Abstract Body

Many neurodegenerative disorders contain neuropathological lesions composed of aggregates of insoluble α-synuclein protein - primary α-synucleinopathies (idiopathic Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) versus secondary α-synucleinopathies (genetic Alzheimer’s disease, lysosomal storage disorders, etc.). In Lewy body disorders astrocytes accumulate α-synuclein, but do not increase their expression of GFAP (definition of reactivity) or many other astrocytic proteins. The damage-associated molecular pattern protein S100B that is secreted by astrocytes is significantly increased in patients with PD, and astrocytes in regions accumulating Lewy pathologies also have increased intracellular α-synuclein. The astrocyte type affected are the grey matter protoplasmic astrocytes whose endfeet form part of the tripartite synapse in addition to the blood-brain barrier. The regional increase in α-synuclein in protoplasmic astrocytes parallels the stages of Lewy body accumulation in Lewy body diseases, with more astrocytes involved than neurons. While neurons but not astrocytes also accumulate α-synuclein in MSA, regional degeneration is related to oligodendroglial cytoplasmnic inclusions (GCIs) rather than neuronal α-synuclein accumulation. The oligodendroglia protein, myelin basic protein (MBP), is reduced in MSA in association with an increase in degraded MBP and an accumulation of p25α and α-synuclein in enlarged cell bodies. There is also a reduction in lipids associated with myelin and an increase in the lipid transporter ABCA8. Oligodendroglial precursors increase their relative numbers in the white matter affected in MSA. These comparisons show that glial accumulation of α-synuclein differentiate Lewy body diseases from MSA, with protoplasmic astrocytes that contact neuronal synapses and cell bodies affected in Lewy body diseases, while oligodendroglia that contact neuronal axons affected in MSA.

Abstract Body

The aggregation of alpha-synuclein (ASYN) in Lewy bodies and Lewy neurites is the typical pathological hallmark of Parkinson’s disease (PD) and other synucleinopathies. Mutations and multiplications in the gene encoding for ASYN are associated with familial and sporadic forms of PD, suggesting this protein plays a central role in the disease. However, the precise contribution of ASYN to neuronal dysfunction and death is still unclear. There is intense debate on the nature of the toxic species of ASYN, and little is still known about the molecular determinants of oligomerization and aggregation of ASYN in the cell.

By harnessing the power of various model organisms, we are making progress towards the understanding of the basic molecular mechanisms underlying PD and other synucleinopathies. In particular, we are ofusing on the effects of different posttranslational modifications (PTMs) on the toxicity and aggregation of ASYN.

Glycation, an age-associated PTM that is also increased in diabetes, is emerging as an important modification that affects ASYN aggregation and toxicity. In our recent work, we are investigating how different sugars affect the aggregation of ASYN, and how quality control mechanisms respond to the accumulation of glycated ASYN.

In conclusion, our data sheds new light into the molecular underpinnings of synucleinopathies, opening novel perspectives for therapeutic intervention.

Abstract Body

Dopaminergic neuronal cell death, associated with intracellular α‐synuclein (α‐syn)-rich inclusions termed Lewy bodies, characterises Parkinson’s disease (PD). Using Lewy bodies extracted from PD brains, we characterized the synucleinopathy in non-human primates (NHPs), by comparing the effects of these large human aggregates with solutions of soluble and smaller α-syn aggregates. To our biggest surprise, while these small α-syn aggregates did not led to neuronal death in mice, NHPs showed neurodegeneration after small aggregates injection; to the same extent of big aggregates. Such aggregates differed by their size but also by their folding properties, suggesting that, within human brains, conformational strain diversity exists. In vitro, various α-syn fibril polymorphs have been obtained from distinct fibrillization conditions and were selected by amyloid monitoring using the probe Thioflavin T (ThT). We report that, concurrently with classical ThT-positive products, fibrillization in saline also gives rise to polymorphs that are invisible to ThT (τ^-), and that crowd the fibril preparations. The emergence of τ^- polymorphs has been ignored so far or mistaken for fibrillization inhibitions/failures. Compared to their ThT-positive counterparts, these new τ^- polymorphs present a yet undescribed atomic organization, an exacerbated propensity towards self-replication in cortical neurons and spreading of brain pathology in living mice. That the human brain aggregate diversity finds an in vitro counterpart where different polymorphs emerge in physiological conditions, strongly suggests that the search for therapeutic solutions must incorporate all adopted structures for hoping to have an impact upon disease progression.

