A mutation in vesicular acetylcholine transporter increases tubulin acetylation compromising axonal transport

Abstract

Kinesin-3 UNC-104(KIF1A) is the major anterograde axonal transporter of synaptic vesicles and is expressed pan-neuronally. Genetic defects in this molecular motor are linked to KIF1A-associated neurological disorders (KAND) encompassing Charcot-Marie-Tooth (CMT) disease and hereditary spastic paraplegia (HSP). From a candidate screen for genes causing neurotransmission defects in C. elegans and simultaneously affecting post-translational modification of tubulin, we identified allele unc-17(e245) significantly elevating tubulin acetylation in vitro and in vivo. UNC-17 encodes for a VAChT (vesicle acetylcholine transporter) and its human ortholog SLC18A3 is implicated in Alzheimers and Huntingtons disease. To exclude secondary effects of the unc-17 mutation, we tracked UNC-104 and RAB-3 motility in the non-cholinergic ALM neuron. With upregulated tubulin acetylation in ALM (anterior lateral microtubule) neurons in unc-17(e245) strains (visualized by immunostaining), motility of both, motor and its cargo, is significantly compromised. However, motility of UNC-104 improves when knocking down alpha-tubulin acetyltransferase MEC-17(ATAT1) in unc-17(e245) strains and, conversely, is negatively affected when overexpressing MEC-17 in wild type animals. UNC-17 and UNC-104 are co-expressed and colocalize in cholinergic head neurons, suggesting a functional motor-cargo relationship. Strikingly, mec-17 knockdown significantly enhances their colocalization, while unc-17 knockdown reduces UNC-104/MEC-17 colocalization in ALM neurons. Direct interactions were confirmed by bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP): MEC-17 depletion strengthens UNC-104/UNC-17 association and stabilizes the motor-cargo complex, whereas unc-17 knockdown attenuates UNC-104/MEC-17 interactions, as evidenced by diminished Co-IP signals. We propose that unc-17 knock-down releases MEC-17 from the transport complex, thereby enhancing its tubulin acetyltransferase activity. This elevated acetylation may disrupt motor motility, ultimately impairing synaptic vesicle trafficking. This dynamic interplay, modulated by tubulin acetylation, highlights a regulatory axis involving UNC-104, UNC-17, and MEC-17 across cholinergic and glutamatergic neurons, offering new insights into axonal transport defects relevant to KIF1A-associated neurological disorder (KAND).