Fragile X syndrome (FXS) is associated with alterations in alpha-band activity recorded using electroencephalography (EEG), including reports of alpha slowing, that may reflect impaired inhibitory regulation and network-level dysfunction. However, it remains unclear whether these abnormalities represent a global shift in oscillatory frequency or dissociable disruptions across distinct systems. In the present study, we leveraged alpha reactivity dynamics, defined as eyes-open to eyes-closed modulation, to probe alpha function in 23 individuals with FXS and 23 typically developing controls using both absolute and relative spectral measures. In FXS, absolute alpha reactivity showed a robust, region-specific reduction localized to occipital cortex, consistent with impaired sensory-inhibitory modulation. While relative alpha reactivity revealed more widespread attenuation across regions, indicating broader network alterations. Peak reactivity frequency showed no evidence of global slowing but was consistent with localized shifts toward lower frequencies. These effects were region- and sex-specific, with females showing reduced frontal relative peak frequency and males showing associations between lower relative peak frequency in non-occipital regions and higher IQ. These findings support a multi-system model of alpha dysfunction in FXS, characterized by posterior inhibitory deficits and distributed network alterations, and highlight alpha reactivity dynamics as a promising translational biomarker.

Contactin-associated protein-like 2 (CNTNAP2) is a transmembrane protein that mediates neuron-glia interactions and regulates dendritic spine growth and neuronal migration. Mutations in the CNTNAP2 gene are linked to autism and epilepsy. Younger Cntnap2 KO mice mimic autism phenotypes, while older mice are a model for epilepsy. Thus, comparing behavioral phenotypes across different ages is needed to better understand the age dependent development of disordered brain networks in Cntnap2 mutants. Male and female Cntnap2 KO and WT controls were tested across different age groups (4, 5, 7, 9, and ∼11 months) using digging, stimulus (reactivity), and nesting assays. Older Cntnap2 KO mice (7, 9, and ∼11 months) showed a significant increase in home cage reactivity (stimulus) assay compared to younger mice at 4 and 5 months of age. Similar trends were observed in male and female Cntnap2 KO mice. No significant differences were observed in WT controls. A significant difference in digging assay was observed in KO female mice between younger (4 month) and older mice post nest removal. An age-dependent significant reduction in nesting behavior was observed in female KO mice; however, no difference was observed in the WT controls. Immunohistochemical analysis showed age dependent change interneuron and microglial network in Cntnap2 KO mice. Our findings suggest disruption in home cage behavior and reactivity in older pre-epileptic Cntnap2 KO mice indicating an age-dependent network alteration and behavior deficits. Significance Statement:This study investigates the age-dependent behavioral changes in Cntnap2 KO mice due to underlying changes in the neuronal network. It has been shown that younger Cntnap2 KO mice display autistic behaviors and that older Cntnap2 KO mice have epilepsy, but it is unknown how behavior is affected during the intervening period of epileptogenesis. We find that female Cntnap2 KO mice at 11 months of age have increased reactivity and decreased motor activity compared to younger age groups, whereas WT mice show no relationship between age and behavior. Overall, the loss of Cntnap2 alters behavior in an age-dependent and sex-specific manner, indicating progressive dysregulation of the neuronal network.

Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability. It is caused by a trinucleotide expansion in the 5’ UTR of the Fragile X messenger ribonucleoprotein 1 (FMR1) gene leading to loss of expression of Fragile X messenger ribonucleoprotein (FMRP). There is currently no cure for FXS. We developed an FMR1 gene therapy based on an adeno-associated viral vector designed with strong translational potential for future clinical testing. The viral vector was tested in Fmr1 knockout mice using two translationally relevant delivery routes and ages corresponding to in utero, toddler, and adolescent ages in humans. Functional studies showed that the FMR1 gene therapy improved select translational FXS phenotypes spanning three critical domains: sensory hyperexcitability, adaptation to change, and altered brain activity. Expression after intracerebroventricular injection was most prominent in the forebrain, whereas intravenous delivery predominantly led to expression across midbrain and brainstem, suggesting that a dual route may be needed to achieve full brain coverage. Biodistribution analyses further suggested that FMRP expression must be titrated carefully for optimal rescue. In summary, we show that FMR1 gene therapy using delivery routes and vehicles approved for clinical use improves core phenotypes in a mouse model for FXS.

