EP1771578A2 - Diagnostische und therapeutische verwendung von slim3 für neurodegenerative erkrankungen - Google Patents

Diagnostische und therapeutische verwendung von slim3 für neurodegenerative erkrankungen

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Publication number
EP1771578A2
EP1771578A2 EP05777722A EP05777722A EP1771578A2 EP 1771578 A2 EP1771578 A2 EP 1771578A2 EP 05777722 A EP05777722 A EP 05777722A EP 05777722 A EP05777722 A EP 05777722A EP 1771578 A2 EP1771578 A2 EP 1771578A2
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EP
European Patent Office
Prior art keywords
slim3
disease
protein
seq
gene
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EP05777722A
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English (en)
French (fr)
Inventor
Heinz Von Der Kammer
Johannes Pohlner
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Evotec Neurosciences GmbH
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Evotec Neurosciences GmbH
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Publication of EP1771578A2 publication Critical patent/EP1771578A2/de
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • 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 Alzheimer's disease in a subject. Furthermore, methods of therapy control and screening for modulating agents of Alzheimer's disease and related diseases are provided.
  • the invention also discloses pharmaceutical compositions, the use of a kit, recombinant animal models and the use of said recombinant animal models.
  • 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 (Vickers et al., Progress in Neurobiology 2000, 60: 139-165; Walsh and Selkoe, Neuron 2004, 44:181-193).
  • AD Alzheimer's disease
  • amyloid- ⁇ protein evolves from the cleavage of the amyloid precursor protein (APP) by different kinds of proteases (Selkoe and Kopan, Annu Rev Neurosci 2003, 26:565-597; Ling et al., lnt J Biochem Cell Biol 2003, 35:1505-1535).
  • Two types of plaques, diffuse plaques and neuritic plaques can be detected in the brain of AD patients. They are primarily found in the cerebral cortex and hippocampus. 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.
  • AD neurofibrillary tangles
  • abnormal neurites described as neuropil threads
  • AD is a progressive disease that is associated with early deficits in memory formation and ultimately leads to the complete erosion of higher cognitive function.
  • the cognitive disturbances include among other things memory impairment, aphasia, agnosia and the loss of executive functioning.
  • a characteristic feature of the pathogenesis of AD is the selective vulnerability of particular brain regions and subpopulations of nerve cells to the degenerative process. Specifically, the temporal lobe region and the hippocampus are affected early and more severely during the progression of the disease.
  • neurons within the frontal cortex, occipital cortex, and the cerebellum remain largely intact and are protected from neurodegeneration (Terry et al., Annals of Neurology 1981 , 10: 184-92).
  • AD 1 there is no cure for AD 1 nor is there an effective treatment to halt the progression of AD or even to diagnose AD ante-mortem with high probability.
  • epsilon 4 allele of the three different existing alleles epsilon 2, 3, and 4 of the apolipoprotein E gene (ApoE) (Strittmatter et al., Proc Natl Acad Sci USA 1993, 90: 1977-81 ; Roses, Ann NY Acad Sci 1998, 855: 738-43).
  • the present invention is based on the detection of dysregulated, differential expression of a gene coding for the skeletal muscle LIM protein 3 (SLIM3) in human Alzheimer's disease brain samples.
  • LIM3 skeletal muscle LIM protein 3
  • the LIM protein family was named for three identified members namely lin-11 (Freyd et al., Nature 1990, 344: 876-879), isl-1 (Karlsson et al., Nature 1990, 344: 879-882), and mec-3 (Way and Chalfie, Cell 1988, 54: 5-16).
  • LIM proteins are involved in a wide range of cellular functions like transcription, oncogenic transformation, signal-transduction and in cell adhesion. This may be achieved through protein-protein interactions as the LIM domain contains zinc-finger structures.
  • SLIM protein 3 which is also known under the name of four and a half LIM protein 2 (FHL2) or down-regulated in rhabdomyosarcoma LIM protein (DRAL), was initially deduced by aligning overlapping cDNA sequences after searching sequence databases with a partial human SLIM1/FHL1 cDNA.
  • SLIM3/FHL2, SLIM1/FHL1 and SLIM2/FHL3 belong to the family of LIM domain proteins.
  • SLIM1 and SLIM2 have 48 and 52% sequence identity to SLIM3, respectively, and exhibit a similar genomic organization (Morgan and Madgwick, Bioch ⁇ m. Biophys. Res. Commun. 1996, 225: 632-638).
  • the SLIM protein 3 was subsequently cloned from a human fetal heart cDNA library and further characterized (Chan et al., Gene 1998, 210: 345-350).
  • the full-length clone consists of 1416 base pairs encoding a 279 amino acid polypeptide with a mass of 32 kDa and was mapped to chromosome 2q12-q13. It is encoded by 5 exons which are distributed over an entire gene sequence of 26 kb (Genini et al., DNA Cell Biol. 1997, 16: 433-442; Waye and Lee, Gene 1998, 210: 345-350; Morgan and Madgwick, Bioch ⁇ m. Biophys. Res. Commun.
  • SLIM3 SLIM1 and SLIM2 each contain 4 complete LIM domains and the second half of a fifth LIM domain (FHL, four and a half Lim protein). Thus, they belong to the FHL-LIM domain protein family. They are members of the LIM-only class of proteins, which consist primarily of LIM domains.
  • the LIM domain is a cysteine-rich zinc-finger motif harbouring the following consensus sequence: CX2CX16-23HX2-CX2CX2CX16-21CX(C,H,D).
  • the domain is responsible for protein-protein interactions (e.g. PDZ-domains, ankyrin repeats, SH3-domain, cytoskeletal proteins and transcription factors) and binds two zinc ions.
  • LIM domains can associate with other LIM domains, thus forming homo- and heterodimers (Feuerstein et al., Proc. Natl Acad. Science USA 1994, 91 : 10655-10659).
  • SLIM3/FHL2/DRAL itself interacts with ⁇ - and li-subunits of integrins and is located in focal adhesion complexes (Wrixler et al., J. Biol. Chem. 2000, 275: 33669-33678).
  • SLIM3/FHL2 has also been found to be located not only in the cytoplasm but also in the nucleus: it is hypothesized that down-regulation of SLIM3/FHL2 may play a role in tumor development and heart function (Scholl et al., J. Cell Biol. 2000, 151 : 495-505).
