Abstract
Parkinson's disease (PD) is the most common neurodegenerative movement disorder, characterized by loss of dopominergic (DA) neurons in substantia nigra pars compacta (SNpc), and can be experimentally mimicked by the neurotoxin MPP+ in vitro models. In this study, we investigated the potential protective effect of SKF-96365, a non-specific inhibitor of SOCE (store-operated calcium entry), on MPP+ induced cytotoxicity in PC12 cells. We found that pretreatment with SKF-96365 (10 µM and 50 µM) 30 min before injury significantly increased cell viability, decreased LDH release, prevented nuclear damage, and inhibited apoptotic cell death in MPP+ stressed PC12 cells. The results of calcium image using the ratiometric calcium indicator Fura-2-AM also showed that SKF-96365 reduced the intracellular calcium overload induced by MPP+ in PC12 cells. In addition, SKF-96365 decreased the expression of Homer1, a more recently discovered postsynaptic scaffolding protein with calcium modulating function, following MPP+ administration in PC12 cells, while had no statistically significant effects on endoplasmic reticulum (ER) calcium concentration. Furthermore, overexpression of Homer1 by using recombinant lentivirus partly reversed protective effects of SKF-96365 against MPP+ injury. The ER Ca2+ release was further amplified and ER calcium recovery was delayed by Homer1 upregulation in PC12 cells following MPP+ insult. Taken together, these data suggest that SKF-96365 protects PC12 cells against MPP+ induced cytotoxicity, and this protection may be at least in part on the inhibition of intracellular calcium overload and suppression of Homer1-mediated ER Ca2+ release.
Highlights
Parkinson’s disease (PD), the second most common chronic neurodegenerative disorder after Alzheimer’s disease, is characterized primarily by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and the formation of cytoplasmic ‘‘Lewy body’’ inclusions in some of the surviving neurons [1,2]
Increasing evidence has demonstrated that abnormal calcium homeostasis is one of the fundamental processes contributing to DA neuronal death, and inhibition of calcium dysfunction through pharmacological or genetic methods could be a critical strategy for PD [5,6,7]
Effect of SKF-96365 on MPP+-induced cytotoxicity To investigate whether SKF-96365 could protect PC12 cells from injury induced by MPP+ insult, cultured PC12 cells were pretreated with SKF-96365 in different concentrations (1 mM, 10 mM or 50 mM) 30 min before MPP+ addition
Summary
Parkinson’s disease (PD), the second most common chronic neurodegenerative disorder after Alzheimer’s disease, is characterized primarily by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and the formation of cytoplasmic ‘‘Lewy body’’ inclusions in some of the surviving neurons [1,2]. Cytosolic calcium acts as a ubiquitous second messenger and Ca2+-mediated cellular stress has long been thought to be important in neurodegenerative disease including PD [8]. The unusual reliance of DA neurons on calcium in autonomous pacemaking suggests that stress of storage organelles (such as mitochondria and endoplasmic reticulum) created by calcium homeostasis dysfunction could be responsible for their selective vulnerability, rather than a late-stage consequence [9,10]. The entry of extracellular calcium generally results from depletion of intracellular stores in a process referred to as capacitative calcium entry or store-operated calcium entry (SOCE), which ensures optimal refilling of the endoplasmic reticulum, and leads to a prolonged increase in cytosolic Ca2+ [11,12]
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