Selective Deletion of Astroglial FMRP Dysregulates Glutamate Transporter GLT1 and Contributes to Fragile X Syndrome Phenotypes In Vivo

Abstract

How the loss of fragile X mental retardation protein (FMRP) in different brain cell types, especially in non-neuron glial cells, induces fragile X syndrome (FXS) phenotypes has just begun to be understood. In the current study, we generated inducible astrocyte-specific Fmr1 conditional knock-out mice (i-astro-Fmr1-cKO) and restoration mice (i-astro-Fmr1-cON) to study the in vivo modulation of FXS synaptic phenotypes by astroglial FMRP. We found that functional expression of glutamate transporter GLT1 is 40% decreased in i-astro-Fmr1-cKO somatosensory cortical astrocytes in vivo, which can be fully rescued by the selective re-expression of FMRP in astrocytes in i-astro-Fmr1-cON mice. Although the selective loss of astroglial FMRP only modestly increases spine density and length in cortical pyramidal neurons, selective re-expression of FMRP in astrocytes significantly attenuates abnormal spine morphology in these neurons of i-astro-Fmr1-cON mice. Moreover, we found that basal protein synthesis levels and immunoreactivity of phosphorylated S6 ribosomal protein (p-s6P) is significantly increased in i-astro-Fmr1-cKO mice, while the enhanced cortical protein synthesis observed in Fmr1 KO mice is mitigated in i-astro-Fmr1-cON mice. Furthermore, ceftriaxone-mediated upregulation of surface GLT1 expression restores functional glutamate uptake and attenuates enhanced neuronal excitability in Fmr1 KO mice. In particular, ceftriaxone significantly decreases the growth rate of abnormally accelerated body weight and completely corrects spine abnormality in Fmr1 KO mice. Together, these results show that the selective loss of astroglial FMRP contributes to cortical synaptic deficits in FXS, presumably through dysregulated astroglial glutamate transporter GLT1 and impaired glutamate uptake. These results suggest the involvement of astrocytemediated mechanisms in the pathogenesis of FXS.

Astrocytic Contributions to Synaptic and Learning Abnormalities in a Mouse Model of Fragile X Syndrome

“…Loss of function or over-expression of these molecules has been shown to affect spine pathology. A recent study showed that Fmr1 deletion in astrocytes in vivo reduced expression of the major glutamate transporter, GLT-1, which normally functions to control extracellular synaptic glutamate levels (47). Loss of GLT-1-mediated glutamate uptake elevated the level of extracellular glutamate and consequently increased the excitability of layer V pyramidal neurons (19, 47).…”

“…A recent study showed that Fmr1 deletion in astrocytes in vivo reduced expression of the major glutamate transporter, GLT-1, which normally functions to control extracellular synaptic glutamate levels (47). Loss of GLT-1-mediated glutamate uptake elevated the level of extracellular glutamate and consequently increased the excitability of layer V pyramidal neurons (19, 47). As raised local glutamate concentration may promote de novo spine growth (48), dysregulated glutamate homeostasis induced by the loss of FMRP in astrocytes may partially account for the increased spine formation observed in global and astrocyte-specific Fmr1 KO mice.…”

The interplay between neurons and glia in synapse development and plasticity

“…Since glia play crucial roles in synapse development and plasticity, it is not surprising that misregulated astrocytes and microglia underlie the pathological mechanisms at work in many neural disorders. Inclusively, glial cells may regulate the pathology of Rett syndrome [55], Down syndrome [56], Spinal Muscular Atrophy [57], Fragile X syndrome [58] and others [59]. Surprisingly, abnormal phenotypes can be rescued or alleviated by wild type glial cells, even when the neurons still harbor the detrimental mutations.…”

FMR1loss results in early changes to intrinsic membrane excitability in human cellular models

“…This suggests that additional cell types or 152 conditions may be required to unmask synaptic phenotypes in vitro. For example, our inclusion 153 of mouse glia increased the health and viability of our neuronal cultures, but could also reduce 154 phenotypic severity if glia contribute to electrophysiological phenotypes in FXS, as reported in 155 other experimental contexts (29)(30)(31). While our experiments allow us to sensitively reveal 156 phenotypes specific to excitatory cortical neurons, they cannot capture the complex relationship 157 with inhibitory neurons and how this may drive network perturbations.…”