Wild-Type Monomeric α-Synuclein Can Impair Vesicle Endocytosis and Synaptic Fidelity via Tubulin Polymerization at the Calyx of Held

Abstract

α-Synuclein is a presynaptic protein the function of which has yet to be identified, but its neuronal content increases in patients of synucleinopathies including Parkinson's disease. Chronic overexpression of α-synuclein reportedly expresses various phenotypes of synaptic dysfunction, but the primary target of its toxicity has not been determined. To investigate this, we acutely loaded human recombinant α-synuclein or its pathological mutants in their monomeric forms into the calyces of Held presynaptic terminals in slices from auditorily mature and immature rats of either sex. Membrane capacitance measurements revealed significant and specific inhibitory effects of WT monomeric α-synuclein on vesicle endocytosis throughout development. However, the α-synuclein A53T mutant affected vesicle endocytosis only at immature calyces, whereas the A30P mutant had no effect throughout. The endocytic impairment by WT α-synuclein was rescued by intraterminal coloading of the microtubule (MT) polymerization blocker nocodazole. Furthermore, it was reversibly rescued by presynaptically loaded photostatin-1, a photoswitcheable inhibitor of MT polymerization, in a light-wavelength-dependent manner. In contrast, endocytic inhibition by the A53T mutant at immature calyces was not rescued by nocodazole. Functionally, presynaptically loaded WT α-synuclein had no effect on basal synaptic transmission evoked at a low frequency, but significantly attenuated exocytosis and impaired the fidelity of neurotransmission during prolonged high-frequency stimulation. We conclude that monomeric WT α-synuclein primarily inhibits vesicle endocytosis via MT overassembly, thereby impairing high-frequency neurotransmission. Abnormal α-synuclein abundance is associated with synucleinopathies including Parkinson's disease, but neither the primary target of α-synuclein toxicity nor its mechanism is identified. Here, we loaded monomeric α-synuclein directly into mammalian glutamatergic nerve terminals and found that it primarily inhibits vesicle endocytosis and subsequently impairs exocytosis and neurotransmission fidelity during prolonged high-frequency stimulation. Such α-synuclein toxicity could be rescued by blocking microtubule polymerization, suggesting that microtubule overassembly underlies the toxicity of acutely elevated α-synuclein in the nerve terminal.

Role of the endolysosomal system in Parkinson’s disease

“…A similar observation was made in mammalian Calyx of Held synapse, where acute elevation of monomeric and mutant a-synuclein levels decreased SV endocytosis with a frequency-dependent effect on SV exocytosis. This was also seen upon transgenic expression of a-synuclein (Xu et al 2016;Eguchi et al 2017). Overexpression of mutant a-synuclein also affects dopamine transporter trafficking on the pre-synaptic membrane, leading to dopamine dyshomeostasis (Kisos et al 2014).…”

The physiological role of α‐synuclein and its relationship to Parkinson’s Disease

“…The acute addition of α‐synuclein is a strength of these studies since it suggests a direct effect on these cellular processes. In one study, mutations were found to disrupt the effect on endocytosis (Eguchi et al ), providing an important negative control. We note however that the direct injection of recombinant protein also raises the possibility of toxicity that may not occur with expression by the cell.…”

“…The acute addition of asynuclein is a strength of these studies since it suggests a direct effect on these cellular processes. In one study, mutations were found to disrupt the effect on endocytosis (Eguchi et al 2017), providing an important negative syn syn Fig. 1 The Role of Synuclein in Exocytosis.…”

Hemithioindigos for Cellular Photopharmacology: Desymmetrised Molecular Switch Scaffolds Enabling Design Control over the Isomer‐Dependency of Potent Antimitotic Bioactivity

“…The requirement for correctly functional microtubule dynamics during mitosis has driven extensive work on targeted and selective microtubule‐binding agents as “antimitotic” cancer chemotherapeutics, including paclitaxel, epothilone and vinca alkaloids . In research, the sheer breadth and spatiotemporal complexity of microtubule‐dependent biological roles presents an excellent opportunity for photopharmacological intervention, and microtubule‐targeting photopharmaceuticals such as PST‐1 have quickly moved on from chemical proof‐of‐concept to find diverse cellular and animal research applications . Due to the massive clinical utility of microtubule‐binding agents in cancer therapy, as well as the systemic side‐effects of conventional antimitotics, it is also of extensive interest to find more potent photoswitchable microtubule inhibitors and evaluate whether they can be guided by localised tumour illumination to act as tumour‐specific antimitotics that spare healthy tissues from chemotherapeutic damage.…”