EP1514118A2 - Diagnostische und therapeutische verwendung von ras-gtpase-aktivierendem sh3-bindendem protein 2 (g3bp2) für neurodegenerative erkrankungen - Google Patents

Diagnostische und therapeutische verwendung von ras-gtpase-aktivierendem sh3-bindendem protein 2 (g3bp2) für neurodegenerative erkrankungen

Info

Publication number
EP1514118A2
EP1514118A2 EP03740295A EP03740295A EP1514118A2 EP 1514118 A2 EP1514118 A2 EP 1514118A2 EP 03740295 A EP03740295 A EP 03740295A EP 03740295 A EP03740295 A EP 03740295A EP 1514118 A2 EP1514118 A2 EP 1514118A2
Authority
EP
European Patent Office
Prior art keywords
g3bp2
gene
disease
activity
level
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03740295A
Other languages
English (en)
French (fr)
Inventor
Heinz Von Der Kammer
Johannes Pohlner
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Evotec Neurosciences GmbH
Original Assignee
Evotec Neurosciences GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Evotec Neurosciences GmbH filed Critical Evotec Neurosciences GmbH
Priority to EP03740295A priority Critical patent/EP1514118A2/de
Publication of EP1514118A2 publication Critical patent/EP1514118A2/de
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention relates to methods of diagnosing, prognosticating, and monitoring the progression of neurodegenerative diseases in a subject. Furthermore, methods of therapy control and screening for modulating agents of neurodegenerative diseases are provided. The invention also discloses pharmaceutical compositions, kits, and recombinant animal models.
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • these diseases constitute an enormous health, social, and economic burden.
  • AD is the most common neurodegenerative disease, accounting for about 70% of all dementia cases, and it is probably the most devastating age-related neurodegenerative condition affecting about 10% of the population over 65 years of age and up to 45% over age 85 (for a recent review see Vickers et al., Progress in Neurobiology 2000, 60: 139-165).
  • AD Alzheimer's disease
  • amyloid- ⁇ (A ⁇ ) protein evolves from the cleavage of the amyloid precursor protein (APP) by different kinds of proteases.
  • the cleavage by the ⁇ / ⁇ -secretase leads to the formation of A ⁇ peptides of different lengths, typically a short more soluble and slow aggregating peptide consisting of 40 amino acids and a longer 42 amino acid peptide, which rapidly aggregates outside the cells, forming the characteristic amyloid plaques (Selkoe, Physiological Rev 2001 , 81 : 741 -66; Greenfield et al., Frontiers Bioscience 2000, 5: D72-83).
  • the neuritic plaques have a diameter of 50 ⁇ m to 200 ⁇ m and are composed of insoluble fibrillar amyloids, fragments of dead neurons, of microglia and astrocytes, and other components such as neurotransmitters, apolipoprotein E, glycosaminoglycans, ⁇ 1 -antichymotrypsin and others.
  • the generation of toxic A ⁇ deposits in the brain starts very early in the course of AD, and it is discussed to be a key player for the subsequent destructive processes leading to AD pathology.
  • NFTs neurofibrillary tangles
  • abnormal neurites described as neuropil threads
  • a loss of neurons can be observed. It is discussed that said neuron loss may be due to a damaged microtubule-associated transport system (Johnson and Jenkins, J Alzheimers Dis 1996, 1 : 38-5 ' 8; Johnson and Hartigan, J Alzheimers Dis 1999, 1 : 329-351 ).
  • AD neurofibrillary tangles and their increasing number correlates well with the clinical severity of AD (Schmitt et al., Neurology 2000, 55: 370-376).
  • AD is a progressive disease that is associated with early deficits in memory formation and ultimately leads to the complete erosion of higher cognitive function.
  • the cognitive disturbances include among other things memory impairment, aphasia, agnosia and the loss of executive functioning.
  • a characteristic feature of the pathogenesis of AD is the selective vulnerability of particular brain regions and subpopuiations of nerve cells to the degenerative process. Specifically, the temporal lobe region and the hippocampus are affected early and more severely during the progression of the disease.
  • AD Alzheimer's disease
  • AD apolipoprotein E gene
  • the polymorphic plasmaprotein ApoE plays a role in the intercellular cholesterol and phospholipid transport by binding low-density lipoprotein receptors, and it seems to play a role in neurite growth and regeneration. Efforts to detect further susceptibility genes and disease-linked polymorphisms, lead to the assumption that specific regions and genes on human chromosomes 10 and 12 may be associated with late-onset AD (Myers et al., Science. 2000, 290: 2304-5; Bertram et al., Science 2000, 290: 2303; Scott et al., Am J Hum Genet 2000, 66: 922-32).
  • NF- ⁇ B transcription factors which play important roles during inflammatory reponses.
  • these proteins are inactive in the cytoplasm because they are tightly bound to the l ⁇ B-class of inhibitory proteins.
  • NF- ⁇ B shuttles into the nucleus where it regulates more than 60 proinflammatory genes. This is accomplished by a rapid ubiquitin-dependent degradation of the l ⁇ B-protein after phosphorylation of N-terminal serine-residues and a C-terminal nuclear localization sequence of NF- ⁇ B.
  • the protein After subsequent re- synthesis of IKB the protein has been reported to enter the nucleus where it binds to NF- ⁇ B thereby initiating the re-transport of the inactive NF- ⁇ B-l ⁇ B-complex into the cytoplasm.
  • This is mediated by nuclear export sequences located at the C-terminus of B and results in consequence in the inactivation of NF- ⁇ B. Therefore, the regulation of l ⁇ b's ability to translocate into the nucleus and to initiate the export of NF- ⁇ B regulates the transcriptional activity of NF- ⁇ B and, hence, the capability to respond to inflammatory processes.
  • a key factor in this regulation is to ensure the cytoplasmic localization of the inactive NF- ⁇ B-l ⁇ B-complex.
  • RasGAP SH3-binding protein 2 RasGAP SH3-binding protein 2
  • G3BP2 RasGAP SH3-binding protein 2
  • An N-terminal so called cytoplasmic retention signal (CRS) in l ⁇ B is thereby recognized by G3BP2 through an acidic stretch, and it is hypothesized that this interaction leads to the retention of NF ⁇ B-I ⁇ B in the cytosol.
  • CRS cytoplasmic retention signal
  • G3BP2 contains a putative mRNA binding domain which is speculated to be involved in RNA binding and/or RNA degradation the above interaction might serve as control in RNA stability and translational efficiency (Pringent et al., supra).
  • G3BP1 The first member of the family of highly homologous RasGap SH3-binding proteins was cloned in 1996 (Parker et al., 1996, Mol. Cell Biol 16:2561 -2569) and termed G3BP (G3BP1 in the following). Subsequently, G3BP2 has been cloned from mouse and human (Kennedy et al., 1996, Biomed. Peptides Proteins Nucleic Acids 2:93- 99). Additionally, the nucleotide sequence of the G3BP2 gene and a corresponding polypeptide sequence were deposited in the NCBI GenBank Database by Ishikawa et al.
  • G3BP2-a Two alternatively spliced isoforms, G3BP2-a and G3BP2-b, were identified and cloned from human fetal brain. They differ with respect to an insertion of 99 base pairs in the central region of G3BP2-a giving rise to the presence of five SH3-binding domains in G3BP2-b compared to 4 domains in G3BP2-a.
  • the open reading frame of G3BP2-a spans 1449 nucleotides (Database entry AF145284; AB014560) which code for 482 amino acids with a calculated molecular weight of 54.1 kDa (GenBank Protein ID: Q9UN86).
  • the open reading frame of G3BP2-b comprises 1350 nucleotides coding for a polypeptide of 449 amino acids with a calculated molecular weight of 50.7 kDa.
  • the gene was mapped to chromosome 4q12-4q24.
  • Both G3BP2-a and G3BP2-b are expressed in lung, liver, kidney, stomach, colon, pancreas, and testis, whereas the sole expression of G3BP2-a seems to be restricted to brain, muscle, and heart.
  • G3BP2-a In brain the expression of G3BP2-a is neuronal but not glial. G3BP2 shares an overall identity of -60% with G3BP1 (-82% identity in the N-terminal region). Both proteins feature an NTF2 (nuclear transport factor 2)-homologous domain, which has been described as an RNA-binding sequence, a proline-rich PXXP-motif, which binds to SH3 (src homology)-regions, and a RNP1 (ribonuclein particle)- and RNP2-domain which is common to hnRNPs.
