Lewy Body-like Inclusions Research Articles

NEDL1, a Novel Ubiquitin-protein Isopeptide Ligase for Dishevelled-1, Targets Mutant Superoxide Dismutase-1

Approximately 20% of familial amyotrophic lateral sclerosis (FALS) arises from germ-line mutations in the superoxide dismutase-1 (SOD1) gene. However, the molecular mechanisms underlying the process have been elusive. Here, we show that a neuronal homologous to E6AP carboxyl terminus (HECT)-type ubiquitin-protein isopeptide ligase (NEDL1) physically binds translocon-associated protein-delta and also binds and ubiquitinates mutant (but not wild-type) SOD1 proportionately to the disease severity caused by that particular mutant. Immunohistochemically, NEDL1 is present in the central region of the Lewy body-like hyaline inclusions in the spinal cord ventral horn motor neurons of both FALS patients and mutant SOD1 transgenic mice. Two-hybrid screening for the physiological targets of NEDL1 has identified Dishevelled-1, one of the key transducers in the Wnt signaling pathway. Mutant SOD1 also interacted with Dishevelled-1 in the presence of NEDL1 and caused its dysfunction. Thus, our results suggest that an adverse interaction among misfolded SOD1, NEDL1, translocon-associated protein-delta, and Dishevelled-1 forms a ubiquitinated protein complex that is included in potentially cytotoxic protein aggregates and that mutually affects their functions, leading to motor neuron death in FALS.

Conditional control of human wild-type and Parkinson's disease-associated mutant alpha-synuclein in transgenic mouse brain

The presynaptic protein alpha-synuclein has been implicated in the pathophysiology of many neurodegenerative disorders, including Parkinson's Disease and dementia with Lewy bodies, collectively referred to as synucleinopathies. To advance the study of the function of alpha-synuclein in these diseases, we have used the tTA-system to generate transgenic mice, which express human wildtype or mutated [A30P] alpha-synuclein in a conditional manner. To obtain reversible expression of human alpha-synuclein in the brain of these mice, we combined the tTA-system with both the PrP promotor and CaMKIIalpha promotor. We investigated the conditional overexpression by western blot analysis and immunostaining of paraffin-embedded brains. Western blot analysis revealed that double-transgenic mice express alpha-synuclein at different levels in specific brain regions. The expression-pattern and level of human alpha-synuclein in the brain of double-transgenic mice depends on both the neuron-specific promotor and the integration site of the human alpha-synuclein construct. Histological analysis of the brain of transgenic mice showed aberrant expression of the protein in cell soma. However, Lewy body-like alpha-synuclein inclusions have not yet been identified. Administration of doxycycline down-regulates alpha-synuclein expression to basal levels in the brain of double-transgenic mice. We currently investigate if one transgenic mouse-line which highly expresses mutated [A30P] alpha-synuclein specifically in the olfactory bulb shows any loss of dopaminergic neurons and an impaired sense of smell. We also perform microarray expression analysis to gain insight in the pathomechanism underlying over-expression of human alpha-synuclein. Our conditional mouse-model may help to define the role of human alpha-synuclein in synucleinopathies and might be used to demonstrate whether neuropathological symptoms of these diseases are reversible.

Expression of peripherin in ubiquinated inclusions of amyotrophic lateral sclerosis

We evaluated the expression of the type III intermediate filament (IF) protein, peripherin (PRP), in ubiquinated inclusions of motor neurons in amyotrophic lateral sclerosis (ALS). A previous study showed that overexpression of PRP in transgenic mice induces motor neuron disease with formation of PRP-containing inclusions before onset of symptoms [J. Cell Biol. 147 (3) (1999) 531]. To determine whether PRP inclusions occur in the human disease, we applied doublelabeling immunofluorescence to paraffin sections of the spinal cord obtained by autopsy of 40 ALS patients with sporadic disease and 39 controls. Inclusions that expressed immunoreactive ubiquitin and peripherin were recorded by video camera, and the sections were stained by hematoxylin and eosin (H&E) to define morphology. Lewy body-like inclusions (LBLIs) were seen in motor neuron perikarya of 9 of 40 ALS cases and none in controls; all LBLIs expressed peripherin. Skein-like inclusions (SLIs) were identified by ubiquitin, but did not express PRP with rare exceptions. Neither skein-like inclusions nor LBLIs expressed αB-crystallin, neurofilament protein (NF-L, NF-M and NF-H subunits), α-internexin, actin or α-synuclein. Immunoblot of the whole spinal cord exhibited a single 57-kDa band of peripherin in ALS patients and controls. Our data document the expression of peripherin in LBLIs, which may provide a clue to the pathogenesis of neurodegeneration in ALS.

