Effects of the C57BL/6 strain background on tauopathy progression in the rTg4510 mouse model

Abstract

Background: Cross-breeding of transgenic mice is commonly used to assess gene-gene interactions, particularly in the context of disease. Strain background changes can influence the phenotype of mouse models and can confound crossbreeding studies. We sought to determine if changing the strain background of a commonly used mouse model of tauopathy (rTg4510) would significantly impact the originally reported phenotype. On the original F1 FVB/N x 129S6 background, rTg4510 mice present with progressive cognitive decline, increased insoluble tau, robust tau pathology and age-dependent neurodegeneration. One of the most common strains in mouse modeling is C57BL/6. We and others have previously reported that this strain background alters the phenotypes of various models, including the JNPL3 model of tauopathy. To determine if the phenotype of rTg4510 mice was similarly affected by the introduction of the C57BL/6 background, we compared rTg4510 mice on the original F1 FVB/N x 129S6 background to rTg4510 mice on an F1 FVB/N x C57BL/6NTac (B6/NTac) background, herein termed rTg4510B6.

Results: Despite a small, but significant increase in soluble human tau levels, young rTg4510B6 mice had equivalent levels of tau phosphorylation, aggregation and cognitive impairments as age-matched rTg4510 mice. At 6.5 months of age, rTg4510B6 mice displayed hyperphosphorylated insoluble tau and robust cortical tau neuropathology that was equivalent to age-matched rTg4510 mice; however, 10.5-month-old rTg4510B6 mice had greater amounts of phospho-tau in the cortex and hippocampus when compared to age-matched rTg4510 mice. Non-transgenic (NT) littermates of rTg4510B6 (NTB6) mice also had greater amounts of cortical and hippocampal phospho-tau at 10.5 months of age when compared to NT littermates of rTg4510 mice. Additionally, older rTg4510B6 mice had gross forebrain neurodegeneration that was equivalent to age-matched rTg4510 mice.

Conclusions: Overall, our data shows that introduction of the C57BL/6 strain into the rTg4510 mouse background modestly alters the tau pathology that was originally reported in rTg4510 on the F1 FVB/129 background. In contrast, behavioral and neurodegenerative outcomes were not altered. These studies support the use of the rTg4510 mouse model on a partial C57BL/6 strain background without losing fidelity of the phenotype and suggest that the C57BL/6 background does not inherently protect against tauopathy.

Figure 1

Breeding scheme for comparison of rTg4510 mice on different strain backgrounds. Breeding scheme used to produce rTg4510 mice on an F1 FVB/N x C57BL/6 (FVB/B6) strain background (rTg4510_B6) (Left) compared to the original rT4510 mice on an F1 FVB/N x 129S6 (FVB/129) strain background (Right). Mice used in these studies include rTg4510_B6 and rTg4510 mice that carry both the tau responder and the tTA activator transgenes and non-transgenic (NT_B6 and NT) mice. Single transgenic mice generated from these F1 crosses were not analyzed in these studies.

Figure 2

2.5-month-old rTg4510_B6 mice have increased human tau expression compared to rTg4510 mice. (A-B) Western blot analyses of the soluble fractions of whole brain lysates from rTg4510_B6 and rTg4510 mice at 2.5 months of age. (A) Representative western blots of soluble human tau (E1 antibody), human and mouse tau (Tau5 antibody), and phosphorylated tau (CP13 and PHF1 antibodies) with a NT littermate shown as a negative control. (B) Densitometric quantification of E1 (human tau), Tau5 (human and mouse tau), CP13 (pS202 tau) and PHF1 (pS396/404 tau) normalized to GAPDH. rTg4510_B6 mice had increased soluble human tau, indicating greater tau transgene expression than age-matched rTg4510 mice. No differences in soluble phospho-tau levels were detected using CP13 and PHF1 antibodies. Each dot represents an individual mouse with the mean indicated by the black line. n = 9-10 per cohort. *P < 0.05 (Student’s t-test).

