Alteration in amino acids in motor neurons of the spinal cord in amyotrophic lateral sclerosis.

Little is known concerning the changes of amino acid composition in different regions of the spinal cord in patients with amyotrophic lateral sclerosis (ALS). We performed quantitative amino acid analyses in the posterior funiculus, the lateral corticospinal tract, and the anterior horn of cervical enlargement of the spinal cord from seven ALS patients, and the results were compared with those of seven patients with other neurologic diseases (control A) and seven patients without neurologic diseases (control B). The levels of collagen-associated amino acids, hydroxyproline, proline, glycine, and hydroxylysine, were markedly lower in the lateral corticospinal tract and the anterior horn of ALS patients than in controls A and B. The contents of the acidic amino acids glutamate and aspartate were also significantly decreased in the lateral corticospinal tract and the anterior horn of ALS patients as compared with those of controls A and B. These data suggest that decreased contents of collagen-associated amino acids and excitatory amino acids are related to the degeneration of the upper and lower motor neurons in the spinal cord in ALS.     

运动神经元疾病患者颈髓弥散张量成像研究

目的 研究运动神经元疾病颈髓弥散张量成像(DTI)特点,探讨DTI在运动神经元疾病中的诊断价值.方法 选取临床诊断为肌萎缩侧索硬化(ALS)患者16例、肯尼迪病(KD)患者12例及健康志愿者15名.分别行颈髓常规MRI及横断面DTI检查,测定各组C3、C5椎体水平层面颈髓前索区、后索区及舣侧皮质脊髓侧束区4个区域内感兴趣区(ROI)的表观弥散系数(apparent diffusion coefficient,ADC)和部分各向异性比值(fractional anisotropy,FA),并分析影像学参数与其病程、ALSFRS-R评分、肺功能等之间的关系.结果 ALS 组患者在C3与C5水平,前索、双侧皮质脊髓侧束的FA值较KD组、健康对照组显著降低(P<0.05),而KD组与健康对照组之间差异无统计学意义(P>0.05).3组在各不同位置的ADC值之间差异无统计学意义(P>0.05).ALS组在C3水平右侧皮质脊髓束FA值与患者ALSFRS-R评分呈正相关(r=0.52,P=0.041);各水平前索、双侧皮质脊髓束的FA值、ADC值与患者病程、肺功能之间无相关性.结论 FA值可反映颈髓皮质脊髓束的功能异常,可用于评估ALS患者上运动神经元的损伤程度,有利于运动神经元疾病的鉴别诊断.     

Spinal cord neurofibrillary tangles of Guamanian amyotrophic lateral sclerosis and parkinsonism-dementia complex: an immunohistochemical study

We studied the topographic distribution and immunohistochemical characteristics of spinal cord neurofibrillary tangles (NFTs) in 6 patients with Guamanian amyotrophic lateral sclerosis (ALS) and 6 patients with parkinsonismdementia complex (PD) on Guam, using antibodies to tau protein and ubiquitin. The NFTs were immunoreactive with both antibodies, but staining for tau was more pronounced. As identified by this reactivity, all the Guamanian ALS and PD cases examined showed spinal cord NFTs. The posterior horn had the most and the anterior horn the least. In the posterior horn the NFTs were located mainly in the marginal areas. Large anterior horn cells showed few, if any, NFTs. In addition to perikaryal NFTs, we observed tau-reactive neurites. Our results provide evidence that spinal cord NFTs are not uncommon in Guamanian ALS and PD on Guam and that they are more numerous than previously found with conventional methods.     

