Synaptic activity controls local exposure of an 'eat-me' signal via ANO3-ITPR1 signaling

Abstract

Neuronal synapses are eliminated during brain development and disease through pruning by glial cells. Individual synapses are marked for engulfment by eat-me signals, which include externalized phosphatidylserine. In apoptotic cells, phosphatidylserine externalization is driven by caspase-dependent activation of Xkr scramblases1 and inactivation of specific flippases2. Localized caspase activation at neuronal synapses can mediate spatially-restricted synaptic phosphatidylserine exposure leading to synapse pruning by glial cells during development and neurodegeneration3-6. It is unknown which caspase-regulated flippases and scramblases promote synaptic phosphatidylserine exposure and whether there are any caspase-independent mechanisms of phosphatidylserine exposure relevant for synapse elimination. To address this question, we here develop a scalable CRISPR screening approach, COMPASS-seq (compartment-anchored sgRNA screen sequencing), to uncover the genetic underpinnings of subcellular phenotypes. COMPASS-seq is compatible with in vitro and in vivo systems and a wide range of subcellular compartments; we here apply it to the neuronal synapse. We discover that inhibition of the caspase-independent, calcium-activated anoctamin ANO3 (TMEM16C) is sufficient to increase synapse numbers in vivo. ANO3 co-localizes with IP3 receptor 1 (ITPR1), a calcium channel in the endoplasmic reticulum, to form a postsynaptic signaling platform that drives spatially restricted phosphatidylserine exposure at synapses. Activation of the ITPR1 calcium channel activity is sufficient to drive synaptic phosphatidylserine exposure via an ANO3-dependent but caspase-independent mechanism. Our results suggest a mechanism for integrating synaptic activity information to control synaptic pruning. The role of ANO3 in regulating synapses could shed light on the mechanisms underlying its numerous associations with both dementia7 and other neurological diseases8,9.