  • NTF2 nuclear transport factor 2
  • PXXP-motif proline-rich PXXP-motif
  • RNP1 ribonuclein particle
  • G3BP1 RNA transport or RNA metabolism and in the regulation of nuclear transport processes through binding of small GTPase Ran is discussed (Prigent et al., supra). Differences in the RNA-binding regions between G3BP1 and G3BP2 suggest that the two proteins bind to different RNAs (Kennedy et al., supra). Despite the high homology between G3BP1 and G3BP2 it has been reported that there is so far no convincing evidence that G3BP2 could bind to RasGAP and, vice versa, G3BP1 could bind to l ⁇ B (Prigent et al., supra).
  • level as used herein is meant to comprise a gage of, or a measure of the amount of, or a concentration of a transcription product, for instance an mRNA, or a translation product, for instance a protein or polypeptide.
  • activity shall be understood as a measure for the ability of a transcription product or a translation product to ' produce a biological effect or a measure for a level of biologically active molecules.
  • activity also refers to enzymatic activity.
  • level and/or “activity” as used herein further refer to gene expression levels or gene activity. Gene expression can be defined as the utilization of the information contained in a gene by transcription and translation leading to the production of a gene product.
  • “Dysregulation” shall mean an upregulation or downregulation of gene expression.
  • a gene product comprises either RNA or protein and is the result of expression of a gene. The amount of a gene product can be used to measure how active a gene is.
  • the term "gene” as used in the present specification and in the claims comprises both coding regions (exons) as well as non-coding regions (e.g. non-coding regulatory elements such as promoters or enhancers, introns, leader and trailer sequences).
  • the term “ORF” is an acronym for "open reading frame” and refers to a nucleic acid sequence that does not possess a stop codon in at least one reading frame and therefore can potentially be translated into a sequence of amino acids.
  • regulatory elements shall comprise inducible and non-inducible promoters, enhancers, operators, and other elements that drive and regulate gene expression.
  • fragment as used herein is meant to comprise e.g. an alternatively spliced, or truncated, or otherwise cleaved transcription product or translation product.
  • derivative as used herein refers to a mutant, or an RNA-edited, or a chemically modified, or otherwise altered transcription product, or to a mutant, or chemically modified, or otherwise altered translation product.
  • a “derivative” may be generated by processes such as altered phosphorylation, or glycosylation, or acetylation, or lipidation, or by altered signal peptide cleavage or other types of maturation cleavage. These processes may occur post-translationally.
  • the term "modulator” as used in the present invention and in the claims refers to a molecule capable of changing or altering the level and/or the activity of a gene, or a transcription product of a gene, or a translation product of a gene.
  • a “modulator” is capable of changing or altering the biological activity of a transcription product or a translation product of a gene.
  • Said modulation may be an increase or a decrease in enzyme activity, a change in binding characteristics, or any other change or alteration in the biological, functional, or immunological properties of said translation product of a gene.
  • agent refers to any substance, chemical, composition or extract that have a positive or negative biological effect on a cell, tissue, body fluid, or within the context of any biological system, or any assay system examined. They can be agonists, antagonists, partial agonists or inverse agonists of a target.
  • agents, reagents, or compounds may be nucleic acids, natural or synthetic peptides or protein complexes, or fusion proteins.
  • oligonucleotide primer or “primer” refer to short nucleic acid sequences which can anneal to a given target polynucleotide by hybridization of the complementary base pairs and can be extended by a polymerase. They may be chosen to be specific to a particular sequence or they may be randomly selected, e.g. they will prime all possible sequences in a mix. The length of primers used herein may preferably vary from 10 nucleotides to 80 nucleotides.
  • Probes are short nucleic acid sequences of the nucleic acid sequences described and disclosed herein or sequences complementary therewith. They may comprise full length sequences, or fragments, derivatives, isoforms, or variants of a given sequence. The identification of hybridization complexes between a "probe” and an assayed sample allows the detection of the presence of other similar sequences within that sample.
  • homolog or homology is a term used in the art to describe the relatedness of a nucleotide or peptide sequence to another nucleotide or peptide sequence, which is determined by the degree of identity and/or similarity between said sequences compared.
  • variant refers to any polypeptide or protein, in reference to polypeptides and proteins disclosed in the present invention, in which one or more amino acids are added and/or substituted and/or deleted and/or inserted at the N-terminus, and/or the C-terminus, and/or within the native amino acid sequences of the native polypeptides or proteins of the present invention.
  • variant shall include any shorter or longer version of a polypeptide or protein.
  • Variants shall also comprise sequences that have at least about 80% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity with the amino acid sequences of the native polypeptides or proteins, of G3BP2, SEQ ID NO.1 .
  • Proteins and polypeptides of the present invention include variants, fragments and chemical derivatives of a protein comprising the amino acid sequences of G3BP2, SEQ ID NO. 1. They can include proteins and polypeptides which can be isolated from nature or be produced by recombinant and/or synthetic means. Native proteins or polypeptides refer to naturally-occurring truncated or secreted forms, naturally occurring variant forms (e.g. splice-variants) and naturally occurring allelic variants.
  • isolated as used herein is considered to refer to molecules that are removed from their natural environment, i.e.
  • sequences encoding such molecules can be linked by the hand of man to polynucleotides, to which they are not linked in their natural state, and that such molecules can be produced by recombinant and/or synthetic means. Even if for said purposes those sequences may be introduced into living or non-living organisms by methods known to those skilled in the art, and even if those sequences are still present in said organisms, they are still considered to be isolated.
  • the terms "risk”, “susceptibility”, and “predisposition” are tantamount and are used with respect to the probability of developing a neurodegenerative disease, preferably Alzheimer's disease.
  • the term 'AD' shall mean Alzheimer's disease.
  • AD-type neuropathology refers to neuropathological, neurophysiologicai, histopathological and clinical hallmarks as described in the instant invention and as commonly known from state- of-the-art literature (see: Iqbal, Swaab, Winblad and Wisniewski, Alzheimer ' s Disease and Related Disorders (Etiology, Pathogenesis and Therapeutics), Wiley & Sons, New York, Weinheim, Toronto, 1999; Scinto and Daffner, Early Diagnosis of Alzheimer ' s Disease, Humana Press, Totowa, New Jersey, 2000; Mayeux and Christen, Epidemiology of Alzheimer ' s Disease: From Gene to Prevention, Springer Press, Berlin, Heidelberg, New York, 1999; Younkin, Tanzi and Christen, Presenilins and Alzheimer's Disease, Springer Press, Berlin, Heidelberg, New York, 1998).
  • Neurodegenerative diseases or disorders according to the present invention comprise Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Pick's disease, fronto-temporal dementia, progressive nuclear palsy, corticobasal degeneration, cerebro-vascular dementia, multiple system atrophy, argyrophilic grain dementia and other tauopathies, and mild- cognitive impairment.
