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

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

University of North Carolina at Chapel Hill
UNC Chapel Hill
University of North Carolina

https://doi.org/10.7554/eLife.85921.1

Open access
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Figures and data

Overexpression of acetylation-mimic TDP-43^K145Q enhances TDP-43 stress-induced nuclear foci formation
A Representative images of immunofluorescence evaluation 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). Stick arrows highlight nuclei with small TDP-43+ puncta and arrowheads highlight nuclei with large TDP-43+ foci. Scale bar = 20µm. B Quantification of percentage of neurons with TDP-43+ foci in vehicle-treated neurons [One-way ANOVA followed by Tukey’s multiple comparisons testing; main effect of treatment between TDP-43 variants F (3,184) = 66.35, p<0.0001]. C Quantification of the average number of TDP-43+ foci per neuron [Two-way ANOVA followed by Tukey’s multiple comparisons testing; main effect of TDP-43 variant F (3, 343) = 36.23, p<0.0001]. Data shown as mean ± SEM. Each data point represents the average value within neurons in a single field of view with 10-100 neurons per field and n=48 fields per condition across 3 biological replicates. Statistical significance is represented by asterisks ****p<0.0001. DIV= Day in vitro

An endogenously encoded acetylation-mimic TDP-43^K145Q mutation causes altered TDP-43 localization and functional impairments in mouse and human 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 and immunolabeled for endogenous TDP-43 (green), NeuN (magenta), and DAPI (blue). B-C Quantification of the number of TDP-43+ neurons and the cytoplasmic:nuclear ratio of TDP-43 fluorescence intensity in TDP-43^KQ/KQ compared to TDP-43^wt neurons [Data shown as mean ± SEM, with data point representing the average value per neuron in a single field of view, 10-110 neurons per field, n=72 fields across 3 biological replicates; analysis by two-way ANOVA followed by Tukey’s multiple comparisons testing; B main effect of genotype F (1, 281) = 109.1, p<0.0001; C main effect of genotype F (1, 274) = 34.05, p<0.0001; TDP-43^wt vs. TDP-43^KQ/KQ +NaAsO² p=0.0363; TDP-43^wt Vehicle vs +NaAsO² p=0.0158]. D Schematic of TDP-43 dependent CFTR splicing reporter construct^68,153. E Gel electrophoresis following RT-PCR shows the splicing patterns of CFTR reporter in TDP-43^wt and TDP-43^KQ/KQ neurons at DIV14 and DIV28. F Quantification of band intensity in e to yield splicing efficiency values [Data shown as mean ± SD, n=3 biological replicates per condition; analysis by two-way ANOVA followed by Tukey’s multiple comparisons testing; main effect of genotype F (1, 8) = 196.6, p<0.0001; *p=0.0133]. G Representative confocal images of CRISPR-modified human iPSC-derived cortical neurons harboring homozygous TDP-43^K145Q (clonal line 12), TDP-43^K145R (clonal line 2) knock-in mutations or no TARDBP modifications (wt) as a control. Additional clones displayed in Supplementary figure 4. 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. Statistical significance is represented by asterisks *p<0.05, ***p<0.001, ****p<0.0001. DIV= Day in vitro

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)=5.285, p=0.0324] and 18-months-old. Filled bars represent presence of auditory cue (tone). 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 3 Source Data 1 file.

Neuronal loss in aged TDP-43^KQ/KQ mice with retention of predominantly nuclear TDP-43.
A Representative confocal images of immunofluorescent labelling of NeuN+ neurons in the neocortex of 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. C Representative widefield images of neocortex sections following Cresyl Violet (CV) labelling of endoplasmic reticulum Nissl substance. E Quantification of total brain cell density within the neocortex. E,H Representative confocal images of neocortex (E) and hippocampus (H) 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,I Quantification of TDP-43 fluorescence intensity within NeuN+ neurons in the neocortex (F) or hippocampal CA3 region (I). G,J Quantification of the nuclear:cytoplasmic ratio of TDP-43 fluorescence intensity within NeuN+ neurons in the neocortex (G) and hippocampus (J). Scale bars = 100 µm (A), 500 µm (B), 20 µm (E,H). SuperPlots⁸⁷ 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. Individual datapoints in F-G and I-J 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. Genotype comparisons by unpaired student’s t-test. Statistical significance is represented by asterisks, **p<0.01; ns = not significant; unmarked = not significant.

TDP-43 is hyper-phosphorylated and mislocalized 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]. 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. 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 located Figure 5 Source Data 1 file.

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.⁹³, “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 LeafCutter¹⁸⁰ (visualized with LeafViz¹⁸¹) demonstrates reduction in exclusion of a late cryptic 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 [neocortex Sort1-ex17b F (1, 23) = 35.64 p<0.0001, Sort1-WT F (1, 24) = 15.22 p=0.0007, Sort1 total F (1, 25) = 0.01856 p=0.8927; hippocampus Sort1-ex17b F (1, 14) = 44.33 p<0.0001, Sort1-WT F (1, 15) = 9.798 p=0.0069, 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]. Neocortex n=5 TDP-43^wt, n=6 TDP-43^KQ/KQ; hippocampus n=4 TDP-43^wt, n=5 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 7 Source Data 1 file.

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