肌萎缩侧索硬化患者SOD1基因突变检测及突变与临床表型的关系

应用PCR技术结合DNA直接测序方法对8例临床确诊为家族性肌萎缩侧索硬化(Familiar amyotrophic lateral sclerosis,FALS)家系的先证者进行铜锌超氧化物歧化酶基因(SOD1)的突变筛查,在3例先证者中检出2种SOD1基因突变,其中,2例携带了位于4号外显子的错义突变Cys111Tyr(c.332G>A),另1例携带了位于5号外显子的错义突变Gly147Asp(c.440G>A),这2种突变在中国ALS患者中属首次报道。该结果扩大了中国FALS患者的SOD1基因突变谱,对研究中国FALS患者SOD1基因突变特点和分布规律有一定帮助。分析携带这2个突变患者的临床特点,提示Cys111Tyr突变导致的临床表型相对温和,而Gly147Asp突变可导致病情进展较快。该结果有待在更多的病例中进行证实。     

肌萎缩性侧索硬化症相关的Cu,Zn-SOD

超氧化物歧化酶（superoxide dismutase,SOD）被称为生物体内自由基的清洁剂,其主要形式Cu,Zn-SOD称SOD1. SOD1突变体可引起致死性运动神经元疾病肌萎缩性侧索硬化症(ALS).但是,SOD1的毒性机理尚未完全清楚.本文概述了SOD1、Cu分子伴侣（copper chaperone for SOD1,CCS）的分子结构和CCS活化SOD1的机理,重点分析了突变体SOD1构象变化的原因及其在ALS中的可能致病机制的最新研究进展.     

肌萎缩性侧索硬化症动物模型的应用进展

肌萎缩性侧索硬化症（ALS）是运动神经元选择性死亡而导致运动功能障碍的神经性疾病,是成年人运动神经元病中最常见的疾病。已有很多学说讨论其发病机制,并且建立了ALS动物模型。随着现代生物学的发展和不同学科间的相互渗入,各种治疗策略在ALS模型实验中得到实践并有望用于临床。简要综述了ALS治疗方法在转基因动物模型中的研究进展。     

肌萎缩侧索硬化脊髓器官型培养模型的建立

利用谷氨酸转运体抑制剂苏—羟天冬氨酸(THA)制备选择性运动神经元凋亡的肌萎缩侧索硬化(ALs)脊髓器官型培养模型。取出生后8天乳鼠腰段脊髓组织切成脊髓薄片,在培养液中分别加入不同浓度THA,用SMI—32免疫组化染色对脊髓腹角α运动神经元进行鉴定,calretinin免疫组化染色对背角中间神经元进行鉴定,测定培养液中谷氨酸(Glu)、乳酸脱氢酶(LDH)的含量,并与对照组比较。结果显示对照组α运动神经元数目恒定;THA引起培养液中剂量依赖性Glu、LDH含量增高和SMI—32阳性的α运动神经元数目减少,脊髓背角的中间神经元损伤相对较轻;100μmol／L THA组在体外培养4周后,细胞外Glu含量增高,SMI—32阳性的α运动神经元数目较对照组明显减少,背角的中间神经元数目无显著变化,可以制成ALS脊髓器官型培养模型。     

稳定表达hSOD1^G93A基因的肌萎缩侧索硬化体外细胞培养模型的建立与鉴定

目的建立并鉴定稳定表达G93A型突变人超氧化物歧化酶（hSOD1^G93A）基因的肌萎缩侧索硬化体外细胞培养模型。方法利用活化的树突状聚合物将空质粒、hSOD1^WT、hSOD1^G93A基因转染入VSC4.1细胞内,G418抗性筛选,从而建立稳定的肌萎缩侧索硬化体外细胞模型。用免疫荧光技术检测VSC4.1细胞系运动神经元标志物。蛋白印迹实验鉴定hSOD1^WT蛋白、hSOD1^G93A蛋白的表达。MTT法检测细胞模型生长曲线。结果VSC4.1细胞分化前表达ClassⅢβ-Tubulin、MNR2,分化后表达ClassⅢβ-Tubulin、MNR2、NF200、MAP2等运动神经元标志物。VSC4.1-hSOD1^WT、VSC4.1-hSOD1^G93A细胞均过表达人来源的SOD1,而VSC4.1-mock则不表达。与VSC4.1-mock、VSC4.1-hSOD1^WT相比,VSC4.1-hSOD1^G93A生长缓慢,在48、72 h细胞活力均低于VSC4.1-mock（P=0.031,P=0.000）、VSC4.1-hSOD1^WT（P=0.001,P=0.000）,其余时间点无明显差异（P〉0.05）。结论成功建立稳定表达hSOD1^WT、hSOD1^G93A基因的VSC4.1细胞系,为进一步研究肌萎缩侧索硬化的发病与治疗奠定了良好的基础。     

