运动神经元病的中医药研究现状

<正>运动神经元病是由于运动神经元受到损伤,逐渐导致全身瘫痪,并丧失吞咽和语言功能,最后因呼吸衰竭而死亡。本病90%以上为散发病例,成人通常在30~60岁发病,男性多于女性,有部分患者有阳性家族史,通常为常染色体显性遗传〔1〕。它是世界卫生组织列的五大绝症之一,与癌症和艾滋病齐名。1西医对运动神经元病的认识运动神经元病的病因和发病机制目前尚无定论,可能包括自由基氧化学说、兴奋性氨基酸的毒性作用、遗传因素、神经微     

运动神经元病治疗的研究进展

运动神经元病是一种病因未明,选择性侵犯脊髓、脑干及皮层运动神经元的进行性变性疾病。当前无实质性减缓疾病进展的治疗手段。近年来基于对发病机制的新理解,提出了许多神经保护性治疗措施,本文对相关治疗研究的进展作一综述。     

易与运动神经元病混淆的疾病

运动神经元病是累及上、下运动神经元的进行性变性病,临床主要表现为不可逆转的进行性肌无力、萎缩和锥体束征.由于其缺乏有效的治疗手段,诊断需要非常慎重,特别要除外其他临床表现相似的疾病.本文重点阐述了需要鉴别的颈椎病、平山病、多灶性运动神经病、单克隆丙球蛋白病伴周围神经病、副肿瘤性运动神经元综合征、脊髓灰质炎后综合征、脊肌萎缩症、肯尼迪病和糖尿病性肌萎缩,希望对临床医生的诊断有所帮助.     

以呼吸衰竭为首发表现的运动神经元病1例

患者男性，68岁。活动后气短伴咳嗽、咳痰3个月，2005年3月22日收入院，既往10年吸烟史，无慢性咳嗽咳痰史。人院查体：血压110／70mmHg（1mmHg：0．133kPa），呼吸浅速、口唇紫绀、双肺下界移动度减低、双肺呼吸低、左下肺少许湿哕音、腹部正常及双下肢不肿。血气分析：P0259．5mmHg，     

大肠癌组织中Cap43蛋白的表达变化及意义

目的观察人大肠癌组织中Cap43蛋白的表达变化。方法采用免疫组化SABC法检测28例大肠正常组织、41例大肠腺瘤及4l例大肠癌组织中的Cap43蛋白。结果大肠癌组织中Cap43蛋白阳性表达率为23．46％,明显低于正常组织的75．00％和大肠腺瘤组织的78．03％（P均〈0．05）。大肠癌组织中Cap43蛋白的表达与性别、分化程度、部位及病理类型均无相关性（P均〉0．05）。结论大肠癌组织中Cap43蛋白表达降低,呈低表达或无表达。     

去卵巢大鼠骨细胞中间隙连接蛋白43表达的研究

目的　研究去卵巢致骨质疏松大鼠骨细胞中间隙连接蛋白 43(Cx43)的表达及意义。方法　采用 1 0月龄未孕产 Wistar雌性大鼠 30只 ,随机分为去卵巢组、假手术组和尼尔雌醇治疗组。于术后 8w末测量三组大鼠全身及腰椎骨密度 (BMD) ,采用放免法测定血清雌二醇水平 ,采用SABC免疫组化法观察 Cx43在成骨细胞和破骨细胞中的表达情况。结果　术后 8w末去卵巢组全身及腰椎 BMD和血清雌二醇水平明显低于假手术组和尼尔雌醇治疗组 (P<0 .0 1 )。去卵巢组成骨细胞内 Cx43阳性表达率 (1 3.8%± 1 .1 4 % )低于假手术组 (63.6%± 2 .46% )和尼尔雌醇治疗组(63.6%± 2 .1 2 % ) (P<0 .0 1 ) ,而破骨细胞内去卵巢组 Cx43阳性表达率 (66.1 %± 1 .37% )高于假手术组 (42 .2 %± 1 .93% )和尼尔雌醇治疗组(41 .8%± 1 .81 % ) (P<0 .0 1 )。上述指标假手术组和尼尔雌醇治疗组差异无显著性。结论　去卵巢大鼠 Cx43在成骨细胞中表达下降 ,而在破骨细胞中表达增加 ,在成骨和破骨细胞中 Cx43表达的变化可能是去卵巢大鼠发生骨质疏松的机制之一。     

