JP2006518199A - Diagnostic and therapeutic use of SCN2B protein for neurodegenerative diseases - Google Patents

Abstract

The present invention discloses differential expression of a gene encoding SCN2B in specific brain regions of Alzheimer's disease patients. Based on this discovery, the present invention provides a method of diagnosing or determining the prognosis of a neurodegenerative disease, particularly Alzheimer's disease in a subject, or determining whether a subject is at increased risk of developing the disease. . Furthermore, the present invention provides therapeutic and prophylactic methods for treating or preventing Alzheimer's disease and related neurodegenerative diseases using the SCN2B gene and the corresponding gene product. A method for screening for a modulator of a neurodegenerative disease is also disclosed.

Description

  The present invention relates to a method for diagnosing progression of a neurodegenerative disease in a subject, determining its prognosis, and monitoring. Further provided are methods of therapeutic control and screening for modulators of neurodegenerative diseases. The present invention also discloses pharmaceutical compositions, kits, and recombinant non-human animal models.

  Neurodegenerative diseases, particularly Alzheimer's disease (AD), have a strongly debilitating effect on patient life. In addition, these diseases represent a very large health, social and economic burden. AD is the most common neurodegenerative disease, accounting for about 70% of all dementia cases, affecting about 10% of the population over the age of 65 and up to 45% of the population over the age of 85 Probably the most devastating age-related neurodegenerative condition (for a recent review, see Vickers et al., Progress in Neurobiology 2000, 60: 139-165). Currently, an estimated 12 million cases are reached in the United States, Europe, and Japan. This situation will inevitably worsen as the number of older people grows demographically in developed countries (the aging of “baby boomers”). Prominent neuropathological features occurring in the brain of individuals suffering from AD are senile plaques composed of amyloid-β protein, cytoskeletal changes and neurofibrillary tangles that coincide with the appearance of abnormal filamentous structures. Formation.

  Amyloid-β (Aβ) peptides are produced by cleavage of amyloid precursor protein (APP) by various types of proteases. Cleavage by secretase β / γ forms Aβ peptides of different lengths, typically short, more soluble, slowly aggregated peptides consisting of 40 amino acids, and rapidly aggregated extracellular peculiar amyloid A long 42 amino acid peptide is formed that forms a plaque (Selkoe, Physiological Rev 2001, 81: 741-66; Greenfield et al., Frontiers Bioscience 2000, 5: D72-83). Two types of diffuse plaques and neuritic plaques can be detected in the brains of AD patients, the latter being classic and the most common type. They are mainly found in the cerebral cortex and hippocampus. Senile plaques have a diameter of 50-200 μm and are insoluble fibrillar amyloid, fragments and other components of dead neurons of microglia and astrocytes, such as neurotransmitters, apolipoprotein E, glycosami It is composed of noglycan and α1-antichymotrypsin. It is argued that the formation of toxic Aβ deposits in the brain plays a major role in the subsequent destructive processes that begin very early in the course of AD and lead to AD symptoms. Other pathological hallmarks of AD are neurofibrillary tangles (NFT) and abnormal neurites described as neurotropic threads (Braak and Braak, Acta Neuropathol 1991, 82: 239-259). NFTs consist of chemically altered tau proteins that appear inside neurons and form paired helical filaments that are more closely fitted around each other. As NFTs form, neuronal loss is observed. It has been argued that the neuronal loss may be due to microtubule-associated transport systems (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 the increase in their number correlate well with the clinical importance of AD (Schmitt et al., Neurology 2000, 55: 370-376).

  AD is a progressive disease associated with an early deficit in memory formation, and ultimately higher cognitive functions are completely compromised. Cognitive impairment includes, among other things, memory impairment, aphasia, agnosia, and reduced executive functioning. A unique feature of the pathogenesis of AD is the selective vulnerability of specific brain regions and neuronal subpopulations to the degenerative process. Specifically, during the course of the disease, the temporal lobe area and the hippocampus are affected early and more severely. On the other hand, neurons in the frontal cortex, occipital cortex, and cerebellum remain largely intact and protected from neurodegeneration (Terry et al., Annals of Neurology 1981, 10: 184-92). ). The age of onset of AD varies within a range of 50 years, with early onset AD occurring in humans under age 65 and late onset AD occurring in humans over 65 years old. About 10% of all AD cases suffer from early onset AD, and only 1-2% are familial and hereditary cases. There is currently no cure for AD, and there is no effective treatment to stop AD progression, or even a high probability of diagnosing AD prematurely. Several risk factors that predispose individuals to developing AD, most notably the epsilon 4 allele of three different existing alleles of the apolipoprotein E gene (ApoE) (epsilon 2, 3, and 4) The gene has been identified (Strittmatter et al., Proc Natl Acad Sci USA 1993, 90: 1977-81; Roses, Ann NY Acad Sci 1998, 855: 738-43). The polymorphic plasma protein ApoE appears to play a role in the transport of cholesterol and phospholipids between cells by binding to low density lipoprotein receptors and in neurite growth and regeneration. Efforts to detect additional susceptibility genes and disease-related polymorphisms have led to the hypothesis that specific regions and genes on human chromosomes 10 and 12 are 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).

  Rare examples of early-onset AD due 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. Although, the dominant form of late-onset sporadic AD has been derived from an unknown etiology to date. The late onset and complex pathogenesis of neurodegenerative diseases pose challenges to the development of therapeutic and diagnostic agents. It is important to expand the pool of potential drug targets and diagnostic markers. Accordingly, the object of the present invention is to provide methods, materials, drugs, compositions, and non-human animal models that provide insight into the etiology of neurological diseases and that are particularly suitable for developing diagnostics and therapeutics for these diseases. Is to provide. This object is solved by the features of the independent claims. The subclaims define preferred embodiments of the invention.

  The present invention is based on differential expression of genes encoding voltage-gated sodium channel type II β subunits (beta-2 subunit, β2-subunit, β2 or SCN2B) in Alzheimer's disease human brain samples. Thus, the SCN2B gene and corresponding transcripts and / or translation products thereof may have a causative role in local selective neuronal degeneration commonly found in AD. Based on these disclosures, the present invention has utility for diagnostic evaluation and prognosis of neurodegenerative diseases, particularly AD, and identification of its predisposition. Furthermore, the present invention provides a method for diagnostically monitoring patients undergoing treatment for such diseases.

  Voltage-gated ion channels play an important role by generating a conducted action potential. Cation (sodium, calcium, potassium ions) and anion (chlorine ion) ion-conducting membrane channels have been described (Lehmann-Horn et al., Physiological Reviews 1999, 79: 1317-1358). The transport of ions across the cell membrane transmits electrical impulses at high speed throughout the cell network. Thereby, the channel switches between three functionally different states: quiescent state, active state and inactive state. Both stationary and inactive states are non-conductive and the channel is closed. As the membrane potential increases from below −60 mV, the channel begins to open its pores (ie, activation). The influx of ions (eg, sodium) further increases the membrane potential until an action potential is generated. By closing the pores within 1 millisecond (ie, rapid inactivation) or within seconds to minutes (ie, slow inactivation), the channel quickly returns to the inactivated state. Ion conductance is very selective and efficient so that processes such as memory, movement and cognition can be fine-tuned (Lehmann-Horn et al., Physiological Reviews 1999, 79: 1317-1358). Molecular cloning of voltage-gated ion channels reveals subtype diversity, particularly improved understanding of the structure and function underlying the sodium channel (Noda et al., Nature 1986, 322: 826- 828; Schaller et al., Journal of Neuroscience 1995, 15: 3231-3242; Isom et al., Neuron 1994, 12: 1183-1194; Isom et al., Cell 1995, 83: 443-445).

  The voltage-gated sodium channel is composed of a pore-forming α-subunit of about 260 kDa and two small auxiliary β-subunits that form a heteromeric complex, about 36 kDa β1 and about 33 kDa β2. Is done. The α-subunit and both β-subunits are highly glycosylated (deglycosylated subunits having about 220 kDa, about 23 kDa, about 21 kDa for α, β1, and β2, respectively). To date, three different genes encoding the β-subunit have been identified. The β1-subunit is non-covalently bound to the α-subunit, whereas the β2-subunit is covalently bound to the α-subunit via a disulfide bridge. A third β-subunit isoform (β3), similar to the β1-subunit, was recently discovered (Morgan et al., Proceedings National Academy of Science USA 2000, 97: 2308-2313). The α-subunit appears to be necessary and sufficient for sodium channel functionality. The β-subunit modulates sodium channel function by altering the voltage dependence by increasing the rate of activation by accelerating the rate of inactivation, increasing the Na + amplitude peak current (Patton et al., Journal Biological Chemistry 1994, 269: 17649-17655). The β2-subunit appears to be less effective than the β1-subunit. Thus, both can cooperate in regulating the function of the α-subunit.

  The human β2-subunit is a glycoprotein with a 29 amino acid signal peptide sequence that is cleaved during protein maturation. In addition, the protein contains a large intracellular amino-terminal extracellular, containing a conserved cysteine residue that can form a disulfide bond with a short intracellular C-terminal segment, a single transmembrane region, and an α-subunit. Domain. The β-subunit represents an immunoglobulin-like motif, ie, an immunoglobulin-like C2-type domain, that has structural similarity to a cell adhesion molecule (CAM), such as contactin. Thus, the β2-subunit can interact with extracellular matrices such as tenascin-C and tenascin-R (Isom et al., Cell 1995, 83: 443-445). Cell adhesion molecules such as neuronal CAM (NCAM) and myelin-linked glycoprotein (MAG) have the same type of immunoglobulin fold as the β2-subunit. β2-subunits differ from other β-subunits in that they feature a CAM motif and in the ability to locally expand cell surface membranes. Thus, in addition to being sodium channel modifiers, they function in cell-cell adhesion interactions as regulators of sodium channel expression, and in certain regions of the plasma membrane Localization and immobilization can be regulated. Human β2 has similarities to the major structural components of myelin protein zero (p0), transmembrane glycoproteins and peripheral nerve myelin (Eubanks et al., Neuroreport 1997, 8: 2775-2779).

  The genomic structure of the SCN2B gene was reported in 1998 (GenBank accession number AF049496; AF049497; Bolino et al., European Journal of Human Genetics 1998, 6: 629-634; and GenBank accession number AF049898; complete coding sequence and equivalent Protein sequence). Mapping studies have mapped SCN2B to chromosome 11q23 (Eubanks et al., Neuroreport 1997, 8: 2775-2779). SCN2B spans approximately 14 kb of genomic DNA, with four exons and three introns, encoding a 186 amino acid mature SCN2B protein and a 215 amino acid SCN2B protein containing a 29 amino acid putative signal peptide (protein sequence and Putative domain: GenBank accession number O60939). Human and rat SCN2B proteins are equally long and highly conserved (89% homology and 93% similarity).

