FOXP1 regulates the development of excitatory synaptic inputs onto striatal neurons and induces phenotypic reversal with reinstatement.

Long-range glutamatergic inputs originating from the cortex and thalamus are indispensable for striatal development, providing the foundation for motor and cognitive functions. Despite their significance, transcriptional regulation governing these inputs remains largely unknown. We investigated the role of a transcription factor encoded by a high-risk autism-associated gene, FOXP1, in sculpting glutamatergic inputs onto spiny projection neurons (SPNs) within the striatum. We find a neuron subtype-specific role of FOXP1 in strengthening and maturing glutamatergic inputs onto dopamine receptor 2-expressing SPNs (D2 SPNs). We also find that FOXP1 promotes synaptically driven excitability in these neurons. Using single-nuclei RNA sequencing, we identify candidate genes that mediate these cell-autonomous processes through postnatal FOXP1 function at the post-synapse. Last, we demonstrate that postnatal FOXP1 reinstatement rescues electrophysiological deficits, cell type-specific gene expression changes, and behavioral phenotypes. Together, this study enhances our understanding of striatal circuit development and provides proof of concept for a therapeutic approach for FOXP1 syndrome and other neurodevelopmental disorders.

Female-specific dysfunction of sensory neocortical circuits in a mouse model of autism mediated by mGluR5 and estrogen receptor α.

Little is known of the brain mechanisms that mediate sex-specific autism symptoms. Here, we demonstrate that deletion of the autism spectrum disorder (ASD)-risk gene, Pten, in neocortical pyramidal neurons (NSEPten knockout [KO]) results in robust cortical circuit hyperexcitability selectively in female mice observed as prolonged spontaneous persistent activity states. Circuit hyperexcitability in females is mediated by metabotropic glutamate receptor 5 (mGluR5) and estrogen receptor α (ERα) signaling to mitogen-activated protein kinases (Erk1/2) and de novo protein synthesis. Pten KO layer 5 neurons have a female-specific increase in mGluR5 and mGluR5-dependent protein synthesis. Furthermore, mGluR5-ERα complexes are generally elevated in female cortices, and genetic reduction of ERα rescues enhanced circuit excitability, protein synthesis, and neuron size selectively in NSEPten KO females. Female NSEPten KO mice display deficits in sensory processing and social behaviors as well as mGluR5-dependent seizures. These results reveal mechanisms by which sex and a high-confidence ASD-risk gene interact to affect brain function and behavior.

Female specific dysfunction of sensory neocortical circuits in a mouse model of autism mediated by mGluR5 and Estrogen Receptor α.

Autism manifests differently in males and females and the brain mechanisms that mediate these sex-dependent differences are unknown. Here, we demonstrate that deletion of the ASD-risk gene, Pten, in neocortical pyramidal neurons (NSE Pten KO) results in robust hyperexcitability of local neocortical circuits in female, but not male, mice, observed as prolonged, spontaneous persistent activity states (UP states). Circuit hyperexcitability in NSE Pten KO mice is mediated by enhanced and/or altered signaling of metabotropic glutamate receptor 5 (mGluR5) and estrogen receptor α (ERα) to ERK and protein synthesis selectively in Pten deleted female neurons. In support of this idea, Pten deleted Layer 5 cortical neurons have female-specific increases in mGluR5 and mGluR5-driven protein synthesis. In addition, mGluR5-ERα complexes are elevated in female cortex and genetic reduction of ERα in Pten KO cortical neurons rescues circuit excitability, protein synthesis and enlarged neurons selectively in females. Abnormal timing and hyperexcitability of neocortical circuits in female NSE Pten KO mice are associated with deficits in temporal processing of sensory stimuli and social behaviors as well as mGluR5-dependent seizures. Female-specific cortical hyperexcitability and mGluR5-dependent seizures are also observed in a human disease relevant mouse model, germline Pten +/- mice. Our results reveal molecular mechanisms by which sex and a high impact ASD-risk gene interact to affect brain function and behavior.

Clinicopathologic Dissociation: Robust Lafora Body Accumulation in Malin KO Mice Without Observable Changes in Home-cage Behavior.

