Distinct ultrastructural phenotypes of glial and neuronal alpha-synuclein inclusions in multiple system atrophy.

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.

Altered TFEB subcellular localization in nigral neurons of subjects with incidental, sporadic and GBA-related Lewy body diseases.

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.

The subcellular arrangement of alpha-synuclein proteoforms in the Parkinson's disease brain as revealed by multicolor STED microscopy.

Various post-translationally modified (PTM) proteoforms of alpha-synuclein (aSyn)-including C-terminally truncated (CTT) and Serine 129 phosphorylated (Ser129-p) aSyn-accumulate in Lewy bodies (LBs) in different regions of the Parkinson's disease (PD) brain. Insight into the distribution of these proteoforms within LBs and subcellular compartments may aid in understanding the orchestration of Lewy pathology in PD. We applied epitope-specific antibodies against CTT and Ser129-p aSyn proteoforms and different aSyn domains in immunohistochemical multiple labelings on post-mortem brain tissue from PD patients and non-neurological, aged controls, which were scanned using high-resolution 3D multicolor confocal and stimulated emission depletion (STED) microscopy. Our multiple labeling setup highlighted a consistent onion skin-type 3D architecture in mature nigral LBs in which an intricate and structured-appearing framework of Ser129-p aSyn and cytoskeletal elements encapsulates a core enriched in CTT aSyn species. By label-free CARS microscopy we found that enrichments of proteins and lipids were mainly localized to the central portion of nigral aSyn-immunopositive (aSyn+) inclusions. Outside LBs, we observed that 122CTT aSyn+ punctae localized at mitochondrial membranes in the cytoplasm of neurons in PD and control brains, suggesting a physiological role for 122CTT aSyn outside of LBs. In contrast, very limited to no Ser129-p aSyn immunoreactivity was observed in brains of non-neurological controls, while the alignment of Ser129-p aSyn in a neuronal cytoplasmic network was characteristic for brains with (incidental) LB disease. Interestingly, Ser129-p aSyn+ network profiles were not only observed in neurons containing LBs but also in neurons without LBs particularly in donors at early disease stage, pointing towards a possible subcellular pathological phenotype preceding LB formation. Together, our high-resolution and 3D multicolor microscopy observations in the post-mortem human brain provide insights into potential mechanisms underlying a regulated LB morphogenesis.

Toll-like Receptor 4 Is Upregulated in Parkinson's Disease Patients and Co-Localizes with pSer129αSyn: A Possible Link with the Pathology.

Growing evidence suggests a crucial role of neuroinflammation in the pathophysiology of Parkinson's disease (PD). Neuroinflammation is linked to the accumulation and aggregation of a-synuclein (αSyn), the primary pathological hallmark of PD. Toll-like receptors 4 (TLR4) can have implications in the development and progression of the pathology. In this study, we analyzed the expression of TLR4 in the substantia nigra (SN) and medial temporal gyrus (GTM) of well-characterized PD patients and age-matched controls. We also assessed the co-localization of TLR4 with pSer129 αSyn. Using qPCR, we observed an upregulation of TLR4 expression in the SN and GTM in PD patients compared to controls, which was accompanied by a reduction in αSyn expression likely due to the depletion of dopaminergic (DA) cells. Additionally, using immunofluorescence and confocal microscopy, we observed TLR4-positive staining and co-localization with pSer129-αSyn in Lewy bodies of DA neurons in the SN, as well as in pyramidal neurons in the GTM of PD donors. Furthermore, we observed a co-localization of TLR4 and Iba-1 in glial cells of both SN and GTM. Our findings provide evidence for the increased expression of TLR4 in the PD brain and suggest that the interaction between TLR4 and pSer129-αSyn could play a role in mediating the neuroinflammatory response in PD.

Contactin-1 Is Reduced in Cerebrospinal Fluid of Parkinson's Disease Patients and Is Present within Lewy Bodies.

Synaptic degeneration is an early phenomenon in Parkinson's disease (PD) pathogenesis. We aimed to investigate whether levels of synaptic proteins contactin-1 and contactin-2 in cerebrospinal fluid (CSF) of PD patients are reduced compared to dementia with Lewy bodies (DLB) patients and controls and to evaluate their relationship with α-synuclein aggregation. Contactin-1 and -2 were measured in CSF from PD patients (n = 58), DLB patients (n = 72) and age-matched controls (n = 90). Contactin concentration differences between diagnostic groups were assessed by general linear models adjusted for age and sex. Contactin immunoreactivity was characterized in postmortem substantia nigra, hippocampus and entorhinal cortex tissue of PD patients (n = 4) and controls (n = 4), and its relation to α-syn aggregation was evaluated using confocal laser scanning microscopy. Contactin-1 levels were lower in PD patients (42 (36-49) pg/mL) compared to controls (52 (44-58) pg/mL, p = 0.003) and DLB patients (56 (46-67) pg/mL, p = 0.001). Contactin-2 levels were similar across all diagnostic groups. Within the PD patient group, contactin-1 correlated with t-α-syn, tTau and pTau (r = 0.30-0.50, p < 0.05), whereas contactin-2 only correlated with t-α-syn (r = 0.34, p = 0.03). Contactin-1 and -2 were observed within nigral and cortical Lewy bodies and clustered within bulgy Lewy neurites in PD brains. A decrease in CSF contactin-1 may reflect synaptic degeneration underlying Lewy body pathology in PD.

Lewy pathology in Parkinson's disease consists of crowded organelles and lipid membranes.

