Pumping the brakes: A noncanonical RNA-binding domain in FMRP stalls elongating ribosomes.

Loss of function of the RNA-binding protein FMRP causes fragile X syndrome, the most common inherited form of intellectual disability and autism spectrum disorders. FMRP is suggested to modulate synaptic plasticity by regulating the synthesis of proteins involved in neuronal and synaptic function; however, the mechanism underlying FMRP mRNA targeting specificity remains unclear. Intriguing recent work published in JBC by Scarpitti and colleagues identifies and characterizes a noncanonical RNA-binding domain that is required for FMRP-mediated translation regulation, shedding light on FMRP function.

G-quadruplex RNA Based PROTAC Enables Targeted Degradation of RNA Binding Protein FMRP for Tumor Immunotherapy.

Fragile X mental retardation protein (FMRP), an RNA binding protein (RBP), is aberrantly hyper-expressed in human tumors and plays an essential role in tumor invasion, metastasis and immune evasion. However, there is no small-molecule inhibitor for FMRP so far. In this study, we developed the first FMRP-targeting degrader based on PROteolysis TArgeting Chimera (PROTAC) technology and constructed a heterobifunctional PROTAC through linking a FMRP-targeting G-quadruplex RNA (sc1) to a von Hippel-Lindau (VHL)-targeting ligand peptide (named as sc1-VHLL). Sc1-VHLL specifically degraded endogenous FMRP via ubiquitination pathway in both mouse and human cancer cells. The FMRP degradation significantly changed the secretion pattern of cancer cells, resulting in higher expression of pro-inflammatory cytokine and smaller amounts of immunomodulatory contents. Furthermore, sc1-VHLL, when encapsulated into ionizable liposome nanoparticles (LNP) efficiently targeted tumor site and degraded FMRP in cancer cells. In CT26 tumor-bearing mouse model, FMRP degradation within tumors substantially promoted the infiltration of lymphocytes and CD8 T cells and reduced the proportion of Treg cells, reshaping the proinflammatory tumor microenvironment and accordingly transforming cold tumor into hot tumor. When combined with immune checkpoint blockade (ICB) therapy, sc1-VHLL based treatment remarkably inhibited the tumor growth.

Aggression Results in the Phosphorylation of ERK1/2 in the Nucleus Accumbens and the Dephosphorylation of mTOR in the Medial Prefrontal Cortex in Female Syrian Hamsters.

Like many social behaviors, aggression can be rewarding, leading to behavioral plasticity. One outcome of reward-induced aggression is the long-term increase in the speed in which future aggression-based encounters is initiated. This form of aggression impacts dendritic structure and excitatory synaptic neurotransmission in the nucleus accumbens, a brain region well known to regulate motivated behaviors. Yet, little is known about the intracellular signaling mechanisms that drive these structural/functional changes and long-term changes in aggressive behavior. This study set out to further elucidate the intracellular signaling mechanisms regulating the plasticity in neurophysiology and behavior that underlie the rewarding consequences of aggressive interactions. Female Syrian hamsters experienced zero, two or five aggressive interactions and the phosphorylation of proteins in reward-associated regions was analyzed. We report that aggressive interactions result in a transient increase in the phosphorylation of extracellular-signal related kinase 1/2 (ERK1/2) in the nucleus accumbens. We also report that aggressive interactions result in a transient decrease in the phosphorylation of mammalian target of rapamycin (mTOR) in the medial prefrontal cortex, a major input structure to the nucleus accumbens. Thus, this study identifies ERK1/2 and mTOR as potential signaling pathways for regulating the long-term rewarding consequences of aggressive interactions. Furthermore, the recruitment profile of the ERK1/2 and the mTOR pathways are distinct in different brain regions.

Neuron-Specific FMRP Roles in Experience-Dependent Remodeling of Olfactory Brain Innervation during an Early-Life Critical Period.

