Rett Syndrome Research Articles

GM1 Oligosaccharide Ameliorates Rett Syndrome Phenotypes In Vitro and In Vivo via Trk Receptor Activation

Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily caused by mutations in the methyl-CpG binding protein 2 (MECP2) gene. Despite advancements in research, no cure exists due to an incomplete understanding of the molecular effects of MeCP2 deficiency. Previous studies have identified impaired tropomyosin receptor kinase (Trk) neurotrophin (NTP) signaling and mitochondrial redox imbalances as key drivers of the pathology. Moreover, altered glycosphingolipid metabolism has been reported in RTT. GM1 ganglioside is a known regulator of the nervous system, and growing evidence indicates its importance in maintaining neuronal homeostasis via its oligosaccharide chain, coded as GM1-OS. GM1-OS directly interacts with the Trk receptors on the cell surface, triggering neurotrophic and neuroprotective pathways in neurons. In this study, we demonstrate that GM1-OS ameliorates RTT deficits in the Mecp2-null model. GM1-OS restored synaptogenesis and reduced mitochondrial oxidative stress of Mecp2-knock-out (ko) cortical neurons. When administered in vivo, GM1-OS mitigated RTT-like symptoms. Our findings indicate that GM1-OS effects were mediated by Trk receptor activation on the neuron’s plasma membrane. Overall, our results highlight GM1-OS as a promising candidate for RTT treatment.

Gastrointestinal manifestations of Rett syndrome: An updated analysis using the Gastrointestinal Health Questionnaire.

We conducted a nationwide survey using a validated Gastrointestinal Health Questionnaire (GHQ) for Rett syndrome (RTT) to provide an updated and accurate baseline assessment of the prevalence of common gastrointestinal (GI) issues in RTT, based on parental reporting. Parents and caregivers of females with RTT or normally developing, unaffected, age-matched controls completed the GHQ survey. The prevalence of GI symptoms and personality and mood symptoms due to stomach or intestinal problems, as well as GI medication usage and surgical interventions, were assessed in females with RTT and unaffected controls. The relation between GI symptoms and medication usage, surgical status, age, and genetic mutation were analyzed. Parents of 118 females with RTT and 27 unaffected females completed the GHQ. GI symptoms were common in females with RTT, including constipation (81%), gas and bloating (70%), issues with eating, chewing and swallowing (73%), and irritability because of stomach or intestinal problems (53%). Females with RTT commonly used proton pump inhibitors (52%) and laxatives (64%). Medication usage was associated with significantly higher GHQ symptom scores. Parents of individuals with RTT reported a significantly higher prevalence of GI symptoms affecting their daughters in all symptom categories compared with unaffected females. GI problems are common in RTT and pose a significant medical burden to caregivers. The GHQ is a useful tool to assess GI issues in individuals with RTT. Improved recognition of these issues may allow for improved treatment and enhanced quality of life for girls and women affected by RTT.

Associations between genotype, phenotype and behaviours measured by the Rett syndrome behaviour questionnaire in Rett syndrome

