MFN2 and BAG6 overexpression protected experimental stroke models by lowering , stabilizing mitochondria, and modulating .
Evidence
This preclinical in vivo and in vitro study used C57BL/6 mice with middle cerebral artery occlusion and SY5Y cells under oxygen-glucose deprivation/reoxygenation with MFN2 and BAG6 gain- and loss-of-function experiments.
Caveat
The evidence is limited to mouse and cell models of ischemia/reperfusion injury, so therapeutic relevance in human stroke remains untested.
Simplified
BACKGROUND: MFN2 (mitofusin-2), a transmembrane dynamin-like protein located on the outer mitochondrial membrane, plays a key role in regulating mitochondrial fusion and . In vitro studies suggested that MFN2 may exert neuroprotective effects postischemia. In gain-of-function and loss-of-function experiments, we investigated MFN2'·s roles in regulating neuronal ischemia/reperfusion injury in vivo and in vitro.
METHODS: MFN2 was knocked down by neuron-specific conditional knockout or siRNA-mediated knockdown and overexpressed by adeno-associated viral vectors or plasmid vectors in C57BL/6 mice of both sexes (10-12 weeks) exposed to middle cerebral artery occlusion and SY5Y cells exposed to oxygen-glucose deprivation/reoxygenation. Neurological deficits were examined using a 48-point score and rotarod tests. Infarct volume was assessed by 2,3,5-triphenyltetrazolium chloride staining. A RealTime Apoptosis and Necrosis Assay was used to measure apoptotic and necrotic cell death. (ROS) formation and autophagic flux were analyzed by functional assays. Protein expression and interaction were evaluated using Western blots, immunoaffinity chromatography, mass spectrometry, and immunoprecipitation analysis. To assess the role of MFN2's interaction partner BAG6, BAG6 was overexpressed in middle cerebral artery occlusion mice and overexpressed or knocked down in SY5Y cells.
RESULTS: Neuron-specific MFN2 deletion exacerbated cerebral ischemia/reperfusion injury, while MFN2 overexpression reduced it. MFN2 deficiency elevated mitochondrial ROS levels and inhibited autophagy, whereas MFN2 overexpression decreased ROS levels. In immunoprecipitation studies, we found a direct interaction between MFN2 and BAG6. Of note, BAG6 overexpression mimicked the effect of MFN2 overexpression on cerebral ischemia/reperfusion injury. Combined MFN2 and BAG6 overexpression synergistically reduced ischemia/reperfusion injury by drastically decreasing cerebral ROS levels, stabilizing mitochondrial function, and modulating autophagy.
CONCLUSIONS: Our study suggests that MFN2 enhances stroke outcome through 2 pathways: by decreasing ROS levels and modulating autophagy via interaction with BAG6. BAG6 potentiates the ROS-lowering, cytoprotective MFN2 actions. The MFN2-BAG6 axis represents a promising target for stroke therapy.
Key numbers
n=7
Increase in
measured in mice vs. control mice post-.
n=3
Decrease in Levels
levels significantly reduced in treated mice compared to controls post-.
n=6
Combined Overexpression Effect
Co-overexpression of and resulted in smaller infarct volumes compared to alone.
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