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Mechanisms of calcium independent but voltage dependent secretion(CiVDS) in somata of mammalian primary sensory neurons

Mechanisms of calcium independent but voltage dependent secr...

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第六届亚太神经科学联合会学术会议暨中国神经科学学会第十一届全国学术会议

作者： Zuyin Chai Xiaoyu Zhang Rong Huang Yuan Wang Rong Zhou Qihui Wu Meiqin Hu Xi Wu Haowen Liu Tao Liu Yeshi Wang Wei Guo Hui Zheng Wei Xiong Chen Zhang Liang-Hong Zheng Claire Xi Zhang Jiu-Ping Ding Changhe Wang Zhuan Zhou State Key Laboratory of Biomembrane and Membrane Biotechnology Institute of Molecular MedicinePeking University Institute of Biophysics and Biochemistry School of Life SciencesHuazhong University of Science and Technology

The somata of primary sensory neurons, including dorsal root ganglion(DRG) neurons, release neurotransmitters and neuropeptides. Following physiological action potentials, in addition to Ca2+-dependent secretion, we have discovered and studied Ca2+-independent but voltage-dependent secretion(Ci VDS) in somata of freshly isolated DRG neurons(Zhang et al, 2002, 2004;Zheng et al, 2009, Liu et al, 2011). Major open question of CiV DS is molecular mechanism, including 3 components: fusion pore machinery(FP), voltage sensor(VS) and the FP-VS linker(LK). Here we report, by using exocytosis assays of patch-clamp recording of membrane capacitance, and single vesicle imaging(EM and TIRF),(1) FP is jointly contributed by 2 components of FP-complex SNARE, SNAP25 and syntaxin;(2) VS is jointly contributed by 2 voltage-gated Ca channels(VGCCs), Cav2.2(N-type VGCC) and Cav1.3(L-type VGCC);(3) LK is the "synprint", Cav-S2-S3 intracellular loop718-963aa(Catterall, 1999);(4) following automatic knockdown of CiV DS by 3d-culture of DRG neurons, CiV DS is rescued by overexpressing any component of FP(SNAPE25or syntaxin) or VS(Cav2.2 or Cav1.3);(5) CiV DS is inhibited by blockers against FP(SNAP25 or sytaxin), Cav(2.2 or 1.3) and FL(synprint-truncated);(6) finally, CiV DS is blocked by Cav2.2-RNAi-KD in DRG pre-transfected in vivo.

关键词： DRG CiVDS Mechanisms of calcium independent but voltage dependent secretion in somata of mammalian primary sensory neurons

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L-Stepholidine Rescues Memory Deficit and Synaptic Plasticity in Models of Alzheimer's Disease via Activating Dopamine D1 Receptor/PKA Signaling Pathway

L-Stepholidine Rescues Memory Deficit and Synaptic Plasticit...

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第六届亚太神经科学联合会学术会议暨中国神经科学学会第十一届全国学术会议

作者： Jing-Ru Hao Nan Sun Lei Lei Xing-Yu Li Bin Yao Kai Sun Xue Zhang Rui Hu Xiao-Dong Shi Can Gao Jiangsu Key Laboratory of Anesthesiology Xuzhou Medical College Jiangsu Key Laboratory of Anesthesia and Analgesia Application Xuzhou Medical College

