Browsing by Subject "Receptors, Metabotropic Glutamate"
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Item An Examination of the Mechanisms of Neocortical Network Excitability in a Mouse Model of Fragile X Syndrome(2012-07-16) Hays, Seth Alanson; Huber, Kimberly M.; Gibson, Jay R.; Smith, Dean P.; Cannon, Stephen C.; Kavalali, Ege T.Fragile X Syndrome (FXS) is the most common heritable form of mental retardation. FXS is caused by loss of function mutations in the product of the Fmr1 gene, the Fragile X Mental Retardation Protein (FMRP). Many FXS patients display symptoms that are indicative of hyperexcitable circuitry, including epilepsy, bursting patterns in their EEG, and sensory hypersensitivity. Similarly, the mouse model of FXS, the Fmr1 KO mouse, displays a propensity for audiogenic seizures and altered sensory processing, recapitulating many of the symptoms observed in human patients. An imbalance in excitation to inhibition ratio is thought to underlie many autism spectrum disorders. The hyperexcitability in the Fmr1 KO may reflect a shift in E/I balance. However, despite the evidence for hyperexcitability, no studies have previously examined basal circuit function in the Fmr1 KO. In this study, I report that neocortical networks are hyperexcitable in the Fmr1 KO. This hyperexcitability is manifest as prolonged persistent activity states, or UP states. UP states are cyclic periods of depolarization that occur synchronously throughout local neocortical neurons. UP states arise from local recurrent connections and are regulated by intrinsic neuronal properties. As such, measurement of UP states provides insight into overall circuit properties. Furthermore, I observe that the increase in UP state duration in the Fmr1 KO is intrinsic to neocortical excitatory neurons. Deletion of FMRP in layer 4 or layers 5 and 6 fractionally increases UP state duration, suggesting that the hyperexcitability does not arise from one particular neuronal subtype, but rather all neurons partially contribute to the network hyperexcitation. Disruption of mGluR5 interactions with the scaffolding protein Homer, which results in consitituive mGluR5 signaling, is sufficient to prolong UP states. Futhermore, restoration of Homer-mGluR complexes in the Fmr1 KO reduces UP state duration to wildtype levels. Pharmacological or genetic reduction of metabotropic glutamate receptor 5 (mGluR5) signaling reduces UP state duration in the Fmr1 KO to normal levels, suggesting a potential therapy for Fragile X patients. These data characterize the novel phenotype of hyperexcitable cortical circuitry in the Fmr1 KO. In addition, this study provides support for an mGluR-Homer dependent mechanism underlying the network hyperexcitability that may be useful in developing additional treatments for FXS.Item Rapid Protein Translation Governs Persistent Changes in AMPAR Trafficking in a Form of Long-Term Synaptic Plasticity(2010-05-14) Waung, Maggie Wai-Ming; Huber, Kimberly M.Activation of group 1 metabotropic glutamate receptors (mGluRs) induces long-term depression of glutamatergic synapses (mGluR-LTD). Postsynaptic endocytosis of ionotropic α-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid receptors (AMPARs) accompanies mGluR-LTD, and long-term decreases in AMPAR surface expression most likely mediate this form of synaptic plasticity. In support of this idea, both mGluR-LTD and decreases in AMPA receptors require rapid protein synthesis in dendrites. To understand how newly synthesized proteins maintain decreases in AMPAR surface expression, we examined how mGluRs persistently alter AMPAR trafficking. Using biochemical and immunocytochemical methods in dissociated rat hippocampal cultures, we find that brief activation of mGluRs by the group 1 mGluR selective agonist, DHPG, results in a rapid (10 min) increase in AMPAR endocytosis rate that persists for at least one hour after the removal of agonist. This persistent increase in endocytosis rate is blocked by the protein synthesis inhibitor anisomycin, suggesting that components of the endocytosis machinery are synthesized and necessary for mGluR-LTD. In contrast, treatment of cultures with NMDA, which induces NMDA receptor-dependent LTD causes a long-term (60 min) decrease in AMPAR surface expression, but does not persistently increase endocytosis rate. Recent work has implicated activity-regulated cytoskeletal associated protein (Arc) in the regulation of AMPAR endocytosis through its interactions with endophilin and dynamin, and Arc mRNA is induced in hippocampal CA1 dendrites following behavioral activity. However, little is known about how Arc is locally synthesized at synapses or whether its local synthesis contributes to synaptic plasticity. We find that DHPG induces rapid increases in local and synaptic dendritic Arc protein expression within 10 minutes in hippocampal neurons. Knockdown of Arc by lentiviral delivery of short-hairpin RNA increases basal surface AMPAR expression and synaptic transmission as measured