Persistent cleavage and nuclear translocation of apoptosis-inducing factor in motor neurons in the spinal cord of sporadic amyotrophic lateral sclerosis patients

Mounting evidence suggests that glutamate excitotoxicity induces both enzymatic cleavage and nuclear translocation of apoptosis-inducing factor (AIF), which is involved in apoptosis-like programed cell death characterized by nuclear condensation without appearance of apoptotic bodies. Given the lack of apoptotic bodies in motor neurons in the spinal cord of patients with amyotrophic lateral sclerosis (ALS), the aim of the present study was to determine the role for AIF in this disease. We investigated the expression of AIF in spinal cords obtained at autopsy from ten sporadic ALS patients and ten age-matched, control subjects, using morphological and quantitative techniques. Immunohistochemical analysis showed that AIF immunoreactivity was localized in the nucleus as well as the cytoplasm of a subset of affected motor neurons and reactive astrocytes in the ALS cases, while it was restricted to the cytoplasm of these cells in the control cases. Immunoblot analysis disclosed immunoreactivity for cleaved AIF in both cytoplasmic and nuclear protein extracts at a 57-kDa mobility. Densitometric analysis revealed significant increases in the cytoplasmic cleaved AIF/cytoplasmic β-actin ratio and the nuclear cleaved AIF/nuclear histone H1 ratio in the ALS group compared with the control group. There was no significant link between the cytoplasmic and nuclear cleaved AIF levels in the ALS spinal cords and the clinical features such as phenotypes, age at death, and disease duration. Our results provide evidence for persistent cleavage and nuclear translocation of AIF in ALS spinal cord, suggesting implications for the AIF-mediated motor neuron death in this disease.     

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.     

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.     

Altered expression of bcl-2 and bax mRNA in amyotrophic lateral sclerosis spinal cord motor neurons

One of the primary neurodegenerative events occurring in amyotrophic lateral sclerosis (ALS) is the selective loss of spinal cord α motor neurons. To study the potential role of apoptosis in the degeneration of these motor neurons, in situ hybridization was used to measure the expression of two apoptotic cell death genes, bcl-2 and bax, in control and ALS lumbar spinal cord sections. The strongest hybridization signal for bcl-2 mRNA in neurological and nonneurological control spinal cords was found primarily in lamina IX α motor neurons, while a weaker hybridization signal was found in neurons of Clarke's nucleus and the proper sensory nucleus of the dorsal horn. Surviving lamina IX motor neurons in ALS spinal cord sections also expressed bcl-2 mRNA, but at levels that were significantly and selectively decreased (4.7-fold) compared with control. bax mRNA hybridization signal was detected in several cells throughout the gray matter in control and ALS lumbar spinal cord, but was significantly and selectively increased (2.8-fold) in ALS motor neurons. Given the proposed interactive roles of these genes in apoptosis, the present findings favor a scenario in which this mode of cell death would contribute to spinal cord motor neuron degeneration in ALS.     

Mitochondrial alterations in the spinal cord of patients with sporadic amyotrophic lateral sclerosis

Little information is available about morphologic changes of mitochondria in sporadic amyotrophic lateral sclerosis (ALS). We examined the anterior horns of the lumbar spinal cord in 14 patients with sporadic ALS and 15 age-matched controls by electron microscopy to illuminate the subject. In the controls, one patient showed occasional swollen mitochondria with markedly increased cristae and marked accumulation of mitochondria in the somata of anterior horn neurons. Another patient had periodic, stubby protrusions on the outer membrane. Among the patients with ALS, 7 showed filamentous structures in the inner compartment of the mitochondria mainly of the somata and only occasionally of the axons. The structures were composed of a stack of multilayered cristae consisting of linear structures on a longitudinal section. Other abnormal structures were periodic transverse processes like rungs of a ladder predominantly in somata and only occasionally in the axons, marked accumulation of mitochondria in the somata, dendrites or proximal axons (axon hillock and initial segment), stubby protrusions on the outer membrane, and swollen mitochondria with markedly increased cristae in the somata. The findings in this study may reflect the metabolic disturbance of mitochondria, probably associated with the pathomechanism of degenerative processes of anterior horn neurons in sporadic ALS.     

