Myosin IIA-related Actomyosin Contractility Mediates Oxidative Stress-induced Neuronal Apoptosis

Abstract

Oxidative stress-induced neuronal apoptosis plays an important role in the progression of central nervous system (CNS) diseases. In our study, when neuronal cells were exposed to hydrogen peroxide (H2O2), an exogenous oxidant, cell apoptosis was observed with typical morphological changes including membrane blebbing, neurite retraction and cell contraction. The actomyosin system is considered to be responsible for the morphological changes, but how exactly it regulates oxidative stress-induced neuronal apoptosis and the distinctive functions of different myosin II isoforms remain unclear. We demonstrate that myosin IIA was required for neuronal contraction, while myosin IIB was required for neuronal outgrowth in normal conditions. During H2O2-induced neuronal apoptosis, myosin IIA, rather than IIB, interacted with actin filaments to generate contractile forces that lead to morphological changes. Moreover, myosin IIA knockout using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) reduced H2O2-induced neuronal apoptosis and the associated morphological changes. We further demonstrate that caspase-3/Rho-associated kinase 1 (ROCK1) dependent phosphorylation of myosin light chain (MLC) was required for the formation of the myosin IIA-actin complex. Meanwhile, either inhibition of myosin II ATPase with blebbistatin or knockdown of myosin IIA with siRNA reversely attenuated caspase-3 activation, suggesting a positive feedback loop during oxidative stress-induced apoptosis. Based on our observation, myosin IIA-actin complex contributes to actomyosin contractility and is associated with the positive feedback loop of caspase-3/ROCK1/MLC pathway. This study unravels the biochemical and mechanistic mechanisms during oxidative stress-induced neuronal apoptosis and may be applicable for the development of therapies for CNS diseases.

<p>NMMHC IIA Inhibition Ameliorates Cerebral Ischemic/Reperfusion-Induced Neuronal Apoptosis Through Caspase-3/ROCK1/MLC Pathway</p>

“…32 Additionally, NMMHC IIA also has been identified to participate in BBB dysfunction in ischemia stroke, and the NMMHC IIA-actin interaction mediates H 2 O 2 -induced neuronal apoptosis. 8,12 NMMHC IIA is also an attractive therapeutic target in ischemic stroke. Zhao et al found that TRPM7 kinase modulates OGD/R-induced neuronal apoptosis via annexin 1 carried by NMMHC IIA, blocking NMMHC IIA function by its antagonist blebbistatin was found to improve learning and memory in rats after MCAO and could also improve cell viability after OGD/R.…”

“…38,39 Our previous studies have confirmed that morphological changes in apoptotic cells are dependent on actomyosin cytoskeleton remodeling in H 2 O 2 -treated neurons. 12 When neurons are stimulated by H 2 O 2 , the interaction between NMMHC IIA and F-actin increased, which provided a basis for generating contractile forces, and ultimately leading to membrane blebbing and neuronal apoptosis. However, little is known about the specific function of NMMHC IIA in neuronal apoptosis under ischemic attack.…”

“…It has been reported that NMMHC IIA-actin interaction is required to initiate actomyosin contractility, which further facilitates cellular apoptosis. 12 We next investigated if AAV-shMyh9 attenuated NMMHC IIA-actin interaction induced by I/R. According to an immunofluorescence assay, we identified an increased interaction between NMMHC IIA and F-actin under ischemic attack when compared with that of the sham group.…”

“…9,10 We have previously illustrated the caspase-3/ROCK1/MLC feedback loop in hydrogen peroxide (H 2 O 2 )-induced PC12 cell apoptosis, and the non-muscle myosin heavy chain IIA (NMMHC IIA)-actin interaction regulates caspase-3/ROCK1/MLC signal cascade by a feedback mechanism upon oxidative stress-induced neuronal apoptosis. 11,12 NMMHC IIA, a member of the cytoskeleton, which is encoded by myosin heavy 9 (Myh9) gene and interacts with actin filaments to form a contractile unit. [13][14][15] Additionally, NMMHC IIA has been identified to participate in blood-brain barrier (BBB) dysfunction in ischemia stroke, 16 and the NMMHC IIA-actin interaction mediates oxidative stress-induced neuronal apoptosis.…”

“…[13][14][15] Additionally, NMMHC IIA has been identified to participate in blood-brain barrier (BBB) dysfunction in ischemia stroke, 16 and the NMMHC IIA-actin interaction mediates oxidative stress-induced neuronal apoptosis. 12 However, the correlation between NMMHC IIA and the caspase-3/ROCK1/MLC positive feedback loop in ischemic stroke remains unknown. Moreover, it is also not clear whether breaking the positive feedback loop by interfering with NMMHC IIA can improve cerebral ischemic injury.…”