Fragile X syndrome (FraX), caused by the loss-of-function of one gene (FMR1), is the most common inherited form of both mental retardation and autism spectrum disorders. The FMR1 product (FMRP) is an mRNA-binding translation regulator that mediates activity-dependent control of synaptic structure and function. To develop any FraX intervention strategy, it is essential to define when and where FMRP loss causes the manifestation of synaptic defects, and whether the reintroduction of FMRP can restore normal synapse properties. In the Drosophila FraX model, dFMRP loss causes neuromuscular junction (NMJ) synapse overelaboration (overgrowth, overbranching, excess synaptic boutons), accumulation of development-arrested satellite boutons, and altered neurotransmission. We used the Gene-Switch method to conditionally drive dFMRP expression to define the spatiotemporal requirements in synaptic mechanisms. Constitutive induction of targeted neuronal dFMRP at wild-type levels rescues all synaptic architectural defects in Drosophila Fmr1 (dfmr1)-null mutants, demonstrating a presynaptic requirement for synapse structuring. By contrast, presynaptic dFMRP expression does not ameliorate functional neurotransmission defects, indicating a postsynaptic dFMRP requirement. Strikingly, targeted early induction of dFMRP effects nearly complete rescue of synaptic structure defects, showing a primarily early-development role. In addition, acute dFMRP expression at maturity partially alleviates dfmr1-null defects, although rescue is not as complete as either early or constitutive dFMRP expression, showing a modest capacity for late-stage structural plasticity. We conclude that dFMRP predominantly acts early in synaptogenesis to modulate architecture, but that late dFMRP introduction at maturity can weakly compensate for early absence of dFMRP function.

Proper brain function requires stringent balance of excitatory and inhibitory synapse formation during neural circuit assembly. Mutation of genes that normally sculpt and maintain this balance results in severe dysfunction, causing neurodevelopmental disorders including autism, epilepsy and Rett syndrome. Such mutations may result in defective architectural structuring of synaptic connections, molecular assembly of synapses and/or functional synaptogenesis. The affected genes often encode synaptic components directly, but also include regulators that secondarily mediate the synthesis or assembly of synaptic proteins. The prime example is Fragile X syndrome (FXS), the leading heritable cause of both intellectual disability and autism spectrum disorders. FXS results from loss of mRNA-binding FMRP, which regulates synaptic transcript trafficking, stability and translation in activity-dependent synaptogenesis and plasticity mechanisms. Genetic models of FXS exhibit striking excitatory and inhibitory synapse imbalance, associated with impaired cognitive and social interaction behaviors. Downstream of translation control, a number of specific synaptic proteins regulate excitatory versus inhibitory synaptogenesis, independently or combinatorially, and loss of these proteins is also linked to disrupted neurodevelopment. The current effort is to define the cascade of events linking transcription, translation and the role of specific synaptic proteins in the maintenance of excitatory versus inhibitory synapses during neural circuit formation. This focus includes mechanisms that fine-tune excitation and inhibition during the refinement of functional synaptic circuits, and later modulate this balance throughout life. The use of powerful new genetic models has begun to shed light on the mechanistic bases of excitation/inhibition imbalance for a range of neurodevelopmental disease states.

Compartmentalized signaling involving cholesterol-rich, liquid-ordered membrane domains occurs during cell activation triggered by receptor cross-linking, growth factors, or other extracellular stimuli (1-3). The redistribution of similar liquidordered domains, called lipid ''rafts,'' accompanies and is required for cell polarization and directed migration (4 -8). Although we do not know the precise molecular mechanisms by which the redistributions of plasma membrane domains occur, an active role of the actin-based membrane skeleton has long been postulated (reviewed in Ref. 9).Redistributions of activated or cross-linked receptors are accompanied by corresponding changes in the localizations of actin, nonmuscle myosin II, spectrin, and associated cytoskeletal proteins (9). Furthermore, disruption of actin filament integrity inhibits many lipid raft-mediated processes, including epidermal growth factor receptor capping in A431 cells (10), insulin receptor capping in lymphocytes (11), activation of fibroblasts (12), polarization of T lymphocytes (5), and downregulation of Fc⑀RI-mediated signaling in mast cells (13). A requirement for myosin II is shown by the greatly diminished receptor redistributions and/or developments of cell polarity that have been observed in cells that either lack myosin II (14, 15) or express a dominant-negative mutant of myosin II function (16 -18). Erythrocyte spectrin (19) and the nonerythroid spectrin called fodrin 1 (20, 21) also have been implicated in lipid raft-mediated processes. Taken together, these observations suggest that actin filaments, perhaps as components of a spectrin-based membrane skeleton, are required for the

IMPORTANCE Awake prone positioning may improve hypoxemia among patients with COVID-19, but whether it is associated with improved clinical outcomes remains unknown.OBJECTIVE To determine whether the recommendation of awake prone positioning is associated with improved outcomes among patients with COVID-19-related hypoxemia who have not received mechanical ventilation.

