S100钙结合蛋白β与α突触核蛋白与帕金森病患者抑郁及运动障碍的关系

目的研究帕金森病(PD)患者血清S100钙结合蛋白β(S100β)及α突触核蛋白(α-syn)水平与患者抑郁及运动障碍的关系。方法选择2022年1月至12月聊城市人民医院收治的PD患者194例,根据汉密尔顿抑郁量表评分分为非抑郁组102例(0~13分)及抑郁组92例(≥14分)。运动障碍采用Hoehn-Yahr(H-Y)分级及统一帕金森病评定量表Ⅲ(UPDRS-Ⅲ)进行评分,用多因素logistic回归分析PD患者抑郁的独立危险因素,用Spearman相关性分析血清S100β及α-syn水平与患者抑郁及运动障碍的关系。结果抑郁组体质量指数低于非抑郁组,H-Y分级、UPDRS-Ⅲ评分、S100β、α-syn均高于非抑郁组,差异有统计学意义(P<0.05,P<0.01)。多因素logistic回归分析显示,H-Y分级、UPDRS-Ⅲ评分、S100β、α-syn为PD患者抑郁的独立危险因素(P<0.05)。Spearman相关性分析显示,PD患者S100β水平与H-Y分级、UPDRS-Ⅲ评分呈正相关(r=0.698,P=0.005;r=0.637,P=0.011);α-syn水平与H-Y分级、UPDRS-Ⅲ评分呈正相关(r=0.654,P=0.021;r=0.611,P=0.035)。ROC曲线显示,S100β、α-syn诊断PD患者抑郁的截断值分别为486.65μg/L、3894.27 ng/L,曲线下面积分别为0.889(95%CI:0.812~0.923)、0.761(95%CI:0.714~0.828),S100β曲线下面积显著优于α-syn(P<0.05)。结论PD患者血清S100β、α-syn水平与其抑郁发生及运动功能障碍密切相关。

帕金森病血浆神经退行性蛋白与非运动症状的关系

目的探讨血浆神经退行性蛋白与帕金森病(PD)非运动症状的关系。方法连续收集就诊于江苏省人民医院(南京医科大学第一附属医院)神经内科门诊的PD患者84例为PD组,同期招募年龄相匹配的健康体检者54例为HC组。PD组患者用系列临床量表评估非运动症状严重程度,采用ELISA法测定血浆tau、磷酸化tau181(p-tau181)、β淀粉样蛋白42(Aβ-42)和α-突触核蛋白(α-syn)水平,采用Spearman's相关性分析和二元logistic回归分析。结果PD组疲劳严重程度量表(fatigue severity scale,FSS)评分较HC组明显升高[(3.22±1.68)分vs(1.89±1.16)分,P=0.000]。PD组血浆α-syn水平较HC组明显升高[(320.00±64.91)ng/L vs (277.78±52.75)ng/L,P=0.000],Aβ-42水平较HC组明显降低[(267.61±77.75)ng/L vs (321.80±49.41)ng/L,P=0.001]。2组tau和p-tau181水平比较,无显著差异(P>0.05)。PD组血浆α-syn水平与FSS评分呈正相关(r=0.237,P=0.030),且血浆α-syn水平是FSS评分的影响因素(OR=1.019,95%CI:1.006～1.032,P=0.004)。结论血浆神经退行性蛋白与PD非运动症状相关。血浆α-syn可能是PD疲劳的一种外周生物标志物。

Aβ1-42寡聚体对α-syn过表达SHSY5YA53T细胞自噬功能的影响

目的构建人A53T突变型α-突触核蛋白过表达的SHSY5Y细胞模型,观察Aβ1-42寡聚体对细胞的毒性作用和自噬功能的影响。方法利用慢病毒稳转方法构建A53T突变型α-突触核蛋白过表达的SHSY5Y细胞及空载体对照细胞,RT-qPCR方法检测SHSY5Y细胞中α-突触核蛋白mRNA的表达。用Aβ1-42寡聚体干预两组细胞24h,CCK-8法检测Aβ1-42寡聚体对细胞增殖的影响,WesternBlot检测细胞自噬相关蛋白的表达水平。结果慢病毒转染SHSY5Y细胞后,过表达组细胞内α-突触核蛋白表达水平较正常细胞组及开载体对照组增加,差异有统计学意义(P<0.001);人A53T突变型α-突触核蛋白过表达不影响细胞的增殖;不同浓度Aβ1-42寡聚体(0、0.5μmol/L、1.25μmol/L、2.5μmol/L、5μmol/L、10μmol/L)处理细胞24h后,细胞增殖抑制率成浓度依赖性;Aβ1-42寡聚体处理后,α-突触核蛋白过表达组细胞LC3-Ⅱ,Beclin-1自噬蛋白表达水平较对照组细胞显著降低(P<0.05)。结论人A53T突变型α-突触核蛋白过表达不影响的SHSY5Y细胞增殖,Aβ1-42寡聚体对α-突触核蛋白过表达细胞具有显著毒性,对细胞的损伤机制可能通过抑制细胞自噬功能。

