Investigation of UCH-L1 interactions and functions.

Ubiquitin C-Terminal Hydrolase L1 (UCH-L1) is a highly stable, massively abundant protein in neurons that acts as a deubiquitinating enzyme (DUB) and plays a key role in monoubiquitin homeostasis. UCH-L1 accounts for 1-5% of total cellular protein in the brain and, despite its relatively small size, has one of the most complicated three-dimensional structures yet discovered. The precise functions of UCH-L1 remain enigmatic but the knotted backbone has been suggested to protect against proteasomal unfolding and degradation, suggesting a possible role in protecting and recycling of ubiquitin released from degraded proteins after proteasomal destruction.

The major phenotype of UCH-L1 loss or dysfunction is axonal dystrophy (so called 'dying back'), particularly in spinal neurons. Consistent with this, gracile axonal dystrophy (gad) mice, that lack functional UCH-L1 develop normally but show ataxia and progressive paralysis as they age and die prematurely.

The aims of this PhD project are to understand the molecular basis of UCH-L1 function in axonal maintenance and ageing by using combinations of wild-type and gad mice, and knockdown and molecular replacement strategies (with UCH-L1 mutants we already have) in spinal cord dorsal root ganglion (DRG) neuronal cultures to identify:

1. How manipulating UCH-L1 impacts on axonal maintenance (e.g. blebbing, occlusion and spheroid formation as well as morphological signs of 'dying back'). These experiments will use confocal imaging and microfluidics chambers to isolate and directly image axonal stability and maintenance by assaying for spheroids/occlusions that mark dying axons. Our objective will be to correlate these phenotypes to transport deficits.

2. The proteins that UCH-L1 interacts with and/or indirectly regulates using quantitative proteomics approaches in collaboration with the Proteomic facility using TMT post-labelling proteomics approaches to i) compare protein abundance in WT and UCH-L1 lacking neurons. This will identify proteins which are regulated by UCH-L1 and ii) performing immunoprecipitation of native UCH-L1 and using GFP-Trap of exogenous virally expressed GFP-UCH-L1 to define proteins that bind to UCH-L1, again using TMT to identify the retained interacting proteins. The protein hits will be validated in separate pull-down assays and then prioritised according to their potential roles in axons. The precise roles of the selected candidate proteins in processes involved in axonal maintenance and/or degradation will then be defined using biochemical, imaging and functional assays.

Because we already have all of the necessary UCH-L1 tools and have a wealth of expertise in these approaches the student will be ideally placed to make significant progress towards unravelling the mechanisms of action, and physiological and pathophysiological roles of UCH-L1 regulation of ubiquitin homeostasis.