DOI: 10.5061/dryad.v41ns1s4f
title: The nuclear import receptor Kapβ2 modifies neurotoxicity mediated by poly(GR) in C9orf72-linked ALS/FTD

description: Expanded intronic G4C2 repeats in the C9ORF72 gene cause amyotrophic
 lateral sclerosis (ALS) and frontotemporal dementia (FTD). These intronic
 repeats are translated through a non-AUG-dependent mechanism into five
 different dipeptide repeat proteins (DPRs), including
 poly-glycine-arginine (GR), which is aggregation-prone and neurotoxic.
 Here, we report that Kapβ2 and GR interact, co-aggregating, in cultured
 neurons in vitro and CNS tissue in vivo. Importantly,
 this interaction significantly decreased the risk of death of cultured
 GR-expressing neurons. Downregulation of Kapβ2 is detrimental to their
 survival, whereas increased Kapβ2 levels mitigated GR-mediated
 neurotoxicity. As expected, GR-expressing neurons displayed TDP-43 nuclear
 loss. Raising Kapβ2 levels did not restore TDP-43 into the nucleus, nor
 did alter the dynamic properties of GR aggregates. Overall, our findings
 support the design of therapeutic strategies aimed at up-regulating Kapβ2
 expression levels as a potential new avenue for contrasting
 neurodegeneration in C9orf72-ALS/FTD.
description: <em>Human tissues: </em>Human post-mortem CNS tissue
 from healthy controls and patients carrying
 the <em>C9ORF72</em> intronic nucleotide repeat expansion was
 obtained from the Target ALS and the Jefferson Weinberg ALS center
 biobanks. Information and demographics of patients and controls are
 presented in Table 4. Human post-mortem tissues (fresh frozen) were stored
 at -80 C. <em>Cell cultures: </em>HEK293
 cells were cultured in DMEM medium (Cytiva Cat.# SH30243.LS ) supplemented
 with 10% FBS (Cytiva Cat.# SH30071.01HI), penicillin, and streptomycin
 (Thermo Fisher Scientific Cat.# SV30010). Cells were passaged every 3-4
 days using 0.05% trypsin (Corning Cat.# 25-051-C).
 <em>Primary cortical neurons: </em>After meninges
 removal, brains were dissected from embryonic day 16 (E16) rat embryos.
 Cortices and midbrain regions were cut into small pieces and incubated on
 a shaker at 80 rcf for 45 min at 37°C in 0.2% trypsin in HBSS without
 Ca<sup>2+</sup> and Mg<sup>2+</sup> (Cytiva Cat.#
 SH30588.01). FBS (Cytiva Cat.# SH30071.01HI) was added, and the cell
 suspension was centrifuged at 800 rcf for 10 min. at 4°C. Cells were
 washed with Ca<sup>2+</sup> and Mg<sup>2+</sup>
 free HBSS and centrifuged at 800 rcf for 10 min. at 4°C. The cell
 suspension was then passed through a 70 mm strainer (Foxx Life Sciences
 Cat.# 410-0002-OEM) to remove undigested connective tissue and large cell
 clumps. Dispersed cells were then counted and plated in poly-D
 lysine-coated plates. The following plating densities were used: 150,000
 cells/well in 24 well plates, 300,000 cells/well in 12 well plates, and
 5,000,000 cells/dish in 10 cm dishes. Arsenite 0.5mM was applied for 30
 min. immediately before processing the neurons for analysis. DSP
 (dithiobis(succinimidyl propionate)) was added to the neurons before
 harvesting at a concentration of 0.1mM to the neurons in culture before
 processing them for western blot analysis.
 <em>Animals: </em>B6.C-Tg(CMV-cre)1Cgn/J::B6.C-GR50
 (GR<sub>50</sub>) or B6.C-Tg(CMV-cre)1Cgn/J::B6.C-GFP (GFP)
 mice were generated as described. At 12 months, male mice were sacrificed,
 and cortices and spinal cords were collected and frozen in liquid
 N<sub>2</sub> or embedded in OCT. Frozen samples were stored
 at -80°C till the time of processing. All animal procedures were compliant
 with the ARRIVE guidelines and approved by IACUC. We have complied with
 all relevant ethical regulations for animal use.