  The four sodium channels in the brain, SCN1A, SCN2A, SCN3A, and SCN8A, show major expression in the central nervous system. The mRNAs of the β1 and β2-subunits (approximately 2 kb and approximately 4 kb transcripts, respectively) have a wide but unique local expression pattern. Unlike the β1-subunit encoding the SCN1B gene, the SCN2B gene is expressed only in the central nervous system. Predominant expression levels were found in Purkinje cells in the parahippocampal, cortex, granule layer, and cerebellum. In rat brain, β2 expression begins at 9 days of fetal age, parallel to neurogenesis, and increases rapidly with axonal outgrowth and synapse formation (Isom et al., Cell 1995, 83: 443-445). .

  For the neuronal sodium channel subunit genes, SCN1A and SCN1B, disease-causing mutations have been identified that cause systemic epilepsy with febrile seizure plus (GEFS +). Thus, it is speculated that mutations in the SCN2B gene may also result in a common subtype of idiopathic systemic epilepsy. Haug and co-workers have identified a novel single nucleotide polymorphism (SNP) and a missense mutation (Asn209Pro) in the SCN2B gene, but have excluded the SCN2B gene as a major susceptibility gene in tantrum (Haug et al. , Neuroreport 2000, 11: 2687-2689). International Publication Nos. WO02 / 19966, WO02 / 087419, and WO02 / 090532 associate sodium channel β-subtypes with cardiovascular, inflammatory and myopathies, cancer and moxibustion. Based on protein homology and chromosomal positioning, SCN2B is also a candidate gene suitable for demyelinating autosomal recessive motility and sensory neuropathy, called Charcot-Marie-Tooth disease (CMT4B) It is regarded. This view, however, was advocated by Bolino and coworkers (Bolino et al., European Journal of Human Genetics 1998, 6: 629-634). Nevertheless, International Publication No. WO 01/79547 describes a single nucleotide polymorphism in the SCN2B gene that may be associated with demyelinating disease. At present, there is no report on the relationship between voltage-gated ion channel type II β2-subunit and neurodegenerative diseases such as Alzheimer's disease.

  Sodium channels are useful targets for a variety of drugs as local anesthetics, anticonvulsants, and antiarrhythmic drugs to treat neuropathic pain, jerking, and stroke. Many toxins, drugs, and inorganic cations are used in the pharmaceutical industry as blockers of sodium channel α-subunits in diseases related to the central nervous system. Inhibitors of voltage-gated ion channels are already commercially available despite their low potency, relatively non-specificity, and their therapeutic potential has not been fully exploited. Of note, there are currently no known drugs that interact with the β-subunit of the sodium channel. Accordingly, it is desirable to find drugs specific for selective subunits of voltage-gated sodium channels such as the β2-subunit.

  The present invention discloses dysregulation of voltage-gated sodium channel β-2 subunit (β2-subunit, SCN2B) gene expression in brain samples from Alzheimer's disease patients. Such dysregulation is not observed in samples obtained from age-matched, healthy controls. To date, no experiments have been described that demonstrate a link between dysregulation of β2-subunit gene expression and Alzheimer's disease and related neurodegenerative diseases. Similarly, it has not been described that mutations in the β2-subunit gene are associated with the disease. As disclosed in the present invention, associating the β2-subunit gene with Alzheimer's disease provides a novel method for diagnosing and treating in particular said disease.

  As used in the specification and claims, the singular forms “a”, “an”, and “the” include the plural unless the context clearly dictates otherwise. For example, “(one) cell” includes a plurality of cells. As used herein and in the claims, the term “and / or” means that the phrase before and after this term should be considered as an alternative or in combination. For example, the phrase “determining level and / or activity” indicates that only level, or only activity, or both level and activity are determined. As used herein, the term “level” is meant to include the gage, measure, amount, or concentration of a transcript, eg, mRNA, or translation product, eg, protein or polypeptide. . The term “activity” as used herein should be understood as a measure of the ability of a transcript or translation product to produce a biological effect or the level of a bioactive molecule. The term “activity” also means enzymatic activity. The terms “level” and / or “activity” as used herein further mean gene expression level or gene activity. Gene expression can be defined as the use of information contained in a gene by transcription and translation that causes the production of the gene product. “Dysregulation” should mean upregulation or downregulation of gene expression. A gene product includes either RNA or protein and is the result of expression of a gene. The amount of gene product can be used to measure how active the gene is. The term “gene” as used herein and in the claims includes both coding regions (exons) and non-coding regions (eg, promoters or enhancers, introns, leaders and trailer sequences). The term “ORF” is an acronym for “open reading frame” and means a nucleic acid sequence that does not carry a stop codon in at least one reading frame and therefore can potentially be translated into a sequence of amino acids. “Regulators” include inducible and non-inducible promoters, enhancers, operators and other elements that manipulate and regulate gene expression. The term “fragment” as used herein is meant to include, for example, alternatively spliced, truncated, or cleaved transcripts or translation products. As used herein, the term “derivative” is a mutation, RNA editing, or chemical modification, or otherwise altered transcript, or mutation, chemical modification, or otherwise altered. Means translated product. For example, “derivatives” are produced by processes such as modified phosphorylation or glycosylation, or acetylation, lipidation, or by modified signal peptide cleavage or other types of mature cleavage. These processes occur after transfer. The term “modifier” as used in the present invention and claims means a molecule capable of altering or altering the level and / or activity of a gene, or a transcription product of a gene, or a translation product of a gene. . “Modifiers” are preferably capable of altering or altering the biological activity of a transcription or translation product of a gene. For example, the modulation can be an increase or decrease in enzyme activity, a change in binding properties, or other changes or modifications of the biological, functional, or immunological properties of the translation product of a gene. The term “agent”, “reagent” or “compound” refers to a positive or negative biological effect on a cell, tissue, body fluid, or in the context of a biological system, or in the assay system being examined. Means a substance, chemical, composition or extract. They can be target agonists, antagonists, partial agonists or inverse agonists. Such agents, reagents, or compounds can be nucleic acids, natural or synthetic peptides or protein complexes, or fusion proteins. They may be antibodies, organic or inorganic molecules or compositions, small molecules, drugs and any combination of the aforementioned agents. They can be used for testing for diagnostic or therapeutic purposes. The term “oligonucleotide primer” or “primer” means a short nucleic acid sequence that can be annealed to a given target polynucleotide by complementary base pair hybridization and extended by a polymerase. They may be selected to be specific for a particular sequence or they may be selected randomly. For example, they will prime all possible sequences in the mixture. Primer lengths used herein vary from 10 nucleotides to 80 nucleotides. A “probe” is a short nucleic acid sequence of the nucleic acid sequences described and disclosed herein, or a sequence complementary thereto. They can include full-length sequences of a given sequence, or fragments, derivatives, isoforms, or variants. By identifying the hybridization complex between the “probe” and the sample being assayed, it is possible to detect the presence of other similar sequences within the sample. As used herein, “homolog or homology” is a term used in the art to describe the relationship between a nucleotide or peptide sequence and other nucleotides or peptide sequences, compared Determined by the degree of identity and / or similarity between the sequences. In the art, the terms “identity” and “similarity” refer to polypeptides or preferably determined by matching each other a query sequence and preferably another sequence (nucleic acid or protein sequence) of the same type. Means the degree of relatedness of polynucleotide sequences. Preferred computer program methods for calculating and determining “identity” and “similarity” include, but are not limited to, the Basic Local Alignment Search Tool (GCG BLAST) (Altschul et al., J. Mol. Biol. 1990, 215). : 403-410; Altschul et al., Nucleic Acids Res. 1997, 25: 3389-3402; Devereux et al., Nucleic Acids Res. 1984, 12: 387), BLASTN 2.0 (Gish W., http: //blast.wustl. edu, 1996-2002), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 1988, 85: 2444-2448), and GCG GelMerge to determine and align a set of contigs with the longest overlap ( Wilbur and Lipman, SIAM J. Appl. Math. 1984, 44: 557-567; Needleman and Wunsch, J. Mol. Biol. 1970, 48: 443-453). The term “variant” as used herein refers to polypeptides and proteins disclosed in the present invention in which one or more amino acids are at the N-terminus and / or C-terminus, And / or any polymorphism that is added and / or substituted and / or deleted and / or inserted within the natural amino acid sequence of the natural polypeptide or protein of the invention, but retains its essential properties. Means peptide or protein. Furthermore, the term “variant” includes shorter or longer types of polypeptides or proteins. A “variant” is at least about 80% sequence identity, more preferably at least about 90% sequence identity, most preferably a voltage-gated sodium channel β subunit protein, particularly SCN2B, SEQ ID NO: 1. Also includes sequences having at least about 95% sequence identity. “Mutants” of the protein molecule shown in SEQ ID NO: 1 include, for example, proteins having conservative amino acid substitutions in a highly conserved region. “Proteins” and “polypeptides” of the present invention include SCN2B, variants, fragments and chemical derivatives of proteins comprising the amino acid sequence of SEQ ID NO: 1. They can include proteins and polypeptides that can be isolated from nature or can be produced by recombinant and / or synthetic means. Naturally occurring proteins or polypeptides refer to naturally occurring truncated or secreted forms, naturally occurring variants (eg, splice variants) and naturally occurring allelic variants. The term “isolated” is found to have been altered and / or removed from their natural environment, ie, isolated from the cells or organism in which they normally reside, or they are associated with it in nature. It is taken to mean a molecule or substance that has been separated or essentially purified from any coexisting components. This concept further means that sequences encoding such molecules can be bound by hand to polynucleotides that they do not bind in their natural state and that such molecules are produced by recombinant and / or synthetic means. Means you can. For these purposes, even if these sequences are introduced into living organisms or non-living organisms by methods known to those skilled in the art, these sequences are still present in the organism, but are still isolated. Considered. In the present invention, the terms “risk”, “susceptibility”, and “predisposition” are used with respect to the probability of developing a neurodegenerative disease, preferably Alzheimer's disease. The term “AD” refers to Alzheimer's disease. As used herein, “AD-type neuropathology” refers to neuropathology, neurophysiology, tissue disease, as described herein and generally known from the state of the art literature. Refers to physical and clinical features (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 include Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Pick's disease, frontotemporal dementia, progressive supranuclear palsy, cortical basal ganglia degeneration, Including cerebrovascular dementia, multiple system atrophy, addiction-granular dementia and other tauopathy, and mild cognitive impairment. Other diseases involving neurodegenerative processes are, for example, ischemic stroke, age-related macular degeneration, narcolepsy, motor neuron disease, prion disease, traumatic nerve injury and repair, and multiple sclerosis.

