自噬在舞蹈病蛋白huntingtin代谢和致病机制中的作用

亨廷顿氏舞蹈病（Huntington’s Disease，HD）是人类中枢神经系统的一种遗传性疾病。该病以舞蹈样动作、进行性痴呆为主要临床特征。病理特点为尾核萎缩，神经细胞变性、脱失，其中以纹状体投射性γ-氨基丁酸能神经元和大脑皮层大锥体神经元丧失最为明显。HD在人群中的发病率约为5／10万，通常在中年发病，而后病情将逐渐加重，因无法治疗．10—20年后死亡。研究发现，HD基因突变是导致亨廷顿氏舞蹈病的原因，并于1983年在人体第4条染色体中确定了HD基因的位置，

Onjisaponin B derived from Radix Polygalae enhances autophagy and accelerates the degradation of mutant α-Synuclein and Huntingtin in PC-12 cells

OBJECTIVE To investigate the autophagic effect of the compounds from the Chinese medicinal herbs,Radix polygalae as a potential neuroprotective agent that enhances the clearance of mutant Huntingtin andα-synuclein in PC-12 cells.METHODS Radix polygalae was extracted with 75%ethanol using refluxing method,and its quality was assayed by UHPLC-TOF-MS.The autophagic effect of Radix polygalae extract,and its major components including polygalacic acid,senegenin and onjisaponin B were investigated using the green fluorescent protein-light chain 3(GFP-LC3)autophagy detection and Western blot platforms for detecting the autophagic markers,GFP-LC3 puncta formation and LC3Ⅱ expression.The degradation of A53Tα-synuclein and the inhibition of α-synuclein oligo merization related to the Parkinson disease were assayed using Western blot and flow cytometer analysis,respectively.While the cytotoxicity and the degradation of the mutant Huntingtin associated with the Huntingtin disease were investigated using MTT method and flow cytometer analysis.RESULTS Radix polygalae ethanol extract and onjisaponin B improved the GFP-LC3 puncta formation and expression of LC3Ⅱ with time and dose manner,and this induction was activated via AMPK-m TOR and Atg 7 gene pathway.Furthermore,the clearance ofα-synuclein and mutant Huntingtin was enhanced via autophagy induction with the treatment of Radix polygalae ethanol extract and onjisaponin B.CONCLUSION Findings in the current study provide detailed insights into the protective mechanism of a novel autophagy inducer,onjisaponin B,which is valuable for further investigation as a new candidate agent for modulating neurodegenerative disorders through the reduction of toxicity and clearance of mutant proteins in the cellular level.

The role of post-translational modifications of huntingtin in the pathogenesis of Huntington's disease

Post-translational modifications are rapid, effective and reversible ways to regulate protein stability, localization, function, and their interactions with other molecules. Post-translational modifications usually occur as chemical modifications at amino acid residues, including SUMOylation, phosphorylation, palmitoylation, acetylation, etc. These complex biochemical modifications tightly regulate and control a variety of cellular processes. Several forms of post-translational modifications of huntingtin （Htt） have been described. These modifications affect Htt metabolism, protein-protein interactions and cellular toxicity. Cleavage and clearance of mutant Htt, and the interactions between mutant Htt and other cellular proteins are important biochemical events leading to Huntington＇s disease （HD）. Therefore, identifying signaling pathways of Htt modification and evaluating the significance of Htt modifications would lead to a better understanding of the normal function of wild-type Htt and the pathogenic mechanisms of mutant Htt.

Essential sequence of the N-terminal cytoplasmic localization-related domain of huntingtin and its effect on huntingtin aggregates

Huntington＇s disease （HD） is caused by abnormal CAG repeat expansion in the 5＇-end of the Huntingtin （HTT） gene. In addition to the canonical C-terminal full-length huntingtin （htt） nuclear export signal, a cytoplasmic localization-related domain （CLRD） in the N-terminus of htt has recently been reported. Here, we analyzed this domain by introducing deletion and substitution mutations in a truncated N-terminal htt protein and subsequently monitored htt expression, aggregation and subcellular localization by immunocytochemistry and Western blot analysis. We demonstrated that Htt1-17 was the essential sequence for htt cytoplasmic localization. We also found that the subcellular distribution of htt was altered when Htt1_17 was mutated to contain amino acids of different charges, suggesting a structural requirement of Htt1-17 for the cytoplasmic localization of htt. Deletion of the first three amino acids did not affect its association with mitochondria. We observed that defective cytoplasmic localization resulted in a reduction of total htt aggregates and increased nuclear aggregates, indicating that the subcellular distribution of the protein might influence the aggregation process. These studies provide new insight into the molecular mechanism of htt aggregation in HD.