IntroductionRett syndrome (RTT) is a rare neurodevelopmental disorder with developmental impairments, comorbidities, and abnormal behaviours such as hand stereotypies and emotional features. The Rett Syndrome Behaviour Questionnaire (RSBQ) was developed to describe the behavioural and emotional features of RTT. Little is known how RSBQ scores are associated with genetic and clinical characteristics in RTT. This study investigated relationships between genotype, age, walking, hand function, sleep, and RSBQ total and subscale scores in RTT.MethodsThis is a cross-sectional analysis of data collected in the Australian Rett Syndrome Database and the International Rett Syndrome Phenotype Database. Parent caregivers completed the RSBQ and Sleep Disturbance Scale for Children [subscales for disorders of initiating and maintaining sleep (DIMS), disorders of excessive somnolence (DOES)], and provided information on age, variant type, functional abilities (mobility, hand function), seizure frequency and gastrointestinal problems. Associations between the RSBQ scores and the independent variables were modelled using linear regression.ResultsData were available for 365 individuals with RTT [median (range) age 17.8 (2.9–51.9) years, 2 males]. Compared to adults, 2- to 12-year-old children had higher mean Total, Night-time Behaviour and Fear/Anxiety scores. Compared to individuals with a C-terminal deletion, individuals with the p.Arg255* variant had higher mean Total and Night-time Behaviours scores, whereas the p.Arg294* variant had higher mean Mood scores. Individuals with intermediate mobility and hand function abilities had a higher mean Total score. Total RSBQ and subscale scores were similar across categories for seizures, constipation, and reflux, but were higher with abnormal DIMS and abnormal DOES scores.ConclusionExcept for associations with sleep, the RSBQ measures the behavioural phenotype rather than clinical severity in RTT, as traditionally conceptualised in terms of functional abilities and comorbidities. When designing clinical trials, the RSBQ needs to be complemented by other outcome measures to assess specific core functions and associated comorbidities in RTT.

RettDb: the Rett syndrome omics database to navigate the Rett syndrome genomic landscape.

Rett syndrome (RTT) is a neurodevelopmental disorder occurring almost exclusively in females and leading to a variety of impairments and disabilities from mild to severe. In >95% cases, RTT is due to mutations in the X-linked gene MECP2, but the molecular mechanisms determining RTT are unknown at present, and the complexity of the system is challenging. To facilitate and provide guidance to the unraveling of those mechanisms, we developed a database resource for the visualization and analysis of the genomic landscape in the context of wild-type or mutated Mecp2 gene in the mouse model. Our resource allows for the exploration of differential dynamics of gene expression and the prediction of new potential MECP2 target genes to decipher the RTT disorder molecular mechanisms. Database URL: https://biomedinfo.di.unipi.it/rett-database/.

MiR126-mediated impaired vascular integrity in Rett syndrome.

Rett syndrome (RTT) is a neurodevelopmental disorder that is caused by mutations in melty-CpG binding protein 2 (MeCP2). MeCP2 is a non-cell type-specific DNA binding protein, and its mutation influences not only neural cells but also non-neural cells in the brain, including vasculature associated with endothelial cells. Vascular integrity is crucial for maintaining brain homeostasis, and its alteration may be linked to the pathology of neurodegenerative disease, but a non-neurogenic effect, especially the relationship between vascular alternation and Rett syndrome pathogenesis, has not been shown. Here, we recapitulate a microvascular network using Rett syndrome patient-derived induced pluripotent stem (iPS) cells that carry MeCP2[R306C] mutation to investigate early developmental vascular impact. To expedite endothelial cell differentiation, doxycycline (DOX)-inducible ETV2 expression vectors were inserted into the AAVS1 locus of Rett syndrome patient-derived iPS cells and its isogenic control by CRISPR/Cas9. With these endothelial cells, we established a disease microvascular network (Rett-dMVNs) and observed higher permeability in the Rett-dMVNs compared to isogenic controls, indicating altered barrier function by MeCP2 mutation. Furthermore, we unveiled that hyperpermeability is involved in the upregulation of miR126-3p in Rett syndrome patient-derived endothelial cells by microRNA profiling and RNAseq, and rescue of miR126-3p level can recover their phenotype. We discover miR126-3p-mediated vascular impairment in Rett syndrome patients and suggest the potential application of these findings for translational medicine.

