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.
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.
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