The Golgi apparatus is fragmented in spinal cord motor neurons of amyotrophic lateral sclerosis with basophilic inclusions

The mechanisms of neuronal death in amyotrophic lateral sclerosis (ALS) are not known. A pathological aggregation of cytoplasmic constituents in the form of variety of inclusions may play a role in the pathogenesis of neuronal death. Cytoplasmic basophilic inclusions (BIs) in motor neurons are commonly found in sporadic juvenile ALS. The functional significance of these inclusions is not known, i.e., whether they represent a protective reaction for the isolation of abnormal products from the cytoplasm, or a sign of irreversible neuronal damage. To gain insights on the significance of BIs we asked whether neurons with BIs had an intact or fragmented Golgi apparatus (GA), a sign of neuronal degeneration reported not only in sporadic and familial ALS with mutations of the Cu/Zn superoxide dismutase gene (SOD1), but also in transgenic mice expressing the G93A mutation of SOD1. In these mice fragmentation of the GA of spinal cord motor neurons was found months before the onset of paralysis. We report here that all neurons bearing the inclusions showed fragmentation and reduced number of GA. These results suggest that common pathogenetic mechanisms are involved in the production of BIs and in the fragmentation of the GA.     

Comparative study of spinal cord ubiquitin expression in post-poliomyelitis and sporadic amyotrophic lateral sclerosis

This study addresses the suggested possible pathogenetic relationship between the late-onset muscular atrophy in patients with the prior diagnosis of poliomyelitis and amyotrophic lateral sclerosis (ALS). For this purpose we applied immunohistochemical techniques to determine the presence of pathological structures that were stained for ubiquitin (a protein involved in degenerative processes) in the spinal cords of patients with a history of poliomyelitis and compared the results with those of ALS, a condition in which cytoplasmic ubiquitin-positive inclusions are invariably found in the anterior horn cells. Our results indicate that post-poliomyelitis patients have no ubiquitinreactive inclusion bodies in these cells; however, some immunopositive globular and cord-shaped structures are seen in less-affected areas. Similar structures were also found in the spinal cords from patients with ALS and from normal individuals. Our findings would suggest that the pathogenesis of late muscular atrophy in post-poliomyelitis patients is dissimilar to that of ALS.Supported in part by a grant from the Amyotrophic Lateral Sclerosis Association     

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.     

IgG reactivity in the spinal cord and motor cortex in amyotrophic lateral sclerosis

The spinal cord and motor cortex of patients with amyotrophic lateral sclerosis (ALS) were examined with immunohistochemical methods for the presence of IgG. In 13 of 15 spinal cords, a population of motoneurons stained positively for IgG in a granular pattern, characteristic of binding to the rough endoplasmic reticulum. In 6 of 11 motor cortices, a proportion of pyramidal cells also stained positively for IgG. No such reactivity was noted in motoneurons of control human tissues, although positive IgG staining was present in astrocytes of ALS and control specimens. Reactive microglia and/or macrophages were detected in the territory of degenerating pyramidal tracts and ventral horns. The surface of most of these cells stained positively for IgG, and 50% stained positively for HLA-DR. The accumulation of IgG in motoneurons and the presence of immunologically active macrophages provide additional evidence for the participation of immunologic factors in the pathogenesis of ALS.     

