α-突触核蛋白对线粒体膜造成孔道样损伤

α-突触核蛋白(α-synuclein,α-syn）作为第一个发现的帕金森病（Parkinson’s disease, PD）致病基因,在PD发生发展过程中具有重要作用。尽管有研究发现,α-syn对线粒体功能有损伤作用,但其对线粒体膜的损害机制尚不明确。本实验旨在探究α-syn对线粒体膜形态的影响,并找到更加微观的方式观察线粒体膜的变化。Thy-1-α-syn转基因动物与野生型动物相比,线粒体膜电势降低17%（P<0.01）,膜通透性增加20.5%（P<0.01）,转基因组线粒体细胞色素C释放增多64%（P＜0.01),这有可能引起线粒体自噬和细胞凋亡。原子力显微镜结果显示,野生型小鼠脑组织线粒体表面光滑,α-syn转基因小鼠脑组织线粒体表面发现有锯齿状改变,而且在过表达α-syn的原代神经元线粒体膜表面有许多小凹陷,出现口径60 ～ 200 nm,深达20 ～ 60 nm的孔道。这可能是α-syn对线粒体膜造成的孔道样损伤。过表达α-syn全长组和N端组的原代神经元电镜结果显示,线粒体膜被破坏,并且出现空泡样线粒体和自噬小泡。本研究发现,过表达α-syn可能在线粒体表面形成孔道样改变,α-syn的N端是引起线粒体膜损伤主要结构域。     

蛋白质/多肽片段互补分析法检测alpha-突触核蛋白在细胞中的聚集

Alpha-突触核蛋白（α-synuclein, α-syn）聚集是引起帕金森病（Parkinson’s disease, PD）发生发展主要原因。本文用蛋白质/多肽片段互补分析法（protein-fragment complementation assays, PCAs）检测α-syn在细胞内的聚集。分别构建融合α-syn与人工改造的荧光素酶human Gaussiaprinceps luciferase（hGLuc）蛋白N端或C端蛋白的质粒,共转入人神经母细胞瘤SK-N-SH细胞,通过检测酶活性来确定野生型（wild type,WT）及A53T突变体α-syn在细胞中的聚集情况。结果表明WT和A53T突变α-syn都能使荧光素酶活性增强,而且与野生型α-syn相比,突变体A53T的荧光素酶活性更强,说明二者都能聚集,而且A53T聚集程度高于WT。PCAs法具有高灵敏度,不仅能检测α-syn在细胞内的聚集,而且能反映其聚集的程度,为研究帕金森病提供了研究思路和相应药物筛选的有效工具。     

