“…Here we provide evidence of direct interactions between members of the GABARAP/LC3 subfamily of autophagosomal proteins and oligomeric/fibrillar α-SYN. Impairment of the formation of autophagosomes by α-SYN aggregates has been shown by several studies, resulting in traffic jams of proteins, organelles and lipid membranes [64][65][66] . Previous proteomics studies have provided contradictory results as to whether LC3 proteins actually interact with α-SYN 67 and our data suggest that such interactions can only occur when aggregation is under way.…”
The aggregation of alpha-synuclein (α-SYN) follows a cascade of oligomeric, prefibrillar and fibrillar forms, culminating in the formation of Lewy Bodies (LB), the pathological hallmarks of Parkinson’s Disease. Although LB contain over 70 proteins, the potential for interactions along the aggregation pathway of α-SYN is unknown. Here we propose a map of interactions of 65 proteins against different species of α-SYN. We measured binding to monomeric α-SYN using AlphaScreen, a sensitive nano-bead luminescence assay for detection of protein interactions. To access oligomeric species, we used the pathological mutants of α-SYN (A30P, G51D and A53T) which form oligomers with distinct properties. Finally, we generated amyloid fibrils from recombinant α-SYN. Binding to oligomers and fibrils was measured by two-color coincidence detection (TCCD) on a single molecule spectroscopy setup. Overall, we demonstrate that LB components are recruited to specific steps in the aggregation of α-SYN, uncovering future targets to modulate aggregation in synucleinopathies.
“…Here we provide evidence of direct interactions between members of the GABARAP/LC3 subfamily of autophagosomal proteins and oligomeric/fibrillar α-SYN. Impairment of the formation of autophagosomes by α-SYN aggregates has been shown by several studies, resulting in traffic jams of proteins, organelles and lipid membranes [64][65][66] . Previous proteomics studies have provided contradictory results as to whether LC3 proteins actually interact with α-SYN 67 and our data suggest that such interactions can only occur when aggregation is under way.…”
The aggregation of alpha-synuclein (α-SYN) follows a cascade of oligomeric, prefibrillar and fibrillar forms, culminating in the formation of Lewy Bodies (LB), the pathological hallmarks of Parkinson’s Disease. Although LB contain over 70 proteins, the potential for interactions along the aggregation pathway of α-SYN is unknown. Here we propose a map of interactions of 65 proteins against different species of α-SYN. We measured binding to monomeric α-SYN using AlphaScreen, a sensitive nano-bead luminescence assay for detection of protein interactions. To access oligomeric species, we used the pathological mutants of α-SYN (A30P, G51D and A53T) which form oligomers with distinct properties. Finally, we generated amyloid fibrils from recombinant α-SYN. Binding to oligomers and fibrils was measured by two-color coincidence detection (TCCD) on a single molecule spectroscopy setup. Overall, we demonstrate that LB components are recruited to specific steps in the aggregation of α-SYN, uncovering future targets to modulate aggregation in synucleinopathies.
“…78 ) have largely relied on immunohistochemical staining at the light microscopic level, providing limited resolution. 79 Thus, a bias might have been created in the past for areas that showed the expected rod-like or fibrous structures (see a commentary by Bartels 79 ). The important new findings of Shahmoradian et al 13 are based on state-of-the-art imaging techniques and, if confirmed by others, have the potential to change the ways in which we conceptualize and model PD pathogenesis and design therapeutics.Fig.…”
Section: Membrane-associated αS Aggregation In Lewy Pathologymentioning
Lipids play a more significant role in Parkinson’s disease and its related brain disorders than is currently recognized, supporting a “lipid cascade”. The 14 kDa protein α-synuclein (αS) is strongly associated with Parkinson’s disease (PD), dementia with Lewy bodies (DLB), other synucleinopathies such as multiple system atrophy, and even certain forms of Alzheimer’s disease. Rigorously deciphering the biochemistry of αS in native systems is the key to developing treatments. αS is highly expressed in the brain, the second most lipid-rich organ, and has been proposed to be a lipid-binding protein that physiologically interacts with phospholipids and fatty acids (FAs). αS-rich cytoplasmic inclusions called Lewy bodies and Lewy neurites are the hallmark lesions of synucleinopathies. Excess αS–membrane interactions may trigger proteinaceous αS aggregation by stimulating its primary nucleation. However, αS may also exert its toxicity prior to or independent of its self-aggregation, e.g., via excessive membrane interactions, which may be promoted by certain lipids and FAs. A complex αS-lipid landscape exists, which comprises both physiological and pathological states of αS. As novel insights about the composition of Lewy lesions occur, new lipid-related PD drug candidates emerge, and genome-wide association studies (GWAS) increasingly validate new hits in lipid-associated pathways, it seems timely to review our current knowledge of lipids in PD and consider the roles for these pathways in synucleinopathies.Fig. 1αS ↔ lipid interplay: aspects of cellular αS homeostasis (blue oval), aspects of lipid homeostasis (green oval), and overlapping aspects.Pathological states are labeled in red. Simplified schematic of both select αS and select lipid species. Several existing publications suggest αS effects on lipids and vice versa, as indicated by arrows. DG diglyceride, ER endoplasmic reticulum, FA fatty acid, LD, lipid droplet, TG triglyceride.
“…These findings resonated with reports 13 that native α-Syn binds lipids such as synaptic vesicle membranes 14 , observations that lipids can catalyze α-Syn aggregation in vitro 15 , and that α-Syn expression in cells is associated with membrane abnormalities 9 . Thus, the disease relevance of fibrillar (amyloid-like) α-Syn aggregation has been questioned, leading to a model in which the main role of α-Syn in Lewy bodies is to cluster cellular membranes 8,9 . Cryo-ET is ideally positioned to test these new ideas, as it can reveal the molecular architecture of protein aggregates at high resolution within neurons pristinely preserved by vitrification 16-18 .…”
Alpha-Synuclein (alpha-Syn) aggregation is a hallmark of devastating neurodegenerative disorders including Parkinsons disease (PD) and multiple systems atrophy (MSA). alpha-Syn aggregates spread throughout the brain during disease progression, suggesting mechanisms of intercellular seeding. Formation of alpha-Syn amyloid fibrils is observed in vitro and fibrillar alpha-Syn has been purified from patient brains, but recent reports questioned whether disease-relevant alpha-Syn aggregates are fibrillar in structure. Here we use cryo-electron tomography (cryo-ET) to image neuronal Lewy body-like alpha-Syn inclusions in situ at molecular resolution. We show that the inclusions consist of alpha-Syn fibrils crisscrossing a variety of cellular organelles such as the endoplasmic reticulum (ER), mitochondria and autophagic structures, without interacting with membranes directly. Neuronal inclusions seeded by recombinant or MSA patient-derived alpha-Syn aggregates have overall similar architecture, although MSA-seeded fibrils show higher structural flexibility. Using gold-labeled seeds we find that aggregate nucleation is predominantly mediated by alpha-Syn oligomers, with fibrils growing unidirectionally from the seed. Our results conclusively demonstrate that neuronal alpha-Syn inclusions contain alpha-Syn fibrils intermixed with cellular membranes, and illuminate the mechanism of aggregate nucleation.
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