All ETDs from UAB

Advisory Committee Chair

Scott Wilson

Advisory Committee Members

Robin Lester

Guillermo Marques

Kevin Roth

Anne Theibert

Document Type

Dissertation

Date of Award

2010

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The ubiquitin proteasome system (UPS) plays critical roles in regulating multiple cellular functions, including protein abundance, trafficking, and cell signaling. In the nervous system, the UPS has been implicated in controlling a wide diversity of cellular process such as synaptic transmission, axon outgrowth, axon targeting, and synapse development. Although impairment of the UPS is observed in both neurodegenerative and developmental diseases, the mechanisms underlying how UPS dysregulation contributes to disease pathogenesis are not known. The proteasome, an approximate 2.5 megadaltons protein complex composed of 19S and 20S assemblies, is the site of ubiquitin-dependent protein degradation. Recent studies have demonstrated dysfunction of 20S core particle in many neurological diseases, however, there have been no studies linking alterations in 19S regulatory particle function to disease. The studies described in this thesis focus on the role of ubiquitin specific protease 14 (Usp14), a deubiquitinating enzyme (DUB) associated with the 19S regulatory particle, in the regulation of the postnatal development of the nervous system. In the first part of this thesis, we show that loss of Usp14 does not cause loss of motor neurons in the spinal cord, but does lead to synaptic developmental deficits at the neuromuscular junctions (NMJs) of the ataxia (axJ) mice. Pre-synaptic defects include phosphorylated neurofilament accumulation, nerve terminal sprouting and poor arborization of the motor nerve terminal, while post-synaptic acetylcholine receptor clusters display immature plaque-like morphology. These structural changes in the NMJs correlate with ubiquitin loss in the spinal cord and sciatic nerve as well as in the synaptosome fraction. Transgenic restoration of Usp14 in the nervous system corrects the levels of monomeric ubiquitin and deficits in both synaptic function and development of the axJ mice. This study identifies an essential role for Usp14 in mediating the postnatal development of the NMJ. In the second part of this study, we demonstrate that ubiquitin pools maintained by the catalytic activity of Usp14 are required for proper synaptic function and development at the NMJs. Neuronal overexpression of ubiquitin by the Thy1-ubiquitin transgene restores ubiquitin levels due to loss of Usp14 in the axJ mice. Increasing ubiquitin levels in the axJ mice significantly improved their viability, growth and locomotor motor activities. The Thy1-ubiquitin transgene also corrects the sprouting and immature endplates observed at the axJ NMJs. In addition, the deficits of synaptic transmission at the axJ NMJs were also corrected by the ubiquitin transgene. Furthermore, local inhibition of Usp14 by injecting a Usp14-specific inhibitor into the wild type muscles results in NMJ deficits similar to those seen in the axJ mice. Together, these data indicate that Usp14 regulates NMJ development by maintaining ubiquitin pools required for the postnatal maturation of motor endplates. Our study reveals a crucial role of proteasome in regulating synaptic function and development at NMJs and provides avenues for developing therapeutic targets of neuromuscular diseases contributed by the impairment of the UPS.

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