Lafora Disease (LD) is a syndrome of progressive myoclonic epilepsy and cumulative neurocognitive deterioration caused by recessively inherited genetic lesions of EPM2A (laforin) or NHLRC1 (malin). Neuropsychiatric symptomatology in LD is thought to be directly downstream of neuronal and astrocytic polyglucosan aggregates, termed Lafora bodies (LBs), which faithfully accumulate in an age-dependent manner in all mouse models of LD. In this study, we applied home-cage monitoring to examine the extent of neurobehavioral deterioration in a model of malin-deficient LD, as a means to identify robust preclinical endpoints that may guide the selection of novel genetic treatments. At 6 weeks, ~6-7 months and ~12 months of age, malin deficient mice ("KO") and wild type (WT) littermates underwent a standardized home-cage behavioral assessment designed to non-obtrusively appraise features of rest/arousal, consumptive behaviors, risk aversion and voluntary wheel-running. At all timepoints, and over a range of metrics that we report transparently, WT and KO mice were essentially indistinguishable. In contrast, within WT mice compared across timepoints, we identified age-related nocturnal hypoactivity, diminished sucrose preference and reduced wheel-running. Neuropathological examinations in subsets of the same mice revealed expected age dependent LB accumulation, gliosis and microglial activation in cortical and subcortical brain regions. At 12 months of age, despite the burden of neocortical LBs, we did not identify spontaneous seizures during an electroencephalographic (EEG) survey, and KO and WT mice exhibited similar spectral EEG features. Using an in vitro assay of neocortical function, paroxysmal increases in network activity (UP states) in KO slices were more prolonged at 3 and 6 months of age, but were similar to WT at 12 months. KO mice displayed a distinct response to pentylenetetrazole, with a greater incidence of clonic seizures and a more pronounced post-ictal suppression of movement, feeding and drinking behavior. Together, these results highlight a stark clinicopathologic dissociation in a mouse model of LD, where LBs accrue substantially without clinically meaningful changes in overall wellbeing. Our findings allude to a delay between LB accumulation and neurobehavioral decline: one that may provide a window for treatment, and whose precise duration may be difficult to ascertain within the typical lifespan of a laboratory mouse.

Excitatory neuron-specific suppression of the integrated stress response contributes to autism-related phenotypes in fragile X syndrome.

Dysregulation of protein synthesis is one of the key mechanisms underlying autism spectrum disorder (ASD). However, the role of a major pathway controlling protein synthesis, the integrated stress response (ISR), in ASD remains poorly understood. Here, we demonstrate that the main arm of the ISR, eIF2α phosphorylation (p-eIF2α), is suppressed in excitatory, but not inhibitory, neurons in a mouse model of fragile X syndrome (FXS; Fmr1-/y). We further show that the decrease in p-eIF2α is mediated via activation of mTORC1. Genetic reduction of p-eIF2α only in excitatory neurons is sufficient to increase general protein synthesis and cause autism-like behavior. In Fmr1-/y mice, restoration of p-eIF2α solely in excitatory neurons reverses elevated protein synthesis and rescues autism-related phenotypes. Thus, we reveal a previously unknown causal relationship between excitatory neuron-specific translational control via the ISR pathway, general protein synthesis, and core phenotypes reminiscent of autism in a mouse model of FXS.

KDM6B cooperates with Tau and regulates synaptic plasticity and cognition via inducing VGLUT1/2.

The excitatory neurotransmitter glutamate shapes learning and memory, but the underlying epigenetic mechanism of glutamate regulation in neuron remains poorly understood. Here, we showed that lysine demethylase KDM6B was expressed in excitatory neurons and declined in hippocampus with age. Conditional knockout of KDM6B in excitatory neurons reduced spine density, synaptic vesicle number and synaptic activity, and impaired learning and memory without obvious effect on brain morphology in mice. Mechanistically, KDM6B upregulated vesicular glutamate transporter 1 and 2 (VGLUT1/2) in neurons through demethylating H3K27me3 at their promoters. Tau interacted and recruited KDM6B to the promoters of Slc17a7 and Slc17a6, leading to a decrease in local H3K27me3 levels and induction of VGLUT1/2 expression in neurons, which could be prevented by loss of Tau. Ectopic expression of KDM6B, VGLUT1, or VGLUT2 restored spine density and synaptic activity in KDM6B-deficient cortical neurons. Collectively, these findings unravel a fundamental mechanism underlying epigenetic regulation of synaptic plasticity and cognition.

