Additional file 1 of Nuclear import receptors are recruited by FG-nucleoporins to rescue hallmarks of TDP-43 proteinopathy
Creators: name: Khalil, Bilal
name: Chhangani, Deepak
name: Wren, Melissa C.
name: Smith, Courtney L.
name: Lee, Jannifer H.
name: Li, Xingli
name: Puttinger, Christian
name: Tsai, Chih-Wei
name: Fortin, Gael
name: Morderer, Dmytro
name: Gao, Junli
name: Liu, Feilin
name: Lim, Chun Kim
name: Chen, Jingjiao
name: Chou, Ching-Chieh
name: Croft, Cara L.
name: Gleixner, Amanda M.
name: Donnelly, Christopher J.
name: Golde, Todd E.
name: Petrucelli, Leonard
name: Oskarsson, Björn
name: Dickson, Dennis W.
name: Zhang, Ke
name: Shorter, James
name: Yoshimura, Shige H.
name: Barmada, Sami J.
name: Rincon-Limas, Diego E.
name: Rossoll, Wilfried
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Computational Biology

description: Additional file 1: Supplementary Fig. 1. KPNB1 specifically reduces insoluble protein levels of TDP-CTF. a, (Top) Immunofluorescence (IF) of SH-SY5Y cells co-expressing mCherry-TDP-CTF with GFP or GFP-KPNB1. mCherry-TDP-CTF forms cytoplasmic aggregates but shows diffuse localization in the presence of GFP-KPNB1. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. (Bottom) Western blot analysis and quantification of insoluble mCherry-TDP-CTF and endogenous TDP-43 protein levels in SH-SY5Y cells expressing GFP or GFP-KPNB1. KPNB1 significantly reduced insoluble TDP-CTF levels, while endogenous insoluble TDP-43 levels were unaffected. β-tubulin was used as a loading control. Statistical analysis was performed using two-sided Student’s t-test (***p &lt; 0.001, n = 10). b, Western blot analysis of soluble and insoluble GFP-TDP-CTF co-expressed with mCherry, mCherry-KPNB1 or untagged KPNB1 in HEK293T cells. KPNB1 only reduces insoluble TDP-CTF levels. β-tubulin was used as a loading control. c, IF of HEK293T cells co-expressing GFP-TDP-CTF or GFP-TDP-43mNLS with mCherry or mCherry-KPNB1. TDP-CTF and TDP-43mNLS form cytoplasmic aggregates positive for phospho-TDP-43S409/410 but show diffuse localization with no pathological hyperphosphorylation in the presence of mCherry-KPNB1. Arrowheads point to colocalization. Scale bar: 5 μm. d, Western blot analysis and quantification of insoluble GFP-tagged TDP-CTFWT, TDP-CTFQ331K, TDP-CTFM337V and TDP-CTFA382T in HEK293T cells expressing mCherry or mCherry-KPNB1. KPNB1 similarly reduced insoluble wild-type and ALS-derived mutant TDP-CTF protein levels. β-tubulin was used as a loading control. Statistical analysis was performed using two-sided Student’s t-test (***p &lt; 0.001, n = 3). e, Western blot analysis of total protein levels of GFP-tagged TDP-CTF or TDP-43 in HEK293T cells expressing mCherry, mCherry-KPNB1 or untagged KPNB1. Cells were lysed in 7 M urea buffer to collect total protein. KPNB1 reduces total protein levels of TDP-CTF, but not full-length TDP-43. β-tubulin was used as a loading control. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (*p &lt; 0.05, ***p &lt; 0.001, n = 4). f, TDP-CTF and TDP-43 transcript levels were quantified in HEK293T cells co-expressing GFP-tagged TDP-CTF or TDP-43 with mCherry, mCherry-KPNB1, or untagged KPNB1. GAPDH was used as a reference to normalize the transcript levels. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (n = 3). Supplementary Fig. 2. Expression of full-length KPNB1 and its N-terminal half do not reduce endogenous insoluble TDP-43 levels. Quantification of endogenous insoluble TDP-43 protein levels in SH-SY5Y cells co-expressing mCherry-TDP-CTF with GFP, GFP-KPNB1 full-length (FL), H1–9 or H10–19. β-tubulin was used as a loading control. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (n = 4). Supplementary Fig. 3. Cytoplasmic GFP-(GR)100 aggregates sequester endogenous TDP-43 and Nup62. a, Immunofluorescence of HEK293T cells expressing GFP-(GR)100 and stained for endogenous TDP-43 and Nup62. Only cytoplasmic GFP-(GR)100 aggregates are immunopositive for TDP-43 and Nup62. White arrowheads point to cytoplasmic poly(GR) aggregates. Nuclear GFP-(GR)100 aggregates render TDP-43 staining diffuse (purple arrowhead), while Nup62 accumulates as puncta in the cytoplasm (red arrowhead). Hoechst staining was used to outline nuclei. Scale bar: 5 μm. b, Quantification of the percentage of cytoplasmic GFP-(GR)100 aggregates positive for TDP-43 or Nup62 in HEK293T cells. Almost all cells are positive for both proteins. Statistical analysis was performed using two-sided Student’s t-test (three independent experiments; n = 239–249 cells per group). Supplementary Fig. 4. Untagged KPNB1 interacts with both the RRM2 and PrLD of TDP-CTF. a, (Left) Lysates from HEK293T cells expressing GFP alone, or untagged KPNB1 with GFP, GFP-TDP-CTF or GFP-TDP-43 were subjected to immunoprecipitation with GFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to western blot analysis using indicated antibodies. (Right) Quantification of relative KPNB1 levels in IP normalized by KPNB1 levels in input. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (**p &lt; 0.01, ***p &lt; 0.001, n = 3). b, Schematic domain structure of TDP-CTF full-length (208–414). TDP-CTF contains part of the RRM2 (non-PrLD, 208–274) and the C-terminal PrLD (275–414). c, (Left) Lysates from HEK293T cells expressing GFP alone, or untagged KPNB1 with GFP, GFP-TDP-CTF, GFP-TDP-CTFnon-PrLD or GFP-TDP-PrLD were subjected to immunoprecipitation with GFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to western blot analysis using indicated antibodies. (Right) Quantification of relative KPNB1 levels in IP normalized by KPNB1 levels in input. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (*p &lt; 0.05, **p &lt; 0.01, n = 3). *cleavage products. Supplementary Fig. 5. KPNB1 reduces TDP-CTF aggregation independent of its interaction with the RRM2 region. a, Schematic domain structures of full-length TDP-CTF (208–414), and its C-terminal and N-terminal deletion constructs used for co-IP experiments. b, Lysates from HEK293T cells co-expressing mCherry-KPNB1 with GFP, GFP-TDP-CTF or GFP-TDP-CTF C-terminal deletion constructs were subjected to immunoprecipitation with RFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to western blot analysis using indicated antibodies. Deletion of amino-acids 230–240 in TDP-CTF reduced its binding to KPNB1. c, Lysates from HEK293T cells co-expressing mCherry-KPNB1 with GFP, GFP-TDP-CTF or GFP-TDP-CTF N-terminal deletion constructs were subjected to immunoprecipitation with RFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to western blot analysis using indicated antibodies. Deletion of amino-acids 230–240 in TDP-CTF reduces its binding to KPNB1. TDP-PrLD weakly interacts with KPNB1. d, (Left) Lysates from HEK293T cells co-expressing mCherry-KPNB1 with GFP, GFP-TDP-CTFWT or GFP-TDP-CTFΔ230–240 were subjected to immunoprecipitation with RFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to western blot analysis using indicated antibodies. KPNB1 interacts less with TDP-CTFΔ230–240. (Right) Quantification of relative GFP levels in IP normalized by GFP levels in input confirm that GFP-TDP-CTFΔ230–240 interacts less with mCherry-KPNB1. Statistical analysis was performed using two-sided Student’s t-test (**p &lt; 0.01, n = 3). e, Immunofluorescence of HEK293T cells co-expressing GFP-tagged TDP-CTFWT or TDP-CTFΔ230–240 with mCherry or mCherry-KPNB1. TDP-CTFΔ230–240 forms small nuclear and big cytoplasmic aggregates. KPNB1 reduces both TDP-CTFWT and TDP-CTFΔ230–240 aggregates and makes TDP-CTFΔ230–240 more nuclear. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. f, Western blot analysis and quantification of insoluble GFP-tagged TDP-CTFWT or TDP-CTFΔ230–240 in HEK293T cells expressing mCherry or mCherry-KPNB1. KPNB1 equally reduced insoluble TDP-CTFWT and TDP-CTFΔ230–240 protein levels. β-tubulin was used as a loading control. Statistical analysis was performed using two-way ANOVA and Bonferroni’s post hoc test (***p &lt; 0.001, n = 3). Supplementary Fig. 6. The PrLD of TDP-43 interacts with FG-Nups and KPNB1. a, Immunofluorescence (IF) of HEK293T cells expressing GFP or GFP-TDP-PrLD and stained for endogenous Nup98 and Nup62. TDP-PrLD forms small cytoplasmic foci positive for Nup62. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. b, IF of HEK293T cells expressing GFP or GFP-TDP-PrLD and stained for endogenous KPNB1. KPNB1 colocalizes with TDP-PrLD foci in the cytoplasm. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. c, Lysates from HEK293T cells expressing GFP or GFP-TDP-PrLD were subjected to immunoprecipitation with GFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to western blot analysis using indicated antibodies. The PrLD of TDP-43 interacts with Nup62, Nup98 and KPNB1, but not with Ran. Supplementary Fig. 7. Colocalization of Nup62 with TDP-43 constructs depends on the PrLD. Colocalization between Nup62-mCherry and GFP or GFP-tagged TDP-43 constructs was measured using the Mander’s overlap coefficient. Values close to 0 and 1 indicate a weak and strong colocalization, respectively. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (***p &lt; 0.001, n = 21–23 cells per group). Supplementary Fig. 8. The NES of sTDP does not prevent it from binding to Nup62 aggregates. Immunofluorescence of HEK293T cells co-expressing Nup62-mCherry with either sTDP or sTDPmNLS ΔNES. Nup62 aggregates do not colocalize with sTDP even after mutating its NLS and NES. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. Supplementary Fig. 9. TDP-CTF PrLD mutations that reduce its association with Nup62 abrogate its interaction and reduced aggregation by KPNB1. a, Immunofluorescence (IF) of HEK293T cells co-expressing Nup62-mCherry with either GFP-tagged TDP-CTFWT or TDP-CTF constructs harboring different mutations in the PrLD. All TDP-CTF mutant variants colocalize with Nup62 aggregates, except for TDP-CTFVLIM-F (green arrowhead) which accumulates in close proximity to Nup62 (red arrowhead). Scale bar: 5 μm. b, Colocalization between Nup62-mCherry and GFP-tagged TDP-CTFWT or TDP-CTFVLIM-F was measured using the Mander’s overlap coefficient. Values close to 0 and 1 indicate a weak and strong colocalization, respectively. Statistical analysis was performed using two-sided Student’s t-test (***p &lt; 0.001, n = 21–22 cells per group). c, IF of HEK293T cells co-expressing Nup62-mCherry with either GFP-tagged wild-type or mutant TDP-43mNLS constructs. RNA-binding deficient TDP-43mNLS (TDP-43mNLS 5F-L) can still colocalize with Nup62. Only TDP-43mNLS VLIM-F aggregates (green arrowhead) do not contain Nup62 (red arrowhead). Scale bar: 5 μm. d, IF of HEK293T cells co-expressing Nup62-mCherry with GFP-FUSΔ14. Cytoplasmic FUS aggregates (green arrowhead) do not colocalize with Nup62 (red arrowhead). Scale bar: 5 μm. e, Protein sequences of TDP-CTFWT and TDP-CTFVLIM-F showing FG repeats (underlined) and VLIM-F mutations (in red) in the PrLD. f, (Left) Lysates from HEK293T cells co-expressing mCherry-KPNB1 with GFP, GFP-tagged TDP-CTFWT or TDP-CTFVLIM-F were subjected to immunoprecipitation with RFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to western blot analysis using indicated antibodies. Introducing VLIM-F mutations in TDP-CTF significantly reduces its binding to KPNB1. (Right) Relative GFP levels in IP normalized by GFP levels in input confirm that GFP-TDP-CTFVLIM-F interacts less with mCherry-KPNB1. Statistical analysis was performed using two-sided Student’s t-test (*p &lt; 0.05, n = 3). g, IF of HEK293T cells co-expressing GFP-tagged TDP-CTFWT or TDP-CTFVLIM-F with mCherry or mCherry-KPNB1. KPNB1 only reduces the size of TDP-CTFVLIM-F aggregates. