Extracellular Alpha-Synuclein Oligomers Induce Parkin S-Nitrosylation: Relevance to Sporadic Parkinson’s Disease Etiopathology

Abstract

α-Synuclein (ASN) and parkin, a multifunctional E3 ubiquitin ligase, are two proteins that are associated with the pathophysiology of Parkinson's disease (PD). Excessive release of ASN, its oligomerization, aggregation, and deposition in the cytoplasm contribute to neuronal injury and cell death through oxidative-nitrosative stress induction, mitochondrial impairment, and synaptic dysfunction. In contrast, overexpression of parkin provides protection against cellular stresses and prevents dopaminergic neural cell loss in several animal models of PD. However, the influence of ASN on the function of parkin is largely unknown. Therefore, the aim of this study was to investigate the effect of extracellular ASN oligomers on parkin expression, S-nitrosylation, as well as its activity. For these investigations, we used rat pheochromocytoma (PC12) cell line treated with exogenous oligomeric ASN as well as PC12 cells with parkin overexpression and parkin knock-down. The experiments were performed using spectrophotometric, spectrofluorometric, and immunochemical methods. We found that exogenous ASN oligomers induce oxidative/nitrosative stress leading to parkin Snitrosylation. Moreover, this posttranslational modification induced the elevation of parkin autoubiquitination and degradation of the protein. The decreased parkin levels resulted in significant cell death, whereas parkin overexpression protected against toxicity induced by extracellular ASN oligomers. We conclude that lowering parkin levels by extracellular ASN may significantly contribute to the propagation of neurodegeneration in PD pathology through accumulation of defective proteins as a consequence of parkin degradation.

P2X7 Receptor is Involved in Mitochondrial Dysfunction Induced by Extracellular Alpha Synuclein in Neuroblastoma SH-SY5Y Cells

“…Moreover, α-Syn and ATP-induced free radicals generation was significantly prevented by pretreatment with nonselective and selective P2X7R antagonists. To verify that the cytosolic redox environment was affected by the increase in ROS, SH-SY5Y cells were transiently transfected with a reporter gene coding for a redox-sensitive green fluorescent protein (roGFP, [27]) and treated with α-Syn for 24 h. The results indicated that α-Syn significantly deregulates cellular redox state in SH-SY5Y cells. Moreover, this effect by α-Syn was significantly ameliorated by PPADS and AZ 11645373 pretreatment (Figure 2b).…”

“…Moreover, this effect by α-Syn was significantly ameliorated by PPADS and AZ 11645373 pretreatment (Figure 2b). Since the activation of P2X7R induced the increased phosphorylation of oxidative stress-related proteins, it is thus possible that the mechanisms of α-Syn neurotoxicity are related to the enhancement of the reactive oxygen species (ROS) level [27]. In addition, P2X7R activation induces ROS generation in various cells including neurons [28,29].…”

The interplay between parkin and alpha-synuclein; possible implications for the pathogenesis of Parkinson’s disease

“…This relatively easy to manipulate mechanism influences the neuroprotective activity of parkin and the tendency of α-syn to aggregate, thus offering important insights into pathophysiological mechanisms leading to PD and potential therapeutic value. Importantly, α-syn also alters parkin levels and activity (Wilkaniec et al, 2019), potentially creating a cycle of self-fueling pathology.…”

“…Recent studies indicate that α-syn is secreted into the extracellular space, and secretion appears to be a precisely regulated, physiological process (Jęśko et al, 2017). However, in PD, abnormal excess release of monomeric and/or aggregated α-syn might lead to free radical stress (Wilkaniec et al, 2019) and trigger immunological response of surrounding astrocytes and microglia, thereby accelerating the neurotoxic insult (Allen Reish and Standaert, 2015). Moreover, the internalization of secreted α-syn by neurons and glia spreads the neurodegenerative pathology and involves other cell types, including cells that do not typically express significant levels of α-syn (Jęśko et al, 2017).…”

“…1). Indeed, parkin undergoes an α-syn-induced S-nitrosylation (Wilkaniec et al, 2019), which can lead to parkin autoubiquitination and degradation in PC12 cells (Wilkaniec et al, 2019). Other research has shown that covalent binding of DA reduces parkin solubility and inhibits its activity as an E3 ubiquitin ligase (LaVoie et al, 2005).…”