Mitochondrial alterations in dorsal root ganglion cells in sporadic amyotrophic lateral sclerosis

Little information is available regarding the morphological changes in the mitochondria in amyotrophic lateral sclerosis (ALS). In particular, mitochondrial changes in dorsal root ganglion cells have not yet been examined. We therefore conducted an electron microscopic examination of the mitochondria in dorsal root ganglion cells in 11 sporadic ALS patients, and 12 age-matched, non-neurological control individuals in order to determine whether or not they are affected in ALS. In both the controls and ALS patients, unusual inclusion bodies were frequently observed in the mitochondria in the somata of the ganglion cells. The inclusions consisted of an aggregate of tubules measuring approximately 40 nm in diameter varying in size and number. Such inclusions were frequently present in the cristae and/or intermembrane space, often expanding to form large bundles in the dilated intermembrane space. These structures quite frequently protruded outward unilaterally or bilaterally and were partially surrounded by the outer membrane of the mitochondria. The number of inclusions was significantly higher in the ALS patients than in the controls (P < 0.0001). Regularly spaced transverse processes similar to the rungs of a ladder were occasionally observed in the intermembrane space, along with infrequent but markedly increased cristae and stubby mitochondria. We concluded that mitochondrial abnormalities may be involved in the degenerative processes in the dorsal root ganglion cells in sporadic ALS. These findings therefore suggest that ALS is a widespread, more generalized disorder than previously thought, and that the degeneration is not confined to the motor neuron system.     

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.     

Neuronal nitric oxide synthase (nNOS) immunoreactivity in the spinal cord of transgenic mice with G93A mutant SOD1 gene

Immunohistochemical and quantitative analyses were used to examine the evolution of neuronal nitric oxide synthase (nNOS) with time in spinal motor neurons of transgenic mice with a G93A mutant Cu/Zn superoxide dismutase (SOD1) gene. Specimens from age-matched non-transgenic wild-type mice served as controls. In the controls, the anterior horn including the anterior horn neurons was not immunostained for nNOS. In the transgenic mice, at the age of 24 weeks (early presymptomatic), when no pathological change was observed in the spinal cord, anterior horn neurons were only occasionally immunostained for nNOS (0.3%). At the age of 28 weeks (late presymptomatic), nNOS-positive anterior horn neurons and their neuronal processes were occasionally observed (7.6%), and at the age of 32 weeks (early symptomatic), nNOS-positive anterior horn cells, including degenerated ones showing central chromatolysis, were frequently demonstrated (27.6%) and nNOS-positive cord-like swollen proximal axons were also observed in the anterior horns. nNOS expression in the anterior horn neurons was almost always observed in the somata. At the age of 35 weeks (end stage), neuronal loss of the anterior horn cells was severe, and nNOS-positive anterior horn neurons and cord-like swollen axons in the anterior horns were less prominent compared to those at the age of 32 weeks (33.8%), but many reactive astrocytes were immunostained for nNOS. Thus, nNOS immunoreactivity in the anterior horn neurons is observed as early as the presymptomatic stage and varies with the progression of the disease. The selective localization of positive nNOS immunoreactivity in the anterior horn neurons and degenerated ones in particular, and swollen proximal axons suggests that nNOS immunoreactivity may be involved in the degeneration of anterior horn neurons in this SOD1 transgenic mouse model.     

Slow component of axonal transport is impaired in the proximal axon of transgenic mice with a G93A mutant SOD1 gene