右美托咪啶通过抑制NADPH氧化酶2缓解氧化应激小鼠模型神经元的毒性和认知障碍的机制

摘要 目的：探讨右美托咪啶通过抑制NADPH氧化酶2缓解氧化应激小鼠模型神经元的毒性和认知障碍的机制。方法：10只野生型以及20只Sod1KO雄性BALB/c小鼠,12月龄,根据实验目的分为3组：对照组（野生型小鼠）,模型组（氧化应激小鼠模型）和DEX组（氧化应激小鼠模型+50 μg/kg DEX治疗）,每组10只。通过MWM 测试检测小鼠的空间学习和记忆能力。通过免疫染色检测海马中Neu-N+细胞数和PSD-95表达水平。通过蛋白质印迹检测海马中Neu-N、PSD-95、TH、总α-突触核蛋白和Ser129-磷酸化α-突触核蛋白表达水平。通过ROS、MDA和SOD检测试剂盒分别检测ROS、MDA和SOD水平。通过 ELISA试剂盒检测NOX2水平。通过RT-qPCR检测IL-1β、IL-6和TNF-α水平。结果：对照小鼠表现出正常的空间学习功能,与对照组小鼠相比,模型组小鼠逃避潜伏期和游泳距离增加（P<0.05）,而DEX治疗能够降低模型组小鼠逃避潜伏期和游泳距离（P<0.05）。三组小鼠平均游泳速度没有统计性差异（P>0.05）。与对照组小鼠相比,模型组小鼠小鼠海马中Neu-N+细胞数和PSD-95表达水平降低（P<0.05）,而DEX治疗能够增加小鼠海马中Neu-N+细胞数和PSD-95表达水平（P<0.05）。与对照组小鼠相比,模型组小鼠小鼠海马中Neu-N、PSD-95和TH蛋白表达水平降低（P<0.05）,总α-突触核蛋白和Ser129-磷酸化α-突触核蛋白表达水平升高（P<0.05）,而DEX治疗能够增加小鼠海马中Neu-N、PSD-95和TH蛋白表达水平（P<0.05）,降低总α-突触核蛋白和Ser129-磷酸化α-突触核蛋白表达水平（P<0.05）。与对照组小鼠相比,模型组小鼠ROS和MDA水平增加,SOD水平降低（P<0.05）,而DEX治疗能够降低ROS和MDA水平,增加SOD水平（P<0.05）。与对照组小鼠相比,模型组小鼠NOX2水平增加（P<0.05）,而DEX治疗能够降低NOX2水平（P<0.05）。与对照组小鼠相比,模型组小鼠IL-1β、IL-6和TNF-α水平增加（P<0.05）,而DEX治疗能够降低IL-1β、IL-6和TNF-α水平（P<0.05）。结论：DEX对NOX2的抑制可通过抑制小鼠模型中的氧化应激和神经炎症来阻断学习和记忆障碍以及海马神经变性。     

CD36基因缺失改善高脂饮食诱导的糖代谢异常并促进肝脏脂质积聚

本研究旨在探讨在高脂饮食状态下CD36基因缺失对小鼠糖脂代谢的影响及作用机制。根据基因型将小鼠分为野生型小鼠(wild type, WT)及CD36基因敲除(CD36~(-/-))小鼠,给予高脂饮食喂养14周。小鼠腹腔注射葡萄糖(1 g/kg)或胰岛素(5units/kg)进行葡萄糖耐量或胰岛素耐量测试。HE染色观察肝脏脂质变性,全自动生化分析仪测定小鼠血清甘油三酯(triglyceride, TG)、血清游离脂肪酸(free fatty acid, FFA)、天门冬氨酸转氨酶(aspartate aminotransferase, AST)和丙氨酸转氨酶(alanine aminotransferase, ALT)浓度。Real-time PCR和Western blot检测小鼠肝脏、肌肉组织胰岛素信号通路。Real-time PCR检测小鼠原代肝细胞中磷酸烯醇式丙酮酸羧激酶(phosphoenolpyruvate carboxykinase, PEPCK)的mRNA水平,葡萄糖检测试剂盒检测糖异生能力。免疫共沉淀(co-immunoprecipitation, Co-IP)及ELISA检测肌肉胰岛素受体β(insulin receptorβ, IRβ)酪氨酸磷酸化水平。Real-time PCR和免疫荧光染色检测小鼠肌肉葡萄糖转运蛋白4 (glucose transporter 4, GLUT4)的表达和定位。结果显示,在高脂喂养后,CD36~(-/-)小鼠血清FFA、TG、AST及ALT水平较WT小鼠明显升高(P 0.05),CD36~(-/-)小鼠肝脏外观呈脂肪样变性,HE染色结果显示肝脏脂质积聚加重,提示CD36缺失促进脂肪肝的发生。然而,相对于WT小鼠,CD36~(-/-)小鼠的空腹血糖水平降低、糖耐量升高,胰岛素耐量降低(P 0.05),提示在高脂饮食喂养条件下,CD36缺失并不会损害小鼠的糖耐量和胰岛素耐量。与WT小鼠相比,CD36~(-/-)小鼠肝脏IR/IRS/AKT胰岛素信号通路无显著差异,两组小鼠原代肝细胞PEPCK表达水平及糖异生能力均无显著差异。而在CD36~(-/-)小鼠肌肉组织中,Co-IP及ELISA实验显示IRβ酪氨酸磷酸化水平显著升高,p-AKT水平显著升高(P 0.05)。免疫荧光染色实验提示肌肉GLUT4在细胞膜的定位增强,表明CD36~(-/-)小鼠肌肉胰岛素敏感性及葡萄糖利用能力增强。以上结果提示,CD36基因缺失加重高脂饮食诱导的肝脏脂质积聚,对高脂饮食诱导的肝脏糖代谢无显著影响;CD36缺失主要通过提高肌肉组织胰岛素敏感性,促进GLUT4介导的葡萄糖利用以改善高脂饮食诱导的小鼠糖代谢异常。     