  • Further conditions involving neurodegenerative processes are, for instance, age-related macular degeneration, narcolepsy, motor neuron diseases, prion diseases, traumatic nerve injury and repair, and multiple sclerosis.
  • the invention features a method of diagnosing or prognosticating a neurodegenerative disease in a subject, or determining whether a subject is at increased risk of developing said disease.
  • the method comprises: determining a level, or an activity, or both said level and said activity of (i) a transcription product of the G3BP2 gene, and/or of (ii) a translation product of the G3BP2 gene, and/or of (iii) a fragment, or derivative, or variant of said transcription or translation product in a sample from said subject and comparing said level, and/or said activity to a reference value representing a known disease or health status, thereby diagnosing or prognosticating said neurodegenerative disease in said subject, or determining whether said subject is at increased risk of developing said neurodegenerative disease.
  • the invention also relates to the construction and the use of primers and probes which are unique to the nucleic acid sequences, or fragments, or variants thereof, as disclosed in the present invention.
  • the oligonucleotide primers and/or probes can be labeled specifically with fluorescent, bioluminescent, magnetic, or radioactive substances.
  • the invention further relates to the detection and the production of said nucleic acid sequences, or fragments and/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 with said primer combinations to amplify said gene specific nucleic acid sequences from a sample containing nucleic acids. Such sample may be derived either from healthy or diseased subjects.
  • the invention provides nucleic acid sequences, oligonucleotide primers, and probes of at least 10 bases in length up to the entire coding and gene sequences, useful for the detection of gene mutations and single nucleotide polymorphisms in a given sample comprising nucleic acid sequences to be examined, which may be associated with neurodegenerative diseases, in particular Alzheimer's disease.
  • This feature has utility for developing rapid DNA-based diagnostic tests, preferably also in the format of a kit.
  • the invention features a method of monitoring the progression of a neurodegenerative disease in a subject.
  • a level, or an activity, or both said level and said activity, of (i) a transcription product of the G3BP2 gene, and/or of (ii) a translation product of the G3BP2 gene, and/or of (iii) a fragment, or derivative, or variant of said transcription or translation product in a sample from said subject is determined.
  • Said- level and/or said activity is compared to a reference value representing a known disease or health status. Thereby the progression of said neurodegenerative disease in said subject is monitored.
  • the invention features a method of evaluating a treatment for a neurodegenerative disease, comprising determining a level, or an activity, or both said level and said activity of (i) a transcription product of the G3BP2 gene, and/or of (ii) a translation product of the G3BP2 gene, and/or of (iii) a fragment, or derivative, or variant of said transcription or translation product in a sample obtained from a subject being treated for said disease. Said level, or said activity, or both said level and said activity are compared to a reference value representing a known disease or health status, thereby evaluating the treatment for said neurodegenerative disease.
  • said gene coding for a protein of the family of SH3-domain-binding and RNA-binding proteins is the gene coding for the Ras-GTPase-activating protein SH3-domain-binding protein, also termed GAP SH3-domain binding protein, or Gap SH3-binding protein (G3BP), particularly for the Ras-GTPase-activating protein SH3-domain-binding protein 2, also termed GAP SH3-domain binding protein 2, or Gap SH3-binding protein 2 (G3BP2), or KIAA0660 (GenBank Database K1AA0660, Ishikawa et al., DNA Res, 1998, 5:169-176) (Database entry AB014560; AF145284; AF053535; BC01 1731 ).
  • said Gap SH3-binding protein 2 also called G3BP2 is represented by SEQ ID NO.1 (GenBank Protein ID: Q9UN86).
  • the gene coding for said Gap SH3-binding protein 2 is also generally referred to as the G3BP2 gene, or G3BP2
  • said Gap SH3-binding protein 2 is also generally referred to as the G3BP2 protein, or G3BP2.
  • said neurodegenerative disease or disorder is Alzheimer's disease, and said subjects suffer from Alzheimer's disease.
  • the present invention discloses the detection and differential expression and regulation of the G3BP2 gene in specific brain regions of AD patients. Consequently, the G3BP2 gene and its corresponding transcription and/or translation products may have a causative role in the regional selective neuronal degeneration typically observed in AD. Alternatively, G3BP2 may confer a neuroprotective function to the remaining surviving nerve cells. Based on these disclosures, the present invention has utility for the diagnostic evaluation and prognosis as well as for the identification of a predisposition to a neurodegenerative disease, in particular AD. Furthermore, the present invention provides methods for the diagnostic monitoring of patients undergoing treatment for such a disease.
  • said sample to be analyzed and determined is selected from the group comprising brain tissue, or other tissues, or other body cells.
  • the sample can also comprise cerebrospinal fluid or other body fluids including saliva, urine, serum plasma, or mucus.
  • the methods of diagnosis, prognosis, monitoring the progression or evaluating a treatment for a neurodegenerative disease, according to the instant invention can be practiced ex corpore, and such methods preferably relate to samples, for instance, body fluids or cells, removed, collected, or isolated from a subject or patient.
  • said reference value is that of a level, or an activity, or both said level and said activity of (i) a transcription product of the G3BP2 gene, and/or of (ii) a translation product of the G3BP2 gene, and/or of (iii) a fragment, or derivative, or variant of said transcription or translation product in a sample from a subject not suffering from said neurodegenerative disease.
  • an alteration in the level and/or activity of a transcription product of the G3BP2 gene and/or of a translation product of the G3BP2 gene and/or of a fragment, or derivative, or variant thereof in a sample cell, or tissue, or body fluid from said subject relative to a reference value representing a known health status indicates a diagnosis, or prognosis, or increased risk of becoming diseased with a neurodegenerative disease, particularly AD.
  • measurement of the level of transcription products of the G3BP2 gene is performed in a sample from a subject using a quantitative PCR- analysis with primer combinations to amplify said gene specific sequences from cDNA obtained by reverse transcription of RNA extracted from a sample of a subject.
  • a Northern blot with probes specific for said gene can also be applied. It might further be preferred to measure transcription products by means of chip-based micro-array technologies. These techniques are known to those of ordinary skill in the art (see Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001 ; Schena M., Microarray Biochip Technology, Eaton Publishing, Natick, MA, 2000).
  • An example of an immunoassay is the detection and measurement of enzyme activity as disclosed and described in the patent application WO 02/14543.
  • a level and/or an activity of a translation product of the G3BP2 gene and/or of a fragment, or derivative, or variant of said translation product, and/or a level of activity of said translation product of the G3BP2 gene and/or of a fragment, or derivative, or variant thereof can be detected using an immunoassay, an activity assay, and/or a binding assay.
  • assays can measure the amount of binding between said protein molecule and an anti-protein antibody by the use of enzymatic, chromodynamic, radioactive, magnetic, or luminescent labels which are attached to either the anti-protein antibody or a secondary antibody which binds the anti-protein antibody.
