Results
We used the tet-off transgene system to express a double mutant version of chimeric mo/huAPP695 (swe/ind KM570, 571NL, and V617F) from a tetracycline-responsive promoter [12,13]. Transgenic APP expression was activated by crossing the APPswe/ind mice to animals producing tTA under control of the CaMKIIα promoter [16]. After initial screening of founders, we identified four lines of tet-APPswe/ind mice that produced very high levels of transgene product in offspring coexpressing tTA (Figures 1 and Figure S1). Compared to a standard APP transgenic line used for previous amyloid studies by our laboratory (line C3–3; [15,26,27]), we estimated that the four controllable lines produce transgenic APP protein at 10- to 30-fold over endogenous levels (Figure S1). This estimate was confirmed by direct comparison of APP levels in nontransgenic and tet-off APP mice using an antibody that recognizes both endogenous APP (and amyloid precursor-like protein 2) and the transgenic protein (monoclonal antibody 22C11; Figure 1D).
Figure 1  Control of Transgenic APP Expression by Dox
(A) Western blotting for transgenic APP using the human-specific 6E10 antibody shows expression of full-length protein in forebrain tissue from young predeposit double transgenic animals (line 107) and its suppression following dox treatment. Untreated animals show high levels of transgene expression; protein levels drop dramatically in animals acutely treated with dox for 2 wk. A faint, but detectable band of full-length protein remains in acutely treated animals that can be eliminated in mice born and raised on dox.
(B) Immunoblotting with the N-terminal antibody 22C11 to detect both transgenic and endogenous protein shows that dox treatment reduces APP/APLP to levels found in nontransgenic mice.
(C) Measurement of signal intensity from the Western blot in (A) shows transgenic APP levels are decreased more than 95% by dox in both acutely and chronically treated animals (97.2% for 4 wk + 2 wk dox, 98.0% for reared on dox versus 4 wk untreated; ANOVA effect of treatment group F 4,15 = 85.55, p < 0.001). APP levels in 4 wk + 2 wk dox, reared on dox, and nontransgenic (NTg) were not significantly different (p > 0.9, Tukey post-hoc test).
(D) Measurement of signal intensity from the Western blot in (B) shows total APP/APLP levels in dox-treated tTA/APP mice are significantly lower than in 4-wk-old untreated mice (ANOVA effect of treatment group F 4,15 = 84.41, p < 0.001) and indistinguishable from those of nontransgenic animals (p > 0.9, Tukey post-hoc test). *, p < 0.001 versus untreated 4-wk-old mice, Tukey post-hoc test applied to significant effect of group ANOVA. Importantly, all four new lines of tet-off APP mice showed nearly complete suppression of the transgene following dox treatment (Figures 1 and Figure S1). We focused on one of the four lines, line 107, to examine in more detail the time dependence and extent of transgene suppression following either acute or chronic treatment with dox. Two dox-treated groups were compared to two untreated groups: one group of mice was born and raised on dox, a second group was treated with dox for 2 wk starting at 1 mo of age (4 wk + 2 wk dox); two untreated groups kept on normal chow were harvested at either 4 or 6 wk of age. Animals born and raised on dox harbored no transgenic APP (Figure 1A). Following as little as 2 wk of dox treatment, transgenic APP expression was reduced by more than 95% compared to pre-dox levels. The residual expression remaining in acutely treated mice represents less than 4% of the transgenic protein produced in the absence of dox (Figure 1C), and likely results from slight leakage at the level of transcription (data not shown). Importantly, the total amount of APP (endogenous plus transgenic) and related APLPs in both acute and chronically treated animals was statistically indistinguishable from that in nontransgenic mice (Figure 1D; statistical analyses for experiments throughout the study are presented in the accompanying figure legends).
