Alpha-synuclein is a major component of intra-neuronal inclusions found in Lewy bodies in Parkinson?s disease patients.
The mitochondria represent a highly promising target for the development of novel therapeutic agents as they are seats
of many metabolic processes including oxidative energy metabolism. In the present study we report mechanisms underlying
cellular toxicity of alpha-synuclein overexpression under conditions of inactivation of mitochondrial respiratory chain complex
A two-step PCR strategy was performed to create alpha-synuclein A53T and A30P expression vectors. To create wild type
alpha-synuclein expression vector, a regular PCR was performed using N- and C-terminal human alpha-synuclein primers and
subcloned into the p-TARGET, while a self-ligated plasmid of the p-TARGET was used as control vector. Wild type and A53T
and A30P alpha-synuclein mutants were transfected to human dopaminergic neuroblastoma SHSY cells. Cellular aggregations
of alpha-synuclein, reactive oxygen species (ROS), protein tyrosine nitration, mitochondrial transmembrane potential, and
cytochrome c release were determined by immunofluorescence, immunogold electron microscopy and immuno-blotting
Results show that over expression of both A53T and A30P mutants or the wild-type alpha-synuclein augmented the aggregation
of alpha-synuclein and that the aggregates were localized within the mitochondria of overexpressing cells. Over expressing cells
show increase in cellular and mitochondrial ROS, nitration of proteins, decreased mitochondrial transmembrane potential and
cellular respiration, and release of cytochrome c from the mitochondria. The degree of ROS production, cellular respiration
and mitochondrial dysfunctioning was increased in A53T and A30P mutants or the wild-type alpha-synuclein overexpressed
cells when complex I was inactivated.
These findings suggest a direct role of mitochondrial respiratory chain complex I in alpha-synuclein toxicity in neuronal cells
and suggest possible therapeutic intervention in Parkinson?s disease involving prevention of aggregation, translocation of
alpha-synuclein into mitochondria and inactivation of complex I.
Dr. Mordhwaj S. Parihar is a professor in Biochemistry & Molecular Biology at Vikram University, Ujjain, India. He completed his PhD from Vikram
University Ujjain, India and postdoctoral studies from Southern Illinois University School of Medicine, Springfield, Illinois, USA. He worked on
molecular and biochemical mechanisms of pathogenesis of Alzheimer?s and Parkinson?s Diseases employing mutations, gene silencing and
transcriptional changes. His current interest includes epigenetic and metabolic factors that contribute to various neurodegenerative and other human
diseases. He has published more than 55 papers in international journals and serving as an ad hock editorial members member of reputed journals
and member international grant review committees.
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