Strain background influences neurotoxicity and behavioral abnormalities in mice expressing the tetracycline transactivator

Abstract

The tet-off system has been widely used to create transgenic models of neurological disorders including Alzheimer's, Parkinson's, Huntington's, and prion disease. The utility of this system lies in the assumption that the tetracycline transactivator (TTA) acts as an inert control element and does not contribute to phenotypes under study. Here we report that neuronal expression of TTA can affect hippocampal cytoarchitecture and behavior in a strain-dependent manner. While studying neurodegeneration in two tet-off Alzheimer's disease models, we unexpectedly discovered neuronal loss within the dentate gyrus of single transgenic TTA controls. Granule neurons appeared most sensitive to TTA exposure during postnatal development, and doxycycline treatment during this period was neuroprotective. TTA-induced degeneration could be rescued by moving the transgene onto a congenic C57BL/6J background and recurred on reintroduction of either CBA or C3H/He backgrounds. Quantitative trait analysis of B6C3 F2 TTA mice identified a region on Chromosome 14 that contains a major modifier of the neurodegenerative phenotype. Although B6 mice were resistant to degeneration, they were not ideal for cognitive testing. F1 offspring of TTA C57BL/6J and 129X1/SvJ, FVB/NJ, or DBA/1J showed improved spatial learning, but TTA expression caused subtle differences in contextual fear conditioning on two of these backgrounds, indicating that strain and genotype can interact independently under different behavioral settings. All model systems have limitations that should be recognized and mitigated where possible; our findings stress the importance of mapping the effects caused by TTA alone when working with tet-off models.

Figure 1.

Hippocampal neurodegeneration is apparent in both the TTA/APP transgenic mouse model of Alzheimer's disease and in TTA single-transgenic controls. Cresyl violet-stained sections taken from mice ranging in age from 2 weeks to 12 months showed significant neuronal loss within the hippocampus of double-transgenic TTA/APP mice starting at 2 months of age. Single-transgenic TTA siblings on the same hybrid strain background also exhibited progressive neurodegeneration in the dentate gyrus. In both cases, the granule cell layer appears normal at 2 weeks of age. In contrast, single-transgenic APP mice showed no abnormalities and appeared similar to NTG at all time points examined. Scale bar, 400 μm.

Figure 2.

TTA single-transgenic siblings of theTg4510 tet-off Tau transgenic model also exhibit dentate granule neuron degeneration. Another tet-off model of Alzheimer's disease, the rTg4510 Tau mouse, has significant neurodegeneration throughout the neocortex, including severe atrophy of the hippocampus. Although clearly not as severe as the rTg4510 bigenic mice, single-transgenic TTA siblings display neuronal loss in the dentate granule cell layer similar to that observed in TTA animals from the tet-off APP model. Scale bar, 300 μm.

Figure 3.

The C57BL/6 background protects against TTA-induced dentate granule cell loss. a, The hippocampus of congenic B6 CaMKIIα-TTA mice appeared identical to NTG at all ages tested. b, The width of the dentate granule cell layer was used to estimate the extent of degeneration in hybrid TTA transgenic mice and in mice backcrossed to B6. On the B6/CBA/C3 hybrid background, the width of the dentate granule cell layer was significantly thinner in TTA-expressing animals than in NTG siblings at both 2–4 months and 6–9 months of age. Moreover, the width decreased with age and was significantly thinner in TTA transgenic mice at 6–9 months than it was at 2–4 months. In contrast, the width of the granule cell layer in congenic B6 TTA mice (backcross generation N19–N24) was no different from NTG at either age tested. n = 5–7 mice per genotype for each age group. Open circle, NTG; filled circles, TTA. *p < 0.05, ***p < 0.001, two-way ANOVA with Bonferroni post hoc test. Scale bar, 300 μm.

Figure 4.

Both C3H/He and CBA contribute to dentate granule cell loss of the original hybrid background. a, As expected from our initial analyses, the dentate granule cell layer was unaffected by TTA expression on the congenic B6 background. b–e, However, on several F1 hybrid backgrounds derived from the B6 congenic line, TTA expression significantly decreased the width of the granule cell layer between 14% (B6C3) and 30% (B6CBA). f, Dentate degeneration was also present in TTA-expressing offspring from the B6C3 F1 intercross, where 15 of 20 F2 TTA mice exhibited thinning of the dentate granule cell layer compared with NTG siblings. One-quarter of the cohort (5 of 20) appeared indistinguishable from NTG. Representative images from both groups are shown. Scale bar, 300 μm. g, The average width of the granule cell layer was measured for each background (n = 6–20 per genotype). One outlier, determined by Grubb's test, was removed from the B6C3 × CBA TTA group before analysis and appears near the mean for NTG animals of that background. Note that the y-axis starts at 30 μm. h, Cell counts performed in C3B6 × C3 mice confirm that the width of the granule cell layer is linearly related to the number of granule cells remaining. Data points from each microscopic field are shown in gray (6–9 fields per mouse) and average values from each animal are shown in black (n = 7–8 per genotype). Solid line indicates the best-fit linear regression line for the average values, dashed lines indicate the 95% confidence interval. Note the x-axis starts at 20 μm. i, Multipoint interval mapping was used to locate genetic modifiers of the neurodegenerative phenotype in the B6C3 F2 TTA cohort. LOD scores are plotted as a function of marker location in centimorgans, with the chromosome number designated. Black line represents the LOD significance threshold for α = 0.05, determined by Haley–Knott permutations. Odd-numbered chromosomes are shaded in light gray. *p < 0.05, ***p < 0.001, two-way ANOVA with Bonferroni post hoc test.

