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ATP-dependent chromatin remodeling protein ATRX is an essential regulator involved in maintenance of DNA structure and chromatin state and regulation of gene expression during development. ATRX was originally identified as the monogenic cause of X-linked α-thalassemia mental retardation (ATR-X) syndrome. Affected individuals display a variety of developmental abnormalities and skeletal deformities. Studies from others investigated the role of ATRX in skeletal development by tissue-specific Atrx knockout. However, the impact of ATRX during early skeletal development has not been examined. Using preosteoblast-specific Atrx conditional knockout mice, we observed increased trabecular bone mass and decreased osteoclast number in bone. In vitro coculture of Atrx conditional knockout bone marrow stromal cells (BMSCs) with WT splenocytes showed impaired osteoclast differentiation. Additionally, Atrx deletion was associated with decreased receptor activator of nuclear factor κ-B ligand ( Rankl )/ osteoprotegerin ( Opg ) expression ratio in BMSCs. Notably, Atrx -deficient osteolineage cells expressed high levels of the neuropeptide cocaine- and amphetamine-regulated transcript prepropeptide ( Cartpt ). Mechanistically, ATRX suppresses Cartpt transcription by binding to the promoter, which is otherwise poised for Cartpt expression by RUNX2 binding to the distal enhancer. Finally, Cartpt silencing in Atrx conditional knockout BMSCs rescued the molecular phenotype by increasing the Rankl / Opg expression ratio. Together, our data show a potent repressor function of ATRX in restricting Cartpt expression during skeletal development.

ATR-X syndrome, caused by mutations in the ATRX gene, leads to intellectual disability and neurodevelopmental deficits, with previous mouse models implicating forebrain ATRX loss in cognitive impairment. However, the region-specific requirements of neuronal ATRX for cognitive function remain unclear. Here, we generated conditional knockout mice with predominant deletion of ATRX in hippocampal CA1 pyramidal neurons in both pure C57Bl/6J and hybrid C57Bl/6J + 129S2/Sv genetic backgrounds. Immunofluorescence confirmed efficient ATRX loss in CA1 neurons, with mosaic expression throughout other forebrain structures. Behavioral analyses revealed that T29-1 CaMKIIα-Cre ATRX knockout mice exhibited significant hypoactivity and increased anxiety traits, particularly in the open field, but retained normal hippocampal-dependent contextual fear memory and spatial learning and memory. In contrast, we confirmed that mice with robust forebrain-wide ATRX ablation in excitatory neurons (R1ag#5 CaMKIIα-Cre-mediated) displayed deficits in these cognitive domains. Our findings demonstrate that ATRX-related intellectual disability requires disruption of broader hippocampal or forebrain circuits to elicit cognitive impairments in learning and memory.

Mutations in the ATRX genes cause alpha-thalassemia X-linked intellectual disability (ATR-X) syndrome. Here, we show that ATRX influences the fate of human neural progenitor cells (hNPCs) by forming condensates through liquid-liquid phase separation (LLPS). The intrinsically disordered region (IDR) of ATRX is essential for LLPS and enables ATRX to form dynamic condensates that recruit co-activators. These condensates are necessary for ATRX localization at super-enhancers (SEs) in hNPCs, linking its compartmentalization to transcriptional regulation. Disruption of ATRX condensates alters gene expression and impairs neuronal differentiation. Our findings support a model in which ATRX phase separation regulates gene networks required for hNPC identity. These findings extend current understanding of ATRX function beyond its roles in chromatin structure and suggest that LLPS is a key regulatory mechanism by which ATRX supports neurodevelopment. This study opens avenues for further investigation into how dysregulation of ATRX and its phase-separation ability may contribute to the pathogenesis of ATR-X syndrome and related neurodevelopmental disorders. Tomooka et al . demonstrate that phase-separated ATRX condensates play an essential role in maintaining human neural progenitor identity through chromatin regulation and transcriptional control.

