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.

Project description:Myotonic dystrophy type 1 is a dominantly inherited multisystemic disease caused by CTG tandem repeat expansions in the DMPK 3' untranslated region. These expanded repeats are transcribed and produce toxic CUG RNAs that sequester and inhibit activities of the MBNL family of developmental RNA processing factors. Although myotonic dystrophy is classified as a muscular dystrophy, the brain is also severely affected by an unusual cohort of symptoms, including hypersomnia, executive dysfunction, as well as early onsets of tau/MAPT pathology and cerebral atrophy. To address the molecular and cellular events that lead to these pathological outcomes, we recently generated a mouse Dmpk CTG expansion knockin model and identified choroid plexus epithelial cells as particularly affected by the expression of toxic CUG expansion RNAs. To determine if toxic CUG RNAs perturb choroid plexus functions, alternative splicing analysis was performed on lateral and hindbrain choroid plexi from Dmpk CTG knockin mice. Choroid plexus transcriptome-wide changes were evaluated in Mbnl2 knockout mice, a developmental-onset model of myotonic dystrophy brain dysfunction. To determine if transcriptome changes also occurred in the human disease, we obtained post-mortem choroid plexus for RNA-seq from donors without neurologically unaffected (two females, three males; ages 50-70) and myotonic dystrophy type 1 donors (one female, three males; ages 50-70). To test that choroid plexus transcriptome alterations resulted in altered CSF composition, we obtained CSF via lumbar puncture from patients with myotonic dystrophy type 1 (five females, five males; ages 35-55) and non-myotonic dystrophy patients (three females, four males; ages 26-51) and Western blot and osmolarity analyses were used to test CSF alterations predicted by choroid plexus transcriptome analysis. We determined that CUG RNA induced toxicity was more robust in the lateral choroid plexus of Dmpk CTG knockin mice due to comparatively higher Dmpk and lower Mbnl RNA levels. Impaired transitions to adult splicing patterns during choroid plexus development were identified in Mbnl2 knockout mice, including mis-splicing previously found in Dmpk CTG knockin mice. Whole transcriptome analysis of myotonic dystrophy type 1 choroid plexus revealed disease-associated RNA expression and mis-splicing events. Based on these RNA changes, predicted alterations in ion homeostasis, secretory output, and CSF composition were confirmed by analysis of myotonic dystrophy type 1 CSF. Our results implicate choroid plexus spliceopathy and concomitant alterations in CSF homeostasis as an unappreciated contributor to myotonic dystrophy type 1 CNS pathogenesis.

Dataset Information

Whole transcriptome analysis and functional studies reveal that senescence plays a pivotal role in Myotonic Dystrophy type 1

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets