PHLDA1 deficiency limits microglia activation and protects dopaminergic neurons through attenuating auto-ubiquitination of TRAF6

OBJECTIVE Pleckstrin homologylike domain family A member 1(PHLDA1),also known as TDAG51,was first identified as a potential transcription factor required for Fas expression and activation-induced apoptosis in mouse T cell hybridomas.Subsequent research showed that PHILDA1 plays crucial roles in cell proliferation and apoptosis,PHLDA1 deficiency caused several phenotypic changes in macrophages that increased cytoprotection against oxidative and endoplasmic reticulum(ER) stress.However,little is known about the critical role of PHLDA1 in the neuroinflammation and neuronal apoptosis.METHODS Mouse Parkinson disease(PD) model was induced by intraperitoneal injection(ip) of MPTP for 7 d.Immunohistofluorescence staining was used to observe TH,Iba-1 and PHLDA1 in brain slides.Primary microglia and BV2 microglial cell lines were exposed to lipopolysacchrides(LPS) at different time points to determine PHLDA1 mRNA and protein level in vitro.BV2 cells with PHLDA1 knockdown using lentivirus experessing shRNA were exposed to LPS for the detection of proinflammatory genes expression by q-PCR or Western blotting.The NF-κB and MAPK pathways were detected to reveal the underlying mechanisms of PHLDA1 regulate microglia activation.Furthermore,to determine the effect of PHLDA1 deletion in the substantia nigra(SN) on motor dysfunction,the mice were previously infected with AAV-GFP(control) or AAV-shP HLDA1 in the SN in PD model,then were administered with behavior assessment.RESULTS PHLDA1 was expressed in microglia.PHLDA1 was increased in primary microglia or BV2 microglia cell lines by LPS treatment,acted as a positive regulator of TLR4 signaling.PHLDA1 deficiency suppressed LPS-induced microglia activation.The underlying mechanisms of PHLDA1 might be through directly interacting with TRAF6 to promote its auto-ubiquitination consequently enhanced neuroinflammatory.In PD model,PHLDA1 deletion in the SN limits microglial activation and protects dopaminergic neurons.PHLDA1 deficiency mice have a good behavioral score.CONCLUSION PHLDA1 deficiency in SN limits microglial inflammatory responses and protects dopaminergic neurons through attenuating auto-ubiquitination of TRAF6 in PD model.

Small-molecule inhibitor of smoothend suppress neuroinflammation through hedgehog signaling independent mechanisms

OBJECTIVE Microglial activation mediated neuroinflammation plays an important role in the progression of neurodegerative diseases.The purpose of this study was to investigate the effect of small-molecule inhibitors of smoothend(Smo) on microglial activation mediate neuroinflammation.METHODS To search for the novel anti-neuroinflammatory agents,the BV-2 cel-based nitric oxide(NO) assay was used to screen a series of small-molecule inhibitors of Smo.The production of NO and cell viability were detected by Griess and MTT assay respectively.The expression of inflammatory factors were evaluated by Real-time quantitative PCR or Western blotting or ELISA assay,and activation of signal molecules were detected by Western blotting.The dopaminergic neuronal cell apoptosis were measured by flow cytometry.The stereotaxic injection of LPS into the mice Substantia Nigra(SN) were employed to establish animal model of neuroinflammation and immunofluorescence staining on brain slices sections were used to verify microglial activation and dopaminergic cell loss.RESULTS To search for the compounds that inhibit microglial activation,we have screened a series of Smo inhibitors(0.01-40 μmol·L^(-1)) for the activity to inhibit NO production in BV-2 microglia cells.Among the Smo inhibitors tested,Hh-079 strongly inhibited LPS-induced microglial NO production without affecting the cell viability.Hh-079 significantly inhibited the expression of proinflammatory factors in LPSstimulated BV-2 microglial cells.Hh-079 inhibited phosphorylation of IKKα/β,phosphorylation and degradation of IκBα,phosphorylation of P65 subunit of NF-κB and NF-κB transcriptional activity in LPS-stimulated BV-2 microglial cells.Hh-079 reduced cytotoxicity of conditioned media of activated microglia toward HT-22 neuroblastoma cells in a co-culture system.Mechanistic studies demonstrated that among the Shh-Ptch-Gli pathway,microglia cells did not express detectable levels of Shh,Smo and Gli.Furthermore,neither Smo inhibitor cyclopamine no Gli inhibitor GANT61 exhibited inhibitory properties on proinflammatory genes expression in LPS activated microglia cells.In addition,co-treatment of recombinant Shh or Smo agonist SAG did not block inhibitory effects of Hh-079 on proinflammatory gene expression in LPS activated microglia cells.In vivo study demonstrated that pretreatment of Hh-079(25 and 50 mg·kg-1) significantly reduced TH-positive cells loss and microglia cells activation in the LPS induced neuroinflammation mouse model.CONCLUSION Smo inhibitor Hh-079 suppress neuroinflammation through hedgehog signaling independent mechanisms.

