Glial Ca2+signaling links endocytosis to K+ buffering around neuronal somas to regulate excitability

Abstract

Glial-neuronal signaling at synapses is widely studied, but how glia interact with neuronal somas to regulate their activity is unclear. Drosophila cortex glia are restricted to brain regions devoid of synapses, providing an opportunity to characterize interactions with neuronal somas. Mutations in the cortex glial NCKXzydeco elevate basal Ca2+, predisposing animals to seizure-like behavior. To determine how cortex glial Ca2+ signaling controls neuronal excitability, we performed an in vivo modifier screen of the NCKXzydeco seizure phenotype. We show that elevation of glial Ca2+ causes hyperactivation of calcineurin-dependent endocytosis and accumulation of early endosomes. Knockdown of sandman, a K2P channel, recapitulates NCKXzydeco seizures. Indeed, sandman expression on cortex glial membranes is substantially reduced in NCKXzydeco mutants, indicating enhanced internalization of sandman predisposes animals to seizures. These data provide an unexpected link between glial Ca2+ signaling and the well-known role of glia in K+ buffering as a key mechanism for regulating neuronal excitability.

SIK3 suppresses neuronal hyperexcitability by regulating the glial capacity to buffer K+ and water

“…Hence, SIK3 is required for an effective glial response to a K + stress. K + dynamics play a critical role in modulating neuronal activity, and dysfunctional glial K + regulation promotes neuronal hyperexcitability in seizure patients and in mouse and fly models of seizure (Bedner et al, 2015;Weiss et al, 2019). Here we tested whether glial SIK3 has a non-cell autonomous impact on axonal excitability at the larval neuromuscular junction (NMJ).…”

Disruption of Glial Ca2+Oscillations at theDrosophilaBlood-Brain Barrier Predisposes to Seizure-Like Behavior

“…To explore in vivo Ca 2+ dynamics in PG cells, we expressed a myristoylated variant of Ca 2+ -sensitive GFP (myr::GCaMP6s) to monitor Ca 2+ in fine processes 24,27 with the PG driver GMR85G01-gal4 28 (hereafter referred to as PG-gal4). We first performed imaging experiments in live, undissected 2 nd instar larvae as previously described 27 .…”

“…We recently found that chronic Ca 2+ increase in Drosophila cortex glia predispose animals to stimulation-induced seizures 24 , while acute increases in intracellular Ca 2+ in Drosophila astrocyte-like glia drives neuronal silencing 22 . To identify glial signaling pathways that may modulate neuronal excitability, we performed a genetic screen using the pan-glial driver repo-gal4 to drive expression of RNA interference (RNAi) targeting ~850 genes encoding membrane receptors, secreted ligands, ion channels and transporters, vesicular trafficking proteins and known cellular Ca 2+ homeostasis and Ca 2+ signaling pathway components 24 . This screen revealed that pan-glial knockdown of dStim led to severe HS-induced seizures-like episodes (Video 4, middle and Figure 4A, S2A), similar to those we previously identified in NCKX zyd (zyd) mutants that disrupt a Ca 2+ ion exchanger in cortex glia 24,27 .…”

“…Several Drosophila glial subtypes have been found to influence neuronal function via distinct mechanisms downstream of their glial Ca 2+ dynamics. Astrocytic Ca 2+ regulates neurotransmitter uptake 22 and secretion of neuromodulators 23 , while disruption of Ca 2+ signaling in Drosophila cortex glia impairs K + buffering capacity 24 . Synchronized Ca 2 + waves in Drosophila SPG cells control nutrient-dependent reactivation of neural stem cells and subsequent brain growth 25,26 .…”

“…In addition, we find that these Ca 2+ dynamics are independent of extracellular Ca 2+ , originate from internal ER stores and spread as waves through gap junctions. In a genetic screen for glial pathways that modulate neuronal excitability 24 , we found that pan-glial knockdown of dStim, the Drosophila homologue of mammalian Stromal Interaction Molecule 1, leads to severe heatshock (HS) and mechanically-induced seizurelike episodes (hereafter referred to as seizures, see methods for definition). Here we demonstrate that dStim is primarily required in PG cells for seizure induction.…”