Ubiquitination and proteasomal degradation in lesioned neurons
The ubiquitin-proteasome system (UPS) is responsible for regulated protein degradation in all eukaryotic cells. In nerve cells, this pathway determines protein abundance and protein quality control in spatially restricted neuronal compartments, such as the nucleus, dendrites and spines, axons and in synaptic boutons. Therefore, the UPS regulates neuronal gene expression, synaptic and spine functions, axonal regeneration and neuronal degeneration by tagging and eliminating key proteins required for morphological and chemical neuroplasticity. Recent progress in the elucidation of the pathomechanisms leading to various neurological disorders clearly revealed the importance of the UPS for neuron survival and maintenance. The significance of ubiquitin-mediated proteolysis in neuronal physiology and pathophysiology has now become firmly established and brings this pathway into the focus of many neuroscience laboratories worldwide (Klimaschewski, 2003).
Neurite outgrowth is accompanied by increased levels of high molecular weight ubiquitin conjugates and decreased levels of free ubiquitin. The search for enzymes responsible for increased utilization of ubiquitin revealed the ubiquitin conjugating enzyme, HR6B (yeast UBC2/RAD6), increased on mRNA level in rat pheochromocytoma (PC12) cells after treatment with nerve growth factor (NGF). HR6B participates in ‘N-end rule degradation’ that is implicated in the cleavage of proteins with destabilizing N-terminal residues (bulky hydrophobic or basic amino acids) and requires E3alpha, the ubiquitin ligase binding N-end rule target proteins. Down-regulation of HR6B or E3alpha mRNA by small interfering RNA and treatment with Leu-Ala, a dipeptide-inhibitor of E3alpha, inhibit neurite outgrowth of PC12 cells. Furthermore, axonal regeneration of adult sensory neurons, which express prominent nuclear and membrane-associated HR6 immunoreactivity, is reduced by Leu-Ala in vitro. Therefore, N-end rule ubiquitination is required for neuronal differentiation of PC12 cells and may be involved in axonal regeneration of peripheral neurons (Kavakebi et al., 2005; Tomasino and Klimaschewski, 2005).
Inhibition of the proteasome by lactacystin, a specific blocker of the catalytic beta-subunits, results in transient neurite outgrowth of neuronal cell lines. Vice versa, as demonstrated in this study, treatment of pheochromocytoma (PC12) cells with nerve growth factor (NGF) or other differentiating agents reduces proteasomal activity. This is accompanied by an increase in mRNA and protein levels of the catalytically active subunits beta1, beta2 and beta5, but not of their inducible counterparts, indicating changes in subunit composition of the proteasome during neuronal differentiation. In contrast to neuronal cell lines, however, pretreatment of primary neurons with proteasome inhibitors completely prevents axon formation, and lower concentrations of lactacystin (0.5-5 µM) significantly reduce axonal elongation and branching in vitro. Furthermore, established axonal networks degenerate rapidly and long-term survival of peripheral neurons is impaired in the presence of proteasome inhibitors. Axonal pathology is reminiscent of the morphological changes observed in neurodegenerative disorders and supports a crucial role of the constitutive catalytic subunits in axon initiation, maintenance and regeneration (Klimaschewski et al., 2006).
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