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Propofol Attenuates a-synuclein Aggregation and Neuronal Damage in a Mouse Model of Ischemic Stroke

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Neuroscience Bulletin 2020年第3期36卷 289-298页

作者： Yuzhu Wang Dan Tian Changwei Wei Victoria Cui Huan Wang Yanbing Zhu Anshi Wu Yun Yue Department of Anesthesiology Beijing Chaoyang HospitalCapital Medical UniversityBeijing 100020China Experimental and Translational Research Center Beijing Friendship HospitalCapital Medical UniversityBeijing 100050China Washington University School of Medicine St.LouisMIUSA

a-synuclein is a soluble monomer abundant in the central nervous system. Aggregates of a-synuclein,consisting of higher-level oligomers and insoluble fibrils,have been observed in many chronic neurological diseases and are implicated in neurotoxicity and neurodegeneration.a-synuclein has recently been shown to aggregate following acute ischemic stroke, exacerbating neuronal damage.Propofol is an intravenous anesthetic that is commonly used during intravascular embolectomy following acute ischemic stroke. While propofol has demonstrated neuroprotective properties following brain injury, the mechanism of protection in the setting of ischemic stroke is unclear. In this study, propofol administration significantly reduced the neurotoxic aggregation of a-synuclein, decreased the infarct area, and attenuated the neurological deficits after ischemic stroke in a mouse model. We then demonstrated that the propofol-induced reduction of a-synuclein aggregation was associated with increased mammalian target of rapamycin/ribosomal protein S6 kinase beta-1 signaling pathway activity and reduction of the excessive autophagy occurring after acute ischemic stroke.

关键词： Propofol a-synuclein Autophagy Stroke Neuroprotection

Neuroprotective effects of genistein on SH-SY5Y cells overexpressing A53T mutant α-synuclein

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Neural Regeneration Research 2018年第8期13卷 1375-1383页

作者： Huan-Cheng Wu Qun-Liang Hu Shi-Jun Zhang Yan-Min Wang Zhan-Kui Jin Ling-Fu Lv Sai Zhang Zhen-Lin Liu Hong-Lian Wu Ou-Mei Cheng Graduate School Tianjin Medical University Tianjin China Tianjin Beichen Hospital Tianjin China Tianjin Key Laboratory of Neurotrauma Repair Institute of Traumatic Brain Injury and Neuroscience Center for Neurology and Neurosurgery of Affiliated Hospital Logistics University of Chinese People's Armed Police Force Tianjin China Department of Clinical Medicine Chongqing Medical University Chongqing China

Genistein, a potent antioxidant compound, protects dopaminergic neurons in a mouse model of Parkinson＇s disease. However, the mecha- nism underlying this action remains unknown. This study investigated human SH-SYSY cells overexpressing the A53T mutant of α-synuclein. Four groups of cells were assayed： a control group （without any treatment）, a genistein group （incubated with 20 μM genistein）, a rote- none group （treated with 50 μM rotenone）, and a rotenone ＋ genistein group （incubated with 20 μM genistein and then treated with 50μM rotenone）. A lactate dehydrogenase release test confirmed the protective effect of genistein, and genistein remarkably reversed mitochondrial oxidative injury caused by rotenone. Western blot assays showed that BCL-2 and Beclin ! levels were markedly higher in the genistein group than in the rotenone group. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling revealed that genistein inhibited rotenone-induced apoptosis in SH-SYSY cells. Compared with the control group, the expression of NFE2L2 and HMOX1 was significantly increased in the genistein ＋ rotenone group. However, after treatment with estrogen receptor and NFE2L2 channel blockers （ICI-182780 and ML385, respectively）, genistein could not elevate NFE2L2 and HMOX1 expression. ICI-182780 effectively prevented genistein-mediated phosphorylation of NFE2L2 and remarkably suppressed phosphorylation of AKT, a protein downstream of the estrogen receptor. These findings confirm that genistein has neuroprotective effects in a cell model of Parkinson＇s dis- ease. Genistein can reduce oxidative stress damage and cell apoptosis by activating estrogen receptors and NFE2L2 channels.

关键词： nerve regeneration genistein neuroprotection SH-SYSY cells Parkinson＇s disease rotenone estrogen receptor NFE2L2 A53T a-synuclein oxidative stress neurodegeneration neural regeneration

百草枯通过α-synuclein介导CD11b/PI3K/NOX2通路在神经炎症中的作用机制

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百草枯通过α-synuclein介导CD11b/PI3K/NOX2通路在神经炎症中的...