Gedunin Degrades Aggregates of Mutant Huntingtin Protein and Intranuclear Inclusions via the Proteasomal Pathway in Neurons and Fibroblasts from Patients with Huntington’s Disease

Huntington's disease(HD) is a deadly neurodegenerative disease with abnormal expansion of CAG repeats in the huntingtin gene. Mutant Huntingtin protein(m HTT) forms abnormal aggregates and intranuclear inclusions in specific neurons, resulting in cell death. Here,we tested the ability of a natural heat-shock protein 90 inhibitor, Gedunin, to degrade transfected m HTT in Neuro-2 a cells and endogenous m HTT aggregates and intranuclear inclusions in both fibroblasts from HD patients and neurons derived from induced pluripotent stem cells from patients. Our data showed that Gedunin treatment degraded transfected m HTT in Neuro-2 a cells, endogenous m HTT aggregates and intranuclear inclusions in fibroblasts from HD patients, and in neurons derived from induced pluripotent stem cells from patients in a dose-and time-dependent manner, and its activity depended on the proteasomal pathway rather than the autophagy route. These findings also showed that although Gedunin degraded abnormal m HTT aggregates and intranuclear inclusions in cells from HD patient, it did not affect normal cells, thus providing a new perspective for using Gedunin to treat HD.

Oral administration of the cannabigerol derivative VCE-003.2 promotes subventricular zone neurogenesis and protects against mutant huntingtin-induced neurodegeneration

Background:The administration of certain cannabinoids provides neuroprotection in models of neurodegenerative diseases by acting through various cellular and molecular mechanisms.Many cannabinoid actions in the nervous system are mediated by CB1 receptors,which can elicit psychotropic effects,but other targets devoid of psychotropic activity,including CB2 and nuclear PPARγreceptors,can also be the target of specific cannabinoids.Methods:We investigated the pro-neurogenic potential of the synthetic cannabigerol derivative,VCE-003.2,in striatal neurodegeneration by using adeno-associated viral expression of mutant huntingtin in vivo and mouse embryonic stem cell differentiation in vitro.Results:Oral administration of VCE-003.2 protected striatal medium spiny neurons from mutant huntingtin-induced damage,attenuated neuroinflammation and improved motor performance.VCE-003.2 bioavailability was characterized and the potential undesired side effects were evaluated by analyzing hepatotoxicity after chronic treatment.VCE-003.2 promoted subventricular zone progenitor mobilization,increased doublecortin-positive migrating neuroblasts towards the injured area,and enhanced effective neurogenesis.Moreover,we demonstrated the proneurogenic activity of VCE-003.2 in embryonic stem cells.VCE-003.2 was able to increase neuroblast formation and striatal-like CTIP2-mediated neurogenesis.Conclusions:The cannabigerol derivative VCE-003.2 improves subventricular zone-derived neurogenesis in response to mutant huntingtin-induced neurodegeneration,and is neuroprotective by oral administration.

Using Huntingtin Knock-In Minipigs to Fill the Gap Between Mouse Models and Patients with Huntington's Disease

Huntington＇s disease （HD） is an inherited autosomal dominant neurodegenerative disease characterized by pro- gressive motor deficits, cognitive decline, and psychiatric symptoms. It is caused by a pathological expansion of CAG trinucleotide repeats in exon 1 of the HD gene, resulting in the translation of a mutant form of huntingtin protein （mutant Htt） with an expanded polyglutamine domain in the N-terminal region [1 ]. Despite great progress in understanding the pathogenesis of HD using multiple mouse models, the exact mechanisms by which mutant Htt induces neuronal dysfunction and death are still not completely clear, and there is no curative treatment for this disease. An important reason is that the mouse, which is the most widely used animal model in HD research, differs from the human in many aspects, including the physiology, drug metabolism, blood-brain barrier, life span, brain volume, and neuroanatomical organization [2]. Thus, it is necessary to establish HD models with higher species than rodents, such as the dog, pig, and non- human primate, so as to bridge the gap between preclinical mouse models and clinical studies.