Evidence of endoplasmic reticular stress in the spinal motor neurons exposed to CSF from sporadic amyotrophic lateral sclerosis patients

We have earlier reported that intrathecal injection of cerebrospinal fluid (CSF) from sporadic Amyotrophic Lateral Sclerosis patients (ALS-CSF) into neonatal rats and supplementation of rat spinal cord cultures with ALS-CSF induces motor neuron degeneration via aberrant neurofilament phosphorylation and Golgi apparatus fragmentation. Intracellular aggregates immunoreactive to ubiquitin, phosphorylated neurofilaments and choline acetyl transferase (ChAT) were prominently seen in NSC-34 cells exposed to ALS-CSF. Protein aggregation could cause stress on endoplasmic reticulum (ER) and may precede Golgi fragmentation. Here we assessed the effect of ALS-CSF on the expression of GRP-78 and caspase-12 proteins, the markers of ER stress responses, in NSC-34 cells and rat spinal cords by immunochemistry and immunoblotting. Both in vitro and in vivo, increased expression of these proteins accompanied elevated active caspase-12 levels. Apoptotic nuclei and nuclear translocation of caspase-12 were noted in some cells. In vitro, the occurrence of ER stress was supported by electron microscopic observations of numerous free polyribosomes and fragmented ER cisternae. Aggregated mSOD1 protein causes ER stress in familial ALS. ER stress is also reported in the autopsy samples of sporadic ALS. Thus our observation of ER stress may be linked to the protein aggregation, viz. phosphorylated neurofilaments and ChAT, reported earlier.     

Characterizations of heterotopic neurons in the spinal cord of amyotrophic lateral sclerosis patients

This report concerns a comparative immunocytochemical, ultrastructural and morphometric investigation on heterotopic neurons in the white matter of the spinal cords of 19 patients with amyotrophic lateral sclerosis (ALS) and 18 age-matched neurologically normal individuals. The study revealed that the heterotopic neurons were scattered in the white matter, often adjacent to gray matter, that they immunoreacted with the antibody to synaptophysin, and that there were synaptic apparatuses on the surface of their somata and their neuronal processes. Bunina bodies and ubiquitin-positive inclusions such as Lewy body-like inclusions and skein-like inclusions, characteristic of anterior horn neurons of ALS, were present in the cytoplasm of the patients’ heterotopic neurons in the anterior or lateral column of the white matter. These findings suggest that heterotopic neurons in the anterior or lateral column have the characteristics of alpha motor neurons. The average number of heterotopic neurons observed in ALS patients was generally less than in normal subjects. This reduction was correlated with the severity of neuronal loss. The heterotopic neurons in ALS were less susceptible to the degenerative process as compared with spinal cord anterior horn cells. We assume that in this disease the heterotopic neurons may be degenerated and their number diminished after or concomitantly with the depletion of anterior horn neurons. Received: 18 August 1997 / Revised, accepted: 20 October 1997     

Characterization of neuronal intermediate filament protein expression in cervical spinal motor neurons in sporadic amyotrophic lateral sclerosis (ALS)

Because transgenic mice expressing an altered stoichiometry of neurofilament proteins develop a motor neuron degeneration associated with neurofilamentous aggregate formation similar to that found in amyotrophic lateral sclerosis (ALS), we studied the expression of intermediate filament proteins in sporadic ALS. Archival cervical spinal cord paraffin-embedded sections from 11 disease and 11 control cases were studied by either in situ hybridization using 35S-labeled riboprobes or immunohistochemically using specific antibodies for the individual neurofilament subunit proteins, alpha-internexin, nestin, peripherin, vimentin, beta-actin, or Talpha1-tubulin. Median NFL, alpha-internexin, and peripherin steady-state mRNA levels were significantly reduced in the lateral motor neuron cell column (p < 0.05) of ALS cases, while neither NFM nor NFH mRNA levels were altered. ALS cases demonstrated an elevation of beta-actin mRNA levels (p < 0.01) with no increase in Talpha1-tubulin mRNA levels. No motor neuronal expression of nestin or vimentin was observed. Ubiquitin-immunoreactive perikaryal aggregates were immunoreactive for NFH or beta-actin, but not for peripherin, alpha-internexin, vimentin, or nestin. In contrast, neuroaxonal spheroids were strongly immunoreactive for NFH and peripherin, but not for beta-actin, alpha-internexin, vimentin, or nestin. These findings suggest that the stoichiometry of cytoskeletal protein expression in ALS spinal motor neurons is significantly altered in a pattern conducive to the formation of neurofilamentous aggregates.     