To ensure that Aβ production was suppressed in concert with the dox-mediated inhibition of its precursor APPswe/ind, we measured Aβ40 and Aβ42 levels by ELISA in forebrain homogenates from young tet-off animals. At 1 mo of age, the mice lacked visible amyloid aggregates that might act as an intractable reservoir of peptide remaining in the brain after the transgene had been suppressed. To further ensure we could detect any such insoluble aggregates that might bias our measure of changes in peptide synthesis, we performed a sequential three-step extraction with PBS, 2% SDS, and 70% FA that would separate peptides by solubility. We compared the levels of human transgene-derived Aβ40 and Aβ42 in untreated mice at 4 and 6 wk of age to animals that had either been born and raised on dox or that had been left untreated for 4 wk and then placed on dox chow for 2 wk prior to harvest (the same groups described above for immunoblot analysis of APPswe/ind levels, line 107). Consistent with the reduction in full-length APPswe/ind synthesis shown by immunoblot (see Figure 1), we found that transgene-derived Aβ levels were completely suppressed in animals born and raised on dox, and were sharply reduced following acute (2 wk) antibiotic treatment. Compared to the levels in untreated 4-wk-old mice, PBS-soluble Aβ42 dropped by 95.2% following 2 wk of dox treatment and by 99.2% with chronic treatment (Figure 2A). Similarly, SDS-soluble Aβ42 decreased by 75.2% and 94.8% following 2-wk or lifelong dox treatment (Figure 2B). Only the FA fraction revealed a small dox-resistant pool of peptide in acutely treated animals that we believe represents stable predeposit aggregates that have already accumulated by 4 wk of age when treatment was begun (Figure 2C). Indeed, animals that were born and raised on dox did not harbor this reservoir of treatment-resistant peptide, with 96.3% less Aβ42 than untreated 4-wk-old mice. Measurement of total Aβ in chronically treated mice, including endogenous and transgene-derived peptide, demonstrated that Aβ levels in tet-off APP mice were reduced to the level of endogenous peptide found in nontransgenic animals (Figure 2D). Taken together with the immunoblotting data for full-length APPswe/ind, the ELISA measurements indicate that dox-mediated suppression of transgenic APPswe/ind synthesis leads to parallel reduction of Aβ levels.
Figure 2  Aβ Levels Are Dramatically Reduced by Transgene Suppression
Cortical homogenates from young, predeposit tTA/APP mice used for Western blot in Figure 1 (line 107) were fractionated by sequential multi-step extraction with PBS, 2% SDS, and 70% FA followed by human-specific Aβ ELISA to measure transgene-derived peptide in each fraction. Aβ40 is shown in white, Aβ42 in black.
(A) PBS-soluble Aβ levels are substantially reduced by both acute and chronic dox treatment (ANOVA, effect of treatment group F 4,24 = 137.10 and 386.01, p < 0.001, for Aβ40 and Aβ42, respectively). Aβ levels in treated animals are indistinguishable from nontransgenic (NTg) animals (p > 0.3, Tukey post-hoc test).
(B) In the young animals tested here prior to the formation of visible amyloid deposits, most Aβ is extracted into the SDS fraction (84% and 76% of all transgene-derived Aβ40 and Aβ42, respectively). As in the PBS-soluble fraction, Aβ levels in the SDS fraction are significantly lowered by dox treatment compared to untreated animals (ANOVA effect of group F 4,24 = 197.57 and 163.48, p < 0.001, for Aβ40 and Aβ42, respectively). Acutely treated animals retained a small (although significant) amount of residual peptide (p < 0.001 compared to nontransgeinc, Tukey post-hoc test), whereas Aβ levels in mice born and raised on dox were reduced to levels indistinguishable from nontransgenic (p > 0.8, Tukey post-hoc test).
(C) The FA-soluble fraction already contains a small but significant pool of aggregated Aβ42 in untreated animals by 4 wk of age (p < 0.05 versus nontransgenic; Tukey post-hoc test applied to significant effect of group ANOVA F 4,24 = 17.11, p < 0.001). By 6 wk of age, the amount of Aβ in the FA fraction is increased significantly preceding the appearance of visible deposits 2 wk later. The FA pool is the only peptide fraction not lowered by acute dox treatment (4 wk untreated = 4 wk + 2 wk dox, p > 0.9, Tukey post-hoc test), consistent with poor turnover of aggregated Aβ species.
