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The formation of misfolded protein aggregates is one of the pathological hallmarks of neurodegenerative diseases. We have previously demonstrated the cytoplasmic aggregate formation of adenovirally expressed transactivation response DNA-binding protein of 43 kDa (TDP-43), the main constituent of neuronal cytoplasmic aggregates in cases of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), in cultured neuronal cells under the condition of proteasome inhibition. The TDP-43 aggregate formation was markedly suppressed by co-infection of adenoviruses expressing heat shock transcription factor 1 (HSF1), a master regulator of heat shock response, and Praja1 RING-finger E3 ubiquitin ligase (PJA1) located downstream of the HSF1 pathway. In the present study, we examined other reportedly known E3 ubiquitin ligases for TDP-43, i.e. Parkin, RNF112 and RNF220, but failed to find their suppressive effects on neuronal cytoplasmic TDP-43 aggregate formation, although they all bind to TDP-43 as verified by co-immunoprecipitation. In contrast, PJA1 also binds to adenovirally expressed wild-type and mutated fused in sarcoma, superoxide dismutase 1, α-synuclein and ataxin-3, and huntingtin polyglutamine proteins in neuronal cultures and suppressed the aggregate formation of these proteins. These results suggest that PJA1 is a common sensing factor for aggregate-prone proteins to counteract their aggregation propensity, and could be a potential therapeutic target for neurodegenerative diseases that include ALS, FTLD, Parkinson's disease and polyglutamine diseases., (© 2022 The Authors. Neuropathology published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Neuropathology.)

Electrophysiological methods to detect the degeneration of the upper motor neuron system have not been fully established in patients with amyotrophic lateral sclerosis (ALS). This may be partly because the parallel demonstration of electrophysiology and a corresponding pathological abnormality is insufficient, and because a substantial number of patients with ALS do not exhibit upper motor neuron degeneration. Recently, we encountered 2 patients with ALS who had been examined for abnormal central motor conduction time (CMCT) using transcranial magnetic stimulation within a 20-day period prior to their death. Autopsy revealed that 1 patient had marked pyramidal degeneration with prolonged CMCT; in contrast, the other patient had no obvious pyramidal degeneration and showed normal CMCT. Both the patients with contrasting clinicopathological differences contributed to the identification that the prolongation of CMCT was possibly linked to the degeneration of the corticospinal tract. This report indicates that CMCT is useful for predicting the severity of upper motor neuron degeneration in patients with ALS.

A vast body of evidence implicates increased oxidative stress and extracellular glutamate accumulation in the pathomechanism of sporadic amyotrophic lateral sclerosis (ALS). Cystine/glutamate antiporter (xCT) carries extracellular cystine uptake and intracellular glutamate release (cystine/glutamate exchange) in the presence of oxidative stress. The aim of the present study was to determine the involvement of xCT in ALS. Immunohistochemical observations in the spinal cord sections demonstrated that xCT was mainly expressed in astrocytes, with staining more intense in 12 sporadic ALS patients as compared to 12 age-matched control individuals. Western blot and densitometric analyses of the spinal cord samples revealed that the relative value of xCT/β-actin optical density ratio was significantly higher in the ALS group as compared to the control group. Next, we conducted cell culture experiments using a human astrocytoma-derived cell line (1321N1) and a mouse motor neuron/neuroblastoma hybrid cell line (NSC34). In 1321N1 cells, the normalized xCT expression levels in cell lysates were significantly increased by H 2 O 2 treatment. Glutamate concentrations in 1321 N1 cell culture-conditioned media were significantly elevated by H 2 O 2 treatment, and the H 2 O 2 -driven elevations were completely canceled by the xCT inhibitor erastin pretreatment. In motor neuron-differentiated NSC34 cells (NSC34d cells), both the normalized xCT expression levels in the cell lysates and glutamate concentrations in the cell-conditioned media were constant with or without H 2 O 2 treatment. The present results provide in vivo and in vitro evidence that astrocytes upregulate xCT expression to release glutamate in response to increased oxidative stress associated with ALS, contributing to extracellular glutamate accumulation., (© 2020 Japanese Society of Neuropathology.)

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., (© 2019 Japanese Society of Neuropathology.)

We report the neuropathology of a patient with a family history of amyotrophic lateral sclerosis (ALS) and a p.N345K mutation in the transactivation response DNA-binding protein 43 kDa (TDP-43) gene (TARDBP). A 62-year-old man had bulbar palsy with progressive weakness in the extremities. Neurological examination revealed evident upper motor neuron signs and lower motor neuron involvement corroborated by needle electromyography. The patient was diagnosed as having probable ALS according to the revised El Escorial diagnostic criteria and was eventually diagnosed with familial ALS. At 65 years of age, respiratory failure became critical, and artificial ventilation was initiated. At 70 years of age, the patient died from a urinary tract infection. Histopathological investigation showed Bunina bodies in the remaining motor neurons and anterolateral funicular myelin pallor in the spinal cord. TDP-43-positive cytoplasmic inclusions were quite rare in the spinal cord motor neurons, being predominantly present in the glial cells (especially astrocytes) of the spinal cord anterior horn. Although the reason for the preferential vulnerability of spinal glial cells to TARDBP mutations remains unclear, our findings indicate that TARDBP p.N345K mutation could have an influence on the topography of TDP-43 aggregation., (© 2019 Japanese Society of Neuropathology.)