作者：田甜宁夏医科大学

学位级别：硕士

目的本研究通过百草枯（PQ）腹腔注射小鼠构建PD样模型,首先观察PQ对小鼠神经行为学及DA能神经元的损伤作用。其次探讨α-synuclein通过CD11b/PI3K通路调控NOX2在PQ诱导小胶质细胞异常激活中的作用机制。最后讨论牛磺酸（Tau）通过抑制... 详细信息

目的本研究通过百草枯（PQ）腹腔注射小鼠构建PD样模型,首先观察PQ对小鼠神经行为学及DA能神经元的损伤作用。其次探讨α-synuclein通过CD11b/PI3K通路调控NOX2在PQ诱导小胶质细胞异常激活中的作用机制。最后讨论牛磺酸（Tau）通过抑制小胶质细胞活化对PQ诱导的神经毒性的保护作用。为环境化学物PQ诱导的PD分子机制和治疗研究提供理论依据。方法1.PD样小鼠模型建立:选用48只SPF级雄性C57BL/6小鼠,随机分为对照组（n=12）和PQ染毒组（n=36）。染毒组小鼠每周腹腔注射15 mg/kg PQ两次,对照组注射同等容量0.9%Saline,连续注射8周。2.Tau保护模型建立:选用48只SPF级C57BL/6雄性小鼠,随机分为4组:对照组（Con）:腹腔注射等容量0.9%Saline;Tau对照组（Tau）:腹腔注射150 mg/kg Tau;PQ染毒组（PQ）:腹腔注射15 mg/kg PQ;Tau干预组（Tau+PQ）:注射PQ前1 h腹腔注射150mg/kg Tau。每组12只小鼠,每周注射2次,连续注射8周。3.神经行为学观察:每周记录一次小鼠体重。观察各组小鼠的精神状态等一般行为变化。于末次给药后第二天取各组小鼠进行神经行为学测试。首先采用爬杆实验和步态测量实验检测各组小鼠的运动协调能力和步态改变,然后采用旷场实验和新物体识别实验测试各组小鼠的认知和学习记忆能力,最后采用悬尾实验和高架十字迷宫实验评价各组小鼠的抑郁和焦虑样行为。4.尼氏染色分析:制备中脑组织石蜡切片,检测黑质区神经元尼氏体数量及形态变化。5.免疫组织化学染色（IHC）分析:观察黑质区和纹状体区神经元核Neu N、DA能神经元TH的形态及数量变化;检测小胶质细胞Iba-1的活化水平和α-突触核蛋白（α-syn）的表达水平。6.免疫荧光双标染色（IF）分析:检测黑质区α-syn与TH的共表达情况以及黑质区Iba-1与TH的共表达情况。7.免疫印迹（Western blot）分析:测定中脑神经元核Neu N、DA能神经元TH、Iba-1、α-syn、纹状体区DA转运蛋白DAT、炎症因子（TNF-α、IL-1β、IL-6、HMGB1、i NOS）和信号通路关键蛋白CD11b、PI3K、PDK1、AKT、p-AKT以及细胞质和细胞膜中P47phox的蛋白表达变化。8.超氧化物阴离子荧光探针（DHE）分析:测定黑质区ROS的表达水平。结果（一）百草枯通过α-synuclein介导CD11b/PI3K通路调控NOX2在神经炎症中的作用机制1.各组小鼠体重及一般行为变化:对照组小鼠在实验过程中一般情况良好。染毒组小鼠腹腔注射PQ后未立即出现明显行为学变化。但随着染毒时间的增加,出现毛发干燥杂乱、逃避反应慢。个别小鼠出现歪头、后背上弓、静止性震颤等症状。各组小鼠体重均呈稳步上升趋势,变化无明显差异。2.PQ对小鼠运动能力的影响:步态分析结果显示,PQ染毒组小鼠的步长较对照组无明显变化（P＞0.05）,但步距逐渐减小,尤其染毒8周组变化最为明显（P＜0.05）。爬杆实验结果显示,6周和8周染毒组小鼠的爬杆时间较对照组有增加趋势（P＜0.05）。3.PQ对小鼠认知、记忆能力的影响:旷场实验结果显示,与对照组比较,PQ染毒组小鼠的移动距离减少,中央区域停留时间降低（P＜0.05）。新物体识别实验中,随着染毒时间和剂量的增加,染毒组小鼠对新物体的触碰时间和次数均呈时间依赖性减少（P＜0.05）。4.PQ对小鼠情绪的影响:悬尾实验结果显示,小鼠悬尾不动时间较对照组呈逐渐增加趋势（P＜0.05）。高架十字迷宫实验中,PQ染毒组小鼠进入开放臂的时间和次数较对照组呈逐渐减少趋势（P＜0.05）。5.PQ对小鼠黑质、纹状体DA能神经元的损伤效应:尼氏染色结果显示,PQ染毒组黑质区尼氏体数量较对照组明显减少（P＜0.05）。IHC显示,PQ染毒组黑质区神经元胞体缩小、轴突纤维散乱、突起末端出现断裂,数量较对照组明显减少（P＜0.05）。纹状体中DA神经元的轴突纤维阳性表达量也明显降低（P＜0.05）。Western blot结果显示,与对照组比较,染毒组小鼠中脑神经元核Neu N、DA能神经元TH以及纹状体DA转运蛋白DAT的蛋白相对表达量均随PQ染毒时间的增加而降低（P＜0.05）。6.PQ对α-synuclein表达水平的影响:IHC结果显示,PQ染毒组小鼠黑质区出现α-syn逐渐聚集增多,阳性染色呈棕黄色,表达水平较对照组呈增高趋势,尤其在染毒8周时最为明显。IF观察到PQ染组小鼠黑质区仅可见少量、稀疏的TH神经元（红色）,染色强度明显减弱并伴随α-syn沉积（绿色）,且α-syn荧光强度明显增强。Western blot结果显示,PQ染毒组小鼠中脑组织中二聚体和寡聚体形式的α-syn及S129-p-α-syn的蛋白相对表达量均呈时间依赖性升高（P＜0

