A Mutation in Vesicular Acetylcholine Transporter Increases Tubulin Acetylation Compromising Synaptic Vesicle...

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), a spectrum of severe neurological conditions 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 (vesicular acetylcholine transporter) and its human ortholog SLC18A3 is implicated in Alzheimer's and Huntington's 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 α-tubulin acetyltransferase MEC-17(ATAT1) in unc-17(e245) strains and, conversely, is negatively affected when overexpressing MEC-17 in wild type animals. Similar effects were observed in cholinergic sublaterals. UNC-17 and UNC-104 colocalize in cholinergic head neurons, consistent with a motor-cargo relationship. Strikingly, mec-17 knockdown significantly decreases their colocalization, while unc-17 knockdown reduces UNC-104/MEC-17 colocalization in head neurons. Direct protein-protein interactions were validated through bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assays. In both assays, mec-17 knockdown significantly reduced the UNC-104/UNC-17 associations, whereas unc-17 knockdown significantly diminished UNC-104/MEC-17 interactions. These findings indicate a tripartite regulatory complex UNC-104/UNC-17/MEC-17. We propose that unc-17 knockdown disrupts sequestration of MEC-17 within this complex and that the release of MEC-17 results in increased tubulin acetylation. Resulting elevated tubulin acetylation suppresses UNC-104 motor processivity and cargo transport efficiency.