Multiple System Atrophy Brains Research Articles

α-synuclein fibrils constitute the central core of oligodendroglial inclusion filaments in multiple system atrophy

Multiple system atrophy (MSA) belongs to synucleinopathies and is characterized pathologically by oligodendroglial inclusions (GCIs) composed of 20- to 30-nm tubular filaments. α-Synuclein fibrils formed in vitro, however, range between 10 and 12 nm in diameter. To understand the relationship between α-synuclein and GCI filaments, we conducted structural analyses of GCIs in fixed brain sections and isolated from fresh-frozen MSA brains. In fixed brain sections, GCIs were composed of amorphous material-coated filaments up to 30 nm in size. The filaments were often organized in parallel bundles extending into oligodendroglial processes. In freshly isolated GCIs, progressive buffer washes removed amorphous material and revealed that GCI filaments consisted of 10-nm-sized central core fibrils that were strongly α-synuclein immunoreactive. Image analysis revealed that each core fibril was made of two subfibrils, and each subfibril was made of a string of 3- to 6-nm-sized particles probably α-synuclein oligomers. Immunogold labeling demonstrated that epitopes encompassing entire α-synuclein molecule were represented in the core fibrils, with the N-terminal 11–26 and C-terminal 108–131 amino acid residues most accessible to antibodies, probably exposed on the surface of the fibril. Our study indicates that GCI filaments are multilayered in structure, with α-synuclein oligomers forming the central core fibrils of the filaments.

Analysis of the expression level of alpha-synuclein mRNA using postmortem brain samples from pathologically confirmed cases of multiple system atrophy.

To determine whether multiple system atrophy (MSA) is associated with altered expression levels of the alpha-synuclein messenger RNA (mRNA), we performed quantitative reverse transcription polymerase chain reaction for alpha-synuclein mRNA using postmortem brain samples from 11 cases of MSA and 14 age-matched control subjects. The brain specimens used in this study contained both the gray matter and white matter, which were dissected from the frontal, temporal or occipital lobe. The expression levels of alpha-synuclein mRNA in the brain specimens of MSA cases were not different from those of the control subjects. These results suggest that the transcriptional regulation of the alpha-synuclein gene is unlikely to be affected in MSA brains.

Midkine, a new neurotrophic factor, is present in glial cytoplasmic inclusions of multiple system atrophy brains.

The glial cytoplasmic inclusion (GCI) is a histological hallmark for multiple system atrophy (MSA): these inclusions are found in oligodendrocytes and consist of abnormal granule-coated fibrils of approximately 24- to 40-nm diameter. To clarify the significance of the presence of midkine (MK) in these GCIs, we carried out immunohistochemical, electron and immunoelectron microscopical, and Western blot analyses of MSA brains using a monoclonal antibody against the C-terminal region of human MK. Immunohistochemically, most of the GCIs were intensely stained by the antibody to MK. Electron and immunoelectron microscopy showed that the GCIs were composed of MK-positive granule-coated fibrils that were essential constituents of these inclusions. No significant MK immunoreactivity was observed in oligodendrocytes, astrocytes and neurons of the normal control subjects. The presence of MK in MSA brain but not in normal brain was confirmed by Western blotting. Together with the fact that MK is associated with fetal morphogenesis during the midgestation period, the presence of MK immunoreactivity in oligodendroglial GCIs may suggest the existence of a repair mechanism on the basis of morphogenesis in the degenerated oligodendrocytes themselves as well as the affected neurons and their axons through the oligodendrocyte-axon-neuron relationship.

Oxidative damage linked to neurodegeneration by selective alpha-synuclein nitration in synucleinopathy lesions.

Aggregated alpha-synuclein proteins form brain lesions that are hallmarks of neurodegenerative synucleinopathies, and oxidative stress has been implicated in the pathogenesis of some of these disorders. Using antibodies to specific nitrated tyrosine residues in alpha-synuclein, we demonstrate extensive and widespread accumulations of nitrated alpha-synuclein in the signature inclusions of Parkinson's disease, dementia with Lewy bodies, the Lewy body variant of Alzheimer's disease, and multiple system atrophy brains. We also show that nitrated alpha-synuclein is present in the major filamentous building blocks of these inclusions, as well as in the insoluble fractions of affected brain regions of synucleinopathies. The selective and specific nitration of alpha-synuclein in these disorders provides evidence to directly link oxidative and nitrative damage to the onset and progression of neurodegenerative synucleinopathies.

