Myoclonus refers to sudden, involuntary jerking of a muscle or group of muscles. Dystonia is defined as a syndrome of sustained muscle contractions, frequently causing twisting and repetitive movements, or abnormal postures. Dystonia is generally believed to be a disorder of the central nervous system. Inherited myoclonus- dystonia (M-D), previously referred to as hereditary essential myoclonus or hereditary (alcohol-responsive) myoclonic dystonia, is an autosomal dominant dystonia with incomplete penetrance. Zimprich and colleagues first identified loss-of-function mutations in a gene coding for ?-sarcoglycan (?-SG;gene name: SGCE in human and Sgce in mouse). SGCE is widely expressed in embryonic development and in adults. SGCE is expressed in almost all brain regions tested. We have established a Sgce knockout model of DYT11 dystonia. We found that the Sgce knockout mice showed myoclonus, motor deficits indicative of dystonia, anxiety, depression, and changes in the dopaminergic and serotonergic systems. Furthermore, we have developed a monoclonal antibody specific for ?-SG. While the knockout mice replicated most of the DYT11 symptoms, the function of ?-SG and the role of its mutated forms in causing M-D are largely unknown. Specifically, at molecular level, whether ?-SG forms a complex in the central synapse, if yes, the nature of this complex is not known. These unknowns hamper efforts to adequately understand the pathogenesis of M-D, thus preventing the development of effective therapeutic strategies for patients. Further detailed analysis of the ?-SG complex should provide a unique opportunity for clarifying the functional role of ?-SG and the pathophysiological roles of SGCE mutations. Detailed understanding of pathophysiology of DYT11 dystonia would also accelerate the drug discovery process to develop treatment for myoclonus-dystonia and related dystonia and myoclonus disorders. The broad, long-term objective of our research is to use transgenic mice to determine: 1) the functional role of ?-SG in vivo 2) how the loss of 5-SG protein leads to M-D. The objective of this application is to further characterize ?-SG protein complex using the Sgce knockout mice and monoclonal antibody that we have already made that will enable us to answer these questions. We hypothesize that ?-SG interacts with other proteins and functions at the synapse. Identification of these interacting proteins may elucidate the function of ?-SG in the synaptic transmission. We further hypothesize that ?-SG interacts with other proteins and loss of ?-SG leads to the reduction of the complex at the synapse. The reduction of the complex leads to altered synaptic transmission and plasticity at circuit level, and myoclonus, motor deficits indicative of dystonia, or both, at system level. The rationale for the proposed research is that once the roles of SGCE in causing dysfunction of movement control in the brain are determined, possible interventions to correct M-D can be developed. We are particularly well prepared to undertake the proposed research because we have created a line of Sgce mutant mice that mimic the DYT11 patients. Furthermore, we have developed other genetic and antibody tools that can critically test the above hypothesis. Our other strength is the multidisciplinary approaches we are able to use, which include molecular, genetic, anatomical, biochemical, neurophysiologic, and behavioral techniques. The work will be conducted in a research environment with many NIH-funded investigators and shared NIH-funded core resources that are focused on using animal models of neurological disorders. We plan to test our hypothesis with the following Specific Aims:
Specific Aim 1 : To identify the interacting proteins, we will prepare the synaptosomal fractions from WT and Sgce KO mouse brains. Immunoprecipitation of the ?-SG complex will be performed using specific monoclonal antibody against ?-SG followed by separation of these proteins by gel electrophoresis. The bands only appeared in the WT mouse brain samples should be ?-SG and the interacting proteins and the bands appears in the Sgce KO mouse brain samples should be false positive bands that non-specifically precipitated in the immunoprecipitation experiment. Protein bands will be isolated and sequenced by Mass Spectrometry.
Specific Aim 2 : To analyze the effect of loss of ?-SG on the expression levels of the interacting proteins, we will prepare synaptosomal fractions from WT and Sgce KO mouse brains and analyze the levels of the interacting proteins by Western blot with antibodies against the identified proteins.
Specific Aim 3 : To analyze the effect of loss of ?-SG on the synaptic transmission and plasticity, we will determine input output relationship, paired pulse ratio, and long-term potentiation of hippocampal CA1 Schaffer collateral pathway. The successful completion of the above Specific Aims will produce a list of candidate synaptic proteins that interact with ?-SG and the effects of loss of ?-SG on their expression levels and synaptic transmission. The results should help us to determine the function of ?-SG in vivo and how the mutation of Sgce causes M-D. The results should significantly increase our understanding of the pathophysiology of M-D, which can ultimately aid the development of therapeutic treatments for M-D patients and other dystonia and myoclonus patients.

Public Health Relevance

Dystonia affects more than half of a million people in US alone. Myoclonus could be commonly associated with epilepsy, and sometimes with Alzheimer's and Parkinson's disease, and multiple sclerosis. The total number of myoclonus patients could be over 1 million. The project is to study how ?-sarcoglycan functions in the cell and how mutation of this protein could lead to DYT11 dystonia or myoclonus dystonia. The results from the proposed research should significantly increase the understanding of the pathophysiology of DYT11 dystonia, which can ultimately aid the development of therapeutic treatments for DYT11 dystonia patients and other dystonia and myoclonus patients.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Small Research Grants (R03)
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Synapses, Cytoskeleton and Trafficking Study Section (SYN)
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Sieber, Beth-Anne
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University of Florida
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DeAndrade, Mark P; Trongnetrpunya, Amy; Yokoi, Fumiaki et al. (2016) Electromyographic evidence in support of a knock-in mouse model of DYT1 Dystonia. Mov Disord 31:1633-1639
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