Journal: Communications Biology

Article Title: The nuclear import receptor Kapβ2 modifies neurotoxicity mediated by poly(GR) in C9orf72-linked ALS/FTD

doi: 10.1038/s42003-024-06071-2

Figure Lengend Snippet: a qPCR analysis of Kapβ2 levels in cortical neurons transduced with GFP or GR 50 . Data are represented as mean ± S.E.M. (n = 6 biological replicates, Student t -test, n.s. not significant). b Homogenates of cortical neurons transduced with GFP or GR 50 were resolved on western blot (WB) and probed for GFP and Kapβ2. Total protein staining was used as the loading control. For cortical neuron transduction experiments, we employed a lentivirus expression vector that encodes GR 50 with Flag and GFP tags fused to the dipeptide N- and C-terminus, respectively. c The graph bar shows the quantification of the WB of cortical neuron extracts for Kapβ2 protein. Total protein staining was used to normalize. Data are represented as mean ± S.E.M. (n = 3 biological replicates, Student t -test, n.s. not significant). d qPCR analysis of Kapβ2 transcript levels in the cortex and spinal cord of GFP and GR 50 -GFP mice. Data are represented as mean ± S.E.M. (n = 3 biological replicates, One Way-ANOVA, n.s.) e Western blot of cortex and spinal cord homogenates from GFP and GR 50 mice. GR 50 is Flag and GFP tagged at the N- and C-termini, respectively. Blots were probed for GFP and Kapβ2. Total protein staining was used as the loading control. f The graph bar shows Kapβ2 protein quantification of the WB of cortex and spinal cord homogenates. Total protein staining was used as a loading control and normalizer. Data are represented as mean ± S.E.M. (n = 3 biological replicates, One Way-ANOVA, n.s.). g Western blot of controls and C9orf72 patient post-mortem cortex extracts. Total protein staining was used as the loading control. h Quantification of Kapβ2 expression levels in controls and C9orf72 patient post-mortem cortex extracts as normalized protein fold change. Data are represented as mean ± S.E.M. (n = 3 biological replicates, Student t -test, n.s. not significant).

Article Snippet: We then performed Kapβ2 immunofluorescence staining in cerebral cortex and spinal cord sections of the GR 50 transgenic mouse employing Imaris software to perform 3D rendering and visualize the Kapβ2 localization in the cortex and spinal cord neurons.

Techniques: Transduction, Western Blot, Staining, Expressing, Plasmid Preparation

Journal: Communications Biology

Article Title: The nuclear import receptor Kapβ2 modifies neurotoxicity mediated by poly(GR) in C9orf72-linked ALS/FTD

doi: 10.1038/s42003-024-06071-2

Figure Lengend Snippet: a Confocal images of rat cortical neurons in culture transduced with GFP and GR 50 and stained for Kapβ2 and Map2. Imaris colocalization channel shows the amount of GFP colocalized with Kapβ2. Scale bar = 10 μm. b Pearson’s coefficient measured in each neuron transduced with the lentivirus GR 50 construct (Flag-GR 50 -GFP). Data are represented as mean ± S.E.M. (n = 3 biological replicates, m > 5 neurons, Student t -test, * P < 0.05). c Confocal images of the cortex and spinal cord tissues of GR 50 mice stained for Kapβ2. Green is GFP reporting for GR 50 , red is Kapβ2, and magenta is NeuN. Imaris colocalization shows the amount of GFP colocalized with Kapβ2. Scale bar = 30 μm. d Pearson’s coefficient measured in the cortex and spinal cord neurons of GR 50 mice. Data are represented as mean ± S.E.M. (n = 3 biological replicates, m > 15 neurons). e Western blot analysis of the immunoprecipitation assay was performed in rat cortical neurons transduced with GR 50 and treated with arsenite (0.5 mM; 30 min). Blots were probed for GFP and Kapβ2. Total protein staining was used as the loading control. SE short exposure time, LE long exposure time. f Western blot of the immunoprecipitation assay performed in the cortex or spinal cord tissue of nTg or GR 50 mice. Blots were probed for GFP and Kapβ2. Total protein staining was used as the loading control. SE short exposure time, LE long exposure time.

