Molecular and Behavioural Analysis of D. melanogaster as an alternative model of Alternating Hemiplegia of Childhood

Alternating Hemiplegia of Childhood (AHC) is a rare and complex neurodevelopmental disorder hallmarked by infantile onset (<18 months) of recurrent temporal paralysis affecting either one (Hemiplegia) or both sides (Quadriplegia) of the body. These episodes are spontaneous but are also triggered by external stressors and are alleviated during sleep. They can present alone or alongside movement, cognitive, and behavioural symptoms that become more apparent with age. The majority of AHC cases are caused by heterozygous de novo mutations in the ATP1A3 gene, encoding the catalytic α3-subunit of the neuronal Na+/K+ ATPase (NKA) pump. The NKA α3 subunit is responsible for the rapid restoration of basal intracellular Na+ in neurons following sustained discharge. A Drosophila melanogaster mutant carrying the fly equivalent (p.Gly744Ser) of the human AHC p.Gly755Ser mutation within its NKA α-subunit (ATPα) showcased mechanical-stress induced paralysis, suggesting conservation of NKA α-subunit function between species thus the potential to be used as an alternative animal model for in vivo studies of AHC. Consistent with this, further characterisation of the heterozygous p.Gly744Ser and null NKA α flies showed AHC-relevant phenotypes including defective walking speed, heightened sensitivity to cold stress and sleep disturbances. Utilization of the Gal4/UAS system to ubiquitously express a copy of the wildtype human ATP1A3, confirmed by qualitative RT-PCR, rescues sensitivity to cold stress in both p.Gly744Ser and null NKA α flies. Hemizygous transgenic flies expressing the p.Asp801Asn ATP1A3 transgene via the Gal4/UAS system did not exhibit AHC-relevant phenotypes, potentially due to the presence of functional endogenous wildtype ATPα alleles. Overall, the capability of NKA α-modified flies to display other AHC-relevant phenotypes and evidence of functional equivalence between species showcases the fly as a suitable in vivo model for AHC research. Thus, a step in the right direction to successfully implement the replacement principle of the 3Rs.