Loss of Neurofascin 186 and Ankyrin G in Purkinje Cells Causes Disruption of Axon Initial Segments, Neurodegeneration and Cerebellar Ataxia

The axon initial segment (AIS) is essential for initiating action potentials and maintaining neuronal polarity, yet the developmental roles of its core molecular components—Neurofascin 186 (NF186) and Ankyrin G (AnkG)—remain incompletely defined in cerebellar Purkinje cells. Here, we generated Purkinje cell-specific NF186/AnkG double knockout mice to investigate how these adhesion and scaffolding proteins cooperatively regulate AIS formation, ion channel localization, synaptic targeting, and neuronal survival. We found that combined deletion of NF186 and AnkG markedly disrupted assembly and maintenance of the AIS cytoskeleton during postnatal development. Other AIS-enriched proteins, including βIV Spectrin (βIVSpec), voltage-gated sodium (Nav) and potassium (Kv1.2) channels, failed to properly localize to the AIS and progressively disintegrated between postnatal days 10 and 30. Notably, Kv1.2 clustering in the pinceau synapse was disrupted, and basket cell axons showed misaligned terminal organization, indicating defective inhibitory synapse innervation. By two months of age, degeneration of Purkinje cells was evident, accompanied by cerebellar dysfunction. In addition, our RNA-seq analysis revealed that Purkinje cell-specific loss of NF186 predominantly activated immune-inflammatory pathways, AnkG loss significantly disrupted neuronal developmental and metabolic processes, and the dual loss of NF186/AnkG produced transcriptional changes that were distinct from, and in part intermediate to, those observed in every single knockout. Collectively, our results show that NF186 and AnkG have complementary, non-redundant roles to establish and maintain the Purkinje cell AIS, and that their loss disrupts synaptic organization at the AIS. These findings advance our understanding of AIS development in cerebellar neurons and have implications for diseases involving AIS dysfunction, including cerebellar ataxia and demyelinating neuropathies.