Tsc1 Deletion in Purkinje Neurons Disrupts the Axon Initial Segment, Impairing Excitability and Cerebellar Function

Abstract

Loss-of-function mutations in tuberous sclerosis 1 (TSC1) are prevalent monogenic causes of autism spectrum disorder (ASD). Selective deletion of Tsc1 from mouse cerebellar Purkinje neurons has been shown to cause several ASD-linked behavioral impairments, which are linked to reduced Purkinje neuron repetitive firing rates. We used electrophysiology methods to investigate why Purkinje neuron-specific Tsc1 deletion (Tsc1mut/mut) impairs Purkinje neuron firing. These studies revealed a depolarized shift in action potential threshold voltage, an effect that we link to reduced expression of the fast-transient voltage-gated sodium (Nav) current in Tsc1mut/mut Purkinje neurons. The reduced Nav currents in these cells was associated with diminished secondary immunofluorescence from anti-pan Nav channel labeling at Purkinje neuron axon initial segments (AIS). Interestingly, anti-ankyrinG immunofluorescence was also found to be significantly reduced at the AIS of Tsc1mut/mut Purkinje neurons, suggesting Tsc1 is necessary for the organization and functioning of the Purkinje neuron AIS. An analysis of the 1st and 2nd derivative of the action potential voltage-waveform supported this hypothesis, revealing spike initiation and propagation from the AIS of Tsc1mut/mut Purkinje neurons is impaired compared to age-matched control Purkinje neurons. Heterozygous Tsc1 deletion resulted in no significant changes in the firing properties of adult Purkinje neurons, and slight reductions in anti-pan Nav and anti-ankyrinG labeling at the Purkinje neuron AIS, revealing deficits in Purkinje neuron firing due to Tsc1 haploinsufficiency are delayed compared to age-matched Tsc1mut/mut Purkinje neurons. Together, these data reveal the loss of Tsc1 impairs Purkinje neuron firing and membrane excitability through the dysregulation of proteins necessary for AIS organization and function.