C-erbB-2蛋白和缝隙连接蛋白43在胃癌组织中的表达及意义

目的探讨C-erbB-2癌蛋白和缝隙连接蛋白43（connexin,Cx43）在腺型胃癌中的表达并分析其临床意义。方法应用免疫组织化学SP法分别检测C-erbB-2癌蛋白、Cx43在48例腺型胃癌和26例胃炎组织中的表达,并分析其与临床病理参数的关系。结果C-erbB-2癌蛋白、Cx43阳性呈棕黄色颗粒定位于细胞膜或细胞质;C-erbB-2癌蛋白在胃癌中的阳性表达率（50.0%）显著高于胃炎黏膜组织的阳性率（2χ=6.70,P〈0.05）;Cx43在胃癌中阳性率（43.8%）显著低于胃炎黏膜组织的阳性率（χ2=23.03,P〈0.05）;C-erbB-2癌蛋白的表达与胃癌的大小、分化程度及淋巴结转移呈显著相关（2χ=6.00、9.41、5.49,P值均〈0.05）;Cx43蛋白表达降低或缺失同样与胃癌的分化程度及淋巴结转移呈显著相关（χ2=7.49、6.86,P值均〈0.05）;C-erbB-2癌蛋白和Cx43蛋白呈负相关（χ2=0.01,r=-0.378,P〈0.05）。结论C-erbB-2癌蛋白的高表达、Cx43表达降低或缺失与胃癌分化程度、浸润转移密切相关,联合检测C-erbB-2和Cx43有助于判断胃癌的生物学行为。     

运动神经元病所致吞咽障碍的症状管理

运动神经元病(MND)是一组累及上、下运动神经元的神经系统变性疾病,进展迅速,缺乏有效治疗。大多数病人可于不同时期出现吞咽障碍,导致营养障碍、吸入性肺炎,是MND病人死亡的主要原因之一。现对MND病人吞咽功能系统评估和管理的最新进展做一综述。     

左归丸与右归丸对EAE大鼠APP及GAP-43表达的影响

目的 观察左归丸和右归丸对实验性变态反应性脑脊髓炎(EAE)大鼠淀粉样前体蛋白(APP)及生长相关蛋白[GAP-43)表达的影响,探讨滋阴与温阳补肾法治疗EAE的可能机制.方法 应用髓鞘碱性蛋白(MBP)68-86免疫诱导Lewis大鼠建立EAE模型.免疫后第7天、第14天、第28天取大鼠脑白质与脊髓,采用Western-blot和实时荧光定量RT-PCR技术检测APP及GAP-43蛋白及mRNA的表达.结果 免疫后第7天,激素和左归丸均能升高EAE大鼠脊髓中APP和脑白质中APP mRNA的过低表达(P<0.05或P<0.01),且在脊髓中二者对APP的调节优于右归丸(P<0.05);第14天,左归丸显著调节脑白质中APP mRNA的过高表达、脊髓中APP mRNA的过低表达(P<0.05或P<0.01),作用优于右归丸(P<0.05).免疫后第7天,激素与左归丸显著上调EAE大鼠脑白质和脊髓中GAP-43水平(P<0.05或P<0.01),在脊髓中二者作用优于右归丸(P<0.01),右归丸显著下调脑白质中GAP-43 mRNA的表达(P<0.05);第14天,激素与左归丸显著抑制脑白质中GAP-43的过高表达(P<0.05);第28天,激素组与左归丸组脑白质中GAP-43水平低于模型组与右归丸组(P<0.01).结论 左右归丸对EAE大鼠脑白质和脊髓中APP和GAP-43蛋白与基因的表达均具有一定调节作用,且左归丸的作用明显优于右归丸.     

