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.     

Autophagy in spinal cord motor neurons in sporadic amyotrophic lateral sclerosis

To assess the potential role of autophagy in amyotrophic lateral sclerosis (ALS), lumbar spinal cords in a total of 19 sporadic ALS cases and 27 age-matched controls were investigated. Immunohistochemical analysis using antibodies to the markers of autophagy microtubule-associated protein light chain 3 (LC3) and p62 was performed on samples from 12 ALS and 15 controls. Electron microscopy was performed on samples from 16 ALS and 15 controls, including overlapping cases. In the ALS cases, the somata of normal-appearing and degenerated motor neurons and round bodies were occasionally immunostained for LC3; round bodies and skein-like inclusions were immunostained for p62. By electron microscopy, all 16 ALS patients showed features of autophagy in the cytoplasm of normal-appearing motor neurons and, more frequently, in degenerated motor neurons. Autophagosomes surrounded by a double-membrane and autolysosomes isolated by a single membrane contained sequestered cytoplasmic organelles, such as mitochondria and ribosome-like structures. These autophagy features were also found in close association with the characteristic inclusions of ALS(i.e. round bodies, skein-like inclusions, and Bunina bodies); honeycomb-like structures also occasionally showed autophagy-associated features. Normal-appearing anterior horn neurons in control patients showed no autophagy features. Thus, autophagy seems to be activated and upregulated in the cytoplasm of motor neurons and may be involved in the mechanisms of neurodegeneration of motor neurons in sporadic ALS.     

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     

Deficient DNA repair in amyotrophic lateral sclerosis cells

We studied survival and DNA repair capacity in cultured sporadic ALS and control skin fibroblasts after treatment with DNA damaging agents producing different types of lesions. Mean survival in ALS and control fibroblasts was similar after exposure to ultraviolet (UV) light, x-rays and mitomycin C (MMC). Both mean survival and mean unscheduled (repair) DNA synthesis (UDS) were significantly reduced in ALS fibroblasts following treatment with the alkylating agent methyl methane sulfonate (MMS). These data suggest that ALS cells are relatively deficient in the repair of alkylation damage, possibly of apurinic/apyrimidinic sites, and that they are not unduly sensitive to DNA damage produced by UV light, x-rays and MMC. Normal survival and UDS seen in some patients' cells after MMS treatment indicate a spectrum of repair efficiency, and suggest heterogeneity of the biochemical defect 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.     

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.     

Golgi apparatus of the motor neurons in patients with amyotrophic lateral sclerosis and in mice models of amyotrophic lateral sclerosis

We examined the Golgi apparatus (GA) of motor neurons of patients with ALS and in mice models of ALS by immunohistological method using antiserum against MG160 and against components of the trans‐Golgi network (TGN46). The GA of half of the remaining spinal cord motor neurons of patients with sporadic ALS showed fragmentation, where the GA were dispersed or fragmented into numerous small, isolated elements. The GA of Betz cells in sporadic ALS were fragmented similar to that of anterior horn cells, and the GA of spinal cord motor neurons of those with familial ALS and of those with ALS with basophilic inclusions were fragmented or diminished. The GA in the majority of the motor neurons contained Bunina bodies, basophilic inclusions and superoxide dismutase 1 (SOD1)‐positive aggregates were fragmented. The motor neurons in transgenic mice expressing G93A mutation of the SOD1 gene showed the fragmentation of the GA months before the onset of paralysis. These findings suggest that the fragmentation of GA may be related to the neuronal degeneration in patients with 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.     

Mending the broken in amyotrophic lateral sclerosis: DNA damage and repair in motor neuron degeneration

正Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that causes paralysis and respiratory failure (Petrov et al.,2017).The driving mechanisms are unknown,and there are no effective treatments(Petrov et al.,2017).Aging and a few gene mutations,a common one being missense mutations in superoxide dismutase-1 (SOD1),are risk factors for ALS (Figure 1).The recent Food and Drug Administration approval of edaravone for the treatment of ALS putatively supports a role for oxidative and nitrative stresses in the disease processes(Figure 1A).DNA damage,abnormalities in     

Accumulation of chondroitin sulfate proteoglycans in the microenvironment of spinal motor neurons in amyotrophic lateral sclerosis transgenic rats

