skilled in the art, can be performed with said primer combinations to amplify said gene specific nucleic acid sequences from a sample containing nucleic acids. Such sample may be derived either from healthy or diseased subjects. Whether an amplification results in a specific nucleic acid product or not, and whether a fragment of different length can be obtained or not, may be indicative for a neurodegenerative disease, in particular Alzheimer's disease. Thus, the invention provides nucleic acid sequences, oligonucleotide primers, and probes of at least 10 bases in length up to the entire coding and gene sequences, useful for the detection of gene mutations and single nucleotide polymorphisms in a given sample comprising nucleic acid sequences to be examined, which may be associated with neurodegenerative diseases, in particular Alzheimer's disease. This feature has utility for developing rapid DNA-based diagnostic tests, preferably also in the format of a kit.

In a further aspect, the invention features a method of monitoring the progression of a neurodegenerative disease in a subject. A level, or an activity, or both said level and said activity, of (i) a transcription product of a gene coding for PLP, and/or of (ii) a translation product of a gene coding for PLP, and/or of (iii) a fragment, or derivative, or variant of said transcription or translation product in a sample from said subject is determined. Said level and/or said activity is compared to a reference value- representing a known disease or health status. Thereby the progression of said neurodegenerative disease in said subject is monitored.

In still a further aspect, the invention features a method of evaluating a treatment for a neurodegenerative disease, comprising determining a level, or an activity, or both said level and said activity of (i) a transcription product of a gene coding for PLP, and/or of (ii) a translation product of a gene coding for PLP, and/or of (iii) a fragment, or derivative, or variant of said transcription or translation product in a sample obtained from a subject being treated for said disease. Said level, or said activity, or both said level and said activity are compared to a reference value representing a known disease or health status, thereby evaluating the treatment for said neurodegenerative disease.

In a preferred embodiment of the herein claimed methods, kits, recombinant animals, molecules, assays, and uses of the instant invention, said gene coding for a PLP and/or DM20 protein is the gene coding for the proteolipid protein, also termed myelin proteolipid protein, PLP, PLP-1 , or lipophilin, or SPG2, herein also referred to as PLP splice variant 1 (amino acid sequence of SEQ ID NO. 1 , nucleotide sequence of SEQ ID NO. 3, GenBank accession number: M271 10), and coding for the isoform DM20, also referred to as PLP splice variant or myelin PLP splice variant, herein also referred to as PLP splice variant 2 or DM20 (amino acid sequence of SEQ ID NO. 2, nucleotide sequence of SEQ ID NO. 4). In the instant invention, the gene coding for said PLP and DM20 protein is also generally referred to as the PLP gene, or PLP/DM20 gene, and said PLP or DM20 protein is also generally referred to as the PLP/DM20 protein, or PLP/DM20.

In a further preferred embodiment of the herein claimed methods, kits, recombinant animals, molecules, assays, and uses of the instant invention, said neurodegenerative disease or disorder is Alzheimer's disease, and said subjects suffer from Alzheimer's disease.

The present invention discloses the detection, differential expression and regulation of a gene coding for PLP/DM20 in specific brain regions of AD patients. Consequently, the PLP gene and its corresponding transcription and translation products may have a causative role in the regional selective neuronal degeneration typically observed in AD. Alternatively, PLP may confer a neuroprotective function to the remaining surviving nerve cells. Based on these disclosures, the present invention has utility for the diagnostic evaluation and prognosis as well as for the identification of a predisposition to a neurodegenerative disease, in particular AD. Furthermore, the present invention provides methods for the diagnostic monitoring of patients undergoing treatment for such a disease.

It is particularly preferred that said sample to be analyzed and determined is selected from the group comprising brain tissue or other tissues or body cells. The sample can also comprise cerebrospinal fluid or other body fluids including saliva, urine, blood, serum plasma, or mucus. Preferably, the methods of diagnosis, prognosis, monitoring the progression or evaluating a treatment for a neurodegenerative disease, according to the instant invention, can be practiced ex corpore, and such methods preferably relate to samples, for instance, body fluids or cells, removed, collected, or isolated from a subject or patient.

In further preferred embodiments, said reference value is that of a level, or an activity, or both said level and said activity of (i) a transcription product of a gene, coding for PLP, and/or of (ii) a translation product of a gene coding for PLP, and/or of (iii) a fragment, or derivative, or variant of said transcription or translation product in a sample from a subject not suffering from said neurodegenerative disease.

In preferred embodiments, an alteration in the level and/or activity of a transcription product of the gene coding for PLP and/or of a translation product of the gene coding for PLP, and/or of a fragment, or derivative, or variant thereof in a sample cell, or tissue, or body fluid from said subject relative to a reference value representing a known health status indicates a diagnosis, or prognosis, or increased risk of becoming diseased with a neurodegenerative disease, particularly AD.

In preferred embodiments, measurement of the level of transcription products of a gene coding for PLP is performed in a sample from a subject using a quantitative PCR-analysis with primer combinations to amplify said gene specific sequences from cDNA obtained by reverse transcription of RNA extracted from a sample of a subject. A Northern blot with probes specific for said gene can also be applied. It might further be preferred to measure transcription products by means of chip-based micro-array technologies. These techniques are known to those of ordinary skill in the art (see Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001 ; Schena M., Microarray Biochip Technology, Eaton Publishing, Natick, MA, 2000). An example of an immunoassay is the detection and measurement of enzyme activity as disclosed and described in the patent application WO 02/14543.

Furthermore, a level and/or an activity of a translation product of a gene coding for PLP and/or of a fragment, or derivative, or variant of said translation product, and/or a level of activity of said translation product and/or of a fragment, or derivative, or variant of said translation product, can be detected using an immunoassay, an activity assay, and/or a 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 which binds the anti-protein antibody. In addition, other high affinity ligands may be used. Immunoassays which can be used include e.g. ELISAs, Western blots and other techniques known to those of ordinary skill in the art (see Hariow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999 and Edwards R, Immunodiagnostics: A Practical Approach, Oxford University Press, Oxford; England, 1999). All these detection techniques may also be employed in the format of microarrays, protein-arrays, antibody microarrays, tissue microarrays, electronic biochip or protein-chip based technologies (see Schena M., Microarray Biochip Technology, Eaton Publishing, Natick, MA, 2000).

In a preferred embodiment, the level, or the activity, or both said level and said activity of (i) a transcription product of a gene coding for PLP, and/or of (ii) a translation product of a gene coding for PLP, and/or of (iii) a fragment, or derivative, or variant of said transcription or translation product in a series of samples taken from said subject over a period of time is compared, in order to monitor the progression of said disease. In further preferred embodiments, said subject receives a treatment prior to one or more of said sample gatherings. In yet another preferred embodiment, said level and/or activity is determined before and after said treatment of said subject.

In another aspect, the invention features a kit for diagnosing or prognosticating neurodegenerative diseases, in particular AD, in a subject, or determining the propensity or predisposition of a subject to develop a neurodegenerative disease, in particular AD, said kit comprising:

(a) at least one reagent which is selected from the group consisting of (i) reagents that selectively detect a transcription product of a gene coding for PLP (ii) reagents that selectively detect a translation product of a gene coding for PLP; and

(b) instruction for diagnosing, or prognosticating a neurodegenerative disease, in particular AD, or determining the propensity or predisposition of a subject to develop such a disease by - detecting a level, or an activity, or both said level and said activity, of said transcription product and/or said translation product of a gene coding for PLP, in a sample from said subject; and

- diagnosing or prognosticating a neurodegenerative disease, in particular AD, or determining the propensity or predisposition of said subject to develop such a disease, wherein a varied level, or activity, or both said level and said activity, of said transcription product and/or said translation product compared to a reference value representing a known health status; or a level, or activity, or both said level and said activity, of said transcription product and/or said translation product similar or equal to a reference value representing a known disease status, indicates a diagnosis or prognosis of a neurodegenerative disease, in particular AD, or an increased propensity or predisposition of developing such a disease. The kit, according to the present invention, may be particularly useful for the identification of individuals that are at risk of developing a neurodegenerative disease, in particular AD. Consequently, the kit, according to the invention, may serve as a means for targeting identified individuals for early preventive measures or therapeutic intervention prior to disease onset, before irreversible damage in the course of the disease has been inflicted. Furthermore, in preferred embodiments, the kit featured in the invention is useful for monitoring a progression of a neurodegenerative disease, in particular AD in a subject, as well as monitoring success or failure of therapeutic treatment for such a disease of said subject.