Sex-specific single cell-level transcriptomic signatures of Rett syndrome disease progression

Dominant X-linked diseases are uncommon due to female X chromosome inactivation (XCI). While random XCI usually protects females against X-linked mutations, Rett syndrome (RTT) is a female neurodevelopmental disorder caused by heterozygous MECP2 mutation. After 6-18 months of typical neurodevelopment, RTT girls undergo a poorly understood regression. We performed longitudinal snRNA-seq on cerebral cortex in a construct-relevant Mecp2e1 mutant mouse model of RTT, revealing transcriptional effects of cell type, mosaicism, and sex on progressive disease phenotypes. Across cell types, we observed sex differences in the number of differentially expressed genes (DEGs) with 6x more DEGs in mutant females than males. Unlike males, female DEGs emerged prior to symptoms, were enriched for homeostatic gene pathways in distinct cell types over time and correlated with disease phenotypes and human RTT cortical cell transcriptomes. Non-cell-autonomous effects were prominent and dynamic across disease progression of Mecp2e1 mutant females, indicating that wild-type-expressing cells normalize transcriptional homeostasis. These results advance our understanding of RTT progression and treatment.

Site-Blocking Antisense Oligonucleotides as a Mechanism to Fine-Tune MECP2 Expression.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by loss-of-function mutations in the methyl CpG binding protein 2 (MECP2) gene. Despite its severe phenotype, studies in mouse models suggest that restoring MeCP2 levels can reverse RTT symptomology. Nevertheless, traditional gene therapy approaches are hindered by MECP2's narrow therapeutic window, complicating the safe delivery of viral constructs without overshooting the threshold for toxicity. The 3' untranslated region (3'UTR) plays a key role in gene regulation, where factors like miRNAs bind to pre-mRNA and fine-tune expression. Given that each miRNA's contribution is modest, blocking miRNA binding may represent a potential therapeutic strategy for diseases with high dosage sensitivity, like RTT. Here, we present a series of site-blocking antisense oligonucleotides (sbASOs) designed to outcompete repressive miRNA binding at the MECP2 3'UTR. This strategy aims to increase MECP2 levels in patients with missense or late-truncating mutations, where the hypomorphic nature of the protein can be offset by increased abundance. Our results demonstrate that sbASOs can elevate MECP2 levels in a dose-dependent manner in SH-SY5Y and patient fibroblast cell lines, plateauing at levels projected to be safe. Confirming in vivo functionality, sbASO administration in wild-type mice led to significant MeCP2 upregulation and the emergence of phenotypes associated with MeCP2 overexpression. In a T158M neural stem cell model of RTT, sbASO treatment significantly increased MECP2 expression and levels of the downstream effector protein, brain-derived neurotrophic factor (BDNF). These findings highlight the potential of sbASO-based therapies for MECP2-related disorders and advocate for their continued development.

USP15 inhibits hypoxia-induced IL-6 signaling by deubiquitinating and stabilizing MeCP2.

Methyl-CpG binding protein 2 (MeCP2) is an important X-linked DNA methylation reader and a key heterochromatin organizer. The expression level of MeCP2 is crucial, as indicated by the observation that loss-of-function mutations of MECP2 cause Rett syndrome, whereas an extra copy spanning the MECP2 locus results in MECP2 duplication syndrome, both being progressive neurodevelopmental disorders. Our previous study demonstrated that MeCP2 protein expression is rapidly induced by renal ischemia-reperfusion injury (IRI) and protects the kidney from IRI through transcriptionally repressing the interleukin-6 (IL-6)/signal transducer and activator of transcription 3 signaling pathway. However, the mechanisms underlying the upregulation of MeCP2 have remained elusive. Here, by using two hypoxia cell models, hypoxia and reoxygenation and cobalt chloride stimulation, we confirmed that the removal of lysine 48-linked ubiquitination from MeCP2 prevented its proteasome-dependent degradation under hypoxic conditions. Through unbiased screening based on a deubiquitinating enzymes library, we identified ubiquitin-specific protease 15 (USP15) as a stabilizer of MeCP2. Further studies revealed that USP15 could attenuate hypoxia-induced MeCP2 degradation by cleaving lysine 48-linked ubiquitin chains from MeCP2, primarily targeting its C-terminal domain. Consistently, USP15 inhibited hypoxia-induced signal transducer and activator of transcription 3 activation, resulting in reduced transcription of IL-6 downstream genes. In summary, our study reveals an important role for USP15 in the maintenance of MeCP2 stability and the regulation of IL-6 signaling.

