Fig 1.
Densitometric analysis of tTDP-43, CTF-35 and hTDP-43 expression in ntg, TAR6 and TAR6/6 mice.
(A) Brain homogenates, (E) hippocampal and (I) spinal cord homogenates from ntg, TAR6 and TAR6/6 mice at the age of 1.5, 3 and 6 months were analyzed by Western blotting and probed with the indicated antibodies (long exp. = long time ECL exposure for 30 min). One representative example of 3 is shown. Densitometric analysis of tTDP-43, CTF-35 and hTDP-43 levels normalized to β-tubulin levels of brain (B- D), hippocampal (F-H) and spinal cord (J-L) homogenates. Two-way ANOVA followed by Bonferroni‘s post-hoc test. Mean+SEM. *significances between genotypes, #significances between age groups. *p<0.05, **p<0.01, ***p<0.001.
Fig 2.
Quantification of hTDP-43 expression in ntg, TAR6 and TAR6/6 mice.
Quantitative hTDP-43 expression in hypothalamus (A), medulla oblongata (B) and spinal cord (C) analyzed by immunofluorescent labeling of CNS samples. (D, E) Representative overview image and magnifications of hTDP-43 labeling of a brain section (olfactory bulb (1), cortex (2), hippocampus (3), thalamus (4), hypothalamus (5) and medulla oblongata (6)) of 3 months old TAR6/6 (D) and ntg (E) mice. Scale bars: Overview images = 1000 μm; magnification images = 20 μm. (A, B) 1.5 months: ntg: n = 4; TAR6: n = 10; TAR6/6: n = 5; 3 months: ntg: n = 5; TAR6: n = 8; TAR6/6: n = 5; 6 months: ntg: n = 3; TAR6: n = 3; TAR6/6: n = 3. (C) n as in A, B exempt: 1.5 months: ntg: n = 3; TAR6: n = 11. (A-C) Two-way ANOVA followed by Bonferroni‘s post-hoc test. Mean+SEM. *significances between genotypes, #significances between age groups. *p<0.05, **p<0.01, ***p<0.001.
Fig 3.
Posttranslational changes in TAR6/6 mice.
(A-D) Localization of tTDP-43, CTF-35 and hTDP-43 after cellular fractionation of 3 months old TAR6/6 mice. (A) Cell fractions of midbrain and cortex samples of 3 months old ntg, TAR6 and TAR6/6 mice were analyzed by Western blotting and probed with the indicated antibodies (GAPDH as cytoplasmic and HDAC3 as nuclear marker; long exp. = long time ECL exposure for 30 min). One representative example of 4 is shown (C: cytoplasm, N: nucleus). (B- D) Densitometric analysis of tTDP-43, CTF-35 and hTDP-43 levels in cell fractions (cyt: cytoplasm, nuc: nucleus). Nuclear tTDP-43 and CTF-35 levels of ntg and hTDP-43 of TAR6/6 were set as 100%. (E-H) Soluble and insoluble TDP-43 protein levels in brains of 3 months old TAR6/6 mice. Brain samples were separated into RIPA soluble (R) and RIPA insoluble / UREA fractions (U). Three representative examples of 5 are shown. Fractions were analyzed by Western blotting and probed with the indicated antibodies (long exp. = long time ECL exposure for 30 min). Coomassie Blue staining was performed as loading control for UREA fraction. (F-H) Densitometric analysis of tTDP-43, CTFs and hTDP-43 levels in RIPA and UREA fractions. For tTDP-43 and CTF evaluation, RIPA fraction of ntg was set as 100%, whereas for hTDP-43 analysis, RIPA sample of TAR6/6 was set as 100%. (B-D; F-H) Two-way ANOVA followed by Bonferroni‘s post-hoc test. Mean+SEM. *significances between genotypes, #significances between fractions. *p<0.05, **p<0.01, ***p<0.001.
Fig 4.
Immunofluorescent double-labeling of human and total TDP-43.
Cortical and hippocampal tissue of three-months old ntg (A) and TAR6/6 (B) mice was labeled with antibodies against human TDP-43 (green) and total TDP-43 (red). Tissue was additionally labeled with NeuN antibody (white) and DAPI (blue) to visualize neuronal somata and nuclei, respectively. The merged images in the right column show human TDP-43, total TDP-43 and DAPI labeling. (C) The preferred nuclear localization of both human TDP-43 and total TDP-43 is obvious when the outlines of NeuN-positive somata are projected on the TDP-43 channels. Scale bars: Overview images = 500 μm; magnification images = 20 μm; (C) = 20 μm.