运动神经元病临床神经电生理检查的研究进展

运动神经元病（motor neuron disease,MND）是以损害脊髓前角、脑干脑神经运动核和锥体束为主的一组慢性进行性变性疾病,包括了肌萎缩侧索硬化症（ALS）、进行性脊髓性肌萎缩症（PSMA）、脊髓性肌萎缩症（SMA）、进行性延髓麻痹（PBP）、原发性侧索硬化症（PLS）。发病率为2～3/10万,目前没有确实有效的治疗手段,从有症状开始,平均仅能存活3～5年。临床诊断主要依靠临床表现、神经电生理检查和神经影像学检查。     

运动神经元病患者认知功能筛查

目的 调查中国运动神经元病患者认知功能异常及额颞叶功能异常的发生情况.方法 对100例运动神经元病的患者行简易智能状态量表(MMSE),神经精神科问卷,汉密尔顿抑郁量表及汉密尔顿焦虑量表检查.并调查患者一般资料及功能等级评分(FRS)等情况.结果 MMSE结果示轻度认知障碍者占24.2%,MMSE正常与异常之间比较FRS总分及抑郁情况,两者差异有统计学意义.抗抑郁治疗3个月后随访,发现2例患者可能存在额颞叶功能受损.结论 运动神经元病患者认知功能可能轻度受损,部分存在精神行为表现异常,2例患者可能有额颞叶功能受损.     

运动神经元病误诊为COPD 2例并文献复习

目的 提高临床医师对以呼吸系统症状为主要表现的运动神经元病的诊断意识,减少误诊.方法 报道广州医科大学附属第一医院呼吸内科收治的2例被误诊为COPD的运动神经元病患者的临床表现及诊治经过并文献复习,以“运动神经元病”或“运动神经元疾病”为检索词检索中国知网、万方数据库的运动神经元病的误诊病例,检索时间为1990年1月至2016年2月.结果 2例患者均为男性,平均年龄64.5岁,均以气促进行性加重为主要表现,肺功能提示为限制性通气功能障碍.血气分析表现为低氧血症,无二氧化碳潴留.肌电图表现为神经源性损害,经神经内科会诊,诊断为运动神经元病.数据库共检索出运动神经元病误诊病例共341例,以颈椎病206例、脑血管疾病46例、腰椎病27例、慢性支气管炎10例居前四位.误诊为呼吸系统疾病文献共12篇,26例.早期误诊为慢性支气管炎、肺内感染、COPD、肺动脉高压、肺间质纤维化、支气管扩张并感染、过敏性哮喘.结论 运动神经元病临床表现不特异,以呼吸系统症状为主要表现时更加容易漏诊,临床医师应详细询问病史、系统查体,并结合肺功能、血气分析等辅助检查,全面考虑,早做排除,以防误诊.     

The Cellular Prion Protein Increases the Uptake and Toxicity of TDP-43 Fibrils

Cytoplasmic aggregation of the primarily nuclear TAR DNA-binding protein 43 (TDP-43) affects neurons in most amyotrophic lateral sclerosis (ALS) and approximately half of frontotemporal lobar degeneration (FTLD) cases. The cellular prion protein, PrP^C, has been recognized as a common receptor and downstream effector of circulating neurotoxic species of several proteins involved in neurodegeneration. Here, capitalizing on our recently adapted TDP-43 real time quaking induced reaction, we set reproducible protocols to obtain standardized preparations of recombinant TDP-43 fibrils. We then exploited two different cellular systems (human SH-SY5Y and mouse N2a neuroblastoma cells) engineered to express low or high PrP^C levels to investigate the link between PrP^C expression on the cell surface and the internalization of TDP-43 fibrils. Fibril uptake was increased in cells overexpressing either human or mouse prion protein. Increased internalization was associated with detrimental consequences in all PrP-overexpressing cell lines but was milder in cells expressing the human form of the prion protein. As described for other amyloids, treatment with TDP-43 fibrils induced a reduction in the accumulation of the misfolded form of PrP^C, PrP^Sc, in cells chronically infected with prions. Our results expand the list of misfolded proteins whose uptake and detrimental effects are mediated by PrP^C, which encompass almost all pathological amyloids involved in neurodegeneration.     

