Comparative Analysis of TDP-43-Controlled RNA Processing in Neuronal and Muscle Cells in Mouse and Human

Šušnjar, Urša (2022). Comparative Analysis of TDP-43-Controlled RNA Processing in Neuronal and Muscle Cells in Mouse and Human. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.000141fd

Abstract

TDP-43 (TAR DNA-binding protein, encoded by TARDBP gene) is a DNA/RNA-binding protein that participates in various steps of RNA metabolism. First identified as a component of cytoplasmic inclusions in motor neurons of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), its aberrant aggregation later became recogcnized in non-neuronal tissues, in particular skeletal muscles of patients with inclusion body myositis (IBM). Despite its ubiquitous expression and pleiotropic functions, it was unclear to what extent TDP-43 participates in basic cellular processes that are uniform across tissues and whether it exhibits tissue-specific functions in cells of different backgrounds.

By silencing TDP-43 in (mouse) neuronal and muscle cell lines we mimicked loss of TDP-43’s function, a pathomechanism commonly believed to underly disease development. RNA-seq allowed transcriptome-wide detection of transcripts regulated by TDP-43 at the level of their overall abundance (differential expression DEG) or splicing (alternative splicing AS).

In this work we discuss similarities and differences concerning the activity of TDP-43 across cell-types. We identified subsets of unique cell-type-characteristic mRNA targets and those that are commonly mediated by TDP-43 in muscles and neurons, whose tight regulation might underlie functions crucial for cell survival. Based on this, we investigated functional consequences of TDP-43 loss in either cell-type, linking TDP-43 pathology to previously described hallmarks of neuro- and myodegeneration.

We further compared alternative splicing and transcript abundance control as two distinct regulatory mechanisms governed by TDP-43 that are not equally exploited by cells of different types. In addition, we investigated how cell-type-characteristic environments shape TDP-43’s function and render it tissue-specific.

Among splicing events that occur in a muscle-specific manner, we started to characterize a TDP-43-dependent switch in Tbc1d1 splicing, as this gene encodes for a GTPase involved in translocation of glucose transporters and thereby mediates glucose uptake by muscle cells.

With those results we set the ground for future studies investigating the function of TDP-43 in muscles and a putative contribution of TDP-43 redistribution to IBM development. Furthermore, our findings stress the importance to select an appropriate cell model to study tissue-characteristic features.

Finally, we identified two novel TDP-43 targets, PPFIBP1 and ASAP2 consistently detected to undergo TDP-43-dependent isoform switch not only in mouse but also in humans. We have shown that the splicing of those is indeed perturbed in brains and skeletal muscles affected by TDP-43 pathology and we therefore believe these two splicing events could make a universal readout of TDP-43 dysfunction across tissues.

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