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Title: Effects of mitochondrial dysfunction on neurofilament turnover and distribution in human neuroblastoma cells
Author: Hanes, A.
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2010
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A common feature of neurodegenerative conditions including Parkinson’s disease (PD) is the presence of intracytoplasmic proteinacious inclusions. In PD these inclusions are called Lewy bodies (LBs) and contain a number of proteins including α-synuclein, ubiquitin and neurofilaments (NFs). NFs, the intermediate filaments expressed in neuronal cells are responsible for the maintenance of axonal structure. Although NFs were the first proteins identified in LBs their role in PD pathogenesis has not been fully explored. The work presented here attempts to address some of the gaps in the current knowledge concerningNF turnover and the role of NFs in PD using the human SH-SY5Y neuroblastoma cell line, commonly used as a cellular model of neurodegeneration. Mitochondrial dysfunction, dopamine (DA) mediated oxidative stress and impaired protein degradation have all been implicated in PD pathogenesis. The complex I inhibitor MPTP and its active metabolite (MPP+) induce Parkinsonism in humans and other primates and have been extensively used as PD mimetics in both cellular and animal models. Addition of specific protease inhibitors in the presence of cycloheximide (an inhibitor of new protein synthesis) revealed that NF-heavy chains are degraded by macroautophagy and cathepsin D, possibly with some involvement of cysteine cathepsin, but not calpain or the ubiquitin proteasome system (UPS). This is in contrast to α-synuclein which was degraded by macroautophagy, the UPS and calpain. Treatment with MPP+ did not increase NF halflife despite a reduction in the activity of the 20S proteasome, cathepsin D and macroautophagy. However an activation of cysteine cathepsin activity was observed with MPP+ treatment, suggesting a role for these cathepsins in NF turnover following complex I inhibition. Inhibition of cysteine cathepsins in MPP+ treated cells resulted in increased cell death suggesting that activation of cysteine cathepsins is protective in this context. Following treatment with 100 μM DA, NF turnover appeared to be reduced, accompanied by a reduction in 20S proteasome activity and macroautophagy but activation of calpain. To allow analysis of the effects of mitochondrial dysfunction on the distribution of NFs in live cells, Green Fluorescent Protein (GFP) tagged NFs were produced. Live imaging of cells treated with a high dose of MPP+ displayed a retraction of axonal processes which occurred within the first 20 - 30 minutes of treatment. Analysis of the rate of retraction of GFP fluorescence and the remaining axonal structure indicated that NF retraction was not an early event in axonal retraction, at least with this concentration of MPP+. The effect of mitochondrial dysfunction and/or proteasome inhibition on protein aggregation and inclusion formation was investigated in differentiated SH-SY5Y cells. Treatment with MPP+, epoxomicin (proteasome inhibitor, epoxo) or MPP+/epoxo resulted in an enrichment of NFs in a detergent insoluble fraction and accumulation of NF-H in a perinuclear location together with p62 and LC3, suggesting the attempted degradation of accumulated NF-H by macroautophagy. Further analysis of GFP-tagged NFs in MPP+ treated cells should assist in the elucidation of the role of NFs in the formation of inclusions and will allow analysis of protein partners during complex I dysfunction and other cellular states linked to neurodegeneration.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available