Genetic correction of MeCP2 levels largely reversed the behavioural, molecular and physiological deficits associated with MECP2 duplication syndrome in a transgenic mouse model; similarly, reduction of MeCP2 levels using an antisense oligonucleotide strategy resulted in phenotypic rescue in adult transgenic mice, and dose-dependently corrected MeCP2 levels in cells from patients with MECP2 duplication. Potential reversal of a developmental disorder MECP2 duplication syndrome is a childhood disorder caused by duplication of the MECP2 gene and, consequently, increased MECP2 protein levels. Huda Zoghbi and colleagues report that genetic correction of MECP2 levels largely reverses the behavioural, molecular and physiological deficits in a transgenic mouse model. Reducing MECP2 levels using an antisense oligonucleotide (ASO) strategy—which has greater potential for therapeutic application—similarly resulted in phenotypic rescue in adult transgenic mice and dose-dependently corrected MECP2 levels in cells from patients with MECP2 duplication. These findings suggest that a disorder caused by copy number variation can be reversed after symptoms have emerged. Copy number variations have been frequently associated with developmental delay, intellectual disability and autism spectrum disorders 1 . MECP2 duplication syndrome is one of the most common genomic rearrangements in males 2 and is characterized by autism, intellectual disability, motor dysfunction, anxiety, epilepsy, recurrent respiratory tract infections and early death 3 , 4 , 5 . The broad range of deficits caused by methyl-CpG-binding protein 2 (MeCP2) overexpression poses a daunting challenge to traditional biochemical-pathway-based therapeutic approaches. Accordingly, we sought strategies that directly target MeCP2 and are amenable to translation into clinical therapy. The first question that we addressed was whether the neurological dysfunction is reversible after symptoms set in. Reversal of phenotypes in adult symptomatic mice has been demonstrated in some models of monogenic loss-of-function neurological disorders 6 , 7 , 8 , including loss of MeCP2 in Rett syndrome 9 , indicating that, at least in some cases, the neuroanatomy may remain sufficiently intact so that correction of the molecular dysfunction underlying these disorders can restore healthy physiology. Given the absence of neurodegeneration in MECP2 duplication syndrome, we propose that restoration of normal MeCP2 levels in MECP2 duplication adult mice would rescue their phenotype. By generating and characterizing a conditional Mecp2- overexpressing mouse model, here we show that correction of MeCP2 levels largely reverses the behavioural, molecular and electrophysiological deficits. We also reduced MeCP2 using an antisense oligonucleotide strategy, which has greater translational potential. Antisense oligonucleotides are small, modified nucleic acids that can selectively hybridize with messenger RNA transcribed from a target gene and silence it 10 , 11 , and have been successfully used to correct deficits in different mouse models 12 , 13 , 14 , 15 , 16 , 17 , 18 . We find that antisense oligonucleotide treatment induces a broad phenotypic rescue in adult symptomatic transgenic MECP2 duplication mice ( MECP2 -TG) 19 , 20 , and corrected MECP2 levels in lymphoblastoid cells from MECP2 duplication patients in a dose-dependent manner.

Creatine is a critical metabolite used to buffer cellular energy demands in highly energetic tissues such as the brain and muscle. Genetic defects in endogenous creatine synthesis or transport across cellular membranes lead to a common set of phenotypes referred to as Cerebral Creatine Deficiency Syndrome (CCDS). The most common form of CCDS is Creatine Transporter 1 (CT1) Deficiency (CTD). It accounts for ~ 70% of cases and results from loss-of-function mutations in the X-linked gene SLC6A8 . Affected individuals suffer from intellectual disability, autistic-like behaviors, and epilepsy. There are currently no effective therapies for this disorder, but gene therapy has emerged as a potential approach. The two enzymes which comprise the endogenous creatine synthetic pathway (AGAT and GAMT) are selectively expressed by specific cell types throughout the body. However, after synthesized, creatine uptake relies on the protein product of SLC6A8 , CT1, to transport creatine into target cell types. We hypothesized that gene delivery of GATM (encoding AGAT) and GAMT into end-user cell types would bypass the need for CT1, allowing for intracellular synthesis of creatine. We tested this strategy in two human cell types: HEK293T cells and primary fibroblasts. Co-delivery of GATM and GAMT increased internal creatine concentrations by 7.6-fold in HEK293T cells and 12.3-fold in healthy control fibroblasts. We then employed this approach to primary fibroblasts from patients with CTD. This resulted in an up to 11.6-fold increase in intracellular creatine concentrations, far exceeding the intracellular concentration of creatine in healthy control fibroblasts. Importantly, overexpression of AGAT and GAMT resulted in proper targeting of these enzymes to their natural cellular compartment and did not impair the growth of patient fibroblasts. These findings establish gene therapy with GATM and GAMT as a potential strategy for patients with CTD.

Objective This study is aimed at investigating the genetic defects and clinical features of Chinese children with SLC16A2 variants and at exploring the effects of mutant MCT8 on protein expression and subcellular localization through in vitro experiments. Methods Children with intellectual disability and abnormal serum thyroid hormone levels were screened using whole‐exome sequencing (WES). We collected patients′ clinical data and assessed their cognitive, linguistic, and motor abilities. Candidate variants were verified by Sanger sequencing, and their pathogenicity and evolutionary conservation were analyzed using in silico prediction tools. Protein expression and subcellular localization of mutant MCT8 were evaluated by Western blotting and immunofluorescence microscopy. Results Exome sequencing identified seven previously uncharacterized SLC16A2 variants in 10 unrelated male patients (nine families). These included three missense mutations (p.Ala150Thr,p.Gly208Arg, and p.Gly208Asp), three frameshift mutations (p.Leu168fs, p.Ser243Cysfs, and p.Val485fs), one deletion–insertion mutation (p.Ile328_Ala329delinsThr), and one balanced translocation, t(X,12) (q13.2; q13.13). All patients exhibited global developmental delay, axial hypotonia, dystonia, and abnormal thyroid hormone profiles. In vitro experiments demonstrated significantly reduced expression of mutant proteins compared with the wild‐type. Immunofluorescence showed that, in addition to residual plasma membrane localization, mutant proteins were also partially retained in the cytoplasm, whereas the wild‐type protein localized exclusively to the plasma membrane. Conclusion Our findings expand the genotypic and phenotypic spectrum of MCT8 deficiency. The results suggest that SLC16A2 variants lead to a loss of function through decreased protein expression and defective plasma membrane trafficking.

