RNA-binding deficient TDP-43 drives cognitive decline in a mouse model of TDP-43 proteinopathy

Julie Necarsulmer author has email address
Jeremy Simon
Baggio Evangelista
Youjun Chen
Xu Tian
Sara Nafees
Ariana Marquez Gonzalez
Huijun Jiang
Ping Wang
Deepa Ajit
Viktoriya Nikolova
Kathryn Harper
Jennifer Ezzell
Feng-Chang Lin
Adriana Beltran
Sheryl Moy
Todd Cohen author has email address

University of North Carolina at Chapel Hill

https://doi.org/10.7554/eLife.85921.2

Open access
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Figures and data

Ectopic expression of acetylation-mimic TDP-43^K145Q, an RNA-binding deficient mutant, shows accelerated formation of stress-induced nuclear TDP-43 foci
A Representative immunofluorescence images of TDP-43 in DIV14 mouse primary cortical neurons overexpressing TDP-43^wt, TDP-43^K145Q, or TDP-43^K145R after vehicle or 200μM NaAsO₂ treatment followed by labeling of TDP-43 (green), NeuN (magenta), and DAPI (blue). Arrows highlight nuclei with TDP-43+ foci. Scale bar = 20μm. B Quantification of percentage of neurons with TDP-43+ foci in vehicle-treated neurons. C Quantification of the average number of TDP-43+ foci per neuron. Data shown as Superplots⁸⁹; solid color bordered symbols and error bars indicate mean value of each biological replicate ± SEM; semi-transparent data points represent the average value per neuron in a single field of view, 10-110 neurons per field, 48 fields across n=2-4 biological replicates. Statistical analysis completed using a linear mixed-effect model. Statistical significance is represented by asterisks *p<0.05, **p<0.01, ***p<0.001. DIV= Day in vitro

An endogenously encoded acetylation-mimic TDP-43^K145Q mutation causes altered TDP-43 localization and functional splicing deficits in mouse primary cortical neurons
A Representative images of primary cortical neurons derived from TDP-43^wt or TDP-43^KQ/KQ mice that were treated with vehicle or 200μM NaAsO₂ at DIV14 and immunolabeled for endogenous TDP-43 (green), NeuN (magenta), and DAPI (blue). B-C Quantification of the number of TDP-43+ foci (B) and the cytoplasmic:nuclear ratio of TDP-43 fluorescence intensity (C) in TDP-43^KQ/KQ compared to TDP-43^wt neurons. D-G RT-qPCR analysis of DIV14 and DIV28 untreated mouse primary cortical neurons using primers specific for mouse (D) Tardbp [F (1, 8) = 3.034, p=0.1197]; and Sort1 splice variants: (E) ratio Sort1+ex17b:Sort1-WT [F (1, 8) = 23.02, p=0.0014] and (F) total Sort1 mRNA [F (1, 8) = 5.086, p=0.0541]. B-C Data shown as Superplots⁸⁹; solid color bordered symbols and error bars indicate mean value of each biological replicate ± SEM; semi-transparent data points represent the average value per neuron in a single field of view, 10-110 neurons per field, 72 fields across n=2-4 biological replicates; one color represents one biological replicate. Statistical analysis performed using a linear mixed-effect model. D-F Analysis by two-way ANOVA followed by Šídák’s multiple comparisons testing. F statistics represent main effect of genotype. Statistical significance is represented by asterisks, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant.

RNA-binding deficient TDP-43^K145Q in human iPSC-derived mature cortical neurons alters TDP-43 distribution and causes disease-relevant splicing dysregulation
(A) Representative confocal images of CRISPR-modified human iPSC-derived cortical neurons harboring homozygous TDP-43^K145Q (clonal lines 12, 18) or TDP-43^K145R (clonal lines 2, 12) knock-in mutations or an isogenic wild-type TDP-43^wt control lacking TARDBP modifications. Differentiated cortical neurons were treated with vehicle or 200μM NaAsO₂ and then immunolabeled for endogenous TDP-43 (green), Map2 (magenta), and DAPI (blue). Scale bars = 20μm. Arrows highlight nuclei with few, bright TDP-43+ foci. Arrowheads highlight nuclei with relative depletion of TDP-43 intensity. B-I RT-qPCR analysis of hiPSC-derived mature cortical neuron samples using primers specific for (B) human Tardbp [F=43.79, p<0.0001], (C) SORT1+ex17b, (D) SORT1-WT [F=2.975, p=0.0516], (E) total SORT1 mRNA [F=3.461, p=0.0321], (F) truncated STMN2 [F=79.06, p<0.0001], (G) STMN2 WT [F=63.93, p<0.0001], (H) UNC13A cryptic exon; and (I) UNC13A WT mRNA [F=2.116, p=0.1262]. RT-qPCR results compared via ordinary one-way ANOVA (F statistics and p-values shown in brackets above) followed by Tukey’s multiple comparisons testing. Statistical significance of multiple comparisons testing is represented by asterisks *p<0.05, ***p<0.001, ****p<0.0001. † Non-detectable levels of UNC13A cryptic exon- and SORT1-ex17b - containing transcripts in some or all control TDP-43^wt and TDP-43^K145R neurons prevented statistical comparisons among groups and therefore no statistical significance was reported and graphs are provided for visualization purposes only.

TDP-43^KQ/KQ mice develop age-dependent cognitive and behavioral defects.
A Body weight of TDP-43^wt and TDP-43^KQ/KQ mice in males [left panel; F(1,48)=17.52, p=0.0001] and females [right panel; F(1,36)=14.17, p=0.0006] at different ages. B Quantification of time in the center region of an open field in mice at 12 months [left panel; F(1,20)=6.118, p=0.0225] and 18 months [right panel; F(1,17)=9.622, p=0.0065]. C Quantification of time spent frozen (immobile) following context-dependent conditioned fear testing 18-months-old [right panel; F(1,17)=5.402, p=0.0328]. D Quantification of time spent frozen following cue-dependent conditioned fear testing at 12-months-old [left panel; F(1,20)=6.136, p=0.0223] and 18-months-old. Filled bars represent presence of auditory cue (tone) and period of statistical analysis. E-F Morris Water Maze (MWM) analysis displaying time to find a hidden platform (escape latency), quantified as daily trial means per animal (E) or average across all days (F). G-H Quantification of escape latencies during MWM reversal learning trials in daily trials (G) [F(1,16)=5.273, p=0.0355] and comparing averages per mouse across all days (H).
Bar and scatter plots shown as mean ± SD. Box and whiskers show line at median, ‘+’ at mean, and whiskers run min to max. a Two-way ANOVA followed by Šídák’s multiple comparisons test. B-D,E,G Two-way repeated measures ANOVA followed by Holm-Šídák’s multiple comparisons tests; F statistics and p-values in legend represent main effect of genotype. F, H Unpaired student’s t-test. Sample sizes as follows unless otherwise indicated: 12-month TDP-43^wt n=15; 12-month TDP-43^KQ/KQ n=7; 18-month TDP-43^wt n=10; 18-month TDP-43^KQ/KQ n=9; one 18-month TDP-43^KQ/KQ extreme outlier removed for MWM analysis (E,F). Statistical significance is represented by asterisks *p<0.05, **p<0.01, ***p<0.001; ns=not significant. Further statistical information is located Figure 4 Source Data 1 file.