Rotenone increases glutamate-induced dopamine release but does not affect hydroxyl-free radical formation in rat striatum.

Impairment of the mitochondrial complex I has been found in Parkinson's disease and recently long-term treatment with the complex I inhibitor rotenone led to neurodegeneration and Lewy body-like inclusions in rats. To investigate the relationship of free radical formation, complex I inhibition, and dopamine release, rotenone (15 mg/kg s.c.) was injected in male Sprague Dawley rats. Complex I inhibition was measured in the striatum and substantia nigra using the lactate accumulation assay. Dopamine release and free radical formation was determined using striatal microdialysis in combination with the salicylate hydroxylation assay. In a second experiment, glutamate (10 mM) stimulation via the microdialysis probe was used to provoke hydroxyl radical formation and dopamine release 60 min after rotenone or vehicle pretreatment. Rotenone significantly increased striatal and nigral lactate levels. However, rotenone did not produce a significant increase in hydroxyl radical formation and dopamine release, but led to a pronounced hypokinesia. In contrast, rotenone in comparison to vehicle pretreatment produced a significant augmentation of glutamate-induced dopamine release (67-fold and 31-fold increase, respectively) and did not affect the glutamate-induced hydroxyl free radical formation (23-fold and 21-fold increase, respectively). The present study demonstrates that a single systemic rotenone administration does not lead to neurotoxicity, but rather to enhanced glutamate-induced dopamine release with no further increase of hydroxyl free radical formation. Thus, acute complex I inhibition in the presence or absence of high extracellular dopamine and glutamate levels is not critically involved in the formation of hydroxyl free radicals.

Association of the Cytoskeletal GTP-binding Protein Sept4/H5 with Cytoplasmic Inclusions Found in Parkinson's Disease and Other Synucleinopathies

alpha-Synuclein-positive cytoplasmic inclusions are a pathological hallmark of several neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Here we report that Sept4, a member of the septin protein family, is consistently found in these inclusions, whereas five other septins (Sept2, Sept5, Sept6, Sept7, and Sept8) are not found in these inclusions. Sept4 and alpha-synuclein can also be co-immunoprecipitated from normal human brain lysates. When co-expressed in cultured cells, FLAG-tagged Sept4 and Myc-tagged alpha-synuclein formed detergent-insoluble complex, and upon treatment with a proteasome inhibitor, they formed Lewy body-like cytoplasmic inclusions. The tagged Sept4 and alpha-synuclein synergistically accelerated cell death induced by the proteasome inhibitor, and this effect was further enhanced by expression of another Lewy body-associated protein, synphilin-1, tagged with the V5 epitope. Moreover, co-expression of the three proteins (tagged Sept4, alpha-synuclein, and synphilin-1) was sufficient to induce cell death. These data raise the possibility that Sept4 is involved in the formation of cytoplasmic inclusions as well as induction of cell death in alpha-synuclein-associated neurodegenerative disorders.

Expression of hepatocyte growth factor and c-Met in the anterior horn cells of the spinal cord in the patients with amyotrophic lateral sclerosis (ALS): immunohistochemical studies on sporadic ALS and familial ALS with superoxide dismutase 1 gene mutation.