Discovery of a Novel 5-HT_(2A) Inhibitor by Pharmacophore-based Virtual Screening

The serotonin 2A（5-HT2A） receptor has been implicated in several neurological conditions and potent 5-HT2A antagonists have therapeutic effects in the treatment of schizo phrenia and depression.In this study,a potent novel 5-HT2A inhibitor 05245768 with a Ki value of （593.89±34.10） nmol/L was discovered by integrating a set of computational approaches and experiments（protein structure prediction,pharmacophore-based virtual screening,automated molecular docking and pharmacological bioassay）.The 5-HT2A receptor showed a negatively charged bin-ding pocket.The binding mode of compound 05245768 with 5-HT2A was obtained by GOLD docking procedure,which revealed the conserved interaction between protonated nitrogen in compound 05245768 and carboxylate group of D3.32 at the active site of 5-HT2A.

Inhibition of glycolysis mitigate microglial-activation mediated neuroinflammation in vitro and in vivo

OBJECTIVE Microglial activation-mediated neuroinflammation plays an important pathological basis in the progression of many neurodegenerative diseases.Activated microglia cells show a metabolic shift from oxidative phos⁃phorylation to aerobic glycolysis.However,the molecular mechanism underlying the role of glycolysis in microglial activation and progres⁃sion of neuroinflammatory diseases have not yet been fully understood.METHODS The anti-inflammatory effects and its underlying mecha⁃nisms of glycolytic inhibition in vitro were exam⁃ined in lipopolysaccharide(LPS)activated BV-2 microglial cells or primary microglial cells by enzyme-linked immunosorbent assay(ELISA),quantitative reverse transcriptase polymerase chain reaction(RT-PCR),Western blotting,immunoprecipitation,Flow cytometry and nuclear factor kappa B(NF-κB)luciferase reporter assays.In vivo,the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-or LPS-induced Par⁃kinson disease(PD)models were constructed to explored the anti-inflammatory and neuropro⁃tective effects of glycolytic inhibitor.RESULTS Inhibition of glycolysis by specific inhibitors[2-DG and 3-bromopyruvic acid(3-BPA)],knockdown of glucose transporter type 1(Glut-1)or hexoki⁃nase(HK)Ⅱabolished LPS-induced expres⁃sion of proinflammatory genes in microglia cells.Mechanistic studies demonstrated that glyco⁃lytic inhibitors significantly inhibited LPS-induced mTOR phosphorylation,IKKβphosphorylation,IκB phosphorylation,IκB degradation,nuclear translocation of P65 and NF-κB luciferase activity.Furthermore,LPS-induced P65 acetyla⁃tion on lysine 310,which is mediated by NAD-dependent protein deacetylase sirtuin-1 and is critical for NF-kB activation,were inhibited by glycolytic inhibitors.A coculture study revealed that 2-DG reduced the cytotoxicity of activated microglia toward MES23.5 dopaminergic neuron cells with no direct protective effect.In vivo,2-DG significantly ameliorated MPTP or LPS induced DA neuron loss and glial cell activation.CONCLUSION Glycolysis is actively involved in microglial activation.Inhibition of glycolysis can ameliorate microglial activation-related neuroinflammatory diseases.

PDE10A inhibitor in addiction

Chronic exposure to drugs of abuse will give rise to persistent structural and functional changes in the central nervous system. These phenomena are usually referred as ′drug-induced neuroplasticity′ and depend on changes in gene expression. The cAMP response element binding protein(CREB),as a downstream molecule in mediating the actions of cAMP is an important transcriptional factor in establishing and maintaining addiction to drugs of abuse. Application of a PDE4 inhibitor attenuates the rewarding properties of cocaine and morphine. Given the fact that PDE10A is specifically located in striatum,an important structure involved in the reward circuit,we thus investigated the PDE10A inhibitormodulated the behavioral reinforcement exerted by morphine. The results show that MP-10 2.5 mg·kg^(-1),administered subcutaneously,significantly inhibited the acquisition of morphine-induced CPP. Moreover,MP-10 did not alter the expression of morphine-induced CPP,but did accelerate the extinction of morphine-induced CPP. Additionally,chronic treatment with MP-10 2.5 mg·kg^(-1)decreased expression of phosphorylated CREB(pC REB) in dorsomedial striatum,in shell of NAc,and in anterior cingulate cortex(ACC) as well as decreased expression of ΔFos B in the shell of NAc and ACC. These data indicate that PDE10A inhibition may have a potential therapeutic effect on addiction. Since the MP-10 has relative short metabolic Stability,we also developed a few novel potent new PDE10A selective inhibitor with improved stability and brain exposure. The new compound exhibited promising potential in schizophrenia and addiction treatment.

S4B-3 Targeted to Serotonergic and Adenosine Systems in Parkinsons Disease Drug Development

Parkinson’s disease(PD)is a progressive neurodegenerative disorder.While dopaminergic agents are effective in reliving the motor symptoms,however,chronic dopamine replacement treatment could also induce motor fluctuation and dyskinesia.In addition,there is still a clinical unmet need for novel medications that can treat the non-motor symptoms.Recent evidence suggests that the serotonergic nervous system may be an important target for both motor and non-motor symptoms for PD Meanwhile,adenosine A2A receptors enriched in basal ganglia areas and their antagonistic role towards dopamine receptor stimulation,have positioned A2A receptor antagonists as an a promising target to improve the motor deficits.We have developed a series of dual dopamine receptor and 5-HT1A receptor ligands and A2A receptor antagonists either through natural resource or rational design.We pharmacologically characterized those compounds and provided clear evidences that dual 5-HT/dopamine or A2A receptor antagonist exhibited excellent anti-Parkinsonian effect while it reduced the development of dyskinesia and effectively relieved some of the non-motor symptoms in PD animal models.