SPINOCEREBELLAR ATAXIA TYPE 2--GENE AND GENE PRODUCT
Stefan M Pulst, Warschaw Chair And Professor
Cedars-sinai Medical Center Los Angeles, Ca 90048
Grant 5R01NS033123-04 from National Institute Of Neurological Disorders And Stroke IRG: MGN
Abstract: Spinocerebellar degenerations are neurodegenerative diseases which involve the cerebellum and its connections and affect as many individuals in the US as multiple sclerosis. Among the hereditary forms, the dominant spinocerebellar ataxis (SCAs) represent clinically and genetically complex disorders leading to severe ataxia, oculo-motor abnormalities and variable degrees of motor disturbance and neuropathy. The genetic defect for SCA type 2 has been localized to chromosome (CHR) 12. Marked anticipation in a large pedigree with SCA2 has recently been documented suggesting that SCA2-like SCA2 and SCA3 - is caused by expansion of a DNA trinucleotide repeat (TNR). A unique resource of four SCA2 families with greater than 500 affected individuals, genetic markers and physical mapping tools has been established by our group and used to narrow the SCA2 region to approximately 1cM genetically and less than 1 Mb physically. A YAC contig including the flanking markers AFM240we1 and AFM291xe9 has been constructed and a contig of P1 artificial chromosome (PAC) clones is being developed. Using these resources, four specific aims are proposed with the goal to isolate and characterize the SCA2 gene 1) the SCA2 region will be further narrowed using a panel of meiotic breakpoints on disease chromosomes and novel microsatellite markers. These will be generated as part of aim 2. 2) The existing contigs of YAC and PAC clones will be expanded to provide a detailed physical map of the SCA2 region. Contigs will b e used to saturate the candidate region with di- and trinucleotide repeats. 3) Candidate SCA2 genes will be identified by evaluating trinucleotide repeats for expansion by cDNA selection, and by exon amplification. Mutations in each of the SCA2 families will be evaluated by Southern blot, SSCP and repeat expansion analysis. 4) SCA2 transcript(s) and protein(s) will be characterized to determine cellular and tissue distribution and embryonic expression using Northern blotting, reverse-transcribed PCR, in situ hybridization, Western blotting and immunocytochemistry. Identification of the SCA2 gene will enhance classification of ataxias and permit presymptomatic diagnosis. It will also contribute to our knowledge of mechanisms underlying late-onset neuronal degeneration.
Keywords: cerebellar ataxia /dyskinesia, gene expression, gene mutation, genetic marker, hereditary cerebellar degeneration, family genetics, gene frequency, genetic mapping, neural degeneration, neurogenetics, nucleic acid repetitive sequence, phenotype, animal genetic material tag, artificial chromosome, human genetic material tag, human tissue, immunocytochemistry, in situ hybridization, northern blotting, nucleic acid sequence, polymerase chain reaction, pulsed field gel electrophoresis, single strand conformation polymorphism, southern blotting, western blotting
Project start date: 1996-02-01
Project end date: 2000-02-29
5R01NS033123-04 (1999): $303447
Sponsored Links Excellgen http://Excellgen.com
SPINOCEREBELLAR ATAXIA TYPE 2 GENE AND GENE PRODUCT
Stefan M Pulst, Dr.
University Of Utah, 75 South 2000 East, Salt Lake City, Ut 84112
Grant 5R01NS033123-11 from National Institute Of Neurological Disorders And Stroke
Abstract: The inherited ataxias comprise a group of genetically distinct diseases with an overall prevalence of approximately 10/100,000. Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder leading to degeneration of neurons in the cerebellum and in other cell populations in the CNS. It belongs to the group of neurodegenerative disorders caused by expansion of a polyglutamine (polyQ) repeat. Neither symptomatic nor neuronprotective agents have been identified for the treatment of human neurodegenerative ataxias. In the previous funding period we defined the calcium channel subunit CACNA1A (Cav2.1) and the parkin E3 ubiquitin ligase as modifiers of the SCA2 phenotype in vitro and in humans. Based on these findings, we will test the following hypotheses making use of mouse lines expressing knockout or knockin alleles for the two genes 1) Cav2. 1 modulates the motor and morphologic phenotypes of SCA2-transgenic mice. 2) The molecular mechanism is related to loss of function and sequestration of Cav2.1 subunits into aggregates or due to altered calcium channel function in the presence of mutant ataxin-2. We hypothesize that different modifier mechanisms may underlie neuronal dysfunction and neuronal death. 