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Oligodendrocytes (OLs), the myelinating cells in the central nervous system (CNS), are differentiated from OL progenitor cells (OPCs). The proliferation of existing OPCs is indispensable for myelination during CNS development and remyelination in response to demyelination stimulation. The transcription factor Olig2 is required for the specification of OLs and is expressed in the OL lineage. However, the post-translational modification of Olig2 in the proliferation of OPCs is poorly understood. Herein, we identified that c-Abl directly phosphorylates Olig2 mainly at the Tyr137 site, and that Olig2 phosphorylation is essential for OPC proliferation. The expression levels of c-Abl gradually decreased with brain development; moreover, c-Abl was highly expressed in OPCs. OL-specific c-Abl knockout at the developmental stage led to an insufficient proliferation of OPCs, a decreased expression of myelin-related genes, and myelination retardation. Accordingly, a c-Abl-specific kinase inhibitor suppressed OPC proliferation in vitro. Furthermore, we observed that OL-specific c-Abl knockout reduced OPC proliferation and remyelination in a cuprizone model of demyelination. In addition, we found that nilotinib, a clinically used c-Abl inhibitor, decreased the expression of myelin basic protein (Mbp) and motor coordination in mice, indicating a neurological side effect of a long-term administration of the c-Abl inhibitor. Thus, we identified the important role of c-Abl in OLs during developmental myelination and remyelination in a disease model.

Background: Major depression is one of the most common psychiatric disorders worldwide, inflicting suffering, significant reduction in life span, and financial burdens on families and society. Mounting evidence implicates that exposure to chronic stress can induce the dysregulation of the immune system, and the activation of brain-resident innate immune cells, microglia, leading to depression-like symptoms. However, the specific mechanisms need to be further elucidated.Method: Animal models of depression were established by chronic restraint stress (CRS), and depression-like behavior was assessed by sucrose preference test (SPT), open field test (OFT), tail suspension test (TST) and forced swimming test (FST). Microglial activation was visualized by immunofluorescent and immunohistochemical staining, and microglial morphological changes were further analyzed by skeleton analysis. The levels of inflammatory cytokines were detected by western blotting and qPCR.Result: Microglial Dlg1 knockout ameliorates CRS-induced mice depression-like behavior. In contrast to the effect of Dlg1 in the LPS-induced mouse model, Dlg1 knockout had little effect on microglial density, but significantly decreased the number of activated microglia and reversed microglia morphological changes in mice challenged with CRS. Moreover, the upregulation of inflammatory cytokines following CRS exposure was partially reversed by Dlg1 deletion.Conclusion: Our study provides the evidence that Dlg1 ablation in microglia remarkedly reverses microglial activation and depression-like behavior in mice exposed to CRS, implicating a potential target for the treatment of clinical depression.

Microglia-mediated neuroinflammation is widely perceived as a contributor to numerous neurological diseases and mental disorders including depression. Discs large homolog 1 (Dlg1), an adaptor protein, regulates cell polarization and the function of K+ channels, which are reported to regulate the activation of microglia. However, little is known about the role of Dlg1 in microglia and the maintenance of central nervous system homeostasis. In this study, we found that Dlg1 knockdown suppressed lipopolysaccharide (LPS)-induced inflammation by down-regulating the activation of nuclear factor-κB signaling and the mitogen-activated protein kinase pathway in microglia. Moreover, using an inducible Dlg1 microglia-specific knockout (Dlg1flox/flox; CX3CR1CreER) mouse line, we found that microglial Dlg1 knockout reduced the activation of microglia and alleviated the LPS-induced depression-like behavior. In summary, our results demonstrated that Dlg1 plays a critical role in microglial activation and thus provides a potential therapeutic target for the clinical treatment of depression.

N6-methyladenosine (m6A) modification regulates RNA translation, splicing, transport, localization, and stability at the post-transcriptional level. The m6A modification has been reported to have a wide range of effects on the nervous system, including neurogenesis, cerebellar development, learning, cognition, and memory, as well as the occurrence and development of neurological disorders. In this review, we aim to summarize the findings on the role and regulatory mechanism of m6A modification in the nervous system, to reveal the molecular mechanisms of neurodevelopmental processes, and to promote targeted therapy for nervous system-related diseases.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline and currently there are no available treatments. Alongside the conventional Aβ and tau hypotheses, neuroinflammation and metabolism disruption have also been regarded as crucial hallmarks of AD. In this study, a novel Chinese formula Nao Tan Qing (NTQ) was developed and shown to improve AD. In vivo experiments showed that NTQ significantly mitigated cognitive impairment, Aβ burden and neuroinflammation in a transgenic AD mouse model (5×FAD). Network pharmacology results revealed that the active components of NTQ could target inflammatory and metabolic pathways. In addition, hippocampal transcriptomics suggested that NTQ regulated signaling pathways related to inflammation and lipid metabolism. Consistently, serum metabolomics further indicated that NTQ could modulate glycolipid metabolism. In summary, a combination of systems pharmacology analysis and biological validation study demonstrates that NTQ could alleviate behavioral abnormality and pathological alterations of AD by targeting glycolipid metabolism and neuroinflammation, and is accordingly a potential therapeutic agent for AD.