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. h, Western blot analysis and quantification of insoluble GFP-tagged TDP-CTFWT or TDP-CTFVLIM-F in HEK293T cells expressing mCherry or mCherry-KPNB1. KPNB1 strongly decreased protein levels of insoluble TDP-CTFWT but not TDP-CTFVLIM-F. β-tubulin was used as a loading control. Statistical analysis was performed using two-way ANOVA and Bonferroni’s post hoc test (***p &lt; 0.001, n = 4). Supplementary Fig. 10. Nup98 strongly associates with TDP-CTFWT, but not TDP-CTFVLIM-F and sTDP. Immunofluorescence of HEK293T cells co-expressing GFP-Nup98 with mCherry or mCherry-tagged TDP-43mNLS, TDP-43mNLS 1–265, TDP-PrLD, sTDP, TDP-CTFWT or TDP-CTFVLIM-F. Arrowheads point to co-aggregation. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. Supplementary Fig. 11. KPNB1 H1–8 reduces cytoplasmic Nup62 aggregates. Immunofluorescence of HEK293T cells co-expressing Nup62-GFP with mCherry, mCherry-tagged KPNB1 H1–8WT or H1–8mNIS. KPNB1 H1–8WT suppresses Nup62 aggregates, whereas NIS mutations abolished this activity. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. Supplementary Fig. 12. Multiple Ketel RNAi lines enhance hTDP-43M337V toxicity in the fly eye when expressed under the control of the Gmr-Gal4 driver. Supplementary Fig. 13. Ketel overexpression does not change total levels of human TDP-43 protein in flies. Western blot analysis of hTDP-43 expression in fly brains. hTDP-43 is expressed upon administration of RU486, and Ketel overexpression had no effect on total protein levels of TDP-43. β-actin was used as a loading control. Statistical analysis was performed using one-way ANOVA and Sidak’s post hoc test (n = 3). Supplementary Fig. 14. Survival Kaplan-Meier curves of Elav-GS flies carrying the indicated transgenes in the absence of RU486. Note that all the negative controls yield similar results. Supplementary Fig. 15. The EP-Ketel insertion P{EPgy2}Fs(2)KetEY06666 rescues mutant hTDP-43 toxicity in flies. a, EP-Ketel suppresses TDP-43M337V toxicity in the Drosophila eye. b, EP-Ketel reduces phospho-TDP-43 staining in the adult brain when co-expressed using the Elav-GS driver and exposed to RU486. c, RU486-treated flies carrying the Elav-GS driver, mutant TDP-43 and EP-Ketel (green line) live longer compared to flies carrying the control LacZ transgene (red line). The blue line corresponds to LacZ; Elav-GS Gal4 flies without drug (solvent only) as a negative control. Survival analysis was performed using the OASIS online tool, and p-values were calculated using Fisher’s exact test (n = 100, ****p &lt; 0.0001 for all the genotypes). Supplementary Fig. 16. Downregulation of Ketel exacerbates toxicity of hTDP-43ΔNLS in the fly eye, but Ketel overexpression alleviates it. a, Eye phenotypes from flies expressing the indicated transgenes via the eye-specific Gmr-Gal4 driver. Arrow points to necrotic areas. b, Quantitative analysis of eye severity in flies co-expressing hTDP-43ΔNLS and the indicated transgenes via the eye-specific Gmr-Gal4 driver. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (***p &lt; 0.001, n = 5). Supplementary Fig. 17. The NIR cdm/IPO13 rescues severe eye degeneration in hTDP-43ΔNLS flies. a, Eye phenotypes from flies expressing the indicated transgenes via the eye-specific Gmr-Gal4 driver. Cdm overexpression (OE) rescues hTDP-43-induced neurodegeneration. b, Quantitative analysis of eye severity in flies co-expressing hTDP-43ΔNLS and the indicated transgenes via the eye-specific Gmr-Gal4 driver. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (*p &lt; 0.05, ***p &lt; 0.001, n = 16–23 flies per group). Supplementary Fig. 18. KPNB1 increases the N-to-C ratio of different TDP-43 constructs in rodent primary neurons. Quantitative analysis of the N-to-C ratio of GFP or GFP-tagged TDP-43WT, TDP-43mNLS or TDP-CTF in primary neurons expressing mScarlet or mScarlet-KPNB1 24 h (a) or 48 h (b) post-transfection. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (***p &lt; 0.001, n = 52–264 neurons per group). Supplementary Fig. 19. KPNB1 reduces cytoplasmic aggregation of TDP-CTF and promotes nuclear localization in BSCs. a, Immunofluorescence of mouse BSCs (DIV12) co-expressing AAV-mScarlet-TDP-CTF with AAV-GFP, AAV-GFP-KPNB1 H1–9WT, AAV-FLAG-KPNB1 H1–9WT, H1–9mNIS, H1–8WT, H1–8mNIS or H10–19. KPNB1 H1–9WT and H1–8WT reduce TDP-CTF cytoplasmic aggregates (arrows) and render it very nuclear (arrowheads), whereas NIS mutations strongly reduce this function. Hoechst staining was used to outline nuclei. Scale bar: 50 μm. b, Quantification of the percentage of neurons exhibiting nuclear (N), nucleocytoplasmic (NC) or cytoplasmic (C) mScarlet-TDP-CTF distribution upon expression of different AAV-KPNB1 constructs. Statistical analysis was performed using two-way ANOVA and Bonferroni’s post hoc test (three independent experiments; n = 150–178 neurons per group; statistics are summarized in Supplementary Table 2). Supplementary Fig. 20. Nup62 colocalizes with pTDP-43 aggregates in the spinal cord of TARDBP ALS case. High resolution co-immunofluorescence staining of pTDP-43 (magenta) and Nup62 (green) with Hoechst (blue) was conducted in fixed spinal cord of a neuropathologically diagnosed TARDBP ALS case. pTDP-43 was stained with a 647/Cy5 secondary antibody and Nup62 with a 488/FITC antibody to ensure there is no bleed through between channels. The image is shown as a panel of projected optical sections from each z-series for magenta and green channels and merged with Hoechst DNA staining. Volume rendered z-series of all channels are shown as the 3D inset. Scale bar: 5 μm. Supplementary Fig. 21. KPNB1 expression does not dysregulate nucleocytoplasmic transport. a, Immunofluorescence (IF) of SH-SY5Y cells co-expressing the transport reporter NES-tdTomato-NLS with GFP or GFP-tagged KPNB1 full-length (FL), H1–9 or H1–8. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. b, Quantification of the N-to-C ratio of the NES-tdTomato-NLS reporter. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (*p &lt; 0.05, n = 21–29 cells per group). c, IF of SH-SY5Y cells expressing GFP or GFP-tagged full-length KPNB1 (FL), H1–9 or H1–8 and stained for endogenous Ran and Nup98. Both proteins remain mostly nuclear in presence of KPNB1. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. Supplementary Fig. 22. Expression of the N-terminal half of KPNB1 does not affect the nuclear localization of endogenous TDP-43 in cells and BSCs. a, Immunofluorescence (IF) of HEK293T cells co-expressing GFP with an empty plasmid (ctrl), FLAG-tagged KPNB1 H1–8WT or H1–8mNIS and stained for endogenous TDP-43. TDP-43 remains nuclear in presence of KPNB1 H1–8. Scale bar: 5 μm. b, IF of mouse BSCs (DIV15) expressing AAV-FLAG-KPNB1 H1–9 and stained for endogenous TDP-43. TDP-43 remains nuclear in presence of KPNB1 H1–9. Scale bar: 25 μm. Supplementary Fig. 23. Nup62 and TDP-CTF aggregates are positive for amyloid staining and their co-expression promotes TDP-CTF aggregation. a, Immunofluorescence of HEK293T cells expressing mCherry-TDP-CTF or Nup62-mCherry which form Amylo-Glo-positive aggregates. DRAQ5 staining was used to outline nuclei. Scale bar: 5 μm. b, Western blot analysis of insoluble mCherry-TDP-CTF in HEK293T cells expressing GFP or Nup62-GFP. Nup62 strongly increases insoluble levels of TDP-CTF. Supplementary Table 1. Demographic information of human post-mortem cases used for Nup62 and KPNB1 co-immunostaining with pTDP-43S409/410. Supplementary Table 2. Summary of statistical analyses. Supplementary Table 3. Summary of DNA constructs and sources. Supplementary Table 4. Summary of Drosophila strains and sources. Supplementary Table 5. Summary of reagents and sources.