鱼藤酮诱导细胞内泛素化α-synuclein聚集选择性损伤多巴胺神经元

目的探讨鱼藤酮对多巴胺能神经元内泛素化-αsynuclein聚集的影响及其细胞损伤作用。方法应用鱼藤酮处理经NGF诱导的神经元样分化的PC12细胞株(多巴胺能神经元)与N2a细胞株(非多巴胺能神经元)4、8、16、24 h以及用利血平预处理PC12细胞4 h再加入鱼藤酮处理16 h;采用免疫荧光双标记方法在共聚焦显微镜下观察细胞内泛素化-αsynuclein聚集,以MTT法和流式细胞术分别检测PC12细胞活力及凋亡率。结果单用鱼藤酮处理16 h后,PC12细胞株与N2a细胞株相比较,免疫荧光双标记显示PC12细胞内泛素化-αsynu-clein发生明显聚集,并且鱼藤酮对PC12细胞株的作用具有时间依赖性,而N2a细胞内泛素化-αsynuclein聚集不明显。利血平耗竭PC12细胞内多巴胺后再经鱼藤酮处理,PC12细胞内泛素化-αsynuclein聚集不明显;经不同浓度鱼藤酮处理后,细胞活力呈剂量依赖性下降;与对照组相比,经20 nmol/L鱼藤酮处理4、16、24 h后细胞存活率分别为(81.6±12.3)%、(59.8±6.7)%和(52.2±7.4)%(P<0.01)。鱼藤酮处理后出现早期凋亡细胞,随着处理时间的延长细胞凋亡率逐渐上升(P<0.01)。结论鱼藤酮选择性作用于多巴胺能神经元,使细胞内泛素化-αsynuclein发生聚集,而且这种变化具有时间依赖性,最终导致细胞发生凋亡,然而鱼藤酮对非多巴胺能神经元作用不明显。同时,鱼藤酮诱导泛素化-αsynuclein发生聚集的作用与神经元的特性存在密切关系。     

泛素-蛋白酶体抑制剂诱导细胞内泛素化α-synuclein聚集选择性损伤多巴胺神经元

目的 探讨多巴胺(DA)神经元诱变剂对细胞内泛素化α-synuclein聚集的影响及其细胞损伤作用.方法 应用MPP+、特异性泛素-蛋白酶体系统(UPS)抑制剂lactacystin及H2O2处理神经生长因子(NGF)诱导的PC12细胞株与N2a细胞株16 h后,采用免疫荧光双标记的方法在共聚焦显微镜下观察细胞内泛素化α-synuclein聚集,比较三种诱变剂对这两种细胞株作用的差异.在PC12细胞中加入不同终浓度的lactacystin处理24 h,MTT方法检测PC12细胞活力.10μmol/Llactacystin处理PC12细胞不同时间,流式细胞术检测PC12细胞的早期凋亡率.结果 经三种诱变剂处理后,MPP+和lactacystin选择性地诱发PC12细胞内泛素化α-synuclein聚集且以lactacystin的作用更为显著,N2a细胞则无明显的泛素化α-synuclein聚集.H2O2作用于PC12细胞的效应与MPP+相近,但仅引起N2a细胞内少量泛素化α-synuclein聚集.经lactacystin处理后的PC12细胞活力呈剂量依赖性下降;5 μmol/L、10μmol/L和20 μmol/L lactacystin处理24 h后细胞存活率分别为79.5%±2.1%、49.3%±3.2%和31.2%±2.8%(与对照组相比,均P＜0.01).流式细胞术显示lactacystin处理后PC12细胞早期出现凋亡细胞,并且随着处理时间的延长,细胞凋亡率逐渐增加.结论 特异性UPS和线粒体呼吸链抑制剂选择性作用于DA神经元,诱导细胞内泛素化α-synuclein聚集,终使细胞发生凋亡,而以氧化应激为主要损伤途径的诱变剂的作用则不具有选择性.     

溶酶体自噬途径降解α-synuclein蛋白作用研究

目的 探讨α-synuclein蛋白细胞内溶酶体途径降解机制.方法 用神经生长因子NGF诱导分化PC12细胞作为研究多巴胺能神经元的细胞载体,应用鱼藤酮处理PC12细胞建立α-synuclein蛋白细胞模型.使用溶酶体途径降解抑制剂E64处理神经元样分化的PC12细胞,应用免疫荧光双标方法观察PC12细胞内硫黄素S、α-synuclein蛋白阳性聚集包涵体形成情况,比较各组的差异.结果 用E64处理鱼藤酮预处理过的PC12细胞后α-synuclein蛋白聚集且较多包涵体形成(15.36±0.85)%,与对照组相比差异有统计学意义(P＜0.05).结论 溶酶体自噬途径可能在α-synuclein蛋白降解、聚集和多巴胺神经元死亡过程中发挥重要作用.     

