Christopher Manuel Gomez
University Of Chicago
Project start date: 1995-04-01
Project end date: 2015-01-31
Sponsored Links Excellgen http://Excellgen.com
Acetylcholine Receptor Genes In Slow-Channel Syndrome
Christopher Manuel Gomez, Professor
University Of Minnesota Twin Cities 450 Mcnamara Alumni Center Minneapolis, Mn 554552070
Grant 5R01NS033202-12 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Abstract: The long-range goal of this research is to understand the pathophysiology and molecular mechanisms involved in the impairment of neuromuscular transmission in the slow-channel congenital myasthenic syndrome (SCS). Up to 14 distinct missense mutations in the genes coding for the four subunits of the muscle acetylcholine receptor (nAChR) are responsible for a dominantly-inherited syndrome of varied clinical severity and pathological findings. Although the common effect of each mutation identified in the SCS to date is to prolong the duration of the acetylcholine-induced channel bursts and endplate currents, differences in gating kinetics, ion permeability, desensitization rate, activation of cell death pathways, and sensitivity to choline and ethanol may affect pathogenesis. In this project we propose to explore specific, novel aspects of the molecular and cellular phenotype of each SCS mutation and to assess the relative contribution of each to neuromuscular weakness. Specifically, we propose to 1) Determine whether pathogenicity in SCS correlates with activation of cell death pathways in vitro. This will be accomplished with cultured mammalian cells expressing SCS mutant AChRs using assays of cell lysis and of activation of cellular proteases. 2) Compare the capacity of ethanol as a model "environmental agent" to modulate activity of mutant AChRs in SCS. This will be accomplished by studying the effect of ethanol on channel activity of AChRs bearing SCS mutations. 3) Determine whether pathogenicity in SCS correlates with differential response to serum choline or spontaneous activity. This will be accomplished by direct comparison of transgenic mice expressing choline-sensitive mutations with varied channel properties. 4) Determine whether reduced AChR channel opening rate ( alone can lead to significant weakness and impairment of synaptic responses. This will be accomplished using an AChR mutation that selectively alters rate of channel opening in transgenic mice. These studies extend the focus of the project by exploring newly recognized SCS mutant channel phenotypes in vitro and by using prototypic AChR subunit mutations to study pathogenic mechanisms in vivo. In addition, to extend the study of genetic and molecular mechanisms, we begin to investigate the influence of the environment on disease phenotype, and whether this influence may vary according to SCS mutation.
Keywords: acetylcholine, apoptosis, congenital neuromuscular disorder, neuromuscular junction, neuromuscular transmission, neurotransmitter receptor, nicotinic receptor, point mutation, choline, genotype, molecular pathology, neuropathology, nucleic acid sequence, phenotype, receptor expression, receptor sensitivity, structural gene, clinical research, genetically modified animal, human genetic material tag, human subject, laboratory mouse, tissue /cell culture
Project start date: 1995-04-01
Project end date: 2006-06-30
5R01NS033202-12 (2006): $307464
5R01NS033202-11 (2005): $321209
ACETYLCHOLINE RECEPTOR GENES IN SLOW CHANNEL SYNDROME
Christopher Manuel Gomez, Professor
University Of Minnesota Twin Cities 450 Mcnamara Alumni Center Minneapolis, Mn 554552070
Grant 5R01NS033202-09 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Abstract: Applicant s ) The long range of this research is to understand the pathophysiology and molecular mechanisms involved in the impairment of neuromuscular transmission in the slow-channel congenital myasthenic syndrome (SCCMS). The rapid advances in the molecular genetics of the SCCMS have established that it is associated with a variety of missense mutations in the genes coding for the subunits of the muscle acetylcholine receptor (nAChR). Although the common effect of each mutation identified in the SCCMS to date is to prolong the duration of the acetylcholine- induced channel bursts and consequently the endplate currents the severity and pattern of the clinical and pathological findings vary greatly. This suggests that each SCCMS-associated mutation has a differential effect on other aspects of nAChR function, and that the combined effect the properties of each nAChR mutation determines the clinicopathological phenotype. In this project we propose to explore specific, novel aspects of the molecular phenotype of each SCCMS mutation and to assess the relative contribution of each to neuromuscular weakness. Specially, we propose to 1) Identify the all the mutations in the genes encoding the a,b,d and e subunits of the nAChR for patients with slow-channel syndrome and other candidate nAChR disorders. This will be accomplished by nucleotide sequence analysis. 2)Confirm the effect of each new mutation on nAChR function and compare the effects of all pathogenic nAChR mutations on nAChR channel gating and desensitization. Aims 2-4 will be accomplished using in vitro expression systems. 3)Compare the effects of all pathogenic nAChR mutations on Ca2+ permeability. 4)Compare the effects of all pathogenic nAChR mutation on receptor assembly and stability. 5)Compare the pathogenicity in vivo of SCCMS-associated mutations differing in nAChR desensitization and calcium permeability. This will be accomplished by generation and systematic comparison of additional transgenic mouse models for the SCCMS. These studies extend the focus of this project from the first phase, the identification of mutations responsible for the SCCMS, to the second phase in which the molecular pathogenesis and genotype-phenotype correlation in the SCCMS are investigated by expression of the mutations in vitro and in vivo.
