Sarbanes, S. | Search bioRxiv

bioRxiv2026

Tubulin autoregulation mediator TTC5 regulates neuronal morphology and migration

Microtubule dynamics regulation is critical for neuronal development, yet how neurons regulate tubulin levels remains poorly understood. Tetratricopeptide repeat domain 5 (TTC5) mediates the co-translational degradation of tubulin transcripts in response to excess soluble tubulin, a.k.a. "tubulin autoregulation", and TTC5 mutations are associated with cerebral atrophy, speech and motor impairment. Despite clinical relevance, the role of TTC5 and tubulin autoregulation in neurons has not been established. Using human induced pluripotent stem cells (iPSCs)-derived cortical-like neurons, we demonstrate that tubulin autoregulation is active in neurons and fully TTC5-dependent. Loss of TTC5 function suppresses microtubule polymerization and impairs axonal outgrowth and arborization, while enhancing cellular motility. These phenotypes recapitulate in vivo where TTC5 loss in mouse cerebral cortex projection neurons disrupts axon and dendrite arborization and drives aberrant hypermigration. Patient disease mutants phenocopy this motility dysregulation, directly linking TTC5 dysfunction to clinically relevant neurological deficits. Together, our findings establish tubulin autoregulation and its mediators as essential regulators of neuronal morphogenesis and connectivity and provides a mechanistic framework for understanding TTC5-associated neurodevelopmental disease.

Cell Biology↗

bioRxiv2025

A system for high-throughput axonal imaging of induced pluripotent stem cell-derived human i3Neurons

Neurons have long, thin axons and branched dendritic processes which rely on an extensive microtubule network that functions as a cellular scaffold and substrate for cargo transport. Microtubule defects are a defining pathological feature of neurological disorders. The highly arborized, long, polarized neuronal processes pose challenges for imaging-based assays. Available methods use either dispersed cultures, which are inefficient for compartment-specific analyses, or microfluidic chambers, which allow clear separation of somatodendritic and axonal compartments but are expensive and difficult to maintain. Here, we introduce an "i3Neurosphere" culture model of induced pluripotent stem cell (iPSC)-derived human cortical i3Neurons that enables high-throughput imaging of hundreds of axons without specialized equipment. We characterize neurite outgrowth, polarization, microtubule dynamics, and motility of diverse cargo, providing a reference for future work on microtubule processes in this system. The high-throughput compartment-specific imaging we present, combined with facile genetic engineering in i3Neurons provides a powerful tool to study human neurons. SIGNIFICANCE STATEMENTHuman neurons are difficult to study due to limited access to tissue and technical challenges in existing in vitro models of axonal transport. We developed i3Neurospheres, a simple and scalable 3D culture system of human iPSC-derived neurons that enables high-throughput imaging of axonal outgrowth, microtubule dynamics, and intracellular transport. This platform provides an accessible, reproducible method for investigating neuronal function and disease mechanisms, offering broad utility for neuroscience research and preclinical drug screening.

neuroscience↗