Molecular and functional characterisation of the neuronal specific de-ubiquitinase UCH-L1 and its role in neuronal polarity and axonal outgrowth

Nerves cell communicate by passing information across synapses. Synapses are structures formed between axons, which relay information out of the nerve cell, and dendrites, which receive information into the cell. In general, synapse formation requires the growth and elongation of axons to find dendrites of other cells. This process sounds simple but, in fact, it requires a highly regulated and coordinated series of events. One of the first things that need to happen is that the cell has to specify which protrusion will be an axon and which will be dendrites. This process is known as polarization and usually results in a single long axon and much smaller dendrites.

The molecular events that regulate neuronal polarization and subsequent axonal outgrowth are not well understood. However, it is clear that the targeted destruction of proteins is an important factor. For example, the main function of some proteins is to block the effects of other proteins. It is only when the first protein is destroyed that the second protein can exert its effects. A major way cells destroy proteins is by tagging them with a small marker protein called ubiquitin. The ubiquitin tag acts as an identifier that allows these proteins to be recruited to the proteasome, a structure that breaks down and recycles unwanted proteins.

We have recently discovered that UCH-L1, a protein that is only present in neurons, plays a key role in regulating the ubiquitin tagging of proteins and that one of its actions is to enhance axonal growth. Our aim is to understand how UCH-L1 does this, what other proteins are involved and what happens when it goes wrong. We believe that this is important because it will provide information about how nerve cells connect and how they form networks, which are the basis of how the brain works. In addition, UCH-L1 is associated with a number of important diseases, such as Parkinson's and Alzheimer's diseases, so better understanding of UCH-L1 function could provide a ways to design drugs to help treat these conditions.

Gracile axonal dystrophy (gad) mice lack UCH-L1, a de-ubiquitinating (DUB) enzyme and one of the most abundant proteins in the brain. The defining phenotype of these mice is extensive axonal degeneration in the gracile tract of the spinal cord suggesting that UCH-L1 likely plays a major role in axonal morphogenesis and maintenance. Consistent with this we have shown that UCH-L1 regulates axonal length in cultured neurons. Moreover, using proteomic approaches we have identified an array of UCH-L1 interacting proteins, including the ubiquitin Cullin Ring E3 Ligase (CRL) Cul1. UCH-L1 binds directly to Cul1 and appears to alter its ligase function by inhibiting auto-ubiquitination. We also have preliminary data suggesting that UCH-L1 binding regulates other post-translational modifications of Cul1 including NEDDylation.

Based on our experiments and the available literature we propose that UCH-L1 modulates Cul1 ubiquitin ligase activity, which in turn regulates substrate proteins are that involved in neuronal morphology and axon specification. Interestingly, our proteomic assays also isolated several other putative UCH-L1 protein-interactors that may participate in this pathway. Thus, we aim to test the hypothesis that protein ubiquitination controlled by UCH-L1 modulation of Cul1 regulates target proteins for degradation by the UPS and that this pathway is a key determinant for expression of neuronal polarity and neuronal connectivity.

We believe that this is an important and timely project that will increase understanding of neuronal development. Further, the function and dysfunction of ubiquitination pathways in general and UCH-L1 in particular have been implicated as fundamentally important factors in neurodegenerative diseases. Therefore, increased knowledge of the interaction partners and specific functions of UCH-L1 will provide valuable insight into the mechanisms of these diseases and may provide targets for drug design and development.

Enhancement and transfer of academic knowledge: A key objective of our work is the effective and timely dissemination of results. We have a good track record of making freely available knowledge, reagents and resources. Phil Rubin, our grant funded lab manager/technician promptly distributes requested tools and reagents, and we are acknowledged in many papers for these efforts. We will continue to publish in highly regarded journals as soon as the data permit. We shall also continue our regular participation in and organization of national and international conferences. For example, the PI is on the organizing committee of the ISN meeting in 2013. The applicants routinely present, communicate, and discuss their findings and establish new collaborations. The PI mainly accepts regular invitations to speak at international conferences. Progress summaries and links to original publications will be posted on the webpages of the School of Biochemistry.

Academic collaboration: We enjoy an extensive and active network of collaborators in Bristol and world-wide. Our findings impact on the work other labs at Bristol working on synaptic processes (e.g. ZI Bashir, J. Mellor, G. Collingridge, M. Ashby) and protein trafficking (e.g. J. Hanley, P. Cullen, G. Banting). Internationally, we have close links with S. Martin in Nice and C Mulle in Bordeaux for collaborations and access to transgenic animals, high-resolution light microscopy and specialised electrophysiology.

Information exchange with industry: We have long-standing collaborations with GSK and currently have a joint EU-funded student soon to spend 6 months at GSK in Singapore. In addition, we have had recent collaborations with UCB (Belgium) specifically on presynaptic proteins, Neurosearch and now Lundbeck on protein trafficking, and we are presently talking to Shionogi (Japan) regarding our work on SUMOylation and neuroprotection. We intend to expand such collaborations through regular networking, including with former members of the lab working in Pharma (e.g. Medimune, Novartis, Eli Lilly, GSK, Lundbeck).

Potential commercial exploitation: We do not currently foresee likely discoveries appropriate for commercialization but, if appropriate, we will consult with the Research and Enterprise Development office.
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Research skills: This project will benefit researchers in our group and associated with our lab by collaborations and/or shared facilities and resources. There will be opportunities to develop their molecular, biochemistry, imaging and (through collaboration) electrophysiology skills. All eligible lab members are encouraged to attend BBSRC, University and faculty training courses to develop their general and specific skill sets resulting in enhanced career prospects.

Teaching and training: There is a skills training programme within our school and the applicants participate in formal and informal training of PhD students in molecular, biochemical and imaging techniques. Both applicants are involved in UG teaching and tutorials. The success of our teaching is assessed by student feedback and internal peer review in which we are highly ranked. This project provides excellent opportunities for research projects for final year undergraduate BSc students as well as Wellcome Trust and RCUK PhD students doing lab rotations. We regularly host overseas students, e.g. in the last two years we have had MSc students for 6 month internships from Germany and Holland and 3 month placements for PhD students from Italy and Portugal.

Public engagement: The PI has routinely engaged in public lectures and outreach activities via
Bristol Neuroscience and the University's Centre for Public Engagement. Lab members are active in Brain Awareness Week and University open days. Press releases of our findings are, and will continue to be posted on university websites. The PIs are registered on the University's Directory of Experts.