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

bioRxiv [Preprint]. 2026 Jun 30:2026.06.26.734915. doi: 10.64898/2026.06.26.734915.

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 neurodegeneration^3-6. 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 diseases^8,9.

PMID:42427633 | PMC:PMC13344984 | DOI:10.64898/2026.06.26.734915