The purpose of this study was to determine whether slow axonal transport of neurofilaments (NFs) is impaired in the spinal cord of G93A Cu/Zn superoxide dismutase (SOD1) mutant transgenic mice expressing a relatively low mutant protein (gene copy 10) and, if so, how the impairment occurs in this animal model. Transgenic mice were killed at the ages of 24, 28 and 32 weeks, and the cervical and lumbar spinal cords were examined under an electron microscope. Age-matched non-transgenic wild-type mice served as controls. At 24 weeks (early presymptomatic stage), anterior horn cells were well preserved. The earliest morphological changes were mild vacuolar changes in the neuronal processes, particularly in proximal axons. At 28 weeks (late presymptomatic stage), mild neuronal loss of anterior horn neurons was observed. Vacuolar changes were more prominent in the proximal axons, including swollen axons (spheroids) and neuropils of the anterior horns. Vacuoles in the axons were frequently large enough to occupy almost the entire axonal caliber. The anterior roots were degenerative, showing vacuolar changes and myelin ovoids. Lewy body-like inclusions (LIs) consisting of filaments thicker than NFs (about 1.5 times larger in diameter) were frequently demonstrated in the neuronal processes including swollen axons (spheroids) and occasionally in the somata. At 32 weeks (symptomatic stage), the anterior horns showed a moderate to severe neuronal loss accompanied by prominent astrogliosis. Cord-like swollen axons consisting of accumulated NFs and many neurofilamentous accumulations were frequently observed in the anterior horn. Vacuolar changes were less prominent or disappeared in the neuropils of the anterior horns and the anterior roots, whereas LIs were frequently demonstrated within the neuronal processes including the cord-like swollen axons. In the anterior roots, degenerative changes such as marked fiber loss and frequent myelin ovoids were remarkable. No accumulation of NFs or mitochondrial vacuolation was detected in somata or proximal dendrites at any stage. These findings suggest that the slow component of axonal transport in the proximal axons is impaired at an early stage in this transgenic mouse model, and that the impairment is probably caused by a mechanical impediment of NFs, or by the accumulation of NFs in the proximal axon, as a result of the obstruction of the axonal flow that initially occurs by vacuolar changes, and is later exacerbated by accumulation of LIs.     

Protein‐bound crotonaldehyde accumulates in the spinal cord of superoxide dismutase‐1 mutation‐associated familial amyotrophic lateral sclerosis and its transgenic mouse model

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 semiquantitative 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, suggesting implications for CRA in the pathomechanism common to these forms of ALS.     

Impairment of axonal transport in the axon hillock and the initial segment of anterior horn neurons in transgenic mice with a G93A mutant SOD1 gene

Impaired axonal transport of the fast or slow component has been reported in patients with sporadic amyotrophic lateral sclerosis (ALS), animal models for ALS, and familial ALS-linked mutant Cu/Zn superoxide dismutase (SOD1) transgenic mice. However, little is known about the impairment of axonal transport in mutant SOD1 transgenic mice. This is the first electron microscopic investigation of the axon hillock (AH) and the initial segment (IS) of anterior horn cells in the spinal cord of transgenic mice expressing the G93A mutant human SOD1, and it was launched with a view toward examining whether the axonal transport is impaired in this region. Six transgenic mice were killed at ages ranging from the presymptomatic to symptomatic stages. Six age-matched non-transgenic wild-type mice served as controls. In the non-transgenic mice, 91 AH and IS were observed, but those with increased neurofilaments or mitochondria were rarely found. In the transgenic mice, 95 AH and IS directly emanating from normal-looking large anterior horn cells were seen. AH and IS with increased neurofilaments or, to a lesser extent, increased mitochondria, and round-shaped mitochondria in particular, were more frequently observed, even at the early presymptomatic stage, than in the controls, and the frequency increased with time through the presymptomatic stages. On the other hand, the somata of large motor neurons directly connected with the axons did not exhibit any abnormal accumulation of neurofilaments or mitochondria. These findings suggest that both the slow axonal transport of neurofilaments and the fast axonal transport of mitochondria are impaired in AH and IS before the onset of disease in this animal model.     

Neuronal nitric oxide synthase immunoreactivity in the spinal cord in amyotrophic lateral sclerosis

