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Brain organoids represent a powerful tool for studying human neurological diseases, particularly those that affect brain growth and structure. However, many diseases manifest with clear evidence of physiological and network abnormality in the absence of anatomical changes, raising the question of whether organoids possess sufficient neural network complexity to model these conditions. Here, we explore the network-level functions of brain organoids using calcium sensor imaging and extracellular recording approaches that together reveal the existence of complex network dynamics reminiscent of intact brain preparations. We demonstrate highly abnormal and epileptiform-like activity in organoids derived from induced pluripotent stem cells from individuals with Rett syndrome, accompanied by transcriptomic differences revealed by single-cell analyses. We also rescue key physiological activities with an unconventional neuroregulatory drug, pifithrin-α. Together, these findings provide an essential foundation for the utilization of brain organoids to study intact and disordered human brain network formation and illustrate their utility in therapeutic discovery. This paper explores neural network and cellular complexity within human cortical and subcortical fusion organoids. The platform is used to model network dysfunction associated with Rett syndrome and to identify new therapeutic candidates.

Mutations in the X-linked MECP2 gene, which encodes the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2), cause Rett syndrome and several neurodevelopmental disorders including cognitive disorders, autism, juvenile-onset schizophrenia and encephalopathy with early lethality. Rett syndrome is characterized by apparently normal early development followed by regression, motor abnormalities, seizures and features of autism, especially stereotyped behaviours. The mechanisms mediating these features are poorly understood. Here we show that mice lacking Mecp2 from GABA (γ-aminobutyric acid)-releasing neurons recapitulate numerous Rett syndrome and autistic features, including repetitive behaviours. Loss of MeCP2 from a subset of forebrain GABAergic neurons also recapitulates many features of Rett syndrome. MeCP2-deficient GABAergic neurons show reduced inhibitory quantal size, consistent with a presynaptic reduction in glutamic acid decarboxylase 1 ( Gad1 ) and glutamic acid decarboxylase 2 ( Gad2 ) levels, and GABA immunoreactivity. These data demonstrate that MeCP2 is critical for normal function of GABA-releasing neurons and that subtle dysfunction of GABAergic neurons contributes to numerous neuropsychiatric phenotypes. The GABAergic system in Rett syndrome Rett syndrome, a neurodevelopmental disorder with autistic features, is caused by mutations in the methyl-CpG-binding protein 2 gene ( MECP2 ). A number of mouse models with full and cell-type specific deletions of Mecp2 have been generated, but show only a subset of the signs of Rett syndrome. Now Huda Zoghbi and colleagues report that mice with selective deletion of MeCP2 in GABAergic neurons show not only impaired GABAergic function, but capitulate many of the key features of Rett syndrome. The finding that disturbance of inhibitory neurons causes a variety of neuropsychiatric phenotypes suggests that the GABAergic system may be a promising target for therapeutic intervention. Mutations in the methyl-CpG-binding protein 2 (MeCP2) gene cause Rett syndrome, a neurodevelopmental disorder with features of autism. Multiple mouse models of MeCP2 have been generated, but show only a subset of the symptoms of Rett syndrome. These authors find that mice with selective deletion of MeCP2 in GABA-mediated neurons show not only impaired GABA-mediated function, but capitulate multiple key features of Rett, further suggesting a role of inhibitory function in neuropsychiatric disease.

Key Points Methyl-CpG-binding protein 2 (MeCP2) functions throughout the brain. Inactivation of MeCP2 in various brain regions and neuronal subtypes has defined the role of MeCP2 in these areas. MeCP2 is a protein that associates with chromatin. The methyl-CpG-binding domain (MBD) is the primary determinant of DNA binding by MeCP2, but other DNA-binding modules are also reported in the molecule. There is evidence that MeCP2 can positively and negatively regulate gene expression at transcriptional and post-transcriptional levels. Mutations in patients with Rett syndrome (RTT) highlight critical regions of MeCP2 (the MBD, an AT-hook and the NCOR–SMRT interaction domain (NID) that determine the presence and severity of RTT pathology. Different models of MeCP2 function (chromatin compaction or recruitment of nuclear receptor co-repressor (NCOR)–SMRT (silencing mediator of retinoic acid and thyroid hormone receptor)) might be consistent with the RTT mutation spectrum. Rett syndrome is a neurological disorder associated with mutations in the X-linked gene MECP2 (methyl-CpG-binding protein 2). This Review details emerging insights into the link between the functions of MeCP2 and the pathogenesis of Rett syndrome. Rett syndrome (RTT) is a severe neurological disorder caused by mutations in the X-linked gene MECP2 (methyl-CpG-binding protein 2). Two decades of research have fostered the view that MeCP2 is a multifunctional chromatin protein that integrates diverse aspects of neuronal biology. More recently, studies have focused on specific RTT-associated mutations within the protein. This work has yielded molecular insights into the critical functions of MeCP2 that promise to simplify our understanding of RTT pathology.

Analysis of the minimal functional unit for MeCP2 protein shows that its function is to recruit the NCoR/SMRT co-repressor complex to methylated sites on chromatin, which may have use in designing strategies for gene therapy of Rett syndrome. MeCP2 to the Rett-scue Rett syndrome is a neurological disorder caused by mutations in the MECP2 gene, which tend to be clustered in two discrete regions of the protein (MeCP2). In this report, the authors parse the minimal form of MeCP2 that is required to retain its functionality, and interrogate which of its many proposed roles is relevant for Rett syndrome progression. The identification of a minimal functional unit for MeCP2 could be helpful in the design of therapeutic strategies for gene therapy for Rett syndrome. Heterozygous mutations in the X-linked MECP2 gene cause the neurological disorder Rett syndrome 1 . The methyl-CpG-binding protein 2 (MeCP2) protein is an epigenetic reader whose binding to chromatin primarily depends on 5-methylcytosine 2 , 3 . Functionally, MeCP2 has been implicated in several cellular processes on the basis of its reported interaction with more than 40 binding partners 4 , including transcriptional co-repressors (for example, the NCoR/SMRT complex 5 ), transcriptional activators 6 , RNA 7 , chromatin remodellers 8 , 9 , microRNA-processing proteins 10 and splicing factors 11 . Accordingly, MeCP2 has been cast as a multi-functional hub that integrates diverse processes that are essential in mature neurons 12 . At odds with the concept of broad functionality, missense mutations that cause Rett syndrome are concentrated in two discrete clusters coinciding with interaction sites for partner macromolecules: the methyl-CpG binding domain 13 and the NCoR/SMRT interaction domain 5 . Here we test the hypothesis that the single dominant function of MeCP2 is to physically connect DNA with the NCoR/SMRT complex, by removing almost all amino-acid sequences except the methyl-CpG binding and NCoR/SMRT interaction domains. We find that mice expressing truncated MeCP2 lacking both the N- and C-terminal regions (approximately half of the native protein) are phenotypically near-normal; and those expressing a minimal MeCP2 additionally lacking a central domain survive for over one year with only mild symptoms. This minimal protein is able to prevent or reverse neurological symptoms when introduced into MeCP2-deficient mice by genetic activation or virus-mediated delivery to the brain. Thus, despite evolutionary conservation of the entire MeCP2 protein sequence, the DNA and co-repressor binding domains alone are sufficient to avoid Rett syndrome-like defects and may therefore have therapeutic utility.

Background Rett syndrome (RTT), a neurodevelopmental disorder that primarily affects girls, is characterised by a period of apparently normal development until 6–18 months of age when motor and communication abilities regress. More than 95% of individuals with RTT have mutations in methyl-CpG-binding protein 2 (MECP2), whose protein product modulates gene transcription. Surprisingly, although the disorder is caused by mutations in a single gene, disease severity in affected individuals can be quite variable. To explore the source of this phenotypic variability, we propose that specific MECP2 mutations lead to different degrees of disease severity. Methods Using a database of 1052 participants assessed over 4940 unique visits, the largest cohort of both typical and atypical RTT patients studied to date, we examined the relationship between MECP2 mutation status and various phenotypic measures over time. Results In general agreement with previous studies, we found that particular mutations, such as p.Arg133Cys, p.Arg294X, p.Arg306Cys, 3° truncations and other point mutations, were relatively less severe in both typical and atypical RTT. In contrast, p.Arg106Trp, p.Arg168X, p.Arg255X, p.Arg270X, splice sites, deletions, insertions and deletions were significantly more severe. We also demonstrated that, for most mutation types, clinical severity increases with age. Furthermore, of the clinical features of RTT, ambulation, hand use and age at onset of stereotypies are strongly linked to overall disease severity. Conclusions We have confirmed that MECP2 mutation type is a strong predictor of disease severity. These data also indicate that clinical severity continues to become progressively worse regardless of initial severity. These findings will allow clinicians and families to anticipate and prepare better for the needs of individuals with RTT.