Fragile X syndrome (FXS), the leading genetic cause of intellectual disability, arises from FMR1 gene silencing and the subsequent loss of the RNA-binding protein FMRP. N6-methyladenosine (m6A) is a prevalent mRNA modification essential for post-transcriptional regulation. FMRP binds and regulates the stability of m6A-containing transcripts. However, how FMRP deficiency impacts transcriptome-wide m6A modifications in FXS remains unknown. To address this, we generated cortical neurons from induced pluripotent stem cells (iPSCs) derived from healthy individuals and FXS patients. Electrophysiology recordings revealed synaptic and neuronal network defects in FXS iPSC-derived neurons. Transcriptome-wide analysis revealed striking m6A hypermethylation predominantly affecting synapse-associated transcripts. Mechanistically, we demonstrated that FMRP deficiency drives the aberrant translational upregulation of core m6A writers, a causal relationship definitively validated using CGG-corrected isogenic control lines. Targeted genetic knockdown of the m6A writer METTL3 successfully rescued synaptic phenotypes in FXS neurons, whereas its overexpression in control neurons phenocopied these synaptic defects, confirming the causal role of m6A dysregulation in FXS pathology. Notably, pharmacological intervention with the METTL3 inhibitor STM-2457 normalized methylation on synapse-associated transcripts and restored synaptic transmission in FXS neurons. Together, our findings uncover an FMRP-dependent epitranscriptomic mechanism contributing to FXS pathogenesis and suggest a promising avenue for m6A-targeted therapies.

Mechanistic target of rapamycin (mTOR) signaling is mediated through mTORC1 and mTORC2. mTORC1 signaling requires the regulatory protein Raptor, while mTORC2 signaling requires Rictor. mTOR signaling is increased during epileptogenesis, and manipulations to inhibit mTOR have been shown to reduce seizure incidence in some epilepsy models. Inhibiting mTOR signaling is hypothesized to prevent epileptogenic changes. To test this hypothesis and to assess how mTORC1 and mTORC2 might modulate epileptogenesis, we deleted Raptor or Rictor from a subset of hippocampal dentate granule cells in male and female mice to cell-autonomously inhibit mTORC1 or mTORC2, respectively. Gene deletion effects were examined in healthy mice and following status epilepticus, which leads to the development of epilepsy. Raptor and Rictor knock-out (KO) cells had fewer dendritic spines than neighboring wild-type cells, and Raptor KO cells had reduced presynaptic terminal volume and contributed less to mossy fiber axon sprouting. Raptor deletion decreased somatic contact with parvalbumin inhibitory neuron puncta and reduced soma area, while Rictor KO cells were more likely to be c-Fos immunoreactive. Findings demonstrate that Raptor and Rictor deletion exert mixed effects on morphological changes associated with epilepsy, implying that mTORC1 and mTORC2 have both overlapping and distinct neuroanatomical targets. In addition, the magnitude of gene deletion effects was similar in saline and SE-exposed animals. The observation implies that rather than specifically blocking epileptogenic circuit rewiring in acquired epilepsy, mTOR inhibition acts similarly on granule cells in healthy and epileptic mice to produce mixed changes on structures underlying excitatory and inhibitory synaptic transmission.

Background:Fragile X Syndrome (FXS) is a rare, neurodevelopmental disorder caused by a mutation to the Fragile X messenger ribonucleoprotein 1 (Fmr1) gene and characterized by sensory processing abnormalities and sensitivities, including neural auditory oscillatory disruptions and reduced neural entrainment to chirp stimuli. The present study aims to evaluate the 40 Hz auditory steady state response (ASSR) in FXS to evaluate stimulus representation maintenance in FXS. Methods:Adolescents and adults (N = 67; 34 FXS and 33 age, sex-matched typically developed controls (TDC)) completed a 40 Hz auditory steady state task during electroencephalography (EEG). Time-frequency analyses using Morlet wavelets were completed to evaluate intertrial phase coherence (ITC) and event-related spectral perturbation (ERSP), including characterization of the transient and sustained components of the 40 Hz ASSR. Results:Both ITC (p = .003) and ERSP (p = .004) at 40 Hz were reduced for FXS compared to TDC. Interestingly, TDC exhibited a significantly elevated early, transient component (100 - 400 ms) which reduced in both ITC and ERSP during transition to the sustained component (650 - 3000 ms) whereas FXS were consistently reduced across the ASSR suggesting a reduced ability for FXS to mount a transient response. Conclusions:Individuals with FXS exhibit robust reductions in magnitude and temporal precision of neural entrainment to the steady state stimulus. The reduced ability to mount a transient response may represent reduced GABAergic modulation where the overall reduction in ITC and ERSP may reflect reduced excitatory/inhibitory balance between NMDA and GABAergic input.

