Jul 2026· Brain : a journal of neurology· 0 citations
Medicine
TL;DR
A model in which WWOX mutations impair neurogenesis via RG through cell-type specific dysregulation of the MYC and Wnt signaling pathways is proposed, highlighting potential therapeutic strategies for WWOX-related disorders and open avenues for interventions targeting these key molecular pathways.
Abstract
WOREE and SCAR12 syndromes are rare neurodevelopmental disorders caused by WWOX mutations, severely impairing brain development. The pleiotropic nature of WWOX complicates identifying specific mechanisms, thus, the specific molecular pathways affected by WWOX deficiency and how they contribute to disease pathogenesis remain largely unknown. Using neural organoids derived from a broad iPSC cohort, including wildtype iPSCs, CRISPR-edited isogenic WWOX-knockout lines, and patient-derived lines, we applied molecular profiling and single-cell transcriptomics to map the early neurodevelopmental pathways disrupted upon loss of WWOX. We identified radial glial cells (RGs) as preferentially affected, with disrupted cell cycle dynamics leading to an accumulation of cells in the G2/M and S phases, overexpression of the proto-oncogene MYC, and concomitant reduction in neuronal generation. Patient-derived organoids exhibited milder phenotypes compared to knockout organoids, showing functional neuronal impairments like hyperexcitability and delayed differentiation rather than RG dysfunction. Remarkably, gene therapy restored neuronal function, normalizing hyperexcitability and promoting maturation, without disturbing RG populations. We propose a model in which WWOX mutations impair neurogenesis via RG through cell-type specific dysregulation of the MYC and Wnt signaling pathways. These insights highlight potential therapeutic strategies for WWOX-related disorders and open avenues for interventions targeting these key molecular pathways.
Myotonic dystrophy type 1 (DM1) is caused by an expansion of a CTG repeat in the 3′ untranslated region of the DMPK gene, leading to accumulation of toxic CUG-repeat RNAs, sequestration of MBNL proteins and widespread splicing dysregulation. Congenital DM1 (CDM), the most severe form of the disease, is associated with profound muscular and neurodevelopmental defects, yet the mechanisms underlying early human brain involvement remain poorly understood. Here, we generated cortical organoids from patient-derived pluripotent stem cells carrying >1000 CTG repeats, an expansion typically associated with CDM, to model early human neurodevelopment. DM1 molecular and cellular hallmarks were detected at early developmental stages, including nuclear DMPK RNA foci in neural progenitor cells and reduced proliferative capacity. As organoids matured, CDM cultures displayed altered cortical composition, with reduced CTIP2⁺ and SATB2⁺ neuronal populations and increased NFIA⁺/GFAP⁺ glial cells. In parallel, 120-day-old organoids recapitulated splicing abnormalities previously identified in DM1 patient brain tissue. To assess the contribution of MBNL dysfunction, we analyzed cortical organoids derived from MBNL2 and MBNL1/2/3 knockout induced pluripotent stem cells, which reproduced key neurodevelopmental phenotypes observed in CDM organoids, supporting a central role for MBNL loss of function in impaired corticogenesis. Finally, we evaluated the translational relevance of this model using tideglusib and erythromycin, two compounds currently under clinical evaluation in DM1 patients. Both treatments reduced DMPK RNA foci and restored proliferation defects in SOX2⁺ neural progenitors. Together, these findings establish cortical organoids as a robust human model of CDM-associated neurodevelopmental defects, uncover MBNL-dependent mechanisms underlying early corticogenesis impairment and demonstrate the utility of this platform for translational therapeutic discovery in DM1.
Azania Abatan, Jérôme Polentes, M. Bouquier et al.· bioRxiv· 0 citations
Human brain development depends on tightly coordinated gene-regulatory programs and the emergence of complex tissue architecture, making large scale functional interrogation difficult using conventional screen models. To overcome this challenge, we used a pooled CRISPR screening approach. Guided by neuro-specific whole-genome screens in Drosophila, we tested 129 poorly characterised human orthologs and found 8 that modify cerebral organoid development. Candidates were validated using individual CRISPR knockouts and mosaic competition assays. Among these candidates we describe FBXW11, a substrate-recognition component of the SCF E3 ubiquitin ligase complex, as a potent negative regulator of cerebral organoid expansion. FBXW11 loss increases radial glial abundance, expands ventricular-like domains, and impairs neuronal maturation. Mechanistically, FBXW11 associates with β-catenin and alters WNT signalling. FBXW11 mutations cause the autosomal-dominant Mendelian syndrome Neurodevelopmental, Jaw, Eye and Digital syndrome (NEDJED), and we found that disease-associated variants mapped preferentially to WD40 substrate-binding repeats and β-catenin contact regions, linking impaired substrate recognition to neurodevelopmental disease. Together, these findings identify FBXW11 as a conserved negative regulator of β-catenin-dependent radial glial expansion and neuronal maturation during human cerebral brain development.