  In one aspect, the invention features a method of diagnosing or determining a prognosis for a neurodegenerative disease in a subject, or determining whether a subject is at increased risk for developing the disease. This method comprises: (i) a transcript of a gene encoding SCN2B and / or (ii) a translation product of a gene encoding SCN2B in a sample from said subject, and / or (iii) of said transcript or translation product Determining the level or activity of a fragment, or derivative, or variant, or both said level and said activity, and comparing said level and / or said activity to a reference value representing a known disease or health condition, Diagnosing or determining the prognosis of said neurodegenerative disease in said subject or determining whether said subject is at increased risk of developing said neurodegenerative disease.

  The present invention also relates to the generation and use of primers and probes that are unique to the nucleic acid sequences, or fragments or variants thereof, disclosed in the present invention. Oligonucleotide primers and / or probes can be specifically labeled with fluorescent, bioluminescent, magnetic, or radioactive material. The invention further relates to the detection and production of said nucleic acid sequences, or fragments or 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 using the combination of primers to amplify the gene-specific nucleic acid sequence from a sample containing nucleic acid. Such samples are taken from healthy or diseased subjects. Whether amplification results in specific nucleic acid products and different length fragments can be indicative of neurodegenerative diseases, particularly Alzheimer's disease. Thus, the present invention provides an entire coding sequence useful for the detection of genetic mutations and single nucleotide polymorphisms in a given sample containing a nucleic acid sequence to be examined, associated with neurodegenerative diseases, particularly Alzheimer's disease. Nucleic acid sequences of at least 10 bases in length up to the gene sequence, oligonucleotide primers, and probes are provided. This feature has utility in the development of rapid DNA-based diagnostic tests, preferably also in kit formats.

  In another aspect, the invention features a method of monitoring the progression of a neurodegenerative disease in a subject. (I) a transcript of a gene encoding SCN2B and / or (ii) a translation product of a gene encoding SCN2B and / or (iii) a fragment or derivative of said transcript or translation product in a sample from said subject Or the level or activity of the variant, or both the level and the activity. Said level and / or said activity is compared to a reference value representing a known disease or condition. Thereby the progression of the neurodegenerative disease in the subject is monitored.

  In yet another aspect, the present invention provides a method for evaluating treatment of a neurodegenerative disease, wherein (i) a transcript of a gene encoding SCN2B in a sample taken from the subject treated for the disease, and And / or (ii) determining the level or activity of a translation product of the gene encoding SCN2B, and / or (iii) a fragment or derivative or variant of the transcript or translation product, or both the level and the activity A method comprising the steps of: The level, or the activity, or both the level and the activity, are compared to a reference value representing a known disease or condition, thereby assessing treatment of the neurodegenerative disease.

  In a preferred embodiment of the method, kit, recombinant non-human animal, molecule, assay, use of the invention claimed herein, said gene encoding a human voltage-gated sodium channel β subunit protein comprises: Genbank Genbank accession number O60939 (protein ID) deduced from accession numbers AF049498, AF049496, AF049497, AF107028, AX376000, and mRNA corresponding to the EST cDNA sequence from the Genbank database (SEQ ID NO: 2), represented by SEQ ID NO: 1. Is a gene encoding human voltage-gated sodium channel type II β subunit (SCN2B), also called β-2 subunit protein or β2-protein. In the present invention, SCN2B also means the nucleic acid sequence of SEQ ID NO: 2 that encodes the protein of SEQ ID NO: 1 (Genbank accession number O60939). In the present invention, the sequence is “isolated” as that term is used herein. Furthermore, in the present invention, the gene encoding the SCN2B protein is generally also referred to as SCN2B gene or simply SCN2B, and the protein of SCN2B is generally also referred to as SCN2B protein or simply SCN2B.

  In other preferred embodiments of the methods, kits, recombinant non-human animals, molecules, assays, uses of the invention claimed herein, the neurodegenerative disease or disorder is Alzheimer's disease and the subject is Alzheimer's. I have a disease.

  The present invention discloses the detection and differential expression and regulation of the gene encoding SCN2B in specific brain regions of AD patients. As a result, the SCN2B gene and its corresponding transcripts and / or translation products have a causative role in locally selective neuronal degeneration normally found in AD. Instead, SCN2B provides a neuroprotective function to the remaining neurons. Based on these disclosures, the present invention has utility in diagnostic evaluation and prognosis of neurodegenerative diseases, particularly AD, and identification of its predisposition. Furthermore, the present invention provides a method for diagnostically monitoring patients undergoing treatment for such diseases.

  The sample to be analyzed and measured is preferably selected from the group comprising brain tissue or other tissues, or somatic cells. The sample can include cerebrospinal fluid or other body fluid, such as saliva, urine, blood, serum plasma, or mucus. According to the present invention, the method of diagnosis of neurodegenerative disease, prognosis determination, monitoring of progression of the disease or evaluation of treatment of the disease can preferably be performed ex vivo, preferably such a subject. Or a sample, eg, body fluid or cell, removed, collected, or isolated from a patient.

  In another preferred embodiment, the reference value is (i) a transcript of a gene encoding SCN2B and / or (ii) a gene encoding SCN2B in a sample from a subject not suffering from the neurodegenerative disease. A translation product, and / or (iii) a level or activity of the transcript or translation product fragment, or derivative, or a variant, or a value of both the level and the activity.

  In a preferred embodiment, a transcription product of a gene encoding SCN2B and / or a translation product of a gene encoding SCN2B, and / or a fragment thereof, in a sample cell, tissue, or body fluid collected from the subject A change in the level or activity of the derivative or variant relative to a reference value that represents a known health condition indicates a diagnosis or prognosis of, or a high risk of suffering from, a neurodegenerative disease, in particular AD.

  In a preferred embodiment, the SCN2B gene is used using quantitative PCR analysis using a combination of primers to amplify the gene specific sequence from cDNA obtained by reverse transcription of RNA extracted from a sample of a subject. The transcript level is measured on a sample from the subject.

  Northern blotting using a probe specific for the gene can also be applied. More preferably, transcripts are measured by chip-based microarray technology. These techniques are known to those skilled in the art (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 oxygen activity as disclosed and described in International Publication No. WO 02/14543.

  Furthermore, the translation product of the gene encoding SCN2B, and / or the level and / or activity of the translation product fragment or derivative, or mutant, and / or the translation product and / or fragment, derivative, or mutation thereof The level of body activity can be detected using immunoassays, activity assays and / or binding assays. In these assays, the protein molecule and the anti-protein antibody are bound to the anti-protein antibody by use of an enzyme, chromodynamic, radioactive, magnetic, or luminescent label that binds to either the anti-protein antibody or a secondary antibody that binds to the anti-protein antibody. The amount of binding to the protein antibody can be measured. Still other high affinity ligands may be used. Immunoassays that can be used include, for example, ELISA, Western blot, and other techniques known to those skilled in the art (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). All these detection techniques can also be used in microarray, protein-array, antibody microarray, tissue microarray, electronic biochip or protein-chip based technology formats (Schena M., Microarray Biochip Technology, Eaton Publishing, Natick, MA , 2000).

  In a preferred embodiment, (i) a transcription product of a gene encoding SCN2B and / or (ii) a translation product of a gene encoding SCN2B and / or (iii) said transcription in a series of samples taken from said subject The level or activity of a product or translation product fragment, or derivative, or variant, or both the level and the activity, are compared over time to monitor the progression of the disease. In another preferred embodiment, the subject receives treatment prior to one or more of the sampling. In yet another preferred embodiment, the level and / or activity is determined before and after the treatment of the subject.

In other embodiments, the present invention determines a subject's propensity or predisposition for diagnosing or determining the prognosis of a neurodegenerative disease, particularly AD in a subject, or for developing a neurodegenerative disease, particularly AD. A kit for
(A) selected from the group consisting of (i) a reagent that selectively detects the transcript of the gene encoding SCN2B, and (ii)) a reagent that selectively detects the translation product of the gene encoding SCN2B, At least one reagent;
(B)-determining in the sample from said subject the level or activity of said transcript and / or said translation product of the gene encoding SCN2B, or both said level and said activity;
Diagnosing or determining the prognosis of a neurodegenerative disease, particularly AD, or determining the propensity or predisposition of a subject to develop such a disease; diagnosing or prognosing a neurodegenerative disease, particularly AD Instructions for determining or a tendency or predisposition for a subject to develop such a disease, the transcript and / or the translation product compared with a reference value representing a known health condition Different levels or activities of both, or both of the levels and the activities; or the levels or activities of the transcript and / or the translation product similar to or equal to a reference value representing a known health condition, or the levels and the activities Both of the diagnosis or prognosis of neurodegenerative diseases, particularly AD, or the propensity or predisposition to develop such diseases Increased is shown, wherein the kit. The kit according to the invention is particularly useful for identifying individuals at risk for developing neurodegenerative diseases, particularly AD. As a result, the kit according to the present invention serves as a means to target the identified individual for early prevention or therapeutic intervention prior to the onset of the disease and before irreversible damage during the disease. . Furthermore, in a preferred embodiment, the kit featured in the invention monitors the progress of neurodegenerative diseases, particularly AD, in a subject and the success or failure of therapeutic treatment of such diseases in the subject. Useful for.

  In another aspect, the invention provides a method for treating or preventing a neurodegenerative disease, particularly AD, in a subject, comprising (i) a gene encoding SCN2B, and / or (ii) a transcript of the gene encoding SCN2B And / or (iii) the translation product of the gene encoding SCN2B, and / or the level or activity of (iv) (i) to (iii) fragments, or derivatives, or variants, or said level and said activity Characterized in that it comprises administering to said subject a therapeutically or prophylactically effective amount of agent (s) that directly or indirectly affect both. Said agent comprises a small molecule or it may also comprise a peptide, oligopeptide or polypeptide. Said peptide, oligopeptide or polypeptide may comprise the translation product of the gene encoding SCN2B protein, or a fragment or derivative thereof, or the amino acid sequence of a variant. In accordance with the present invention, an agent for treating or preventing neurodegenerative diseases, particularly AD, may also consist of nucleotides, oligonucleotides, or polynucleotides. Said oligonucleotide or polynucleotide may comprise the nucleotide sequence of the gene encoding the SCN2B protein in either the sense or antisense direction.