A sound-driven cortical phase-locking change in the Fmr1 KO mouse requires Fmr1 deletion in a subpopulation of brainstem neurons.

BACKGROUND: Sensory impairments commonly occur in patients with autism or intellectual disability. Fragile X syndrome (FXS) is one form of intellectual disability that is often comorbid with autism. In electroencephalographic (EEG) recordings obtained from humans with FXS, the ability of cortical regions to consistently synchronize, or "phase-lock", to modulated auditory stimuli is reduced compared to that of typically developing individuals. At the same time, less time-locked, "non-phase-locked" power induced by sounds is higher. The same changes occur in the Fmr1 knockout (KO) mouse - an animal model of FXS. We determined if Fmr1 deletion in a subset of brainstem auditory neurons plays any role in these EEG changes in the mouse. METHODS: We reinstated FMRP expression in a subpopulation of brainstem auditory neurons in an otherwise Fmr1 KO control (conditional on; cON Fmr1) mouse and used EEG recordings to determine if reinstatement normalized, or "rescued", the phase-locking phenotype observed in the cON Fmr1 mouse. In determining rescue, this also meant that Fmr1 deletion in the same neuron population was necessary for the phenotype to occur. RESULTS: We find that Fmr1 reinstatement in a subset of brainstem neurons rescues certain aspects of the phase-locking phenotype but does not rescue the increase in non-phase-locked power. Unexpectedly, not all electrophysiological phenotypes observed in the Fmr1 KO were observed in the cON Fmr1 mouse used for the reinstatement experiments, and this was likely due to residual expression of FMRP in these Fmr1 KO controls. CONCLUSIONS: Fmr1 deletion in brainstem neurons is necessary for certain aspects of the decreased phase-locking phenotype in the Fmr1 KO, but not necessary for the increase in non-phase-locked power induced by a sound. The most likely brainstem structure underlying these results is the inferior colliculus. We also demonstrate that low levels of FMRP can rescue some EEG phenotypes but not others. This latter finding provides a foundation for how symptoms in FXS individuals may vary due to FMRP levels and that reinstatement of low FMRP levels may be sufficient to alleviate particular symptoms.

Experience-dependent weakening of callosal synaptic connections in the absence of postsynaptic FMRP.

Reduced structural and functional interhemispheric connectivity correlates with the severity of Autism Spectrum Disorder (ASD) behaviors in humans. Little is known of how ASD-risk genes regulate callosal connectivity. Here, we show that Fmr1, whose loss-of-function leads to Fragile X Syndrome (FXS), cell autonomously promotes maturation of callosal excitatory synapses between somatosensory barrel cortices in mice. Postnatal, cell-autonomous deletion of Fmr1 in postsynaptic Layer (L) 2/3 or L5 neurons results in a selective weakening of AMPA receptor- (R), 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. In contrast to callosal inputs, synapses originating from local L4 and L2/3 circuits are normal, revealing an input-specific role for postsynaptic Fmr1 in regulation of synaptic connectivity within local and callosal neocortical circuits. These results suggest direct cell autonomous and postnatal roles for FMRP in development of specific cortical circuits and suggest a synaptic basis for long-range functional underconnectivity observed in FXS patients.

GABAA Alpha 2,3 Modulation Improves Select Phenotypes in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability. FXS is caused by functional loss of the Fragile X Protein (FXP), also known as Fragile X Mental Retardation Protein (FMRP). In humans and animal models, loss of FXP leads to sensory hypersensitivity, increased susceptibility to seizures and cortical hyperactivity. Several components of the GABAergic system, the major inhibitory system in the brain, are dysregulated in FXS, and thus modulation of GABAergic transmission was suggested and tested as a treatment strategy. However, so far, clinical trials using broad spectrum GABAA or GABAB receptor-specific agonists have not yielded broad improvement of FXS phenotypes in humans. Here, we tested a more selective strategy in Fmr1 knockout (KO) mice using the experimental drug BAER-101, which is a selective GABAA α2/α3 agonist. Our results suggest that BAER-101 reduces hyperexcitability of cortical circuits, partially corrects increased frequency-specific baseline cortical EEG power, reduces susceptibility to audiogenic seizures and improves novel object memory. Other Fmr1 KO-specific phenotypes were not improved by the drug, such as increased hippocampal dendritic spine density, open field activity and marble burying. Overall, this work shows that BAER-101 improves select phenotypes in Fmr1 KO mice and encourages further studies into the efficacy of GABAA-receptor subunit-selective agonists for the treatment of FXS.