3) Decreasing parkin expression will enhance pathogenicity of mutant ataxin-2 by increasing ataxin-2 steady-state levels. To test these hypotheses, we will use our SCA2 transgenic model, which targets expression of mutant ataxin-2[Q58] to cerebellar Purkinje cells and replicates salient features of the human disease such as progressive functional loss and morphologic alterations in the cerebellum. We will also generate novel mouse lines expressing ataxin-2 under control of SCA2 regulatory elements. In the first aim, we will cross SCA2 transgenic mice with lines that express Cacna1a knock-out or Cacna1a[Q30] or [Q84]- knockin alleles. Mice will be evaluated by functional testing, morphologic analysis, and physiology using the cerebellar slice preparation. We will differentiate effects on disease initiation and on progression by determining age of onset as well as parameters of morphologic and behavioral progression in the resulting crosses. In the second aim, we will cross parkin null mice with SCA2 transgenic mice and examine whether loss of parkin in vivo accelerates motor dysfunction and morphologic changes. We will also determine whether overexpression of parkin delays disease onset by crossing SCA2 transgenic mice with mice overexpressing parkin in Purkinje cells. The proposed experiments address two significant questions relating to SCAs the importance of ion channels in cerebellar dysfunction and neuronal death in the presence of mutant polyQ proteins and feasibility and effects of down-regulating ataxin-2 by modulating E3 ligase function. Neurodegenerative diseases are imposing an increasing health and financial burden given the rapid increase of the aging segment of the population. The spinocerebellar ataxias are inherited neurodegenerative disorders affecting brain circuits involved in movement coordination. In the previous funding cycle, we have identified modifiers of SCA neurodegeneration. We are now proposing to test the precise mechanisms underlying disease modification making use of mouse models. This will allow us to test the effects of specific proteins on nerve cell dysfunction and cell death in live animals with the ultimate goal of identifying new therapeutic avenues for treatment of human neurodegenerative diseases
Keywords: Address; Affect; Age; Age of Onset; Aging; Alleles; Allelomorphs; Animals; Ataxia; Ataxy; Behavioral; Blood Coagulation Factor IV; Brain; Ca(v)2.1; Ca++ element; Calcium; Calcium Channel; Calcium Channel Antagonist Receptor; Cav2.1; Cell Culture Techniques; Cell Death; Cells; Cerebellar Diseases; Cerebellar Disorders; Cerebellar Dysfunction; Cerebellar Syndromes; Cerebellum; Cerebellum Diseases; Cessation of life; Coagulation Factor IV; Collaborations; Complex; Coordination Impairment; DISSEC; Death; Degenerative Diseases, Nervous System; Degenerative Neurologic Disorders; Disease; Disease Progression; Disorder; Dissection; Dysfunction; Dyssynergia; E3 Ligase; E3 Ubiquitin Ligase; Elements; Encephalon; Encephalons; Factor IV; Functional disorder; Funding; Gene Transfer Clinical; Gene Transfer Procedure; Gene-Tx; Genes; Genetic; Genetic Intervention; Goals; Health; Hereditary; Human; Human, General; In Vitro; Inherited; Intervention; Intervention Strategies; Intervention, Genetic; Ion Channel; Ion Channels, Calcium; Ionic Channels; Knock-in; Knock-in Mouse; Knock-out; Knockout; Knockout Mice; Life; Mammals, Mice; Man (Taxonomy); Man, Modern; Mediating; Membrane Channels; Mice; Mice, Knock-out; Mice, Knockout; Modeling; Modification; Molecular; Molecular Biology, Gene Therapy; Morphology; Motor; Mouse Strains; Movement; Murine; Mus; Nerve Cells; Nerve Degeneration; Nerve Unit; Nervous System, Brain; Neural Cell; Neurocyte; Neurodegenerative Diseases; Neurodegenerative Disorders; Neurologic Degenerative Conditions; Neurologic Diseases, Degenerative; Neuron Degeneration; Neuronal Dysfunction; Neurons; Null Mouse; Onset of illness; P-Q type VDCC; Parkin; Pathogenicity; Pathway interactions; Performance; Phenotype; Physiology; Physiopathology; Poly Q; Population; Preparation; Prevalence; Process; Proteins; Purkinje Cells; Purkinje`s Corpuscles; Receptors, Calcium Channel Blocker; Regulatory Element; RegulatoryElement; Role; SCA2 protein; Senescence; Severity of illness; Slice; Spinocerebellar Ataxia-2; Spinocerebellar Ataxias; Spinocerebellar Ataxias, Dominantly-Inherited; Spinocerebellar Atrophies; Staging; Symptoms; Testing; Therapy, DNA; Touch; Touch sensation; Transgenic Mice; Transgenic Model; Type 2 Spinocerebellar Ataxia; Ubiquitin-Protein Ligase E3; VDCC; Variant; Variation; Voltage-Dependent Calcium Channels; ataxin-2; base; body movement; calcium flux; calcium mobilization; cerebellar Purkinje cell; d-Numb; disease onset; disease severity; disease/disorder; disorder onset; dosage; experiment; experimental research; experimental study; functional loss; gene product; gene therapy; genetic therapy; human disease; improved functioning; in vivo; interventional strategy; loss of function; mouse model; mutant; necrocytosis; neural degeneration; neurodegeneration; neurodegenerative illness; neuronal; neuronal degeneration; new therapeutics; next generation therapeutics; novel; novel therapeutics; numb protein; omega-agatoxin-IVA-sensitive VDCC; overexpression; parkin gene/protein; pathophysiology; pathway; poly(glutamine); polyQ; polyglutamine; public health relevance; release of sequestered calcium ion into cytoplasm; research study; senescent; social role; spinocerebellar ataxia type 2 gene product; tissue culture; ubiquitin-protein ligase; voltage-dependent calcium channel (P-Q type)
Project start date: 1996-02-01
Project end date: 2013-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
PFA/PA: PA-07-070
5R01NS033123-11 (2010): $328337
5R01NS033123-10 (2009): $342701
SPINOCEREBELLAR ATAXIA TYPE 2--GENE AND GENE PRODUCT
Stefan M Pulst, Warschaw Chair And Professor
Cedars-sinai Medical Center Los Angeles, Ca 90048
Grant 5R01NS033123-03 from National Institute Of Neurological Disorders And Stroke IRG: MGN