Discrete mitochondrial aberrations in the spinal cord of sporadic ALS patients

Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease characterized by progressive motor neuron degeneration in the brain and spinal cord leading to muscle atrophy, paralysis, and death. Mitochondrial dysfunction is a major contributor to motor neuron degeneration associated with ALS progression. Mitochondrial abnormalities have been determined in spinal cords of animal disease models and ALS patients. However, molecular mechanisms leading to mitochondrial dysfunction in sporadic ALS (sALS) patients remain unclear. Also, segmental or regional variation in mitochondrial activity in the spinal cord has not been extensively examined in ALS. In our study, the activity of mitochondrial electron transport chain complex IV was examined in post‐mortem gray and white matter of the cervical and lumbar spinal cords from male and female sALS patients and controls. Mitochondrial distribution and density in spinal cord motor neurons, lateral funiculus, and capillaries in gray and white matter were analyzed by immunohistochemistry. Results showed that complex IV activity was significantly decreased only in gray matter in both cervical and lumbar spinal cords from ALS patients. In ALS cervical and lumbar spinal cords, significantly increased mitochondrial density and altered distribution were observed in motor neurons, lateral funiculus, and cervical white matter capillaries. Discrete decreased complex IV activity in addition to changes in mitochondria distribution and density determined in the spinal cord in sALS patients are novel findings. These explicit mitochondrial defects in the spinal cord may contribute to ALS pathogenesis and should be considered in development of therapeutic approaches for this disease.     

Widespread spinal cord involvement in progressive supranuclear palsy

We describe the histopathologic features of spinal cord lesions in 10 cases of progressive supranuclear palsy (PSP) and review the literature. Histologic examination revealed atrophy with myelin pallor in the anterior funiculus and anterolateral funiculus in the cervical and thoracic segments in eight of the 10 cases, whereas the posterior funiculus was well preserved. The degrees of atrophy of the anterior funiculus and the anterolateral funiculus correlated with that of the tegmentum of the medulla oblongata. Myelin pallor of the lateral corticospinal tract was observed in two of the 10 cases. Microscopic observation of the spinal white matter, particularly the cervical segment, revealed a few to several neuropil threads, particularly in the white matter surrounding the anterior horn after Gallyas‐Braak (GB) staining or AT‐8 tau immunostaining. However, the posterior funiculus was completely preserved from the presence of argyrophilic or tau‐positive structures. In the spinal gray matter, widespread distribution of neurons with cytoplasmic inclusions and neuropil threads was observed, particularly in the medial division of the anterior horn and intermediate gray matter, especially in the cervical segment. Globose‐type neurofibrillary tangles and pretangles were found. The distribution of GB‐ or AT‐8 tau‐positive small neurons and neuropil threads resembled that of the spinal interneurons. In conclusion, the spinal cord, especially the cervical segment, is constantly involved in the pathologic process of PSP. We speculate that spinal interneurons and their neuronal processes, particularly in the medial division of the anterior horn and intermediate gray matter of the cervical segment, are most severely damaged in the PSP spinal cord.     

Numbers of axons in lateral and ventral funiculi of rat sacral spinal cord

The present study determines the numbers of myelinated and unmye-linated axons in the ventral and lateral funiculi of rat sacral spinal cord. On average, there are 55,000 myelinated and 110,000 unmyelinated axons in the lateral funiculus and 26,000 myelinated and 9,000 unmyelinated axons in the ventral funiculus at these levels. These figures combined with data from earlier studies of the posterior funiculus and the tract of Lissauer give approximate figures of 88,500 myelinated and 131,500 unmyelinated axons for the entire white matter of one side of the rat sacral spinal cord. Thus unmyelinated axons predominate in the white matter of the rat sacral spinal cord. The majority of axons, particularly the unmyelinated axons, are located in the lateral funiculus. The axons are concentrated in the dorsalateral part of the lateral funiculus, and so the dorsal part of the lateral funiculus, of ten referred to as the dorsolateral funiculus, contains more than half the fibers in the white matter of the spinal cord. A small nick in the dorsal and lateral part of the lateral funiculus, which is often done for various experimental reasons, could thus remove 40% of the axons in the white matter of rat sacral spinal cord. The data reported in the present paper will serve as a basis for future studies on the white matter of the isolated spinal cord.     

