Fisetin alleviates cerebral ischemia/reperfusion injury by regulating Sirt1/Foxc1/Ubqln1 pathway-mediated proteostasis.

BACKGROUND: Cerebral ischemia/reperfusion injury (IRI) is pathologically associated with protein damage. The flavonoid fisetin has good therapeutic effects on cerebral IRI. However, the role of fisetin in regulating protein damage during cerebral IRI development remains unclear. This study investigated the pharmacological effects of fisetin on protein damage during cerebral IRI progression and defined the underlying mechanism of action. METHODS: In vivo and in vitro models of cerebral IRI were established by middle cerebral artery occlusion/reperfusion (MACO/R) and oxygen-glucose deprivation/reperfusion (OGD/R) treatment, respectively. Triphenyl tetrazolium chloride staining was performed to detect cerebral infarct size, and the modified neurologic severity score was used to examine neurological deficits. LDH activity and protein damage were assessed using kits. HT22 cell vitality and apoptosis were examined using CCK-8 assay and TUNEL staining, respectively. Interactions between Foxc1, Ubqln1, Sirt1, and Ezh2 were analyzed using CoIP, ChIP and/or dual-luciferase reporter gene assays. RESULTS: Fisetin alleviated protein damage and ubiquitinated protein aggregation and neuronal death caused by MCAO/R and OGD/R. Ubqln1 knockdown abrogated the inhibitory effect of fisetin on OGD/R-induced protein damage, ubiquitinated protein aggregation, and neuronal death in HT22 cells. Further experiments demonstrated that Foxc1 functions as a transcriptional activator of Ubqln1 and that Sirt1 promotes Foxc1 expression by deacetylating Ezh2 and inhibiting its activity. Furthermore, Sirt1 knockdown abrogated fisetin-mediated biological effects on OGD/R-treated HT22 cells. CONCLUSION: Fisetin improved proteostasis during cerebral IRI by regulating the Sirt1/Foxc1/Ubqln1 signaling axis. Our findings strongly suggest that fisetin-mediated inhibition of protein damage after ischemic stroke is a part of the mechanism through which fisetin is neuroprotective in cerebral IRI.

Autophagy promotes membrane trafficking of NR2B to alleviate depression by inhibiting AQP4 expression in mice.

Depression is a high-incidence mental illness that seriously affects human health. AQP4 has been reported to be closely associated with depression, while the underlying mechanism is still unclear. This work aimed to investigate the functional role of AQP4 in depression. Depression mouse model was constructed by administration of chronic social defeat stress (CSDS). We found that AQP4 was highly expressed in the hippocampal tissues of CSDS mice. AQP4 knockdown alleviated depression and enhanced the expression of NR2B and PSD95 in CSDS mice. Moreover, primary hippocampal neurons were treated with N-methyl-d-aspartate (NMDA) to induce neuron injury. AQP4 overexpression repressed cell viability and promoted apoptosis of NMDA-treated primary hippocampal neurons. AQP4 up-regulation repressed the expression of NR2B (surface), and enhanced the expression of NR2B (intracellular), P-NR2B, CaMK II and CK2 in the NMDA-treated primary hippocampal neurons. The influence conferred by AQP4 up-regulation was abolished by KN-93 (CaMK II inhibitor) or TBB (CK2 inhibitor) treatment. Rapamycin treatment enhanced the expression of NR2B (surface), and repressed the expression of AQP4, NR2B (intracellular) and P-NR2B in the primary hippocampal neurons by activating autophagy. The activated autophagy alleviated depression in CSDS mice by repressing AQP4 expression. In conclusion, our data demonstrated that autophagy ameliorated depression by repressing AQP4 expression in mice, and AQP4 knockdown promoted membrane trafficking of NR2B and inhibited phosphorylation of NR2B via CaMK II/CK2 pathway. Thus, our work suggests that AQP4 may be a promising molecular target for the development of antidepressant drugs.

Safinamide protects against amyloid ß (Aß)-induced oxidative stress and cellular senescence in M17 neuronal cells.