Loss of fragile X mental retardation 1 (FMR1) gene function is the most common cause of inherited mental retardation and autism spectrum disorders, characterized by attention disorder, hyperactivity and disruption of circadian activity cycles. Pursuit of effective intervention strategies requires determining when the FMR1 product (FMRP) is required in the regulation of neuronal circuitry controlling these behaviors. In the well-characterized Drosophila disease model, loss of the highly conserved dFMRP causes circadian arrhythmicity and conspicuous abnormalities in the circadian clock circuitry. Here, a novel Sholl Analysis was used to quantify over-elaborated synaptic architecture in dfmr1-null small ventrolateral neurons (sLNvs), a key subset of clock neurons. The transgenic Gene-Switch system was employed to drive conditional neuronal dFMRP expression in the dfmr1-null mutant background in order to dissect temporal requirements within the clock circuit. Introduction of dFMRP during early brain development, including the stages of neurogenesis, neuronal fate specification and early pathfinding, provided no rescue of dfmr1 mutant phenotypes. Similarly, restoring normal dFMRP expression in the adult failed to restore circadian circuit architecture. In sharp contrast, supplying dFMRP during a transient window of very late brain development, wherein synaptogenesis and substantial subsequent synaptic reorganization (e.g. use-dependent pruning) occur, provided strong morphological rescue to reestablish normal sLNvs synaptic arbors. We conclude that dFMRP plays a developmentally restricted role in sculpting synaptic architecture in these neurons that cannot be compensated for by later reintroduction of the protein at maturity.

Nodes of Ranvier are specialized, highly polarized axonal domains crucial to the propagation of saltatory action potentials. In the peripheral nervous system, axo–glial cell contacts have been implicated in Schwann cell (SC) differentiation and formation of the nodes of Ranvier. SC microvilli establish axonal contact at mature nodes, and their components have been observed to localize early to sites of developing nodes. However, a role for these contacts in node formation remains controversial.Using a myelinating explant culture system, we have observed that SCs reorganize and polarize microvillar components, such as the ezrin-binding phosphoprotein 50 kD/regulatory cofactor of the sodium-hydrogen exchanger isoform 3 (NHERF-1), actin, and the activated ezrin, radixin, and moesin family proteins before myelination in response to inductive signals. These components are targeted to the SC distal tips where live cell imaging reveals novel, dynamic growth cone–like behavior. Furthermore, localized activation of the Rho signaling pathway at SC tips gives rise to these microvillar component–enriched “caps” and influences the efficiency of node formation.

Fragile X syndrome (FXS), caused by loss of FMR1 gene function, is the most common heritable cause of intellectual disability and autism spectrum disorders. The FMR1 protein (FMRP) translational regulator mediates activity-dependent control of synapses. In addition to the metabotropic glutamate receptor (mGluR) hyperexcitation FXS theory, the GABA theory postulates that hypoinhibition is causative for disease state symptoms. Here, we use the Drosophila FXS model to assay central brain GABAergic circuitry, especially within the Mushroom Body (MB) learning center. All 3 GABAA receptor (GABAAR) subunits are reportedly downregulated in dfmr1 null brains. We demonstrate parallel downregulation of glutamic acid decarboxylase (GAD), the rate-limiting GABA synthesis enzyme, although GABAergic cell numbers appear unaffected. Mosaic analysis with a repressible cell marker (MARCM) single-cell clonal studies show that dfmr1 null GABAergic neurons innervating the MB calyx display altered architectural development, with early underdevelopment followed by later overelaboration. In addition, a new class of extra-calyx terminating GABAergic neurons is shown to include MB intrinsic α/β Kenyon Cells (KCs), revealing a novel level of MB inhibitory regulation. Functionally, dfmr1 null GABAergic neurons exhibit elevated calcium signaling and altered kinetics in response to acute depolarization. To test the role of these GABAergic changes, we attempted to pharmacologically restore GABAergic signaling and assay effects on the compromised MB-dependent olfactory learning in dfmr1 mutants, but found no improvement. Our results show that GABAergic circuit structure and function are impaired in the FXS disease state, but that correction of hypoinhibition alone is not sufficient to rescue a behavioral learning impairment.

Fragile X syndrome (FXS), caused by loss of fragile X mental retardation 1 (FMR1) gene function, is the most common heritable cause of intellectual disability and autism spectrum disorders. The FMR1 product (FMRP) is an RNA-binding protein best established to function in activity-dependent modulation of synaptic connections. In the Drosophila FXS disease model, loss of functionally-conserved dFMRP causes synaptic overgrowth and overelaboration in pigment dispersing factor (PDF) peptidergic neurons in the adult brain. Here, we identify a very different component of PDF neuron misregulation in dfmr1 mutants: the aberrant retention of normally developmentally-transient PDF tritocerebral (PDF-TRI) neurons. In wild-type animals, PDF-TRI neurons in the central brain undergo programmed cell death and complete, processive clearance within days of eclosion. In the absence of dFMRP, a defective apoptotic program leads to constitutive maintenance of these peptidergic neurons. We tested whether this apoptotic defect is circuit-specific by examining crustacean cardioactive peptide (CCAP) and bursicon circuits, which are similarly developmentally-transient and normally eliminated immediately post-eclosion. In dfmr1 null mutants, CCAP/bursicon neurons also exhibit significantly delayed clearance dynamics, but are subsequently eliminated from the nervous system, in contrast to the fully persistent PDF-TRI neurons. Thus, the requirement of dFMRP for the retention of transitory peptidergic neurons shows evident circuit specificity. The novel defect of impaired apoptosis and aberrant neuron persistence in the Drosophila FXS model suggests an entirely new level of “pruning” dysfunction may contribute to the FXS disease state.