3 例以延髓麻痹为首发表现的肌萎缩侧索硬化的病例报告

目的:探讨以延髓麻痹为首发症状的肌萎缩侧索硬化的临床特点。方法:报告3例以构音障碍等延髓麻痹症状起病的肌萎缩侧索硬化少见病例,结合文献复习,分析其临床特点。结果:在ALS患者中,以延髓麻痹为首发症状发生率相对较低,多见于老年人,最易表现为构音障碍,患者的生存期缩短,预后不良。结论:对于以延髓麻痹表现起病的ALS患者,要引起重视,及时针对延髓麻痹对症治疗,改善患者生活质量。     

肌萎缩侧索硬化患者肌电图F波和束颤波的特点及其相关性*

目的:研究肌萎缩侧索硬化(ALS)患者肌电图检查中异常F波与束颤波的特点及其相关性。方法:收集2016年9月至2018年2月就诊于浙江大学医学院附属第二医院神经内科的54例ALS患者,进行常规肌电图检查和F波测定,记录216条正中神经、胫后神经的F波以及324块肌肉的束颤波相关参数,计算F波出现率、巨大F波、束颤波的出现率及其异常率,分析巨大F波、束颤波与病程的关系以及巨大F波和束颤波之间的关联性。结果:F波出现率的异常率88.89%,巨大F波出现率55.56%,束颤波出现率48.15%;有束颤波的病程与没有束颤波病程比较具有明显差异(P＜0.01),有巨大F波的病程与未出现巨大F波病程比较无明显差异(P＞0.05);上肢正中神经巨大F波出现与束颤波的出现无关联(P＞0.05),下肢胫后神经巨大F波出现与束颤波的出现比较具有相关性(P=0.05)。结论:以上肢为主的F波出现率异常、以下肢为主的巨大F波和束颤波的出现可作为ALS电生理诊断阳性指标。有束颤波或巨大F波时可考虑疾病相对较早,且有较好的神经再支配及代偿,进展相对较慢。     

肌萎缩侧索硬化患者血清SOD、GSH-Px、MIP-1α、VEGF水平与肌电图特征及病程的关系研究

摘要 目的：研究肌萎缩侧索硬化（ALS）患者血清超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、巨噬细胞炎性蛋白-1α（MIP-1α）、血管内皮生长因子（VEGF）水平与肌电图特征及病程的关系。方法：将从2018年12月起直至2020年12月,我院收治的ALS患者86例纳入研究,记作病变组。另取同期90例于我院进行体检的健康人员作为对照组。检测并比较两组血清SOD、GSH-Px、MIP-1α、VEGF水平及肌电图特征。将所有病变组患者根据病程的差异分为病程较长组42例以及病程较短组44例,比较两组血清SOD、GSH-Px、MIP-1α、VEGF水平以及肌萎缩侧索硬化症功能评分量表（ALSFRS-r）评分。采用Pearson相关性分析ALS患者血清SOD、GSH-Px、MIP-1α、VEGF水平与肌电图特征及病程的关系。结果：病变组血清SOD、GSH-Px水平均低于对照组,而MIP-1α、VEGF水平均高于对照组（均P＜0.05）。病变组各项肌电图参数水平均低于对照组（均P＜0.05）。病程较长组血清SOD、GSH-Px水平以及ALSFRS-r评分均低于病程较短组,而MIP-1α、VEGF水平均高于病程较短组（均P＜0.05）。经Pearson相关性分析可得：ALS患者血清SOD、GSH-Px水平与肌电图各神经符合肌肉动作电位（CMAP）、ALSFRS-r评分均呈正相关,与病程呈负相关（均P＜0.05）;MIP-1α、VEGF水平则与肌电图各神经CMAP、ALSFRS-r评分均呈负相关,与病程呈正相关（均P＜0.05）。结论：ALS患者血清SOD、GSH-Px水平较低,MIP-1α、VEGF水平较高,且和肌电图特征以及病程密切相关,值得临床关注。     