Critical periods are developmental windows during which neural circuits effectively adapt to the new sensory environment. Animal models of fragile X syndrome (FXS), a common monogenic autism spectrum disorder (ASD), exhibit profound impairments of sensory experience-driven critical periods. However, it is not known whether the causative fragile X mental retardation protein (FMRP) acts uniformly across neurons, or instead manifests neuron-specific functions. Here, we use the genetically-tractable Drosophila brain antennal lobe (AL) olfactory circuit of both sexes to investigate neuron-specific FMRP roles in the odorant experience-dependent remodeling of the olfactory sensory neuron (OSN) innervation during an early-life critical period. We find targeted OSN class-specific FMRP RNAi impairs innervation remodeling within AL synaptic glomeruli, whereas global dfmr1 null mutants display relatively normal odorant-driven refinement. We find both OSN cell autonomous and cell non-autonomous FMRP functions mediate odorant experience-dependent remodeling, with AL circuit FMRP imbalance causing defects in overall glomerulus innervation refinement. We find OSN class-specific FMRP levels bidirectionally regulate critical period remodeling, with odorant experience selectively controlling OSN synaptic terminals in AL glomeruli. We find OSN class-specific FMRP loss impairs critical period remodeling by disrupting responses to lateral modulation from other odorant-responsive OSNs mediating overall AL gain control. We find that silencing glutamatergic AL interneurons reduces OSN remodeling, while conversely, interfering with the OSN class-specific GABAA signaling enhances remodeling. These findings reveal control of OSN synaptic remodeling by FMRP with neuron-specific circuit functions, and indicate how neural circuitry can compensate for global FMRP loss to reinstate normal critical period brain circuit remodeling.SIGNIFICANCE STATEMENT Fragile X syndrome (FXS), the leading monogenic cause of intellectual disability and autism spectrum disorder (ASD), manifests severe neurodevelopmental delays. Likewise, FXS disease models display disrupted neurodevelopmental critical periods. In the well-mapped Drosophila olfactory circuit model, perturbing the causative fragile X mental retardation protein (FMRP) within a single olfactory sensory neuron (OSN) class impairs odorant-dependent remodeling during an early-life critical period. Importantly, this impairment requires activation of other OSNs, and the olfactory circuit can compensate when FMRP is removed from all OSNs. Understanding the neuron-specific FMRP requirements within a developing neural circuit, as well as the FMRP loss compensation mechanisms, should help us engineer FXS treatments. This work suggests FXS treatments could use homeostatic mechanisms to alleviate circuit-level deficits.

Up-regulation of cell division cycle 20 expression alters the morphology of neuronal dendritic spines in the nucleus accumbens by promoting FMRP ubiquitination.

The nucleus accumbens (NAc) is the key area of the reward circuit, but its heterogeneity has been poorly studied. Using single-cell RNA sequencing, we revealed a subcluster of GABAergic neurons characterized by cell division cycle 20 (Cdc20) mRNA expression in the NAc of adult rats. We studied the coexpression of Cdc20 and Gad1 mRNA in the NAc neurons of adult rats and assessed Cdc20 protein expression in the NAc during rat development. Moreover, we microinjected AAV2/9-hSyn-Cdc20 with or without the dual-AAV system into the bilateral NAc for sparse labeling to observe changes in the synaptic morphology of mature neurons and assessed rat behaviors in open field and elevated plus maze tests. Furthermore, we performed the experiments with a Cdc20 inhibitor, Cdc20 over-expression AAV vector, and Cdc20 conditional knockout primary striatal neurons to understand the ubiquitination-dependent degradation of fragile X mental retardation protein (FMRP) in vitro and in vivo. We confirmed the mRNA expression of Cdc20 in the NAc GABAergic neurons of adult rats, and its protein level was decreased significantly 3 weeks post-birth. Up-regulated Cdc20 expression in the bilateral NAc decreased the dendritic spine density in mature neurons and induced anxiety-like behavior in rats. Cdc20-APC triggered FMRP degradation through K48-linked polyubiquitination in Neuro-2a cells and primary striatal neurons and down-regulated FMRP expression in the NAc of adult rats. These data revealed that up-regulation of Cdc20 in the bilateral NAc reduced dendritic spine density and led to anxiety-like behaviors, possibly by enhancing FMRP degradation via K48-linked polyubiquitination.

ICAM5 as a Novel Target for Treating Cognitive Impairment in Fragile X Syndrome.

