Mutations in the MECP2 gene cause the severe neurological disorder Rett syndrome. A cluster of frameshift-causing C-terminal deletions (CTDs) removes ~100 amino acids and accounts for approximately 10% of RTT-causing mutations. Their pathogenicity is unexpected because this C-terminal domain is dispensable in mice. Analysis of pathogenic and benign human MECP2 variants reveals that some individuals with apparently typical CTDs do not develop Rett syndrome, confirming that C-terminal truncations are not intrinsically pathogenic. Using human sequence data and mouse models we show that pathogenicity results from a marked reduction in MeCP2 levels and depends on the presence of a proline proline stop motif (-PPX) generated by a shift to the +2 reading frame. CTDs that shift to the +1 frame avoid this motif and are benign. Replacing the stop codon of the PPX motif with tryptophan restores MeCP2 expression and rescues RTT-like phenotypes in a CTD mouse model. An adenine base editor efficiently introduces this substitution in cultured cells. These findings define a reliable prognostic distinction between benign and pathogenic CTDs and establish a potential editing strategy for correcting disease-causing CTD mutations.

Many neurological diseases impact specific brain regions despite widespread expression of the disease-related protein. Spinocerebellar ataxia type 1 (SCA1) primarily affects the cerebellum, though Ataxin-1 (ATXN1) is widely expressed. We previously showed that intensified interaction between mutant ATXN1 and Capicua (CIC) drives SCA1 pathogenesis in the cerebellum, whereas ATXN1 loss augments amyloid β production in the hippocampus and cortex. CIC, however, forms a complex with ATXN1 and its paralog, Ataxin-1-like (ATXN1L), yet knockout of either yields completely different phenotypes. To determine whether this could be due to CIC having two isoforms, we generated mice bearing either the long (CIC-L) or short (CIC-S) isoform. Loss of CIC-L led to cognitive deficits, whereas loss of CIC-S caused early postnatal lethality, phenocopying ATXN1 and ATXN1L knockout mice, respectively. Furthermore, CIC-L preferentially interacts with ATXN1, and CIC-S with ATXN1L. Our data underscore the importance of isoform-paralog interplay in studying regional vulnerability in neurodegenerative diseases.

Rett syndrome (RTT) is a postnatal neurological disorder caused by loss-of-function mutations in the gene that encodes methyl-CpG binding protein 2. RTT is characterized by initially normal development, followed by developmental regression at 6 to 18 months of age. Individuals with RTT subsequently develop motor deficits, impaired learning and memory, and breathing abnormalities. In this review, we summarize recent findings on how neuronal circuitry is impaired in RTT and how deep brain stimulation and presymptomatic, task-specific training significantly improve learning and memory in RTT mice. Translating these findings to clinical applications, interventional studies have shown initial evidence that structured behavioral exercises can benefit individuals with RTT. Based on the converging evidence from preclinical and clinical research, we advocate for early-stage intensive behavioral training to supplement other therapeutic approaches and enhance treatment outcomes.

Rett syndrome (RTT) is an X-linked neurological disorder caused by MECP2 mutations, creating distinct cellular environments in females (mosaic) versus males (nonmosaic). Despite female patients representing most cases, how mosaicism contributes molecularly to RTT pathogenesis, particularly in presymptomatic stages, remains poorly understood. To address this question, we profiled hippocampal transcriptomes of young female and male RTT mice using bulk and single-nucleus RNA sequencing. We identified a core disease signature of consistently dysregulated genes only in MeCP2- cells across RTT models. Moreover, we uncovered non-cell autonomous effects exclusively in female MeCP2+ excitatory neurons, suggesting that these circuits are more vulnerable early in the mosaic RTT environment. The single-nuclei data also revealed an underappreciated MeCP2- interneuron subtype that had the most transcriptional dysregulation in both male and female RTT hippocampi. Together, these data highlight the different effects of MeCP2 loss on excitatory and inhibitory circuits between the mosaic and nonmosaic environments in early RTT pathogenesis.

To better understand large-effect pathogenic variation associated with autism, we generated long-read sequencing (LRS) data to construct phased and near-complete genome assemblies (average contig N50 = 43 Mbp, QV = 56) for 189 individuals from 51 families with unsolved cases. We applied read- and assembly-based strategies to facilitate comprehensive characterization of de novo mutations, structural variants (SVs), and DNA methylation. Using LRS pangenome controls, we efficiently filtered >97% of common SVs exclusive to 87 offspring. We find no evidence of increased autosomal SV burden for probands when compared to unaffected siblings yet observe a suggestive trend toward an increased SV burden on the X chromosome among affected females. We establish a workflow to prioritize potential pathogenic variants by integrating autism risk genes and putative noncoding regulatory elements defined from ATAC-seq and CUT&Tag data from the developing cortex. In total, we identified three pathogenic variants in TBL1XR1, MECP2, and SYNGAP1, as well as nine candidate de novo and biallelic inherited homozygous SVs, most of which were missed by short-read sequencing. Our work highlights the potential of phased genomes to discover complex more pathogenic mutations and the power of the pangenome to restrict the focus on an increasingly smaller number of SVs for clinical evaluation.