肌萎缩侧索硬化患者血清鸢尾素、热休克蛋白70检测及其临床意义

摘要 目的：探讨肌萎缩侧索硬化（ALS）患者血清鸢尾素（Irisin）、热休克蛋白70（HSP70）水平及其临床意义。方法：选取2017年1月-2020年12月中国人民解放军联勤保障部队第904医院神经内科收治的ALS患者62例,根据E1 Escorial诊断标准分为ALS早期组（n=30）和ALS晚期组（n=32）,另选取同期在我院体检的健康志愿者58例为对照组,比较三组血清Irisin、HSP70水平,分析ALS患者血清Irisin、HSP70水平与氧化应激指标、改良后肌萎缩侧索硬化功能（ALSFRS-R）评分、爱丁堡认知行为量表（ECAS）评分和肌电图参数的相关性,采用受试者工作特征（ROC）曲线分析血清Irisin、HSP70对ALS的诊断效能。结果：与对照组比较,ALS早期组、ALS晚期组患者血清Irisin、HSP70、谷胱甘肽过氧化酶（GPX）、超氧化物歧化酶（SOD）水平明显降低,且ALS晚期组低于ALS早期组（P<0.05）。与对照组比较,ALS早期组、ALS晚期组患者血清8-羟基脱氧鸟苷酸(8-OHDG)、丙二醛（MDA）水平明显升高,且ALS晚期组高于ALS早期组（P<0.05）;ALS晚期组ECAS评分、ALSFRS-R评分低于ALS早期组（P<0.05）;与ALS早期组比较,ALS晚期组正中神经、尺神经、腓总神经以及胫神经的复合肌肉动作电位（CMAP）水平明显降低（P<0.05）;Pearson 检验结果显示,血清Irisin、HSP70水平与血清8-OHDG、MDA呈负相关（P<0.05）,与血清GPX、SOD水平、ECAS评分、ALSFRS-R评分以及正中神经CMAP、尺神经CMAP、腓总神经CMAP、胫神经CMAP呈正相关（P<0.05）;ROC曲线分析显示,Irisin、HSP70联合应用时曲线下面积（AUC）（0.95CI）为0.850（0.722～0.976）,诊断效能较高。结论：ALS患者血清Irisin、HSP70水平与患者体内氧化应激的过度激活、认知功能及神经功能的损害相关,血清Irisin、HSP70有可能成为ALS诊断和病情评估的生物标记物之一。     

不同剂量MOG抗原免疫诱导小鼠自身免疫性脑脊髓炎模型的比较

目的比较不同剂量髓鞘少突胶质细胞糖蛋白(myelin oligodendrocyte glycoprotein,MOG35-55)免疫诱导C57BL/6小鼠实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)的作用。方法将C57BL/6小鼠分为正常组和三组不同剂量MOG35-55诱导的EAE模型组,共4组。模型组分别以每只200、100、50μg的MOG35-55与完全弗氏佐剂(complete Freund s adjuvant,CFA)混合的乳化抗原皮下注射免疫诱导EAE模型,正常组以生理盐水替代。观察不同剂量MOG35-55对C57BL/6小鼠体重、发病率以及神经功能评分等影响,同时取小鼠脑和脊髓,利用光镜和透射电镜观察小鼠病理组织学改变。结果三组不同剂量MOG35-55均能诱导EAE模型,发病率为100%,呈慢性单相病程,病理学观察发现小鼠脑和脊髓有炎性细胞浸润、脱髓鞘及轴突损伤等改变。但小剂量组在体重减轻、临床症状评分及病理学改变等方面均较中、大剂量组明显。结论用MOG35-5550μg剂量免疫诱导的C57BL/6小鼠EAE模型稳定,可在今后的研究中应用。     

Abnormal exocytotic release of glutamate in a mouse model of amyotrophic lateral sclerosis