甘草酸调控AMPK/ACC/SREBP信号通路改善胰岛素抵抗的机制

为了观察甘草酸对胰岛素抵抗的作用,探究其可能的机制,将50只C57BL/6J雄性小鼠随机分成正常组、模型组、甘草酸高剂量组、甘草酸低剂量组和二甲双胍组,每组10只。除正常组外,其余小鼠采用长期高脂饮食法,复制胰岛素抵抗模型,并给予相应药物进行干预。采用全自动生化仪测定小鼠血清中总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)和高密度脂蛋白胆固醇(HDL-C);检测小鼠血糖和胰岛素的水平,观察其糖耐量和胰岛素耐量变化;Western blotting法检测肝脏腺苷酸活化蛋白激酶(AMPK)、磷酸化乙酰辅酸A羟化酶(ACC)和固醇调节元件结合蛋白(SREBP)的表达。结果表明:长期高脂饮食的小鼠TC、TG和LDL-C明显升高,空腹血糖、血清胰岛素水平均明显升高,糖耐量和胰岛素耐量出现异常,肝脏AMPK和ACC的磷酸化水平和SREBP表达下降,与正常组小鼠相比,具有极显著性差异(P0.01)。经药物干预后,甘草酸高、低剂量组和二甲双胍组小鼠体质量下降,TC、TG和LDL-C明显降低,空腹血糖和血清胰岛素降低,糖耐量和胰岛素耐量异常得到改善,AMPK和ACC的磷酸化水平和SREBP表达升高,与模型组小鼠相比,具有显著性差异(P0.01,P0.05)。因此,本研究表明:甘草酸能够改善长期高脂饮食诱导的胰岛素抵抗,其机制可能与其调节肝脏AMPK/ACC/SREBP通路有关。     

AAV介导的α-synuclein基因过表达致多巴胺能神经元损伤-一种制作轻度帕金森病大鼠模型的新方法

目的：研究过表达α-synuclein基因是否导致大鼠黑质纹状体选择性损伤,为帕金森病（parkinson’s disease, PD）大鼠模型的制备提供一种新的方法。方法:用腺相关病毒(adeno-associated virus, AAV)做载体,将人野生型α突触核蛋白(α-synuclein, NACP)引入大鼠脑内,观察大鼠行为学的改变,通过免疫组织化学染色观察其对黑质多巴胺能神经元细胞的影响,高效液相色谱（HPLC）检测纹状体多巴胺（DA）的含量。结果:α-synuclein基因过表达后大鼠出现自发性活动减少、爬行活动减慢、暂时性躯干震颤、竖毛等类似PD初期的症状和体征;大鼠脑黑质TH阳性神经元细胞随时间的延长出现数目减少,并且纹状体DA含量也出现减少,并且出现α-synuclein的积聚。结论:上述结果表明α-synuclein基因的过表达引起黑质多巴胺能神经元细胞的死亡,对大鼠的运动行为有一定的影响,产生类似于PD早期的症状与体征,与化学毒素（如6-OHDA, MPTP）诱导的动物模型相比,此法制作的动物模型可模拟PD缓慢发展的进程,为研究PD的病程进展及发病机制提供一个理想的动物模型。     