Article Snippet: We then performed Kapβ2 immunofluorescence staining in cerebral cortex and spinal cord sections of the GR 50 transgenic mouse employing Imaris software to perform 3D rendering and visualize the Kapβ2 localization in the cortex and spinal cord neurons.

Techniques: Transduction, Staining, Construct, Western Blot, Immunoprecipitation

Journal: Communications Biology

Article Title: The nuclear import receptor Kapβ2 modifies neurotoxicity mediated by poly(GR) in C9orf72-linked ALS/FTD

doi: 10.1038/s42003-024-06071-2

Figure Lengend Snippet: a Western blot showing Kapβ2 expression in neurons treated with Smart pool siRNA (100 μM). Blots were probed for Kapβ2. Total protein staining was used as the loading control. b Kapβ2 levels in primary neurons treated with Smart pool siRNA 100 μM or scramble. Data are represented as mean ± S.E.M. (n = 3 biological replicates, Mann-Whitney non parametric test, P < 0.05). c The plot depicts the probability of neuronal death in each group via a cumulative risk of death plot. Time-lapse experiments on rat primary cortical neurons transfected with siRNA Kapβ2 100 μM or scramble and GFP, GR 50 -GFP, or GR 100 -GFP. (n = 3 biological replicates, m > 50 neurons, Log-rank Mantel-Cox test * p < 0.05, *** p < 0.001). d Quantification of the mean fluorescence of GFP expressing neurons 48 h after transfection in cells previously treated with Kapβ2 siRNA or scramble. Data are represented as mean ± S.E.M. (n = 3 biological replicates, m > 10 neurons, One-Way ANOVA, p = n.s. not significant). e Quantification of the mean nuclear fluorescence of GFP expressing cells 48 h after transfection in cells previously treated with Kapβ2 siRNA or scramble. Data are represented as mean ± S.E.M. (n = 3 biological replicates, m > 10 neurons, One-Way ANOVA, p = n.s. not significant).

Article Snippet: We then performed Kapβ2 immunofluorescence staining in cerebral cortex and spinal cord sections of the GR 50 transgenic mouse employing Imaris software to perform 3D rendering and visualize the Kapβ2 localization in the cortex and spinal cord neurons.

Techniques: Western Blot, Expressing, Staining, MANN-WHITNEY, Transfection, Fluorescence

Journal: Communications Biology

Article Title: The nuclear import receptor Kapβ2 modifies neurotoxicity mediated by poly(GR) in C9orf72-linked ALS/FTD