(D) Measurements of total Aβ, including both endogenous and transgene-derived peptides, show that animals born and raised on dox harbor Aβ levels identical to nontransgenic animals (p > 0.9, Tukey post-hoc test, effect of group ANOVA F 4,24 = 39.13 and 35.29, p < 0.001, for Aβ40 and Aβ42, respectively). Whereas chronic transgene suppression fully prevents synthesis of both peptides, acute dox treatment fully suppresses Aβ40 levels (p > 0.8, Tukey post-hoc test), but leaves a small amount of nonsuppressed Aβ42. The residual Aβ42 observed in acutely treated young animals derives from uncleared aggregates extracted in the SDS and FA fractions. *, p < 0.05; **, p < 0.005; ***, p < 0.001 versus 4-wk-old untreated mice, Tukey post-hoc applied to significant effect of group ANOVA. The ELISA data also confirmed that incorporation of the Swedish and Indiana mutations led to high levels of Aβ42, which we predicted would induce rapid plaque formation in untreated animals. Histological characterization of double transgenic (CaMKIIα-tTA × tet-APPswe/ind) mice revealed early-onset amyloid formation in all four new lines. Amyloid plaques were seen in mice as young as 8 wk of age (data not shown). Plaques were limited to the forebrain, including the cortex, hippocampus, olfactory bulb, and striatum, where the CaMKIIα promoter is known to be most active [16,28] (Figure S2). By 6 mo of age, amyloid burden became severe, covering large areas of the cortex and hippocampus (Figure S3). No lesions were seen in the cerebellum or brain stem even at late ages, consistent with CaMKIIα-controlled transgene expression. Unlike what is thought to occur in the human disease, the first visible plaques in the tet-off APP mice are fibrillar-cored deposits. We have noted the same early appearance of cored deposits in other lines of APP transgenic mice that harbor the Swedish mutation [27]. Diffuse plaques were apparent in 6-mo-old tTA/APP mice, and became relatively abundant by 9 mo of age. At older ages (9–12 mo) amyloid deposits were visible in the thalamus, which has also been observed in mice expressing mutant APP via the Thy-1 promoter. The presence of amyloid pathology in this region has been attributed to axonal transport of APP/Aβ to the terminals of cortical neurons in the thalamus [29]. Most importantly, only double transgenic mice, expressing both the tTA and APP transgenes, developed amyloid lesions. Single transgenic mice up to 15 mo of age showed no sign of pathology (Figure S3). Similarly, amyloid pathology can be completely prevented in double transgenic animals born and raised on dox. Animals from our highest expressing line (line 885) maintained on dox for up to 1 y harbored no amyloid pathology (data not shown), indicating that residual leakage of transgene expression in the presence of dox does not provide sufficient Aβ peptide to induce amyloid formation even over long periods.
To mimic therapeutic intervention with inhibitors of Aβ production, we raised a group of 25 double transgenic mice (CaMKIIα-tTA × APP line 107) on normal food until 6 mo of age, when we knew amyloid formation was already well underway in the brain. At 6 mo, half of the animals were switched from normal chow to food containing dox at 200 mg/kg until they were sacrificed at 9 or 12 mo of age. The remaining control animals were kept on standard chow (untreated). In all, four cohorts were created: 6 mo untreated (n = 7), 9 mo untreated (n = 5), 6 mo + 3 mo treated (n = 8), and 6 mo + 6 mo treated (n = 5). Full suppression (>95%) of transgenic APPswe/ind levels in the dox-treated animals was confirmed by immunoblot (Figure 3). To ensure that the transgene could be suppressed as rapidly in 6-mo-old mice with fulminant pathology as it can in young, predeposit animals, we treated an additional set of 6-mo-old animals with dox for 1 wk prior to harvest. Importantly, both APPswe/ind and APP–C-terminal fragment levels were fully suppressed after only 1 wk of treatment, indicating that the in vivo half-life of APPswe/ind and its processed C-terminal fragments are relatively short (Figure 3D).
Figure 3  Robust Transgene Suppression in Older Mice with Preexisting Amyloid Pathology
(A) Cortical homogenates from 6- to 12-mo-old animals used for pathology studies described below (line 107) were immunoblotted with human-specific antibody 6E10 to examine transgene suppression following 3 or 6 mo of dox treatment. The blot was co-immunostained for endogenous superoxide dismutase 1 (SOD1) as a control for loading.
(B) Quantitation of signal intensity from the Western blot shown in (A). Transgenic APP levels are significantly suppressed following 3 or 6 mo of dox treatment (96.9% and 97.6%, respectively). *, p < 0.001 compared to 6-mo-old untreated animals, Tukey post-hoc test applied to significant effect of group ANOVA F 3,12 = 107.22, p < 0.001. These data demonstrate that strong transgene suppression is attained both before and after the onset of amyloid pathology (see Figure 1 for predeposit experiments).