关键词：百草枯牛磺酸帕金森病 a-synuclein 神经炎症

Decreasing a-synuclein aggregation by methanolic extract of Centella asiatica in zebrafish Parkinson's model

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Asian Pacific Journal of Tropical Biomedicine 2015年第11期5卷 948-954页

作者： Husnul Khotimah Mulyohadi Ali Sutiman Bambang Sumitro Mochamad Aris Widodo Medical Faculty Brawijaya University Laboratory of Pharmacology Medical FacultyBrawijaya University Department of Biology Faculty of ScienceBrawijaya University

Objective: To observe the effects of Centella asiatica(C. asiatica) methanolic extract on a-synuclein aggregation and its expression in rotenone-exposed zebra fish.Methods: Zebra fish(Danio rerio) were exposed to 5 m g/L rotenone for 28 days and coincubated with 2.5, 5.0 and 10.0 m g/mL of C. asiatica methanolic extract. The medium was changed every 48 h for maintain the concentration of rotenone and extract. After 28 days zebra fish were sacrificed on the ice block and protein was isolated from zebra fish brain for ELISA of dopamine and Western blotting of a-synuclein. Immunohistochemistry was conducted to observe the a-synuclein expressions from histopathological preparation of zebra fish brain. The head were soaked in 10% formaline for less than 24 h and embedded onto paraffin block, then sliced for immunohistochemistry using anti a-synuclein antibody. We also measured zebra fish motility for 5 min in each week.Results: C. asiatica has important bioactive compounds such as asiaticoside that has antiin flammatory and antioxidant properties. It may inhibit cascade reaction due to oxidative stress induced by rotenone. Decreasing reactive oxygen species proposed probability of radical attack to a-synuclein protein that caused aggregation and increase of its expression.The motility of zebra fish was also maintained in C. asiatica groups due to the increasing dopamine level in rotenone-induced zebra fish. High level of reactive oxygen species inactivated enzyme for dopamine synthesis such as tyrosine hydroxylase, and oxidized dopamine itself. Oxidized dopamine increased a-synuclein aggregation. Thus, the dopamine level decreased in rotenone-induced zebra fish, but C. asiatica increased dopamine level.Conclusions: C. asiatica has a potential to be developed as an anti-Parkinson's disease treatment due to its capability for minimized the sign of Parkinson's such as a-synuclein aggregation and expression, increasing motility and dopamine as well.

关键词： a-synuclein Centella asiatica Rotenone Parkinson’s

GBA L444P突变对α-synuclein蛋白影响的研究

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临床医药文献电子杂志 2019年第31期6卷 31-32页

作者：毛雪晔马莎李珊杨帅峰云南省第一人民医院

目的探讨GBA L444P突变对α-syn蛋白的影响。方法构建稳定表达野生型和突变型GBA(L444P)基因的细胞模型,根据转染目的片段不同,将其分为四组:GBA WT组、GBA L444P组、Mock组、Control组,每组取5例。使用western blot检测α-syn蛋白... 详细信息