 <em>Plasmids and siRNA: </em>The following plasmids
 were used: pCDNA3_hSyn_Td_Tomato; pCDNA3_hU6_Flag_mCherry;
 pCDNA3_hU6_Flag_GR<sub>50</sub>_mCherry;
 pCDNA3_hU6_Flag_GR<sub>100</sub>_mCherry; pCDNA3_CMV_eGFP;
 pCDNA3_CMV_eGFP_Kapβ2. eGFP and eGFP_Kapβ2 were co-transfected with
 Td_Tomato at a DNA concentration ration of 1:2 (200ng/400ng) or 1:4
 (200ng/800ng). eGFP and eGFP_Kapβ2 were co-transfected with mCherry,
 mCherry_GR<sub>50</sub> and
 mCherry_GR<sub>100</sub> at 1:1 ratio (400ng/400ng). Human
 TDP-43 and TDP-43-GFP were subcloned into pE-SUMO (LifeSensors, Malvern,
 PA) as described (McGurk et al., 2018). All plasmid inserts were sequenced
 and confirmed to be correct. To silence Kapβ2, 50 mM of ON-TARGETplus
 siRNA-TNPO1-Smart Pool (Horizon Discovery, L-086861-02-0005) was used.
 50mM of AllStars Negative Control scramble siRNA (Qiagen Cat. #1027280)
 was used as control. <em>Transfection:
 </em>Lipofectamine 2000 (Fisher Scientific Cat. #11-668-019) was
 used to transfect HEK cells and cortical neurons (1 mL Lipo2000/500ng
 total D.N.A.). HiPerfect Transfection Reagent (Qiagen Cat. #301705) was
 used to transfect siRNA (8mL HiPerfect/50nM siRNA). siRNA was transfected
 24 hours before plasmids transfection. For cortical neurons, Lipo/DNA or
 RNA mix was incubated with the neurons for 1h, and then cell media were
 changed with fresh ones. For HEK293 cells, the mixture was incubated with
 the cells overnight. <em>Lentivirus constructs
 production: </em>HEK293 cells were transfected with the following
 plasmids: pLenti_hSyn_Flag_GR<sub>50</sub>_eGFP, psPAX2
 (Addgene Plasmid #12260), pMD2.G (Addgene Plasmid #12259). Plasmids were
 diluted in DMEM, and PEI MAX was added as a transfection reagent. The mix
 was incubated with HEK293 cells confluent at 70% for 5h; the media was
 changed with a fresh one. Cells supernatant was collected 48h after
 transfection and centrifuged for 10 min at 2,000 rcf at 4°C. Lentivirus
 Concentrator (OriGene Technologies Cat. #TR30025) was added to the
 supernatant and incubated overnight at 4°C. To pellet the virus, the media
 was centrifuged at 500 rcf for 45 min at 4°C. Viral particles were then
 resuspended in Ca<sup>2+</sup> and Mg<sup>2+</sup>
 free PBS (Cytiva Cat. #SH30028. L.S.), aliquoted, and immediately frozen
 in liquid N<sub>2</sub>.
 <em>Purification of Recombinant Proteins:
 </em>His<sub>6</sub>-SUMO1 N-terminally tagged TDP-43-WT
 and TDP-43-GFP were purified as described (McGurk et al., 2018) and
 overexpressed in BL21(DE3) RIL <em>E.coli</em> cells. These
 cells were then sonicated on ice in 50 mM HEPES (pH 7.5), 2% TritonX-100,
 300 mM NaCl, 30 mM imidazole, 5% glycerol, 2 mM β-mercaptoethanol, and
 protease inhibitors (cOmplete, EDTA-free, Roche). The recombinant TDP-43
 proteins were purified over Ni-NTA agarose beads (Qiagen) and eluted using
 50 mM HEPES (pH 7.5), 150 mM NaCl, 300 mM imidazole, 5% glycerol, and 5 mM
 DTT. The buffer was exchanged with the same buffer without imidazole. The
 eluate containing the recombinant TDP-43 proteins was then frozen in
 liquid N<sub>2</sub> and stored as aliquots at -80°C until
 use. Kapβ2 was purified as described (Guo et al., 2018). In
 brief, <em>E. coli</em> BL21-CodonPlus(DE3)-RIL cells
 (Agilent) were transformed with GST-Tev-Kapβ2 plasmid, and expression was
 induced overnight at 25°C with 1 mM IPTG. Cells were pelleted and
 resuspended in Tris buffer (50 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA, 20%
 (v/v) glycerol, 2 mM DTT, supplemented with protease inhibitors), then
 lysed by sonication. Cell lysate was then loaded onto glutathione
 Sepharose<sup>TM</sup> 4 Fast Flow resin (GE Healthcare) and
 washed with Tris buffer, followed by ATP buffer (50 mM Tris pH 7.5, 100 mM
 NaCl, 1 mM EGTA, 0.5 mM MgCl<sub>2</sub>, 5 mM ATP, 20%
 glycerol, 2 mM DTT, supplemented with protease inhibitors), then washed
 and eluted with Buffer A (20 mM imidazole, 75 mM NaCl, 1 mM EDTA, 20%
 (v/v) glycerol, 2 mM DTT). Finally, the protein was cleaved with Tev