Therapeutic advances in neural regeneration for Huntington’s disease

Huntington’s disease is a neurodegenerative disease caused by the expansion mutation of a cytosine-adenine-guanine triplet in the exon 1 of the HTT gene which is responsible for the production of the huntingtin (Htt) protein. In physiological conditions, Htt is involved in many cellular processes such as cell signaling, transcriptional regulation, energy metabolism regulation, DNA maintenance, axonal trafficking, and antiapoptotic activity. When the genetic alteration is present, the production of a mutant version of Htt (mHtt) occurs, which is characterized by a plethora of pathogenic activities that, finally, lead to cell death. Among all the cells in which mHtt exerts its dangerous activity, the GABAergic Medium Spiny Neurons seem to be the most affected by the mHtt-induced excitotoxicity both in the cortex and in the striatum. However, as the neurodegeneration proceeds ahead the neuronal loss grows also in other brain areas such as the cerebellum, hypothalamus, thalamus, subthalamic nucleus, globus pallidus, and substantia nigra, determining the variety of symptoms that characterize Huntington’s disease. From a clinical point of view, Huntington’s disease is characterized by a wide spectrum of symptoms spanning from motor impairment to cognitive disorders and dementia. Huntington’s disease shows a prevalence of around 3.92 cases every 100,000 worldwide and an incidence of 0.48 new cases every 100,000/year. To date, there is no available cure for Huntington’s disease. Several treatments have been developed so far, aiming to reduce the severity of one or more symptoms to slow down the inexorable decline caused by the disease. In this context, the search for reliable strategies to target the different aspects of Huntington’s disease become of the utmost interest. In recent years, a variety of studies demonstrated the detrimental role of neuronal loss in Huntington’s disease condition highlighting how the replacement of lost cells would be a reasonable strategy to overcome the neurodegeneration. In this view, numerous have been the attempts in several preclinical models of Huntington’s disease to evaluate the feasibility of invasive and non-invasive approaches. Thus, the aim of this review is to offer an overview of the most appealing approaches spanning from stem cell-based cell therapy to extracellular vesicles such as exosomes in light of promoting neurogenesis, discussing the results obtained so far, their limits and the future perspectives regarding the neural regeneration in the context of Huntington’s disease.

亨廷顿病发病机制的研究进展

亨廷顿病是由于IT15基因突变而产生,其导致huntingtin蛋白(Htt)的N端大量多聚谷氨酰胺重复扩张。重复扩张的多聚谷氨酰胺持续改变着正常Htt功能。并且这种变异的蛋白(Htt)本身是有毒性的。本文从Htt在神经元中不能够被泛素蛋白酶体系统所溶解的异常聚集、神经元基因转录水平失调导致显性改变、神经元线粒体和新陈代谢的异常导致能量代谢水平下调、轴突传输改变和突触失衡等角度阐述了其发病机制的研究进展。

HIP1在维生素K3诱导Hela细胞损伤中的表达及意义

目的:观察Huntingtin相关蛋白1(HIP1)在维生素K3诱导Hela细胞损伤中的表达及意义。方法:对数生长期Hela细胞随机分为对照组(Control)、VK3处理组(VK3)、VK3与NAC联合作用组(VK3+NAC)及NAC单独应用组(NAC);MTT法检测各组细胞存活率;Western blotting法检测各组HIP1、NFκ-B蛋白表达水平;RT-PCR方法检测各组Cyclin D1 mRNA表达水平。结果:与对照组相比,VK3组Hela细胞存活率降低(P<0.01),HIP1蛋白、NF-κB蛋白表达量降低(P<0.05),Cyclin D1 mRNA表达量明显下降(P<0.05);与VK3组相比,VK3与NAC联合应用组细胞存活率增加,能够明显拮抗HIP1蛋白,NFκ-B蛋白表达量和Cyclin D1 mRNA表达量均增加(P<0.05)。结论:VK3具有抑制细胞增殖作用,可能与降低HIP1的蛋白表达水平有关。

亨廷顿舞蹈病的分子病理研究进展

亨廷顿舞蹈病 (Huntington’sdisease ,HD)是一种常染色体显性遗传性神经退行性疾病。该病的发生是由一个编码亨廷顿蛋白 (Huntingtin ,Htt)的基因突变引起的。Htt氨基端有一段重复的谷氨酰胺序列 ,它的长度在正常人少于 35个 ,而在HD患者则超过 37个。突变后Htt的致病机制是当今神经退行性病变中研究的热门课题之一。