目的探讨GBA L444P突变对α-syn蛋白的影响。方法构建稳定表达野生型和突变型GBA(L444P)基因的细胞模型,根据转染目的片段不同,将其分为四组:GBA WT组、GBA L444P组、Mock组、Control组,每组取5例。使用western blot检测α-syn蛋白的表达,比较不同组的α-syn蛋白表达情况。结果GBA WT组和GBA L444P组GBA蛋白的表达均高于Mock组、Control组,提示细胞模型构建成功。GBA L444P组的细胞α-syn蛋白的表达量高于GBA WT组、Mock组和Control组,差异有统计学意义(P0.05)。结论 GBA L444P突变可增加α-syn蛋白的表达。

关键词： GBA L444P 突变 a-synuclein 蛋白表达

The Concentration of Hydrogen Peroxide Generated during Aggregation of a-synuclein in vitro Is Lower than 5 nmol/L

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Chinese Journal of Chemistry 2004年第12期22卷 1440-1443页

作者：吉丽娜李洪涛罗晓莹张峰胡红雨胡钧 Shanghai Institute of Applied Physics Chinese Academy of Sciences Shanghai 201800 China Key Laboratory of Proteomics Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sci-ences Chinese Academy of Sciences Shanghai 200031 China Shanghai Institute of Applied Physics Chinese Academy of Sciences Shanghai 201800 ChinaBio-X Research Center Shanghai Jiaotong University Shanghai 200030 China

Using a fluorometric method with a detection limit of 5 nmol/L, here it is reported that albeit positive results were got from bovine serum albumin (BSA) and chicken ovalbumin (OVA) as published in literature, no detectable amount of hydrogen peroxide (H2O2) was generated during a-synuclein (a-Syn) aggregation in vitro even in the presence of transition metal ions Cu(II) or Fe(III). The results suggest that the concentration of H2O2 generated during aggregation of a-Syn in vitro be lower than 5 nmol/L beyond the detection limit of the adopted method and it is far too poor to be responsible for the cytotoxicity of a-Syn aggregates, thus allowing people to extensively elu-cidate the mechanism underlying neurotoxicities of the aggregates formed by some amyloidogenic proteins.

关键词： a-synuclein hydrogen peroxide aggregation cytotoxicity

人a-synuclein及其突变体的克隆、细菌表面展示与筛选

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人a-synuclein及其突变体的克隆、细菌表面展示与筛选

作者：熊莉丽西南交通大学

学位级别：硕士

帕金森病（Parkinson’s disease,PD）是最为广泛的神经退行性疾病之一,近年来对此疾病的研究越来越受到人们的重视。a-synuclein是synuclein蛋白家族成员之一,由140个氨基酸编码组成,主要在脑组织中大量表达,是Parkinson病中特征性路... 详细信息