 protease and purified on a HiTrap Q HP column (GE Healthcare) using a salt
 gradient. Purified protein was concentrated, flash-frozen, and stored at
 -80°C. Before the aggregation assay, the protein was thawed and
 centrifuged at 16,100 x<em> g</em> for 10 min to remove any
 preformed aggregates. Protein concentration was determined by Bradford
 assay (Bio-Rad, Hercules, CA). Equimolar concentrations of Kapβ2, TDP-43,
 and GR<sub>20</sub> (5mM) were used. 200nM
 HIS<sub>6</sub>-SUMO-TDP-43-GFP and 100nM
 TAMRA-GR<sub>20</sub> were added to visualize TDP-43 and
 GR<sub>20</sub>. <em>Peptides:
 </em>Chemically synthesized GR<sub>20</sub> and
 Tetramethylrhodamine (TAMRA)-tagged GR<sub>20 </sub>were
 obtained as a lyophilized powder from Peptide2.0 and dissolved in
 PBS. <em>Sample preparation: </em>100 mg of
 fresh frozen lumbar spinal cord samples were homogenized in 1% SDS using a
 15ml Dounce Homogenizer. The homogenate was centrifuged at 3000 rcf for 20
 minutes at 4°C to remove tissue debris. Protein estimation was performed
 on the clear homogenate using a BCA assay (Pierce BCA kit #23225). 30 mg
 of protein samples are loaded for Western blot analysis.
 For mouse tissues: 50 mg of CNS tissues were chopped into small
 pieces, incubated in RIPA for 2 h, and homogenized by pipetting every
 20/25 minutes. Samples were then sonicated using Bioruptor
 Pico-Diagenode. For cell cultures: Cells were harvested
 in PBS and centrifuged at 500 rcf for 3 minutes. PBS was removed by
 suction, and samples were resuspended in 80 mL of RIPA with protease
 inhibitor (Sigma-Aldrich Cat# P2714-1BTL). Cells were incubated for 15
 minutes at 4°C on an orbital shaker and then sonicated. After sonication,
 samples were centrifuged at 16,000 rcf for 5 minutes at 4°C. The
 supernatant was kept, and BCA was used to measure the protein content of
 each sample. <em>Western blot: </em>Cell
 cultures and animal tissues: 15mg of protein extracts were loaded on
 pre-cast Stain Free gels (Bio-Rad Cat.#4568034) and run for 1hr at 100V.
 Gels were then transferred on PVDF membrane (Millipore Cat.#IPVH00010)
 overnight at 4°C at 30V and developed with primary and secondary
 antibodies at the indicated concentration. Before transfer, gels were
 cross-linked by UV light exposure for 5 minutes, and total protein content
 was then revealed on the membrane by UV light exposure. Membranes were
 developed with SuperSignal™ West Femto Maximum Sensitivity Substrate
 (Thermo Fisher Scientific Cat#34094) and imaged using ChemiDOC XRS+ System
 (Bio-Rad). Human samples: The gels were UV-activated
 for 5 minutes before transferring onto the 0.22 mm nitrocellulose
 membrane. The semi-dry transfer was done using a Trans-Blot Turbo Transfer
 System (Bio-Rad) at 25mV for 10 minutes. After the transfer, a stain-free
 blot image was captured and used as total protein content. The blot was
 processed as previously described. The quantification was done using Image
 Lab Software (Bio-Rad); the intensity of the Kapβ2-positive band is
 normalized to its total proteins. Quantification was performed using Image
 Lab software. Adjusted volume was measured for each band and divided by
 the adjusted volume calculated, considering the entire lane of total
 proteins. The abundance of the protein of interest was normalized to the
 total amount of proteins in each lane, therefore removing variations
 associated with comparing abundance to a single protein arbitrarily chosen
 as housekeeping protein (e.g. GAPDH). Total protein normalization is more
 compatible with detecting and quantifying proteins of lower
 abundance. <em>Immunoprecipitation:
 </em>Flag: Immunoprecipitation was carried out using anti-Flag
 agarose beads (mouse IgG agarose beads were used as control). Rat primary
 cortical neurons and tissues were extracted in IP buffer containing 50 mM
 Tris-HCl (pH 7.5), 150 mM NaCl, 1mM EDTA, 0.5% NP-40, 100 mM EDTA (pH 8.0)
 supplemented with protease inhibitor (Sigma-Aldrich Cat.# P2714-1BTL).
