Tau isoforms modulate the axon initial segment controlling axonal trafficking and neuronal excitability
The axon initial segment (AIS) is a specialized neuronal compartment integrating action potential initiation with selective control of axonal trafficking. The microtubule associated protein tau is a central regulator of cytoskeletal organization and transport, yet how distinct tau isoforms contribute to AIS development and function remains unclear. Here, we examined the role of tau isoform relative abundance in regulating AIS establishment, maturation, excitability, and transport selectivity using murine primary neurons and human induced pluripotent stem cell (hiPSC)-derived neurons combined with super resolution imaging, electrophysiology, and live trafficking assays. We found that tau expression levels and isoform content modulate the timing and robustness of AIS maturation. In murine neurons, tau deficiency or predominance of 3 repeat (3R) tau delays Ankyrin G accumulation and AIS stabilization without preventing AIS formation. hiPSC-derived neurons display an intrinsic AIS developmental program accompanied by progressive changes in tau isoform content. Super resolution DNA PAINT reveals that endogenous tau decorates axonal microtubules in discrete nanoclusters with compartment specific distributions. Modulation of the endogenous 3R/4R tau balance in hiPSC-derived neurons shows that isoform composition, independently of tau levels, regulates AIS positioning and Ankyrin G organization. Functionally, shifts towards 3R-tau reduce sodium currents, impair action potential firing, and alter lysosomal transport dynamics within the AIS. Together, these findings identify tau isoform balance as a developmental regulator of AIS maturation, linking cytoskeletal organization to neuronal excitability and transport gating. Because the aberrant alternative tau splicing of exon 10 is a defining feature of primary tauopathies, our results provide mechanistic insight into how imbalanced tau isoforms may contribute to neuronal dysfunction.