帕金森病（Parkinson’s disease,PD）是最为广泛的神经退行性疾病之一,近年来对此疾病的研究越来越受到人们的重视。a-synuclein是synuclein蛋白家族成员之一,由140个氨基酸编码组成,主要在脑组织中大量表达,是Parkinson病中特征性路易小体的主要组成部分。a-synuclein蛋白的聚集状态的变化可能是影响帕金森病发生一个关键因素,而a-synuclein蛋白结构的转变受到许多遗传学与细胞学因素的影响。确定影响a-synuclein蛋白聚集的因素,明确a-synuclein存在的不同状态以及在蛋白聚集过程中的相互关系,有助于人们最终在分子细胞水平上揭示帕金森病及其它神经退行性疾病的发生机制,寻找治疗帕金森病更为有效的途径。细菌表面展示技术的提出和广泛应用为a-synuclein的细菌表面正确折叠展示和靶分子的筛选及研究提供了重要的技术平台和条件。本文首先采用生物信息学手段对不同物种synuclein蛋白进行序列比对,构建分子进化树,亲缘关系分析;同时对人a-synuclein野生型蛋白及突变体A30P、E46K和A53T的二级结构,三级结构以及疏水性进行分析;在此基础之上,采用叠套PCR（overlap extension PCR）介导的点突变方法,以野生型a-synuclein为模板,克隆a-synuclein A30P、E46K和A53T 3个突变型基因和采用PCR介导的连接方法克隆a-synuclein剪接变体98,112和126;利用细菌亲密素展示系统paskint100对野生型a-synuclein及其突变体A30P、E46K和A53T进行细菌表面的展示,并采用SDS-PAGE凝胶电泳和Western Blot对外源蛋白a-synuclein进行检测和确定;最后利用展示在大肠杆菌细胞表面的a-synuclein筛选噬菌体随机12肽库,筛选与a-synuclein高度亲和的特异性多肽分子。进化分析结果表明:synuclein家族蛋白可以聚为3类,各物种的a、β和γ各聚成一类。a-synuclein、β-synuclein和γ-synuclein属于直系同源基因;而相同物种的a、β和γ-synuclein由基因复制而产生,属于旁系同源基因,与直系同源的a-synuclein、β-synuclein和γ-synuclein基因相比则表现出较远的距离。通过生物信息学分析发现人类a-synuclein蛋白可以从序列上分为3个区域:N端（1-60）的两性a螺旋区,主要包括膜结合区和疏水性NAC区,主要由6个“KTKEGV”非完全重复序列组成;中间NAC（61-95）区域表现出较强的疏水性;C端（96-140）则主要由酸性氨基酸组成。a-synuclein突变体A30P、E46K和A53T的突变位点位于a-synuclein基因的不同外显子上。突变体A30P的88位突变碱基位于外显子2上;而突变体E46K和A53T的突变位点则位于外显子3。a-synuclein外显子的异位拼接产生4种异构体。5个外显子都参与编码一个由140个氨基酸组成的14.5 kDa的蛋白,这是a-synuclein在人体内存在的主要形式;同时外显子3和5在体内存在选择性剪接的机制,外显子3或5的缺失,分别形成与140个氨基酸a-synuclein同框的126个氨基酸（13.1 kDa）,112个氨基酸（11.4 kDa）的剪接变体;而外显子3和5的同时缺失则形成一个只编码98个氨基酸的a-synuclein。其次,采用叠套PCR介导的点突变方法和连接方法,以野生型a-synuclein为模板,成功获得了a-synuclein A30P、E46K和A53T 3个突变型基因以及a-synuclein剪接变体98,112和126。第三,以GFP为模型蛋白通过paskin100重组质粒的构建和细菌表面展示、GFP蛋白诱导表达和绿色荧光显示确认细菌亲密素展示系统的正确性。在此基础上将野生型a-synuclein及突变体A30P、E46K和A53T成功展示在大肠杆菌的细胞表面,SDS-PAGE凝胶电泳和Western Blot检测到外源a-synuclein蛋白目的带的高效表达。第四,利用展示在大肠杆菌表面a-synuclein筛选噬菌体随机12肽库,筛选与a-synuclein高度亲和的特异性短肽,经前两轮的筛选,已收获与a-synuclein结合的噬菌体肽库,正用于进一步的筛选和富集。综上,通过本研究得到如下初步结论:1)synuclein直系同源基因a-synuclein、β-synuclein和γ-synuclein基因表现为较近的亲缘关系,而相同物种由基因复制产生的旁系同源基因a、β和γ-synuclein则表现出较远的距离;2)生物信息学分析发

关键词： a-synuclein 突变体细菌表面展示噬菌体肽库筛选

The mechanism of interaction between copper transporters and α-synuclein

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The mechanism of interaction between copper transporters and...

HDAC6 regulates aggresome-autophagy degradation pathway of α-synuclein in response to MPP+-induced stress

中国神经科学学会第十二届全国学术会议

作者： De-Hai GOU Ting-Ting HUANG Long-Hai LI Chen-Hao SUO Xin-Di GAO Chen DING Jia-Yi LI Institute of Neuroscience College of Life and Health Sciences Northeastern University

Previous studies showed that Copper and Copper transport 1（Ctr1） were decreased in the substantia nigra compacta of the midbrain and the striatum of patients with Parkinson’s disease（PD）. Copper is related to increased oxidative stress, α-synuclein oligomerization, and Lewy-body formation. In addition, Copper regulates neurotransmission when it is released after neuronal stimulus and the metal is able to modulate the function of NMDA and GABA receptors. At present, there is limited information about the pathological mechanism of copper and it’s related proteins in PD. Therefore, it is important to study the pathology of copper in PD. The present project aims to investigate the interaction between copper, copper related proteins（Ctr1, CCS, COX4, etc.） and α-synuclein to seek for new therapeutic targets for PD. Using HEK293 or SH-SY5 Y cell lines stably expressing human wildtype α-synuclein tagged with GFP or Ds Red, we detected that copper can promote α-synuclein aggregation in a dosedependent manner. In order to study the role of Ctr1 on α-synuclein accumulation and aggregation and subsequent effects on neuronal function, we have established transgenic mouse model overexpressing Ctr1 and knockout Ctr1 models at specific brain regions, such as the hippocampus and the substantia nigra. Open field test and Morris water maze test showed little changes in the Ctr1 knockout mice in the hippocampus compared to the WT mice. In on-going experiments, we do stereotaxic injection of copper or α-synuclein fibrils to the mice to detect copper concentration, protein expression of copper relative protein and the distribution and expression of α-synuclein.

关键词： copper Ctr1 a-synuclein PD

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HDAC6 regulates aggresome-autophagy degradation pathway of ...