In another aspect, the invention features a method of treating or preventing a neurodegenerative disease, in particular AD, in a subject comprising the administration to said subject in 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 gene coding for PLP, and/or (ii) a transcription product of a gene coding for PLP, and/or (iii) a translation product of a gene coding for PLP, and/or (iv) a fragment, or derivative, or variant of (i) to (iii). Said agent may comprise a small molecule, or it may also comprise a peptide, an oligopeptide, or a polypeptide. Said peptide, oligopeptide, or polypeptide may comprise an amino acid sequence of a translation product of the PLP gene, SEQ ID NO. 1 and/or SEQ ID NO. 2, or a fragment, or derivative, or a variant thereof. An agent for treating or preventing a neurodegenerative disease, in particular AD, according to the instant invention, may also consist of a nucleotide, an oligonucleotide, or a polynucleotide. Said oligonucleotide or polynucleotide may comprise a nucleotide sequence of the PLP gene coding for PLP/DM20, either in sense orientation or in antisense orientation.

In preferred embodiments, the method comprises the application of per se known methods of gene therapy and/or antisense nucleic acid technology to administer said agent or agents. In general, gene therapy includes several approaches: molecular replacement of a mutated gene, addition of a new gene resulting in the synthesis of a therapeutic protein, and modulation of endogenous cellular gene expression by recombinant expression methods or by drugs. Gene-transfer techniques are described in detail (see e.g. Behr, Ace Chem Res 1993, 26: 274-278 and Mulligan, Science 1993, 260: 926-931 ) and include direct gene-transfer techniques such as mechanical microinjection of DNA into a cell as well as indirect techniques employing biological vectors (like recombinant viruses, especially retroviruses) or model liposomes, or techniques based on transfection with DNA coprecipitation with polycations, cell membrane pertubation by chemical (solvents, detergents, polymers, enzymes) or physical means (mechanic, osmotic, thermic, electric shocks). The postnatal gene transfer into the central nervous system has been described in detail (see e.g. Wolff, Curr Opin Neurobiol 1993, 3: 743-748).

In particular, the invention features a method of treating or preventing a neurodegenerative disease by means of antisense nucleic acid therapy, i.e. the down-regulation of an inappropriately expressed or defective gene by the introduction of antisense nucleic acids or derivatives thereof into certain critical cells (see e.g. Gillespie, DN&P 1992, 5: 389-395; Agrawai and Akhtar, Trends Biotechnol 1995, 13: 197-199; Crooke, Biotechnology 1992, 10: 882-6). Apart from hybridization strategies, the application of ribozymes, i.e. RNA molecules that act as enzymes, destroying RNA that carries the message of disease has also been described (see e.g. Barinaga, Science 1993, 262: 1512-1514). In preferred embodiments, the subject to be treated is a human, and therapeutic antisense nucleic acids or derivatives thereof are directed against transcripts of a gene coding for PLP. It is preferred that cells of the central nervous system, preferably the brain, of a subject are treated in such a way. Cell penetration can be performed by known strategies such as coupling of antisense nucleic acids and derivatives thereof to carrier particles, or the above described techniques. Strategies for administering targeted therapeutic oligo-deoxynucleotides are known to those of skill in the art (see e.g. Wickstrom, Trends Biotechnol 1992, 10: 281 -287). In some cases, delivery can be performed by mere topical application. Further approaches are directed to intracellular expression of antisense RNA. In this strategy, cells are transformed ex vivo with a recombinant gene that directs the synthesis of an RNA that is complementary to a region of target nucleic acid. Therapeutical use of intracellularly expressed antisense RNA is procedurally similar to gene therapy. A recently developed method of regulating the intracellular expression of genes by the use of double-stranded RNA, known variously as RNA interference (RNAi), can be another effective approach for nucleic acid therapy (Hannon, Nature 2002, 418: 244-251 ).

In further preferred embodiments, the method comprises grafting donor cells into the central nervous system, preferably the brain, of said subject, or donor cells preferably treated so as to minimize or reduce graft rejection, wherein said donor cells are genetically modified by insertion of at least one transgene encoding said agent or agents. Said transgene might be carried by a viral vector, in particular a retroviral vector. The transgene can be inserted into the donor cells by a nonviral physical transfection of DNA encoding a transgene, in particular by microinjection. Insertion of the transgene can also be performed by electroporation, chemically mediated transfection, in particular calcium phosphate transfection or liposomal mediated transfection (see Mc Celland and Pardee, Expression Genetics: Accelerated and High-Throughput Methods, Eaton Publishing, Natick, MA, 1999).

In preferred embodiments, said agent for treating and preventing a neurodegenerative disease, in particular AD, is a therapeutic protein which can be administered to said subject, preferably a human, by a process comprising introducing subject cells into said subject, said subject cells having been treated in vitro to insert a DNA segment encoding said therapeutic protein, said subject cells expressing in vivo in said subject a therapeutically effective amount of said therapeutic protein. Said DNA segment can be inserted into said cells in vitro by a viral vector, in particular a retroviral vector.

Methods of treatment, according to the present invention, comprise the application of therapeutic cloning, transplantation, and stem cell therapy using embryonic stem cells or embryonic germ cells and neuronal adult stem cells, combined with any of the previously described cell- and gene therapeutic methods. Stem cells may be totipotent or pluripotent. They may also be organ-specific. Strategies for repairing diseased and/or damaged brain cells or tissue comprise (i) taking donor cells from an adult tissue. Nuclei of those cells are transplanted into unfertilized egg cells from which the genetic material has been removed. Embryonic stem cells are isolated from the blastocyst stage of the cells which underwent somatic cell nuclear transfer. Use of differentiation factors then leads to a directed development of the stem cells to specialized cell types, preferably neuronal cells (Lanza et al., Nature Medicine 1999, 9: 975-977), or (ii) purifying adult stem cells, isolated from the central nervous system, or from bone marrow (mesenchymal stem cells), for in vitro expansion and subsequent grafting and transplantation, or (iii) directly inducing endogenous neural stem cells to proliferate, migrate, and differentiate into functional neurons (Peterson DA, Curr. Opin. Pharmacol. 2002, 2: 34-42). Adult neural stem cells are of great potential for repairing damaged or diseased brain tissues, as the germinal centers of the adult brain are free of neuronal damage or dysfunction (Colman A, Drug Discovery World 2001 , 7: 66-71 ).

In preferred embodiments, the subject for treatment or prevention, according to the present invention, can be a human, an experimental animal, e.g. a mouse or a rat, a domestic animal, or a non-human primate. The experimental animal can be an animal model for a neurodegenerative disorder, e.g. a transgenic mouse and/or a knock-out mouse with an AD-type neuropathology.

In a further aspect, the invention features a modulator of an activity, or a level, or both said activity and said level of at least one substance which is selected from the group consisting of (i) a gene coding for PLP, and/or (ii) a transcription product of a gene coding for PLP, and/or (iii) a translation product of a gene coding for PLP, and/or (iv) a fragment, or derivative, or variant of (i) to (iii).

In an additional aspect, the invention features a pharmaceutical composition comprising said modulator and preferably a pharmaceutical carrier. Said carrier refers to a diluent, adjuvant, excipient, or vehicle with which the modulator is administered.

In a further aspect, the invention features a modulator of an activity, or a level, or both said activity and said level of at least one substance which is selected from the group consisting of (i) a gene coding for PLP, and/or (ii) a transcription product of a gene coding for PLP, and/or (iii) a translation product of a gene coding for PLP, and/or (iv) a fragment, or derivative, or variant of (i) to (iii) for use in a pharmaceutical composition.

In another aspect, the invention provides for the use of a modulator of an activity, or a level, or both said activity and said level of at least one substance which is, selected from the group consisting of (i) a gene coding for PLP, and/or (ii) a transcription product of a gene coding for PLP and/or (iii) a translation product of a gene coding for PLP, and/or (iv) a fragment, or derivative, or variant of (i) to (iii) for a preparation of a medicament for treating or preventing a neurodegenerative disease, in particular AD.