 After incubating at 4⁰C for 30 min., the samples were sonicated and then
 centrifuged for 10 min. at 10,000 rcf. Bradford assay was used to
 determine protein concentration. The agarose beads were added to 600 mg of
 cell lysates and incubated at 4°C for 24h under constant shaking. Cell
 lysates were centrifuged for 1 min. at 1,000 rcf to collect the
 supernatant. The pellets were washed five times, but only the first wash
 was collected. After the last centrifugation, the samples were eluted with
 SDS-PAGE loading dye at 95°C for 5 min. The entire eluate was run for
 Western blot. 2% of total lysate was used as input. GFP
 and Kapβ2: Following manufacturer instructions, immunoprecipitation was
 carried out using magnetic beads (Thermo Fisher Scientific Cat.# 88804).
 Briefly, 25 mL of magnetic beads were conjugated with 10 mg of the desired
 antibody overnight and then crosslinked for 30 minutes using DSS
 (disuccinimidyl suberate). The obtained beads-antibody complex was
 incubated with cell or tissue lysates overnight. The next day, beads were
 washed, and the bound protein was eluted. The entire eluate was run for
 Western blot. 2% of total lysate was used as input. For Kapβ2 IP, a double
 volume of beads was used. Quantification: The signal
 specifically corresponding to Kapβ2 and GFP bands was measured with
 ImageLab and the ratio was calculated between the respective adjusted
 volumes. <em>Immunofluorescence:
 </em>Primary cortical neurons were fixed for 20 minutes at 37°C with
 4% PFA and incubated in PermBlock solution (2% BSA, 0.3% Triton-x, 5%
 donkey serum in PBS). Primary antibodies were diluted in 0.1% BSA cells
 and were incubated overnight at 4°C on an orbital shaker. The following
 day, cells were washed two times with PBS at room temperature for 10
 minutes and incubated with the appropriate secondary antibody for 1h at
 room temperature. Cells were washed twice in PBS and incubated with
 Hoechst solution (Thermo Fisher Scientific Cat.# 62249) for 10 minutes at
 room temperature to stain the nuclei. After a final rinse in PBS,
 coverslips were mounted on glass slides using AquaMount (Lerner
 Laboratories Cat.# 13800). Tissues were cryo-sectioned using Cryostar
 NX50. 20 mm thick sections were placed on glass slides and incubated for
 10 minutes in 4% PFA. Slides were then washed in TBS-T for 10 minutes and
 incubated for 1h at room temperature in PermBlock solution (2% BSA, 0.3%
 Triton-x, 5% donkey serum in PBS) and then incubated with primary antibody
 diluted in 1% BSA, 0.3% Triton-x and 1% donkey serum overnight at 4°C in a
 humidified chamber. The following day, slides were washed with TBS-T,
 incubated with the appropriate fluorescent secondary antibody for 1 hour
 at room temperature, rinsed twice in TBS-T, incubated for 10 minutes with
 Hoechst (Thermo Fisher Scientific Cat.# 62249), and mounted on coverslips
 using AquaMount (Lerner Laboratories Cat.# 13800) for imaging
 analysis. <em>Antibodies and dilutions:
 </em>Primary antibodies: anti-Kapβ2 (RRID:AB_2206884, Santa Cruz
 Biotechnology, Inc Cat.# sc-101539; WB dilution 1:1000); anti-Kapβ2
 (RRID:AB_262123, Sigma Cat.# T0825; IF dilution 1:500); anti-Map2
 (RRID:AB_2138178 Novus Cat.#NB300-213; IF dilution 1:2000); anti-GFP
 (RRID:AB_11042881, Proteintech Cat.#50430-2-AP, WB dilution 1:1000);
 anti-TDP-43 (RRID:AB_615042, Proteintech Cat.#10782-2-AP; IF dilution