20-HETE对小鼠葡萄糖刺激胰岛素分泌反应的影响

为了考察20-羟基二十碳四烯酸(20-hydroxyeicosatetraenoic acids, 20-HETE)对葡萄糖刺激胰岛素分泌反应的影响,本研究选择CYP4F2转基因小鼠和小鼠胰岛素瘤INS-1E细胞作为研究材料,通过LCMS/MS检测WT和TG小鼠的胰腺20-HETE水平。通过IPGTT测定小鼠葡萄糖耐量,通过ELISA测定小鼠血浆C肽水平来检测胰岛素分泌。通过Western blotting、Real time PCR、免疫组化和免疫荧光来检测小鼠胰腺或INS-1E细胞中Glut2、GSK-3β(Ser9点)和AKT (Ser473点)的磷酸化水平。TG小鼠的20-HETE水平((7.26±2.03) ng/mg蛋白)显著高于WT小鼠((2.14±0.76) ng/mg蛋白)。在用20-HETE合成的选择性抑制剂HET0016处理后,TG小鼠((0.33±0.07) ng/mg蛋白)和WT小鼠((0.27±0.06) ng/mg蛋白)胰腺组织中的20-HETE水平均急剧降低。给予葡萄糖处理30 min后,TG小鼠的血糖水平均显著高于WT小鼠,而血浆C肽水平显著低于WT小鼠(p<0.05)。与WT小鼠相比,TG小鼠的胰腺组织中Glut2 m RNA和蛋白水平显著降低。与WT小鼠相比,CYP4F2转基因小鼠的GSK-3β和AKT磷酸化均显著降低。20-HETE处理可导致INS-1E细胞中AKT/GSK-3β磷酸化水平和Glut2表达水平显著降低(p<0.05)。此外,用17 mmol/L葡萄糖处理INS-1E细胞1 h,20-HETE处理组的胰岛素分泌显著降低。应用GSK-3β选择性抑制剂TWS119预处理INS-1E细胞3 h后,TWS119 (一种GSK-3β选择性抑制剂)预处理显著逆转了Glut2表达水平的降低以及胰岛素分泌的减少。20-HETE主要通过AKT/GSK-3β信号通路来下调Glut2的表达,进而减弱胰岛素分泌,导致胰岛素分泌功能障碍。     

帕金森病患者血浆alpha-synuclein、Abeta及tau蛋白变化特点

目的：探索帕金森病（Parkinson''s disease,PD）患者血浆中alpha- 突触核蛋白、Abeta及tau 蛋白变化情况。方法：募集2014 年4 月至 2015 年4 月来我院就诊的PD 患者62 例,正常对照人群59 例,采集两组人群的基本临床信息,测定血浆中琢- 突触核蛋白、 Abeta40、Abeta42、pT181-tau 蛋白、pT231-tau 蛋白和总tau 蛋白浓度,比较两组之间的差异,同时进行相关性分析。结果：PD患者血浆 alpha- 突触核蛋白和pT181-tau 蛋白浓度显著高于对照组(P 值分别为0.001,0.019),而两组间Abeta40、Abeta42、pT231-tau 蛋白和总tau 蛋白浓度无明显差异（P>0.05）。相关性分析提示PD 患者血浆alpha-突触核蛋白和pT181-tau 蛋白浓度与患者年龄、性别、教育程度、 病程、高血压、糖尿病、Hoehn/ Yahr 分级及Schwab &England 评分无相关性（P>0.05）。结论：虽然PD患者血浆琢- 突触核蛋白和 pT181-tau 蛋白高于正常对照组,但尚不适宜作为PD 的生物标志物。     

