Progressive motor neuron impairment in an animal model of familial amyotrophic lateral sclerosis

Mutations of Cu,Zn superoxide dismutase cause an autosomal dominant form of familial amyotrophic lateral sclerosis. An animal model of the disease has been produced by expressing mutant human SOD1 in transgenic mice (G93A). In order to quantify the dysfunction of the motor unit in transgenic mice, electromyographic recordings were performed during the course of the disease. The first alterations in neuromuscular function appeared between P63 and P90. The deficits became even more striking after P100; compound muscle action potentials in the hindlimb decreased by 80% of initial value. Spontaneous fibrillation potentials were measured in more than 50% of transgenic mice. The number of motor units in the gastrocnemius muscle was progressively reduced over time, down to 18% of the control value at P130. Moreover, distal motor latencies increased after P120. These data suggest that the initial dysfunctions of motor unit are related to a severe motor axonal degeneration, which is followed at later periods by myelin alteration. © 1997 John Wiley & Sons, Inc.     

Motor unit firing in amyotrophic lateral sclerosis and other upper and lower motor neurone disorders

Objective

Motor unit recruitment order and firing rate was investigated in healthy subjects, and in small numbers of patients 50 years ago. We aimed to investigate firing rate in different disorders, testing the same target muscle with normal strength, to evaluate possible application in diagnosing upper motor neuron (UMN) lesion.

Methods

We studied motor unit firing in the tibialis anterior muscle in six groups of subjects; normal subjects (n = 45), patients with amyotrophic lateral sclerosis (ALS) (n = 36), primary lateral sclerosis (PLS) (n = 21), progressive muscular atrophy (PMA) (n = 14), various upper motor neurone lesions (n = 16) and polyneuropathy (n = 42). In all these subjects the tibialis anterior muscle was of normal strength. Motor units were recruited during slight contraction in order to study 2–5 motor units at each recording site, using a standard concentric needle electrode, so that 20–22 motor units were recorded in each muscle. We analysed the coefficient of variation (CV) for amplitude, area, duration and firing rate in these motor units, and the correlation between motor unit potential size and recruitment order.

Results

The mean MU firing rate in this task was similar in each group. No recruitment order was disclosed within the limits of the study task. The CV of firing rate was decreased in UMN and PLS groups. ALS patients with marked spasticity showed a lower CV of motor unit firing rate. The CV of amplitude, area and duration was similar between groups.

Conclusions

These results in tibialis anterior indicate that physiological modulation of lower motor neuron (LMN) firing rate is decreased in patients with lower limb spasticity. The variability of MU discharges tends to be greater in diseases affecting the LMN.

Significance

These results suggest that, notwithstanding the simplicity of the task we have used, the physiological variability of motor unit firing may be a useful variable in assessing UMN involvement in motor system disorders.     

Ifenprodil对肌萎缩侧索硬化离体模型中运动神经元的保护作用

目的 探讨Ifenprodil对苏-羟天冬氨酸(THA)诱导的肌萎缩侧索硬化(ALS)离体模型中运动神经元的保护作用.方法 在离体培养的脑组织片中加入THA造成慢性谷氨酸损伤,模拟肌萎缩侧索硬化的特征性病理改变.将脑片分为6组:(1)对照组,不添加THA; (2)THA损伤组,细胞换液时加入100 μmol/L THA,持续1周;(3)不同剂量Ifenprodil组(分别在THA损伤基础上添加0.1、1、10、100 μmol/L Ifenprodil);每组6个培养皿.Ifenprodil 于THA损伤前2h加入到各组脑片中.根据PI染色的荧光亮度测定Ifenprodil保护作用的最佳浓度.在损伤的不同时间点用丙二醛(MDA)试剂盒测定培养体系中MDA的含量,Western blotting测定运动皮层中半胱氨酸天冬氨酸酶-3(caspase-3)蛋白表达量.结果 PI染色显示10 μmol/L Ifenprodil对运动神经元有明显保护作用.THA损伤后,培养体系中MDA含量明显增加,运动皮层中caspase-3活化亚基(相对分子量为17 000)表达量增高,与其他组别比较差异有统计学意义(P＜0.05).10μmol/L Ifenprodil能有效阻止MDA和活化caspase-3的增加.结论 Ifenprodil能够通过减轻运动神经元的氧化应激反应,抑制caspase-3活化,进而有效保护ALS离体模型中运动神经元的损伤.     

