Progressive accumulation of ubiquitin and disappearance of α-synuclein epitope in multiple system atrophy-associated glial cytoplasmic inclusions: triple fluorescence study combined with Gallyas-Braak method

-Synuclein (S) and ubiquitin (Ub) are shared constituents of glial cytoplasmic inclusions (GCIs) and Lewy bodies (LBs), both composed of fibrillary structures. Staining profiles of GCIs were investigated with triple immunofluorescence involving immunostaining for S and Ub, both amplified with catalyzed reporter deposition, and a fluorochrome, thiazin red (TR) that has an affinity to fibrillary structures. After observation for the triple-fluorescent images, the sections were subsequently stained with the Gallyas-Braak method. Sections of putamen, cerebellar white matter and motor cortex from patients suffering from multiple system atrophy (MSA) with varying duration of the disease (4–15 years) were quantified for these staining profiles of Gallyas-positive GCIs. Although most of GCIs were positive for Ub and variably positive for S, they were consistently negative for TR. The result was opposite in LBs in Lewy body disease with variable affinity to TR, suggesting that the construction of GCIs is different from that of LBs. These four staining features (S, Ub, TR and Gallyas) alone failed to exhibit apparent correlation with disease duration, lesion site or severity of degeneration as reported previously. The fraction of S-negative and Ub-positive GCIs, however, linearly increased along the disease progression, while that of S-positive and Ub-negative GCIs decreased in contrast. This reciprocal change suggests that S immunoreactivity in GCIs is being replaced by Ub immunoreactivity during the disease progression, which resulted in the ultimate predominance of S-negative and Ub-positive GCIs in the most advanced case. Interestingly, this predominance of S-negative and Ub-positive GCIs was a feature of motor cortex, where degeneration usually remains mild in spite of robust appearance of Gallyas-positive GCIs. Another fraction, S-positive and Ub-positive GCIs were frequent in cerebellar white matter, suggesting that GCI evolution is heterogeneous and dependent also on area examined. Progressive accumulation of Ub with concomitant disappearance of S epitope and their colocalization, partly shared with LBs, may represent a process of GCI formation, possibly linked to an aspect of degeneration in MSA.     

Different conformation of neuronal tau deposits distinguished by double immunofluorescence with AT8 and thiazin red combined with Gallyas method

Different kinds of tau deposits were quantitatively investigated with thiazin red (TR), a fluorochrome that binds to fibrillary structures like neurofibrillary tangles (NFTs), in brains obtained at autopsy from patients with Alzheimer's disease (AD), Pick body (PB) disease, corticobasal degeneration (CBD) or diffuse NFTs with calcification (DNTC). After recording double-labeling fluorescence images with anti-paired helical filament tau (AT8) and TR, the sections were subjected to Gallyas method (GAL). This enabled three different staining properties to be compared on the identical neuron. AT8-positive neocortical neurons of AD and DNTC were fibrillary and uniformly positive for TR and GAL, consistently forming NFTs. NFTs lacking AT8 immunoreactivity (IR) were more frequent in DNTC than in AD, suggesting that evolution of NFTs is more accelerated in DNTC. Scarce TR staining in tau-positive neocortical neurons of CBD suggests their paucity of fibrillary structure. Since the affinity of TR for PB was not consistent, this may be dependent not only on the amount but also the characteristics of fibrillary structures. PBs were further characterized by the scarcity of GAL staining. This approach, which quantitatively clarifies differences between AT8-IR, TR and GAL, provides a morphological basis for further investigations of the different conformational states of tau from its deposition to fibril formation of various types.     

Morphogenesis of Lewy bodies: dissimilar incorporation of alpha-synuclein, ubiquitin, and p62

