Effects of overexpression of GFP-TDP-43, GFP-TDP-43(86-414) or GFP in human H4 cells, in the presence or absence of the EIF2B activator DNL343.

Neuronal TDP-43 aggregates are a hallmark ALS pathology. The integrated stress response (ISR) occurs downstream of TDP-43 pathology and may exacerbate TDP-43 aggregation and neurodegeneration. Here we tested the effects of overexpression of GFP-taggted TDP-43 proteins in H4 cells, in the presence or ascence of the EIF2B activiting small molecule DNL343. Cytoplasmic TDP-43 expression caused upregulation of ISR gene transcripts, including MTHFD2, GDF15, SLC7A11, and ATF3, relative to the GFP expressing sample. Transcript level changes induced by GFP-TDP-43(86-414) were prevented by DNL343 treatment, with significant downregulation of ISR genes, including CHAC1, ATF4, and DDIT3. Overall design: H4 cells were engineered to overexpress either GTF-TDP-43 (full length), GFP-TDP-43 (cytomplasmic domain) or GFP alone. After inducing protein expression for either 8 or 24 hours, cells were treated with either DMSO or 1 uM DNL343 for 2.5 hours before harvesting total RNA. The experiment was performed in 4 replicates, e.g. cells were treated independently on 4 different days. One sample failed quality control, leaving 47 samples in total for 3-tag RNA-sequencing to quantify transcriptome-wide gene expression levels.