  • other high affinity ligands may be used.
  • Immunoassays which can be used include e.g. ELISAs, Western blots, and other techniques known to those of ordinary skill in the art (see 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 may also be employed in the format of microarrays, protein-arrays, antibody microarrays, tissue microarrays, electronic biochip or protein-chip based technologies (see Schena M., Microarray Biochip Technology, Eaton Publishing, Natick, MA, 2000).
  • the level, or the activity, or both said level and said activity of (i) a transcription product of the G3BP2 gene, and/or of (ii) a translation product of the G3BP2 gene, and/or of (iii) a fragment, or derivative, or variant of said transcription or translation product in a series of samples taken from said subject over a period of time is compared, in order to monitor the progression of said disease.
  • said subject receives a treatment prior to one or more of said sample gatherings.
  • said level and/or activity is determined before and after said treatment of said subject.
  • the invention features a kit for diagnosing or prognosticating neurodegenerative diseases, in particular AD, in a subject, or determining the propensity or predisposition of a subject to develop a neurodegenerative disease, in particular AD, said kit comprising: (a) at least one reagent which is selected from the group consisting of (i) reagents that selectively detect a transcription product of the G3BP2 gene (ii) reagents that selectively detect a translation product of the G3BP2 gene; and
  • a neurodegenerative disease in particular AD
  • determining the propensity or predisposition of said subject to develop such a disease wherein a varied level, or activity, or both said level and said activity, of said transcription product and/or said translation product compared to a reference value representing a known health status; or a level, or activity, or both said level and said activity, of said transcription product and/or said translation product similar or equal to a reference value representing a known disease status, indicates a diagnosis or prognosis of a neurodegenerative disease, in particular AD, or an increased propensity or predisposition of developing such a disease.
  • the kit, according to the present invention may be particularly useful for the identification of individuals that are at risk of developing a neurodegenerative disease, in particular AD. Consequently, the kit, according to the invention, may serve as a means for targeting identified individuals for early preventive measures or therapeutic intervention prior to disease onset, before irreversible damage in the course of the disease has been inflicted. Furthermore, in preferred embodiments, the kit featured in the invention is useful for monitoring a progression of a neurodegenerative disease, in particular AD, in a subject, as well as monitoring success or failure of therapeutic treatment for such a disease of said subject.
  • the invention features a method of treating or preventing a neurodegenerative disease, in particular AD, in a subject comprising the administration to said subject in a therapeutically or prophylactically effective amount of an agent or agents which directly or indirectly affect a level, or an activity, or both said level and said activity, of (i) the G3BP2 gene, and/or (ii) a transcription product of the G3BP2 gene, and/or (iii) a translation product of the G3BP2 gene, and/or (iv) a fragment, or derivative, or variant of (i) to (iii).
  • Said agent may comprise a small molecule, or it may also comprise a peptide, an oligopeptide, or a polypeptide.
  • Said peptide, oligopeptide, or polypeptide may comprise an amino acid sequence of a translation product of a gene coding for G3BP2, or a fragment, or derivative, or a variant thereof.
  • An agent for treating or preventing a neurodegenerative disease, in particular AD, according to the instant invention may also consist of a nucleotide, an oligonucleotide, or a polynucleotide.
  • Said oligonucleotide or polynucleotide may comprise a nucleotide sequence of the gene coding for G3BP2 protein, either in sense orientation or in antisense orientation.
  • the method comprises the application of per se known methods of gene therapy and/or antisense nucleic acid technology to administer said agent or agents.
  • gene therapy includes several approaches: molecular replacement of a mutated gene, addition of a new gene resulting in the synthesis of a therapeutic protein, and modulation of endogenous cellular gene expression by recombinant expression methods or by drugs. Gene-transfer techniques are described in detail (see e.g.
  • the invention features a method of treating or preventing a neurodegenerative disease by means of antisense nucleic acid therapy, i.e. the down-regulation of an inappropriately expressed or defective gene by the introduction of antisense nucleic acids or derivatives thereof into certain critical cells (see e.g. Gillespie, DN&P 1992, 5: 389-395; Agrawal and Akhtar, Trends Biotechnol 1995, 13: 197-199; Crooke, Biotechnology 1992, 10: 882-6).
  • ribozymes i.e. RNA molecules that act as enzymes, destroying RNA that carries the message of disease has also been described (see e.g.
  • the subject to be treated is a human, and therapeutic antisense nucleic acids or derivatives thereof are directed against transcripts of a gene coding for G3BP2. It is preferred that cells of the central nervous system, preferably the brain, of a subject are treated in such a way. Cell penetration can be performed by known strategies such as coupling of antisense nucleic acids and derivatives thereof to carrier particles, or the above described techniques. Strategies for administering targeted therapeutic oligodeoxynucleotides are known to those of skill in the art (see e.g. Wickstrom, Trends Biotechnol 1992, 10: 281 -287). In some cases, delivery can be performed by mere topical application.
  • RNA interference RNA interference
  • the method comprises grafting donor cells into the central nervous system, preferably the brain, of said subject, or donor cells preferably treated so as to minimize or reduce graft rejection, wherein said donor cells are genetically modified by insertion of at least one transgene encoding said agent or agents.
  • Said transgene might be carried by a viral vector, in particular a retroviral vector.
  • the transgene can be inserted into the donor cells by a nonviral physical transfection of DNA encoding a transgene, in particular by microinjection.
  • Insertion of the transgene can also be performed by electroporation, chemically mediated transfection, in particular calcium phosphate transfection, or liposomal mediated transfection (see Mc Celland and Pardee, Expression Genetics: Accelerated and High-Throughput Methods, Eaton Publishing, Natick, MA, 1999).
  • said agent for treating and preventing a neurodegenerative disease is a therapeutic protein which can be administered to said subject, preferably a human, by a process comprising introducing subject cells into said subject, said subject cells having been treated in vitro to insert a DNA segment encoding said therapeutic protein, said subject cells expressing in vivo in said subject a therapeutically effective amount of said therapeutic protein.
  • Said DNA segment can be inserted into said cells in vitro by a viral vector, in particular a retroviral vector.
  • Methods of treatment comprise the application of therapeutic cloning, transplantation, and stem cell therapy using embryonic stem cells or embryonic germ cells and neuronal adult stem cells, combined with any of the previously described cell- and gene therapeutic methods.
  • Stem cells may be totipotent or pluripotent. They may also be organ-specific. Strategies for repairing diseased and/or damaged brain cells or tissue comprise (i) taking donor cells from an adult tissue. Nuclei of those cells are transplanted into unfertilized egg cells from which the genetic material has been removed. Embryonic stem cells are isolated from the blastocyst stage of the cells which underwent somatic cell nuclear transfer.