  • DRAL interacted with a hydrophilic loop region (amino acids 269-298) in the endoproteolytic N-terminal fragment of PS2, but not that of PS1.
  • a hydrophilic loop region amino acids 269-298
  • PS2 point mutations within a region from residues 275 to 296 abolished the binding with DRAL.
  • SLIM3/FHL2 may regulate gene expression by transmitting rho-GTPase signals from the cell membrane into the nucleus (M ⁇ ller et al., EMBO J. 2002, 4: 736-748).
  • SLIM3/FHL2 has been described as a tissue-specific coactivator of the androgen receptor and may play a role in the effects of androgens on heart and prostate (M ⁇ ller et al., EMBO J. 2000, 19: 359-369). It has been shown that LIM family proteins may selectively activate CREB- and CREM- transcription factors (Fimia et al., MoI. Cell. Biol. 2000, 20: 8613-8622). A role in mediating osteoregulatory effects in osteoblasts by interacting with Insulin-like Growth Factor-binding Protein 5 (IGFBP-5) has been speculated (Amaar et al., J. Biol. Chem.
  • 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 or to biological activity and/or pharmacological activity which refers to binding, antagonization, repression, blocking, neutralization or sequestration of an ion channel or ion channel subunit and which refers to activation, agonization, upregulation of an ion channel or ion channel subunit.
  • Biological activity includes but is not limited to the transmembrane transport of ions and/or transmembrane ion flow and/or the regulation thereof.
  • “Pharmacological activity” includes but is not limited to the ability of an ion channel or an ion channel subunit to bind a ligand, a compound, an agent, a modulator and/or another ion channel subunit.
  • 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.
  • 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).
  • 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 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 transcript for instance, refers to a transcript having alterations in the nucleic acid sequence such as single or multiple nucleotide deletions, insertions, or exchanges.
  • a derivative translation product for instance, 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.
  • modulator 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 the biological activity and/or pharmacological activity, 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.
  • a “modulator” refers to a molecule which has the capacity to either enhance or inhibit, thus to “modulate” a functional property of an ion channel subunit or an ion channel, to “modulate” binding, antagonization, repression, blocking, neutralization or sequestration of an ion channel or ion channel subunit and to “modulate” activation, agonization and upregulation.
  • Module will be also used to refer to the capacity to affect the biological activity of a cell.
  • the terms “agent”, “reagent”, or “compound” refer to any substance, chemical, composition, or extract that have a positive or negative biological effect on a cell, tissue, body fluid, or within the context of any biological system, or any assay system examined.
  • agents 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. They may also be antibodies, organic or anorganic molecules or compositions, small molecules, drugs and any combinations of any of said agents above. They may be used for testing, for diagnostic or for therapeutic purposes.
  • 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.
  • primers 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.
  • identity and “similarity” mean the degree of polypeptide or polynucleotide sequence relatedness which are determined by matching a query sequence and other sequences of preferably the same type (nucleic acid or protein sequence) with each other.
  • Preferred computer program methods to calculate and determine "identity” and “similarity” include, but are not limited to GCG BLAST (Basic Local Alignment Search Tool) (Altschul et al., J.
  • 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, but retains its essential properties.
  • variant shall include any shorter or longer version of a polypeptide or protein.
  • “Variants” shall also comprise a sequence that has at least about 80% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity with the amino acid sequences of a SLIM protein, in particular SLIM3, SEQ ID NO: 1. "Variants” include, for example, proteins with conservative amino acid substitutions in highly conservative regions. "Proteins and polypeptides” of the present invention include variants, fragments and chemical derivatives of the protein comprising the amino acid sequences of a SLIM protein, SEQ ID NO: 1 , SLIM3. Sequence variations shall be included wherein a codon are replaced with another codon due to alternative base sequences, but the amino acid sequence translated by the DNA sequence remains unchanged.
  • Proteins and polypeptides can be included 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 or substances which have been changed and/or 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 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 “AD pathology” as used herein refers to neuropathological, neurophysiological, histopathological and clinical hallmarks, signs and symptoms 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,.
  • Brain stage or "Braak staging” refers to the classification of brains according to the criteria proposed by Braak and Braak (Braak and Braak, Acta Neuropathology 1991 , 82: 239-259; Braak and Braak, J Neural Transm 1998, 53: 127-140).
  • the neuropathologic progression of AD is divided into six stages (stage 0 to 6).
  • low Braak stages (0-3) may represent persons which are not considered to suffer from Alzheimer disease signs and symptoms ("controls")
  • Braak stages 4 to 6 may represent persons already suffering from Alzheimer's disease ("AD patients”).
  • the values obtained from "controls” are the “reference values” representing a "known health status” and the values obtained from "AD patients” are the “reference values” representing a "known disease status”.
  • a recommendation is given by Braak H. (www. alzforum.org).
  • 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, cerebrovascular dementia, multiple system atrophy, argyrophilic grain dementia and other tauopathies, and mild-cognitive impairment.
  • Further conditions involving neurodegenerative processes are, for instance, ischemic stroke, age-related macular degeneration, narcolepsy, motor neuron diseases, prion diseases, traumatic nerve injury and repair, and multiple sclerosis.
  • the present invention discloses the identification, the differential expression, regulation, a dysregulation of skeletal muscle LIM protein 3 (SLIM3) gene expression, in specific brain regions of Alzheimer's disease patients in comparison with each other and/or in comparison to age-matched control tissue samples of frontal cortex, of temporal cortex and of hippocampus, respectively. No such dysregulation is observed in samples derived from age-matched, healthy controls.
  • the present invention discloses that the gene expression for SLIM3 is varied, is dysregulated in AD-affected brains, in that SLIM3 mRNA levels are decreased, are down-regulated in the temporal cortex as compared to the frontal cortex, or are elevated, are up-regulated in the frontal cortex as compared to the temporal cortex or the hippocampus.