We investigated the spinal cords of 15 patients with sporadic amyotrophic lateral sclerosis (ALS) immunohistochemically using an anti-human neuronal nitric oxide synthase (nNOS) antibody to examine whether there is increased nNOS immunoreactivity in anterior horn neurons. Specimens from 16 patients without any neurological disease served as controls. In the controls, nNOS immunoreactivity of large anterior horn neurons was detected in 10 out of 16 cases. However, there were few nNOS-positive neurons, and most of large anterior horn neurons were spared. In the ALS patients, the mean number of nNOS-positive anterior horn neurons per transverse section of L4 and L5 was significantly larger (16.2 +/- 10.9) than that in the controls (7.0 +/- 9.2) (P < 0.0001). Moreover, 41.4% of large anterior horn neurons in ALS showed nNOS immunoreactivity in remarkable contrast to 7.6% in the controls. All ALS patients, whether showing mild, moderate or severe depletion of anterior horn neurons, displayed a higher percentage of nNOS-positive anterior horn neurons than the control patients showing nNOS immunoreactivity (P < 0.01). Most of the remaining anterior horn neurons in ALS showed more intense nNOS immunoreactivity on the surface of the neurons and their neuronal processes compared with the controls. Degenerated anterior horn neurons frequently demonstrated more intense nNOS immunoreactivity on the surface of the neurons than normal-appearing neurons. Some anterior horn cells displayed nNOS immunoreactivity in the somata. Dot-like nNOS deposits on anterior horn neurons were also positively immunoreactive with anti-synaptophysin antibody. Thus, increased nNOS expression is located mainly at the synaptic sites on the anterior horn neurons in sporadic ALS, which may be related to the degeneration of anterior horn neurons in this disease. Further studies are needed to determine whether the increased nNOS immunoreactivity plays a neuroprotective or neurotoxic role in the anterior horn neurons, and to show nitric oxide production in ALS.     

Mitochondria covered with a net of parallel and latticed filaments in nigral neurons of monkeys with experimental parkinsonism

Summary Nigral neurons of crab-eating monkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) showed a peculiar configuration occasionally in mitochondria. The outer membrane of mitochondria was covered with a net of fine parallel or latticed filaments, which turned spirally about the long axis of the mitochondria. The filaments were approximately 8 nm in diameter: parallel filaments were arranged at intervals of about 13 nm from center to center; and latticed filaments intersected each other at an angle of almost 135°. When mitochondria were present in groups, the intermitochondrial gap was occasionally filled with the same parallel filaments. The netted mitochondria were frequently associated with intramitochondrial abnormalities such as small flocculent inclusions and disintegrated cristae. Only one or two netted mitochondria were counted in the perikaryon of one section of an injured neuron. They appeared in about one-third of mildly or moderately injured neurons in three of six MPTP-treated monkeys, and not in normal surviving and recovering neurons of treated animals, or in neurons of control animals. We consider the netted mitochondria to be a pathological configuration related to a metabolite of oxidation of MPTP, and to be different from the stubby mitochondria reported in amyotrophic lateral sclerosis (ALS) and a non-ALS case.Supported in part by a grant from the CNS Degenerative Diseases Research Committee, the Ministry of Health and Welfare of Japan     

Anterior horn changes of motor neuron disease associated with demyelinating radiculopathy

Morphologic study of the spinal cord of a patient with generalized motor deficits revealed changes in the anterior horns characterized by the selective loss of large motor neurons, gliosis and the abnormal accumulation of 10 nm filaments which appeared as argyrophilic spheroids in the perikarya and axons of motor neurons. The ventral roots were predominantly affected and showed a variable loss of axons. The remaining axons displayed prominent onion-bulb formations, frequent axonal sprouting and occasionally evidence of active demyelination. The coexistence of a demyelinating motor radiculopathy and anterior horn changes simulating those of amyotrophic lateral sclerosis (ALS) may contribute to our understanding of the unresolved question of whether the neuronal perikaryon or its axon is the primary target in the pathogenesis of ALS. These observations also indicate that a rigid separation of pathogenetic mechanisms into neuronopathy, axonopathy and myelinopathy may not be always possible.     

Ultrastructural study of mitochondria in the spinal cord of transgenic mice with a G93A mutant SOD1 gene