hNT neurons delay onset of motor deficits in a model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease that manifests as a progressive muscular weakness leading to paralysis and death. Because of the diffuse nature of the motor neuron death, this disease is not considered a good candidate for treatment through neural transplantation. The purpose of this study was to show that transplantation of human neuron-like cells (hNT neurons) into the spinal cord of a transgenic ALS mouse model would improve motor deficits. The hNT neurons were transplanted bilaterally into L4-L5 spinal cord of the transgenic mice ( approximately 8 weeks of age), and the animals were evaluated on health and behavioral measures. The animals were perfused, and immunohistochemistry was performed to identify the transplanted cells. Transplantation of the hNT neurons into the spinal cord delayed the onset of motor behavioral symptoms. This was the first demonstration that even localized transplantation of neural cells directly into the parenchyma could improve motor function in an ALS model. Further study is needed to delineate the mechanism underlying these effects. This therapeutic approach has the potential to restore neural transmission, thereby improving quality of life for the ALS patient and possibly extend life expectancy.     

Quantification of upper motor neuron loss in amyotrophic lateral sclerosis.

OBJECTIVE: To quantitatively estimate upper motor neuron (UMN) loss in ALS. METHODS: We used the recently developed triple stimulation technique (TST) to study corticospinal conduction to 86 abductor digiti minimi muscles of 48 ALS patients. This method employs a collision technique to estimate the proportion of motor units activated by a transcranial magnetic stimulus. At the same time, it yields an estimate of lower motor neuron (LMN) integrity. RESULTS: The TST disclosed and quantified central conduction failures attributable to UMN loss in 38 sides of 24 patients (subclinical in 15 sides), whereas conventional motor evoked potentials detected abnormalities in only 18 sides of 12 patients (subclinical in two sides). The increased sensitivity of the TST to detect UMN dysfunction was particularly observed in early cases. Increased central motor conduction times (CMCT) occurred exclusively in sides with conduction failure. In sides with clinical UMN syndromes, the TST response size (but not the CMCT) correlated with the muscle weakness. In sides with clinical LMN syndromes, the size of the peripherally evoked compound muscle action potentials correlated with the muscle weakness. CONCLUSION: The TST is a sensitive method to detect UMN dysfunction in ALS. It allows a quantitative estimate of the UMN loss, which is related to the functional deficit. Therefore, the TST has a considerable impact on diagnostic certainty in many patients. It will be suited to follow the disease progression and therapeutic trials.     

Intravenous immunoglobulin therapy in amyotrophic lateral sclerosis

Seven consecutive patients with amyotrophic lateral sclerosis (ALS) were treated with intravenous immunoglobulins (IVIg; 0.4 g/kg per day for 5 consecutive days followed by monthly 2-day infusions at the same daily dosage) continued with oral cyclophosphamide (1–2 mg/kg per day), for 4–13 months (mean 8.1). Response to treatment was assessed by means of the Medical Research Council (MRC) rating scale for muscle strength on 40 muscles (10 per limb), a clinical scale for bulbar function and a modified Rankin disability scale. All patients continued to deteriorate during treatment on as regards both their MRC score and either their bulbar or Rankin score or both. The progression of the disease during treatment, expressed as the monthly variation in MRC score (mean=−2.71; SD=1.36), was no slower than that estimated before therapy (mean=−1.81; SD=0.93). Even if the results of this small, uncontrolled study do not permit the exclusion of an effect of IVIg on the progression of ALS, they also do not provide any evidence that this expensive form of therapy consistently slows the course of the disease.     

