Abstract
α-Synuclein is a presynaptic protein that regulates synaptic vesicle (SV) trafficking. In Parkinson’s disease (PD) and several other neurodegenerative disorders, aberrant oligomerization and aggregation of α-synuclein lead to synaptic dysfunction and neurotoxicity. Despite evidence that α-synuclein oligomers are generated within neurons under physiological conditions, and that altering the balance of monomers and oligomers contributes to disease pathogenesis, how each molecular species of α-synuclein impacts SV trafficking is currently unknown. To address this, we have taken advantage of lamprey giant reticulospinal (RS) synapses, which are accessible to acute perturbations via axonal microinjection of recombinant proteins. We previously reported that acute introduction of monomeric α-synuclein inhibited SV recycling, including effects on the clathrin pathway. Here, we report the effects of α-synuclein dimers at synapses. Similar to monomeric α-synuclein, both recombinant α-synuclein dimers that were evaluated bound to small liposomes containing anionic lipids in vitro, but with reduced efficacy. When introduced to synapses, the α-synuclein dimers also induced SV recycling defects, which included a build up of clathrin-coated pits (CCPs) with constricted necks that were still attached to the plasma membrane, a phenotype indicative of a vesicle fission defect. Interestingly, both α-synuclein dimers induced longer necks on CCPs as well as complex, branching membrane tubules, which were distinct from the CCPs induced by a dynamin inhibitor, Dynasore. In contrast, monomeric α-synuclein induced a buildup of free clathrin-coated vesicles (CCVs), indicating an inhibition of clathrin-mediated endocytosis at a later stage during the clathrin uncoating process. Taken together, these data further support the conclusion that excess α-synuclein impairs SV recycling. The data additionally reveal that monomeric and dimeric α-synuclein produce distinct effects on clathrin-mediated endocytosis, predicting different molecular mechanisms. Understanding what these mechanisms are could help to further elucidate the normal functions of this protein, as well as the mechanisms underlying PD pathologies.
Highlights
IntroductionLamprey reticulospinal (RS) synapses are large, glutamatergic synapses that reside within the giant axons of the lamprey spinal cord, and they have been instrumental in elucidating the molecular mechanisms of synaptic vesicles (SVs) trafficking (Pieribone et al, 1995; Shupliakov et al, 1997; Gad et al, 2000; Bloom et al, 2003; Brodin and Shupliakov, 2006)
Reviewed by: Ling-Gang Wu, National Institute of Neurological Disorders and Stroke (NIH), United States Jacob Bendor, University of California, San Francisco, United States
Its physiological function is still under investigation, overexpression or mutation of the α-synuclein gene, which lead to misfolding and aggregation of the protein, are linked to familial Parkinson’s disease (PD; Krüger et al, 1998; Spillantini et al, 1998; Lee and Trojanowski, 2006; Bendor et al, 2013; Dettmer et al, 2016; Lautenschläger et al, 2017). α-Synuclein overexpression and aggregation are commonly observed in other neurodegenerative disorders, including Lewy body dementia (LBD), multiple systems atrophy and some variants of Alzheimer’s disease (Schulz-Schaeffer, 2010; Moussaud et al, 2014; Ingelsson, 2016)
Summary
Lamprey reticulospinal (RS) synapses are large, glutamatergic synapses that reside within the giant axons of the lamprey spinal cord, and they have been instrumental in elucidating the molecular mechanisms of SV trafficking (Pieribone et al, 1995; Shupliakov et al, 1997; Gad et al, 2000; Bloom et al, 2003; Brodin and Shupliakov, 2006) Using this preparation, we previously demonstrated that acute introduction of monomeric α-synuclein directly to presynapses severely inhibited SV endocytosis (Busch et al, 2014). Unlike monomeric α-synuclein, which inhibits the uncoating of CCVs, the α-synuclein dimers selectively inhibited clathrin-mediated SV recycling at an earlier stage during the vesicle fission step These effects were distinct from those produced by an inhibitor of dynamin, the GTPase that drives vesicle scission from the PM. These data further corroborate the finding that excess α-synuclein impairs SV endocytosis and illustrate a novel concept that α-synuclein α-Synuclein Dimers Inhibit Vesicle Endocytosis monomers and dimers affect different steps in the endocytic pathway
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