It's accepted that amyloid β-derived diffusible ligands(ADDLs) play a prominent role in triggering the early cognitive deficits that constitute Alzheimer's disease(AD). But there is still no effective treatment for preventing or reversing the progression of the disease. Targeting AMPA receptors trafficking and its regulation is a new strategy for AD early treatment. Here, we investigate the effect and mechanism of L-Stepholidine(L-SPD), which elicits dopamine D1-type receptor agonistic activity while acts as D2-type receptor antagonist on cognition and synaptic plasticity in amyloid precursor protein(APP) and presenilin 1(PS1) double-transgenic(APP/PS1) mice and hippocampal cultures or slices treated with ADDLs. L-SPD could improve the hippocampus-dependent memory, surface expression of Glu A1-containing α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid(AMPA) receptors and spine density in hippocampus of APP/PS1 transgenic mice. L-SPD not only rescued decreased phosphorylation and surface expression of Glu A1 in hippocampal cultures but also protected the LTP in hippocampal slices induced by ADDLs. Protein kinase A(PKA) agonist Sp-c AMPS or D1-type receptor agonist SKF81297 had similar effects, while PKA antagonist Rp-c AMPS or D1-type receptor antagonist SCH23390 abolished the effect of L-SPD on Glu A1 trafficking. This was mediated mainly by PKA, which could phosphorylate serine residue at 845 of the Glu A1. L-SPD may be explored as a potential therapeutic drug for AD through a mechanism that improves AMPA receptors trafficking and synaptic plasticity via activating D1/PKA signaling pathway.

关键词： Amyloid β-derived diffusible ligands Alzheimer's disease α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid(AMPA) receptors trafficking cognitive deficits L-Stepholidine synaptic plasticity

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The critical period of TRP channels in pain development

The critical period of TRP channels in pain development

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第六届亚太神经科学联合会学术会议暨中国神经科学学会第十一届全国学术会议

作者： Lei-Jun Xue Wei Zhu Jia Li Qiu-Hang Ran Wen-Tao Shi Qing-Hu Yang Xia Liu Zhi-Hong Sun Zhan-Tao Bai College of Life Sciences Yanan University Shaanxi Engineering & Technological Research Center for Conversation & Utilization of Regional Biological Resources Shaanxi Provincial Key Lab of Red Dates

Transient receptor potential(TRP) proteins are a family of ion channels central for sensory signaling. These receptors and, in particular, those involved in cold /hot sensing are also involved in pain signaling. Multiple TRP channels are present in all cells and are involved in diverse aspects of cellular function, including sensory perception and signal transduction. However, rat behavioral responses and molecule mechanism of TRP channel participating in pain sensation, especially during development, are still far from clear. Here we showed that with differential temperature stimulation, rat behaviors including paw flinching, moving, head scratching, body shaking paw licking and sex excitation were distinctly evoked. During development, rat tolerance to cold /hot temperature was increased. What's more, our results showed that there was an insensitive period of pain development during the second to fourth weeks. The expression of TRP channels, such as TRPV1, TRPV2, TRPV3, TRPV4, TRPM8 and TRPA1, highly expressed in 2 to 4 weeks. Those demonstrated that rat TRP channels-related cold/hot detection demonstrates age- and temperature-related differentiation, and the second to fourth weeks after postnatal maybe the critical period for maturation of somatic pain sensation. These would further us understanding the mechanisms of TRP-related pain and sound child rearing during development.

关键词： TRP channels Critical period Development Pain

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SUMO-specific protease 1 is critical for neuroprotection in transient brain ischemia/reperfusion

SUMO-specific protease 1 is critical for neuroprotection in ...

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第六届亚太神经科学联合会学术会议暨中国神经科学学会第十一届全国学术会议

作者： Huijun Zhang Yan Wang Dehua Huang Hao Sun Chao Huang Jinke Cheng Michael X.Zhu Yong Li Department of Biochemistry and Molecular Cell Biology Shanghai Key Laboratory for Tumor Microenvironment and InflammationInstitute of Medical SciencesShanghai Jiao Tong University School of Medicine Department of Integrative Biology and Pharmacology The University of Texas Health Science Center at Houston

