Prion-like propagation of α-synuclein in gutbrain axis

Parkinson disease(PD) is a progressive degenerative disease of the nervous system,which is characterized by movement disorders,such as static tremor,rigidity,and bradykinesia in advanced patients.Gastrointestinal(GI) dysfunction,such as gastric dysmotility,constipation,and anorectic dysfunction,is common non-motor symptom in the early stage of PD.The progression of PD includes the degenerative loss of dopaminergic neurons and aggregation ofα-synuclein in the substantia nigra.Interestingly,both of them are also present in the enteric nervous system of PD patients.In this review,we describe the relationship between non-motor symptoms particularly GI dysfunction and the pathogenesis of PD,aiming to show the powerful evidences about the prion-like propagation of α-synuclein and support the hypothesis of gut-brain axis in PD.We then summarize the mechanism of the gut-brain axis and confirmα-synuclein as a potential target for drug design or new clinical treatment.

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.

Decoding the Cellular Trafficking of Prion-like Proteins in Neurodegenerative Diseases

The accumulation and spread of prion-like proteins is a key feature of neurodegenerative diseases(NDs)such as Alzheimer’s disease,Parkinson's disease,or Amyotrophic Lateral Sclerosis.In a process known as‘seeding’,prion-like proteins such as amyloid beta,microtubule-associated protein tau,α-synuclein,silence superoxide dismutase 1,or transactive response DNA-binding protein 43 kDa,propagate their misfolded conformations by transforming their respective soluble monomers into fibrils.Cellular and molecular evidence of prion-like propagation in NDs,the clinical relevance of their‘seeding’capacities,and their levels of contribution towards disease progression have been intensively studied over recent years.This review unpacks the cyclic prion-like propagation in cells including factors of aggregate internalization,endo-lysosomal leaking,aggregate degradation,and secretion.Debates on the importance of the role of prion-like protein aggregates in NDs,whether causal or consequent,are also discussed.Applications lead to a greater understanding of ND pathogenesis and increased potential for therapeutic strategies.

克—雅氏病患者叠朊蛋白基因多态性的研究

目的探讨叠朊蛋白基因(PRND)与克—雅氏病(CJD)易感性的关系。方法应用聚合酶链式反应—限制性片段长度多态性(PCR-RFLP)方法,对15例CJD患者(观察组)和15例查体健康者(对照组)的PRND第56位点进行基因多态性分型,统计学方法分析多态性与CJD发病的关系;应用PCR法扩增观察组PRND基因,进行测序。结果对一个家系的2例疑诊家族性CJD患者行PRND基因测序发现,其均有一少见突变,观察组PRND第56位点脯氨酸/亮氨酸杂合子的基因型频率明显高于与对照组(P<0.05)。结论 PRND56位点基因多态性可能与CJD有关。

人成熟叠朊在大肠埃希菌中高效表达

以含有人叠朊编码基因Prnd的ORF的质粒HoPrnd-T为模板,PCR扩增出成熟叠朊编码基因片段homDpl,并将其插入原核表达载体pGEX-6P-1的EcoRⅠ和XhoⅠ位点之间,构建出重组表达质粒pGEX-homDpl。将pGEX-homDpl电转化到宿主菌株BL21(DE3)中,以1.0 mmol/LIPTG诱导表达,SDS-PAGE结果显示人成熟叠朊被大肠埃希菌高效表达。

