Nuclear MBNL1-containing foci increase in size upon cell-cycle exit in fibroblasts from DM2 patients

Myotonic dystrophies (DM1 and DM2) are multisystemic diseases that belong to non-coding repeat expansion disorders. The presence of the CTG or CCTG expansion respectively, leads to nuclear accumulation of the expanded RNAs that sequester essential proteins (such as MBLN1) and disrupt their normal function in the cells of different tissues. The cytochemical detection of nuclear foci of expanded RNAs or MBNL1 is an accepted diagnostic marker for DM1. It is apparent that in DM patients the most significant pathological features are found in the skeletal muscle, heart and the central nervous system where non-cycling cell populations are mainly found; on the contrary, cells from self-renewing tissues (such as skin fibroblasts or layering epithelial cells) appear much less affected. The mechanisms that underlie the nuclear retention of expanded RNAs and its aggregation into nuclear foci are not currently well understood. A recent study on dynamics of CUG repeat foci in living myoblasts has revealed that these are constantly aggregating and disaggregating structures, and that MBNL1 is directly involved in the stochastic process of foci2. In this investigation, we aimed to elucidate whether in DM tissues the exit of cells from the cell cycle may lead to the increase in size of intranuclear foci due to the progressive sequestration of protein factors (namely MBNL1); to do this, we investigated by immunocytochemical and morphometric techniques the fate of MBNL1-containing foci in proliferating cells during the cell cycle, and in non-cycling cells. Cultured skin fibroblasts from DM2 patients were chosen as a model cell system. We found that nuclear MBNL1-containing foci do not associate with chromosomes at mitosis, and remain in the cytoplasm at cytodieresis, being disassembled in early G1 and re-formed in the nucleus, at each cell cycle. After cells had spontaneously stopped dividing in senescing fibroblast cultures, the nuclear foci were observed to increase in number and size. Interestingly, morphometric measurements of nuclear MBNL1-containing foci in muscle biopsies taken from the same DM2 patients at different ages demonstrated that they become larger with increasing patient’s age.