Phospholipid scramblase Xkr8 is required for developmental axon pruning via phosphatidylserine exposure

The mature mammalian brain connectome emerges during development via the extension and pruning of neuronal connections. Glial cells have been identified as key players in the phagocytic elimination of neuronal synapses and projections. Recently, phosphatidylserine has been identified as neuronal “eat‐me” signal that guides elimination of unnecessary input sources, but the associated transduction systems involved in such pruning are yet to be described. Here, we identified Xk‐related protein 8 (Xkr8), a phospholipid scramblase, as a key factor for the pruning of axons in the developing mammalian brain. We found that mouse Xkr8 is highly expressed immediately after birth and required for phosphatidylserine exposure in the hippocampus. Mice lacking Xkr8 showed excess excitatory nerve terminals, increased density of cortico‐cortical and cortico‐spinal projections, aberrant electrophysiological profiles of hippocampal neurons, and global brain hyperconnectivity. These data identify phospholipid scrambling by Xkr8 as a central process in the labeling and discrimination of developing neuronal projections for pruning in the mammalian brain. Synopsis Phosphatidylserine exposure is required for neuronal circuit refinement during mammalian development, but the upstream mechanisms causing its externalization are unclear. This study demonstrates that phospholipid scramblase Xkr8 is developmentally regulated in the mouse brain and required for postnatal axonal pruning. Emx1 ::Cre allows to conditionally delete Xkr8 in excitatory cortical and hippocampal neurons. Lack of Xkr8 leads to increased density of vGluT1 + particles and reduced microglial uptake of synaptic and axonal material in developing somatosensory cortex. Xkr8 cKO mice have increased axonal density in the corpus callosum and in the corticospinal tract indicating impaired axonal pruning. Xkr8 deficiency has long‐term post‐developmental effects on synaptic function and global connectivity observed in the adult brain. Graphical Abstract Xkr8 regulates phospholipid exposure on neurons to promote the labeling and discrimination of developing neuronal projections for pruning in developing mammalian brain.