 1:500); anti-GAPDH (RRID:AB_2107436, Proteintech Cat.#60004-1-Ig; WB
 dilution 1:1000); Neun (D4G40) XP (RRID:AB_2651140, Cell Signaling
 Technology Cat.#24307; IF dilution 1:500): FUS (RRID:AB_2247082,
 Proteintech Cat.#11570-1-AP; IF:1:500) Secondary
 HRP-conjugated antibodies: HRP-Conjugated anti-rabbit (RRID:AB_772191,
 Cytiva Cat.# NA9340-1ML; WB dilution 1:10,000); HRP-Conjugated anti-mouse
 (RRID:AB_772193, Cytiva Cat.# NA9310-1ML; WB dilution 1:10,000);
 HRP-Conjugated anti-rat (RRID:AB_2936877, Sigma Cat.# AP136P; WB dilution
 1:3,000); Secondary fluorescent antibodies:
 AlexaFluor-546 anti-mouse (RRID:AB_2534012, Thermo Fisher Scientific Cat.#
 A10036; IF dilution 1:1,000); AlexaFluor-647 anti-rabbit (RRID:AB_2536183,
 Thermo Fisher Scientific Cat.# A31573; IF dilution 1:1,000);
 AlexaFluor-647 anti-chicken (RRID:AB_2762845, Thermo Fisher Scientific
 Cat.# A32933; IF dilution 1:1,000).
 <em>Extraction of total RNA and quantitative Real-Time PCR:
 </em>Total RNA was extracted using Trizol Reagent (Ambion Cat.#
 15596026) following the manufacturer’s instructions. Briefly, cells were
 lysed in Trizol Reagent and homogenized using a syringe for insulin. RNA
 was then extracted in isopropanol. Samples were centrifuged for 15 min. at
 12,000 rcf. The RNA-containing pellet was washed with 75% ethanol and
 centrifuged for 5 min. at 7,500 rcf. Dried pellets were resuspended in
 DEPC water, and concentration was assessed with Nanodrop. 500ng of total
 RNA was first treated with DNAse I (Thermo Fisher Scientific Cat.# EN0521)
 and retrotranscribed using SuperScript™ IV Reverse Transcriptase (Thermo
 Fisher Scientific Cat.# 18090010). The cDNA was diluted 1:2 and used for
 qRT-PCR, which was performed using the following TaqMan assay: GAPDH-VIC
 (Thermo Fisher Scientific Cat# 4352338E) and Kapβ2-FAM (Thermo Fisher
 Scientific Cat.# 4331182 Assay# Rn01489969_m1). 50ng of cDNA was used per
 reaction. Samples were analyzed using the Design and Analysis tool by
 Thermo Fisher Scientific. Results are shown as fold change using the
 2<sup>DDCt</sup> method. <em>Confocal
 Imaging Analysis: </em>Confocal images were taken on a Nikon A1R
 confocal microscope with a 60X objective. Z-stacks were taken for each
 image. 0.2 mm Z-stacks were taken to perform colocalization studies and
 puncta counting using a built-in function in Imaris. One mm stacks were
 taken for TDP-43 nuclear intensity studies. Maximum intensity projection
 was then generated through a built-in function of the A1R analysis
 software. The nuclei of each co-transfected cell were selected as the
 region of interest, and TDP-43 intensity was assessed. The mask was made
 on the mCherry channel to analyze aggregate content, and the TDP-43
 fluorescence was subsequently evaluated. For Kapb2 nuclear and cytoplasmic
 quantification maximum intensity projections were generated, and ROI were
 drawn around nucleus and cytoplasm and intensity referred to the Kapβ2
 chnnel was measured. The ratio between nuclear and cytoplasmic intensities
 in the same cell was then calculated. FRAP experiment was performed
 in<em> </em>green (GFP or GFP-Kap2) and red (mCherry,
 GR50-mCherry, GR100-mCherry) fluorescent cells. Aggregates were selected
 as regions of interest and photobleached for 2 sec. with blue light with
 laser power at 25% power. Following photobleaching, images were taken
 every 2 seconds for 3 minutes and analyzed with Nikon A1R software.