  In a preferred embodiment, this method comprises the application of methods known per se of gene therapy and / or antisense nucleic acid technology for administering said agent (s). In general, gene therapy has several approaches: molecular replacement of mutated genes, addition of new genes from the synthesis of therapeutic proteins, and regulation of endogenous cellular gene expression by recombinant expression methods or by drugs including. Gene transfer techniques have been described in detail (see, eg, Behr, Acc Chem Res 1993, 26: 274-278 and Mulligan, Science 1993, 260: 926-931), and direct gene transfer techniques such as Mechanical microinjection of DNA and indirect techniques using biological vectors (such as recombinant viruses, especially retroviruses) or model liposomes, or DNA co-precipitation with polycations, chemical means (solvents, detergents, Polymer, enzyme) or transfection based techniques using cell membrane pertubation by physical means (mechanical, osmotic, thermal, electric shock). Postnatal gene transfer into the central nervous system has been described in detail (see, eg, Wolff, Curr Opin Neurobiol 1993, 3: 743-748).

  In particular, the present invention provides a method for treating or preventing neurodegenerative diseases by antisense nucleic acid therapy, ie, inappropriately expressed or defective by introducing antisense nucleic acid or its derivatives into certain critical cells. Characterized by a method of down-regulating genes (see eg Gillespie, DN & P 1992, 5: 389-395; Agrawal and Akhtar, Trends Biotechnol 1995, 13: 197-199; Crooke, Biotechnology 1992, 10: 882-6) ). Apart from hybridization methods, the application of RNA molecules acting as enzymes that destroy ribozymes, ie RNAs carrying disease messages, has also been described (see, for example, Barinaga, Science 1993, 262: 1512-1514). In a preferred embodiment, the subject to be treated is a human and a therapeutic antisense nucleic acid or derivative thereof is made to a transcript of the gene encoding SCN2B. It is preferred to treat the central nervous system, preferably the cells of the subject's brain, in this way. Cell penetration can be performed by known methods such as binding of antisense nucleic acids and derivatives thereof to carrier particles, or the techniques described above. Methods of administering therapeutic target oligodeoxynucleotides are well known to those skilled in the art (see, eg, Wickstrom, Trends Biotechnol 1992, 10: 281-287). In some cases, delivery can be accomplished by simple topical application. Other approaches are directed to intracellular expression for topical application. In this method, cells are transformed ex vivo with a recombinant gene that directs the synthesis of RNA complementary to the region of the target nucleic acid. The therapeutic use of antisense RNA expressed in cells is procedurally similar to gene therapy. A recently developed method of modulating the intracellular expression of a gene through 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 other preferred embodiments, compounds to SCN2B that co-express with voltage-gated sodium channel SCN2A or other voltage-gated sodium channel proteins in suitable cells, such as CHO cells or HEK293 cells, or other neural cells and / or Alternatively, a method for investigating the action of an agent is provided. Thereby, the electrophysiological effects of compounds and / or agents on sodium current mediated by SCN2A or by SCN2B co-expressed with other voltage-gated sodium channel proteins are investigated. To perform the test, the cDNA encoding the human gene product SCN2B is cloned into an appropriate expression vector. The cDNA encoding SCN2A (Genbank accession numbers AF327224 to AF327246) or cDNA encoding other voltage-gated sodium channels is cloned into other appropriate expression vectors. Suitable cell lines as described above, preferably using DMRIE-C (a cationic lipid 1,2-dimysteroxypropyl-3-dimethyl-hydroxyethylammonium bromide-cholesterol liposome formation) such as a reagent Are transfected with the plasmid. Patch clamp experiments can be performed in voltage clamp mode (Hamill et al., Pflugers Arch. 1981, 391: 85-100), the total cell current is recorded, and the resulting signal is generated using appropriate software such as Pulse / Pulsefit ( HEKA, Lambrecht, Germany) is amplified, digitized, stored and analyzed. Current “run-dow” or “run-u” (Varnum et al., Pro. Natl. Acad. Sci. USA 1993, 90: 11528-11532) is still a compound and / or agent effect. If too strong for evaluation, studies on the mediated currents of the voltage-gated sodium channel can be performed with a perforated patch clamp method to prevent non-specific current amplitude changes (Dart et al., J. Physiol 1995, 483: 29-39; Dinesh & Hablitz, Brain Res. 1990, 535: 318-322). An example of a stimulation protocol for studying the effects and reversibility of test compounds on SCN2B co-expressed with SCN2A or other sodium channel proteins is shown below. Cells that co-express SCN2B with SCN2A or other sodium channels will be fixed, for example, at a holding potential of −80 mV. The pulse cycle rate is 10 seconds. For compound and / or agent testing, stably transfected cells are hyperpolarized from a holding potential of, for example, −80 mV, for example, to −100 mV or −120 mV, for example for 100 milliseconds, followed by, for example, −60 mV to It can be depolarized to a test potential of +60 mV for 1 second. The peak current amplitude of the test pulse will be used for the analysis. This method is a compound and / or action that can open, close, activate, inactivate, or modify the biophysical properties of SCN2B co-expressed with SCN2A or other voltage-gated sodium channels It is also suitable for identifying and testing substances. The voltage-gated sodium channel modulators identified and tested in this way can affect learning and memory function and can be used, for example, in therapeutic approaches for neurodegenerative diseases, particularly Alzheimer's disease.

  In other preferred embodiments, the method is preferably performed in the subject's central nervous system, preferably the brain, to treat donor cells, or donor cells, to minimize or reduce graft rejection. A method wherein the donor cells are genetically modified by inserting at least one transgene encoding the agent (s). Said transgene is carried by a viral vector, in particular a retroviral vector. The transgene can be inserted into the donor cell by non-viral physical transfection of the DNA encoding the transgene, particularly microinjection. Transgene insertion can also be performed by electroporation, chemically mediated transfection, particularly calcium phosphate transfection, or liposome-mediated transfection (Mc Celland and Pardee, Expression Genetics: Accelerated and High-Throughput Methods, Eaton Publishing, Natick, MA, 1999).

  In a preferred embodiment, said agent for treating and preventing neurodegenerative diseases, particularly AD, is a process comprising introducing subject cells into said subject, said subject cells encoding said therapeutic protein Can be administered to the subject, preferably a human, by a process that is treated in vitro to insert a DNA segment to be expressed and the subject cells express a therapeutically effective amount of the therapeutic protein in the subject in vivo. It is a therapeutic protein. Said DNA segment can be inserted into said cells in vitro by viral vectors, in particular retroviral vectors.

  Therapeutic methods according to the present invention include therapeutic cloning, transplantation, and application of stem cell therapy using embryonic stem cells or embryonic germ cells and adult neural stem cells, in combination with any of the previously described cell and gene therapy methods. Stem cells are totipotent or pluripotent. They can also be organ specific. A method of repairing diseased and / or damaged brain cells or tissues comprises (i) harvesting donor cells from adult tissue. The nuclei of these 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 cells that have undergone somatic cell nuclear transfer. The use of differentiation factors then results in the directional development of stem cells into specialized cell types, preferably neurons (Lanza et al., Nature Medicine 1999, 9: 975-977), or ( ii) Purify adult stem cells isolated from the central nervous system or bone marrow (mesenchymal stem cells) for subsequent growth in vitro, followed by grafting and transplantation, or (iii) directing endogenous neural stem cells directly Induces, proliferates, migrates and differentiates into functional neurons (Peterson DA, Curr. Opin. Pharmacol. 2002, 2: 34-42). Adult neural stem cells have great potential for the repair of damaged or diseased brain tissue because the germinal center of the adult brain is free of nerve damage or dysfunction (Colman A, Drug Discovery World 2001, 7 : 66-71).

  In a preferred embodiment, the subject to be treated or prevented according to the present invention is a human, a laboratory animal, such as a mouse or rat, a domestic animal, or a non-human primate. The experimental animals are non-human animal models for neurodegenerative diseases, such as transgenic mice and / or knockout mice with AD type neuropathology.

  In other embodiments, the invention provides (i) a gene encoding SCN2B, and / or (ii) a transcription product of a gene encoding SCN2B, and / or (iii) a translation product of a gene encoding SCN2B, and / or Or (iv) the activity or level of at least one substance selected from the group consisting of fragments, derivatives, or variants of (i) to (iii), or a modulator of both the activity and the level And

  In another aspect, the invention features a pharmaceutical composition comprising the modifier and preferably a pharmaceutical carrier. The carrier means a diluent, adjuvant, excipient, or vehicle with which the modifier is administered.

  In another aspect, the invention relates to (i) a gene encoding SCN2B and / or (ii) a transcript of the gene encoding SCN2B and / or (iii) SCN2B used in a pharmaceutical composition A translation product of the gene and / or the activity or level of at least one substance selected from the group consisting of (iv) (i) to (iii) fragments, derivatives or mutants, or the activity and Characterized by both levels of modifiers.

  In another aspect, the present invention provides (i) a gene encoding SCN2B and / or (ii) a transcript of a gene encoding SCN2B for the manufacture of a medicament for treating or preventing neurodegenerative diseases, particularly AD. And / or (iii) at least one substance selected from the group consisting of a translation product of a gene encoding SCN2B, and / or a fragment, derivative, or variant of (iv) (i) to (iii) Activity or level, or use of both said activity and said level modulators.

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

  In another aspect, the invention features a recombinant non-human animal comprising a non-native gene sequence encoding SCN2B, or a fragment, derivative or variant thereof. Production of the recombinant non-human animal comprises (i) providing a gene targeting construct containing the gene sequence and a selectable marker sequence; and (ii) introducing the targeting construct into stem cells of the non-human animal; Introducing the non-human animal stem cells into a non-human embryo; (iv) transplanting the embryo into a pseudopregnant non-human animal; (v) developing the embryo until the next month; and (vi) its genome. Identifying a genetically modified non-human animal comprising a modification of said gene sequence in both alleles, and (vii) breeding the genetically modified non-human animal of step (vi), wherein the genome is said endogenous gene Obtaining a genetically modified non-human animal comprising the modification of: wherein the gene is misexpressed, underexpressed, or overexpressed, and The corrupted or modified, neurodegenerative disease, said non-human animal, particularly showing a predisposition to developing symptoms similar neuropathology and AD obtained. Methods and techniques for producing and constructing such animals are known to those skilled in the art (eg, Capecchi, Science 1989, 244: 1288-1292; 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 Transgenetics: A Practical Approach, Oxford University Press, Oxford, England, 1999). It is preferable to use such recombinant non-human animals as animal models for studying neurodegenerative diseases, particularly AD. Such animals are useful for screening, testing and validating compounds, agents and modifiers in the development of diagnostics and therapeutics to treat neurodegenerative diseases, particularly Alzheimer's disease. In a preferred embodiment, the recombinant non-human animal comprises a non-native gene sequence encoding SCN2B protein, or a fragment or derivative thereof, or a variant thereof.