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder. Despite its severity, there are no effective treatments because of the complexity of its pathogenesis. As one of the underlying mechanisms of Cu, Zn superoxide dismutase (SOD1) gene mutation-induced ALS, SOD1 mutants (SOD1 mut ) commonly interact with an endoplasmic reticulum-resident membrane protein Derlin-1, triggering motoneuron death. However, the importance of SOD1-Derlin-1 interaction in in vitro human model and in vivo mouse model remains to be elucidated. Here, we identify small-molecular-weight compounds that inhibit the SOD1-Derlin-1 interaction by screening approximately 160,000 compounds. The inhibitor prevents 122 types of SOD1 mut from interacting with Derlin-1, and significantly ameliorates the ALS pathology both in motoneurons derived from patient induced pluripotent stem cells and in model mice. Our data suggest that the SOD1-Derlin-1 interaction contributes to the pathogenesis of ALS and is a promising drug target for ALS treatment.

The immunolocalization of the 14-3-3 eta isoform in the anterior horn cells (AHCs) of patients with sporadic amyotrophic lateral sclerosis (ALS) and controls was examined. Compared with the immunolocalization of other 14-3-3 isoforms, the immunolocalization of the 14-3-3 eta isoform was either synaptic at the periphery of AHCs, spindle-shaped in neurites, or granular in the cytoplasm. By double labeling with phosphorylated (p-)TDP-43, the transactivation response DNA binding protein of 43kDa (TDP-43) demonstrated frequent colocalization of the 14-3-3 eta isoform in granular structures (90%) and spindle-shaped structures (85.4%), but not in p-TDP-43-positive round inclusions. It is speculated that the 14-3-3 eta isoform is associated with not only a synaptic pathology of ALS but also TDP-positive small lesions in the cytoplasm and neurites. The absence of eta-like immunoreactivity in p-TDP-43-positive large inclusions suggests the restricted relevance of the 14-3-3 eta isoform during ALS pathogenesis to some phases of the p-TDP pathology., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Emerging evidence suggests that innate immunity and increased oxidative stress contribute to pathomechanisms in amyotrophic lateral sclerosis (ALS). The aim of the present study was to verify the involvement of monocyte chemoattractant protein-1 (MCP-1) and its specific CC chemokine receptor 2 (CCR2) in the disease progression of ALS. We here demonstrate the expression state of MCP-1 and CCR2 in lumbar spinal cords of mice overexpressing a transgene for G93A mutant human superoxide dismutase 1 (SOD1) (ALS mice) as a mouse model of ALS as well as the involvement of MCP-1/CCR2-mediated signaling in behavior of cultured astrocytes derived from those mice., Results: Quantitative polymerase chain reaction analysis revealed that MCP-1 and CCR2 mRNA levels were significantly higher in ALS mice than those in nontransgenic littermates (control mice) at the presymptomatic stage. Immunoblot analysis disclosed a significantly higher CCR2/β-actin optical density ratio in the postsymptomatic ALS mouse group than those in the age-matched control mouse group. Immunohistochemically, MCP-1 determinants were mainly localized in motor neurons, while CCR2 determinants were exclusively localized in reactive astrocytes. Primary cultures of astrocytes derived from ALS mice showed a significant increase in proliferation activity under recombinant murine MCP-1 stimuli as compared to those from control mice., Conclusions: Our results provide in vivo and in vitro evidence that MCP-1 stimulates astrocytes via CCR2 to induce astrocytosis in ALS with SOD1 gene mutation. Thus, it is likely that MCP-1/CCR2-mediated sigaling is involved in the disease progression of ALS.

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Signal transducer and activator of transcription-3 (STAT3) is a member of the proinflammatory transcription factor STAT family. Several studies have documented implications for neuroinflammation in amyotrophic lateral sclerosis (ALS). We recently demonstrated activation of STAT3 in spinal cords obtained at autopsy from sporadic ALS patients. To determine the involvement of STAT3 and effects of pioglitazone on STAT3 activity in familial ALS with superoxide dismutase-1 (SOD1) mutation, we performed immunoblot and immunohistochemical analyses of the active form of STAT3 (p-STAT3) in spinal cords from mice overexpressing mutant SOD1 (ALS mice) and nontransgenic littermates (control mice). Immunoblot analysis delineated significant increases in nuclear p-STAT3 levels in non-treated ALS mice as compared with pioglitazone-treated ALS mice and non-treated and pioglitazone-treated control mice. Immunohistochemical analysis revealed prominent p-STAT3 accumulations in the nucleus of motor neurons, reactive astrocytes and activated microglia in non-treated ALS mice but not pioglitazone-treated ALS mice and non-treated and pioglitazone-treated control mice. The present results provide in vivo evidence for increased phosphorylative activation and nuclear translocation of STAT3 in motor neurons and glia in mouse motor neuron disease, suggesting a common pathological process between sporadic and SOD1-mutated familial forms of ALS. Moreover, it is likely that pioglitazone may exert inhibitory effects on STAT3-mediated proinflammtory mechanisms in this disease.