The purpose of this study was to examine mitochondrial changes in the spinal cord of transgenic mice of a relatively low transgenic copy number (gene copy 10) expressing a G93A mutant human Cu/Zn superoxide dismutase (SOD1) that were generated in our own laboratories by electron and immunoelectron microscopy from presymptomatic to symptomatic stages. Age-matched non-transgenic mice served as controls at each stage. Ultrastructurally, at the early presymptomatic stage, many mitochondria in large myelinated axons exhibited swelling with an increased number of cristae, and bore small vacuoles in the matrix, cristae or both, in the anterior root exit zone, anterior root, and in the neuropils of the ventral portion of the anterior horn. At the late presymptomatic stage, vacuoles of various sizes (including large ones) were observed in the same regions as in the previous stage. The intermembrane space of mitochondria was also vacuolated. In mitochondria with advanced vacuolation, the vacuolar space was filled with a granular or amorphous substance. At the symptomatic stage, mitochondrial vacuolation seen in the late presymptomatic stage persisted, although to a lesser extent. These vacuolated mitochondria were predominantly seen in the axons, but not in the somata of normal-looking neurons or dendrites at any stage, which differs from that described in other reports. Non-transgenic littermates occasionally exhibited vacuolar changes in the axons of anterior horns. However, they were smaller both in size and number than those in transgenic mice. By immunoelectron microscopy using an immunogold labeling method, at the presymptomatic and symptomatic stages both SOD1 and ubiquitin determinants were localized in vacuolated mitochondria, particularly in the granular or amorphous substance of large vacuoles, but were not detected in most normal-appearing mitochondria. The SOD1-immunoreactive mitochondria were exclusively observed in the axons, and not in proximal dendrites or somata. These findings suggest that the toxicity of mutant SOD1 directly affects mitochondria in the axons and increases with the disease progression. Thus, the mutant SOD1 toxicity might disrupt axonal transport of substrates needed for neuronal viability, leading to motor neuron degeneration. The localization of both ubiquitin and SOD1 in vacuolated mitochondria indicates that protein degradation by ubiquitin-proteasomal system may be also disrupted by several pathomechanisms, such as decreased processing of ubiquitinated proteins due to impairment of mitochondrial function or of proteasomal function, both of which are caused by mutant SOD1. Moreover, giant mitochondrial vacuoles occupying almost the entire axonal caliber could be another contributing factor in motor neuron degeneration, in that they could physically block axonal transport.     

The mitochondrial permeability transition pore in motor neurons: Involvement in the pathobiology of ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons (MNs) that causes paralysis. Some forms of ALS are inherited, caused by mutations in the superoxide dismutase-1 (SOD1) gene. The mechanisms of human mutant SOD1 (mSOD1) toxicity to MNs are unresolved. Mitochondria in MNs might be key sites for ALS pathogenesis, but cause–effect relationships between mSOD1 and mitochondriopathy need further study. We used transgenic mSOD1 mice to test the hypothesis that the mitochondrial permeability transition pore (mPTP) is involved in the MN degeneration of ALS. Components of the multi-protein mPTP are expressed highly in mouse MNs, including the voltage-dependent anion channel, adenine nucleotide translocator (ANT), and cyclophilin D (CyPD), and are present in mitochondria marked by manganese SOD. MNs in pre-symptomatic mSOD1-G93A mice form swollen megamitochondria with CyPD immunoreactivity. Early disease is associated with mitochondrial cristae remodeling and matrix vesiculation in ventral horn neuron dendrites. MN cell bodies accumulate mitochondria derived from the distal axons projecting to skeletal muscle. Incipient disease in spinal cord is associated with increased oxidative and nitrative stress, indicated by protein carbonyls and nitration of CyPD and ANT. Reducing the levels of CyPD by genetic ablation significantly delays disease onset and extends the lifespan of G93A-mSOD1 mice expressing high and low levels of mutant protein in a gender-dependent pattern. These results demonstrate that mitochondria have causal roles in the disease mechanisms in MNs in ALS mice. This work defines a new mitochondrial mechanism for MN degeneration in ALS.     

Accumulation of Neurofilaments in a Sporadic Case of Amyotrophic Lateral Sclerosis

Abstract: We report a case of sporadic amyotrophic lateral sclerosis (ALS) characterized by a marked accumulation of neurofilaments in the cytoplasm of neurons. The neurofilament was identified by immunohistochemical and electron microscopic studies. The distribution of the accumulation in this case was unique, not only in the motoneurons of the anterior horn but also in the neurons of the other areas of the spinal gray matter, some nuclei in the brain stem, pontine reticular formation, substantia nigra, dentate nucleus in the cerebellum and pyramidal cells in the motor cortex. These observations shed light on the pathogenesis of ALS.     