  • stem cells preferably neuronal cells (Lanza et al., Nature Medicine 1999, 9: 975-977), or (ii) purifying adult stem cells, isolated from the central nervous system, or from bone marrow (mesenchymal stem cells), for in vitro expansion and subsequent grafting and transplantation, or (iii) directly inducing endogenous neural stem cells to proliferate, migrate, and differentiate into functional neurons (Peterson DA, Curr. Opin. Pharmacol. 2002, 2: 34-42).
  • Adult neural stem cells are of great potential for repairing damaged or diseased brain tissues, as the germinal centers of the adult brain are free of neuronal damage or dysfunction (Colman A, Drug Discovery World 2001 , 7: 66-71 ).
  • the subject for treatment or prevention can be a human, an experimental animal, e.g. a mouse or a rat, a domestic animal, or a non-human primate.
  • the experimental animal can be an animal model for a neurodegenerative disorder, e.g. a transgenic mouse and/or a knock-out mouse with an AD-type neuropathology.
  • the invention features a modulator of an activity, or a level, or both said activity and said level of at least one substance which is selected from the group consisting of (i) the G3BP2 gene, and/or (ii) a transcription product of the G3BP2 gene and/or (iii) a translation product of the G3BP2 gene, and/or (iv) a fragment, or derivative, or variant of (i) to (iii).
  • the invention features a pharmaceutical composition
  • a pharmaceutical carrier refers to a diluent, adjuvant, excipient, or vehicle with which the modulator is administered.
  • the invention features a modulator of an activity, or a level, or both said activity and said level of at least one substance which is selected from the group consisting of (i) the G3BP2 gene, and/or (ii) a transcription product of the G3BP2 gene, and/or (iii) a translation product of the G3BP2 gene, and/or (iv) a fragment, or derivative, or variant of (i) to (iii) for use in a pharmaceutical composition.
  • the invention provides for the use of a modulator of an activity, or a level, or both said activity and said level of at least one substance which is selected from the group consisting of (i) the G3BP2 gene, and/or (ii) a transcription product of the G3BP2 gene and/or (iii) a translation product of the G3BP2 gene, and/or (iv) a fragment, or derivative, or variant of (i) to (iii) for a preparation of a medicament for treating or preventing a neurodegenerative disease, in particular AD.
  • the present invention also provides a kit comprising one or more containers filled with a therapeutically or prophylactically effective amount of said pharmaceutical composition.
  • the invention features a recombinant, non-human animal comprising a non-native gene sequence coding for G3BP2, or a fragment, or a derivative, or a variant thereof.
  • the generation of said recombinant, non-human animal comprises (i) providing a gene targeting construct containing said gene sequence and a selectable marker sequence, and (ii) introducing said targeting construct into a stem cell of a non-human animal, and (iii) introducing said non- human animal stem cell into a non-human embryo, and (iv) transplanting said embryo into a pseudopregnant non-human animal, and (v) allowing said embryo to develop to term, and (vi) identifying a genetically altered non-human animal whose genome comprises a modification of said gene sequence in both alleles, and (vii) breeding the genetically altered non-human animal of step (vi) to obtain a genetically altered non-human animal whose genome comprises a modification of said endogenous gene, wherein said gene is mis-ex
  • said recombinant, non-human animal comprises a non- native gene sequence coding for G3BP2 protein, or a fragment, or derivative, or variant thereof.
  • the invention features an assay for screening for a modulator of neurodegenerative diseases, in particular AD, or related diseases and disorders of one or more substances selected from the group consisting of (i) the G3BP2 gene, and/or (ii) a transcription product of the G3BP2 gene, and/or (iii) a translation product of the G3BP2 gene, and/or (iv) a fragment, or derivative, or variant of (i) to (iii).
  • a modulator of neurodegenerative diseases in particular AD, or related diseases and disorders of one or more substances selected from the group consisting of (i) the G3BP2 gene, and/or (ii) a transcription product of the G3BP2 gene, and/or (iii) a translation product of the G3BP2 gene, and/or (iv) a fragment, or derivative, or variant of (i) to (iii).
  • This screening method comprises (a) contacting a cell with a test compound, and (b) measuring the activity, or the level, or both the activity and the level of one or more substances recited in (i) to (iv), and (c) measuring the activity, or the level, or both the activity and the level of said substances in a control cell not contacted with said test compound, and (d) comparing the levels of the substance in the cells of step (b) and (c), wherein an alteration in the activity and/or level of said substances in the contacted cells indicates that the test compound is a modulator of said diseases and disorders.
  • the invention features a screening assay for a modulator of neurodegenerative diseases, in particular AD, or related diseases and disorders of one or more substances selected from the group consisting of (i) the G3BP2 gene, and/or (ii) a transcription product of the G3BP2 gene, and/or (iii) a translation product of the G3BP2 gene, and/or (iv) a fragment, or derivative, or variant of (i) to (iii), comprising (a) administering a test compound to a test animal which is predisposed to developing or has already developed symptoms of a neurodegenerative disease or related diseases or disorders, and (b) measuring the activity and/or level of one or more substances recited in (i) to (iv), and (c) measuring the activity and/or level of said substances in a matched control animal which is equally predisposed to developing or has already developed symptoms of said diseases and to which animal no such test compound has been administered, and (d) comparing the activity and/or level of the substance in the animals of
  • test animal and/or said control animal is a recombinant, non-human animal which expresses a gene coding for G3BP2, or a fragment, or a derivative, or a variant thereof, under the control of a transcriptional regulatory element which is not the native G3BP2 gene transcriptional control regulatory element.
  • the present invention provides a method for producing a medicament comprising the steps of (i) identifying a modulator of neurodegenerative diseases by a method of the aforementioned screening assays and (ii) admixing the modulator with a pharmaceutical carrier.
  • said modulator may also be identifiable by other types of screening assays.
  • the present invention provides for an assay for testing a compound, preferably for screening a plurality of compounds, for inhibition of binding between a ligand and a translation product of the gene coding for G3BP2, or a fragment, or derivative, or variant thereof.
  • Said screening assay comprises the steps of (i) adding a liquid suspension of said G3BP2 translation product, or a fragment, or derivative, or variant thereof, to a plurality of containers, and (ii) adding a compound or a plurality of compounds to be screened for said inhibition to said plurality of containers, and (iii) adding a detectable, preferably a fluorescently labelled ligand to said containers, and (iv) incubating said G3BP2 translation product, or said fragment, or derivative, or variant thereof, and said compound or plurality of compounds, and said detectable, preferably fluorescently labelled ligand, and (v) measuring the amounts of preferably fluorescence associated with said G3BP2 translation product, or with said fragment, or derivative, or variant thereof, and (vi) determining the degree of inhibition by one or more of said compounds of binding of said ligand to said G3BP2 translation product, or said fragment, or derivative, or variant thereof.
  • Said method may be useful for the identification of novel compounds as well as for evaluating compounds which have been improved or otherwise optimized in their ability to inhibit the binding of a ligand to an G3BP2 translation product, or a fragment, or derivative, or variant thereof.