Alzheimer's disease (AD) is a neurodegenerative disorder that is pathologically related to oxidative stress and cellular senescence. Safinamide is one of the clinically prescribed monoamine oxidase B (MAOB) inhibitors. It has been reported to possess therapeutic potential in neurological disorders. However, the therapeutic potential of safinamide in AD is still under investigation. In this study, we explored the effect of safinamide in amyloid (Aß)1-42 oligomers-stimulated M17 neuronal cells. We established the in vitro model with M17 cells by treating them with 1 µM Aß1-42 oligomers with or without safinamide (100 or 200 nM). The results show that safinamide ameliorated Aß1-42 oligomers-induced oxidative stress in M17 cells as revealed by the decreased reactive oxygen species (ROS) production and reduced glutathione (GSH) content. Safinamide treatment significantly ameliorated senescence-associated-ß-galactosidase (SA-ß-gal)-positive cells and telomerase activity. Further, we show that safinamide treatment resulted in decreased mRNA and protein expressions of p21 and plasminogen activator inhibitor-1 (PAI-1). Moreover, silencing of Sirtuin1 (SIRT1) abolished the effects of safinamide on the mRNA levels of p21 and PAI-1, as well as SA-ß-gal-positive cells in Aß1-42 oligomers-induced M17 cells. In conclusion, we reveal that safinamide exerted a protective function on M17 cells from Aß1-42 oligomers induction-caused oxidative stress and cellular senescence through SIRT1 signaling. These present results provide meaningful evidence that safinamide may be medically developed for the prevention and therapy of AD.

IKKß Alleviates Neuron Injury in Alzheimer's Disease via Regulating Autophagy and RIPK1-Mediated Necroptosis.

Alzheimer's disease (AD), featured with memory loss and multiple cognitive impairments, is a devastating neurodegenerative disease that affects millions of people in the world, especially the elder people. IKKß plays important role in the development of neurodegenerative diseases. However, the molecular mechanism of IKKß, especially related with autophagy and necroptosis, in AD, is still unclear. Here, we studied the function of IKKß in regulating autophagy and RIPK1-induced necroptosis in SH-SY5Y cells and APP/PS1 mice. By silencing IKKß in the SH-SY5Y cells, we found that inhibition of IKKß could promote the RIPK1-induced necroptosis caused by Aß accumulation as well as suppress the autophagy of SH-SY5Y cells. Furthermore, we discovered that autophagy was significantly enhanced, and RIPK1-induced necroptosis was inhibited when IKKß was constitutively activated in SH-SY5Y cells. Then, using APP/PS1 mouse model, we demonstrated that silencing IKKß could significantly enhance the accumulation of Aß but have not impact on the mice behavior and cognitive ability. Even the controversial results about the role of IKKß in AD is not fully understood, our results might provide an important potential therapeutic target for slowing AD. .

MicroRNA-155-5p Targets SKP2, Activates IKKß, Increases Aß Aggregation, and Aggravates a Mouse Alzheimer Disease Model.

The nuclear factor kappa B (NF-κB) pathway and inhibitor of NF-κB kinase ß (IKKß) are involved in Alzheimer disease (AD) pathogenesis. This study explored the mechanisms underlying IKKß-mediated Aß aggregation and neuron regeneration in APP.PS1 mice. Adenoviral transduction particles were injected into the hippocampal CA1 region of the mice to knock down or inhibit target genes. Morris water maze was performed to evaluate the cognitive function of the mice. Aß deposition was determined by histological examination. sh-IKKß plasmids and microRNA (miR)-155-5p inhibitor were transfected into Aß1-42-induced N2a cells. The expressions of AD-related proteins were detected by Western blot. The interaction between S-phase kinase-associated protein 2 (SKP2) and IKKß was assessed by co-immunoprecipitation. IKKß knockdown (KD) and miR-155-5p inhibition ameliorated cognitive impairment, improved neuron regeneration, and attenuated Aß deposition in APP/PS1 mice. SKP2 KD aggravated cognitive impairment, inhibited neuron regeneration, and promoted Aß deposition in the mice. SKP2 regulated the stability of IKKß protein via ubiquitination. MiR-155-5p regulates Aß deposition and the expression of Aß generation-related proteins in N2a cells via targeting SKP2. These results indicate that the miR-155-5p/SKP2/IKKß axis was critical for pathogenesis in this AD model and suggest the potential of miR-155-5p as a target for AD treatment.

Caspase-1/IL-1ß represses membrane transport of GluA1 by inhibiting the interaction between Stargazin and GluA1 in Alzheimer's disease.