  In another aspect, the invention provides a modulator of a neurodegenerative disease, particularly AD, or related diseases and disorders, wherein (i) a gene encoding SCN2B and / or (ii) a gene encoding SCN2B One or more selected from the group consisting of a transcript and / or (iii) a translation product of a gene encoding SCN2B, and / or a fragment, or derivative, or variant of (iv) (i)-(iii) or Featuring assays that screen for modifiers of multiple substances. This screening method comprises (a) contacting a cell with a test compound, and (b) the activity or level of one or more substances described in (i) to (iv), or both the activity and level. Measuring (c) the activity or level of the substance in a control cell not contacted with the test compound, or both the activity and level, and (d) steps (b) and (c) A change in the activity and / or level of the substance in the contacted cell indicates that the test compound is a modulator of the disease and disorder.

  In another aspect, the present invention provides a modulator of a neurodegenerative disease, particularly AD, or related diseases and disorders, wherein (i) a gene encoding SCN2B and / or (ii) a gene encoding SCN2B And / or (iii) a translation product of a gene encoding SCN2B, and / or (iv) a fragment, derivative, or variant of (i) to (iii) Or a screening assay for modifiers of a plurality of substances, wherein (a) a test compound is administered to a non-human test animal that is predisposed to, or has already developed, a neurodegenerative disease or related disease or disorder Measuring the activity and / or level of one or more substances according to (b) (i)-(iv) And (c) a matched non-human control animal that is equally given a predisposition to develop said symptom or has already developed said symptom and has not been administered such a test compound to that non-human Measuring the activity and / or level of said substance in an animal; and (d) comparing the activity and / or level of the substance in the animal of steps (b) and (c), A screening assay is characterized in that a change in the activity and / or level of the substance in indicates that the test compound is a modulator of the disease and disorder.

  In a preferred embodiment, the non-human test animal and / or the non-human control animal is a gene encoding SCN2B, or a fragment thereof, or a derivative thereof under the control of a transcriptional regulatory factor that is not a natural SCN2B gene transcriptional regulatory factor. Or a recombinant non-human animal that expresses the variant.

  In another embodiment, the invention comprises (i) identifying a modulator of neurodegenerative disease by the method of screening assay described above, and (ii) mixing the modifier with a drug carrier. A method for producing a drug is provided. However, the modifier can also be identified by other types of screening assays.

  In another aspect, the present invention provides a method for testing a compound for inhibiting the binding of a translation product of a gene encoding SCN2B, or a fragment, derivative or variant thereof to a ligand, preferably a plurality of Assays for screening the compounds are provided. The screening assay comprises (i) adding a liquid suspension of the SCN2B translation product, or a fragment or derivative thereof to a plurality of containers; and (ii) a compound to be screened for the inhibition (one or (Iii) adding a plurality of species to the plurality of containers; (iii) adding a detectable ligand, preferably a fluorescently labeled ligand, to the container; and (iv) the SCN2B translation product, or the fragment thereof, or Incubating a derivative or variant, the compound (s), the detectable ligand, preferably a fluorescently labeled ligand, (v) the SCN2B translation product, or a fragment thereof, or Measuring the amount of fluorescence, preferably binding to a derivative or variant; (vi) said compound By one or more out, including the steps of determining the degree of inhibition of binding of the ligand of the SCN2B translation product or said fragment thereof, or derivative thereof, or to variants. Preferably, the SCN2B translation product, or a fragment, derivative or variant thereof is reconstituted into an artificial liposome to produce the corresponding proteoliposome, and inhibition of binding between the ligand and the SCN2B translation product is determined. Methods for reconstitution of SCN2B translation products from detergent into liposomes have been described in detail (Schwarz et al., Biochemistry 1999, 38: 9456-9464; Krivosheev and Usanov, Biochemistry-Moscow 1997, 62: 1064-1073). Instead of utilizing a fluorescently labeled ligand, in some embodiments it is preferred to use other detectable labels known to those skilled in the art, such as radioactive labels, and detect it accordingly. The methods have been improved in identifying new compounds and in their ability to inhibit the binding of a ligand to the gene product of the gene encoding SCN2B, or a fragment or derivative or variant thereof, or It can be useful for evaluating optimized compounds. An example of a fluorescence binding assay in this case based on the use of carrier particles is disclosed and described in International Publication No. WO 00/52451. Another example is the competitive assay described in International Publication No. WO02 / 01226. Preferred signal detection methods for the screening assays of the present invention are described in the following International Publication Nos. WO96 / 13744, WO98 / 16814, WO98 / 23942, WO99 / 17086, WO99 / 34195, WO00 / 66985, WO01 / 59436, WO01 / 59416. Has been.

  In another embodiment, the present invention provides (i) identifying a compound as an inhibitor of binding of a ligand to the gene product of the gene encoding SCN2B by the inhibitory binding assay described above; (ii) Mixing the compound with a pharmaceutical carrier. However, the compounds can also be identified by other types of screening assays.

  In another embodiment, the present invention tests a compound to determine the extent to which the compound binds to the translation product of the gene encoding SCN2B, or a fragment, derivative or variant thereof, preferably a plurality Features an assay that screens The screening assay comprises: (i) adding a liquid suspension of the SCN2B translation product, or a fragment or derivative or variant thereof to a plurality of containers; and (ii) being screened for the binding; Adding a type of detectable, preferably fluorescently labeled compound, or a plurality of types of detectable, preferably fluorescently labeled compounds, to the plurality of containers; (iii) the SCN2B translation product, or the Incubating said fragments, derivatives or variants thereof, and said detectable, preferably fluorescently-labeled compound (s), (iv) said SCN2B translation product, or said thereof Preferably measuring the amount of fluorescence bound to a fragment, derivative or variant, and (v Including the steps of determining the degree of binding by one or more of the SCN2B translation product, or fragment or derivative thereof, of the or the compound to mutants,. In this type of assay, it is preferred to use fluorescent labels. However, other types of detectable labels can also be used. Also in this type of assay, it is preferable to reconstitute the SCN2B translation product or a fragment, derivative or mutant thereof in the artificial liposome described in the present invention. The assay is useful for the identification of new compounds and for the evaluation of compounds that have been improved or optimized in their ability to bind to SCN2B translation products, or fragments or derivatives or variants thereof.

  In another embodiment, the present invention provides (i) identifying a compound as a binder to the gene product of SCN2B by the binding assay described above, mixing the compound with a drug carrier, A method for producing a drug is provided. However, the compounds can also be identified by other types of screening assays.

  In other embodiments, the present invention provides a drug obtainable by any of the screening assay methods claimed herein. In another embodiment, the present invention provides a drug obtainable by any of the screening assay methods claimed herein.

  The present invention features a protein molecule represented by SEQ ID NO: 1, wherein the protein molecule is a translation product of a gene encoding SCN2B used as a diagnostic target for detecting a neurodegenerative disease, preferably Alzheimer's disease, Or a fragment, derivative or mutant thereof.

  The present invention is further characterized by the protein molecule shown in SEQ ID NO: 1, which is used as a screening target for reagents or compounds that prevent, treat, or ameliorate a neurodegenerative disease, preferably Alzheimer's disease. It is the translation product of the gene encoding SCN2B, or a fragment, derivative or mutant thereof.

  The present invention is characterized by an antibody that is specifically immunoreactive with an immunogen, which is a translation product of a gene encoding SCN2B, SEQ ID NO: 1, or a fragment, derivative, or variant thereof . The immunogen includes an immunogenic or antigenic epitope or portion of a translation product of the gene, the immunogenic or antigenic portion of the translation product is a polypeptide, and the polypeptide elicits an antibody response in an animal And the polypeptide is immunospecifically bound by the antibody. Methods for producing 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). As used herein, the term “antibody” refers to polyclonal antibodies, monoclonal antibodies, chimeric antibodies, recombinant antibodies, anti-idiotype antibodies, humanized antibodies, single chain antibodies, and fragments thereof, and the like. Includes all forms of antibodies known in the art (Dubel and Breitling, Recombinant Antibodies, Wiley-Liss, New York, NY, 1999). The antibodies of the present invention are useful in a variety of diagnostic and therapeutic methods based on current technology such as, for example, enzyme immunoassays (eg, enzyme immunoassays, ELISA), radioimmunoassays, chemiluminescent immunoassays, western blots, immunoprecipitations and antibody microarrays. (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) . These methods include detection of the translation product of the SCN2B gene, or a fragment, derivative or variant thereof.

  In a preferred embodiment of the invention, the antibody can be used to detect the pathological state of cells in a sample from a subject, comprising immunocytochemically staining the cells with the antibody. The pathological state of the cell is indicated by a change in the degree of staining in the cell or a change in staining pattern compared to a cell representing a known health condition. Preferably, the pathological condition relates to a neurodegenerative disease, in particular AD. Immunocytochemical staining of cells can be performed by a number of different experimental methods that are well known in the art. However, it is preferred to apply an automated method for the detection of antibody binding, in which the determination of the degree of staining of the cells, or the determination of cellular or subcellular staining of the cells, or the cell surface Determination of the phase distribution of the antigen above or between the organelles and other intracellular structures in the cell is performed according to the method described in US Pat. No. 6,150,173.

  Other features and advantages of the present invention will be apparent from the following description of the drawings and examples, which are merely exemplary and in no way limit the remainder of the disclosure.

  FIG. 1 illustrates brain regions that have selective vulnerability to neuronal loss and degeneration in AD. Primarily, neurons in the inferior temporal lobe, entorhinal cortex, hippocampus, and amygdala are susceptible to degenerative processes in AD (Terry et al., Annals of Neurology 1981, 10: 184-192). Most of these brain regions are involved in processing learning and memory functions. In contrast, neurons in the frontal cortex, occipital cortex, and cerebellum remain largely undamaged and are protected from neurodegenerative processes in AD. Brain tissue from frontal cortex (F), temporal cortex (T) and hippocampus (H) of AD patients and healthy, age-matched control individuals were used in the examples disclosed herein. For illustrative purposes, normal healthy brain images were picked from a publication by Stranger (Brain Biochemistry and Brain Disorders, Oxford University Press, Oxford, 1992, p. 4).