Abstract: Spinocerebellar degenerations are neurodegenerative diseases which involve the cerebellum and its connections and affect as many individuals in the US as multiple sclerosis. Among the hereditary forms, the dominant spinocerebellar ataxis (SCAs) represent clinically and genetically complex disorders leading to severe ataxia, oculo-motor abnormalities and variable degrees of motor disturbance and neuropathy. The genetic defect for SCA type 2 has been localized to chromosome (CHR) 12. Marked anticipation in a large pedigree with SCA2 has recently been documented suggesting that SCA2-like SCA2 and SCA3 - is caused by expansion of a DNA trinucleotide repeat (TNR). A unique resource of four SCA2 families with greater than 500 affected individuals, genetic markers and physical mapping tools has been established by our group and used to narrow the SCA2 region to approximately 1cM genetically and less than 1 Mb physically. A YAC contig including the flanking markers AFM240we1 and AFM291xe9 has been constructed and a contig of P1 artificial chromosome (PAC) clones is being developed. Using these resources, four specific aims are proposed with the goal to isolate and characterize the SCA2 gene 1) the SCA2 region will be further narrowed using a panel of meiotic breakpoints on disease chromosomes and novel microsatellite markers. These will be generated as part of aim 2. 2) The existing contigs of YAC and PAC clones will be expanded to provide a detailed physical map of the SCA2 region. Contigs will b e used to saturate the candidate region with di- and trinucleotide repeats. 3) Candidate SCA2 genes will be identified by evaluating trinucleotide repeats for expansion by cDNA selection, and by exon amplification. Mutations in each of the SCA2 families will be evaluated by Southern blot, SSCP and repeat expansion analysis. 4) SCA2 transcript(s) and protein(s) will be characterized to determine cellular and tissue distribution and embryonic expression using Northern blotting, reverse-transcribed PCR, in situ hybridization, Western blotting and immunocytochemistry. Identification of the SCA2 gene will enhance classification of ataxias and permit presymptomatic diagnosis. It will also contribute to our knowledge of mechanisms underlying late-onset neuronal degeneration.
Keywords: cerebellar ataxia /dyskinesia, gene expression, gene mutation, genetic marker, hereditary cerebellar degeneration, family genetics, gene frequency, genetic mapping, neural degeneration, neurogenetics, nucleic acid repetitive sequence, phenotype, animal genetic material tag, artificial chromosome, human genetic material tag, human tissue, immunocytochemistry, in situ hybridization, northern blotting, nucleic acid sequence, polymerase chain reaction, pulsed field gel electrophoresis, single strand conformation polymorphism, southern blotting, western blotting
Project start date: 1996-02-01
Project end date: 2000-01-31
5R01NS033123-03 (1998): $296713
5R01NS033123-02 (1997): $315094
SPINOCEREBELLAR ATAXIA TYPE 2 - GENE AND GENE PRODUCT
Stefan M Pulst, Warschaw Chair And Professor
Cedars-sinai Medical Center Los Angeles, Ca 90048
Grant 5R01NS033123-08 from National Institute Of Neurological Disorders And Stroke IRG: MGN
Abstract: Expansion of a polyglutamine (polyQ) tract is the basis of several inherited neurodegenerative diseases including spinocerebellar ataxia type 2 (SCA2). Polyglutamine diseases share several features, most importantly, the presence of intranuclear neuronal aggregates in disease brains. However, pathogenesis leading to neurodegeneration in SCA2 may differ in that the pathologic polyQ tracts in ataxin-2, the SCA2 gene product, are relatively short and intranuclear aggregates have not been found. Four hypotheses will be tested 1) the ER exist motif, clathrin mediated sorting signal and self-association domain in ataxin-2 are functional and are important for pathogenesis. 2) Expression of full- length ataxin-2 in mouse Purkinje cells in vivo causes dysfunction and neurodegeneration. 3) The interaction of ataxin-2 with a novel binding protein, A2BP1, is important for pathogenesis. 4) Proteolytic cleavage of ataxin-2 increases pathogenesis. To evaluate these hypotheses two specific aims are proposed In the first aim, cultured cells and human tissues will be used to determine the subcellular localization and proteolytic processing of normal and mutated ataxin-2. The consequences of deleting specific domains will be analyzed. The interaction of A2BP1with ataxin-2 will be characterized, especially as it relates to the self-interaction of ataxin-2. Studies in the second aim will determine which domains in ataxin-2 are necessary for pathogenesis in vivo. Based on the results in aim 1, transgenic mice expressing ataxin-2 with normal and pathologic polyQ tracts will be generated, as well as mice expressing ataxin-2 with deletions or point mutations in specific domains. Mice will be compared on tests of motor ability. The deletion of point mutations in specific domains. Mice will be compared on tests of motor ability. The extent of changes in cerebellar Purkinje cells will be examined with anatomical techniques. The ultimate goal of this proposal is to define the necessary molecular components of SCA2 pathogenesis and to contribute to the understanding of neurodegenerative diseases in general.