In one aspect, the present invention also provides a kit comprising one or more, containers filled with a therapeutically or prophylactically effective amount of said pharmaceutical composition.

In a further aspect, the invention features a recombinant, non-human animal comprising a non-native gene sequence coding for PLP, or a fragment, or derivative, or variant thereof. The generation of said recombinant, non-human animal comprises (i) providing a gene targeting construct containing said gene sequence and a selectable marker sequence, and (ii) introducing said targeting construct into a stem cell of a non-human animal, and (iii) introducing said non-human animal stem cell into a non-human embryo, and (iv) transplanting said embryo into a pseudopregnant non-human animal, and (v) allowing said embryo to develop to term, and (vi) identifying a genetically altered non-human animal whose genome comprises a modification of said gene sequence in both alleles, and (vii) breeding the genetically altered non-human animal of step (vi) to obtain a genetically altered non-human animal whose genome comprises a modification of said endogenous gene, wherein said gene is mis-expressed, or under-expressed, or over-expressed, and wherein said disruption or alteration results in said non-human animal exhibiting a predisposition to developing symptoms of neuropathology similar to a neurodegenerative disease, in particular AD. Strategies and techniques for the generation and construction of such an animal are known to those of ordinary skill in the art (see e.g. Capecchi, Science 1989, 244: 1288-1292 and Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1994 and Jackson and Abbott, Mouse Genetics and Transgenics: A Practical Approach, Oxford University Press, Oxford, England, 1999). It is preferred to make use of such a recombinant non- human animal as an animal model for investigating neurodegenerative diseases, in particular Alzheimer's disease. Such an animal may be useful for screening, testing and validating compounds, agents and modulators in the development of diagnostics and therapeutics to treat neurodegenerative diseases, in particular Alzheimer's disease.

In another aspect, the invention features an assay for screening for a modulator of neurodegenerative diseases, in particular AD, or related diseases and disorders of one or more substances selected from the group consisting of (i) a gene coding for PLP, and/or (ii) a transcription product of a gene coding for PLP, and/or (iii) a translation product of a gene coding for PLP, and/or (iv) a fragment, or derivative, or variant of (i) to (iii). This screening method comprises (a) contacting a cell with a test compound, and (b) measuring the activity, or the level, or both the activity and the level of one or more substances recited in (i) to (iv), and (c) measuring the activity, or the level, or both the activity and the level of said substances in a control cell not contacted with said test compound, and (d) comparing the levels of the substance in the cells of step (b) and (c), wherein an alteration in the activity and/or level of said substances in the contacted cells indicates that the test compound is a modulator of said diseases and disorders.

In one further aspect, the invention features a screening assay for a modulator of neurodegenerative diseases, in particular AD, or related diseases and disorders of one or more substances selected from the group consisting of (i) a gene coding for PLP, and/or (ii) a transcription product of a gene coding for PLP, and/or (iii) a translation product of a gene coding for PLP, and/or (iv) a fragment, or derivative, or variant of (i) to (iii), comprising (a) administering a test compound to a test animal which is predisposed to developing or has already developed symptoms of a neurodegenerative disease or related diseases or disorders, in particular AD, and (b) measuring the activity and/or level of one or more substances recited in (i) to (iv), and (c) measuring the activity and/or level of said substances in a matched control animal which is equally predisposed to developing or has already developed said symptoms of said diseases and to which animal no such test compound has been administered, and (d) comparing the activity and/or level of the substance in the animals of step (b) and (c), wherein an alteration in the activity and/or level of substances in the test animal indicates that the test compound is a modulator of said diseases and disorders.

In a preferred embodiment, said test animal and/or said control animal is a recombinant, non-human animal which expresses a gene coding for PLP, or a fragment, or a derivative, or a variant thereof, under the control of a transcriptional regulatory element which is not the native PLP gene transcriptional control regulatory element.

In another embodiment, the present invention provides a method for producing a medicament comprising the steps of (i) identifying a modulator of neurodegenerative diseases by a method of the aforementioned screening assays and (ii) admixing the modulator with a pharmaceutical carrier. However, said modulator may also be identifiable by other types of screening assays.

in another aspect, the present invention provides for an assay for testing a compound, preferably for screening a plurality of compounds, for inhibition of binding between a ligand and PLP protein, or a fragment, or derivative, or variant thereof. Said screening assay comprises the steps of (i) adding a liquid suspension of said PLP protein, or a fragment, or derivative, or variant thereof, to a plurality of containers, and (ii) adding a compound or a plurality of compounds to be screened for said inhibition to said plurality of containers, and (iii) adding a detectable, preferably a fluorescently labeled ligand to said containers, and (iv) incubating said PLP protein, or said fragment, or derivative, or variant thereof, and said compound or plurality of compounds, and said detectable, preferably fluorescently labeled ligand, and (v) measuring the amounts of fluorescence associated with said PLP protein, or with said fragment, or derivative, or variant thereof, and (vi) determining the degree of inhibition by one or more of said compounds of binding of said ligand to said PLP protein, or said fragment, or derivative, or variant thereof. It might be preferred to reconstitute said PLP translation product, or fragment, or derivative, or variant thereof into artificial liposomes to generate the corresponding proteoliposomes to determine the inhibition of binding between a ligand and said PLP translation product. Methods of reconstitution of PLP translation products from detergent into liposomes have been detailed (Schwarz et al., Biochemistry 1999, 38: 9456-9464; Krivosheev and Usanov, Biochemistry-Moscow 1997, 62: 1064-1073). Instead of utilizing a fluorescently labeled ligand, it might in some aspects be preferred to use any other detectable label known to the person skilled in the art, e.g. radioactive labels, and detect it accordingly. Said method may be useful for the identification of novel compounds as well as for evaluating compounds which have been improved or otherwise optimized in their ability to inhibit the binding of a ligand to a gene product of a gene coding for PLP, or a fragment, or derivative, or variant thereof. One example of a fluorescent binding assay, in this case based on the use of carrier particles, is disclosed and described in patent application WO 00/52451. A further example is the competitive assay method as described in patent WO 02/01226. Preferred signal detection methods for screening assays of the instant invention are described in the following patent applications: WO 96/13744, WO 98/16814, WO 98/23942, WO 99/17086, WO 99/34195, WO 00/66985, WO 01/59436, WO 01/59416.

In one further embodiment, the present invention provides a method for producing a medicament comprising the steps of (i) identifying a compound as an inhibitor of binding between a ligand and a gene product of a gene coding for PLP by the aforementioned inhibitory binding assay and (ii) admixing the compound with a pharmaceutical carrier. However, said compound may also be identifiable by other types of screening assays.

In another aspect, the invention features an assay for testing a compound, preferably for screening a plurality of compounds to determine the degree of binding of said compounds to PLP protein, or to a fragment, or derivative, or variant thereof. Said screening assay comprises (i) adding a liquid suspension of said PLP protein, or a fragment, or derivative, or variant thereof, to a plurality of containers, and (ii) adding a detectable, preferably a fluorescently labeled compound or a plurality of detectable, preferably fluorescently labeled compounds to be screened for said binding to said plurality of containers, and (iii) incubating said PLP protein, or said fragment, or derivative, or variant thereof, and said detectable, preferably said fluorescently labeled compound or fluorescently labeled compounds, and (iv) measuring the amounts of preferably the fluorescence associated with said PLP protein, or with said fragment, or derivative, or variant thereof, and (v) determining the degree of binding by one or more of said compounds to said PLP protein, or said fragment, or derivative, or variant thereof. In this type of assay it might be preferred to use a fluorescent label. However, any other type of detectable label might also be employed. Also in this type of assay it might be preferred to reconstitute a PLP translation product or fragment, or derivative, or variant thereof into artificial liposomes as described in the present invention. Said method may be useful for the identification of novel compounds as well as for evaluating compounds which have been improved or otherwise optimized in their ability to bind to PLP, or a fragment, or derivative, or variant thereof.

In one further embodiment, the present invention provides a method for producing a medicament comprising the steps of (i) identifying a compound as a binder to a gene product of a gene coding for PLP by the aforementioned binding assays and (ii) admixing the compound with a pharmaceutical carrier. However, said compound may also be identifiable by other types of screening assays.