  • the present invention discloses that the SLI M3 expression differs between the frontal cortex and the temporal cortex of healthy age-matched control subjects compared to the frontal cortex and the temporal cortex of AD patients in that the level of SLIM3 is downregulated in the temporal and frontal cortex of AD patients. No such dysregulation is observed comparing samples with each other obtained from age-matched, healthy controls. This dysregulation presumably relates to a pathologic alteration of SLIM3 in AD- affected brains. To date, no experiments have been described that demonstrate a relationship between the dysregulation of SLIM3 gene expression and the pathology of neurodegenerative diseases, in particular AD. Likewise, no mutations in the SLIM3 gene or protein have been described to be associated with said diseases. Linking the SLIM3 gene and protein to such diseases, as disclosed in the instant invention, offers new ways, inter alia, for the diagnosis and treatment of said diseases.
  • the present invention discloses a dysregulation of a gene coding for SLIM3 in specific brain regions of AD patients.
  • 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 and display a selective vulnerability to neuronal loss and degeneration in AD.
  • neurons within the frontal cortex, the occipital cortex, and the cerebellum remain largely intact and preserved from neurodegenerative processes.
  • 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.
  • 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 or of monitoring the effect of a treatment administered to a subject having a neurodegenerative disease.
  • the method comprises: determining a level, an expression or an activity, or both said level, expression and said activity of (i) a transcription product of the gene coding for the skeletal muscle LIM protein 3 (SLIM3 protein), and/or of (ii) a translation product of the gene coding for the skeletal muscle LIM protein 3 (SLIM3 protein), and/or of (iii) a fragment, or derivative, or variant of said transcription or translation product in a sample obtained from said subject and comparing said level, expression and/or said activity to a reference value representing a known disease status (patient), and/or to a reference value representing a known health status (control), and/or to a reference value representing a known Braak stage and analysing whether said level, expression and/or said activity is varied, is altered compared to a reference value representing a known health status, and/or is similar or equal to a reference value representing a known disease status and/or is similar compared to a reference value representing a known Braak stage which is an indication that said subject has a
  • the invention also relates to the construction and the use of primers and probes which are unique to the nucleic acid sequences, or fragments, or variants thereof, as disclosed in the present invention.
  • the oligonucleotide primers and/or probes can be labeled specifically with fluorescent, bioluminescent, magnetic, or radioactive substances.
  • the invention further relates to the detection and the production of said nucleic acid sequences, or fragments and variants thereof, using said specific oligonucleotide primers in appropriate combinations. PCR- analysis, a method well known to those skilled in the art, can be performed with said primer combinations to amplify said gene specific nucleic acid sequences from a sample containing nucleic acids.
  • Such sample may be derived either from healthy or diseased subjects or subjects with defined ⁇ braak stages. Whether an amplification results in a specific nucleic acid product or not, and whether a fragment of different length can be obtained or not, may be indicative for a neurodegenerative disease, in particular Alzheimer's disease.
  • 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, expression or an activity, or both said level, expression and said activity, of (i) a transcription product of the gene coding for the skeletal muscle LIM protein 3 (SLIM3 protein), and/or of (M) a translation product of the gene coding for the skeletal muscle LIM protein 3 (SLIM3 protein), and/or of (iii) a fragment, or derivative, or variant of said transcription or translation product in a sample obtained from said subject is determined.
  • Said level, expression and/or said activity is compared to a reference value representing a known disease or health status or a known Braak stage. 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, expression or an activity, or both said level, expression and said activity of (i) a transcription product of the gene coding for the skeletal muscle LIM protein 3 (SLIM3 protein), and/or of (ii) a translation product of the gene coding for the skeletal muscle LIM protein 3 (SLIM3 protein), 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, expression or said activity, or both said level, expression and said activity are compared to a reference value representing a known disease or health status or a known Braak stage, thereby evaluating the treatment for said neurodegenerative disease.
  • said SLIM gene coding for a LIM protein is the SLIM3 gene and protein, also referred to as the skeletal muscle LIM protein 3 (SLIM3) or synonymously four and a half LIM protein 2 (FHL2), also named as down-regulated in rhabdomyosarcoma LIM protein (DRAL), is represented by the SLIM3 gene coding for the protein of SEQ ID NO: 1 , SLIM3 (Genbank accession number U29332).
  • the amino acid sequence of said protein is deduced from the mRNA sequence corresponding to SEQ ID NO: 2, SLIM3 cDNA which corresponds to the cDNA sequence of Genbank accession number BC014397 (SLIM3 mRNA, Genbank accession number L42176).
  • SLIM3 also refers to the nucleic acid sequences of SEQ ID NO: 2, coding for the protein of SEQ ID NO: 1.
  • SLIM3 also refers to the nucleic acid sequence SEQ ID NO: 4 representing the coding sequence (cds) of human SLI M3.
  • said sequences are "isolated" as the term is employed herein.
  • the gene coding for said SLIM3 protein is also generally referred to as the SLIM3 gene or simply SLIM3.
  • the protein of SLIM3 is also generally referred to as the SLIM3 protein or simply SLIM3.
  • said neurodegenerative disease or disorder is Alzheimer's disease (AD), and said subjects suffer from signs and symptoms of Alzheimer's disease.
  • AD Alzheimer's disease
  • the sample to be analyzed and determined is selected from the group comprising brain tissue or other tissues, or body cells.
  • the sample can also comprise cerebrospinal fluid or other body fluids including saliva, urine, stool, blood, 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 corpor ⁇ , they are practiced "in vitro" 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, of expression or of an activity, or both said level and said activity of (i) a transcription product of the gene coding for SLIM3 protein, and/or of (ii) a translation product of the gene coding for SLIM3 protein, and/or of (iii) a fragment, or derivative, or variant of said transcription or translation product in a sample obtained from a subject not suffering from said neurodegenerative disease (control sample, control) or in a sample obtained from a subject suffering from a neurodegenerative disease, in particular Alzheimer's disease (patient sample, patient, AD sample) or from a person with a defined Braak stage.
  • an alteration in the level, expression and/or activity, a varied level, expression and/or activity of a transcription product of the gene coding for SLIM3 protein and/or of a translation product of the gene coding for SLIM3 protein and/or of a fragment, or derivative, or variant thereof in a sample cell, or tissue, or body fluid obtained from said subject (patient sample) relative to a reference value representing a known health status (control sample) 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 gene coding for SLIM3 protein is performed in a sample obtained 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.
  • Primer combinations SEQ ID NO: 5, SEQ ID NO: 6 are given in Example (vi) of the instant invention, but also other primers generated from the sequences as disclosed in the instant invention can be used.
