Title:
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In Vitro Model Systems of a-Synuclein Over-Expression
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Parkinson's disease (PD) is the second most common neurodegenerative
disease in humans. Pathologically, it is characterised by an accumulation of
proteinaceous intraneuronal inclusions termed Lewy bodies (LBs). a-Synuclein is
the major protein found at the core of LBs. However, it is not known what stimulus
causes inclusion formation, or whether LBs are cytotoxic. I wished to address these
questions using cell-based model systems. Consequently, stable transfected nonneuronal
(HEK293) and neuronal (SH-SY5Y) cell lines were generated and cells
could be induced to express wild type or mutants of a-synuc1ein. Moreover, the
Parkin inducible cell lines were generated and used as a comparison.
The characterisation of a-synuclein in HEK293 cells was demonstrated to have
a relatively long half-life, and over-expression had no effect on cell death. In the
absence of exogenous stimuli, over-expression of a-synuclein did not form
inclusions. Only after the inhibition of proteasomal activity, inclusions were
observed a relatively small number of cells. The mutated A53T form appeared to
make the protein more prone to aggregate, and caused a greater level of cell· death.
This data suggested that additional factors are required to provide an initial seed for
inclusion formation in the presence of high concentrations of a-synuclein.
A proteomic approach was then applied to determine if the over-expression asynuclein
caused a wide-ranging effect on the cellular proteome. Few changes were
observed. However, of particular interest, one of the most significant changes was a
decreased level of the proteasome a7 subunit. Although this observation suggested
that the levels of proteasomal activity may be altered in cells, I went on to demonstrate that over-expression of a-synuclein did not lead to an inhibition of total
cellular proteasomal activity.
One observation that may explain the long-term effect of a-synuclein in cells
was that its over-expression caused the induction of the unfolded protein response
followed by the activation of the endoplasmic reticulum-associated protein
degradation (ERAD) pathway. Chronic activation of this pathway could induce
apoptosis cells, eventually causing cell death.
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