Accumulation of neurofilaments in a sporadic case of amyotrophic lateral sclerosis

We report a case of sporadic amyotrophic lateral sclerosis (ALS) characterized by a marked accumulation of neurofilaments in the cytoplasm of neurons. The neurofilament was identified by immunohistochemical and electron microscopic studies. The distribution of the accumulation in this case was unique, not only in the motoneurons of the anterior horn but also in the neurons of the other areas of the spinal gray matter, some nuclei in the brain stem, pontine reticular formation, substantia nigra, dentate nucleus in the cerebellum and pyramidal cells in the motor cortex. These observations shed light on the pathogenesis of ALS.     

Ultrastructural study of aggregates in the spinal cord of transgenic mice with a G93A mutant SOD1 gene

The ultrastructural features of SOD1-positive aggregates were determined to clarify whether these aggregates are associated with the pathogenesis of SOD1 mutant mice. We examined the spinal cord of transgenic mice expressing a G93A mutant human SOD1 gene with fewer copies (gene copy 10). At the early presymptomatic stage (age 24 weeks), SOD1- and ubiquitin-positive granular, linear, or round deposits were found occasionally in the neuropil of the anterior horns. Ultrastructurally, small filamentous aggregates were observed occasionally in the neuronal processes including the axons in the anterior horns. At the late presymptomatic stage (28 weeks), SOD1- and ubiquitin-positive deposits and Lewy body-like inclusions (LIs) were frequently demonstrated in the neuronal processes including cord-like swollen axons and in some remaining anterior horn neurons. Ultrastructurally, larger filamentous aggregates were frequent, predominating in the neuronal processes of the anterior horns including the proximal axons, but were rare in the somata and dendrites. The aggregates usually consisted of interwoven intermediate filaments (about 10–15 nm in diameter) and frequently contained electron-dense cores in the center resembling LIs. Occasionally the aggregates consisted mainly of granular, amorphous, or vesicular substance, showing fewer filamentous structures. At the symptomatic stages (32 and 35 weeks), LIs were frequently demonstrated within the neuronal processes in the anterior horns, particularly in the cord-like swollen axons. Many more prominent SOD1- and ubiquitin-positive deposits were observed over the whole white matter columns and in the gray matter of the anterior and posterior horns than at the previous stage. Ultrastructurally, aggregates frequently contained electron-dense cores, and were frequently observed in cord-like swollen axons consisting of accumulated neurofilaments. A high level of human SOD1-and ubiquitin-immunogold labeling was present in small to large aggregates even at the presymptomatic stages, and the aggregates increased in size and frequency with time. Compactly packed filaments and electron-dense cores of aggregates showed SOD1-and ubiquitin-immunogold labeling more prominently than in loosely packed filaments. These findings suggest that the accumulation of SOD1-positive aggregates in the neuronal processes, predominantly in the axons, constitutes an important determinant of neurotoxicity and the pathogenesis of this animal model, probably causing impairment of axonal transport by the sequestration of mutant SOD1 protein within aggregates, or in part by physically blocking the axonal transport.     

Increase in diameter of the axonal initial segment is an early change in amyotrophic lateral sclerosis

We measured the diameter of the most distal portion of the axonal initial segment, the neuronal size of anterior horn cells, and the length of the axon hillock plus the initial segment (AH+IS) in the lumbar spinal cord in motor neuron disease. Three patients with amyotrophic lateral sclerosis (ALS) and one with lower motor neuron disease (LMND) were compared with 11 controls. Serial plastic sections stained with toluidine blue and electron micrographs were studied. A total of 214 axons directly emanating from the somata (n = 207) and the primary dendrites (n = 7) were observed in the patients. Approximately 19% of the proximal myelinated axons (24 axons out of 155 in ALS, and 17 axons out of 59 in LMND) were swollen at the first internode, and most of the swellings extended to the middle portion of the initial segment. Electron microscopy showed that the swellings of the proximal axons (the initial segment and the first internode) directly connected with their somata consisted mainly of accumulations of 10-nm neurofilaments. The average diameter of the most distal initial segment was markedly larger in ALS (n = 155) (P < 0.0001) and LMND (n = 59) (P < 0.0001) than in the controls (n = 258). Moreover, the average diameter of the most distal portion of even normal-appearing initial segments of the non-swollen axons was larger in ALS (n = 131) (P < 0.0001) than in the controls. The perikarya and axon hillocks connected with the normal-appearing and swollen proximal axons and their dendrites almost always appeared normal. These findings suggest that increase in diameter of the axonal initial segment which reflects the abnormal accumulation of neurofilaments represents an early pathological change in motor neuron disease and that the slow axonal transport of neurofilaments is probably impaired in this portion of the axon at an early stage in the disease process. The average size of the normal-appearing cell bodies from which axons emanated was smaller in ALS (P < 0.0001) and LMND (P < 0.0001) than in the controls. There was no significant difference in the AH+IS length among ALS having normal-appearing initial segment, LMND, and controls.     