Chondroitin sulfate proteoglycans (CSPGs) are the major components of extracellular matrix in the central nervous system. In the spinal cord under various types of injury, reactive gliosis emerges in the lesion accompanied by CSPG up-regulation. Several types of CSPG core proteins and their side chains have been shown to inhibit axonal regeneration in vitro and in vivo. In the present study, we examined spatiotemporal expression of CSPGs in the spinal cord of transgenic (Tg) rats with His46Arg mutation in the Cu/Zn superoxide dismutase gene, a model of amyotrophic lateral sclerosis (ALS). Immunofluorescence disclosed a significant up-regulation of neurocan, versican, and phosphacan in the ventral spinal cord of Tg rats compared with age-matched controls. Notably, Tg rats showed progressive and prominent accumulation of neurocan even at the presymptomatic stage. Immunoblotting confirmed the distinct increase in the levels of both the full-length neurocan and their fragment isoforms. On the other hand, the up-regulation of versican and phosphacan peaked at the early symptomatic stage, followed by diminishment at the late symptomatic stage. In addition, double immunofluorescence revealed a colocalization between reactive astrocytes and immunoreactivities for neurocan and phosphacan, especially around residual large ventral horn neurons. Thus, reactive astrocytes are suggested to be participants in the CSPG accumulation. Although the possible neuroprotective involvement of CSPG remains to be investigated, the present results suggest that both the reactive astrocytes and the differential accumulation of CSPGs may create a nonpermissive microenvironment for neural regeneration in neurodegenerative diseases such as 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.     

Synaptophysin expression in motor neurons of transgenic mice with amyotrophic lateral sclerosis

BACKGROUND: Affected signal convection of synaptophysin on motor neurons may cause injury of motor neurons and then induce neurodegeneration and cell death in the end. OBJECTIVE: To investigate the number and density of synaptophysin on motor neurons in the anterior horn of lumbar spinal cord and sensorimotor cortex of the transgenic mouse model of amyotrophic lateral sclerosis (ALS). DESIGN: Randomized controlled animal study. SETTING: Brain Injury and Repair Group, HFI Institute of Melbourne University. MATERIALS: Transgenic mice expressing a mutated human superoxide dismutase 1 (SOD-1) were taken as ALS group (n =36), while those derived from the B6SJL-TgN gene line were taken as control group (n =36), according to the difference of gender and three postnatal time points (postnatal 60, 90 and 120 days), twelve mice of either gender were allocated in each subgroup. METHODS: The experiment was carried out in Brain Injury and Repair Group, HFI Institute of Melbourne University from November 2003 to June 2004. ① Fluorogold labeling was used for the motor neurons in the lumbar and sensorimotor cortex. ② Immunofluorescence was applied for the labeling of synaptophysin; positive control sections were represented by adding the synaptophysin antibody and the staining, showing a positive result. For negative controls, the synaptophysin antibody was omitted. ③ Stereological counting system was adopted in the statistical analysis. MAIN OUTCOME MEASURES: ① Fluorogold labeling of motor neurons; ② number of synaptophysin on the motor neurons. RESULTS: ① Fluorogold labeling of motor neurons: The motor neurons in the lumbar and sensorimotor cortex were clearly labeled by fluorogold under the detection of fluorescent microscope. ② The number of synaptophysin on the motor neurons: The number statistically decreased at the mid stage (postnatal 90 days) and late stage (postnatal 120 days) [motor neuron somas at lumbar spinal cord: (0.75±0.06), (0.59±0.09)/μm; motor neuron dendrite at lumbar spinal cord: (0.71±0.06), (0.55±0.03)/μm; motor neuron somas at sensorimotor cortex: (0.79±0.03), (0.63±0.08)/μm; motor neuron dendrite at sensorimotor cortex: (0.76±0.07), (0.61±0.08)/μm, P < 0.01]. The reduction of synaptophysin was parallel between the gender differences. CONCLUSION: Loss of synaptophysin on motor neurons is directly related to the progression of ALS.     