7027 A Rare And Multifactorial Etiology Of Severe Parathyroid Hormone Independent Hypercalcemia In An Adult Female With Rett Syndrome

Abstract Disclosure: M.I. Foo: None. M. Thompson: None. Background: Symptomatic hypercalcemia is a rare, but potentially life-threatening disorder. Although dehydration is an unusual cause of severe hypercalcemia, in the appropriate clinical setting dehydration may amplify underlying mild or moderate hypercalcemia to dangerous levels.Case: A 46-year-old non-verbal, non-mobile Caucasian female with Rett Syndrome (RS) with Stage III Chronic Kidney Disease (CKD3) who is dependent on percutaneous endoscopic gastrostomy (PEG) tube for nutrition and known PTH independent hypercalcemia of unknown etiology presented to the hospital with intractable vomiting and constipation, an outpatient serum calcium level of 16.0 mg/dL, and serum creatinine 4.03 mg/dL (baseline 1.2 mg/dL). Initial laboratory assessment revealed elevated corrected serum calcium levels (15.7 mg/dL), with appropriately suppressed PTH levels (12.5 pg/mL) and hypernatremia (146 mmol/L). Thyroid function tests, both Vitamin D 1,25(OH)2D and D 25(OH)D levels and PTH-RP were normal. Computed tomography of the abdomen and pelvis revealed bilateral nephrolithiasis and moderate stool burden, but no evidence of malignancy or granulomatous disease. Skeletal scintigraphy was also without evidence of osseous metastatic disease or Paget’s disease of bone. An in-depth interview with the patient’s caregiver uncovered that the patient had a relative intolerance to enteral hydration and had been receiving minimal water flushes prior to admission, contributing to severe dehydration. Hypercalcemia was ultimately ascribed to prolonged immobilization due to RS compounded by inadequate fluid intake and auto-diuresis secondary to hypercalciuria in the setting of CKD (prior baseline eGFR 45 mL/min/1.73m2).Outcome: The patient's hypercalcemia improved after a multidisciplinary and multi-directed approach involving intravenous and enteral fluid resuscitation, calcitonin, furosemide, and ultimately zoledronic acid following improvement in creatinine with fluid resuscitation. The dietary service employed a strategy of 24-hour enteral feeding, increasing both the frequency and amount of enteral water, which the patient tolerated well, and provided her with a new pump for home use. Education on monitoring tube feed tolerance and providing adequate enteral hydration was provided to both the caregiver and group home. On discharge, eGFR stabilized at 33 mL/min/1.73m2 and corrected calcium stabilized at 10.0–10.4 mg/dl. She was provided with an endocrinology follow-up to continue bisphosphonate therapy.Discussion: Hypercalcemia of immobility should remain a differential diagnosis in severe hypercalcemia, especially PEG tube dependent patients with underlying kidney disease. This case highlights the importance of thorough history taking and a multidisciplinary strategy to guarantee optimal care and mitigate potential adverse outcomes in medically complex patients. Presentation: 6/2/2024