by mEPSC amplitude. Arc knockdown blocks mGluR-induced decreases in surface AMPARs, AMPAR endocytosis as well as mGluR-LTD. Acute inhibition of new Arc translation with antisense nucleotides also blocks mGluR-induced persistent changes in AMPAR trafficking and mGluR-LTD. The involvement of rapid Arc synthesis in mGluR regulation of synaptic function provides a link between behavior-driven neuronal activity and plasticity at the synapse.Item The Role of Homer Scaffolding to Metabotropic Glutamate Receptor 5 in the Mouse Models of Neurodevelopement Disorders(2014-04-16) Collins, Katie Anne; Kavalali, Ege T.; Albanesi, Joseph P.; Parada, Luis F.; Huber, Kimberly M.Autism is a neurological disorder characterized by repetetive behaviors, social anxiety and verbal and non-verbal communication. Fragile X Syndrome (FXS) is the most common genetic cause of autism and inheritable form of intellectual disability. FXS is caused by the transcriptional silencing of the Fmr1 gene, which encodes for the Fragile X Mental Retardation Protein (FMRP), which is a Ribonucleic acid (RNA) binding protein. FMRP binds to messenger RNA (mRNA) and suppresses their translation. FMRP regulates hundreds of mRNAs, making it a complex disease with several possible dysfunctions causing the many symptoms, like audiogenic seizures or hypersensitivity. While there are several studies which rescue phenotypes, there is little known about what causes the abnormalities, and if it is possible to replicate the symptoms with a single genetic manipulation. There is also little know about common links between different genetic causes of autism. In this study, I manipulate the interaction between metabotropic glutamate receptor 5 (mGluR5) and Homer, and report how these interactions are important in causing some of the phenotypes in FXS. By rescuing disrupted mGluR5-Homer interactions with a Homer 1a knock out I can rescue several phenotypes, and by disrupting mGluR5-Homer interactions with an mGluR5 knock-in mouse that is mutated so it cannot bind to Homer, I was able to mimic them. I was able to rescue/mimic increased basal translation, altered mGluR-signaling, increased neocortical excitability, decreased anxiety, and partial rescue/mimic audiogenic seizures seen in Fragile X mice. However, I was unable to rescue/mimic the enhanced mGluR-LTD. In this study, I also report how the disrupted mGluR5-Homer interactions are caused by Homer being phosphorylation by CaMKIIα. CaMKIIα is an FMRP target and elevated in the Fmr1 KO mice, causing increased phosphorylation of Homer. Inhibiting CaMKIIα, rescues mGluR5-Homer interactions, basal protein synthesis rates and increased neocortical excitability. Lastly, I report how in PTEN conditional KO (cKO) mice, another autism model, also have disrupted mGluR5-Homer interactions. The PTEN cKO mice have increased neocortical excitability, which can be rescued by inhibiting mGluR5. This suggests a common mGluR5 dysfunction in multiple autism models, which could lead to a common treatment.Item A Study on an FMRP-Mediated Translational Switch in the MGluR-Triggered Translation of Arc and Synaptic Plasticity(2012-07-16) Niere, Farr; Huber, Kimberly M.The group 1 metabotropic glutamate receptor (mGluR)-stimulated protein synthesis and long-term synaptic depression (mGluR-LTD) are altered in a mouse model of Fragile X Syndrome, Fmr1 knockout (KO) mouse. Fmr1 encodes the Fragile X mental retardation protein (FMRP), a dendritic RNA-binding protein that functions, in part, as a translational suppressor. It is unknown if and how FMRP acutely regulates LTD and/or the rapid synthesis of new proteins required for LTD, such as the activity-regulated cytoskeletal-associated protein (Arc). The protein phosphatase PP2A dephosphorylates FMRP, which contributes to the translational activation of some target mRNAs. Here, I report that PP2A and the dephosphorylation of FMRP at S500 are required for an mGluR-induced, rapid increase in dendritic Arc protein and LTD in rat and mouse hippocampal neurons. In the Fmr1 KO neurons, basal, dendritic Arc protein levels and mGluR-LTD are enhanced, and the mGluR-triggered Arc synthesis is absent. A lentiviral-mediated expression of the wildtype FMRP in Fmr1 KO neurons suppresses basal, dendritic Arc levels and mGluR-LTD, and restores the rapid mGluR-triggered Arc synthesis. A phosphomimic of FMRP (S500D) suppresses steady state dendritic Arc levels but does not rescue the mGluR-induced Arc synthesis. A dephosphomimic of FMRP (S500A) neither suppresses the basal, dendritic Arc levels nor supports the mGluR-induced Arc synthesis. Accordingly, expressing the S500D-FMRP in Fmr1 KO neurons suppresses mGluR-LTD, whereas the S500A-FMRP has no effect. These data support a model whereby a phosphorylated FMRP at S500 functions to suppress the steady state and the mGluR-induced translation of Arc and mGluR-LTD. However, upon mGluR activation of PP2A, FMRP is rapidly dephosphorylated which contributes to the rapid, new synthesis of Arc and mGluR-LTD.