TDP-43 is ubiquitously expressed in the nucleus of motor neurons and is closely associated with the pathogenesis of amyotrophic lateral sclerosis (ALS). However, little is known about alterations in the subcellular or intracellular localization of TDP-43, either under normal conditions or in ALS. We examined the anterior horn neurons of the spinal cord in patients with sporadic ALS and age-matched controls immunohistochemically and immunoelectron-microscopically using anti-TDP-43 antibody. Immunohistochemically, the present study showed a decrease in TDP-43 immmunoreactivity in the nucleus and, by contrast, an increase in the cytoplasm in ALS patients. Immunoelectron-microscopically, we demonstrated the consistent presence of TDP-43-immunogold-labeled deposits primarily in the nucleus, particularly in the nucleolus, and frequently in the rough endoplasmic reticulum (rER), and, to a lesser extent, in the mitochondria and the synaptic vesicles of the presynaptic terminals on the surface of anterior horn neurons both in controls and ALS subjects. In ALS, a reduced number of TDP-43-immunogold-labeled deposits were observed in the nuclei, particularly in the nucleoli of even normal-looking motor neurons. In contrast, the number of TDP-43-immunogold-labeled deposits in the rER of the normal-appearing motor neurons was significantly larger in ALS than in the controls (p=0.0036). These findings suggest that TDP-43 is synthesized in the rER and translocates to the nucleus, particularly to the nucleolus, and in ALS, TDP-43 trafficking between the nucleus and the cytoplasm is disturbed, resulting in an accumulation of TDP-43 in the cytoplasm in the form of insoluble aggregates., (Copyright 2010. Published by Elsevier Ireland Ltd.)

Compelling evidence identifies a link between cytotoxic effects of cytosolic phospholipase A2 (cPLA2) activity and neuron death in cell cultures. cPLA2 catalyzes the hydrolysis of membrane phospholipids to produce and release arachidonate, leading to plasma membrane injury, inflammatory response and subsequent cell death. To assess a role for cPLA2 in the pathomechanism of amyotrophic lateral sclerosis (ALS), we performed immunohistochemical, immunoblot, and densitometric analyses of cPLA2 and its active form phosphorylated at S505 (p-cPLA2) on spinal cords obtained at autopsy from ten sporadic ALS patients and ten age-matched controls. On sections, immunoreactivities for cPLA2 and p-cPLA2 were distinct and localized in almost all of the motor neurons, reactive astrocytes, and activated microglia in the ALS cases, while immunoreactivities were only weak or not at all observed in neurons and glia in the control cases. On immunoblots, both the cPLA2/β-actin density ratio and the p-cPLA2/cPLA2 density ratio were significantly increased in the ALS group compared to the control group. There was no significant link between the densitometric data and the clinical phenotypes, age at death or disease duration of the ALS patients. These results provide in vivo evidence for increased expression and activation of cPLA2 in motor neurons, reactive astrocytes, and activated microglia in ALS, suggesting occurrence of arachidonate cascade-induced motor neuron death via cell-autonomous and/or non-cell-autonomous mechanisms.

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.

Background: Neuroinflammation has been implicated in the pathomechanism of amyotrophic lateral sclerosis (ALS). It is known that signal transducer and activator of transcription-3 (STAT3) is a proinflammatory transcription factor. However, it remains to be determined whether STAT3 is involved in ALS., Objective: To test the hypothesis that STAT3 may be upregulated, activated, or both in the spinal cord of ALS patients., Methods: We performed immunohistochemical, immunoblot and densitometric analyses of total STAT3 (t-STAT3) or phosphorylated active form of STAT3 (p-STAT3) in spinal cords obtained at autopsy from 10 sporadic ALS patients and 10 age-matched control subjects., Results: On sections, p-STAT3 immunoreactivity was localized in the nucleus as well as the cytoplasm of almost all activated microglia in the ALS cases, while it was detectable in a few resting microglia in the control cases. On blots, densitometric p-STAT3 levels in nuclear protein extracts significantly increased in the ALS group compared with the control group, although there was no significant difference in densitometric t-STAT3 levels in cytosolic protein extracts between the two groups. Additionally, there was no significant relationship between the nuclear p-STAT3 levels in the ALS cases and the clinical phenotypes, age at death, or disease duration., Conclusion: The present results suggest that persistent activation and nuclear translocation but not upregulation of STAT3 occurs in ALS spinal cord microglia, which may regulate inflammatory activity.