8428 Rare Variants in the MECP2 Gene in Two Boys with Central Precocious Puberty

Abstract Disclosure: A.P. Canton: None. J.B. Mebarak: None. M. Magnuson: None. S. Roberts: None. N. Mauras: None. M. Benson: None. S. Witchel: None. R.S. Carroll: None. A. Latronico: None. U.B. Kaiser: None. A. Abreu: None. Introduction: Central precocious puberty (CPP) occurs more frequently in girls and is usually labelled as idiopathic; however, in boys organic causes are more frequent. Identification of imprinted genes causing CPP have revealed epigenetic mechanisms underlying puberty. MECP2, an X-linked gene, encodes a methylated DNA reader protein with a role in gene transcription. MECP2 loss-of-function mutations usually cause Rett syndrome, a severe neurodevelopmental disorder that may be associated with early pubertal development. Recently, rare variants in MECP2 have been recognized in girls with sporadic CPP with or without mild neurodevelopmental disorders. In our cohort of 78 patients with idiopathic CPP, no MECP2 mutations were identified in 73 girls. In this study, five boys with CPP were evaluated for potential MECP2 sequence variants. Methods and Results: Five boys with CPP were screened for MECP2 sequence variants using Sanger sequencing. At the time of CPP diagnosis, they had median (interquartile range) chronological age 9.2 yr (4.3), bone age advancement 2.1 yr (3.5), height SDS 2.3 (1.5), basal LH levels 1.6 IU/L (0.9), and testosterone levels 370 ng/dL (570). Organic causes of CPP were excluded. No MKRN3 or DLK1 mutations were identified. Familial segregation analysis was performed when appropriate. We identified a hemizygous MECP2 variant in a boy (Patient 1) who presented at age 2.7 yr with sporadic CPP. In addition, he had speech delay and behavioral changes, defined as autism spectrum disorder by neuropsychological assessment. He harbored an extremely rare (gnomAD AF=0.000004956) missense variant (p.Val312Ile) in exon 3 of MECP2, encoding the transcriptional repression domain, critical for protein function. The p.Val312Ile mutation was classified as likely pathogenic (ACMG criteria) with a potential association with the phenotype. Another hemizygous MECP2 variant was identified in a boy (Patient 2) who presented at age 8.0 yr with sporadic CPP; he had no neurodevelopmental conditions. He harbored a rare (gnomAD AF=0.00008) missense variant (p.Arg366Cys) in exon 3, encoding the C-terminal domain, classified as a variant of uncertain significance (ACMG criteria). Familial segregation analysis revealed that both boys inherited the MECP2 variants from their unaffected mothers, consistent with a pattern of clinical variability described in women with defects in X-linked genes.Conclusions: We identified rare MECP2 variants in two boys with sporadic CPP without classic features of Rett syndrome, expanding the phenotype of patients with MECP2 mutations, as described in girls. The MECP2 p.Val312Ile variant was likely pathogenic, whereas the functional significance of the p.Arg366Cys variant in still indeterminate. These findings provide additional evidence for a role of MECP2, an epigenetic factor, in the hypothalamic control of pubertal timing. Presentation: 6/1/2024

Rett Syndrome and the Broader Implications of the Use of Eponyms in Medicine.

Rett Syndrome and the Broader Implications of the Use of Eponyms in Medicine.

Trofinetide treatment for Rett syndrome: Lessons to learn

The US FDA approval of trofinetide as the first pharmacological treatment to improve Rett syndrome's symptomatology marks a significant milestone with broad implications for various disorders. The LILAC trials demonstrate long-term safety and efficacy of trofinetide.1,2 While further research is needed to fully resolve the condition, insights from trofinetide trials can inform strategies for future treatments and trials.

Boosting Serotonin Synthesis Is Not Sufficient to Improve Motor Coordination of Mecp2 Heterozygous Mouse Model of Rett Syndrome

Motor deficit is a core symptom of Rett syndrome, a rare neurological disease caused in most cases by mutations of the methyl-CpG-binding protein2 (MECP2) gene. Serotonin reuptake inhibitors improve motor coordination in Mecp2 heterozygous (Het) mice and serotonin depletion prevented this effect. Here, we assess alterations in indole levels in various brain regions and whether boosting brain serotonin synthesis with the serotonin precursors tryptophan, 5-hydroxytryptophan and α-lactalbumin rescued motor coordination deficit of Mecp2 Het mice. Motor coordination was assessed in the accelerated rotarod during and after systemic administration of serotonin precursors for 2–3 weeks. Since no data are available, the effect of α-lactalbumin on tryptophan, serotonin and 5-hydroxyindoleacetic acid levels was evaluated in various brain regions in order to identify the dose of ALAC to evaluate on motor coordination. As compared to WT, Mecp2 Het mice show reduced levels of serotonin in the whole brain, hippocampus, brainstem and cerebral cortex, but not the striatum. Reduced levels of 5-hydroxyindoleacetic acid were observed in the hippocampus and brainstem. Doses of serotonin precursors increasing brain tryptophan and/or serotonin production and metabolism had no effect on motor coordination. The results indicate that boosting serotonin synthesis is not sufficient to improve motor coordination of Mecp2 Het mice.

