Role of MAST3 kinase in developmental and epileptic encephalopathy

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Epilepsy is one of the most common neurological disorders globally, estimated to affect 3.4 million people in the United States and 50 million people globally. Alarmingly, even in high-income countries, epilepsy in one- third of patients resists conventional drug intervention, highlighting the need to further understand the pathogenesis and molecular changes associated with this disease. Developmental and epileptic encephalopathy (DEE) is a relatively new concept in epilepsy research that describes genetic disorders in which a variation in a gene, such as a mutation, causes increased epileptiform activity and slowing of mental development. Recently, several developmental neurological conditions, such as Rett syndrome, cerebral palsy, and mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations have been associated with mutations in the microtubule-associated serine/threonine (MAST) protein kinase family. Specifically, our colleagues have discovered seven individuals with DEE with de novo missense mutations in the MAST3 gene: G510S, G515S, L516P, and V551L. Our group recently showed in brain that MAST3 phosphorylates ARPP-16, a small heat- and acid-stable protein enriched in striatum and cortex, at Ser46, converting it into a potent inhibitor of the serine/threonine phosphatase PP2A. Preliminary results suggest the DEE mutations in MAST3 cause a gain-of-function increase in MAST3 activity, promoting ARPP-16 phosphorylation at Ser46 which would lead to subsequent inhibition of specific forms of PP2A. Interestingly, other recent studies have identified loss of function mutations in the catalytic C, and substrate targeting B subunits, of PP2A in individuals with intellectual disability and developmental delay, the majority of which also have epilepsy. We hypothesize that increased MAST3 activity, and selective perturbation of PP2A signaling, may in part play a causative role in the development of childhood neurological conditions. The proposed research contains two specific aims to test the hypothesis that the DEE MAST3 mutations increase kinase activity and may change its interactome, causing detrimental changes in neuronal development. Aim 1 will determine which proteins and substrates interact with MAST3, how this interactome changes when the DEE MAST3 mutations are introduced, and how this affects kinase activity. This aim will be achieved using in vitro biochemical techniques, such as kinase assays with recombinant proteins, and pulldown assays in primary neuron cultures to identify interacting proteins and substrates. Aim 2 will investigate the role of the DEE MAST3 mutations in neuronal development and function using primary neuron cultures to visualize dendritic arborization and spines, morphological changes, and electrophysiological properties. Results gained from this study will further elucidate the potential for MAST3 to be a prominent force in the development of several neurological conditions early in life. Understanding this mechanism should lead to new diagnostic tools and interventions aimed at improving the quality of life of individuals affected by these disorders.