Lactacystin诱导PC12细胞致帕金森病的实验研究

目的应用蛋白酶体抑制剂Lactacystin构建帕金森病细胞模型,从泛素-蛋白酶体功能角度探讨帕金森病的发病机制。方法Lactacystin(0、5、10、15和20μmol/L)分别处理PC12细胞24h,MTT法检测细胞活力;HE染色观察包涵体生成;免疫组织化学法观察α-synuclein在胞内聚集情况;AO/EB双染及电镜检测细胞凋亡。结果10μmol/L Lactacystin作用24h后细胞活力开始显著低于对照组(P<0.01),随着浓度大,细胞活力进一步下降,呈浓度依赖性(P<0.01);HE染色显示胞浆核周出现圆形或椭圆形嗜伊红的包涵体;免疫组化显示包涵体α-synuclein染色呈强阳性;10μmol/L Lactacystin作用细胞24h后AO/EB双染提示细胞早期凋亡;电镜显示细胞核变小偏位,部分胞核固缩、趋边、凝聚。结论Lactacystin对多巴胺能神经元有毒性作用,可形成胞浆内包涵体并诱导细胞凋亡。蛋白酶体功能异常可能在帕金森病发病机制中发挥重要作用。     

蛋白酶体抑制剂诱导多巴胺能神经元变性伴包涵体形成

目的 观察蛋白酶体抑制剂诱导黑质多巴胺(DA)能神经元变性伴胞浆内包涵体形成，探讨蛋白酶体功能在帕金森病发病机制中的作用。方法将蛋白酶体抑制剂Lactacystin立体定向注射至大鼠黑质部位。免疫荧光观察黑质区DA神经元变性缺失及胶质细胞变化。免疫荧光双标法观察DA能神经元内蛋白聚集的包涵体及其主要成分α-共核蛋白(α-synuclein)、Parkin和泛素(ubiquitin)的表达。同时观察。DA能神经元发生细胞凋亡。结果注射Lactacystin第7天大鼠开始出现自发性活动减少，阿扑吗啡可诱导出旋转行为；3周后黑质部位酪氨酸羟化酶(TH)阳性细胞减少，呈剂量依赖性；小胶质细胞增生明显。TH与硫磺素、硫磺素与α-synuclein、硫磺素与Parkin、以及硫磺素与ubiquitin复合染色呈阳性；TH与TUNEL双染亦呈阳性。结论Lactacystin对多巴胺能神经元具有毒性作用，且导致蛋白聚集，包涵体形成。蛋白酶体功能异常可能在帕金森发病机制中起重要作用。     

鱼藤酮致大鼠脑内α-突触核蛋白的表达分布变化(英文)

目的研究鱼藤酮所致的帕金森病大鼠的脑内α-突触核蛋白(α-synuclein, ASN)分布。方法 Wistar大鼠随机分成两组,分别给予鱼藤酮和/ 或溶剂(对照组)皮下注射,4 周后取脑组织,对黑质部位HE 染色,光镜下观察Lewy小体形态;对黑质、海马、纹状体等脑区进行酪氨酸羟化酶(tyrosine hydroxylase, TH)、ASN 免疫组织化学染色。结果在对照组大鼠脑内,ASN 广泛分布于各脑区,尤其在皮质、纹状体、海马等纤维投射丰富的区域。鱼藤酮处理的大鼠脑中,黑质TH阳性多巴胺能神经元数目减少、纹状体区TH阳性纤维脱失,黑质部位可见Lewy小体样结构;ASN阳性染色在各个脑区均有增强但各个脑区增强程度不一,黑质部位神经元胞浆和胞核内均有ASN明显聚集,纹状体可见ASN聚集围绕在细胞周围。海马部位偶见ASN在胞浆中点状聚集,胞核中无明显改变。结论在鱼藤酮皮下注射导致的帕金森病大鼠的脑内,ASN在多个脑区中表达增加,而在黑质纹状体部位聚集最为明显,蛋白分布由多巴胺能神经元的突触末端向胞浆和胞核扩展。     

α-synuclein和帕金森病的关系研究进展

近年来,大量研究显示SNCA基因突变等多方面的原因可导致α-synuclein异常聚集,而其和家族型和散发型帕金森病有密切关系,但是具体机制还不清楚.病理性的α-synuclein是路易小体和路易轴突的主要成分,而后两者在脑内的沉积是帕金森病的标志.帕金森病相关基因突变引起α-synuclein的聚集,其低聚物和聚集物可能干扰正常细胞功能和促进多巴胺神经元变性.本文综述了近年来关于病理性α-synuclein参与帕金森病病理生理过程及相应干预措施的研究进展以及针对α-synuclein的治疗.     