(C) Experimental design. To examine the effects of chronic Aβ suppression on amyloid pathology after the onset of deposition, we compared untreated controls harvested at 6 and 9 mo of age to animals placed on dox at 6 mo of age and harvested after 3 or 6 mo of treatment.
(D) Dox treatment leads to rapid transgene suppression even in 6-mo-old tTA/APP mice. Immunostaining with 6E10 shows APPswe/ind levels are dramatically reduced in 6-mo-old mice treated for 1 wk with dox (upper panel). A separate blot was immunostained for APP C-terminal fragments with CT15 antibody to show that the precursors to Aβ cleavage are decreased in parallel with the full-length protein (middle panel). Costaining for superoxide dismutase 1 was used as an internal control for loading (lower panel, taken from bottom half of 6E10 blot). Tissue sections from each animal in the four treatment groups were stained for amyloid pathology by Hirano silver, Campbell-Switzer silver, thioflavin-S, and Aβ immunohistochemistry. As expected, the 6 mo untreated cohort displayed moderate amyloid pathology, and the 9 mo untreated cohort progressed to a severe amyloid burden. In contrast, the extent of amyloid pathology in mice from the 6 mo + 3 mo treated or 6 mo + 6 mo treated cohorts closely resembled that of the 6 mo untreated cohort, despite the significant age difference between the treated and untreated groups (Figures 4 and Figure S3). Well-formed plaques remained in the treated animals after 6 mo of transgene suppression, even though as much time was given to clear the lesions as they had taken to form. Moreover, both types of amyloid, diffuse and fibrillar, remained intact throughout treatment. Using the Campbell-Switzer silver stain to distinguish different forms of amyloid, we found diffuse plaques were as persistent as cored deposits (Figure S4). It was nevertheless clear that dox-induced suppression of transgenic APP had completely halted the progression of pathology.
Figure 4  Suppression of Transgenic APP Arrests Progression of Amyloid Pathology
(A) Aggregated Aβ was quantified in cortical tissue from dox-treated and control tTA/APP mice (line 107) using a filter trap assay. Serial dilutions of protein homogenate were passed through a cellulose acetate filter; protein aggregates larger than the pore size were trapped and immunostained for Aβ.
(B) Quantitation of signal intensity in the linear range of each filter trap dilution series (arrow in [A]) was used to compare aggregate load across treatment groups. Aggregated Aβ increased significantly between 6 and 9 mo of age in untreated mice (significant effect of group ANOVA F 3,18 = 7.85, p < 0.002). This progression of pathology was completely prevented by transgene suppression. The amount of aggregated Aβ was identical in untreated mice at 6 mo of age to that in 9- or 12-mo-old animals treated with dox (p > 0.9, Tukey post-hoc test). Single transgenic tTA samples were included as negative controls and showed no signal above background. *, p < 0.01; **, p < 0.005 versus 9-mo-old untreated mice, Tukey post-hoc test; ***, p < 0.001 versus 9-mo-old untreated mice, Student's t-test.
(C) Amyloid pathology in the hippocampus of representative mice from each treatment group: Hirano silver stain (top row), thioflavin-S (middle row), and Aβ immunohistochemistry (bottom row). Amyloid burden increases dramatically between 6 and 9 mo of age in untreated animals, but remains stable in transgene-suppressed mice over the same period (6 mo + 3 mo dox and 6 mo + 6 mo dox). Single transgenic animals (tTA only shown here) show no sign of amyloid pathology at any age tested. To confirm that the arrest of plaques without any sign of clearance was not unique to the line 107 mice, we repeated the dox-suppression experiment in a second line of tet-off APP mice (CaMKIIα-tTA × tet-APPswe/ind line 18; n = 22). Again, long-term dox treatment was begun at 6 mo of age, and mice were harvested after 3 mo of transgene suppression (6 mo untreated, n = 8; 9 mo untreated, n = 6; 6 mo + 3 mo treated, n = 8). Immunoblotting for APP confirmed full transgene suppression in the treated animals (Figure S5). As in the line 107 mice described above, amyloid burden worsened substantially in the untreated mice between 6 and 9 mo of age. Suppression of transgene expression abruptly arrested progression of pathology (Figure S6), but again without any sign of reduction. Both silver- and thioflavin-S-positive plaques could still be found in each of the dox-treated animals.