People with epilepsy may experience sudden death due to respiratory failure through mechanisms that are currently not well understood. Epilepsy causing mutations are thought to elicit seizures due to altered function of forebrain circuits, yet breathing is controlled largely by the brainstem. To investigate how altered forebrain activity could impact breathing, we examined respiratory and seizure phenotypes in a mouse epilepsy model with a forebrain-specific deletion of the phosphatase and tensin homolog (Pten) gene. Using chronic diaphragm electromyography and cortical electroencephalography, we monitored Pten conditional knock-out (PTEN-cKO) mice (six males and four females) and control littermates (six males and three females) continuously from preseizure onset through end-stage disease. PTEN-cKO mice develop spontaneous seizures that progress in frequency with age, accompanied by gradual changes in respiratory function, even during interictal periods. As seizure burden increases, PTEN-cKO mice experience an increased frequency of interictal apneas, slowing of respiratory rhythm, prolongation of inspiratory bursts, and elevation of inspiratory effort. All animals experienced a terminal apnea prior to cardiac arrest. These findings demonstrate that P ten deletion in the forebrain disrupts the control of breathing and leads to terminal respiratory failure.

Somatic mutations in genes regulating mechanistic target of rapamycin (mTOR) pathway signaling can cause epilepsy, autism, and cognitive dysfunction. Research has predominantly focused on mTOR regulation of excitatory neurons in these conditions; however, dysregulated mTOR signaling among interneurons may also be critical. In this review, we discuss clinical evidence for interneuron involvement, and potential mechanisms, known and hypothetical, by which interneurons might come to directly harbor pathogenic mutations. To understand how mTOR hyperactive interneurons might drive dysfunction, we review studies in which mTOR signaling has been selectively disrupted among interneurons and interneuron progenitors in mouse model systems. Complex cellular mosaicism and dual roles for mTOR (hyper)activation in mediating disease pathogenesis and homeostatic responses raise challenging questions for effective treatment of these disorders.

Background:Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and is caused by reduced or absent Fragile X messenger ribonucleoprotein (FMRP). Cognitive and adaptive skills widely vary among individuals with FXS, and these individual phenotypic differences are not fully accounted for by individual differences in FMRP expression. Social-environmental factors, including social determinants of health, may help further explain these individual differences, but these environmental factors have been under-studied in FXS. Methods:175 participants with FXS (123 males; age range: 4-72 years) completed the Stanford-Binet, Fifth Edition to estimate IQ and a blood draw to quantify peripheral FMRP levels. Caregivers from a subset of participants also completed the Vineland Adaptive Behavior Scales. Neighborhood-level social-environmental information was extracted by linking participants' home addresses to rankings of neighborhood resources (e.g., household income, pollution, healthcare access) from the Child Opportunity Index (COI). We calculated the unique variance in IQ and adaptive behaviors accounted for by these neighborhood-level social-environmental factors from the COI while covarying for FMRP expression. Results:Even after accounting for individual differences in FMRP, numerous neighborhood factors were associated with greater IQ in males with FXS, including social resources and indicators of healthcare access. Different social-environment factors were associated with stronger adaptive skills in males with FXS, including economic and educational resources. Almost no neighborhood factors were associated with clinical outcomes in females. Discussion:Our finding of stronger links between neighborhood resources and clinical outcomes in males with FXS is consistent with previous work and may reflect increased reliance on social-environmental supports in males who typically have more significant intellectual and adaptive deficits than females. Consistent associations between greater social resources, higher IQ, and stronger adaptive skills suggest social support (e.g., social cohesion, resource and knowledge sharing) may be a particularly salient target for intervention. Associations between economic resources and adaptive communication skills also highlight the benefits of targeted economic supports for families affected by FXS. Together, our findings underscore the role of social determinants of health as key contributors to individual differences and the importance of considering these factors in clinical studies of FXS.

