Aims

The nature and formation of Lewy bodies in synucleinopathies is under intense debate. While initially only amyloid formation was investigated as a central motif of synuclein related neurodegeneration, recent human data implies that aberrant lipid binding of alpha-synuclein is an elemental part of the disease mechanism leading to the pathological hallmark, the Lewy Body. Here we are trying to dissect the relationship of aberrant synuclein-lipid binding to the formation of amyloid rich inclusions in neuronal cell models and human tissue.

Methods

Here we analyze the membrane binding capacity of synuclein in human tissue at different stages of PD pathology. Furthermore, using cellular models that modulate the formation of mixed lipid-protein phases through either pathogenic synuclein mutations or manipulations of lipid metabolism we investigate the mechanism of amyloid formation via biochemical fractionation and life cell FRAP microscopy.

Results

Here we can demonstrate that enhanced lipid interaction during disease progression facilitate amyloid formation through lowering the aggregation resistance of physiological protein. Furthermore, we can demonstrate that alpha-synuclein forms cytoplasmic inclusions together with lipids while remaining highly mobile. The dynamic of the inclusions decreased over time, suggesting that the lipid-synuclein condensates undergo a subsequent maturation into amyloid fibrils further implying that lipid binding is directly tied to the formation of amyloid fibrils.

Conclusions

Our data provide evidence that lipid binding is an important event during the development of cellular synuclein pathology and that protein-lipid phases are a part of the amyloid aggregation pathway.

Aims

α-Synuclein has been shown to associate with membranes and to bind acidic phospholipids. However, the significance of these associations for the cell biology of α-Syn is not fully clear.

Methods

We analyzed α-Syn associations with the acidic phosphoinositides (PIPs) and asked whether these associations may provide answers to ongoing controversies in the field. That is, 1) α-Syn role in synaptic vesicles and clathrin mediated endocytosis (CME), and 2) α-Syn role in axonal growth, plasticity and integrity.

Results

Our data show that α-Syn associates with phosphatidylinositol 4,5-bisphosphate (PI4,5P₂) and phosphatidylinositol 3,4-bisphosphate (PI3,4P₂). We find that α-Syn involvement in endocytosis is dependent on the availability of PI4,5P₂ levels on the plasma membrane. In accord with their effects on PI4,5P₂ levels, the PD associated A30P, E46K and A53T mutations in α-Syn further enhance CME in neuronal and non-neuronal cells. We further show that the rate of synaptic vesicle (SV) endocytosis is affected by the α-Syn mutations and associates with their effects on PI4,5P₂ levels, however, with the exception of the A30P mutation.

Our results also show that through regulating PI4,5P₂ levels, α-Syn acts to elongate the main axon and collaterals, resulting in a higher density of axons in the striatal white matter tracts (WMTs). In human brains, our data link α-Syn- enhanced axonal growth with evidence for axonal injury and accumulation of Lewy pathology.

Conclusions

Based on the data, we suggest that α-Syn associations with PIPs provide insights to its physiology in the healthy brain and pathology in PD.

Aims

Parkinson's disease (PD) is a neurodegenerative disorder characterized by defects in membrane trafficking. SNCA, encoding α-synuclein (α-syn), is a major genetic determinant of PD pathogenesis involved in membrane trafficking. α-syn has recently emerged as regulator of SNARE (Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor)-dependent vesicle fusion. Recently the vesicular SNARE protein VAMP4 emerged as novel PD-risk factor and VAMP7 as a mediator of ER-phagy secretion. Here we proposed to investigate the potential functional interactions between α-syn and VAMPs 4 and 7 as well as the aggregation profile of α-syn and its release in cell medium

Methods

The co-localization and interaction between α-syn and VAMPs was analyzed by immunocytochemistry and Proximity Ligation Assay. The released α-syn exocytosis was measured at different time points upon α-syn over-expression in cells. Extracellular vesicles isolation from cell culture media was performed in VAMP2, -4, -7 KO PC12 cells. Pharmacological treatment was performed in order to analyze the clearance of VAMP2, VAMP-4, VAMP-7, upon lysosomal inhibition in cells.

