CDK5/p35-Dependent Microtubule Reorganization Contributes to Homeostatic Shortening of the Axon Initial Segment

Abstract

The structural plasticity of the axon initial segment (AIS) contributes to the homeostatic control of activity and optimizes the function of neural circuits; however, the underlying mechanisms are not fully understood. In this study, we prepared a slice culture containing nucleus magnocellularis from chickens of both sexes that reproduces most features of AIS plasticity in vivo, regarding its effects on characteristics of AIS and cell-type specificity, and revealed that microtubule reorganization via activation of CDK5 underlies plasticity. Treating the culture with a high-K⁺ medium shortened the AIS and reduced sodium current and membrane excitability, specifically in neurons tuned to high-frequency sound, creating a tonotopic difference in AIS length in the nucleus. Pharmacological analyses revealed that this AIS shortening was driven by multiple Ca²⁺ pathways and subsequent signaling molecules that converge on CDK5 via the activation of ERK1/2. AIS shortening was suppressed by overexpression of dominant-negative CDK5, whereas it was facilitated by the overexpression of p35, an activator of CDK5. Notably, p35(T138A), a phosphorylation-inactive mutant of p35, did not shorten the AIS. Moreover, microtubule stabilizers occluded AIS shortening during the p35 overexpression, indicating that CDK5/p35 mediated AIS shortening by promoting disassembly of microtubules at distal AIS. This study highlights the importance of microtubule reorganization and regulation of CDK5 activity in structural AIS plasticity and the tuning of AIS characteristics in neurons.SIGNIFICANCE STATEMENT The structural plasticity of AIS has a strong impact on the output of neurons and plays a fundamental role in the physiology and pathology of the brain. However, the mechanisms linking neuronal activity to structural changes in AIS are not well understood. In this study, we prepared an organotypic culture of avian auditory brainstem, reproducing most AIS plasticity features in vivo, and we revealed that activity-dependent AIS shortening occurs through the disassembly of microtubules at distal AIS via activation of CDK5/p35 signals. This study emphasizes the importance of microtubule reorganization and regulation of CDK5 activity in structural AIS plasticity and tonotopic differentiation of AIS structures in the brainstem auditory circuit.

Keywords: CDK5; Ca2+; axon initial segment; microtubules; p35; plasticity.