Survival of motor neuron protein downregulates miR-9 expression in patients with spinal muscular atrophy

Spinal muscular atrophy (SMA) is a lethal hereditary disease caused by homozygous absence of the survival of the motor neuron (SMN) 1 gene (SMN1), and it is the leading genetic cause of infant mortality. The severity of SMA is directly correlated with SMN protein levels in affected patients; however, the cellular regulatory mechanisms for SMN protein expression are not completely understood. In this study, we investigated the regulatory effects between SMN expression and miR-9a, a downstream noncoding small RNA. Using an inducible SMN short hairpin RNA interference (shRNAi) system in NSC 34 and human skin fibroblast cells, cellular miR-9 levels and SMN protein repression were time-dependently upregulated. Conversely, cellular miR-9 levels decreased when HeLa cells were transfected with SMN protein fused with green fluorescent protein. In SMA-like mice spinal cords and human primary skin fibroblasts isolated from patients with different degrees of SMA, human SMN exhibited a disease severity-dependent decrease, whereas cellular miR-9 levels increased. These results clearly suggested that cellular SMN proteins regulated miR-9 expression and that miR-9 expression was related to SMA severity. Thus, miR-9 may be a marker for SMA prognosis.     

骨化三醇对周围神经损伤后大鼠脊髓运动神经元的保护作用

目的 探讨骨化三醇[1,25(OH)_2D_3]对周围神经损伤后脊髓前角运动神经元的保护作用. 方法 48只SD大鼠随机分为安慰剂组和骨化三醇组,每组24只,切断右侧坐骨神经,近端结扎,远端埋入股二头肌.术后骨化三醇组给予骨化三醇2μg·kg~(-1)·d~(-1)灌胃,安慰剂组给予脂肪乳剂2 ml·kg~(-1)·d~(-1)灌胃,灌胃7 d.术后1、2、4周分别处死动物,取腰膨大(L4～L6节段)脊髓,分别行尼氏染色,胆碱酯酶(CHE)染色和酸性磷酸酶(ACP)染色,并结合计算机进行图像分析. 结果术后1、2、4周,骨化三醇组脊髓前角运动神经元存活率分别为(90.8±3.5)%、(84.1±2.3)%、(76.2±2.7)%,高于安慰剂组((84.3±6.5)%、(76.3±2.7)%、(73.4±1.9)%](t=-5.442、-6.443、-2.286,P<0.01或P<0.05);骨化三醇组脊髓CHE染色阳性面积百分率分别为(69.1±1.4)%、(74.5±3.4)%、(78.9±6.6)%,高于安慰剂组[(59.5±1.0)%、(65.8±4.0)%、(66.8±5.3)%](t=-15.787、-10.781、-11.954,P<0.01);骨化三醇组脊髓ACP染色阳性面积百分率分别为(182.8±5.5)%、(115.0±12.5)%、(108.7±8.4)%,低于安慰剂组[(206.5±14.7)%、(138.2±9.9)%、(115.1±9.0)%],第1、2周t=4.275、4.113,P<0.01;第4周t=1.458,P=0.167. 结论骨化三醇对周围神经损伤所致的脊髓前角运动神经元损伤有一定的保护作用.     

