Caspase-1 deletion reveals pyroptosis participates in neural damage induced by cerebral ischemia/reperfusion in tMCAO model mice.

Pyroptosis, a form of programmed cell death, drives inflammation in the context of cerebral ischemia/reperfusion. The molecular mechanism of pyroptosis underlying ischemia/reperfusion, however, is not fully understood. The transient middle cerebral artery occlusion was applied to wild-type and caspase-1 knockout mice. 2,3,5-Triphenyltetrazolium chloride-staining and immunohistochemistry were used to identify the ischemic region, and western blot and immunofluorescence for the examination of neuronal pyroptosis. The expression of inflammatory factors and the behavioral function assessments were further conducted to examine the effects of caspase-1 knockout on protection against ischemia/reperfusion injury. Ischemia/reperfusion injury increased pyroptosis-related signals represented by the overexpression of pyroptosis-related proteins including caspase-1 and gasdermin D (GSDMD). Meanwhile, the number of GSDMD positive neurons increased in penumbra by immunofluorescence staining. Compared with wild-type mice, those with caspase-1 knockout exhibited decreased levels of pyroptosis-related proteins following ischemia/reperfusion. Furthermore, ischemia/reperfusion attack-induced brain infarction, cerebral edema, inflammatory factors, and neurological outcomes were partially improved in caspase-1 knockout mice. The data indicate that pyroptosis participates in ischemia/reperfusion induced-damage, and the caspase-1 might be involved, it provides some new insights into the molecular mechanism of ischemia.

Ischemic accumulation of succinate induces Cdc42 succinylation and inhibits neural stem cell proliferation after cerebral ischemia/reperfusion.

Ischemic accumulation of succinate causes cerebral damage by excess production of reactive oxygen species. However, it is unknown whether ischemic accumulation of succinate affects neural stem cell proliferation. In this study, we established a rat model of cerebral ischemia/reperfusion injury by occlusion of the middle cerebral artery. We found that succinate levels increased in serum and brain tissue (cortex and hippocampus) after ischemia/reperfusion injury. Oxygen-glucose deprivation and reoxygenation stimulated primary neural stem cells to produce abundant succinate. Succinate can be converted into diethyl succinate in cells. Exogenous diethyl succinate inhibited the proliferation of mouse-derived C17.2 neural stem cells and increased the infarct volume in the rat model of cerebral ischemia/reperfusion injury. Exogenous diethyl succinate also increased the succinylation of the Rho family GTPase Cdc42 but repressed Cdc42 GTPase activity in C17.2 cells. Increasing Cdc42 succinylation by knockdown of the desuccinylase Sirt5 also inhibited Cdc42 GTPase activity in C17.2 cells. Our findings suggest that ischemic accumulation of succinate decreases Cdc42 GTPase activity by induction of Cdc42 succinylation, which inhibits the proliferation of neural stem cells and aggravates cerebral ischemia/reperfusion injury.

Momordica charantia Exosome-Like Nanoparticles Exert Neuroprotective Effects Against Ischemic Brain Injury via Inhibiting Matrix Metalloproteinase 9 and Activating the AKT/GSK3ß Signaling Pathway.

Plant exosome-like nanoparticles (ELNs) have shown great potential in treating tumor and inflammatory diseases, but the neuroprotective effect of plant ELNs remains unknown. In the present study, we isolated and characterized novel ELNs from Momordica charantia (MC) and investigated their neuroprotective effects against cerebral ischemia-reperfusion injury. In the present study, MC-ELNs were isolated by ultracentrifugation and characterized. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) and MC-ELN injection intravenously. The integrity of the blood-brain barrier (BBB) was examined by Evans blue staining and with the expression of matrix metalloproteinase 9 (MMP-9), claudin-5, and ZO-1. Neuronal apoptosis was evaluated by TUNEL and the expression of apoptotic proteins including Bcl2, Bax, and cleaved caspase 3. The major discoveries include: 1) Dil-labeled MC-ELNs were identified in the infarct area; 2) MC-ELN treatment significantly ameliorated BBB disruption, decreased infarct sizes, and reduced neurological deficit scores; 3) MC-ELN treatment obviously downregulated the expression of MMP-9 and upregulated the expression of ZO-1 and claudin-5. Small RNA-sequencing revealed that MC-ELN-derived miRNA5266 reduced MMP-9 expression. Furthermore, MC-ELN treatment significantly upregulated the AKT/GSK3ß signaling pathway and attenuated neuronal apoptosis in HT22 cells. Taken together, these findings indicate that MC-ELNs attenuate ischemia-reperfusion-induced damage to the BBB and inhibit neuronal apoptosis probably via the upregulation of the AKT/GSK3ß signaling pathway.

Astrocytic Kir6.1 deletion aggravates neurodegeneration in the lipopolysaccharide-induced mouse model of Parkinson's disease via astrocyte-neuron cross talk through complement C3-C3R signaling.