Glutamate-mediated excitotoxicity plays a major role in the degeneration of motor neurons in amyotrophic lateral sclerosis and reduced astrocytary glutamate transport, which in turn increases the synaptic availability of the amino acid neurotransmitter, was suggested as a cause. Alternatively, here we report our studies on the exocytotic release of glutamate as a possible source of excessive glutamate transmission. The basal glutamate efflux from spinal cord nerve terminals of mice-expressing human soluble superoxide dismutase (SOD1) with the G93A mutation [SOD1/G93A(+)], a transgenic model of amyotrophic lateral sclerosis, was elevated when compared with transgenic mice expressing the wild-type human SOD1 or to non-transgenic controls. Exposure to 15 mM KCl or 0.3 μM ionomycin provoked Ca(2+)-dependent glutamate release that was dramatically increased in late symptomatic and in pre-symptomatic SOD1/G93A(+) mice. Increased Ca(2+) levels were detected in SOD1/G93A(+) mouse spinal cord nerve terminals, accompanied by increased activation of Ca(2+)/calmodulin-dependent kinase II and increased phosphorylation of synapsin I. In line with these findings, release experiments suggested that the glutamate release augmentation involves the readily releasable pool of vesicles and a greater capability of these vesicles to fuse upon stimulation in SOD1/G93A(+) mice.     

Comparative study of behavioural tests in the SOD1G93A mouse model of amyotrophic lateral sclerosis

In preclinical trials, a sensitive functional test is required to detect changes in the motor behaviour of the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS). We evaluated changes in body weight and motor impairment in behavioural tests, such as the rotarod, the hanging-wire test and the treadmill, of transgenic and wild type mice. We found differences in detection of the onset of symptoms and progression of the disease between the different tests assessed. Moreover, the data showed significant gender differences in the motor behaviour of this mouse model. The rotarod and the hanging-wire test were more sensitive to detect early motor impairment. Moreover, the results suggested that the rotarod and hanging-wire became the most accurate tests rather than treadmill to characterise the ALS disease phenotype.     

Inducible superoxide dismutase 1 aggregation in transgenic amyotrophic lateral sclerosis mouse fibroblasts

High molecular weight detergent-insoluble complexes of superoxide dismutase 1 (SOD1) enzyme are a biochemical abnormality associated with mutant SOD1-linked familial amyotrophic lateral sclerosis (FALS). In the present study, SOD1 protein from spinal cords of transgenic FALS mice was fractionated according to solubility in saline, zwitterionic, non-ionic or anionic detergents. Both endogenous mouse SOD1 and mutant human SOD1 were least soluble in SDS, followed by NP-40 and CHAPS, with an eight-fold greater detergent resistance of mutant protein overall. Importantly, high molecular weight mutant SOD1 complexes were isolated with SDS-extraction only. To reproduce SOD1 aggregate pathology in vitro, primary fibroblasts were isolated and cultured from neonatal transgenic FALS mice. Fibroblasts expressed abundant mutant SOD1 without spontaneous aggregation over time with passage. Proteasomal inhibition of cultures using lactacystin induced dose-dependent aggregation and increased the SDS-insoluble fraction of mutant SOD1, but not endogenous SOD1. In contrast, paraquat-mediated superoxide stress in fibroblasts promoted aggregation of endogenous SOD1, but not mutant SOD1. Treatment of cultures with peroxynitrite or the copper chelator diethyldithiocarbamate (DDC) alone did not modulate aggregation. However, DDC inhibited lactacystin-induced mutant SOD1 aggregation in transgenic fibroblasts, while exogenous copper slightly augmented aggregation. These data suggest that SOD1 aggregates may derive from proteasomal or oxidation-mediated oligomerisation pathways from mutant and endogenous subunits respectively. Furthermore, these pathways may be affected by copper availability. We propose that non-neural cultures such as these transgenic fibroblasts with inducible SOD1 aggregation may be useful for rapid screening of compounds with anti-aggregation potential in FALS.     