SUMO-specific protease 1(SENP1) deconjugates SUMO from modified proteins. Although post-ischemic activation of SUMO conjugation was suggested to be neuroprotective against ischemia/reperfusion(I/R) injury, the function of SENP1 in this process remained unclear. Here we show that transient middle cerebral artery occlusion(t MCAO) in mice followed by 6, 12 and 24 h reperfusion significantly enhanced SENP1 levels in the affected brain area, independent of transcription. Conditional ablation of SENP1 gene in principal neurons of adult mice, using a SENP1flox/flox;Cam KII-cre(SENP1 c KO) line, resulted in a significant increase in infarct volume in cerebral cortex and more severe motor impairment in response to I/R as compared to the wild type littermates. Cortical neurons from I/R injured SENP1 c KO mice became more apoptotic than wild type neurons, as indicated by both TUNEL staining and caspase-3 activation. Moreover, consistent with the increase in SENP1 levels, I/R induced an overall decrease in SUMO1-conjugation of cortical neuron proteins including the pro-apoptotic protein, dynamin-related protein 1(Drp1), and a decrease in Drp1 protein level. These changes were absent in SENP1 c KO neurons. We conclude that SENP1 plays a neuroprotective role in I/R injury by inhibiting apoptosis through decreasing SUMO1 conjugation and destabilization of Drp1. These findings reveal a novel mechanism of neuroprotection by protein desumoylation, which may have important implications for stroke therapy.

关键词： transient middle cerebral artery occlusion(tMCAO) SENP1flox/flox CamKll-cre(SENP1 cKO) SUMO-specific protease 1(SENP1) neurological deficits apoptosis Drp1

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MicroRNA-132 and early growth response-1 in Nucleus Basalis of Meynert during the course of Alzheimer's disease

MicroRNA-132 and early growth response-1 in Nucleus Basalis ...

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第六届亚太神经科学联合会学术会议暨中国神经科学学会第十一届全国学术会议

作者： Qiong-Bin Zhu Unga Unmehopa Koen Bossers Ronald Verwer Rawien Balesar Juan Zhao Ai-Min Bao Dick Swaab Department of Neurobiology Key Laboratory of Medical Neurobiology of Ministry of Health of China Zhejiang Province Key Laboratory of Neurobiology Zhejiang University School of Medicine Netherlands Institute for Neuroscience an Institute of the Royal Netherlands Academy of Arts and Sciences

The cholinergic Nucleus Basalis of Meynert(NBM), which is important for memory functions, shows neuronal activation(‘up-phase') during the early stages of Alzheimer's disease(AD) and neurodegeneration(‘down-phase') in later stages of AD. Micro RNA-132(mi R-132) and the transcription factor early growth response-1(Egr-1) were proposed as possible candidate molecules regulating such an up-down activity pattern of the NBM during the course of AD, since they both show this up-down pattern of expression in the prefrontal cortex during the course of AD. These two molecules not only stimulate synaptic activity and plasticity, but are also involved in AD pathology and might, in addition, affect cholinergic function. In the NBM we investigated the expression of mi R-132 and Egr-1 along the entire course of AD. Forty-nine postmortem NBM samples were studied, ranging from non-demented controls(Braak stage = 0) to late AD patients(Braak stage = VI). Each stage contained 7 samples, all well-matched for confounding factors of age(range 58～91), sex, postmortem delay, cerebrospinal fluid p H(as a measure for agonal state), ApoE genotype, clock time of death, tissue fixation time, and tissue storage *** alterations of these two molecules were studied over the course of AD in relation to the expression of β-amyloid(Aβ), hyperphosphorylated tau, neuronal fibrillary tangles and neuropil threads, ApoE ε4 and in relation to alterations in choline acetyltransferase(Ch AT). We found that the expression of mi R-132 and Egr-1 in the NBM was quite stable during the early stages of AD and decreased significantly only during late AD stages. In addition, mi R-132 and Egr-1 respectively showed a significant positive correlation with Ch AT expression, while Ch AT expression showed a significantly negative correlation with hyperphosphorylated tau. From the functional changes of mi R-132 and Egr-1 along the course of AD we conclude i) that these two molecules may play a role in keeping the chol

关键词： Alzheimer's disease,microRNA-132,Early growth response-1,Nucleus Basalis of Meynert

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The influence of maternal stress-induced dysregulation of neuroinflammatory responses on neurodevelopment and behaviors in offspring

The influence of maternal stress-induced dysregulation of ne...

