Project Summary:
Proper development of the human cortex is essential for brain function and depends on the synchronization of
complex molecular and cellular processes. Malformations of cortical development may occur in the setting of
genetic mutations altering the activity of genes essential for this synchrony. Polymicrogyria (PMG) is a
malformation of cortical development characterized by abnormal folding of the cerebral cortex and is often
associated with epilepsy and intellectual disability. Our lab has performed whole exome sequencing (WES) of
over 200 families affected with PMG and no previously identified diagnosis, establishing the genetic landscape
of PMG pathogenesis. From our cohort, nearly 20% of mutations found in solved cases occur in ion channels
with high in utero expression, implicating developmental channelopathies as a key pathway in PMG
pathogenesis. However, the role of ion channels in cortical histogenesis—and their contribution to disease—
remains poorly understood. Through our exome analysis, we identified three affected individuals with de novo
missense variants in the gene PANX1, encoding Pannexin 1. PANX1 forms a heptameric ion channel of seven
PANX1 subunits and is expressed in early development in neurons and glia. Opening of the heptameric ion
channel releases small anions and ATP into the extracellular milieu, participating in purinergic signaling. The
channel is further speculated to contribute to the propagation of calcium waves—a form of developmental
electrical signaling that drives cell proliferation and migration—yet its definitive contribution in the fetal cortex is
unknown. Thus, in this proposal, I aim to 1) characterize our novel missense PANX1 variants and their
impact on channel activity and 2) channel structure, and 3) leverage the gyrencephalic ferret as an
animal model to study PANX1’s role in cortical development. In Aim 1, I will investigate how our newly
identified PMG-associated PANX1 variants alter protein function. By overexpressing mutant constructs in
heterologous cells, I will assess expression and localization using western blotting. Furthermore, using both
heterologous cells and induced pluripotent stem (iPSC)-derived neurons, I will determine alterations to ATP
release and channel conductance with patch clamping. In Aim 2, I will perform cryo-electron microscopy on
purified wildtype and mutant channels, assessing how mutations alter structural properties and gap junction
formation. These two aims will provide a molecular, cellular, and structural level mechanism for PANX1
perturbation in disease. In Aim 3, I will provide functional evidence for the association between PANX1 activity
and cortical histogenesis in ferrets. While mice are lissencephalic, ferrets develop gyri, enabling investigation
of disordered gyrification. I will also use calcium imaging to characterize the circuit level impact of PANX1
disruption on developmental calcium signaling. Functional investigation of human PANX1 variants will provide
key insight into how genes associated with PMG influence foundational mechanisms of cortical organization
and will further elucidate our understanding of how electrical activity shapes fetal cortex development.