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Updated: Dec 29, 2025
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Published on: January 3, 2025
NMMHC IIA Inhibition Ameliorates Cerebral Ischemic/Reperfusion-Induced Neuronal Apoptosis Through Caspase-3/ROCK1/MLC
Guang-Yun Wang1, Tie-Zheng Wang1, Yuan-Yuan Zhang1
1State Key Laboratory of Natural Products, Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Pharmacology of Chinese Material Medica, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, People's Republic of China.
Purpose:
Our previous studies have indicated that non-muscle myosin heavy chain IIA (NMMHC IIA) is involved in H2O2-induced neuronal apoptosis, which is associated with the positive feedback loop of caspase-3/ROCK1/MLC pathway. However, the neuroprotective effect of NMMHC IIA inhibition with an adeno-associated virus (AAV) vector after transient middle cerebral artery occlusion (MCAO) and its role in caspases-3/ROCK1/MLC pathway remain blurred.
Methods:
Green fluorescent protein (GFP) and a small hairpin RNA targeting Myh9 (encoding NMMHC IIA) were cloned and packaged into the AAV9 vector. AAV-shMyh9 or control vector were injected into C57BL/6J mice four weeks prior to 60 min MCAO. Twenty-four hours after reperfusion, functional and histological analyses of the mice were performed.
Results:
In this study, AAV-shMyh9 was used to down-regulate NMMHC IIA expression in mice. We found that down-regulation of NMMHC IIA could improve neurological scores and histological injury in ischemic mice. Ischemic attack also activated neuronal apoptosis, and this effect was partially attenuated when NMMHC IIA was inhibited by AAV-shMyh9. In addition, AAV-shMyh9 significantly reduced cerebral ischemic/reperfusion (I/R)-induced NMMHC IIA-actin interaction, caspase-3 cleavage, Rho-associated kinase1 (ROCK1) activation and myosin light-chains (MLC) phosphorylation.
Conclusion:
Consequently, we showed that AAV-shMyh9 inhibits I/R-induced neuronal apoptosis linked with caspase-3/ROCK1/MLC/NMMHC IIA-actin cascade, which has also been confirmed to be a positive feedback loop. These findings put some insights into the neuroprotective effect of AAV-shMyh9 associated with the regulation of NMMHC IIA-related pathway under ischemic attack and provide a therapeutic strategy for ischemic stroke.
Insights
Inhibiting non-muscle myosin heavy chain IIA (NMMHC IIA) with AAV-shMyh9 reduces neuronal apoptosis and improves outcomes after ischemic stroke. This neuroprotection involves the caspase-3/ROCK1/MLC pathway, offering a potential therapeutic strategy.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Non-muscle myosin heavy chain IIA (NMMHC IIA) is implicated in H2O2-induced neuronal apoptosis via the caspase-3/ROCK1/MLC pathway.
- The neuroprotective role of NMMHC IIA inhibition post-ischemic stroke remains unclear.
Purpose of the Study:
- To investigate the neuroprotective effect of NMMHC IIA inhibition using an adeno-associated virus (AAV) vector after transient middle cerebral artery occlusion (MCAO).
- To elucidate the role of NMMHC IIA in the caspase-3/ROCK1/MLC pathway during ischemic stroke.
Main Methods:
- Adeno-associated virus serotype 9 (AAV9) vectors carrying GFP or shRNA targeting Myh9 (AAV-shMyh9) were constructed.
- AAV-shMyh9 or control vector was administered to C57BL/6J mice four weeks before 60-minute MCAO.
- Functional and histological analyses were performed 24 hours after reperfusion.
Main Results:
- Down-regulation of NMMHC IIA by AAV-shMyh9 improved neurological scores and reduced histological injury in ischemic mice.
- AAV-shMyh9 partially attenuated ischemia-induced neuronal apoptosis.
- NMMHC IIA inhibition reduced NMMHC IIA-actin interaction, caspase-3 cleavage, ROCK1 activation, and MLC phosphorylation.
Conclusions:
- AAV-shMyh9 inhibits ischemic/reperfusion-induced neuronal apoptosis by modulating the caspase-3/ROCK1/MLC/NMMHC IIA-actin cascade.
- This pathway forms a positive feedback loop, and its inhibition offers neuroprotection.
- NMMHC IIA inhibition presents a therapeutic strategy for ischemic stroke.
