Identifying modifiers of seed-induced TDP-43 loss of function

Both gain of toxicity and loss of normal function of the RNA-binding protein TDP-43 contribute to neurodegeneration in ALS and FTD, but their mechanistic connection remains unclear. Increasing evidence suggests that TDP-43 aggregates act as self-templating seeds, propagating pathology through the central nervous system via a prion-like cascade. We developed a robust TDP-43 seeding platform for quantitative, high-throughput assessment of TDP-43 aggregate uptake, cell-to-cell spreading and direct quantification of TDP-43 nuclear function within living cells, while they progress towards pathology. We show that both patient-derived and recombinant TDP-43 pathological aggregates are abundantly internalized in human neuron-like cells, efficiently recruit endogenous TDP-43 and form cytoplasmic inclusions reminiscent of ALS/FTD pathology. These neoaggregates progressively drive the nuclear egress of TDP-43 leading to its loss of function. The scope of this project is to identify the signatures involved in the progressive egress of TDP-43 from the nucleus to the cytoplasm. We generated a cell line which incorporates a sensor (TDP-REG) capable of reporting for TDP-43 loss of function in living cells. Specifically, TDP-REG encodes for the fluorescent protein mScarlett, but contains a TDP-43-inhibited cryptic exon essential for the complete mScarlett transcript. In cells with functional TDP- 43, this cryptic exon is repressed and the presence of a downstream premature termination codon results in nonsense mediated decay of the transcript, and therefore, no mScarlett protein. Conversely, in the absence of functional TDP-43, the cryptic exon is derepressed and included in the mature mRNA, leading to the translation of mScarlett fluorescent protein. We observed that after treatment with fluorescently labeled (Atto-488) TDP-43 aggregates, a small percentage of cells (0.1-0.2% of the total) shows the activation of the reporter.