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability, resulted from the silencing of the Fmr1 gene and the subsequent loss of fragile X mental retardation protein (FMRP). Spine dysgenesis and cognitive impairment have been extensively characterized in FXS; however, the underlying mechanism remains poorly understood. As an important regulator of spine maturation, intercellular adhesion molecule 5 (ICAM5) mRNA may be one of the targets of FMRP and involved in cognitive impairment in FXS. Here we show that in Fmr1 KO male mice, ICAM5 was excessively expressed during the late developmental stage, and its expression was negatively correlated with the expression of FMRP and positively related with the morphological abnormalities of dendritic spines. While in vitro reduction of ICAM5 normalized dendritic spine abnormalities in Fmr1 KO neurons, and in vivo knockdown of ICAM5 in the dentate gyrus rescued the impaired spatial and fear memory and anxiety-like behaviors in Fmr1 KO mice, through both granule cell and mossy cell with a relative rate of 1.32 ± 0.15. Furthermore, biochemical analyses showed direct binding of FMRP with ICAM5 mRNA, to the coding sequence of ICAM5 mRNA. Together, our study suggests that ICAM5 is one of the targets of FMRP and is implicated in the molecular pathogenesis of FXS. ICAM5 could be a therapeutic target for treating cognitive impairment in FXS.SIGNIFICANCE STATEMENT Fragile X syndrome (FXS) is characterized by dendritic spine dysgenesis and cognitive dysfunctions, while one of the FMRP latent targets, ICAM5, is well established for contributing both spine maturation and learning performance. In this study, we examined the potential link between ICAM5 mRNA and FMRP in FXS, and further investigated the molecular details and pathological consequences of ICAM5 overexpression. Our results indicate a critical role of ICAM5 in spine maturation and cognitive impairment in FXS and suggest that ICAM5 is a potential molecular target for the development of medication against FXS.

Fragile X mental retardation protein-regulated proinflammatory cytokine expression in the spinal cord contributes to the pathogenesis of inflammatory pain induced by complete Freund's adjuvant.

Studies have verified that Fragile X mental retardation protein (FMRP), an RNA-binding protein, plays a potential role in the pathogenesis of formalin- and (RS)-3,5-dihydroxyphenylglycine-induced abnormal pain sensations. However, the role of FMRP in inflammatory pain has not been reported. Here, we showed an increase in FMRP expression in the spinal dorsal horn (SDH) in a rat model of inflammatory pain induced by complete Freund's adjuvant (CFA). Double immunofluorescence staining revealed that FMRP was mainly expressed in spinal neurons and colocalized with proinflammatory cytokines [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)]. After consecutive intrathecal injection of fragile X mental retardation 1 small interfering RNA for 3 days post-CFA injection, FMRP expression in the SDH was reduced, and CFA-induced hyperalgesia was decreased. In addition, the CFA-induced increase in spinal TNF-α and IL-6 production was significantly suppressed by intrathecal administration of fragile X mental retardation 1 small interfering RNA. Together, these results suggest that FMRP regulates TNF-α and IL-6 levels in the SDH and plays an important role in inflammatory pain.

Reciprocal regulation of spontaneous synaptic vesicle fusion by Fragile X mental retardation protein and group I metabotropic glutamate receptors.

Fragile X mental retardation protein (FMRP) is a neuronal protein mediating multiple functions, with its absence resulting in one of the most common monogenic causes of autism, Fragile X syndrome (FXS). Analyses of FXS pathophysiology have identified a range of aberrations in synaptic signaling pathways and plasticity associated with group I metabotropic glutamate (mGlu) receptors. These studies, however, have mostly focused on the post-synaptic functions of FMRP and mGlu receptor activation, and relatively little is known about their presynaptic effects. Neurotransmitter release is mediated via multiple forms of synaptic vesicle (SV) fusion, each of which contributes to specific neuronal functions. The impacts of mGlu receptor activation and loss of FMRP on these SV fusion events remain unexplored. Here we combined electrophysiological and fluorescence imaging analyses on primary hippocampal cultures prepared from an Fmr1 knockout (KO) rat model. Compared to wild-type (WT) hippocampal neurons, KO neurons displayed an increase in the frequency of spontaneous excitatory post-synaptic currents (sEPSCs), as well as spontaneous SV fusion events. Pharmacological activation of mGlu receptors in WT neurons caused a similar increase in spontaneous SV fusion and sEPSC frequency. Notably, this increase in SV fusion was not observed when spontaneous activity was blocked using the sodium channel antagonist tetrodotoxin. Importantly, the effect of mGlu receptor activation on spontaneous SV fusion was occluded in Fmr1 KO neurons. Together, our results reveal that FMRP represses spontaneous presynaptic SV fusion, whereas mGlu receptor activation increases this event. This reciprocal control appears to be mediated via their regulation of intrinsic neuronal excitability.

