3. Results
3.1. Behavioral characterization of the mouse lines
3.1.1. Disease onset determination
3.1.1.3. Muscle coordination and strength
I used two tests, the accelerating rotarod and grip strength, to assess muscle coordination and strength.
The accelerating rotarod test was used to determine both motor coordination and strength [168]. This A
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test focuses on the hindlimbs and measures the latency to fall of an animal. The animals were left to run on a rotarod for 300sec at an increasing speed. The longer an animal can run on the rotarod, the better its motor coordination capacity is and stronger its hindlimbs are. The average of three replicates per animal was calculated, followed by calculation of the average of each group, per time point.
The forelimb grip strength test aims to determine the strength on the forelimbs of the mice. The strength is measured upon release of the forelimbs from a metal detector bar when the mice are gently pulled by the tail. It has been previously demonstrated that in rodent models of ALS, the animals develop a focal disease onset that starts in the hindlimbs and only in later stages of the disease spreads to the forelimbs [169]. I performed this test to determine if and when there is a spread of muscle weakness to the forelimbs. Here, the average of three replicates per animal was calculated, followed by calculation of the average of each group, per time point.
3.1.1.3.1. Accelarating rotarod test
hTDP-43 control mice showed WT behavior on the rotarod (Figure 12 A). For both sexes and in all genotypes, the animals could not stay longer than 200sec on average, on the rotarod. For this mouse cohort no differences between genotypes were observed between or within each time point, as expected.
In contrast, A315T males showed clear deficits in rotarod performance relative to control littermates already at 8 weeks of age and their performance worsened with time (Figure 12 B). In comparison to control littermates, female mice carrying the A315T mutation performed equally well at 8 weeks and started to show significantly reduced performance at 12 weeks of age. Moreover, relative performance of the A315T female mice did not decline from 12 weeks to 16 weeks (Figure 12 B). As for neurological score and body weight, the presence of the Chat::L10a-GFP transgene had no influence on rotarod phenotypes.
3. Results
33 Figure 12. The A315T mice show clear deficits in motor coordination and muscle weakness.
(A) Latency to fall of the hTDP-43 mice and littermate controls throughout time for both male and female cohorts.
(B) Latency to fall of the A315T mice and littermate controls throughout time for both male and female cohorts.
Male cohorts show ALS-like phenotypes earlier than female cohorts. The average of three replicates per animal was calculated, followed by calculation of the average of each group, per time point. n= 8-12; dash line (---): represents the maximum time the animals can run on the rotarod; error bars: ± SEM; 2-way RM ANOVA with Bonferroni´s post hoc test; significance: *p<0.05; **p<0.01 and ***p<0.001.
3.1.1.3.2. Forelimb grip strength test
In the case of the hTDP-43 mouse cohorts it was observed that both sexes gained significant strength throughout time with no differences between genotypes (Figure 13 A).
Male A315T mice showed no increase in strength at 16 weeks of age, when comparing with the control littermates (Figure 13 B). For female A315T mice, no differences between genotypes within each time point were observed. All groups showed a similar increase in forelimb strength from 12 to 16 weeks. The presence of Chat::L10a-GFP transgene also had no significant influence on this test.
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Figure 13. A315T female and male cohorts show different strength patterns throughout time in the forelimbs.
Forelimb strength was measured at the indicated timepoints for littermate controls vs. hTDP-43 (A) or A315T (B) cohorts. The average of three replicates per animal was calculated, followed by calculation of the average of each group, per time point. N.S.: not significant. n= 8-12; error bars: ±SEM; 2-way RM ANOVA with Bonferroni´s post hoc test; Significance: *p<0.05 and **p<0.01.
Figure 14 summarizes the results from all behavioral tests and the information that they provide with respect to establishing timing of disease onset for A315T mice. Males had visible rotarod deficits and their peak body weight was already at 9 weeks of age. This means that their pre-symptomatic phase is presumably earlier and remains undefined. In contrast, females showed no phenotypes in any assays at 8 weeks, but clear motor phenotypes in both rotarod and neurological score at 12 weeks of age. Neither body weight nor grip strength differed from controls, even at 16 weeks, suggesting that these tests do not reveal disease onset in females. Thus, females, but not males, carrying the A315T mutation show a clear transition between pre-symptomatic to early symptomatic phases of the disease in the 8-12 week time window.
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3. Results
35 Figure 14. Time of ALS disease onset in the A315T mice.
Schematic representation of the behavioral results obtained and described above. Four independent tests were applied to determine disease onset: neurological score, peak body weight, accelerating rotarod and grip strength.
Bars indicate the first point where phenotypes are manifest. Solid coloring reflects a constant phenotype. Gradient shading reflects a progressively stronger phenotype. Results for male and female cohorts are represented in blue and red, respectively. While males already show a rotarod phenotype at 8 weeks of age, females show a clear transition between pre-symptomatic to early symptomatic phases of the disease for the two most important behavioral tests in the 8-12 week period.
The behavioral tests revealed that the A315T male mice develop ALS-like phenotypes earlier than female mice. Because of this difference could be driven by different expression levels of mutant TDP-43 protein, I next examined this explicitly.