Aims

Neuronal aggregates of alpha-synuclein (αSyn) (Lewy bodies and neurites) are a pathological hallmark of Dementia with Lewy Bodies (DLB) and Parkinson’s disease (PD). We investigate here what determines the brain area specificity of αSyn pathology and what leads to the differences in pattern of pathology and neurodegeneration in DLB and PD.

Methods

We use here human brain tissue from 9 different regions and various structural assays to characterize the different functional forms of αSyn dependent on brain region and disease condition. Mechanistically, we then use the neuroblastoma cell line M17D expressing different forms of αSyn and the exposure to pre-aggregated amyloid to investigate what makes pathology spread possible and explains higher resistance of some brain regions over others.

Results

We can demonstrate that DLB and sPD patients exhibit a region-specific reduction of αSyn helical multimers in brain tissue according to the classical Braak staging scheme, indicating their destabilization in the course of the disease. We show here that the main physiological form is not only resistant to time-dependent self-aggregation but also shows increased resistance towards "prion-like" aggregation.

Conclusions

The results indicate the vulnerability of early affected brain regions, the importance of a balance of aSYN multimers and monomers and the functional reserve of different brain regions. A factor governing the stabilization of multimers seems to be lipid composition of the cell specific membranes since transient lipid contact acts as a catalyst for multimer formation, meaning that lipid vesicles might act as a “liposomal chaperone” capable of conferring aggregation resistance to the cytosolic αSyn.

Aims

Axons are crucial for impulse transmission, transportation of organelles, and clearance of proteins necessary for neuronal and synaptic survival. In Parkinson’s disease (PD), axonal degeneration occurs early and contributes to spread of pathology. The aim of this study is to assess features of axonal degeneration and protein aggregation in the anterior insular sub-regions in PD and dementia with Lewy bodies (DLB).

Methods

The post-mortem anterior insula was collected from 25 PD, PD dementia (PDD), and DLB donors. Axonal loss was evaluated using modified Bielschowsky silver staining and unbiased stereology. Pathology load was semi-quantified on sections stained for α-synuclein, hyperphosphorylated (HPF)-tau, and amyloid-β proteins. For cytoskeletal damage, immunofluorescent multi-labelling with neurofilament (NFL), myelin, and α-synuclein was analyzed using confocal laser-scanning microscopy. Parametric tests and linear mixed model were used for data analysis.

Results

The anterior agranular insula showed a significantly higher load of α-synuclein (t(21)=5.3;p<0.001), HPF-tau pathology (t(19)=5.1;p<0.001) and axonal loss (t(23)=-5.7;p<0.001) compared to the anterior dysgranular insula. The dysgranular insula alternatively showed a significant higher load of amyloid-β pathology (t(9)=-2.5;p=0.03). In mixed model analysis, HPF-tau significantly contributed to axonal loss (b=-3.45x10^-5;F(1,42)=4.2;p=0.046). NFL showed fragmentation and swellings; while myelin showed axon-myelin disruption in both sub-regions. The DLB group showed most severe axonal loss, cytoskeletal damage and protein aggregates.

Conclusions

The agranular and dysgranular insular sub-regions were differentially vulnerable to axonal loss and pathological aggregates, most pronounced in DLB. Our results highlight the selective vulnerability of the anterior insula to various converging pathologies disrupting axonal integrity, potentially contributing to non-motor deficits in PD and DLB.

Aims

The involvement of the enteric nervous system in idiopathic Parkinson’s disease (PD) has long been established but underlying pathophysiologic mechanisms have not yet been fully understood. Here, we investigated changes in the expression of microRNAs (miRNAs) in routine colonic biopsies from patients with PD to evaluate their potential as diagnostic biomarkers and gain further insight into the pathophysiology underlying PD.

Methods

Patients with PD (n=13) and healthy controls (n=17) undergoing routine colonoscopy for cancer screening were prospectively recruited and colonic biopsies were obtained. Total RNA was extracted from the biopsy material and expression of miRNAs was quantified by Ilumina High-Throughput-Sequencing.

Results

The miRNA hsa-miR-486-5p was significantly enriched in the submucosa of colonic biopsies from PD patients compared to healthy controls. Expression levels correlated with age and disease severity measured by the UPDRS and Hoehn & Yahr scale. miRNA gene target analysis identified 301 gene targets that are affected by miR-486-5p. A follow-up associated target identification and pathway enrichment analysis further determined their role in distinct biological processes in the enteric nervous system (ENS).

Conclusions

In this work we demonstrate an enrichment of submucosal miR-486-5p in colonic biopsies from PD patients compared to healthy controls. The expression levels of miR-486-5p correlated with age and disease severity as measured by the UPDRS-III. In summary, our results will support the investigation of colonic miRNAs as a disease biomarker in PD.

Aims

To identify the possible effect of downregulated platelet-derived miRNAs in dementia with Lewy bodies (DLB) on brain function-related mRNAs expressed in platelets and to compare expression pattern with Alzheimer disease (AD).

Methods

Total RNA was purified from platelets obtained from DLB, AD and control cases. Eight samples of each group were analyzed with nCounter Neuropathology panel including 770 mRNAs, by subjecting 75 ng of total RNA to profiling by NanoString-technology. Expression changes of 49 mRNAs were validated in 20 independent DLB, AD and control samples by a custom nCounter panel. Results were evaluated by nSolver software (NanoString), deltadeltaCt method, String and Reactome Databases.

Results

Of the 770 mRNAs included in the panel, 457 were expressed in DLB. Ninety-six mRNAs were overexpressed and related to neuronal plasticity and platelet activation. Of 78 down-regulated genes, 31 were related the immune system and 9 to transcription factor binding. Validation of the 26 most up-regulated and 23 most down-regulated genes revealed overexpression of APP, ACTN1, CD9, DGKE, EGF and P2RY12 (platelet activation), and RTN4, APP, NPTN, CA2, SLC6A4, SLC18A2, HSPB1 (neuronal plasticity). CCR5, IL13RA1, HSPA6, NFKBIB, ITGAX and TNFRSF1B (immune system) and JUN, PCNA, TAF4 and SPI1 (transcription factor binding) were down-regulated in DLB. Platelet-activation-related genes overexpressed in DLB were down-regulated in AD indicating that abnormal platelet activation may be a key event in DLB pathogenesis.

Conclusions

The expression of platelet-derived mRNAs is altered in DLB and differs from AD. Involvement of platelet activation in DLB pathogenesis should be further explored.

Aims

The aim of this study is to investigate whether the liver is susceptible to α-syn accumulation in Parkinson’s disease (PD).

Methods

We assessed whether α-syn assemblies could be internalized by cultured primary human and HuH-7 hepatocytes. We then characterized human α-syn in liver tissue samples from transgenic models modeling PD, multiple system atrophy and Alzheimer’s disease (AD). Finally, we corroborated our results in human PD using imaging techniques.

Results

We demonstrate that human hepatocytes internalize oligomeric α-syn assemblies mediated, at least in part, by the gap junction protein connexin 32 (Cx32). Moreover, we identified a time dependent accumulation of α-syn pathology within the liver of three different transgenic mouse models overexpressing human α-syn under CNS-specific promoters. Such a brain to liver transmission route could be corroborated by detection of α-syn pathology within the liver of wild type mice one-month after a striatal α-syn injection. Notably, accumulation of Aß pathology in mice modeling AD was relatively absent. Consistent with our models, we identified the presence of α-syn pathology in a subset of human liver tissues from cases neuropathologically diagnosed with α-syn in the brain.

Conclusions

Our preliminary observations reveal α-syn pathology accumulation within the liver in a subset of human PD cases and mice modeling PD, a pathological process unique tothe synucleinopathies as pathology associated with AD is relatively absent. These results suggest that α-syn liver accumulation is likely a consequence of brain to liver or peripheral-liver transmission. Thus, the liver may be involved in the clearance and detoxification of pathological proteins in PD and related synucleinopathies.