Results

We present preliminary results of PLA and co-localization testing the interaction between α-syn and VAMP4/7. Effects of inhibition of lysosomal activity on both VAMPs and α-syn levels were also tested. The α-syn release in cell medium is detectable up to 48h after transfection. We observed a reduced α-syn release in extracellular vesicles of PC12 KO for VAMP4 and VAMP7 compared to WT PC12 cells

Conclusions

α-syn and VAMPs are both involved in vesicular and membrane trafficking and our preliminary results may suggest an involvement of the same pathway.

Aims

Objectives. Receptors of the low-density lipoprotein (LDLR) family are involved in endocytosis and APP (amyloid precursor protein) processing. ApoER2/LRP8, a member of this family, is cleaved after binding of the agonist reelin. This generates an intracellular domain (ApoER2-ICD) that regulates reelin expression. We aim to analyse whether ApoER2-ICD regulates the expression of LRP3, the most unknown LDLR. We analyzed LRP3 expression in middle-aged individuals (MA) and Alzheimer’s disease (AD)-related pathology individuals, and the relation of LRP3 with APP.

Methods

Methods. Full-length ApoER2 and ApoER2-ICD were overexpressed in SH-SY5H cells, and recombinant reelin was used to induce ApoER2 cleavage. LRP3 expression in human frontal cortex extracts was analysed from MA cases (n=11) and AD-related pathology cases (Braak NFT stages I-II, n=14; III-IV, n=14; V-VI, n=12). LRP3 was overexpressed in CHO-PS70 cells to analyse APP protein levels.

Results

Results. Both ApoER2 and ApoER2-ICD overexpression increased LRP3 expression, this being enhanced by reelin treatment. In human frontal cortex extracts LRP3 co-immunoprecipitated with apolipoprotein E and APP. In AD-related pathology cases, the levels of LRP3 mRNA and protein were lower at Braak I-II than those in MA subjects. Interestingly, LRP3 transfection in CHO-PS70 cells induced a decrease of full-length APP levels, APP-CTF, soluble APP fragments (sAPPa and sAPPβ) and Aβ peptides. When lysosomal/autophagy function was impaired by chloroquine, full-length APP and sAPPα levels increased significantly, respect to non-treated cells.

Conclusions

Conclusion. LRP3 expression is regulated via ApoER2/reelin signaling, and its levels are affected at early AD-related pathology. LRP3 is involved in APP expression likely by inducing its endocytosis.

Aims

The etiology of late-onset Alzheimer’s disease (LOAD) is multifactorial, with aging being the most significant risk factor and genetic predisposition accelerating the disease onset. While dominant mutations in the rare early-onset familial AD lead to excessive neuronal production of the longest form of beta-amyloid (Abeta42), in LOAD, it remains to be established if Ab42 production increases. Abeta is produced intracellularly upon APP processing in endosomes and controlled by the trafficking of APP and its secretases. We hypothesize that endosomal trafficking dysfunction is a causal mechanism of Abeta accumulation in LOAD.

We aim to dissect the mechanisms whereby LOAD risk factors and neuronal aging alter endocytic trafficking to potentiate Abeta42 production.

Methods

We analyzed wild-type primary mouse cortical neurons matured or aged in culture using a sensitive cell biological and neurobiological approach to determine APP and BACE1 trafficking alterations and their impact on the Abeta42 production.

Results

We have discovered that AD patients’ mutations in Bin1 and CD2AP (LOAD genetic risk factors) increase Abeta42 endocytic production, recapitulating the impact of Bin1 and CD2AP loss of function on BACE1 recycling and APP sorting to lysosomal degradation, indicating that the mutations may be pathological. Importantly, we discovered that neuronal aging alone potentiates clathrin and actin-mediated APP endocytosis, increasing intracellular Abeta42.

Conclusions

Our results highlight the importance of endocytic trafficking defects, driven by LOAD genetic risk factors and aging, as a central LOAD mechanism.