Keywords: cerebellar ataxia /dyskinesia, gene expression, genetic marker, hereditary cerebellar degeneration, pathologic process, cerebellar Purkinje cell, clathrin, neural degeneration, neurogenetics, phenotype, protein protein interaction, confocal scanning microscopy, density gradient ultracentrifugation, human tissue, immunocytochemistry, immunoprecipitation, laboratory mouse, site directed mutagenesis, tissue /cell culture, transgenic animal, western blotting, yeast two hybrid system
Project start date: 1996-02-01
Project end date: 2006-02-28
5R01NS033123-08 (2003): $296268
5R01NS033123-07 (2002): $289155
5R01NS033123-06 (2001): $281130
Grants awarded to Stefan M Pulst
NEUROFIBROMATOSIS--MOLECULAR GENETIC APPROACH
Stefan M Pulst, Warschaw Chair And Professor
Cedars-sinai Medical Center
los Angeles, Ca 90048
Grant 5K08NS001428-05 from National Institute Of Neurological Disorders And Stroke IRG: NSPB
Abstract: Von Recklinghausen-Neurofibromatosis (NF1) is one of the most common autosomal dominant Mendelian disorders affecting man. It is also the most common inherited predisposition toward tumor formation in man. The gene for NF1 has recently been the location further refined by the observation of NF patients with translocations involving chromosome 17. The study of patients with chromosome deletions may greatly aid int he identification of the NF1 gene. These patients are clinically recognized by the occurrence of several inherited conditions in one individual often accompanied by mental retardation. Three such patients have been identified. Two patients are mentally retarded and have a de novo occurrence of NF1 and achondroplasia (ACH). Because the chance association of these two diseases would be approximately 1 in 700 million, this suggests that these two diseases may be caused by a small deletion on chromosome 17. A third mentally retarded patient has NF1 and a cytogenetically visible deletion of chromosome 17. The proposed research has four specific aims directed towards analysis of the NF1 locus 1) Identification of missing DNA sequences from patient with putative deletions by quantitative Southern blotting and pulsed-field gel electrophoresis using single copy DNA sequences tightly linked to the NF1 locus. 2) Elucidation of possible mechanisms of tumor formation in tumor cell lines established from NF1 benign and malignant tumors by analysis for loss of chromosomal material and loss of heterozygosity. 3) Development of novel techniques for identifying small chromosomal deletions using the analysis of hybrid cell lines containing a single chromosome 17 with interspersed repeated DNA sequences. This strategy could have major implications for the analysis of constitutional or somatic deletions in other diseases, for example NF2. 4) Molecular analysis of the mutation(s) causing Familial Spinal Neurofibromatosis by analysis of two families with autosomal dominant spinal neurofibromas. These families have shown linkage to the NF1 locus in preliminary studies. The sequence analysis of these mutation(s) may uniquely contribute to our understanding of tumorigenesis in NF1. These studies will contribute towards elucidating the molecular basis of NF1 mutations
Keywords: autosomal dominant trait, chromosome deletion, linkage mapping, molecular pathology, neurofibromatosis achondroplasia, allele, chromosome translocation, gene expression, gene rearrangement, genetic disorder diagnosis, genetic mapping, genetic recombination, human population genetics, hybrid cell, mental retardation, messenger RNA, neoplastic growth DNA, RNA, electrophoresis, gel electrophoresis, human genetic material tag, human subject, in situ hybridization, karyotype, neoplastic cell culture for noncancer research, nucleic acid hybridization, nucleic acid sequence, polymerase chain reaction, restriction fragment length polymorphism
Project start date: 1990-09-01
Project end date: 1995-08-31
5K08NS001428-05 (1994): $75276
NEUROFIBROMATOSIS--MOLECULAR-GENETIC APPROACH
