Studies on myotonic dystrophy type 1 (DM1) have led to the RNAmediated disease model for hereditary disorders caused by noncoding microsatellite expansions. This model proposes that DM1 disease manifestations are caused by a reversion to fetal RNA processing patterns in adult tissues due to the expression of toxic CUG RNA expansions (CUG exp ) leading to decreased muscleblind-like, but increased CUGBP1/ETR3-like factor 1 (CELF1), alternative splicing activities. Here, we test this model in vivo, using the mouse HSA LR poly(CUG) model for DM1 and recombinant adeno-associated virus (rAAV)-mediated transduction of specific splicing factors. Surprisingly, systemic overexpression of HNRNPA1, not previously linked to DM1, also shifted DM1-relevant splicing targets to fetal isoforms, resulting in more severe muscle weakness/myopathy as early as 4 to 6 wk posttransduction, whereas rAAV controls were unaffected. Overexpression of HNRNPA1 promotes fetal exon inclusion of representative DM1-relevant splicing targets in differentiated myoblasts, and HITS-CLIP of rAAV-mycHnrnpa1-injected muscle revealed direct interactions of HNRNPA1 with these targets in vivo. Similar to CELF1, HNRNPA1 protein levels decrease during postnatal development, but are elevated in both regenerating mouse muscle and DM1 skeletal muscle. Our studies suggest that CUG exp RNA triggers abnormal expression of multiple nuclear RNA binding proteins, including CELF1 and HNRNPA1, that antagonize MBNL activity to promote fetal splicing patterns.M icrosatellite expansions in the noncoding regions of several human genes are associated with hereditary neurological diseases, including fragile X mental retardation (FRAXA) and myotonic dystrophy (DM) (1). In DM type 1 (DM1), the transcription of a CTG microsatellite expansion (CTG exp ) in the 3′ untranslated region (3′ UTR) of the DMPK gene results in the expression of toxic CUG exp RNA, which sequesters muscleblindlike (MBNL) proteins and blocks their splicing activity (2, 3). MBNL inhibition in DM1 tissues leads to a shift from adult to fetal splicing events for MBNL-targeted RNAs and results in tissuespecific disease manifestations, including muscle hyperexcitability (myotonia) and insulin resistance. In addition, CUG exp RNA also activates protein kinase C (PKC), resulting in the hyperphosphorylation and elevated levels of CUGBP1/ETR3-like factor 1 (CELF1) in DM1 muscle and heart (4). In contrast to MBNL, CELF1 induces fetal splicing events in embryonic and early neonatal stages, and increased CELF1 levels in adult DM1 tissues causes a reversion to fetal isoforms. Thus, the current DM1 disease model is that coordinate MBNL sequestration and CELF1 accumulation caused by CUG exp RNA expression synergistically promote aberrant fetal splicing patterns in DM1 (3, 5). Support for both MBNL loss of function and CELF gain of function has come from mouse knockout and transgenic models (6-8). Mbnl1 knockout adults develop muscle myotonia and myopathy, subcapsular dust-like cataracts, and alternative splicing changes ch...

Short tandem repeats (STRs) are prone to expansion mutations that cause multiple hereditary neurological and neuromuscular diseases. To study pathomechanisms using mouse models that recapitulate the tissue specificity and developmental timing of an STR expansion gene, we used rolling circle amplification and CRISPR/Cas9-mediated genome editing to generate Dmpk CTG expansion (CTG exp ) knockin models of myotonic dystrophy type 1 (DM1). We demonstrate that skeletal muscle myoblasts and brain choroid plexus epithelial cells are particularly susceptible to Dmpk CTG exp mutations and RNA missplicing. Our results implicate dysregulation of muscle regeneration and cerebrospinal fluid homeostasis as early pathogenic events in DM1.

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.