  • a Northern blot or a ribonuclease protection assay (RPA) with probes specific for said gene can also be applied. It might further be preferred to measure transcription products by means of chip-based microarray technologies.
  • a level and/or an activity of a translation product of the gene coding for SLIM3 protein and/or of a fragment, or derivative, or variant of said translation product, and/or the level of activity of said translation product, 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 1 Western blots and other techniques known to those of ordinary skill in the art (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999 and Edwards R, Immunodiagnostics: A Practical Approach, Oxford University Press, Oxford; England, 1999). All these detection techniques may also be employed in the format of microarrays, protein-arrays, antibody microarrays, tissue microarrays, electronic biochip or protein-chip based technologies (see Schena M., Microarray Biochip Technology, Eaton Publishing, Natick, MA, 2000).
  • the level, expression or the activity, or both said level, expression and said activity of (i) a transcription product of the gene coding for SLIM3 protein, and/or of (ii) a translation product of the gene coding SLIM3 protein, 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 the use of 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, or of monitoring the effect of a treatment administered to a subject having a neurodegenerative disease, in particular AD 1 said kit comprising:
  • reagents that selectively detect a transcription product of the gene coding for a SLIM3 protein and/or for fragments, derivatives, variants thereof
  • reagents that selectively detect a translation product of the gene coding for a SLIM3 protein and/or for fragments, derivatives, variants thereof;
  • SLIM3 protein skeletal muscle LIM protein 3
  • kits may be particularly useful for the identification of individuals that are at risk of developing a neurodegenerative disease, in particular AD.
  • Reagents that selectively detect a transcription product and/or a translation product of the gene coding for SLIM protein can be sequences of various length, fragments of sequences, antibodies, aptamers, siRNA, microRNA, ribozymes.
  • the invention features the use of a kit in a method of diagnosing or prognosticating a neurodegenerative disease, in particular Alzheimer's disease, in a subject, and in a method of determining the propensity or predisposition of a subject to develop such a disease, and in a method of monitoring the effect of a treatment administered to a subject having a neurodegenerative disease, particularly AD.
  • the kit 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.
  • 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 1 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 gene coding for SLIM3 protein, and/or (ii) a transcription product of the gene coding for SLIM3 protein, and/or (iii) a translation product of the gene coding for SLIM3 protein, and/or (iv) a fragment, or derivative, or variant of (i) to (iii).
  • an agent or agents which directly or indirectly affect a level, or an activity, or both said level and said activity, of (i) the gene coding for SLIM3 protein, and/or (ii) a transcription product of the gene coding for SLIM3 protein, and/or (iii) a translation product of the gene coding for SLIM3 protein, and/or (iv
  • Said agent may comprise a small molecule, or it may also comprise a peptide, an oligopeptide, or a polypeptide.
  • Said peptide, oligopeptide, or polypeptide may comprise an amino acid sequence of a translation product of the gene coding for SLIM3 protein, or a fragment, or derivative, or a variant thereof.
  • An agent for treating or preventing a neurodegenerative disease, in particular AD 1 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 SLIM3 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 transcription products of the gene coding for SLIM3 protein. It is preferred that cells of the central nervous system, preferably the brain, of a subject are treated in such a way. Cell penetration can be performed by known strategies such as coupling of antisense nucleic acids and derivatives thereof to carrier particles, or the above described techniques. Strategies for administering targeted therapeutic oligo- deoxynucleotides are known to those of skill in the art (see e.g. Wickstrom, Trends Biot ⁇ chnol 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 1 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 non-human animal, e.g. a mouse or a rat, a domestic animal, or a non-human primate.
  • the experimental non- human 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 an agent, a selective antagonist or agonist or a modulator of an activity, or a level, or both said activity and said level, and/or of expression of at least one substance which is selected from the group consisting of (i) the gene coding for SLIM3 protein, and/or (ii) a transcription product of the gene coding for SLIM3 protein, and/or (iii) a translation product of the gene coding for SLIM3 protein, and/or (iv) a fragment, or derivative, or variant of (i) to (iii), and said agent, selective antagonist or agonist, or said modulator has a potential activity in the treatment of neurodegenerative diseases, in particular
  • the invention features a pharmaceutical composition
  • a pharmaceutical composition comprising said modulator and preferably a pharmaceutical carrier.
  • Said carrier refers to a diluent, adjuvant, excipient, or vehicle with which the modulator is administered.
  • 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 gene coding for S L I M 3 protein, and/or (ii) a transcription product of the gene coding SLIM3 protein, and/or (iii) a translation product of the gene coding for SLIM3 protein, 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 an agent, an antibody, a selective antagonist or agonist, or a modulator of an activity, or a level, or both said activity and said level, and/or of expression of at least one substance which is selected from the group consisting of (i) the gene coding for SLIM3 protein, and/or (ii) a transcription product of the gene coding for SLIM3 protein, and/or (iii) a translation product of the gene coding for SLIM3 protein, and/or (iv) a fragment, or derivative, or variant of (i) to (iii) in the manufacture of a medicament for treating or preventing a neurodegenerative disease, in particular AD.
  • Said antibody may be specifically immunoreactive with an immunogen which is a translation product of a gene coding for SLIM3 having SEQ ID NO: 1.
  • 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, genetically modified non- human animal comprising a non-native SLIM3 gene sequence coding for a SLIM3 protein having SEQ ID NO: 1 , or a fragment, or a derivative, or variant thereof under the control of a transcriptional element which is not the native SLIM3 gene transcriptional control element.
  • the generation of said recombinant, non-human animal comprises (i) providing a gene targeting construct containing said gene sequence and a selectable marker sequence, and (ii) introducing said targeting construct into a stem cell of a non-human animal, and (iii) introducing said non- human animal stem cell into a non-human embryo, and (iv) transplanting said embryo into a pseudopregnant non-human animal, and (v) allowing said embryo to develop to term, and (vi) identifying a genetically altered non-human animal whose genome comprises a modification of said gene sequence in both alleles, and (vii) breeding the genetically altered non-human animal of step (vi) to obtain a genetically altered non-human animal whose genome comprises a modification of said endogenous gene, wherein said gene is mis-expressed, or under-expressed, or over-expressed, and wherein said disruption or alteration results in said non- human animal exhibiting a predisposition to developing signs and symptoms of a neurodegenerative disease, which signs and
  • Such an animal may be useful for screening, testing and validating compounds, agents and modulators in the development of diagnostics and therapeutics to treat neurodegenerative diseases, in particular Alzheimer's disease.
  • the use of such a genetically modified animal in a screening method is disclosed in the instant invention.
  • the invention makes use of a cell, in which a gene sequence coding for a SLIM3 protein having SEQ ID NO: 1 , or a fragment, or derivative, or variant thereof is mis-expressed, under-expressed, non-expressed or over- expressed, or disrupted or in another way alterated for screening, testing and validating compounds, agents and modulators in the development of diagnostics and therapeutics to treat neurodegenerative diseases, in particular Alzheimer's disease.
  • the use of such a cell in a screening method is disclosed in the instant invention.
  • the invention features method of screening for an agent, a modulator, a selective antagonist or agonist for use in the treatment of neurodegenerative diseases, in particular AD, or related diseases and disorders, which agents, modulators or selective antagonists or agonists have an ability to alter expression and/or level and/or activity of one or more substances selected from the group consisting of (i) the gene coding for SLIM3 protein having SEQ ID NO: 1 , and/or (ii) a transcription product of the gene coding for SLIM3 protein having SEQ ID NO: 1 , and/or (iii) a translation product of the gene coding for SLIM3 protein having SEQ ID NO: 1 , 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 and/or the level, or both the activity and the level, and/or the expression of one or more substances recited in (i) to (iv), and (c) measuring the activity and/or the level, or both the activity and the level and/or the expression of said substances in a control cell not contacted with said test compound, and (d) comparing the levels and/or activities and/or the expression of the substance in the cells of step (b) and (c), wherein an alteration in the activity and/or level and/or expression of said substances in the contacted cells indicates that the test compound is an agent, modulator, selective antagonist or agonist for use in the treatment of neurodegenerative diseases and disorders.
  • Said cells may be cells as disclosed in the instant invention.
  • the invention features a method of screening for an agent, a modulator, a selective antagonist or agonist for use in the treatment of neurodegenerative diseases, in particular AD 1 or related diseases and disorders which agents, modulators or selective antagonists or agonists have an ability to alter expression and/or level and/or activity of one or more substances selected from the group consisting of (i) the gene coding for SLIM3 protein having SEQ ID NO: 1 , and/or (ii) a transcription product of the gene coding for SLI M3 protein having SEQ ID NO: 1 , and/or (iii) a translation product of the gene coding for SLIM3 protein having SEQ ID NO: 1 , and/or (iv) a fragment, or derivative, or variant of (i) to (iii), comprising (a) administering a test compound to a non-human test animal which is predisposed to developing or has already developed signs and symptoms of a neurodegenerative disease or related diseases or disorders, said animal may be an animal model as disclosed in the group consisting of
  • 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 methods and 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 SLIM3 protein having SEQ ID NO: 1 , or a fragment, or derivative, or variant thereof.
  • Said screening assay comprises the steps of (i) adding a liquid suspension of said SLIM3 protein having SEQ ID NO: 1 , 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 SLIM3 protein, 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 the fluorescence associated with said SLIM3 protein, or with said fragment, or derivative, or variant thereof, and (vi) determining the degree of inhibition by one or more of said compounds of binding of said ligand to said SLIM3 protein, 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 a gene product of the gene coding for SLIM3 protein, 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 SLIM3 protein 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 SLIM3 protein 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 SLIM3 protein having SEQ ID NO: 1 , or to a fragment, or derivative, or variant thereof.
  • Said screening assay comprises (i) adding a liquid suspension of said SLIM3 protein, 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 SLIM3 protein, or said fragment, or derivative, or variant thereof, and said detectable, preferably fluorescently labelled compound or detectable, preferably fluorescently labelled compounds, and (iv) measuring the amounts of preferably the fluorescence associated with said SLIM3 protein, or with said fragment, or derivative, or variant thereof, and (v) determining the degree of binding by one or
  • SLI M3 translation product or a fragment, or derivative, or variant thereof into artificial liposomes as described in the present invention.
  • 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 SLIM3 protein, or a 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 gene coding for SLIM3 protein 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 the use of protein molecules of SEQ ID NO: 1 , said protein molecules SLIM3 being translation products of the gene coding for SLIM3, or a fragment, or derivative, or variant thereof, as diagnostic targets for detecting a neurodegenerative disease, in particular Alzheimer's disease.
  • the present invention further features the use of protein molecules of SEQ ID NO: 1 , said protein molecules SLIM3 being translation products of the gene coding for SLIM3, or a fragment, or derivative, or variant thereof, as screening targets for agents, modulators, selective antagonists, agonists, reagents or compounds preventing, or treating, or ameliorating a neurodegenerative disease, in particular Alzheimer's disease.
  • the present invention features antibodies which are specifically immunoreactive with an immunogen, wherein said immunogen is a translation product of the gene coding for SLIM3 protein, SEQ ID NO: 1 , or fragments, or derivatives, or variants thereof.
  • the immunogen may comprise immunogenic or antigenic epitopes or portions of a translation product of said gene, wherein said immunogenic or antigenic portion of a translation product is a polypeptide, and wherein said polypeptide elicits an antibody response in an animal, and wherein said polypeptide is immunospecifically bound by said antibody.
  • Methods for generating antibodies are well known in the art (see Harlow et al., Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988).
  • antibody 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) 1 radioimmuno assays, chemoluminescence-immuno assays, Western-blot, immunoprecipitation and antibody microarrays. These methods involve the detection of translation products of the SLIM3 gene, or fragments, or derivatives, or variants thereof.
  • enzyme-immuno assays e.g. enzyme-linked immunosorbent assay, ELISA
  • chemoluminescence-immuno assays chemoluminescence-immuno assays
  • said antibodies can be used for detecting the pathological state of a cell in a sample obtained 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.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 discloses the initial identification of the differential expression of the gene coding for SLIM3 proteins 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 SLIM3 gene derived from frontal cortex and from the temporal cortex of AD patients as compared to healthy controls exist.
  • the differential expression reflects a down-regulation of human SLIM3 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.