doi: 10.1038/s42003-024-06071-2

Figure Lengend Snippet: a Left: Confocal imaging of primary neurons transfected with GFP-Kapβ2. Arrows indicate cytoplasmic puncta. Green is GFP, and blue is DAPI. Scale bar = 5 μm. Center: Imaris 3D rendering of GFP-Kapβ2 expression in primary rat cortical neurons. Right: Magnification of Imaris’ rendering of GFP-Kapβ2 puncta around the nucleus. Scale bar = 3 μm. b Probability of neuronal death via a cumulative risk of death plot of rat primary cortical neurons transfected with 200/400 ng of Tm + / GFP-Kapβ2 + (GFP used as control) per 150,000 cells. Neurons double positive Tm + /GFP + were counted (n = 3 biological replicates, m > 150 neurons, Log-rank Mantel-Cox test: n.s. not significant). c Representative images of neurons over time transfected with 200/400 ng of Tm + /GFP + . Green is Kapβ2; red is Td-Tomato. Scale bar = 20 μm. d Probability of neuronal death via a cumulative risk of death plot of rat primary cortical neurons transfected with 400/400 ng of GFP-Kapβ2 (GFP alone used as control) and GR 50 -mCherry (mCherry alone used as control) per 150,000 cells. Neurons double positive GFP + /mCherry + were counted (n = 3 biological replicates, m > 150 neurons, Log-rank Mantel-Cox test: mCherry vs GR 50 -mCherry p < 0.0001; GR 50 -mCherry vs GR 50 -mCherry /GFP-Kapβ2 p < 0.0001). Inset: bar graph showing quantification of GR 50 -mCherry and GR 50 -mCherry /GFP-Kapβ2 overall survival normalized to mCherry 7 days post-transfection, n = 3 biological replicates. Data are represented as mean ± S.E.M. e Probability of neuronal death via a cumulative risk of death plot of rat primary cortical neurons transfected with 400/400 ng of GFP-Kapβ2 + (GFP alone used as control) and GR 100 -mCherry (mCherry alone used as control) per 150,000 cells. Neurons double positive GFP + /mCherry + were counted (n = 3 biological replicates, m > 150 neurons, Log-rank Mantel-Cox test: mCherry vs GR 100 -mCherry p < 0.0001; GR 100 -mCherry vs GR 100 -mCherry/Kapβ2 p = 0.005). Inset: bar graph showing quantification of GR 100 -mCherry and GR 100 -mCherry /GFP-Kapβ2 overall survival normalized to mCherry at 7 days post-transfection, n = 3 biological replicates. Data are represented as mean ± S.E.M.

Article Snippet: We then performed Kapβ2 immunofluorescence staining in cerebral cortex and spinal cord sections of the GR 50 transgenic mouse employing Imaris software to perform 3D rendering and visualize the Kapβ2 localization in the cortex and spinal cord neurons.

Techniques: Imaging, Transfection, Expressing

Journal: Communications Biology

Article Title: The nuclear import receptor Kapβ2 modifies neurotoxicity mediated by poly(GR) in C9orf72-linked ALS/FTD

doi: 10.1038/s42003-024-06071-2

Figure Lengend Snippet: a Confocal analysis of neurons overexpressing GFP or Kapβ2 and GR 50 -mCherry or GR 100 -mCherry. The top row shows an x-y-z view, while the bottom row shows the maximum intensity projection of the Z plans acquired. Green is GFP, red is mCherry, and blue is DAPI. The white line represents the length over which GFP and mCherry fluorescence were analyzed. Scale bar = 10 μm. b Graph showing intensity profiles of GFP or mCherry fluorescence (a.u.) over distance (μm). c The bar graph shows quantification of GR 50 -mCherry and GR 100 -mCherry nuclear intensity Data are represented as mean ± S.E.M. Only double positive cells were considered. (n = 3 biological replicates, m = at least 30 neurons/group, One-Way ANOVA, Tukey’s multiple comparisons test, n.s. not significant). d The bar graph shows the quantification of GR 50 -mCherry and GR 100 -mCherry mean fluorescence intensity in GR-positive granules. Data are represented as mean ± S.E.M. Only double positive cells were considered. (n = 3 biological replicates, m = at least 30/group neurons, One-Way ANOVA, Tukey’s multiple comparisons test, n.s. not significant). e The bar graph shows the quantification of the GR 50 -mCherry and GR 100 -mCherry aggregates per cell. Data are represented as mean ± S.E.M. Only double positive cells were considered. (n = 3 biological replicates, m = at least 30 neurons/group, One-Way ANOVA, Tukey’s multiple comparisons test, n.s. not significant). f Pearson’s coefficient analysis of GFP-Kapβ2 and GR 50 -mCherry or GR 100 -mCherry. Data are represented as mean ± S.E.M. (n = 3 biological replicates, m = 9–10 neurons/group, Student t -test, P < 0.05).

Article Snippet: We then performed Kapβ2 immunofluorescence staining in cerebral cortex and spinal cord sections of the GR 50 transgenic mouse employing Imaris software to perform 3D rendering and visualize the Kapβ2 localization in the cortex and spinal cord neurons.