The role of selected postsynaptic scaffolding proteins at glutamatergic synapses in autism-related animal models.

Pathological changes in synapse formation, plasticity, and development are caused by altered trafficking and assembly of postsynaptic scaffolding proteins at sites of glutamatergic and gamma-aminobutyric acid (GABA)ergic synapses, suggesting their involvement in the etiology of neurodevelopmental disorders, including autism. Several autism-related mouse models have been developed in recent years for studying molecular, cellular, and behavioural defects in order to understand the etiology of autism and test the potential treatment strategies. In this review, we explain the role of alterations in selected postsynaptic scaffolding proteins in relevant transgene autism-like mouse models. We also provide a summary of selected animal models by paying special attention to interactions between guanylate kinases or membrane-associated guanylate kinases (MAGUKs), as well as other synapse protein components which form functional synaptic networks. The study of early developmental stages of autism-relevant animal models can help us understand the origin and development of diverse autistic symptomatology.

Tuning Specific Translation in Cancer Metastasis and Synaptic Memory: Control at the MNK-eIF4E Axis.

The eukaryotic translation initiation factor (eIF) 4E, which binds to the 5'-cap of mRNA, undergoes phosphorylation on a single conserved serine, executed by the mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs). However, the functional consequences and physiological roles of MNK signalling have remained obscure. Now, new pharmacological and genetic tools have provided unprecedented insights into the function of MNKs and eIF4E phosphorylation. The studies suggest that MNKs control the translation of specific mRNAs in cancer metastasis and neuronal synaptic plasticity by a novel mechanism involving the regulation of the translational repressor, cytoplasmic fragile-X protein-interacting protein 1 (CYFIP1). These recent breakthroughs go a long way to resolving the longstanding enigma and controversy surrounding the function of the MNK-eIF4E axis in cancer cell biology and neurobiology.

The RNA-binding protein FMRP facilitates the nuclear export of N6-methyladenosine-containing mRNAs.

N6-Methyladenosine (m6A) is the most abundant post-transcriptional mRNA modification in eukaryotes and exerts many of its effects on gene expression through reader proteins that bind specifically to m6A-containing transcripts. Fragile X mental retardation protein (FMRP), an RNA-binding protein, has previously been shown to affect the translation of target mRNAs and trafficking of mRNA granules. Loss of function of FMRP causes fragile X syndrome, the most common form of inherited intellectual disability in humans. Using HEK293T cells, siRNA-mediated gene knockdown, cytoplasmic and nuclear fractions, RNA-Seq, and LC-MS/MS analyses, we demonstrate here that FMRP binds directly to a collection of m6A sites on mRNAs. FMRP depletion increased mRNA m6A levels in the nucleus. Moreover, the abundance of FMRP targets in the cytoplasm relative to the nucleus was decreased in Fmr1-KO mice, an effect also observed in highly methylated genes. We conclude that FMRP may affect the nuclear export of m6A-modified RNA targets.

The RNA binding protein fragile X mental retardation protein promotes myelin sheath growth.

During development, oligodendrocytes in the central nervous system extend a multitude of processes that wrap axons with myelin. The highly polarized oligodendrocytes generate myelin sheaths on many different axons, which are far removed from the cell body. Neurons use RNA binding proteins to transport, stabilize, and locally translate mRNA in distal domains of neurons. Local synthesis of synaptic proteins during neurodevelopment facilitates the rapid structural and functional changes underlying neural plasticity and avoids extensive protein transport. We hypothesize that RNA binding proteins also regulate local mRNA regulation in oligodendrocytes to promote myelin sheath growth. Fragile X mental retardation protein (FMRP), an RNA binding protein that plays essential roles in the growth and maturation of neurons, is also expressed in oligodendrocytes. To determine whether oligodendrocytes require FMRP for myelin sheath development, we examined fmr1-/- mutant zebrafish and drove FMR1 expression specifically in oligodendrocytes. We found oligodendrocytes in fmr1-/- mutants developed myelin sheaths of diminished length, a phenotype that can be autonomously rescued in oligodendrocytes with FMR1 expression. Myelin basic protein (Mbp), an essential myelin protein, was reduced in myelin tracts of fmr1-/- mutants, but loss of FMRP function did not impact the localization of mbpa transcript in myelin. Finally, expression of FMR1-I304N, a missense allele that abrogates FMRP association with ribosomes, failed to rescue fmr1-/- mutant sheath growth and induced short myelin sheaths in oligodendrocytes of wild-type larvae. Taken together, these data suggest that FMRP promotes sheath growth through local regulation of translation.

