α-Synuclein facilitates clathrin assembly in synaptic vesicle endocytosis

Abstract

40 word limit): Synucleins can sense and generate membrane 20 curvature. We previously showed that synuclein null mice exhibit deficits in 21 synaptic vesicle endocytosis. Here, Vargas et al. provide evidence that α-synuclein 22 functions specifically in clathrin assembly during early steps of synaptic vesicle 23 endocytosis. 24 25 Abstract (160 word limit): 26 27 α-Synuclein plays a central role in Parkinson's disease (PD); hence, 28 elucidating its normal physiological function(s) is important. α-Synuclein and family 29 members β-, and γ-synuclein, are presynaptically enriched proteins. Synucleins 30 sense and generate membrane curvature, properties consistent with their 31 described roles in synaptic vesicle (SV) cycling. We have previously shown SV 32 endocytosis (SVE) deficits in αβγ-synuclein knockout (KO) neurons. Here, we 33 investigate which steps of SVE are regulated by α-synuclein. Immuno-electron 34 microscopy (EM) of synaptosomes reveals that α-synuclein relocalizes from SVs 35to the synaptic membrane upon stimulation, allowing α-synuclein to function there 36 during or after stimulation. Using membrane recruitment assays, we show that α-37 synuclein is co-localized with clathrin patches. We also observe that recruitment 38 of clathrin and its adaptor, AP180, to synaptic membranes is altered in the absence 39 of synucleins. Visualizing clathrin assembly on membranes in an in vitro 40reconstitution system reveal that synucleins increase clathrin patch size and 41 curvature, facilitating clathrin coated pit maturation during the early steps of SVE. 42 43Introduction 44 45α-Synuclein became a principal focus of neurodegenerative research when 46it was identified as the major constituent of Lewy Bodies, the pathological protein 47aggregates found in the brains of PD patients [1]. The importance of α-synuclein 48 was further underscored by the identification of families with Mendelian forms of 49 PD arising from causal point mutations and gene multiplications of SNCA, the α-50 synuclein gene [2-7]. Genome-wide association studies have shown that 51 sequence variants in SNCA are also associated with sporadic PD [8, 9]. Based on 52these observations, many current therapeutic strategies for PD are focused on 53 eliminating or reducing α-synuclein levels in the brain. Therefore, there is a growing 54interest in understanding α-synuclein's physiological functions and how loss of α-55 synuclein impacts neuronal functions. 56 57Since its discovery as a SV-associated protein in the electric organ of 58Torpedo [10], several distinct approaches have been used to determine the 59 physiological function(s) of α-synuclein. Structural studies have revealed that α-60 synuclein can adopt several conformations, principally, unfolded in solution, but 61 also α-helical on phospholipid membranes. In α-helical conformations, the N-62terminus folds into either a single elongated amphipathic helix on flatter 63 membranes or a 'broken' helix when bound to curved lipid membranes [11, 12]. α-64Synuclein can switch between the two ...

α-Synuclein plasma membrane localization correlates with cellular phosphatidylinositol polyphosphate levels

“…PM-specific αSyn-lipid interactions were additionally confirmed by ‘unroofing’ experiments in related SK-MEL-28 cells ( Kaur and Lee, 2020 ), where endogenous protein pools colocalized with members of the exocytosis machinery including the known αSyn binding partners Rab3A ( Chen et al, 2013 ) and synaptobrevin-2/VAMP2 ( Burré et al, 2010 ). Two other studies implicated αSyn and αSyn-PIP 2 interactions in clathrin assembly and clathrin-mediated endocytosis, respectively ( Vargas et al, 2020 ; Schechter et al, 2020b ), which further strengthens the notion that phosphatidylinositol polyphosphates contribute to αSyn functions at the PM.…”

α-Synuclein plasma membrane localization correlates with cellular phosphatidylinositol polyphosphate levels

“…PM-specific aSyn-lipid interactions were additionally confirmed by 'unroofing' experiments in related SK-MEL-28 cells 55 , where endogenous protein pools co-localized with members of the exocytosis machinery including the known aSyn binding partners Rab3A 56 and synaptobrevin-2/VAMP2 57 . Two other studies implicated aSyn and aSyn-PIP2 interactions in clathrin assembly and clathrin-mediated endocytosis, respectively 58,59 , which further strengthens the notion that phosphatidylinositol polyphosphates contribute to aSyn functions at the plasma membrane. Table 1. 1 µg of plasmids was used in all cases.…”

MOBP and HIP1 in multiple system atrophy: New α‐synuclein partners in glial cytoplasmic inclusions implicated in the disease pathogenesis

“…Our protein–protein network analysis revealed common interactors between HIP1 and SNCA, several of which are involved in clathrin‐mediated endocytosis signalling. Recent studies have implicated SNCA in clathrin assembly and in changes in clathrin‐mediated endocytosis 37,38 . We previously found significant downregulation of SNCA mRNA in MSA cerebellar white matter, and very low levels of SNCA mRNA in microdissected oligodendrocytes 18 .…”

“…Recent studies have implicated SNCA in clathrin assembly and in changes in clathrin-mediated endocytosis. 37,38 We previously found significant downregulation of SNCA mRNA in MSA cerebellar white matter, and very low levels of SNCA mRNA in microdissected oligodendrocytes. 18 These observations support the hypothesis that the accumulation of SNCA in oligodendrocytes in MSA is contributed to by SNCA uptake from neurons or from the extracellular environment through a clathrin-dependent internalization mechanism, 30,39 a process in which HIP1 may be involved.…”