In another embodiment, the present invention provides for a medicament obtainable by any of the methods according to the herein claimed screening assays. In one further embodiment, the instant invention provides for a medicament obtained by any of the methods according to the herein claimed screening assays.

The present invention features protein molecules as shown in SEQ ID NO. 1 and SEQ ID NO. 2, said protein molecules being translation products of the PLP gene, or fragments, or derivatives, or variants thereof, for use as diagnostic targets for detecting a neurodegenerative disease, in particular Alzheimer's disease.

The present invention further features protein molecules as shown in SEQ ID NO.1 and SEQ I D NO. 2, said protein molecules being translation products of the PLP gene, or fragments, or derivatives, or variants thereof, for use as screening targets for reagents or compounds preventing, or treating, or ameliorating a neurodegenerative disease, in particular Alzheimer's disease.

The present invention features an antibody which is specifically immunoreactive with an immunogen, wherein said immunogen is a translation product of a gene coding for PLP, or a fragment, or derivative, or variant thereof. The immunogen may comprise immunogenic or antigenic epitopes or portions of a translation product of said gene, wherein said immunogenic or antigenic portion of a translation product is a polypeptide, and wherein said polypeptide elicits an antibody response in an animal, and wherein said polypeptide is immunospecifically bound by said antibody. Methods for generating antibodies are well known in the art (see Harlow et al., Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988). The term "antibody", as employed in the present invention, encompasses all forms of antibodies known in the art, such as polyclonal, monoclonal, chimeric, recombinatorial, anti-idiotypic, humanized, or single chain antibodies, as well as fragments thereof (see Dubel and Breitling, Recombinant Antibodies, Wiley-Liss, New York, NY, 1999). Antibodies of the present invention are useful, for instance, in a variety of diagnostic and therapeutic methods, based on state-in-the-art techniques (see Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999 and Edwards R., Immunodiagnostics: A Practical Approach, Oxford University Press, Oxford, England, 1999) such as enzyme-immuno assays (e.g. enzyme-linked immunosorbent assay, ELISA), radioimmuno assays, chemoluminescence-immuno assays, Western-blot, immunoprecipitation and antibody microarrays. These methods involve the detection of translation products of the PLP gene, or fragments, or derivatives, or variants thereof.

In a preferred embodiment of the present invention, said antibodies can be used for detecting the pathological state of a cell in a sample from a subject, comprising immunocytochemical staining of said cell with said antibody, wherein an altered degree of staining, or an altered staining pattern in said cell compared to a cell representing a known health status indicates a pathological state of said cell. Preferably, the pathological state relates to a neurodegenerative disease, in particular to AD. Immunocytochemical staining of a cell can be carried out by a number of different experimental methods well known in the art. It might be preferred, however, to apply an automated method for the detection of antibody binding, wherein the determination of the degree of staining of a cell, or the determination of the cellular or subcellular staining pattern of a cell, or the topological distribution of an antigen on the cell surface or among organelles and other subcellular structures within the cell, are carried out according to the method described in US patent 6150173.

Other features and advantages of the invention will be apparent from the following description of figures and examples which are illustrative only and not intended to limit the remainder of the disclosure in any way. Figure 1 depicts the brain regions with selective vulnerability to neuronal loss and degeneration in AD. Primarily, neurons within the inferior temporal lobe, the entorhinal cortex, the hippocampus, and the amygdala are subject to degenerative processes in AD (Terry et al., Annals of Neurology 1981 , 10:184-192). These brain regions are mostly involved in the processing of learning and memory functions. In contrast, neurons within the frontal cortex, the occipital cortex, and the cerebellum remain largely intact and preserved from neurodegenerative processes in AD. Brain tissues from the frontal cortex (F), the temporal cortex (T), and the hippocampus (H) of AD patients and healthy, age-matched control individuals were used for the herein disclosed examples. For illustrative purposes, the image of a normal healthy brain was taken from a publication by Strange {Brain Biochemistry and Brain Disorders, Oxford University Press, Oxford, 1992, p.4).

Figures 2 and 3 illustrate the verification of the differential expression of the human PLP gene, in particular of the PLP splice variant 1 (PLP), in AD brain tissues by quantitative RT-PCR analysis. Quantification of RT-PCR products from RNA samples collected from the frontal cortex (F) and the temporal cortex (T) of AD patients (Figure 2a) and samples from the frontal cortex (F) and the hippocampus (H) of AD patients (Figure 3a) was performed by the LightCycler rapid thermal cycling technique. Likewise, samples of healthy, age-matched control individuals were compared (Figure 2b for frontal cortex and temporal cortex, Figure 3b for frontal cortex and hippocampus). The data were normalized to the combined average values of a set of standard genes which showed no significant differences in their gene expression levels. Said set of standard genes consisted of genes for cyclophilin B, the ribosomal protein S9, the transferrin receptor, GAPDH, and beta- actin. The figures depict the kinetics of amplification by plotting the cycle number against the amount of amplified material as measured by its fluorescence. Note that the amplification kinetics of PLP splice variant 1 cDNAs from both, the frontal and temporal cortices of a normal control individual, and from the frontal cortex and hippocampus of a normal control individual, respectively, during the exponential phase of the reaction are juxtaposed (Figures 2b and 3b, arrowheads), whereas in Alzheimer's disease (Figures 2a and 3a, arrowheads) there is a significant separation of the corresponding curves, indicating a differential expression of the gene coding for PLP, in particular of the PLP splice variant 1 , in the respective analyzed brain regions, preferably an up-regulation of a transcription product of the human PLP gene, in particular of the PLP splice variant 1 , in the temporal cortex relative to frontal cortex, and in the hippocampus relative to the frontal cortex, respectively.

Figures 4 and 5 illustrate the verification of the differential expression of the human PLP gene, in particular of the PLP splice variant 2 (DM20), in AD brain tissues by quantitative RT-PCR analysis. Quantification of RT-PCR products from RNA samples collected from the frontal cortex (F) and the temporal cortex (T) of AD patients (Figure 4a) and samples from the frontal cortex (F) and the hippocampus (H) of AD patients (Figure 5a) was performed by the LightCycler rapid thermal cycling technique. Likewise, samples of healthy, age-matched control individuals were compared (Figure 4b for frontal cortex and temporal cortex, Figure 5b for frontal cortex and hippocampus). The data were normalized to the combined average values of a set of standard genes which showed no significant differences in their gene expression levels. Said set of standard genes consisted of genes for cyclophilin B, the ribosomal protein S9, the transferrin receptor, GAPDH, and beta- actin. The figures depict the kinetics of amplification by plotting the cycle number against the amount of amplified material as measured by its fluorescence. Note that the amplification kinetics of PLP splice variant 2 cDNAs from both, the frontal and temporal cortices of a normal control individual, and from the frontal cortex and hippocampus of a normal control individual, respectively, during the exponential phase of the reaction are juxtaposed (Figures 4b and 5b, arrowheads), whereas in Alzheimer's disease (Figures 4a and 5a, arrowheads) there is a significant separation of the corresponding curves, indicating a differential expression of the gene coding for PLP, in particular of the PLP splice variant 2, in the respective analyzed brain regions, preferably an up-regulation of a transcription product of the human PLP gene, in particular of the PLP splice variant 2, in the temporal cortex relative to frontal cortex, and in the hippocampus relative to the frontal cortex, respectively.

Figure 6 discloses SEQ ID NO. 1 , the amino acid sequence of the PLP protein (PLP splice variant 1 ) (NCBI GenBank accession number: P06905). The full length human PLP protein comprises 276 amino acids.

Figure 7 discloses SEQ ID NO. 2, the amino acid sequence of the DM20 protein (PLP splice variant 2) (NCBI GenBank accession number: AAH02665). The full length human DM20 protein comprises 242 amino acids. Figure 8 represents SEQ ID NO. 3, the nucleotide sequence of the human PLP cDNA (PLP splice variant 1 ). The length of the PLP cDNA according to NCBI GenBank entry M271 10 is 2777 base pairs.

Figure 9 represents SEQ ID NO. 4, the nucleotide sequence of the human PLP cDNA (PLP splice variant 2). The PLP cDNA, constructed from nucleotides 1 to 894 of GenBank entry BC002665 following nucleotides 972 to 2777 of NCBI GenBank entry M271 10, comprises 2700 base pairs.