No evidence of neuronal loss in aged TDP-43^KQ/KQ mice with retention of predominantly nuclear TDP-43.
A Representative confocal immunofluorescence images of NeuN+ neurons in the neocortex of 12- and 18-month TDP-43^wt or TDP-43^KQ/KQ mice. B Quantification of NeuN+ neurons per square millimeter (mm) in TDP-43^wt and TDP-43^KQ/KQ mice neocortex. C Quantification of the percent of neocortex area that is occupied by cells with NeuN immunoreactivity. D,G Representative confocal images of neocortex (D) and hippocampus (G) sections from 18-month-old TDP-43^wt and TDP-43^KQ/KQ mice immunolabeled with TDP-43 (green), NeuN (magenta), and DAPI (blue). F,H Quantification of TDP-43 fluorescence intensity within NeuN+ neurons in the neocortex (F) or hippocampal CA3 region (H). F,I Quantification of the nuclear:cytoplasmic ratio of TDP-43 fluorescence intensity within NeuN+ neurons in the neocortex (F) and hippocampus (I). Scale bars = 100 μm (A), 20 μm (D,G). SuperPlots⁸⁹ show average value per animal in solid color bordered symbol (as mean ± SEM) over top of semi-transparent individual values from each animal. One color represents one animal. Individual datapoints in (B) and (C) represent density in a single field of view, with 10-16 fields across 4 brain sections per animal, n = 3-4 mice per genotype of 18-month-old animals and n=6 mice per genotype of 12-month-old animals. Individual datapoints in E-F and H-I represent values from a single neuron within one field of view, with 1000-5000 neurons per animal across 12-16 fields from 4 (neocortex) or 2 (hippocampus) brain sections of n = 3-4 mice per genotype of 18-month-old animals. Statistical analyses performed using linear mixed-effect models. Omission of asterisks indicates no statistical significance.

Hyperphosphorylated and mislocalized TDP-43 protein and autoregulated induction of the Tardbp transcript is found in the neocortex and hippocampus of aged TDP-43^KQ/KQ mice
A-F Western blot images comparing soluble and insoluble protein fractions in neocortex (A) and hippocampus (D) from TDP-43^wt and TDP-43^KQ/KQ mice at 12- and 18-months of age. Quantification of soluble TDP-43 protein levels in neocortex and hippocampus tissue relative to total transferred protein (TTP) [neocortex (B) F (1, 14) = 97.67, p<0.0001; hippocampus (E) main effect of genotype F (1, 13) = 3.699, p=0.0766 and main effect of genotype*age interaction F (1,13) = 10.21, p=0.0070). Quantification of insoluble phosphorylated p(409/410)-TDP-43 levels in neocortex and hippocampus tissue [neocortex (C) F (1, 14) = 18.81, p=0.0007; hippocampus (F) F (1, 13) = 23.07, p=0.0003]. G-I Western blot images following isolation of soluble nuclear and cytoplasmic protein fractions from neocortex (G) and hippocampus (J) of TDP-43^wt and TDP-43^KQ/KQ mice at 12- and 18-months of age. J-L Quantification of cytoplasmic TDP-43 measured as percent of total TDP-43 in neocortex and hippocampus [neocortex (H) F (1, 8) = 69.38, p<0.0001; hippocampus (K) F (1, 8) = 22.40, p=0.0015] and relative cytoplasmic mislocalization of TDP-43 after normalizing to cytoplasmic/nuclear ratio of the nuclear Sp1 protein in neocortex and hippocampus [neocortex (I) F (1, 8) = 19.79, p=0.0021; hippocampus (L) F (1, 8) = 29.94, p=0.0006]. M-N Tardbp expression was analyzed by qPCR in the (M) hippocampus [F (1, 16) = 76.86; p<0.0001] and (N) neocortex [F (1, 23) = 80.74; p<0.0001] of TDP-43^KQ/KQ and TDP-43^wt mice at 12- and 18-months-old. Relative expression (RQ) values in each sample were determined using the Plaff method and B-actin and Pgk1 as housekeeping genes. A-F n=4 TDP-43^wt and n=5 TDP-43^KQ/KQ at all ages and regions except 18-month hippocampus n=4 TDP-43^KQ/KQ. F-L n=4 all ages and regions. M Neocortex n=4 12-month TDP-43^wt, n=5 12-month TDP-43^KQ/KQ, n=10 18-month TDP-43^wt, n=9 18-month TDP-43^KQ/KQ. N Hippocampus n=4 12-month TDP-43^wt, n=5 12-month TDP-43^KQ/KQ, n=6 18-month TDP-43^wt, n=6 18-month TDP-43^KQ/KQ. Data are presented as mean ± SD. Two-way ANOVA followed by Šídák’s multiple comparisons test. F statistics represent the main effect of genotype unless otherwise stated. Statistical significance represented by asterisks, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Further statistical information is available in the Figure 6 Source Data 1 file. Figures showing uncropped western blot images are available in Figure 6 Source Data 2 files. Full images of western blots are available in Figure 6 Source Data 3 files.

