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Central angiotensin II-induced Alzheimer-like tau phosphorylation in normal rat brains
FEBS Letters 586 (2012) 3737–3745
journal homepage: www.FEBSLetters.org
Central angiotensin II-induced Alzheimer-like tau phosphorylation in normal rat brains Minjie Tian 1, Donglin Zhu 1, Wei Xie, Jingping Shi ⇑ Department of Neurology, Nanjing Brain Hospital Affiliated to Nanjing Medical University, Nanjing, PR China
a r t i c l e
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Article history: Received 22 July 2012 Revised 29 August 2012 Accepted 1 September 2012 Available online 13 September 2012 Edited by Barry Halliwell Keywords: Angiotensin II Glycogen synthase kinase 3b Tau Alzheimer disease
a b s t r a c t Growing evidence suggests that Alzheimer disease (AD) origins in vascular lesions. As the crucial mediator of vascular pathology, angiotensin II-induced significant amyloid production in our laboratory, although amyloid neurotoxicity depended on phosphorylated tau (p-tau) in recent studies. In the present study, p-tau levels were significantly elevated by central angiotensin II via glycogen synthase kinase 3b (GSK 3b) and other tau kinases. Moreover, angiotensin II-induced cognitive impairment and tau phosphorylation was attenuated by losartan and a GSK 3b inhibitor. These findings implicate Ang II as a crucial mediator of AD pathology and a link between cardiovascular events and AD. Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Alzheimer disease (AD) is one of the most important unmet needs in neurology, with oligomer-prone b-amyloid (Ab) and phosphorylated tau (p-tau) as its pathological hallmarks [1]. Glycogen synthase kinase 3b (GSK 3b), the main tau kinase, are thus also involved in AD pathogenesis. More recently, growing evidence has indicated that AD is initiated by vascular factors that precede the neurodegenerative process, leading to AD being termed a vascular disorder [2]. As an intrinsic vasoactive hormone and the main actor in the renin–angiotensin system (RAS), brain angiotensin II (Ang II) is always present in abnormally high quantities in many cardiovascular diseases [3]. More recently, it has also been implicated in AD [4,5]. Excess Ang II was linked to increased blood pressure, decreased cerebrovascular compliance and enhanced brain inflammation, all recognized risk factors for AD, while Ang II type 1 receptor (AT1R) blockade suppressed such processes [6,7]. Moreover, application of AT1R blockers to rodents led to attenuated neurotoxic accumulation and cognitive deficits [8–12]. In our laboratory, central infusion of Ang II significantly promoted Ab production via enhanced amyloidogenic processing [13]. These findings implicated Ang II as a crucial mediator in AD, just as it has been implicated in cardiovascular pathology [14]. ⇑ Corresponding author. Fax: +86 025 83719457. 1
E-mail address: [email protected] (J. Shi). These authors contributed equally to this work.
However, Ab plaques correlate poorly with cognition decline [15] and plaque removal did not prevent neurodegeneration [16], suggesting that other entities may be required for Ab-induced neurotoxicity. As noted in AD rodent models, the presence of tau is required for Ab-induced neuronal and synaptic damage [1]. Reducing endogenous tau ameliorates Ab-induced neuronal dysfunction [17], while dendritic function of tau mediates Ab-associated synaptotoxicity [18]. In older individuals at risk for dementia, Ab-associated brain volume loss and longitudinal clinical decline occurs only in the presence of p-tau [19,20]. It thus has been proposed that Ab neurotoxicity is dependent on tau, especially p-tau. It is therefore intriguing to determine the levels of ptau in response to Ang II stimulation, because our findings have confirmed that Ang II could induce Ab production. In the present study, graded doses of Ang II were delivered into rat brain ventricles via osmotic pumps, as a model of increased brain Ang II in vascular-related events. The levels of AD-like ptau were then assayed and the signaling pathways involved were also uncovered. 2. Materials and methods 2.1. Animals and groups Male Sprague–Dawley (SD) rats, Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were obtained from the Shanghai branch of the National Rodent Laboratory Animal Resources of China and housed under standard environmental
0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.09.004
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conditions (temperature 25 ± 1 °C; relative humidity 70%; 12-h light/dark cycle with lights on at 6:00 am; and ad libitum access to food and water). Two months old (280–310 g) was qualified for these experiments. Animal care and experiment protocols were carried out with adherence to the Guide for the Care and Use of Laboratory Animals of Nanjing Medical University and were approved by the Biological Research Ethics Committee of Nanjing Medical University. All surgeries were performed with the aim of limiting the animals’ suffering. To investigate the effects of central Ang II on tau phosphorylation, SD rats (n = 12) were intracerebroventricularly subjected to denaturized Ang II (24 ng/ll, the control group), low-dose Ang II (24 ng/ll, Sigma, Germany), medium-dose Ang II (240 ng/ll), high-dose Ang II (2400 ng/ll), medium-dose Ang II + losartan (800 ng/ll), or losartan (800 ng/ll, Sigma, Germany). To investigate the effects of blood pressure on GSK 3b activation, SHR (n = 12) were administrated Propranolol hydrochloride (50 mg/kg or 100 mg/kg, Sigma, Germany) orally with a gastric tube once daily for 1 week, while SHR control and WKY rats were administrated saline (1 ml/kg) in the same way. To investigate the effects of GSK 3b inhibition on tau phosphorylation, SD rats (n = 12) were intracerebroventricularly subjected to: denaturized Ang II, high-dose Ang II, high-dose Ang II + SB (SB216763, 20 ng/ll, a highly selective and cell permeable GSK3 inhibitor, Sigma, Germany) or SB (20 ng/ll). Because the pumps work at a rate of 0.25 ll/h, the actual doses delivered into rat brains were actually 1/4 of those mentioned above (about 1.67, 16.7, and 167 pg/s, respectively for Ang II). Moreover, the high dose was arbitrarily chosen to be approximately one tenth of the highest dose reported by Porter et al. [21]. Systolic blood pressure (SBP) was measured between 08:00 and 12:00 am on day 0 (pre-surgery), day 3, day 5 and day 7 in SD rats and before sacrifice in SHR and WKY rats. The assays of Morris water maze were performed between 02:00 and 06:00 pm on day 4–7. Then, all animals were sacrificed and the hippocampi were removed. Hippocampal Ang II contents, p-tau levels and GSK3b activities were then assayed. In our preliminary experiments, 800 ng/ll losartan could completely cancel the pressor effects of 240 ng/ll Ang II and denaturalized Ang II infusion was not accompanied with alteration in SBP. 2.2. Blood pressure measurements In the present study, SBP was measured by the tail cuff method using a non-invasive blood