BackgroundNon-motor symptoms, including sleep disorders and depression, are common in Parkinson’s disease (PD). The purpose of our study is to explore the effect of sleep disorders, including the probable rapid eye movement (REM) sleep behavior disorder (pRBD) and the daytime sleepiness, on depressive symptoms in patients with early and prodromal PD.MethodsA total of 683 participants who obtained from the Parkinson Progression Markers Initiative (PPMI) were included, consisting of 423 individuals with early PD, 64 individuals with prodromal PD, and 196 healthy controls (HCs), who were followed up to 5 years from baseline. Multiple linear regression models and linear mixed-effects models were conducted to explore the relationship between sleep disorders and depression at baseline and longitudinally, respectively. Multiple linear regression models were used to further investigate the association between the change rates of daytime sleepiness score and depression-related score. Mediation analyses were also performed.ResultsAt baseline analysis, individuals with early and prodromal PD, who had higher RBD screening questionnaire (RBDSQ) score, or who were considered as pRBD, or who manifested specific behaviors of RBD (things falling down when sleep or disturbance of sleep), showed significantly the higher score of depression-related questionnaires. Our 5-year follow-up study showed that sleep disorders, including pRBD and daytime sleepiness, were associated with the increased depressive-related score in individuals with early and prodromal PD. Interestingly, we also found that the increased possibilities of daytime sleepiness were associated with depressive-related score. Finally, mediation analysis demonstrated that the relationship between RBD and depressive symptoms was partially mediated by autonomic symptoms, such as postural hypertension, salivation, dysphagia, and constipation.ConclusionOur study shows that sleep disorders, including pRBD and daytime sleepiness, are associated with depression at baseline and longitudinally, which is partially mediated by the autonomic dysfunction in early and prodromal PD, with an implication that sleep management is of great value for disease surveillance.

N6-methyladenosine (m

The proline-rich coiled-coil 2A (PRRC2A) gene has been reported to underlie risk of various autoimmune diseases. However, no data reveal the risk susceptibility of PRRC2A to neuromyelitis optica spectrum disorder (NMOSD) and multiple sclerosis (MS) so far. To explore the association between PRRC2A variants and NMOSD and MS susceptibility in Han Chinese population. Totally, 207 NMOSD (98 AQP4+ and 109 AQP4−) patients, 141 MS and 196 healthy controls (HC) were enrolled. Candidate tagging single nucleotide polymorphisms (tag-SNPs) were selected from the 1000G database based on the Chinese data. SNP genotyping was performed using MassArray and Sanger sequencing. PRRC2A variants rs2736171, rs2736157, rs2844470 alter susceptibility to AQP4+ NMOSD, while rs2242659 to MS. Genotype AT of rs2844470 and AG of rs2242659 increased risk susceptibility for AQP4+ NMOSD and MS, respectively. AQP4+ NMOSD exhibited a higher frequency of genotype AG of rs2736157 compared with AQP4− NMOSD. Haplotype TCAAGGTAG was conferred risk susceptibility to AQP4+ NMOSD and haplotype TTAGAGTAG had a protective effect on both AQP4+ and AQP4− NMOSD. Further, we identified various gene expression levels in disease-related regions that are significantly modulated by three cis-eQTL SNPs rs2736157, rs2736171 and rs2242659 (p

Microglial cells consume adenosine triphosphate (ATP) during phagocytosis to clear neurotoxic β-amyloid in Alzheimer's disease (AD). However, the contribution of energy metabolism to microglial function in AD remains unclear. Here, we demonstrate that hexokinase 2 (HK2) is elevated in microglia from an AD mouse model (5xFAD) and AD patients. Genetic deletion or pharmacological inhibition of HK2 significantly promotes microglial phagocytosis, lowers the amyloid plaque burden and attenuates cognitive impairment in male AD mice. Notably, the ATP level is dramatically increased in HK2-deficient or inactive microglia, which can be attributed to a marked upregulation in lipoprotein lipase (LPL) expression and subsequent increase in lipid metabolism. We further show that two downstream metabolites of HK2, glucose-6-phosphate and fructose-6-phosphate, can reverse HK2-deficiency-induced upregulation of LPL, thus supporting ATP production and microglial phagocytosis. Our findings uncover a crucial role for HK2 in phagocytosis through regulation of microglial energy metabolism, suggesting a potential therapeutic strategy for AD by targeting HK2.

Microglial activation-induced neuroinflammation is closely associated with the development of Parkinson disease (PD). Macroautophagy/autophagy regulates many biological processes, but the role of autophagy in microglial activation during PD development remains largely unclear. In this study, we showed that deletion of microglial Atg5 caused PD-like symptoms in mice, characterized by impairment in motor coordination and cognitive learning, loss of tyrosine hydroxylase (TH) neurons, enhancement of neuroinflammation and reduction in dopamine levels in the striatum. Mechanistically, we found that inhibition of autophagy led to NLRP3 (NLR family pyrin domain containing 3) inflammasome activation via PDE10A (phosphodiesterase 10A)–cyclic adenosine monophosphate (cAMP) signaling in microglia, and the sequential upregulation of downstream IL1B/IL-1β in turn increased the expression of MIF (macrophage migration inhibitory factor [glycosylation-inhibiting factor]), a pro-inflammatory cytokine. Inhibition of NLRP3 inflammasome activation by administration of MCC950, a specific inhibitor for NLRP3, decreased MIF expression and neuroinflammatory levels, and rescued the loss of TH neurons in the substantial nigra (SN). Interestingly, we found that serum MIF levels in PD patients were significantly elevated. Taken together, our results reveal an important role of autophagy in microglial activation-driven PD-like symptoms, thus providing potential targets for the clinical treatment of PD. Abbreviations: ATG: autophagy related; cAMP: cyclic adenosine monophosphate; cKO: conditional knockout; NOS2/INOS: nitric oxide synthase 2, inducible; IL1B: interleukin 1 beta; ITGAM/CD-11b: integrin alpha M/cluster of differentiation molecule 11B; MAP1LC3: microtubule-associated protein 1 light chain 3; MIF: macrophage migration inhibitory factor (glycosylation-inhibiting factor); NLRP3: NLR family pyrin domain containing 3; PBS: phosphate-buffered saline; PD: parkinson disease; PDE10A: phosphodiesterase 10A; SN: substantial nigra; TH: tyrosine hydroxylase; TNF: tumor necrosis factor; WT: wild type.

