Microglial P2Y6 receptor calcium signaling as a core regulator of epileptogenesis

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Project Summary/Abstract Microglia, resident immune cells of the central nervous system, utilize G-protein signaling to accomplish different tasks. Gi-signaling is used to perform process outgrowth, while Gs signaling is engaged during periods of neuronal hypoactivity (such as anesthesia). However, very little is known about microglial Gq calcium signaling. Partially, this is due to the fact that microglia rarely display calcium transients at rest. On the other hand, microglia greatly elevate their calcium signaling in the period that follows status epilepticus—a prolonged seizure state predictive of later epilepsy development. The purpose of microglial calcium signaling during epilepsy development is not known, but is hypothesized to be a component of microglial phagocytosis. Most microglial calcium signaling in epilepsy development is attributable to P2Y6 receptor signaling—a Gq-calcium receptor activated by a key purine. P2Y6 is best described for its role in phagocytosis, but that has not been fully explored in epilepsy development. (Innovation) The current proposal will utilize a novel mouse line that allows for the simultaneous examination of microglial calcium activity and process movement in the living animal (using two- photon microscopy). (Aim 1) This line will be used to test the role of P2Y6 in microglial phagocytosis, after neurons are selectively killed through excitotoxicity using optogenetic techniques. In parallel, tissue studies will be conducted across key time points in epilepsy development to determine if the loss of the P2Y6 pathway prevents proper clearance of dead/dying neurons after status epilepticus. The prolonged presence of dying neurons is pro-inflammatory and hypothesized to negatively affect neuronal network dynamics in the long term. (Aim 2) For these reasons, we will test multiple aspects of long-term P2Y6 signaling loss during epilepsy development. We will determine the long-term pro-inflammatory effects of P2Y6 signaling loss. Additionally, we will use miniscope technology and 24/7 video EEG to determine whether the loss of P2Y6 calcium signaling and its putative phagocytosis alters network dysregulation or epilepsy risk. The proposed research will enhance the candidate’s experience in using advanced techniques to probe neuronal circuit function. Such training is directly related to the candidate’s goal of independently studying key glial pathways and how they influence complex neuronal circuits during epilepsy development. These studies will be performed at the Mayo Clinic under the supervision of experts in glia (Dr. Long-Jun Wu), epilepsy (Dr. Greg Worrell and Dr. Peyman Golshani), and miniscope technology (Dr. Luis Lujan and Dr. Peyman Golshani).