RNA sequencing of aged TDP-43^KQ/KQ mouse brain reveals dysregulation of neurodegeneration and FTLD-TDP-associated pathways
Genes differentially expressed between TDP-43^KQ/KQ (TDP43-KQ) and TDP-43^wt (TDP43-WT) cortex and hippocampus were compared and their intersections were defined as 6 gene groups, as represented by an UpSet plot (left). Normalized gene expression values, centered around the mean of TDP-43^wt samples for each brain region, were then hierarchically clustered and plotted as a heatmap (center, red indicates expression higher than that of WT, blue indicates expression lower than WT). Genes that were significantly altered in human FTLD-TDP temporal and/or frontal cortex (Hasan et. al.⁹², “FTLD-TDP Cortex” bar), or striatal mouse brain TDP-43-knockdown (Polymenidou et al.⁹⁷, “TDP-43 knockdown” bar) were demarcated with tick marks. Each gene group was assessed for over-enrichment of Gene Ontology terms and these results were summarized as word clouds (right). Neocortex n=5 TDP-43^wt, n=6 TDP-43^KQ/KQ; hippocampus n=4 TDP-43^wt, n=5 TDP-43^KQ/KQ.

Dysfunctional splicing regulation of the Sort1 transcript in TDP-43^KQ/KQ mice
A Differential splicing analysis of TDP-43^wt and TDP-43^KQ/KQ neocortex (“Ctx”) and hippocampus (“Hpc”) using LeafCutter¹⁷⁷ (visualized with LeafViz¹⁷⁸) demonstrates reduction in exclusion of a 3′ exon within the Sort1 transcript. Diagram in (A) shows full-length Sort1 gene in upper panel and highlights the differentially spliced region in gray. Chromosomal location, intron start and end points, annotation status, and Δ percent spliced in (dPSI) of the intronic region is listed in the lower table. B-C qPCR analysis of additional WT and TDP-43^KQ/KQ neocortex (B) and hippocampus (C) samples using primers specific for Sort1 splice variants to show the ratio of Sort1-ex17b:Sort1-WTmRNA levels and total Sort1 mRNA levels [B neocortex; Sort1-ex17b:Sort1-WT ratio, F (1, 22) = 51.85, p<0.0001; Sort1 total, F (1, 25) = 0.01856 p=0.8927; C hippocampus; Sort1-ex17b:Sort1-WT ratio, F (1, 14) = 39.54, p<0.0001, Sort1 total F (1, 16) = 2.086 p=0.1679]. D-G Images of western blots probed for Sort1 protein from neocortex (D) and hippocampus (E) lysates from 12- and 18-month-old mice. The Sort1 protein band intensity is plotted relative to total transferred protein (TTP) and quantified in F-G [neocortex, F(1,14) =9.308, p=0.0086; hippocampus, F(1,13) =6.117, p=0.0280]. A Neocortex n=5 TDP-43^wt, n=6 TDP-43^KQ/KQ; hippocampus n=4 TDP-43^wt, n=5 TDP-43^KQ/KQ. B Neocortex n=3 12-month TDP-43^wt, n=5 12-month TDP-43^KQ/KQ, n=9 18-month TDP-43^wt, n=9 18-month TDP-43^KQ/KQ. C Hippocampus n=3 12-month TDP-43^wt, n=5 12-month TDP-43^KQ/KQ, n=5 18-month TDP-43^wt, 5=9 18-month TDP-43^KQ/KQ. D-G n=4 12-month TDP-43^wt, n=5 12-month TDP-43^KQ/KQ, n=4 18-month TDP-43^wt, n=4 (hippocampus) or n=5 (neocortex) 18-month TDP-43^KQ/KQ. Data are presented as mean ± SD. Two-way ANOVA followed by Šídák’s multiple comparisons test. F statistics represent main effect of genotype, unless otherwise stated. Statistical significance is represented by asterisks, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Further statistical information is located Figure 8 Source Data 1 file. Figures showing uncropped western blot images are available in Figure 8 Source Data 2 files. Full images of western blots are available in Figure 8 Source Data 3 files.

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