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

Abstract

Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability 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 RNA binding protein involved in protein synthesis regulation at synapses. Many evidences suggest a central role of the Group-I metabotropic glutamate receptor subtype 5 (mGluR5) in the FXS pathophysiology. In particular, an exaggerated signaling response following mGluR5 activation may underlie synaptic dysfunction in this disorder. Although much work has focused on the dysregulation of synaptic protein synthesis as a consequence of this enhanced mGluR5 signaling, it becomes clear that in FXS there is also an altered balance of mGluR5 association with Homer scaffolding proteins, which are postsynaptic density (PSD) partners of mGluR5. Although an extensive literature describes the mGluR5/Homer association, very little is known about the consequences of the disruption of this interaction in the FXS context. Therefore, the goal of my thesis was to study the consequences of mGluR5/Homer crosstalk disruption in the Fmr1 knockout (KO) mouse model of FXS in term of properties and functions of mGluR5, such as expression during development, surface expression and axonal/dendritic targeting, agonist-induced internalization, surface dynamics and mGluR5-mediated modulation of NMDA receptor (NMDAR) currents. In a first set of experiments we investigated the mGluR5 surface expression in cultured hippocampal neurons from WT and Fmr1 KO mice by using immunofluorescence techniques and biotinylation assay. We found that mGluR5 was more expressed on the neuronal surface and was differently distributed in dendrites and axons of Fmr1 KO cultured neurons. We then hypothesized that these alterations were a direct consequence of the mGluR5/Homer crosstalk disruption. We demonstrated that these altered expression and targeting of mGluR5 were critically dependent on mGluR5/Homer crosstalk disruption. We also observed that mGluR5 did not undergo internalization upon sustained mGluR5 activation with DHPG in Fmr1 KO neurons. This latter phenotype, however, was not dependent on the disruption of the mGluR5/Homer crosstalk. Altogether, these results demonstrate that mGluR5/Homer crosstalk disruption contributes to the pathophysiology of FXS altering expression and targeting of mGluR5 on the surface of Fmr1 KO neurons. In the second part of my study we investigated the consequences of the disrupted mGluR5/Homer crosstalk for the mGluR5 surface dynamics, and consequently for NMDAR function in Fmr1 KO neurons. Using a combination of live-cell imaging and single-molecule tracking, we found that mGluR5/Homer crosstalk disruption specifically increased the mGluR5 lateral diffusion at the synapse of cultured Fmr1 KO hippocampal neurons. The higher mGluR5 mobility resulted in an increased probability of transient physical interaction with NMDAR in the PSD of Fmr1 KO. This interaction altered the mGluR5-mediated modulation of NMDAR currents as evidenced by the two following changes. First, using patch-clamp recordings from CA1 pyramidal neurons, we found that NMDAR-mediated excitatory postsynaptic currents (NMDAR-EPSCs) evoked by Schaffer collateral stimulation showed lower amplitudes in Fmr1 KO neurons. Second, the postsynaptic expression of mGluR5 mediated long term depression (LTD) of NMDAR-EPSCs was reduced in Fmr1 KO neurons. Finally, we demonstrated that these defects in NMDA currents were strongly dependent on the mGluR5/Homer crosstalk disruption and altered mGluR5 dynamics. Our results show that mGluR5/Homer disruption contributes to the mGluR5 dysregulation in Fmr1 KO neurons. This study might have implication for the treatment of mGluR5 synaptic dysfunctions in FXS by targeting mGluR5/Homer interaction and provide new suggestions to correct the defective signaling underlying cognitive impairment and autism.