Understanding Tissue Specificity in Repeat Expansion Disorders

The instability of short tandem repeats and consequent somatic expansion underlies an entire class of neurological disorders. In DM1, the interaction between expanded repeat transcripts and the RNA binding protein/splicing regulator MBNL determines subsequent pathogenic events. Although the inherited (progenitor) repeat length is independent of cell/tissue type, tissue specificity in somatic expansion defines both the timing and severity of organ systems impacted by DM1. Questions of the spatial and temporal patterns of disease pathophysiology are difficult to ask and answer in studies of patients. Yet, most model organisms used to study DM1 to date are not capable of interrogating mechanisms that underlie such cell type- and tissue-specific effects.

New Mouse Models to DM1 Study Tissue Specificity

New technologies, rolling circle amplification and CRISPR/Cas9 genome editing, have been used by Dr Maury Swanson (University of Florida) and colleagues to develop novel Dmpk 3’UTR CTG^exp knock-in mouse models of DM1. Mice expressing 170 (Dmpk^170/170) and 480 (Dmpk^480/480) repeats were selected. These new models enable study of repeat expansion, MBNL sequestration, mis-splicing, and pathophysiology on specific cell and tissue type levels. Dr. Curtis Nutter, recipient of a 2018 MDF Fellowship, was lead author on this work.

Dmpk^exp mice exhibited nuclear foci and reduced Dmpk mRNA and protein levels, but disrupted splicing regulation was not observed and downstream muscle phenotypic changes (myotonia, central nuclei) were absent. Similar reductions in Dmpk mRNA and protein were seen in cultured myoblasts and myotubes. In contrast to absent splicing changes in adult mice, MBNL sequestration and mis-splicing were seen in Dmpk^480/480 but not in Dmpk^170/170 mouse myotubes. To reconcile their findings, the authors found that MBNL activity might be more effectively damped in myoblasts/myotubes than in mature muscle—reinforcing the notion that pathology reflects both tissue and developmental stage specificity due to interactions of relative repeat load (expansion length and gene expression level) with MBNL protein levels. Crosses resulting in Dmpk^480/480 mice haploinsufficient for MBNL supported this idea.

Given the severity of the CNS phenotype in DM1, the research team also evaluated choroid plexus (a CNS tissue expressing high Dmpk levels) in the Dmpk^exp mice. Nuclear foci were prominent and DM1-related splicing anomalies detected in choroid plexus of these mice.

Insights into Cell Type Specificity and Pathogenesis of DM1

 The Dmpk^exp mice have not, to date, shown evidence of repeat instability—in keeping with the absence of an in vivo phenotype. Larger repeat loads may be necessary to trigger mouse phenotypes. Perhaps more importantly, these data help establish a link between DMPK CTG^exp length and the cell/tissue patterns of pathology in DM1. That choroid plexus may be affected earlier (i.e., at lower repeat load) than skeletal muscle helps solidify understanding of the spatial and temporal patterning of DM1. It’s now highly likely that the repeat load in other cell/tissue types can be more directly linked to their disease phenotype. Finally, these results also make it clear that there is a need to explore potential involvement of affected choroid plexus function (CSF production) in the CNS consequences of DM1.

Reference:

Cell-type-specific dysregulation of RNA alternative splicing in short tandem repeat mouse knockin models of myotonic dystrophy.
Nutter CA, Bubenik JL, Oliveira R, Ivankovic F, Sznajder ŁJ, Kidd BM, Pinto BS, Otero BA, Carter HA, Vitriol EA, Wang ET, Swanson MS.
Genes Dev. 2019 Oct 17. doi: 10.1101/gad.328963.119. [Epub ahead of print]