Mice lacking MBNL1 and MBNL2 exhibit sudden cardiac death and molecular signatures recapitulating myotonic dystrophy.

Myotonic dystrophy (DM) is caused by expansions of C(C)TG repeats in the non-coding regions of the DMPK and CNBP genes, and DM patients often suffer from sudden cardiac death due to lethal conduction block or arrhythmia. Specific molecular changes that underlie DM cardiac pathology have been linked to repeat-associated depletion of Muscleblind-like (MBNL) 1 and 2 proteins and upregulation of CUGBP, Elav-like family member 1 (CELF1). Hypothesis solely targeting MBNL1 or CELF1 pathways that could address all the consequences of repeat expansion in heart remained inconclusive, particularly when the direct cause of mortality and results of transcriptome analyses remained undetermined in Mbnl compound knockout (KO) mice with cardiac phenotypes. Here, we develop Myh6-Cre double KO (DKO) (Mbnl1-/-; Mbnl2cond/cond; Myh6-Cre+/-) mice to eliminate Mbnl1/2 in cardiomyocytes and observe spontaneous lethal cardiac events under no anesthesia. RNA sequencing recapitulates DM heart spliceopathy and shows gene expression changes that were previously undescribed in DM heart studies. Notably, immunoblotting reveals a nearly 6-fold increase of Calsequestrin 1 and 50% reduction of epidermal growth factor proteins. Our findings demonstrate that complete ablation of MBNL1/2 in cardiomyocytes is essential for generating sudden death due to lethal cardiac rhythms and reveal potential mechanisms for DM heart pathogenesis.

Muscleblind-like 2 knockout shifts adducin 1 isoform expression and alters dendritic spine dynamics of cortical neurons during brain development.

AIMS: Muscleblind-like 2 (MBNL2) plays a crucial role in regulating alternative splicing during development and mouse loss of MBNL2 recapitulates brain phenotypes in myotonic dystrophy (DM). However, the mechanisms underlying DM neuropathogenesis during brain development remain unclear. In this study, we aim to investigate the impact of MBNL2 elimination on neuronal development by Mbnl2 conditional knockout (CKO) mouse models. METHODS: To create Mbnl2 knockout neurons, cDNA encoding Cre-recombinase was delivered into neural progenitors of Mbnl2flox/flox mouse brains by in utero electroporation. The morphologies and dynamics of dendritic spines were monitored by confocal and two-photon microscopy in brain slices and live animals from the neonatal period into adulthood. To investigate the underlying molecular mechanism, we further detected the changes in the splicing and molecular interactions of proteins associated with spinogenesis. RESULTS: We found that Mbnl2 knockout in cortical neurons decreased dendritic spine density and dynamics in adolescent mice. Mbnl2 ablation caused the adducin 1 (ADD1) isoform to switch from adult to fetal with a frameshift, and the truncated ADD1 failed to interact with alpha-II spectrin (SPTAN1), a critical protein for spinogenesis. In addition, expression of ADD1 adult isoform compensated for the reduced dendritic spine density in cortical neurons deprived of MBNL2. CONCLUSION: MBNL2 plays a critical role in maintaining the dynamics and homeostasis of dendritic spines in the developing brain. Mis-splicing of downstream ADD1 may account for the alterations and contribute to the DM brain pathogenesis.

Deprivation of Muscleblind-Like Proteins Causes Deficits in Cortical Neuron Distribution and Morphological Changes in Dendritic Spines and Postsynaptic Densities.

Myotonic dystrophy (Dystrophia Myotonica; DM) is the most common adult-onset muscular dystrophy and its brain symptoms seriously affect patients' quality of life. It is caused by extended (CTG)n expansions at 3'-UTR of DMPK gene (DM type 1, DM1) or (CCTG)n repeats in the intron 1 of CNBP gene (DM type 2, DM2) and the sequestration of Muscleblind-like (MBNL) family proteins by transcribed (CUG)n RNA hairpin is the main pathogenic mechanism for DM. The MBNL proteins are splicing factors regulating posttranscriptional RNA during development. Previously, Mbnl knockout (KO) mouse lines showed molecular and phenotypic evidence that recapitulate DM brains, however, detailed morphological study has not yet been accomplished. In our studies, control (Mbnl1 +/+; Mbnl2 cond/cond; Nestin-Cre -/-), Mbnl2 conditional KO (2KO, Mbnl1 +/+; Mbnl2 cond/cond; Nestin-Cre +/-) and Mbnl1/2 double KO (DKO, Mbnl1 ΔE3/ΔE3; Mbnl2 cond/cond; Nestin-Cre +/-) mice were generated by crossing three individual lines. Immunohistochemistry for evaluating density and distribution of cortical neurons; Golgi staining for depicting the dendrites/dendritic spines; and electron microscopy for analyzing postsynaptic ultrastructure were performed. We found distributional defects in cortical neurons, reduction in dendritic complexity, immature dendritic spines and alterations of postsynaptic densities (PSDs) in the mutants. In conclusion, loss of function of Mbnl1/2 caused fundamental defects affecting neuronal distribution, dendritic morphology and postsynaptic architectures that are reminiscent of predominantly immature and fetal phenotypes in DM patients.