Non-prophylactic resveratrol-mediated protection of neurite integrity under chronic hypoxia is associated with reduction of Cav1.2 channel expression and calcium overloading

Abstract

Cellular hypoxia is a major cause of oxidative stress, culminating in neuronal damage in neurodegenerative diseases. Numerous ex vivo studies have implicated that hypoxia episodes leading to disruption of Ca²⁺ homeostasis and redox status contribute to the progression of various neuropathologies and cell death. Isolation and maintenance of primary cell culture being cost-intensive, the details of the time course relationship between Ca²⁺ overload, L-type Ca²⁺ channel function, and neurite retraction under chronic and long-term hypoxia remain undefined. In order to explore the effect of oxidative stress and Ca²⁺ overload on neurite length, first, we developed a 5-day-long neurite outgrowth model using N2a cell line. Second, we propose a chronic hypoxia model to investigate the modulation of the L-type Ca²⁺ channel (Cav1.2) and oxidative resistance gene (OXR1) expression level during the process of neurite retraction and neuronal damage over 32 h. Thirdly, we developed a framework for quantitative analysis of cytosolic Ca²⁺, superoxide formation, neurite length, and constriction formation in individual cells using live imaging that provides an understanding of molecular targets. Our findings suggest that an increase in cytosolic Ca²⁺ is a feature of an early phase of hypoxic stress. Further, we demonstrate that augmentation in the L-type channel leads to amplification in Ca²⁺ overload, ROS accumulation, and a reduction in neurite length during the late phase of hypoxic stress. Next, we demonstrated that non-prophylactic treatment of resveratrol leads to the reduction of calcium overloading under chronic hypoxia via lowering of L-type channel expression. Finally, we demonstrate that resveratrol-mediated reduction of Cav1.2 channel and STAT3 expression are associated with retention of neurite integrity. The proposed in vitro model assumes significance in the context of drug designing and testing that demands monitoring of neurite length and constriction formations by imaging before animal testing.

Keywords: Ca(2+); Cav1.2; Chemical hypoxia; LSCM; Live imaging; N2a cells; Neurodegeneration; ROS.