To clarify the trophic mechanism of residual anterior horn cells affected by sporadic amyotrophic lateral sclerosis (SALS) and familial ALS (FALS) with superoxide dismutase 1 (SOD1) mutations, we investigated the immunohistochemical expression of hepatocyte growth factor (HGF), a novel neurotrophic factor, and its receptor, c-Met. In normal subjects, immunoreactivity to both anti-HGF and anti-c-Met antibodies was observed in almost all anterior horn cells, whereas no significant immunoreactivity was observed in astrocytes and oligodendrocytes. Histologically, the number of spinal anterior horn cells in ALS patients decreased along with disease progression. Immunohistochemically, the number of neurons negative for HGF and c-Met increased with ALS disease progression. However, throughout the course of the disease, certain residual anterior horn cells co-expressed both HGF and c-Met with the same, or even stronger intensity in comparison with those of normal subjects, irrespective of the reduction in the number of immunopositive cells. Western blot analysis revealed that c-Met was induced in the spinal cord of a patient with SALS after a clinical course of 2.5 years, whereas the level decreased in a SALS patient after a clinical course of 11 years 5 months. These results suggest that the autocrine and/or paracrine trophic support of the HGF-c-Met system contributes to the attenuation of the degeneration of residual anterior horn cells in ALS, while disruption of the neuronal HGF-c-Met system at an advanced disease stage accelerates cellular degeneration and/or the process of cell death. In SOD1-mutated FALS patients, Lewy body-like hyaline inclusions (LBHIs) in some residual anterior horn cells exhibited co-aggregation of both HGF and c-Met, although the cytoplasmic staining intensity for HGF and c-Met in the LBHI-bearing neurons was either weak or negative. Such sequestration of HGF and c-Met in LBHIs may suggest partial disruption of the HGF-c-Met system, thereby contributing to the acceleration of neuronal degeneration in FALS patients.

Alpha-synuclein pathology affecting Bergmann glia of the cerebellum in patients with alpha-synucleinopathies.

We carried out immunohistochemical examinations of the brains (cerebella) of patients who had suffered from Parkinson's disease (PD), diffuse Lewy body disease (DLBD) or multiple system atrophy (MSA), using antibodies specific for alpha-synuclein. Alpha-synuclein-positive doughnut-shaped structures were found occasionally in the cerebellar molecular layer in some of these patients. Double-labeling immunofluorescence and immunoelectron microscopy studies revealed that these alpha-synuclein-positive doughnut-shaped structures were located in the glial fibrillary acidic protein-positive radial processes of Bergmann glia, corresponding to the outer area of Lewy body-like inclusions, and consisted of granulo-filamentous structures. These findings indicate that, although not frequently, Bergmann glia of the cerebellum are also the targets of alpha-synuclein pathology in alpha-synucleinopathies such as PD, DLBD and MSA.

Motor dysfunction and gliosis with preserved dopaminergic markers in human α-synuclein A30P transgenic mice

α-Synuclein is a major component of Lewy bodies (LBs) in the substantia nigra and cortex in Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), and in glial inclusions in multiple systems atrophy (MSA). Mutations in α-synuclein have been associated with autosomal dominant forms of PD. We investigated the clinical and neuropathological effects of overexpression of human α-synuclein, α-synuclein A30P, and α-synuclein A53T under the control of the hamster prion protein (PrP) promoter; 5–15× endogenous levels of protein expression were achieved with widespread neuronal, including nigral, transgene expression. High expression of α-synuclein A30P in the Tg5093 line was associated with a progressive motor disorder with rigidity, dystonia, gait impairment, and tremor. Histological analysis of this line showed aberrant expression of the protein in cell soma and progressive CNS gliosis, but no discrete Lewy body-like α-synuclein inclusions could be identified. Biochemical analysis demonstrated α-synuclein fragmentation. Despite strong expression of the transgene in the nigra, there was no specific deterioration of the nigrostriatal dopaminergic system as assessed by quantitation of nigral tyrosine hydroxylase (TH) containing neurons, striatal TH immunoreactivity, dopamine levels, or dopamine receptor number and function. Lower expressing lines had no specific behavioral or histopathological phenotype. Thus, high expression of mutant human α-synuclein resulted in a progressive motor and widespread CNS gliotic phenotype independent of dopaminergic dysfunction in the Tg5093 line.

Clinical features and neuropathological findings of familial amyotrophic lateral sclerosis with a His46Arg mutation in Cu/Zn superoxide dismutase

We examined the characteristic clinical features of one family of familial amyotrophic lateral sclerosis (FALS) with a His46Arg mutation in the enzyme Cu/Zn superoxide dismutase-1 (SOD1). The disease duration for this family was 18.1±13.2 (mean±S.D.) years, with the age at onset being 39.7±10.5 years old (mean±S.D.). The initial sign was distal weakness of the unilateral lower limb, extending to the lower limb of the other side. A wheel chair became necessary at 9.8±3.2 years after the onset. Upper limb weakness started at 15.5±8.9 years following from the onset. An autopsy was performed on a 71-year-old woman of the family with the mutation. Her disease duration was 47 years, and she died of pneumonia. She had no clear upper motor neuron involvement. Bulbar sign and respiratory muscle weakness had developed 2 years before her death. Neuropathological findings showed degeneration of corticospinal tracts, anterior/posterior spinocerebellar tracts, posterior columns, and Clarke's columns. There were few anterior horn cells in the lumbar spinal cord and no Lewy body-like hyaline inclusion bodies in these remaining anterior horn neurons. This is the first autopsy report of FALS with a His46Arg mutation in the SOD1 enzyme.