  • Figures 2 and 3 illustrate the verification of the differential expression of the SLIM3 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 2a) and samples from the frontal cortex (F) and the hippocampus (H) of AD patients ( Figure 3a) was performed by the LightCycler rapid thermal cycling technique. Likewise, samples of healthy, age- matched control individuals were compared ( Figure 2b for frontal cortex and temporal cortex, Figure 3b for frontal cortex and hippocampus). The data were normalized to the combined average values of a set of standard genes which showed no significant differences in their gene expression levels.
  • Said set of standard genes consisted of genes for cyclophilin B 1 the ribosomal protein S9, the transferrin receptor, GAPDH 1 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 4 shows the analysis of absolute mRNA expression of SLIM3 by comparison of control and AD stages using statistical method of the median at 98%-confidence level.
  • the data were calculated by defining control groups including subjects with either Braak stages 0 to 1 , Braak stages 0 to 2, or Braak stages 0 to 3 which are compared with the data calculated for the defined AD patient groups including Braak stages 2 to 6, Braak stages 3 to 6 and Braak stages 4 to 6, respectively. Additionally, three groups including subjects with either Braak stages 0 to 1 , Braak stages 2 to 3 and Braak stages 4 to 6, respectively, were compared with each other. A significant difference was detected comparing frontal cortex (F) and inferior temporal cortex (T) of AD patients and of healthy age-matched control persons with each other.
  • F frontal cortex
  • T inferior temporal cortex
  • Said difference reflects a down-regulation of SLI M3 in the temporal cortex and the frontal cortex of AD patients relative to the temporal cortex and frontal cortex of control persons and a down-regulation of SLIM3 in the temporal cortex of AD patients compared to their frontal cortices.
  • Figure 5 discloses SEQ ID NO: 1 , the amino acid sequence of the SLIM3 protein.
  • the full length human SLIM3 protein comprises 279 amino acids.
  • Figure 6 shows SEQ ID NO: 2, the nucleotide sequence of the human SLIM3 cDNA (Genbank accession number BC014397), comprising 1892 nucleotides.
  • Figure 7 depicts SEQ ID NO: 3, the nucleotide sequence of the 477 bp SLIM3 cDNA fragment, identified and obtained by differential display and subsequent cloning (sequence in 5' to 3' direction).
  • Figure 8 shows the nucleotide sequence of SEQ ID NO: 4, the coding sequence (cds) of the human SLIM3 gene, comprising 840 nucleotides, harbouring nucleotides 580 to 1419 of SEQ ID NO: 2.
  • Figure 9 outlines the sequence alignment of SEQ ID NO: 3 to the nucleotide sequence of the human SLIM3 cDNA, SEQ ID NO: 2 (GenBank accession number BC014397).
  • Figure 10 depicts the sequence alignment of the primers used for SLIM3 transcription level profiling (primer A, SEQ ID NO: 5 and primer B, SEQ ID NO:6) by quantitative RT-PCR with the corresponding clippings of SEQ ID NO: 2, SLIM3 cDNA.
  • Figure 11 schematically charts the alignment of the SLIM3 cDNA sequence SEQ ID NO: 2, the identified cDNA fragment sequence SEQ ID NO: 3, the coding sequence of SLIM3, SEQ ID NO:4 and both primer sequences SEQ ID NO: 5 and SEQ ID NO: 6 used for SLIM3 transcription level profiling (primer A, primer B) and the coding sequence (cds) of SLIM3.
  • the sequence positions are indicated on the right side.
  • Figure 12 lists SLIM3 gene expression levels in the temporal cortex relative to the frontal cortex in fifteen AD patients, herein identified by internal reference numbers P010, P011 , P012, P014, P016, P017, P019, P038, P040, P041 , P042, P046, P047, P048, P049 and twentyfive age-matched control individuals, herein identified by internal reference numbers C005, C008, C011 , C012, C014, C025, C026, C027, C028, C029, C030, C031, C032, C033, C034, C035, C036, C038, C039, C041 , C042, DE02, DE03, DE05, DE07.
  • the values shown are calculated according to the formula described herein (see below) and in case of an up-regulation in the frontal cortex the reciprocal values are calculated, respectively.
  • the bar diagram visualizes individual natural logarithmic values of the temporal to frontal cortex, ln(IT/IF), and of the frontal to temporal cortex regulation factors, ln(IF/IT), in different Braak stages (0 to 6).
  • Figure 13 lists the gene expression levels in the hippocampus relative to the frontal cortex for the SLIM3 gene in six Alzheimer's disease patients, herein identified by internal reference numbers P010, P011 , P012, P014, P016, P019 (0.37 to 1.04 fold) and three healthy, age-matched control individuals, herein identified by internal reference numbers C004, C005, C008 (0.46 to 0.78 fold).
  • the values shown are calculated according to the formula described herein (see below).
  • the scatter diagram visualizes individual logarithmic values of the hippocampus to frontal cortex regulation ratios, log(ratio HC/IF), in control samples (dots) and in AD patient samples (triangles).
  • Figure 14 depicts a Western blot image of total cell protein extracts labeled with polyclonal anti-SLIM3 antibodies (LTE-2, 1 :1000).
  • Lanes 1 and 2 total protein extract of human brain (1 ) and of H4APPsw cells stably expressing SLIM3 (2).
  • Lane 3 H4APPsw cells (negative control).
  • the arrow indicates a major band at about 36 kDa (lane 1 and 2), which corresponds to the predicted molecular weight of the SLIM3 protein.
  • Figure 15 shows the immunofluorescence analysis of H4APPsw control cells and H4APPsw cells stably over-expressing the myc-tagged SLIM3 protein (H4APPsw- SLIM3cds-myc).
  • the SLIM3-myc protein was detected with polyclonal anti-myc antibodies (MBL) and a Cy3-conjugated anti-rabbit antibody (Amersham) ( Figures 15A and 15B).
  • the cellular nucleus was stained with DAPI ( Figures 15C and 15D).
  • the overlay analysis indicates that the SLIM3cds-myc protein is mainly localized within the cytoplasma and at the plasma membrane ( Figure 15E).
  • Figure 16 exemplarily depicts micrographs (magnification 10x) from SLIM3- immunofluorescence-labeled sections of the inferior temporal gyrus from a control donor (Control T) and from an Alzheimer patient (Patient T).