Deep brain stimulation (DBS) of the fimbria–fornix—a region that provides input to the hippocampus—is shown to restore hippocampus-dependent memory and hippocampal long-term potentiation and neurogenesis in a mouse model of Rett syndrome, suggesting that DBS, which is already used in the treatment of several neurological conditions, could be a viable approach to mitigating cognitive impairment in Rett syndrome and other disorders of childhood intellectual disability. Deep brain stimulation in Rett syndrome Rett syndrome is a genetic disorder that causes profound intellectual disability and other impairments. Huda Zoghbi and colleagues now show that in a mouse model of the disorder, a two-week course of daily deep-brain stimulation of the fimbria-fornix — part of the brain that provides input to the hippocampus — restored hippocampal-dependent memory when tested three weeks after the end of the treatment. It also restored hippocampal long-term potentiation and neurogenesis. These findings indicate that deep-brain stimulation, which is already used in the treatment of motor diseases such as Parkinson's disease and dystonia, could be a viable approach to mitigating cognitive impairment in Rett syndrome and other disorders of childhood intellectual disability. Deep brain stimulation (DBS) has improved the prospects for many individuals with diseases affecting motor control, and recently it has shown promise for improving cognitive function as well. Several studies in individuals with Alzheimer disease and in amnesic rats have demonstrated that DBS targeted to the fimbria–fornix 1 , 2 , 3 , the region that appears to regulate hippocampal activity, can mitigate defects in hippocampus-dependent memory 3 , 4 , 5 . Despite these promising results, DBS has not been tested for its ability to improve cognition in any childhood intellectual disability disorder. Such disorders are a pressing concern: they affect as much as 3% of the population and involve hundreds of different genes. We proposed that stimulating the neural circuits that underlie learning and memory might provide a more promising route to treating these otherwise intractable disorders than seeking to adjust levels of one molecule at a time. We therefore studied the effects of forniceal DBS in a well-characterized mouse model of Rett syndrome (RTT), which is a leading cause of intellectual disability in females. Caused by mutations that impair the function of MeCP2 (ref. 6 ), RTT appears by the second year of life in humans, causing profound impairment in cognitive, motor and social skills, along with an array of neurological features 7 . RTT mice, which reproduce the broad phenotype of this disorder, also show clear deficits in hippocampus-dependent learning and memory and hippocampal synaptic plasticity 8 , 9 , 10 , 11 . Here we show that forniceal DBS in RTT mice rescues contextual fear memory as well as spatial learning and memory. In parallel, forniceal DBS restores in vivo hippocampal long-term potentiation and hippocampal neurogenesis. These results indicate that forniceal DBS might mitigate cognitive dysfunction in RTT.

Rett syndrome (RTT) is a severe neurological disorder caused by mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2). Almost two decades of research into RTT have greatly advanced our understanding of the function and regulation of the multifunctional protein MeCP2. Here, we review recent advances in understanding how loss of MeCP2 impacts different stages of brain development, discuss recent findings demonstrating the molecular role of MeCP2 as a transcriptional repressor, assess primary and secondary effects of MeCP2 loss and examine how loss of MeCP2 can result in an imbalance of neuronal excitation and inhibition at the circuit level along with dysregulation of activity-dependent mechanisms. These factors present challenges to the search for mechanism-based therapeutics for RTT and suggest specific approaches that may be more effective than others.

Perineuronal nets (PNNs), a specialized form of extracellular matrix, are abnormal in the brains of people with Rett syndrome (RTT). We previously reported that PNNs function to restrict synaptic plasticity in hippocampal area CA2, which is unusually resistant to long-term potentiation (LTP) and has been linked to social learning in mice. Here we report that PNNs appear elevated in area CA2 of the hippocampus of an individual with RTT and that PNNs develop precociously and remain elevated in area CA2 of a mouse model of RTT (Mecp2-null). Further, we provide evidence that LTP could be induced at CA2 synapses prior to PNN maturation (postnatal day 8-11) in wild-type mice and that this window of plasticity was prematurely restricted at CA2 synapses in Mecp2-null mice. Degrading PNNs in Mecp2-null hippocampus was sufficient to rescue the premature disruption of CA2 plasticity. We identified several molecular targets that were altered in the developing Mecp2-null hippocampus that may explain aberrant PNNs and CA2 plasticity, and we discovered that CA2 PNNs are negatively regulated by neuronal activity. Collectively, our findings demonstrate that CA2 PNN development is regulated by Mecp2 and identify a window of hippocampal plasticity that is disrupted in a mouse model of RTT.