Figure 3

Equivalent accumulation and phosphorylation of insoluble tau in 2.5-month-old rTg4510_B6 and rTg4510 mice. (A-B) Western blot analyses of the sarkosyl-insoluble fractions of whole brain lysates from rTg4510_B6 and rTg4510 mice at 2.5 months of age. (A) Representative western blots of insoluble human tau (E1 antibody) and phosphorylated tau (CP13 and PHF1 antibodies) with a NT littermate shown as a negative control. (B) Densitometric quantification of E1 (human tau), CP13 (pS202 tau) and PHF1 (pS396/404 tau). Phospho-tau is normalized to the amount of human tau aggregated in that fraction. Insoluble tau accumulation and phosphorylation were similar between rTg4510_B6 and rTg4510 mice. Each dot represents an individual mouse with the mean indicated by the black line. n = 9-10 per cohort.

Figure 4

Strain background does not alter swim speed or search path in the MWM. (A-B) Performance in the cued MWM task was equivalent amongst rTg4510 and NT littermates on either strain background at 2.5 months of age. (A) Swim speeds to the visible platform were equivalent across all groups. (B) All groups improved performance over training (p < 0.001) with the search paths to reach the visible platform longer for rTg4510 mice on days 1 and 2, but comparable to NT mice by day 3. No differences between strain backgrounds were detected. Data expressed as mean ± SEM and analyzed via multifactorial (genotype x strain) RM ANOVA with post hoc Bonferonni’s multiple comparisons test. n = 5 per cohort.

Figure 5

rTg4510_B6 and rTg4510 mice have equivalent cognitive deficits at 2.5 months of age. Spatial learning and memory was equivalently impaired in rTg4510_B6 and rTg4510 mice at 2.5 months of age. (A) rTg4510 mice took significantly more time to find the hidden platform than NT mice (p < 0.001), with no difference between the strain backgrounds. (B) The search paths to reach the hidden platform for both rTg4510_B6 and rTg4510 mice were equivalent and significantly longer than the search paths of NT mice (p < 0.001). (C) rTg4510_B6 and rTg4510 mice also had similar thigmotaxic swimming that was significantly longer than that of NT mice (p < 0.001). (D) Spatial memory was equally impaired in rTg4510_B6 and rTg4510 mice compared to age-matched NT mice as indicated by the decreased percentage of distance traveled in the target quadrant (TQ) during the final probe trial. (A-C) Data expressed as mean ± SEM and analyzed via multifactorial (genotype x strain) RM ANOVA. (D) Each dot represents an individual mouse with the mean indicated by the black line. ****p < 0.0001 [two-way ANOVA (genotype x strain): main effect of genotype indicated]. n = 5 per cohort.

Figure 6

Older rTg4510_B6 mice have increased soluble Tau5 and PHF1 levels compared to rTg4510 mice. (A-B) Western blot analyses of the soluble fractions of whole brain lysates from 6.5- and 10.5-month-old rTg4510_B6 and rTg4510 mice. (A) Representative western blots of soluble human tau, human and mouse tau, and phosphorylated tau and with a NT littermate shown as a negative control. (B) Densitometric quantification of E1 (human tau), Tau5 (human and mouse tau), CP13 (pS202 tau) and PHF1 (pS396/404 tau) normalized to GAPDH. rTg4510_B6 mice had equivalent levels of soluble CP13 and human tau, and increased soluble total tau (human and moues) and PHF1 tau compared to rTg4510 mice on the original strain background. Each dot represents an individual mouse with the mean indicated by the black line. n = 3-4 per cohort. **p < 0.01 [two-way ANOVA (strain x age): main effect of strain indicated].

Figure 7

Accumulation and phosphorylation of insoluble tau is not significantly changed in older rTg4510_B6 mice. (A-B) Western blot analyses of the sarkosyl-insoluble fractions of whole brain lysates from rTg4510_B6 and rTg4510 mice at 6.5 and 10.5 months of age. (A) Representative western blots of insoluble human and phosphorylated tau with a NT littermate shown as a negative control. (B) Densitometric quantification of E1 (human tau), CP13 (pS202 tau) and PHF1 (pS396/404 tau). Phospho-tau is normalized to the amount of human tau aggregated in that fraction. Each dot represents an individual mouse with the mean indicated by the black line. n = 3-4 per cohort.

