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Fig. 6 | Molecular Neurodegeneration

Fig. 6

From: Nuclear import receptors are recruited by FG-nucleoporins to rescue hallmarks of TDP-43 proteinopathy

Fig. 6

KPNB1 promotes nuclear localization of TDP-43mNLS in primary neurons and mouse brain tissue. a Immunofluorescence (IF) of primary cortical neurons co-expressing NLS-mTagBFP (nuclear marker), HaloTag (cell marker), GFP or GFP-tagged TDP-43WT/TDP-43mNLS/TDP-CTF and mScarlet/mScarlet-KPNB1. KPNB1 increased the nuclear localization of TDP-43mNLS and TDP-CTF. Scale bar: 25 μm. b 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 72 h post-transfection. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (**p < 0.01, ***p < 0.001, n = 52–264 neurons per group). c IF of HEK293T cells co-expressing GFP-TDP-43mNLS with mCherry or mCherry-tagged KPNB1 full-length (FL) or its fragments. KPNB1 FL, H1–9 and H1–8 constructs increase the nuclear localization of TDP-43mNLS. Arrowheads point to co-aggregation. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. d Quantification of the percentage of HEK293T cells exhibiting nuclear (N), nucleocytoplasmic (NC) or cytoplasmic (C) GFP-TDP-43mNLS distribution upon expression of different mCherry-KPNB1 constructs. Statistical analysis was performed using two-way ANOVA and Bonferroni’s post hoc test (three independent experiments; n = 66–81 cells per group; statistics are summarized in Supplementary Table 2). e IF of mouse brain slice cultures (DIV12) co-expressing AAV-GFP-TDP-43mNLS with AAV-FLAG-mCherry or AAV-FLAG-KPNB1 H1–9WT, H1–9mNIS, H1–8WT, H1–8mNIS or H10–19. KPNB1 H1–9WT and H1–8WT increase the nuclear localization of TDP-43mNLS, whereas NIS mutations abolished this effect. Hoechst staining was used to outline nuclei. Scale bar: 50 μm. f Quantification of the percentage of neurons exhibiting nuclear (N), nucleocytoplasmic (NC) or cytoplasmic (C) GFP-TDP-43mNLS 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 = 150–154 neurons per group; statistics are summarized in Supplementary Table 2)

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