NMMHC IIA triggers neuronal autophagic cell death by promoting F-actin-dependent ATG9A trafficking in cerebral ischemia/reperfusion.

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.

The Traditional Chinese Medicine Compound, GRS, Alleviates Blood-Brain Barrier Dysfunction.

INTRODUCTION: Traditional Chinese medicine (TCM) provides unique advantages for treatment of ischemic stroke, an aging-related vascular disease. Shengmai powder (GRS) is composed of three active components, specifically, ginsenoside Rb1, ruscogenin and schisandrin A, at a ratio of 6:0.75:6. The main objective of this study was to evaluate the effects of GRS on blood-brain barrier (BBB) dysfunction under conditions of middle cerebral artery occlusion/reperfusion (MCAO/R). METHODS: C57BL/6J mice subjected to MCAO/R were used as a model to assess the protective effects of varying doses of GRS (6.4, 12.8, and 19.2 mg/kg) on BBB dysfunction. RESULTS: GRS reduced cerebral infarct volume and degree of brain tissue damage, improved behavioral scores, decreased water content and BBB permeability, and restored cerebral blood flow. Moreover, GRS promoted expression of zona occludens-1 (ZO-1) and claudin-5 while inhibiting matrix metalloproteinase 2/9 (MMP-2/9) expression and myosin light chain (MLC) phosphorylation. In vitro, GRS (1, 10, and 100 ng/mL) enhanced the viability of bEnd.3 cells subjected to oxygen glucose deprivation/reoxygenation (OGD/R) and decreased sodium fluorescein permeability. CONCLUSION: Consistent with in vivo findings, ZO-1 and claudin-5 were significantly upregulated by GRS in bEnd.3 cells under OGD/R and MMP-2/9 levels and MLC phosphorylation reduced through the Rho-associated coil-forming protein kinase (ROCK)/cofilin signaling pathway. Based on the collective findings, we propose that the TCM compound, GRS, plays a protective role against I/R-induced BBB dysfunction.