以胸髓前角先受累的肌萎缩侧索硬化的临床特点

目的探讨胸髓前角先受累的肌萎缩侧索硬化(ALS)患者的临床特征。方法回顾性分析3例以胸髓前角先受累的ALS患者的临床资料。结果3例患者均为男性,发病年龄分别为52岁、66岁、62岁;均以呼吸困难为首发表现,有明显的肋间肌和腹直肌萎缩,而肢体肌无力及肌萎缩的出现相对较轻、较迟;肌电图显示上肢及椎旁肌的神经源性损害。结论以胸髓前角先受累的ALS患者以男性多见,起病年龄晚于ALS的平均发病年龄,呼吸困难明显,呼吸肌萎缩早于肢体肌无力及肌萎缩,肌电图检查可以确诊。     

FALS with Gly72Ser mutation in SOD1 gene: report of a family including the first autopsy case

Clinical information on familial amyotrophic lateral sclerosis (FALS) with Gly72Ser mutation in the Cu/Zn superoxide dismutase-1 (SOD1) gene has been limited and autopsy findings remain to be clarified. We describe one Japanese family with ALS carrying Gly72Ser mutation in the SOD1 gene, in which autopsy was performed on one affected member. The autopsied female patient developed muscle weakness of the left thigh at age 66 and showed transient upper motor neuron signs. She died of respiratory failure 13 months after onset without artificial respiratory support. There were no symptoms suggesting bladder or rectal dysfunction throughout the clinical course. Her brother with ALS was shown to have Gly72Ser mutation in the SOD1 gene. Histopathologically, motor neurons were markedly decreased throughout the whole spinal cord, whereas corticospinal tract involvement was very mild and was demonstrated only by CD68 immunohistochemistry. Degeneration was evident in the posterior funiculus, Clarke's nucleus, posterior cerebellar tract, and Onuf's nucleus. Neuronal hyaline inclusions were rarely observed in the neurons of the spinal cord anterior horn including Onuf's nucleus, and were immunoreactive for SOD1. To date, neuron loss in Onuf's nucleus has hardly been seen in ALS, except in the patients showing prolonged disease duration with artificial respiratory support. Involvement of Onuf's nucleus may be a characteristic pathological feature in FALS with Gly72Ser mutation in the SOD1 gene.     

Beta-amyloid 42 accumulation in the lumbar spinal cord motor neurons of amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) is characterized by a progressive loss of large motor neurons in the brain and spinal cord. Amyloid precursor protein (APP), the transmembrane precursor of beta-amyloid (A beta), accumulates in the anterior horn motor neurons of ALS patients with mild lesions. APP undergoes an alternative proteolysis mediated by caspase-3, which is activated in motor neurons in a mouse model of ALS. The ALS spinal cord motor neurons also show evidence of increased oxidative damage, which is thought to alter APP processing. We sought to determine whether A beta42, the more pathogenic A beta species, accumulates in the postmortem lumbar spinal cord of ALS patients. While there was little or no A beta42 labeling in control spinal cord tissues, elevated A beta42 immunoreactivity occurred in ALS motor neuronal perikarya and axonal swellings in the anterior horn. A few A beta42-positive neurons exhibited thioflavine S staining. No extracellular A beta42 deposits were found. A beta42 coexisted with the oxidative damage markers malondialdehyde, 8-hydroxydeoxyguanosine, heme oxygenase-1, and nitrotyrosine in abnormal neurons. The neurons with intracellular A beta42 accumulation also displayed robust cleaved caspase-3 immunoreactivity. Very little A beta40 immunoreactivity occurred in motor neurons of both control and ALS. These results suggest that aberrant accumulation of A beta42 in ALS spinal cord motor neurons is associated with oxidative stress, and may play a role in the pathogenesis of neurodegeneration in ALS.     

