Analysis of mouse models to understand disease mechanisms and test potential therapies in ATP1A3-related disorders

Background and Objective: The ATP1A3 gene encodes the α3 subunit of the Na+,K+-ATPase, a critical ion pump responsible for maintaining electrochemical gradients across the membrane, particularly in neurons, and supporting action potential recovery. Mutations in ATP1A3 cause several neurological disorders, including Alternating Hemiplegia of Childhood (AHC), Rapid-onset Dystonia-Parkinsonism (RDP), and Cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) syndrome. In this study, we characterised two novel mouse models, Atp1a3D923Y/+ (AHC-associated) and Atp1a3E818K/+ (CAPOS), to evaluate their phenotypic relevance to human disease and explore the models’ utility for understanding ATP1A3-related pathophysiology. As there are currently no widely effective treatments for ATP1A3-related disorders, we generated a double mutant mouse model, Atp1a3E309D;D923Y/+, to assess the in vivo rescuing effect of a second-site mutation (E309D), which was predicted by in silico structural modelling to disrupt a salt bridge at the extracellular surface of the Na+,K+-ATPase.

Methods: A comprehensive set of behavioural, sensory, and physiological assessments was conducted on Atp1a3E818K/+, Atp1a3D923Y/+, and Atp1a3E309D;D923Y/+ mice in comparison with wild-type littermates. Phenotyping included evaluation of general locomotor activity and anxiety-related behaviours, motor coordination and balance, learning and memory performance, visual function, and auditory function. Molecular and cellular analyses were also performed to investigate underlying disease-related mechanisms.

Results: Atp1a3E818K/+ mice (8-24 weeks of age) recapitulated several core features of CAPOS syndrome, including cerebellar ataxia, cognitive deficits, and features consistent with optic atrophy. Atp1a3D923Y/+ mice (8 weeks of age) exhibited locomotor hyperactivity, inattention, cognitive impairment, and impaired motor coordination, reflecting non-paroxysmal phenotypes observed in AHC patients. Importantly, these behavioural abnormalities were partially rescued in Atp1a3E309D;D923Y/+ double-mutant mice, supporting the therapeutic potential of second-site modification.

Interpretation: This study establishes and validates novel mouse models of AHC and CAPOS syndrome that recapitulate key and overlapping of their respective human conditions. Furthermore, the phenotypic rescue observed in Atp1a3E309D;D923Y/+ mice demonstrates a modus operandi by which future pharmacological interventions might restore Na+,K+-ATPase function and have therapeutic effects in patients with AHC and other ATP1A3-related disorders.