Fig 5.
Quantification of GFAP and CD11b expression in the central nervous system of ntg, TAR6 and TAR6/6 mice.
(A-C) GFAP expression levels labeling reactive astrocytes in the cortex (A), medulla oblongata (B) and spinal cord (C); and CD11b expression levels labeling reactive microglia in the cortex (D) and medulla oblongata (E) as IR area in percent. (A) 1.5 months: ntg: n = 4; TAR6: n = 9; TAR6/6: n = 5; 3 months: ntg: n = 4; TAR6: n = 9; TAR6/6: n = 5; 6 months: ntg: n = 3; TAR6: n = 3; TAR6/6: n = 3. (D) n as in A except: 1.5 months: TAR6: n = 10; 3 months: ntg: n = 5; TAR6: n = 8. (B, E) n as in D except: 3 months: TAR6/6: n = 7. C) n as in B except: 1.5 months: TAR6: n = 5; TAR6/6: n = 3; 3 months: ntg: n = 3; TAR6: n = 7; TAR6/6: n = 4. (A-E) Two-way ANOVA followed by Bonferroni‘s post-hoc test. Mean+SEM. *significances between genotypes. *p<0.05, **p<0.01, ***p<0.001.
Fig 6.
Quantification of neuron loss in the spinal cord of 3 months old TAR6/6 mice.
(A, C) Number of ChAT+ neurons in the ventral horn of the cervical (A) and lumbar (C) spinal cord grey matter. (B, D) Representative images of ChAT labeling in the cervical (B) and lumbar (D) spinal cord of 3 months old TAR6/6 mice compared to ntg animals. Nuclei of cells were stained by DAPI. (A) ntg: n = 4, TAR6/6: n = 3. (C) ntg: n = 3, TAR6/6: n = 3. Unpaired Student’s t-test followed by Welch’s correction. Mean+SEM. *p<0.05.
Fig 7.
Motor impairments and reduced anxiety in TAR6/6 mice.
(A) Clasping behavior scored from 1–3 of 1.5 and 3 months old TAR6/6 mice compared to ntg littermates (score of 1: normal clasping). (B) Latency to fall in the wire suspension test in seconds of 1.5 and 3 months old TAR6/6 mice compared to ntg littermates. (C) Latency to fall from the rotating rod in the Rota Rod test of 1.5 to 4.25 months old TAR6/6 mice compared to ntg littermates. (D) Nesting behavior scored from 1–5 of 3 months old TAR6/6 mice compared to ntg littermates (high score: perfect nest). (E) Time in seconds of 1.5 and 4.5 months old TAR6/6 mice spent in the open arms of the elevated plus maze compared to ntg littermates. F: Body weight in gram of 1.5 to 4.25 months old TAR6/6 mice compared to ntg mice of the same age. (A, B) 1.5 months: TAR6/6: n = 5, ntg: n = 7; 3 months: TAR6/6: n = 13, ntg: n = 16. (C) 1.5 months: TAR6/6: n = 5, ntg: n = 7; 3.5 months: TAR6/6: n = 8, ntg: n = 7; 3.75 months: TAR6/6: n = 12, ntg: n = 16; 4 months: TAR6/6: n = 10, ntg: n = 16; 4.25 months: TAR6/6: n = 9, ntg: n = 16. (D) TAR6/6: n = 16, ntg: n = 12. E: 1.5 months: TAR6/6: n = 6, ntg: n = 4; 4.5 months: TAR6/6: n = 8, ntg: n = 16. (F) 1.5 months: TAR6/6: n = 5, ntg: n = 7; 3.25 months: TAR6/6: n = 7, ntg: n = 12; 3.5 months: TAR6/6: n = 12, ntg: n = 18; 3.75 months: TAR6/6: n = 10, ntg: n = 18; 4.0 months: TAR6/6: n = 8, ntg: n = 18; 4.25 months: TAR6/6: n = 8, ntg: n = 18. (A-C, E, F). Two-way ANOVA followed by Bonferroni‘s post-hoc test. D: Mann Whitney U-test. A,B,E: Mean+SEM; C,D,F: Mean±SEM. *p<0.05, **p<0.01, ***p<0.001.
Table 1.
Summary of pathological features of TAR6/6 mice.