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5-hydroxymethylcytosine (5hmC) occurs at maximal levels in postmitotic neurons, where its accumulation is cell-specific and correlated with gene expression. Here we demonstrate that the distribution of 5hmC in CG and non-CG dinucleotides is distinct and that it reflects the binding specificity and genome occupancy of methylcytosine binding protein 2 (MeCP2). In expressed gene bodies, accumulation of 5hmCG acts in opposition to 5mCG, resulting in “functional” demethylation and diminished MeCP2 binding, thus facilitating transcription. Non-CG hydroxymethylation occurs predominantly in CA dinucleotides (5hmCA) and it accumulates in regions flanking active enhancers. In these domains, oxidation of 5mCA to 5hmCA does not alter MeCP2 binding or expression of adjacent genes. We conclude that the role of 5-hydroxymethylcytosine in postmitotic neurons is to functionally demethylate expressed gene bodies while retaining the role of MeCP2 in chromatin organization.

Epigenetic mechanisms are gene regulatory processes that control gene expression and cellular identity. Epigenetic factors include the “writers”, “readers”, and “erasers” of epigenetic modifications such as DNA methylation. Accordingly, the nuclear protein Methyl-CpG-Binding Protein 2 (MeCP2) is a reader of DNA methylation with key roles in cellular identity and function. Research studies have linked altered DNA methylation, deregulation of MeCP2 levels, or MECP2 gene mutations to different types of human disease. Due to the high expression level of MeCP2 in the brain, many studies have focused on its role in neurological and neurodevelopmental disorders. However, it is becoming increasingly apparent that MeCP2 also participates in the tumorigenesis of different types of human cancer, with potential oncogenic properties. It is well documented that aberrant epigenetic regulation such as altered DNA methylation may lead to cancer and the process of tumorigenesis. However, direct involvement of MeCP2 with that of human cancer was not fully investigated until lately. In recent years, a multitude of research studies from independent groups have explored the molecular mechanisms involving MeCP2 in a vast array of human cancers that focus on the oncogenic characteristics of MeCP2. Here, we provide an overview of the proposed role of MeCP2 as an emerging oncogene in different types of human cancer.

Chronic stress causes maladaptive changes in the brain that lead to depressive behavior. In the present study, we investigate whether chronic stress alters gut microbiota compositions that are related to stress-induced maladaptive changes in the brain. Mice treated with daily 2-h restraint for 14 days (CRST) exhibit depressive-like behavior. Sequence readings of 16S rRNA genes prepared from fecal samples taken from CRST-treated mice suggest that chronic stress induces gut microbiota changes that are pronounced in the post-stress period, relative to those that occur in the 14-day stress phase. The genus Lactobacillus is one such microbiota substantially changed following chronic stress. In contrast, intraperitoneal injection of extracellular vesicles (EVs) isolated from culture media of the Gram-positive probiotic Lactobacillus plantarum is sufficient to ameliorate stress-induced depressive-like behavior. Interestingly, EVs from the Gram-positive probiotic Bacillus subtilis and EVs from the Gram-negative probiotic Akkermansia muciniphila also produce anti-depressive-like effects. While chronic stress decreases the expression of MeCP2, Sirt1, and/or neurotrophic factors in the hippocampus, EVs from the three selected probiotics differentially restore stress-induced changes of these factors. These results suggest that chronic stress produces persistent changes in gut microbiota composition, whereas purified EVs of certain probiotics can be used for treatment of stress-induced depressive-like behavior.

De novo loss‐of‐function mutations in methyl‐CpG‐binding protein 2 (MeCP2) lead to the neurodevelopmental disorder Rett syndrome (RTT). Despite promising results from strategies aimed at increasing MeCP2 levels, additional studies exploring how hypomorphic MeCP2 mutations impact the therapeutic window are needed. Here, we investigated the consequences of genetically introducing a wild‐type MECP2 transgene in the Mecp2 R133C mouse model of RTT. The MECP2 transgene reversed the majority of RTT‐like phenotypes exhibited by male and female Mecp2 R133C mice. However, three core symptom domains were adversely affected in female Mecp2R133C/+ animals; these phenotypes resemble those observed in disease contexts of excess MeCP2. Parallel control experiments in Mecp2Null/+ mice linked these adverse effects to the hypomorphic R133C mutation. Collectively, these data provide evidence regarding the safety and efficacy of genetically overexpressing functional MeCP2 in Mecp2 R133C mice and suggest that personalized approaches may warrant consideration for the clinical assessment of MeCP2‐targeted therapies. Introducing a functional MeCP2 in a Rett syndrome mouse model with a hypomorphic MECP2 mutation is contingent on mutation and symptom domain.