Figure 8

Older rTg4510_B6 mice have region-specific increases in CP13 compared to rTg4510 mice. rTg4510_B6 mice had increased CP13 (pS202 tau) staining in the cortex and hippocampus, but not the brainstem regions of the brain compared to rTg4510 mice at 10.5 months of age. (A) Representative images of cortical, hippocampal and brainstem regions of 6.5- and 10.5-month-old rTg4510_B6 and rTg4510 mice stained with the CP13 antibody. (B) Quantitative analyses of the stained sections showed that the CP13 burden in the cortex of rTg4510_B6 mice was significantly greater than the corresponding regions of rTg4510 mice at 10.5 months, but not 6.5 months of age, and increased in the hippocampus of rTg4510_B6 at both age points. Each dot represents an individual mouse with the mean indicated by the black line, n = 3-5 per cohort. ***p < 0.001 [two-way ANOVA (strain x age): main effect of strain indicated]. ##p < 0.01 (post hoc Bonferonni’s multiple comparisons test). Scale bar represents 200 μm.

Figure 9

Increased PHF1 tau staining in multiple brain regions of older rTg4510_B6 mice. Older rTg4510_B6 mice had increased PHF1 (pS396/404 tau) staining compared to rTg4510 mice in the cortex, hippocampus and brainstem regions of the brain. (A) Representative images of cortical, hippocampal and brainstem regions from 6.5-and 10.5-month-old rTg4510_B6 and rTg4510 mice stained with the PHF1 antibody. (B) Quantitative analyses of the stained sections showed that the PHF1 burden was significantly greater in the cortex of rTg4510_B6 mice at 10.5 months and in the hippocampus and brainstem at both 6.5 and 10.5 months of age compared to the corresponding regions of rTg4510 mice. Each dot represents an individual mouse with the mean indicated by the black line, n = 3-5 per cohort. ***p < 0.001 [two-way ANOVA (strain x age): main effect of strain indicated]. ##p < 0.01 (post hoc Bonferonni’s multiple comparisons test). Scale bars represent 200 μm. CC = Corpus callosum; 7 N = 7^th Cranial nerve.

Figure 10

Increased PHF1 staining in older NT_B6 mice compared to NT mice. NT mice on an F1 FVB/B6 background (NT_B6) had increased PHF1 (pS396/404 tau) staining in the cortex, hippocampus and brainstem regions of the brain compared to NT mice on an F1 FVB/129 background (NT). (A) Representative images from the primary sensory cortex, hippocampal formation and brainstem of NT_B6 and NT mice stained with PHF1. (B) Quantification of PHF1 burden in the cortex, hippocampus, and brainstem of 6.5- and 10.5-month-old NT_B6 and NT mice. Cortical PHF1 staining was significantly increased in NT_B6 mice compared to NT mice only at 10.5 months of age, while at 6.5 and 10.5 months of age PHF1 burden was increased in the hippocampal formation and brainstem. Each triangle represents an individual mouse with the mean indicated by the black line, n = 3-7 per cohort. ***p < 0.0001 [two-way ANOVA (strain x age): main effect of strain indicated]. ###p < 0.001 (post hoc Bonferonni’s multiple comparisons test). Scale bars represent 200 μm. CC = Corpus callosum; 7 N = 7^th Cranial nerve.

Figure 11

Older rTg4510_B6 mice have equivalent gross neurodegeneration. Gross brain and hippocampal atrophy was equivalent in rTg4510_B6 and rTg4510 mice at 6.5 and 10.5 months of age. (A) All mice on an F1 FVB/B6 background had smaller hemi-brains than all mice on an F1 FVB/129S background (p < 0.05). Independent of strain background, hemi-brain weights for rTg4510 mice were significantly decreased compared to NT mice. (B) Hippocampal atrophy, as assessed by decreased hippocampal area, was significantly increased in age-matched rTg4510 mice compared to NT littermates, with no difference between rTg4510 mice on different strain backgrounds. (C) rTg4510_B6 and rTg4510 mice had similar CA1 index values that were significantly less than that of NT mice, indicating increased neuronal loss in the CA1 hippocampal sub-region. (D)  Overall, rTg4510 mice had gross atrophy of the forebrain and ventricle enlargement compared to NT mice that was indistinguishable between age-matched rTg4510 mice on different strain backgrounds. In rTg4510 mice, the hippocampal formation was visibly reduced in size with substantial thinning of the neuronal layers in the CA1 and dentate gyrus sub-regions. (A-C) Each dot represents an individual mouse with the mean indicated by the black line. Data was analyzed via multifactorial (age x genotype x strain) ANOVA. ***p < 0.001 (main effect of genotype indicated). n = 2-7 per cohort. Scale bars represent 200 μm.