Rationale: It is known that neuroinflammation plays a critical and detrimental role in the development of cerebral ischemia/reperfusion (I/R), but the regulation of the cyclic GMP-AMP synthase (cGAS)-mediated innate immune response in I/R-induced neuroinflammation is largely unexplored. This study aimed to investigate the function and regulatory mechanism of cGAS in I/R-induced neuroinflammation and brain injury, and to identify possible strategies for the treatment of ischemic stroke. Methods: To demonstrate that microglial histone deacetylase 3 (HDAC3) regulates the microglial cGAS-stimulator of interferon genes (cGAS-STING) pathway and is involved in I/R-induced neuroinflammation and brain injury, a series of cell biological, molecular, and biochemical approaches were utilized. These approaches include transient middle cerebral artery occlusion (tMCAO), real-time polymerase chain reaction (PCR), RNA sequencing, western blot, co-immunoprecipitation, chromosome-immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dual-luciferase reporter assay, immunohistochemistry, and confocal imaging. Results: The microglial cGAS- STING pathway was activated by mitochondrial DNA, which promoted the formation of a pro-inflammatory microenvironment. In addition, we revealed that HDAC3 transcriptionally promoted the expression of cGAS and potentiated the activation of the cGAS-STING pathway by regulating the acetylation and nuclear localization of p65 in microglia. Our in vivo results indicated that deletion of cGAS or HDAC3 in microglia attenuated I/R-induced neuroinflammation and brain injury. Conclusion: Collectively, we elucidated that the HDAC3-p65-cGAS-STING pathway is involved in the development of I/R-induced neuroinflammation, identifying a new therapeutic avenue for the treatment of ischemic stroke.

Abstract Background. Transcutaneous electrical nerve stimulation (TENS) is generally applied for tactile feedback in the field of prosthetics. The distinct mechanisms of evoked tactile perception between stimulus patterns in conventional TENS (cTENS) and neuromorphic TENS (nTENS) are relatively unknown. This is the first study to investigate the neurobiological effect of nTENS for cortical functional mechanism in evoked tactile perception. Methods. Twenty-one healthy participants were recruited in this study. Electroencephalogram (EEG) was recorded while the participants underwent a tactile discrimination task. One cTENS pattern (square pattern) and two nTENS patterns (electromyography and single motor unit patterns) were applied to evoke tactile perception in four fingers, including the right and left index and little fingers. EEG was preprocessed and somatosensory-evoked potentials (SEPs) were determined. Then, source-level functional networks based on graph theory were evaluated, including clustering coefficient, path length, global efficiency, and local efficiency in six frequency bands. Main results. Behavioral results suggested that the single motor units (SMUs) pattern of nTENS was the most natural tactile perception. SEPs results revealed that SMU pattern exhibited significant shorter latency in P1 and N1 components than the other patterns, while nTENS patterns have significantly longer latency in P3 component than cTENS pattern. Cortical functional networks showed that the SMU pattern had the lowest short path and highest efficiency in beta and gamma bands. Conclusion. This study highlighted that distinct TENS patterns could affect brain activities. The new characteristics in tactile manifestation of nTENS would provide insights for the application of tactile perception restoration.

Background and purpose Ageing is associated with progressive metabolic dysregulation. Rutin is a metabolic regulator with a poor solubility. Using soluble sodium rutin we investigating the effect and mechanisms of rutin in ageing process. Experimental approach Wild type male mice were treated with or without sodium rutin ( 0.2 mg·ml-1 in drinking water from 8-month-old until end of life. Kaplan-Meier survival curve was used for lifespan assay, ageing-related histopathology analysis and metabolic analysis were performed to determine the effects of chronic sodium rutin on the longevity. Serological test, liver tissue metabolomics and transcriptomics were used for liver function assay. SiRNA knockdown Angptl8 and autophagy flux assay in HepG2 cell lines explored the mechanism through which sodium rutin might impact the function of hepatocyte. Key results Sodium rutin treatment extends the lifespan of mice by 10%. Sodium rutin supplementation alleviates ageing-related pathological changes and promotes behaviour performance in ageing mice. Sodium rutin supplementation altered the whole-body metabolism in mice, which exhibited increased energy expenditure and lower respiratory quotient. Transcriptomics analysis showed that Sodium rutin affected the expression of metabolic genes. Metabolomics analysis showed that Sodium rutin reduced liver steatosis through increased lipid β-oxidation. Sodium rutin treatment increased the autophagy level both in vivo and in vitro. The inhibition of autophagy partially abolished the sodium rutin-mediated effect on lipolysis in HepG2 cells. Conclusion and implications Sodium rutin treatment extends the lifespan and health span of mice with beneficial effects on metabolism, which were achieved by enhancing the autophagy activity in hepatocytes.

Activation of the NLRP3 inflammasome results in caspase 1 cleavage, which subsequently leads to IL-1β and IL-18 secretion, as well as pyroptosis, and aberrant activation of the inflammasome is involved in several diseases such as type 2 diabetes, atherosclerosis, multiple sclerosis, Parkinson’s disease, and Alzheimer’s disease. NLRP3 activity is regulated by various kinases. Genetic and pharmacological inhibition of the hematopoietic cell kinase (HCK), a member of the Src family of non-receptor tyrosine kinases (NRTKs) primarily expressed in myeloid cells, has previously been shown to ameliorate inflammation, indicating that it may be involved in the regulation of microglia function. However, the underlying mechanism is not known. Hence, in this study, we aimed to investigate the role of HCK in NLRP3 inflammasome activation. We demonstrated that HCK silencing inhibited NLRP3 inflammasome activation. Furthermore, the HCK-specific inhibitor, A419259, attenuated the release of IL-1β and caspase 1(P20) from the macrophages and microglia and reduced the formation of the apoptosis-associated speck-like protein with a CARD domain (ASC) oligomer. We also observed that HCK binds to full length NLRP3 and its NBD(NACHT) and LRR domains, but not to the PYD domain. In vivo, the HCK inhibitor attenuated the LPS-induced inflammatory response in the liver of LPS-challenged mice. Collectively, these results suggested that HCK plays a critical role in NLRP3 inflammasome activation. Our results will enhance current understanding regarding the effectiveness of HCK inhibitors for treating acute inflammatory diseases.