Inputs from the ventral hippocampus (vHIP) to the medial prefrontal cortex (mPFC) are implicated in several neuropsychiatric disorders. Here, we show that the vHIP-mPFC projection is hyperactive in the Mecp2 knockout mouse model of the autism spectrum disorder Rett syndrome, which has deficits in social memory. Long-term excitation of mPFC-projecting vHIP neurons in wild-type mice impaired social memory, whereas their long-term inhibition in Rett mice rescued social memory deficits. The extent of social memory improvement was negatively correlated with vHIP-evoked responses in mPFC slices, on a mouse-per-mouse basis. Acute manipulations of the vHIP-mPFC projection affected social memory in a region and behavior selective manner, suggesting that proper vHIP-mPFC signaling is necessary to recall social memories. In addition, we identified an altered pattern of vHIP innervation of mPFC neurons, and increased synaptic strength of vHIP inputs onto layer five pyramidal neurons as contributing factors of aberrant vHIP-mPFC signaling in Rett mice.

Calling structural variations (SVs) is technically challenging, but using long reads remains the most accurate way to identify complex genomic alterations. Here we present Sniffles2, which improves over current methods by implementing a repeat aware clustering coupled with a fast consensus sequence and coverage-adaptive filtering. Sniffles2 is 11.8 times faster and 29% more accurate than state-of-the-art SV callers across different coverages (5–50×), sequencing technologies (ONT and HiFi) and SV types. Furthermore, Sniffles2 solves the problem of family-level to population-level SV calling to produce fully genotyped VCF files. Across 11 probands, we accurately identified causative SVs around MECP2 , including highly complex alleles with three overlapping SVs. Sniffles2 also enables the detection of mosaic SVs in bulk long-read data. As a result, we identified multiple mosaic SVs in brain tissue from a patient with multiple system atrophy. The identified SV showed a remarkable diversity within the cingulate cortex, impacting both genes involved in neuron function and repetitive elements. Sniffles2 detects mosaic structural variation from bulk long-read sequencing data.

Purpose Besides their developmental and neurological phenotype, most patients with MECP2/IRAK1 duplication syndrome present with recurrent and severe infections, accompanied by strong inflammation. Respiratory infections are the most common cause of death. Standardized pneumological diagnostics, targeted anti-infectious treatment, and knowledge of the underlying pathomechanism that triggers strong inflammation are unmet clinical needs. We investigated the influence of IRAK1 overexpression on the canonical NF-κB signaling as a possible cause for excessive inflammation in these patients. Methods NF-κB signaling was examined by measuring the production of proinflammatory cytokines and evaluating the IRAK1 phosphorylation and degradation as well as the IκBα degradation upon stimulation with IL-1β and TLR agonists in SV40-immortalized fibroblasts, PBMCs, and whole blood of 9 patients with MECP2/IRAK1 duplication syndrome, respectively. Results Both, MECP2/IRAK1 -duplicated patients and healthy controls, showed similar production of IL-6 and IL-8 upon activation with IL-1β and TLR2/6 agonists in immortalized fibroblasts. In PBMCs and whole blood, both patients and controls had a similar response of cytokine production after stimulation with IL-1β and TLR4/2/6 agonists. Patients and controls had equivalent patterns of IRAK1 phosphorylation and degradation as well as IκBα degradation upon stimulation with IL-1β. Conclusion Patients with MECP2/IRAK1 duplication syndrome do not show increased canonical NF-κB signaling in immortalized fibroblasts, PBMCs, and whole blood. Therefore, we assume that these patients do not benefit from a therapeutic suppression of this pathway.

Neurodevelopmental spectrum disorders like autism (ASD) are diagnosed, on average, beyond age 4 y, after multiple critical periods of brain development close and behavioral intervention becomes less effective. This raises the urgent need for quantitative, noninvasive, and translational biomarkers for their early detection and tracking. We found that both idiopathic (BTBR) and genetic (CDKL5- and MeCP2-deficient) mouse models of ASD display an early, impaired cholinergic neuromodulation as reflected in altered spontaneous pupil fluctuations. Abnormalities were already present before the onset of symptoms and were rescued by the selective expression of MeCP2 in cholinergic circuits. Hence, we trained a neural network (ConvNetACh) to recognize, with 97% accuracy, patterns of these arousal fluctuations in mice with enhanced cholinergic sensitivity (LYNX1-deficient). ConvNetACh then successfully detected impairments in all ASD mouse models tested except in MeCP2-rescued mice. By retraining only the last layers of ConvNetACh with heart rate variation data (a similar proxy of arousal) directly from Rett syndrome patients,we generated ConvNetPatients, a neural network capable of distinguishing them from typically developing subjects. Even with small cohorts of rare patients, our approach exhibited significant accuracy before (80% in the first and second year of life) and into regression (88% in stage III patients). Thus, transfer learning across species and modalities establishes spontaneous arousal fluctuations combined with deep learning as a robust noninvasive, quantitative, and sensitive translational biomarker for the rapid and early detection of neurodevelopmental disorders before major symptom onset.