Rett syndrome (RTT) is a neurological disorder caused by loss-of-function mutations in methyl-CpG-binding protein 2 (MECP2), which encodes a transcriptional regulator essential for maintenance of normal neuronal function. The current US Food and Drug Administration-approved treatment for RTT, trofinetide, mildly alleviates some symptoms. In contrast, reintroducing MeCP2 or increasing its amount through transgenesis in mouse RTT models improves most neurological phenotypes and enhances survival. Here, we devised a therapeutic strategy to moderately increase MeCP2 protein by modulating the alternative splicing of MECP2 to switch the less efficiently translated e2 to the more efficiently translated e1 isoform. We deleted Mecp2 exon 2 (unique to e2), leading to production of only e1 mRNA, and showed that this up-regulated MeCP2 by 50 to 60% in mice. Next, we investigated the consequences of isoform switching in two independent RTT induced pluripotent stem cell (iPSC)-derived neuron models harboring mutations that reduce both MeCP2 expression and function. Exon 2 deletion in neurons derived from patients with MeCP2-G118E up-regulated MeCP2, ameliorated morphological and electrophysiological changes, and corrected the dysregulated transcriptome in these neurons. Isoform switching in neurons derived from patients with MeCP2-G118E, modeling a severe RTT mutation, only modestly affected MeCP2 protein abundance and, despite this, led to a partial transcriptomic rescue. Last, an exon 2-skipping morpholino up-regulated MeCP2-E1 in vivo in mice. These data set the stage for a potential therapeutic strategy using antisense oligonucleotides to promote isoform switching in patients with RTT who carry partially functioning alleles of MECP2.

Most Mendelian disorders caused by a deficiency or excess of one gene product lack targeted therapies. Since these disorders can be modeled with a gene overexpression, knockout, or knockdown, drugs that oppose the transcriptomic effects of such perturbations may be promising therapeutic candidates. RNA-Sequencing (RNA-Seq) studies can fuel this drug-prioritization, but their labels, written in plain language, must be annotated manually. Hence, we introduce Signature-based Networks from Automatically Curated Knockout, Knockdown, and Small-molecule Studies (SNACKKSS), which automatically curates gene-disruption and drug studies from the Gene Expression Omnibus and, in partnership with uniformly computed read count datasets, feeds the labels and RNA-Seq data directly into regulatory relationship predictions. Through cross-validation, we show that SNACKKSS' predictions (specifically, from a variation called "SA4") make a unique contribution to finding protein-inhibiting compounds, even alongside existing predictors. We demonstrate the benefit of aggregating multiple predictive tools, and provide this powerful ensemble alongside SNACKKSS. Importantly, we advise researchers to test complex machine learning models on multiple devices. Even with code packages kept consistent, they can run deterministically within a machine, but inconsistently on different ones. Nonetheless, the downstream predictive ability was striking, and leveraging multiple sources of information, RNA-Seq data included, will vastly improve drug-repurposing screens.

Parkinson's disease is characterized by dopaminergic neuron loss and accumulation of α-synuclein aggregates in the brain. G51D α-synuclein knock-in mice provide a genetically and clinically relevant model of disease, exhibiting early olfactory deficits, age-dependent motor impairment, and progressive phospho-α-synuclein accumulation. In multiple Parkinson's disease models, striatal cholinergic and parvalbumin interneurons, as well as astrocytes, lose primary cilia and the neurotrophic signaling needed to sustain dopaminergic neurons. We show here that G51D α-synuclein mice share these phenotypes. Phospho-Ser129 α-synuclein accumulation correlates with cilia loss in cholinergic interneurons but not in spiny projection neurons that accumulate higher phospho-α-synuclein levels. In the piriform cortex, parvalbumin neurons lose primary cilia and downregulate Neurturin, potentially contributing to olfactory dysfunction. Within the peripheral olfactory epithelium, horizontal basal cells lose cilia, whereas multiciliated olfactory sensory neuron cilia remain intact. These findings reveal convergent cellular vulnerabilities across Parkinson's disease models and highlight a pathogenic role for impaired ciliary signaling.

