Molecular mechanisms of glial-mediated synapse alterations in Fragile X syndrome

SUMMARY Fragile X Syndrome (FXS) is the most common single-gene cause of both autism and intellectual disability and is caused by the transcriptional silencing of Fmr1. While neuronal mechanisms underlying aspects of FXS have been identified, therapeutic strategies targeting these mechanisms have been largely unsuccessful, suggesting that our current focus is missing an essential piece of the puzzle. One such missing piece may be glia, or the non-neuronal cells of the brain, which express FMRP and which our lab and others have recently identified as involved in FXS-associated phenotypes. We are leveraging the unique advantages of the developing visual (retinogeniculate) system, a classic model for studying synapse growth and refinement, to test the hypothesis that loss of FMRP alters glia-dependent mechanisms of synapse refinement. Retinal ganglion cells (RGCs) extend from the retina and synapse onto the dorsal lateral geniculate nucleus (dLGN) of the brain, initially forming an excess of synaptic connections that are subsequently eliminated in a stereotyped manner to segregate inputs from each eye. This refinement process requires astrocytic and microglial engulfment of synapses. In preliminary data, we found that RGC segregation is enhanced in the Fmr1 KO dLGN at P7, a time of peak refinement. To identify the molecular mechanisms underlying this enhanced refinement, we performed differential single-nucleus RNA sequencing (snSeq) of the P7 Fmr1 KO and WT dLGN in collaboration with Drs. Michael Greenberg and Lucas Cheadle (Harvard) and identified 15 different cell type clusters, each with distinct sets of differentially expressed genes in KO vs WT. Notably, this unsupervised analysis identified two distinct clusters of astrocytes in both KO and WT, suggesting two astrocyte subpopulations. It also identified an overexpression of the phagocytic TAM receptors, MerTK and Axl, as well as their binding partners and downstream effectors in astrocytes and microglia. MerTK and Axl, not previously studied in the context of autism, are known mediators of astrocyte and microglial engulfment. Indeed, we find that both astrocyte and microglial engulfment of RGC synapses is enhanced in the P7 Fmr1 KO dLGN when compared with WT littermates. Taken together, these data are consistent with the hypothesis that loss of FMRP increases synaptic elimination via glial-mediated phagocytosis. Here we will investigate whether pharmacological inhibition of the glial phagocytic TAM receptors MerTK and Axl will alter Fmr1 KO developmental phenotypes (Aim 1) and whether other systemic changes in gene expression might contribute to FXS-related defects at this stage of development (Aim 2). Importantly, TAM receptors are a novel potential therapeutic autism target, and an effective pharmacological dual-inhibitor of MerTK and Axl (ONO-7475) is already in FDA-approved Phase I Clinical Trials as a cancer therapeutic. Because changes in synapse structure are a general feature of autism, the mechanisms identified here may be applicable to other brain regions and other types of autism.