Role of fragile X mental retardation protein in chronic pain.

Chronic pain has detrimental effects on one's quality of life. However, its treatment options are very limited, and its underlying pathogenesis remains unclear. Recent research has suggested that fragile X mental retardation protein is involved in the development of chronic pain, making it a potential target for prevention and treatment. The current review of literature will examine the function of fragile X mental retardation protein and its associated pathways, through which we hope to gain insight into how fragile X mental retardation protein may contribute to nociceptive sensitization and chronic pain.

MiR-315 is required for neural development and represses the expression of dFMR1 in Drosophila melanogaster.

AIM: The fragile X mental retardation protein (FMRP), the product of the FMR1 gene, is responsible for the fragile X syndrome (FXS). FMRP regulates miRNA expression and is involved in miRNA-mediated gene silencing. However, the question of whether FMRP is, in turn, regulated by miRNAs remains unanswered. MAIN METHODS: We detected the FMRP expression pattern by in situ hybridization. MiR-315 overexpression and knockout models were generated by germ-line transformation and ends-out homologous recombination, respectively. Western blotting and immunohistochemistry were used to detect Drosophila FMRP (dFMRP) and a Luciferase reporter assay was used to confirm the regulation of dfmr1 mRNA by mir-315. Synaptic structural quantification and electrophysiological methods were used to compare synaptic functions among groups. KEY FINDINGS: Here, we determined that the transcription product of dFMR1, the Drosophila homologue of FMR1, is a direct target of miR-315. MiR-315 is mainly expressed in the nervous system of Drosophila. Flies overexpressing miR-315 showed pupation defects and reduced hatching rates. A homozygous miR-315 knockout status is embryonic lethal in flies. These observations indicate that miR-315 is a key regulator of the Drosophila nervous system. Furthermore, computational prediction and cell-based luciferase and in vivo assays demonstrated that dfmr1 is directly targeted by miR-315. Lastly, using the neuromuscular junction as a model, we found that miR-315 regulates synaptic structure and transmission by targeting dfmr1. SIGNIFICANCE: These findings provide compelling evidence that miR-315 targets dfmr1 in the Drosophila nervous system, acting as a regulatory factor for the fine-tuned modulation of FMRP expression.

Loss of fragile X mental retardation protein precedes Lewy pathology in Parkinson's disease.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) and the gradual appearance of α-synuclein (α-syn)-containing neuronal protein aggregates. Although the exact mechanism of α-syn-mediated cell death remains elusive, recent research suggests that α-syn-induced alterations in neuronal excitability contribute to cell death in PD. Because the fragile X mental retardation protein (FMRP) controls the expression and function of numerous neuronal genes related to neuronal excitability and synaptic function, we here investigated the role of FMRP in α-syn-associated pathological changes in cell culture and mouse models of PD as well as in post-mortem human brain tissue from PD patients. We found FMRP to be decreased in cultured DA neurons and in the mouse brain in response to α-syn overexpression. FMRP was, furthermore, lost in the SNc of PD patients and in patients with early stages of incidental Lewy body disease (iLBD). Unlike fragile X syndrome (FXS), FMR1 expression in response to α-syn was regulated by a mechanism involving Protein Kinase C (PKC) and cAMP response element-binding protein (CREB). Reminiscent of FXS neurons, α-syn-overexpressing cells exhibited an increase in membrane N-type calcium channels, increased phosphorylation of ERK1/2, eIF4E and S6, increased overall protein synthesis, and increased expression of Matrix Metalloproteinase 9 (MMP9). FMRP affected neuronal function in a PD animal model, because FMRP-KO mice were resistant to the effect of α-syn on striatal dopamine release. In summary, our results thus reveal a new role of FMRP in PD and support the examination of FMRP-regulated genes in PD disease progression.

Postsynaptic FMRP Regulates Synaptogenesis In Vivo in the Developing Cochlear Nucleus.