Cesar L. Moreno, Helen E. King, Sophia Trabish et al.· bioRxiv· 0 citations
BACKGROUND
Pontocerebellar hypoplasia type 6 (PCH6) is caused by biallelic pathogenic variants in RARS2, encoding mitochondrial arginyl-tRNA synthetase. Although mitochondrial dysfunction is a recognised feature, how RARS2 deficiency disrupts neural lineage development remains unclear.
METHODS
We generated rars2-deficient zebrafish using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system and performed single-cell RNA sequencing (scRNA-seq) at 48 hours postfertilisation, complemented by immunofluorescence, in situ hybridisation, behavioural assays and ultrastructural analyses. Neural lineage composition, developmental trajectories, intercellular communication and transcriptional programmes were systematically examined.
RESULTS
rars2 -/- zebrafish displayed impaired survival, locomotor deficits, early mitochondrial ultrastructural damage and marked disruption of neurogenesis. scRNA-seq revealed reduced neuronal populations and expansion of neural progenitor and glial-like cells. Key neurogenic regulators (neurod4, her6 and pou3f1) were downregulated, whereas glial and stress-associated markers (hmgb1a, fabp7a and foxp1b) were upregulated. Developmental pathways including Notch and non-canonical Wnt were attenuated while extracellular matrix (ECM), adhesion and inflammatory programmes were activated. Additional trajectory-based analyses supported dysregulated lineage progression characterised by glial programme activation and impaired maintenance of neurogenic differentiation.
CONCLUSION
RARS2 deficiency disrupts mitochondrial integrity and reprograms neural lineage development through coordinated suppression of neurogenic transcriptional networks and activation of glial/ECM programmes. These findings provide mechanistic insight into loss-of-function RARS2 deficiency and highlight candidate molecular pathways for future therapeutic investigation.
Xing Wei, Jing Wang, Yanyun Wang et al.· Journal of Medical Genetics· 0 citations
This study provides substantial evidence for the vital role of trip12 in the early stages of development, as homozygous individuals exhibited early mortality by Day 23 post-fertilization, while a substantial mortality rate was observed by Day 35 in ‘heterozygous’ mutants.
Maider Roibás-Santos, P. Suarez‐Bregua, J. Rotllant et al.· Brain Communications· 0 citations
Methyltransferase-like 5 (METTL5) catalyzes N6-methyladenosine (m6A) modification on 18S rRNA. In humans, loss-of-function mutations in METTL5 cause severe microcephaly and intellectual disability, whereas Mettl5 knockout (KO) animal models display inconsistent and milder phenotypes. To better model human disease, we generated METTL5-KO human-induced pluripotent stem cell (hiPSC)-derived cortical organoids, which exhibit impaired neural progenitor cell (NPC) proliferation and differentiation, leading to reduced ventricle-like structures and significant reductions in cortical organoid diameter. Mechanistically, Ribo-seq analysis revealed broad translational changes in METTL5-KO NPCs consistent with cellular stress responses rather than transcript-specific translational changes. Single-cell RNA-seq identified downregulation of coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a mitochondrial regulator of oxidative metabolism. Overexpression of CHCHD2 in METTL5-KO NPCs rescued proliferation and partially rescued oxidative metabolism in NPCs and ventricle formation in organoids. This highlights a previously uncharacterized connection between CHCHD2, oxidative metabolism, and METTL5-mediated regulation of human neurogenesis.
Elena M Turkalj, Gugene Kang, I. Liu et al.· Stem Cell Reports· 0 citations
Fragile X syndrome (FXS), the leading genetic cause of intellectual disability, arises from FMR1 gene silencing and the subsequent loss of the RNA-binding protein FMRP. N6-methyladenosine (m6A) is a prevalent mRNA modification essential for post-transcriptional regulation. FMRP binds and regulates the stability of m6A-containing transcripts. However, how FMRP deficiency impacts transcriptome-wide m6A modifications in FXS remains unknown. To address this, we generated cortical neurons from induced pluripotent stem cells (iPSCs) derived from healthy individuals and FXS patients. Electrophysiology recordings revealed synaptic and neuronal network defects in FXS iPSC-derived neurons. Transcriptome-wide analysis revealed striking m6A hypermethylation predominantly affecting synapse-associated transcripts. Mechanistically, we demonstrated that FMRP deficiency drives the aberrant translational upregulation of core m6A writers, a causal relationship definitively validated using CGG-corrected isogenic control lines. Targeted genetic knockdown of the m6A writer METTL3 successfully rescued synaptic phenotypes in FXS neurons, whereas its overexpression in control neurons phenocopied these synaptic defects, confirming the causal role of m6A dysregulation in FXS pathology. Notably, pharmacological intervention with the METTL3 inhibitor STM-2457 normalized methylation on synapse-associated transcripts and restored synaptic transmission in FXS neurons. Together, our findings uncover an FMRP-dependent epitranscriptomic mechanism contributing to FXS pathogenesis and suggest a promising avenue for m6A-targeted therapies.
Lu Lu, Avijite Kumer Sarkar, Lan Dao et al.· Molecular Psychiatry· 0 citations