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Abstract

Calcium regulates neuronal signaling and viability through a vast range of cellular and molecular mechanisms. However, many details of these mechanisms still remain to be elucidated. It is believed that calcium-related pathways may comprise promising therapeutic targets for the treatment of many human neurologic conditions, including those involving neurodegeneration. Therefore, achieving a better understanding of calcium-related signaling and survival pathways may lead to concrete advances in the development of new therapeutics for such conditions. In the present study we investigate the potential involvement of two such calcium-related mechanisms in the regulation of neuronal viability. In the first part of the project we assessed the potential role of the neuronal calcium sensor hippocalcin in mitigating striatal neuron death associated with Huntington's disease (HD). The rationale for these experiments comprised previous evidence for neuroprotective effects of hippocalcin in other neuronal cell types as well as data from our laboratory showing decreased hippocalcin expression in HD brain. In order to obtain basic information on hippocalcin's range of normal functions in striatal neurons, we assessed its protein-protein interactions and studied its compartmentalization and calcium-dependent trafficking in living striatal cells. Cellular trafficking experiments demonstrated the calcium-dependent translocation of hippocalcin from the nuclear and cytoplasmic compartments to the trans-Golgi network. Through protein-protein interaction studies, we identified three novel hippocalcin mitochondrial interactor proteins, thereby providing a novel potential link between hippocalcin and energy metabolism. Surprisingly, experiments designed to assess neuroprotective properties of hippocalcin revealed no effects of hippocalcin overexpression either alone or in combination with its potential effectors. These included its assessment in three models of HD-associated neurotoxicity comprised of striatal neurons exposed to mutant huntingtin, excitotoxic doses of glutamate, or mitochondrial succinate dehydrogenase inhibitors. While studying hippocalcin binding to one of the potential interactors, the β2-subunit of clathrin adaptor protein complex 2 (AP-2), we serendipitously discovered that AP-2 is hydrolyzed by the calcium-activated protease calpain. The second part of the thesis was subsequently devoted to the investigation of the role of calpain's proteolysis of AP-2 and other clathrin adaptors in regulating clathrin-mediated endocytosis (CME) and promoting neurodegeneration. Biochemical studies first confirmed that both the α- and β2-subunits of AP-2 (α- and β2-adaptins) were substrates for calpain both in vitro and in vivo. By immunopurification and amino acid sequencing of its C-terminal cleavage fragment, we subsequently defined the precise endogenous calpain cleavage site in β2-adaptin. We further demonstrated calpain cleavage of adaptins occurred in living neuronal cells exposed to glutamate. We then showed that adaptin hydrolysis was accompanied by a decrease in clathrin-mediated endocytosis of plasma membrane receptors. Truncated forms of β2-adaptin corresponding to the calpain cleavage products were demonstrated to mimic the inhibitory effects of calpain hydrolysis on CME of plasma membrane receptors, and moreover, neurons expressing these fragments showed increased sensitivity to glutamate receptor-mediated toxicity. Accessory clathrin adaptors epsin 1, adaptor protein 180 (AP180) and the clathrin assembly lymphoid myeloid leukemia protein (CALM) were also shown to be substrates of calpains in vitro. Finally, cleavage fragments of α- and β2-adaptin, epsin 1, AP180 and CALM were shown to be present in an animal model of focal brain ischemia and in postmortem samples of human Alzheimer's disease cortex. These findings show that calpain hydrolysis of clathrin adaptors comprises an important regulator of CME and suggest that excessive calpain activation may promote excitotoxic neurodegeneration through the decreased internalization of surface receptors.

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