FOXP1 negatively regulates intrinsic excitability in D2 striatal projection neurons by promoting inwardly rectifying and leak potassium currents.

Heterozygous loss-of-function mutations in the transcription factor FOXP1 are strongly associated with autism. Dopamine receptor 2 expressing (D2) striatal projection neurons (SPNs) in heterozygous Foxp1 (Foxp1+/-) mice have higher intrinsic excitability. To understand the mechanisms underlying this alteration, we examined SPNs with cell-type specific homozygous Foxp1 deletion to study cell-autonomous regulation by Foxp1. As in Foxp1+/- mice, D2 SPNs had increased intrinsic excitability with homozygous Foxp1 deletion. This effect involved postnatal mechanisms. The hyperexcitability was mainly due to down-regulation of two classes of potassium currents: inwardly rectifying (KIR) and leak (KLeak). Single-cell RNA sequencing data from D2 SPNs with Foxp1 deletion indicated the down-regulation of transcripts of candidate ion channels that may underlie these currents: Kcnj2 and Kcnj4 for KIR and Kcnk2 for KLeak. This Foxp1-dependent regulation was neuron-type specific since these same currents and transcripts were either unchanged, or very little changed, in D1 SPNs with cell-specific Foxp1 deletion. Our data are consistent with a model where FOXP1 negatively regulates the excitability of D2 SPNs through KIR and KLeak by transcriptionally activating their corresponding transcripts. This, in turn, provides a novel example of how a transcription factor may regulate multiple genes to impact neuronal electrophysiological function that depends on the integration of multiple current types - and do this in a cell-specific fashion. Our findings provide initial clues to altered neuronal function and possible therapeutic strategies not only for FOXP1-associated autism but also for other autism forms associated with transcription factor dysfunction.

Audiogenic Seizures in the Fmr1 Knock-Out Mouse Are Induced by Fmr1 Deletion in Subcortical, VGlut2-Expressing Excitatory Neurons and Require Deletion in the Inferior Colliculus.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and the leading monogenetic cause of autism. One symptom of FXS and autism is sensory hypersensitivity (also called sensory over-responsivity). Perhaps related to this, the audiogenic seizure (AGS) is arguably the most robust behavioral phenotype in the FXS mouse model-the Fmr1 knock-out (KO) mouse. Therefore, the AGS may be considered a mouse model of sensory hypersensitivity. Hyperactive circuits are hypothesized to underlie dysfunction in a number of brain regions in patients with FXS and Fmr1 KO mice, and the AGS may be a result of this. But the specific cell types and brain regions underlying AGSs in the Fmr1 KO are unknown. We used conditional deletion or expression of Fmr1 in different cell populations to determine whether Fmr1 deletion in those cells was sufficient or necessary, respectively, for the AGS phenotype in males. Our data indicate that Fmr1 deletion in glutamatergic neurons that express vesicular glutamate transporter 2 (VGlut2) and are located in subcortical brain regions is sufficient and necessary to cause AGSs. Furthermore, the deletion of Fmr1 in glutamatergic neurons of the inferior colliculus is necessary for AGSs. When we demonstrate necessity, we show that Fmr1 expression in either the larger population of VGlut2-expressing glutamatergic neurons or the smaller population of inferior collicular glutamatergic neurons-in an otherwise Fmr1 KO mouse-eliminates AGSs. Therefore, targeting these neuronal populations in FXS and autism may be part of a therapeutic strategy to alleviate sensory hypersensitivity.SIGNIFICANCE STATEMENT Sensory hypersensitivity in fragile X syndrome (FXS) and autism patients significantly interferes with quality of life. Audiogenic seizures (AGSs) are arguably the most robust behavioral phenotype in the FXS mouse model-the Fmr1 knockout-and may be considered a model of sensory hypersensitivity in FXS. We provide the clearest and most precise genetic evidence to date for the cell types and brain regions involved in causing AGSs in the Fmr1 knockout and, more broadly, for any mouse mutant. The expression of Fmr1 in these same cell types in an otherwise Fmr1 knockout eliminates AGSs indicating possible cellular targets for alleviating sensory hypersensitivity in FXS and other forms of autism.