Erratum to: 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.

Copper chaperone for superoxide dismutase co-aggregates with superoxide dismutase 1 (SOD1) in neuronal Lewy body-like hyaline inclusions: an immunohistochemical study on familial amyotrophic lateral sclerosis with SOD1 gene mutation.

The copper chaperone for superoxide dismutase (CCS) interacts with Cu/Zn-binding superoxide dismutase 1 (SOD1) specifically and delivers copper to SOD1. To determine the role of the CCS-SOD1 interaction in the pathogenesis of SOD1-mutated familial amyotrophic lateral sclerosis (FALS) patients, we produced an affinity-purified rabbit antibody against CCS and investigated the immunohistochemical localization of both CCS and SOD1 in neuronal Lewy body-like hyaline inclusions (LBHIs) in the spinal cords of two FALS patients with a two-base pair deletion at codon 126 in the SOD1 gene and three FALS patients with an Ala to Val substitution at codon 4. The LBHIs in anterior horn cells from the five FALS patients showed identical immunoreactivities for CCS: the reaction product deposits with the antibody against CCS were generally restricted to the periphery of the core and halo-type LBHIs. The localizations of the immunoreactivities for CCS and SOD1 were similar in the inclusions: both CCS and SOD1 colocalized in neuronal LBHIs in the five mutant SOD1-linked FALS patients. Our results suggest that the specific interaction and aggregation of CCS-SOD1 (probably CCS-mutant SOD1) in SOD1-mutated FALS patients may amplify the formation of inclusions and emphasize a more marked mutant SOD1-mediated toxicity.

Induction of Neuronal Cell Death by Rab5A-dependent Endocytosis of α-Synuclein

The presynaptic alpha-synuclein is a prime suspect for contributing to Lewy pathology and clinical aspects of diseases, including Parkinson's disease, dementia with Lewy bodies, and a Lewy body variant of Alzheimer's disease. Here we examined the pathogenic mechanism of neuronal cell death induced by alpha-synuclein. The exogenous addition of alpha-synuclein caused a marked decrease of cell viability in primary and immortalized neuronal cells. The neuronal cell death appeared to be correlated with the Rab5A-specific endocytosis of alpha-synuclein that subsequently caused the formation of Lewy body-like intracytoplasmic inclusions. This was further supported by the fact that the expression of GTPase-deficient Rab5A resulted in a significant decrease of its cytotoxicity as a result of incomplete endocytosis of alpha-synuclein.

Lack of Nigral Pathology in Transgenic Mice Expressing Human α-Synuclein Driven by the Tyrosine Hydroxylase Promoter

α-Synuclein has been identified as a major component of Lewy body inclusions, which are one of the pathologic hallmarks of idiopathic Parkinson's disease. Mutations in α-synuclein have been found to be responsible for rare familial cases of Parkinsonism. To test whether overexpression of human α-synuclein leads to inclusion formation and neuronal loss of dopaminergic cells in the substantia nigra, we made transgenic mice in which the expression of wild-type or mutant (A30P and A53T) human α-synuclein protein was driven by the promoter from the tyrosine hydroxylase gene. Even though high levels of human α-synuclein accumulated in dopaminergic cell bodies, Lewy-type-positive inclusions did not develop in the nigrostriatal system. In addition, the number of nigral neurons and the levels of striatal dopamine were unchanged relative to non-transgenic littermates, in mice up to one year of age. These findings suggest that overexpression of α-synuclein within nigrostriatal dopaminergic neurons is not in itself sufficient to cause aggregation into Lewy body-like inclusions, nor does it trigger overt neurodegenerative changes.