pressure analyzer (BP-2000, Visitech Systems, Inc., Apex, NC, USA). Seven days prior to treatment, SBP was measured to make sure that animals were acclimated to the BP-2000 Blood Pressure System. Then, measurements were performed on day 0 (before surgery), day 3 (three days after surgery), day 5 (five days after surgery) and day 7 (seven days after surgery) in SD rats and before sacrifice in SHR between 08:00 and 12:00 am. Each measurement was performed 10 times to acquire a mean SBP. 2.3. Intracerebroventricular infusion After one-week of house acclimation, AD rats were subjected to unilateral intracerebroventricular (ICV) infusion via osmotic pumps (Alzet, model 2004, CA, USA) for another one-week. The pumps were incubated in sterile saline at 37 °C for at least 40 h before they were implanted into animals. ICV implantation was performed following procedures wellestablished in our laboratory [13]. Briefly, after being anesthetized with 10% chloral hydrate (0.35 ml/100 g, Amresco, OH, USA), rats were placed in a stereotactic frame (David Kopf, CA, USA). Stainless steel cannulas from the brain infusion kit were stereotaxically implanted in the right lateral ventricle of the brain (anteroposterior,
0.8 mm; dorsoventral, 3.5 mm; mediolateral, 1.4 mm from bregma as a reference). The pumps were then implanted subcutaneously on the backs of the rats. 2.4. Morris water maze Cognitive and behavioral performances of all rats were assayed by Morris water maze. The protocols were published anywhere. Briefly, a circular pool, 150 cm in diameter, was filled up with tap water and maintained at 22 ± 1 °C. The escape platform (15 cm in diameter) was placed in the pool so that it was submerged 2 cm below the water surface. Each rat was given four trials per day over three consecutive days (day 4, 5 and 6). For each trial, the rat was placed in the pool (facing pool wall) at one of four selected starting points (North, South, East or West pole). On locating the platform, the rat was allowed to remain there for 30 s before being returned to its cage. If the rat did not find the platform within 120 s, the time was recorded as 120 s. Then, the rat was guided to the platform by hand and allowed to remain there for 30 s. The escape latency and swim speed were measured by a video tracking system for the analysis of performance. After the last training trial, rats were subjected to a probe trial in which the platform was removed. Animals were placed in the pool at the same pole and allowed to swim for 2 min. The time that each animal spent in the quadrant that had previously contained the hidden platform was recorded. 2.5. Enzyme immunoassays (EIAs) for hippocampal Ang II Ang II levels in rat brain extracts were measured using commercial EIA kits (Bachem, Switherland). Briefly, hippocampus was dissected and homogenized in 5 ml/g lysis buffer (10 mM Tris, pH 7.4). After mixing and centrifugation (1,600g for 15 min at 4 °C), the supernatant was loaded on an equilibrated SEP-Pak C18 cartridge (Millipore, CA, USA). Equilibration was performed by washing once with 1 ml of buffer B (60% acetonitrile in 1% trifluoroacetic acid) and three times with 3 ml of buffer A (1% trifluoroacetic acid). Peptides were eluted with 3 ml of buffer B and collected in a 15-ml polystyrol tube, evaporated to dryness, and the residue was dissolved in 250 ll of EIA buffer. For EIA, 50 ll of each sample was used. All samples were analyzed in duplicate. Concentrations are expressed in pg per 1 g of brain tissue. 2.6. Western blotting The hippocampi were homogenized in ice-cold lysis buffer consisting of 50 mmol/l Tris–HCl, 150 mmol/l sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, protease inhibitors phenylmethylsulfonyl fluoride (Sigma) and a phosphatase inhibitor cocktail (Roche, Switzerland) using an ultrasound homogenizer (IKA Laboratory, NC, USA). Tissue homogenates were then centrifuged for 15 min at 12,000 rpm at 4 °C (Beckman, CA, USA). Supernatants were carefully collected. Protein concentrations were determined using the Bradford method and were stored at 80 °C until analysis. Equal amounts of protein (about 60 lg) were electrophoresed on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore). After blocking with buffer containing 50 mmol/l Tris–HCl, pH 7.4, 200 mmol/l sodium chloride and 0.5 mmol/l Tween 20 (TBST buffer) and 5% non-fat milk powder, the membranes were incubated overnight at 4 °C with primary antibodies (Table 1) in blocking buffer. Then, after washing in several volumes TBST buffer, the blots were incubated with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies in TBST buffer mixed with 10% non-fat dried milk. The blots were then soaked in
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Table 1 Characteristics of antibodies. Antibody
Type
Specificity
Tau-5 pSer396 pSer404 pSer356 pSer235 pThr205 pThr231 GSK3 pGSK3b
MonoPolyPolyPolyPolyPolyPolyMonoPoly-
Tau p-tau p-tau p-tau p-tau p-tau p-tau GSK3 P-GSK3b
Phosphorylation sites Ser396 Ser235 Ser356 Thr231 Ser404 Thr205 Tyr216
Reference/source Invitrogen Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz Abcam Santa Cruz
enhanced chemiluminescent substrate (Pierce, IL. USA) and exposed to films (Kodak, Japan) for various periods of time (0.05– 5 min). The blots were then incubated with 30% (w/v) hydrogen peroxide for 15 min at 37 °C, reblocked, and reprobed with other antibodies. Intensities from X-ray film were quantified using Densitometer Quantity One software (Bio-Rad, CA, USA) after ascertaining linearity. 2.7. Enzyme-linked immunosorbent assay (ELISA) for Ab For Ab concentration assays, tissue extracts were prepared as described by us [13]. Briefly, hippocampus were homogenized in 10 volumes of high-salt buffer (100 mmol/l Tris–HCl, pH 7.0, 1 mmol/l EGTA, 0.5 mmol/l magnesium sulfate, 750 mmol/l sodium chloride, 20 mmol/l sodium fluoride) containing protease inhibitors (Sigma). Soluble Ab was extracted using diethyl acetate 0.4% in 100 mmol/l sodium chloride and the sample was centrifuged for 60 min at 100,000g at 4 °C (Beckman). The supernatants were carefully collected and then neutralized by adding 500 mmol/ l Tris–HCl (pH 6.8). Protein quantification was performed using the Bradford method. The levels of soluble Ab40 and Ab42 in rat hippocampus extracts were detected using sandwich ELISA kits (Wako, Japan), according to the manufacturer’s instructions. All samples were analyzed in duplicate. Concentrations are expressed in pg per 100 lg of brain tissue.
Fig. 1. Effects of central Ang II or SB on SBP in rats. The alterations of SBP by Ang II (A), losartan (A) and SB (B) was tracked by the tail cuff method. Measurements were performed on day 0 (before surgery), day 3 (three days after surgery), day 5 (five days after surgery) and day 7 (seven days after surgery). Each measurement was performed 10 times to acquire a mean SBP. All results are presented as mean ± S.D., n = 12 for each group, ⁄P < 0.05 or ⁄⁄P < 0.01 versus the control rats. ++P < 0.01 the high-dose AngII + SB group versus the control group.