Nutritional support teams increase percutaneous endoscopic gastrostomy uptake in motor neuron disease

AIM: To examine factors influencing percutaneous endoscopic gastrostomy (PEG) uptake and outcomes in motor neuron disease (MND) in a tertiary care centre.METHODS: Case notes from all patients with a confirmed diagnosis of MND who had attended the clinic at the Repatriation General Hospital between January 2007 and January 2011 and who had since died, were audited. Data were extracted for demographics (age and gender), disease characteristics (date of onset, bulbar or peripheral predominance, complications), date and nature of discussion of gastrostomy insertion, nutritional status [weight measurements, body mass index (BMI)], date of gastrostomy insertion and subsequent progress (duration of survival) and quality of life (QoL) [Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R)]. In addition, the type of clinician initiating the discussion regarding gastrostomy was recorded as Nutritional Support Team (involved in providing nutrition input viz Gastroenterologist, Speech Pathologist, Dietitian) and other (involved in non-nutritional aspects of patient care). Factors affecting placement and outcomes including length of survival, change in weight and QoL were determined.RESULTS: Case records were available for all 86 patients (49 men, mean age at diagnosis 66.4 years). Thirty-eight patients had bulbar symptoms and 48 had peripheral disease as their presenting feature. Sixty-six patients reported dysphagia. Thirty-one patients had undergone gastrostomy insertion. The major indications for PEG placement were dysphagia and weight loss. Nine patients required immediate full feeding, whereas 17 patients initially used the gastrostomy to supplement oral intake, 4 for medication administration and 1 for hydration. Initially the PEG regime met 73% ± 31% of the estimated total energy requirements, increasing to 87% ± 32% prior to death. There was stabilization of weight in patients undergoing gastrostomy [BMI at 3 mo (22.6 ± 2.2 kg/m²) and 6 mo (22.5 ± 2.0 kg/m²) after PEG placement compared to weight at the time of the procedure (22.5 ± 3.0 kg/m²)]. However, weight loss recurred in the terminal stages of the illness. There was a strong trend for longer survival from diagnosis among MND in PEG recipients with limb onset presentation compared to similar patients who did not undergo the procedure (P = 0.063). Initial discussions regarding PEG insertion occurred earlier after diagnosis when seen by nutrition support team (NST) clinicians compared to other clinicians. (5.4 ± 7.0 mo vs 11.9 ± 13.4 mo, P = 0.028). There was a significant increase in PEG uptake (56% vs 24%, P = 0.011) if PEG discussions were initiated by the NST staff compared to other clinicians. There was no change in the ALSFRS-R score in patients who underwent PEG (pre 34.1 ± 8.6 vs post 34.8 ± 7.4), although in non-PEG recipients there was a non-significant fall in this score (33.7 ± 7.9 vs 31.6 ± 8.8). Four patients died within one month of the procedure, 4 developed bacterial site infection requiring antibiotics and 1 required endoscopic therapy for gastric bleeding. Less serious complications attributed to the procedure included persistent gastrostomy site discomfort, poor appetite, altered bowel function and bloating.CONCLUSION: Initial discussion with NST clinicians increases PEG uptake in MND. Gastrostomy stabilizes patient weight but weight loss recurs with advancing disease.     

S-nitrosylated TDP-43 triggers aggregation,cell-to-cell spread,and neurotoxicity in hiPSCs and in vivo models of ALS/FTD

Rare genetic mutations result in aggregation and spreading of cognate proteins in neurodegenerative disorders; however, in the absence of mutation (i.e., in the vast majority of “sporadic” cases), mechanisms for protein misfolding/aggregation remain largely unknown. Here, we show environmentally induced nitrosative stress triggers protein aggregation and cell-to-cell spread. In patient brains with amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), aggregation of the RNA-binding protein TDP-43 constitutes a major component of aberrant cytoplasmic inclusions. We identify a pathological signaling cascade whereby reactive nitrogen species cause S-nitrosylation of TDP-43 (forming SNO-TDP-43) to facilitate disulfide linkage and consequent TDP-43 aggregation. Similar pathological SNO-TDP-43 levels occur in postmortem human FTD/ALS brains and in cell-based models, including human-induced pluripotent stem cell (hiPSC)-derived neurons. Aggregated TDP-43 triggers additional nitrosative stress, representing positive feed forward leading to further SNO-TDP-43 formation and disulfide-linked oligomerization/aggregation. Critically, we show that these redox reactions facilitate cell spreading in vivo and interfere with the TDP-43 RNA-binding activity, affecting SNMT1 and phospho-(p)CREB levels, thus contributing to neuronal damage in ALS/FTD disorders.