Keywords: congenital neuromuscular disorder, neuromuscular junction, neuromuscular transmission, nicotinic receptor, point mutation, genotype, molecular pathology, neuropathology, nucleic acid sequence, phenotype, receptor expression, receptor sensitivity, structural gene, clinical research, human genetic material tag, human subject, laboratory mouse, tissue /cell culture, transgenic animal
Project start date: 1995-04-01
Project end date: 2004-02-29
5R01NS033202-09 (2003): $297771
5R01NS033202-08 (2002): $307996
5R01NS033202-07 (2001): $278916
5R01NS033202-06 (2000): $275319
5R01NS033202-04 (1998): $111328
5R01NS033202-03 (1997): $107044
Acetylcholine Receptor Genes In Slow-Channel Syndrome
Christopher Manuel Gomez, Professor
Neurologyuniversity Of Chicago
5801 S Ellis Ave
chicago, Il 60637
Grant 5R01NS033202-14 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Keywords: acetylcholine, apoptosis, congenital neuromuscular disorder, neuromuscular junction, neuromuscular transmission, neurotransmitter receptor, nicotinic receptor, point mutation choline, genotype, molecular pathology, neuropathology, nucleic acid sequence, phenotype, receptor expression, receptor sensitivity, structural gene clinical research, genetically modified animal, human genetic material tag, human subject, laboratory mouse, tissue /cell culture
Project start date: 1995-04-01
Project end date: 2009-02-28
5R01NS033202-14 (2007): $316941
ACETYLCHOLINE RECEPTOR GENES IN SLOW CHANNEL SYNDROME
Christopher Manuel Gomez, Professor
University Of Minnesota Twin Cities 450 Mcnamara Alumni Center Minneapolis, Mn 554552070
Grant 5R01NS033202-02 from National Institute Of Neurological Disorders And Stroke IRG: NLS
Project start date: 1995-04-01
Project end date: 1999-01-31
5R01NS033202-02 (1996): $101591
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Christopher Manuel Gomez
AIM2010, 3RD ATAXIA INVESTIGATORS MEETING
Christopher Manuel Gomez, Professor
University Of Chicago, 5801 S Ellis Ave, Chicago, Il 60637
Grant 1R13NS070391-01 from National Institute Of Neurological Disorders And Stroke
Abstract: The Third Ataxia Investigators´ Meeting will focus on the multi-disciplinary nature of ataxia research by assembling an international roster of clinical investigators, diagnosticians, pathologists, geneticists and molecular biologists. In addition to focusing on the most recent scientific advances, the goals of the meeting include 1) To enhance the open exchange of information related to ataxia research; 2) To stimulate the initiation of collaborative research between investigators worldwide; 3) to improve the understanding of human diseases related to ataxia and establish international protocols for the common investigation and storage of data related to ataxia and its treatment; 4) To provide junior investigators with an opportunity to present their work interact with more established scientists in the field, and to provide them with the opportunity to interact with patients and support groups so that they can see the clinical impact and importance of their work. This meeting has been an important stepping stone for collaboration and discussion on ataxia research and therapeutic approaches, and is of great importance now that the field is at the brink of meaningful clinical trials. The Third Ataxia Investigators´ Meeting will focus on the most recent advances in ataxia research, and therapeutic approaches for ataxic disorders. Ataxia, which is defined as the loss of motor control, can affect all aspects of human movement - gait, dexterity, speech, swallowing, eye movements and more - and afflicts approximately 1 in every 2000 individuals worldwide. An expanding genetic understanding of ataxias over the last 15 years has recently led investigators to propose several possible therapeutic approaches, which demand an increase in international communication, collaboration, and standardization of research and clinical methods, all of which will be specifically addressed at the 3rd Ataxia Investigators´ Meeting. This meeting will also provide a forum for recruiting new investigators to this field of biomedical research, which is an essential element for achieving the rapid success that appears increasingly possible for treating these diseases
Keywords: Address; Affect; Ataxia; Ataxy; Biliary or Urinary Stones; Biomedical Research; Calculi; Clinical; Clinical Investigator; Clinical Trials; Clinical Trials, Unspecified; Collaborations; Communication; Coordination Impairment; Data Storage and Retrieval; Deglutition; Disease; Disorder; Dyssynergia; Elements; Eye Movements; Gait; Genetic; Goals; Human; Human, General; Individual; International; Investigation; Investigators; Man (Taxonomy); Man, Modern; Methods; Molecular; Movement; Nature; Pathologist; Patients; Protocol; Protocols documentation; Recruitment Activity; Research; Research Personnel; Researchers; Scientific Advances and Accomplishments; Scientist; Self-Help Groups; Speech; Standardization; Stone; Support Groups; Swallowing; Therapeutic; Therapeutic Human Experimentation; Therapeutic Research; Work; ing; body movement; clinical investigation; data retrieval; data storage; disease/disorder; human disease; improved; meetings; motor control; public health relevance; recruit; scientific accomplishments; scientific advances; self help organization; success