Hereditary pure lower motor neuron disease with adult onset and rapid progression

We describe three members each of two families presenting with a hereditary form of lower motor neuron disease with adult onset and rapid progression and compare their pathological and clinical features with hereditary lower motor neuron disease with adult onset, as described in the literature. No involvement of upper motor neurons was found either clinically or pathologically. Disease progression was rapid, and the majority of patients died from respiratory failure within 1–5 years after onset of disease. On pathological examination of the spinal cord we found ballooned neurons, neuronophagia and gliosis in family A, which have been regarded as characteristic pathological features of infantile-onset spinal muscular atrophy (SMA). In family B specific neuronal changes were observed that also occur in patients with amyotrophic lateral sclerosis (ALS). An autosomal dominant mode of inheritance would seem likely in both families. In family A the pathological findings and the clinical presentation with symmetrical proximal limb weakness show similarities with autosomal dominant SMA. Based on the finding of pathological features in family B that also occur in ALS, together with the distal asymmetrical muscle weakness and bulbar signs and a high age at onset we hypothesize that the members of family B suffered from familial ALS. The disease forms in both families in our opinion further broaden the spectrum of motor neuron disease Received: 2 June 2000, Received in revised form: 1 September 2000, Accepted: 23 October 2000     

The electrophysiology of the motor neuron diseases

The motor neuron diseases are a set of disorders associated with the selective degeneration of motor neurons. Amyotrophic lateral sclerosis (ALS) is the most common and confers the gravest prognosis. Although ALS occurs with known genetic causes in a small minority, other motor neuron disorders have well-defined genetic mutations. Electrodiagnostic testing is important to distinguish these various disorders. Electrodiagnostic testing is also crucial for distinguishing potential mimic syndromes, such as multifocal motor neuropathy and inclusion body myositis. Newer neurophysiology techniques have been developed in the past several years. What role these techniques will play in clinical practice is currently unknown.     

三重经颅磁刺激技术对肌萎缩侧索硬化上运动神经元损害的评价及其作用

目的 研究三重经颅磁刺激技术(triple stimulation technique,TST)在肌萎缩侧索硬化(amyotrophic lateral sclerosis,ALS)患者上运动神经元(upper motor neuron,UMN)损害的评价和诊断中的作用.方法 收集我院门诊和病房收治的ALS患者50例和健康志愿者22名进行右上肢小指展肌TST测定、中枢运动传导时间(central motor conduction time,CMCT)测定、运动诱发电位(motor evoked potential,MEP)潜伏期、静息运动阈值(resting motor threshold,RMT)、复合肌肉动作电位(compound muscle action potential,CMAP)测定及对TST与改良的Ashworth评分(Modified Ashworth Scale,MAS)、医学研究委员会评分、修订的ALS功能评分、病情进展速度等进行相关性分析.结果 根据EI Escorial的ALS诊断标准,临床确诊5例,临床拟诊19例,实验室辅助拟诊24例,临床可能2例.ALS患者右上肢有UMN体征组[28例,62.0%(40.7%,75.9%)]与无UMN体征组[22例,95.6%(85.4%,100.0%)],健康对照组(96.9%±2.6%)比较,TST波幅比[M50(M25,M75)]差异均有统计学意义(Z=-4.827、-5.435,均P=0.000).有UMN体征组(89.3%)与无UMN体征组(27.3%)、健康对照组(9.1%)TST波幅比异常出现率差异均有统计学意义(χ2=20.109、31.897,均P=0.000),无UMN体征组与健康对照组差异无统计学意义(χ2=1.375,P=0.241).TST波幅比、MEP潜伏期、易化状态下MEP潜伏期、CMCT、RMT对ALS患者UMN体征的阳性检出率分别为89.3%、64.3%、53.6%、64.3%、78.6%.TST波幅比与右上肢腱反射(r=0.690)、MAS评分(r=-0.772)、诊断分级(r=0.483)存在相关性(均P=0.000),与RMT(r=-0.774,P=0.000)、MEP潜伏期(r=-0.444,P=0.005)、MEP/CMAPerb(r=0.685,P=0.000)、易化状态下MEP/CMAPerb(r=0.770,P=0.000)存在相关性.结论 TST为ALS患者UMN损害提供了敏感的检测指标,能够发现临床下UMN受累,较传统经颅磁刺激方法 提高了诊断的敏感性,能够对UMN损害进行半定量评价,提示其有可能成为监测病情变化的客观评价指标.     