Local cortical circuit correlates of altered EEG in the mouse model of Fragile X syndrome.

Electroencephalogram (EEG) recordings in Fragile X syndrome (FXS) patients have revealed enhanced sensory responses, enhanced resting "gamma frequency" (30-100 Hz) activity, and a decreased ability for sensory stimuli to modulate cortical activity at gamma frequencies. Similar changes are observed in the FXS model mouse - the Fmr1 knockout. These alterations may become effective biomarkers for diagnosis and treatment of FXS. Therefore, it is critical to better understand what circuit properties underlie these changes. We employed Channelrhodopsin2 to optically activate local circuits in the auditory cortical region in brain slices to examine how changes in local circuit function may be related to EEG changes. We focused on layers 2/3 and 5 (L2/3 and L5). In Fmr1 knockout mice, light-driven excitation of L2/3 revealed hyperexcitability and increased gamma frequency power in both local L2/3 and L5 circuits. Moreover, there is increased synchrony in the gamma frequency band between L2/3 and L5. Hyperexcitability and increased gamma power were not observed in L5 with L5 light-driven excitation, indicating that these changes were layer-specific. A component of L2/3 network hyperexcitability is independent of ionotropic receptor mediated synaptic transmission and may be mediated by increased intrinsic excitability of L2/3 neurons. Finally, lovastatin, a candidate therapeutic compound for FXS that targets ERK signaling did not normalize changes in gamma activity. In conclusion, hyperactivity and increased gamma activity in local neocortical circuits, together with increased gamma synchrony between circuits, provide a putative substrate for EEG alterations observed in both FXS patients and the FXS mouse model.

Distinct stages of synapse elimination are induced by burst firing of CA1 neurons and differentially require MEF2A/D.

Experience and activity refine cortical circuits through synapse elimination, but little is known about the activity patterns and downstream molecular mechanisms that mediate this process. We used optogenetics to drive individual mouse CA1 hippocampal neurons to fire in theta frequency bursts to understand how cell autonomous, postsynaptic activity leads to synapse elimination. Brief (1 hr) periods of postsynaptic bursting selectively depressed AMPA receptor (R) synaptic transmission, or silenced excitatory synapses, whereas more prolonged (24 hr) firing depressed both AMPAR and NMDAR EPSCs and eliminated spines, indicative of a synapse elimination. Both synapse silencing and elimination required de novo transcription, but only silencing required the activity-dependent transcription factors MEF2A/D. Burst firing induced MEF2A/D-dependent induction of the target gene Arc which contributed to synapse silencing and elimination. This work reveals new and distinct forms of activity and transcription-dependent synapse depression and suggests that these processes can occur independently.

Experience-Dependent and Differential Regulation of Local and Long-Range Excitatory Neocortical Circuits by Postsynaptic Mef2c.

Development of proper cortical circuits requires an interaction of sensory experience and genetic programs. Little is known of how experience and specific transcription factors interact to determine the development of specific neocortical circuits. Here, we demonstrate that the activity-dependent transcription factor, Myocyte enhancer factor-2C (Mef2c), differentially regulates development of local versus long-range excitatory synaptic inputs onto layer 2/3 neurons in the somatosensory neocortex in vivo. Postnatal, postsynaptic deletion of Mef2c in a sparse population of L2/3 neurons suppressed development of excitatory synaptic connections from all local input pathways tested. In the same cell population, Mef2c deletion promoted the strength of excitatory inputs originating from contralateral neocortex. Both the synapse promoting and synapse suppressing effects of Mef2c deletion required normal whisking experience. These results reveal a role of Mef2c in experience-dependent development of specific sensory neocortical circuits.