中心粒周蛋白对胰岛素分泌的调节作用及其机制

目的:探讨中心粒周蛋白(pericentrin,PCNT)对胰岛素双相分泌的调节作用及其机制。方法:构建在小鼠胰岛β细胞中PCNT表达减少的转基因鼠(△PCNTβ小鼠),检测△PCNTβ小鼠与正常对照小鼠在给予糖耐量试验后第一时相和第二时相血糖和胰岛素分泌情况。检测两组小鼠胰岛内PCNT、胰岛素、纤维肌动蛋白(F-actin)变化情况,以及相关蛋白表达情况。结果:Western blot和RT-PCR显示△PCNTβ小鼠胰尾组织PCNT表达较对照组明显减低,免疫荧光提示△PCNTβ小鼠胰岛内PCNT、胰岛素表达较对照组明显减低。行腹腔注射葡萄糖耐量试验(Intraperitoneal glucose tolerance tests,IPGTT)△PCNTβ小鼠第一时相血糖曲线下面积(Quantification of area under the curve,AUC)显著高于对照组,第二时相血糖AUC两组小鼠间无统计学差异。△PCNTβ小鼠空腹胰岛素水平与对照组比较明显升高,葡萄糖刺激胰岛素分泌(Glucose stimulated insulin secretion,GSIS)后15min胰岛素增加值显著低于对照组,30 min和120 min时胰岛素水平与对照组无显著差异。Western blot显示△PCNTβ小鼠与对照组比较F-actin表达明显减低,ERK、p-ERK表达明显升高。RT-PCR测定△PCNTβ小鼠与对照组比较ETV4表达显著升高。免疫荧光提示△PCNTβ小鼠胰岛内F-actin和突触融合蛋白4(Syntaxin4,Syn-4)表达较对照组明显减低。结论:抑制小鼠胰岛β细胞内PCNT表达后,其通过抑制F-actin和Syn-4表达影响胰岛素分泌,导致空腹时胰岛素过度分泌和第一时相胰岛素分泌受损。     

SH-SY5Y细胞α-突触核蛋白的过表达可部分抵抗鱼藤酮诱导的氧化应激

帕金森病（Parkinson’s disease,PD）的发病机制涉及到遗传和环境因素。环境因素通过线粒休导致氧化应激和α-突触核蛋白（α—synuclein）聚集,但其确切的作用机制尚不明确。本文利用过表达α-突触核蛋白-增强型绿色荧光蛋白（enhanced green fluorescent protein．EGFP）的人多巴胺能神经母细胞瘤细胞株SH—SY5Y为模型,研究α-突触核蛋白对鱼藤酮诱导氧化应激的影响,从而进一步了解α-突触核蛋白和细胞存活之间的关系。（1）用荧光显微镜观察融合绿色荧光蛋白的α-突触核蛋白的表达情况;（2）用实时定量PCR检测α-突触核蛋白基因的表达;（3）用免疫细胞化学测定α-突触核蛋白的分布;（4）用不同浓度的鱼藤酮作用细胞后,以MTT法测细胞的活力、DCF法检测细胞的氧化应激状态、黄嘌呤氧化酶法检测超氧化物歧化酶的活力,并用流式细胞仪分析细胞的凋亡。实时定量PCR结果显示,α-突触核蛋白基因表达量在α-突触核蛋白过表达的细胞要高于SH—SY5Y细胞,在荧光显微镜下可见绿色荧光蛋白和α-突触核蛋白的表达。鱼藤酮可使细胞活力下降、线粒体complex Ⅰ的活性降低,诱导细胞内氧化应激,而过表达α-突触核蛋白的细胞可以部分抵抗鱼藤酮的毒性作用,表现为细胞抗氧化能力迅速增高（P〈0．05）和鱼藤酮诱导的细胞凋亡数目明显降低。本研究证明α-突触核蛋白对鱼藤酮产生的氧化应激有部分抵抗作用,而使过表达α-突触核蛋白的SH—SY5Y细胞对鱼藤酮的毒性作用表现出一定的耐受性。这种耐受性也可能是细胞对外界损害的一种代偿反应,从而促进细胞的存活。     