A global loss of the fragile X mental retardation protein (FMRP; encoded by the Fmr1 gene) leads to sensory dysfunction and intellectual disabilities. One underlying mechanism of these phenotypes is structural and functional deficits in synapses. Here, we determined the autonomous function of postsynaptic FMRP in circuit formation, synaptogenesis, and synaptic maturation. In normal cochlea nucleus, presynaptic auditory axons form large axosomatic endbulb synapses on cell bodies of postsynaptic bushy neurons. In ovo electroporation of drug-inducible Fmr1-shRNA constructs produced a mosaicism of FMRP expression in chicken (either sex) bushy neurons, leading to reduced FMRP levels in transfected, but not neighboring nontransfected, neurons. Structural analyses revealed that postsynaptic FMRP reduction led to smaller size and abnormal morphology of individual presynaptic endbulbs at both early and later developmental stages. We further examined whether FMRP reduction affects dendritic development, as a potential mechanism underlying defective endbulb formation. Normally, chicken bushy neurons grow extensive dendrites at early stages and retract these dendrites when endbulbs begin to form. Neurons transfected with Fmr1 shRNA exhibited a remarkable delay in branch retraction, failing to provide necessary somatic surface for timely formation and growth of large endbulbs. Patch-clamp recording verified functional consequences of dendritic and synaptic deficits on neurotransmission, showing smaller amplitudes and slower kinetics of spontaneous and evoked EPSCs. Together, these data demonstrate that proper levels of postsynaptic FMRP are required for timely maturation of somatodendritic morphology, a delay of which may affect synaptogenesis and thus contribute to long-lasting deficits of excitatory synapses.SIGNIFICANCE STATEMENT Fragile X mental retardation protein (FMRP) regulates a large variety of neuronal activities. A global loss of FMRP affects neural circuit development and synaptic function, leading to fragile X syndrome (FXS). Using temporally and spatially controlled genetic manipulations, this study provides the first in vivo report that autonomous FMRP regulates multiple stages of dendritic development, and that selective reduction of postsynaptic FMRP leads to abnormal development of excitatory presynaptic terminals and compromised neurotransmission. These observations demonstrate secondary influence of developmentally transient deficits in neuronal morphology and connectivity to the development of long-lasting synaptic pathology in FXS.

Dysregulation of mRNA Localization and Translation in Genetic Disease.

RNA-binding proteins (RBPs) acting at various steps in the post-transcriptional regulation of gene expression play crucial roles in neuronal development and synaptic plasticity. Genetic mutations affecting several RBPs and associated factors lead to diverse neurological symptoms, as characterized by neurodevelopmental and neuropsychiatric disorders, neuromuscular and neurodegenerative diseases, and can often be multisystemic diseases. We will highlight the physiological roles of a few specific proteins in molecular mechanisms of cytoplasmic mRNA regulation, and how these processes are dysregulated in genetic disease. Recent advances in computational biology and genomewide analysis, integrated with diverse experimental approaches and model systems, have provided new insights into conserved mechanisms and the shared pathobiology of mRNA dysregulation in disease. Progress has been made to understand the pathobiology of disease mechanisms for myotonic dystrophy, spinal muscular atrophy, and fragile X syndrome, with broader implications for other RBP-associated genetic neurological diseases. This gained knowledge of underlying basic mechanisms has paved the way to the development of therapeutic strategies targeting disease mechanisms.

Developmental experience-dependent plasticity in the first synapse of the Drosophila olfactory circuit.

Evidence accumulating over the past 15 years soundly refutes the dogma that the Drosophila nervous system is hardwired. The preponderance of studies reveals activity-dependent neural circuit refinement driving optimization of behavioral outputs. We describe developmental, sensory input-dependent plasticity in the brain olfactory antennal lobe, which we term long-term central adaption (LTCA). LTCA is evoked by prolonged exposure to an odorant during the first week of posteclosion life, resulting in a persistently decreased response to aversive odors and an enhanced response to attractive odors. This limited window of early-use, experience-dependent plasticity represents a critical period of olfactory circuit refinement tuned by initial sensory input. Consequent behavioral adaptations have been associated with changes in the output of olfactory projection neurons to higher brain centers. Recent studies have indicated a central role for local interneuron signaling in LTCA presentation. Genetic and molecular analyses have implicated the mRNA-binding fragile X mental retardation protein and ataxin-2 regulators, Notch trans-synaptic signaling, and cAMP signal transduction as core regulatory steps driving LTCA. In this article, we discuss the structural, functional, and behavioral changes associated with LTCA and review our current understanding of the molecular pathways underlying these developmental, experience-dependent changes in the olfactory circuitry.