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第六届亚太神经科学联合会学术会议暨中国神经科学学会第十一届全国学术会议

作者： Qiu-Ying Zhao Jin-Qiang Zhang Shuo Yan Zi-Li You School of Life Science and Technology University of Electronic Science and Technology of China

Objective Clinical investigation indicates that prenatal stress could result in adverse neuroanatomical, biological and behavioral consequences in the offspring. Chronic stress results in an increased neuroinflammatory responses via increases in pro-inflammatory molecules and decreases in anti-inflammatory cytokines(You et al., 2011), which contributed to an inflammatory environment on the fetal development. Microglia play a central role as modulators of the neural stem cells niches and have inhibitory or promote regulation on neurogenesis dependent on pro-inflammatory(M1) or anti-inflammatory(M2) microglial activation. In this study, we detect the effects of maternal sleep deprivation(MSD) on neurodevelopment and behaviors in offspring and its underlying inflammatory pathway. Methods Pregnant rats were sleep-deprived for 72 hours by modified water-platforms. The male offspring at 21 days old were used in this study. Hippocampus-dependent spatial learning and memory were estimated by the Morris Water Maze and anhedonia was determined in sucrose preference. Neurogenesis was confirmedby Immunohistochemistry(IHC)(Brd U and DCX label) in hippocampus of brain section. Inflammatory cytokines(IL-1β, IL-6, TNFα, i NOS and IL-10) and microglial activated phenotype(classical activation: M1 phenotype;alternative activation: M2 phenotype) were determined by Real Time PCR, ELISA and IHC. Minocycline was injected into intraperitoneal at 50 mg/kg for 5 days to inhibit microglial activation. Results(1) The male offspring with MSD showed impaired spatial learning and memory and anhedonia, decreased neurogenesis(Zhao et al., 2014).(2) Microglia showed predominant M1 phenotype with a dysregulation between pro- and anti-inflammatory cytokines in MSD offspring. After minocycline injection, pro-inflammatory cytokines decreased and anti-inflammatory cytokines increased released from activated microglia.(3) Minocycline enhanced neurogenesis and improved behavioral deficits in the MSD offsp

关键词： neuroinflammation microglial phenotype minocycline neurogenesis maternal sleep deprivation

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Neuropathic pain affected P2X3 receptor expression and function in DRG neurons of neonatal and adult rats

Neuropathic pain affected P2X3 receptor expression and funct...

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第六届亚太神经科学联合会学术会议暨中国神经科学学会第十一届全国学术会议

作者： Hui-Zhong Wen Lu Du Xi-Gui Wu Shi-Hao-Gao Shao-Jun Wang Jian Cui Yan-Dong Zhao Huai-Zhen Ruan Department of Neurobiology Chongqing Key Laboratory of NeurobiologyThird Military Medical University

Nervous system of the neonatal is characterized by greater plasticity and reorganization, therefore, is particularly susceptible to exogenous and endogenous insults. Neonatal pain experiences may exert a profound and long-lasting influence on nervous system. Up-regulation of P2X3 receptor in dorsal root ganglion(DRG) has impacts on behavioral responses to pain of adult rats. While relatively little is known about the influence of neonatal pain on DRG P2X3 receptor. In this study, immunohistochemistry and western blotting results suggested that the sciatic nerve chronic constriction injury(CCI) has lead to down-regulation of P2X3 receptor expressed in DRG neurons of neonatal rats(p<0.01 compare to control). Patch clamp results reflect that the electrophysiological functions of P2X3 receptors were increased in both neonatal and adult CCI rat models. Chronic neuropathic pain is triggered and maintained by peripheral and central mechanisms. Although neonatal rats have clear reflex withdrawal to brief noxious stimulation, their ability to build a significant and maintained hyperalgesic response to inflammatory stimuli is weaker at birth, and is established until the second postnatal week. Electrophysiological function of P2X3 receptors of adult and neonatal rats showed the same tendency, the EPSCs amplitude of CCI of neonatal group and adult group were increased than controls(p<0.01 compare to control). While the P2X3 receptors expression was decreased in CCI models. These findings suggest that early traumatic and painful injuries have profound impact on the development of pain-processing circuitry through effect the expression of P2X3 receptor. Peripheral nerve injury causes structural and functional alterations in peripheral sensory pathways and these differences obviously in neonate than *** neurons of different mature stage respond differently to the same damage. If there was any mechanisms for regulating functions and expressions of P2X3 receptor to mainta