Parkinson's disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites: neuronal inclusions immunopositive for the protein α-synuclein. In-depth ultrastructural analysis of Lewy pathology is crucial to understanding pathogenesis of this disease. Using correlative light and electron microscopy and tomography on postmortem human brain tissue from Parkinson's disease brain donors, we identified α-synuclein immunopositive Lewy pathology and show a crowded environment of membranes therein, including vesicular structures and dysmorphic organelles. Filaments interspersed between the membranes and organelles were identifiable in many but not all α-synuclein inclusions. Crowding of organellar components was confirmed by stimulated emission depletion (STED)-based super-resolution microscopy, and high lipid content within α-synuclein immunopositive inclusions was corroborated by confocal imaging, Fourier-transform coherent anti-Stokes Raman scattering infrared imaging and lipidomics. Applying such correlative high-resolution imaging and biophysical approaches, we discovered an aggregated protein-lipid compartmentalization not previously described in the Parkinsons' disease brain.

Gender-related changes in increase of dopaminergic neurons in the olfactory bulb of Parkinson's disease patients.

Gender differences in dopaminergic related neurodegenerative diseases have hardly been studied until now. It is generally accepted that more men than women suffer from Parkinson's disease. One of the most prevalent symptoms in Parkinson's patients, hyposmia, does not show gender differences, while normally the sense of smell is better developed in females. Whether the change in dopamine in the olfactory bulb contributes equally to hyposmia in male and female Parkinson's patients is the subject of the present study. In a stereological study the total number of tyrosine hydroxylase immunoreactive neurons in the olfactory bulbs of male and female Parkinson's patients and age-matched controls has been estimated. The present stereological study shows that the number of tyrosine hydroxylase positive cells in control females is significantly lower than those in control males. The number of dopaminergic cells in the olfactory bulbs of both male and female Parkinson's patients equals that of healthy males of the same age group. We therefore conclude that the hyposmia in Parkinson's disease patients cannot simply be ascribed to dopamine in the olfactory bulb.

Distribution and Load of Amyloid-ß Pathology in Parkinson Disease and Dementia with Lewy Bodies.

Parkinson disease (PD), Parkinson disease with dementia (PDD), and Dementia with Lewy bodies (DLB) differ clinically with regard to the presence and timing of dementia. In this postmortem study, we evaluated whether the burden and distribution pattern of amyloid-ß (Aß) pathology differs among these disease entities. We assessed Aß phases and neuritic plaque scores in 133 patients fulfilling clinical diagnostic criteria for PD, PDD, and DLB, and determined the presence and load of Aß pathology in 5 cortical and 4 subcortical regions in a subset of patients (n = 89) using a multispectral imaging system. Aß phases and neuritic plaque scores were higher in DLB versus PDD (both p < 0.001) and in PDD vs PD patients (p = 0.020 and 0.022, respectively). Aß pathology was more often observed in the entorhinal cortex, amygdala and putamen in DLB versus PDD patients; Aß load was higher in both cortical and subcortical regions. PDD patients had more frequent Aß pathology in temporal cortex and higher Aß load in cortical regions and striatum versus PD patients. Our findings suggest that the load and extent of Aß pathology may contribute to cognitive dysfunction in PDD and the early-stage severe dementia in DLB.

The proteome of the locus ceruleus in Parkinson's disease: relevance to pathogenesis.

The locus ceruleus is among the earliest affected brain regions in Parkinson's disease (PD) showing Lewy body pathology and neuronal loss. To improve our understanding of the pathogenesis of PD, we performed the first proteomic analysis ever of post-mortem locus ceruleus tissue of six pathologically confirmed PD patients, and six age- and gender-matched non-neurological controls. In total 2495 proteins were identified, of which 87 proteins were differentially expressed in the locus ceruleus of PD patients compared with controls. The majority of these differentially expressed proteins are known to be involved in processes that have been implicated in the pathogenesis of PD previously, including mitochondrial dysfunction, oxidative stress, protein misfolding, cytoskeleton dysregulation and inflammation. Several individual proteins were identified that have hitherto not been associated with PD, such as regucalcin, which plays a role in maintaining intracellular calcium homeostasis, and isoform 1 of kinectin, which is involved in transport of cellular components along microtubules. In addition, pathway analysis suggests a pathogenetic role for aminoacyl-tRNA-biosynthesis. These findings indicate that the proteome of the locus ceruleus of PD patients and non-neurological controls provides data that are relevant to the pathogenesis of PD, reflecting both known and potentially novel pathogenetic pathways.

A 100% increase of dopaminergic cells in the olfactory bulb may explain hyposmia in Parkinson's disease.

Hyposmia is one of the most prevalent symptoms of Parkinson's disease. It may occur even before the motor symptoms start. To determine whether the olfactory dysfunctions, like the motor symptoms, are associated with a loss of dopamine, the number of dopaminergic cells in the olfactory bulb of Parkinson's disease patients was studied using tyrosine hydroxylase immunohistochemistry. The quantitative analysis reveals that the total number of tyrosine hydroxylase-immunoreactive neurons in the olfactory bulb is twice as high in Parkinson patients compared to age and gender-matched controls. Because dopamine is known to inhibit olfactory transmission in the olfactory glomeruli, we suggest that the increase of dopaminergic neurons in the olfactory bulb is responsible for the hyposmia in Parkinson patients. The increase of dopamine in the olfactory bulb explains why olfaction does not improve with levodopa therapy.