2.8. Statistical analysis
3.2. High-dose Ang II-induced cognitive impairment was attenuated by losartan or SB
Data were analyzed using analysis of variance (ANOVA), followed by least significant difference post hoc tests or independent samples t tests performed using SPSS 13.0 software (SPSS Inc., IL, USA). All results are expressed as mean ± standard deviation (S.D.). 3. Results 3.1. Low-dose and medium-dose Ang II as well as SB did not alter rat SBP The alterations in SBP caused by continuous Ang II infusion were tracked in male SD rats by the tail cuff method. Measurements were performed on each of four intermittent days to avoid depressing the animals. As shown in Fig. 1A, high-dose Ang II treatment was accompanied by significantly increased SBP (P < 0.01), whereas low-dose and medium-dose Ang II caused no statistically significant alteration in SBP (both P > 0.05). Moreover, the baseline levels of SBP were significantly reduced by combined or single losartan (both P < 0.05) and no significant difference between them was shown (P > 0.05). Measurements were also performed in SD rats receiving SB infusion and SHR with or without oral Propranolol. As shown in Fig. 1B, the increases in SBP by high-dose Ang II and its baseline
levels were not altered by SB (all P > 0.05). As compared to WKY rats, SHR possessed significantly elevated SBPs (Fig. S1, P < 0.01). After one-week oral administration of 50 or 100 mg/kg Propranolol, the SBPs in SHR were dose-dependently reduced (both P < 0.01). More reduction was achieved by 50 mg/kg Propranolol rather than 100 mg/kg Propranolol (P < 0.05).
Cognitive function of rats was herein tested by Morris water maze and escape latency as well as the time spent in global area was recorded. As shown in Fig. 2, in rats receiving high-dose Ang II infusion, escape latency increased while the time spent in global area declined (all P < 0.01), suggesting that the functions of learning and memory were impaired. However, no similar alterations were shown in low-dose and medium-dose Ang II infusion (P > 0.05). When infusion combined with losartan or SB, high-dose Ang II-induced cognitive impairment was canceled. However, single losartan or single SB caused no alteration in performance. Swim speed reflecting movement function was also recorded. However, no remarkable difference among all groups was shown, suggesting that ICV infusion did not interfere in rat movement. 3.3. Hippocampal Ang II levels were elevated by exogenous infusion Hippocampal distribution of Ang II after ICV infusion was assessed by measurements of Ang II, using commercial enzymatic immunoassay (EIA) kits. As shown in Fig. S2, the basal levels of hippocampal Ang II in normal rat brains were approximately 55 pg/g. After one-week infusion of doses of exogenous Ang II, the levels were increased to 74, 98 and 178 pg/g, respectively. Moreover, its
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Fig. 2. High-dose Ang II-induced cognitive impairment was attenuated by losartan or SB. Effects of Ang II, losartan or SB on cognitive performance were assayed by Morris water maze on day 4–7. Escape latency (A and D), time spent in global area (B and E) and swim speed (C and F) were recorded. All results are presented as mean ± S.D., n = 12 for each group, ⁄⁄P < 0.01 versus the control rats.
distribution was not interfered by losartan, a blocker of Ang II signaling as well as SB, the inhibitor of GSK3b. 3.4. Central Ang II significantly induced tau phosphorylation Using commercially available antibodies, the levels of tau phosphorylated at different sites could be semi-quantitated by western blotting analyses. According to a newly recommended protocol [22], at least five different sequential detections without losing significant amounts of blotted proteins can be achieved, given that the horseradish peroxidase-conjugated secondary antibody is irreversibly inhibited by excess hydrogen peroxide. Therefore, the films presented herein represent blots using antibodies against tau phosphorylated at specific sites. All antibodies used herein were listed in Table 1. As shown in Fig. 3, after one-week infusion of central Ang II or blockers of its signaling, the levels of total tau protein (anti-Tau 5 antibody),
including unphosphorylated and phosphorylated tau, were not remarkably changed (P > 0.05, data not shown). In contrast, the levels of p-tau at six sites were notably increased by one-week stimulation with central Ang II. Specifically, a nearly 1.5-fold increase in p-tau at Thr 205 and Ser 356 (both P < 0.05), and at Thr231, Ser235, Ser396, and Ser404 (all P < 0.01), were achieved in low-dose and medium-dose Ang II-infused rat brains, compared with denaturalized Ang II-treated ones (the control group). Furthermore, in high-dose Ang II-treated animals, the increase was extended to 2.5-fold for p-tau at Thr 205, Ser235, Ser 356, Ser396 and Ser404, and to approximately 3-fold in p-tau at Thr231 (all P < 0.01). However, some slight but not statistically significant alterations in the levels of p-tau were found when Ang II was administered in combination with losartan (P > 0.05). Because losartan exerts specific blockade of AT1R, the results herein confirmed the involvement of AT1R in central Ang II-induced tau phosphorylation.
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Fig. 3. Central Ang II infusion significantly induced tau phosphorylation. The hippocampal levels of tau phosphorylated at six sites were measured by western blotting and subsequent quantitative analysis. (A) Representative enhanced chemiluminescence radiographs of immunoblots showing tau phosphorylated at various sites. Density analysis showing levels of tau phosphorylated at Thr205 (B), Thr231 (C), Ser235 (D), Ser356 (E), Ser396 (F), and Ser404 (G). Data in (B–G) were normalized to the level of total tau (anti-tau 5), whereas the intensity of tau 5 was normalized to the level of b actin. All data are represented as mean ± S.D., n = 12 for each group, ⁄P < 0.05 or ⁄⁄P < 0.01 versus the control group.
3.5. Central Ang II promoted the activation of GSK3b Because the action of GSK 3b is regulated by activity-dependent phosphorylation at Tyr216, the activity of GSK 3b is represented by the levels of phosphorylated GSK 3 b (Fig. 4A and B). After a oneweek Ang II infusion, the levels of p-Tyr216-GSK 3b was significantly unregulated (P < 0.05 or 0.01), suggesting the activation of GSK 3b. However, when normalized to the levels of b actin, the levels of total GSK 3 were not affected (P > 0.05, data not shown). The alterations in GSK 3b were all prevented by a combination of Ang II with losartan (P > 0.05), suggesting that activation of GSK 3b is derived from stimulated AT1Rs. 3.6. Activated GSK 3b in SHR hippocampus was attenuated by Propranolol In hippocampus of SHR, a genetic hypertensive model, the levels of p-Tyr216-GSK 3b were also significantly elevated, as compared to its normotensive control, the WKY rat (Fig. 4C and D). Then, the increases were attenuated by daily administration of 50 or 100 mg/kg Propranolol (P < 0.01), a non-RAS inhibitor
anti-hypertensive drug. However, as compared to 50 mg/kg Propranolol, a rebound increase was shown in 100 mg/kg Propranolol (P < 0.05). 3.7. A GSK 3b inhibitor attenuated Ang II-induced tau phosphorylation, but didn’t alter Ab production SB216763 (SB) has been widely accepted as an inhibitor of GSK 3b. When 20 ng/ll SB was co-administered with high-dose Ang II, the levels of p-tau at six sites were significantly attenuated (P < 0.01 or 0.05, Fig. 5), compared with animals receiving highdose Ang II. However, compared with the control group, only the levels of p-tau at Ser 356 were normalized to the basal level (P > 0.05). However, high-dose Ang II-induced Ab (including Ab40 and Ab42) production was not altered by SB, and single SB infusion was not accompanied with Ab production (Fig. 6). 4. Discussion According to the vascular hypothesis, AD is triggered by progressing vascular abnormalities [2], and vascular risk factor
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Fig. 4. The activation of GSK3b was promoted by central Ang II and hypertension. The activity of GSK3b is represented by the levels of the active form of GSK 3b (pGSK3b-Tyr 216), using western blotting. (A and C) Representative immunoblots showing the immunoreactivity for total and phosphorylated GSK3b. (B and D) quantitative analysis of the levels of phosphorylated GSK3b. The levels of total GSK 3b were normalized to those of b actin (data not shown) and the levels of phosphorylated GSK 3b were normalized to those of total GSK 3b. All data are represented as mean ± S.D., n = 12 for each group, ⁄⁄P < 0.01 versus the control group or the WKY rats, ##P < 0.01 versus the high-dose Ang II group, &P < 0.05 versus the 50 mg/kg Propranolol group.