Stefan M Pulst, Warschaw Chair And Professor
Cedars-sinai Medical Center Los Angeles, Ca 90048
Grant 5K08NS001428-04 from National Institute Of Neurological Disorders And Stroke IRG: NSPB
Abstract: Von Recklinghausen-Neurofibromatosis (NF1) is one of the most common autosomal dominant Mendelian disorders affecting man. It is also the most common inherited predisposition toward tumor formation in man. The gene for NF1 has recently been the location further refined by the observation of NF patients with translocations involving chromosome 17. The study of patients with chromosome deletions may greatly aid int he identification of the NF1 gene. These patients are clinically recognized by the occurrence of several inherited conditions in one individual often accompanied by mental retardation. Three such patients have been identified. Two patients are mentally retarded and have a de novo occurrence of NF1 and achondroplasia (ACH). Because the chance association of these two diseases would be approximately 1 in 700 million, this suggests that these two diseases may be caused by a small deletion on chromosome 17. A third mentally retarded patient has NF1 and a cytogenetically visible deletion of chromosome 17. The proposed research has four specific aims directed towards analysis of the NF1 locus 1) Identification of missing DNA sequences from patient with putative deletions by quantitative Southern blotting and pulsed-field gel electrophoresis using single copy DNA sequences tightly linked to the NF1 locus. 2) Elucidation of possible mechanisms of tumor formation in tumor cell lines established from NF1 benign and malignant tumors by analysis for loss of chromosomal material and loss of heterozygosity. 3) Development of novel techniques for identifying small chromosomal deletions using the analysis of hybrid cell lines containing a single chromosome 17 with interspersed repeated DNA sequences. This strategy could have major implications for the analysis of constitutional or somatic deletions in other diseases, for example NF2. 4) Molecular analysis of the mutation(s) causing Familial Spinal Neurofibromatosis by analysis of two families with autosomal dominant spinal neurofibromas. These families have shown linkage to the NF1 locus in preliminary studies. The sequence analysis of these mutation(s) may uniquely contribute to our understanding of tumorigenesis in NF1. These studies will contribute towards elucidating the molecular basis of NF1 mutations.
Keywords: autosomal dominant trait, chromosome deletion, linkage mapping, molecular pathology, neurofibromatosis, achondroplasia, allele, chromosome translocation, gene expression, gene rearrangement, genetic disorder diagnosis, genetic mapping, genetic recombination, human population genetics, hybrid cell, mental retardation, messenger RNA, neoplastic growth, DNA, RNA, electrophoresis, gel electrophoresis, human genetic material tag, human subject, in situ hybridization, karyotype, neoplastic cell culture for noncancer research, nucleic acid hybridization, nucleic acid sequence, polymerase chain reaction, restriction fragment length polymorphism
Project start date: 1990-09-01
Project end date: 1995-08-31
5K08NS001428-04 (1993): $86292
SCA2 GENE AND GENE REPLACEMENT
Stefan M Pulst, Warschaw Chair And Professor
La Biomed Res Inst/ Harbor Ucla Med Ctr Torrance, Ca 90502
Grant 3M01RR000425-30S10743 from National Center For Research Resources
Abstract: The spinocerebellar ataxias are inherited disorders associated with unsteadiness of gait, speech problems, and motor incoordination. The goal of this study is to evaluate and identify the genes responsible for SCA2, by looking for a speci-fic type of mutation which the investigator s hypothesis claims is reponsible for this disease. The GCRC is assisting in the collection, processing, storage of samples, and establishment of lymphoblastoid cell lines obtained from SCA2 patients both here in Los Angeles as well as across the globe.