Emerging evidence suggests the involvement of programmed cell death and inflammation in amyotrophic lateral sclerosis (ALS). To assess molecular pathological effects of the anti-inflammatory peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist pioglitazone in ALS, we verified changes in the population of neurons, astrocytes, and microglia in the ventral horns of spinal cord lumbar segments from the pioglitazone-treated and non-treated groups of mice carrying a transgene for G93A mutant human superoxide dismutase-1 (SOD1) (ALS mice) and non-transgenic littermates (control mice), performed immunohistochemical and immunoblot analyses of PPARgamma, active form of phosphorylated p38 mitogen-activated protein kinase (p-p38) and inhibitor of nuclear factor-kappaB (NF-kappaB)-alpha (IkappaBalpha) in the spinal cords, and compared the results between the different groups. Image analysis revealed that optical density of NeuN-immunoreactive neurons was significantly lower in the non-treated groups of presymptomatic and advanced ALS mice than in the non-treated groups of age-matched control mice and was recovered with pioglitazone treatment, and that optical densities of GFAP-immunoreactive astrocytes and Iba1-immunoreactive microglia were significantly higher in the non-treated group of advanced ALS mice than in the non-treated group of control mice and were recovered with pioglitazone treatment. Immunohistochemical analysis demonstrated that immunoreactivities for PPARgamma and p-p38 were mainly localized in neurons, and that IkappaBalpha immunoreactivity was mainly localized in astrocytes and microglia. Immunoblot analysis showed that pioglitazone treatment resulted in no significant change in nuclear PPARgamma-immunoreactive density, a significant decrease in cytosolic p-p38-immunoreactive density, and a significant increase in cytosolic IkappaBalpha-immunoreactive density. Our results suggest that pioglitazone protects motor neurons against p38-mediated neuronal death and NF-kappaB-mediated glial inflammation via a PPARgamma-independent mechanism.

We describe a patient with familial amyotrophic lateral sclerosis (FALS) in whom we identified a substitution of G for CGTTTA at codon 144 in the Cu/Zn superoxide dismutase 1 (SOD1) gene, causing amino acid changes from leucine to phenylalanine, valine and a stop codon (L144FVX). This mutation is novel, and so we report the clinical and neuropathological features of this case compared with those of other FALS cases with SOD1 mutations. A 39-year-old woman developed muscle weakness and atrophy in the hands, which rapidly progressed and expanded to other muscles, resulting in respiratory insufficiency and death at only 10 months after the onset Her grandmother, father and uncle had also been diagnosed as having ALS. The most noticeable neuropathological findings in the present case were marked loss of large motor neurons in the anterior horns associated with the frequent appearance of cord-like swollen, partially SOD 1- and ubiquitin-immunopositive axons. These findings together with the absence of Bunina bodies are compatible with the neuropathology of FALS with SOD1 gene mutation, although Lewy body-like inclusions characteristic for this condition were not observed.

Growing evidence documents oxidative stress involvement in ALS. We previously demonstrated accumulation of a protein-bound form of the highly toxic lipid peroxidation product crotonaldehyde (CRA) in the spinal cord of sporadic ALS patients. In the present study, to the determine the role for CRA in the disease processes of superoxide dismutase-1 (SOD1) mutation-associated familial ALS (FALS), we performed immunohistochemical and semi-quantitative cell count analyses of protein-bound CRA (P-CRA) in the spinal cord of SOD1-mutated FALS and its transgenic mouse model. Immunohistochemical analysis revealed increased P-CRA immunoreactivity in the spinal cord of the FALS patients and the transgenic mice compared to their respective controls. In the FALS patients, P-CRA immunoreactivity was localized in almost all of the chromatolytic motor neurons, neurofilamentous conglomerates, spheroids, cordlike swollen axons, reactive astrocytes and microglia, and the surrounding neuropil in the affected areas represented by the anterior horns. In the transgenic mice, P-CRA immunoreactivity was localized in only a few ventral horn glia in the presymptomatic stage, in almost all of the vacuolated motor neurons and cordlike swollen axons and some of the ventral horn reactive astrocytes and microglia in the onset stage, and in many of the ventral horn reactive astrocytes and microglia in the advanced stage. Cell count analysis on mouse spinal cord sections disclosed a statistically significant increase in the density of P-CRA-immunoreactive glia in the ventral horns of the young to old G93A mice compared to the age-matched control mice. The present results indicate that enhanced CRA formation occurs in motor neurons and reactive glia in the spinal cord of SOD1-mutated FALS and its transgenic mouse model as well as sporadic ALS, sug- gesting implications for CRA in the pathomechanism common to these forms of ALS.

