Dataset Information

Transplantation studies reveal internuclear transfer of toxic RNA in engrafted muscles of myotonic dystrophy 1 mice.

ABSTRACT:

Background

Stem cell transplantation represents a potential therapeutic option for muscular dystrophies (MD). However, to date, most reports have utilized mouse models for recessive types of MD. Here we performed studies to determine whether myotonic dystrophy 1 (DM1), an autosomal dominant type of MD, could benefit from cell transplantation.

Methods

We injected human pluripotent stem (PS) cell-derived myogenic progenitors into the muscles of a novel mouse model combining immunodeficiency and skeletal muscle pathology of DM1 and investigated transplanted mice for engraftment as well as for the presence of RNA foci and alternative splicing pattern.

Findings

Engraftment was clearly observed in recipient mice, but unexpectedly, we detected RNA foci in donor-derived engrafted myonuclei. These foci proved to be pathogenic as we observed MBNL1 sequestration and abnormal alternative splicing in donor-derived transcripts.

Interpretation

It has been assumed that toxic CUG repeat-containing RNA forms foci in situ in the nucleus in which it is expressed, but these data suggest that CUG repeat-containing RNA may also exit the nucleus and traffic to other nuclei in the syncytial myofiber, where it can exert pathological effects. FUND: This project was supported by funds from the LaBonte/Shawn family and NIH grants R01 AR055299 and AR071439 (R.C.R.P.). R.M-G. was funded by CONACyT-Mexico (#394378).

SUBMITTER: Mondragon-Gonzalez R

PROVIDER: S-EPMC6796515 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Transplantation studies reveal internuclear transfer of toxic RNA in engrafted muscles of myotonic dystrophy 1 mice.

Mondragon-Gonzalez Ricardo R Azzag Karim K Selvaraj Sridhar S Yamamoto Ami A Perlingeiro Rita C R RCR

EBioMedicine 20190821

<h4>Background</h4>Stem cell transplantation represents a potential therapeutic option for muscular dystrophies (MD). However, to date, most reports have utilized mouse models for recessive types of MD. Here we performed studies to determine whether myotonic dystrophy 1 (DM1), an autosomal dominant type of MD, could benefit from cell transplantation.<h4>Methods</h4>We injected human pluripotent stem (PS) cell-derived myogenic progenitors into the muscles of a novel mouse model combining immunode ...[more]

PMID: 31446083

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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.

Project description:Myotonic dystrophy type 1 (DM1, MIM #160900) is an autosomal dominant disorder, clinically characterized by progressive muscular weakness and multisystem degeneration. The broad phenotypes observed in DM1 patients resemble the appearance of a multisystem accelerated aging process. However, the molecular mechanisms underlying these phenotypes remain largely unknown. Transcriptomic analysis of fibroblasts derived from DM1 patients and healthy individuals revealed a decrease in cell cycle activity, cell division, and DNA damage response in DM1, all of which were required for the accumulation of cellular senescence. Serial passage studies in vitro confirmed the accelerated increase in senescence and the acquisition of a senescence-associated secretory phenotype in DM1 fibroblasts. Moreover, functional studies highlighted the impact of BMI-1/p16INK4a pathway dysregulation in DM1-associated cellular phenotypes. Importantly, treatment with the senolytic compounds, quercetin, dasatinib, or navitoclax, reversed the accelerated aging phenotypes in both DM1 fibroblasts in vitro and in fruit flies in vivo. Our results identified the accumulation of senescence-related processes as a major driver of DM1 pathophysiology and therefore, demonstrated the efficacy of senolytic compounds in a pre-clinical setting. This approach warrants further assessment as a novel therapeutic strategy for DM1. Cell isolation and culture: For the isolation of primary fibroblasts from healthy donors and DM1 patients (age range, 34 to 71), punch skin biopsies were cut into 2–3 mm3 fragments and placed on a surface moistened with modified Eagle’s medium, containing 13% newborn calf serum, 0.4% penicillin/streptomycin (Gibco, Waltham, MA, USA) and 2 mM L-glutamine (Gibco). Flasks were incubated vertically for 3–6 h at 37ºC under 5% CO2 and then returned to the horizontal position. Human fibroblasts were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco) containing 10% fetal bovine serum (Sigma-Aldrich, St Louis, MO, USA), 1% L-glutamine (Gibco), and 1% penicillin–streptomycin (Gibco). Fibroblasts were tested regularly for mycoplasma contamination. Seven cultures from different DM1 patients and five from healthy controls were established. Transcriptomic study and data analysis: RNA was extracted from the fibroblasts from six DM1 patients and four age- and sex-matched controls at an early passage using TRIzol (Life Technologies, Carlsbad, CA, USA). Large-scale gene expression analysis was performed using the Human Clariom D microarray (Affymetrix, Santa Clara, CA, USA). RNA integrity was confirmed using an RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA). Those samples with an RNA integrity value above 9 were used in the analysis. A total of 300 ng of RNA from each sample was used for microarray analysis, following the manufacturer's instructions. Microarray data analysis: The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was performed using the log2 scaling, and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab (MathWorks™). The GO terms were taken from the AMIGO gene ontology database. The significance of the GO terms of the DEGs was addressed calculating the P-values using an enrichment approach based on the hypergeometric distribution. All the sets of GO terms were back-propagated from the final term appearing in the gene annotation to the root term of each GO.

Dataset Information

Transplantation studies reveal internuclear transfer of toxic RNA in engrafted muscles of myotonic dystrophy 1 mice.

Background

Methods

Findings

Interpretation

Publications

Transplantation studies reveal internuclear transfer of toxic RNA in engrafted muscles of myotonic dystrophy 1 mice.

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