Epilepsy and mental retardation limited to females (EFMR) is a disorder with an X-linked mode of inheritance and an unusual expression pattern. Disorders arising from mutations on the X chromosome are typically characterized by affected males and unaffected carrier females. In contrast, EFMR spares transmitting males and affects only carrier females. Aided by systematic resequencing of 737 X chromosome genes, we identified different protocadherin 19 ( PCDH19 ) gene mutations in seven families with EFMR. Five mutations resulted in the introduction of a premature termination codon. Study of two of these demonstrated nonsense-mediated decay of PCDH19 mRNA. The two missense mutations were predicted to affect adhesiveness of PCDH19 through impaired calcium binding. PCDH19 is expressed in developing brains of human and mouse and is the first member of the cadherin superfamily to be directly implicated in epilepsy or mental retardation.

Polyamines are tightly regulated polycations that are essential for life. Loss-of-function mutations in spermine synthase (SMS), a polyamine biosynthesis enzyme, cause Snyder-Robinson syndrome (SRS), an X-linked intellectual disability syndrome; however, little is known about the neuropathogenesis of the disease. Here we show that loss of dSms in Drosophila recapitulates the pathological polyamine imbalance of SRS and causes survival defects and synaptic degeneration. SMS deficiency leads to excessive spermidine catabolism, which generates toxic metabolites that cause lysosomal defects and oxidative stress. Consequently, autophagy–lysosome flux and mitochondrial function are compromised in the Drosophila nervous system and SRS patient cells. Importantly, oxidative stress caused by loss of SMS is suppressed by genetically or pharmacologically enhanced antioxidant activity. Our findings uncover some of the mechanisms underlying the pathological consequences of abnormal polyamine metabolism in the nervous system and may provide potential therapeutic targets for treating SRS and other polyamine-associated neurological disorders. Mutations in spermine synthase lead to Snyder-Robinson syndrome, a form of intellectual disability syndrome. Here the authors develop a Drosophila model of this disease, and show that lysosomal dysfunction and oxidative stress contribute to the morphological phenotype in these flies, as well as to cellular deficits in cells derived from patients.

Tarpey et al . carry out a large-scale systematic sequencing of the majority of X-chromosome coding exons from 208 families with multiple individuals with mental retardation and a pattern of transmission compatible with X linkage in order to identify XLMR-causative mutations. They find several mutations that appear to be causative in loci already known to be involved in XLMR, as well as new data about those loci, and make inferences about the role of the different classes of variants in these diseases. Large-scale systematic resequencing has been proposed as the key future strategy for the discovery of rare, disease-causing sequence variants across the spectrum of human complex disease. We have sequenced the coding exons of the X chromosome in 208 families with X-linked mental retardation (XLMR), the largest direct screen for constitutional disease-causing mutations thus far reported. The screen has discovered nine genes implicated in XLMR, including SYP , ZNF711 and CASK reported here, confirming the power of this strategy. The study has, however, also highlighted issues confronting whole-genome sequencing screens, including the observation that loss of function of 1% or more of X-chromosome genes is compatible with apparently normal existence.

Creatine transporter deficiency (CTD) caused by mutations in SLC6A8 encoding the creatine transporter (CRT), leads to cerebral creatine deficiency syndromes; however, the cellular impact of CRT loss remains unclear. In this study, we investigated the consequences of the G561R mutation by examining fibroblasts using proteomics and functional assays. We observed severe intracellular creatine deficiency (> 90% reduction), leading to impaired energy metabolism (low ATP and high ADP/ATP). Proteomic analysis revealed significant alterations in the mitochondrial and extracellular vesicle pathways. Our investigation revealed impaired mitochondrial oxidative phosphorylation, reduced spare respiratory capacity, elevated oxidative stress, and significant alterations in amino acid transporter activity. Protein misfolding associated with G561R exacerbated these deficits compared to the deletion model. These findings elucidate the key pathological mechanisms induced by the CRT-G561R mutation—including energy metabolic reprogramming, mitochondrial dysfunction, and cellular stress—which significantly contribute to our understanding of the pathogenesis of creatine transporter deficiency and suggest potential therapeutic targets.