不同亚细胞定位的alpha-突触核蛋白对SK-N-SH细胞的毒性影响

在帕金森病中,alpha-突触核蛋白(α-synuclein)累积与聚集所产生的细胞毒性是发病的重要原因。本文目的是探求不同亚细胞定位的α-突触核蛋白对于细胞的毒性影响。分别在α-突触核蛋白前插入核输出序列(nuclear export sequence,NES)或核定位序列(nuclear localization sequence,NLS),使其特定地表达在细胞质或细胞核中。构建成功的质粒分别在神经母细胞瘤SK-N-SH细胞中表达,通过免疫荧光与Western印迹法检测蛋白质的表达情况。结果显示,NES-α-synuclein特异性地在细胞质中表达,NLS-α-synuclein特异性地在细胞核中表达。乳酸脱氢酶法检测结果表明,相较于WT-α-synuclein组,NES-α-synuclein组的乳酸脱氢酶的释放量减少26. 54%,NLS-α-synuclein组的乳酸脱氢酶释放量增加12. 85%。CCK8法检测细胞活性结果表明,相较于WT-α-synuclein组,NES-α-synuclein组的细胞活力提高35. 51%,NLS-α-synuclein组的细胞活力减少7. 93%。上述结果提示,胞质内表达α-synuclein对于细胞的毒性更小,而细胞核内表达的α-synuclein对细胞有更强的毒性作用。这些发现为研究帕金森病的分子机制提供了新的研究思路。     

Interaction between the polyol pathway and non-enzymatic glycation on aortic smooth muscle cell migration and monocyte adhesion

We investigated for the interaction between the polyol pathway and enhanced non-enzymatic glycation, both implicated in the pathogenesis of diabetic atherosclerosis, in the activation of aortic smooth muscle cell (SMC) function. Mouse aortas and primary cultures of SMCs from wildtype (WT) mice and transgenic (TG) mice expressing human aldose reductase (AR) were studied regarding changes in AR activity, and SMC gene activation, migration and monocyte adhesion, in response to advanced glycation end-product modified BSA (AGE-BSA). Results showed that AGE-BSA increased AR activity in both WT and TG aortas, with greater increments (p < 0.05) in TG aortas which, basally, had elevated AR activity (2.8 fold of WT). These increments were attenuated by zopolrestat, an AR inhibitor. Similar AGE-induced increments in AR activity were observed in primary cultures of aortic SMCs from WT and TG mice (60% and 100%, respectively, P < 0.01). Such increments were accompanied by increases in intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) mRNA levels (both P < 0.05), activation of membrane-associated PKC-beta1 (P < 0.05) as well as increased SMC migration and Tamm-Horsfall protein (THP)-1 monocyte adhesion to SMCs (both p < 0.01), with all changes being significantly greater in TG SMCs (P < 0.05) and suppressible by either zopolrestat or transfection with an AR antisense oligonucleotide. Our findings suggest that the effects of AGEs on SMC activation, migration and monocyte adhesion are mediated partly through the polyol pathway and, possibly, PKC activation. The greater AGE-induced changes in the TG SMCs have provided further support for the dependency of such changes on polyol pathway hyperactivity.     