4-Hydroxy-2-hexenal (HHE) is a toxic, reactive aldehydic intermediate formed by nonenzymatic peroxidation of n-3 polyunsaturated fatty acids. The aim of this study was to determine the implication for HHE in the pathomechanism of amyotrophic lateral sclerosis (ALS) by immunohistochemical and enzyme-linked immunosorbent assay (ELISA) techniques using a mouse monoclonal IgG(1) antibody mAbHHE53 specific for protein-bound HHE. Immunohistochemical analysis on formalin-fixed, paraffin-embedded sections and frozen sections of spinal cords obtained at autopsy from 10 sporadic ALS patients and 10 age-matched control subjects demonstrated that protein-bound HHE immunoreactivity was seen and was prominent in the entire gray matter in the ALS cases and localized in the neurons, reactive astrocytes, microglial cells, and the surrounding neuropil, while the immunoreactivity was obscure or undetectable in the control cases. No significant protein-bound HHE immunoreactivity was seen in sections processed with omission of mAbHHE53 or in sections incubated with the antibody with an excess of the respective antigen. Competitive ELISA analysis on trypsin-digested protein extracts of fresh-frozen spinal cord samples disclosed a significant increase in protein-bound HHE level in the ALS cases compared with the control cases. Our results indicate that enhanced HHE formation occurs in the entire gray matter of sporadic ALS spinal cords and suggest that the selective vulnerability of motor neurons to HHE mediates the pathomechanism of this disease.

Recent studies have documented carbonyl stress involvement in the pathogenesis of sporadic amyotrophic lateral sclerosis (ALS). The aim of the present study was to assess a role for carbonyl stress in motor neuron degeneration associated with superoxide dismutase-1 (SOD1) mutant familial ALS and its transgenic mouse model, using an immunohistochemical investigation of advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs). In the spinal cords from six familial ALS patients with SOD1 A4V mutation and six transgenic mice expressing G93A mutant human SOD1, immunoreactivities for N(epsilon)-(carboxyethyl)lysine, argpyrimidine, pyrraline and N(epsilon)-(carboxymethyl)lysine as AGEs were distinct in almost all of the reactive astrocytes and obscure in the residual neurons, whereas no immunoreactivity for pentosidine as an AGE, or 4-hydroxy-2-nonenal-histidine, malondialdehyde-lysine or acrolein-lysine as ALEs was detectable. Spinal cords from age-matched control humans and mice exhibited no significant immunoreactivities for the examined products. Our results indicate that protein glycation, but not lipid peroxidation, is enhanced in ALS patients with an SOD1 mutation and mutant SOD1 transgenic mice, in which certain AGEs are selectively formed in the spinal cord astrocytes.

For determining whether both the spinal cord motor neurons and glial cells are exposed to increased oxidative stress in amyotrophic lateral sclerosis (ALS), we performed an immunohistochemical investigation of end products of lipid peroxidation and protein glycoxidation in spinal cords from seven sporadic ALS patients and seven age-matched control individuals. In the ALS spinal cords, immunoreactivities for adducts of 4-hydroxy-2-nonenal-histidine and crotonaldehyde-lysine as markers of lipid peroxidation, N(epsilon)-(carboxymethyl)lysine as a marker of lipid peroxidation or protein glycoxidation, and pentosidine as a marker of protein glycoxidation were localized in the gray matter neuropil and almost all of the motor neurons, reactive astrocytes and microglia/macrophages, whereas none of the immunoreactivities for N(epsilon)-(carboxyethyl)lysine or argpyrimidine as markers of protein glycoxidation or enzymatic glycolysis, or pyrraline or imidazolone as markers of nonoxidative protein glycation were detectable. The control spinal cords displayed no significant immunoreactivities for any of these examined products. Our results indicate that in sporadic ALS, both lipid peroxidation and protein glycoxidation are enhanced in the spinal cord motor neurons and glial cells, and suggest that the formation of certain products in these abnormal reactions is implicated in motor neuron degeneration.

Familial amyotrophic lateral sclerosis (ALS) with mutations in the gene for superoxide dismutase-1 (SOD1) is clinicopathologically reproduced by transgenic mice expressing mutant forms of SOD1 detectable in familial ALS patients. Motor neuron degeneration associated with SOD1 mutation has been thought to result from a novel neurotoxicity of mutant SOD1, but not from a reduction in activity of this enzyme, based on autosomal dominant transmission of SOD1 mutant familial ALS and its transgenic mouse model, clinical severity of the ALS patients independent to enzyme activity, no ALS-like disease in SOD1 knockout or wild-type SOD1-overexpressing mice, and clinicopathological severity of mutant SOD1 transgenic mice dependent on transgene copy numbers. Proposed mechanisms of motor neuron degeneration such as oxidative injury, peroxynitrite toxicity, cytoskeletal disorganization, glutamate excitotoxicity, disrupted calcium homeostasis, SOD1 aggregation, carbonyl stress and apoptosis have been discussed. Intracytoplasmic vacuoles, indicative of increased oxidative damage to the mitochondria and endoplasmic reticulum, in the neuropil and motor neurons appear in high expressors of mutant SOD1 transgenic mice but not in low expressors of the mice or familial ALS patients, suggesting that overexpression of mutant SOD1 in mice may enhance oxidative stress generation from this enzyme. Thus, transgenic mice carrying small transgene copy numbers of mutant SOD1 would provide a beneficial animal model for SOD1 mutant familial ALS. Such a model would contribute to elucidating the pathomechanism of this disease and establishing new therapeutic agents.

