Many neurodegenerative diseases, including Parkinson's disease (PD), show deficits in axonal transport. However, it is not clear how these defects contribute to pathogenesis. The primary motor driving long-distance retrograde axonal transport is cytoplasmic dynein, along with its required activator dynactin (dynein activator). Dynactin is a multi-protein complex that directly binds the dynein motors as well as the microtubule plus-end binding proteins EB1 and EB3 (EBs) and CLIP-170. The p150Glued subunit of dynactin directly interacts with EBs and CLIP-170 via the Cytoskeletal Associated Protein-Glycine Rich domain (CAP-Gly). Human mutations in the CAP-Gly domain of p150Glued reduce retrograde transport initiation from the distal axon and cause a rare, genetic form of Parkinsonism known as Perry syndrome. The precise mechanism and regulation of transport initiation however, are not known. In addition, there are no established mechanisms linking reduced retrograde transport initiation to neurodegeneration. I will address these questions in the following specific aims:
In Aim 1, I will determine how Perry mutations affect the binding of p150Glued to CLIP-170 using cellular and biochemical approaches. Then, I will reconstitute the retrograde transport initiation complex in vitro and visualize transport initiatio of purified brain-derived vesicles using TIRF microscopy with single-molecule resolution. I predict that Perry syndrome mutations abrogate the ability of p150Glued to bind CLIP-170, thus preventing the CLIP-170 dependent recruitment of dynactin for transport initiation.
In Aim 2, I will test how Perry mutations affect two major functions of retrograde transport, which are to facilitate autophagic clearance of proteins from the distal axon and to transport neurotrophic signals to the cell body. I hypothesize that Perry syndrome mutations will reduce autophagosome transport and/ or neurotrophic factor signaling. Disruption of either process individually, or both together, may explain the pathogenesis of Perry syndrome. I will test this hypothesis using live-cell imaging of transfected cortical neurons in microfluidic chambers. Finally, I will use the mechanistic insight gained from our biochemical studies to test whether small molecules can increase retrograde transport initiation and rescue the defects seen in neurons expressing Perry mutant p150Glued. Understanding the mechanism and regulation of transport initiation is crucial to define the pathogenic mechanisms in Perry syndrome. In addition, the new insights from this work may suggest novel approaches to modulate axonal transport with small molecules. Finally, since Perry syndrome shares many clinical and pathological hallmarks of Parkinson's, the knowledge gained from these studies may be relevant for sporadic Parkinson's disease.
Neurons have long, branched processes that require active transport to remain healthy. Many neurodegenerative diseases, including Parkinson's disease, have impaired axonal transport; however, it is not known how defects in transport cause neurodegeneration. This work focuses on understanding the pathogenic mechanisms of neurodegeneration in Perry syndrome, a genetic form of Parkinsonism.
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