Alpha-Synuclein (ASN), a small cytosolic protein enriched in synaptic terminals, was implicated in the pathomechanism of several neurodegenerative disorders called alpha-synucleinopathies. ASN was shown to be a main component of characteristic intraneuronal protein aggregates called Lewy bodies (LB) and Lewy neurites (LN), observed i.a. in Parkinson’s disease, dementia with LBs and in the LB variant of Alzheimer’s disease. Recent studies demonstrated that ASN may exist also in the extracellular space. Low-molecular ASN aggregates distributed in the brain parenchyma likely may be more toxic than ASN in LB, however, the exact mechanism of cytotoxicity of extracellular ASN is not fully understood. Our previous studies demonstrated the significant impact of extracellular ASN on calcium homeostasis. ASN evoked deregulation of intracellular calcium concentration leading in consequence to enhancement of nitric oxide synthesis. Deregulation of calcium homeostasis affects other calcium-dependent enzymes, including Calpains. The aim of the present study was to investigate the involvement of Calpaindependent activation of Cyclin Dependent Kinase 5 (Cdk5) in molecular mechanism of extracellular ASN cytotoxicity. The activation of Cdk5 is regulated by binding of regulatory subunits p35 and p39. Deregulation of calcium homeostasis may induce the Calpainmediated breakdown of Cdk5/p35 into Cdk5/p25 leading to overactivation of Cdk5. In our studies we used rat Pheochromocytoma PC12 cells incubated with exogenous ASN (10 µM) in the presence of Calpain inhibitor Calpeptin (10 µM) and Cdk5 inhibitors Roscovitine (10 µM) and BML-259 (10 µM). Our results indicated that incubation of PC12 cells in the presence of extracellular ASN (10 µM) for 48 h evoked cell death, and Cdk5 inhibitors efficiently prevented ASN toxicity, indicating an important role of Cdk5 in molecular mechanism of ASN toxicity. The level of Cdk5 protein was unchanged, but phosphorylation of Cdk5 at Tyr15 was significantly increased, suggesting that the enzymatic activity of Cdk5 is increased in ASN-treated cells. The presence of p25 protein was observed, what suggests that Calpain-dependent proteolysis of p35 occurred in ASN-treated cells. Calpeptin, an inhibitor of Calpains, prevented ASN-induced cell death, confirming the important role of Calpain activation in mechanism of ASN toxicity. In summary, our results demonstrated that alteration of calcium homeostasis evoked by extracellular ASN induce Calpain-dependent overactivation of Cdk5. These molecular processes may be involved in ASN-evoked cell death in vitro and probably also in neurodegenerative disorders.
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