We biochemically measured the amount of aggregated Aβ in the brains of our mice before and after transgene suppression using filter trap analysis of cortical tissue from each animal. In this assay, serial dilutions of protein homogenate are passed through a cellulose acetate filter; particles larger than the pore size of the filter become trapped in the membrane and are revealed by immunoblotting [22]. Consistent with our visual analysis of the histological sections, line 107 tTA/APP mice treated with dox for 3 or 6 mo had the same amount of aggregated Aβ as when they started treatment at 6 mo of age (Figure 4A and 4B). In contrast, untreated 9-mo-old mice had almost twice as much aggregated Aβ as either of the treated groups. Filter trap analysis of line 18 tTA/APP mice yielded similar results: the increase in aggregated Aβ observed in untreated animals between 6 and 9 mo of age was completely arrested by transgene suppression (Figure S5C and S5D).
We next used ELISA to measure total Aβ in the brains of each group to determine whether any change in the amount or solubility of peptide occurred while APPswe/ind expression was suppressed. Cortical homogenates were sequentially extracted to separate peptide into PBS-, SDS-, and FA-soluble fractions, then transgene-derived Aβ40 and Aβ42 were measured by human-specific ELISA [23]. In all animals harboring amyloid deposits, we found that the vast majority of Aβ (>99%) was extracted into the SDS and FA fractions (Figure 5A and 5B). Consistent with the filter trap results presented above, there were no significant differences in SDS- or FA-soluble Aβ between the 6 mo untreated cohort and either the 6 mo + 3 mo treated or 6 mo + 6 mo treated cohorts. However, brains of both 6 mo + 3 mo and 6 mo + 6 mo treated cohorts contained roughly twice as much PBS-soluble Aβ40 as untreated 6-mo-old mice (Figure 5C). Levels of Aβ42 showed a similar trend, but did not reach statistical significance. In fact, levels of PBS-soluble Aβ40 and Aβ42 in the 6 mo + 3 mo and 6 mo + 6 mo treated cohorts were most similar to that of the 9 mo untreated cohort, suggesting that age, as opposed to synthetic rate (which would be negligible in the treated animals), may determine the fraction of PBS-soluble Aβ in these animals.
Figure 5  Aβ ELISA Confirms Arrest of Progression without Clearance of Peptide in Mice with Preexisting Aggregates
Aβ levels in untreated 6- and 9-mo-old tTA/APP line 107 mice (shown in Figure 4) were compared to those in 9- and 12-mo-old animals treated with dox from the age of 6 mo. Single transgenic APP samples were included as negative controls. Cortical homogenates were fractionated by sequential multi-step extraction with PBS, 2% SDS, and 70% FA followed by human-specific Aβ ELISA to measure transgene-derived peptide in each fraction. Aβ40 is shown in white, Aβ42 in black.
(A and B) Most Aβ in the brains of plaque-bearing mice is extracted into the FA and SDS fractions. Consistent with amyloid burden (Figures 4 and Figure S3), SDS- and FA-extracted Aβ levels in untreated 9-mo-old mice were significantly higher than in untreated 6-mo-old mice (Tukey post-hoc test applied to significant effect of group ANOVA for SDS and FA fractions F 3,18 = 4.72–12.92, p < 0.02). In contrast, 3 or 6 mo of transgene suppression held Aβ at levels equivalent to those harbored when treatment was started (p > 0.2 compared to 6 mo untreated mice, Tukey post-hoc test). *, p < 0.05; **, p < 0.005; ***, p < 0.001 versus 9-mo-old untreated mice, Tukey post-hoc test. Significance for APP versus 9-mo-old untreated mice is based on Student's t-test.