Increased stem cell proliferation in the spinal cord of adult amyotrophic lateral sclerosis transgenic mice

Harnessing the regenerative potential of the central nervous system to repopulate depleted cellular populations from endogenous stem cells would be a novel approach for the treatment of neurological diseases resulting from cell death. Consequently, understanding if and how the central nervous system is capable of such regeneration would determine if such an approach is feasible. In this report, we provide evidence of widespread regenerative response in the spinal cord of amyotrophic lateral sclerosis transgenic mice. However, this regenerative response appears to be largely unproductive. We demonstrate that there is significantly increased gliogenesis, but an absence of convincing neurogenesis. The fact that the neurodegenerative process stimulates a regenerative response suggests that the adult spinal cord has at least limited ability for regeneration. Further studies will determine if this endogenous regenerative process can be enhanced and directed so as to slow or even reverse the natural progression of this devastating disease.     

Selective loss of neurofilament expression in Cu/Zn superoxide dismutase (SOD1) linked amyotrophic lateral sclerosis

Neurofilament pathology is a hallmark of sporadic and familial amyotrophic lateral sclerosis (SALS and FALS). The disease mechanisms underlying this pathology are presently unclear, but recent evidence in SALS patients suggest that reductions in neurofilament light subunit (NFL) mRNA may contribute to the death of motor neurones. Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) represent the best-studied cause of FALS, and a number of laboratory models of SOD1-mediated disease exist. Here we have used microdissected lumbar spinal cord motor neurones from human SOD1 FALS patients as well as G93A SOD1 transgenic mice and demonstrated that reduced NFL mRNA levels are seen in both. To probe the molecular mechanisms underpinning these observations, we generated NSC34 motor neurone-like cell lines expressing wild-type and mutant SOD1. NSC34 cells expressing G37R or G93A SOD1 showed selective reductions in NFL and NFM mRNA and protein. These data suggest that NFL mRNA reductions are common to SALS and FALS patients, and that cells and mice expressing mutant SOD1 may enable us to characterize the molecular mechanism(s) responsible for the loss of neurofilament mRNA.     

The CB2 cannabinoid agonist AM-1241 prolongs survival in a transgenic mouse model of amyotrophic lateral sclerosis when initiated at symptom onset

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron loss, paralysis and death within 2-5 years of diagnosis. Currently, no effective pharmacological agents exist for the treatment of this devastating disease. Neuroinflammation may accelerate the progression of ALS. Cannabinoids produce anti-inflammatory actions via cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2), and delay the progression of neuroinflammatory diseases. Additionally, CB2 receptors, which normally exist primarily in the periphery, are dramatically up-regulated in inflamed neural tissues associated with CNS disorders. In G93A-SOD1 mutant mice, the most well-characterized animal model of ALS, endogenous cannabinoids are elevated in spinal cords of symptomatic mice. Furthermore, treatment with non-selective cannabinoid partial agonists prior to, or upon, symptom appearance minimally delays disease onset and prolongs survival through undefined mechanisms. We demonstrate that mRNA, receptor binding and function of CB2, but not CB1, receptors are dramatically and selectively up-regulated in spinal cords of G93A-SOD1 mice in a temporal pattern paralleling disease progression. More importantly, daily injections of the selective CB2 agonist AM-1241, initiated at symptom onset, increase the survival interval after disease onset by 56%. Therefore, CB2 agonists may slow motor neuron degeneration and preserve motor function, and represent a novel therapeutic modality for treatment of ALS.     

Cell-permeable peptide antioxidants as a novel therapeutic approach in a mouse model of amyotrophic lateral sclerosis

Reactive oxygen species (ROS) play a major role in the pathogenesis of neurodegenerative diseases. They are important contributors to necrotic and apoptotic cell death. A major proportion of cellular ROS is generated at the inner mitochondrial membrane by the respiratory chain. In the present study, we investigated a novel peptide antioxidant (SS-31) targeted to the inner mitochondrial membrane for its therapeutic effects both in vitro and in vivo in the G93A mouse model of amyotrophic lateral sclerosis (ALS). SS-31 protected against cell death induced by hydrogen peroxide in vitro in neuronal cells stably transfected with either wild-type or mutant Cu/Zn superoxide dismutase (SOD1). Daily intraperitoneal injections of SS-31 (5 mg/kg), starting at 30 days of age, led to a significant improvement in survival and motor performance. In comparison with vehicle-treated G93A mice, SS-31-treated mice showed a decreased cell loss and a decrease in immunostaining for markers of oxidative stress in the lumbar spinal cord. This further enhances the concept that pharmacological modification of oxidative stress is a therapeutic option for the treatment of ALS.