detection and control may prevent AD [23]. Accordingly, several dozen heterogeneous vascular risk factors for AD have been reported, including ischemic stroke, atherosclerosis, hypertension, diabetes and cardiac disease [24]. Mild but persistent vascular lesions are associated with serial sequential events, including Ab upregulation or deposition [25,26]. Moreover, recent evidence has documented that the toxicity of Ab and the severity of AD is dependent on elevated p-tau [1], suggesting the necessity of ptau in AD pathogenesis. The RAS, one of the most important cardiovascular management systems, has been implicated in AD [27]. Ang II, the principal mediator of the RAS, acts as an amplifier of cardiovascular risk, mainly through AT1R [28]. Moreover, the use of RAS blockers in animal models or human beings affected by AD or at risk for AD was linked to reduced AD incidence, attenuated neurotoxic pathology or rescued cognitive impairment. In our laboratory, central Ang II stimulation promoted brain Ab production in normal rodents, and this was canceled by AT1R blockers [13]. However, the levels of p-tau were not assayed, despite being of crucial importance in Ab neurotoxicity and subsequent AD-related neurodegeneration. In the present study, graded doses of Ang II were delivered continuously into rat brain ventricles via osmotic pumps. Although Ang II acts as a pressor peptide, ICV administration of low- or medium-dose Ang II caused no remarkable increases in SBP, whereas high-dose Ang II did (Fig. 1A). This suggested that central Ang II-induced pressor effects are dose-dependent. The increases in SBP by high-dose Ang II were then canceled by losartan. Moreover, no statistical significance was shown between combined and single losartan, inferring that anti-hypertensive effects of losartan are not dependent on Ang II. We then assayed the cognitive performance of rats using Morris water maze. Similar to alterations in SBP, cognitive impairment was induced only by high-dose Ang II and prevented by losartan (Fig. 2A and B). However, when combined with SB, a specific GSK 3b inhibitor, high-dose Ang II-induced cognitive impairment was also prevented without decline in SBP (Figs. 1B, 2D and E).
Therefore, these findings suggested that the impairment by Ang II was not dependent on blood pressure. As noted herein, no movement dysfunction was induced by Ang II (Fig. 2C and F). Hippocampus is crucial to cognition. In the present study, hippocampal levels of Ang II after one-week infusion were assayed by EIA. As expected, hippocampal Ang II contents were remarkably elevated by doses of exogenous Ang II. It may be due to simple diffusion and enhanced synthesis by itself. Interestingly, losartan, the blocker of Ang II signaling, caused no alteration in Ang II distribution, although abundant AT1R was localized in hippocampus [29]. Similar results were shown in Ang II + SB animals, suggesting that the GSK3b signaling was not involved in Ang II levels. Although tau phosphorylation was also crucial to many physiological processes including axonal growth [30], phosphorylation at certain sites was involved in AD pathogenesis. Among the 85 putative phosphorylation sites in tau, tau phosphorylated at six representative sites accumulates in AD brains [31], and these sites were selected for analysis herein. As shown in Fig. 3, the levels of p-tau at these six sites were dose-dependently elevated after one-week Ang II infusion, although the levels of total tau were not altered. Moreover, significant tau phosphorylation was induced by lowand medium-dose Ang II without detected cognitive impairment. This implied that pathological alteration preceded visible phenotypes in AD, which was in accordance with previous reports [30]. Then, Ang II-induced tau phosphorylation was significantly attenuated by blockade of AT1R, suggesting that Ang II-induced tau phosphorylation is AT1R-mediated. As noted, losartan can also block the endogenous Ang II signaling, which may contribute to the slight decrease in single losartan-treated rats herein (Fig. 3). However, due to complicated regulatory network or alternative complementary mechanisms in body, the basal p-tau levels were not significantly altered. GSK3b, a proline-directed serine-threonine kinase, has been identified as the main tau kinase [32]. Its expression and activity is induced in AD brains [33]. Interestingly, the actions of GSK3b are also phosphorylation dependent. It can be activated by
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Fig. 5. A GSK 3b inhibitor attenuated Ang II-induced tau phosphorylation. Ang II was delivered into rat brains in combination with SB, a widely-studied GSK 3 inhibitor. (A) Representative immunoblots showing tau phosphorylated at various sites. Density analysis showing the levels of tau phosphorylated at Thr205 (B), Thr231 (C), Ser235 (D), Ser356 (E), Ser396 (F), and Ser404 (G). Data in (B–G) were normalized to total tau (anti-Tau 5), whereas the intensity of Tau 5 was normalized to that of b actin (data not shown). All data are represented as mean ± S.D., n = 12 for each group, ⁄P < 0.05 or ⁄⁄P < 0.01 versus the control group. #P < 0.05 or ##P < 0.01 versus high-dose Ang II-infused group.
Fig. 6. Ang II-induced Ab production was not altered by the GSK3b inhibitor, SB. Hippocampal Ab levels were measured in the high-dose Ang II group with or without SB, using sandwich ELISA. Assays in Ab40 (A) and Ab42 (B) were performed in duplicate. All data are represented as mean ± S.D., n = 12 for each group, ⁄P < 0.05 or ⁄⁄P < 0.01 versus the control group.
