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Reducing Endogenous Tau Ameliorates Amyloid ß-Induced Deficits in an Alzheimer's Disease Mouse Model

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Science  04 May 2007:
Vol. 316, Issue 5825, pp. 750-754
DOI: 10.1126/science.1141736

Abstract

Many potential treatments for Alzheimer's disease target amyloid-β peptides (Aβ), which are widely presumed to cause the disease. The microtubule-associated protein tau is also involved in the disease, but it is unclear whether treatments aimed at tau could block Aβ-induced cognitive impairments. Here, we found that reducing endogenous tau levels prevented behavioral deficits in transgenic mice expressing human amyloid precursor protein, without altering their high Aβ levels. Tau reduction also protected both transgenic and nontransgenic mice against excitotoxicity. Thus, tau reduction can block Aβ- and excitotoxin-induced neuronal dysfunction and may represent an effective strategy for treating Alzheimer's disease and related conditions.

Deposits of amyloid-β peptide (Aβ) and tau are the pathological hallmarks of Alzheimer's disease (AD). Treatments aimed at Aβ production, clearance, or aggregation are all in clinical trials. However, interest in tau as a target has been muted, partly because tau pathology seems to occur downstream of Aβ (14), making it uncertain whether tau-directed therapeutics would prevent Aβ-induced impairments. Also, tau is posttranslationally modified in AD (58), and debate continues about which modifications should be targeted. Reducing overall tau levels might be an alternative approach (9). As tau haplotypes driving slightly higher tau expression increase AD risk (10), reducing tau levels might be protective. Therefore, we determined the effect of reducing endogenous tau expression on cognitive deficits in transgenic mice expressing human amyloid precursor protein (hAPP) with familial AD mutations that increase Aβ production.

We crossed hAPP mice (11) with Tau–/– mice (12) and examined hAPP mice with two (hAPP/Tau+/+), one (hAPP/Tau+/–), or no (hAPP/Tau–/–) endogenous tau alleles, compared with Tau+/+, Tau+/–, and Tau–/– mice without hAPP (13). Tau reduction did not affect hippocampal hAPP expression, and conversely, hAPP did not affect hippocampal tau levels (fig. S1). The six genotypes showed no differences in weight, general health, basic reflexes, sensory responses, or gross motor function.

To test learning and memory, we used the Morris water maze. In the cued version, mice learn to find the target platform using a conspicuous marker placed directly above it. At 4 to 7 months of age, Tau+/+, Tau+/–, and Tau–/– mice learned quickly, but as expected (14, 15), hAPP/Tau+/+ mice took longer to master this task (Fig. 1A; P < 0.001). In contrast, hAPP/Tau+/– and hAPP/Tau–/– mice performed at control levels.

Fig. 1.

Tau reduction prevented water maze deficits in hAPP mice (n = 7 to 11 mice per genotype, age 4 to 7 months). (A) Cuedplatform learning curves. Day 0 indicates performance on the first trial, and subsequent points represent average of all daily trials. Performance differed by genotype (repeated measures analysis of variance (RMANOVA): P < 0.001; hAPP by tau interaction, P = 0.058). In post-hoc comparisons, only hAPP/Tau+/+ differed from groups without hAPP (P < 0.001). (B) Hidden platform learning curves differed by genotype (RMANOVA: P < 0.001; hAPP by Tau interaction, P < 0.02). In post-hoc comparisons, hAPP/Tau+/+ differed from all groups without hAPP (P < 0.001); hAPP/Tau+/– differed from hAPP/Tau+/+ (P < 0.02) and groups without hAPP (P <0.01); hAPP/Tau–/– differed from hAPP/Tau+/+ (P < 0.001) but not from any group without hAPP. (C and D) Probe trial 24 hours after completion of 3 days of hidden-platform training. (C) Representative path tracings. (D) Number of target platform crossings versus crossings of the equivalent area in the three other quadrants differed by genotype (target crossing by genotype interaction, P < 0.001). In post-hoc comparisons, all genotypes except hAPP/Tau+/+ and hAPP/Tau+/– exhibited a preference for the target location over equivalent areas in the other three quadrants (*P <0.05; **P < 0.01; ***P < 0.001). (E) Probe trial 72 hours after completion of 5 days of hidden-platform training. Target platform preference differed by genotype (target crossing by genotype interaction, P < 0.001; target crossing by hAPP by tau interaction, P <0.05). In post-hoc comparisons, all genotypes except hAPP/Tau+/+ exhibited a preference for the target location (**P < 0.01; ***P < 0.001). Error bars show SEM.