BACKGROUND: Alzheimer's disease is a neurodegenerative disease. Previous study has reported that caspase-1/IL-1ß is closely associated with Alzheimer's disease. However, the biological role of caspase-1/IL-1ß in Alzheimer's disease has not been fully elucidated. This study aimed to explore the mechanism of action of caspase-1/IL-1ß in Alzheimer's disease. METHODS: Mouse hippocampal neurones were treated with Aß1-42 to induce Alzheimer's disease cell model. APP/PS1 mice and Aß1-42-induced hippocampal neurones were treated with AC-YVAD-CMK (caspase-1 inhibitor). Spatial learning and memory ability of mice were detected by morris water maze. Flow cytometry, TUNEL staining, Thioflavin S staining and immunohistochemistry were performed to examine apoptosis and senile plaque deposition. Enzyme linked immunosorbent assay and western blot were performed to assess the levels of protein or cytokines. Co-Immunoprecipitation was performed to verify the interaction between Stargazin and GluA1. RESULTS: AC-YVAD-CMK treatment improved spatial learning and memory ability and reduced senile plaque deposition of APP/PS1 mice. Moreover, AC-YVAD-CMK promoted membrane transport of GluA1 in APP/PS1 mice. In vitro, Aß1-42-induced hippocampal neurones exhibited an increase in apoptosis and a decrease in the membrane transport of GluA1, which was abolished by AC-YVAD-CMK treatment. In addition, Stargazin interacted with GluA1, which was repressed by caspase-1. Caspase-1/IL-1ß inhibited membrane transport of GluA1 by inhibiting the interaction between Stargazin and GluA1. CONCLUSIONS: Our data demonstrate that caspase-1/IL-1ß represses membrane transport of GluA1 by inhibiting the interaction between Stargazin in Alzheimer's disease. Thus, caspase-1/IL-1ß may be a target for Alzheimer's disease treatment.

Comprehensive Landscape of Immune Infiltration and Aberrant Pathway Activation in Ischemic Stroke.

Ischemic stroke (IS) is a multifactorial disease caused by the interaction of multiple environmental and genetic risk factors, and it is the most common cause of disability. The immune microenvironment and inflammatory response participate in the whole process of IS occurrence and development. Therefore, the rational use of relevant markers or characteristic pathways in the immune microenvironment will become one of the important therapeutic strategies for the treatment of IS. We collected peripheral blood samples from 10 patients diagnosed with IS at the First Affiliated Hospital of Gannan Medical University and First Affiliated Hospital, Jinan" University, and from 10 normal people. The GSE16561 dataset was downloaded from the Gene Expression Omnibus (GEO) database. xCell, gene set enrichment analysis (GSEA), single-sample GSEA (ssGSEA) and immune-related gene analysis were used to evaluate the differences in the immune microenvironment and characteristic pathways between the IS and control groups of the two datasets. xCell analysis showed that the IS-24h group had significantly reduced central memory CD8+ T cell, effector memory CD8+ T cell, B cell and Th1 cell scores and significantly increased M1 macrophage and macrophage scores. GSEA showed that the IS-24h group had significantly increased inflammation-related pathway activity(myeloid leukocyte activation, positive regulation of tumor necrosis factor biosynthetic process, myeloid leukocyte migration and leukocyte chemotaxis), platelet-related pathway activity(platelet activation, signaling and aggregation; protein polymerization; platelet degranulation; cell-cell contact zone) and pathology-related pathway activity (ERBB signaling pathway, positive regulation of ERK1 and ERK2 cascade, vascular endothelial growth factor receptor signaling pathway, and regulation of MAP kinase activity). Immune-related signature analysis showed that the macrophage signature, antigen presentation-related signature, cytotoxicity-related signature, B cell-related signature and inflammation-related signature were significantly lower in the IS-24h group than in the control group. In this study, we found that there were significant differences in the immune microenvironment between the peripheral blood of IS patients and control patients, as shown by the IS group having significantly reduced CD8+ Tcm, CD8+ Tem, B cell and Th1 cell scores and significantly increased macrophage and M1 macrophage scores. Additionally, inflammation-related, pathological, and platelet-related pathway activities were significantly higher in the IS group than in the control group.

Inhibitor Kappa B Kinase ß, Modulated by DJ-1/p-VHL, Reduces Phosphorylated Tau (p-Tau) Accumulation via Autophagy in Alzheimer's Disease Model.