Myostatin inhibition slows muscle atrophy in rodent models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease leading to motor neuron cell death, but recent studies suggest that non-neuronal cells may contribute to the pathological mechanisms involved. Myostatin is a negative regulator of muscle growth whose function can be inhibited using neutralizing antibodies. In this study, we used transgenic mouse and rat models of ALS to test whether treatment with anti-myostatin antibody slows muscle atrophy, motor neuron loss, or disease onset and progression. Significant increases in muscle mass and strength were observed in myostatin-antibody-treated SOD1(G93A) mice and rats prior to disease onset and during early-stage disease. By late stage disease, only diaphragm muscle remained significantly different in treated animals in comparison to untreated controls. Myostatin inhibition did not delay disease onset nor extend survival in either the SOD1(G93A) mouse or rat. Together, these results indicate that inhibition of myostatin does not protect against the onset and progression of motor neuron degenerative disease. However, the preservation of skeletal muscle during early-stage disease and improved diaphragm morphology and function maintained through late stage disease suggest that anti-myostatin therapy may promote some improved muscle function in ALS.     

Interplay of upper and lower motor neuron degeneration in amyotrophic lateral sclerosis

Objective

We studied motor unit recruitment to test a new method to identify motor unit firing rate (FR) variability.

Diffusion tensor MRI as a diagnostic tool of upper motor neuron involvement in amyotrophic lateral sclerosis

BACKGROUND: Clinical identification of upper motor neuron (UMN) dysfunction in amyotrophic lateral sclerosis (ALS) is often difficult, particularly early in the course of the disease, or when lower motor neuron (LMN) dysfunction is prominent. Diffusion tensor MR imaging (DTI) can provide unique information on axonal organization by measuring diffusion anisotropy and the directionally independent diffusion. OBJECTIVE: The purpose of this study was to assess water diffusion changes along pyramidal tracts of the brainstem in patients with ALS and to investigate possible correlations between changes of diffusion properties and various clinical parameters. METHODS: We studied 16 patients (M:F=9:7, 50.5+/-12.4 years) with ALS as defined by clinical and electrophysiological examinations. These patients were compared with 11 healthy, age and sex-matched controls (M:F=5:6, 54.5+/-9.9 years). DTI was performed using a single shot SE-EPI with 25 noncollinear diffusion gradient directions (b=1000 s/mm(2)) and with no diffusion gradient on a 3.0-T MR system. RESULTS: By multifactorial ANOVA, the effects of group (patient versus control) and anatomical level on fractional anisotropy (FA) and mean diffusivity (MD) were significant (p<0.001 for both parameters), whereas the effect of side (left versus right) and interactions between factors (group by side and group by anatomical level) were not (p>0.05). In all subjects, FA and MD varied greatly depending on the anatomical level, and FA was highly variable even between contiguous slices in the pons and medulla, whereas relatively constant FA values were noted at the level of the midbrain. Cerebral peduncle was the only area that showed significant differences of diffusion properties between patients and controls (p<0.001 for FA, p=0.001 for MD). Correlation analysis revealed a significant inverse relationship between the FA value and the extent of UMN signs (r=-0.81, p<0.001). CONCLUSIONS: Alteration of diffusion properties in the cerebral peduncle in ALS may reflect pathological changes in structures rather than regional architectural variations of the corticospinal tracts or experimental artifacts.     