Techniques: Fluorescence

Journal: Communications Biology

Article Title: The nuclear import receptor Kapβ2 modifies neurotoxicity mediated by poly(GR) in C9orf72-linked ALS/FTD

doi: 10.1038/s42003-024-06071-2

Figure Lengend Snippet: a Representative images of the GR 50 -mCherry bleached aggregates at the start (0 s), bleaching (5 s), and end (180 s). Scale bar = 10 μm. b Representative images of the GR 100 -mCherry bleached aggregates at the start (0 s), bleaching (5 s), and end (180 s). Scale bar = 10 μm. c The graph shows fluorescence recovery over time for GR 50 -mCherry and GR 100 -mCherry. Data are represented as mean ± S.E.M. (n = 3 biological replicates, m > 5 neurons, Student t -test, P < 0.001). d Representative images of the GFP-Kapβ2/GR 50 -mCherry bleached aggregates at the start (0 s), bleaching (5 s), and end (180 s). Scale bar = 10 μm. e Representative images of the GFP-Kapβ2/ GR 100 -mCherry bleached aggregates at the start (0 s), bleaching (5 s), and end (180 s). Scale bar = 10 μm. f The graph shows fluorescence recovery over time for GR 50 -mCherry and GFP-Kapβ2/GR 50 -mCherry. Data are represented as mean ± S.E.M. (n = 3 biological replicates, m > 5 neurons). g The graph shows fluorescence recovery over time for GR 100 -mCherry and GFP-Kapβ2/GR 100 -mCherry. Data are represented as mean ± S.E.M. (n = 3 biological replicates, m > 5 neurons). h A table summarizing mean and S.E.M. of the mobile fraction, t1/2 (tau), and the significance level for the FRAP curve profiles presented in ( f ) and ( g ); n.s. not significant.

Article Snippet: We then performed Kapβ2 immunofluorescence staining in cerebral cortex and spinal cord sections of the GR 50 transgenic mouse employing Imaris software to perform 3D rendering and visualize the Kapβ2 localization in the cortex and spinal cord neurons.

Techniques: Fluorescence

Journal: Communications Biology

Article Title: The nuclear import receptor Kapβ2 modifies neurotoxicity mediated by poly(GR) in C9orf72-linked ALS/FTD

doi: 10.1038/s42003-024-06071-2

Figure Lengend Snippet: a Epifluorescence images of condensate formation in vitro in a test tube containing equimolar concentration (5 μM) ofTDP-43, GR 20, and Kapβ2. Green is TDP-43, and red is GR 20 . Scale bar = 15 μm. b Quantification of the size of TDP-43/GR 20 aggregates in the presence or absence of Kapβ2. Data are represented as mean ± S.E.M. (n = 3 biological replicates, Student t -test, P < 0.01). c Ratio between the mean GFP intensity inside and outside TDP-43/GR 20 aggregates in the presence or absence of Kapβ2. Data are represented as mean ± S.E.M. (n = 3 biological replicates, Student t -test, P < 0.0001). d Confocal analysis of neurons overexpressing GFP or Kapβ2 and GR 50 -mCherry (top row) or GR 100 -mCherry (bottom row). Scale bar = 5 μm. e Single-nuclei TDP-43 staining quantification. Data are represented as mean ± S.E.M. Only double positive cells were considered. (n = 3 biological replicates, m = at least 9 neurons/group, Student t -test, P < 0.01; P < 0.001). f TDP-43 quantification into GR aggregates. Data are represented as mean ± S.E.M. Only double positive cells were considered. (n = 3 biological replicates, m = at least 30 neurons/group, Student t -test, P < 0.01; P < 0.001).

Article Snippet: We then performed Kapβ2 immunofluorescence staining in cerebral cortex and spinal cord sections of the GR 50 transgenic mouse employing Imaris software to perform 3D rendering and visualize the Kapβ2 localization in the cortex and spinal cord neurons.

Techniques: In Vitro, Concentration Assay, Staining