It has been demonstrated Inhibitor Kappa B Kinase ß (IKKß) facilitates autophagy, which in turn mediates p-Tau protein clearance. However, the specific regulatory mechanism in Alzheimer's disease (AD) remains unclear. Firstly, AD model was generated by the intracerebroventricular (ICV) injection of the Β-amyloid 1-42 (Aß1-42) peptide. Subsequently, mice were injected with shRNA adenoviral transduction particles designed to target DJ-1 or Aß1-42 or Aß1-42 + shNC or Aß1-42 + shRNA against DJ-1. shRNA against DJ-1 were injected into hippocampus of mice (8 × 104 viral particles for each mice) for seven consecutive days. Immunohistochemistry was performed to detect the accumulation of Aß in the hippocampus of mice, and Hematoxylin-Eosin (HE) staining assay was carried to detect pathological changes in the hippocampus of mice. Further, sh-IKKß, shDJ-1, pcDNA-IKKß and pcDNA-DJ-1 plasmids were transfected into HT-22 cells, MTT assay, TUNEL staining and Hoechst staining were performed to detect cell viability and apoptosis, respectively. Western blotting was carried to measure the relative expression of proteins. Findings indicated that Aß1-42 inhibited autophagy and up-regulated p-Tau protein expression; Overexpression of IKKß and DJ-1 all rescued the autophagy inhibited by Aß1-42 and down-regulated p-Tau protein expression induced by Aß1-42; DJ-1 up-regulated IKKß via p-VHL, further promoted autophagy and reduced the expression of p-Tau protein; DJ-1 knockdown inhibited autophagy and up-regulated p-Tau protein expression, resulting in delayed behavior in mice. In conclusion, IKKß, modulated by DJ-1/p-VHL, reduces p-Tau accumulation via autophagy in AD's disease model. This study may provide theoretical basis for the treatment of AD.

Long non-coding RNA uc.80- overexpression promotes M2 polarization of microglias to ameliorate depression in rats.

Microglia polarization is associated with the pathogenesis of depression. A previous study shows that long non-coding RNA uc.80- is down-regulated in the hippocampus of depressed rats. Thus, this article aims to investigate the role of uc.80- in microglia polarization in depression. We first established depression model rats by chronic unpredictable mild stress (CUMS) regiment. We found that hippocampus of depressed rats exhibited an increase of M1 microglias and a decrease of M2 microglias. uc.80- was down-regulated in hippocampus of depressed rats. Furthermore, the detection of behaviouristics of depressed rats showed that uc.80- overexpression alleviated depression of rats. In addition, uc.80- overexpression promoted M2 polarization of microglias in vivo and in vitro. uc.80- overexpression led to a decrease in apoptosis of hippocampal neurons in vivo and in vitro. In conclusion, our study confirms that lncRNA uc.80- overexpression ameliorates depression in rats by promoting M2 polarization of microglias. Thus, our work suggests that uc.80- may be a target gene for depression treatment.

Pinocembrin mitigates depressive-like behaviors induced by chronic unpredictable mild stress through ameliorating neuroinflammation and apoptosis.

BACKGROUND: The majority of patients with chronic fatigue have a risk of comorbidity with depression. Pinocembrin (PB) is a kind of flavonoid molecule isolated from honey and propolis and has antimicrobial, anti-inflammatory, antioxidant, and anticancer function. The purpose of the current study was to determine the possible function of PB on treatment of depression. METHODS: A chronic unpredictable mild stress (CUMS) mouse model was established to mimic the depressive-like behaviors in vivo. The depressive-like behaviors of CUMS mice were measured by sucrose preference test (SPT), open field test (OFT), forced swim test (FST) and tail suspension test (TST). The concentration of reactive oxygen species (ROS), malondialdehyde (MDA) and the activity or superoxide dismutase (SOD) were detected by commercial kit. The inflammatory factor including interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-10 and transforming growth factor (TGF)-ß were examined. RESULTS: We found that PB alleviated the decreasing of sucrose preference and body weight. CUMS mice significantly increased the immobility time but decreased latency to abandon in FST, increased the immobility time in TST, and reduced crossing score and rearing score in OFT, whereas these changes were reversed by PB treatment. More importantly, PB decreased the concentration of ROS and MDA, but increased the SOD activity, suggesting that it could protected against oxidative stress in CUMS mice. Interestingly, PB inhibited cell apoptosis and regulated inflammatory factors expression in hippocampus of CUMS mice. Moreover, PB activated Nrf2/HO-1 signal pathway but inhibited the phosphorylation of NF-kB. CONCLUSIONS: In conclusion, PB mitigated CUMS-induced depressive-like behaviors through ameliorating neuroinflammation and apoptosis. TRIAL REGISTRATION: Not Applicable.

Cystathionine-ß-synthase-derived hydrogen sulfide is required for amygdalar long-term potentiation and cued fear memory in rats.