Enhanced Fos expression in rat lumbar spinal cord cultured with cerebrospinal fluid from patients with amyotrophic lateral sclerosis.

The etiology of amyotrophic lateral sclerosis (ALS) remains unknown although an existence of neurotoxic substances in cerebrospinal fluid (CSF) from ALS patients have been postulated. In order to investigate a possible effect of CSF from ALS patients on cellular signaling in spinal neurons, we compared Fos-like immunoreactivity (Fos-LI) in organotypic cultures of rat lumbar spinal cord after addition of CSF from ALS patients or another neurologic disease. Fos-LI was normally present predominantly in dorsal horn neurons, whereas only a few ventral horn neurons were positive for Fos-LI. The number of Fos-LI positive neurons significantly increased in dorsal horn with addition of CSF from ALS patients as well as glutamate at 100 microM. However, the increase was not observed with addition of CSF from other neurologic diseases. The increase in Fos-LI positive neurons in dorsal horn was reversed by a further supplement of MK801, an N-methyl-D-aspartate (NMDA) receptor antagonist, but not of CNQX, an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate antagonist. These results indicate that there may be substances in CSF from ALS patients that stimulate Fos expression in certain populations of spinal neurons via the NMDA receptors.     

Novel intracytoplasmic inclusions immunoreactive for phosphorylated‐TDP43 and cystatin C in anterior horn cells in a case of sporadic amyotrophic lateral sclerosis

Novel intracytoplasmic inclusions immunoreactive for phosphorylated transactivation response DNA‐binding protein 43 (p‐TDP43), cystatin C, and transferrin were found in anterior horn cells in a case of sporadic amyotrophic lateral sclerosis (ALS). The patient was a 59‐year‐old woman, who died of ALS after a clinical course of 8 years. She had been receiving mechanical support for respiration for 6 years and in a “totally locked‐in” state for 4 years prior to death. The spinal cord showed severe degeneration involving the anterior and lateral funiculi, whereas the posterior funiculus was preserved. Neurons in the anterior horn and Clarke's column were markedly lost, and many Bunina bodies and a few skein‐like inclusions were found. Some remaining anterior horn cells had round and densely eosinophilic or amphophilic intracytoplasmic inclusions. They were immunoreactive for ubiquitin, p‐TDP43, cystatin C and transferrin. On confocal laser microscopy, cystatin C was found to consistently surround p‐TDP43 within the inclusions. The inclusions ultrastructurally consisted of granule‐associated fibrils and, in the central portion, dense aggregates of fibrils were associated with masses of electron‐dense, coarsely granular or amorphous material. Although their pathogenesis remains unknown, these unique inclusions may have been formed under a specific condition whereby p‐TDP43 and cystatin C interacted with each other.     

Fragmentation of the Golgi apparatus of the anterior horn cells in patients with familial amyotrophic lateral sclerosis with SOD1 mutations and posterior column involvement

The Golgi apparatus (GA) of the anterior horn cells in the spinal cord was examined by immunohistological methods with an antibody against the MG-160 protein, a conserved intrinsic membrane sialoglycoprotein of the medial cisternae of the GA, in three patients with familial amyotrophic lateral sclerosis (FALS) with posterior column involvement. Large motor neurons in the anterior horns were markedly reduced in number and 10 of total 14 remaining large motor neurons showed fragmentation and a reduction in the number of the elements of the GA. The fragmentation of the GA was identical to that previously reported in motor neurons of the spinal cord and motor cortex from patients with sporadic ALS and in transgenic mice expressing the G93A mutation of the gene encoding the Cu/Zn superoxide dismutase months before the onset of paralysis. This is the first report of fragmented GA of the anterior horn cells in patients with FALS with posterior column involvement. The findings suggest that the GA is a common target in the neuronal degeneration in sporadic and FALS.     