 Normalization and curve fitting were performed using the online tool
 EasyFRAP (normalization was performed using double normalization, and
 curve fitting was performed using double equation). The fluorescence
 recovery intensity curves were represented over time using Prism GraphPad
 9.0. The mobile fraction in FRAP experiments represents the proportion of
 molecules or particles that can freely move within the sample after
 photobleaching. The mobile fraction was calculated using the following
 formula: Mobile Fraction=Mobile Fluorescence Recovery\Initial Fluorescence
 Loss where the mobile fluorescence recovery is the increase in
 fluorescence signal after photobleaching, indicating the recovery of
 mobile molecules, and the initial fluorescence Loss is the initial
 decrease in fluorescence signal due to photobleaching.
 <em>Time-lapse microscopy: </em>Cells were imaged
 every two days after transfection. 20´ objective was used, and 25
 fields/wells were acquired. Only cells positives for both Kapβ2 and
 mCherry were counted. Cells were scored dead when they disappeared from
 the field of view or clear signs of neurite fragmentation appeared. Kaplan
 Meier curves were then constructed in Prism GraphPad 9.0.
 <em>Visualization of TDP-43 Condensates:
 </em>His<sub>6</sub>-SUMO1-TDP-43 (5 mM) was incubated
 with an equimolar concentration of (GR)<sub>20</sub> for 1
 hour at room temperature in PBS. 200 nM
 His<sub>6</sub>-SUMO1-TDP-43-GFP and 100 nM
 TAMRA-(GR)<sub>20</sub> were added to visualize TDP-43 and
 (GR)<sub>20</sub>. The reaction mixture was incubated at room
 temperature for 1h, and samples were spotted onto a coverslip and imaged
 by DIC and fluorescent microscopy using a Leica DMi8 Inverted microscope.
 To inhibit condensation, 5mM Kapβ2 was added at the beginning of the
 assay. <em>Proteomics analysis:
 </em>Publicly available data sets from Liu, F., Morderer, D., Wren,
 M.C. et al. were downloaded from massive.ucsd.edu (MassIVE MSV000088581).
 Analysis and statistical comparison of protein abundance were performed
 using basic packages (ggplot, ggplot2) of R (version 4.0.5) and Rstudio
 (version 2022.07.2). <em>Transcriptomics
 analysis: </em>In this study, we performed a transcriptomic analysis
 using publicly available datasets to investigate Kapβ2 expression patterns
 in iPS-derived cortical neurons and ALS patients' cerebral
 tissue. For the AnswerALS data: The raw sequencing data were processed
 using quality control metrics and aligned to a reference genome using
 Kallisto. Differential gene expression analysis was performed using
 DESeq2. Kapβ2 expression levels across samples were analyzed with ggplo2,
 and statistical analysis was carried out using GGPUBR. For the human
 tissue data, raw counts were downloaded from https://github.com/NathanSkene/ALS_Human_EWCE. Kapβ2 expression levels across samples were analyzed with ggplo2; statistical analysis was done using GGPUBR. <em>Statistics and Reproducibility: </em>All experiments were performed at least 3 times (n³3). At least 3 mice/group were used. For live-cell imaging, &gt;150 neurons/condition were counted for each n. For colocalization, puncta counting, and TDP-43 intensity, ³10 neurons/condition were counted for each n. For the FRAP experiment, ³5 cells/conditions were analyzed for each n. When two groups were compared, the Kolmogorov-Smirnov test assessed the normality of each population. If the two populations' results were normally distributed, then the unpaired t-student test was performed using the built-in function in Prism GraphPad 9.0. If one or both populations were not normally distributed, then the Mann-Whitney test was used to compare ranks, or the Kolmogorov-Smirnov test was used using Prism GraphPad 9.0. The log-rank and Cox proportional hazards tests compared the Kaplan Meier curves and the statistical significance between curves was assessed with Cox (GraphPad software). For the FRAP experiment, statistical differences between the curves were evaluated by curve analysis (one-way ANOVA test) in Prism GraphPad 9.0.
