Figure 1.

Expression of an expanded DMPK transgene induces foci accumulation and splicing dysregulation in the CNS. (A) Fluorescent in situ hybridization and immunofluorescence revealed nuclear foci of transgenic DMPK messenger RNA in both NeuN-positive neurons and GFAP-positive astrocytes, in multiple regions of the brain of 1-month-old DMSXL homozygotes. (B) MBNL1 and MBNL2 co-localized with nuclear RNA foci. No RNA foci were observed in DM20 and wild-type control animals (data not shown). Punctuated MBNL2 staining was occasionally detected and did not correspond to RNA aggregates. Diamidino-2-phenylindole was used for nuclear staining. Scale bar = 5 µm. (C) Quantification of the percentage of astrocytes and neurons exhibiting nuclear RNA aggregates (±SEM) throughout the brain of 1-month-old DMSXL homozygotes (n = 2) (**P < 0.01, χ² test). (D) Percentage of inclusion of alternative exons in messenger RNA transcripts encoding: GRIN1/NMDAR1 glutamate receptor, ATP2A1/SERCA1 endoplasmic calcium ATPase, microtubule-associated protein tau (MAPT/TAU), amyloid beta precursor protein (APP), insulin receptor (INSR), MBNL1 and MBNL2 splicing regulators, LDB3/CYPHER cytoskeleton-interacting protein and FXR1 RNA-binding protein. The analysis was performed in frontal cortex and brainstem of homozygous DMSXL mice (n = 9) and wild-type control mice (n = 9) at age 1 month. Splicing profiles were compared with those of wild-type newborns collected as post-natal Day 1 (n = 3). The graphs show the average fractional inclusion of the specified exon in triplicate assays (±SEM). (E) Quantification of CELF proteins in homozygous DMSXL (n = 3) and wild-type mice (n = 3) at age 1 month. (F) Quantification of CELF proteins in frontal cortex of adult myotonic dystrophy type 1 individuals (n = 9) and non-myotonic dystrophy control subjects (n = 3). The graphs in E and F show the average steady-state levels relative to normalized control subjects. *P < 0.05, **P < 0.01, ***P < 0.001. WT = wild-type.