The proline-rich coiled-coil 2A (PRRC2A) gene has been reported to underlie risk of various autoimmune diseases. However, no data reveal the risk susceptibility of PRRC2A to neuromyelitis optica spectrum disorder (NMOSD) and multiple sclerosis (MS) so far.To explore the association between PRRC2A variants and NMOSD and MS susceptibility in Han Chinese population.Totally, 207 NMOSD (98 AQP4PRRC2A variants rs2736171, rs2736157, rs2844470 alter susceptibility to AQP4PRRC2A variants are first reported to be associated with NMOSD and MS. The identified PRRC2A variants may shed light on the pathogenesis of NMOSD and MS and potentially lead to an individualized therapeutic approach for both distinct disease entities.

While N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic mRNA, is linked to cell differentiation and tissue development, the biological significance of m6A modification in mammalian glial development remains unknown. Here, we identify a novel m6A reader, Prrc2a (Proline rich coiled-coil 2 A), which controls oligodendrocyte specification and myelination. Nestin-Cre-mediated knockout of Prrc2a induces significant hypomyelination, decreased lifespan, as well as locomotive and cognitive defects in a mouse model. Further analyses reveal that Prrc2a is involved in oligodendrocyte progenitor cells (OPCs) proliferation and oligodendrocyte fate determination. Accordingly, oligodendroglial-lineage specific deletion of Prrc2a causes a similar phenotype of Nestin-Cre-mediated deletion. Combining transcriptome-wide RNA-seq, m6A-RIP-seq and Prrc2a RIP-seq analysis, we find that Olig2 is a critical downstream target gene of Prrc2a in oligodendrocyte development. Furthermore, Prrc2a stabilizes Olig2 mRNA through binding to a consensus GGACU motif in the Olig2 CDS (coding sequence) in an m6A-dependent manner. Interestingly, we also find that the m6A demethylase, Fto, erases the m6A modification of Olig2 mRNA and promotes its degradation. Together, our results indicate that Prrc2a plays an important role in oligodendrocyte specification through functioning as a novel m6A reader. These findings suggest a new avenue for the development of therapeutic strategies for hypomyelination-related neurological diseases.

The transcriptional co-activator Yes-associated protein (YAP) has been implicated as an oncogene and is found to promote breast cancer metastasis. However, the pro-metastatic mechanism of YAP remains unclear. Here, we demonstrated that YAP functions as a transcriptional repressor of growth differentiation factor-15 (GDF15), a divergent member of the transforming growth factor superfamily, in several breast cancer cell lines. Functionally, knockdown of YAP decreased, whereas knockdown of GDF15 increased, the metastatic potential of breast cancer cells. More than that, the reduced metastasis in YAP-depleted cells could be reversed by simultaneous knockdown of GDF15. Mechanistically, the repressive effect of YAP on GDF15 requires its transcriptional factor TEAD (TEA domain family). In addition, YAP recruits polycomb repressive complex 2 (PRC2) to tri-methylate histone H3 lysine 27 in the promoter region of GDF15. Co-immunoprecipitation experiments demonstrated that YAP and enhancer of zeste 2 PRC2 subunit (EZH2) physically interact with each other. In conclusion, our data reveal that YAP promotes metastasis of breast cancer cells by repressing GDF15 transcription and present a novel molecular mechanism underlying the pro-metastasis function of YAP oncoprotein, with the implication of a therapeutic avenue for breast cancer treatment.

Ferroptosis is a regulated form of cell death characterized by the iron-dependent accumulation of lipid hydroperoxides. Ceruloplasmin (CP) is a glycoprotein that plays an essential role in iron homeostasis. However, whether CP regulates ferroptosis has not been reported. Here, we show that CP suppresses ferroptosis by regulating iron homeostasis in hepatocellular carcinoma (HCC) cells. Depletion of CP promoted erastin- and RSL3-induced ferroptotic cell death and resulted in the accumulation of intracellular ferrous iron (Fe2+) and lipid reactive oxygen species (ROS). Moreover, overexpression of CP suppressed erastin- and RSL3-induced ferroptosis in HCC cells. In addition, a novel frameshift mutation (c.1192-1196del, p.leu398serfs) of CP gene newly identified in patients with iron accumulation and neurodegenerative diseases lost its ability to regulate iron homeostasis and thus failed to participate in the regulation of ferroptosis. Collectively, these data suggest that CP plays an indispensable role in ferroptosis by regulating iron metabolism and indicate a potential therapeutic approach for hepatocellular carcinoma.

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Postoperative cognitive dysfunction (POCD) is a common postoperative complication observed in patients following. Here we tested the molecular mechanisms of memory loss in hippocampus of rat POCD model. We found that high-dose propofol anesthesia significantly alleviated spatial memory loss. The proteomes and transcriptomes in hippocampus showed that hippocampal cytoskeleton related pathways were abnormal in low group while not in high group. The protein assays confirmed that hippocampal actin cytoskeleton was depolymerized in low group while maintained in high group. This study confirms that high-dose propofol anesthesia could mitigate the development of POCD and provides evidences for actin cytoskeleton associated with this syndrome.