松果菊苷对6-OHDA帕金森病模型大鼠海马α-Synuclein、β-actin蛋白表达影响随机平行对照研究

[目的]观察松果菊苷对6-OHDA帕金森病模型大鼠海马α-Synuclein、β-actin蛋白表达影响。[方法]使用随机平行对照方法,将72只SPF级SD雄性大鼠适应性饲养14d,按随机数字表法分为6组,空白对照组、假手术组、模型组、松果菊苷(低、中、高剂量)组,12只/组。各组大鼠腹腔注射10%水合氯醛(30mg/kg)麻醉,固定于颅脑定位仪,消毒,沿颅顶正中线切开皮肤,钝性分离颅骨外膜,暴露大鼠颅骨前囟门;以前囟为坐标原点(大鼠脑定位立体图谱),确定前脑内侧束(MFB)坐标,MFB:前囟后4.8mm,矢状缝右侧1.2mm,硬脑下7.8mm;颅骨钻钻孔,立体定向靶点微量注射6-OHDA(2ug/uL,溶于0.1%抗坏血酸生理盐水),注射速度1uL/min,留针15min;骨蜡封闭大鼠脑部颅骨孔,消毒缝合皮肤,复制帕金森病模型,松果菊苷(低、中、高剂量)组(8 ug)、模型组(2ug);空白对照组、假手术组(与模型组等量生理盐水)。灌胃干预:复制成功模型,松果菊苷组(低20mg/kg、中40mg/kg,高80mg/kg),余各组均等体积生理盐水,1次/d,连续10d。PAGE胶蛋白电泳;免疫印迹(Western Blot,WB)法检测α-Synuclein、β-actin蛋白。[结果]模型复制48只大鼠,麻醉过度死亡3只,模型复制不成功2只,最终纳入结果分析43只,模型组10只,松果菊苷组低、中、高剂量组各11只。α-Synuclein蛋白水平假手术组、松果菊苷各组(低中高)均低于空白对照组(P<0.01);β-actin各组间无明显差异(P>0.05);α-Synuclein/β-actin假手术组、松果菊苷低剂量组、松果菊苷中剂量组、松果菊苷高剂量组均低于空白对照组(P<0.01)。[结论]松果菊苷干预帕金森病模型大鼠,可降低海马α-Synuclein表达。

Unfolded annealing molecular dynamics conformers for wild-type and disease-associated variants of alpha-synuclein show no propensity for beta-sheetformation

Aggregation of alpha-synuclein leads to the formation of Lewy bodies in the brains of patients affected by Parkinson's disease (PD). Native human alpha-synuclein is unfolded in solution but assumes a partial alpha-helical conformation upon transient binding to lipid membranes. Annealing Molecular Dynamics (AMD) was used to generate a diverse set of unfolded conformers of free monomeric wild-type alpha-synuclein and PD-associated mutants A30P and A53T. The AMD conformers were compared in terms of secondary structure, hydrogen bond network, solvent-accessible surface per residue, and molecular volume. The objective of these simulations was to identify structural properties near mutation sites and the non-amyloid component (NAC) region that differ between wild- type and disease-associated variants and may be associated to aggregation of alpha- synuclein. Based on experimental evidence, a hypothesis exists that aggregation involves the formation of intermolecular beta sheets. According to our results, disease-associated mutants of alpha-synuclein are no more propense to contain extended beta regions than wild-type alpha-synuclein. Moreover, extended beta structures (necessary for beta sheet formation) were not found at or around positions 30 and 53, or the NAC region in any unfolded conformer of wild-type, A30P or A53T alpha-synuclein, under the conditions of the simulations. These results do not support the hypothesis that the mutant's higher propensity to aggregation results solely from changes in amino acid sequence leading to changes in secondary structure folding propensity.

Targeting prion-like protein spreading in neurodegenerative diseases

The infectious template-mediated protein conversion is a unique mechanism for the onset of rare and fatal neurodegenerative disorders known as transmissible spongiform encephalopathies, or prion diseases, which affect humans and other animal species. However, emerging studies are now demonstrating prion-like mechanisms of self-propagation of protein misfolding in a number of common, non-infectious neurodegenerative diseases such as Alzheimer＇s disease and Parkinson＇s disease. It has been proposed that distinct and unrelated proteins（beta-amyloid, tau, α-synuclein, TAR DNA-binding protein 43 and huntingtin, etc.） associated with common neurodegenerative disorders can seed conversion and spread via cellto-cell transfer, sustaining the transmission of neurotoxic agents along a stereotypic route, sharing features at the heart of the intrinsic nature of prions. Here we review the most recent development on both the molecular mechanisms underlying the pathogenesis of prion-like neurodegenerative diseases as well as innovative methods and strategies for potential therapeutic applications.