Transplanting bone marrow from wild-type mice into MECP2-lacking mice results in wild-type microglial engraftment, extends lifespan and reduces symptoms of disease such as breathing and locomotor abnormalities, implicating microglia in the pathophysiology of Rett syndrome. Marrow implants in Rett syndrome The X-linked autism spectrum disorder known as Rett syndrome is predominantly linked to mutations in the MECP2 gene. It is typically associated with neuronal dysfunction, almost exclusively in girls, but new evidence suggests that restoring MECP2 function in other cell types may also arrest disease development. Here, the authors show in a mouse model that transplanting bone marrow from wild-type mice into mice lacking Mecp2 results in an invasion of donor-derived microglial cells into the brain, accompanied by increased lifespan and reduced signs of disease, including improved breathing and locomotion. The donor cells expressed normal MECP2 and high levels of the neurotrophic factor IGF-1. These results point to a crucial role for microglia in Rett syndrome, and open the possibility that bone-marrow implants might be of therapeutic benefit. Rett syndrome is an X-linked autism spectrum disorder. The disease is characterized in most cases by mutation of the MECP2 gene, which encodes a methyl-CpG-binding protein 1 , 2 , 3 , 4 , 5 . Although MECP2 is expressed in many tissues, the disease is generally attributed to a primary neuronal dysfunction 6 . However, as shown recently, glia, specifically astrocytes, also contribute to Rett pathophysiology. Here we examine the role of another form of glia, microglia, in a murine model of Rett syndrome. Transplantation of wild-type bone marrow into irradiation-conditioned Mecp2 -null hosts resulted in engraftment of brain parenchyma by bone-marrow-derived myeloid cells of microglial phenotype, and arrest of disease development. However, when cranial irradiation was blocked by lead shield, and microglial engraftment was prevented, disease was not arrested. Similarly, targeted expression of MECP2 in myeloid cells, driven by Lysm cre on an Mecp2 -null background, markedly attenuated disease symptoms. Thus, through multiple approaches, wild-type Mecp2 -expressing microglia within the context of an Mecp2 -null male mouse arrested numerous facets of disease pathology: lifespan was increased, breathing patterns were normalized, apnoeas were reduced, body weight was increased to near that of wild type, and locomotor activity was improved. Mecp2 +/− females also showed significant improvements as a result of wild-type microglial engraftment. These benefits mediated by wild-type microglia, however, were diminished when phagocytic activity was inhibited pharmacologically by using annexin V to block phosphatydilserine residues on apoptotic targets, thus preventing recognition and engulfment by tissue-resident phagocytes. These results suggest the importance of microglial phagocytic activity in Rett syndrome. Our data implicate microglia as major players in the pathophysiology of this devastating disorder, and suggest that bone marrow transplantation might offer a feasible therapeutic approach for it.

In this study, the authors show that MeCP2 interacts with the NCoR/SMRT co-repressor complex and that a discrete cluster of Rett syndrome–causing mutations in the C-terminal domain of MeCP2 disrupts this interaction, impairing transcriptional repression. Knock-in mice expressing one of these MeCP2 missense mutations exhibit severe motor phenotypes. Rett syndrome (RTT) is a severe neurological disorder that is caused by mutations in the MECP2 gene. Many missense mutations causing RTT are clustered in the DNA-binding domain of MeCP2, suggesting that association with chromatin is critical for its function. We identified a second mutational cluster in a previously uncharacterized region of MeCP2. We found that RTT mutations in this region abolished the interaction between MeCP2 and the NCoR/SMRT co-repressor complexes. Mice bearing a common missense RTT mutation in this domain exhibited severe RTT-like phenotypes. Our data are compatible with the hypothesis that brain dysfunction in RTT is caused by a loss of the MeCP2 'bridge' between the NCoR/SMRT co-repressors and chromatin.