Parkin involvement in misfolded protein responses and neurodegeneration associated with age-related disorders of the brain.

The accumulation of misfolded proteins has been implicated in a number of age-related neurodegenerative disorders of the brain including Parkinson¿s, Alzheimer¿s and Huntington¿s diseases. Of these, Parkinson¿s disease (PD) is a common (1-2% of the population over 65), age-associated neurodegenerative disorder. Most cases of PD appear as sporadic and of unknown cause. However, there are rare familial autosomal dominant forms of PD caused by mutations in the alpha-synuclein and C-terminal hydrolase genes, as well as an early-onset autosomal recessive juvenile Parkinsonism (AR-JP) which is caused by mutations in the parkin gene.
Parkin is a ubiquitin E3 ligase involved in the ubiquitin-proteasome degradation pathway. Mutations in the parkin gene result in loss of ubiquitin E3 ligase function and accumulation of parkin substrates. Other aspects of the mechanism of action of parkin and its mutated derivatives include a role in oxidative stress responses and possibly also apoptotic cell death. Over-expression of mutant AR-JP parkin
increases oxidative stress responses as shown by levels of protein carbonyls and lipid peroxidation. Recently, parkin was shown to be a substrate for caspase-mediated proteolysis during apoptosis. Previous studies on the effects of wild type and mutated parkin have used agents that directly and effectively induce unfolded proteins and associated stress responses. Little is known about the effects of parkin on unfolded protein stress in response to upstream perturbations more relevant to PD pathogenesis and neurodegeneration, such as glutamate excitoxicity, disrupted calcium homeostasis and oxidative damage. To address this question, we will use human M17 neuroblastoma cells transiently transfected with wild-type parkin and AR-JP mutants to test parkin involvement on unfolded protein stress in response to disrupted ER calcium homeostasis and both oxidative and apoptotic stress. Wild type Parkin and AR-JP cells will be stressed and the effects on calcium homeostasis will be determined using Fluo-3. ER unfolded protein stress will be assayed as the induction of heat shock protein (Hsp)-70 and GRP78 (BiP). The number of cells undergoing eventual apoptosis will be determined by propidium iodide staining and TUNEL labelling.
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