Urinary collagen metabolite excretion in amyotrophic lateral sclerosis

Collagen abnormalities of the spinal cord and the skin have been reported in patients with amyotrophic lateral sclerosis (ALS). The urinary concentrations of the hydroxylysine glycosides, i.e., glucosylgalactosyl hydroxylysine (glu-gal Hyl) and galactosyl hydroxylysine (gal Hyl), indicate the tissue origin of the collagen metabolites and the rate of the degradation of collagen. We measured the urinary levels of glu-gal Hyl and gal Hyl in 12 ALS patients, 10 diseased control subjects with other neurologic or muscular diseases (Control Group A), and 10 healthy control subjects (Control Group B). The urinary level of glu-gal Hyl in ALS patients was significantly lower than in the two control groups. In addition, a significant negative relationship between glu-gal Hyl urinary level and duration of illness was found in ALS patients. There was no marked difference in the urinary level of gal Hyl between ALS patients and the control groups. Our data suggest that the decreased urinary level of glu-gal Hyl may be useful in assessing the alteration in collagen metabolism in ALS and may have a relationship with the progression of ALS.     

Determination of manganese concentrations in the spinal cords from amyotrophic lateral sclerosis patients by inductively coupled plasma emission spectroscopy

Continuing analytical studies on environmental factors in the foci of amyotrophic lateral sclerosis (ALS) in the Kii Peninsula of Japan and Guam, and metal analysis using neutron activation analysis in central nervous system (CNS) tissues from ALS cases indicate that chronic exposure to metals such as aluminum and manganese, together with a deficiency of minerals such as calcium and magnesium, may play a causative role in the neurodegeneration seen in ALS. An accurate and simple method for detecting minerals and trace metals in small, fresh samples of CNS tissue is necessary in order to follow the pathogenetic behavior of these elements. In this paper, we describe a method for measuring manganese (Mn) content in CNS tissue samples taken from mouse brain using inductively coupled plasma (ICP) spectroscopy and present results of the determination of Mn contents in spinal cord samples from ALS cases using the same method. ICP emission spectroscopy is considered to have great advantage for the simultaneous determination of elements in small, fresh CNS samples because of its simplicity and convenience, and the elimination of the necessity to use potentially dangerous acids. Results showed that the mean concentration of Mn in a mouse brain (0.56 microgram/g) and in human spinal cords (0.39 microgram/g wet weight in the anterior horn, 0.37 in the lateral fasciculus, 0.39 in the posterior horn and 0.28 in the posterior fasciculus) were compatible with results previously reported using other methods. In ALS spinal cords, the mean content of Mn was similar to that of controls, but the distribution differed. In the ALS cases, Mn contents were higher in the anterior horn and lateral fasciculus than in the posterior horn.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced Fos expression in rat lumbar spinal cord cultured with cerebrospinal fluid from patients with amyotrophic lateral sclerosis

Abstract

School, Okayama, Japan The etiology of amyotrophic lateral sclerosis (ALS) remains unknown although an existence of neurotoxic substances in cerebrospinal fluid (CSF) from ALS patients have been postulated. In order to investigate a possible effect of CSF from ALS patients on cellular signaling in spinal neurons, we compared Fos-like immunoreactivity (Fos-LI) in organotypic cultures of rat lumbar spinal cord after addition of CSF from ALS patients or another neurologic disease. Fos-LI was normally present predominantly in dorsal horn neurons, whereas only a few ventral horn neurons were positive for Fos-LI. The number of Fos-LI positive neurons significantly increased in dorsal horn with addition of CSF from ALS patients as well as glutamate at 100 μM. However, the increase was not observed with addition of CSF from other neurologic diseases. The increase in Fos-LI positive neurons in dorsal horn was reversed by a further supplement of MK801, an N-methyl-D-aspartate (NMDA) receptor antagonist, but not of CNQX, an a-amino-3-hydroxy-5-methyl-4- isoxazole propionic acid (AMPA)/kainate antagonist. These results indicate that there may be substances in CSF from ALS patients that stimulate Fos expression in certain populations of spinal neurons via the NMDA receptors. [Neurol Res 1999; 21: 309-312]     