Relevance: 7. Narrative The AIM2010 Third Ataxia Investigators´ Meeting will focus on the most recent advances in ataxia research, and therapeutic approaches for ataxic disorders. This meeting will also provide a forum for recruiting new investigators to this field of biomedical research, which is an essential element for achieving the rapid success that appears increasingly possible for treating these diseases
Project start date: 2010-03-01
Project end date: 2011-02-28
Budget start date: 1-MAR-2010
Budget end date: 28-FEB-2011
PFA/PA: PA-08-149
1R13NS070391-01 (2010): $43000
ACETYLCHOLINE RECEPTOR GENES IN SLOW-CHANNEL SYNDROME
Christopher Manuel Gomez, Professor
University Of Chicago, 5801 S Ellis Ave, Chicago, Il 60637
Grant 2R01NS033202-15A2 from National Institute Of Neurological Disorders And Stroke
Abstract: The slow channel syndrome (SCS) is a disorder congenital and progressive weakness due to mutations in the muscle acetylcholine receptor (AChR) that cause Ca2+ overload and degeneration of the neuromuscular junction (NMJ). The long-range goal of this project is to define the pathways and explore therapies for the progressive synaptic dysfunction seen in SCS. Detailed in vitro and in vivo expression studies in this laboratory and elsewhere have identified several consequences of mutations in the SCS that may contribute to disease pathogenesis, with Ca2+ overload of the junctional sarcoplasm being the most critical. Recently we have recognized the combined participation of two separate, but interrelated protease pathways that impair neuromuscular transmission at pre and post-synaptic levels. Our goals are to Aim 1. Test and compare strategies for NMJ protection in SCS by eliminating Ca2+ overload in intact synapses. We will test the hypothesis that elimination of sources of Ca2+ overload through pharmacological blockade of mutant AChRs or IP3Rs will reduce activation of proteases and correct the functional deficit and pathological changes of SCS mice. Second, we will attempt to identify new potential long duration AChR ion channel blockers for treating SCS by screening a panel of candidate channel blockers to identify those that normalize mutant SCS AChR channel openings, shorten synaptic currents recorded in vitro and block endplate Ca2+ overload in muscle from transgenic mice expressing diverse set of SCS mutations. Aim 2. Characterize the basis for the postsynaptic impairment and the role of caspase activation in SCS. We test the hypothesis that the defect in AChR density is due to a metabolic or synthetic defect in AChRs. Furthermore, we will use direct expression of recombinant caspase inhibitors to test the hypothesis that inhibition of caspases in muscle will increase in AChR density and improve synaptic function. Aim 3. Characterize the role of calpain in the presynaptic impairment of neuromuscular transmission in SCS. We test the hypothesis that muscle calpain, acts through a Cdk5 pathway to cause reduced ACh vesicle release and impaired synaptic strength, to increase nNOS enzymatic activity and synaptic NO production. We will test this hypothesis using transfection and transgenic expression of recombinant Cdk5 and nNOS proteins and testing the effect on presynaptic function and the activity of these pathways. RELEVANCE These studies will lead to practical strategies for therapeutic intervention with long-term benefit for myasthenic syndromes. They may also provide new insights into modulation of both pre and post synaptic impairment of neuromuscular transmission that are relevant to both synaptic plasticity and synaptic diseases. By assessing the relative contribution of different enzymatic and signaling pathways to neuromuscular weakness and synaptic degeneration in vivo, our studies of an animal model of SCS may lead to a practical strategy for therapeutic intervention with long-term benefit in SCS. These studies will also help identify pathways for postsynaptic influence on presynaptic function that may be relevant for neuromuscular disease and synaptic plasticity
Keywords: Animal Model; Animal Models and Related Studies; Apoptosis; Apoptosis Pathway; Axotomy; Bungarotoxins; Ca2+-Activated Protease; Calcium-Activated Neutral Protease; Calcium-Activated Neutral Proteinase; Calcium-Activated Protease; Calcium-Dependent Neutral Protease; Calcium-Dependent Neutral Proteinase; Calpain; Caspase Inhibitor; Cell Death, Programmed; Cell Division Kinase 5; Cell Division Protein Kinase 5; Cell-Death Protease; Chinidin; Cholera Toxin B Subunit; Cholera Toxin Protomer B; Choleragenoid; Cholinergic Receptors; Cholinoceptive Sites; Cholinoceptors; Cinchonan-9-ol, 6`-methoxy-, (9S)-; Color; Congenital Disorders; Cyclin-Dependent Kinase 5; Data; Defect; Desminase; Disease; Disease Pathway; Disease Progression; Disorder; Disorders, Congenital; Dysfunction; Effects, Longterm; Esteroproteases; Extremities; Fluoxetin; Fluoxetine; Frequencies (time pattern); Frequency; Functional disorder; Genetic Alteration; Genetic Change; Genetic defect; Goals; ICE-like protease; Impairment; In Vitro; Ions; Laboratories; Lead; Limb structure; Limbs; Long-Term Effects; Mammals, Mice; Measures; Mediating; Messenger RNA; Metabolic; Mice; Mice, Transgenic; Modeling; Murine; Mus; Muscle; Muscle Fibers; Muscle Tissue; Mutation; Myasthenic Syndrome; Myoneural Junction; Myotubes; N-Methyl-gamma-(4-(trifluoromethyl)phenoxy)benzenepropanamine; Neural