Immunocytochemical and ultrastructural studies of upper motor neurons in amyotrophic lateral sclerosis

Summary The pathological alterations in upper motor neurons were investigated in 27 cases of adult-onset sporadic amyotrophic lateral sclerosis (ALS). No signficant cytoskeletal alterations were found in the Betz cells of any of the cases except one, although cytoskeletal pathology was consistently present in lower motor neurons. The one case had severe circumscribed atrophy of the precentral gyrus and, microscopically, had argentophilic intracytoplasmic inclusions in Betz cells and other pyramidal neurons in the primary motor area as eell as in the lower motor neurons. Immunocytochemically these inclusions contained the epitope of phosphorylated neurofilament and ubiquitin and ultrastructurally consisted of granule-associated filaments with neurofilaments. This is the first demonstration of alterations of cytoskeleton and ubiquitination in the giant cells of Betz, an established subset of upper motor neurons in ALS. Thus, although uncommon, cytoskeletal changes can be found in upper motor neurons in some ALS cases.Supported in part by USPHS grants NS24453, HD03110 and ES01704     

Base composition of RNA obtained from motor neurons in amyotrophic lateral sclerosis

The base composition of RNA obtained from the large motor neurons of the cervical and lumbar swelling was examined in amyotrophic lateral sclerosis (ALS) patients and a similar number of age-matched controls. Spinal cords were obtained at autopsy and immediately fixed in buffered formalin. The single cell technique of Edstr?m was employed to extract, hydrolyze, and electrophoresis the RNA. The base composition obtained for the controls was 17.47% adenine, 28.88% guanine, 28.50% cytidylic acid, and 25.14% uridylic acid. The cervical intumescence revealed higher levels of uridylic acid than the lumbar, 27.23% in the cervical and 23.31% in the lumbar intumescence. The motor neuron cell bodies isolated from patients having had ALS revealed a lower percentage of adenine in both the cervical (13%) and lumbar (10%) intumescences. When the data for these areas were combined, the percentage of adenine was 15.52, compared to 17.47% in the controls (p less than 0.01). The A/U ratio was also significantly reduced in the ALS group. The composition of the remaining bases in ALS appeared to be similar to the controls. The significant change in adenine, coupled with the quantitative reduction in total neuronal RNA, suggests that a disorder of nucleic acid metabolism may relate to the pathogenesis of ALS.     

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     

Immunocytochemical and ultrastructural studies of lower motor neurons in amyotrophic lateral sclerosis

Neuronal inclusions in lower motor neurons in 23 cases of adult-onset sporadic amyotrophic lateral sclerosis were studied immunocytochemically and ultrastructurally. Monoclonal and polyclonal antiubiquitin antibodies recognized four structures in the neuronal perikarya: (1) all Lewy body-like inclusions in 6 cases with a relatively short clinical course, (2) a small percentage of Bunina bodies in 4 cases with abundant Bunina bodies, (3) ill-defined structures closely associated with Bunina bodies (Bunina body-related structures) in 15 cases, and (4) a focally aggregated meshwork of fine filamentous structures not associated with Bunina bodies in all cases. These four structures were not recognized by the antibodies raised against cytoskeletal proteins (neurofilament, tubulin, microtubule-associated protein 2, and phosphorylated tau). Electron microscopy revealed Lewy body-like inclusions to be accumulations of randomly oriented filaments, approximately 15 nm in diameter, covered by fine granules. Bundles of coated filaments 12 nm in diameter that sometimes formed Bunina body-like structures were also observed in the perikarya. Immunoelectron microscopy showed the reaction product with antiubiquitin to be on the filaments, 15 nm in diameter, of Lewy body-like inclusions. Our study revealed the existence of two types of filaments in lower motor neurons of patients with amyotrophic lateral sclerosis: (1) ubiquitin-positive, granule-associated filaments, approximately 15 nm in diameter, that form Lewy body-like inclusions; and (2) 12 nm coated filaments that may be a candidate for another ubiquitin-positive structure and possibly a precursor of Bunina bodies. These two types of filaments may represent early pathological changes of lower motor neurons in amyotrophic lateral sclerosis.     

Effects of amyotrophic lateral sclerosis serum on cultured chick spinal neurons

We used a quantitative immunoassay to examine the effects of human serum and immunoglobulins on neurofilament protein expression in cultures of chick spinal neurons. Compared with cultures grown in the presence of serum from healthy controls or patients with other neurologic disorders, ALS serum lowered the level of neurofilament proteins. Effects were similar with or without muscle-derived neurotrophic factors; there was no specificity for motor neurons. No neurotoxic activity was found in immunoglobulin fractions, and there was no evidence of circulating antibodies that might neutralize muscle-derived neurotrophic factors or induce cytolysis of spinal neurons.     

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.