The more difficult hidden-platform version of the water maze demands spatial learning. Mice without hAPP learned this task over 3 days of training regardless of tau genotype, whereas hAPP/Tau+/+ mice showed no evidence of learning until days 4 and 5 (P < 0.001; Fig. 1B). Notably, hAPP/Tau+/– mice were less impaired than hAPP/Tau+/+ mice (P < 0.02), and hAPP/Tau–/– mice did not differ from controls without hAPP (Fig. 1B). Probe trials, in which the platform was removed and mice were given 1 min to explore the pool, confirmed the beneficial effect of tau reduction (Fig. 1, C to E). In an initial probe trial 24 hours after 3 days of training, hAPP/Tau+/+ mice had no apparent spatial memory of the platform location, crossing the target platform location no more than they crossed equivalent areas in nontarget quadrants (Fig. 1D). However, hAPP/Tau–/– mice, similar to mice without hAPP, did cross the target platform location more often (P < 0.01; Fig. 1D). After two additional days of training, hAPP/Tau+/– mice also had more target than nontarget crossings (P < 0.01), whereas hAPP/Tau+/+ mice still showed no spatial learning and memory (Fig. 1E). Thus, the tau reduction gene dose-dependently ameliorates Aβ-dependent water maze learning and memory deficits.

Increased exploratory locomotor activity is seen after entorhinal cortex lesions and may reflect deficits in spatial information processing (16); hAPP mice show similar hyperactivity (15). hAPP/Tau+/+ mice were hyperactive in the Y maze (P < 0.001; Fig. 2A), a new cage (P < 0.05; Fig. 2B), and the elevated plus maze (P < 0.001; Fig. 2C). In contrast, these abnormalities were not seen in hAPP/Tau+/– and hAPP/Tau–/– mice (Fig. 2, A to C). To determine whether the benefits afforded by tau reduction were sustained, we examined older mice. Hyperactivity persisted in hAPP/Tau+/+ mice and remained absent in hAPP/Tau–/– mice at 12 to 16 months (P < 0.05; Fig. 2D).

Fig. 2.

Tau reduction prevented behavioral abnormalities and premature mortality in hAPP mice. (A) Total arm entries during a 6-min exploration of the Y maze (n = 49 to 58 mice per genotype; age 4 to 7 months; ANOVA: genotype effect, P < 0.0001; hAPP by tau interaction, P < 0.0001; ***P <0.001 versus groups without hAPP). (B) Percentage of time spent active during a 5-min exploration of a new cage (n = 7 to 14 mice per genotype; age 4 to 7 months; ANOVA: genotype effect, P < 0.01; hAPP by tau interaction, P < 0.05; *P < 0.05 versus groups without hAPP). (C) Total distance traveled in both open and closed arms during a 10-min exploration of the elevated plus maze (n = 49 to 59 mice per genotype; age 4 to 7 months; ANOVA: genotype effect, P < 0.0001; hAPP by tau interaction, P < 0.05; ***P < 0.001 versus groups without hAPP). (D) Total distance traveled during exploration of elevated plus maze (n = 6 to 13 mice per genotype; age 12 to 16 months; ANOVA: hAPP effect, P < 0.01; hAPP by tau interaction, P = 0.079; *P < 0.05 versus groups without hAPP). Error bars in (A) to (D) show SEM. (E) Kaplan-Meier survival curves showing effect of tau reduction on premature mortality in hAPP mice. All genotyped mice in the colony (n = 887) were included in the analysis. By log-rank comparison, only hAPP/Tau+/+ mice differed from all other groups (P <0.005).

Premature death of unclear etiology was also observed in hAPP mice (P < 0.005; Fig. 2E) (17, 18). Again, both hAPP/Tau–/– and hAPP/Tau+/– mice were protected from this early mortality. Thus, tau reduction prevented major Aβ-dependent adverse effects in hAPP mice. We examined several plausible mechanisms by which tau reduction might exert protective effects and we eventually discovered an unexpected role for tau.

We first ruled out the possibility that tau reduction altered Aβ levels or aggregation. Tau reduction did not alter hAPP expression (fig. S1), soluble Aβ1-x or Aβ1-42 levels, or the Aβ1-42/Aβ1-x ratio (fig. S2). In addition, hAPP/Tau+/+, hAPP/Tau+/–, and hAPP/Tau–/– mice had similar plaque load at 4 to 7 months (fig. S3) and 14 to 18 months (Fig. 3, A and B). We also found no effect of tau reduction on levels of Aβ*56, a specific Aβ assembly linked to memory deficits (19) (fig. S4). Thus, the beneficial effects of reducing tau were observed without detectable changes in Aβ burden, suggesting that tau reduction uncouples Aβ from downstream pathogenic mechanisms.

