Selective Disruption of Metabotropic Glutamate Receptor 5-Homer Interactions Mimics Phenotypes of Fragile X Syndrome in Mice

Abstract

Altered function of the Gq-coupled, Group 1 metabotropic glutamate receptors, specifically mGlu5, is implicated in multiple mouse models of autism and intellectual disability. mGlu5 dysfunction has been most well characterized in the fragile X syndrome mouse model, the Fmr1 knock-out (KO) mouse, where pharmacological and genetic reduction of mGlu5 reverses many phenotypes. mGlu5 is less associated with its scaffolding protein Homer in Fmr1 KO mice, and restoration of mGlu5-Homer interactions by genetic deletion of a short, dominant negative of Homer, H1a, rescues many phenotypes of Fmr1 KO mice. These results suggested that disruption of mGlu5-Homer leads to phenotypes of FXS. To test this idea, we examined mice with a knockin mutation of mGlu5 (F1128R; mGlu5 R/R ) that abrogates binding to Homer. Although FMRP levels were normal, mGlu5 R/R mice mimicked multiple phenotypes of Fmr1 KO mice, including reduced mGlu5 association with the postsynaptic density, enhanced constitutive mGlu5 signaling to protein synthesis, deficits in agonist-induced translational control, protein synthesis-independent LTD, neocortical hyperexcitability, audiogenic seizures, and altered behaviors, including anxiety and sensorimotor gating. These results reveal new roles for the Homer scaffolds in regulation of mGlu5 function and implicate a specific molecular mechanism in a complex brain disease.

Activity-dependent changes in synaptic protein complex composition are consistent in different detergents despite differential solubility

“…Alternatively, widespread dissociation of scaffold proteins may allow for the competitive capture of dendritic proteins, expansion of the synapse, and an increase in synaptic strength (22). Decreased association of Homer_mGluR5 itself has been crucially implicated in both normal mGluR5 signaling and the pathophysiology of Fragile X syndrome (19,29,44), further supporting the physiological relevance of our findings in NP-40 and Triton conditions. Collectively, our data support a model in which strong synaptic stimulation elicits widespread dissociation of multiprotein complexes localized to the PSD.…”

“…We initially examined the effect of lysis buffer reagents on Homer_mGluR5, a well characterized synaptic protein interaction known to play an important role in glutamatergic signaling (19,26,29,35). Significantly more Homer_mGluR5 co-association was detected in NP-40 and Triton compared to DOC ( Figure 3B, C).…”

“…We find this unlikely for three reasons: first, we confirmed by SEC fractionation that both NP-40 and Triton solubilize high MW complexes that contain 4 well known PSD associated proteins: Homer, mGluR5, PSD-95, and SynGAP. Second, we recapitulated two well-known activity dependent interactions that have been localized to within the PSD, dissociation of Homer_mGluR5 (29) and SynGAP_PSD95 (22). We find it unlikely that the high MW multiprotein complexes observed by SEC-western in NP-40 or Triton conditions are both composed of PSD-associated proteins, and exhibit activity-dependent dynamics similar to the PSD, but are not actually part of the PSD.…”

“…However, because the PSD, as it is traditionally defined, is not soluble in NP40, it is unclear if these dissociations represent proteins localized to the PSD, or a group of peri-synaptic proteins. The Huber group showed that levels of mGluR5 found in a PSD fraction are also reduced following activity, suggesting direct relevance of the mGluR-Homer dissociation (29). Similarly, Frank et al (11) reported that PSDassociated protein super-complexes were consistent across five different detergents.…”

“…While many large protein complexes do survive DOC solubilization and have been extensively characterized by mass spectrometry and other approaches (7,11,26,27), interactions that might be present at the native PSD but that do not survive DOC solubilization may remain biochemically uncharacterized. Recently, we and others have reported activitydependent changes in protein co-associations involving presumably synaptic proteins using the relatively gentle, non-ionic detergent NP-40 (18,19,28,29). For example, Ronesi et.…”