FMRP Mediates Chronic Ethanol-Induced Changes in NMDA, Kv4.2, and KChIP3 Expression in the Hippocampus.

BACKGROUND: Exposure to chronic ethanol (EtOH) results in changes in the expression of proteins that regulate neuronal excitability. This study examined whether chronic EtOH alters the hippocampal expression and function of fragile X mental retardation protein (FMRP) and the role of FMRP in the modulation of chronic EtOH-induced changes in the expression of NMDA receptors and Kv4.2 channels. METHODS: For in vivo studies, C57BL/6J mice underwent a chronic intermittent EtOH (CIE) vapor exposure procedure. After CIE, hippocampal tissue was collected and subjected to immunoblot blot analysis of NMDA receptor subunits (GluN1, GluN2B), Kv4.2, and its accessory protein KChIP3. For in vitro studies, hippocampal slice cultures were exposed to 75 mM EtOH for 8 days. Following EtOH exposure, mRNAs bound to FMRP was measured. In a separate set of studies, cultures were exposed to an inhibitor of S6K1 (PF-4708671 [PF], 6 µM) in order to assess whether EtOH-induced homeostatic changes in protein expression depend upon changes in FMRP activity. RESULTS: Immunoblot blot analysis revealed increases in GluN1 and GluN2B but reductions in Kv4.2 and KChIP3. Analysis of mRNAs bound to FMRP revealed a similar bidirectional change observed as reduction of GluN2B and increase in Kv4.2 and KChIP3 mRNA transcripts. Analysis of FMRP further revealed that while chronic EtOH did not alter the expression of FMRP, it significantly increased phosphorylation of FMRP at the S499 residue that is known to critically regulate its activity. Inhibition of S6K1 prevented the chronic EtOH-induced increase in phospho-FMRP and changes in NMDA subunits, Kv4.2, and KChIP3. In contrast, PF had no effect in the absence of alcohol, indicating it was specific for the chronic EtOH-induced changes. CONCLUSIONS: These findings demonstrate that chronic EtOH exposure enhances translational control of plasticity-related proteins by FMRP, and that S6K1 and FMRP activities are required for expression of chronic EtOH-induced homeostatic plasticity at glutamatergic synapses in the hippocampus.

Fragile X Syndrome FMRP Co-localizes with Regulatory Targets PSD-95, GABA Receptors, CaMKIIα, and mGluR5 at Fiber Cell Membranes in the Eye Lens.

Fmr1 and FMRP underlie Fragile X Syndrome (FXS) and are linked with related autism spectrum disorders (ASD). Fmr1 also has an essential role in eye and lens development. Lenses express FMRP along with γ-aminobutyric acid (GABA) receptors (GABARs), post-synaptic density protein 95 (PSD-95), Tyr-phosphatase STEP, CaMKIIα and Alzheimer's disease Aß precursor protein, which are verified targets of FMRP regulation in neurons and outline major topics in FXS/ASD research. PSD-95 as well as CaMKIIα transcripts undergo polypryimidine tract binding protein dependent alternative splicing in lens, consistent with PSD-95 translation in lens. At least 13 GABAR subunits and GAD25/65/67 GABA metabolism enzymes are expressed in lenses beginning in embryonic development, matching neural development. Interestingly, GABAergic drugs (e.g. baclofen) studied as FXS/ASD therapeutics are shown to resolve developmental vision defects in experimental myopia. Here, we demonstrated that FMRP co-localizes at fiber cell membranes with PSD-95, GABAAδ, GABAAß3, GABBR1, STEP, CaMKIIα, and mGluR5 in young adult lenses. GAD65 and GABA detection was greatest at the peri-nuclear lens region where fiber cell terminal differentiation occurs. These findings add to an extensive list of detailed parallels between fiber cell and neuron morphology and their lateral membrane spine/protrusions, also reflected in the shared expression of genes involved in the morphogenesis and function of these membrane structures, and shared use of associated regulatory mechanisms first described as distinguishing the neuronal phenotype. Future studies can determine if GABA levels currently studied as a FXS/ASD biomarker in the brain, and generated by GAD25/65/67 in a comparable cell environment in the lens, may be similarly responsive to Fmr1 mutation in lens. The present demonstration of FMRP and key regulatory targets in the lens identifies a potential for the lens to provide a new research venue, in the same individual, to inform about Fmr1/FMRP pathobiology in brain as well as lens.