Project Summary/Abstract (max. 30 lines) Loss of corneal sensory nerves develops secondary to herpes infections, stroke, or trauma; treatments are often only partially successful and lead to suffering and vision loss. The long-term objective of our research is to identify the factors that permit the corneal epithelium to reform a stable barrier after trauma so we can intervene to prevent corneal erosions from forming. Our studies of the cornea have recently focused on the intraepithelial corneal sensory nerves (ICNs) which consist of the intraepithelial corneal basal nerves (ICBNs) and the intraepithelial corneal nerve terminals (ICNTs). Mitomycin C (MMC) applied topically at the time of debridement injury acts as a neuroprotective agent. Corneal epithelial and resident dendritic cells phagocytose axonal mitochondria (aMito) from severed axons after injury. Apical cell desquamation, ICN growth and ICNT shedding are under diurnal control. We hypothesize that the ocular surface barrier and the extent of corneal tissue damage in response to injury and irritants varies with the time of day. We will test this hypothesis in Aim A by answering the following questions: 1. Does shedding of the ICNTs and the desquamation of squames occur spontaneously when lights turn on or does it persist in the dark? 2. Do differences in the corneal barrier and ICN density in the resting and active phase in the mouse impact the extent of damage done to the cornea and signaling by the ICNs to the trigeminal ganglion in response to injury and after exposure to ocular irritants The daily shedding and severing the ICNs leads to accumulation of aMito within corneal epithelial cell lysosomes followed by their degradation via transmitophagy; by contrast to retinal pigment epithelial (RPE) cells, the mechanisms that regulate phagocytosis of axonal debris in the corneal epithelium are not known. Data from our lab and others lead us to hypothesize that corneal epithelial cells internalize aMito via LC3B associated phagocytosis (LAP) and, after internalization, corneal epithelial cells regulate whether aMito undergo transmitophagy or are retained in their cytoplasm to function metabolically. We test this hypothesis in Aim B by answering the following questions: 1. Is phagocytosis of aMito and severed axons by corneal epithelial cells mediated by LAP? 2. Are functional aMito retained in the cytoplasm of epithelial cells after being transferred from axons into corneal epithelial cells during homeostasis and in response to injury? The experiments proposed use quantitative in vitro and in vivo cellular and molecular approaches to answer these questions. The knowledge gained from these studies will not only inform clinicians about the optimal time of day to deliver treatments for ocular surface disorders, they will also clarify the role that mitochondrial transfer has on enhancing wound recovery after injury in the cornea.