Involvement of mGluR5/Homer crosstalk disruption in the pathophysiology of Fragile X Syndrome

Fragile X Syndrome (FXS) is the most common inherited form of intellectualdisability and autism. FXS is caused by a mutation in the fragile X mental retardation 1(Fmr1) gene which leads to the lack of the encoded FMRP protein. FMRP is an RNAbinding protein involved in protein synthesis regulation at synapses. Many evidencessuggest a central role of the Group-I metabotropic glutamate receptor subtype 5(mGluR5) in the FXS pathophysiology. In particular, an exaggerated signaling responsefollowing mGluR5 activation may underlie synaptic dysfunction in this disorder.Although much work has focused on the dysregulation of synaptic protein synthesis as aconsequence of this enhanced mGluR5 signaling, it becomes clear that in FXS there isalso an altered balance of mGluR5 association with Homer scaffolding proteins, whichare postsynaptic density (PSD) partners of mGluR5. Although an extensive literaturedescribes the mGluR5/Homer association, very little is known about the consequences ofthe disruption of this interaction in the FXS context. Therefore, the goal of my thesis wasto study the consequences of mGluR5/Homer crosstalk disruption in the Fmr1 knockout(KO) mouse model of FXS in terms of properties and functions of mGluR5, such asexpression during development, surface expression and axonal/dendritic targeting,agonist-induced internalization, surface dynamics and mGluR5-mediated modulation ofNMDA receptor (NMDAR) currents.In a first set of experiments we investigated the mGluR5 surface expression incultured hippocampal neurons from WT and Fmr1 KO mice by usingimmunofluorescence techniques and biotinylation assay. We found that mGluR5 wasmore expressed on the neuronal surface and was differently distributed in dendrites andaxons of Fmr1 KO cultured neurons. We then hypothesized that these alterations were adirect consequence of the mGluR5/Homer crosstalk disruption. We demonstrated thatthe altered expression and targeting of mGluR5 were critically dependent onmGluR5/Homer crosstalk disruption. We also observed that mGluR5 did not undergointernalization upon sustained mGluR5 activation with DHPG in Fmr1 KO neurons.This latter phenotype, however, was not dependent on the disruption of themGluR5/Homer crosstalk. Altogether, these results demonstrate that mGluR5/Homercrosstalk disruption contributes to the pathophysiology of FXS altering expression andtargeting of mGluR5 on the surface of Fmr1 KO neurons.In the second part of my study we investigated the consequences of the disruptedmGluR5/Homer crosstalk for the mGluR5 surface dynamics, and consequently forNMDAR function in Fmr1 KO neurons. Using a combination of live-cell imaging andsingle-molecule tracking, we found that mGluR5/Homer crosstalk disruption specificallyincreased the mGluR5 lateral diffusion at the synapse of cultured Fmr1 KO hippocampalneurons. The higher mGluR5 mobility resulted in an increased probability of transientphysical interaction with NMDAR in the PSD of Fmr1 KO. This interaction altered themGluR5-mediated modulation of NMDAR currents as evidenced by the two followingchanges. First, using patch-clamp recordings from CA1 pyramidal neurons, we foundthat NMDAR-mediated excitatory postsynaptic currents (NMDAR-EPSCs) evoked bySchaffer collateral stimulation showed lower amplitudes in Fmr1 KO neurons. Second,the postsynaptic expression of mGluR5 mediated long term depression (LTD) ofNMDAR-EPSCs was reduced in Fmr1 KO neurons. Finally, we demonstrated that thesedefects in NMDA currents were strongly dependent on the mGluR5/Homer crosstalkdisruption and altered mGluR5 dynamics.Altogether, our results show that mGluR5/Homer disruption contributes to themGluR5 dysregulation in Fmr1 KO neurons. This study might have implication for thetreatment of mGluR5 synaptic dysfunctions in FXS by targeting mGluR5/Homerinteraction and provide new suggestions to correct the defective signaling underlyingcognitive impairment and autism.