Figure 10 depicts SEQ I D NO. 5, the nucleotide sequence of the 213 bp PLP cDNA fragment, identified and obtained by suppressive subtractive hybridization on DNA biochips and subsequent cloning.

Figure 1 1 outlines the sequence alignment of SEQ ID NO. 5, the 213 bp human PLP cDNA fragment, with SEQ ID NO. 3, the nucleotide sequence of the PLP cDNA (nucleotides 2522 to 2734, GenBank accession number M271 10).

Figure 12 charts the schematic alignment of SEQ ID NO. 5 with the PLP and the DM20 cDNA. The region of the coding sequences (eds) of the PLP and the DM20 cDNAs are represented. Dots indicate where the sequence differences between the PLP and the DM20 gene products are located. Thin bars represent the 5' and 3' untranslated regions (UTRs).

Table 1 discloses the initial identification of differential expression of the human PLP gene by subtractive suppressive microarray hybridization experiments. Identical biochips containing cDNA clones of subtracted AD and control brain cDNA libraries were co-hybridized with different Cyanine3 and Cyanineδ labeled cDNA probes, designated as probes A, B, or C, respectively. Cy3 and Cy5 labeled cDNA probes (A) were generated by labeling cDNAs from frontal or temporal cortex of AD patients and control persons, respectively, refer to section (vi-a). Cy3 and Cy5 labeled SMART probes (B) were generated from cDNAs, derived from frontal or temporal cortex of AD patients and control persons, respectively, refer to section (vi-b). Cy3 and Cy5 labeled SSH probes (C) were derived from cDNA populations after suppressive subtractive hybridization of brain cDNAs from frontal and temporal cortex of AD patients and of control individuals, respectively, refer to section (vi-c) (PFssHfD = AD patients frontal cortex cDNA after subtraction of AD patients temporal cortex cDNA; PT_SSH₍2) = AD patients temporal cortex cDNA after subtraction of AD patients frontal cortex cDNA). The table lists the gene expression level of PLP indicated as the ratio of fluorescence intensity measured for the temporal cortex relative to the frontal cortex of AD patients. The ratios of fluorescence intensity reflect a differential regulation of human PLP RNA expression in temporal and frontal cortex of AD patients.

Table 2 lists the gene expression levels in the temporal cortex relative to the frontal cortex for the PLP gene (splice variant 1 , PLP) in seven AD patients, herein identified by internal reference numbers P010, P01 1 , P012, P014, P016, P017, P019 (0.78 to 5.38 fold) and five healthy, age-matched control individuals, herein identified by internal reference numbers C005, C008, C011 , C012, C014 (0.48 to 1 .54 fold). The scatter diagram visualizes individual values of the temporal to frontal cortex regulation ratios in control samples (dots) and in AD patient samples (triangles), respectively. The values have been calculated according to the formula disclosed herein (see below).

Table 3 lists the gene expression levels in the hippocampus relative to the frontal cortex for the PLP gene (splice variant 1 , PLP) in six Alzheimer's disease patients, herein identified by internal reference numbers P010, P011 , P012, P014, P016, P019 (0.86 to 3.90 fold) and three healthy, age-matched control individuals, herein identified by internal reference numbers C004, C005, C008 (0.85 to 1 .08 fold). The scatter diagram visualizes individual values of the hippocampus to frontal cortex regulation ratios in control samples (dots) and in AD patient samples (triangles). The values have been calculated according to the formula disclosed herein (see below).

Table 4 lists the gene expression levels in the temporal cortex relative to the frontal cortex for the PLP gene (splice variant 2, DM20) in seven AD patients, herein identified by internal reference numbers P010, P01 1 , P012, P014, P016, P017, P019 (0.78 to 5.71 fold) and five healthy, age-matched control individuals, herein identified by internal reference numbers C005, C008, C01 1 , C012, C014 (0.64 to 1 .33 fold). The scatter diagram visualizes individual values of the temporal to frontal cortex regulation ratios in control samples (dots) and in AD patient samples (triangles), respectively. The values have been calculated according to the formula disclosed herein (see below). Table 5 lists the gene expression levels in the hippocampus relative to the frontal cortex for the PLP gene (splice variant 2, DM20) in six Alzheimer's disease patients, herein identified by internal reference numbers P010, P01 1 , P012, P014, P016, P019 (0.77 to 3.85 fold) and three healthy, age-matched control individuals, herein identified by internal reference numbers C004, C005, C008 (0.90 to 1 .53 fold). The scatter diagram visualizes individual values of the hippocampus to frontal cortex regulation ratios in control samples (dots) and in AD patient samples (triangles). The values have been calculated according to the formula disclosed herein (see below).

EXAMPLE I:

(i) Brain tissue dissection from patients with AD:

Brain tissues from AD patients and age-matched control subjects were collected within 6 hours post-mortem and immediately frozen on dry ice. Sample sections from each tissue were fixed in paraformaldehyde for histopathological confirmation of the diagnosis. Brain areas for differential expression analysis were identified (see Figure 1) and stored at -80 °C until RNA extractions were performed.

(ii) Isolation of total mRNA:

Total RNA was extracted from post-mortem brain tissue by using the RNeasy kit (Qiagen) according to the manufacturer's protocol. The accurate RNA concentration and the RNA quality were determined with the DNA LabChip system using the Agilent 2100 Bioanalyzer (Agilent Technologies). For additional quality testing of the prepared RNA, i.e. exclusion of partial degradation and testing for DNA contamination, specifically designed intronic GAPDH oligonucleotides and genomic DNA as reference control were used to generate a melting curve with the LightCycler technology as described in the manufacturer's protocol (Roche).

(iii) cDNA synthesis and Rsa I digestion

In order to identify changes in gene expression in different tissues, a screening method combining cDNA synthesis, suppressive subtractive hybridization (SSH) and screening of microarray chips with a diversity of cDNA probes from SSH was employed. This technique compares different populations of mRNA and provides clones of genes that are expressed in one population of cells but not, or at lower level, in the other population of cells. In the present invention, RNA populations from selected post-mortem brain tissues (frontal and temporal cortex) of AD patients and age-matched control subjects were compared.

As a starting material for the suppressive subtractive microarray analysis total RNA was extracted as described above (ii). For production of preferably full-length cDNAs, the polymerase chain reaction (PCR)-based method 'SMART cDNA Synthesis' was performed according to the manufacturer's protocol (Clontech). The principle of 'SMART cDNA synthesis' has been described in detail (Chenchik et al., in Gene Cloning and Analysis by RT-PCR, Eds. Siebert and Larrick, Biotechniques Books, Natick, MA 1998: 305-320). For SMART cDNA synthesis, four RNA pools, each consisting of 8 μg total RNA, were prepared. Each pool contained 2 μg of each of four different samples, i.e. from inferior frontal cortex (CF) and from inferior temporal cortex (CT) of control brains, from inferior frontal cortex (PF) and from inferior temporal cortex (PT) of patient brains, respectively. An amount of 1 μg of total RNA mix was utilized in a reaction volume of 50 μl (PCR cycler: Multi Cycler PTC 200, MJ Research). The second SMART PCR step was performed using 19 cycles. Superscript II RNaseH Reverse Transcriptase and 5x first-strand buffer (Invitrogen) were used.

After extraction and purification of the PCR products, restriction digestions were carried out with 30 U Rsa I (MBI Fermentas) for 2.5 hours at 37°C. Rsa I restriction sites are located within the universal priming sites of the double stranded (ds) cDNA. The quality of the digestions was analyzed by agarose gel electrophoresis, the digested samples were purified (QIAquick PCR Purification Kit, Qiagen), and the cDNA concentrations were determined by UV spectrophotometry (Biorad).

(iv) Suppressive subtractive hybridization (SSH)

Four SMART cDNA pools (iii), were compared using suppressive subtractive hybridization. A pool of cDNA containing differentially expressed genes is thereby designated as "tester", the reference cDNA pool as "driver". The two pools are hybridized, and all cDNAs, present in both pools, will be eliminated, i.e. the driver- pool will be subtracted from the tester-pool. Thus, clones of genes that are predominantly expressed in the tester population are obtained.