Transmission; Neuromuscular Diseases; Neuromuscular Junction; Non-Trunk; Papain-Like Cysteine Protease; Pathogenesis; Pathway interactions; Pb element; Peptidases; Peptide Hydrolases; Phase; Physiopathology; Production; Proteases; Proteinase inhibitor, calpastatin; Proteinases; Proteins; Proteolytic Enzymes; Quinidine; RNA, Messenger; Receptor Gene; Receptor Protein; Receptors, ACh; Receptors, Acetylcholine; Recombinants; Recycling; Relative; Relative (related person); Rhabdomyocyte; Role; Sarcoplasm; Screening procedure; Serine/Threonine-Protein Kinase PSSALRE; Serotonergic Agents; Serotonergic Drugs; Serotonin Agents; Serotonin Drugs; Signal Pathway; Skeletal Fiber; Skeletal Muscle Cell; Skeletal Muscle Fiber; Skeletal Myocytes; Source; Synapses; Synaptic; Synaptic Transmission; Synaptic plasticity; Syndrome; TPKII Catalytic Subunit; Tau Protein Kinase II Catalytic Subunit; Testing; Therapeutic; Therapeutic Intervention; Toxin; Transfection; Transgenic Mice; Transgenic Organisms; VESCL; Vesicle; base; calpastatin; caspase; cdc2-related kinase PSSALRE; channel blockers; cholinergic; cystein protease; cystein proteinase; cysteine endopeptidase; disease/disorder; gene product; genome mutation; heavy metal Pb; heavy metal lead; improved; in vivo; insight; intervention therapy; ion channel blocker; mRNA; model organism; mutant; myoneural disorder; neuromuscular; neuromuscular disorder; neuromuscular transmission; novel; pathophysiology; pathway; postsynaptic; presynaptic; public health relevance; receptor; receptor density; retrograde transport; screening; screenings; social role; synapse function; synaptic function; transgenic; uptake
Relevance: 7. : By assessing the relative contribution of different enzymatic and signaling pathways to neuromuscular weakness and synaptic degeneration in vivo, our studies of an animal model of SCS may lead to a practical strategy for therapeutic intervention with long-term benefit in SCS. These studies will also help identify pathways for postsynaptic influence on presynaptic function that may be relevant for neuromuscular disease and synaptic plasticity
Project start date: 1995-04-01
Project end date: 2015-01-31
Budget start date: 1-FEB-2010
Budget end date: 31-JAN-2011
PFA/PA: PA-07-070
2R01NS033202-15A2 (2010): $430305
Calcium Channels And Hereditary Ataxia
Christopher Manuel Gomez, Professor
Neurologyuniversity Of Minnesota Twin Cities
450 Mcnamara Alumni Center
minneapolis, Mn 554552070
Grant 1R01NS038332-01A2 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Abstract: The longterm objective of this study is to define the role of the P/Q-type voltage-gated channel in Purkinje-cell degeneration and hereditary ataxia. P/Q channels are membrane proteins that play a crucial role in membrane excitability and triggering synaptic transmission. The P/Q a subunit (a1A) encoded by the gene, CACNA1A, has been implicated in familial migraine, hereditary episodic ataxia and spinocerebellar ataxia type 6 (SCA6). SCA6, a form of autosomal dominant progressive ataxia associated with nearly selective Purkinje cell degeneration, bears the putative pathogenic mutation in the long C terminus of a novel spliceform of the a1A mRNA. This study proposes to investigate the distribution of this a1A subunit spliceform and the pathogenicity of the CAG repeat expansion to which the disease is attributed. Using antisera specific for the long splice form, fusion proteins composed of the long C terminus and chimeric full length a1A subunits bearing the long tail with and without expanded CAG repeats we propose to 1) Characterize the distribution and localization of the wild type and SCA6 mutant long splice form of the a1A subunit of the P/Q-type voltage-gated Ca2+ channel in brain and transfected cells and in relation to b subunit subtypes. 2) Characterize the effect of the SCA6 P/Q channel mutation and b subunit subtype on P/Q channel kinetics, and on Ca2+ overload and calcium-coupled pathways. 3) Develop mice that express the a1A subunit with SCA6-associated polyglutamine expansions to investigate the role of these mutations in the development of ataxia and Purkinje cell dysfunction in transgenic mice. These studies will provide new information about the biology of Ca+2 channels and may aid in understanding the basis for selective vulnerability of Purkinje cells in SCA6. Identification of an effect of an elongated polyglutamine tract in the a1A C terminus on specific Ca+2 channel properties or on Purkinje cell viability would help elucidate the pathogenesis of SCA6 and increase our understanding of P/Q-type Ca+2 channel function
Keywords: Friedreich`s ataxia, RNA splicing, calcium channel, cerebellar Purkinje cell, gene mutation, neural degeneration, neuropathology, neurophysiology, protein structure function calcium flux, cerebral cortex, chemical kinetics, gene targeting, polyglutamate, protein isoform, protein localization, psychomotor function, transgenic animal, voltage gated channel cell line, chimeric protein, confocal scanning microscopy, fluorescence microscopy, immunocytochemistry, immunoelectron microscopy, laboratory mouse
Project start date: 2001-04-20
Project end date: 2005-03-31
1R01NS038332-01A2 (2001): $325287
5R01NS038332-04 (2004): $330282
5R01NS038332-03 (2003): $330316
Acetylcholine Receptor Genes In Slow-Channel Syndrome
Christopher Manuel Gomez, Professor