Morphometric and topographical studies of small neurons in sporadic amyotrophic lateral sclerosis spinal gray matter

Little attention has been paid to the degeneration of small neurons in ALS spinal gray matter. The purpose of the present paper was to undertake morphometric and quantitative analysis of the spinal gray matter of 15 ALS patients and compare findings to those of five controls. A significant reduction of small neurons in the anteromedial and intermediate parts of the gray matter were detected in ALS spinal cords with diffuse myelin pallor in the ventral aspects of the anterolateral columns outside the corticospinal tracts, and the number of small neurons in these areas was decreased significantly depending on the intensity of the myelin pallor. There were no significant alterations in the number of small neurons in the corresponding areas of ALS spinal cords without diffuse myelin pallor or in those of controls. In the posterior parts of the gray matter, there were no significant differences in the number of small neurons among ALS patients and controls. These findings strongly suggest that diffuse myelin pallor in the ventral aspects of anterolateral columns in ALS spinal cords is derived from the degeneration of small neurons in the anteromedial and intermediate parts of the gray matter.     

Decrease of protein kinase C in the spinal motor neurons of amyotrophic lateral sclerosis

The expression of protein kinase C (PKC), a calcium- and phospholipid-dependent signaling molecule, was studied immunohistochemically in the spinal motor neurons of cases of sporadic amyotrophic lateral sclerosis (SALS). In the normal spinal cord, intense PKC immunoreactivity was found in subsets of large motor neurons. PKC immunoreactivity was markedly decreased in the spinal motor neurons of SALS. The result suggests that down-regulation of PKC is associated with the degeneration of spinal motor neurons in SALS. Received: 7 April 1997 /Revised, accepted: 18 December 1997     

Soluble iron accumulation induces microglial glutamate release in the spinal cord of sporadic amyotrophic lateral sclerosis

Previous studies on sporadic amyotrophic lateral sclerosis (SALS) demonstrated iron accumulation in the spinal cord and increased glutamate concentration in the cerebrospinal fluid. To clarify the relationship between the two phenomena, we first performed quantitative and morphological analyses of substances related to iron and glutamate metabolism using spinal cords obtained at autopsy from 12 SALS patients and 12 age-matched control subjects. Soluble iron content determined by the Ferrozine method as well as ferritin (Ft) and glutaminase C (GLS-C) expression levels on Western blots were significantly higher in the SALS group than in the control group, while ferroportin (FPN) levels on Western blots were significantly reduced in the SALS group as compared to the control group. There was no significant difference in aconitase 1 (ACO1) and tumor necrosis factor-alpha (TNFα)-converting enzyme (TACE) levels on Western blots between the two groups. Immunohistochemically, Ft, ACO1, TACE, TNFα, and GLS-C were proven to be selectively expressed in microglia. Immunoreactivities for FPN and hepcidin were localized in neuronal and glial cells. Based on these observations, it is predicted that soluble iron may stimulate microglial glutamate release. To address this issue, cell culture experiments were carried out on a microglial cell line (BV-2). Treatment of BV-2 cells with ferric ammonium citrate (FAC) brought about significant increases in intracellular soluble iron and Ft expression levels and conditioned medium glutamate and TNFα concentrations. Glutamate concentration was also significantly increased in conditioned media of TNFα-treated BV-2 cells. While the FAC-driven increases in glutamate and TNFα release were completely canceled by pretreatment with ACO1 and TACE inhibitors, respectively, the TNFα-driven increase in glutamate release was completely canceled by GLS-C inhibitor pretreatment. Moreover, treatment of BV-2 cells with hepcidin resulted in a significant reduction in FPN expression levels on Western blots of the intracellular total protein extracts. The present results provide in vivo and in vitro evidence that microglial glutamate release in SALS spinal cords is enhanced by intracellular soluble iron accumulation-induced activation of ACO1 and TACE and by increased extracellular TNFα-stimulated GLS-C upregulation, and suggest a positive feedback mechanism to maintain increased intracellular soluble iron levels, involving TNFα, hepcidin, and FPN.     

