Cognitive Deficit in Amyloid-β–Injected Mice Was Improved by Pretreatment With a Low Dose of Telmisartan Partly Because of Peroxisome Proliferator-Activated Receptor-γ Activation
The pathological hallmark of Alzheimer disease is deposition of amyloid-β protein (Aβ) in the brain. Telmisartan is a unique angiotensin II receptor blocker with peroxisome proliferator-activated receptor-γ (PPAR-γ)–stimulating activity. Activation of PPAR-γ is expected to prevent inflammation and Aβ accumulation in the brain. We investigated the possible preventive effect of telmisartan on cognitive decline in an Alzheimer disease mouse model via PPAR-γ activation. Here, male ddY mice underwent ICV injection of Aβ 1-40. Cognitive function was evaluated by the Morris water maze test. A low dose of telmisartan (0.35 mg/kg per day) was administered in drinking water with or without GW9662, a PPAR-γ antagonist. Cerebral blood flow was evaluated by laser speckle flowmetry. Inflammatory cytokine levels were measured by quantitative RT-PCR. Aβ 1-40 ICV injection significantly impaired cognitive function. Pretreatment with telmisartan improved this cognitive decline to a similar level to that in control mice. Cotreatment with GW9662, a PPAR-γ antagonist, attenuated this telmisartan-mediated improvement of cognition. Treatment with telmisartan enhanced cerebral blood flow and attenuated the Aβ-induced increase in expression of cytokines, such as tumor necrosis factor-α and inducible NO synthase in the brain. Interestingly, coadministration of GW9662 cancelled these beneficial effects of telmisartan. Aβ 1-40 concentration in the brain was significantly decreased by treatment with telmisartan, whereas administration of GW9662 attenuated the decrease in telmisartan-mediated Aβ 1-40 concentration. Taken together, our findings suggest that even a low dose of telmisartan had a preventive effect on cognitive decline in an Alzheimer disease mouse model, partly because of PPAR-γ activation.
Alzheimer disease is the most common cause of cognitive deficit and results in impaired social interaction and quality of life for patients. Treatment of cognitive impairment attributed to Alzheimer disease is complex because of its multifactorial pathology, which includes increased amyloid-β (Aβ) deposition, neuroinflammation, and oxidative damage in the brain.1–3⇓⇓ Increased Aβ deposition is believed to have a central role in the pathogenesis of Alzheimer disease.4 Recently, peroxisome proliferator-activated receptor-γ (PPAR-γ) activation has been reported to induce clearance of the Aβ peptide5 and repression of β-secretase (β-site amyloid precursor protein cleaving enzyme)6; therefore, PPAR-γ agonists have been expected to be effective for the prevention of Alzheimer disease. Several studies using genetic mouse models of Alzheimer disease have shown that treatment with rosiglitazone, a PPAR-γ agonist, attenuated learning and memory deficits because of the glucocorticoid regulation.7,8⇓ In fact, it has been reported that treatment with rosiglitazone prevented cognitive impairment in patients with early Alzheimer disease in a preliminary study.9,10⇓ This beneficial effect of PPAR-γ agonists in Alzheimer disease was mediated by controlling neuroinflammation and reducing neuronal death.11,12⇓
On the other hand, although it is well known that hypertension is a direct risk factor for vascular dementia, recent studies have suggested that hypertension also impacts on the prevalence of Alzheimer disease13; however, little is known about the effects of antihypertensive drugs on Alzheimer disease. Telmisartan, an angiotensin II type 1 receptor blocker (ARB), uniquely possesses therapeutic effectiveness, particularly in patients with metabolic syndrome, with a partial PPAR-γ agonistic property in addition to its antihypertensive effect.14 Therefore, we speculate that telmisartan may have a preventive effect on cognitive impairment caused by Alzheimer disease. Very recently, we reported that telmisartan had a preventive effect on cognitive impairment evaluated by the shuttle avoidance test in an ICV Aβ-injected mouse model, involving PPAR-γ activation15; however, the dose of telmisartan that was used in that study was high (100 mg/kg per day) for clinical use, and cognitive function was assessed 2 weeks after Aβ ICV injection. Therefore, we examined the long-term effect of a lower dose of telmisartan and more specific mechanisms of improvement of cognitive function, focusing on hippocampal activity. We investigated the possibility that a lower dose (0.35 mg/kg per day) of telmisartan could improve cognitive function evaluated by the Morris water maze test 4 weeks after Aβ ICV injection. Moreover, we also investigated neuroinflammation and cerebral blood flow (CBF) after treatment with a lower dose of telmisartan and the roles of PPAR-γ stimulation by telmisartan.
