Vargas, K. J. | bioRxiv Science

bioRxiv2020

α-Synuclein facilitates clathrin assembly in synaptic vesicle endocytosis

-Synuclein and family members {beta}-, and {gamma}-synuclein, are presynaptic proteins that sense and generate membrane curvature, properties important for synaptic vesicle (SV) cycling. {beta}{gamma}-synuclein triple knockout (KO) neurons exhibit SV endocytosis (SVE) deficits. Here, we investigate how SVE is regulated by -synuclein. Immuno-electron microscopy (EM) of synaptosomes reveals that -synuclein relocalizes from SVs to the synaptic membrane upon stimulation, allowing -synuclein to function on presynaptic membranes during or after stimulation. On cell membranes, we observe that -synuclein is colocalized with clathrin and its adaptor AP180. Clathrin patches that contain both -synuclein and AP180 were significantly larger than clathrin patches containing either protein alone. We also find that recruitment of clathrin and AP180 recruitment to membranes are altered in the absence of synucleins. Visualizing clathrin assembly on membranes using an in vitro endocytosis reconstitution system reveals that -synuclein increases clathrin patch size and enhances clathrin lattice curvature, facilitating normal clathrin coated pit maturation. Thus, -synuclein is an endocytic accessory protein that acts at early stages of SVE to controls the size and curvature of clathrin structures on the membrane.

neuroscience↗

bioRxiv2020

α-Synuclein-112 impairs synaptic vesicle recycling consistent with its enhanced membrane binding properties

Synucleinopathies are neurological disorders associated with -synuclein overexpression and aggregation. While it is well established that overexpression of wild type -synuclein (-syn-140) leads to cellular toxicity and neurodegeneration, much less is known about other naturally occurring -synuclein splice isoforms. In this study we provide the first detailed examination of the synaptic effects caused by one of these splice isoforms, -synuclein-112 (-syn-112). -Syn-112 is produced by an in-frame excision of exon 5, resulting in deletion of amino acids 103-130 in the C-terminal region. -Syn-112 is upregulated in the substantia nigra, frontal cortex, and cerebellum of parkinsonian brains and is correlated with susceptibility to sporadic Parkinsons disease (PD), dementia with Lewy bodies (DLB) and multiple systems atrophy (MSA). We report here that -syn-112 binds strongly to anionic phospholipids when presented in highly-curved liposomes, similar to -syn-140. However, -syn-112 bound significantly stronger to all phospholipids tested, including the phosphoinositides. -Syn-112 also dimerized and trimerized on isolated synaptic membranes, while -syn-140 remained largely monomeric. When introduced acutely to lamprey synapses, -syn-112 robustly inhibited synaptic vesicle recycling. Interestingly, -syn-112 produced effects on the plasma membrane and clathrin-mediated synaptic vesicle endocytosis that were phenotypically intermediate between those caused by monomeric and dimeric -syn-140. These findings indicate that -syn-112 exhibits enhanced phospholipid binding and oligomerization in vitro and consequently interferes with synaptic vesicle recycling in vivo in ways that are consistent with its biochemical properties. This study provides additional evidence suggesting that impaired vesicle endocytosis is a cellular target of excess -synuclein and advances our understanding of potential mechanisms underlying disease pathogenesis in the synucleinopathies.

neuroscience↗