Formation of advanced glycation end-product-modified superoxide dismutase-1 (SOD1) is one of the mechanisms responsible for inclusions common to familial amyotrophic lateral sclerosis patients with SOD1 gene mutation, and transgenic mice expressing human SOD1 gene mutation.

Neuronal Lewy body-like hyaline inclusions (LBHI) and astrocytic hyaline inclusions (Ast-HI) are morphological hallmarks of certain familial amyotrophic lateral sclerosis (FALS) patients with superoxide dismutase-1 (SOD1) gene mutations, and transgenic mice expressing the human SOD1 gene mutation. The ultrastructure of inclusions in both diseases is identical: the essential common constituents are granule-coated fibrils approximately 15-25nm in diameter and granular materials. Detailed immunohistochemical analyses have shown that the essential common protein of the inclusions in both diseases is an SOD1 protein. This finding, together with the immunoelectron microscopy finding that the abnormal granule-coated fibrils comprising the inclusions are positive for SOD1, indicates that these granule-coated fibrils containing SOD1 are important evidence for mutant SOD1-linked disease in human and mouse. For immunoelectron microscopy, the granule-coated fibrils are modified by advanced glycation endproducts (AGE) such as N(epsilon)-carboxymethyl lysine, pyrraline and pentosidine (Maillard reaction). Based on the fact that AGE themselves are insoluble molecules with direct cytotoxic effects, the granule-coated fibrils and granular materials are not digested by the lysosomal and ubiquitin systems. The neurons and astrocytes of the normal individuals and non-transgenic mice show no significant immunoreactivity for AGE. Considered with the mutant-SOD1 aggregation toxicity, a portion of the SOD1 comprising both types of the inclusion is modified by the AGE, and the formation of the AGE-modified SOD1 (probably AGE-modified mutant SOD1) is one of the mechanisms responsible for the aggregation (i.e. granule-coated fibril formation).

Advanced glycation endproduct-modified superoxide dismutase-1 (SOD1)-positive inclusions are common to familial amyotrophic lateral sclerosis patients with SOD1 gene mutations and transgenic mice expressing human SOD1 with a G85R mutation.

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.

Nonoxidative protein glycation is implicated in familial amyotrophic lateral sclerosis with superoxide dismutase-1 mutation.

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.

Aggregates of mutant protein appear progressively in dendrites, in periaxonal processes of oligodendrocytes, and in neuronal and astrocytic perikarya of mice expressing the SOD1 G93A mutation of familial amyotrophic lateral sclerosis

Mice expressing the G93A and other mutations of Cu,Zn superoxide dismutase (SOD1 G93A) are valid models for the familial form of amyotrophic lateral sclerosis (FALS) with SOD1 mutations and, probably, for sporadic ALS. Adult mice become progressively paralyzed and show most of the histopathological lesions reported in sporadic ALS, i.e. neuronal loss, astrogliosis, ubiquitin and Lewy body-like inclusions, dystrophic axons and fragmentation of the Golgi apparatus (GA) of motor neurons. In transgenic mice, the mutant protein and ubiquitin aggregate within pathological 13 nm thick filaments [Stieber A, Gonatas JO, Gonatas NK. J Neurol Sci 2000;173:53–62]. This immunocytochemical and quantitative study of mice expressing SOD1 G93A establishes the chronological order and cellular localization of aggregates of SOD1 and their correlation with fragmentation of the GA. Young asymptomatic mice expressing SOD1 G93A showed aggregates of mutant SOD1 within neurites, prior to the detection of SOD1 in the perikarya of spinal cord motor neurons and astrocytes. Both dendrites and the periaxonal oligodendroglial cytoplasm, surrounding atrophic axons, contained SOD1 as revealed by immunoelectron microscopy The perikarya of a small percentage of spinal cord motor neurons contained both fragmented GA and aggregates of SOD1; however, about 50% of motor neurons with fragmented GA did not contain SOD1 in the perikaryon, suggesting that aggregates of mutant protein may not directly cause fragmentation of the GA. The mechanism of the putative toxic effect by the mutant protein remains to be clarified. The isolation and biochemical characterization of the filamentous aggregates of mutant protein and ubiquitin from spinal cords of transgenic mice expressing mutations of the SOD1 gene may offer clues on pathogenetic mechanisms.