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phosphorylation at Tyr 216 (p-Tyr 216-GSK3b) [34]. As shown in Fig. 4, despite no change in the levels of total GSK3b after continuous Ang II stimulation, the levels of p-Tyr 216-GSK3b were significantly elevated. However, these alterations were canceled by losartan, suggesting that the expression of GSK3b and its activities can be upregulated by Ang II binding to AT1R. As noted, brain GSK 3b phosphorylation can be also induced by Ab [35]. Therefore, the elevated production of Ab-induced by Ang II, as confirmed in our previous studies, could be an important mediator of Ang II-induced tau phosphorylation, which is now being investigated using Ab immunization in our laboratory. As noted herein, the activities of GSK 3b were increased by Ang II but reduced by losartan, accompanied with alterations in SBP. We then investigated the roles of blood pressure in GSK 3b activation using a genetic hypertensive model, SHR. Since Ang II exerts many effects unrelated to its pressor action, doses of a non-RAS inhibitor anti-hypertensive drug, Propranolol, were orally administrated to SHR. Elevated SBPs in SHR were confirmed by the tail cuff method (Fig. S1), while unregulated p-Tyr 216-GSK3b levels were also shown in SHR hippocampus. The SBPs in SHR were reduced by daily Propranolol, accompanied by attenuated GSK 3b activation. However, more significant decrease in GSK 3b activities was achieved in 50 mg/kg although lower SBP was shown in 100 mg/kg. These findings suggested that hypertension is an important but not the only signaling in Ang II-induced GSK 3b activation. Some other kinases, including cyclin-dependent kinase 5, casein kinase 1, protein kinase A and mitogen-activated protein kinases (MAPKs) have been implicated in tau phosphorylation [36] and in neuronal Ang II signaling [37]. The involvement of GSK3b in the Ang II-induced tau phosphorylation observed herein was confirmed using SB, a widely-studied inhibitor specific for GSK3b. As expected, the phosphorylation of all sites induced by central Ang II was significantly attenuated by inhibition of GSK3b. P-tau at Ser356 was normalized to the basal level by SB, implying that the dose of SB used herein was sufficient. However, phosphorylation at other sites was not blocked to the same extent. This inconsistency suggested that GSK3b might phosphorylate tau at specific sites only, and that it may not be the only kinase mediating Ang IIinduced tau phosphorylation. Further studies are thus warranted to elucidate the roles of other kinases, for example, MAPKs, in Ang II-induced tau phosphorylation. Ab production was also stimulated by Ang II in our previous studies [13]. The role of the GSK3b signaling in Ang II-induced Ab production was thus investigated herein. As shown in Fig. 6, inhibition of GSK3b by SB didn’t alter the levels of Ab-induced by highdose Ang II. This is in accordance with a recent study, in which in vivo Ab production was not controlled by GSK 3b [38]. Taken together, these findings suggested that Ang II, the crucial mediator of cardiovascular pathology, induced significant cognitive impairment and significant tau phosphorylation via activating GSK3b and other kinases. Attenuated tau phosphorylation protected against cognitive impairment, confirmed the crucial role of p-tau in AD pathogenesis. Given the roles of Ang II in cardiovascular events, these findings combined with our previous report will promote the establishment of the Ang II-Ab-GSK3b-tau cascade, and provide evidences for the vascular origin of AD pathogenesis. Since the blood–brain carrier is permeable to most AT1R blockers including losartan, blockade of brain Ang II signaling represents a promising strategy for anti-AD therapies. Acknowledgment This work was supported by the Medical Science and Technology Development Foundation, Nanjing Department of Health (No. ZKX 08035, No. YKK11031).
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(2005) Angiotensin II AT1 receptor blockade abolishes brain microvascular inflammation and heat shock protein responses in hypertensive rats. J. Cereb. Blood Flow Metab. 25, 878–886. [7] Ito, T., Yamakawa, H., Bregonzio, C., Terron, J.A., Falcon-Neri, A. and Saavedra, J.M. (2002) Protection against ischemia and improvement of cerebral blood flow in genetically hypertensive rats by chronic pretreatment with an angiotensin II AT1 antagonist. Stroke 33, 2297–2303. [8] Pelisch, N. et al. (2011) Blockade of AT1 receptors protects the blood–brain barrier and improves cognition in Dahl salt-sensitive hypertensive rats. Am. J. Hypertens. 24, 362–368. [9] Mechaeil, R., Gard, P., Jackson, A. and Rusted, J. (2011) Cognitive enhancement following acute losartan in normotensive young adults. Psychopharmacology (Berl). [10] Dong, Y.F. et al. (2011) Perindopril, a centrally active angiotensin-converting enzyme inhibitor, prevents cognitive impairment in mouse models of Alzheimer’s disease. 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(1992) Neurofibrillary tangles but not senile plaques parallel duration and severity of Alzheimer’s disease. Neurology 42, 631–639. [16] Holmes, C. et al. (2008) Long-term effects of Abeta42 immunisation in Alzheimer’s disease: follow-up of a randomised, placebo-controlled phase I trial. Lancet 372, 216–223. [17] Roberson, E.D. et al. (2007) Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer’s disease mouse model. Science 316, 750–754. [18] Ittner, L.M. et al. (2010) Dendritic function of tau mediates amyloidbeta toxicity in Alzheimer’s disease mouse models. J. Cell. Mol. Med. 142, 387–397. [19] Desikan, R.S. et al. (2011) Amyloid-beta associated volume loss occurs only in the presence of phospho-tau. Ann. Neurol. 70, 657–661. [20] Desikan, R.S., McEvoy, L.K., Thompson, W.K., Holland, D., Brewer, J.B., Aisen, P.S., Sperling, R.A. and Dale, A.M. (2012) Amyloid-beta-associated clinical decline occurs only in the presence of elevated P-tau. Arch. 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Central angiotensin II-induced Alzheimer-like tau phosphorylation in normal rat brains Minjie Tian 1, Donglin Zhu 1, Wei Xie, Jingping Shi ⇑ Department of Neurology, Nanjing Brain Hospital Affiliated to Nanjing Medical University, Nanjing, PR China
a r t i c l e
i n f o
Article history: Received 22 July 2012 Revised 29 August 2012 Accepted 1 September 2012 Available online 13 September 2012 Edited by Barry Halliwell Keywords: Angiotensin II Glycogen synthase kinase 3b Tau Alzheimer disease
a b s t r a c t Growing evidence suggests that Alzheimer disease (AD) origins in vascular lesions. As the crucial mediator of vascular pathology, angiotensin II-induced significant amyloid production in our laboratory, although amyloid neurotoxicity depended on phosphorylated tau (p-tau) in recent studies. In the present study, p-tau levels were significantly elevated by central angiotensin II via glycogen synthase kinase 3b (GSK 3b) and other tau kinases. Moreover, angiotensin II-induced cognitive impairment and tau phosphorylation was attenuated by losartan and a GSK 3b inhibitor. These findings implicate Ang II as a crucial mediator of AD pathology and a link between cardiovascular events and AD. Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Alzheimer disease (AD) is one of the most important unmet needs in neurology, with oligomer-prone b-amyloid (Ab) and phosphorylated tau (p-tau) as its pathological hallmarks [1]. Glycogen synthase kinase 3b (GSK 3b), the main tau kinase, are thus also involved in AD pathogenesis. More recently, growing evidence has indicated that AD is initiated by vascular factors that precede the neurodegenerative process, leading to AD being termed a vascular disorder [2]. As an intrinsic vasoactive hormone and the main actor in the renin–angiotensin system (RAS), brain angiotensin II (Ang II) is always present in abnormally high quantities in many cardiovascular diseases [3]. More recently, it has also been implicated in AD [4,5]. Excess Ang II was linked to increased blood pressure, decreased cerebrovascular compliance and enhanced brain inflammation, all recognized risk factors for AD, while Ang II type 1 receptor (AT1R) blockade suppressed such processes [6,7]. Moreover, application of AT1R blockers to rodents led to attenuated neurotoxic accumulation and cognitive deficits [8–12]. In our laboratory, central infusion of Ang II significantly promoted Ab production via enhanced amyloidogenic processing [13]. These findings implicated Ang II as a crucial mediator in AD, just as it has been implicated in cardiovascular pathology [14]. ⇑ Corresponding author. Fax: +86 025 83719457. 1
E-mail address: [email protected] (J. Shi). These authors contributed equally to this work.