Mechanism-based anti-anxiety effects of polysaccharides extracted from Shudihuang (Radix Rehmanniae Preparata) by two-dimensional electrophoresis analysis in rat hippocampus proteins

OBJECTIVE: To investigate mechanism-based anti-anxiety effects of Shudihuang (Radix Rehmanniae Preparata) polysaccharides (RRPPs) through two-dimensional electrophoresis (2-DE) analysis with mass spectrometry (MS) of hippocampus proteins in rats treated with monosodium L-glutamate (MSG).METHODS: MSG (4 g/kg) or normal saline (NS) was injected subcutaneously into infant male rats on days 2, 4, 6, 8, 10 after birth. MSG-treated rats at 8 weeks old were given NS, diazepam, or RRPPs daily for seven consecutive days by intragastric administration, while NS-treated rats given the same volume of NS. Elevated plus maze (EPM) and light/ dark transition (LDT) tests were used to observe anti-anxiety effects of RRPPs at 1 h after the last administration. After EPM and LDT tests, hippocampus tissues were excised on ice rapidly from the brains of rats. Thereafter, 2-DE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) were used for detecting differential proteins in hippocampus of rats so as to explore the potential mechanisms. RESULTS: RRPPs (200, 400 mg/kg) could significantly inhibit MSG-induced decrease of time and entries percentages in open zones in EPM test and numbers of light-dark transition in LDT test. Further analysis of 2-DE and MALDI-TOF/MS indicated that β-synuclein, protein DJ-1, peroxiredoxin-2, peroxiredoxin-6, dimethylarginine dimethylaminohydrolase 1 (DDAH-1) and iron-sulfur proteins were all found to be down-regulated significantly in MSG-treated rats, while such down-regulation was significantly inhibited after treatment with RRPPs. CONCLUSION: RRPPs showed anti-anxiety effects and potential mechanisms might be related to inhibiting MSG-induced down-regulation of β-synuclein, DJ-1, peroxiredoxin-2, peroxiredoxin-6, DDAH-1 and iron-sulfur proteins in hippocampus of rats.

β-synuclein对Ⅱ型囊泡单胺转移体表达的促进作用

目的探讨β-突触核蛋白(β-synuclein)对Ⅱ型囊泡单胺转移体(VMAT2)表达的影响。方法通过2',7'-二氯荧光黄双乙酸盐(DCFH-DA)染色检测β-synuclein稳定表达对细胞内ROS水平的影响;采用免疫荧光双标法、免疫组织化学法及Western blotting检测VMAT2在稳定转染β-synuclein的SH-SY5Y细胞及正常SH-SY5Y细胞中的表达情况。结果 DCFH-DA荧光染色显示,稳定表达β-synuclein的细胞的荧光强度较正常SH-SY5Y细胞明显减弱。VMAT2在稳定转染β-synuclein的SH-SY5Y细胞中的表达水平较正常SH-SY5Y细胞明显升高。结论β-synuclein能够促进VMAT2的表达,减少胞浆内DA含量,抑制ROS产生,在帕金森病多巴胺神经元变性过程中对其起到保护作用。

血清α-syn、Aβ1-42、SSA在帕金森病患者中的表达及与认知功能损害的关系

目的:探讨血清α-突触核蛋白(α-synuclein,α-syn)、β淀粉样蛋白1-42(β-amyloid 1-42,Aβ1-42)、淀粉样蛋白A(serum amyloid A,SSA)在帕金森病患者中的表达及与认知功能损害的关系。方法:收集本院2018年7月2020年11月收治的136例帕金森病患者为病例组,依据Hoehn-Yahr(H-Y)分级分为早期组(n=76)与中晚期组(n=60);依据蒙特利尔认知评估量表(Montreal Cognitive Assessment Scale,Mo CA)分为认知功能正常组(n=94)与认知功能损害组(n=42)。另选同期本院体检正常者105例纳入对照组。对比各组血清α-syn、SSA、Aβ1-42水平及Mo CA,并用Pearson相关系数分析病例组患者血清α-syn、SSA、Aβ1-42水平与Mo CA的相关性,用受试者工作特征曲线(receiver operating characteristic curve,ROC)分析血清α-syn、SSA、Aβ1-42水平对帕金森病和认知功能损害的诊断价值。结果:病例组血清α-syn、SSA水平高于对照组,差异有统计学意义(P<0.05);病例组Aβ1-42、Mo CA评分低于对照组(P<0.05)。病例组晚期患者血清α-syn、SSA水平高于早期患者(P<0.05);病例组晚期患者Aβ1-42、Mo CA评分低于早期患者,差异有统计学意义(P<0.05)。病例组有认知功能损害患者血清α-syn、SSA水平高于认知功能正常患者(P<0.05);病例组有认知功能损害患者Aβ1-42、Mo CA评分低于认知功能正常患者(P<0.05)。病例组患者血清α-syn、SSA与Mo CA均呈正相关(P<0.05),Aβ1-42与Mo CA呈正相关(P<0.05)。血清α-syn、SSA、Aβ1-42水平对帕金森病诊断的曲线下面积分别为0.858、0.821、0.785;血清α-syn、SSA、Aβ1-42水平对帕金森病认知功能损害预测的曲线下面积分别为0.877、0.825、0.783。结论:帕金森病患者血清α-syn、SSA、Aβ1-42水平较健康者变化明显,且可能和帕金森病的诊断、病情进展和认知功能损害有一定关系。