Caspase-12 is Expressed in Purkinje Neurons and Prevents Psychiatric-Like Behavior in Mice.

Caspase-12 is a caspase family member for which functions in regulating cell death and inflammation have previously been suggested. In this study, we used caspase-12 lacZ reporter mice to elucidate the expression pattern of caspase-12 in order to obtain an idea about its possible in vivo function. Strikingly, these reporter mice showed that caspase-12 is expressed explicitly in Purkinje neurons of the cerebellum. As this observation suggested a function for caspase-12 in Purkinje neurons, we analyzed the brain and behavior of caspase-12 deficient mice in detail. Extensive histological analyses showed that caspase-12 was not crucial for establishing cerebellum structure or for maintaining Purkinje cell numbers. We then performed behavioral tests to investigate whether caspase-12 deficiency affects memory, motor, and psychiatric functions in mice. Interestingly, while the absence of caspase-12 did not affect memory and motor function, caspase-12 deficient mice showed depression and hyperactivity tendencies, together resembling manic behavior. Next, suggesting a possible molecular mechanistic explanation, we showed that caspase-12 deficient cerebella harbored diminished signaling through the brain-derived neurotrophic factor/tyrosine kinase receptor B/cyclic-AMP response binding protein axis, as well as strongly enhanced expression of the neuronal activity marker c-Fos. Thus, our study establishes caspase-12 expression in mouse Purkinje neurons and opens novel avenues of research to investigate the role of caspase-12 in regulating psychiatric behavior.

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.

Lepidium meyenii Walp (Maca)-derived extracellular vesicles ameliorate depression by promoting 5-HT synthesis via the modulation of gut-brain axis.

Depression is a common and debilitating condition for which effective treatments are needed. Lepidium meyenii Walp (Maca) is a plant with potential medicinal effects in treating depression. Recently, there has been growing interest in plant-derived extracellular vesicles (EVs) due to their low toxicity and ability to transport to human cells. Targeting the gut-brain axis, a novel strategy for depression management, may be achieved through the use of Maca-derived EVs (Maca-EVs). In this study, we successfully isolated Maca-EVs using gradient ultracentrifugation and characterized their shape, size, and markers (CD63 and TSG101). The in vivo imaging showed that the Dil-labeled Maca-EVs crossed the brain-blood barrier and accumulated in the brain. The behavioral tests revealed that Maca-EVs dramatically recovered the depression-like behaviors of unpredictable chronic mild stress (UCMS) mice. UCMS mice fecal were characterized by an elevated abundance of g_Enterococcus, g_Lactobacillus, and g_Escherichia_Shigella, which were significantly restored by administration of Maca-EVs. The effects of Maca-EVs on the altered microbial and fecal metabolites in UCMS mice were mapped to biotin, pyrimidine, and amino acid (tyrosine, alanine, aspartate, and glutamate) metabolisms, which were closely associated with the serotonin (5-HT) production. Maca-EVs were able to increase serum monoamine neurotransmitter levels in UCMS mice, with 5-HT showing the most significant changes. We further demonstrated that 5-HT improved the expression of brain-derived neurotrophic factor, a key regulator of neuronal plasticity, and its subsequent activation of TrkB/p-AKT signaling by regulating the GTP-Cdc42/ERK pathway. These findings suggest that Maca-EVs enhance 5-HT release, possibly by modulating the gut-brain axis, to improve depression behavior. Our study sheds light on a novel approach to depression treatment using plant-derived EVs.

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.

Promotion of Momordica Charantia polysaccharides on neural stem cell proliferation by increasing SIRT1 activity after cerebral ischemia/reperfusion in rats.

The deacetylase SIRT1 has been reported to play a critical role in regulating neurogenesis, which may be an adaptive processes contributing to recovery after stroke. Our previous work showed that the antioxidant capacity of Momordica charantia polysaccharides (MCPs) could protect against cerebral ischemia/reperfusion (I/R) after stroke. However, whether the protective effect of MCPs on I/R injury is related to neural stem cell (NSC) proliferation remains unclear. In the present study, we designed invivo and invitro experiments to elucidate the underlying mechanisms by which MCPs promote endogenous NSC proliferation during cerebral I/R. Invivo results showed that MCPs rescued the memory and learning abilities of rats after I/R damage and enhanced NSC proliferation in the rat subventricular zone (SVZ) and subgrannular zone (SGZ) during I/R. Invitro experiments demonstrated that MCPs could stimulate the proliferation of C17.2 cells under oxygen-glucose deprivation (OGD) conditions. Further studies revealed that the proliferation-promoting mechanism of MCPs relied on increasing the activity of SIRT1, decreasing the level of acetylation of ß-catenin in the cytoplasm, and then triggering the translocation of ß-catenin into the nucleus. These data provide experimental evidence that the up-regulation of SIRT1 activity by MCPs led to an increased cytoplasmic deacetylation of ß-catenin, which promoted translocation of ß-catenin to the nucleus to participate in the signaling pathway involved in NSC proliferation. The present study reveals that MCPs function as a therapeutic drug to promote stroke recovery by increasing the activity of SIRT1, decreasing the level of acetylated ß-catenin, promoting the nuclear translocation of ß-catenin and thereby increasing endogenous NSC proliferation.

