Fig. 5

The Drosophila KPNB1 ortholog Ketel reduces human TDP-43 pathology and toxicity in fly models of TDP-43 proteinopathy. a Eye phenotypes from flies expressing the indicated transgenes via the eye-specific Gmr-Gal4 driver. Ketel knock-down (RNAi) enhances TDP-43-induced neurodegeneration whereas its overexpression (V5-Ketel) rescues it. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (**p < 0.01, ***p < 0.001, n ≥ 4). b Immunostaining of phosphorylated TDP-43^M337V (pTDP-43) in adult fly brains treated with RU486 (40 μg/ml). This conditional Drosophila model of TDP-43 proteinopathy is based on expression of mutant human TDP-43^M337V under the control of the Elav-GS driver in adult flies upon administration of RU486. Ketel RNAi enhances and extends the accumulation of pTDP-43-positive aggregates in the fly brain (arrowheads), compared to LacZ control (arrows), whereas overexpression of V5-Ketel dramatically reduces the pTDP-43 staining. c Locomotion assays on flies expressing mutant TDP-43^M337V under the control of the Elav-GS driver in the presence or absence of RU486. Ketel downregulation enhances mutant TDP-43-induced locomotor dysfunction in adult flies, whereas Ketel overexpression rescues it. Locomotion was assessed with the DAM2 activity monitor from TriKinetics, Inc. Statistical analysis was performed using two-way ANOVA and Bonferroni’s post hoc test (***p < 0.001, n = 13). d Survival curves of flies expressing the indicated transgenes under the control of the Elav-GS driver in the presence of the drug RU486. Flies co-expressing TDP-43^M337V with the innocuous transgene LacZ have a dramatic lifespan reduction in the presence of RU486 (blue line) compared to flies lacking mutant TDP-43 (gray line). Co-expression of Ketel (green line) improves this phenotype, whereas a Ketel RNAi transgene (red line) exacerbates it. Survival analysis was performed using the OASIS online tool, and p-values were calculated using Fisher’s exact test (**p < 0.01 and ****p < 0.0001, n = 80). e Immunofluorescence (IF) of HEK293T cells co-expressing GFP or GFP-TDP-43^mNLS with mScarlet or mScarlet-tagged Ketel full-length (FL), H1–9 or H10–19. Both full-length and the N-terminal half of Ketel reduce human TDP-43^mNLS aggregation and cytoplasmic mislocalization, whereas the C-terminal half of Ketel co-aggregates with TDP-43^mNLS. Arrowheads indicate co-aggregation. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. f IF of HEK293T cells co-expressing GFP-Ketel with mCherry or Nup62-mCherry. While Ketel is mostly nuclear with mCherry, it strongly colocalizes with Nup62-mCherry aggregates in the cytoplasm. Arrowheads indicate co-aggregation. Hoechst staining was used to outline nuclei. Scale bar: 5 μm