关键词： P2X3 receptor Neuropathic pain Neonate Adult

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The toxicity effect of ALLN on primary rat retinal neurons

The toxicity effect of ALLN on primary rat retinal neurons

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第六届亚太神经科学联合会学术会议暨中国神经科学学会第十一届全国学术会议

作者： Na Li Lei Shang Shu-Chao Wang Lu-Shuang Liao Dan Chen Kun Xiong Ju-Fang Huang Department of Anatomy and Neurobiology School of Basic Medical SciencesCentral South University

Objective N-acetyl-L-leucyl-L-leucyl-L-Norleucina(ALLN), a tripeptidic inhibitor of proteasomes and calpain, is used to reduce proteasomes or calpain-mediated cell death in several neurodegenerative diseases. However, ALLN is toxic to retinal neurons to some extent. The optimal concentration of ALLN for protecting retinal neurons is unclear, and the precise mechanism of cell death induced by ALLN remains elusive. This present study is to investigate the toxicity effect of ALLN on primary retinal neurons. Methods Primary rat retinal neurons were cultured and the neurons were treated with different concentrations(1, 2.5, 5 and 7.5 μM) of ALLN for 6, 12 and 24 hours. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay was used to test cell viability. Necrosis was examined by lactate dehydrogenase(LDH) release and propidium iodide(PI) staining. Apoptosis was measured by the protein levels of caspase 3, the ratio of Bax/Bcl-2 and Annexin V-FITC staining. Autophagy was detected by the ratio of microtubule-associated protein 1 light chain 3(LC3) II/LC3 I and monodansylcadaverine(MDC) staining. Results(1) MTT assay showed no significant difference at the concentration of 1 μM(p>0.05) but decreased cell viability at the concentration of 2.5, 5 and 7.5 μM(p<0.01) after all treatment time compared with control group.(2) LDH release was highly elevated and PI positive cells were significantly increased at the concentration of 2.5, 5 μM(p<0.01) and 7.5 μM(p<0.05) after all treatment time.(3) Western blot showed that protein levels of caspase 3 were up-regulated at the concentration of 2.5, 5 and 7.5 μM after 12 hours(p<0.01) and 24 hours(p<0.05) of ALLN treatment. The ratio of Bax/Bcl-2 was raised and Annexin V positive cells were increased at the concentration of 5 and 7.5 μM after 12 and 24 hours of ALLNtreatment(p<0.05).(4) There is no significant difference in both the ratio of LC3 II/LC3 I and MDC staining between ALLN treatment groups and control

关键词： ALLN toxicity retinal neuron necrosis apoptosis

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Methylene Blue improves the disruption of blood brain barrier and attenuates astroglia activation after cerebral ischemia/reperfusion injury in rats

Methylene Blue improves the disruption of blood brain barrie...

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第六届亚太神经科学联合会学术会议暨中国神经科学学会第十一届全国学术会议

作者： Min Wu Qing Fang Zhong-Fang Shi Li-Xin Xu Li-Ping Dong Xu Yan Shao-Hua Yang Fang Yuan Department of Pathophysiology Beijing Neurosurgical InstituteCapital Medical University Department of Pharmacology and Neuroscience University of North Texas Health Science Center