IGF-1对PC12细胞PRNP表达及APP代谢的影响

目的·探讨胰岛素样生长因子-1(IGF-1)对肾上腺嗜铬细胞瘤细胞(PC12)中朊蛋白编码基因(PRNP)表达及淀粉样蛋白前体(APP)代谢的影响。方法·建立阿尔茨海默病(AD)细胞模型;用不同浓度的IGF-1(20、40、80 ng/mL)作用于PC12细胞24 h,采用real-time PCR检测PRNP m RNA的表达水平;Western blotting检测AKT/p AKT、ERK/p ERK蛋白的表达水平;ELISA检测细胞上清液中β-淀粉样蛋白42(Aβ42)的水平。结果·与空白对照组相比,AD模型组PRNP m RNA的表达量明显升高(P<0.01);不同浓度IGF-1处理细胞24 h后,40 ng/mL及80 ng/mL IGF-1组PRNP m RNA的表达量显著升高(P<0.01)。模型组及IGF-1各浓度组中APP蛋白的表达均无显著变化(P>0.05)。与对照组相比,AD模型组上清液中Aβ42的水平显著下降,其他各组随着IGF-1浓度的增加而降低,其中40 ng/mL及80 ng/mL IGF-1组Aβ42的表达水平降低显著(P<0.05)。与对照组相比,各组AKT/p AKT、ERK/p ERK蛋白的表达量均明显升高,且随着IGF-1剂量的增加而上升(P<0.05)。结论·IGF-1降低PRNP基因表达的同时影响APP代谢,此过程可能与PI3K/AKT和MAPK/ERK1/2信号通路有关。

肌萎缩侧索硬化症发病机制的遗传学研究进展

肌萎缩侧索硬化症(amyotrophic lateral sclerosis,ALS)是一种罕见的累及上、下运动神经元的神经退行性疾病,临床表现为肌肉进行性无力、萎缩,患者最终因吞咽、呼吸困难而死亡。ALS的病因众多,其中遗传因素相关性极大。神经元中蛋白质内稳态失衡、异常蛋白质的朊病毒样增殖和传播、线粒体功能障碍、谷氨酸介导的兴奋性毒性、神经元内物质运输障碍是目前公认的发病机制。对发病机制相关基因突变的研究将搭起ALS分子水平研究和细胞水平研究的桥梁,从而加深对ALS的发生和发展及基因突变在其中扮演角色的了解,并为疾病的治疗提供新的思路与启示。

人源α-synuclein跨细胞转运特性及对神经细胞突起的影响

目的确定人源α-synuclein是否能够跨细胞转运,过表达人源α-synuclein对神经细胞突起有何影响。方法建立稳定表达人源α-synuclein的N2a细胞株,通过接触和非接触两种方法观察人源α-synuclein的转运现象,通过测量神经突起长度,评价人源α-synuclein过表达对神经细胞突起生长的影响。结果接触和非接触两种方法均能够观察到人源α-synuclein跨细胞转运,但总体转运率低。过表达人源α-synuclein导致神经细胞突起生长不良。结论人源α-synuclein跨细胞转运现象存在,转运可能受到多因素影响,效率较低。过表达人源α-synuclein蛋白对神经突起有损伤作用。

Protein amyloid aggregate:Structure and function

Protein amyloid aggregation has been widely observed to occur and plays impor-tant roles in both physiological processes and pathological diseases.Remarkably,amyloid aggregates assembled by native proteins gain a variety of different biolog-ical activities,which cannot be adopted by the unassembled protein alone.Thus,it is important to investigate the molecular basis of self-assembly of protein amyloid aggregates and how the aggregated protein structure determines its function.In the review,wefirstly introduce our structural knowledge on how different amyloid pro-teins undergo conformational transition and assemble into amyloid aggregate,with the main focus on amyloidfibril,which is the major species of amyloid aggregate.Then,we elaborate how different structures of amyloidfibrils enable them to fulfill highly diverse functions in either physiological or pathological condition.Further-more,we discuss the structural polymorph which is a very unique feature of amyloidfibril,and its implication in understanding the structure-function relationship of amy-loidfibrils.Finally,we point out the importance of applying and integrating new approaches for deepening the structure-function study of amyloidfibrils and high-light the potential of designing amyloidfibril-based functional bio-nanomaterials for application.

Heat shock cognate 71 (HSC71) regulates cellular antiviral response by impairing formation of VISA aggregates

In response to viral infection, RIG-I-like RNA helicases detect viral RNA and signal through the mitochondrial adapter protein VISA. VISA activation leads to rapid activation of transcription factors IRF3 and NF-κB, which collaborate to induce transcription of type I interferon (IFN) genes and cellular antiviral response. It has been demonstrated that VISA is activated by forming prion-like aggregates. However, how this process is regulated remains unknown. Here we show that overexpression of HSC71 resulted in potent inhibition of virus-triggered transcription of IFNB1 gene and cellular antiviral response. Consistently, knockdown of HSC71 had opposite effects. HSC71 interacted with VISA, and negatively regulated virus-triggered VISA aggregation. These findings suggest that HSC71 functions as a check against VISA-mediated antiviral response.