Recurrent spontaneous seizures in epilepsy cause a myriad of structural, circuit-related, and molecular modifications in the brain. The multifaceted molecular changes suggest that wide-reaching epigenetic mechanisms are altered in epilepsy. Indeed, it has been known for more than 15 years that a class of epigenetic regulators called microRNAs-short, noncoding RNAs that control the translation and stability of sometimes hundreds of mRNA targets-are dysregulated after seizures and in epilepsy in human patients and rodent models. Epilepsy-associated microRNAs regulate many different molecular contributors to epilepsy, including ion channels, neuroinflammatory modulators, and proteins critical for neuronal and synaptic structure. In recent years, it has become clear that microRNAs are important at every phase of epilepsy-from the onset of the first seizure to the latent phase and chronic epilepsy. In line with these findings, manipulation of a subset of microRNAs has been shown to alter seizure susceptibility, reduce epileptogenesis, and/or decrease the frequency of spontaneous recurrent seizures in animal models of epilepsy. These studies illustrate the promise of microRNAs as future therapeutic targets in epilepsy and show that specific microRNAs play different regulatory roles depending on the phase and type of epilepsy. In this concise review, we summarize recent findings of microRNAs in epilepsy, emphasizing novel approaches that advance the field. We discuss the insight on underlying mechanisms and disease etiology that can be drawn from these studies and highlight the importance of timing when developing microRNA-based therapeutic strategies.

Fragile X syndrome (FXS) is the most common inherited intellectual disability. FXS is caused by a trinucleotide repeat expansion in the 5′ untranslated region of the FMR1 gene, which leads to gene methylation, transcriptional silencing, and lack of expression of Fragile X Messenger Riboprotein (FMRP). Currently available FXS therapies are inefficient, and the disease severity is highly variable, making it difficult to predict disease trajectory and treatment response. We and others have recently shown that a subset of full-mutation, fully-methylated (FM–FM) males with FXS express low amounts of FMRP which could contribute to phenotypic variability. To better understand the underlying mechanisms, we developed a sensitive qRT-PCR assay to detect FMR1 mRNA in blood. This assay reproducibly detects trace amounts of FMR1 mRNA in a subset of FM–FM males, suggesting that current Southern Blot and PCR determination of FM–FM status is not always associated with complete transcriptional silencing. The functional relevance of trace-level FMR1 mRNA is confirmed by showing a positive correlation with cognitive function; however, phenotypic variability is not fully explained by FMR1 expression. These results corroborate the need for better molecular assays for FXS diagnosis and encourage studies to elucidate the factors contributing to the phenotypic variability of FXS.

MicroRNAs are emerging as crucial regulators within the complex, dynamic environment of the synapse, and they offer a promising new avenue for the treatment of neurological disease. These small noncoding RNAs modify gene expression in several ways, including posttranscriptional modulation via binding to complementary and semicomplementary sites on target mRNAs. This rapid, finely tuned regulation of gene expression is essential to meet the dynamic demands of the synapse. Here, we provide a detailed review of the multifaceted world of synaptic microRNA regulation. We discuss the many mechanisms by which microRNAs regulate gene expression at the synapse, particularly in the context of neuronal plasticity. We also describe the various factors, such as age, sex, and neurological disease, that can influence microRNA expression and activity in neurons. In summary, microRNAs play a crucial role in the intricate and quickly changing functional requirements of the synapse, and context is essential in the study of microRNAs and their potential therapeutic applications.

OBJECTIVE:Extracellular signal-regulated kinase (ERK1/2) is a conserved central intracellular signaling cascade involved in many aspects of neuronal development and plasticity. Converging evidence support investigation of ERK1/2 activity in autism spectrum disorder (ASD). We previously reported enhanced baseline lymphocytic ERK1/2 activation in autism, and now we extend our work to investigate the early phase kinetics of lymphocytic ERK1/2 activation in idiopathic ASD. METHOD:Study participants included 67 individuals with ASD (3-25 years of age), 65 age- and sex-matched typical developing control (TDC) subjects, and 36 age-, sex-, and IQ-matched developmental disability control (DDC) subjects matched to those with ASD and IQ <90. We completed an additional analysis comparing results from ASD, TDC, and DDC groups with data from 37 individuals with Fragile X syndrome (FXS). All subjects had blood lymphocyte samples analyzed by flow cytometry following stimulation with phorbol ester and sequentially analyzed for ERK1/2 activation (phosphorylation) at several time points. RESULTS:The ASD group (mean = 5.81 minutes; SD = 1.5) had a significantly lower (more rapid) mean ERK1/2 T1/2 activation value than both the DDC group (mean = 6.78 minutes; SD = 1.6; p = .00078) and the TDC group (mean = 6.4 minutes; SD = 1.5; p = .025). More rapid ERK1/2 T1/2 activation times did correlate with increased social impairment across all study groups including the ASD cohort. Differences in ERK1/2 T1/2 activation were more pronounced in younger than in older individuals in the primary analysis. The ASD group additionally had more rapid activation times than the FXS group, and the FXS group activation kinetics did not differ from those of the TDC and DDC groups. CONCLUSION:Our findings indicate that lymphocytic ERK1/2 activation kinetics are dysregulated in persons with ASD, marked by more rapid early phase activation. Group differences in ERK1/2 activation kinetics appear to be driven by findings from the youngest children analyzed. DIVERSITY & INCLUSION STATEMENT:We worked to ensure sex and gender balance in the recruitment of human participants. We actively worked to promote sex and gender balance in our author group. The author list of this paper includes contributors from the location and/or community where the research was conducted who participated in the data collection, design, analysis, and/or interpretation of the work.

