TSC1 regulation of the purkinje neuron axon initial segment and cerebellar function

The National Institute of Neurological Disorders and Stroke awarded Miami University $539,920 on September 5, 2026, under the Extramural Research Programs in the Neurosciences and Neurological Disorders program (CFDA 93.853) to investigate how TSC1 loss disrupts Purkinje neuron axon initial segment organization and cerebellar function in autism spectrum disorder.

The research examines the cellular mechanisms by which TSC1 deletion in Purkinje neurons alters axon initial segment function and intrinsic firing through three specific aims. Aim 1 tests whether TSC1 deletion impairs Purkinje neuron firing through axon initial segment dysfunction using patch clamp electrophysiology, dynamic clamp rescue strategies, and quantitative immunofluorescence of axon initial segment proteins. Aim 2 determines whether Purkinje neuron excitability and axon initial segment phenotypes caused by TSC1 deletion are mediated by mTORC1 rather than mTORC2 signaling using neuron-targeted viral knockdown approaches and assessing molecular, electrophysiological, and behavioral outcomes. Aim 3 defines the mechanism underlying ankyrin-G loss by distinguishing among reduced ANK3 transcription, increased axon initial segment scaffold turnover, or increased proteolysis through transcript profiling from sorted Purkinje neurons and quantitative analysis.

Performance of work occurs in Oxford, Ohio. The period of performance extends from the award date through August 31, 2029. This is a project grant, an assistance type under NINDS extramural research funding.

Abstract

Autism spectrum disorder (ASD) is strongly linked to cerebellar dysfunction. Multiple lines of evidence indicate that impaired cerebellar purkinje neuron (PN) activity can contribute directly to ASD-related behaviors. Loss-of-function mutations in TSC1 cause tuberous sclerosis complex (TSC), a multisystem disorder frequently associated with ASD. In a mouse model with PN-specific TSC1 deletion (L7/PCP2-CRE (+) ; TSC1FLOX/flox), PNS exhibit reduced repetitive firing, decreased axon initial segment (AIS) sodium channel expression, and a marked reduction of the AIS scaffolding protein ankyrin-g (ankg). These observations suggest that loss of TSC1 results in a disruption of the molecular organization at the purkinje neuron AIS, and thereby, impairs intrinsic excitability and cerebellar function. The overall goal of this project is to extend this work and define the cellular mechanisms by which TSC1 loss in PNS alters AIS function, intrinsic firing, and cerebellum-dependent behaviors. Aim 1 will test the hypothesis that TSC1 deletion impairs PN firing through AIS dysfunction, extending beyond the loss of AIS NAV channels, by testing if AIS potassium channel mechanisms that shape repolarization are also disrupted. Aim 1 will use patch clamp electrophysiology, dynamic clamp rescue strategies, and quantitative immunofluorescence of AIS proteins. Aim 2 will directly determine whether the PN excitability and AIS phenotypes caused by TSC1 deletion are mediated by MTORC1, rather than MTORC2 signaling, using PN-targeted viral knockdown approaches and assessing molecular, electrophysiological, and behavioral outcomes. Aim 3 will define the mechanism underlying ankg loss by distinguishing among reduced ANK3 transcription, increased AIS scaffold turnover, or increased proteolysis, integrating transcript profiling from sorted PNS with pharmacologic perturbations and quantitative analysis of the AIS structure. These studies will advance our understanding of how TSC1-mtor pathways regulate neuronal excitability at the AIS and will provide mechanistic insight into cerebellar contributions to ASD and TSC. As an area project, this work will provide intensive training for several undergraduate and graduate student researchers in electrophysiology, imaging, cell-type specific molecular profiling, and computational approaches for dynamic clamp experiments.