Identification of genetic, pathological, and clinical signatures of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) suggests a continuum of disease on a single ALS/FTD spectrum of disorders. At a genetic level, mutations in specific genes, including TARDBP (encoding the RNA-binding protein TDP-43), fused-in-sarcoma (FUS), and valosin containing protein (VCP), or hexanucleotide repeat expansion, as in C9ORF72, have been linked to ALS and/or FTD (1). Even in the absence of mutation, TDP-43 proteinopathy, involving cytoplasmic aggregation and consequent nuclear clearance of TDP-43 in affected cells, represents a major pathological hallmark appearing in 97% of ALS and 45% of FTD cases (2, 3). The high prevalence of TDP-43 proteinopathy in the face of rare genetic mutations in the TARDBP gene (representing only ∼4% of ALS cases) points to the possibility that other factors related to age or environment contribute to TDP-43 aggregation in the vast majority of patients with ALS/FTD spectrum disorders.Under normal conditions, TDP-43 resides predominantly in the nucleus, functioning as an RNA-binding protein for regulation of mRNA processing and stabilization (4, 5). In contrast, in ALS/FTD, TDP-43 becomes highly phosphorylated, ubiquitinated, and insoluble and mislocalizes to the cytosol to form stress granules (SGs) (6). For example, enhanced activity of casein kinases and possibly other kinases, such as glycogen synthase kinase 3 and cyclin-dependent kinases, lead to hyperphosphorylation of TDP-43, promoting aggregation of TDP-43 in cytosolic SGs (7, 8). SGs are nonmembrane-bound organelles that can sequester specific mRNAs via phase separation (9) to inhibit the initiation of translation. Additionally, emerging evidence suggests that cell-to-cell spreading of pathological aggregates of TDP-43 contributes to the propagation of the proteinopathy (10–12). Prior reports support the notion that disease progression in TDP-43 proteinopathy can be mediated by aggregation-related loss-of-normal function (e.g., RNA binding in the nucleus) or gain-of-toxic function, or possibly both (13). Studies have also implicated microglia-dependent pathways in TDP-43 proteinopathy (14). Furthermore, recent studies have revealed that TDP-43 pathology occurs in a wide variety of other neurodegenerative disorders, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD) (15), suggesting a wider role for TDP-43 proteinopathy in neurodegeneration. However, mechanistic insight into the development and execution of TDP-43 pathology remains incompletely understood.Two risk factors that have been strongly implicated in the pathogenesis of neurodegenerative disorders are the aging process and environmental toxins (16–19). Both of these risk factors engender dramatic increases in chemically reactive species, including reactive oxygen and nitrogen species (ROS/RNS) such as nitric oxide (NO), and it has been suggested that this may play a role in the degenerative process as observed in ALS/FTD (18–22). Along these lines, we and others have demonstrated that protein S-nitrosylation, resulting from posttranslational modification of cysteine thiol groups by NO-related species, contributes to protein misfolding, mitochondrial dysfunction, synaptic impairment, and eventually neuronal cell loss (19). Several chemical mechanisms for in vivo formation of protein S-nitrosylation have been proposed (23, 24). For example, cysteine thiol (or more properly thiolate anion) can perform a reversible nucleophilic attack on a nitroso nitrogen to form a protein S-nitrosothiol via transnitrosation or transnitrosylation (24, 25). Mechanism notwithstanding, protein S-nitrosylation is now well recognized as a major contributor to both the physiological and pathophysiological activity (19, 20).Interestingly, the Food and Drug Administration (FDA)-approved drug edaravone (MCI-186) delays disease progression in some cases of ALS (26), possibly via scavenging RNS/ROS related to NO and hydroxyl radical groups (27, 28). While exogenous addition of ROS-generating agents has been reported to decrease the solubility of TDP-43 in vitro in cell-based models (29–31), endogenous reactive chemical species have not been previously reported to do this in a pathophysiologically relevant manner. Accordingly, in the present study, we report that not only exogenous but also endogenous RNS can trigger TDP-43 aggregation via S-nitrosylation and consequent disulfide bond formation; in models of FTD and ALS, we identify endogenous SNO-TDP-43 formation as a critical effector of pathological signaling, leading to its aggregation, altered RNA-binding activity, and neurotoxicity. Moreover, we find that increased expression of TDP-43 protein, as found in ALS, FTD, and other neurodegenerative disorders, results not only in misfolded/aggregated protein but also in dramatically increased NO production and additional protein misfolding/aggregation, representing a positive feed-forward loop to enhance nitrosative stress and thus protein misfolding. Additionally, we report that, in conjunction with NO, mutation in the VCP gene, as observed in some cases of ALS/FTD, triggers a dramatic increase in misfolded/aggregated TDP-43 in human-induced pluripotent stem cell (hiPSC)-derived motoneurons. Our results thus place TDP-43 at a unique node of intersection between genetic mutations associated with ALS/FTD and aging/environmental risk factors mediated by NO-related species, which together contribute to neurodegeneration in ALS/FTD spectrum disorders.     

ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43

Transactivating response region DNA binding protein (TDP-43) is the major protein component of ubiquitinated inclusions found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitinated inclusions. Two ALS-causing mutants (TDP-43^Q331K and TDP-43^M337V), but not wild-type human TDP-43, are shown here to provoke age-dependent, mutant-dependent, progressive motor axon degeneration and motor neuron death when expressed in mice at levels and in a cell type-selective pattern similar to endogenous TDP-43. Mutant TDP-43-dependent degeneration of lower motor neurons occurs without: (i) loss of TDP-43 from the corresponding nuclei, (ii) accumulation of TDP-43 aggregates, and (iii) accumulation of insoluble TDP-43. Computational analysis using splicing-sensitive microarrays demonstrates alterations of endogenous TDP-43–dependent alternative splicing events conferred by both human wild-type and mutant TDP-43^Q331K, but with high levels of mutant TDP-43 preferentially enhancing exon exclusion of some target pre-mRNAs affecting genes involved in neurological transmission and function. Comparison with splicing alterations following TDP-43 depletion demonstrates that TDP-43^Q331K enhances normal TDP-43 splicing function for some RNA targets but loss-of-function for others. Thus, adult-onset motor neuron disease does not require aggregation or loss of nuclear TDP-43, with ALS-linked mutants producing loss and gain of splicing function of selected RNA targets at an early disease stage.Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) are progressive, adult-onset neurodegenerative diseases with overlapping clinical and pathological features (1–3). ALS is characterized by the selective loss of upper and lower motor neurons, leading to progressive fatal paralysis and muscle atrophy. A large majority (∼90%) of ALS and FTLD-U cases are without a known genetic cause. Importantly, in these sporadic cases, the appearance of ubiquitinated inclusions within the affected neurons of the nervous system characterizes both ALS and FTLD-U patients, suggesting an overlapping mechanism underlying both diseases. Biochemical characterization of brains and spinal cords from ALS and FTLD-U patients identified transactivating response region (TAR) DNA binding protein (TDP-43) as the major protein component of these ubiquitinated inclusions (4, 5). The discovery of ALS-linked mutations in the glycine-rich C-terminal domain of TDP-43 (6-8) demonstrated a pathological role of TDP-43 in both diseases. The subsequent identification of mutations in a structurally and functionally related nucleic acid binding protein, FUS/TLS (fused in sarcoma/translocated in liposarcoma) (9, 10), further implicated defects in RNA processing in ALS pathogenesis.TDP-43 is a multifunctional nucleic acid binding protein. Within the nervous system, TDP-43 binds to >6,000 pre-mRNAs and affects the levels of ∼600 mRNAs and the splicing patterns of another 950 (11). Structurally, the 414-aa protein consists of two RNA recognition motifs (RRM1 and RRM2) (12, 13), nuclear import and export signal (14), and a glycine-rich region implicated in protein–protein interactions (15, 16) that include components of the RNA splicing