Immunohistochemical and ultrastructural study of basophilic inclusions in adult-onset motor neuron disease

We immunohistochemically and ultrastructurally studied basophilic inclusions (BI) in a patient with adult-onset sporadic motor neuron disease (MND). BI were frequently observed not only in degenerated anterior horn cells, such as central chromatolytic neurons, but also in normal-appearing large anterior horn neurons. They had various shapes, round, elliptical or irregular, and occasionally they had distinct basophilic rims. They also varied in size. There were no halos around them nor core in their centers. Immunohistochemically, some BI were immunostained for ubiquitin or SOD1, but BI were not immunoreactive with anti-phosphorylated neurofilament (SMI 31), phosphorylated tau, cystatin C or Golgi (MG-160) antibodies. Ubiquitin-positive skein-like inclusions (SI) were occasionally observed in the somata of anterior horn neurons. Ultrastructurally, BI consisted of filamentous structures associated with granules, which were attached to thick filaments. The thick filaments were straight without constriction or side arms and their diameter was twice that of the neurofilaments. BI occasionally contained tubular structures among the granule-associated filaments. The granulo-filamentous profiles varied from being compactly arranged to being more loosely packed. The structure of BI resembles that of the Lewy body-like hyaline inclusions (LBHI) observed in sporadic MND patients. Bundles of filaments resembling SI, which were composed of compactly packed filaments without fine granules running parallel to the longitudinal axis, were frequently observed inside or at the periphery of BI, and occasionally clustered in the perikarya. Each filament measured approximately 15-25 nm in diameter, and a bundle of these grouped filaments was sometimes surrounded by a unit membrane. We also occasionally observed in-between structures of BI and bundles of filaments resembling SI. These findings suggest a certain relationship between BI, SI and LBHI in the pathomechanism of BI development. Further studies are needed to elucidate whether sporadic adult-onset MND characterized by BI forms a different subtype of MND.     

Fine structure of anterior horns in patients without amyotrophic lateral sclerosis

Light and electron microscopic study of the anterior horns in nine patients without amyotrophic lateral sclerosis (ALS) disclosed several features usually described in ALS. Central chromatolysis and spheroids with fine structure identical to that seen in ALS were observed in three cases. Rare honeycomb-like structures, dense filamentous aggregates, dense granular deposits, stubby mitochondria and membrane-bound aggregates of spherical and tubular particles were also observed. No typical Bunina bodies or focal argyrophilia of the chromatolytic neurons were found.     

Ubiquitin-immunoreactive filamentous inclusions in anterior horn cells of Guamanian and non-Guamanian amyotrophic lateral sclerosis

Summary Immunohistochemical studies with an antibody to ubiquitin revealed the presence of filamentous inclusions in spinal anterior horn cells in all of six patients with Guamanian amyotrophic lateral sclerosis (ALS) and one of six cases of parkinsonism-dementia complex (PD) on Guam. Similar ubiquitin-reactive filamentous inclusions were found in all of seven non-Guamanian sporadic ALS patients examined. No similar inclusions were seen in six normal controls or in non-ALS patients who had chromatolytic neurons. The filamentous inclusions differed from spinal neurofibrillary tangles, a characteristic feature of Guamanian ALS and PD, since they were restricted to anterior horn cells and did not react with anti-tau antibody. The chromatolytic neurons of non-ALS patients occasionally had weak diffuse immunoreactivity, but no focal inclusions were detected. These results suggest that ubiquitin-reactive focal filamentous inclusions may reflect a characteristic degenerative process of anterior horn cells of motor neuron disease.     

