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MFN2 and BAG6 Synergistically Protect Against Cerebral Reperfusion Injury by Regulating ROS Levels and Autophagic
Dongting Lu1, Yukun Yang2, Guodong Huang3
1Department of Neurology, Guangxi University of Chinese Medicine, Nanning, China (D.L., Y.-C.W.).
Background:
MFN2 (mitofusin-2), a transmembrane dynamin-like protein located on the outer mitochondrial membrane, plays a key role in regulating mitochondrial fusion and autophagy. 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. Reactive oxygen species (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.
Insights
Mitofusin-2 (MFN2) protects against stroke by reducing reactive oxygen species (ROS) and modulating autophagy through interaction with BAG6. This MFN2-BAG6 axis offers a promising therapeutic target for stroke treatment.
Area of Science:
- Neuroscience
- Cell Biology
- Mitochondrial Biology
Background:
- Mitofusin-2 (MFN2) is a mitochondrial outer membrane protein crucial for mitochondrial fusion and autophagy.
- In vitro studies suggest MFN2 possesses neuroprotective properties following ischemic events.
- This study investigates MFN2's role in neuronal ischemia/reperfusion injury in vivo and in vitro.
Purpose of the Study:
- To elucidate the role of MFN2 in neuronal ischemia/reperfusion injury.
- To investigate the mechanisms by which MFN2 exerts neuroprotection.
- To explore the interaction between MFN2 and its partner BAG6 in the context of stroke.
Main Methods:
- MFN2 was manipulated (knockdown/overexpression) in mice and neuronal cells subjected to ischemia/reperfusion models.
- Neurological deficits, infarct volume, apoptosis, necrosis, ROS levels, and autophagic flux were assessed.
- Protein interactions were analyzed using immunoprecipitation, mass spectrometry, and Western blotting.
Main Results:
- Neuron-specific MFN2 deletion worsened ischemia/reperfusion injury, while MFN2 overexpression improved outcomes.
- MFN2 deficiency increased mitochondrial ROS and inhibited autophagy; MFN2 overexpression decreased ROS and modulated autophagy.
- A direct interaction between MFN2 and BAG6 was identified, with BAG6 overexpression mimicking MFN2's protective effects.
Conclusions:
- MFN2 enhances stroke outcomes by reducing ROS and modulating autophagy via interaction with BAG6.
- BAG6 amplifies MFN2's ROS-lowering and cytoprotective actions.
- The MFN2-BAG6 axis presents a potential therapeutic strategy for stroke.
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