University Of Minnesota Twin Cities 450 Mcnamara Alumni Center Minneapolis, Mn 554552070
Grant 2R01NS033202-10 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Abstract: The long-range goal of this research is to understand the pathophysiology and molecular mechanisms involved in the impairment of neuromuscular transmission in the slow-channel congenital myasthenic syndrome (SCS). Up to 14 distinct missense mutations in the genes coding for the four subunits of the muscle acetylcholine receptor (nAChR) are responsible for a dominantly-inherited syndrome of varied clinical severity and pathological findings. Although the common effect of each mutation identified in the SCS to date is to prolong the duration of the acetylcholine-induced channel bursts and endplate currents, differences in gating kinetics, ion permeability, desensitization rate, activation of cell death pathways, and sensitivity to choline and ethanol may affect pathogenesis. In this project we propose to explore specific, novel aspects of the molecular and cellular phenotype of each SCS mutation and to assess the relative contribution of each to neuromuscular weakness. Specifically, we propose to 1) Determine whether pathogenicity in SCS correlates with activation of cell death pathways in vitro. This will be accomplished with cultured mammalian cells expressing SCS mutant AChRs using assays of cell lysis and of activation of cellular proteases. 2) Compare the capacity of ethanol as a model "environmental agent" to modulate activity of mutant AChRs in SCS. This will be accomplished by studying the effect of ethanol on channel activity of AChRs bearing SCS mutations. 3) Determine whether pathogenicity in SCS correlates with differential response to serum choline or spontaneous activity. This will be accomplished by direct comparison of transgenic mice expressing choline-sensitive mutations with varied channel properties. 4) Determine whether reduced AChR channel opening rate ( alone can lead to significant weakness and impairment of synaptic responses. This will be accomplished using an AChR mutation that selectively alters rate of channel opening in transgenic mice. These studies extend the focus of the project by exploring newly recognized SCS mutant channel phenotypes in vitro and by using prototypic AChR subunit mutations to study pathogenic mechanisms in vivo. In addition, to extend the study of genetic and molecular mechanisms, we begin to investigate the influence of the environment on disease phenotype, and whether this influence may vary according to SCS mutation.
Keywords: acetylcholine, apoptosis, congenital neuromuscular disorder, neuromuscular junction, neuromuscular transmission, neurotransmitter receptor, nicotinic receptor, point mutation, choline, genotype, molecular pathology, neuropathology, nucleic acid sequence, phenotype, receptor expression, receptor sensitivity, structural gene, clinical research, genetically modified animal, human genetic material tag, human subject, laboratory mouse, tissue /cell culture
Project start date: 1995-04-01
Project end date: 2008-02-29
2R01NS033202-10 (2004): $335313
Christopher Manuel Gomez, Professor
Institution:
Grant 5P01NS025701-100008 from National Institute Of Neurological Disorders And Stroke
Abstract: The HIV-1 external glycoprotein, gp120, has been implicated in the development of AIDS dementia complex. The goal of this project is to investigate the neurotoxicity of this molecule, as summarized in two specific aims 1) Is gp120 toxic to neurons in vivo? 2) Is the secreted form of gp120 neurotoxic in vitro and, if so, does neurotoxicity vary with the HIV strain of origin? The hypothesis that gp120 is neurotoxic in vivo will be tested using transgenic mice that secrete gp120 from one of the following central nervous system (CNS) cells 1) all neurons; 2) oligodendrocytes; 3) cerebellar Purkinje cells. tissue-specific promoters will be used to expose neurons to gp120 arising from several distinct cellular sources in vivo. These studies will also test whether the effect of gp120 secretion is independent of its site of production. gp120-producing transgenic mice will be systematically analyzed for neuropathological alterations similar to those of ADC. In parallel to the transgenic experiments, gp120-induced neurotoxicity will be determined in primary neuronal cultures allowing direct testing of the hypothesis that gp120-induced neurotoxicity in vitro accurately reflects gp120 actions in vivo. The possibility will be explored that neurotoxicity varies with differences in sequence and cell tropism by using gp120 subunits from different isolates. The subunits of macrophage-tropic and lymphotropic isolates will be expressed in cultured cell lines using recombinant expression vectors. Cells will be co-cultured with primary neuronal cells and the viability of the neurons assessed. In addition, the culture medium of the infected cells will be used as the source of gp120 for the neurotoxicity studies in vitro. The studies proposed in this application are aimed at understanding the role of the HIV-1 envelope glycoprotein in the AIDS dementia complex. The information obtained from these experiments may provide an important model of retrovirus-induced neurodegeneration and contribute to the development of improved therapeutic modalities for this AIDS complication.