链脲佐菌素敏感的胰岛细胞表达亨廷顿蛋白相关蛋白1

目的探讨亨廷顿蛋白相关蛋白1(HAP1)在大鼠胰岛中的定位。方法应用一次性大剂量腹腔注射链脲佐菌素(streptozotocin)的方法选择性破坏大鼠胰岛B细胞复制糖尿病模型,免疫组织化学ABC法显示胰岛内HAP1和胰岛素免疫反应性。结果在正常大鼠胰腺内,HAP1选择性表达于胰岛内,HAP1免疫反应阳性细胞呈短条索状或团状分布在每个胰岛内,主要位于胰岛的中央部,与胰岛B细胞的分布十分相似。注射链脲佐菌素的大鼠,胰岛中含HAP1的细胞数量在注射链脲佐菌素3d后已明显减少,并随着注射后动物存活时间的延长而进一步进行性减少;在注射后4周的大鼠,胰岛中仅有少数散在分布的HAP1免疫反应弱阳性细胞。链脲佐菌素对胰岛中表达HAP1细胞的影响与对表达胰岛素细胞的影响一致。结论HAP1也存在于胰岛内,并主要定位于链脲佐菌素敏感的B细胞内。

大鼠视网膜中HAP1的免疫组织化学定位及不同光照条件对HAP1表达的影响

目的 探讨亨廷顿蛋白相关蛋白 1(huntingtinassociatedprotein 1,HAP1)是否存在于视网膜内及是否与视觉有关。方法 对正常大鼠眼球壁用ABC法进行免疫组织化学染色 ,观察HAP1在视网膜中的定位 ;用半定量免疫印迹方法(Westernblotting)检测不同光照条件对大鼠视网膜中HAP1表达的影响。结果 HAP1较广泛地分布在大鼠视网膜各层 ,但以内核层及外核层中免疫反应较强 ,阳性反应产物主要定位在节细胞层和内核层 /外核层中部分细胞胞体内 ;其余各层中 ,HAP1免疫反应较弱 ,阳性产物呈弥散分布 ,未见明显的阳性胞体。在连续处于黑暗环境中 72小时大鼠视网膜中 ,HAP1表达较常规光照动物明显减少 ,而连续光照 72h大鼠视网膜内HAP1表达无明显变化。结论 HAP1在视网膜中的存在及不同光照条件对视网膜HAP1表达的影响表明 ,HAP1可能与视觉活动有关。

亨廷顿蛋白相关蛋白1在成年大鼠脊髓中的分布

目的 观察亨廷顿蛋白相关蛋白 1(huntingtin associatedprotein 1,HAP1)在成年大鼠脊髓中的分布特点。方法 采用免疫组织化学ABC法和免疫印迹 (Westernblotting)方法。结果 免疫组织化学结果显示 ,在成年大鼠脊髓中 ,以背角灰质浅层 (RexedⅠ ,Ⅱ层 )的HAP1免疫反应性最强 ,阳性细胞最密集 ,免疫反应产物除分布在胞体外 ,还大量弥散分布于胞体间的神经毡内 ;背角深层有部分HAP1免疫反应阳性细胞呈散在分布 ,中央管周围灰质 (RexedX)内阳性胞体密度和免疫反应性强度仅次于后角浅层 ,而在脊髓腹角 ,偶见HAP1免疫反应阳性神经元。此外 ,Westernblotting分析显示 ,脊髓背角内HAP1表达水平明显高于脊髓前角。结论 HAP1主要分布于大鼠脊髓背角灰质浅层和中央管周围灰质神经元内 ,提示其可能与痛觉信息一级传入和 /或调控有关。

亨廷顿蛋白相关蛋白-1在成年大鼠脑内的定位

目的:探讨亨廷顿蛋白相关蛋白1(HAP1)在成年大鼠脑内的分布。方法:健康成年大鼠取脑做连续冠状切片,免疫组织化学方法观察HAP1在脑内的表达。结果:大量的HAP-1表达出现在海马、齿状回、隔核、黑质、红核、连合下核、连合下器、脊髓前庭核、臂旁核、大脑脚基底部、束间核、后屈束、穹窿、被盖腹侧区、嘴侧线形中缝核以及丘脑、下丘脑各区;蜗背侧核、背侧纵束、前庭内侧核、中缝背核背侧部、中缝背核腹侧部、中缝背核腹外侧部、中央灰质等处的表达稍弱;其余像嗅球、大脑皮层各区、小脑皮层等处均有表达但强度极低,Stigmoid小体数目的变化趋势与HAP1基本一致。结论:HAP1广泛分布于脑的各部,推测HAP1可能参与了脑的多种生理功能。