Complement pathway over-activation has been implicated in a variety of neurological diseases. However, the signaling pathways governing astrocytic complement activation in Parkinson's disease (PD) are poorly understood. Kir6.1, a pore-forming subunit of ATP-sensitive potassium (K-ATP) channel, is prominently expressed in astrocytes and exhibits anti-inflammatory effects. Therefore, we hypothesize that Kir6.1/K-ATP channel may regulate astrocytic complement activation in the pathogenesis of PD. In this study, astrocytic Kir6.1 knockout (KO) mice were used to examine the effect of astrocytic Kir6.1/K-ATP channel on astrocytic complement activation triggered by the lipopolysaccharide (LPS). Here, we found that astrocytic Kir6.1 KO mice showed more dopaminergic neuron loss and more astrocyte reactivity in substantia nigra compacta than controls. We also found that astrocytic Kir6.1 KO increased the expression of complement C3 in astrocytes in LPS-induced mouse model of PD. Mechanistically, astrocytic Kir6.1 KO promoted astroglial NF-κB activation to elicit extracellular release of C3, which in turn interacted with neuronal C3aR to induce neuron death. Blocking complement function by NF-κB inhibitor or C3aR antagonist rescued the aggravated neuron death induced by Kir6.1 KO. Collectively, our findings reveal that astrocytic Kir6.1/K-ATP channel prevents neurodegeneration in PD via astrocyte-neuron cross talk through NF-κB/C3/C3aR signaling and suggest that targeting astroglial Kir6.1/K-ATP channel-NF-κB-C3-neuronal C3aR signaling represents a novel therapeutic strategy for PD.

Kir6.1/K-ATP channel on astrocytes protects against dopaminergic neurodegeneration in the MPTP mouse model of Parkinson's disease via promoting mitophagy.

ATP-sensitive potassium (K-ATP) channels, coupling cell metabolism to cell membrane potential, are involved in brain diseases, including Parkinson's disease (PD). Kir6.1, a pore-forming subunit of K-ATP channel, is prominently expressed in astrocytes and participates in regulating its function. However, the precise role of astrocytic Kir6.1-contaning K-ATP channel (Kir6.1/K-ATP) in PD is not well characterized. In this study, astrocytic Kir6.1 knockout (KO) mice were used to examine the effect of astrocytic Kir6.1/K-ATP channel on dopaminergic (DA) neurodegeneration triggered by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Here, we found that astrocytic Kir6.1 KO mice showed more DA neuron loss in substantia nigra compacta (SNc), lower level of dopamine in the striatum, and more severe motor dysfunction than controls. Interestingly, this companied by increased neuroinflammation and decreased autophagy level in SNc in vivo and astrocytes in vitro. Mechanistically, astrocytic Kir6.1 KO inhibited mitophagy which resulted in an increase in the accumulation of damaged mitochondria, production of reactive oxygen species and neuroinflammation in astrocytes. Restoration of astrocytic mitophagy rescued the deleterious effects of astrocytic Kir6.1 ablation on mitochondrial dysfunction, inflammation and DA neuron death. Collectively, our findings reveal that astrocytic Kir6.1/K-ATP channel protects against DA neurodegeneration in PD via promoting mitophagy and suggest that astrocytic Kir6.1/K-ATP channel may be a promising therapeutic target for PD.

Kir6.1/K-ATP channel modulates microglia phenotypes: implication in Parkinson's disease.

Classical activation (M1 phenotype) and alternative activation (M2 phenotype) are the two polars of microglial activation states that can produce either neurotoxic or neuroprotective effects in the immune pathogenesis of Parkinson's disease (PD). Exploiting the beneficial properties of microglia cells by modulating their polarization states provides great potential for the treatment of PD. However, the mechanism that regulates microglia polarization remains elusive. Here we demonstrated that Kir6.1-containing ATP-sensitive potassium (Kir6.1/K-ATP) channel switched microglia from the detrimental M1 phenotype toward the beneficial M2 phenotype. Kir6.1 knockdown inhibited M2 polarization and simultaneously exaggerated M1 microglial inflammatory responses, while Kir6.1 overexpression promoted M2 polarization and synchronously alleviated the toxic phase of M1 microglia polarization. Furthermore, we observed that the Kir6.1 deficiency dramatically exacerbated dopaminergic neuron death companied by microglia activation in mouse model of PD. Mechanistically, Kir6.1 deficiency enhanced the activation of p38 MAPK-NF-κB pathway and increased the ratio of M1/M2 markers in the substantia nigra compacta of mouse model of PD. Suppression of p38 MAPK in vivo partially rescued the deleterious effects of Kir6.1 ablation on microglia phenotype and dopaminergic neuron death. Collectively, our findings reveal that Kir6.1/K-ATP channel modulates microglia phenotypes transition via inhibition of p38 MAPK-NF-κB signaling pathway and Kir6.1/K-ATP channel may be a promising therapeutic target for PD.

[Construction of a baculovirus transfer vector and expression of baculovirus-mediated gfp gene in larvae of Spodoptera litura].

To construct a novel baculovirus expression system of Spodoptera litura multicapsid nucleopolyhedrovirus, the 5' end and 3' end-flanking fragments of ph gene were amplified from the genome DNA of SpltMNPV, Japan-C3 strain using two pairs of primers synthesized according to SpltMNPV China-G2 strain genome DNA sequence published in GenBank. To obtain the transfer vector pSplt-gfp, the fragment of gfp gene was inserted into this vector between two fragments tandem linked into pUC18. The spli cells were cotransfected with pSplt-gfp and the wild SpltMNPV genome DNA. The recombinant virus containing gfp was selected with the limited dilution method. The fluorescence can be observed in the spli cells and the 3rd instar larvae after 24 and 48 hours by infection of the recombinant virus, respectively. The result showed that the recombinant virus was obtained successfully. It will be helpful to establish Spodoptera litura multicapsid nucleopolyhedrovirus expression system and more effective pesticide for Spodoptera litura.