Animal Models of Autistic-like Behavior in Rodents: A Scoping Review and Call for a Comprehensive Scoring System

Appropriate animal models of human diseases are a cornerstone in the advancement of science and medicine. To create animal models of neuropsychiatric and neurobehavioral diseases such as autism spectrum disorder (ASD) necessitates the development of sufficient neurobehavioral measuring tools to translate human behavior to expected measurable behavioral features in animals. If possible, the severity of the symptoms should also be assessed. Indeed, at least in rodents, adequate neurobehavioral and neurological tests have been developed. Since ASD is characterized by a number of specific behavioral trends with significant severity, animal models of autistic-like behavior have to demonstrate the specific characteristic features, namely impaired social interactions, communication deficits, and restricted, repetitive behavioral patterns, with association to several additional impairments such as somatosensory, motor, and memory impairments. Thus, an appropriate model must show behavioral impairment of a minimal number of neurobehavioral characteristics using an adequate number of behavioral tests. The proper animal models enable the study of ASD-like-behavior from the etiologic, pathogenetic, and therapeutic aspects. From the etiologic aspects, models have been developed by the use of immunogenic substances like polyinosinic-polycytidylic acid (PolyIC), lipopolysaccharide (LPS), and propionic acid, or other well-documented immunogens or pathogens, like Mycobacterium tuberculosis. Another approach is the use of chemicals like valproic acid, polychlorinated biphenyls (PCBs), organophosphate pesticides like chlorpyrifos (CPF), and others. These substances were administered either prenatally, generally after the period of major organogenesis, or, especially in rodents, during early postnatal life. In addition, using modern genetic manipulation methods, genetic models have been created of almost all human genetic diseases that are manifested by autistic-like behavior (i.e., fragile X, Rett syndrome, SHANK gene mutation, neuroligin genes, and others). Ideally, we should not only evaluate the different behavioral modes affected by the ASD-like behavior, but also assess the severity of the behavioral deviations by an appropriate scoring system, as applied to humans. We therefore propose a scoring system for improved assessment of ASD-like behavior in animal models.

The Multifaceted Role of L-Type Amino Acid Transporter 1 at the Blood-Brain Barrier: Structural Implications and Therapeutic Potential.

L-type amino acid transporter 1 (LAT1) is integral to the transport of large neutral amino acids across the blood-brain barrier (BBB), playing a crucial role in brain homeostasis and the delivery of therapeutic agents. This review explores the multifaceted role of LAT1 in neurological disorders, including its structural and functional aspects at the BBB. Studies using advanced BBB models, such as induced pluripotent stem cell (iPSC)-derived systems and quantitative proteomic analyses, have demonstrated LAT1's significant impact on drug permeability and transport efficiency. In Alzheimer's disease, LAT1-mediated delivery of anti-inflammatory and neuroprotective agents shows promise in overcoming BBB limitations. In Parkinson's disease, LAT1's role in transporting L-DOPA and other therapeutic agents highlights its potential in enhancing treatment efficacy. In phenylketonuria, studies have revealed polymorphisms and genetic variations of LAT1, which could be correlated to disease severity. Prodrugs of valproic acid, pregabalin, and gabapentin help use LAT1-mediated transport to increase the therapeutic activity and bioavailability of the prodrug in the brain. LAT1 has also been studied in neurodevelopment disorders like autism spectrum disorders and Rett syndrome, along with neuropsychiatric implications in depression. Its implications in neuro-oncology, especially in transporting therapeutic agents into cancer cells, show immense future potential. Phenotypes of LAT1 have also shown variations in the general population affecting their ability to respond to painkillers and anti-inflammatory drugs. Furthermore, LAT1-targeted approaches, such as functionalized nanoparticles and prodrugs, show promise in overcoming chemoresistance and enhancing drug delivery to the brain. The ongoing exploration of LAT1's structural characteristics and therapeutic applications reiterates its critical role in advancing treatments for neurological disorders.

