Impact of the E209K PACS2 Syndrome mutation on neuronal metabolism and neurotransmission. - Project Summary PACS2 syndrome is a recently identified neurodevelopmental disorder caused by a recurrent de novo missense mutation in PACS2 (p.Glu209Lys (PACS2E209K)). Patients carrying this missense mutation suffer from developmental and epileptic encephalopathy-66 (DEE66), and share several deficits, including neonatal seizures, global developmental delay, hypotonia, autism, and cerebellar dysgenesis. The mechanism by which PACS2E209K causes PACS2 syndrome is unknown, and no curative treatment is available. PACS2 is a multifunctional sorting protein that is essential for formation of ER-mitochondria contacts (MAMs), and localizes calcium signaling molecules to the ER, including the calcium-permeable channel PKD2. MAMs are dynamic quasi-synaptic structures that localize mTORC2/Akt, which phosphorylates PACS2 to modulate calcium transfer and lipid metabolism. MAM-localized PACS2 acts as a phosphorylation state-dependent metabolic switch that coordinates SIRT1/PGC-1α-dependent oxidative metabolism with MAM integrity and function. The E209K substitution is located in a critical autoregulatory domain that controls binding of PACS2 to its client proteins. Preliminary studies suggest PACS2 interacts with PKD2 to mediate ER-mitochondria calcium transfer and that the E209K substitution reduces MAM contacts and diverts calcium into the cytosol. Consequently, PACS2E209K increases aerobic glycolysis in both patient cells and the cortex of Pacs2E209K/+ mice. Consistent with these findings, preliminary electrophysiology studies reveal PACS2E209K increases glutamatergic synaptic inputs in L2/3 cortical neurons. Together, these findings suggest PACS2E209K disturbs MAM function, impacting neuronal metabolism, neurotransmission, and behavior. Our long-term goal is to understand how PACS2E209K causes disease and to use this information to develop effective therapies. The objective of this particular application is to determine how PACS2E209K dysregulates ER-mitochondrial calcium transfer to disturb CNS metabolism and behavior. We hypothesize that PACS2E209K disturbs PKD2 function to dysregulate MAM integrity and calcium diffusion dynamics, which consequently switches the brain to glycolytic metabolism and dysregulates neurotransmission resulting in behavioral deficits. Guided by strong preliminary data, we will test our hypothesis by pursuing three specific aims: 1) Determine how PACS2E209K alters ER-mitochondria calcium coupling in isolated neurons and ex vivo L2/3 slice cultures, 2) Determine the impact of PACS2E209K on fuel handling by measuring glucose and lactate dynamics, mitochondrial metabolism and gene expression, and 3) Determine how PACS2E209K alters synaptic activity, learning and behavior. The approach is innovative because we will combine advanced live-cell imaging with metabolic profiling and behavioral assessments of the first mouse model for PACS2 syndrome to elucidate the mechanism by which the recurrent E209K substitution causes neuronal dysfunction. This research is significant because it will advance our mechanistic understanding of a critical cellular process that might be targeted to treat this debilitating disorder.