  • Figures 16B and 16F show the signals resulting from immunostaining with anti-SLIM3 goat polyclonal antiserum.
  • CT cerebral cortex
  • WM white matter
  • Figures 16A and 16E display the immunoreactivity of neurons (neuronal nuclei and somata) stained with an antibody against the neuron-specific marker NeuN.
  • Figures 16C and 16G each display a picture which results from merging the pictures 16A, 16B and DAPI staining and the pictures 16E 1 16F and DAPI staining, respectively, wherein the colors code for the detected immunosignals from NeuN (red), SLIM3 (green) and for the nuclear staining with DAPI (blue). Immunoreactivity of SLIM3 was also observed in astrocytes (mainly localized to the nuclei), microglial cells (mainly localized to the nuclei) and oligodendrocytes (mainly localized to the nuclei).
  • Figure 17 exemplarily depicts micrographs (magnification 10x) from SLIM3- immunofluorescence-labeled sections of the frontal cortex from a control donor (Control F) and from an Alzheimer patient (Patient F).
  • Figures 17B and 17F show the signals resulting from immunostaining with anti-SLIM3 goat polyclonal antiserum.
  • CT cerebral cortex
  • WM white matter
  • Figures 17A and 17E display the immunoreactivity of neurons (neuronal nuclei and somata) stained with an antibody against the neuron-specific marker NeuN.
  • Figures 17C and 17G each display a picture which results from merging the pictures 17A 1 17B and DAPI staining and the pictures 17E, 17F and DAPI staining, respectively, wherein the colors code for the detected immunosignals from NeuN (red), SLIM3 (green) and for the nuclear staining with DAPI (blue). Immunoreactivity of SLIM3 was also observed in astrocytes (mainly localized to the nuclei), microglial cells (mainly localized to the nuclei) and oligodendrocytes (mainly localized to the nuclei).
  • Brain tissues from AD patients and age-matched control subjects were collected, on average, 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 and stored at -80 0 C until RNA extractions were performed.
  • DD screening method In order to identify changes in gene expression in different tissue, a modified and improved differential display (DD) screening method was employed.
  • 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 populations of RNA and provides clones of genes that are expressed in one population but not in the other. Several samples can be analyzed simultaneously and both up- and down-regulated genes can be identified in the same experiment. By adjusting and refining several steps in the DD method as well as modifying technical parameters, e.g.
  • RNA populations from carefully selected post-mortem brain tissues (frontal and temporal cortex) of Alzheimer's disease patients and age-matched control subjects were compared.
  • RNA 1 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 HT11A, HT11G or HT11 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), 1x 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 0 C 1 40 0 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 0 C 1 60°C for 2 min, heating 1°C/sec up to 72°C, 72°C for 1 min. One final step at 72 0 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 DD 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 0 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 conditions were set as follows: one round at 94°C for 30 sec for denaturing, cooling 1°C/sec down to 40 0 C, 40 0 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.
  • 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 DD 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, 6O 0 C for 45 sec, ramp 1 °C/sec to 70 0 C for 45 sec, and one final step at 72°C for 5 min.
  • the mRNA expression profiling between frontal cortex tissue (F) and inferior temporal cortex tissue (T) of SLIM3 has been analyzed in four up to nine tissues per Braak stage. Because of the lack of high quality tissues from one donor with Braak 3 pathology, tissues of one additional donor with Braak 2 pathology were included, and because of the lack of high quality tissues from one donor with Braak 6 pathology, tissue samples of one additional donor with Braak 5 pathology were included. For the analysis of the profiling two general approaches have been applied. Both comparative profiling studies, frontal cortex against inferior temporal cortex as well as control against AD patients, which contribute to the complex view of the relevance of SLIM3 in AD physiology, are shown in detail below.
  • Primer A SEQ ID NO: 5, 5'-AACCCCATCAGCGGACTTG-3 1 and Primer B, SEQ ID NO: 6, 5 I -ACGGCAGTGGCATAACGAC-3 I .
  • PCR amplification (95°C and 1 sec, 56 0 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 3 mM MgCI 2 - Melting curve analysis revealed a single peak at approximately 82.5°C with
  • the PCR protocol was applied to determine the PCR efficiency of a set of reference genes which were selected as a reference standard for quantification.
  • the mean value of five such reference genes was determined: (1) cyclophilin B 1 using the specific primers SEQ ID NO: 7, 5 1 -ACTGAAGCACTACGGGCCTG-3 1 and SEQ ID NO: 8, ⁇ '-AGCCGTTGGTGTCTT- TGCC-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).
  • Ribosomal protein S9 (RPS9), using the specific primers SEQ ID NO: 9, ⁇ '-GGTCAAATTTACCCTG GCCA-3 1 and SEQ ID NO: 10, 5'- TCTCATCAAGCGTCAGCAGTTC-3' (exception: additional 1 mM MgCI 2 was added instead of 3 mM). Melting curve analysis revealed a single peak at approximately 85°C with no visible primer dimers. Agarose gel analysis of the PCR product showed one single band with the expected size (62 bp).
  • beta-actin using the specific primers SEQ ID NO: 11 , ⁇ '-TGGAACGGTGAAGGTGACA-S" and SEQ ID NO: 12, ⁇ '-GGCAAGGGACTTCCTGTAA-S'.
  • PCR product showed one single band with the expected size (81 bp).
  • Transferrin receptor TRR using the specific primers SEQ ID NO: 15, 5 1 - GTCGCTGGTCAGTTCGTGATT-S 1 and SEQ ID NO: 16, 5 1 - AGCAGTTGGCTGTTGTACCTCTC-S 1 .
  • Melting curve analysis revealed a single peak at approximately 83°C with no visible primer dimers.
  • Agarose gel analysis of the PCR product showed one single band with the expected size (80 bp).
  • the logarithm of the cDNA concentration was plotted against the threshold cycle number C t for the gene coding for SLIM3 protein and the five reference standard genes. The slopes and the intercepts of the standard curves (i.e.
  • cDNAs from frontal cortices of AD patients and of healthy control individuals, from temporal cortices of AD patients and of healthy control individuals, from hippocampi of AD patients and of healthy control individuals, and cDNAs from the frontal cortex and the temporal cortex of AD patients and of control individuals and from the frontal cortex and the hippocampus of AD patients and of control individuals, respectively, were analyzed in parallel and normalized to cyclophilin B.