  • a fluorescent binding assay in this case based on the use of carrier particles, is disclosed and described in patent application WO 00/52451.
  • a further example is the competitive assay method as described in patent WO 02/01226.
  • the present invention provides a method for producing a medicament comprising the steps of (i) identifying a compound as an inhibitor of binding between a ligand and a gene product of the gene coding for G3BP2 by the aforementioned inhibitory binding assay and (ii) admixing the compound with a pharmaceutical carrier.
  • a compound as an inhibitor of binding between a ligand and a gene product of the gene coding for G3BP2 by the aforementioned inhibitory binding assay and (ii) admixing the compound with a pharmaceutical carrier.
  • said compound may also be identifiable by other types of screening assays.
  • the invention features an assay for testing a compound, preferably for screening a plurality of compounds to determine the degree of binding of said compounds to a translation product of the gene coding for G3BP2, or to a fragment, or derivative, or variant thereof.
  • Said screening assay comprises (i) adding a liquid suspension of said G3BP2 translation product, or a fragment, or derivative, or variant thereof, to a plurality of containers, and (ii) adding a detectable, preferably a fluorescently labelled compound or a plurality of detectable, preferably fluorescently labelled compounds to be screened for said binding to said plurality of containers, and (iii) incubating said G3BP2 translation product, or said fragment, or derivative, or variant thereof, and said detectable, preferably fluorescently labelled compound or preferably fluorescently labelled compounds, and (iv) measuring the amounts of preferably the fluorescence associated with said G3BP2 translation product, or with said fragment, or derivative, or variant thereof, and (v) determining the degree of binding by one or
  • assay it might be preferred to use a fluorescent label.
  • any other type of detectable label might also be employed.
  • Said assay methods may be useful for the identification of novel compounds as well as for evaluating compounds which have been improved or otherwise optimized in their ability to bind to a G3BP2 translation product, or fragment, or derivative, or variant thereof.
  • the present invention provides a method for producing a medicament comprising the steps of (i) identifying a compound as a binder to a gene product of the G3BP2 gene by the aforementioned binding assays and (ii) admixing the compound with a pharmaceutical carrier.
  • said compound may also be identifiable by other types of screening assays.
  • the present invention provides for a medicament obtainable by any of the methods according to the herein claimed screening assays.
  • the instant invention provides for a medicament obtained by any of the methods according to the herein claimed screening assays.
  • the present invention features a protein molecule shown in SEQ ID NO. 1 , said protein molecule being a translation product of the gene coding for G3BP2 protein, or a fragment, or derivative, or variant thereof, for use as a diagnostic target for detecting a neurodegenerative disease, preferably Alzheimer's disease.
  • the present invention further features a protein molecule shown in SEQ ID NO. 1 , said protein molecule being a translation product of the gene coding for G3BP2 protein, or a fragment, or derivative, or variant thereof, for use as a screening target for reagents or compounds preventing, or treating, or ameliorating a neurodegenerative disease, preferably Alzheimer's disease.
  • the present invention features an antibody which is specifically immunoreactive with an immunogen, wherein said immunogen is a translation product of the G3BP2 gene, or a fragment, or a derivative, or variant thereof.
  • the immunogen may comprise immunogenic or antigenic epitopes or portions of a translation product of said gene, wherein said immunogenic or antigenic portion of a translation product is a polypeptide, and wherein said polypeptide elicits an antibody response in an animal, and wherein said polypeptide is immunospecifically bound by said antibody.
  • antibody as employed in the present invention, encompasses all forms of antibodies known in the art, such as polyclonal, monoclonal, chimeric, recombinatorial, anti-idiotypic, humanized, or single chain antibodies, as well as fragments thereof (see Dubel and Breitling, Recombinant Antibodies, Wiley-Liss, New York, NY, 1999).
  • Antibodies of the present invention are useful, for instance, in a variety of diagnostic and therapeutic methods, based on state-in-the-art techniques (see Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999 and Edwards R., Immunodiagnostics: A Practical Approach, Oxford University Press, Oxford, England, 1999) such as enzyme-immuno assays (e.g. enzyme-linked immunosorbent assay, ELISA), radioimmuno assays, chemoluminescence-immuno assays, Western-blot, immunoprecipitation and antibody microarrays. These methods involve the detection of translation products of a gene coding for a G3BP protein, in particular G3BP2.
  • enzyme-immuno assays e.g. enzyme-linked immunosorbent assay, ELISA
  • radioimmuno assays e.g. enzyme-linked immunosorbent assay, ELISA
  • radioimmuno assays
  • said antibodies can be used for detecting the pathological state of a cell in a sample from a subject, comprising immunocytochemical staining of said cell with said antibody, wherein an altered degree of staining, or an altered staining pattern in said cell compared to a cell representing a known health status indicates a pathological state of said cell.
  • the pathological state relates to a neurodegenerative disease, in particular to AD.
  • Immunocytochemical staining of a cell can be carried out by a number of different experimental methods well known in the art.
  • Figure 1 depicts the brain regions with selective vulnerability to neuronal loss and degeneration in AD.
  • neurons within the inferior temporal lobe, the entorhinal cortex, the hippocampus, and the amygdala are subject to degenerative processes in AD (Terry et al., Annals of Neurology 1981 , 10:184-192). These brain regions are mostly involved in the processing of learning and memory functions.
  • neurons within the frontal cortex, the occipital cortex, and the cerebellum remain largely intact and preserved from neurodegenerative processes in AD.
  • Brain tissues from the frontal cortex (F), the temporal cortex (T), and the hippocampus (H) of AD patients and healthy, age-matched control individuals were used for the herein disclosed examples.
  • the image of a normal healthy brain was taken from a publication by Strange (Brain Biochemistry and Brain Disorders, Oxford University Press, Oxford, 1992, p.4).
  • Figure 2 discloses the initial identification of the differential expression of the gene coding for G3BP2 in a fluorescence differential display screen.
  • the figure shows a clipping of a large preparative fluorescent differential display gel.
  • PCR products from the frontal cortex (F) and the temporal cortex (T) of two healthy control subjects and six AD patients were loaded in duplicate onto a denaturing polyacrylamide gel (from left to right).
  • PCR products were obtained by amplification of the individual cDNAs with the corresponding one-base-anchor oligonucleotide and the specific Cy3 labelled random primers.
  • the arrow indicates the migration position where significant differences in intensity of the signals for a transcription product of the gene coding for G3BP2 derived from frontal cortex as compared to the signals derived from the temporal cortex of AD patients exist.
  • the differential expression reflects an down-regulation of G3BP2 gene transcription in the temporal cortex compared to the frontal cortex of AD patients. Comparing the signals derived from temporal cortex and frontal cortex of healthy non-AD control subjects with each other, no difference in signal intensity, i.e. no altered expression level can be detected.