朊病毒中朊蛋白及朊蛋白现象

朊病毒病是一种由朊病毒侵染动物神经系统并引发神经退行性症状的传染性疾病。朊病毒是由正常朊蛋白PrP^C通过构象转化形成具蛋白酶抗性的异常朊蛋白PrP^Se的病原微生物。最新研究表明，朊蛋白通过构象转变形成新的功能分子的现象在生物界中普遍存在，并与正常生物功能密切相关。通过研究类朊蛋白现象可以有助于揭示朊病毒感染机制以及深化对生物遗传多样性的了解。

α-突触核蛋白致病机制研究进展

细胞内错误折叠的α-突触核蛋白(α-synuclein,α-syn)在中枢神经系统及外周神经系统的沉积导致了帕金森病、路易体痴呆及多系统萎缩等突触核蛋白病的发生,而其病理传播机制尚不完全清楚。最近的研究表明具有神经毒性的寡聚体α-syn的细胞间传播是大脑区域之间疾病传播的主要模式。本综述回顾了不同模式的寡聚体α-syn细胞分泌和再摄取的现有证据,包括细胞间直接转移、朊蛋白样传播、外泌体分泌和内吞作用、纳米隧道及小胶质细胞介导作用,以便更详细地了解突触核蛋白病理学在整个大脑中传播的模式,为防止疾病进展的治疗提供新靶点。

Differential seeding and propagating efficiency ofα-synuclein strains generated in different conditions

Background:Accumulation of alpha-synuclein(α-syn)is a main pathological hallmark of Parkinson’s and related diseases,which are collectively known as synucleinopathies.Growing evidence has supported that the same protein can induce remarkably distinct pathological progresses and disease phenotypes,suggesting the existence of strain difference amongα-syn fibrils.Previous studies have shown thatα-syn pathology can propagate from the peripheral nervous system(PNS)to the central nervous system(CNS)in a“prion-like”manner.However,the difference of the propagation potency from the periphery to CNS among differentα-syn strains remains unknown and the effect of different generation processes of these strains on the potency of seeding and propagation remains to be revealed in more detail.Methods:Three strains of preformedα-syn fibrils(PFFs)were generated in different buffer conditions which varied in pH and ionic concentrations.Theα-syn PFFs were intramuscularly(IM)injected into a novel bacterial artificial chromosome(BAC)transgenic mouse line that expresses wild-type humanα-syn,and the efficiency of seeding and propagation of these PFFs from the PNS to the CNS was evaluated.Results:The three strains ofα-syn PFFs triggered distinct propagation patterns.The fibrils generated in mildly acidic buffer led to the most severeα-syn pathology,degeneration of motor neurons and microgliosis in the spinal cord.Conclusions:The differentα-syn conformers generated in different conditions exhibited strain-specific pathology and propagation patterns from the periphery to the CNS,which further supports the view thatα-syn strains may be responsible for the heterogeneity of pathological features and disease progresses among synucleinopathies.

朊蛋白相关蛋白Shadoo与朊病毒病

朊病毒病，即传染性海绵状脑病（transmissible spongiform encephalopathies, TSEs），是一类传染性、致死性神经退行性疾病。在朊病毒病的病理过程中，细胞正常朊蛋白PrP。转化为异常构象的PrP是至关重要的，但是PrP‘的正常生理功能仍不清楚。国外学者利用比较基因组学发现了-个新的朊蛋白相关蛋白-shadoo（Sho）。Sho与PrP。在氨基酸序列和细胞定位的相似性及主要在脑组织表达，使它成为-个非常值得研究的PrP相关蛋白。对Sho可能存在的与PrP。重叠的功能甚至直接相互作用的研究工作，将对今后揭示PrPc正常生理功能以及揭示Pfion病发病机制具有重要现实意义。