Fig. 3.

Tau reduction did not change Aβ plaque deposition, neuritic dystrophy, or aberrant sprouting. (A) Thioflavin-S staining of hippocampal amyloid plaques in hAPP mice. Percentage of hippocampal area covered by plaques was normalized to the mean value in hAPP/Tau+/+ mice (n = 6 to 11 mice per genotype; age 14 to 18 months). (B) Immunostaining of hippocampal Aβ deposits in hAPP mice. Percentage of hippocampal area covered by plaques was normalized to the mean value in hAPP/Tau+/+ mice (n = 6 to 11 mice per genotype; age 14 to 18 months). (C) Double-labeling of hippocampus for dystrophic neurites (antibody 8E5, red) and amyloid plaques (thioflavin-S, green) in hAPP mice aged 14 to 18 months, with quantification of dystrophic neurites expressed as percentage of thioflavin-S–positive plaques with surrounding neuritic dystrophy (n = 9 to 11 mice per genotype). (D) GAP43 immunostaining of aberrant axonal sprouting in the molecular layer of the dentate gyrus (oml, outer molecular layer; mml, middle molecular layer; iml, inner molecular layer; dgc, dentate granule cells). Bracket highlights GAP43-positive sprouting in the outer molecular layer of hAPP mice. Sprouting was quantified by densitometry and normalized to the mean value in Tau+/+ mice (n = 7 to 14 mice per genotype; age 4 to 7 months; ***P <0.001 versus groups without hAPP). Error bars show SEM.

Next, we looked for abnormal forms of tau that might act as downstream effectors of Aβ in hAPP/Tau+/+ mice. Major AD-related phosphorylation sites in human tau are conserved in murine tau, including those phosphorylated by proline-directed kinases, such as glycon synthase kinase (GSK)–3β and cdk5, or by microtubule affinity–regulating kinase (MARK). Changes in murine tau phosphorylation at these sites are easily detected, for example after brief hypothermia (20) (fig. S4). However, in hippocampal homogenates of 4- to 7-month-old hAPP/Tau+/+ mice, we did not find changes in tau phosphorylation at proline-directed kinase sites, including Thr181, Ser202, Thr231, and Ser396/404, or at the primary site for MARK, Ser262 (fig. S5). Generation of neurotoxic tau fragments has also been implicated as a mechanism of Aβ toxicity (21). Tau-deficient primary neurons are resistant to Aβ-induced degeneration (3, 22), apparently because Aβ toxicity in vitro involves production of a 17-kD tau fragment (21). We confirmed the presence of a 17-kD tau fragment in lysates of Aβ-treated primary neurons, but found no abnormal tau proteolysis in hippocampal homogenates from hAPP mice (fig. S6), suggesting that the neuroprotective effects of tau reduction in the two systems are mechanistically different. The relative lack of modified tau also distinguishes our model from transgenic lines overexpressing tau with mutations that cause frontotemporal dementia, but not AD, in humans (2, 4, 23). In our study, reduction of endogenous, wild-type tau protected hAPP mice against Aβ-dependent cognitive impairments, and this did not involve the elimination of a large pool of tau with typical AD-associated modifications. Our experiments do not rule out the possibility that another type of tau modification, or a small pool of modified tau in a restricted subcellular compartment or cellular population, could play a role downstream of Aβ.

To begin addressing this possibility, we stained brain sections from Tau+/+ and hAPP/Tau+/+ mice with phospho-tau antibodies. We saw little difference overall between Tau+/+ and hAPP/Tau+/+ mice in phospho-tau immunoreactivity, but we did observe scattered phospho-tau–positive punctae in dystrophic neurites surrounding amyloid plaques (fig. S7). We thus wondered whether the benefits of tau reduction in hAPP mice could relate to prevention of neuritic dystrophy, which may contribute to AD-related cognitive decline (24). Despite the differences in their behavior, hAPP/Tau+/+, hAPP/Tau+/–, and hAPP/Tau–/– mice had similar amounts of neuritic dystrophy (Fig. 3C). Thus, tau is not required for the formation of plaque-associated dystrophic neurites. Given that tau reduction prevented behavioral deficits but not neuritic dystrophy, these may represent parallel, rather than causally linked, disease manifestations, or tau reduction may act downstream of neuritic dystrophy.

