Nogo-B deletion mitigates mitochondrial dynamics imbalance in cerebral ischemia/reperfusion injury via the TLR4/ERK pathway.

BACKGROUND

Our previous studies have demonstrated that the downregulation of Nogo-B expression in microglia suppresses neuroinflammatory responses by modulating microglial polarization, thereby attenuating cerebral ischemia/reperfusion (I/R) injury. However, the direct role of Nogo-B in neuronal cells remains unclear.

METHODS

Middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen-glucose deprivation and reoxygenation (ODG/R) models were utilized to mimic ischemic stroke. Various methods, including Nogo-B shRNA transfection, balance beam and corner turn tests, immunofluorescence staining, electron microscopy, Western blot, and TUNEL, were used to investigate the effects of Nogo-B deletion on mitochondrial dynamics imbalance in neurons following cerebral I/R injury, as well as its underlying mechanisms.

RESULTS

Neuron-specific conditional knockout (cko) of Nogo-B significantly reduced cerebral infarction volume and neurological deficit scores in mice subjected to cerebral I/R. Nogo-B deletion enhanced neuronal cell viability, suppressed apoptosis, improved mitochondrial respiratory chain activity, increased mitochondrial membrane potential and ATP production, and lowered ROS levels and oxidative stress markers. Additionally, it decreased the expression of fission-related proteins (p-Drp1, Fis1) and mitochondrial fragmentation while increasing the expression of fusion-related proteins (Opa1, Mfn1, and Mfn2). Mechanistic studies show that Nogo-B knockout can significantly reduce the expression of TLR4 and p-ERK1/2 proteins. Nogo-B downregulation also resulted in a significant reduction in TLR4 and p-ERK1/2, a marked decrease in mitochondrial fission proteins, a substantial increase in fusion proteins, and concomitant improvement in mitochondrial structure, function, and cell viability. Nogo-B downregulation produced effects comparable to TAK-242. However, these protective effects were substantially attenuated by the ERK activator TPA.

CONCLUSION

Nogo-B deletion has a direct protective effect on neurons after cerebral ischemia-reperfusion injury. The mechanism may be related to Nogo-B reducing the expression of TLR4 on the cell membrane through some indirect action, thereby inhibiting the TLR4/ERK pathway and regulating mitochondrial dynamic imbalance.