Mutant antisense <i>DMPK</i> RNA nuclear foci in human DM1 fetal samples.

<p>RNA foci containing (CAG)n expansions were labeled in red, using a 5’-Cy3-labeled (CTG)₅ PNA probe in heart, skeletal muscle and brain samples from 12–13.5 week-old (12–13.5 wk), 16–17 week-old (16–17 wk) and 33–33.5 week-old (33–33.5 wk) DM1 fetuses.</p

Sense and Antisense <i>DMPK</i> RNA Foci Accumulate in DM1 Tissues during Development

<div><p>Myotonic dystrophy type 1 (DM1) is caused by an unstable expanded CTG repeat located within the <i>DMPK</i> gene 3’UTR. The nature, severity and age at onset of DM1 symptoms are very variable in patients. Different forms of the disease are described, among which the congenital form (CDM) is the most severe. Molecular mechanisms of DM1 are well characterized for the adult form and involve accumulation of mutant <i>DMPK</i> RNA forming foci in the nucleus. These RNA foci sequester proteins from the MBNL family and deregulate CELF proteins. These proteins are involved in many cellular mechanisms such as alternative splicing, transcriptional, translational and post-translational regulation miRNA regulation as well as mRNA polyadenylation and localization. All these mechanisms can be impaired in DM1 because of the deregulation of CELF and MBNL functions. The mechanisms involved in CDM are not clearly described. In order to get insight into the mechanisms underlying CDM, we investigated if expanded RNA nuclear foci, one of the molecular hallmarks of DM1, could be detected in human DM1 fetal tissues, as well as in embryonic and neonatal tissues from transgenic mice carrying the human <i>DMPK</i> gene with an expanded CTG repeat. We observed very abundant RNA foci formed by sense <i>DMPK</i> RNA and, to a lesser extent, antisense <i>DMPK</i> RNA foci. Sense <i>DMPK</i> RNA foci clearly co-localized with MBNL1 and MBNL2 proteins. In addition, we studied <i>DMPK</i> sense and antisense expression during development in the transgenic mice. We found that <i>DMPK</i> sense and antisense transcripts are expressed from embryonic and fetal stages in heart, muscle and brain and are regulated during development. These results suggest that mechanisms underlying DM1 and CDM involved common players including toxic expanded RNA forming numerous nuclear foci at early stages during development.</p></div

Sense and antisense <i>DMPK</i> RNA levels in transgenic mice.

<p>qRT-PCR were performed in heart, skeletal muscle and brain from DMSXL and DM20 embryos and neonates at embryonic E14.5 to postnatal P29 stages. Levels of sense <i>DMPK</i> (left panels) and antisense transcripts (right panels) were reported on graphs using 18S as internal control, in arbitrary units (a.u.) with standard deviation of the mean for repeated experiments.</p

Sense and antisense <i>DMPK</i> RNA levels in DM1 fetuses.

<p>qRT-PCR were performed in heart (left panels) and in brain (right panels) using specific primers and standard curves established with a known number of plasmid molecules with the amplicons as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137620#pone.0137620.ref032" target="_blank">32</a>]. Levels of sense and antisense <i>DMPK</i> transcripts were reported on graphs using 18S as internal control, in arbitrary units (a.u.). Cont 18–25: samples from control fetuses aged between 18–25 weeks. DM1 16–20: samples from DM1 fetuses aged between 16–20 weeks. DM1 33: sample from a 33 weeks old DM1 fetus. The mean and SEM are represented respectively, as horizontal and vertical bars. (**, p<0.01, two-tailed Student’s t-test).</p

Simultaneous detection of mutant sense and antisense <i>DMPK</i> RNA nuclear foci.

<p>Localization of sense and antisense <i>DMPK</i> foci was studied using 5’-cy3-(CAG)5 (recognizing sense (CUG)n transcripts in red) and 5’-Alexa 488-(CTG)5 (recognizing antisense (CAG)n transcripts in green) probes in the same experiment. Left panel: human 12 week-old fetus heart sample; right panel: DMSXL E14.5 embryonic muscle from the hind leg.</p

Glutamate dysfunction at extrasynaptic site in hippocampus of mice model of myotonic dystrophy type 1 (DM1) disease

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Mutant sense <i>DMPK</i> RNA nuclear foci in human DM1 fetal samples.

<p>RNA foci containing (CUG)n expansion were labeled in red, using a 5’-Cy3-labeled (CAG)₅ PNA probe in heart, skeletal muscle and brain samples from 12–13.5 week-old (12–13.5 wk), 16–17 week-old (16–17 wk) and 33–33.5 week-old (33–33.5 wk) DM1 fetuses.</p

Mutant sense <i>DMPK</i> RNA nuclear foci in DMSXL embryos and neonates.

<p>RNA foci containing (CUG)n expansion were labeled in red, using a 5’-Cy3-labeled (CAG)₅ PNA probe in E14.5 embryos heart, muscle (hind leg) and brain (cortex) and P7 neonates heart (ventricule), muscle (gastrocnemius) and brain (frontal cortex).</p

DM1 transgenic mice exhibit abnormal neurotransmitter homeostasis and synaptic plasticity in association with RNA mis-splicing in the hippocampus

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DM1 transgenic mice exhibit abnormal neurotransmitter homeostasis and synaptic plasticity in association with RNA mis-splicing in the hippocampus.

International audienceMyotonic dystrophy type 1 (DM1) is a severe neuromuscular disease mediated by a toxic gain of function of mutant RNAs. The neuropsychological manifestations affect multiple domains of cognition and behaviour, but their aetiology remains elusive. Transgenic DMSXL mice carry the human DM1 mutation and show relevant behavioural abnormalities (including altered exploratory behaviour and anhedonia) and express reduced levels of GLT1, a critical regulator of glutamate homeostasis in the brain. However, the impact of glutamate homeostasis dysfunction on neurotransmission in DM1 remains unknown. In the present study, we show an in vivo reduced glutamate uptake in the DMSXL hippocampus compared to wildtype mice, but extracellular glutamate levels sampled in the dorsal hippocampus using zero-flow quantitative microdialysis were unaltered. Patch clamp recordings of CA1 pyramidal neurons and DG granule cells in hippocampal slices from DMSXL mice revealed an increased tonic excitation, likely mediated by higher levels of ambient glutamate at the vicinity of extrasynaptic NMDA receptors. We also found an unexpected elevated extracellular GABA level in DMSXL mice associated with an increase in tonic inhibition and a higher GABA release. Finally, we found evidence of abnormal short-term plasticity in the DG and CA1 area, suggestive of synaptic dysfunction in DMSXL mice. Synaptic dysfunction was accompanied by the accumulation of RNA foci that are more abundant and larger in the DG than in CA1 area, and by the mis-splicing of candidate genes with relevant functions in amino acidergic neurotransmission (ion channels, neurotransmitter receptors and synaptic proteins, as well as proteins involved in neuronal vesicle trafficking). Taken together, molecular and functional changes triggered by the accumulation of toxic RNA may induce synaptic abnormalities in restricted brain areas of the brain, causing neuronal dysfunction