Methylene blue(MB) was first synthesized in 1876 and has been used as treatment for malaria, methemoglobinemia, and cyanide poisoning. Recently, MB has been demonstrated to be neuroprotective in models of ischemic stroke and Parkinson's disease due to its capability as an alternative mitochondria electron carrier. However, the effects of the MB on the blood-brain barrier(BBB) and astrocytes after cerebral ischemia/reperfusion have not been ***, in this study, by using a middle cerebral artery occlusion(MCAO) and reperfusion rat model, the changes of BBB and astrocytes were examined at 48 hours after MCAO with or without MB treatment. Adult male rats were subjected to transient focal cerebral ischemia induced by MCAO for 1 hour, and followed by reperfusion for 47 hours. MB(3mg/kg) was infused intravenously immediately after reperfusion and MB(1.5 mg/kg) again at 2 hours after reperfusion. Normal saline was administered as vehicle control. A similar procedure without MCAO was conducted in normal rats. Successful establishment of MCAO was determined when r CBF decreased to 20% of baseline levels before ischemia. The classic hematoxylin-eosin(HE) staining technique was used to observe the histological injury. Albumin immunohistochemistry was used to evaluate the permeability of the BBB and the changes of astrocytes were assessed by immunohistochemical staining of glial fibrillary acidic protein(GFAP). Compared with the sham-operated group, the HE staining showed that neurons and blood vessels in the vehicle-control group were not intact in the ischemic penumbra, while in the MB-treatment group the integrity of neurons and blood vessels were improved. Immunohistochemical staining showed that there was no macroscopical albumin extravasation in the sham-operated group, but there was massive extravasation of albumin in the vehicle-control groups, suggesting that permeability of the BBB increased. The area and appearance of albumin leakage was much smaller in

关键词： methylene blue transient middle cerebral artery occlusion blood-brain barrier astrocyte

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Neuronal reprogramming and therapeutic application of NG2 glial precursor cells

Neuronal reprogramming and therapeutic application of NG2 gl...

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第六届亚太神经科学联合会学术会议暨中国神经科学学会第十一届全国学术会议

作者： Hai-Jie Yang Pei-Jun Ju Lei Wang Si Zhang Zhi-Wei Feng School of Life Science and Technology Xinxiang Medical University Shanghai Mental Health Center Shanghai Jiao Tong University School of Medicine

NG2-expressing cells in CNS consist of heterogeneous cell populations. They proliferate rapidly in response to injuries, making them a promising cell target for various neural diseases. In order to elucidate their lineage potential and therapeutic value, we firstly establish cell populations by cloning NG2+ cells and evaluated their differentiation capacity. In vitro differentiation analysis revealed that all the clones could differentiate into oligodendrocytes, and some of them were bipotent of differentiating into oligodendrocytes and astrocytes. Interestingly, one clone exhibited a multipotent capacity of differentiating into not only neuronal and glial lineages, but also chondrocytes. These subtypes of NG2+ cells also exhibit phenotypic heterogeneity according to a spectrum of neural progenitor markers(NG2, PDGFαR, Nestin, and A2B5). These results collectively reveal that NG2+ cells contain heterogeneous populations with various differentiation capacities. We then explored the therapeutic role of neurogenesis from NG2+ cells in spinal cord injury. In experimental contusive spinal cord injury(SCI) model, we found that antagonizing function of EGFR with a specific EGFR inhibitor(PD168393) caused significant amount of neuronal phenotypes from NG2+ cells(either in situ or through ex-vivo transplantation) in mouse SCI, which presumably led to accumulation of newly-generated neurons. Additionally, by attenuating EGFR signaling in the injured niche, improved behavioral performance and histological protection were also observed. Lastly, NG2+ produced astrogliosis and microglia/macrophages reduced, and improved myelination from NG2+ cells increased. Our results demonstrated that NG2+ cells, well-known as glial progenitors, could be manipulated for repairing neuronal loss post SCI. These findings support the possibility of evoking endogenous neuronal replacement from NG2+ cells and suggest that EGFR inhibition may be beneficial for treating CNS trauma.

关键词： NG2+ cells,neuronal reprogramming,Spinal cord injury,EGFR

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