APP Causes Hyperexcitability in Fragile X Mice.

Amyloid-beta protein precursor (APP) and metabolite levels are altered in fragile X syndrome (FXS) patients and in the mouse model of the disorder, Fmr1KO mice. Normalization of APP levels in Fmr1KO mice (Fmr1KO /APPHET mice) rescues many disease phenotypes. Thus, APP is a potential biomarker as well as therapeutic target for FXS. Hyperexcitability is a key phenotype of FXS. Herein, we determine the effects of APP levels on hyperexcitability in Fmr1KO brain slices. Fmr1KO /APPHET slices exhibit complete rescue of UP states in a neocortical hyperexcitability model and reduced duration of ictal discharges in a CA3 hippocampal model. These data demonstrate that APP plays a pivotal role in maintaining an appropriate balance of excitation and inhibition (E/I) in neural circuits. A model is proposed whereby APP acts as a rheostat in a molecular circuit that modulates hyperexcitability through mGluR5 and FMRP. Both over- and under-expression of APP in the context of the Fmr1KO increases seizure propensity suggesting that an APP rheostat maintains appropriate E/I levels but is overloaded by mGluR5-mediated excitation in the absence of FMRP. These findings are discussed in relation to novel treatment approaches to restore APP homeostasis in FXS.

FoxP1 orchestration of ASD-relevant signaling pathways in the striatum.

Mutations in the transcription factor Forkhead box p1 (FOXP1) are causative for neurodevelopmental disorders such as autism. However, the function of FOXP1 within the brain remains largely uncharacterized. Here, we identify the gene expression program regulated by FoxP1 in both human neural cells and patient-relevant heterozygous Foxp1 mouse brains. We demonstrate a role for FoxP1 in the transcriptional regulation of autism-related pathways as well as genes involved in neuronal activity. We show that Foxp1 regulates the excitability of striatal medium spiny neurons and that reduction of Foxp1 correlates with defects in ultrasonic vocalizations. Finally, we demonstrate that FoxP1 has an evolutionarily conserved role in regulating pathways involved in striatal neuron identity through gene expression studies in human neural progenitors with altered FOXP1 levels. These data support an integral role for FoxP1 in regulating signaling pathways vulnerable in autism and the specific regulation of striatal pathways important for vocal communication.

Increased Cortical Inhibition in Autism-Linked Neuroligin-3R451C Mice Is Due in Part to Loss of Endocannabinoid Signaling.

A single, maternally inherited, X-linked point mutation leading to an arginine to cysteine substitution at amino acid 451 (R451C) of Neuroligin 3 (NLGN3R451C) is a likely cause of autism in two brothers. Knockin mice expressing the Nlgn3R451C mutation in place of wild-type Nlgn3 demonstrate increased inhibitory synaptic strength in somatosensory cortex, resulting in an excitatory/inhibitory (E/I) imbalance that is potentially relevant for autism-associated behavioral deficits characteristic of these mice. We have replicated the increase in evoked inhibitory postsynaptic currents (eIPSCs) onto layer II/III cortical pyramidal neurons. We also find that increased frequency of spontaneous mIPSCs in Nlgn3R451C mice occurs in the absence of action potential-driven transmission. This suggests the E/I imbalance is due to changes at the synapse level, as opposed to the network level. Next, we use paired whole-cell recordings in an attempt to identify specific interneuron subtypes affected by the Nlgn3R451C mutation. Curiously, we observe no change in the amplitude of cell-to-cell, unitary IPSCs (uIPSCs) from parvalbumin-positive (PV) or somatostatin-positive (SOM) interneurons onto pyramidal neurons. We also observe no change in the number or density of PV and SOM interneurons in LII/III of somatosensory cortex. This effectively rules out a role for these particular interneurons in the increased inhibitory synaptic transmission, pointing to perhaps alternative interneuron subtypes. Lastly, impaired endocannabinoid signaling has been implicated in hippocampal synaptic dysfunction in Nlgn3R451C mice, but has not been investigated at cortical synapses. We find that bath application of the CB1 antagonist, AM 251 in WT mice eliminates the Nlgn3R451C increase in eIPSC amplitude and mIPSC frequency, indicating that increased inhibitory transmission in mutant mice is due, at least in part, to a loss of endocannabinoid signaling through CB1 receptors likely acting at interneurons other than PV or SOM.