The 'PCR-Select cDNA Subtraction Kit' (Clontech) was used to perform the subtractive hybridization. The 'tester' SMART cDNA pools, derived from temporal cortex (CT) of control brains, and from frontal and temporal cortex (PF and PT) of patient brains (iii), were subdivided into two pools each. Each pool was ligated with adaptor 1 or adaptor 2, respectively, thus obtaining 6 different 'tester' cDNA pools. The three 'driver' SMART cDNA pools, CT, PF and PT, remained unligated. In a first hybridization step, used to enrich for differentially expressed sequences, the following three different 'tester' SMART cDNA pools were combined with an excess of the following 'driver' SMART cDNAs: SSH(1 ): PF-'tester' and PT-'driver'; SSH(2): PT-'tester' and PF-'driver'; SSH(3): CT-'tester' and PT-'driver'; SSH(4): PT-'tester' and CT-'driver'. Following a denaturation step for 1 .5 min at 98°C, the hybridization was carried out for 8 hours at 68°C. In a second step, the two corresponding primary hybridization samples of 'tester' SMART cDNA pools ligated to adaptor 1 or 2, respectively, were mixed and re-hybridized at 68°C for 15 hours, with an excess of the 'driver' SMART cDNA pool, as used before. Thus, suitable double stranded cDNAs for subsequent amplification, i.e. with both adaptor sequences at their 5' and 3' ends and therefore with different annealing sites, were generated. The following PCR steps were applied to obtain efficiently amplified specific products and to suppress nonspecific amplification. In the first PCR, missing strands of the adaptors . were filled in by DNA-polymerase activity. 1 μl of the obtained hybridization products each were subjected to PCR using the corresponding 'primer 1 ' (10 μM) (Clontech) along with 1 x PCR reaction buffer (Clontech), 10 mM dNTP-Mix (dATP, dGTP, dCTP, dTTP, Amersham Pharmacia Biotech), and 0.5 μl 50x Advantage cDNA Polymerase Mix (Clontech) in a 25 μl final volume. PCR conditions were set as follows: one round at 75 °C for 5 min, which was followed by 27 or 30 cycles: 94 °C for 30 sec, 64 °C or 66 °C for 30 sec, 72 °C for 1 .5 min. One final step at 72 °C for 5 min was added to the last cycle. A second nested PCR was performed as described for the first PCR, except that instead of 'primer 1 ' the nested primers 'nested primer 1 ' and '2R' were used and an annealing temperature of 66°C or 68°C and 12 or 15 cycles, were applied. PCR-products obtained by different conditions were pooled for subsequent analysis. For the primer sequences used, refer to appendix B of the supplier's user manual (Clontech).

(v) Cloning of subtracted PCR products and production of DNA-biochips The SSH SMART double stranded cDNAs of the four different combinations SSH(1 )- SSH(4), refer to (iv), were ligated into the pCR2.1 -vector and transformed into INValphaF' cells according to the manufacturer's instructions (TA Cloning Kit, Invitrogen). Bacterial colonies were picked and analyzed by colony PCR on MTPs (microtiter plates, 96 well, Abgene), using 'nested primer 1 ' and 'nested primer 2'. Those MTPs showing more than 90% positive clones were subjected to a preparative colony PCR approach. Per well, the following PCR mix was generated: the corresponding oligonucleotides 'nested primer 1 ' and 'nested primer 2' (0.5 μM each), 1 x Titanium PCR buffer (Clontech), 200 μM dNTP-Mix (Amersham Pharmacia Biotech), 0.2 x TitaniumTaq DNA-Polymerase (Clontech) in a 120 μl final volume. PCR conditions were set as follows: one round at 94 °C for 30 sec for denaturing, the next round was followed by 35 cycles: 94 °C for 30 sec and 68°C for 3 min. The quality of the amplified products was analyzed (DNA LabChip system, Agilent 2100 Bioanalyzer, Agilent Technologies), followed by purification (Multiscreen-PCR-Purification system, Millipore).

Additionally, the following standard control samples were generated: three different Arabidopsis thaliana genes, polyA-DNA, salmon sperm DNA, human Cot-1 DNA, and 3xSSC-buffer were used as negative controls (Microarray Validation System, Stratagene); beta-actin and Xenopus cDNA were used as normalizing controls. Several MTPs were made of each of the SSH combinations SSH(1 )-(4), harboring amplification products of 96 different clones per plate. The amplified products were spotted in triplicates onto GAPS glass-slides (CMT-GAPS, Corning) by GeneScan Europe.

(vi) Probe synthesis and identification of differentially expressed genes by screening of DNA biochips a) DNA probe synthesis

As starting material for the generation of Cyanine3 (Cy3) and Cyanineδ (Cy5) labeled cDNA probes total RNA was extracted and used as described above (ii) and (iii). Two samples of a mix of 2 μg of total RNA and additionally 2 ng Xenopus RNA (standard RNA) per labeling reaction, were subjected to a specific reverse transcriptase reaction, whereby the polyA-mRNA is converted into fluorescein-12- dCTP (FL) or biotin-1 1 -dCTP (B) labeled cDNA. The RNA samples derived from frontal cortex (CF) of control brains were labeled with fluorescein, the RNA from the temporal cortex (PT) of patient brains with biotin, respectively. The cDNA reactions were performed according to the Micromax TSA Labeling protocol (NEN Life Science). The purified cDNA probes were resuspended in hybridization buffer and denatured for 7 min at 100°C. Subsequently, half the volume of the fluorescein- labeling reaction (i.e. 1 μg RNA) and half the amount of the biotin-probe were mixed together in 5xSSC, 0.1 % SDS, 25% formamide buffer, and applied evenly onto one prehybridized (5x SSC, 0.1 % SDS, 1 % BSA, 45 min at 42°C) microarray. Array hybridization was performed over night at 42°C. After high stringency washing, the detection of the bound fluorescein- and biotin labeled probes was performed according to the instructions of the TSA Detection Kit protocol (NEN Life Science). Thereby, in a first step, anti-FL-HRP (fluorescein- horseradish peroxidase) binds to the FL-labeled cDNA probe, and HRP catalyzes the deposition of the fluorescent reporter Cy3 tyramide. In a second step, streptavidin-HRP binds to the B-labeled cDNA probe and catalyzes the deposition of the fluorescent reporter molecule Cy5 tyramide. Biochip 3 was hybridized with cDNA mix PF(Cy3) and PT(Cy5). Scanning the microarrays with the appropriate wavelengths (635 nm, 532 nm) allowed detection of both cyanine dyes simultaneously. b) SMART probe synthesis

For the production of Cyanine3 (Cy3) and Cyanineδ (Cy5) labeled SMART cDNA- probes, the PCR-based method 'SMART cDNA Synthesis' was performed as described in section (iii). Here, we used total RNA as starting material, which had been extracted as described above (ii). Four RNA mixtures were prepared as described in section (iii). 1 μg of each RNA mix and 1 ng Xenopus total RNA were subjected to the SMART cDNA reaction. For PCR amplification, extraction and purification of the cDNAs, restriction digestion with Rsa I, and subsequent purification of the digested samples, refer to section (iii).

