RNAi Screening and Analysis of Factors Influencing ER-Associated Degradation and Alpha-Synuclein Aggregation in C. elegans

Failure of proteins to adopt their proper structure is a common cause of cellular dysfunction. Quality control mechanisms within cells serve to promote accurate protein folding. Breakdown of these neuroprotective processes can result in aberrant aggregation or clumping of proteins and is a hallmark of a variety of neurological disorders, including Parkinson's disease (PD). Lewy bodies, a clinical characteristic of post-mortem brains from patients with PD, primarily consist of aggregates of alpha-synuclein, the protein product of a gene that is associated with familiar forms of PD. Molecular analyses of Lewy bodies indicates that these aggregates also contain another protein called torsinA, the product of a gene implicated in another hereditary movement disorder termed early-onset torsion dystonia. We are extending observations from our lab wherein we have demonstrated that torsins function to impede or reduce formation of intracellular protein aggregates in living animals. For these studies, we are employing the nematode roundworm, C. elegans, as an animal model to examine a potential role for torsins in cellular mechanisms that respond to protein misfolding. Since these worms are transparent, we can see into their bodies and directly examine aggregates while they are alive and as they age over time. C. elegans is a genetically tractable, rapidly cultured, microscopic animal with a completely sequenced genome. Despite its simplicity, C. elegans still shares most neurological molecules with humans (including dopamine, serotonin, ion channels, neuromuscular synapses). Whereas the human brain has ~100 billion neurons, this tiny worm contains only 302, exactly â€" and their connectivity has been completely elucidated. Our current work is designed to advance our molecular understanding of PD by investigating the effects of torsins on alpha-synuclein folding and cellular toxicity. Additionally, we are attempting to identify novel molecules that alter alpha-synuclein aggregation via a systematic large-scale genomic screen using worms engineered to contain fluorescently tagged human alpha-synuclein aggregates. The response of cells to protein misfolding represents an undefined biological mechanism with significant consequences for therapeutic intervention; implication of torsins in this process provides a context for better understanding PD and its relation to other disease mechanisms.