(C) The PBS fraction represents less than 0.1% of total Aβ (note the change in y-axis from [A] and [B]), but only here do Aβ levels in the dox-treated mice differ from those in younger untreated mice. Although both peptides appear elevated in the treated groups compared to the untreated 6-mo-old mice, only Aβ40 reaches statistical significance (p < 0.05, Tukey post-hoc test applied to significant effect of group ANOVA for Aβ40 F 3,18 = 4.60, p < 0.02). A similar trend was seen for Aβ42, where ANOVA yielded a significant effect of group for PBS-soluble Aβ42 (F 3,18 = 3.75, p < 0.03), however this was due only to differences between the untreated 6- and 9-mo-old groups. •, p < 0.05 versus 6-mo-old untreated mice, Tukey post-hoc test; •••, p < 0.001 versus 6-mo-old untreated mice, Student's t-test. We also assessed neuritic and glial pathology surrounding the plaques to determine whether there were any changes in nearby tissue following long-term transgene suppression. Both Hirano silver stain and ubiquitin immunostaining showed neuritic pathology in all treatment groups (Figure 6). Similarly, activated astrocytes immunostained for GFAP were found near plaques in all animals (Figure 6). Neuritic and glial pathology were more severe in the older untreated mice. In contrast, transgene suppression prevented the growth of individual deposits apparent in untreated mice, and limited the surrounding pathology to what was already present when treatment began.
Figure 6  Neuritic and Glial Pathology Are Unchanged following Transgene Suppression
Dystrophic neurites and activated astrocytes surround most compact plaques in tet-off APP mice (line 107). Dark-stained, ubiquitin-filled neurites and reactive astrocytes form a halo around cored, fibrillar deposits by 6 mo of age that worsens with time in untreated mice. Both plaque-associated pathologies are arrested, although not reversed, by transgene suppression. Hirano silver stain (top row); GFAP immunohistochemistry (middle row); ubiquitin immunohistochemistry (bottom row). An obvious question we sought to address was whether the deposition of Aβ diminished cognitive ability in untreated mice, and what might happen to cognition when the process was interrupted. Unfortunately, efforts to characterize cognitive behavior were compromised by severe hyperactivity in untreated double transgenic mice. The tTA/APP animals were often seen running in circles around the perimeter of their cages, and a similar swimming pattern was noted when the mice were tested in the Morris water maze. In the radial water maze, repetitive swim patterns were noted with no evidence of choice-motivated actions. Other studies have dealt with similar problems by excluding animals that do not show adequate attention to the task, retaining only those mice that meet certain performance criteria [30]. In our case, the penetrance of hyperactivity was close to 100%, leaving us with no testable animals. This phenotype has not affected previous lines of APPswe mice we have produced, such as lines E1–2 or C3–3, that express lower levels of transgenic protein. Indeed, in past studies where hyperactivity was not a factor, we established a clear relationship between amyloid load and cognitive ability [31]. However, in the current study, we feel that although the poor performance of the tTA/APP mice in the maze tests could technically be scored as cognitive impairment, the animals' severe hyperactivity made interpretation of the cognitive tasks impossible.
In order to understand the nature and extent of hyperactivity in the tTA/APP mice, we quantified daily activity levels in double transgenic animals along with their single transgenic and nontransgenic siblings using four-beam frames designed to monitor ambulation within an enclosed cage. As shown in Figure 7, the double transgenic mice were up to 10-fold more active during the dark phase of the day–night cycle than any of the control groups. Activity levels appeared to follow a relatively normal diurnal cycle, decreasing substantially during the daylight hours. However, even during the light phase, the tTA/APP mice remained many-fold more active than normal controls. This behavior was partially, but not consistently, reversed by 1 mo of transgene suppression beginning at 4–5 mo of age (data not shown). In contrast, hyperactivity was completely abolished by rearing tTA/APP mice on dox. Animals born and raised on dox showed activity levels similar to the untreated controls (Figure 7C). Intriguingly, all of the dox-reared animals, both transgenic and wild-type, showed altered circadian rhythms with far less distinction between their day- and nighttime activity levels.
Figure 7  Transgene Suppression Attenuates Hyperactivity in tTA/APP Mice
(A) A 48-h measure of ambulation records extreme hyperactivity in untreated double transgenic mice compared to single transgenic and nontransgenic controls (line 107). This phenotype is completely eliminated by rearing the double transgenic mice on dox.
(B) The same data shown in (A) are replotted to magnify data from untreated control and dox-treated groups.
(C and D) Activity levels in the combined control groups of (A) and (B) are here separated by genotype. None of the single transgenic or nontransgenic control groups display the hyperactivity present in untreated tTA/APP animals. Again, note the y-axes have been enlarged for detail compared to (A).