To clarify the biological significance of the neuronal Lewy body-like hyaline inclusions and astrocytic hyaline inclusions characteristically found in patients with familial amyotrophic lateral sclerosis with superoxide dismutase-1 (SOD1) gene mutations and in transgenic mice expressing human SOD1 with G85R mutation, the detailed protein composition in both types of inclusions was immunohistochemically analyzed using 45 different antibodies. Both types of inclusions had very strong immunoreactivity for SOD1. The SOD1-positive inclusions in both cell types were also immunoreactive for the insoluble advanced glycation endproducts (AGEs) such as Nepsilon-(carboxymethyl)lysine (CML), pyrraline and pentosidine: both inclusions in both conditions were ultrastructurally composed of the granule-coated fibrils that had immunoreactivities to CML and pyrraline. Both types of inclusions were negative for stress-response proteins (SRPs), 4-hydroxy-2-nonenal (HNE), acrolein, nitric oxide synthases (NOSs) and nitrotyrosine as representative markers of oxidative stress. The neurons and astrocytes of the normal individuals and non-transgenic mice showed no significant immunoreactivity for SOD1, AGEs, SRPs, HNE, acrolein, NOSs or nitrotyrosine. Our results suggest that a portion of the SOD1 composing both type of inclusions, probably toxic mutant SOD1, is modified by the AGEs, and that the formation of the AGE-modified SOD1 is one of the mechanisms responsible for the aggregation involving no significant oxidative mechanisms.

We carried out an immunohistochemical investigation of the spinal cords of 15 patients with sporadic amyotrophic lateral sclerosis (ALS), using antibodies to inducible nitric oxide synthase (iNOS) and nitrotyrosine; our purpose was to search for a possible role of increased oxidative damage in the motor system that may contribute to the neurodegenerative process in this disease. Specimens from 16 patients without any neurological disease served as controls. In the controls, normal-appearing neurons and their dendrites were negatively immunostained for iNOS. In the ALS patients, most of normal-appearing anterior horn neurons did not show iNOS immunoreactivity either in the perikarya or in their dendrites. However, many of the degenerated neurons showing central chromatolysis or simple atrophy demonstrated focally or diffusely positive iNOS immunoreactivity within the perikarya and their neuronal processes. In the neuropil of the anterior horns, the reactive astrocytes were more intensely immunostained for iNOS as compared with the controls. Some of the swollen proximal axons (spheroids) were focally or diffusely immunostained by the antibody. The corticospinal tracts demonstrated positive iNOS immunoreactivity of proliferated reactive astrocytes. The immunostaining pattern of nitrotyrosine in the anterior horn neurons of the spinal cord was similar to that of iNOS. These findings suggest that selective nitric oxide-mediated oxidative damage in the motor system plays a part in the pathomechanism of the neuronal degeneration in the spinal cord of sporadic ALS.

To assess a role for oxidative stress in the pathogenesis of amyotrophic lateral sclerosis (ALS), we analyzed the immunohistochemical localization of 8-hydroxy2'-deoxyguanosine (OHdG) as a nucleic acid oxidation product, acrolein-protein adduct and 4-hydroxy-2-nonenal (HNE)-protein adduct as lipid peroxidation products, Nepsiloncarboxymethyl-lysine (CML) as a lipid peroxidation or protein glycoxidation product, pentosidine as a protein glycoxidation product, and imidazolone and pyrraline as nonoxidative protein glycation products in the spinal cord of three familial ALS patients with superoxide dismutase(SOD 1) A4V mutation, six sporadic ALS patients, and six age-matched control individuals. The spinal cord sections of the control cases did not show any distinct immunoreactivities for these examined products. In the familial ALS cases, intense immunoreactivities for pyrraline and CML were confined to the characteristic Lewy body-like hyaline inclusions, and imidazolone immunoreactivity was located in the cytoplasm of the residual motor neurons. No significant immunoreactivities for other examined products were detected in the familial ALS spinal cords. In the sporadic ALS cases, intense immunoreactivities for pentosidine, CML and HNE-protein adduct were seen in the cytoplasm of the degenerated motor neurons, and OHdG immunoreactivity was located in the cell nuclei of the residual neurons and glial cells. The present results indicate that oxidative reactions are involved in the disease processes of sporadic ALS, while there is no evidence for increased oxidative damage except for CML deposition in the familial ALS spinal cords. Furthermore, it is likely that the accumulation of pyrraline and imidazolone supports a nonoxidative mechanism in SOD1-related motor neuron degeneration.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that primarily involves the motor neuron system. Approximately 5-10% of ALS is familial. Superoxide dismutase 1 (SOD1) gene mutations are shown to be associated with about 20% of familial ALS (FALS) patients. The neuronal Lewy-body-like hyaline inclusion (LBHI) and astrocytic hyaline inclusion (Ast-HI) are morphological hallmarks of certain SOD1-linked FALS patients with SOD1 gene mutant and transgenic mice expressing human SOD1 with G85R mutation. From the detailed immunohistochemical analyses, the essential common protein of both inclusions is SOD1. Ultrastructurally, both inclusions consist of granule-coated fibrils 15-25 nm in diameter. Based on the immuno-electron microscopical finding that these abnormal granule-coated fibrils are positive for SOD1, the formation (or aggregation) of the abnormal fibrils containing SOD1 would be essential evidence in diseases caused by various SOD1 mutations. The granule-coated fibrils are also modified by advanced glycation end products (AGEs). The AGEs themselves are insoluble molecules with direct toxic effects on cells. AGE formation of SOD1 composing the granule-coated fibrils (probable AGE-modified mutant SOD1) may amplify their aggregation and produce a more marked toxicity.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by motor neuron system involvement, and is epidemiologically subclassified into sporadic, familial and endemic forms. About 20% of ALS families are associated with mutations in the gene for superoxide dismutase-1 (SOD1) encoded on chromosome 21q22.1. Several studies have pointed to a variety of functions of mutant SOD1, which has enhanced catalytic activity of the peroxynitrite-mediated tyrosine nitration, readily releases the reactive Cu ions, induces apoptotic cell death, has enhanced peroxidase activity, damages the mitochondria to release Ca2+, and forms SOD1-containing aggregates in the cytoplasm. Many of these studies have obtained evidence for increased oxidative damage in ALS. On the other hand, some reports disagree with oxidative damage involvement in SOD1 mutant ALS. In considering the findings of increased oxidative damage in mutant SOD1-expressing transgenic mice, it should be remembered that overexpression of mutant SOD1 may enhance oxidative stress generation from this enzyme. In this review, we present the clinicopathological features of SOD1 mutant familial ALS and its transgenic mouse model, and also discuss SOD1 mutation-related neurotoxicity, including SOD1 protein aggregation and post-translational protein modification.