中国家族性肌萎缩侧索硬化患者超氧化物歧化酶1基因突变分析

目的 肌萎缩侧索硬化(ALS)是上、下运动神经元退变引起的一种进行性致死性疾病,家族性ALS占10%～20%,铜锌SOD1基因突变存在于20%的家族性患者和少数散发性患者,某些突变还具有地域分布和特殊临床表型的规律性.我们旨在分析我国3个家族性ALS家系SOD1基因突变特点,并与不同国家和地区SOD1突变比较,分析其临床表型的特征性.方法 提取患者外周血基因组DNA,采用SOD1基因的5对引物对其5个外显子进行PCR扩增,产物直接测序.归纳整理患者临床表型资料,进行表型-基因型关联分析.结果 家系1中SOD1基因外显子2的H46R杂合突变,即CAT→CGT,使得46位由编码组氨酸变为编码精氨酸.先证者48岁,女性,43岁起病,主要表现为肢体无力和萎缩.家系中其他3例患者首发症状与先证者相似,病情进展均较缓慢,生存期较长.家系2中先证者20岁,男性,临床表现为延髓性麻痹,病程进展快速,生存期1年,位于3号外显子的杂合突变,即G72C突变.其父亲也出现同样的突变,但无ALS临床表现,其姑姑也有类似病史.家系3中5例患者5号外显子的E133V杂合突变,先证者中年起病,病程逐渐进展,生存期5年.结论 H46R的杂合突变国内未见报道,国外日本人曾多次报道及巴基斯坦有1个家系报道,欧美均未见报道,推测此突变可能为亚裔所特有.G72C突变的家系,临床表型为较早发病,病程进展迅速,生存期1年,突变外显率低导致家族成员不发病,常被诊断为散发病例,因此对于散发性患者及其家族成员同时检测SOD1基因突变十分必要.E133V突变的家系为国际首先报道.     

Assessing the upper motor neuron in amyotrophic lateral sclerosis using the triple stimulation technique: A multicenter prospective study

ObjectiveTo evaluate the relevance of transcranial magnetic stimulation (TMS) using triple stimulation technique (TST) to assess corticospinal function in amyotrophic lateral sclerosis (ALS) in a large-scale multicenter study.MethodsSix ALS centers performed TST and conventional TMS in upper limbs in 98 ALS patients during their first visit to the center. Clinical evaluation of patients included the revised ALS Functional Rating Scale (ALSFRS-R) and upper motor neuron (UMN) score.ResultsTST amplitude ratio was decreased in 62% of patients whereas conventional TMS amplitude ratio was decreased in 25% of patients and central motor conduction time was increased in 16% of patients. TST amplitude ratio was correlated with ALSFRS-R and UMN score. TST amplitude ratio results were not different between the centers.ConclusionsTST is a TMS technique applicable in daily clinical practice in ALS centers for the detection of UMN dysfunction, more sensitive than conventional TMS and related to the clinical condition of the patients.SignificanceThis multicenter study shows that TST can be a routine clinical tool to evaluate UMN dysfunction at the diagnostic assessment of ALS patients.     

Phenotypic variability in amyotrophic lateral sclerosis

Clinically, ALS phenotypes depend on the areas of the body that are affected, the different degrees of involvement of upper and lower motor neurons, the degrees of involvement of other systems, particularly cognition and behavior, and rates of progression. Phenotypic variability of ALS is characteristic and can be declined on the distribution of motor manifestations but also on the presence of extra-motor signs present in a variable manner in ALS patients. Neuropathologically, ALS is defined by the loss of UMN and LMN and the presence of two representative motor neuronal cytoplasmic inclusions, Bunina bodies and 43 kDa Transactivation Response DNA Binding Protein (TDP-43) – positive cytoplasmic inclusions. The distribution of cytopathology and neuronal loss in patients is variable and this variability is directly related to phenotypic variability. Key regulators of phenotypic variability in ALS have not been determined. The functional decrement of TDP-43, and region-specific neuronal susceptibility to ALS, may be involved. Due to the selective vulnerability among different neuronal systems, lesions are multicentric, region-oriented, and progress at different rates. They may vary from patient to patient, which may be linked to the clinicopathological variability across patients.     