  • the C t values were measured and converted to ng total brain cDNA using the corresponding standard curves:
  • the set of reference standard genes was analyzed in parallel to determine the mean average value of the AD patient to control person temporal cortex ratios, of the AD patient to control person frontal cortex ratios, and of the temporal to frontal ratios of AD patients and control persons and the hippocampi to frontal ratios of AD patients and control persons, 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 the gene coding for SLIM3 protein 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 gene coding for SLIM3 protein are shown in Figures 2, 3 and 12.
  • First analysis used cyclophilin values from qPCR experiments of frontal cortex and inferior temporal cortex tissues from three different donors. From each tissue the 39 same cDNA preparation was used in all analyzed experiments. Within this analysis no normal distribution of values was achieved due to small number of data. Therefore the method of median and its 98 %-conficence level was applied. This analysis revealed a middle deviation of 8.7 % from the median for comparison of absolute values and a middle deviation of 6.6 % from the median for relative comparison.
  • Second analysis used cyclophilin values from qPCR experiments of frontal cortex and inferior temporal cortex tissues from two different donors each, but different cDNA preparations from different time points were used. This analysis revealed a middle deviation of 29.2 % from the median for comparison of absolute values and a middle deviation of 17.6 % from the median for relative comparison. From this analysis it was concluded, that absolute values from qPCR experiments can be used, but the middle deviation from median should be taken into further considerations. A detailed analysis of absolute values for SLIM3 was performed. Therefore, absolute levels of SLIM3 were used after relative normalization with cyclophilin.
  • the median as well as the 98 %-confidence level was calculated for the control group (Braak 0 - Braak 3) and the patient group (Braak 4 - Braak 6), respectively. Same analysis was done redefining the control group (Braak 0 - Braak 2) and the patient group (Braak 3 - Braak 6) as well as redefining the control group (Braak 0 - Braak 1) and the patient group (Braak 2 - Braak 6). The latter analysis was aimed to identify early onset of mRNA expression differences between controls and AD patients.
  • Total protein extract was obtained from human brain and of H4APPsw cells stably expressing SLIM3 by homogenization 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.
  • 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
  • H4 cells human neuroglioma cell line
  • H4APPsw cells which stably expresses the human APP695 isoform carrying the Swedish mutation (K670N, M671 L)
  • the H4APPsw cells were transduced with a pFB-Neo vector (Stratagene, #217561 ) containing the coding sequence of SLIM3 (SLIM3 cds, SEQ ID NO: 4) and a myc- tag (pFB-Neo-CMV-SLIMScds-myc, SLIM3-myc vector) under the control of a strong CMV promotor.
  • the SLIM3cds- myc sequence was introduced into the multiple cloning site (MCS) of the pFB-Neo vector.
  • MCS multiple cloning site
  • the retroviral expression system ViraPort from Stratagene was used.
  • the myc-tagged SLIM3 over-expressing cells (H4APPsw-SLIM3-myc) were seeded onto glass cover slips in a 24 well plate (Nunc, Roskilde, Denmark; #143982) at a density of 5x10 4 cells and incubated at 37°C at 5% CO 2 over night.
  • the second antibody (Cy3-conjugated anti-rabbit antibody, 1 :1000, Amersham Pharmacia, Germany) was applied in blocking solution and incubated for 1 hour at room temperature. The cells were washed 3 times in PBS for 5 minutes. Coverslips were mounted onto microscope slides using Permafluor (Beckman Coulter) and stored over night at 4°C to harden the mounting media. Cells were visualized using microscopic dark field epifluorescence and bright field phase contrast illumination conditions (1X81 , Olympus Optical). Microscopic images ( Figure 15) were digitally captured with a PCO SensiCam and analysed using the appropriate software (AnalySiS, Olympus Optical). 41
  • SLIM3 For immunofluorescence staining of SLIM3 in human brain, post-mortem fresh- frozen frontal and temporal forebrain specimens from donors comprising patients with clinically diagnosed and neuropathologically confirmed Alzheimer's disease at various Braak stages as well as age-matched control individuals without Alzheimer, were cut at 14 ⁇ m thickness using a cryostat (Leica CM3050S). The tissue sections were air-dried at room temprature and fixed in acetone for 10 min.
  • the sections were pre-incubated with blocking buffer (10% normal horse serum, 0.2% Triton X-100 in PBS) for 30 min and then incubated with anti-SLIM3 goat polyclonal antiserum (1 :8 diluted in blocking buffer; Santa Cruz, sc-13409) overnight at 4°C. After rinsing three times in 0.1% Triton X-100/PBS, the sections were incubated with FITC-conjugated donkey anti-goat IgG antiserum (Dianova, 705-096-147, 1 :150 diluted in 1% BSA/PBS) for 2 hours at room temperature and then again washed in PBS.
  • blocking buffer 10% normal horse serum, 0.2% Triton X-100 in PBS
  • anti-SLIM3 goat polyclonal antiserum 1 :8 diluted in blocking buffer; Santa Cruz, sc-13409
  • FITC-conjugated donkey anti-goat IgG antiserum Dianova, 705-0
  • Staining of the neuronal cells was performed as described above using a mouse monoclonal antibody against the neuronal specific marker NeuN (Chemicon, MAB377, dilution 1 :200) and a secondary Cy3-conjugated donkey anti-mouse IgG antiserum (Dianova, 115-166- 150, dilution 1 :600). Staining of the nuclei was performed by incubation of the sections with 5 ⁇ M DAPI in PBS for 3 min. In order to block the autofluoresence of lipofuscin in human brain, the sections were treated with 1% Sudan Black B in 70% ethanol for 2-10 min at room temperature and then sequentially dipped in 70% ethanol, destilled water and PBS.
  • the sections were coverslipped with 'Vectashield 1 mounting medium (Vector Laboratories, Burlingame, CA). Microscopic images were obtained using dark field epifluorescence and bright field phase contrast illumination conditions (1X81, Olympus Optical). Microscopic images were digitally captured with a PCO SensiCam and analyzed using the appropriate software (AnalySiS, Olympus Optical) (see Figures 16, 17).

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