Abstract
Previous findings have shown that non-muscle myosin heavy-chain IIA (NMMHC IIA) is involved in autophagy induction triggered by starvation in D. melanogaster; however, its functional contribution to neuronal autophagy remains unclear. The aim of this study is to explore the function of NMMHC IIA in cerebral ischemia-induced neuronal autophagy and the underlying mechanism related to autophagy-related gene 9A (ATG9A) trafficking. Functional assays and molecular mechanism studies were used to investigate the role of NMMHC IIA in cerebral ischemia-induced neuronal autophagy in vivo and in vitro. A middle cerebral artery occlusion (MCAO) model in mice was used to evaluate the therapeutic effect of blebbistatin, a myosin II ATPase inhibitor. Herein, either depletion or knockdown of NMMHC IIA led to increased cell viability in both primary cultured cortical neurons and pheochromocytoma (PC12) cells exposed to oxygen–glucose deprivation/reoxygenation (OGD/R). In addition, NMMHC IIA and autophagic marker LC3B were upregulated by OGD/R, and inhibition of NMMHC IIA significantly reduced OGD-induced neuronal autophagy. Furthermore, NMMHC IIA-induced autophagy is through its interactions with F-actin and ATG9A in response to OGD/R. The NMMHC IIA–actin interaction contributes to ATG9A trafficking and autophagosome formation. Inhibition of the NMMHC IIA–actin interaction using blebbistatin and the F-actin polymerization inhibitor cytochalasin D significantly suppressed ATG9A trafficking and autophagy induction. Furthermore, blebbistatin significantly improved neurological deficits and infarct volume after ischemic attack in mice, accompanied by ATG9A trafficking and autophagy inhibition. These findings demonstrate neuroprotective effects of NMMHC IIA inhibition on regulating ATG9A trafficking-dependent autophagy activation in the context of cerebral ischemia/reperfusion.
Highlights
Ischemic stroke is one of the primary causes of disability and death worldwide[1]
Our study demonstrate that NMMHC IIA inhibition is neuroprotective in cerebral ischemia/reperfusion injury through the coordination of neuronal autophagic cell death
We revealed that NMMHC IIA induces excessive autophagy via interactions with F-actin and autophagy-related gene 9A (ATG9A) during oxygen–glucose deprivation/reoxygenation (OGD/R)
Summary
Ischemic stroke is one of the primary causes of disability and death worldwide[1]. many devastating cascades[2,3,4] have been shown to be associated with ischemicOfficial journal of the Cell Death Differentiation AssociationWang et al Cell Death and Disease (2020)11:4283-methyladenine (3-MA) into the lateral ventricle after cerebral ischemia significantly decreased both neuronal autophagy and the lesion volume[11]. Exploring relevant targets to inhibit neuronal autophagic cell death could potentially aid in the prevention or the treatment of ischemic stroke. ATG9, an autophagy-related gene, is required for transportation of membranous components and autophagosome formation. A pool of Atg[11] mediates the anterograde transport of ATG9 to the preautophagosomal structure (PAS) that is dependent on the actin cytoskeleton during yeast vegetative growth[15]. These results demonstrate that actin filaments are essential for autophagy and the movement of Atg[9] between the peripheral sites and the PAS16. UNC51-like kinase 1 (ULK1/Atg1) promotes phosphorylation-dependent activation of NMMHC II to regulate ATG9A trafficking from the trans-Golgi network (TGN) to PAS, facilitating autophagosome formation[17]
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