determination-mitochondrial-morphology-live-cells-using-confocal

Determination of Mitochondrial Morphology in Live Cells Using Confocal Microscopy

The dynamic balance of mitochondrial fusion and fission directly contributes to mitochondrial homeostasis, which influences numerous cellular functions in ...

Southern Medical University

Given the established role of nuclear receptor corepressor 1 (NCoR1) in sensing environmental cues and the importance of inflammation in neurodegenerative diseases, elucidation of NCoR1 involvement in neuroinflammation has notable implications. Yet, its regulatory mechanism remains largely unclear. Under in vitro conditions, NCoR1 expression peaked and then decreased at 12 h after lipopolysaccharides (LPS) stimulation in BV2 cells, However, NCoR1 knockdown using si-RNA attenuated microglial inflammation, evident by reduced the levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), phosphorylated-JNK and high mobility group box-1 (HMGB1). Furthermore, NCoR1 suppression could counteract the decline in mitochondrial membrane potential while simultaneously enhancing the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). Under in vivo conditions, microglia-specific NCoR1 knockout (MNKO) mice after LPS injections alleviated the symptoms of anhedonia, diminished autonomic activity and cognitive impairment. Additionally, MNKO mice showed attenuation of microglial activation, downregulated HMGB1 and COX2, and upregulated PGC-1α expression in the cortex. In conclusion, these findings suggest that NCoR1 deficiency leads to a modest reduction in neuroinflammation, possibly attributed to the increased expression of PGC-1α.

Microglia nuclear receptor corepressor 1 deficiency alleviates neuroinflammation in mice.

Dynamin-related protein 1 (Drp1) regulates mitochondrial fission and participates in neuronal apoptosis during the pathology of cerebral ischemia. We have previously shown that inhibition of phosphodiesterase-4 (PDE4) protects against neuronal apoptosis in models of ischemic stroke. However, it remains unclear whether PDE4 inhibition affects Drp1-mediated mitochondrial dysfunction and apoptosis under cerebral ischemia conditions. This study aimed to determine whether ZX21011, a novel PDE4 inhibitor synthesized in our laboratory, can act on Drp1 to counteract ischemic brain injury and to elucidate its mechanism of action. We demonstrated that ZX21011 effectively reduced neuronal apoptosis caused by oxygen-glucose deprivation/reoxygenation (OGD/R) in HT22 cells and ameliorated neurological deficits caused by middle cerebral artery occlusion/reperfusion (MCAO/R) in rats. ZX21011 enhanced glycogen synthase kinase-3β (GSK3β) phosphorylation (Ser9), GSK3β(S9A) mutation blocked the protective effects of ZX21011. Simultaneously, ZX21011 reduced the levels of reactive oxygen species (ROS), restored the morphology of mitochondria, and inhibited the phosphorylation of Drp1(Ser616). The Drp1(S616E) mutation blocked the protective effects of ZX21011 on ROS production and mitochondrial morphology function after cerebral ischemia. What's more, co-immunoprecipitation analysis revealed that ZX21011 decreased the binding of GSK3β to Drp1, and GSK3β(S9A) mutation reversed the effects of ZX21011 on Drp1 phosphorylation and cell viability. Moreover, ZX21011 decreased Drp1(Ser616) phosphorylation within the ischemic penumbra of rats following cerebral ischemia/reperfusion. In summary, ZX21011 counteracts ischemic stroke-induced oxidative stress and neuronal death, and its action is related to decreased Drp1 phosphorylation at Ser616. Thus, ZX21011 is a potential compound for the intervention of stroke.

A novel PDE4 inhibitor ZX21011 alleviates neuronal apoptosis by decreasing GSK3β-mediated Drp1 Ser616 phosphorylation in cerebral ischemia reperfusion.

Microglial activation and complement-mediated synaptic pruning are involved in depression development. We previously found that the inhibition of phosphodiesterase 4 (PDE4) inhibits microglial activation and increases synaptic plasticity. However, the role of PDE4 in microglia phagocytosis and complement-mediated synaptic pruning during depression remains unclear. Here, we investigated the effect of PDE4 on the expression of complement component 1q (C1q) and C3. We also designed and synthesized a novel PDE4 inhibitor LS21013A-06 (A06), and examined whether A06 exerts antidepressant-like effects by regulating microglia phagocytosis and complement-mediated synaptic pruning. We found that treatment with high-mobility group box-1 (HMGB1) triggered an inflammatory response, enhanced levels of complement component 1q (C1q) and C3, and promoted microglial phagocytosis both in vitro and in vivo. Notably, PDE4B knockdown reduced the levels of HMGB1, C1q, and C3 in lipopolysaccharide (LPS)-treated BV2 cells. Inhibition of PDE4 by A06 reduced the levels of HMGB1, suppressed neuroinflammation and microglial phagocytosis. In addition, A06 alleviated LPS-induced depressive-like behaviors in mice, reduced the levels of HMGB1, C1q, and C3 in the hippocampus, elevated the level of postsynaptic density protein-95, and reduced excessive microglial phagocytosis and engulfment of synapses. Moreover, C1q overexpression inhibited the effects of A06 on microglial activation and synaptic pruning. In conclusion, we demonstrated for the first time that PDE4 regulates the expression of C1q/C3, and A06 reduces microglial activation and ameliorates depressive-like behavior in mice. This mechanism involves complement C1q/C3-mediated excessive microglia phagocytosis and synaptic pruning.

Article	Year
Microglia nuclear receptor corepressor 1 deficiency alleviates neuroinflammation in mice. Neuroscience letters\| PubMed ID: 38242347	2024
A novel PDE4 inhibitor ZX21011 alleviates neuronal apoptosis by decreasing GSK3β-mediated Drp1 Ser616 phosphorylation in cerebral ischemia reperfusion. Chemico-biological interactions\| PubMed ID: 39889870	2025
PDE4 inhibition alleviates HMGB1/C1q/C3-mediated excessive phagocytic pruning of synapses by microglia and depressive-like behaviors in mice. Brain, behavior, and immunity\| PubMed ID: 39947489	2025

Zihong Xian

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