Enhanced mGluR5 function is causally associated with the pathophysiology of Fragile X Syndrome (FXS), a leading inherited cause of intellectual disability and autism. Here we provide evidence that altered mGluR5-Homer scaffolds contribute to mGluR5 dysfunction and phenotypes in the FXS mouse model, Fmr1 KO. In Fmr1 KO mice mGluR5 is less associated with long Homer isoforms, but more associated with the short Homer1a. Genetic deletion of Homer1a restores mGluR5- long Homer scaffolds and corrects multiple phenotypes in Fmr1 KO mice including altered mGluR5 signaling, neocortical circuit dysfunction, and behavior. Acute, peptide-mediated disruption of mGluR5-Homer scaffolds in wildtype mice mimics many Fmr1 KO phenotypes. In contrast, Homer1a deletion does not rescue altered mGluR-dependent long-term synaptic depression or translational control of FMRP target mRNAs. Our findings reveal novel functions for mGluR5-Homer interactions in the brain and delineate distinct mechanisms of mGluR5 dysfunction in a mouse model of cognitive dysfunction and autism.

SUMMARY A novel experience induces Arc/Arg3.1 gene as well as plasticity of CA1 neural networks. To understand how these are linked, we briefly exposed GFP reporter mice of Arc transcription to a novel environment. Excitatory synaptic function of CA1 neurons with recent in vivo Arc-induction (ArcGFP+) was similar to neighboring non-induced neurons (ArcGFP–). However, in response to group 1 mGluR activation, ArcGFP+ neurons preferentially displayed long-term synaptic depression (mGluR-LTD) and robust increases in dendritic Arc protein. mGluR-LTD in ArcGFP+ neurons required rapid protein synthesis and Arc suggesting that dendritic translation of Arc underlies the priming of mGluR-LTD. In support of this idea, novelty exposure increased Arc mRNA in CA1 dendrites and promoted mGluR-induced translation of Arc in hippocampal synaptoneurosomes. Repeated experience suppressed synaptic transmission onto ArcGFP+ neurons and occluded mGluR-LTD ex vivo. mGluR-LTD priming in neurons with similar Arc activation history may contribute to encoding a novel environment.

Summary Abnormal metabotropic glutamate receptor 5 (mGluR5) function, as a result of disrupted scaffolding with its binding partner Homer, contributes to the pathophysiology of Fragile X Syndrome, a common inherited from of intellectual disability and autism caused by mutations in Fmr1. How loss of Fmr1 disrupts mGluR5-Homer scaffolds is unknown, and little is known about the dynamic regulation of mGluR5-Homer scaffolds in wildtype neurons. Here we demonstrate that brief (minutes) elevations in neural activity cause CaMKIIα-mediated phosphorylation of long Homer proteins and dissociation from mGluR5 at synapses. In Fmr1 knockout cortex, Homers are hyperphosphorylated as a result of elevated CaMKIIα protein. Genetic or pharmacological inhibition of CaMKIIα or replacement of Homers with dephosphomimetics restores mGluR5-Homer scaffolds and multiple Fmr1 KO phenotypes, including circuit hyperexcitability and/or seizures. This work links translational control of an FMRP target mRNA, CaMKIIα, to the molecular, cellular and circuit level brain dysfunction in a complex neurodevelopmental disorder.

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.

BACKGROUND The Activity-regulated cytoskeleton-associated protein, Arc, is an immediate-early gene product implicated in various forms of synaptic plasticity. Arc promotes endocytosis of AMPA type glutamate receptors and regulates cytoskeletal assembly in neuronal dendrites. Its role in endocytosis may be mediated by its reported interaction with dynamin 2 (Dyn2), a 100 kDa GTPase that polymerizes around the necks of budding vesicles and catalyzes membrane scission. METHODS Enzymatic and turbidity assays are used in this study to monitor effects of Arc on dynamin activity and polymerization. Arc oligomerization is measured using a combination of approaches, including size exclusion chromatography, sedimentation analysis, dynamic light scattering, fluorescence correlation spectroscopy, and electron microscopy. RESULTS We present evidence that bacterially-expressed His6-Arc facilitates the polymerization of Dyn2 and stimulates its GTPase activity under physiologic conditions (37°C and 100 mM NaCl). At lower ionic strength Arc also stabilizes pre-formed Dyn2 polymers against GTP-dependent disassembly, thereby prolonging assembly-dependent GTP hydrolysis catalyzed by Dyn2. Arc also increases the GTPase activity of Dyn3, an isoform of implicated in dendrite remodeling, but does not affect the activity of Dyn1, a neuron-specific isoform involved in synaptic vesicle recycling. We further show in this study that Arc (either His6-tagged or untagged) has a tendency to form large soluble oligomers, which may function as a scaffold for dynamin assembly and activation. CONCLUSIONS and GENERAL SIGNIFICANCE The ability of Arc to enhance dynamin polymerization and GTPase activation may provide a mechanism to explain Arc-mediated endocytosis of AMPA receptors and the accompanying effects on synaptic plasticity. This study represents the first detailed characterization of the physical properties of Arc.