Browsing by Subject "Fragile X Mental Retardation Protein"
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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 Experience-Dependent and Input-Specific Regulation of Neocortical Circuit Development by Genes Linked to Neurodevelopmental Disorders(2022-05) Zhang, Zhe; Volk, Lenora J.; Huber, Kimberly M.; Gibson, Jay R.; Roberts, Todd; Chahrour, MariaAbnormal structural and functional brain connectivity has been widely observed in human neuropsychiatric diseases. Specifically, patients with neurodevelopmental disorders like autism often show an imbalance in the local versus long-range connectivity for cerebral cortex. Whether and how genes implicated in neurodevelopmental disorders regulate development of cortical synaptic connectivity in a pathway-specific manner remain largely unknown. Furthermore, environmental sensory experience can determine or significantly remodel the postnatal development of synaptic connections and neural circuits in sensory cortices. Knowledge on what intracellular proteins or mechanisms can mediate experience-dependent development of specific cortical synaptic connections is also lacking. In this work, I studied the roles of two neurodevelopment disease implicated genes, namely, fragile X mental retardation 1 (Fmr1) and myocyte enhancer factor 2c (Mef2c) in the postnatal experience-dependent development of input-specific synaptic connections. I report that postnatal, cell-autonomous deletion of Fmr1 in postsynaptic L2/3 or L5 neurons results in a selective weakening of AMPA receptor-, but not NMDA receptor-, mediated callosal synaptic function, indicative of immature synapses. Sensory deprivation by contralateral whisker trimming normalizes callosal input strength, suggesting that experience-driven activity of postsynaptic Fmr1 KO L2/3 neurons weakens callosal synapses. Unlike callosal inputs, synapses originating from local L4 and L2/3 circuits are normal with postsynaptic Fmr1 deletion, revealing an input-specific role for postsynaptic Fmr1 in regulation of synaptic connectivity within local and callosal neocortical circuits. Opposite to Fmr1 KO, postnatal deletion of Mef2c in L2/3 neurons leads to a cell autonomous and selective weakening of excitatory synapses from L4, whereas ipsilateral or contralateral long-range excitatory synaptic inputs are unaffected. Postsynaptic Mef2c only promotes the development but not the maintenance of L4-to-L2/3 excitatory synaptic connections and Fmr1 is not required for this process, in contrast to predictions from work in CA1 hippocampal neurons. Weakening of L4-L2/3 synaptic strength by sensory deprivation can be rescued by postnatal postsynaptic expression of a transcriptionally active form of MEF2C (MEF2-VP16), suggesting that MEF2C transcriptional activation drives experience-dependent development of L4-L2/3 synapses. Together, my findings on Fmr1 and Mef2c demonstrate an interaction of experience and gene functions in regulation of specific synaptic connections with important implications for neurodevelopmental disorders.Item Fragile X Mental Retardation Protein Induces Synapse Loss Through Acute Postsynaptic Translational Regulation(2009-01-14) Pfeiffer, Brad Erich; Huber, Kimberly M.Fragile X Syndrome (FXS) is the most common form of inherited mental retardation. The root cause of FXS is loss of the function of a single protein: the Fragile X Mental Retardation Protein (FMRP). FMRP is an RNA-binding protein that plays a complex role in translational regulation. FMRP may be an important regulator of dendritic protein synthesis, which occurs at or near synapses in response to synaptic activity. Many types of long-term synaptic change require local protein synthesis for their induction and/or maintenance, and several protein synthesis-dependent forms of synaptic plasticity are altered in the absence of FMRP. Both human FXS patients and mice lacking FMRP (Fmr1-KO mice) display increased numbers of dendritic spines, the primary sites of excitatory synaptic connections. In addition to increased numbers, the spines of FXS patients and Fmr1-KO mice appear morphologically immature. It was unknown whether FMRP plays a direct, cell-autonomous role in the regulation of synapse number or function. Moreover, the mechanisms through which FMRP might govern neuronal function or number were unclear. I report that acute postsynaptic expression of FMRP in Fmr1-KO neurons results in a decrease in the number of functional