Role for the Na⁺/K⁺ATPase pump alpha 3 (ATP1A3) subunit in folding and lamination of the human neocortex

Abstract

Osmotic equilibrium and membrane potential in animal cells depend on concentration gradients of sodium (Na+) and potassium (K+) ions across the plasma membrane, a function that is catalyzed by the Na,K-ATPase alpha subunit. In vertebrates, four paralogous genes, ATP1A1-4, encode distinct alpha subunit isoforms (a1-a4), three of which (a1, a2, a3) are expressed in the brain, and two (a1, a3) predominantly in neurons. The a3 isoform, encoded by ATP1A3, is critical to neuronal physiology, and a growing spectrum of neurological diseases are associated with ATP1A3 pathogenic variants, with ages of onset ranging from early childhood to adulthood. Here, we describe ATP1A3 variants encoding dysfunctional a3 subunits in children affected by polymicrogyria, a developmental malformation of the cerebral cortex characterized by abnormal folding and laminar organization. To gain cell-biological insights into the spatiotemporal dynamics of prenatal ATP1A3 expression, we established a transcriptional atlas of ATP1A3 expression during cortical development using mRNA in situ hybridization and transcriptomic profiling of ~125,000 individual cells with single-cell RNA sequencing (Drop-Seq) from various areas of the midgestational human neocortex. We find that fetal expression of ATP1A3 is restricted to a subset of excitatory neurons carrying transcriptional signatures of neuronal activity and maturation characteristic of the developing subplate. Furthermore, by performing Drop-Seq on ~52,000 nuclei from four different areas of an infant human neocortex, we show that ATP1A3 expression persists throughout early postnatal development, not only within excitatory neurons across all cortical layers, but also and more predominantly in inhibitory neurons, with specific enrichment in fast-spiking basket cells. In addition, we show that ATP1A3 expression, both in fetal and postnatal neurons, tends to be higher in frontal cortical areas than in occipital areas, in a pattern consistent with the rostro-caudal maturation gradient of the human neocortex. Finally, we discover distinct co-expression patterns linking catalytic α subunit isoforms (ATP1A1,2,3) and auxiliary isoforms (ATP1B1,2,3), suggesting the ATPase pump may form non-redundant, cell-type specific α-β combinations. Together, the importance of the developmental phenotypes and dynamic expression patterns of ATP1A3 point to a key role for a3 in the development and function of human cortex.

Exome Sequencing and the Identification of New Genes and Shared Mechanisms in Polymicrogyria

“…Of 90 families in our polymicrogyria cohort with identified genetic explanations, 16 (17.7%) had variants in genes encoding ion-conducting proteins, on par with categories that have previously been considered the main genetic etiologies of polymicrogyria, such as mTORopathies and tubulinopathies. Traditionally associated with epilepsy in the absence of brain malformation, genes encoding ion-conducting proteins have been associated with polymicrogyria in multiple case series 43 , 44 , 45 ; however, even recent cohort studies and reviews of polymicrogyria have discussed channelopathies as an exceptional or emerging, rather than common cause of polymicrogyria, and such genes are not included in most MCD gene panels. 1 , 2 , 3 , 8 , 9 Many ion-conducting proteins are ubiquitously expressed in the developing brain with cell type and temporal specificity, which suggests that control of ionic flux is a key factor in normal cortical folding.…”