Materials and Methods
The detailed Materials and Methods section is available in the online Data Supplement, available at http://hyper.ahajournals.org. This study was performed in accordance with the National Institutes of Health guidelines for the use of experimental animals. All of the animal studies were reviewed and approved by the animal studies committee of Ehime University.
Animals and Treatment
Adult male ddY mice (CLEA, Tokyo, Japan) were used in this study. Telmisartan, an ARB (provided by Boehringer Ingelheim), and GW9662, a PPAR-γ antagonist (Sigma-Aldrich), were administered to 8-week–old mice at a concentration of 0.35 mg/kg per day in drinking water. Aβ 1-40 (Peptide Institute) was injected ICV at 200 pmol in 5 μL of PBS after 2 weeks of drug treatment, as described previously.15,16⇓ For the vehicle control, the same dose of PBS was injected. To verify that injection was carried out properly, the same volume of Evans blue was ICV injected into another group of mice. We confirmed the distribution of the injected Evans blue throughout both sides of the ventricles. Systolic blood pressure was monitored in conscious mice by the tail-cuff method (MK-1030, Muromachi Co), as described previously.17 At 1 and 4 weeks after Aβ injection, the animals were anesthetized and brains were removed. Collected brains were frozen in liquid nitrogen and stored at −80°C until further analysis.
The Morris water maze test18 was performed in mice after 4 weeks of treatment with or without ICV Aβ injection, as described previously.19 Swimming was video tracked, and latency, path length, swim speed, and cumulative distance from the platform were analyzed by AnyMaze (Stoelting Co).
Real-Time PCR Method
mRNA was extracted from brain samples after homogenization in Sepazol (Nacalai Tesque Inc). Real-time quantitative RT-PCR was performed with a SYBR Green I kit (MJ Research, Inc).
Measurement of CBF
At 1 and 4 weeks after Aβ injection, CBF was determined by laser speckle flowmetry (Omegazone, laser speckle blood flow imager, Omegawave), which obtains high-resolution 2D images in a matter of seconds, as described previously.20
Aβ Concentration Analysis
Brain samples were obtained 1 or 4 weeks after Aβ injection. Aβ concentration in the brain was measured by ELISA (Human β Amyloid 1-40 ELISA Kit Wako II, Wako Chemical Industries), according to the manufacturer’s protocol. The amount of Aβ was calculated by comparison with a standard curve of synthetic human Aβ 1-40.
All of the data are expressed as mean±SEM in the text and figures. Data were analyzed by ANOVA. When a statistically significant effect was found, post hoc analysis was performed to detect the difference between the groups. A value of P<0.05 was considered statistically significant.
Telmisartan Improved Cognitive Function in Aβ-Injected Mice Partly Because of PPAR-γ Activation
In the water maze test, control (Aβ [−]) mice exhibited a progressive decrease in escape latency by training (Figure 1). In contrast, Aβ ICV-injected mice showed longer escape latency than Aβ (−) mice (Figure 1). Administration of telmisartan from 8 weeks of age did not affect blood pressure (data not shown)15 by the tail-cuff method, although telemetry is a more reliable method. We expected that these concentrations of drugs used in this experiment would not affect blood pressure. Pretreatment with telmisartan significantly reduced the decline in cognitive function by Aβ injection to a similar level to that in the Aβ (−) group (Figure 1). Interestingly, cotreatment with GW9662, a PPAR-γ antagonist, attenuated this telmisartan-mediated improvement of cognitive function to a level similar to that in the Aβ-injected group (Figure 1). GW9662 treatment alone showed no remarkable effect on Aβ-induced cognitive decline.