machinery (17, 18).Disruption in mice of the highly conserved Tardbp gene is embryonically lethal (19–22). Similarly, postnatal inactivation of Tardbp (by Cre recombinase-mediated gene excision encoded by a ubiquitously-expressed CAG-Cre transgene) results in rapid postnatal death accompanied by defects in fat metabolism (22). TDP-43 autoregulates its own RNA level (11, 23) at least in part by stimulating excision of an intron in its 3′ untranslated region, thereby making the spliced RNA a substrate for nonsense-mediated RNA degradation (11). Furthermore, transgenic rodent models have been used to demonstrate that overriding the autoregulatory mechanism by overexpression of unregulated wild-type (24–28) or disease-linked mutant (26, 28–35) TDP-43 transgenes can produce neurodegeneration in mice.ALS and FTLD-U patient brain and spinal cord samples are characterized by the accumulation of cytoplasmic TDP-43 aggregates accompanied by a distinct clearing of nuclear TDP-43 within affected neurons and glia (36, 37), implicating possible loss of nuclear TDP-43 function in disease pathogenesis. In human disease, TDP-43 has been reported to be abnormally phosphorylated, ubiquitinated, and cleaved to produce C-terminal fragments (4, 5, 38, 39). Ectopic expression of these C-terminal fragments in cell-culture models (40–42) has shown that they are aggregation-prone and confer an intrinsic toxicity. However, the extent of the contribution of these C-terminal fragments to disease pathogenesis is undetermined. Indeed, double-immunofluorescent labeling of ALS patient spinal cords using N-terminal–specific and C-terminal–specific antibodies suggests that inclusions in spinal cord motor neurons are comprised primarily of full-length TDP-43 (37). Importantly, retention of ability to bind RNA by full-length TDP-43 has been demonstrated to be required for toxicity in yeast, fly, and Caenorhabditis elegans models (43–46).Nevertheless, it remains unresolved whether toxicity to motor neurons from mutations in TDP-43 is mediated through a gain of toxic property, loss-of-function, or a combination of both. By generation of transgenic mice encoding levels of wild-type or mutant human TDP-43 comparable to endogenous TDP-43, we demonstrate mutant-dependent, age-dependent motor neuron disease from ALS-linked TDP-43 mutants in the absence of overexpression, cytoplasmic accumulation of a 35 kDa TDP-43 fragment, or insoluble TDP-43 aggregates. Accompanying autoregulation-mediated reduction of endogenous wild-type TDP-43 are splicing alterations previously identified to be TDP-43–dependent (11). Additional splicing alterations are identified by systematic genome-wide analyses of alternative splicing that are indicative of both enhancement and loss-of-function by the TDP-43 mutants for individual RNA substrates, from which we conclude that ALS-linked mutations confer both loss- and gain-of-function properties to TDP-43, and that these act intranuclearly to induce splicing alterations that may underlie age-dependent motor neuron disease.     

胶质微环境与中枢神经系统损伤修复

中枢神经系统(central mervous system,CNS)损伤包括腩损伤和脊髓损伤,致残率和病死率较高.因此,CNS损伤修复一直是神经科学领域的一个研究重点和热点.成年哺乳动物神经元的内在再生能力有限是CNS损伤后再生困难的原因之一,但更为重要的原因是损伤局部抑制性胶质微环境的形成.文章就胶质微环境内星形胶质细胞、小胶质细胞、少突胶质细胞等各类组成细胞在CNS损伤修复中的作用做了综述.