NOD-like receptors (NLRs) are critical cytoplasmic pattern-recognition receptors (PRRs) that play an important role in the host innate immune response and immunity homeostasis. There is a growing body of evidence that NLRs are involved in a wide range of inflammatory diseases, including cancer, metabolic diseases, and autoimmune disorders. Recent studies have indicated that the proteins of the NLR family are linked with the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS), and psychological diseases. In this review, we mainly focus on the role of NLRs and the underlying signaling pathways in central nervous system (CNS) diseases. © 2016 Wiley Periodicals, Inc.

Increasing energy expenditure through activation of brown adipose tissue (BAT) is a critical approach to treating obesity and diabetes. In this study, rutin, a natural compound extracted from mulberry and a drug used as a capillary stabilizer clinically for many years without any side effects, regulated whole-body energy metabolism by enhancing BAT activity. Rutin treatment significantly reduced adiposity, increased energy expenditure, and improved glucose homeostasis in both genetically obese (Db/Db) and diet-induced obesity (DIO) mice. Rutin also induced brown-like adipocyte (beige) formation in subcutaneous adipose tissue in both obesity mouse models. Mechanistically, we found that rutin directly bound to and stabilized SIRT1, leading to hypoacetylation of peroxisome proliferator-activated receptor γ coactivator-1α protein, which stimulated Tfam transactivation and eventually augmented the number of mitochondria and UCP1 activity in BAT. These findings reveal that rutin is a novel small molecule that activates BAT and may provide a novel therapeutic approach to the treatment of metabolic disorders.-Yuan, X., Wei, G., You, Y., Huang, Y., Lee, H. J., Dong, M., Lin, J., Hu, T., Zhang, H., Zhang, C., Zhou, H., Ye, R., Qi, X., Zhai, B., Huang, W., Liu, S., Xie, W., Liu, Q., Liu, X., Cui, C., Li, D., Zhan, J., Cheng, J., Yuan, Z., Jin, W. Rutin ameliorates obesity through brown fat activation.

Cerebral ischemia-reperfusion injury is a major public health concern that causes high rates of disability and mortality in adults. Microglial activation plays a crucial role in ischemic stroke-induced alteration of the immune microenvironment. However, the mechanism underlying the triggering of microglial activation by ischemic stroke remains to be elucidated. Previously, we demonstrated that the protein kinase Hippo/MST1 plays an important role in oxidative stress-induced cell death in mammalian primary neurons and that the protein kinase c-Abl phosphorylates MST1 at Y433, which increases MST1 kinase activity. Microglial activation has been implicated as a secondary detrimental cellular response that contributes to neuronal cell death in ischemic stroke. Here, we are the first, to our knowledge, to demonstrate that MST1 mediates stroke-induced microglial activation by directly phosphorylating IκBα at residues S32 and S36. We further demonstrate that Src kinase functions upstream of MST1-IκB signaling during microglial activation. Specific deletion of MST1 in microglia mitigates stroke-induced brain injury. Therefore, we propose that Src-MST1-IκB signaling plays a critical role in stroke-induced microglial activation. Together with our previous work demonstrating that MST1 is important for oxidative stress-induced neuronal cell death, our results indicate that MST1 could represent a potent therapeutic target for ischemic stroke.

To investigate the polarization effect of Malibatol A on oxygen-glucose deprivation (OGD)-BV-2 cells, and the possible molecular mechanism involved in c-Abl-MST signaling pathway.The OGD BV-2 cell model was established. BV-2 cells were exposed to OGD for 8 h followed by reperfusion for 15 h with Malibatol A at different concentration of 0.5, 1, 2, 4, 8, 16 μM or without it. And then cells, mRNA and protein were harvested respectively. The cell viability and apoptosis were measured by MTT assay and flow cytometry. The mRNA of classical activated microglia (M1) markers (MCP-1, IL-1 and TNF-α) and alternatively activated microglia (M2) markers (Ym-1, CD206, IL-10, TGF-β) in BV-2 cells were measured by RT-PCR. Meanwhile, the proteins of Ym-1 and CD206 was assayed by flow cytometry. Furthermore, the expression of c-Abl and MST was measured by Western blot.Malibatol A significantly decreased apoptosis and increased viability of OGD BV-2 cells in a dose-dependent manner. In the presence of Malibatol A, the mRNA levels of Ym-1, CD206, IL-10 and TGF-β mRNA was significantly increased in OGD-BV-2 cells, while the mRNA levels of MCP-1, IL-1 and TNF-α was obviously down-regulated. Meanwhile, the proteins of Ym-1 and CD206 was raised in OGD BV-2 cells with Malibatol A. Besides, Malibatol A also inhibited OGD-induced p-MST1(Y433) in BV-2 cells.Malibatol A could attenuate OGD-induced BV-2 cell injury and promote M2 microglia polarization. The mechanism may be related to inhibition of MST1 phosphorylation at Y433.

The Hippo/Yki and RB/E2F pathways both regulate tissue growth by affecting cell proliferation and survival, but interactions between these parallel control systems are poorly defined. In this study, we demonstrate that interaction between Drosophila E2F1 and Sd disrupts Yki/Sd complex formation and thereby suppresses Yki target gene expression. RBF modifies these effects by reducing E2F1/Sd interaction. This regulation has significant effects on apoptosis, organ size, and progenitor cell proliferation. Using a combination of DamID-seq and RNA-seq, we identified a set of Yki targets that play a diversity of roles during development and are suppressed by E2F1. Further, we found that human E2F1 competes with YAP for TEAD1 binding, affecting YAP activity, indicating that this mode of cross-regulation is conserved. In sum, our study uncovers a previously unknown mechanism in which RBF and E2F1 modify Hippo signaling responses to modulate apoptosis, organ growth, and homeostasis. Citation Format: Peng Zhang, Chunli Pei, Zengqiang Yuan, Bruce A. Edgar. A balance of yki/sd activator and e2f1/sd repressor complexes controls cell survival and affects organ size [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A15.