Selective role of the catalytic PI3K subunit p110ß in impaired higher order cognition in fragile X syndrome.

Distinct isoforms of the PI3K catalytic subunit have specialized functions in the brain, but their role in cognition is unknown. Here, we show that the catalytic subunit p110ß plays an important role in prefrontal cortex (PFC)-dependent cognitive defects in mouse models of Fragile X syndrome (FXS), an inherited intellectual disability. FXS is caused by loss of function of the fragile X mental retardation protein (FMRP), which binds and translationally represses mRNAs. PFC-selective knockdown of p110ß, an FMRP target that is translationally upregulated in FXS, reverses deficits in higher cognition in Fmr1 knockout mice. Genetic full-body reduction of p110ß in Fmr1 knockout mice normalizes excessive PI3K activity, restores stimulus-induced protein synthesis, and corrects increased dendritic spine density and behavior. Notably, adult-onset PFC-selective Fmr1 knockdown mice show impaired cognition, which is rescued by simultaneous p110ß knockdown. Our results suggest that FMRP-mediated control of p110ß is crucial for neuronal protein synthesis and cognition.

Increased expression of the PI3K enhancer PIKE mediates deficits in synaptic plasticity and behavior in fragile X syndrome.

The PI3K enhancer PIKE links PI3K catalytic subunits to group 1 metabotropic glutamate receptors (mGlu1/5) and activates PI3K signaling. The roles of PIKE in synaptic plasticity and the etiology of mental disorders are unknown. Here, we show that increased PIKE expression is a key mediator of impaired mGlu1/5-dependent neuronal plasticity in mouse and fly models of the inherited intellectual disability fragile X syndrome (FXS). Normalizing elevated PIKE protein levels in FXS mice reversed deficits in molecular and cellular plasticity and improved behavior. Notably, PIKE reduction rescued PI3K-dependent and -independent neuronal defects in FXS. We further show that PI3K signaling is increased in a fly model of FXS and that genetic reduction of the Drosophila ortholog of PIKE, CenG1A rescued excessive PI3K signaling, mushroom body defects, and impaired short-term memory in these flies. Our results demonstrate a crucial role of increased PIKE expression in exaggerated mGlu1/5 signaling causing neuronal defects in FXS.

Vasoactive intestinal polypeptide (VIP)-expressing neurons in the suprachiasmatic nucleus provide sparse GABAergic outputs to local neurons with circadian regulation occurring distal to the opening of postsynaptic GABAA ionotropic receptors.

GABAergic synaptic transmission plays an important role in resetting and synchronizing circadian rhythms in the suprachiasmatic nucleus (SCN). Although the circadian modulation of intrinsic membrane currents and biochemical signaling have been examined in the SCN, the modulation of specific synaptic pathways within the SCN is unexplored. In addition, little is known about the functional properties of these pathways, including which ones involve GABAA receptors (GABAA-Rs). In brain slices obtained from mice, we examined synaptic responses originating from the SCN neurons expressing vasoactive intestinal peptide (VIP+ neurons). Focusing on the local projection within the ventromedial SCN, we found that VIP+ afferents provided input onto 49% of neurons with a preference for VIP-negative (VIP-) neurons. Responses were mediated by GABAA-Rs. The projection was sparsely connected and preferentially targeted a subset of SCN neurons unrelated to postsynaptic VIP expression. For most aspects of VIP+ network output, there was no circadian regulation. Excitability and spontaneous firing of the presynaptic VIP+ neurons were unchanged between day and night, and their network connectivity and synaptic function up through the evoked synaptic conductance were also unchanged. On the other hand, VIP+ input onto VIP- neurons became less inhibitory at night suggesting a postsynaptic alteration in the coupling of GABAA-R conductances to action potential firing. These data suggest that components of the VIP network and its synaptic output up through GABAA-R opening are invariant during the circadian cycle, but the effect on action potential firing is modulated by postsynaptic processes occurring after GABAA-R channel opening.