  • Figure 3 and 4 illustrate the verification of the differential expression of the G3BP2 gene in AD brain tissues by quantitative RT-PCR analysis. Quantification of RT-PCR products from RNA samples collected from the frontal cortex (F) and the temporal cortex (T) of AD patients ( Figure 3a) and samples from the frontal cortex (F) and the hippocampus (H) of AD patients ( Figure 4a) was performed by the LightCycler rapid thermal cycling technique. Likewise, samples of healthy, age-matched control individuals were compared ( Figure 3b for frontal cortex and temporal cortex, Figure 4b for frontal cortex and hippocampus). The data were normalized to the combined average values of a set of standard genes which showed no significant differences in their gene expression levels.
  • Said set of standard genes consisted of genes for the ribosomal protein S9, cyclophilin B, the transferrin receptor, GAPDH, and beta- actin.
  • the figures depict the kinetics of amplification by plotting the cycle number against the amount of amplified material as measured by its fluorescence.
  • Figure 5 discloses SEQ ID NO. 1 ; the amino acid sequence of the G3BP2 protein.
  • the full length human G3BP2 protein comprises 482 amino acids.
  • Figure 6 shows SEQ ID NO. 2, the coding sequence of the human G3BP2 cDNA, comprising 1449 nucleotides.
  • Figure 7 depicts SEQ ID NO. 3, the nucleotide sequence of the 93 bp G3BP2 cDNA fragment, identified and obtained by differential display (sequence in 5' to 3' direction).
  • Figure 8 outlines the exact nucleotide sequence alignment of the G3BP2 primer pair to the nucleotide sequence of the human G3BP2 gene (GenBank accession number AB014560).
  • Figure 9 depicts a Western blot image of total human brain extracts labeled with an affinity-purified rabbit anti-G3BP2 antiserum raised against a peptide corresponding to amino acids 139 to 148 of SEQ ID NO. 1.
  • Lane 1 molecular weight standard
  • Lane 2 total brain extract of a donor AD patient.
  • the arrow indicates a major band just below 55 kD (Figure 9a), which corresponds to the predicted molecular weight of 54.1 kD for G3BP2. All immunoreactive bands could be competed by preincubation of the antiserum with the corresponding peptide ( Figure 9b).
  • Table 1 lists the gene expression levels in the frontal cortex relative to the temporal cortex for the G3BP2 gene in seven AD patients, herein identified by internal reference numbers P010, P01 1 , P012, P014, P016, P017, P019 (1.15 to 1.74 fold) and five healthy, age-matched control individuals, herein identified by internal reference numbers C005, C008, C01 1 , C012, C014 (0.85 to 1.33 fold).
  • the scatter diagram visualizes individual values of the frontal to temporal cortex regulation ratios in control samples (dots) and in AD patient samples (triangles), respectively. The values shown are reciprocal values according to the formula described herein (see below).
  • Table 2 lists the G3BP2 gene expression levels in the frontal cortex relative to the hippocampus in six Alzheimer's disease patients, herein identified by internal reference numbers P010, P01 1 , P012, P014, P016, P019 (0.92 to 1.85 fold) and three healthy, age-matched control individuals, herein identified by internal reference numbers C004, C005, C008 (0.90 to 1.23 fold).
  • the values shown are reciprocal values according to the formula described herein (see below).
  • the scatter diagram visualizes individual values of the frontal cortex to hippocampus regulation ratios in control samples (dots) and in AD patient samples (triangles), respectively.
  • Brain tissue dissection from patients with AD Brain tissues from AD patients and age-matched control subjects were collected within 6 hours post-mortem and immediately frozen on dry ice. Sample sections from each tissue were fixed in paraformaldehyde for histopathological confirmation of the diagnosis. Brain areas for differential expression analysis were identified (see Figure 1 ) and stored at -80°C until RNA extractions were performed.
  • DD differential display
  • DD polymerase chain reactions
  • RNA extracted as described above (ii). Equal amounts of 0.05 ⁇ g RNA each were transcribed into cDNA in 20 ⁇ l reactions containing 0.5 mM each dNTP, 1 ⁇ l Sensiscript Reverse Transcriptase and 1x RT buffer (Qiagen), 10 U RNase inhibitor (Qiagen) and 1 ⁇ M of either one-base- anchor oligonucleotides HTnA, HTn G or HT-n C (Liang et al., Nucleic Acids Research 1994, 22: 5763-5764; Zhao et al., Biotechniques 1995, 18:842-850).
  • PCR polymerase chain reaction
  • a polymerase chain reaction employing the corresponding one-base-anchor oligonucleotide (1 ⁇ M) along with either one of the Cy3 labelled random DD primers (1 ⁇ M), 1 x GeneAmp PCR buffer (Applied Biosystems), 1 .5 mM MgCl2 (Applied Biosystems), 2 ⁇ M dNTP-Mix (dATP, dGTP, dCTP, dTTP Amersham Pharmacia Biotech), 5 % DMSO (Sigma), 1 U AmpliTaq DNA Polymerase (Applied Biosystems) in a 20 ⁇ l final volume.
  • PCR polymerase chain reaction
  • PCR conditions were set as follows: one round at 94 °C for 30 sec for denaturing, cooling 1 °C/sec down to 40 °C, 40 °C for 4 min for low- stringency annealing of primer, heating 1 °C/sec up to 72 °C, 72 °C for 1 min for extension. This round was followed by 39 high-stringency cycles: 94 °C for 30 sec, cooling 1 °C/sec down to 60 °C, 60 °C for 2 min, heating 1 °C/sec up to 72 °C, 72 °C for 1 min. One final step at 72 °C for 5 min was added to the last cycle (PCR cycler: Multi Cycler PTC 200, MJ Research).
  • the obtained preparations were used as templates for reamplification by 15 high-stringency cycles in 25- ⁇ l PCR mixtures containing the corresponding primer pairs as used for the differential display PCR (see above) under identical conditions, with the exception of the initial round at 94 °C for 5 min, followed by 15 cycles of: 94 °C for 45 sec, 60 °C for 45 sec, ramp 1 °C/sec to 70 °C for 45 sec, and one final step at 72 °C for 5 min.
  • PCR amplification (95 °C and 1 sec, 56 °C and 5 sec, and 72 °C and 5 sec) was performed in a volume of 20 ⁇ l containing LightCycler-FastStart DNA Master SYBR Green I mix (contains FastStart Taq DNA polymerase, reaction buffer, dNTP mix with dUTP instead of dTTP, SYBR Green I dye, and 1 mM MgCI 2 ; Roche), 0.5 ⁇ M primers, 2 ⁇ l of a cDNA dilution series (final concentration of 40, 20, 10, 5, 1 and 0.5 ng human total brain cDNA; Clontech) and, depending on the primers used, additional 3mM MgCI 2 .
  • LightCycler-FastStart DNA Master SYBR Green I mix contains FastStart Taq DNA polymerase, reaction buffer, dNTP mix with dUTP instead of dTTP, SYBR Green I dye, and 1 mM M
  • the mean value of five such reference genes was determined: (1 ) cyclophilin B, using the specific primers 5'- ACTGAAGCACTACGGGCCTG-3' and 5'-AGCCGTTGGTGTCTTTGCC-3' except for MgCI 2 (an additional 1 mM was added instead of 3 mM). Melting curve analysis revealed a single peak at approximately 87 °C with no visible primer dimers. Agarose gel analysis of the PCR product showed one single band of the expected size (62 bp).
  • Ratio G3BP2 frontal [ng] / cyclophilin B frontal [ng]
  • Ratio G3BP2 frontal [ng] / cyclophilin B frontal [ng]
  • a third step the set of reference standard genes was analyzed in parallel to determine the mean average value of the temporal to frontal ratios, and of the hippocampal to frontal ratios, respectively, of expression levels of the reference standard genes for each individual brain sample.
  • cyclophilin B was analyzed in step 2 and step 3, and the ratio from one gene to another gene remained constant in different runs, it was possible to normalize the values for G3BP2 to the mean average value of the set of reference standard genes instead of normalizing to one single gene alone. The calculation was performed by dividing the respective ratio shown above by the deviation of cyclophilin B from the mean value of all housekeeping genes. The results of such quantitative RT-PCR analysis for the G3BP2 gene are shown in Figures 3 and 4.
  • Brain tissue (0.1 g) was homogenized in 1 ml RIPA buffer (150 mM sodium chloride, 50 mM tris-HCI, pH7.4, 1 mM ethylenediamine-tetraacetic acid, 1 mM phenylmethylsulfonyl flouride, 1 % Triton X-100, 1 % sodium deoxycholic acid, 1 % sodium dodecylsulfate, 5 ⁇ g/ml of aprotinin, 5 ⁇ g/ml of leupeptin) on ice. After centrifuging for 15 min at 13,000 rpm, the supernatant was diluted five-fold in SDS- loading buffer.
  • RIPA buffer 150 mM sodium chloride, 50 mM tris-HCI, pH7.4, 1 mM ethylenediamine-tetraacetic acid, 1 mM phenylmethylsulfonyl flouride, 1 % Triton X-100, 1 % sodium