Treatment With Telmisartan Increased CBF
To assess the effect of telmisartan treatment on CBF, laser speckle flowmetry was performed after the water maze task. One week after ICV Aβ injection, a slight, but not significant, difference in the mean CBF level was observed; however, mean CBF was significantly increased in the telmisartan-treated group compared with that in the Aβ-injected group 4 weeks after Aβ injection (Figure 2). Interestingly, this telmisartan-induced increase in CBF was not observed in the group cotreated with GW9662 (Figure 2).
Treatment With Telmisartan Decreased Tumor Necrosis Factor-α Expression in the Brain
To assess the changes in inflammatory cytokines in the brain after Aβ ICV injection, brain samples were obtained at 1 week and 4 weeks after Aβ injection. In mice, mRNA expressions of NO synthase (NOS) 2, tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein 1 in the brain tended to increase after Aβ injection compared with that in the Aβ (−) group, but there were no significant differences in the expressions between each group (Figure 3A through 3C). However, in mice 4 weeks after Aβ injection, treatment with telmisartan significantly suppressed TNF-α expression in the brain compared with that in the Aβ-injected group. This reduction was not observed in mice cotreated with GW9662 (Figure 3A). Moreover, treatment with telmisartan tended to decrease NOS2 expression compared with that in the Aβ (−) group; however, cotreatment with GW9662 significantly increased NOS2 expression compared with that after telmisartan treatment (Figure 3B). On the other hand, mRNA expression of monocyte chemoattractant protein 1 was not significantly different between each group (Figure 3C).
Telmisartan Significantly Reduced Aβ Concentration in the Brain
Finally, the cerebrocortical level of Aβ was assessed 1 week after Aβ ICV injection using an ELISA method. Pretreatment with telmisartan significantly reduced the Aβ concentration in the brain (Figure 4). On the other hand, cotreatment with GW9662 inhibited this beneficial effect of telmisartan, and GW9662 treatment alone had no effect on the Aβ concentration compared with that in the telmisartan-treated group. However, 4 weeks after Aβ injection, Aβ concentration in the brain was below the detectable level in each group.
The present study demonstrated that even a low dose of telmisartan prevented cognitive impairment in an Aβ-injection mouse model, at least partly through clearance of Aβ by an increase of CBF, in addition to inhibition of inflammation. This increase in Aβ clearance was induced by partial agonistic effects of PPAR-γ, which were confirmed by coadministration of GW9662. We speculate that telmisartan synergistically acted through angiotensin II type 1 (AT1) receptor blockade and partial agonistic effects of PPAR-γ, thereby resulting in improvement of the cognitive decline after Aβ injection.
Our previous report demonstrated that pretreatment with a higher dose of telmisartan significantly inhibited cognitive decline evaluated by the shuttle avoidance test, together with reduced Aβ deposition.15 To examine the possible synergistic effects of telmisartan on AT1 receptor blockade and PPAR-γ activation on cognitive decline after Aβ injection, we used a lower dose of telmisartan. Moreover, in this study, we used the Morris water maze test to evaluate cognitive function instead of the shuttle avoidance test so that we could focus on hippocampus function. The shuttle avoidance test (also called the 2-way active avoidance test) is considered to measure nondeclarative memory and relies on both classic fear conditioning and instrumental conditioning21 and is often thought to be primarily striatum dependent.22 On the other hand, a spatial working memory task, such as the Morris water maze test, depends on hippocampus function,23–25⇓⇓ and we hypothesized that an even lower dose of telmisartan treatment would moderate hippocampal activity. Moreover, Aβ-induced behavioral impairment varies according to the injected region, and its mechanism in the acquisition phase is totally unknown, because spatial learning ability was impaired by Aβ ICV injection26 and by injection into the hippocampus27 but was not changed by injection into the amygdala28 or the basal forebrain.29 Thus, Aβ ICV injection–induced cognitive impairment would be caused by a decrease in hippocampal activity. Accordingly, we speculated and examined the possibility that treatment with an even lower dose of telmisartan could be effective in preventing mainly hippocampal function through AT1 receptor blockade and PPAR-γ activation.