However, Ab plaques correlate poorly with cognition decline [15] and plaque removal did not prevent neurodegeneration [16], suggesting that other entities may be required for Ab-induced neurotoxicity. As noted in AD rodent models, the presence of tau is required for Ab-induced neuronal and synaptic damage [1]. Reducing endogenous tau ameliorates Ab-induced neuronal dysfunction [17], while dendritic function of tau mediates Ab-associated synaptotoxicity [18]. In older individuals at risk for dementia, Ab-associated brain volume loss and longitudinal clinical decline occurs only in the presence of p-tau [19,20]. It thus has been proposed that Ab neurotoxicity is dependent on tau, especially p-tau. It is therefore intriguing to determine the levels of ptau in response to Ang II stimulation, because our findings have confirmed that Ang II could induce Ab production. In the present study, graded doses of Ang II were delivered into rat brain ventricles via osmotic pumps, as a model of increased brain Ang II in vascular-related events. The levels of AD-like ptau were then assayed and the signaling pathways involved were also uncovered. 2. Materials and methods 2.1. Animals and groups Male Sprague–Dawley (SD) rats, Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were obtained from the Shanghai branch of the National Rodent Laboratory Animal Resources of China and housed under standard environmental
0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.09.004
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conditions (temperature 25 ± 1 °C; relative humidity 70%; 12-h light/dark cycle with lights on at 6:00 am; and ad libitum access to food and water). Two months old (280–310 g) was qualified for these experiments. Animal care and experiment protocols were carried out with adherence to the Guide for the Care and Use of Laboratory Animals of Nanjing Medical University and were approved by the Biological Research Ethics Committee of Nanjing Medical University. All surgeries were performed with the aim of limiting the animals’ suffering. To investigate the effects of central Ang II on tau phosphorylation, SD rats (n = 12) were intracerebroventricularly subjected to denaturized Ang II (24 ng/ll, the control group), low-dose Ang II (24 ng/ll, Sigma, Germany), medium-dose Ang II (240 ng/ll), high-dose Ang II (2400 ng/ll), medium-dose Ang II + losartan (800 ng/ll), or losartan (800 ng/ll, Sigma, Germany). To investigate the effects of blood pressure on GSK 3b activation, SHR (n = 12) were administrated Propranolol hydrochloride (50 mg/kg or 100 mg/kg, Sigma, Germany) orally with a gastric tube once daily for 1 week, while SHR control and WKY rats were administrated saline (1 ml/kg) in the same way. To investigate the effects of GSK 3b inhibition on tau phosphorylation, SD rats (n = 12) were intracerebroventricularly subjected to: denaturized Ang II, high-dose Ang II, high-dose Ang II + SB (SB216763, 20 ng/ll, a highly selective and cell permeable GSK3 inhibitor, Sigma, Germany) or SB (20 ng/ll). Because the pumps work at a rate of 0.25 ll/h, the actual doses delivered into rat brains were actually 1/4 of those mentioned above (about 1.67, 16.7, and 167 pg/s, respectively for Ang II). Moreover, the high dose was arbitrarily chosen to be approximately one tenth of the highest dose reported by Porter et al. [21]. Systolic blood pressure (SBP) was measured between 08:00 and 12:00 am on day 0 (pre-surgery), day 3, day 5 and day 7 in SD rats and before sacrifice in SHR and WKY rats. The assays of Morris water maze were performed between 02:00 and 06:00 pm on day 4–7. Then, all animals were sacrificed and the hippocampi were removed. Hippocampal Ang II contents, p-tau levels and GSK3b activities were then assayed. In our preliminary experiments, 800 ng/ll losartan could completely cancel the pressor effects of 240 ng/ll Ang II and denaturalized Ang II infusion was not accompanied with alteration in SBP. 2.2. Blood pressure measurements In the present study, SBP was measured by the tail cuff method using a non-invasive blood pressure analyzer (BP-2000, Visitech Systems, Inc., Apex, NC, USA). Seven days prior to treatment, SBP was measured to make sure that animals were acclimated to the BP-2000 Blood Pressure System. Then, measurements were performed on day 0 (before surgery), day 3 (three days after surgery), day 5 (five days after surgery) and day 7 (seven days after surgery) in SD rats and before sacrifice in SHR between 08:00 and 12:00 am. Each measurement was performed 10 times to acquire a mean SBP. 2.3. Intracerebroventricular infusion After one-week of house acclimation, AD rats were subjected to unilateral intracerebroventricular (ICV) infusion via osmotic pumps (Alzet, model 2004, CA, USA) for another one-week. The pumps were incubated in sterile saline at 37 °C for at least 40 h before they were implanted into animals. ICV implantation was performed following procedures wellestablished in our laboratory [13]. Briefly, after being anesthetized with 10% chloral hydrate (0.35 ml/100 g, Amresco, OH, USA), rats were placed in a stereotactic frame (David Kopf, CA, USA). Stainless steel cannulas from the brain infusion kit were stereotaxically implanted in the right lateral ventricle of the brain (anteroposterior,
0.8 mm; dorsoventral, 3.5 mm; mediolateral, 1.4 mm from bregma as a reference). The pumps were then implanted subcutaneously on the backs of the rats. 2.4. Morris water maze Cognitive and behavioral performances of all rats were assayed by Morris water maze. The protocols were published anywhere. Briefly, a circular pool, 150 cm in diameter, was filled up with tap water and maintained at 22 ± 1 °C. The escape platform (15 cm in diameter) was placed in the pool so that it was submerged 2 cm below the water surface. Each rat was given four trials per day over three consecutive days (day 4, 5 and 6). For each trial, the rat was placed in the pool (facing pool wall) at one of four selected starting points (North, South, East or West pole). On locating the platform, the rat was allowed to remain there for 30 s before being returned to its cage. If the rat did not find the platform within 120 s, the time was recorded as 120 s. Then, the rat was guided to the platform by hand and allowed to remain there for 30 s. The escape latency and swim speed were measured by a video tracking system for the analysis of performance. After the last training trial, rats were subjected to a probe trial in which the platform was removed. Animals were placed in the pool at the same pole and allowed to swim for 2 min. The time that each animal spent in the quadrant that had previously contained the hidden platform was recorded. 