Astrocyte-microglia signaling controls developmental thalamocortical synapse refinement

Synapse formation and elimination are two crucial processes that concurrently take place in the developing brain. Astrocytes and microglia have been shown to control both processes. However, it is largely unknown how these two major glial cell types of the central nervous system (CNS) communicate to balance synapse formation and elimination. Astrocytes secrete a synaptogenic protein called Hevin/SPARCL1, which induces the formation and plasticity of thalamocortical synapses in the mouse visual cortex. Hevin does so by physically localizing to synaptic clefts and bridging the thalamic axon/cortical dendrite via its interactions with presynaptic Neurexin1a and postsynaptic Neuroligin1b. Here, we found that in addition to this synaptogenic function, Hevin directly signals to microglia cells by interacting with Toll-like Receptors (TLRs) TLR4 and TLR2. This signaling occurs when Hevin is proteolytically cleaved producing an active C-terminal fragment. This fragment is sufficient to upregulate TLR2 expression in microglia and increase microglia phagocytic activity in vivo. This signaling is required for proper refinement of thalamocortical synapses in early postnatal development and for early life ocular dominance plasticity. Overall design: Microglia were isolated from P7-P9 mouse forebrains via Magnetic Activated Cell-Sorting using Cd11b microbeads. 40-80k cells were plated in each well of a 96 well tissue culture plate. Cells were treated with recombinant proteins at 500nM final concentration. LPS treatment was done at a final concentration of 50ng/mL. OxPAPC treated cells was performed at a final concentration of 15ug/mL. Control cells received additional growth media equivalent to the treatment volume. Microglia were cultured for 6 hours after treatment and then collected and pelleted. RNA was extracted from the cell pellets. RNA was sent to MedGenome for RNA-sequencing library generation and sequencing. Libraries were generated using the Illumina TruSeq stranded mRNA kit and then sequenced on a NovaSeq.