Summary Loss of Hippo tumor-suppressor activity and hyperactivation of YAP are commonly observed in cancers. Inactivating mutations of Hippo kinases MST1/2 are uncommon, and it remains unclear how their activity is turned off during tumorigenesis. We identified STRN3 as an essential regulatory subunit of protein phosphatase 2A (PP2A) that recruits MST1/2 and promotes its dephosphorylation, which results in YAP activation. We also identified STRN3 upregulation in gastric cancer correlated with YAP activation and poor prognosis. Based on this mechanistic understanding and aided by structure-guided medicinal chemistry, we developed a highly selective peptide inhibitor, STRN3-derived Hippo-activating peptide, or SHAP, which disrupts the STRN3-PP2Aa interaction and reactivates the Hippo tumor suppressor, inhibits YAP activation, and has antitumor effects in vivo.

Reactive oxygen species (ROS) production in phagocytes is a major defense mechanism against pathogens. However, the cellular self-protective mechanism against such potential damage from oxidative stress remains unclear. Here we show that the kinases Mst1 and Mst2 (Mst1/2) sense ROS and maintain cellular redox balance by modulating the stability of antioxidant transcription factor Nrf2. Site-specific ROS release recruits Mst1/2 from the cytosol to the phagosomal or mitochondrial membrane, with ROS subsequently activating Mst1/2 to phosphorylate kelch like ECH associated protein 1 (Keap1) and prevent Keap1 polymerization, thereby blocking Nrf2 ubiquitination and degradation to protect cells against oxidative damage. Treatment with the antioxidant N-acetylcysteine disrupts ROS-induced interaction of Mst1/2 with phagosomes or mitochondria, and thereby diminishes the Mst-Nrf2 signal. Consistently, loss of Mst1/2 results in increased oxidative injury, phagocyte ageing and death. Thus, our results identify the Mst-Nrf2 axis as an important ROS-sensing and antioxidant mechanism during an antimicrobial response., Immune cells produce reactive oxygen species (ROS) to eliminate pathogens, but cell-spontaneous death and ageing may also be induced. Here the authors show that, upon sensing ROS, Mst1/2 kinases modulate the activity of Nrf2 transcription factor and downstream genetic programs to protect mouse macrophages from death and ageing.

Background Synaptic protein dyshomeostasis and functional loss is an early invariant feature of Alzheimer’s disease (AD), yet the unifying etiological pathway remains largely unknown. Knowing that cyclin-dependent kinase 5 (CDK5) plays critical roles in synaptic formation and degeneration, its phosphorylation targets were reexamined in search of candidates with direct global impacts on synaptic protein dynamics, and the associated regulatory network was also analyzed. Methods Quantitative phosphoproteomics and bioinformatics analyses were performed to identify top-ranked candidates. A series of biochemical assays was used to investigate the associated regulatory signaling networks. Histological, electrochemical, and behavioral assays were performed in conditional knockout, small hairpin RNA–mediated knockdown, and AD-related mice models to evaluate the relevance of CDK5 to synaptic homeostasis and functions. Results Among candidates with known implications in synaptic modulations, BAG3 ranked the highest. CDK5-mediated phosphorylation on S297/S291 (mouse/human) destabilized BAG3. Loss of BAG3 unleashed the selective protein degradative function of the HSP70 machinery. In neurons, this resulted in enhanced degradation of a number of glutamatergic synaptic proteins. Conditional neuronal knockout of Bag3 in vivo led to impairment of learning and memory functions. In human AD and related mouse models, aberrant CDK5-mediated loss of BAG3 yielded similar effects on synaptic homeostasis. Detrimental effects of BAG3 loss on learning and memory functions were confirmed in these mice, and such effects were reversed by ectopic BAG3 reexpression. Conclusions Our results highlight that the neuronal CDK5-BAG3-HSP70 signaling axis plays a critical role in modulating synaptic homeostasis. Dysregulation of the signaling pathway directly contributes to synaptic dysfunction and AD pathogenesis.

Significance Skin pigmentation provides first-line protection against UV radiation (UVR) that increases the risk of skin cancers. However, mechanisms underlying this process remain poorly understood. Here, we identified the autophagic tumor suppressor UVRAG as a bona fide player in melanosome biogenesis by targeting biogenesis of lysosome-related organelles complex 1 (BLOC-1) independently of autophagy. UVRAG maintains the localization and stability of BLOC-1 to facilitate the sorting/delivery of melanogenic cargoes. Reduced levels of UVRAG rendered cells unresponsive to UVR–α-MSH–MITF signaling and defective melanocyte development in vivo. Moreover, UVRAG-mediated melanogenesis and tanning response were impaired in oncogene-driven melanoma. This study represents a description of a noncanonical role of autophagy factor in melanogenic remodeling and also provides mechanistic insights into UVRAG in pigmentation disorder and UV-associated cancer.

Saltified rutin enhances microglial Aβ clearance, providing a potential therapeutic avenue for Alzheimer’s disease treatment., The accumulation of aggregated amyloid-β (Aβ) in the brain is the first critical step in the pathogenesis of Alzheimer’s disease (AD), which also includes synaptic impairment, neuroinflammation, neuronal loss, and eventual cognitive defects. Emerging evidence suggests that impairment of Aβ phagocytosis and clearance is a common phenotype in late-onset AD. Rutin (quercetin-3-rutinoside) has long been investigated as a natural flavonoid with different biological functions in some pathological circumstances. Sodium rutin (NaR), could promote Aβ clearance by increasing microglial by increasing the expression levels of phagocytosis-related receptors in microglia. Moreover, NaR promotes a metabolic switch from anaerobic glycolysis to mitochondrial OXPHOS (oxidative phosphorylation), which could provide microglia with sufficient energy (ATP) for Aβ clearance. Thus, NaR administration could attenuate neuroinflammation and enhance mitochondrial OXPHOS and microglia-mediated Aβ clearance, ameliorating synaptic plasticity impairment and eventually reversing spatial learning and memory deficits. Our findings suggest that NaR is a potential therapeutic agent for AD.

