Modeling Oculopharyngeal Muscular Dystrophy in Myotube Cultures Reveals Reduced Accumulation of Soluble Mutant PABPN1 Protein

Abstract

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease caused by an alanine tract expansion mutation in poly(A) binding protein nuclear 1 (expPABPN1). To model OPMD in a myogenic and physiological context, we generated mouse myoblast cell clones stably expressing either human wild type (WT) or expPABPN1 at low levels. Transgene expression is induced on myotube differentiation and results in formation of insoluble nuclear PABPN1 aggregates that are similar to those observed in patients with OPMD. Quantitative analysis of PABPN1 in myotube cultures revealed that expPABPN1 accumulation and aggregation is greater than that of the WT protein. We found that aggregation of expPABPN1 is more affected than WT PABPN1 by inhibition of proteasome activity. Consistent with this, in myotube cultures expressing expPABPN1, deregulation of the proteasome was identified as the most significantly perturbed pathway. Differences in the accumulation of soluble WT and expPABPN1 were consistent with differences in ubiquitination and rate of protein turnover. This study demonstrates, for the first time to our knowledge, that, in myotubes, the ratio of soluble/insoluble expPABPN1 is significantly lower compared with that of the WT protein. We suggest that this difference can contribute to muscle weakness in OPMD.

Regulated Intron Retention and Nuclear Pre-mRNA Decay Contribute to PABPN1 Autoregulation

“…8B). Although most of the results presented in the current study support an autoregulatory mechanism that acts primarily at the RNA level, we do not exclude that posttranslational mechanisms, such as the formation of insoluble nuclear PABPN1 aggregates (47), also contribute to control PABPN1 protein levels.…”

Nuclear poly(A)-binding protein aggregates misplace a pre-mRNA outside of SC35 speckle causing its abnormal splicing

“…Neither did the addition of a wild-type functional PABPN1 expressing vector in Ala17 cells (data not shown), suggesting that TNNT3 splicing defect is not linked to the level of available PABPN1 itself. To further reinforce the crucial role of the aggregate we checked the splicing defect in H2KB-WTa (Ala10 cells), overexpressing wild-type Flag-tagged PABPN1 at the same level as in Ala17 cells and containing aggregates in differentiation ((47) and Supplementary Figure S3A-B): the splicing defect was confirmed in this differentiated cell line as well (Supplementary Figure S3C and D). Together these results strongly suggest a direct link between the presence of nuclear aggregates and the splicing misregulation of TNNT3 .…”

“…We checked the splicing pattern of Tnnt3 in a murine cell model of OPMD, namely the H2KB-D7e cells (47). Primary H2KB-IM2 cells are conditionally immortalized mouse myoblasts derived from the ImmortoMouse (55) and H2KB-D7e are cells stably derived from the H2KB-IM2 cell line by transduction with an expanded Flag-tagged PABPN1 construct under the control of the human desmin locus control region and promoter.…”

“…For clarity, the H2KB-IM2 and -D7e will be called hereafter ‘control’ and ‘Ala17’ muscle cells respectively. In differentiation conditions, these Ala17 cells contain a large number of nuclear aggregates present in more than 50% of the nuclei ((47) and Figure 2E). In control cells, the Tnnt3 exon 16 isoform is absent at day 1, but progressively increases during differentiation.…”

“…Mutated-PABPN1 (H2KB-D7e named ‘Ala17’ hereafter) and wild-type-PABPN1 (H2KB-WTa named ‘Ala10’) stable cells lines were derived from IM2 cells line by transfection with a plasmid expressing expanded-PABPN1 or wild-type-PABPN1 respectively under the control of the human desmin locus control region and promoter to ensure myotube-specific expression of the human PABPN1 transgene (47). Cells were cultivated on matrigel coated support (Corning) in DMEM, supplemented with 20% foetal bovine serum (Invitrogen), 0.5% chicken embryo extract (Seralab), 100 U/ml penicillin-streptomycin (Thermo Scientific), and 20 U/ml interferon-γ (Millipore) at 33°C in a humidified 5% CO 2 air atmosphere.…”