To determine the role of advanced glycation endproducts (AGE) in the pathogenesis of familial amyotrophic lateral sclerosis (ALS) with superoxide dismutase-1 (SOD1) mutation, we investigated the immunohistochemical localization of N(epsilon)-carboxymethyl-lysine (CML), one of the major AGE structures, in spinal cords from three familial ALS patients with a heterozygous Ala to Val substitution at codon 4 in the gene for SOD1. Neuronal hyaline inclusions (NHIs), the abnormal structures seen in some of the remaining lower motor neurons of familial ALS patients with SOD1 mutation, were intensely stained by a monoclonal antibody specific for CML in contrast to the only weakly stained cytoplasm. Immunoelectron microscopy depicted the CML determinants restricted to the granule-associated thick linear structures that mainly compose the NHIs. The NHIs were also recognized by antibodies to SOD1, phosphorylated neurofilament protein and ubiquitin. No focal collection of either CML or SOD1 was found in neurons of the control individuals. Our results indicate that CML is a component of the NHIs of familial ALS patients with SOD1 mutation, and suggest that the CML formation may be mediated by protein glycoxidation or lipid peroxidation in the presence of oxidative stress from mutant SOD1, in association with motor neuron degeneration.

To clarify the neuropathological significance of the deposition of N(epsilon)-carboxymethyl lysine (CML), an advanced glycation endproduct, in astrocytic hyaline inclusions in familial amyotrophic lateral sclerosis (FALS), autopsy specimens from five members of two different families who had the superoxide dismutase 1 (SOD1) gene mutations were analysed. Immunohistochemically, most of the neuronal and astrocytic hyaline inclusions were intensely stained by the antibody against CML. The distributions and intensities of the immunoreactivities for CML and SOD1 were similar in the inclusions in both cell types. Immunoelectron microscopy showed that both inclusions consisted of CML-positive granule-coated fibrils and granular materials. No significant CML or SOD1 immunoreactivity was observed in the neurons and astrocytes of the normal control subjects. Our results suggest that astrocytic hyaline inclusions contain CML and SOD1 in FALS patients with SOD1 gene mutations, and that the formation of CML-modified protein (probably CML-modified SOD1) is related to the cell degeneration.

This report presents a familial amyotrophic lateral sclerosis (FALS) patient with widespread vacuoles and hyaline inclusions strongly immunostained with the anti-superoxide dismutase (SOD1) antibody. The overall pathologic similarity between our non-SOD1-linked FALS patient and transgenic mice expressing a mutated human SOD1 gene suggests that common pathogenetic mechanisms other than an SOD1 mutation exist in the development of these diseases.