Tau has a well-characterized role in axonal outgrowth (12), so we tested whether tau reduction prevented the aberrant sprouting of hippocampal axons observed in AD (25) and hAPP mice (18). Similar degrees of sprouting were observed, regardless of tau genotype (Fig. 3D). Thus, although tau reduction affected important outcome measures related to Aβ-induced neuronal dysfunction, not all effects of Aβ were blocked.

Excitotoxicity is implicated in the pathogenesis of AD (26, 27). Consistent with the increased incidence of seizures in AD patients (28), TgCRND8 hAPP mice are more susceptible to the γ-aminobutyric acid type A (GABAA) receptor antagonist pentylenetetrazole (PTZ) (29). Using a similar paradigm, we found that hAPP/Tau+/+ mice were also abnormally sensitive to PTZ, with 20% suffering fatal status epilepticus at a dose that was not lethal to mice without hAPP (P < 0.05). Tau reduction prevented this effect, as no hAPP/Tau+/– or hAPP/Tau–/– mice died. Seizures in hAPP/Tau+/– and hAPP/Tau–/– mice were less severe and occurred at longer latencies than in hAPP/Tau+/+ mice (P < 0.01; Fig. 4, A and B).

Fig. 4.

Tau reduction increased resistance to excitotoxin-induced seizures. (A) Tau reduction lowered seizure severity after a single intraperitoneal injection of PTZ (40 mg/kg; n = 10 to 11 mice per genotype; age 4 to 7 months; ANOVA: tau effect, P < 0.0001). Seizures were less severe in hAPP/Tau+/– and hAPP/Tau–/– mice than in hAPP/Tau+/+ mice (**P < 0.01 versus hAPP/Tau+/+). Seizures were also less severe in Tau–/– mice than in Tau+/+ mice (##P <0.01 versus Tau+/+). (B and C) Latency to reach each stage of seizure severity after PTZ administration. (B) PTZ-induced seizures occurred more rapidly in hAPP/Tau+/+ mice than hAPP/Tau+/– and hAPP/Tau–/– mice (RMANOVA: P < 0.01). (C) Tau reduction also slowed the onset of PTZ-induced seizures in mice without hAPP (RMANOVA: P < 0.001). Error bars in (A) to (C) show SEM. (D) After a single intraperitoneal injection of kainate at the doses indicated, occurrence of generalized tonic-clonic seizures was scored. Tau reduction lowered susceptibility to kainate, shifting dose-response curves to the right (n = 19 to 24 mice per genotype; age 2 to 5 months; logistic regression: P <0.05).

Tau reduction also increased resistance to PTZ in hAPP-nontransgenic mice, lowering seizure severity and delaying seizure onset (P < 0.01; Fig. 4, A and C). To confirm that tau reduction could reduce aberrant neuronal overexcitation, we challenged mice with excitotoxic doses of the glutamate receptor agonist kainate. As expected, intraperitoneal injection of kainate dose-dependently induced seizures in Tau+/+ mice (Fig. 4D). In contrast, Tau+/– and Tau–/– mice were resistant to kainate across a range of doses (P < 0.05; Fig. 4D). Thus, tau modulates sensitivity to excitotoxins and may be involved in regulating neuronal activity. The excitoprotective effect of tau reduction in mice without hAPP is more likely related to a physiological function of tau than to the removal of a pathological form of the protein. Sensitization of neurons to Aβ by physiological forms of tau could explain why tau reduction is effective in hAPP/Tau+/+ mice despite their lack of obvious tau modifications.

Our findings raise the possibility that tau reduction could protect against AD and other neurological conditions associated with excitotoxicity. Of course, the therapeutic implications of our findings must be interpreted with caution. First, there are differences between the mouse model and AD, including the absence of substantial neuronal loss or neurofibrillary pathology in hAPP mice. The contribution of these abnormalities to AD-related cognitive impairment, relative to the role of reversible Aβ-induced neuronal dysfunction that is modeled in hAPP mice, remains to be determined (30). Second, microdeletions of chromosome 17q21 encompassing the tau gene are associated with learning disabilities in humans (31), although abnormalities in these individuals may relate to insufficiency of other genes in the region, such as the corticotropin-releasing hormone receptor gene, which is implicated in neuropsychiatric disease (32). We found no adverse effects of tau reduction on health or cognition in mice, and the evidence that even partial tau reduction robustly protected mice from Aβ and excitotoxic agents highlights its potential benefits.

Supporting Online Material

www.sciencemag.org/cgi/content/full/316/5825/750/DC1

Materials and Methods

Figs. S1 to S7

References

References and Notes

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