A2AR antagonists triggered the AMPK/m-TOR autophagic pathway to reverse the calcium-dependent cell damage in 6-OHDA induced model of PD

Abstract

Calcium dyshomeostasis, oxidative stress, autophagy and apoptosis are the pathogenesis of selective dopaminergic neuronal loss in Parkinson's disease (PD). Earlier, we reported that A_2A R modulates IP₃-dependent intracellular Ca²⁺ signalling via PKA. Moreover, A_2A R antagonist has been reported to reduce oxidative stress and apoptosis in PD models, however intracellular Ca²⁺ ([Ca²⁺]_i) dependent autophagy regulation in the 6-OHDA model of PD has not been explored. In the present study, we investigated the A_2A R antagonists mediated neuroprotective effects in 6-OHDA-induced primary midbrain neuronal (PMN) cells and unilateral lesioned rat model of PD. 6-OHDA-induced oxidative stress (ROS and superoxide) and [Ca²⁺]_i was measured using Fluo4AM, DCFDA and DHE dye respectively. Furthermore, autophagy was assessed by Western blot of p-m-TOR/mTOR, p-AMPK/AMPK, LC3I/II, Beclin and β-actin. Apoptosis was measured by Annexin V-APC-PI detection and Western blot of Bcl₂, Bax, caspase3 and β-actin. Dopamine levels were measured by Dopamine ELISA kit and Western blot of tyrosine hydroxylase. Our results suggest that 6-OHDA-induced PMN cell death occurred due to the interruption of [Ca²⁺]_i homeostasis, accompanied by activation of autophagy and apoptosis. A_2A R antagonists prevented 6-OHDA-induced neuronal cell death by decreasing [Ca²⁺]_i overload and oxidative stress. In addition, we found that A_2A R antagonists upregulated mTOR phosphorylation and downregulated AMPK phosphorylation thereby reducing autophagy and apoptosis both in 6-OHDA induced PMN cells and 6-OHDA unilateral lesioned rat model. In conclusion, A_2A R antagonists alleviated 6-OHDA toxicity by modulating [Ca²⁺]_i signalling to inhibit autophagy mediated by the AMPK/mTOR pathway.