Keywords: HIV envelope protein gp120, HIV infection, genetic strain, human immunodeficiency virus 1, neurotoxin, virus cytopathogenic effect, AIDS dementia complex, cell transformation, cerebellar Purkinje cell, gene expression, histopathology, neuron, neuroprotectant, oligodendroglia, secretion, immunocytochemistry, in situ hybridization, laboratory mouse, northern blotting, polymerase chain reaction, recombinant DNA, southern blotting, tissue /cell culture, transfection, transgenic animal, western blotting
TRANSGENE EXPRESSION OF EMBRYONIC ACETYLCHOLINE RECEPTOR
Christopher Manuel Gomez, Professor
University Of Minnesota Twin Cities 450 Mcnamara Alumni Center Minneapolis, Mn 554552070
Grant 5K08NS001540-04 from National Institute Of Neurological Disorders And Stroke IRG: NST
Abstract: The formation of a mature neuromuscular junction (NMJ) requires changes in expression of several muscle genes that are regulated by activation of the nicotinic acetylcholine receptor (ChR). During development, after its insertion into the NMJ the AChR undergoes a series of physiological and structural changes that have been ascribed to the replacement of the gamma (gamma) subunit by the epsilon (epsilon) subunit. These changes may have a role in protecting muscle from potentially excitotoxic effects of excessive stimulation by acetylcholine (ACh) since they lead to a diminution of the ACh-sensitivity of muscle fibers just as the nerve has arrived to establish cholinergic transmission. Failure to make this subunit switch might be the molecular basis for the slow channel syndrome, a hereditary muscle disease. The goal of this study is threefold To investigate 1) the the mode of regulation of the gamma to epsilon AChR subunit switch and its role in the developmental changes of the AChR of the NMJ; 2) the influence these changes may have on regulation of cholinergic genes and other proteins involved in synaptic transmission; 3) the role of these changes in maintaining the integrity and long-term viability of neuromuscular transmission. This latter goal will test the hypothesis that the phenomenon of excitotoxicity can arise as a result of an abnormal response on the part of an excitatory neurotransmitter receptor. For this study several lines of transgenic mice have been generated that constitutively express the mRNA for the embryonic (gamma) subunit of AChR in muscle. By studying the transcription and stability of the mRNA for gamma, the synthesis of the gamma subunit protein, and its assembly into mature AChRs, the control system for regulation of the embryonic phenotype will be clarified. By studying the properties of the gamma-containing AChRs incorporated into adult motor endplates, the role of the subunit switch in the developmental changes of AChR will be determined. The effect of this persistence of fetal properties on both muscle viability and expression of other genes involved in cholinergic transmission will be evaluated. Further understanding of the molecular basis for the developmentally related phenotypic changes in AChRs and the NMJ will broaden insights into the basis for such changes in other neurotransmitter systems and in neural control in general. Pathologic changes demonstrated in the muscle of animals with such abnormal AChRs would implicate genes for excitatory neurotransmitter receptors in the pathogenesis of hereditary neurodegenerative diseases. It may also provide a model of excitotoxicity for investigation of the second messenger systems participating in its pathogenesis and for development of possible drug therapies.
Keywords: cholinergic receptor, developmental genetics, gene expression, neuromuscular junction, neuromuscular transmission, nicotinic receptor, transfection, genetic transcription, histopathology, messenger RNA, neural degeneration, protein kinase C, second messenger, synapse, syndrome, denervation, electromyography, laboratory mouse, microelectrode, northern blotting, radiotracer, transgenic animal
Project start date: 1992-01-15
Project end date: 1996-12-31
5K08NS001540-04 (1995): $82818
5K08NS001540-03 (1994): $91801
Sponsored Links Excellgen http://Excellgen.com
5K08NS001540-02 (1993): $91598
Genetic And Molecular Characterization Of SCA26
Christopher Manuel Gomez, Professor
University Of Minnesota Twin Cities 450 Mcnamara Alumni Center Minneapolis, Mn 554552070
Grant 1R03NS052582-01 from National Institute Of Neurological Disorders And Stroke IRG: GHD
Abstract: The autosomal dominant spinocerebellar ataxias (SCA) are a clinically and genetically heterogeneous group of neurodegenerative diseases. It is clear that a diverse of genes and mutational mechanisms can cause SCA, but the molecular process and mechanism for Purkinje cell degeneration that leads to SCA is still unknown. Further insights into SCA pathogenesis may come from more studies that have demonstrated that not all SCAs are due to expanded DNA repeats in novel genes. We recently identified a large family with a novel dominant ataxia, register as SCA26, and mapped the disease locus to 19p13.3. The long-range goal of this research is to expand our understanding of the pathogenesis of the hereditary ataxias, and specifically the basis for the nearly selective Purkinje cell degeneration. We hypothesize that SCA26 is caused by a mutation in a gene that is vital to neuron survival or specific function in the cerebellum, and have identified a few compelling candidate genes. The objective of this project is to refine the locus map by recruiting more family members, and by seeking a different founder haplotype from unrelated families, and to identify the gene and mutational basis by sequencing all coding regions of top candidate genes, and to survey the prevalence of SCA26. This project is significant because 1) it will directly benefit ataxic patients by providing a new genetic test for diagnosis and genetic consulting; 2) it will provide a new model to study Purkinje cell and cerebellar degeneration in ataxia patients; 3) more broadly, it will establish a new point to inter-connect known factors together, and unveil new insights to delineate the common pathways involved in neurodegeneration or vital to neuron survival and function.