溶酶体自噬途径降解α—synuclein蛋白作用研究

目的探讨α—synuclein蛋白细胞内溶酶体途径降解机制。方法用神经生长因子NGF诱导分化PCI2细胞作为研究多巴胺能神经元的细胞载体,应用鱼藤酮处理PCI2细胞建立α-synuclein蛋白细胞模型。使用溶酶体途径降解抑制剂E64处理神经元样分化的PCI2细胞,应用免疫荧光双标方法观察PCI2细胞内硫黄素S、α—synuclein蛋白阳性聚集包涵体形成情况,比较各组的差异。结果用E64处理鱼藤酮预处理过的PCI2细胞后α—synuclein蛋白聚集且较多包涵体形成（15．36±0．85）％,与对照组相比差异有统计学意义（P〈0．05）。结论溶酶体自噬途径可能在α—synuclein蛋白降解、聚集和多巴胺神经元死亡过程中发挥重要作用。     

阻断泛素-蛋白酶体通路诱导PC12细胞死亡和泛素阳性包涵体生成

目的探讨泛素蛋白酶体通路的功能障碍对于多巴胺能细胞的活力以及胞质内包涵体生成的影响。方法应用蛋白酶体抑制剂lactacystin(5μmol/L、10μmol/L、15μmol/L和20μmol/L)处理PC12细胞24h,MTT方法检测细胞活力,WesternBlot方法测定细胞内泛素化蛋白质水平,免疫荧光细胞化学染色观察泛素免疫阳性包涵体的生成。结果经5μmol/L、10μmol/L、15μmol/L和20μmol/Llactacystin处理24h后,PC12细胞的活力显著降低(细胞存活率分别为81.5%±3.6%、75.4%±2.4%、70.2%±2.7%和60.4%±3.9%),呈现剂量依赖性。WesternBlot证实对照组细胞内未检测到相对高分子质量的泛素化蛋白质;随着lactacystin作用浓度的增加,细胞内相对高分子质量泛素化蛋白质的含量逐渐增高。免疫荧光染色显示对照组中仅有极少数细胞内含有泛素阳性包涵体;20μmol/Llactacystin处理组中含有泛素阳性包涵体的细胞数目显著增多(P<0.01)。结论泛素蛋白酶体通路的功能缺失能诱导多巴胺能细胞死亡,造成细胞内泛素化蛋白质积聚,促进胞质内泛素阳性包涵体的生成,可能在帕金森病黑质多巴胺能神经元变性死亡和Lewy小体形成中发挥重要作用。     

ATP损耗是MPP^＋引起α-synuclein转基因线虫多巴胺能神经元死亡和虫体死亡的主要原因

目的揭示环境神经毒素MPP~ 对线虫的毒性影响并探讨其毒性机理。方法以人源α-synuclein转基因线虫作为动物模型,用MPP~ 处理该线虫,观察MPP~ 对线虫多巴胺能神经元和其生存能力的影响。通过供给OP50以提高线虫体内ATP的水平,对比分析ATP水平、蛋白质异常沉积等重要指标,探讨二者在MPP~ 引起的转基因线虫的病变中所起的作用。结果MPP~ 能够引起线虫多巴胺能神经元和线虫虫体的死亡;尽管进食OP50也会引起α-synuclein的沉积,但进食OP50能够提高体内ATP的水平并缓解MPP~ 的毒性。虽无直接证据证明α-synuclein沉积对神经细胞的影响,但结果提示,在该转基因线虫中,与蛋白质的异常沉积相比,MPP~ 导致的ATP损耗是其毒性作用的最主要诱因。结论MPP~ 可以引起α-synuclein转基因线虫多巴胺能神经元的死亡和虫体的死亡,其毒性的主要原因是ATP损耗而不是α-synuclein的异常聚集(沉积)。     