The formation of Lewy bodies (LBs) and their relationship to other types of nigral inclusions associated with Parkinson disease (PD), such as pale bodies (PBs), remain poorly understood. Known constituents of LBs include alpha-synuclein (alphaS) and ubiquitin (Ub), providing windows to their morphogenesis. Additionally, p62/sequestosome 1 has been identified as a common component of neuropathological and hepatocytic inclusions. To study the formation of PD-associated nigral inclusions, we analyzed the substantia nigra of cases with abundant LBs and PBs in hematoxylin and eosin (H&E) stain, using immunohistochemistry for alphaS, Ub, and p62. We found morphologically diverse alphaS-immunoreactive deposits within neuronal perikarya and neurites. Perikaryal types extended from punctate cytoplasmic staining to variform compact (i.e. PB-type and LB-type) inclusions. Using H&E, only a small subset of the compact deposits could be unambiguously identified. Labeling for p62 was highly similar to alphaS in compact perikaryal inclusions, whereas no punctate staining or intraneuritic inclusions were detected. Ubiquitin antibodies labeled compact deposits both within perikarya and neurites. The data suggest that pathological alphaS is first evident as punctate perikaryal material that, via coalescence and incorporation of p62 and Ub, yields PB-type structures from which LB-type inclusions form in a compaction-like manner. The results also point at dissimilarities in the formation of perikaryal vs intraneuritic inclusions.     

The Lewy body in Parkinson's disease: Molecules implicated in the formation and degradation of α‐synuclein aggregates

The histological hallmark of Parkinson's disease (PD) is the presence of fibrillar aggregates called Lewy bodies (LBs). LB formation has been considered to be a marker for neuronal degeneration, because neuronal loss is found in the predilection sites for LBs. To date, more than 70 molecules have been identified in LBs, in which α‐synuclein is a major constituent of LB fibrils. α‐synuclein immunohistochemistry reveals that diffuse cytoplasmic staining develops into pale bodies via compaction, and that LBs arise from the peripheral portion of pale bodies. This α‐synuclein abnormality is found in 10% of pigmented neurons in the substantia nigra and more than 50% of those in the locus ceruleus in PD. Recent studies have suggested that oligomers and protofibrils of α‐synuclein are cytotoxic, and that LBs may represent a cytoprotective mechanism in PD.     

Quantitative proteomic analysis of mitochondrial proteins: relevance to Lewy body formation and Parkinson's disease

The mechanisms underlying Parkinson's disease (PD) and Lewy body (LB) formation, a pathological hallmark of PD, are incompletely understood; however, mitochondrial dysfunction is likely to be at least partially responsible. To study the processes that might be related to nigral neurodegeneration and LB formation, we employed nonbiased quantitative proteomics with isotope-coded affinity tag (ICAT) to compare the mitochondrial protein profiles in the substantia nigra (SN) between controls and mice treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a potent mitochondrial toxicant, and an adjuvant, probenecid (prob), for 5 weeks, which produced selective nigrostriatal neurodegeneration with formation of LB-like cytoplasmic inclusions in the remaining nigral neurons. This method identified a total of more than 300 proteins; of these proteins, more than 100 displayed significant changes in relative abundance in the MPTP/prob-treated mice compared to the controls. We validated one of these proteins, DJ-1, whose mutation has been implicated in familial PD, with Western blot analysis, followed by immunohistochemical studies of its distribution in the SN in relation to cytoplasmic inclusions in mice, as well as in classical LBs in PD patients. The results demonstrated that DJ-1 was not only colocalized with alpha-synuclein in dopaminergic neurons but also to cytoplasmic inclusions in mice treated with MPTP/prob. In addition, DJ-1 was present in the halo but not in the core of classical LBs in patients with PD. Our findings suggested that DJ-1 might play an important role in mitochondrial dysfunction, as well as LB formation in PD.     

Monoclonal antibodies to purified cortical lewy bodies recognize the mid-size neurofilament subunit

Lewy bodies (LBs) are filamentous intraneuronal inclusions that are hallmark lesions of Parkinson's disease, and LBs have been shown, by immunohistochemistry, to contain cytoskeletal as well as other cellular proteins. Similar LBs also occur in the cortical neurons of a subset of patients with Alzheimer's disease (AD), and cortical LBs are the predominant or sole lesions in the brains of patients with an AD-like dementia known as diffuse Lewy-body disease (DLBD). To gain insight into the biochemical composition of LBs, we generated monoclonal antibodies (mAbs) to LBs purified from the brains of patients with DLBD. Here, we describe three of these new mAbs (LB48, LB202, and LB204) that stained LBs by immunohistochemistry and recognized the medium molecular mass neurofilament (NF) protein in western blots. These results support the hypothesis that NF subunits are integral components of LBs. Continued efforts to clarify the composition of LBs are likely to lead to novel strategies for the antemortem diagnosis of LB disorders as well as to insight into the role LBs play in the degeneration of affected neurons in these disorders.     