Immunocytochemical and ultrastructural study of the motor cortex in patients with lower motor neuron disease

This report conveys the results of an immunocytochemical and ultrastructural study of the motor cortices of six patients with clinically and pathologically-diagnosed lower motor neuron disease (LMND) such as progressive spinal muscular atrophy, progressive bulbar palsy, or both. These patients showed neither upper motor neuron signs nor upper motor neuron system involvement including the corticospinal tract in postmortem tissues after conventional stainings. Specimens from 12 age-matched normal individuals served as controls. All patients showed loss of brainstem motor neurons and anterior horn cells. Betz cells in LMND patients were significantly reduced in number as compared to controls ( P<0.01). However, there was no significant difference in the density of phosphorylated neurofilament (PNF) (200 kDa)-positive Betz cells between LMND patients and controls. The pyramidal cells of layer III were immunostained for PNF in four of six LMND patients, but there was no significant difference in the density of PNF-positive pyramidal cells between LMND patients and controls. The number of astrocytes immunostained for glial fibrillary acidic protein increased in layer III and at the transition between white matter and motor cortex in three out of six patients and one of 12 controls. Ultrastructural examination revealed that the Betz cells of five of six LMND patients had Bunina bodies, Lewy body-like inclusions or skein-like inclusions, all of which are characteristic of amyotrophic lateral sclerosis (ALS). These findings suggest that most patients with clinically and pathologically-diagnosed LMND should be classified into the category of ALS.

Motor neuronal loss and neurofilament-ubiquitin alteration in MoMuLV-ts1 encephalopathy.

A temperature-sensitive mutant of Moloney murine leukemia virus (MoMuLV-ts1) induces immunosuppression and spongiform encephalopathy in susceptible newborn mice. The associated neuronal degeneration primarily involves the motor neurons in specific target areas of the central nervous system (CNS). Neuronal loss occurs in the absence of direct viral infection of neurons and is the most dramatic pathological change in the CNS of infected mice. To quantitatively demonstrate neuronal loss, an unbiased morphometric stereological study was undertaken using the optical disector method. Using highly susceptible FVB/N mice, neuronal loss was quantitated in the tissue sections of brain stem from infected and noninfected mice at 20 and 35 days post inoculation (dpi). Results indicated that there was no significant neuronal loss at 20 dpi, but significant (P < 0.05) at 35 dpi. In addition, histology, transmission electron microscopy and immunohistochemistry revealed Lewy body-like inclusions consisting of aggregates of neurofilaments and cellular organelles. Degenerated neurons and glial cells were heavily ubiquitinated. Together, these results suggest that significant neuronal loss occurs at the end of the disease process and that Lewy body-like formation and protein ubiquitination are part of the pathogenic process in ts1-induced encephalopathy.

Neuropathology of amyotrophic lateral sclerosis with extra-motor system degeneration: characteristics and differences in the molecular pathology between ALS with dementia and Guamanian ALS.

Amyotrophic lateral sclerosis (ALS) is classified into distinct subtypes mainly based on clinicopathological features, in addition to epidemiologic and genetic backgrounds. In addition to sporadic ALS with classical pathology, characteristics in the clinical features, in the histological findings and their topographical distribution, and in the molecular pathology, especially the intracytoplasmic neuronal inclusions, enable us to identify the following subtypes: ALS with dementia (ALS-D), ALS in the Western Pacific, ALS with multi-system degeneration, familial ALS, and superoxide dismutase 1-linked ALS. These subtypes not infrequently exhibit various types of extra-motor system degeneration, and even multi-system pathology. Some of the subtypes (for instance ALS-D or familial ALS) can be deduced, to a certain extent, from characteristic neuronal inclusions such as ALS-type ubiquitinated inclusions, Lewy body-like hyaline inclusions (LBHIs), or ubiquitinated intracytoplasmic neuronal inclusions as typically seen in the dentate fascia. The purpose of this article is to clarify the molecular pathogenesis of the cerebral cortex in ALS-D and Guamanian ALS and parkinsonism-dementia complex (PDC), in order to elucidate the relationship and distinction between these two subtypes. As indicated previously, investigations on ubiquitin-immunoreactivity in the hippocampus further support the view that the pathology of G-ALS/PDC may be that of a tau-related tangle disorder, whilst ALS-D has a feature of the motor neuron disease type-frontotemporal dementia.