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MBNL2 dysfunction in outer radial glial cells is associated with disrupted corticogenesis in congenital myotonic dystrophy [scRNA-Seq]

ABSTRACT: The loss of Muscleblind-like (MBNL) protein function, resulting from its sequestration by toxic CUG-expanded DMPK transcripts, is a central pathological mechanism in myotonic dystrophy type 1 (DM1). However, the role of MBNL in neurodevelopmental disorders associated with severe forms of DM1 remains poorly understood. We performed single-cell RNA sequencing using the 10x Genomics Chromium platform to investigate the cellular composition and transcriptomic alterations in forebrain organoids derived from an unaffected individual, a patient with congenital myotonic dystrophy (CDM) carrying approximately 2,000 CTG repeats or an isogenic control in which the pathogenic repeat expansion was removed. Our analysis revealed that MBNL2, known to play a key role in the pathogenesis of myotonic dystrophy, is expressed in a subset of neural stem cells in mature forebrain organoids. We identified altered expression of genes involved in neuronal migration and axonal projection specifically in upper-layer cortical neurons of CDM organoids when compared with both the isogenic and the unaffected control. These findings suggest a role for MBNL2 during prenatal cortical development and highlight impaired differentiation of late-born neurons as a potential contributor to the neurodevelopmental abnormalities associated with CDM.

ORGANISM(S): Homo sapiens

PROVIDER: GSE315443 | GEO | 2026/03/12

REPOSITORIES: GEO

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Project description:For some neurological disorders, disease is primarily RNA-mediated due to expression of non-coding microsatellite expansion RNAs (RNAexp). Toxicity is thought to result from enhanced binding of proteins to these expansions and depletion from their normal cellular targets. However, experimental evidence for this sequestration model is lacking. Here, we use HITS-CLIP and pre-mRNA processing analysis of human control versus myotonic dystrophy (DM) brains to provide compelling evidence for this RNA toxicity model. MBNL2 binds directly to DM repeat expansions in the brain resulting in depletion from its normal RNA targets with downstream effects on alternative splicing and polyadenylation. Similar RNA processing defects were detected in Mbnl compound knockout mice, highlighted by dysregulation of Mapt splicing and fetal tau isoform expression in adults. These results demonstrate that MBNL proteins are directly sequestered by RNAexp in the DM brain and introduce a powerful experimental tool to evaluate RNA-mediated toxicity in other expansion diseases. HITS-CLIP analysis was performed to identify RNA binding sites of MBNL2 in control, DM type 1 (DM1), and DM type 2 (DM2) autopsy-derived brain (n=3). Two regions of the brain selected for the study included the frontal cortex and hippocampus. Libraries were sequenced and wiggle files were generated for each biological replicate as well as three pooled biological replicates (3BRs) per group (control, DM1, and DM2). In addition, differential CLIP analysis (dCLIP) was performed to normalize binding data between groups and identify statistically significant changes in binding. The dCLIP analysis generated bedgraph files representing normalized binding profiles of MBNL2 in each group (control, DM1, and DM2) for visualization and comparative analysis. PolyA-seq was performed on control, DM1, and DM2 autopsy-derived brain samples (hippocampus and frontal cortex, n=3) as well as wild-type (WT) and Mbnl1; Mbnl2 conditional double knockout (Mbnl1-/-; Mbnl2c/c; Nestin-Cre+/- or DKO) brain (n=3). Libraries were sequenced and the resultant files were processed and aligned to the reference genomes (hg19 and mm10). Further computational processing was performed to remove internal oligo(dT) mis-priming events, identify valid polyA sites, and trim to the exact polyA sites. BedGraph files were generated for each group in human (control, DM1, and DM2) and mouse (WT and Mbnl DKO) for comparative visualization.

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MBNL2 dysfunction in outer radial glial cells is associated with disrupted corticogenesis in congenital myotonic dystrophy [scRNA-Seq]

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