  FIG. 2 discloses the initial identification of differential expression of the gene encoding SCN2B on a fluorescent differential display screen. This figure shows clipping of a large preparative fluorescent differential display gel. PCR products from frontal cortex (F) and temporal cortex (T) of 2 healthy control subjects and 6 AD patients were added in duplicate on a denaturing polyacrylamide gel (left to right). PCR products were obtained by amplifying individual cDNAs using corresponding one-base-anchor oligonucleotides and specific Cy3-labeled random primers. The arrow indicates the position of movement, where there is a significant difference in the signal intensity of the transcript of the gene encoding SCN2B derived from the frontal cortex, compared to the signal derived from the temporal cortex of AD patients To do. Its differential expression reflects down-regulation of SCN2B gene transcription in the temporal cortex compared to the frontal cortex of AD patients. When signals obtained from the temporal cortex and frontal cortex of healthy non-AD control subjects are compared with each other, no difference in signal intensity, ie no change in expression level, can be detected.

  Figures 3 and 4 show the human SCN2B gene in AD brain tissue by quantitative RT-PCR analysis using two different sets of primer pairs, primer set A (Figure 3) and primer set B (Figure 4). The verification of differential expression is shown. Quantification of RT-PCR products from RNA samples collected from frontal cortex (F) and temporal cortex (T) of AD patients (FIGS. 3a; 4a) and age-matched healthy control individuals (FIGS. 3b; 4b) Was performed by the LightCycler rapid thermal cycling technique. Data were normalized to the combined mean of a set of standard genes that did not show significant differences in their gene expression levels. The set of standard genes consisted of genes for ribosomal protein S9, transferrin receptor, GAPDH, cyclophilin B and β-actin. The figure illustrates the kinetics of amplification by plotting the number of cycles against the amount of amplified material measured by its fluorescence. During the log phase of the response, the amplification kinetics of SCN2B cDNA from both frontal and temporal cortex of normal control individuals are close (FIG. 3b; 4b: arrows), whereas in AD (FIG. 3a; Note that there is a significant separation of the corresponding curves indicating the differential expression of SCN2B in the two brain regions analyzed.

  FIG. 5 shows the differential expression of the human SCN2B gene in AD brain tissue by quantitative RT-PCR analysis using primer set A. Quantification of RT-PCR products from RNA samples taken from frontal cortex (F) and hippocampus (H) of AD patients (FIG. 5a) was performed by the light cycler rapid thermal cycling technique. Similarly, samples of age-matched healthy control individuals were compared (FIG. 5b). Data were normalized to the combined mean of a set of standard genes that did not show significant differences in their gene expression levels. The set of standard genes consisted of genes for cyclophilin B, ribosomal protein S9, transferrin receptor, GAPDH, and β-actin. The figure illustrates the kinetics of amplification by plotting the number of cycles against the amount of amplified material measured by its fluorescence. During the log phase of the response, the amplification kinetics of SCN2B cDNA from both the frontal cortex and hippocampus of normal control individuals are close (Figure 5b: arrows), whereas in Alzheimer's disease (Figure 5a, arrows) There is a significant separation of the corresponding curves, differential expression of the gene encoding SCN2B in each brain region analyzed, preferably a transcript of the human SCN2B gene in the frontal cortex to the hippocampus, or a fragment or derivative thereof, Note also that mutant dysregulation has been shown.

  FIG. 6 discloses SEQ ID NO: 1; the amino acid sequence of the SCN2B protein. Full-length human SCN2B protein contains 215 amino acids.

  FIG. 7 shows the nucleotide sequence of human SCN2B cDNA comprising 4903 nucleotides represented by the consensus sequence obtained from the assembly of SEQ ID NO: 2, overlapping large set of overlapping human nucleotide sequence fragments found in the Genbank human genome database (FIG. 7). 10).

  FIG. 8 illustrates the nucleotide sequence of the 99 bp SCN2B cDNA fragment identified and obtained by SEQ ID NO: 3, differential display (sequence from 5 'to 3').

  FIG. 9 shows the sequence alignment of SEQ ID NO: 3 to the nucleotide sequence of human SCN2B cDNA, SEQ ID NO: 2.

  FIG. 10 schematically shows extended and corrected consensus sequences based on and derived from SCN2B consensus cDNA sequences, GenBank accession numbers AF049498, AF107028, AX376000, HSU87555, and other EST sequence fragments shown, and SCN2B 2 illustrates the assembly of SEQ ID NO: 2 from a genomic database sequence fragment that constitutes the sequence obtained from PCR amplification with primers for the gene encoding (ens-PCR1, ens-PCR2). Primer set D (ens-PCR1, nucleotide positions 3204-4182 of SEQ ID NO: 2): 5'-TGAGCGAGTCCAAGCCCATCTGG-3 'and 5'-CCAAAGCCAGTTCCAAGGCACCTC-3'; primer set C (ens-PCR2, nucleotide of SEQ ID NO: 2 Sequence at positions 2138-3230): 5′-GGGAAGCAGAGGTTGCAGGTGAAC-3 ′ and 5′-CATTTCCAGATGGGCTTGAACTCGCTC-3 ′.

  FIG. 11 shows a schematic alignment with the SCN2B cDNA of SEQ ID NO: 3. The open rectangle represents the SCN2B open reading frame and the thin bar represents the 5 'and 3' untranslated region (UTR).

  FIG. 12 illustrates a section from a previous human center labeled with an affinity purified rabbit polyclonal anti-SCN2B antiserum (green signal) produced against a peptide corresponding to amino acids 179-215 of SCN2B. . SCN2B immunoreactivity was observed in both cerebral cortex (CT) and white matter (WM) (FIG. 12a, low magnification). A punctate pattern of SCN2B immune activity (dendritic spines) decorating the neural process and weaker and diffused immune activity of the neuronal cell body were observed (FIG. 12b, high magnification). FIG. 12c shows the punctate immune activity of glial cell bodies and nerve fibers. Blue signal indicates nuclei stained with DAPI.

  The table in FIG. 13 shows 15 AD patients identified herein by internal reference numbers P010, P011, P012, P014, P016, P017, P019, P026, P031, P038, P040, P041, P042, P046, P047. (0.29 to 0.98 times) and internal reference numbers C005, C008, C011, C012, C014, C020, C022, C024, C025, C026, C027, C028, C029, C030, C031, C032, C033, C034 , SCN2B gene expression levels in the temporal cortex relative to the frontal cortex in 19 age-matched healthy control individuals (0.59-2.15 fold) identified herein by C035. Quantitative RT-PCR analysis was performed using primer pair set A. The values shown are calculated by the formulas described herein (see below). The scatter plot visualizes the individual logarithm of the regulation ratio between temporal and frontal cortex, log (ratio IT / IF), in the control sample (dots) and AD patient sample (triangles), respectively.

  The table in FIG. 14 shows seven AD patients identified herein by internal reference numbers P010, P011, P012, P014, P016, P017, P019 (0.20 to 0.91 times), and internal reference number C005. , C008, C011, C012, C014, SCN2B gene expression levels in the temporal cortex relative to the frontal cortex in 5 age-matched healthy control individuals (0.48-1.48 times) Indicates. Quantitative RT-PCR analysis was performed using primer pair set B. The values shown are calculated by the formulas described herein (see below). The scatter plot visualizes the individual logarithm, log (ratio IT / IF) of the temporal to frontal cortex regulation ratio in the control sample (dots) and AD patient sample (triangles), respectively.

  The table in FIG. 15 shows six Alzheimer seconds patients identified herein by internal reference numbers P010, P011, P012, P014, P016, P019 (0.48 to 10.87 times), and internal reference number C004, 3 shows gene expression levels in the hippocampus to the frontal cortex for the SCN2B gene in three age-matched healthy control individuals (0.77-1.26 fold) identified herein by C005, C008. Quantitative RT-PCR analysis was performed using primer pair set A. The values shown are calculated by the formulas described herein (see below). The scatter plot visualizes the individual logarithm of the hippocampal to frontal cortex regulation ratio, log (ratio HC / IF), in the control sample (dots) and AD patient sample (triangles), respectively.

Example I:
(I) Brain tissue dissection from AD patients Brain tissue from AD patients and age-matched control subjects were collected within 6 hours of death and immediately frozen on dry ice. Sample sections from each tissue were fixed with paraformaldehyde to confirm the diagnosis histopathologically. Brain regions for differential expression analysis were identified (see FIG. 1) and stored at −80 ° C. until RNA extraction was performed.

(Ii) Isolation of total mRNA:
Total RNA was extracted from postmortem brain tissue using the RNeasy kit (Qiagen) according to the manufacturer's protocol. The exact RNA concentration and RNA quality was determined on a DNA LabChip system using an Agilent 2100 bioanalyzer (Agilent Technologies). In order to further quality test the generated RNA, i.e. to eliminate partial degradation and to test for DNA contamination, using specifically designed intron GAPDH oligonucleotides and genomic DNA as reference controls, Melting curves were generated using the light cycler technique described in the manufacturer's protocol (Roche).