α‐synuclein is developmentally expressed in cultured rat brain oligodendrocytes

Although a neuronal protein, α-synuclein is a major component of glial cytoplasmic inclusions (GCIs) in oligodendrocytes of multiple system atrophy (MSA) brains. Because α-synuclein has not been identified in oligodendrocytes of normal brains, we examined cultured rat brain oligodendrocytes during in vitro development and showed that α-synuclein mRNA and protein are present in cultured oligodendrocytes. The expression of α-synuclein was developmentally regulated; it increased to peak levels at 2 or 3 days in culture but declined thereafter. Indirect immunofluorescence further shows that α-synuclein was localized predominantly in cell bodies and primary processes of oligodendroglia. Thus, GCIs may be a consequence of altered rather than de novo expression of α-synuclein in MSA oligodendrocytes. J. Neurosci. Res. 62:9–14, 2000. © 2000 Wiley-Liss, Inc.

Characterisation of isolated α-synuclein filaments from substantia nigra of Parkinson's disease brain

The defining neuropathological deposits of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy are strongly immunoreactive for α-synuclein. We have shown previously that isolated filaments from dementia with Lewy bodies and multiple system atrophy brains are labelled in a characteristic fashion by a number of α-synuclein antibodies. Here we have extracted filaments from substantia nigra of patients with idiopathic Parkinson's disease. Antibodies directed against the carboxy-terminal region of α-synuclein labelled isolated filaments along their entire lengths. By contrast, an antibody directed against the amino-terminal region of α-synuclein only labelled one filament end. These characteristics were identical to those of filaments extracted from brains of patients with dementia with Lewy bodies and multiple system atrophy.

Immunohistochemical and Biochemical Studies Demonstrate a Distinct Profile of α-Synuclein Permutations in Multiple System Atrophy

Although alpha-synuclein (alpha-syn) has been implicated as a major component of the abnormal filaments that form glial cytoplasmic inclusions (GCIs) in multiple system atrophy (MSA), it is uncertain if GCIs are homogenous and contain full-length alpha-syn. Since this has implications for hypotheses about the pathogenesis of GCIs, we used a novel panel of antibodies to defined regions throughout alpha-syn in immunohistochemical epitope mapping studies of GCIs in MSA brains. Although the immunostaining profile of GCIs with these antibodies was similar for all MSA brains, there were significant differences in the immunoreactivity of the alpha-syn epitopes detected in GCIs. Notably, carboxy-terminal alpha-syn epitopes were immunodominant in GCIs, but the entire panel of antibodies immunostained cortical Lewy bodies (LBs) in dementia with LBs brain with similar intensity. While the distribution of alpha-syn labeled GCIs paralleled that previously reported using silver stains, antibodies to carboxy-terminal alpha-syn epitopes revealed a previously undescribed burden of GCIs in the MSA hippocampal formation. Finally, Western blots demonstrated detergent insoluble monomeric and high-molecular weight alpha-syn species in GCI rich MSA cerebellar white matter. Collectively, these data indicate that alpha-syn is a prominent component of GCIs in MSA, and that GCIs and LBs may result from cell type specific conformational or post-translational permutations in alpha-syn.

Fiber diffraction of synthetic alpha-synuclein filaments shows amyloid-like cross-beta conformation.

Filamentous inclusions made of alpha-synuclein constitute the defining neuropathological characteristic of Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Rare familial cases of Parkinson's disease are associated with mutations A53T and A30P in alpha-synuclein. We report here the assembly properties and secondary structure characteristics of recombinant alpha-synuclein. Carboxy-terminally truncated human alpha-synuclein (1-87) and (1-120) showed the fastest rates of assembly, followed by human A53T alpha-synuclein, and rat and zebra finch alpha-synuclein. Wild-type human alpha-synuclein and the A30P mutant showed slower rates of assembly. Upon shaking, filaments formed within 48 h at 37 degrees C. The related proteins beta- and gamma-synuclein only assembled after several weeks of incubation. Synthetic human alpha-synuclein filaments were decorated by an antibody directed against the carboxy-terminal 10 amino acids of alpha-synuclein, as were filaments extracted from dementia with Lewy bodies and multiple system atrophy brains. Circular dichroism spectroscopy indicated that alpha-synuclein undergoes a conformational change from random coil to beta-sheet structure during assembly. X-ray diffraction and electron diffraction of the alpha-synuclein assemblies showed a cross-beta conformation characteristic of amyloid.