Keywords: cerebellar ataxia /dyskinesia, gene mutation, molecular pathology, tissue resource /registry, clinical research, human subject, tissue /cell culture
PARKIN BINDERS IN PROGRESSION OF CELLULAR DYSFUNCTION AND DEATH
Stefan M Pulst
University Of California Los Angeles, Office Of Research Administration, Los Angeles, Ca 90095
Abstract: The overall goal of the UCLA UDALL center is to elucidate mechanisms of early dysfunction in cellular and animal models of Parkinson disease (PD) that can eventually be translated into novel neuroprotective treatments for PD in humans. Basic and clinical research evidence is accumulating that synaptic and cellular dysfunctions not only precede dopaminergic cell loss but also affect non-dopaminergic neurons. The identification of genetic mutations responsible for familial forms of PD offers the potential to glean new insight into the mechanisms underlying the more common sporadic forms of the disease. Mutations in the parkin gene cause an early-onset autosomal recessive form of PD, but are also found in patients with late- onset forms of PD indistinguishable from idiopathic PD. This project builds upon our discovery of four novel parkin-binding proteins, synaptotagmin (syt)1 and syt11, a synapsin-like-protein (SLP), and ataxin-2, a polyQ domain protein. SLP, syt11, and ataxin-2 are found in Lewy bodies of PD patients. The interaction of parkin with ataxin-2 is of particular importance in light of recent findings that polyQ expansion in the SCA2 (ataxin-2) gene are found in patients with late-onset tremor-predominant PD without ataxia or slow saccadic eye movements. We will test the following hypotheses 1) Parkin ubiquitinates and facilitates degradation of these parkin interactors. 2) Over-expression of these parkin interactors causes neurotransmitter homeostatic dysfunction that progresses to cell death in vitro. 3) Dysfunction and death are prevented by co-expression of parkin, but not by co-expression of parkin mutants. 4) These effects are not restricted to primary dopaminergic neurons, but are also seen in hippocampal glutamatergic neurons. Three specific aims are proposed to test these hypotheses 1) We will characterize the interaction of parkin with these novel proteins and determine whether parkin accelerates degradation of the proteins in cell-lines. We will also investigate whether mutant parkins lose the ability to bind these proteins or to ubiquitinate them. 2) In cultured cell-lines, we will determine whether exogenous expression of parkin interactors results in alterations in dopamine release and metabolism and cell death and whether co-expression of parkin can prevent these changes. 3) We will examine the mechanism by which these parkin interactors may regulate synaptic transmission and cell survival in primary dopaminergic and glutamatergic neurons utilizing measures of synaptic vesicle recycling. This project is closely integrated with Projects 2 and 3, in which parkin-mediated synaptic dysregulation will be studied in vivo, in brain slices and in primary cultured neurons. Our experiments will be particularly informative for the translation of our basic research findings to the diagnosis and treatment of PD (Project 5), hopefully preventing significant neuronal loss
Budget start date: 1-MAY-2010
Budget end date: 30-APR-2011
5P50NS038367-10_0004 (2010): $544465
5P50NS038367-09_0004 (2009): $544464
Spinocerebellar Ataxia Type 2 Gene And Gene Product
Stefan M Pulst, Warschaw Chair And Professor
Neurologyuniversity Of Utah
75 South 2000 East
salt Lake City, Ut 84112
Grant 2R01NS033123-09A2 from National Institute Of Neurological Disorders And Stroke IRG: CDIN
Abstract: The inherited ataxias comprise a group of genetically distinct diseases with an overall prevalence of approximately 10/100,000. Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder leading to degeneration of neurons in the cerebellum and in other cell populations in the CNS. It belongs to the group of neurodegenerative disorders caused by expansion of a polyglutamine (polyQ) repeat. Neither symptomatic nor neuronprotective agents have been identified for the treatment of human neurodegenerative ataxias. In the previous funding period we defined the calcium channel subunit CACNA1A (Cav2.1) and the parkin E3 ubiquitin ligase as modifiers of the SCA2 phenotype in vitro and in humans. Based on these findings, we will test the following hypotheses making use of mouse lines expressing knockout or knockin alleles for the two genes 1) Cav2. 1 modulates the motor and morphologic phenotypes of SCA2-transgenic mice. 2) The molecular mechanism is related to loss of function and sequestration of Cav2.1 subunits into aggregates or due to altered calcium channel function in the presence of mutant ataxin-2. We hypothesize that different modifier mechanisms may underlie neuronal dysfunction and neuronal death. 