Lack of changes in the PI3K/AKT survival pathway in the spinal cord motor neurons of a mouse model of familial amyotrophic lateral sclerosis

The vulnerability of motor neurons in transgenic SOD1G93A mice, a model of familial amyotrophic lateral sclerosis (ALS), may depend on the failure of these cells to activate survival mechanisms in response to the toxic mutant SOD1. To test this we investigated whether defects in the PI3K/Akt pathway, a survival signal, and of its neuron-specific activator, Rai, were important for motor neuron degeneration in these mice. No substantial changes were found in the levels of Rai, PI3K(p85) or phosphorylated Akt (P-Akt) in the ventral horn of spinal cord of SOD1G93A mice during disease progression. P-Akt immunoreactivity was the same in degenerating and healthy motor neurons. Rai ablation in SOD1G93A mice slightly accelerated the motor dysfunction without affecting their life span. Thus, motor neurons in SOD1G93A mice do not lose the pro-survival PI3K/Akt signal nor increase it in order to suppress the cell death mechanisms.     

Survival motor neuron protein in the nucleolus of mammalian neurons

Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by mutations in the survival motor neuron gene (SMN1). While it has been shown that the SMN protein is involved in spliceosome biogenesis and pre-mRNA splicing, there is increasing evidence indicating that SMN may also perform important functions in the nucleolus. We demonstrate here through the use of a previously characterized polyclonal anti-SMN antibody, abSMN, that the SMN protein shows a striking colocalization with the nucleolar protein, fibrillarin, in both nucleoli and Cajal bodies/gems of primary neurons. Immunoblot analysis with antifibrillarin and two different anti-SMN antibodies reveals that SMN and fibrillarin also cofractionate in the insoluble protein fraction of cultured cell lysates. Immunoprecipitation experiments using whole cell extracts of HeLa cells and cultured neurons revealed that abSMN coprecipitated small amounts of the U3 small nucleolar RNA (snoRNA) previously shown to be associated with fibrillarin in vivo. These studies raise the possibility that SMN may serve a function in rRNA maturation/ribosome synthesis similar to its role in spliceosome biogenesis.     

Electrical impedance myography for monitoring motor neuron loss in the SOD1 G93A amyotrophic lateral sclerosis rat

Objective

Human studies have shown that electrical impedance myography (EIM), a technique based on the surface application of high-frequency, low-intensity electrical current to localized areas of muscle, is sensitive to muscle denervation. In this study, we examined the role of EIM as a potential biomarker for assessing ALS disease progression in the SOD1 transgenic rat by comparing it to motor unit number estimation (MUNE).

Methods

Multi-frequency EIM and MUNE were performed twice weekly in 16 rats from approximately 10 weeks of age onward. Four different EIM measures were evaluated, including the previously studied 50 kHz phase and three condensed multi-frequency parameters.

Results

The rate of deterioration in the multi-frequency phase data from 100–500 kHz had the strongest correlation to survival (ρ = 0.79, p < 0.001), surpassing that of MUNE (ρ = 0.57, p = 0.020). These two measures were also strongly correlated (ρ = −0.94, p < 0.001) to one another.

Conclusions

These findings support that EIM is an effective tool for assessing disease progression in the ALS rat.

Significance

Given its ease of application and ability to assess virtually any superficial muscle, EIM deserves further study as a biomarker in human ALS clinical therapeutic trials.     

Lack of cervical paraspinal muscle involvement in juvenile distal spinal muscular atrophy: an electromyographic study on 15 cases

Electromyography (EMG) of the lower cervical paraspinal muscles was performed in 15 young Chinese males with distal spinal muscular atrophy of the upper extremities. The lack of fibrillation and positive sharp waves in all patients, both in early or active and chronic or steady stages, did not correlate with the EMG status in the affected upper extremity on the same side. This finding is in striking contrast with that in amyotrophic lateral sclerosis.