Borland, C. | Search bioRxiv

bioRxiv2026

Pathogenic KIF1A variants differentially disrupt axonal trafficking and impede synaptic development

The nervous system relies on billions of neurons connected through trillions of synapses to support a vast array of vital functions. Despite the critical importance of this synaptic network, the cellular mechanisms dictating synapse formation during human neurodevelopment remain unclear. Long-distance trafficking of synaptic components is critical for both synaptogenesis and the maintenance of synaptic function across lifespan. The microtubule motor KIF1A has a highly conserved role in the trafficking of synaptic vesicle precursors, while mutations in KIF1A are causal for the neurodevelopmental and neurodegenerative disease KIF1A-Associated Neurological Disorder (KAND). Here, we employ isogenic human induced pluripotent stem cells (iPSCs) gene-edited to express pathogenic KIF1A variants to assess how disparate mutations alter synaptic trafficking and function. We compared the effects of both loss-of-function and gain-of-function mutations on KIF1A motor activity. We found that both null (p.C92*) and hypoactive (p.P305L) mutations induce delayed neurite outgrowth, mislocalization of synaptic cargos, and decreased synapse density. Conversely, the hyperactive KIF1A mutation (p.R350G) supports neurite outgrowth but leads to aberrant motility of synaptic vesicle precursors along the axon. Further, live imaging reveals that hyperactive KIF1A induces deficits in the microtubule-dependent patterning of presynaptic components along the developing axon, suggesting a failure to respond to cytoskeletal cues directing cargo delivery. Functional analysis of neuronal activity via multi-electrode arrays reveals delayed synaptic maturation in loss-of-function mutations (p.P305L, p.C92*). In contrast, the hyperactive p.R350G mutation exhibits accelerated activity maturation and possible excitotoxicity. Together, these data provide insights detailing how pathogenic variants in KIF1A causative for KAND exhibit distinct effects at the molecular level that lead to significant downstream deficits in synaptic function in human neurons.

neuroscience↗

bioRxiv2023

Mass spectrometry and split luciferase complementation assays reveal the MecA protein interactome of Streptococcus mutans

MecA is a highly conserved adaptor protein encoded by prokaryotes from the Bacillota phylum. MecA mutants exhibit similar pleiotropic defects in a variety of organisms, although most of these phenotypes currently lack a mechanistic basis. MecA mediates ClpCP-dependent proteolysis of its substrates, but only several such substrates have been reported in the literature and there are suggestions that proteolysis-independent regulatory mechanisms may also exist. Here, we provide the first comprehensive characterization of the MecA interactome and further assess its regulatory role in Clp-dependent proteolysis. Untargeted coimmunoprecipitation assays coupled with mass spectrometry revealed that the MecA ortholog from the oral pathobiont Streptococcus mutans likely serves as a major protein interaction network hub by potentially complexing with >100 distinct protein substrates, most of which function in highly conserved metabolic pathways. The interactome results were independently verified using a newly developed prokaryotic split luciferase complementation assay (SLCA) to detect MecA protein-protein interactions in vivo. In addition, we further develop a new application of SLCA to support in vivo measurements of MecA relative protein binding affinities. SLCA results were independently verified using targeted coimmunoprecipitation assays, suggesting the general utility of this approach for prokaryotic protein-protein interaction studies. Our results indicate that MecA indeed regulates its interactome through both Clp-dependent proteolysis as well as through an as yet undefined proteolysis-independent mechanism that may affect more than half of its protein interactome. This suggests a significant aspect of MecA regulatory function still has yet to be discovered.

microbiology↗