Alpha-synuclein is developmentally expressed in cultured rat brain oligodendrocytes.

Although a neuronal protein, alpha-synuclein is a major component of glial cytoplasmic inclusions (GCIs) in oligodendrocytes of multiple system atrophy (MSA) brains. Because alpha-synuclein has not been identified in oligodendrocytes of normal brains, we examined cultured rat brain oligodendrocytes during in vitro development and showed that alpha-synuclein mRNA and protein are present in cultured oligodendrocytes. The expression of alpha-synuclein was developmentally regulated; it increased to peak levels at 2 or 3 days in culture but declined thereafter. Indirect immunofluorescence further shows that alpha-synuclein was localized predominantly in cell bodies and primary processes of oligodendroglia. Thus, GCIs may be a consequence of altered rather than de novo expression of alpha-synuclein in MSA oligodendrocytes.

α‐Synuclein Immunoisolation of Glial Inclusions from Multiple System Atrophy Brain Tissue Reveals Multiprotein Components

Journal of NeurochemistryVolume 73, Issue 5 p. 2093-2100 Free Access α-Synuclein Immunoisolation of Glial Inclusions from Multiple System Atrophy Brain Tissue Reveals Multiprotein Components First published: 04 October 2002 https://doi.org/10.1046/j.1471-4159.1999.02093.xCitations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume73, Issue5November 1999Pages 2093-2100 RelatedInformation

Epitope mapping of LB509, a monoclonal antibody directed against human α-synuclein

α-Synuclein is a 140 amino acid protein that forms the major component of the abnormal filaments that make up the Lewy bodies and Lewy neurites of Parkinson's disease and dementia with Lewy bodies. It is also the major component of the filamentous glial cytoplasmic inclusions of multiple system atrophy. Here we have used recombinant α-synucleins and peptide competition to show that the monoclonal anti-α-synuclein antibody LB509 recognizes amino acids 115–122 of human α-synuclein. The antibody strongly labelled filaments extracted from multiple system atrophy brain, showing the presence of residues 115–122 of α-synuclein. LB509 failed to react with mouse, rat and zebra finch α-synuclein, because of amino acid differences with human α-synuclein. Since LB509 recognizes human but not rodent α-synuclein, it will be a useful reagent for the characterization of mouse lines transgenic for human α-synuclein.

Cyclin-dependent kinase 5 and mitogen-activated protein kinase in glial cytoplasmic inclusions in multiple system atrophy.

Glial cytoplasmic inclusions (GCI) characteristically occur in the oligodendrocytes of patients with multiple system atrophy (MSA). However, the molecular mechanisms underlying GCI formation are unknown. To investigate whether these inclusions are related to proline-directed protein kinases that have been associated with neuronal inclusion bodies in some other neurodegenerative diseases, we immunohistochemically probed tissue samples from MSA brains with a panel of antibodies against cyclin-dependent kinases and mitogen-activated protein kinase. We unexpectedly detected cyclin-dependent kinase 5- (cdk5) and mitogen-activated protein kinase- (MAPK) immunoreactivities in GCI. We also found TAU1 immunoreactivity in GCI, and a strong expression of microtubule-associated protein (MAP) 2 immunoreactivity in oligodendrocytes of MSA brains. This immunoreactivity was not observed in the normal or neurological controls. The accumulated evidence suggest a close association between GCI and the microtubular cytoskeleton. Cdk5 phosphorylates tau and MAP2, and MAPK is capable of phosphorylating MAP2. The present results suggest that the aberrant or ectopic expression of cdk5 and MAPK causes abnormal phosphorylation of microtubular cytoskeletal proteins, thus leading to GCI formation in affected oligodendrocytes.

