Cellular Pathophysiology of Neuronal Na/K-ATPase Dysfunction

Heterozygous loss-of-function mutations in ATP1A3, the gene encoding the catalytic (α3) subunit of the neuronal Na/K-ATPase, are associated with a spectrum of neurodevelopmental syndromes including the prototypical disorder Alternating Hemiplegia of Childhood (AHC), which has no effective therapy. These conditions are associated with acute attacks of transient weakness and dystonia, and poor long term outcome with delayed neurodevelopment and brain atrophy believed secondary to chronic neuron loss. Although rare, ATP1A3 mutations evoke neurological dysfunction shared by common disorders such as epilepsy and migraine. While much has been learned about the genetic basis of these disorders, the cellular consequences of ATP1A3 dysfunction in human neurons and fundamental pathophysiological mechanisms are poorly understood. We have modeled the cellular effects of ATP1A3 mutations using neurons differentiated from patient-specific induced pluripotent stem cells (iPSCs). We propose to exploit this model to determine cellular pathophysiological mechanisms associated with impaired Na/K pump activity and the resulting altered ion homeostasis that explain both short term (hemiplegia, dystonia) and long term (developmental delay, chronic neuron loss) manifestation of ATP1A3 dysfunction. In Aim 1, we will test the hypothesis that direct measurement of neuronal pump current can distinguish between haploinsufficiency and dominant-negative mechanisms, and determine if impaired pump activity can be rescued with a viral ATP1A3 transgene. In Aim 2, we will test the hypothesis that a blunted transmembrane K+ concentration gradient causes a depolarized neuronal resting membrane potential as a consequence of lower than normal driving force mediating outward K+ leak current, which impacts neuronal excitability. We will test this hypothesis by determining if potentiating K+ leak channel activity pharmacologically or genetically in ATP1A3 mutant neurons will compensate for the blunted intracellular to extracellular K+ driving force, normalize the resting potential and prevent depolarization block. Separate experiments will investigate susceptibility to and recovery from depolarization block between mutant and non-mutant neurons, and correlate these findings with intracellular Na+ dynamics. In Aim 3, we will investigate potential cellular pathophysiological mechanisms responsible for the long-term manifestations of ATP1A3. We will test the hypothesis that ATP1A3 mutant neurons exhibit a delayed GABA switch and this can be corrected by inhibition or knockdown of the Na/K/2Cl cotransporter (NKCC1). Finally, we will test hypothesis that impaired Na/K-ATPase activity renders neurons susceptible to intracellular Na+ overload, which can trigger cytosolic Ca2+ overload and cytotoxicity. Collectively, this work will reveal important aspects of short- and long-term neuronal pathogenesis associated with ATP1A3 dysfunction, and promote a mechanistically driven approach to finding new therapeutic strategies