Mordhwaj S. Parihar

Mitochondrial association of alpha-synuclein causes oxidative stress

Abstract.α-Synuclein is a neuron-specific protein that contributes to the pathology of Parkinson’s disease via mitochondria-related mechanisms. The present study investigated possible interaction of α-synuclein with mitochondria and consequences of such interaction. Using SHSY cells overexpressing α-synuclein A53T mutant or wild-type, as well as isolated rat brain mitochondria, the present study shows that α-synuclein localizes at the mitochondrial membrane. In both SHSY cells and isolated mitochondria, interaction of α-synuclein with mitochondria causes release of cytochrome c, increase of mitochondrial calcium and nitric oxide, and oxidative modification of mitochondrial components. These findings suggest a pivotal role for mitochondria in oxidative stress and apoptosis induced by α-synuclein.

Inactivation of mitochondrial respiratory chain complex I leads mitochondrial nitric oxide synthase to become pro-oxidative

We recently demonstrated that mitochondrial nitric oxide synthase (mtNOS) functionally couples with mitochondrial respiratory chain complex I to produce nitric oxide [M.S. Parihar, R.R. Nazarewicz, E. Kincaid, U. Bringold, P. Ghafourifar, Association of mitochondrial nitric oxide synthase activity with respiratory chain complex I, Biochem. Biophys. Res. Commun. 366 (2008) 23-28]. The present report shows that inactivation of complex I leads mtNOS to become pro-oxidative. Our findings suggest a crucial role for mtNOS in oxidative stress caused by mitochondrial complex I inactivation.

mAtNOS1 regulates mitochondrial functions and apoptosis of human neuroblastoma cells.

mAtNOS1 is a novel gene recently reported in mammalian cells with functions that are not fully understood. The present study generated human neuroblastoma SHSY cells over- and underexpressing mAtNOS1 and shows that mAtNOS1 is involved in regulating mitochondrial nitric oxide, mitochondrial transmembrane potential, protein tyrosine nitration, cytochrome c release, and apoptosis of those cells.

Detection Assays for Determination of Mitochondrial Nitric Oxide Synthase Activity; Advantages and Limitations

Nitric oxide (NO) is a reactive radical synthesized by members of the NO synthase (NOS) family, including mitochondrial-specific NOS (mtNOS). Some of the assays used for the determination of cytoplasmic NOS activity have been utilized to detect mtNOS activity. However, it seems that many of those assays need to be adjusted and optimized to detect NO in the unique environment of mitochondria. Additionally, most mtNOS detection assays are designed and optimized for isolated mitochondria and may exert inherent pitfalls and limitations once used in living cells. This chapter describes several assays used commonly for mtNOS detection in isolated mitochondria and in mitochondria of live cells. Those include colorimetric and spectrophotometric methods, Griess reaction, radioassay, and polarographic and chemiluminescence assays. It also describes fluorescent-based assays for the detection of mitochondrial NO in live cells. Advantages and limitations of each assay are discussed.

mAtNOS1 induces apoptosis of human mammary adenocarcinoma cells

mAtNOS1 is a novel gene recently reported in mammalian genome with functions that are not fully understood. The present study shows that in human mammary adenocarcinoma MCF-7 cells, mAtNOS1 expression increases mitochondrial nitric oxide and calcium. Our study further shows that overexpression of mAtNOS1 induces apoptosis in MCF-7 cells by increasing mitochondrial protein tyrosine nitration and cytochrome c release. The present study suggests a novel function for mAtNOS1 in regulating mitochondrial nitric oxide and calcium and inducing apoptosis of MCF-7 cells.

Alpha Synuclein and Parkinson’s Disease

α-Synuclein predominantly expressed in the brain is a small acidic protein containing three domains namely N-terminal lipid-binding α-helix, amyloid-binding central domain (NAC), and C-terminal acidic tail. The physiological functions of this protein remain poorly understood. α-Synuclein localizes specifically to the nerve terminal and has been extensively described to take active task in the regulation of release of neurotransmitter at the presynapse in brain. Accumulating evidence suggests that prefibrillar species, and aggregated form of α-synuclein, are accountable for the pathogenicity of Parkinson’s disease (PD). Larger oligomers of α-synuclein exhibited to impair many functions of neuronal cells including: impairment of synaptic functions, impairment of the mitochondrial and the endoplasmic reticulum functions, and the impairment of protein degradation pathways. Oligomers/protofibrils once accumulated inside or outside cells may cause a lethal effect on the synapse, which may cause disruption of the neurotransmission. The oligomeric α-synuclein species has also the property of spreading between neuronal cells, either as drifting proteins or via extracellular vesicles, and thus spreading the toxic effects in different parts of the brain. Although several mutations in α-synuclein gene have been known that causes familial PD in human, the mechanisms that elevate the accumulation and aggregation of α-synuclein protein are not well addressed. Considering of the mechanism of aggregation of α-synuclein protein and targeting the toxic functions of this protein in brain cells including dysregulated mitochondrial functions may lead to novel therapeutic approaches in Parkinson’s disease.