家族性肌萎缩侧索硬化动物模型脊髓侧角神经元特征

目的探讨脊髓侧角神经元在SOD1G93A转基因鼠发病过程中的变化特征。方法利用甲苯胺蓝染色及免疫组化的方法观察SOD1G93A转基因小鼠不同病变时期上、中、下胸髓侧角神经元的数量变化特征。结果甲苯胺蓝染色法定量研究发现不同时期SOD1G93A转基因小鼠上胸髓、下胸髓水平,症状前期和发病期神经元计数较终末期均有显著性差别(P<0.05);与对照组相比,终末期胸髓各段侧角神经元计数明显减少且有统计学意义;免疫组化发现SOD1G93A转基因小鼠上胸髓、中胸髓水平不同时期相比,症状前期和发病期乙酰胆碱酯酶(AchE)阳性神经元计数较终末期均有显著性差别(P<0.05);与对照组相比,发病期上胸髓及终末期中、下胸髓水平侧角AchE阳性神经元数量减少具有统计学意义;结论在SOD1G93A转基因鼠发病过程中,存在脊髓侧角神经元丢失,尤其是在疾病的终末期,上、中、下胸髓节段间无明显差异。     

Expression of ciliary neurotrophic factor is maintained in spinal motor neurons of amyotrophic lateral sclerosis

Ciliary neurotrophic factor (CNTF) was originally identified as a potent survival factor for a variety of neuronal cell types in vitro and in vivo and in particular in spinal motor neurons of embryonic chick and rat. Using a monoclonal antibody against CNTF (clone 4–68) we analysed the expression of CNTF in paraffin sections of seven human brains and spinal cords immunocytochemically using the ABC method and compared these results with sections of the spinal cords of patients suffering from amyotrophic lateral sclerosis (ALS). In normal human tissue of the central nervous system CNTF immunoreactivity was found in most of the motor neurons of the motor cortex and ventral horn, neurons of the nucleus oculomotorius, intermediolateralis, thoracicus, ependymal cells as well as in smooth muscle cells and endothelial cells of small arteries. A reduced number of astrocytes showed a positive immunocytochemical reaction. In peripheral nerves and nerve roots of the spinal cord we also found a positive staining of Schwann cells and some axons. These immunoreactions could be confirmed by Western blot analyses. Next we analysed postmortem paraffin sections of the spinal cord of seven patients suffering from ALS (age range 30–76 years, median age 46 years, female/male = 4:3). We found CNTF immunoreactivity in most of the motor neurons of the ventral horn in 5 cases. In two cases the number of positively stained motor neurons was less. From these results we conclude that CNTF is expressed in a high number of upper and lower motor neurons in the human CNS and that its expression is maintained in ALS patients.     

Clarke's column in sporadic amyotrophic lateral sclerosis

Summary Histological, ultrastructural and morphometrical observations on Clarke's column were carried out in 18 patients with sporadic amyotrophic lateral sclerosis (ALS) and 15 age-matched control subjects. Of the 18 ALS patients 6 had been on a respirator before death. Bunina bodies were found in the neuronal cytoplasm in 7 of the 12 non-respirator-supported ALS patients and in 3 of the 6 respirator-supported patients. The number of spheroids was significantly higher in the non-respirator-supported patients (P<0.01) than in the control subjects; however, the number in the respirator-supported patients was about equal to that in the controls. The number of neurons in Clarke's column in the non-respirator-supported ALS patients was not reduced, but in the respirator-supported patients they tended to disappear with time after respiratory support. These findings suggest that Clarke's column neurons are also involved primarily in the disease process in sporadic ALS. However, they may begin to disappear only after the patients require respiratory support.Supported in part by a research grant for CNS degenerative diseases from the Ministry of Health and Welfare, Japan