Duplication or deficiency of the X‐linked MECP2 gene reliably produces profound neurodevelopmental impairment. MECP2 mutations are almost universally responsible for Rett syndrome (RTT), and particular mutations and cellular mosaicism of MECP2 may underlie the spectrum of RTT symptomatic severity. No clinically approved treatments for RTT are currently available, but human pluripotent stem cell technology offers a platform to identify neuropathology and test candidate therapeutics. Using a strategic series of increasingly complex human stem cell‐derived technologies, including human neurons, MECP2 ‐mosaic neurospheres to model RTT female brain mosaicism, and cortical organoids, we identified synaptic dysregulation downstream from knockout of MECP2 and screened select pharmacological compounds for their ability to treat this dysfunction. Two lead compounds, Nefiracetam and PHA 543613, specifically reversed MECP2‐ knockout cytologic neuropathology. The capacity of these compounds to reverse neuropathologic phenotypes and networks in human models supports clinical studies for neurodevelopmental disorders in which MeCP2 deficiency is the predominant etiology. Synopsis Deficiency of the X‐linked MECP2 gene profoundly impairs neurodevelopment. Clinically, mutations in MECP2 most commonly present as the severe and untreatable disease Rett syndrome. Innovative human pluripotent stem cell (PSC) technology enables the investigation of therapeutic candidates. Neurons differentiated from human MECP2 ‐KO PSCs showed altered expression of synapse‐relevant genes, synaptic morphology, and decreased calcium and network activities compared to control neurons. An in silico neural network simulation affirmed that synaptic structural parameters link with neural network activity, supporting our approach of rescuing synaptic structure and increasing activity. Strategic screening of select drug candidates with mechanisms of action that counteract MECP2 ‐KO deficiencies isolated two lead compounds, Nefiracetam and PHA 543613, for subsequent validation in 3D human cell models. MECP2 ‐mosaic neurospheres, a novel model of Rett female brain mosaicism, showed altered calcium activity that could be reversed by Nefiracetam and/or PHA 543613. Nefiracetam and/or PHA 543613 increased synaptic gene expression and reversed network pathology in human MECP2 ‐KO cortical organoids, arguing for their clinical trial in patients with MeCP2‐deficient neurodevelopmental disorders. Graphical Abstract Deficiency of the X‐linked MECP2 gene profoundly impairs neurodevelopment. Clinically, mutations in MECP2 most commonly present as the severe and untreatable disease Rett syndrome. Innovative human pluripotent stem cell (PSC) technology enables the investigation of therapeutic candidates.

Parvalbumin-positive (PV+) interneurons play a pivotal role in orchestrating windows of experience-dependent brain plasticity during development. Critical period closure is marked by the condensation of a perineuronal net (PNN) tightly enwrapping subsets of PV+ neurons, both acting as a molecular brake on plasticity and maintaining mature PV+ cell signaling. As much of the molecular organization of PNNs exists at length scales near or below the diffraction limit of light microscopy, we developed a superresolution imaging and analysis platform to visualize the structural organization of PNNs and the synaptic inputs perforating them in primary visual cortex. We identified a structural trajectory of PNN maturation featuring a range of net structures, which was accompanied by an increase in Synaptotagmin-2 (Syt2) signals on PV+ cells suggestive of increased inhibitory input between PV+ neurons. The same structural trajectory was followed by PNNs both during normal development and under conditions of critical period delay by total sensory deprivation or critical period acceleration by deletion of MeCP2, the causative gene for Rett syndrome, despite shifted maturation levels under these perturbations. Notably, superresolution imaging further revealed a decrease in Syt2 signals alongside an increase in vesicular glutamate transporter-2 signals on PV+ cells in MeCP2-deficient animals, suggesting weaker recurrent inhibitory input between PV+ neurons and stronger thalamocortical excitatory inputs onto PV+ cells. These results imply a latent imbalanced circuit signature that might promote cortical silencing in Rett syndrome before the functional regression of vision.