Hsc70 Ameliorates the Vesicle Recycling Defects Caused by Excess α-Synuclein at Synapses

Abstract

␣-Synuclein overexpression and aggregation are linked to Parkinson's disease (PD), dementia with Lewy bodies (DLB), and several other neurodegenerative disorders. In addition to effects in the cell body, ␣-synuclein accumulation occurs at presynapses where the protein is normally localized. While it is generally agreed that excess ␣-synuclein impairs synaptic vesicle trafficking, the underlying mechanisms are unknown. We show here that acute introduction of excess human ␣-synuclein at a classic vertebrate synapse, the lamprey reticulospinal (RS) synapse, selectively impaired the uncoating of clathrin-coated vesicles (CCVs) during synaptic vesicle recycling, leading to an increase in endocytic intermediates and a severe depletion of synaptic vesicles. Furthermore, human ␣-synuclein and lamprey ␥-synuclein both interact in vitro with Hsc70, the chaperone protein that uncoats CCVs at synapses. After introducing excess ␣-synuclein, Hsc70 availability was reduced at stimulated synapses, suggesting Hsc70 sequestration as a possible mechanism underlying the synaptic vesicle trafficking defects. In support of this hypothesis, increasing the levels of exogenous Hsc70 along with ␣-synuclein ameliorated the CCV uncoating and vesicle recycling defects. These experiments identify a reduction in Hsc70 availability at synapses, and consequently its function, as the mechanism by which ␣-synuclein induces synaptic vesicle recycling defects. To our knowledge, this is the first report of a viable chaperone-based strategy for reversing the synaptic vesicle trafficking defects associated with excess ␣-synuclein, which may be of value for improving synaptic function in PD and other synuclein-linked diseases.

Effects of Excess Brain-Derived Human α-Synuclein on Synaptic Vesicle Trafficking

“…Next, 2–3 cm segments of spinal cord were dissected and pinned ventral side up in a Sylgard-lined dish containing fresh, oxygenated Lamprey Ringer (100 mM NaCl, 2.1 mM KCl, 1.8 mM MgCl 2 , 4 mM glucose, 2 mM HEPES, 0.5 mM L-glutamine, 2.6 mM CaCl 2 , pH 7.4). Axonal microinjections were performed as previously described ( Medeiros et al, 2017 ; Walsh et al, 2018 ; Banks et al, 2020 ; Soll et al, 2020 ). Briefly, SEC fraction 12 containing human α-synuclein was diluted in lamprey internal solution (180 mM KCl and 10 mM HEPES K+; pH 7.4) to a pipet concentration of 800 nM and subsequently loaded into glass microelectrodes (20–25 MΩ) for microinjection into giant RS axons.…”

“…Using the lamprey giant reticulospinal synapse as a model, our lab previously showed that acute introduction of excess recombinant monomeric or dimeric human α-synuclein led to a loss of synaptic vesicles, expansion of the plasma membrane, and increased numbers of clathrin-coated structures and other endocytic intermediates, indicating impaired synaptic vesicle recycling ( Busch et al, 2014 ; Medeiros et al, 2017 ; Banks et al, 2020 ). An inhibition of endocytosis, but not exocytosis, was also reported at mammalian calyx of Held synapses dialyzed with recombinant human α-synuclein ( Xu et al, 2016 ; Eguchi et al, 2017 ).…”

“…An inhibition of endocytosis, but not exocytosis, was also reported at mammalian calyx of Held synapses dialyzed with recombinant human α-synuclein ( Xu et al, 2016 ; Eguchi et al, 2017 ). Interestingly, at lamprey synapses where clathrin-mediated endocytosis is the dominant mode of synaptic vesicle endocytosis, monomeric and dimeric α-synuclein inhibited different stages of the process ( Busch et al, 2014 ; Medeiros et al, 2017 , 2018 ; Banks et al, 2020 ). While monomeric α-synuclein impaired the uncoating of clathrin-coated vesicles (CCVs), dimeric α-synuclein impaired an earlier stage of vesicle fission ( Medeiros et al, 2017 , 2018 ; Banks et al, 2020 ).…”

“…Interestingly, at lamprey synapses where clathrin-mediated endocytosis is the dominant mode of synaptic vesicle endocytosis, monomeric and dimeric α-synuclein inhibited different stages of the process ( Busch et al, 2014 ; Medeiros et al, 2017 , 2018 ; Banks et al, 2020 ). While monomeric α-synuclein impaired the uncoating of clathrin-coated vesicles (CCVs), dimeric α-synuclein impaired an earlier stage of vesicle fission ( Medeiros et al, 2017 , 2018 ; Banks et al, 2020 ). Together, these studies demonstrated that different molecular species of α-synuclein produce distinct effects on synaptic vesicle endocytosis.…”

Bidirectional, unlike unidirectional transport, allows transporting axonal cargos against their concentration gradient

“…Ref. [42] estimated the concentration of -syn in the presynaptic terminal to be 3.5 M. Monomeric -syn is most likely degraded in proteasomes [43,51].…”