Astrocytes are key regulators of lipid metabolism, and dysregulated astrocytic lipid processing is implicated in Parkinson's disease (PD) pathogenesis. Our prior genome-wide screens identified ACSBG1, an astrocyte-enriched acyl-CoA synthetase, as a candidate regulator of α-synuclein (α-Syn) levels. However, how ACSBG1 links lipid reprogramming to inflammatory astrocyte activation and α-Syn pathology remains unknown. We compared the transcriptomic, cytokine, and lipid secretomes of TNF-α and IL-1α stimulated primary astrocytes from wild-type (WT) and Acsbg1 knockout (KO) mice. In vivo, we crossed Acsbg1 KO mice with a Thy1-α-Syn PD model to assess behavior, neuroinflammation, synaptic integrity, and α-Syn levels. Following cytokine exposure, Acsbg1 KO astrocytes mounted an attenuated inflammatory transcriptional response, secreting significantly fewer inflammatory mediators (e.g., IL-6, RANTES, MIP-3α) and less long-chain Sphingosine 20:1 than WT astrocytes. Importantly, exogenous Sphingosine 20:1 or cytokines from WT reactive astrocytes induced neuronal α-Syn phosphorylation (pS129). In vivo, Acsbg1 deletion in Thy1-α-Syn mice reduced astrogliosis, rescued synaptic and behavioral deficits, and decreased total and pS129-α-Syn. These findings establish ACSBG1 as a key regulator of inflammatory astrocyte signaling that contributes to α-Syn phosphorylation via specific cytokine and lipid mediators, identifying ACSBG1 as a novel therapeutic target for modulating astrocyte-neuron communication in PD. Graphical abstract:

Memory impairment is a hallmark cognitive deficit in Rett syndrome (RTT). Yet, long-term memory deficits in RTT animal models remain poorly understood, largely due to the technical challenges inherent in tracking neural activity over extended periods. Here, we used longitudinal two-photon calcium imaging to follow the same population of hippocampal CA1 neurons as RTT mice and their littermate controls formed cognitive maps of their environment during a spatial learning task. Neural representations in RTT mice were marked by excessive place cell (PC) activity, with individual PCs exhibiting pronounced instability across days. This disrupted single-cell stability propagated to the population level, resulting in unstable ensemble codes that poorly retained previously learned task information. Both excessive PC recruitment and instability could be attributed to a higher incidence of behavioral timescale synaptic plasticity (BTSP) in RTT mice. In wild-type littermates, place-cell consolidation across days is reflected by an increased likelihood of neuron-specific synaptic plasticity at the location of prior PC coding. This cellular mechanism of memory consolidation based on the location of BTSP was disrupted in RTT mice, where excessive and ectopic plasticity reduced PC stability, and degraded long-term stable representations. Backed by theoretical modeling, these results identify a plausible cellular and circuit-level mechanism underlying memory impairments in RTT mice and suggest principles that may be generalized to other neurological disorders involving memory deficits.

The US biomedical research enterprise is renowned for its historical and ongoing scientific breakthroughs and advancements. Yet its capacity to solve complex health issues, bridge health equity gaps, and strengthen public trust is constrained by the lack of an overarching national vision, fragmented coordination for research funding, and critical workforce recruitment and retention challenges. To improve national health outcomes and retain global competitiveness, the sector must embrace new approaches. This article, part of the National Academy of Medicine's Vital Directions for Health and Health Care: Priorities for 2025 initiative, identifies four key opportunities to revitalize the biomedical research enterprise: establishing a national advisory body, bolstering the workforce, prioritizing research to reduce health disparities, and developing approaches to streamlining and coordinating federal research funding. These priorities will help the biomedical research enterprise meet twenty-first-century challenges, promote healthy longevity, and preserve US leadership in the global arena.

Polyglutamine (polyQ) diseases, caused by a CAG repeat expansion encoding a glutamine tract in nine distinct proteins, present a complex molecular puzzle in which each piece contributes to neurodegeneration. While each of the causative proteins has a distinct function, the downstream consequences of polyQ toxicity are often similar, including protein accumulation, transcriptional dysregulation, somatic CAG repeat instability, disrupted energy homeostasis, compromised synaptic function, and selective neuronal death. This review summarizes emerging insights into how proteins with an expanded polyQ tract disrupt distinct cellular functions, and we examine a multitude of discoveries that are inspiring and reshaping novel therapeutic strategies.

