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
]. 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 ( and Figure S1
). Compared to a standard APP transgenic line used for previous amyloid studies by our laboratory (line C3–3; [15
]), 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; D).
Control of Transgenic APP Expression by Dox
Importantly, all four new lines of tet-off APP mice showed nearly complete suppression of the transgene following dox treatment ( 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 (A). 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 (C), 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 (D; 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 ), 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 (A). Similarly, SDS-soluble Aβ42 decreased by 75.2% and 94.8% following 2-wk or lifelong dox treatment (B). 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 (C). 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 (D). 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.
Aβ Levels Are Dramatically Reduced by Transgene Suppression
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
] (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 (). 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 (D).
Robust Transgene Suppression in Older Mice with Preexisting Amyloid Pathology
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 ( 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.
Suppression of Transgenic APP Arrests Progression of Amyloid Pathology
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 (A and B). 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 S5
C and S5
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 (A and B). 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 (C). 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.
Aβ ELISA Confirms Arrest of Progression without Clearance of Peptide in Mice with Preexisting Aggregates
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 (). Similarly, activated astrocytes immunostained for GFAP were found near plaques in all animals (). 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.
Neuritic and Glial Pathology Are Unchanged following Transgene Suppression
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 , 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 (C). 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.
Transgene Suppression Attenuates Hyperactivity in tTA/APP Mice