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Neurology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Neurosurgery (AREA)
  • Biotechnology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
EP03740295A 2002-06-20 2003-06-20 Diagnostische und therapeutische verwendung von ras-gtpase-aktivierendem sh3-bindendem protein 2 (g3bp2) für neurodegenerative erkrankungen Withdrawn EP1514118A2 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP03740295A EP1514118A2 (de) 2002-06-20 2003-06-20 Diagnostische und therapeutische verwendung von ras-gtpase-aktivierendem sh3-bindendem protein 2 (g3bp2) für neurodegenerative erkrankungen

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US38992702P 2002-06-20 2002-06-20
EP02013692 2002-06-20
EP02013692 2002-06-20
US389927P 2002-06-20
PCT/EP2003/006521 WO2004001422A2 (en) 2002-06-20 2003-06-20 Diagnostic and therapeutic use of ras-gtpase-activating sh3-domain-binding protein 2 (g3bp2) for neurodegenerative diseases
EP03740295A EP1514118A2 (de) 2002-06-20 2003-06-20 Diagnostische und therapeutische verwendung von ras-gtpase-aktivierendem sh3-bindendem protein 2 (g3bp2) für neurodegenerative erkrankungen

Publications (1)

Publication Number Publication Date
EP1514118A2 true EP1514118A2 (de) 2005-03-16

Family

ID=56290442

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03740295A Withdrawn EP1514118A2 (de) 2002-06-20 2003-06-20 Diagnostische und therapeutische verwendung von ras-gtpase-aktivierendem sh3-bindendem protein 2 (g3bp2) für neurodegenerative erkrankungen

Country Status (3)

Country Link
EP (1) EP1514118A2 (de)
AU (1) AU2003278497A1 (de)
WO (1) WO2004001422A2 (de)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10303974A1 (de) 2003-01-31 2004-08-05 Abbott Gmbh & Co. Kg Amyloid-β(1-42)-Oligomere, Verfahren zu deren Herstellung und deren Verwendung
GB2419332A (en) 2004-10-22 2006-04-26 Gibbs Tech Ltd Steering arrangement with retractable wheels
PT1954718E (pt) 2005-11-30 2014-12-16 Abbvie Inc Anticorpos anti-globulómeros aβ, suas porções de ligação ao antigénio, correspondentes hibridomas, ácidos nucleicos, vectores, células hospedeiras, métodos de produção dos ditos anticorpos, composições compreendendo os ditos anticorpos, usos dos ditos anticorpos e métodos para uso dos ditos anticorpos
DK1976877T4 (en) 2005-11-30 2017-01-16 Abbvie Inc Monoclonal antibodies to amyloid beta protein and uses thereof
US8455626B2 (en) 2006-11-30 2013-06-04 Abbott Laboratories Aβ conformer selective anti-aβ globulomer monoclonal antibodies
WO2008104386A2 (en) 2007-02-27 2008-09-04 Abbott Gmbh & Co. Kg Method for the treatment of amyloidoses
MX360403B (es) 2010-04-15 2018-10-31 Abbvie Inc Proteinas de union a amiloide beta.
EP2603524A1 (de) 2010-08-14 2013-06-19 AbbVie Inc. Amyloid-beta-bindende proteine
WO2023174838A1 (en) * 2022-03-14 2023-09-21 F. Hoffmann-La Roche Ag Modulators of the g3bp2-tau interaction for the treatment of tau associated diseases

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2780062B1 (fr) * 1998-06-17 2000-07-28 Rhone Poulenc Rorer Sa Anticorps monoclonaux diriges contre la proteine g3bp, et leurs utilisations

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SCHNEIDER MARTIN: "A rational approach to maximize success rate in target discovery", ARCHIV DER PHARMAZIE, vol. 337, no. 12, 1 December 2004 (2004-12-01), Weinheim, pages 625-633, XP002553686, DOI: doi:10.1002/ardp.200400913 *
See also references of WO2004001422A3 *

Also Published As

Publication number Publication date
WO2004001422A2 (en) 2003-12-31
WO2004001422A3 (en) 2004-03-18
AU2003278497A8 (en) 2004-01-06
AU2003278497A1 (en) 2004-01-06

Similar Documents

Publication Publication Date Title
US20080269103A1 (en) Diagnostic and Therapeutic Use of the Kcne4 Gene and Protein for Alzheimer's Disease
EP1776591B1 (de) Diagnostische und therapeutische verwendung einer plasmamembran-atpase
WO2004001422A2 (en) Diagnostic and therapeutic use of ras-gtpase-activating sh3-domain-binding protein 2 (g3bp2) for neurodegenerative diseases
EP1497661A1 (de) Diagnostische und therapeutische verwendung von "ensadin-0477" für neurodegenerative erkrankungen
WO2004038411A2 (en) Diagnostic and therapeutic use of ensadin-0289 gene and protein for neurodegenerative diseases
US20060052280A1 (en) Diagnostic and therapeutic use of a golgi protein for neurodegenerative diseases
US20070269800A9 (en) Diagnostic and therapeutic use of foap-13 polynucleotides and polypeptides for neurodegenerative diseases
EP1565751A1 (de) Diagnostische und therapeutische verwendung des proteins h-rev107 für alzheimersche krankheit
WO2004035823A2 (en) Diagnostic and therapeutic use of the nicotinamide mononucleotide adenylytransferase 2 (nmnat-2) gene and protein for neurodegenerative diseases
EP1490694B1 (de) Camp-reguliertes phosphoprotein für die diagnose und behandlung von neurodegenerativen krankheiten
US20060051757A1 (en) Diagnostic and therapeutic use of ensadin-0477 gene and protein for neurodegenerative diseases
US20060073480A1 (en) Diagnostic and therapeutic use of vault polynucleotides and proteins for neurodegenerative diseases
EP1561117A1 (de) Diagnostische und therapeutische verwendung von arl7 für alzheimer krankheit
US20050153295A1 (en) Diagnostic and therapeutic use of human maguin proteins and nucleic acids for neurodegenerative diseases
US20060160728A1 (en) Diagnostic and therapeutic use of ensandin-0138 gene and protein for neurodegenerative diseases
WO2003098221A1 (en) Diagnostic and therapeutic use of ensadin-0255 gene and protein for neurodegenerative diseases
US20060294602A1 (en) Diagnostic and therapeutic use of a rab family gtp-binding protein for neurodegenerative diseases
EP1534856A2 (de) Diagnostische und therapeutische verwendung von thbp im kontext neurodegenerativer erkrankungen
WO2003080661A1 (en) Diagnostic and therapeutic use of human maguin proteins and nucleic acids for neurodegenerative diseases
WO2006008294A2 (en) Diagnostic and therapeutic use of slim3 for neurodegenerative diseases
EP1478930A2 (de) Diagnostische und therapeutische verwendung von ma onconeuronalen antigenen für neurodegenerative erkrankungen

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20041203

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20061214

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20081202