Camacho et al5 reported that activation of the PPAR-γ pathway decreased Aβ deposition in HEK293 cells stably transfected with amyloid precursor protein and a differentiated human neuroblastoma cell line, primary cultures of murine glia cells, and murine cortical neurons via an increase in Aβ clearance. Although Camacho et al5 suggested that intracellular degradation may be involved in the mechanism of Aβ clearance, the detailed mechanism has not been elucidated. We observed that treatment with telmisartan increased CBF, resulting in an increase in Aβ clearance. This increase in CBF was partially reduced by a PPAR-γ antagonist. Recently, rosiglitazone, a PPAR-γ agonist, was shown to enhance angiogenesis in a focal cerebral ischemia model, with increased endothelial NOS (NOS3) expression around the ischemic margin and downregulation of an apoptotic stimulus, FasL.30 Moreover, endothelial PPAR-γ could act as a critical regulator of endothelial function in the cerebral circulation using endothelial-specific interference with PPAR-γ.31 Furthermore, our previous report demonstrated that treatment with an ARB, valsartan, increased capillary density in the brain.32 Therefore, we speculated that treatment with an even lower dose of telmisartan would increase CBF, with PPAR-γ activation as well as AT1 receptor blockade.
On the other hand, Vinik et al33 reviewed the regulation of NO by PPAR-γ agonistic drugs. Rosiglitazone improved NO production to a normal level, whereas pioglitazone had a marked effect to reduce nitrosative stress. Aβ stimulates NO production in astrocytes through an nuclear factor κB–dependent mechanism34,35⇓ and exerts a synergistic action with cytokines to induce neuronal damage via an NO-dependent pathway.36,37⇓ Our study also showed that the expression level of NOS2 (also called inducible NOS) in the brain was reduced by telmisartan, which was almost totally attenuated by GW9662, suggesting that the partial PPAR-γ agonistic effect of telmisartan could attenuate nitrosative stress in the brain. Moreover, Aβ ICV injection induced the gene expression of inflammatory cytokines, as well as NOS2, in the brain,38 and ARBs are also expected to prevent cardiac and vascular events through an anti-inflammatory effect.39 The reduction of TNF-α expression in the brain by telmisartan treatment and its partial attenuation by GW9662 indicate that telmisartan may protect against neuroinflammation through both PPAR-γ activation and AT1 receptor blockade. On the other hand, the TNF-associated cognitive decline might also relate to changes in synaptic plasticity, as well as neuroinflammation. TNF-α is thought to be released from astrocytes by Aβ-induced neurotoxicity in response to decreased neuronal activity.40 An increase in TNF-α regulates “synaptic scaling” and results in disease progression in Alzheimer disease,41 indicating that inhibition of TNF-α is expected to be a new target for the treatment of Alzheimer disease. Therefore, the present study suggests the therapeutic possibility of administration of telmisartan for Alzheimer disease, focusing on long-term inhibition of Aβ-induced TNF-α.
The beneficial effect of telmisartan on cognition was also observed 4 weeks after Aβ ICV injection in terms of increased CBF and reduced expression of TNF-α despite no change in the Aβ level in the brain. After 4 weeks, ICV-injected Aβ was almost removed from the brain; however, the improvement in neuroinflammation and the increase in CBF persisted. Thus, an even lower dose of telmisartan could be therapeutically useful for preventing neural damage after neuroinflammation and cerebral hypoperfusion.
Although the present study has not been designed to compare the effectiveness of different ARBs to prevent cognitive deficits induced by Aβ injection, it is interesting to use this model to compare the protective effects of ARBs that differ in ability to stimulate PPAR-γ. Moreover, it is interesting to study protective effects of telmisartan in Aβ-injected mice with targeted deficiency of PPAR-γ in brain to further evaluate the more detailed mechanisms of PPAR-γ agonistic effects of telmisartan to prevent cognitive decline in the brain.
Our results suggest that a low dose of telmisartan ameliorates cognitive impairment induced by Aβ injection through partial agonistic activation of PPAR-γ. These results support the notion that treatment with an even lower dose of telmisartan would prevent the onset of Alzheimer disease by reducing Aβ deposition. Additional clinical investigations are necessary to confirm the effect of telmisartan on Alzheimer disease.
Sources of Funding
This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan (to M.H., M.M., and L.-J.M.) and the Takeda Science Foundation (to M.M.).
- Received May 28, 2009.
- Revision received June 9, 2009.
- Accepted July 2, 2009.
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