(Iii) cDNA synthesis and identification of differentially expressed genes by fluorescence differential display (FDD):
A modified and improved differential display (DD) screening method was used to identify changes in gene expression in different tissues. The original DD screening method is known to those skilled in the art (Liang and Pardee, Science 1995, 267: 1186-7). This technique compares two RNA populations of RNA and obtains clones of genes that are expressed in one population but not in the other population. Several samples can be analyzed simultaneously and both up-regulated and down-regulated genes can be identified in the same experiment. Adjusting and refining several steps in the DD method and improving technical parameters, for example, increasing redundancy, evaluating optimized reagents and conditions for reverse transcription of total RNA, Techniques have been developed that can achieve highly reproducible and sensitive results by optimizing the polymerase chain reaction (PCR) and the separation of its products. Applied and improved techniques are described in detail by von der Kammer et al. (Nucleic Acids Research 1999, 27: 2211-2218). A set of 64 specifically designed primers was developed (standard set) to achieve a statistically comprehensive analysis of all possible RNA species. In addition, the method was improved to produce a preparative DD slab gel technology based on the use of fluorescently labeled primers. In the present invention, RNA populations from carefully selected postmortem brain tissues (frontal and temporal cortex) of Alzheimer's disease patients and age-matched control subjects were compared. As a starting material for DD analysis, total RNA extracted as described in (ii) above was used. 0.5 mM each dNTP, Sensscript ™ reverse transcriptase 1 μl and 1 × RT buffer (Qiagen), 10 U RNase inhibitor (Qiagen), and 1 μM single base anchor oligonucleotide HT ₁₁ A, HT ₁₁ G or HT Equal amounts of 0.05 μg of RNA were each transcribed into cDNA in a 20 μl reaction containing any of ₁₁ C (Liang et al., Nucleic Acids Research 1994, 22: 5763-5764; Zhao et al., Biotechniques 1995, 18: 842-850). Reverse transcription was performed at 37 ° C. for 60 minutes, and the final denaturation step was performed at 93 ° C. for 5 minutes. Final volume 20 μl, any one of Cy3-labeled random DD primers (1 μM), 1 × GeneAmp PCR buffer (Applied Biosystems), 1.5 mM MgCl ₂ (Applied Biosystems), 2 μM dNTP-mix (dATP, dGTP, dCTP, dTTP, Amersham Pharmacia Biotech), 5% DMSO (Sigma), 1U AmpliTaq DNA polymerase (Applied Biosystems) together with the corresponding 1 base anchor oligonucleotide (1 μM), resulting cDNA 2 μl Each was subjected to polymerase chain reaction (PCR). PCR conditions were set as follows: one round was performed at 94 ° C. for 30 seconds for denaturation, cooled to 40 ° C. at 1 ° C./second, and 4 ° C. at 40 ° C. to anneal the primers with low stringency. 1 minute / second and heated to 72 ° C. for 1 minute at 72 ° C. for elongation. This round was followed by 39 high stringency cycles: 94 ° C for 30 seconds followed by 1 ° C / second cooling to 60 ° C, 60 ° C for 2 minutes, 1 ° C / second. Heated to 72 ° C. and performed at 72 ° C. for 1 minute. A final stage of 5 min at 72 ° C. was added to the last cycle (PCR cycler: Multi Cycler PTC 200, MJ Research). 8 μl of DNA loading buffer was added to 20 μl of PCR product preparation, denatured for 5 minutes and kept on ice until added onto the gel. 3.5 μl each in a slab gel system (Hitachi Genetic Systems) for 1 hour and 40 minutes at 2000 V, 60 W, 30 mA on a 0.4 mm thick 6% polyacrylamide (Long Ranger) / 7M urea sequencing gel separated. After electrophoresis was completed, the gel was scanned with an FMBIO II fluorescence scanner (Hitachi Genetic Systems) using the appropriate FMBIO II analysis 8.0 software. Full-size photos were printed, differentially expressed bands were marked, removed from the gel, transferred to a 1.5 ml container, overlaid with 200 μl of sterile water and kept at −20 ° C. until extracted.

Elution and re-amplification of differential display products: boil in ₂₀₀ μl H ₂ O for 10 minutes, cool on ice, and use ethanol (Merck) and glycogen / sodium acetate (Merck) at −20 ° C. The differential band was extracted from the gel by sedimentation overnight from followed by centrifugation at 13.000 rpm for 25 minutes at 4 ° C. The pellet was washed twice with ice-cold ethanol (80%), resuspended in 10 mM Tris pH 8.3 (Merck), and 10% glycerol (Merck) on a 0.025 μm VSWP membrane (Millipore). Dialyzed at room temperature for 1 hour. The first round took place at 94 ° C for 5 minutes, followed by 15 cycles of 94 ° C for 45 seconds, 60 ° C for 45 seconds, 1 ° C / second for 45 minutes to 70 ° C, and a final stage of 72 ° C for 5 minutes. Except for reamplification by 15 high stringency cycles in 25 μl PCR mix containing the corresponding primer pair used for differential display PCR (see above) under the same conditions. The preparation was used as a template.

  Cloning and sequencing of differential display products: Re-amplified cDNA was analyzed using the DNA LabChip system (Agilent 2100 Bioanalyzer, Agilent Technologies), ligated into the pCR-Blunt II-TOPO vector and the manufacturer's instructions (Zero Top10F ′ E. coli cells were transformed according to Blunt TOPO PCR cloning kit (Invitrogen). The cloned cDNA fragment was sequenced by a commercially available sequencing facility. The results of such a fluorescent differential display experiment for the human SCN2B gene are shown in FIG.

(Iv) Confirmation of differential expression by quantitative RT-PCR:
A light cycler technology (Roche) was used to provide definitive support for the differential expression of the gene encoding SCN2B. This technique features a rapid thermal cycle for the polymerase chain reaction, as well as real-time measurement of the fluorescent signal during amplification, and thus allows RT-PCR products to be highly optimized by using kinetic readings rather than endpoint readings. Can be accurately quantified. The ratio of SCN2BcDNA from the temporal cortex to SCN2B cDNA from the frontal cortex and the ratio of SCN2B cDNA from the hippocampus to SCN2B cDNA from the frontal cortex were respectively determined (relative quantification).

First, a standard curve was generated to determine the efficiency of PCR using primers specific for the gene encoding CN2B.
Primer set A:
5'-GACTGCTGGGATGTTATCTGCTTT-3 '
5'-TTGTCGCCAGTAGACCCAAAC-3 '.
Primer set B:
5'-CCAAGGCTGGGAAATGAG-3 '
5′-CAAGGGCAACTGGGAGAGTTC-3 ′.

LightCycler-FastStart DNA Master SYBR Green I mix (FastStart Taq DNA polymerase, reaction buffer, dNTP mixture with dUTP instead of dTTP, SYBR Green I dye, 1 mM MgCl ₂ ; Roche Primer containing 0.5 μM primer, cDNA dilution series 2 μl (final human brain cDNA final concentration 40, 20, 10, 5, 1, and 0.5 ng; Clontech) Accordingly, PCR amplification (95 ° C. for 1 second, 56 ° C. for 5 seconds, 72 ° C. for 5 seconds) was further performed in a volume of 20 μl containing 3 mM MgCl ₂ . Analysis of the melting curve showed a single peak at about 86.5 ° C. and no visible primer dimer was observed. The quality and size of the PCR product was determined with the DNA LabChip system (Agilent 2100 Bioanalyzer, Agilent Technologies). A single peak at the expected size of 82 bp was observed in the primer set A, and an expected size of 84 bp in the primer set B. In a similar manner, a PCR protocol was applied to determine the PCR efficiency of a set of reference genes selected as a reference standard for quantification. In the present invention, the average value of five such reference genes was determined: (1) Cyclophilin B; except for MgCl ₂ (added with 1 mM MgCl ₂ instead of 3 mM), the specific primer 5′-ACTGAAGCACTACGGGCTCTG -3 'and 5'-AGCCGTTGGTGTCTTTGCC-3' were used. Melting curve analysis showed a single peak at about 87 ° C. and no visible primer dimer was observed. Agarose gel analysis of the PCR product showed a single band of the expected size (62 bp). (2) Ribosomal protein S9 (RPS9); specific primers 5′-GGTCAAAATTTACCCGCGCCA-3 ′ and 5′-TCTCATCAAGCGTCAGCAGTTC-3 ′ were used (in addition to 3 mM, 1 mM MgCl ₂ was added). Melting curve analysis showed a single peak at about 85 ° C. and no visible primer dimer was observed. Agarose gel analysis of the PCR product showed a single band of the expected size (62 bp). (3) β-actin; specific primers 5′-TGGAACGGGTGAAGGTGACA-3 ′ and 5′-GGCAAGGGACTTCCTGTAA-3 ′ were used. Melting curve analysis showed a single peak at about 87 ° C. and no visible primer dimer was observed. Agarose gel analysis of the PCR product showed a single band of the expected size (142 bp). (4) GAPDH; specific primers 5'-CGTCCATGGGTGTACACCATG-3 'and 5'-GCTAAGCAGTTGGGTGGCAG-3' were used. Melting curve analysis showed a single peak at about 83 ° C. and no visible primer dimer was observed. Agarose gel analysis of the PCR product showed a single band of the expected size (81 bp). (5) Transferrin receptor TRR; specific primers 5′-GTCGCTGGTCAGTTCGTGATT-3 ′ and 5′-AGCAGTTGGCTGTTGTACTCTCTC-3 ′ were used. Melting curve analysis showed a single peak at about 83 ° C. and no visible primer dimer was observed. Agarose gel analysis of the PCR product showed a single band of the expected size (80 bp).

To calculate the values, the logarithm of the cDNA concentration was first plotted against the threshold cycle value Ct for the gene encoding SCN2B and the five reference standard genes. The slope and intercept of the standard curve (ie linear regression) was calculated for all genes. In the second stage, cDNA from frontal cortex and temporal cortex, and cDNA from frontal cortex and hippocampus, respectively, were analyzed in parallel and normalized to cyclophilin B. Ct values were measured and converted to whole brain cDNA (ng) using the corresponding standard curve:
10 ^ ((Ct value-intercept) / slope) [whole brain cDNA (ng)]

Frontal cortex and temporal cortex SCN2B cDNA values and frontal cortex and hippocampal SCN2B cDNA values were normalized to cyclophilin B, respectively, and the ratio was calculated according to the following equation:

  In the third stage, a set of reference standard genes was analyzed in parallel, the average level of the expression level of the reference standard gene for each individual brain sample, and the ratio between the temporal and frontal cortex, and the hippocampus and frontal cortex The average value of the ratio was determined. Cyclophilin was analyzed in steps 2 and 3, and the ratio of one gene to the other remained constant in different implementations, so it was not normalized to a single gene only, but a reference standard It was possible to normalize the value of SCN2B to the average value of the set of genes. Calculations were performed by dividing the respective ratios indicated 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 SCN2B gene are shown in FIGS.

(V) Immunohistochemistry:
In immunofluorescent staining of SCN2B in the human brain, frozen sections were prepared from the previous center after death of the donor using a cryostat (Leica CM3050S) and fixed in acetone at room temperature for 10 minutes. After washing in PBS, the sections were preincubated with blocking buffer (10% normal goat serum in PBS, 0.2% Triton X-100) for 30 minutes and then affinity purified rabbit anti-SCN2B antiserum (blocking buffer). Diluted 1:20 to 1:40 in S; 6811, Sigma) overnight at 4 ° C. After rinsing three times in 0.1% Triton X-100 / PBS, sections were incubated with FITC-conjugated goat anti-rabbit IgG antiserum (diluted 1: 150 in 1% BSA / PBS) for 2 hours at room temperature. Then, it was washed again in PBS. Nuclear staining was performed by incubating sections with 5 μM DAPI in PBS for 3 minutes (blue signal). To block lipofuscin autofluorescence in the human brain, treatment with 1% sudan black B in 70% ethanol for 2-10 minutes at room temperature followed by immersion in 70% ethanol, distilled water and PBS. Sections were covered with a cover glass using “Vectashield” mounting medium (Vector Laboratories, Burlingame, Calif.) And observed with an inverted microscope (IX81, Olympus Optical). Digital images were captured with appropriate software (AnalySiS, Olympus Optical).