and structural synapses without an effect on their synaptic strength or maturational state. Similarly, wild-type neurons endogenously expressing FMRP have fewer synapses than neighboring Fmr1-KO neurons, indicating a clear role for FMRP in the regulation of synapse number. An intact K homology 2 (KH2) RNA-binding domain and dephosphorylation of FMRP at S500 are required for the effects of FMRP on synapse number, indicating that FMRP-dependent translation of mRNA targets of FMRP leads to synapse loss. Furthermore, I demonstrate novel phenotypic interactions of FMRP with the transcription factor MEF2. MEF2 activity in wild-type neurons induces robust synapse loss; however, MEF2 fails to decrease synapse number in Fmr1-KO neurons. A dominant-negative form of MEF2 increases synapse number in WT, but not Fmr1-KO neurons. Finally, when co-expressed with a dominant negative form of MEF2, FMRP fails to induce synapse loss in Fmr1-KO neurons. These data represent novel mechanisms through which FMRP regulates neuronal function and suggest novel therapeutic targets and strategies for FXS treatment.Item Identification and Characterization of Novel Mechanisms of Functional and Structural Synapse Remodeling: Focus on Vav Guanine Nucleotide Exchange Factors and MEF2 Transcription Factors(2014-07-23) Hale, Carly Fenwick; Huber, Kimberly M.; Cowan, Christopher W.; Green, Carla B.; Kim, Tae-KyungProper development of synaptic connectivity is a dynamic process requiring formation, elimination, maintenance, and plasticity of synapses. During early postnatal development, excess synapses are formed in most neural circuits, which are subsequently pruned during adolescence in a sensory- and activity-dependent mechanism. The brain also exhibits experience-dependent synaptic modifications that may enhance or weaken functional synapse strength. Investigation of numerous neurodevelopmental and psychiatric disorders reveals dysfunctions in synapse formation and function; however, underlying molecular mechanisms remain poorly understood. In Part One of this study, I identify a novel role for Vav guanine nucleotide exchange factors (GEFs) in brain-derived neurotrophic factor (BDNF)-dependent synapse plasticity. BNDF and its receptor, TrkB, are well-established positive modulators of hippocampal long-term potentiation (LTP), and increasing evidence suggests that BDNF/TrkB facilitates LTP in part through the stimulation of Rho GTPases and subsequent F-actin remodeling and dendritic spine structural dynamics. I report that Vav-family GEFs are activated by BDNF/TrkB signaling, and are required for BDNF-induced Rac-GTP formation. Vav GEFs, which are enriched at hippocampal glutamatergic synapses, are necessary for rapid BDNF-induced dendritic spine growth and CA3-CA1 LTP. Furthermore, Vav2/3-deficient mice have impaired contextual fear conditioning, as well as reduced anxiety. Together, findings support a role for Vav-dependent F-actin dynamics in BDNF-stimulated dendritic spine head enlargement and LTP, and normal hippocampal-dependent learning and memory and anxiety in mice. Part Two of this study reports the identification of common MEF2 and FMRP mRNA targets that are required for MEF2-induced synapse elimination. The activity-dependent transcription factor myocyte enhancer factor 2 (MEF2) is a key negative regulator of excitatory synapse number, promoting synapse removal in neurons through a complex program of gene expression. The RNA binding protein and translational regulator fragile X mental retardation protein (FMRP) was recently identified as an essential downstream component of MEF2-induced synapse elimination, suggesting that these autism-linked proteins coordinate transcriptional and translational control of common transcripts to mediate proper synaptic connectivity. Using high throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) of FMRP, I find a large overlap of MEF2-induced transcripts and FMRP-associated mRNAs, consistent with their shared roles in synapse elimination. More specifically, protocadherin 17 (Pcdh17) mRNA is induced by MEF2 and exhibits differential binding to FMRP following MEF2 activation. Reducing Pcdh17 alone does not alter basal synapse number, but reducing Pcdh17 levels blocks MEF2-induced dendritic spine elimination of hippocampal neurons. These data suggest that MEF2-induced synapse elimination requires Pcdh17 – a MEF2 target gene and FMRP-associated transcript.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.