Neuropathology of ALS: Spheroids

I briefly review spheroids observed in the anterior horns of the spinal cord in amyotrophic lateral sclerosis (ALS). Spheroids are argentophilic bodies more than 20 μm in diameter. Recently, some connections between the proximal axonal swellings including spheroids and the perikarya have been reported in some ALS patients with a short clinical course or mild depletion of anterior horn neurons. Most of the cell bodies directly connected with the axonal swellings appear normal, and spheroids are considered to be one of the hallmarks of the early histological changes in this disorder. Spheroids are strongly positive with anti-phosphorylated neurofilament antibody, and are also positive with calcitonin gene-related peptide and anti-peripherin antibody. Some spheroids are immunostained with anti-synaptophysin antibody and anti-ubiquitin antibody. Spheroids are not immunostained with anti-phosphorylated tau antibody, or high molecular weight microtubule associated proteins. Electron microscopically, spheroids are usually composed of densely packed accumulation of 10 nm neurofilaments with a variety of orientations, plus vesicles, dense bodies and mitochondria. When the swellings of the initial segment is relatively pronounced, the undercoating is obscured and the neurofilaments become interwoven in some parts. In the first internode of the myelinated axons, as the swellings become larger, the neurofilaments lose their parallel orientation and become intermingled. Large accumulation of neurofilaments resembling spheroids in the perikarya of large anterior horn cells suggests that spheroids could be derived not only from the axon including the proximal portion, but also from the perikarya. Structures apparently identical to axonal spheroids are observed at the light and electron microscopic levels in the proximal portion of axons of anterior horn cells in animal models intoxicated with β, β'-iminodipropionitrile (IDPN), or with aluminum, in hereditary canine spinal muscular atrophy (HCSMA). The pathogenetic mechanism is probably associated with an impairment in slow axonal transport which particularly affects the neurofilaments in IDPN and aluminum intoxication. Impairment of slow axonal transport of neurofilaments also plays an important role in the pathogenesis of ALS. The average diameter of even normalappearing initial segment is larger in ALS than in the controls. The perikarya connected with the swollen proximal axons and their dendrites almost always appear normal. These findings suggest that the slow axonal transport of neurofilaments is probably impaired in this portion of the axon at an early stage in ALS as well as animal models for human ALS. However, techniques to analyze slow axonal transport in humans still remain tobe developed. Recently, overexpression of neurofilament subunits in transgenic mice produces a condition resembling ALS. The transgenic model may offer an interesting perspective not only for testing therapeutic strategies but also for investigating in a systematic way the various genetic and environment factors controlling the onset and progression of the disease and might yield new insights on the etiology of ALS.     

Immunoreactivity of β-amyloid precursor protein in amyotrophic lateral sclerosis

We investigated the localization and extent of β-amyloid precursor protein (β-APP₆₉₅) immunoreactivity as a sensitive marker for impairment of fast axonal transport in the spinal cords of 21 patients with amyotrophic lateral sclerosis (ALS), paying special attention to anterior horn neurons. Specimens from 18 patients without neurological disease served as controls. Increased β-APP immunoreactivity was frequently recognized in the anterior horns of the ALS patients with short clinical courses or with mild depletion of anterior horn cells, while no β-APP immunoreactivity was demonstrated in those with severe depletion of anterior horn neurons or with long-standing clinical courses. Increased β-APP immunoreactivity in the anterior horn neurons was mainly confined to the perikarya and no immunoreactivity was recognized in the dendrites or proximal axons directly emanating from the somata, except some spheroids (proximal axonal swellings) which showed increased immunoreactivity of β-APP. Increased β-APP immunoreactivity was spotted or focally aggregated in the perikarya of normal-looking large anterior horn neurons, while it was frequently diffuse in that of degenerative neurons such as central chromatolytic cells and or those with simple atrophy. On the other hand, the controls showed no immunostaining with β-APP in the spinal cord. These findings suggest that increased immunoreactivity of β-APP in neuronal perikarya of the anterior horn cells and in some proximal axonal swellings is an early change of ALS, and may be a response of the increased synthesis of β-APP resulting from neuronal damage, or the impairment of fast axonal transport. Received: 27 August 1998 / Revised, accepted: 4 November 1998