Momordica charantia polysaccharides modulate the differentiation of neural stem cells via SIRT1/Β-catenin axis in cerebral ischemia/reperfusion.

BACKGROUND: Stroke is the leading cause of long-term motor disability and cognitive impairment. Recently, neurogenesis has become an attractive strategy for the chronic recovery of stroke. It is important to understand the molecular mechanism that promotes neural stem cell (NSC) neurogenesis for future NSC-based therapies. Our previous study showed that Momordica charantia polysaccharides (MCPs) exerted neuroprotective effects on stroke via their anti-oxidant and anti-inflammation activities. However, it remains unknown whether MCPs promote NSC neurogenesis after cerebral ischemic/reperfusion injury (IRI). METHODS: We investigated MCPs' function in differentiation of neural stem cells (NSCs) in vivo and in vitro experiments. Based on a middle cerebral artery occlusion (MCAO) rat model, the effect of MCPs on neuronal differentiation after MCAO was analyzed. Primary NSCs and neural stem cell line C17.2 were cultured and subjected to glutamate stimulation to establish the cell model of IRI. We evaluated the effect of MCPs on NSC differentiation in IRI cell model by Western blot and immunofluorescence staining. The SIRT1 activity of NSCs post glutamate stimulation was also evaluated by CELL SIRT1 COLORIMETRY ASSAY KIT. In addition, molecular mechanism was clarified by employing the activator and inhibitor of SIRT1. RESULTS: MCPs had no effects on the differentiation of neural stem cells under physiological conditions while shifted NSC differentiation potential from the gliogenic to neurogenic lineage under pathological conditions. Activation of SIRT1 with MCPs was responsible for the neuronal differentiation of C17.2-NSCs. The neuronal differentiation effect of MCPs was attributed to upregulation SIRT1-mediated deacetylation of ß-catenin. MCP-induced deacetylation via SIRT1 promoted nuclear accumulation of ß-catenin in NSCs. CONCLUSION: Our findings indicate that the deacetylation of ß-catenin by SIRT1 represents a critical mechanism of action of MCPs in promoting NSC neuronal differentiation. It provides an improved understanding of molecular mechanism underlying neuroprotective effects of MCPs in IRI, indicating its potential role on treating ischemic stroke especially chronic recovery.

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.

Dopamine D2 receptor restricts astrocytic NLRP3 inflammasome activation via enhancing the interaction of ß-arrestin2 and NLRP3.

Astrocytes are involved in the neuroinflammation of neurodegenerative diseases, such as Parkinson's disease (PD). Among the numerous inflammatory cytokines, interleukin-1ß (IL-1ß) produced by astrocytic Nod-like receptor protein (NLRP) inflammasome is crucial in the pathogenesis of PD. ß-arrestin2-mediated dopamine D2 receptor (Drd2) signal transduction has been regarded as a potential anti-inflammatory target. Our previous study revealed that astrocytic Drd2 suppresses neuroinflammation in the central nervous system. However, the role of Drd2 in astrocytic NLRP3 inflammasome activation and subsequent IL-1ß production remains unclear. In the present study, we used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD mouse model to investigate whether Drd2 could suppress astrocytic NLRP3 inflammasome activation. We showed that Drd2 agonist inhibited NLRP3 inflammasome activation, evidenced by decreased caspase-1 expression and reduced IL-1ß release in the midbrain of wild type mice. The anti-inflammasome effect of Drd2 was abolished in ß-arrestin2 knockout and ß-arrestin2 small interfering RNA-injected mice, suggesting a critical role of ß-arrestin2 in Drd2-regulated NLRP3 inflammasome activation. We also found that Drd2 agonists suppressed the upregulation of caspase-1 and IL-1ß expression in primary cultured mouse astrocytes in response to the activation of NLRP3 inflammasome induced by lipopolysaccharide plus adenosine triphosphate. Furthermore, we demonstrated that ß-arrestin2 mediated the inhibitory effect of Drd2 on NLRP3 inflammasome activation via interacting with NLRP3 and interfering the inflammasome assembly. Collectively, our study illustrates that astrocytic Drd2 inhibits NLRP3 inflammasome activation through a ß-arrestin2-dependent mechanism, and provides a new strategy for treatment of 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.