Glucose homeostasis, insulin secretion, and islet phospholipids in mice that overexpress iPLA2beta in pancreatic beta-cells and in iPLA2beta-null mice

Studies with genetically modified insulinoma cells suggest that group VIA phospholipase A(2) (iPLA(2)beta) participates in amplifying glucose-induced insulin secretion. INS-1 insulinoma cells that overexpress iPLA(2)beta, for example, exhibit amplified insulin-secretory responses to glucose and cAMP-elevating agents. To determine whether similar effects occur in whole animals, we prepared transgenic (TG) mice in which the rat insulin 1 promoter (RIP) drives iPLA(2)beta overexpression, and two characterized TG mouse lines exhibit similar phenotypes. Their pancreatic islet iPLA(2)beta expression is increased severalfold, as reflected by quantitative PCR of iPLA(2)beta mRNA, immunoblotting of iPLA(2)beta protein, and iPLA(2)beta enzymatic activity. Immunofluorescence microscopic studies of pancreatic sections confirm iPLA(2)beta overexpression in RIP-iPLA(2)beta-TG islet beta-cells without obviously perturbed islet morphology. Male RIP-iPLA(2)beta-TG mice exhibit lower blood glucose and higher plasma insulin concentrations than wild-type (WT) mice when fasting and develop lower blood glucose levels in glucose tolerance tests, but WT and TG blood glucose levels do not differ in insulin tolerance tests. Islets from male RIP-iPLA(2)beta-TG mice exhibit greater amplification of glucose-induced insulin secretion by a cAMP-elevating agent than WT islets. In contrast, islets from male iPLA(2)beta-null mice exhibit blunted insulin secretion, and those mice have impaired glucose tolerance. Arachidonate incorporation into and the phospholipid composition of RIP-iPLA(2)beta-TG islets are normal, but they exhibit reduced Kv2.1 delayed rectifier current and prolonged glucose-induced action potentials and elevations of cytosolic Ca(2+) concentration that suggest a molecular mechanism for the physiological role of iPLA(2)beta to amplify insulin secretion.     

Hexosamines regulate sensitivity of glucose-stimulated insulin secretion in beta-cells

Hexosamines serve a nutrient-sensing function through enzymatic O-glycosylation of proteins. We previously characterized transgenic (Tg) mice with overexpression of the rate-limiting enzyme in hexosamine production, glutamine:fructose-6-phosphate amidotransferase, in beta-cells. Animals were hyperinsulinemic, resulting in peripheral insulin resistance. Glucose tolerance deteriorated with age, and males developed diabetes. We therefore examined islet function in these mice by perifusion in vitro. Young (2-mo-old) Tg animals had enhanced sensitivity to glucose of insulin secretion. Insulin secretion was maximal at 20 mM and half maximal at 9.9 +/- 0.5 mM glucose in Tg islets compared with maximal at 30 mM and half maximal at 13.5 +/- 0.7 mM glucose in wild type (WT; P < 0.005). Young Tg animals secreted more insulin in response to 20 mM glucose (Tg, 1,254 +/- 311; WT, 425 +/- 231 pg x islet(-1) x 35 min(-1); P < 0.01). Islets from older (8-mo-old) Tg mice became desensitized to glucose, with half-maximal secretion at 16.1 +/- 0.8 mM glucose, compared with 11.8 +/- 0.7 mM in WT (P < 0.05). Older Tg mice secreted less insulin in response to 20 mM glucose (Tg, 2,256 +/- 342; WT, 3,493 +/- 367 pg x islet(-1) x 35 min(-1); P < 0.05). Secretion in response to carbachol was similar in WT and Tg at both ages. Glucose oxidation was blunted in older Tg islets. At 5 mM glucose, islet CO2 production was comparable between Tg and WT. However, WT mice increased islet CO2 production 2.7 +/- 0.4-fold in 20 mM glucose, compared with only 1.4 +/- 0.1-fold in Tg (P < 0.02). Results demonstrate that hexosamines are involved in nutrient sensing for insulin secretion, acting at least in part by modulating glucose oxidation pathways. Prolonged excess hexosamine flux results in glucose desensitization and mimics glucose toxicity.     

Parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and alpha-synuclein mutations promote Tau protein phosphorylation at Ser262 and destabilize microtubule cytoskeleton in vitro

In Parkinson disease (PD) brain, a progressive loss of dopaminergic neurons leads to dopamine depletion in the striatum and reduced motor function. Lewy bodies, the characteristic neuropathological lesions found in the brain of PD patients, are composed mainly of α-synuclein protein. Three point mutations in the α-synuclein gene are associated with familial PD. In addition, genome-wide association studies indicate that α-synuclein and Tau protein synergistically increase disease susceptibility in the human population. To determine the mechanism by which α-synuclein and Tau act together, we have used PD-causing neurotoxin MPTP and pathogenic α-synuclein mutants A30P, E46K, and A53T as models. We found that exposure of human neuroblastoma M17 cells to MPTP enhances the intracellular α-synuclein protein level, stimulates Tau protein phosphorylation at Ser(262), and induces apoptosis. In mouse brain, ablation of α-synuclein function significantly suppresses Tau phosphorylation at Ser(262). In vitro, α-synuclein binds to phosphorylated Ser(214) of Tau and stimulates PKA-catalyzed Tau phosphorylation at Ser(262). PD-associated α-synuclein mutations increase α-synuclein binding to Tau and stimulate Tau phosphorylation at Ser(262). In HEK-293 cells, α-synuclein and its all PD-associated mutants destabilize the microtubule cytoskeleton in a similar extent. In contrast, when co-expressed with Tau, these PD-associated mutants destabilize microtubules with significantly higher potency than WT. Our results demonstrate that α-synuclein is an in vivo regulator of Tau protein phosphorylation at Ser(262) and suggest that PD-associated risk factors such as environmental toxins and α-synuclein mutations promote Tau phosphorylation at Ser(262), causing microtubule instability, which leads to loss of dopaminergic neurons in PD brain.     

N-acetylcysteine reverses cardiac myocyte dysfunction in HIV-Tat proteinopathy

HIV cardiomyopathy remains highly prevalent among the estimated 33 million HIV-infected individuals worldwide. This is particularly true in developing countries. Potential mechanisms responsible for myocardial dysfunction following HIV infection include direct effects of HIV proteins. We have previously reported that cardiac myocyte-specific expression of HIV-Tat (Tat) results in a murine cardiomyopathy model. We now report that Tat exhibits decreased myocardial ATP [wild type (WT) vs. Tat transgenic (TG), P < 0.01] and myocyte GSH levels (WT vs. TG, P < 0.01), decreased GSH/GSSG ratio (WT vs. TG, P < 0.01), increased H(2)O(2) levels (WT vs. TG, P < 0.05), and increased catalase (TG vs. WT, P < 0.05) and GPX1 (glutathione peroxidase 1) activities (WT vs. TG, P < 0.05), blunted cardiac myocyte positive inotropy (% peak shortening, WT vs. TG, P < 0.01; +dl/dt, WT vs. TG, P < 0.01) and negative inotropy (-dl/dt, WT vs. TG, P < 0.01), and blunted inotropic responses to Ca(2+) (P < 0.01, for each) and shortened anatomical and functional survival in vitro (P < 0.01). The sulfhydryl donor, N-acetylcysteine (NAC; 10(-4) M), completely reversed both the positive and negative inotropic defects in Tat; increased GSH (P < 0.01) and GSH/GSSG (P < 0.01); reversed H(2)O(2) level (P < 0.05) and GPX1 activity (P < 0.05); and normalized the blunted inotropic response to Ca(2+) (P < 0.01). NAC (10(-7)) M normalized duration of contractile function from <40 min to >120 min (P < 0.01), with no effect on GSH and GSH/GSSG. NAC (10(-4) M) reverses cardiac myocyte dysfunction and markers of oxidative stress. NAC (10(-7) M) enhances myocyte function independent of changes in glutathione. Elucidating the molecular mechanisms involved in the GSH-dependent and GSH-independent salutary effects of NAC should identify novel therapeutic targets for myocardial proteinopathies recently appreciated in human cardiomyopathies.