The Microbiota-Gut-Brain Axis and Neurological Disorders: A Comprehensive Review.

Microbes have inhabited the earth for hundreds of millions of years longer than humans. The microbiota-gut-brain axis (MGBA) represents a bidirectional communication pathway. These communications occur between the central nervous system (CNS), the enteric nervous system (ENS), and the emotional and cognitive centres of the brain. The field of research on the gut-brain axis has grown significantly during the past two decades. Signalling occurs between the gut microbiota and the brain through the neural, endocrine, immune, and humoral pathways. A substantial body of evidence indicates that the MGBA plays a pivotal role in various neurological diseases. These include Alzheimer's disease (AD), autism spectrum disorder (ASD), Rett syndrome, attention deficit hyperactivity disorder (ADHD), non-Alzheimer's neurodegeneration and dementias, fronto-temporal lobe dementia (FTLD), Wilson-Konovalov disease (WD), multisystem atrophy (MSA), Huntington's chorea (HC), Parkinson's disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), temporal lobe epilepsy (TLE), depression, and schizophrenia (SCZ). Furthermore, the bidirectional correlation between therapeutics and the gut-brain axis will be discussed. Conversely, the mood of delivery, exercise, psychotropic agents, stress, and neurologic drugs can influence the MGBA. By understanding the MGBA, it may be possible to facilitate research into microbial-based interventions and therapeutic strategies for neurological diseases.

Comprehensive High-Depth Proteomic Analysis of Plasma Extracellular Vesicles Containing Preparations in Rett Syndrome.

Backgroud: Rett syndrome is a neurodevelopmental disorder that affects 1 in 10,000 females. Various treatments have been explored; however, no effective treatments have been reported to date, except for trofinetide, a synthetic analog of glycine-proline-glutamic acid, which was approved by the FDA in 2023. Serological biomarkers that correlate with the disease status of RTT are needed to promote early diagnosis and to develop novel agents. Methods: In this study, we performed a high-depth proteomic analysis of extracellular vesicles containing preparations extracted from patient plasma samples to identify novel biomarkers. Results: We identified 33 upregulated and 17 downregulated candidate proteins among a total of 4273 proteins in RTT compared to the healthy controls. Among these, UBE3B was predominantly increased in patients with Rett syndrome and exhibited a strong correlation with the clinical severity score, indicating the severity of the disease. Conclusions: We demonstrated that the proteomics of high-depth extracellular vesicles containing preparations in rare diseases could be valuable in identifying new disease biomarkers and understanding their pathophysiology.

Prediction and design of transcriptional repressor domains with large-scale mutational scans and deep learning.

Regulatory proteins have evolved diverse repressor domains (RDs) to enable precise context-specific repression of transcription. However, our understanding of how sequence variation impacts the functional activity of RDs is limited. To address this gap, we generated a high-throughput mutational scanning dataset measuring the repressor activity of 115,000 variant sequences spanning more than 50 RDs in human cells. We identified thousands of clinical variants with loss or gain of repressor function, including TWIST1 HLH variants associated with Saethre-Chotzen syndrome and MECP2 domain variants associated with Rett syndrome. We also leveraged these data to annotate short linear interacting motifs (SLiMs) that are critical for repression in disordered RDs. Then, we designed a deep learning model called TENet ( T ranscriptional E ffector Net work) that integrates sequence, structure and biochemical representations of sequence variants to accurately predict repressor activity. We systematically tested generalization within and across domains with varying homology using the mutational scanning dataset. Finally, we employed TENet within a directed evolution sequence editing framework to tune the activity of both structured and disordered RDs and experimentally test thousands of designs. Our work highlights critical considerations for future dataset design and model training strategies to improve functional variant prioritization and precision design of synthetic regulatory proteins.