FIG. 1 illustrates brain regions that have selective vulnerability to neuronal loss and degeneration in AD. FIG. 2 discloses the initial identification of differential expression of the gene encoding SCN2B on a fluorescent differential display screen. FIG. 3 shows validation of the differential expression of the human SCN2B gene in AD brain tissue by quantitative RT-PCR analysis using two different sets of primer pairs, primer set A. FIG. 4 shows validation of the differential expression of the human SCN2B gene in AD brain tissue by quantitative RT-PCR analysis using two different sets of primer pairs, primer set B. FIG. 5 shows the differential expression of the human SCN2B gene in AD brain tissue by quantitative RT-PCR analysis using primer set A. FIG. 6 discloses SEQ ID NO: 1; the amino acid sequence of the SCN2B protein. Full-length human SCN2B protein contains 215 amino acids. FIG. 7 shows the nucleotide sequence of human SCN2B cDNA, comprising 4903 nucleotides represented by the consensus sequence obtained from the assembly of overlapping large sets of overlapping human nucleotide sequence fragments found in SEQ ID NO: 2, Genbank human genome database. FIG. 7 shows the nucleotide sequence of human SCN2B cDNA, comprising 4903 nucleotides represented by the consensus sequence obtained from the assembly of overlapping large sets of overlapping human nucleotide sequence fragments found in SEQ ID NO: 2, Genbank human genome database. FIG. 8 illustrates the nucleotide sequence of the 99 bp SCN2B cDNA fragment identified and obtained by SEQ ID NO: 3, differential display (sequence from 5 'to 3'). FIG. 9 shows the sequence alignment of SEQ ID NO: 3 to the nucleotide sequence of human SCN2B cDNA, SEQ ID NO: 2. FIG. 10 schematically shows extended and corrected consensus sequences based on and derived from SCN2B consensus cDNA sequences, GenBank accession numbers AF049498, AF107028, AX376000, HSU87555, and other EST sequence fragments shown, and SCN2B 2 illustrates the assembly of SEQ ID NO: 2 from a genomic database sequence fragment that constitutes the sequence obtained from PCR amplification with primers for the gene encoding (ens-PCR1, ens-PCR2). FIG. 11 shows a schematic alignment with the SCN2B cDNA of SEQ ID NO: 3. The open rectangle represents the SCN2B open reading frame and the thin bar represents the 5 'and 3' untranslated region (UTR). FIG. 12 illustrates a section from a previous human center labeled with an affinity purified rabbit polyclonal anti-SCN2B antiserum (green signal) produced against a peptide corresponding to amino acids 179-215 of SCN2B. . FIG. 13 shows SCN2B gene expression levels in the temporal cortex relative to the frontal cortex in 19 age-matched healthy control individuals (0.59-2.15 times). FIG. 14 shows the SCN2B gene expression level in the temporal cortex relative to the frontal cortex in five age-matched healthy control individuals (0.48 to 1.48 times). FIG. 15 shows the hippocampus for the frontal cortex for the SCN2B gene in 6 Alzheimer seconds patients (0.48 to 10.87 fold) and 3 age-matched healthy control individuals (0.77 to 1.26 fold). The gene expression level in is shown.

Claims (12)

A method of diagnosing or determining the prognosis of a neurodegenerative disease in a subject or determining whether a subject is at increased risk of developing the disease:
In a sample from the subject,
(I) the transcript of the gene encoding SCN2B, and / or (ii) the translation product of the gene encoding SCN2B, and / or (iii) the level of the transcript or translation product fragment or derivative, or variant And / or determining activity and comparing said level and / or said activity to a reference value representing a known disease or health condition thereby diagnosing or determining the prognosis of said neurodegenerative disease in said subject Or determining whether the subject is at increased risk of developing the neurodegenerative disease.   The method of claim 1, wherein the neurodegenerative disease is Alzheimer's disease. A kit for diagnosing or determining the prognosis of a neurodegenerative disease, particularly Alzheimer's disease in a subject, or for determining a propensity or predisposition of a subject to develop such a disease,
(A) (i) a reagent that selectively detects a transcript of a gene encoding SCN2B, and (ii) a reagent that selectively detects a translation product of a gene encoding SCN2B, at least One kind of reagent,
(B) (i) determining, in a sample from the subject, the level or activity of the transcript and / or the translation product of the gene encoding SCN2B, or both the level and the activity; (ii) nerve Diagnosing or prognosing a neurodegenerative disease, particularly Alzheimer's disease, by diagnosing or determining a prognosis of a degenerative disease, particularly Alzheimer's disease, or determining a propensity or predisposition of a subject to develop such a disease; Instructions for determining or a tendency or predisposition for a subject to develop such a disease, the transcript and / or the translation product compared with a reference value representing a known health condition Different levels or activities, or both said levels and said activities; or represents a known health condition Diagnosis or prognosis of a neurodegenerative disease, particularly Alzheimer's disease, or develop such a disease by the level or activity of the transcript and / or the translation product, or both of the level and the activity, which are similar to or equal to the threshold A kit that shows a high tendency or predisposition. (I) a gene encoding SCN2B, and / or (ii) a transcription product of a gene encoding SCN2B, and / or (iii) a translation product of a gene encoding SCN2B, and / or (iv) (i)-( A modifier of the activity and / or level of at least one substance selected from the group consisting of fragments, derivatives or variants of iii). A recombinant non-human animal comprising a non-native gene sequence encoding SCN2B, or a fragment, derivative or variant thereof, wherein the recombinant non-human animal is:
(I) providing a gene targeting construct containing the gene sequence and a selectable marker sequence;
(Ii) introducing the targeting construct into a non-human animal stem cell;
(Iii) introducing the non-human animal stem cells into a non-human embryo;
(Iv) transplanting the embryo into a pseudopregnant non-human animal;
(V) developing the embryo until the final month;
(Vi) identifying a genetically modified non-human animal whose genome contains modifications of said gene sequence in both alleles;
(Vii) breeding the genetically modified non-human animal of step (vi) to obtain a genetically modified non-human animal whose genome comprises a modification of the endogenous gene, wherein said disruption ( A recombinant non-human animal wherein the disruption) results in said non-human animal being predisposed to developing symptoms of a neurodegenerative disease or related disease or disorder. (I) a gene encoding SCN2B, and / or (ii) a transcription product of a gene encoding SCN2B, and / or (iii) a translation product of a gene encoding SCN2B, and / or (iv) (i)-( iii) an assay for screening one or more substances selected from the group consisting of fragments, derivatives or mutants of neurodegenerative diseases, in particular Alzheimer's disease, or related diseases or disorders. And
(A) contacting the cell with a test compound;
(B) measuring the activity and / or level of one or more substances according to (i) to (iv);
(C) measuring the activity and / or level of one or more substances according to (i) to (iv) in a control cell not contacted with the test compound;
(D) comparing the level and / or activity of the substance in the cells of steps (b) and (c), and by changing the activity and / or said level of the substance in the contacted cell, Assay where is shown to be a modifier of said diseases and disorders. (I) a gene encoding SCN2B, and / or (ii) a transcription product of a gene encoding SCN2B, and / or (iii) a translation product of a gene encoding SCN2B, and / or (iv) (i)-( iii) a method for screening one or more substances selected from the group consisting of fragments, derivatives or mutants of neurodegenerative diseases, particularly Alzheimer's disease, or related diseases or disorders. And
(A) With respect to the substances described in (i) to (iv), a test compound is administered to a non-human test animal that has been given or predisposed to develop a neurodegenerative disease or related disease or disorder Stages,
(B) measuring the activity and / or level of one or more substances according to (i) to (iv);
(C) With respect to the substances described in (i) to (iv), in matched non-human control animals that have been given or predisposed to develop neurodegenerative diseases or related diseases or disorders Measuring the activity and / or level of one or more substances according to (i) or (iv) in a non-human animal to which such test compound has not been administered;
(D) comparing the activity and / or level of the substance in the non-human animal of steps (b) and (c), and by changing the activity and / or level of the substance in the non-human test animal, Where is shown to be a modifier of said disease or disorder.   The non-human test animal and / or the non-human control animal express SCN2B, or a fragment thereof, or a derivative or variant thereof under the control of a transcriptional regulatory factor that is not a natural SCN2B gene transcriptional regulatory factor. 8. The method of claim 7, wherein the method is a replacement non-human animal. An assay for testing a compound, preferably one that screens a plurality of compounds to determine the extent of binding of said compound to an SCN2B translation product, or a fragment, derivative or variant thereof:
(I) adding a liquid suspension of the SCN2B translation product, or a fragment or derivative or variant thereof to a plurality of containers;
(Ii) adding a detectable, in particular one or more fluorescently labeled compound to be screened for said binding to said plurality of containers;
(Iii) incubating the SCN2B translation product, or the fragment, derivative or variant thereof, and the detectable, in particular, fluorescently labeled compound (s),
(Iv) measuring the amount of fluorescence, preferably bound to the SCN2B translation product, or the fragment, derivative or variant thereof, preferably;
(V) determining the extent of binding by one or more of the compounds to the SCN2B translation product, or the fragment, derivative or variant thereof.   The protein molecule is SCN2B, a translation product of the gene encoding SEQ ID NO: 1, or a fragment, derivative or variant thereof, used as a diagnostic target for detecting neurodegenerative diseases, preferably Alzheimer's disease , Protein molecules.   SCN2B, a translation product of a gene encoding SEQ ID NO: 1, or a fragment thereof, wherein the protein molecule is used as a screening target for a reagent or compound for preventing or treating or ameliorating a neurodegenerative disease, preferably Alzheimer's disease, Or a protein molecule that is a derivative or variant.   Use of an antibody specifically immunoreactive with an immunogen for detecting a pathological state of a cell in a sample from a subject, wherein the immunogen is a translation of a gene encoding SCN2B, SEQ ID NO: 1 Use of a product, or a fragment, derivative or variant thereof, comprising immunocytochemical staining of the cell with the antibody, and staining in the cell as compared to a cell representing a known health condition Use in which the pathological state of the cells is indicated by a change in the degree of or a change in staining pattern.

Images