The relationship of multiple system atrophy to sporadic olivopontocerebellar atrophy and other forms of idiopathic late-onset cerebellar atrophy.

Multiple system atrophy (MSA) is a sporadic progressive neurodegenerative disease of undetermined cause characterized clinically by combinations of cerebellar, pyramidal, extrapyramidal, and autonomic disorders. 1,2 Many patients with MSA initially develop extrapyramidal symptoms and later experience autonomic disturbance or cerebellar features, or both; others start with autonomic disturbance, and some first manifest cerebellar symptoms and later develop autonomic or extrapyramidal features. Many patients develop only extrapyramidal and autonomic symptoms, but in most of these cases, subsequent neuropathologic studies also demonstrate degenerative changes in the cerebellum and brainstem. 3,4 The term MSA encompasses striatonigral degeneration (SND), the Shy-Drager syndrome (SDS), and many cases of sporadic olivopontocerebellar atrophy (sOPCA). Autopsy examination discloses neuronal loss and gliosis within some or all of the following structures: inferior olives, pons, cerebellum, substantia nigra, locus ceruleus, striatum (mainly putamen), and the intermediolateral columns and Onufs nucleus of the spinal cord. 5 Recently there have been descriptions of distinctive neuropathologic features of MSA consisting of oligodendroglial 6–11 and neuronal 12–14 intracytoplasmic and intranuclear argyrophilic inclusions containing accumulations of tubular structures. The glial cytoplasmic inclusions (GCIs) are present in all MSA brains, regardless of whether the patients were diagnosed in life as having SND, SDS, or sOPCA-type MSA. 6,14,15 In contrast, GCIs were not initially 6 found in a large number of neurologic controls, including patients with Parkinson's disease, progressive supranuclear palsy (PSP), and Machado-Joseph disease. However, a recent report 16 indicates that, although characteristic of MSA, the presence of GCIs is not …

Olivopontocerebellar pathology in multiple system atrophy

Olivopontocerebellar atrophy (OPCA) is widely accepted as part of the neuropathological spectrum of multiple system atrophy (MSA). The distribution of affected sites in the olivopontocerebellar (OPC) system and their interrelationship remain poorly understood due to lack of quantitative studies. To further investigate the OPC pathology in MSA, we performed a morphometric analysis of 20 MSA cases and eight healthy controls. In the MSA cases, mean neuronal cell densities were significantly reduced in (medial and dorsal) accessory and principal inferior olives, pontine nuclei, cerebellar vermis (except nodulus), and hemispheres. Inferior olives and pontine nuclei were more severely affected than cerebellar Purkinje cells in most cases. Cerebellar Purkinje cells were more severely depleted in vermis rather than in hemisphere. There was a poor topographic correlation between neuronal cell loss in inferior olives and cerebellar cortex. These results suggest a primary degeneration of olivopontine nuclei and cerebellar Purkinje cells in OPCA. Inferior olives, pontine nuclei and cerebellar cortex were all significantly more severely affected in cases with a pure or predominating cerebellar syndrome (OPCA type, n = 4) compared to those with pure or predominating parkinsonism (SND type, n = 14). However, although cerebellar signs had been noted in life in only six cases, morphometry revealed OPCA in 17 of the 20 MSA brains.

Oligodendroglial and neuronal inclusions in multiple system atrophy.

Argyrophilic cytoplasmic inclusions of oligodendrocytes have been described in cases of multiple system atrophy (olivopontocerebellar atrophy, striatonigral degeneration, and Shy-Drager syndrome). The oligodendroglial cytoplasmic inclusions are immunolabeled with antiubiquitin antibodies. Ultrastructurally, they appear as granule-associated filaments. They have not been found in other neurological diseases or in normal brains, and are now considered to be early and specific markers of multiple system atrophy. More recently, similar argyrophilic inclusion bodies have been reported in the cytoplasm of neurons and in both oligodendroglial and neuronal nuclei of multiple system atrophy brains. Neuronal and oligodendroglial cytoplasmic inclusions have identical ultrastructural characteristics, but different antigenic properties. The chemical nature of the inclusions is presently unknown and their significance remains controversial: they could be a primary event in the course of the degenerative process or merely an epiphenomenon of some disordered cytoskeletal metabolism.