Involvement of extracellular vesicle microRNA clusters in developing healthy and Rett syndrome brain organoids

Rett syndrome (RTT) is a neurodevelopmental disorder caused by de novo mutations in the MECP2 gene. Although miRNAs in extracellular vesicles (EVs) have been suggested to play an essential role in several neurological conditions, no prior study has utilized brain organoids to profile EV-derived miRNAs during normal and RTT-affected neuronal development. Here we report the spatiotemporal expression pattern of EV-derived miRNAs in region-specific forebrain organoids generated from female hiPSCs with a MeCP2:R255X mutation and the corresponding isogenic control. EV miRNA and protein expression profiles were characterized at day 0, day 13, day 40, and day 75. Several members of the hsa-miR-302/367 cluster were identified as having a time-dependent expression profile with RTT-specific alterations at the latest developmental stage. Moreover, the miRNA species of the chromosome 14 miRNA cluster (C14MC) exhibited strong upregulation in RTT forebrain organoids irrespective of their spatiotemporal location. Together, our results suggest essential roles of the C14MC and hsa-miR-302/367 clusters in EVs during normal and RTT-associated neurodevelopment, displaying promising prospects as biomarkers for monitoring RTT progression.Graphical

Pharmacological inhibition of the CB1 cannabinoid receptor restores abnormal brain mitochondrial CB1 receptor expression and rescues bioenergetic and cognitive defects in a female mouse model of Rett syndrome

BackgroundDefective mitochondria and aberrant brain mitochondrial bioenergetics are consistent features in syndromic intellectual disability disorders, such as Rett syndrome (RTT), a rare neurologic disorder that severely affects mainly females carrying mutations in the X-linked MECP2 gene. A pool of CB1 cannabinoid receptors (CB1R), the primary receptor subtype of the endocannabinoid system in the brain, is located on brain mitochondrial membranes (mtCB1R), where it can locally regulate energy production, synaptic transmission and memory abilities through the inhibition of the intra-mitochondrial protein kinase A (mtPKA). In the present study, we asked whether an overactive mtCB1R-mtPKA signaling might underlie the brain mitochondrial alterations in RTT and whether its modulation by systemic administration of the CB1R inverse agonist rimonabant might improve bioenergetics and cognitive defects in mice modeling RTT.MethodsRimonabant (0.3 mg/kg/day, intraperitoneal injections) was administered daily to symptomatic female mice carrying a truncating mutation of the Mecp2 gene and its effects on brain mitochondria functionality, systemic oxidative status, and memory function were assessed.ResultsmtCB1R is overexpressed in the RTT mouse brain. Subchronic treatment with rimonabant normalizes mtCB1R expression in RTT mouse brains, boosts mtPKA signaling, and restores the defective brain mitochondrial bioenergetics, abnormal peripheral redox homeostasis, and impaired cognitive abilities in RTT mice.LimitationsThe lack of selectivity of the rimonabant treatment towards mtCB1R does not allow us to exclude that the beneficial effects exerted by the treatment in the RTT mouse model may be ascribed more broadly to the modulation of CB1R activity and distribution among intracellular compartments, rather than to a selective effect on mtCB1R-mediated signaling. The low sample size of few experiments is a further limitation that has been addressed replicating the main findings under different experimental conditions.ConclusionsThe present data identify mtCB1R overexpression as a novel molecular alteration in the RTT mouse brain that may underlie defective brain mitochondrial bioenergetics and cognitive dysfunction.