description: # The nuclear import receptor Kapβ2 modifies neurotoxicity mediated by
 poly(GR) in C9orf72-linked ALS/FTD
 [https://doi.org/10.5061/dryad.v41ns1s4f](https://doi.org/10.5061/dryad.v41ns1s4f) These datasets contain all the source files for the graphs in the associated paper. ## Description of the data and file structure Source data for each graph labeled as panel yx (y=number of figure, x=letter of the panel) ### Figure 1. GR does not affect Kapβ2 levels in neurons. * a) qPCR analysis of Kapβ2 levels in cortical neurons transduced with GFP or GR50. Data are represented as mean ± S.E.M. (n=6 biological replicates, Student t-test, n.s. = not significant) * 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 GR50-GFP mice. Data are represented as mean ± S.E.M. (n=3 biological replicates, One Way-ANOVA, n.s.) * f) The graph bar shows Kapβ2 protein quantification of the WB of the cortex and spinal cord homogenates. Tubulin was used to normalize. Data are represented as mean ± S.E.M. (n=3 biological replicates, One Way-ANOVA, n.s.) * 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) ### Figure 2. GR interacts and recruits Kapβ2 in neurons. * b) Pearson’s coefficient measured in each neuron transduced with the lentivirus GR50 construct (Flag-GR50-GFP). Data are represented as mean ± S.E.M. (n=3 biological replicates, m&gt;5 neurons, Student t-test, *P&lt;0.05) * d) Pearson’s coefficient measured in the cortex and spinal cord neurons of GR50 mice. Data are represented as mean ± S.E.M. (n=3 biological replicates, m&gt;15 neurons) ### Figure 3. Silencing of Kapβ2 increases death risk in the presence of GR. * b) Kapβ2 levels in primary neurons treated with Smart pool siRNA 100 mM or scramble. Data are represented as mean ± S.E.M. (n=3 biological replicates, Mann-Whitney non-parametric test, P&lt;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 mM or scramble and GFP, GR50-GFP, or GR100-GFP. (n=3 biological replicates, m&gt;50 neurons, Log-rank Mantel-Cox test * p&lt;0.05, *** p&lt; 0.001) * d) Quantification of the mean fluorescence of GFP expressing neurons 48 hours after transfection in cells previously treated with Kapβ2 siRNA or scramble. Data are represented as mean ± S.E.M. (n=3 biological replicates, m&gt;10 neurons, One-Way ANOVA, p=n.s. = not significant). * e) Quantification of the mean nuclear fluorescence of GFP expressing cells 48 hours after transfection in cells previously treated with Kapβ2 siRNA or scramble. Data are represented as mean ± S.E.M. (n=3 biological replicates, m&gt;10 neurons, One-Way ANOVA, p=n.s. = not significant). ### Figure 4. Increased expression of Kapβ2 does not affect neuronal viability. * b) Probability of neuronal death via a cumulative risk of death plot of rat primary cortical neurons transfected with 200/400ng of Tm+/ GFP-Kapβ2+ (GFP used as control) per 150,000 cells. Neurons double positive Tm+/GFP+ were counted (n=3 biological replicates, m&gt;150 neurons, Log-rank Mantel-Cox test: n.s.=not significant) * d) Probability of neuronal death via a cumulative risk of death plot of rat primary cortical neurons transfected with 400/400ng of GFP-Kapβ2 (GFP alone used as control) and GR50-mCherry (mCherry alone used as control) per 150,000 cells. Neurons double positive GFP+/mCherry+ were counted (n=3 biological replicates, m&gt;150 neurons, Log-rank Mantel-Cox test: mCherry vs GR50-mCherry p&lt;0.0001; GR50-mCherry vs GR50-mCherry /GFP-Kapβ2 p&lt;0.0001). Inset: bar graph showing quantification of GR50-mCherry and GR50-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 400/400ng of GFP-Kapβ2+ (GFP alone used as control) and GR100-mCherry (mCherry alone used as control) per 150,000 cells. Neurons double positive GFP+/mCherry+ were counted (n=3 biological replicates, m&gt;150 neurons, Log-rank Mantel-Cox test: mCherry vs GR100-mCherry p&lt;0.0001; GR100-mCherry vs GR100-mCherry / Kapβ2 p=0.005). Inset: bar graph showing quantification of GR100-mCherry and GR100-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.  ### Figure 5. Increased expression of Kapβ2 mitigates GR neuronal toxicity. * b) Graph showing intensity profiles of GFP or mCherry fluorescence (a.u.) over distance (mm). * c) The bar graph shows the quantification of GR50-mCherry and GR100-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). All the Excel files are provided in this repository. * d) The bar graph shows the quantification of GR50-mCherry and GR100-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 GR50-mCherry and GR100-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 GR50-mCherry or GR100-mCherry. Data are represented as mean ± S.E.M. (n=3 biological replicates, m=9-10 neurons/group, Student t-test, P&lt;0.05). ### Figure 6. Kapβ2 does not change the dynamic properties of GR aggregates. * c) The graph shows fluorescence recovery over time for GR50-mCherry and GR100-mCherry. Data are represented as mean ± S.E.M. (n=3 biological replicates, m&gt;5 neurons, Student t-test, P&lt;0.001). All the Excel files are provided in this repository (TRITC = mCherry/mCherry-GR50/mCherry-GR100, ND.T=time, ND.Z=relative z, ND.M=relative position). * f) The graph shows fluorescence recovery over time for GR50-mCherry and GFP-Kapβ2/GR50-mCherry. Data are represented as mean ± S.E.M. (n=3 biological replicates, m&gt;5 neurons). All the Excel files are provided in this repository (TRITC = mCherry/mCherry-GR50/mCherry-GR100, ND.T=time, ND.Z=relative z, ND.M=relative position). * g) The graph shows fluorescence recovery over time for GR100-mCherry and GFP-Kapβ2/GR100-mCherry. Data are represented as mean ± S.E.M. (n=3 biological replicates, m&gt;5 neurons). All the Excel files are provided in this repository (TRITC = mCherry/mCherry-GR50/mCherry-GR100, ND.T=time, ND.Z=relative z, ND.M=relative position). ### Figure 7. Increasing Kapb2 expression levels does not affect TDP-43 subcellular localization in the presence of GR. * b) Quantification of the size of TDP-43/GR20 aggregates in the presence or absence of Kapβ2. Data are represented as mean ± S.E.M. (n=3 biological replicates, Student t-test, P&lt;0.01). * c) Ratio between the mean GFP intensity inside and outside TDP-43/GR20 aggregates in the presence or absence of Kapβ2. Data are represented as mean ± S.E.M. (n=3 biological replicates, Student t-test, P&lt;0.0001). * 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&lt;0.01; P&lt;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&lt;0.01; P&lt;0.001). ### Supplementary Figure 1 * a) qPCR analysis of GFP transcript levels in cortical neurons transduced with GFP or GR50. Data are represented as mean ± S.E.M. (n=6 biological replicates, Student t-test, n.s.=not significant) * b) qPCR analysis of GFP transcript levels in the cortex and spinal cord of GFP and GR50 mice. Data are represented as mean ± S.E.M. (n=3 biological replicates, One Way-ANOVA, n.s.=not significant) * c) Box plot showing Kapβ2 transcript levels in iPS-derived cortical neurons (iCN) from control (healthy) and C9orf72 (C9-ALS) patients. (n&gt;15 biological replicates, Student t-test, n.s.=not significant). Scripts for the analysis are provided in this repository. * d) Box plot showing Kapβ2 transcript levels in the frontal cortex (fctx) of control (healthy) and C9orf72 (C9-ALS)patients (n&gt;10 biological replicates, Student t-test, n.s.=not significant). Scripts for the analysis are provided in this repository. ### Supplementary Figure 2 * a) Quantification of Kapβ2 nuclear and cytoplasmic fluorescence intensity in cortical neurons transduced with GFP or GR50-GFP and their ratio. Data are represented as mean ± S.E.M. (n=3 biological replicates, m&gt;3 neurons, Student t-test, n.s.=not significant) ### Supplementary Figure 3 * b) The floating bar graph shows the quantification of the intensities of the peptides for Kapβ2 and Kapβ2b in GFP and GR50-GFP. Scripts for the analysis are provided in this repository. * d) Quantification of the ratio between Kapβ2 and GFP signal from Suppl Fig 3C. Data are represented as mean ± S.E.M. (n=3 biological replicates, Student t-test, n.s.) ### Supplementary Figure 5 * b) Quantification of nuclear intensity of FUS. Data are presented as mean ± S.E.M. (n=3 biological replicates, m&gt;20 neurons/group, Student t-test, n.s.=not significant) ### Supplementary Figure 6 * b) Probability of cell death via a cumulative risk of death plot of rat primary cortical neurons transfected with 200ng/800ng of Tm+/GFP-Kapβ2+ (GFP used as control) per 150,000 cells. Neurons double positive Tm+/GFP+ were counted (n=3 biological replicates, m&gt;150 neurons, Log-rank Mantel-Cox test: not significant)