Keywords: Friedreich s ataxia, molecular pathology, neural degeneration, pathologic process, ataxia, cerebellum, cerebral degeneration, diagnosis design /evaluation, diagnostic test, family genetics, gene mutation, genetic mapping, genetic screening, genotype, molecular biology information system, neuron, clinical research, human genetic material tag, human subject, nucleic acid sequence, patient oriented research
Project start date: 2005-08-15
Project end date: 2006-05-31
1R03NS052582-01 (2005): $74750
Christopher Manuel Gomez, Professor
Neurologyuniversity Of Minnesota Twin Cities
450 Mcnamara Alumni Center
minneapolis, Mn 554552070
Grant 5R01NS037211-04 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Abstract: from applicant´s ) The autosomal dominant spinocerebellar ataxias (SCAS) and episodic ataxias (EAs) are a group of adult- and juvenile-onset neurodegenerative diseases characterized by progressive or intermittent dysarthria and incoordination due to degeneration of the cerebellum and brainstem. Advances in the genetic understanding of these diseases have established that, despite similar clinical presentations, there are at least 9 genetically distinct subtypes, SCAI-SCA7, EA-1 and EA-2. Clinical observations suggest that eye movements and postural stability are universally but differentially impaired in the SCAs, presumably due to regional differences in brainstem and cerebellar involvement in the disease. The voluntary and reflexive control of oculomotor and vestibular function rely heavily on the normal function of the cerebellum and its interaction with brainstem neurons. A precise understanding of extraocular movements and vestibular dependent reflexes in SCA may identify both common abnormalities useful for comparative scoring among kindreds, and abnormalities that are unique to a given SCA subtype. In this project s propose to take advantage of a large database of SCA patients, and recent developments in both the genetics of autosomal dominant ataxia, and in the technology for recording and analyzing eye movements and the dynamic control of posture to address the question of whether specific patterns of eye movement abnormalities and postural instability characterize genetically-defined SCAs. These studies will allow them to assess the functional integrity of widespread areas of the brainstem and cerebellum. These measurements are non-invasive, more sensitive than static magnetic resonance imaging, and can be applied to a larger number of genetically defined ataxia patients than could be possible using pathological studies. They propose to 1) Determine the genetic status of all SCA and EA (episodic ataxia) patients in the University of Minnesota Ataxia Database. 2) Determine whether genetically homogeneous forms of SCA manifest unique patterns of oculomotor and vestibular abnomalities. 3) Determine whether the length of CAG repeat expansions in SCA 1, 2, 3 and 6 correlate with the profile of oculomotor and vestibular abnormalities. 4) Define the progression of oculomotor, vestibular and postural abnormalities as a function of disease duration for SCA 1-7. These studies may identify diagnostic features for some SCA types and provide valuable information about selective vulnerability of CNS neurons and the pathogenesis of CAG repeat diseases. They also may identify traits common to all patients with ataxia that will be useful quantitative measures for therapeutic trials. These studies will test the hypothesis that sensitive measures of eye movements and balance can be used to detect and quantify ataxia from its earliest stages
Keywords: ataxia, autosomal dominant trait, brain stem, neurogenetics balance, developmental genetics, eye movement disorder, nucleic acid repetitive sequence, posture, psychomotor disorder, vestibuloocular reflex behavioral /social science research tag, clinical research, human genetic material tag, human subject
Project start date: 1998-08-01
Project end date: 2004-07-31
5R01NS037211-04 (2001): $235210
5R01NS037211-03 (2000): $228360
5R01NS037211-02 (1999): $221707
3R01NS037211-01A1S1 (1999): $85000
1R01NS037211-01A1 (1998): $231798
ACETYLCHOLINE RECEPTOR GENES IN SLOW CHANNEL SYNDROME
Christopher Manuel Gomez, Professor
University Of Minnesota Twin Cities 450 Mcnamara Alumni Center Minneapolis, Mn 554552070
Grant 3R01NS033202-07S1 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Abstract: Applicant s ) The long range of this research is to understand the pathophysiology and molecular mechanisms involved in the impairment of neuromuscular transmission in the slow-channel congenital myasthenic syndrome (SCCMS). The rapid advances in the molecular genetics of the SCCMS have established that it is associated with a variety of missense mutations in the genes coding for the subunits of the muscle acetylcholine receptor (nAChR). Although the common effect of each mutation identified in the SCCMS to date is to prolong the duration of the acetylcholine- induced channel bursts and consequently the endplate currents the severity and pattern of the clinical and pathological findings vary greatly. This suggests that each SCCMS-associated mutation has a differential effect on other aspects of nAChR function, and that the combined effect the properties of each nAChR mutation determines the clinicopathological phenotype. In this project we propose to explore specific, novel aspects of the molecular phenotype of each SCCMS mutation and to assess the relative contribution of each to neuromuscular weakness. Specially, we propose to 1) Identify the all the mutations in the genes encoding the a,b,d and e subunits of the nAChR for patients with slow-channel syndrome and other candidate nAChR disorders. This will be accomplished by nucleotide sequence analysis. 2)Confirm the effect of each new mutation on nAChR function and compare the effects of all pathogenic nAChR mutations on nAChR channel gating and desensitization. Aims 2-4 will be accomplished using in vitro expression systems. 3)Compare the effects of all pathogenic nAChR mutations on Ca2+ permeability. 4)Compare the effects of all pathogenic nAChR mutation on receptor assembly and stability. 5)Compare the pathogenicity in vivo of SCCMS-associated mutations differing in nAChR desensitization and calcium permeability. This will be accomplished by generation and systematic comparison of additional transgenic mouse models for the SCCMS. These studies extend the focus of this project from the first phase, the identification of mutations responsible for the SCCMS, to the second phase in which the molecular pathogenesis and genotype-phenotype correlation in the SCCMS are investigated by expression of the mutations in vitro and in vivo.