Restricted occurrence of Lewy bodies in the dorsal vagal nucleus in a patient with late-onset parkinsonism.

Lewy body (LB) is consistently found in the substantia nigra in Parkinson's disease. We report a 68-year-old woman with late-onset, dopa-responsive parkinsonism. Her parents were first cousins, but no other affected individuals were present in the family. Autopsy revealed moderate loss of pigmented neurons with gliosis, but neither LBs nor neurofibrillary tangles in the substantia nigra. The locus ceruleus showed neuronal loss with scarce LBs. The most striking change was found in the dorsal vagal nucleus, where marked neuronal loss and fibrillary gliosis with many LBs were evident. Despite the use of ubiquitin and alpha-synuclein immunohistochemistry, no further LBs were identified in other brain regions. These findings suggest that this case was an unusual, anatomically restricted manifestation of LB disease.     

Cu/Zn superoxide dismutase-like immunoreactivity is present in Lewy bodies from Parkinson disease: a light and electron microscopic immunocytochemical study

Copper-zinc superoxide dismutase (SOD1)-like immunoreactivity has been demonstrated in Lewy body-like inclusions (LIs) in brain tissues from patients with familial and sporadic amyotrophic lateral sclerosis. Using immunocytochemistry, we studied Lewy bodies (LBs), the original inclusions from which the term LI was derived, in five patients with Parkinson disease (PD). Surprisingly, many LBs were immunostained by an antibody against SOD1. There were two types of staining pattern: a diffuse pattern, and a peripheral pattern with an unstained core. An immunoelectron microscopic study demonstrated that the immunoreactive products were restricted to the fibrillary profiles, sparing the unstructured core. Our results showed that SOD1-like immunoreactivity occurred frequently in LBs and LIs, suggesting that a common cytopathological process is responsible for the formation of LB-type neuronal intracytoplasmic inclusions. Our results also suggest that SOD1 plays a role in the neurodegeneration associated with PD.     

Cyclin-dependent kinase 5 (Cdk5) associated with Lewy bodies in diffuse Lewy body disease

We investigated the cyclin-dependent kinase (Cdk) 5 distribution pattern in diffuse Lewy body disease brains using immunohistochemistry. Cdk5 immunoreactivity was detected in both brainstem-type Lewy bodies (LBs) and cortical LBs. The number of Cdk5-positive LBs was less than that of ubiquitin- or α-synuclein-positive LBs, and more than that of phosphorylated neurofilament-positive LBs. Immunoelectron microscopy revealed Cdk5-immunolabeled granulo-filamentous components in LBs and LB-related neurites. These data suggest that Cdk5 may be associated with LB formation.     

Isopentenyl diphosphate isomerase,a cholesterol synthesizing enzyme,is localized in Lewy bodies

Isopentenyl diphosphate isomerase (IDI) is a cytoplasmic enzyme involved in the biosynthesis of isoprenoids including cholesterols. IDI has two isoforms in humans: IDI1 and IDI2. Since lipids are known to be a component of Lewy bodies (LBs), we investigated the immunohistochemical localization of IDI1 and IDI2 in the brain of patients with LB disease and multiple system atrophy (MSA) and normal control subjects. In normal controls, the cytoplasm of neurons was weakly immunostained with anti‐IDI1 and anti‐IDI2 antibodies throughout the nervous system. In LB disease, brainstem‐type LBs were strongly positive for IDI1 and IDI2, and cortical LBs were unstained or barely immunolabeled. Double immunofluorescence staining revealed co‐localization of phosphorylated α‐synuclein with IDI1 or IDI2. Glial cytoplasmic inclusions in MSA were unstained. Previous studies have shown that levels of cholesterol metabolites are increased in the cerebral cortex of patients with LB disease, and that these metabolites accelerate α‐synuclein aggregation. The present findings suggest that IDI1 and IDI2 may be associated with the production of cholesterol metabolites in neurons, leading to α‐synuclein aggregation during the process of LB formation.