2.5. Enzyme immunoassays (EIAs) for hippocampal Ang II Ang II levels in rat brain extracts were measured using commercial EIA kits (Bachem, Switherland). Briefly, hippocampus was dissected and homogenized in 5 ml/g lysis buffer (10 mM Tris, pH 7.4). After mixing and centrifugation (1,600g for 15 min at 4 °C), the supernatant was loaded on an equilibrated SEP-Pak C18 cartridge (Millipore, CA, USA). Equilibration was performed by washing once with 1 ml of buffer B (60% acetonitrile in 1% trifluoroacetic acid) and three times with 3 ml of buffer A (1% trifluoroacetic acid). Peptides were eluted with 3 ml of buffer B and collected in a 15-ml polystyrol tube, evaporated to dryness, and the residue was dissolved in 250 ll of EIA buffer. For EIA, 50 ll of each sample was used. All samples were analyzed in duplicate. Concentrations are expressed in pg per 1 g of brain tissue. 2.6. Western blotting The hippocampi were homogenized in ice-cold lysis buffer consisting of 50 mmol/l Tris–HCl, 150 mmol/l sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, protease inhibitors phenylmethylsulfonyl fluoride (Sigma) and a phosphatase inhibitor cocktail (Roche, Switzerland) using an ultrasound homogenizer (IKA Laboratory, NC, USA). Tissue homogenates were then centrifuged for 15 min at 12,000 rpm at 4 °C (Beckman, CA, USA). Supernatants were carefully collected. Protein concentrations were determined using the Bradford method and were stored at 80 °C until analysis. Equal amounts of protein (about 60 lg) were electrophoresed on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore). After blocking with buffer containing 50 mmol/l Tris–HCl, pH 7.4, 200 mmol/l sodium chloride and 0.5 mmol/l Tween 20 (TBST buffer) and 5% non-fat milk powder, the membranes were incubated overnight at 4 °C with primary antibodies (Table 1) in blocking buffer. Then, after washing in several volumes TBST buffer, the blots were incubated with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies in TBST buffer mixed with 10% non-fat dried milk. The blots were then soaked in
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Table 1 Characteristics of antibodies. Antibody
Type
Specificity
Tau-5 pSer396 pSer404 pSer356 pSer235 pThr205 pThr231 GSK3 pGSK3b
MonoPolyPolyPolyPolyPolyPolyMonoPoly-
Tau p-tau p-tau p-tau p-tau p-tau p-tau GSK3 P-GSK3b
Phosphorylation sites Ser396 Ser235 Ser356 Thr231 Ser404 Thr205 Tyr216
Reference/source Invitrogen Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz Abcam Santa Cruz
enhanced chemiluminescent substrate (Pierce, IL. USA) and exposed to films (Kodak, Japan) for various periods of time (0.05– 5 min). The blots were then incubated with 30% (w/v) hydrogen peroxide for 15 min at 37 °C, reblocked, and reprobed with other antibodies. Intensities from X-ray film were quantified using Densitometer Quantity One software (Bio-Rad, CA, USA) after ascertaining linearity. 2.7. Enzyme-linked immunosorbent assay (ELISA) for Ab For Ab concentration assays, tissue extracts were prepared as described by us [13]. Briefly, hippocampus were homogenized in 10 volumes of high-salt buffer (100 mmol/l Tris–HCl, pH 7.0, 1 mmol/l EGTA, 0.5 mmol/l magnesium sulfate, 750 mmol/l sodium chloride, 20 mmol/l sodium fluoride) containing protease inhibitors (Sigma). Soluble Ab was extracted using diethyl acetate 0.4% in 100 mmol/l sodium chloride and the sample was centrifuged for 60 min at 100,000g at 4 °C (Beckman). The supernatants were carefully collected and then neutralized by adding 500 mmol/ l Tris–HCl (pH 6.8). Protein quantification was performed using the Bradford method. The levels of soluble Ab40 and Ab42 in rat hippocampus extracts were detected using sandwich ELISA kits (Wako, Japan), according to the manufacturer’s instructions. All samples were analyzed in duplicate. Concentrations are expressed in pg per 100 lg of brain tissue.
Fig. 1. Effects of central Ang II or SB on SBP in rats. The alterations of SBP by Ang II (A), losartan (A) and SB (B) was tracked by the tail cuff method. Measurements were performed on day 0 (before surgery), day 3 (three days after surgery), day 5 (five days after surgery) and day 7 (seven days after surgery). Each measurement was performed 10 times to acquire a mean SBP. All results are presented as mean ± S.D., n = 12 for each group, ⁄P < 0.05 or ⁄⁄P < 0.01 versus the control rats. ++P < 0.01 the high-dose AngII + SB group versus the control group.
2.8. Statistical analysis
3.2. High-dose Ang II-induced cognitive impairment was attenuated by losartan or SB
Data were analyzed using analysis of variance (ANOVA), followed by least significant difference post hoc tests or independent samples t tests performed using SPSS 13.0 software (SPSS Inc., IL, USA). All results are expressed as mean ± standard deviation (S.D.). 3. Results 3.1. Low-dose and medium-dose Ang II as well as SB did not alter rat SBP The alterations in SBP caused by continuous Ang II infusion were tracked in male SD rats by the tail cuff method. Measurements were performed on each of four intermittent days to avoid depressing the animals. As shown in Fig. 1A, high-dose Ang II treatment was accompanied by significantly increased SBP (P < 0.01), whereas low-dose and medium-dose Ang II caused no statistically significant alteration in SBP (both P > 0.05). Moreover, the baseline levels of SBP were significantly reduced by combined or single losartan (both P < 0.05) and no significant difference between them was shown (P > 0.05). Measurements were also performed in SD rats receiving SB infusion and SHR with or without oral Propranolol. As shown in Fig. 1B, the increases in SBP by high-dose Ang II and its baseline
levels were not altered by SB (all P > 0.05). As compared to WKY rats, SHR possessed significantly elevated SBPs (Fig. S1, P < 0.01). After one-week oral administration of 50 or 100 mg/kg Propranolol, the SBPs in SHR were dose-dependently reduced (both P < 0.01). More reduction was achieved by 50 mg/kg Propranolol rather than 100 mg/kg Propranolol (P < 0.05).