Keywords: congenital neuromuscular disorder, neuromuscular junction, neuromuscular transmission, nicotinic receptor, point mutation, genotype, molecular pathology, neuropathology, nucleic acid sequence, phenotype, receptor expression, receptor sensitivity, structural gene, clinical research, human genetic material tag, human subject, laboratory mouse, tissue /cell culture, transgenic animal
Project start date: 1995-04-01
Project end date: 2003-11-30
3R01NS033202-07S1 (2001): $33312
2R01NS033202-05 (1999): $260781
Sponsored Links Excellgen http://Excellgen.com
PATHOLOGICAL MECHANISMS IN TRANSYNAPTIC CALCIUM OVERLOAD
Christopher Manuel Gomez, Professor
University Of Minnesota Twin Cities 450 Mcnamara Alumni Center Minneapolis, Mn 554552070
Grant 3R01NS036809-04S1 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Abstract: Adapted from applicant s ) The goal of this research is to understand the processes involved in excitotoxic degeneration of synapses using an animal model. Animal models for human neurodegenerative disease are of great value for exploring the cellular and biochemical mediators and molecular pathogenesis of a slowly progressive disease process. The slow channel congenital myasthenic syndrome (SCCMS) is caused by mutations that result in delayed closure of the ion channel of the acetylcholine receptor (AM) of the neuromuscular (NMJ). The delayed channel closure is associated with calcium overload and degeneration of the NMJ, AChR loss, and progressive muscle weakness. Thus, the SCCMS is a prototype for a hereditary excitotoxic disorder. Using transgenic mice technology and site-directed mutagenesis of AChR subunit coding sequences, we have developed the slow-channel transgenic mouse, an animal model for the SCCS that manifest all the features seen in the human disease. In this proposal, the investigator proposes to (1) Determine whether slow-channel transgenic mice have reduced expression of neuromuscular synapse-specific genes. This will be accomplished by comparison of mRNA levels for the AChR subunit genes and other NMJ-specific genes between transgenic and control mice and between degenerating NMJ nuclei and remote from the NMJ nuclei; (2) Determine the cause(s) of the organellar damage and endplate myopathy in slow-channel mice. Three likely pathways of intracellular damage activation of calcium-activated proteases, oxidative damage by free radicals, and apoptosis will be explored using a combination of specific antibody probes and stains to look for damaged proteins and DNA at the NMJ and genetic and pharmacological manipulation of these pathways to alter the course of the disease; and (3). Determine if quinidine can protect the slow-channel transgenic mice from endplate degeneration.
Keywords: calcium disorder, molecular pathology, neural degeneration, neurotoxin, synapse, calpain, cholinergic receptor, disease /disorder model, enzyme activity, gene expression, inborn biological transport disorder, model design /development, neuromuscular disorder, neuropharmacology, oxidative stress, quinidine, genetically modified animal, laboratory mouse, site directed mutagenesis
Project start date: 1999-04-15
Project end date: 2003-03-31
3R01NS036809-04S1 (2003): $20000
5R01NS036809-03 (2001): $199231
5R01NS036809-02 (2000): $193428
1R01NS036809-01A2 (1999): $196448
ACETYLCHOLINE RECEPTOR GENES IN SLOW CHANNEL SYNDROME
Christopher Manuel Gomez, Professor
Neurologyuniversity Of Minnesota Twin Cities
450 Mcnamara Alumni Center
minneapolis, Mn 554552070
Grant 3R01NS033202-02S1 from National Institute Of Neurological Disorders And Stroke IRG: NLS
Project start date: 1995-04-01
Project end date: 1999-01-31
3R01NS033202-02S1 (1997): $4350
TRANSGENE EXPRESSION OF EMBRYONIC ACETYLCHOLINE RECEPTOR
Christopher Manuel Gomez, Professor
University Of Minnesota Twin Cities 450 Mcnamara Alumni Center Minneapolis, Mn 554552070
Grant 5K08NS001540-05 from National Institute Of Neurological Disorders And Stroke IRG: NST
Project start date: 1992-01-15
Project end date: 1997-12-31
5K08NS001540-05 (1996): $81001