A significant proportion of neurodevelopmental disorders (NDDs) are caused by gain-of-function (GOF) or loss-of-function (LOF) of specific genes. Strategies to normalize disease gene expression offer therapeutic potential for these disorders. The success and approval of RNA-based therapeutics for various disorders have led to a surge in RNA-based therapeutic research for NDDs with antisense oligonucleotides leading the field. This review discusses recent advances in therapeutic strategies that target pre-mRNA or mRNA for GOF and LOF NDDs that have promising preclinical evidence. These developments highlight important considerations and exciting future avenues for the development of therapies for NDDs.

Mitochondria and nucleotide metabolism are critical for cellular and developmental homeostasis, yet their potential interdependence and role in neurodevelopmental disease remain unclear. In MECP2 Duplication Syndrome (MDS), we identify a conserved correlation between mitochondrial function and purine metabolism that is disrupted across human, organoid, and mouse models. Multiomics integration reveals Complex III as the focal point of mitochondrial collapse, leading to redox stress, DNA damage, and hyperactivation of the de novo purine biosynthesis via purinosome assembly. The breakdown of mitochondria-purinosome coupling compromises genome stability, impairs radial glia proliferation, and delays neuronal maturation. By linking a defined genetic dosage imbalance to metabolic network failure, our study positions the mitochondria-purinosome coordination as a fundamental control axis for neurodevelopment and a therapeutic entry point across metabolic and neurodevelopmental disorders.

Mutations in the MECP2 gene cause the severe neurological disorder Rett syndrome. A cluster of frameshift-causing C-terminal deletions (CTDs) lead to loss of ~100 amino acids at the C-terminus of the MeCP2 protein, and account for approximately 10% of RTT-causing mutations. The pathogenicity of C-terminal deletions (CTDs) is unexpected, as this C-terminal domain is non-essential in mice. Utilising databases of pathogenic and benign human MECP2 mutations, we find that some individuals with apparently typical CTDs do not exhibit Rett syndrome, confirming that C-terminal truncations are not intrinsically pathogenic. Using human DNA sequence data and mouse models, we demonstrate that pathogenicity results from a drastic reduction in MeCP2 levels and is determined by the presence of the short amino acid motif proline-proline-stop (-PPX) at the C-terminus, which results from a shift to the +2 reading frame. Individuals with CTDs that shift to the +1 frame avoid this motif and do not develop Rett syndrome. Mutating the stop codon of the PPX motif to tryptophan rescues MeCP2 expression and RTT-like phenotypes in a CTD mouse model. Finally, we demonstrate that an adenine base editor can efficiently introduce this tryptophan substitution in cultured cells. Overall, our findings uncover a simple and reliable prognostic distinction between benign and pathogenic CTDs and provide proof-of-concept for an editing strategy that potentially corrects all disease-causing CTD mutations.

Mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene cause Rett syndrome, a severe childhood neurological disorder. MeCP2 is a well-established transcriptional repressor, yet upon its loss, hundreds of genes are dysregulated in both directions. To understand what drives such dysregulation, we deleted Mecp2 in adult mice, circumventing developmental contributions and secondary pathogenesis. We performed time series transcriptional, chromatin, and phenotypic analyses of the hippocampus to determine the immediate consequences of MeCP2 loss and the cascade of pathogenesis. We find that loss of MeCP2 causes immediate and bidirectional progressive dysregulation of the transcriptome. To understand what drives gene downregulation, we profiled genome-wide histone modifications and found that a decrease in histone H3 acetylation (ac) at downregulated genes is among the earliest molecular changes occurring well before any measurable deficiencies in electrophysiology and neurological function. These data reveal a molecular cascade that drives disease independent of any developmental contributions or secondary pathogenesis.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by motor and nonmotor symptoms. Its pathological hallmarks include the accumulation of misfolded alpha-synuclein (α-Syn) in Lewy bodies and Lewy neurites. Phosphorylation of α-Syn is a prominent feature of these inclusions, but its role in disease pathogenesis remains unclear. To identify the role of α-Syn phosphorylation in synucleinopathy, we generated two Snca knock-in (KI) mouse models carrying phosphomimetic mutations at SncaY39 or SncaS129 (SncaY39E or SncaS129D ) which manipulated epitopes phosphorylated in the PD brain. Both SncaY39E and SncaS129D KI mice displayed increased α-Syn phosphorylation, enhanced oligomer formation, and a shift of α-Syn localization from membrane-bound to cytoplasm. However, neurodegeneration in the substantia nigra was not observed up to 24 months of age. These findings demonstrate that mimicking the phosphorylation of Y39 or S129 can induce endogenous α-Syn phosphorylation. Still, a single phosphomimetic mutation alone is insufficient to induce PD-like behavior and pathology in the mouse's lifespan. Overall, our study provides a mouse model for investigating the role of phosphorylation at Y39 and S129 α-Syn epitopes in vivo.