Endoplasmic reticulum stress and mitochondrial cell death pathways mediate A53T mutant alpha-synuclein-induced toxicity

TLDR

Parkinson’s disease is a neurodegenerative movement disorder marked by selective loss of dopaminergic neurons and Lewy bodies, with alpha‑synuclein as a major component whose mutations cause autosomal‑dominant hereditary PD. The study aims to elucidate alpha‑synuclein cellular toxicity mechanisms in Parkinson’s disease and to establish a cell model for screening therapeutic agents. The authors generated A53T mutant alpha‑synuclein‑inducible PC12 cell lines via a Tet‑off system. Induction of A53T alpha‑synuclein in differentiated PC12 cells reduced proteasome activity, raised ROS, and caused ~40% cell death accompanied by mitochondrial cytochrome‑C release and caspase‑9/3 activation; this death was partially mitigated by cyclosporine A, z‑VAD, and caspase‑9/3 inhibitors but not caspase‑8 inhibition, while ER stress and caspase‑12 activation also contributed, as knockdown of caspase‑12 or salubrinal partially rescued cells and further reduced toxicity when combined with z‑VAD.

Abstract

Parkinson's disease (PD) is a neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. Alpha-synuclein is a major component of Lewy bodies in sporadic PD, and mutations in alpha-synuclein cause autosomal-dominant hereditary PD. Here, we generated A53T mutant alpha-synuclein-inducible PC12 cell lines using the Tet-off regulatory system. Inducing expression of A53T alpha-synuclein in differentiated PC12 cells decreased proteasome activity, increased the intracellular ROS level and caused up to ∼40% cell death, which was accompanied by mitochondrial cytochrome C release and elevation of caspase-9 and -3 activities. Cell death was partially blocked by cyclosporine A [an inhibitor of the mitochondrial permeability transition (MPT) process], z-VAD (a pan-caspase inhibitor) and inhibitors of caspase-9 and -3 but not by a caspase-8 inhibitor. Furthermore, induction of A53T alpha-synuclein increased endoplasmic reticulum (ER) stress and elevated caspase-12 activity. RNA interference to knock down caspase-12 levels or salubrinal (an ER stress inhibitor) partially protected against cell death and further reduced A53T toxicity after treatment with z-VAD. Our results indicate that both ER stress and mitochondrial dysfunction contribute to A53T alpha-synuclein-induced cell death. This study sheds light into the pathogenesis of alpha-synuclein cellular toxicity in PD and provides a cell model for screening PD therapeutic agents.