Hydrogen sulfide (H2S) is an endogenous gaseous molecule that functions as a neuromodulator in the brain. We previously reported that H2S regulated amygdalar synaptic plasticity and cued fear memory in rats. However, whether endogenous H2S is required for amygdalar long-term potentiation (LTP) induction and cued fear memory formation remains unclear. Here, we show that cystathionine-ß-synthase (CBS), the predominant H2S-producing enzyme in the brain, was highly expressed in the amygdala of rats. Suppressing CBS activity by inhibitor prevented activity-triggered generation of H2S in the lateral amygdala (LA) region. Incubating brain slices with CBS inhibitor significantly prevented the induction of NMDA receptors (NMDARs)-dependent LTP in the thalamo-LA pathway, and intra-LA infusion of CBS inhibitor impaired cued fear memory in rats. Notably, treatment with H2S donor, but not CBS activator, significantly reversed the impairments of LTP and fear memory caused by CBS inhibition. Mechanismly, inhibition of CBS activity led to a reduction in NMDAR-mediated synaptic response in the thalamo-LA pathway, and treatment with H2S donor restored the function of NMDARs. Collectively, these results indicate that CBS-derived H2S is required for amygdalar synaptic plasticity and cued fear memory in rats, and the effects of endogenous H2S might involve the regulation of NMDAR function.

The flavonoid baicalein rescues synaptic plasticity and memory deficits in a mouse model of Alzheimer's disease.

Increasing evidence suggests that disruptions of synaptic functions correlate with the severity of cognitive deficit in Alzheimer's disease (AD). Our previous study demonstrated that baicalein enhances long-term potentiation (LTP) in acute rat hippocampal slices and improves hippocampus-dependent contextual fear conditioning in rats. Given that baicalein possess various biological activities, especially its effects on synaptic plasticity and cognitive function, we examined the effect of baicalein on synaptic function both in vitro and in vivo in AD model. The effect of baicalein on Aß42 oligomer impaired LTP was investigated by electrophysiological methods. Baicalein was administered orally via drinking water to the APP/PS1 mice and sex- and age-matched wild-type mice. Treatment started at 5 months of age and mice were assessed for cognition and AD-like pathology at 7-month-old. Cognition was analyzed by Morris water maze test, fear conditioning test, and novel object recognition test. Changes in hippocampal 12/15 Lipoxygenase (12/15LO) and glycogen synthase kinase 3ß (GSK3ß) activity, Aß production, tau phosphorylation, synaptic plasticity, and dendritic spine density were evaluated. Baicalein prevented Aß-induced impairments in hippocampal LTP through activation of serine threonine Kinase (Akt) phosphorylation. Long-term oral administration of baicalein inhibited 12/15LO and GSK3ß activity, reduced ß-secretase enzyme (BACE1), decreased the concentration of total Aß, and prevented phosphorylation of tau in APP/PS1 mice. Meanwhile, baicalein restored spine number, synaptic plasticity, and memory deficits. Our results strengthen the potential of the flavonoid baicalein as a novel and promising oral bioactive therapeutic agent that prevents memory deficits in AD.

D-Serine in the nucleus accumbens region modulates behavioral sensitization and extinction of conditioned place preference.

BACKGROUND: D-serine, the endogenous co-agonist of N-methyl-D-aspartate receptors (NMDARs), is considered to be essential for learning and memory. The aim of the current investigation was to systematically evaluate the role of D-serine on addiction behaviors considered to be mediated by the nucleus accumbens (NAc). METHODS: D-Serine concentration in the NAc was measured by high-performance liquid chromatography (HPLC). Cocaine-induced behavioral sensitization and conditioned place preference (CPP) models were used to evaluate the relation between changes in serine in the nucleus accumbens and cocaine-induced behavioral effects. The expression of serine racemase (SR), D-amino acid oxidase (DAAO), the cAMP response element-binding protein (CREB) and upstream kinases, and N-methyl-D-aspartate (NMDA) receptors subunits were analyzed by western blot. Long-term depression (LTD) in the NAc was investigated by electrophysiological methods. RESULTS: The NAc slices obtained from the behavioral sensitization rats presented significantly reduced D-serine concentrations, increased expression of DAAO, and down-regulated expression of SR in a dose-dependent manner. Furthermore, D-serine injections into the nucleus accumbens blocked the development of behavioral sensitization and caused extinction of CPP. The ERK-CREB-Fos pathway and the NMDA receptor NR2B subunits in the NAc were involved in the cocaine-induced behavioral sensitization. We also found that D-serine was essential for NMDAR-dependent LTD and D-serine-regulated LTD in a bell-shaped concentration-dependent manner. The disrupted NMDAR-dependent LTD in the NAc of cocaine-treated rats was reversed by D-serine. CONCLUSIONS: Our results provide evidence for a critical role of D-serine in synaptic plasticity relevant to cocaine addiction and indicate that D-serine may be an effective therapeutic agent for cocaine addiction.