Amyotrophic lateral sclerosis. Lack of central chromatolytic response of motor neurocytons corresponding to active axonal degeneration

Ventral spinal roots and anterior horn cells in the lateral nuclear group of the fourth lumbar segment from 21 patients with amyotrophic lateral sclerosis (ALS) and 23 control patients were morphometrically analyzed. The number of large myelinated fibers was remarkably decreased, while small myelinated fibers were well preserved. The population of large myelinated fibers significantly correlated with the population of anterior horn cells. Numerous axonal degenerations were observed in the ventral spinal roots of patients with ALS, even in patients with severe loss of neurons and axons. In spite of this high frequency of active axonal degeneration, the incidence of central chromatolysis of anterior horn cells remained at the control level.     

Alterations of the blood‐spinal cord barrier in sporadic amyotrophic lateral sclerosis

The blood‐spinal cord barrier (BSCB) of the spinal cord capillary consists of non‐fenestrated endothelial cells with tight junctions, basal laminae, pericytes and astrocyte feet processes, referred to as a “neurovascular unit.” The primary function of the BSCB is the maintenance and control of homeostasis of the spinal cord parenchyma by the selective transport of molecules and cells from the systemic compartment. Dysfunction of the BSCB shows important function in the etiology or progression of several pathological conditions of the spinal cord, including amyotrophic lateral sclerosis (ALS). However, the role of BSCB in the pathogenesis of ALS is still unclear. Here the changes of BSCB in sporadic ALS patients were studied by electron microscopy to determine whether the BSCB is disrupted and involved in the pathogenesis of motor neuron degeneration. A total of 358 and 366 cross‐sectioned capillaries were quantitatively examined in controls and ALS patients, respectively. The frequency of degenerated endothelia and pericytes, vacuolar changes of the cytoplasm in the endothelia and pericytes, and the replication of basement membranes was significantly higher in ALS patients than in the controls (P = 0.0175). The areas of the capillaries with diameters of ≤ 5 µm in the ALS patients were significantly smaller than those in the controls (P = 0.0124). The frequency of collagen fiber content of more than a moderate degree around the perivascular space was significantly higher in the ALS patients compared to the controls (P = 0.048), although there was no significant difference in the mild degree of accumulation of collagen fibers. Thus, the BSCB may be disrupted in sporadic ALS patients due to increased permeability and reduced microcirculation, leading to motor neuron degeneration and to the progression of the disease.     

Increased manganese level in spinal cords of amyotrophic lateral sclerosis determined by radiochemical neutron activation analysis

The manganese distribution in the cross-section of the cervical, thoracic and lumbar portion of the spinal cords from 7 autopsied cases with amyotrophic lateral sclerosis (ALS) and 6 control subjects were determined by radiochemical neutron activation analysis. It was possible to determine 1 ng levels of manganese content accurately in the small tissues about 1 mg of dried weight using wet ashing and chemical separation after neutron activation. The dried weight of spinal cord was about $\text{1}\text{3}$ of the wet weight. Manganese concentration in the wet tissue was calculated from the ratio of dried to wet weight.In the anterior horn of the cervical cords of ALS, manganese concentration was the highest; 1.75 ± 0.39 ng/mg of dried weight and 0.59 ± 0.09 ng/mg of wet weight, respectively. These were significantly higher (P < 0.01) compared to those in controls; 1.02 ± 0.12 ng/mg of dried weight and 0.35 ± 0.04 ng/mg of wet weight, respectively. The elevation of manganese level in the spinal cords of ALS was more prominent in the anterior horn and lateral column than in the posterior column both in dried and wet tissues.Since manganese inhibits neuronal transmission, it is likely that neurological degenerative changes occur as a result of local disturbances of manganese metabolism in the spinal cord of ALS.