慢性复合应激对大鼠胰腺HAP1表达的影响

目的探讨慢性复合应激大鼠胰腺内胰岛内分泌细胞HAP1表达的变化及其意义。方法36只大鼠随机分为两组:慢性复合应激组和正常对照组。应激组动物无规律交替暴露于垂直旋转、睡眠剥夺、捆绑(6h/d)和夜间光照等慢性复合性应激,共6周;实验结束后,采用免疫组织化学和Western-blot等方法检测两组大鼠胰腺胰岛内分泌细胞内HAP1蛋白表达的变化。结果HAP1在大鼠胰腺内选择性表达于胰岛内分泌细胞中。与对照组相比,慢性复合应激组大鼠胰腺HAP1的表达明显增强(P<0.05)。结论6周慢性复合性应激可使大鼠胰腺中胰岛内分泌细胞的HAP1表达加强,提示HAP-1在慢性复合应激促进胰腺内分泌功能中可能发挥一定作用。

Micro RNA-124 slows down the progression of Huntington's disease by promoting neurogenesis in the striatum

MicroRNA-124 contributes to neurogenesis through regulating its targets, but its expression both in the brain of Huntington＇s disease mouse models and patients is decreased. However, the effects of microRNA-124 on the progression of Huntington＇s disease have not been reported. Results from this study showed that microRNA-124 increased the latency to fall for each R6/2 Hunting- ton＇s disease transgenic mouse in the rotarod test. 5-Bromo-2＇-deoxyuridine （BrdU） staining of the striatum shows an increase in neurogenesis. In addition, brain-derived neurotrophic factor and peroxisome proliferator-activated receptor gamma coactivator 1-alpha protein levels in the striatum were increased and SRY-related HMG box transcription factor 9 protein level was de- creased. These findings suggest that microRNA-124 slows down the progression of Huntington＇s disease possibly through its important role in neuronal differentiation and survival.

糖皮质激素对大鼠下丘脑亨廷顿蛋白相关蛋白1表达的影响

目的探讨外源性糖皮质激素对大鼠下丘脑亨廷顿蛋白相关蛋白1（HAP1）表达的影响。方法成年雄性Wistar大鼠80只,随机分为实验对照组（注射生理盐水）、皮质酮注射组、皮质酮＋皮质酮受体拮抗剂（RU38486）注射组、饮用氢化可的松组。皮质酮注射[（5mg/（kg.12h）]组又分为注射持续1d、3d、5d、7d组;皮质酮＋皮质酮受体拮抗剂（RU38486）注射[（10mg/（kg.12h）]组注射持续3d;饮用氢化可的松组大鼠饮用水中加氢化可的松（0.01g/L）,连续1个月。应用RT-PCR、免疫印迹法观察大鼠下丘脑HAP1表达的变化。结果注射皮质酮1d和3d后,下丘脑HAP1表达明显减少,其mRNA的表达明显下调,5d后HAP1开始回升,7d后恢复到正常水平,HAP1mRNA表达5d后明显增加,并高于正常水平;长期（一个月）饮用氢化可的松的大鼠,其下丘脑中HAP1的表达明显减少;在皮质酮注射的同时注射RU38486,可以对抗由皮质酮注射所引起的下丘脑HAP1的变化。结论外源性过量糖皮质激素能影响下丘脑HAP1的表达,下丘脑中的HAP1可能参与下丘脑神经元或神经内分泌细胞的功能活动。

胞浆内非聚集型突变亨廷顿蛋白影响神经细胞的分化

目的 :探讨胞浆内非聚集型突变亨廷顿蛋白 (huntingtin ,Htt)对神经细胞分化的影响。 方法 :利用磷酸钙沉淀法在培养的小鼠成神经瘤细胞 (N2a细胞 )瞬时或稳定转染编码正常或突变Htt的氨基末端片段的cDNA ,观察突变Htt对视黄酸 (retinoicacid ,RA)和无血清培养刺激N2a细胞突起生长的影响。结果 :在瞬时转染的N2a细胞中 ,2 0Q的正常Htt氨基末端片段弥散分布在胞浆内 ,1 5 0Q的突变Htt氨基末端片段在多数细胞胞浆内形成聚集物。在稳定表达 1 5 0Q的N2a细胞部分克隆中 ,1 5 0Q弥散分布在胞浆内。无论是含突变Htt聚集物的瞬时转染细胞还是仅在胞浆中表达非聚集型突变Htt的稳定转染细胞 ,其突起生长能力均较表达正常Htt的瞬时转染细胞和稳定转染细胞差。结论 :胞浆内非聚集型突变Htt也能影响神经细胞的分化。