2-DPMP (desoxypipradrol, 2-benzhydrylpiperidine, 2-phenylmethylpiperidine) and D2PM (diphenyl-2-pyrrolidin-2-yl-methanol,diphenylprolinol): A preliminary review

2-DPMP (desoxypipradrol, 2-benzhydrylpiperidine, 2-phenylmethylpiperidine) and D2PM (diphenyl-2-pyrrolidin-2-yl-methanol, diphenylprolinol) are psychoactive substances, sold primarily over the Internet and in ‘head’ shops as ‘legal highs’, ‘research chemicals’ or ‘plant food’. Originally developed in the 1950s for the treatment of narcolepsy and ADHD, 2-DPMP's use soon became very limited. Recreational use of 2-DPMP and D2PM appears to have started in March 2007, but only developed slowly. However, in the UK their popularity grew in 2009, increasing rapidly during summer 2010. At this time, there were many presentations to UK Emergency Departments by patients complaining of undesirable physical and psychiatric effects after taking 2-DPMP. In spring 2011 there were similar presentations for D2PM. Recreational use of these drugs has been reported only occasionally in on-line user fora. There is little scientifically-based literature on the pharmacological, physiological, psychopharmacological, toxicological and epidemiological characteristics of these drugs. Here we describe what is known about them, especially on their toxicity, including what we believe to be the first three deaths involving the use of 2-DPMP in August 2010. There are no international controls imposed on 2-DPMP or D2PM. However, a ban on their UK importation was imposed in November 2011 and they became Class C drugs on 13 June 2012. It is critical that any other cases, including non-fatal overdoses, are documented so that a scientific evidence-base can be established for them.     

Spinocerebellar ataxia 2 (SCA2)

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominantly inherited, neurodegenerative disease. It can manifest either with a cerebellar syndrome or as Parkinson’s syndrome, while later stages involve mainly brainstem, spinal cord and thalamus. This particular atrophy pattern resembles sporadic multi-system-atrophy (MSA) and results in some clinical features indicative of SCA2, such as early saccade slowing, early hyporeflexia, severe tremor of postural or action type, and early myoclonus. For treatment, levodopa is temporarily useful for rigidity/bradykinesia and for tremor, magnesium for muscle cramps, but neuroprotective therapy will depend on the elucidation of pathogenesis. The disease cause lies in the polyglutamine domain of the protein ataxin-2, which can expand in families over successive generations resulting in earlier onset age and faster progression. Genetic testing in SCA2 and other polyglutamine disorders like the well-studied Huntington’s disease is now readily available for family planning. Although these disorders differ clinically and in the affected neuron populations, it is not understood how the different polyglutamine proteins mediate such tissue specificity. The neuronal intranuclear inclusion bodies described in other polyglutamine disorders are not frequent in SCA2. For the quite ubiquitously expressed ataxin-2, a subcellular localization at the Golgi, the endoplasmic reticulum and the plasma membrane, in interaction with proteins of mRNA translation and of endocytosis have been observed. As a first victim of SCA2 degeneration, cerebellar Purkinje neurons may be preferentially susceptible to alterations of these subcellular pathways, and therefore our review aims to portray the particular profile of the SCA2 disease process and correlate it to the specific features of ataxin-2.     

Neurofibromatosis 2

PURPOSE OF REVIEW: Recent clinical and molecular research on neurofibromatosis 2 (NF2) is reviewed, and the implications for clinical practice and research are discussed. RECENT FINDINGS: NF2 patients who are treated in specialty centers have a significantly lower risk of mortality than those who are treated in non-specialty centers. Vestibular schwannoma growth rates in NF2 are generally higher in younger people but are highly variable, even among multiple NF2 patients of similar ages in the same family. Radiation therapy is best reserved for NF2 patients who have particularly aggressive tumors, those who are poor surgical risks, those who refuse surgery, or those who are elderly. In-vivo studies have demonstrated that leptomeningeal cell activation of in mice results in leptomeningeal hyperplasia and meningioma formation. In-vitro studies have identified molecules that interact with the product (merlin or schwannomin), some of which (e.g., CD44 and paxillin) may play critical roles in merlin growth regulation. SUMMARY: NF2 patients should be referred to specialty treatment centers for optimal care. Clinical management of multiple patients in NF2 families cannot be based on the expectation of similar vestibular schwannoma growth rates, even when other clinical aspects of disease severity are similar. The availability of accurate mouse models of human NF2-associated tumors and the identification of molecules involved in merlin growth regulation now provide an opportunity to design targeted treatments for schwannomas and meningiomas.