3) Decreasing parkin expression will enhance pathogenicity of mutant ataxin-2 by increasing ataxin-2 steady-state levels. To test these hypotheses, we will use our SCA2 transgenic model, which targets expression of mutant ataxin-2[Q58] to cerebellar Purkinje cells and replicates salient features of the human disease such as progressive functional loss and morphologic alterations in the cerebellum. We will also generate novel mouse lines expressing ataxin-2 under control of SCA2 regulatory elements. In the first aim, we will cross SCA2 transgenic mice with lines that express Cacna1a knock-out or Cacna1a[Q30] or [Q84]- knockin alleles. Mice will be evaluated by functional testing, morphologic analysis, and physiology using the cerebellar slice preparation. We will differentiate effects on disease initiation and on progression by determining age of onset as well as parameters of morphologic and behavioral progression in the resulting crosses. In the second aim, we will cross parkin null mice with SCA2 transgenic mice and examine whether loss of parkin in vivo accelerates motor dysfunction and morphologic changes. We will also determine whether overexpression of parkin delays disease onset by crossing SCA2 transgenic mice with mice overexpressing parkin in Purkinje cells. The proposed experiments address two significant questions relating to SCAs the importance of ion channels in cerebellar dysfunction and neuronal death in the presence of mutant polyQ proteins and feasibility and effects of down-regulating ataxin-2 by modulating E3 ligase function. Neurodegenerative diseases are imposing an increasing health and financial burden given the rapid increase of the aging segment of the population. The spinocerebellar ataxias are inherited neurodegenerative disorders affecting brain circuits involved in movement coordination. In the previous funding cycle, we have identified modifiers of SCA neurodegeneration. We are now proposing to test the precise mechanisms underlying disease modification making use of mouse models. This will allow us to test the effects of specific proteins on nerve cell dysfunction and cell death in live animals with the ultimate goal of identifying new therapeutic avenues for treatment of human neurodegenerative diseases
Project start date: 1996-02-01
Project end date: 2013-06-30
2R01NS033123-09A2 (2008): $329219
Stefan M Pulst, Warschaw Chair And Professor
Cedars-sinai Medical Center Los Angeles, Ca 90048
Grant 5R21NS048083-02 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Abstract: Mutations in the parkin gene cause PARK2, a predominantly early-onset autosomal recessive form of Parkinsonism. Parkin mutations are also found in patients with tremor-predominant later onset forms of Parkinson disease (PD) indistinguishable from idiopathic Parkinson s disease. Parkin is an E3 ubiquitin ligase that attaches ubiquitin chains to several proteins destined for degradation through the proteasome-dependent protein degradation pathway. Some proteins that interact with parkin, such as a-synuclein and synphilin-1, have themselves been found to cause Parkinson s disease, when mutated. In previous experiments, we identified two members of the synaptotagmin (syt) family as parkin binders. We have now identified a novel parkin-binding protein with homologies to synapsin, designated synapsin-like-protein (SLP). SLP and synaptotagmin XI are both found in Lewy bodies of PD patients. We will test the following hypotheses 1) parkin interacts and regulates a select group of synaptic vesicle associated proteins. 2) Genes encoding these proteins contain causative mutations or predisposing sequence variants in patients with familial or sporadic forms of Parkinsonism. Two Specific Aims are proposed 1) We will further characterize the parkin-SLP interaction, and investigate whether mutant parkins lose the ability to bind SLP or to ubiquitinate SLP. We will determine whether parkin accelerates degradation of SLP. 2) We will explore sequence variants in the SLP, SYT1 and SYT11 genes in two PD patient groups and five matched control groups. We will establish which variants represent rare causative mutations and which variants may constitute susceptibility alleles. Variants will be tested for cell toxicity using an in vitro assay. The ultimate goal of this R21 proposal is to further characterize proteins involved in parkin function and to screen these proteins for mutations or susceptibility alleles in PD patients.
Keywords: Parkinson s disease, disease /disorder onset, gene mutation, parkin gene /protein, protein protein interaction, protein structure function, autosomal recessive trait, binding protein, genetic susceptibility, synapsin, synaptic vesicle, synaptotagmin, clinical research, human genetic material tag, immunoprecipitation, nucleic acid sequence, polymerase chain reaction, transfection, yeast two hybrid system
Project start date: 2005-09-15
Project end date: 2008-06-30
5R21NS048083-02 (2006): $211363
1R21NS048083-01A2 (2005): $180375
Stefan M Pulst, Warschaw Chair And Professor
Cedars-sinai Medical Center
los Angeles, Ca 90048
Grant 5R01NS037883-03 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Abstract: Germline mutations in the NF2 gene predispose to tumors of multiple types as well as ocular features such as cataracts and retinal hamartomas. Although NF2 germline mutations are relatively rare, the NF2 gene is the single most commonly mutated gene in sporadic benign brain tumors. Other than its function as a recessive tumor suppressor based on genetic analyses and sequence homology to ERM proteins, little is known about the function of the NF2 gene product, called merlin or schwannomin. They have identified seven schwannomin/merlin binding proteins (SBkPs) using the yeast two-hybrid system. In addition to BII-spectrin or fodrin, six novel proteins have been identified. Three specific aims are proposed to evaluate these proteins 1) The interaction of schwannomin and SBPs will be verified using in vitro binding assays and in vivo co-immunoprecipitation. Interacting domains will be determined by testing partial fusions of schwannomin and SBPs using filter assays for B-galactosidase and in vitro binding assays. 