SMART cDNA samples were labeled with either Cy3 or Cy5 (Atlas Glass Fluorescent Labeling Kit, Clontech). In the first labeling step, aliphatic amino groups, i.e. aminoallyl-dUTP (Clontech), were incorporated into denatured (100°C, 7 min) Rsa I digested PCR products. The reaction was catalyzed by the Klenow Fragment (MBI Fermentas). In a second labeling step, the fluorescent reporter dyes Cy3 or Cy5 were coupled to the incorporated functionalities. The purified Cy3 and Cy5 labeled SMART cDNA probes (Atlas NucleoSpin Extraction Kit, Clontech) were resuspended in hybridization buffer (5x SSC, 0.1 % SDS, 25% formamide) after denaturation for 7 min at 100°C. Subsequently, the Cy3 labeled SMART probe was mixed with the Cy5 labeled SMART probe and together applied evenly onto one prehybridized (5x SSC, 0.1 % SDS, 1 % BSA, 45 min at 42°C) microarray. Array hybridization was performed over night at 42°C. High stringency washing of the biochips followed according to the instructions of the TSA Detection Kit protocol (NEN Life Science). Biochip 2 was hybridized with SMART cDNA mix PF(Cy3) and PT(Cy5). Scanning the microarrays with the appropriate wavelengths (635 nm, 532 nm) allowed detection of both cyanine dyes simultaneously. c) Subtraction probe synthesis For the production of Cyanine3 (Cy3) and Cyanineδ (Cy5) labeled SSH cDNA- probes, the PCR-based method 'SMART cDNA Synthesis' was performed as described in section (iii). Here, we used total RNA as starting material, which was extracted as described above (ii). Four RNA mixtures were prepared as disclosed in section (iii). 1 μg of each RNA mix was subjected to the SMART cDNA reaction. For PCR amplification, extraction and purification of the cDNAs, restriction digestion with Rsa I, and subsequent purification of the digested samples, refer to section (iii). For subtractive hybridization, the PCR-Select cDNA Subtraction Kit (Clontech) was utilized as described in detail in section (iv). The subtracted PCR products of the combinations SSH(1 ) and SSH(2), and of SSH(3) and SSH(4), respectively, were purified (StrataClean Kit, Stratagene), and adaptor 1 and 2 removed by restriction digest with Rsa I and Sma I (MBI Fermentas). The SSH cDNA pools were labeled with either Cy3 or Cy5 (Atlas Glass Fluorescent Labeling Kit, Clontech). In the first labeling step, aliphatic amino groups, i.e. aminoallyl-dUTP (Clontech), were incorporated into the denatured (100°C, 7 min) Rsa I and Sma I digested SSH cDNA products. The reaction was catalyzed by the Klenow Fragment (MBI Fermentas). In a second labeling step, the fluorescent reporter dyes Cy3 and Cy5 were coupled to the incorporated functionalities.

The purified Cy3 and Cy5 labeled SSH cDNA probes (for purification refer to the Atlas NucleoSpin Extraction Kit, Clontech) were resuspended in hybridization buffer (5x SSC, 0.1 % SDS, 25% formamide) after denaturation for 7 min at 100°C. Subsequently, the Cy3 labeled SSH1 probe was mixed with the Cy5 labeled SSH2 probe, and the Cy3 labeled SSH3 probe with the Cy5 labeled SSH4 probe, respectively. Each combination was applied evenly onto one prehybridized (5x SSC, 0.1 % SDS, 1 % BSA, 45 min at 42°C) microarray. Array hybridization was performed over night at 42°C. High stringency washing of the biochips followed according to the instructions of the TSA Detection Kit protocol (NEN Life Science). Biochip 1 was hybridized with the cDNA mix SSH(1 )(Cy3) and SSH(2)(Cy5). Scanning of the microarrays with the appropriate wavelengths (635 nm, 532 nm) allowed detection of both cyanine dyes simultaneously.

(vii) DNA biochips data evaluation

Fluorescence raw data for Cy3 and Cy5, measured at 635 and 532 nm, respectively, were taken severalfold (for each of the three spots per cDNA). One set of measurements was performed within the spot area (signal) and another set of measurements was taken nearby (background). Subsequently the net fluorescence intensity (Fl₆₃₅, Fl₅₃₂) of the spots was calculated as follows:

Flβ35/532 ⁼ (M FI_Spot - 1 SD FI_Spot) - (M F lbac ground ⁺ 1 S D F lbackground)-

According to the above formula, M defines the median of the replicate measurements per spot, SD the standard deviation of the corresponding mean. Subsequently, only Fl₆₃₅ and FI5₃₂ values of >2 were considered for further evaluation plus those Fl₆35 and FI53₂ values of <2 where the corresponding value for the second wavelength was >3.

In an analogous manner, the corresponding values for the Xenopus cDNA control and the set of standard (housekeeping) genes were evaluated. The Xenopus cDNA was used as an internal calibrator for the efficiency of cDNA synthesis of the disease relevant mRNAs. Then, from the background corrected FI6₃5_/53₂ medians of the three replicate spots, the statistical mean was calculated and the signal ratio R for the cDNA probes was derived using formula:

Rβ35/532 ⁼ F 35, calibrated FI532, calibrated.

In a last step of evaluation, the results of the different hybridizations were considered for logical coherence.

(viii) Confirmation of differential expression by quantitative RT-PCR Positive corroboration of differential expression of the PLP gene, splice variant 1 (PLP) and splice variant 2 (DM20), was performed using the LightCycler technology (Roche). This technique features rapid thermal cyling for the polymerase chain reaction as well as real-time measurement of fluorescent signals during amplification and therefore allows for highly accurate quantification of RT-PCR products by using a kinetic, rather than an endpoint readout. The ratios of PLP cDNA from the temporal cortex and frontal cortex, and from the hippocampus and frontal cortex, respectively, were determined (relative quantification).

First, a standard curve was generated to determine the efficiency of the PCR with specific primers for the PLP gene splice variant 1 (PLP): 5'-GGGATGTCCTAGCCATTTTCC-3' and 5'-TCCAGAGGCCAACATCAAGC-3' and with specific primers for the PLP gene splice variant 2 (DM20): 5'-AGGTGATGCCCACAAACG-3' and 5'-TGCTGGCTGAGGGCTTCTA-3'. PCR amplification (95 °C and 1 sec, 56 °C and 5 sec, and 72 °C and 5 sec) was performed in a volume of 20 μl containing LightCycler-FastStart DNA Master SYBR Green I mix (contains FastStart Taq DNA polymerase, reaction buffer, dNTP mix with dUTP instead of dTTP, SYBR Green I dye, and 1 mM MgCI₂; Roche), 0.5 μM primers, 2 μl of a cDNA dilution series (final concentration of 40, 20, 10, 5, 1 and 0.5 ng human total brain cDNA; Clontech) and, depending on the primers used, additional 3 mM MgCI₂. Melting curve analysis revealed a single peak at approximately 82.5 °C for the PLP splice variant 1 -primers and at approximately 88.5 °C for the PLP splice variant 2-primers with no visible primer dimers. Quality and size of the PCR product were determined with the DNA LabChip system (Agilent 2100 Bioanalyzer, Agilent Technologies). A single peak at the expected size of 72 bp and of 108 bp, respectively, for the PLP gepe was observed in the electropherogram of the samples.

In an analogous manner, the PCR protocol was applied to determine the PCR efficiency of a set of reference genes which were selected as a reference standard for quantification. In the present invention, the mean value of five such reference genes was determined: (1 ) cyclophilin B, using the specific primers 5'- ACTGAAGCACTACGGGCCTG-3' and 5'-AGCCGTTGGTGTCTTTGCC-3' except for MgCI₂ (an additional 1 mM was added instead of 3 mM). Melting curve analysis revealed a single peak at approximately 87 °C with no visible primer dimers. Agarose gel analysis of the PCR product showed one single band of the expected size (62 bp). (2) Ribosomal protein S9 (RPS9), using the specific primers 5'- GGTCAAATTTACCCTGGCCA-3' and 5'- TCTCATCAAGCGTCAGCAGTTC-3' (exception: additional 1 mM MgCI₂ was added instead of 3 mM). Melting curve analysis revealed a single peak at approximately 85°C with no visible primer dimers. Agarose gel analysis of the PCR product showed one single band with the expected size (62 bp). (3) beta-actin, using the specific primers 5'- TGGAACGGTGAAGGTGACA-3' and 5'-GGCAAGGGACTTCCTGTAA-3'. Melting curve analysis revealed a single peak at approximately 87°C with no visible primer dimers. Agarose gel analysis of the PCR product showed one single band with the expected size (142 bp). (4) GAPDH, using the specific primers 5'- CGTCATGGGTGTGAACCATG-3' and 5'-GCTAAGCAGTTGGTGGTGCAG-3'. Melting curve analysis revealed a single peak at approximately 83°C with no visible primer dimers. Agarose gel analysis of the PCR product showed one single band with the expected size (81 bp). (5) Transferrin receptor TRR, using the specific primers 5'- GTCGCTGGTCAGTTCGTGATT-3' and 5'-AGCAGTTGGCTGTTGTACCTCTC-3'. Melting curve analysis revealed a single peak at approximately 83°C with no visible primer dimers. Agarose gel analysis of the PCR product showed one single band with the expected size (80 bp).