Figure 5.

Strain background has a greater influence on open-field activity than does TTA expression. a, Open-field analysis revealed significant differences between strains in the total distance traveled over the 30 min trial but no significant effect of TTA expression within any strain background (n = 10–14 per genotype per strain). b, On average, each group of mice spent between 20 and 30% of their distance traveled in the central third of the arena, with no significant difference between strains. Furthermore, in all strain backgrounds, expression of TTA had no effect on percentage path in center.

Figure 6.

Both TTA expression and background strain influence performance in the Morris water maze. a–d, For all genotypes tested, escape distances decrease over time in the Morris water maze (n = 10–14 per genotype). Within each strain, acquisition by TTA mice was similar to NTG; however, as discussed in the Results, both genotypes performed poorly on the B6 background (NTG: dotted lines, open circles; TTA: solid lines, filled circles). Dotted lines mark 200 cm mean distance traveled. e–h, Long-term probe trials, conducted 24 h after each training session, measured the consolidation of spatial memory for the trained platform location. In general, the percentage of total swim path spent in the target quadrant increases with continued training. However, this trend was not apparent for TTA mice on the B6 background, where TTA mice swam less distance in the target quadrant than their NTG siblings on probe trials 4 and 5. Dotted lines mark chance performance of 25%. Open bars, NTG; filled bars, TTA. Two-way RM ANOVA with Bonferroni post hoc test, *p < 0.05, **p < 0.01.

Figure 7.

Strain-dependent effects of TTA expression on contextual fear conditioning. a, Freezing responses during the 5 min context test varied significantly with strain, but the average percentage time spent freezing did not differ between genotypes on any background tested. b, Minute-by-minute freezing for the four strain backgrounds (NTG: dotted lines, open circles; TTA: solid lines, filled circles). On the B6129 background, there was a noticeable divergence between the two genotypes over time. Similarly, freezing responses in B6DBA TTA and NTG mice diverged significantly over time during testing. In contrast, there was no difference in freezing between genotypes on the B6FVB or congenic C57BL/6 background over time (n = 10–14 per genotype). Two-way RM ANOVA with Bonferroni post hoc test, *p < 0.05.

Figure 8.

TTA expression decreases granule cell layer width on F1 backgrounds. a, Consistent with our previous results, the structure of the dentate gyrus was indistinguishable between TTA and NTG on the congenic C57BL/6 background. b–d, By comparison, TTA expression caused some thinning of the granule cell layer on the B6129, B6FVB, and B6DBA F1 backgrounds. Scale bar, 300 μm. e, B6129, B6FVB, and B6DBA TTA-expressing animals have significantly thinner dentate granule cell layers compared with NTG siblings (n = 6–14 per genotype). Two-way ANOVA with Bonferroni post hoc test, ***p < 0.001.

Figure 9.

Lifelong doxycycline treatment prevents degeneration of the dentate granule cell layer. Animals maintained on dox throughout life show no evidence of granule neuron loss, even in the most severely affected strain background and at ages well beyond when half these cells would be gone from untreated animals. a–c, In the original B6/C3/CBA hybrid background, both TTA and TTA/APP mice at 6–9 months of age exhibited substantial granule cell loss compared with tetO-APP and NTG (data not shown). d–f, Following lifelong dox treatment, the granule cell layer of 24-month-old TTA and TTA/APP mice appears identical to tetO-APP (and NTG, data not shown). Animals shown here were derived from intercrosses of CaMKII-TTA × tetO-APPswe/ind Line 885. Similar results were observed with tetO-APPswe/ind Line 107 (data not shown) (n = 4–6 per genotype). Scale bar, 300 μm.

Figure 10.

Postnatal doxycycline treatment provides long-term protection against granule cell loss. Dox treatment from shortly after birth until 6 weeks of age protects the integrity of the dentate gyrus for many months after the drug is removed. a, b, TTA transgenic mice generated on a B6FVB F1 background would normally show 20% reduction in granule cell layer width by 3 months of age. In contrast, early dox treatment (P1–P3 until P41–P43) protects against overt neuronal loss in the dentate gyrus of TTA mice on this background up to 6 months later. Images show cresyl violet staining from FVBB6 F1 mice at 7.5 months of age following dox rearing until 6 weeks of age. Scale bar, 300 μm. c, Measurements of granule cell layer width confirm that neuronal loss is ameliorated by inactivating TTA during postnatal development (n = 5–6 per genotype, p = 0.62, Student's t test).