While N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic mRNA, is linked to cell differentiation and tissue development, the biological significance of m6A modification in mammalian glial development remains unknown. Here, we identify a novel m6A reader, Prrc2a (Proline rich coiled-coil 2 A), which controls oligodendrocyte specification and myelination. Nestin-Cre-mediated knockout of Prrc2a induces significant hypomyelination, decreased lifespan, as well as locomotive and cognitive defects in a mouse model. Further analyses reveal that Prrc2a is involved in oligodendrocyte progenitor cells (OPCs) proliferation and oligodendrocyte fate determination. Accordingly, oligodendroglial-lineage specific deletion of Prrc2a causes a similar phenotype of Nestin-Cre-mediated deletion. Combining transcriptome-wide RNA-seq, m6A-RIP-seq and Prrc2a RIP-seq analysis, we find that Olig2 is a critical downstream target gene of Prrc2a in oligodendrocyte development. Furthermore, Prrc2a stabilizes Olig2 mRNA through binding to a consensus GGACU motif in the Olig2 CDS (coding sequence) in an m6A-dependent manner. Interestingly, we also find that the m6A demethylase, Fto, erases the m6A modification of Olig2 mRNA and promotes its degradation. Together, our results indicate that Prrc2a plays an important role in oligodendrocyte specification through functioning as a novel m6A reader. These findings suggest a new avenue for the development of therapeutic strategies for hypomyelination-related neurological diseases.

Chromodomain helicase DNA-binding protein 2 (CHD2) has been associated with a broad spectrum of neurodevelopmental disorders, such as autism spectrum disorders and intellectual disability. However, it is largely unknown whether and how CHD2 is involved in brain development. Here, we demonstrate that CHD2 is predominantly expressed in Pax6+ radial glial cells (RGs) but rarely expressed in Tbr2+ intermediate progenitors (IPs). Importantly, the suppression of CHD2 expression inhibits the self-renewal of RGs and increases the generation of IPs and the production of neurons. CHD2 mediates these functions by directly binding to the genomic region of repressor element 1-silencing transcription factor (REST), thereby regulating the expression of REST. Furthermore, the overexpression of REST rescues the defect in neurogenesis caused by CHD2 knockdown. Taken together, these findings demonstrate an essential role of CHD2 in the maintenance of the RGs self-renewal levels, the subsequent generation of IPs, and neuronal output during neurogenesis in cerebral cortical development, suggesting that inactivation of CHD2 during neurogenesis might contribute to abnormal neurodevelopment. Stem Cells 2015;33:1794–1806

The Hippo signaling pathway restricts organ size by inactivating the Yorkie (Yki)/Yes-associated protein (YAP) family proteins. The oncogenic Yki/YAP transcriptional coactivator family promotes tissue growth by activating target gene transcription, but the regulation of Yki/YAP activation remains elusive. In mammalian cells, we identified Brg1, a major subunit of chromatin-remodeling SWI/SNF family proteins, which interacts with YAP. This finding led us to investigate the in vivo functional interaction of Yki and Brahma (Brm), the Drosophila homolog of Brg1. We found that Brm functions at the downstream of Hippo pathway and interacts with Yki and Scalloped (Sd) to promotes Yki-dependent transcription and tissue growth. Furthermore, we demonstrated that Brm is required for the Crumbs (Crb) dysregulation-induced Yki activation. Interestingly, we also found that crb is a downstream target of Yki–Brm complex. Brm physically binds to the promoter of crb and regulates its transcription through Yki. Together, we showed that Brm functions as a critical regulator of Hippo signaling during tissue growth and plays an important role in the feedback loop between Crb and Yki.

Co-ordination of cell proliferation, differentiation, and apoptosis maintains tissue development and homeostasis under normal or stress conditions. Recently, the highly conserved Hippo signaling pathway, discovered in Drosophila melanogaster and mammalian system, has been implicated as a key regulator of organ size control. Importantly, emerging evidence suggests that Hippo pathway is involved in the responses to cellular stresses, including mechanic stress, DNA damage, and oxidative stress, to maintain homeostasis at the cellular and organic levels. The mutation or deregulation of the key components in the pathway will result in degenerative disorder, developmental defects, or tumorigenesis. The purpose of this review is to summarize the recent findings and discuss how Hippo pathway responds to cellular stress and regulates early development events, tissue homeostasis as well as tumorigenesis.

The H3 histone variant CENP-A is an epigenetic marker critical for the centromere identity and function. However, the precise regulation of the spatiotemporal deposition and propagation of CENP-A at centromeres during the cell cycle is still poorly understood. Here, we show that CENP-A is phosphorylated at Ser68 during early mitosis by Cdk1. Our results demonstrate that phosphorylation of Ser68 eliminates the binding of CENP-A to the assembly factor HJURP, thus preventing the premature loading of CENP-A to the centromere prior to mitotic exit. Because Cdk1 activity is at its minimum at the mitotic exit, the ratio of Cdk1/PP1α activity changes in favor of Ser68 dephosphorylation, thus making CENP-A available for centromeric deposition by HJURP. Thus, we reveal that dynamic phosphorylation of CENP-A Ser68 orchestrates the spatiotemporal assembly of newly synthesized CENP-A at active centromeres during the cell cycle.