Dendritic spines are small, dynamic protrusions along the dendrite that comprise more than 90% of excitatory connections in the brain, making them essential sites for neuronal communication. These synaptic sites change throughout the process of development, reducing in density and shifting morphology as synapses are refined. One important class of dendritic spine regulators is microRNA (miRNA), small noncoding RNAs that post-transcriptionally regulate gene expression. Several studies suggest that miRNA-324-5p regulates dendritic spine formation. In addition, we have previously shown that miR-324-5p plays a role in seizure and long-term potentiation, both of which involve dendritic spine changes. In this study, we aimed to characterize the role of miRNA-324-5p in developmental spine regulation by assessing the effect of Mir324 knockout (KO) on dendritic spine density and expression of a subset of dendritic proteins at select developmental time points. We show that miR-324-5p expression is developmentally regulated and peaks at four weeks of age. We demonstrate that loss of miR-324-5p expression leads to differential changes in both target protein expression and spine density at different time points during development, disrupting the pattern of spine density changes and leading to a premature loss of dendritic spines in KO mice, which is compensated later. Our findings indicate that miR-324-5p plays a role in synaptic refinement across development. Additionally, our data illustrate the importance of context in the study of miRNA, as regulation by and/or of miRNA can vary dramatically across development and in disease.

MicroRNAs are an emerging class of synaptic regulators. These small noncoding RNAs post-transcriptionally regulate gene expression, thereby altering neuronal pathways and shaping cell-to-cell communication. Their ability to rapidly alter gene expression and target multiple pathways makes them interesting candidates in the study of synaptic plasticity. Here, we demonstrate that the proconvulsive microRNA miR-324-5p regulates excitatory synapse structure and function in the hippocampus of mice. Both Mir324 knockout (KO) and miR-324-5p antagomir treatment significantly reduce dendritic spine density in the hippocampal CA1 subregion, and Mir324 KO, but not miR-324-5p antagomir treatment, shift dendritic spine morphology, reducing the proportion of thin, “unstable” spines. Western blot and quantitative Real-Time PCR revealed changes in protein and mRNA levels for potassium channels, cytoskeletal components, and synaptic markers, including MAP2 and Kv4.2, which are important for long-term potentiation (LTP). In line with these findings, slice electrophysiology revealed that LTP is severely impaired in Mir324 KO mice, while neurotransmitter release probability remains unchanged. Overall, this study demonstrates that miR-324-5p regulates dendritic spine density, morphology, and plasticity in the hippocampus, potentially via multiple cytoskeletal and synaptic modulators.

Inflammasomes are intracellular protein complexes that promote an inflammatory host defense in response to pathogens and damaged or neoplastic tissues and are implicated in inflammatory disorders and therapeutic-induced toxicity. We investigated the mechanisms of activation for inflammasomes nucleated by NOD-like receptor (NLR) proteins. A screen of a small-molecule library revealed that several tyrosine kinase inhibitors (TKIs)—including those that are clinically approved (such as imatinib and crizotinib) or are in clinical trials (such as masitinib)—activated the NLRP3 inflammasome. Furthermore, imatinib and masitinib caused lysosomal swelling and damage independently of their kinase target, leading to cathepsin-mediated destabilization of myeloid cell membranes and, ultimately, cell lysis that was accompanied by potassium (K + ) efflux, which activated NLRP3. This effect was specific to primary myeloid cells (such as peripheral blood mononuclear cells and mouse bone marrow–derived dendritic cells) and did not occur in other primary cell types or various cell lines. TKI-induced lytic cell death and NLRP3 activation, but not lysosomal damage, were prevented by stabilizing cell membranes. Our findings reveal a potential immunological off-target of some TKIs that may contribute to their clinical efficacy or to their adverse effects.