RESUMO
Multiple System Atrophy is characterized pathologically by the accumulation of alpha-synuclein (aSyn) into glial cytoplasmic inclusions (GCIs). The mechanism underlying the formation of GCIs is not well understood. In this study, correlative light and electron microscopy was employed to investigate aSyn pathology in the substantia nigra and putamen of post-mortem multiple system atrophy brain donors. Three distinct types of aSyn immuno-positive inclusions were identified in oligodendrocytes, neurons and dark cells presumed to be dark microglia. Oligodendrocytes contained fibrillar GCIs that were consistently enriched with lysosomes and peroxisomes, supporting the involvement of the autophagy pathway in aSyn aggregation in multiple system atrophy. Neuronal cytoplasmic inclusions exhibited ultrastructural heterogeneity resembling both fibrillar and membranous inclusions, linking multiple systems atrophy and Parkinson's disease. The novel aSyn pathology identified in the dark cells, displayed GCI-like fibrils or non-GCI-like ultrastructures suggesting various stages of aSyn accumulation in these cells. The observation of GCI-like fibrils within dark cells suggests these cells may be an important contributor to the origin or spread of pathological aSyn in multiple system atrophy. Our results suggest a complex interplay between multiple cell types that may underlie the formation of aSyn pathology in multiple system atrophy brain and highlight the need for further investigation into cell-specific disease pathologies in multiple system atrophy.
RESUMO
Transcription factor EB (TFEB) is a master regulator of genes involved in the maintenance of autophagic and lysosomal homeostasis, processes which have been implicated in the pathogenesis of GBA-related and sporadic Parkinson's disease (PD), and dementia with Lewy bodies (DLB). TFEB activation results in its translocation from the cytosol to the nucleus. Here, we investigated TFEB subcellular localization and its relation to intracellular alpha-synuclein (aSyn) accumulation in post-mortem human brain of individuals with either incidental Lewy body disease (iLBD), GBA-related PD/DLB (GBA-PD/DLB) or sporadic PD/DLB (sPD/DLB), compared to control subjects. We analyzed nigral dopaminergic neurons using high-resolution confocal and stimulated emission depletion (STED) microscopy and semi-quantitatively scored the TFEB subcellular localization patterns. We observed reduced nuclear TFEB immunoreactivity in PD/DLB patients compared to controls, both in sporadic and GBA-related cases, as well as in iLBD cases. Nuclear depletion of TFEB was more pronounced in neurons with Ser129-phosphorylated (pSer129) aSyn accumulation in all groups. Importantly, we observed previously-unidentified TFEB-immunopositive perinuclear clusters in human dopaminergic neurons, which localized at the Golgi apparatus. These TFEB clusters were more frequently observed and more severe in iLBD, sPD/DLB and GBA-PD/DLB compared to controls, particularly in pSer129 aSyn-positive neurons, but also in neurons lacking detectable aSyn accumulation. In aSyn-negative cells, cytoplasmic TFEB clusters were more frequently observed in GBA-PD/DLB and iLBD patients, and correlated with reduced GBA enzymatic activity as well as increased Braak LB stage. Altered TFEB distribution was accompanied by a reduction in overall mRNA expression levels of selected TFEB-regulated genes, indicating a possible early dysfunction of lysosomal regulation. Overall, we observed cytoplasmic TFEB retention and accumulation at the Golgi in cells without apparent pSer129 aSyn accumulation in iLBD and PD/DLB patients. This suggests potential TFEB impairment at the early stages of cellular disease and underscores TFEB as a promising therapeutic target for synucleinopathies.