Proteomic analysis of cellular models of neurodegeneration and mitochondrial dysfunction

Burté, F., 2009. Proteomic analysis of cellular models of neurodegeneration and mitochondrial dysfunction. PhD, Nottingham Trent University.

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Abstract

Mitochondrial dysfunction is thought to contribute to neurodegenerative processes. As an example, dysfunction of complex I of the electron transport chain has been observed in Parkinson’s disease patients and 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), a complex I inhibitor, produces a Parkinsonian state in mammals. The aims of the present study were to determine the effects of MPTP on the mitochondrial proteome in a cellular model using mouse N2a neuroblastoma cells and to identify novel biomarkers of MPTP-induced toxicity.

The enrichment of mitochondria and the presence of cross-contamination from other subcellular components were monitored using a range of molecular markers. Mitochondrial proteins were then fractionated using an optimised 2-dimensional gel electrophoresis (2DE) protocol and the reproducibility of the method was investigated. A preliminary study comparing the mitochondrial proteome profile from two different states of mouse N2a neuroblastoma cells, mitotic and differentiated, was undertaken to establish whether differentiation of cells had major effects on the mitochondrial proteome. Since nine proteins showed changes in levels, which included stress-70 protein and aconitase, it was decided that differentiation did affect the mitochondrial proteome: hence, differentiated cells were used for further studies.

The effects of different concentrations (0 to 5 mM) and time-points (0 to 48 hours) of MPTP on plasma membrane integrity, cellular metabolic activity, cellular ATP concentration, mitochondrial potential, cytochrome c release and a variety of caspase activities were investigated. From this study, sub-cytotoxic and cytotoxic concentrations were defined and 1 mM MPTP for 24 hours was chosen as an example of a sub-cytotoxic concentration for further analysis. Using the previously optimised protocol for 2DE, mitochondrial preparations from differentiated N2a neuroblastoma cells treated with 1 mM MPTP for 24 hours were fractionated and compared to controls. Up to 32 proteins showed changes in protein levels, of which 10 were identified by peptide mass fingerprinting. Increases in the levels of 60 kDa heat shock protein (Hsp60), heat shock cognate 71 kDa protein (Hsc70), glutamate oxaloacetate 2 (GOT2) and voltage-dependent anion channel 1 (VDAC1) were validated as potential markers of MPTP-induced toxicity using western blot analysis. In parallel, a study of the mitochondrial phosphoproteome was undertaken. Despite the limitations of detection methods, a change in the phosphorylation status of a few mitochondrial proteins was observed following MPTP treatment, notably potential increased phosphorylation of Hsc70 and Hsp60.

Further analysis was undertaken in order to gain a better understanding of the increase in VDAC1 levels following sub-cytotoxic treatments with MPTP. Although VDAC1 protein levels were increasing in a dose- and time-dependent manner, no mRNA upregulation was observed. Similarly, the use of other inhibitors of the electron transfer chain led to increased VDAC1 protein levels but no change in mRNA. Finally, modulations in VDAC1 phosphorylation were observed following MPTP-induced toxicity, further implicating the channel in mitochondrial dysfunction.

To conclude, studies with this cellular model implicate the involvement of several mitochondrial pathways in the MPTP-induced Parkinsonian syndrome. In particular, alterations to VDAC1 may represent a novel target of neurodegeneration.

Item Type:	Thesis
Creators:	Burté, F.
Date:	2009
ISBN:	9781369316643
Identifiers:	Number Type PQ10183500 Other
Rights:	This work is the intellectual property of the author, and may also be owned by the research sponsor(s) and/or Nottingham Trent University. You may copy up to 5% of this work for private study, or personal, non-commercial research. Any re-use of the information contained within this document should be fully referenced, quoting the author, title, university, degree level and pagination. Queries or requests for any other use, or if a more substantial copy is required, should be directed in the first instance to the author.
Divisions:	Schools > School of Science and Technology
Record created by:	EPrints Services
Date Added:	09 Oct 2015 09:33
Last Modified:	30 Sep 2020 11:35
URI:	https://irep.ntu.ac.uk/id/eprint/3

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