This report concerns retrospective immunohistochemical and immunoelectron microscopic studies on superoxide dismutase-1 (SOD1) in intracytoplasmic hyaline inclusions (IHIs) of the anterior horn cells of three patients with familial amyotrophic lateral sclerosis (ALS) with posterior column involvement. All of the patients were members of the American "C" family. Almost all of the IHIs, present in the soma and cordlike swollen neurites of some affected neurons of the three patients, were intensely stained by an antibody to human SOD1. By contrast, the cytoplasm of anterior horn cells of the ALS patients and of ten control individuals reacted only weakly with the antibody or not at all. Immunoelectron microscopy revealed that the granule-associated thick linear structures that composed the IHIs were intensely labeled by the antibody to SOD1. The IHIs were also positively stained by antibodies to ubiquitin and phosphorylated neurofilament protein, with the distribution of immunoreactivity resembling that seen with the anti-SOD1 antibody. The DNA analysis disclosed a single-site GCC to GTC substitution at codon 4 (Ala4 --> Val) in the SOD1 gene from the brain samples of the patients and from the peripheral blood of their family members. Our results suggest that SOD1 is a component of IHIs and may interact with Ubiquitin and neurofilament protein, and point to the possibility that the presence of intense SOD1 immunoreactivity in the IHIs may be of relevance in processes involving structurally altered SOD1 molecules encoded by the mutated gene.

A role mutations in the superoxide dismutase (SOD)-1 gene in the pathogenesis of amyotrophic lateral sclerosis (ALS) has been discussed. To investigate immunohistochemical alterations of SOD in the spinal cord affected with the disease, we examined 3 patients with SOD1 mutation-associated family with ALS, 20 patients with sporadic ALS and 10 control individuals. Lewy body-like hyaline inclusions (LBHIs) were seen in the anterior horn cells of all the familial patients and 10 of the 20 sporadic patients, while skein-like inclusions (SIs) and Bunina bodies (BBs) were present in the 20 sporadic patients but not in the familial patients. The primary antibodies used for immunostaining were rabbit antisera raised against human SOD1 and SOD2. The anti-SOD1 antibody reacted strongly with all LBHIs of each familial patient and with some LBHIs of each sporadic patient. The cytoplasm of morphologically intact and degenerated spinal cord neurons as well as spheroids seen in the cases examined was only weakly stained by the antibody to SOD1 or not at all. The reactive astrocytes displayed weak to moderate staining for SOD1. The anti-SOD2 antibody strongly immunolabeled the reactive astrocytes and microglia. LBHIs of both familial and sporadic ALS were negatively stained for SOD2. Spinal cord neurons and spheroids of each case exhibited no significant SOD2 immunoreactivity. Neither antibodies reacted with SIs nor BBs. These results indicate that SOD1 may be involved in the formative process of LBHIs especially in familial ALS but not always in that of SIs or BBs, and imply that SOD2 may have no connection with any of these ALS-related abnormal structures.

This report concerns a case of sporadic amyotrophic lateral sclerosis (ALS) with dementia and Lewy body-like hyaline inclusions (LBHIs). The patient was a 70-year-old woman who initially showed memory disturbance and later developed bulbar palsy, muscle atrophy and weakness. The total clinical course was 51 months. The postmortem examination revealed superficial sponginess and subcortical gliosis in the frontotemporal cortices. Ubiquitin-positive intraneuronal inclusions were found in small cortical neurons of the frontotemporal lobe. Neuronal loss was marked in the spinal anterior horn with degeneration of the pyramidal tracts. The anterior horn cells had ubiquitin-immunoreactive skein-like inclusions and Bunina bodies. LBHIs were present in the lumbar horn; ultrastructurally they were composed of randomly arranged thick filamentous structures studded with granules. The LBHIs were intensely stained with anti-ubiquitin antibody. As in familial ALS and in certain cases of sporadic ALS, some of these inclusions reacted with an antibody against Cu/Zn superoxide dismutase, the enzyme whose gene was recently found to be mutated in some forms of familial ALS.

Cu/Zn superoxide dismutase (SOD)-like immunoreactivity (LI) was found within Lewy body-like inclusions (LBIs) in the spinal cords of patients with sporadic amyotrophic lateral sclerosis (ALS) by using an antibody to human Cu/ZnSOD. LBIs were detected in the anterior horn cells in 10 of 20 patients with sporadic ALS. In each of these patients, 7 to 60% of LBIs showed Cu/ZnSOD-LI. No Cu/ZnSOD-LI was detected in intact neurons and glia in the 20 ALS patients, as well as in these cells in 10 normal control individuals. The skein-like inclusions and Bunina bodies, which were found in all of the 20 ALS cases, showed no Cu/ZnSOD-LI. Thus, Cu/ZnSOD appears to play roles in the production and/or degradation process of LBIs.

This report concerns the demonstration and distribution of neurofibrillary tangles (NFTs), immunoreactive neuropil thread-like structures and dots in the spinal cord gray and white matter of six patients with parkinsonism-dementia complex on Guam and five patients with Guamanian amyotrophic lateral sclerosis (ALS). A monoclonal antibody to Alzheimer NFTs was used. NFTs were found in the spinal cord gray matter and white matter and all patients had immunoreactive neuropil thread-like structures and dots in the gray matter as well as in the white matter. They were particularly numerous in the lateral funiculus of patients with Guamanian ALS.