2) Full length cDNAs for SBkPs will obtained and mapped to human chromosome regions using FISH and radiation hybrid mapping. tissue and subcellular distribution of SBPs by Northern and Western blot analyses as well as immunocytochemistry will be analyzed. Subcellular co-localization will be determined by confocal microscopy. 3) The functional importance of SBPs will be determined by analyzing decrease of binding to schwannomins containing naturally occurring missense mutations. After transfection of SBP cDNAs into cell lines and human schwannoma cells, changes of cell morphology, proliferation and schwannomin intracellular localization will be observed. Based on nucleolar localization of schwannomin in STS26T Schwann cells, its effect on RNA polymerase I transcription will be assayed in vitro using nuclear extracts, purified transcription factors and recombinant proteins
Keywords: DNA binding protein, spectrin, tumor suppressor gene, tumor suppressor protein chimeric protein, gene mutation, genetic mapping, oncoprotein Schwann cell, beta galactosidase, confocal scanning microscopy, fluorescent in situ hybridization, immunoprecipitation, northern blotting, western blotting, yeast two hybrid system
Project start date: 1998-09-30
Project end date: 2002-08-31
5R01NS037883-03 (2000): $211749
Sponsored Links Excellgen http://Excellgen.com
1R01NS037883-01 (1998): $140502
SPINOCEREBELLAR ATAXIA TYPE 2 - GENE AND GENE PRODUCT
Stefan M Pulst, Warschaw Chair And Professor
Cedars-sinai Medical Center Los Angeles, Ca 90048
Grant 3R01NS033123-08S1 from National Institute Of Neurological Disorders And Stroke IRG: MGN
Abstract: Expansion of a polyglutamine (polyQ) tract is the basis of several inherited neurodegenerative diseases including spinocerebellar ataxia type 2 (SCA2). Polyglutamine diseases share several features, most importantly, the presence of intranuclear neuronal aggregates in disease brains. However, pathogenesis leading to neurodegeneration in SCA2 may differ in that the pathologic polyQ tracts in ataxin-2, the SCA2 gene product, are relatively short and intranuclear aggregates have not been found. Four hypotheses will be tested 1) the ER exist motif, clathrin mediated sorting signal and self-association domain in ataxin-2 are functional and are important for pathogenesis. 2) Expression of full- length ataxin-2 in mouse Purkinje cells in vivo causes dysfunction and neurodegeneration. 3) The interaction of ataxin-2 with a novel binding protein, A2BP1, is important for pathogenesis. 4) Proteolytic cleavage of ataxin-2 increases pathogenesis. To evaluate these hypotheses two specific aims are proposed In the first aim, cultured cells and human tissues will be used to determine the subcellular localization and proteolytic processing of normal and mutated ataxin-2. The consequences of deleting specific domains will be analyzed. The interaction of A2BP1with ataxin-2 will be characterized, especially as it relates to the self-interaction of ataxin-2. Studies in the second aim will determine which domains in ataxin-2 are necessary for pathogenesis in vivo. Based on the results in aim 1, transgenic mice expressing ataxin-2 with normal and pathologic polyQ tracts will be generated, as well as mice expressing ataxin-2 with deletions or point mutations in specific domains. Mice will be compared on tests of motor ability. The deletion of point mutations in specific domains. Mice will be compared on tests of motor ability. The extent of changes in cerebellar Purkinje cells will be examined with anatomical techniques. The ultimate goal of this proposal is to define the necessary molecular components of SCA2 pathogenesis and to contribute to the understanding of neurodegenerative diseases in general.
Keywords: cerebellar ataxia /dyskinesia, gene expression, genetic marker, pathologic process, cerebellar Purkinje cell, clathrin, neural degeneration, neurogenetics, phenotype, protein protein interaction, confocal scanning microscopy, density gradient ultracentrifugation, genetically modified animal, human tissue, immunocytochemistry, immunoprecipitation, laboratory mouse, site directed mutagenesis, tissue /cell culture, western blotting, yeast two hybrid system
Project start date: 1996-02-01
Project end date: 2006-02-28
3R01NS033123-08S1 (2003): $30000
2R01NS033123-05 (2000): $2
3R01NS033123-05S1 (2000): $2
Parkin Binders In Progression Of Cellular Dysfunction And Death
Stefan M Pulst, Warschaw Chair And Professor
University Of California Los Angeles Office Of Research Administration Los Angeles, Ca 90095
Grant 5P50NS038367-070004 from National Institute Of Neurological Disorders And Stroke IRG: ZNS1
Keywords: Parkinson s disease, cell death, death, gene mutation, genetics, model, neuron, protein
SCA2 GENE AND GENE REPLACEMENT
Stefan M Pulst, Warschaw Chair And Professor
La Biomed Res Inst/ Harbor Ucla Med Ctr Torrance, Ca 90502
Grant 5M01RR000425-340743 from National Center For Research Resources
Keywords: cerebellar ataxia /dyskinesia, gene mutation, molecular pathology, tissue resource /registry, clinical research, human subject, tissue /cell culture
SPINOCEREBELLAR ATAXIA TYPE 2--GENE AND GENE PRODUCT
Stefan M Pulst, Warschaw Chair And Professor
Cedars-sinai Medical Center
los Angeles, Ca 90048
Grant 3R01NS033123-01A2S1 from National Institute Of Neurological Disorders And Stroke IRG: MGN
Project start date: 1996-02-01
Project end date: 2000-01-31
3R01NS033123-01A2S1 (1997): $5435
3R01NS033123-01A2S2 (1997): $28075
Sponsored Links Excellgen http://Excellgen.com
1R01NS033123-01A2 (1996): $234401