For calculation of the values, first the logarithm of the cDNA concentration was plotted against the threshold cycle number C_t for PLP, i.e. for the PLP splice variant 1 and the PLP splice variant 2, respectively, and the five reference standard genes. The slopes and the intercepts of the standard curves (i.e. linear regressions) were calculated for all genes. In a second step, cDNAs from temporal cortex and frontal cortex, and from hippocampus and frontal cortex, respectively, were analyzed in parallel and normalized to cyclophilin B. The C_t values were measured and converted to ng total brain cDNA using the corresponding standard curves:

10 ^{Λ '}( (C_t value - intercept) / slope ) [ng total brain cDNA]

The values for temporal and frontal cortex cDNAs of PLP, and the values for hippocampus and frontal cortex PLP cDNAs, i.e. of the PLP splice variant 1 and of the PLP splice variant 2, respectively, were normalized to cyclophilin B, and the ratios were calculated according to formulas:

PLP temporal [ng] / cyclophilin B temporal [ng]

Ratio = PLP frontal [ng] / cyclophilin B frontal [ng]

PLP hippocampus [ng] / cyclophilin B hippocampus [ng]

Ratio = PLP frontal [ng] / cyclophilin B frontal [ng]

In a third step, the set of reference standard genes was analyzed in parallel to determine the mean average value of the temporal to frontal ratios, and of the hippocampal to frontal ratios, respectively, of expression levels of the reference standard genes for each individual brain sample. As cyclophilin B was analyzed in step 2 and step 3, and the ratio from one gene to another gene remained constant in different runs, it was possible to normalize the values for PLP, i.e. for the PLP splice variant 1 (PLP) and for the PLP splice variant 2 (DM20), respectively, to the mean average value of the set of reference standard genes instead of normalizing to one single gene alone. The calculation was performed by dividing the respective ratio shown above by the deviation of cyclophilin B from the mean value of all housekeeping genes. The results of such quantitative RT-PCR analysis for the PLP gene, for the PLP splice variant 1 (PLP) and for the PLP splice variant 2 (DM20), are shown in Figures 2, 3 and in Figures 4 and 5, respectively.

Claims

1. A method of diagnosing or prognosticating a neurodegenerative disease in a subject, or determining whether a subject is at increased risk of developing said disease, comprising: determining a level and/or an activity of

(i) a transcription product of a gene coding for PLP, and/or

(ii) a translation product of a gene coding for PLP and/or

(iii) a fragment, or derivative, or variant of said transcription or translation product, in a sample from said subject and comparing said level and/or said activity to a reference value representing a known disease or health status, thereby diagnosing or prognosticating said neurodegenerative disease in said subject, or determining whether said subject is at increased risk of developing said neurodegenerative disease.

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

3. A kit for diagnosing or prognosticating a neurodegenerative disease, in particular Alzheimer's disease, in a subject, or determining the propensity or predisposition of a subject to develop such a disease, said kit comprising:

(a) at least one reagent which is selected from the group consisting of (i) reagents that selectively detect a transcription product of a gene coding for PLP and (ii) reagents that selectively detect a translation product of a gene coding for PLP and

(b) an instruction for diagnosing, or prognosticating a neurodegenerative disease, in particular Alzheimer's disease, or determining the propensity or predisposition of a subject to develop such a disease by (i) detecting a level, or an activity, or both said level and said activity, of said transcription product and/or said translation product of a gene coding for PLP, in a sample from said subject; and (ii) diagnosing or prognosticating a neurodegenerative disease, in particular Alzheimer's disease, or determining the propensity or predisposition of said subject to develop such a disease, wherein a varied level, or activity, or both said level and said activity, of said transcription product and/or said translation product compared to a reference value representing a known health status; or a level, or activity, or both said level and said activity, of said transcription product and/or said translation product similar or equal to a reference value representing a known disease status indicates a diagnosis or prognosis of a neurodegenerative disease, in particular Alzheimer's disease, or an increased propensity or predisposition of developing such a disease.

4. A modulator of an activity and/or of a level of at least one substance, which is selected from the group consisting of

(i) a gene coding for PLP and/or

(ii) a transcription product of a gene coding for PLP and/or

(iii) a translation product of a gene coding for PLP, and/or

(iv) a fragment, or derivative, or variant of (i) to (iii).

5. An assay for screening for a modulator of neurodegenerative diseases, in particular Alzheimer's disease, or related diseases or disorders of one or more substances selected from the group consisting of

(i) a gene coding for PLP, and/or

(ii) a transcription product of a gene coding for PLP, and/or

(iii) a translation product of a gene coding for PLP, and/or

(iv) a fragment, or derivative, or variant of (i) to (iii), said method comprising:

(a) contacting a cell with a test compound;

(b) measuring the activity and/or level of one or more substances recited in (i) to

(iv);

(c) measuring the activity and/or level of one or more substances recited in (i) to (iv) in a control cell not contacted with said test compound; and

(d) comparing the levels and/or activities of the substance in the cells of step (b) and (c), wherein an alteration in the activity and/or level of substances in the contacted cells indicates that the test compound is a modulator of said diseases or disorders.

6. A method of screening for a modulator of neurodegenerative diseases, in particular Alzheimer's disease, or related diseases or disorders of one or more substances selected from the group consisting of

(i) a gene coding for PLP, and/or

(ii) a transcription product of a gene coding for PLP, and/or (iii) a translation product of a gene coding for PLP, and/or (v) a fragment, or derivative, or variant of (i) to (iii), said method comprising:

(a) administering a test compound to a test animal which is predisposed to developing or has already developed symptoms of a neurodegenerative disease or related diseases or disorders, in particular Alzheimer's disease, in respect of the substances recited in (i) to (iv);

(b) measuring the activity and/or level of one or more substances recited in (i) to (iv);

(c) measuring the activity and/or level of one or more substances recited in (i) or (iv) in a matched control animal which is predisposed to developing or has already developed symptoms of a neurodegenerative disease or related diseases or disorders, in particular Alzheimer's disease, in respect to the substances recited in (i) to (iv) and to which animal no such test compound has been administered;

(d) comparing the activity and/or level of the substance in the animals of step (b) and (c), wherein an alteration in the activity and/or level of substances in the test animal indicates that the test compound is a modulator of said diseases or disorders.

7. The method according to claim 6 wherein said test animal and/or said control animal is a recombinant animal which expresses a gene coding for PLP, or a fragment, or a derivative, or a variant thereof, under the control of a transcriptional control element which is not the native PLP gene transcriptional control element.

8. An assay for testing a compound, preferably for screening a plurality of compounds to determine the degree of binding of said compounds to a PLP translation product, or to a fragment, or derivative, or variant thereof, said assay comprising the steps of:

(i) adding a liquid suspension of said PLP translation product, or a fragment, or derivative, or variant thereof, to a plurality of containers; (ii) adding a detectable, in particular a fluorescently labeled compound or a plurality of detectable, in particular fluorescently labeled compounds to be screened for said binding to said plurality of containers; (iii) incubating said PLP translation product, or said fragment, or derivative, or variant thereof, and said detectable, in particular fluorescently labeled compound or fluorescently labeled compounds; (iv) measuring amounts of preferably fluorescence associated with said PLP translation product, or with said fragment, or derivative, or variant thereof; and (v) determining the degree of binding by one or more of said compounds to said

PLP translation product, or said fragment, or derivative, or variant thereof.

9. A protein molecule, said protein molecule being a translation product of the gene coding for PLP, SEQ ID NO. 1 , SEQ ID NO. 2, or a fragment, or derivative, or variant thereof, for use as a diagnostic target for detecting a neurodegenerative disease, in particular Alzheimer's disease.

10. A protein molecule, said protein molecule being a translation product of the gene coding for PLP, SEQ ID NO. 1 , SEQ ID NO. 2, or a fragment, or derivative, or variant thereof, for use as a screening target for reagents or compounds preventing, or treating, or ameliorating a neurodegenerative disease, in particular Alzheimer's disease.

11. Use of an antibody specifically immunoreactive with an immunogen, wherein said immunogen is a translation product of a gene coding for PLP, SEQ ID NO. 1 , SEQ ID NO. 2, or a fragment, or derivative, or variant thereof, for detecting the pathological state of a cell in a sample from a subject, comprising immunocytochemical staining of said cell with said antibody, wherein an altered degree of staining, or an altered staining pattern in said cell compared to a cell representing a known health status indicates a pathological state of said cell.