The mammalian Ste20-like kinase 1 (Mst-1) is a serine-threonine kinase and a component of the Hippo tumor suppressor pathway, which reacts to pathologically relevant stress and regulates cell death. However, little is known about its role in spinal cord injury. Here, we found that p-Mst-1, the activated form of Mst-1, was induced in the post-traumatic spinal motor neurons. In vivo evidence demonstrated that Mst-1 deficiency promoted post-traumatic spinal motor neuron survival, Basso mouse scale scores, and synapse survival. Moreover, we found that autophagosome formation and autolysosome degradation enhanced by Mst-1 deficiency were crucial to attenuate the death of injured spinal motor neurons. Taken together, our findings demonstrate that Mst-1 deficiency promotes post-traumatic spinal motor neuron survival via enhancement of autophagy flux.

The Hippo signaling pathway is highly conserved from Drosophila to mammals and plays a central role in maintaining organ size and tissue homeostasis. The blood-brain barrier (BBB) physiologically isolates the brain from circulating blood or the hemolymph system, and its integrity is strictly maintained to perform sophisticated neuronal functions. Until now, the underlying mechanisms of subperineurial glia (SPG) growth and BBB maintenance during development are not clear. Here, we report an miR-285-Yorkie (Yki)/Multiple Ankyrin repeats Single KH domain (Mask) double-negative feedback loop that regulates SPG growth and BBB integrity. Flies with a loss of miR-285 have a defective BBB with increased SPG ploidy and disruptive septate junctions. Mechanistically, miR-285 directly targets the Yki cofactor Mask to suppress Yki activity and down-regulates the expression of its downstream target cyclin E, a key regulator of cell cycle. Disturbance of cyclin E expression in SPG causes abnormal endoreplication, which leads to aberrant DNA ploidy and defective septate junctions. Moreover, the expression of miR-285 is increased by knockdown of yki or mask and is decreased with yki overexpression, thus forming a double-negative feedback loop. This regulatory loop is crucial for sustaining an appropriate Yki/Mask activity and cyclin E level to maintain SPG ploidy and BBB integrity. Perturbation of this signaling loop, either by dysregulated miR-285 expression or Yki activity, causes irregular SPG ploidy and BBB disruption. Furthermore, ectopic expression of miR-285 promotes canonical Hippo pathway-mediated apoptosis independent of the p53 or JNK pathway. Collectively, these results reveal an exquisite regulatory mechanism for BBB maintenance through an miR-285-Yki/Mask regulatory circuit.

Neuroinflammation plays a critical role in ischemia-induced brain injury. Mib2, an E3 ubiquitin ligase, has been reported to regulate Notch signaling and participate in the peripheral immune system. However, the roles of Mib2 in the nervous system are not well characterized. In this study, we show that Mib2 is involved in lipopolysaccharide (LPS)- and oxygen-glucose deprivation (OGD)-induced microglial activation. Mechanistically, Mib2 interacts with the IKK complex and regulates the activation of NF-κB signaling, thus modulating Notch1 transcription in the microglia. Furthermore, we generated a microglia-specific Mib2 knockout mice and found that microglia-specific deletion of Mib2 significantly alleviates ischemia-induced neuroinflammation and brain injury. Taken together, our results reveal a critical role of Mib2 in microglial activation and ischemia-induced brain injury, thus providing a potential target for the treatment of stroke.

E2F1 and FOXO3 are two transcription factors that have been shown to participate in cellular senescence. Previous report reveals that E2F1 enhanced cellular senescence in human fibroblast cells, while FOXO transcription factors play against senescence by regulation reactive oxygen species scavenging proteins. However, their functional interplay has been unclear. Here we use E2F1 knock-out murine Embryonic fibroblasts (MEFs), knockdown RNAi constructs, and ectopic expression of E2F1 to show that it functions by negatively regulating FOXO3. E2F1 attenuates FOXO3-mediated expression of MnSOD and Catalase without affecting FOXO3 protein stability, subcellular localization, or phosphorylation by Akt. We mapped the interaction between E2F1 and FOXO3 to a region including the DNA binding domain of E2F1 and the C-terminal transcription-activation domain of FOXO3. We propose that E2F1 inhibits FOXO3-dependent transcription by directly binding FOXO3 in the nucleus and preventing activation of its target genes. Moreover, knockdown of the Caenorhabditis elegans E2F1 ortholog efl-1 significantly extends lifespan in a manner that requires the activity of the C. elegans FOXO gene daf-16. We conclude that there is an evolutionarily conserved signaling connection between E2F1 and FOXO3, which regulates cellular senescence and aging by regulating the activity of FOXO3. We speculate that drugs and/or therapies that inhibit this physical interaction might be good candidates for reducing cellular senescence and increasing longevity.

Reactive oxygen species (ROS) are key regulators in cell proliferation, survival, tumor initiation and development. However, the role of ROS in tumor metastasis is less clear. Here, we show that oxidative stress inhibited tumor metastasis via activation of Hippo kinase MST1/2, which led to the phosphorylation and nuclear accumulation of FoxO3a, resulting in upregulation of ΔNp63α expression and suppression of cell migration independent of YAP. Strikingly, while loss of MST1 led to and disruption of cell-cell junction exemplified by reduced E-cadherin expression, resulting in scattered cell growth, loss of MST2 led to disruption of cell-matrix adhesion as evidenced by reduced integrin β4, resulting in increased cell migration and tumor metastasis. Furthermore, expression of MST1 and MST2 was down-regulated in human breast carcinoma. Furthermore, oxidative stress inhibited HER2-or PI3K-mediated tumor metastasis via the MST2-FoxO3a-ΔNp63α pathway. Together, these results that this noncanonical Hippo MST2-FoxO3a-ΔNp63α pathway may play a critical role in ROS-mediated regulation of cell migration and tumor metastasis. Keywords: Hippo kinase, Metastasis, ΔNp63α, Cell-cell junction, Cell-matrix adhesion