Cognitive function of rats was herein tested by Morris water maze and escape latency as well as the time spent in global area was recorded. As shown in Fig. 2, in rats receiving high-dose Ang II infusion, escape latency increased while the time spent in global area declined (all P < 0.01), suggesting that the functions of learning and memory were impaired. However, no similar alterations were shown in low-dose and medium-dose Ang II infusion (P > 0.05). When infusion combined with losartan or SB, high-dose Ang II-induced cognitive impairment was canceled. However, single losartan or single SB caused no alteration in performance. Swim speed reflecting movement function was also recorded. However, no remarkable difference among all groups was shown, suggesting that ICV infusion did not interfere in rat movement. 3.3. Hippocampal Ang II levels were elevated by exogenous infusion Hippocampal distribution of Ang II after ICV infusion was assessed by measurements of Ang II, using commercial enzymatic immunoassay (EIA) kits. As shown in Fig. S2, the basal levels of hippocampal Ang II in normal rat brains were approximately 55 pg/g. After one-week infusion of doses of exogenous Ang II, the levels were increased to 74, 98 and 178 pg/g, respectively. Moreover, its
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Fig. 2. High-dose Ang II-induced cognitive impairment was attenuated by losartan or SB. Effects of Ang II, losartan or SB on cognitive performance were assayed by Morris water maze on day 4–7. Escape latency (A and D), time spent in global area (B and E) and swim speed (C and F) were recorded. All results are presented as mean ± S.D., n = 12 for each group, ⁄⁄P < 0.01 versus the control rats.
distribution was not interfered by losartan, a blocker of Ang II signaling as well as SB, the inhibitor of GSK3b. 3.4. Central Ang II significantly induced tau phosphorylation Using commercially available antibodies, the levels of tau phosphorylated at different sites could be semi-quantitated by western blotting analyses. According to a newly recommended protocol [22], at least five different sequential detections without losing significant amounts of blotted proteins can be achieved, given that the horseradish peroxidase-conjugated secondary antibody is irreversibly inhibited by excess hydrogen peroxide. Therefore, the films presented herein represent blots using antibodies against tau phosphorylated at specific sites. All antibodies used herein were listed in Table 1. As shown in Fig. 3, after one-week infusion of central Ang II or blockers of its signaling, the levels of total tau protein (anti-Tau 5 antibody),
including unphosphorylated and phosphorylated tau, were not remarkably changed (P > 0.05, data not shown). In contrast, the levels of p-tau at six sites were notably increased by one-week stimulation with central Ang II. Specifically, a nearly 1.5-fold increase in p-tau at Thr 205 and Ser 356 (both P < 0.05), and at Thr231, Ser235, Ser396, and Ser404 (all P < 0.01), were achieved in low-dose and medium-dose Ang II-infused rat brains, compared with denaturalized Ang II-treated ones (the control group). Furthermore, in high-dose Ang II-treated animals, the increase was extended to 2.5-fold for p-tau at Thr 205, Ser235, Ser 356, Ser396 and Ser404, and to approximately 3-fold in p-tau at Thr231 (all P < 0.01). However, some slight but not statistically significant alterations in the levels of p-tau were found when Ang II was administered in combination with losartan (P > 0.05). Because losartan exerts specific blockade of AT1R, the results herein confirmed the involvement of AT1R in central Ang II-induced tau phosphorylation.
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Fig. 3. Central Ang II infusion significantly induced tau phosphorylation. The hippocampal levels of tau phosphorylated at six sites were measured by western blotting and subsequent quantitative analysis. (A) Representative enhanced chemiluminescence radiographs of immunoblots showing tau phosphorylated at various sites. Density analysis showing levels of tau phosphorylated at Thr205 (B), Thr231 (C), Ser235 (D), Ser356 (E), Ser396 (F), and Ser404 (G). Data in (B–G) were normalized to the level of total tau (anti-tau 5), whereas the intensity of tau 5 was normalized to the level of b actin. All data are represented as mean ± S.D., n = 12 for each group, ⁄P < 0.05 or ⁄⁄P < 0.01 versus the control group.
3.5. Central Ang II promoted the activation of GSK3b Because the action of GSK 3b is regulated by activity-dependent phosphorylation at Tyr216, the activity of GSK 3b is represented by the levels of phosphorylated GSK 3 b (Fig. 4A and B). After a oneweek Ang II infusion, the levels of p-Tyr216-GSK 3b was significantly unregulated (P < 0.05 or 0.01), suggesting the activation of GSK 3b. However, when normalized to the levels of b actin, the levels of total GSK 3 were not affected (P > 0.05, data not shown). The alterations in GSK 3b were all prevented by a combination of Ang II with losartan (P > 0.05), suggesting that activation of GSK 3b is derived from stimulated AT1Rs. 3.6. Activated GSK 3b in SHR hippocampus was attenuated by Propranolol In hippocampus of SHR, a genetic hypertensive model, the levels of p-Tyr216-GSK 3b were also significantly elevated, as compared to its normotensive control, the WKY rat (Fig. 4C and D). Then, the increases were attenuated by daily administration of 50 or 100 mg/kg Propranolol (P < 0.01), a non-RAS inhibitor
anti-hypertensive drug. However, as compared to 50 mg/kg Propranolol, a rebound increase was shown in 100 mg/kg Propranolol (P < 0.05). 3.7. A GSK 3b inhibitor attenuated Ang II-induced tau phosphorylation, but didn’t alter Ab production SB216763 (SB) has been widely accepted as an inhibitor of GSK 3b. When 20 ng/ll SB was co-administered with high-dose Ang II, the levels of p-tau at six sites were significantly attenuated (P < 0.01 or 0.05, Fig. 5), compared with animals receiving highdose Ang II. However, compared with the control group, only the levels of p-tau at Ser 356 were normalized to the basal level (P > 0.05). However, high-dose Ang II-induced Ab (including Ab40 and Ab42) production was not altered by SB, and single SB infusion was not accompanied with Ab production (Fig. 6). 4. Discussion According to the vascular hypothesis, AD is triggered by progressing vascular abnormalities [2], and vascular risk factor
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Fig. 4. The activation of GSK3b was promoted by central Ang II and hypertension. The activity of GSK3b is represented by the levels of the active form of GSK 3b (pGSK3b-Tyr 216), using western blotting. (A and C) Representative immunoblots showing the immunoreactivity for total and phosphorylated GSK3b. (B and D) quantitative analysis of the levels of phosphorylated GSK3b. The levels of total GSK 3b were normalized to those of b actin (data not shown) and the levels of phosphorylated GSK 3b were normalized to those of total GSK 3b. All data are represented as mean ± S.D., n = 12 for each group, ⁄⁄P < 0.01 versus the control group or the WKY rats, ##P < 0.01 versus the high-dose Ang II group, &P < 0.05 versus the 50 mg/kg Propranolol group.
detection and control may prevent AD [23]. Accordingly, several dozen heterogeneous vascular risk factors for AD have been reported, including ischemic stroke, atherosclerosis, hypertension, diabetes and cardiac disease [24]. Mild but persistent vascular lesions are associated with serial sequential events, including Ab upregulation or deposition [25,26]. Moreover, recent evidence has documented that the toxicity of Ab and the severity of AD is dependent on elevated p-tau [1], suggesting the necessity of ptau in AD pathogenesis. The RAS, one of the most important cardiovascular management systems, has been implicated in AD [27]. Ang II, the principal mediator of the RAS, acts as an amplifier of cardiovascular risk, mainly through AT1R [28]. Moreover, the use of RAS blockers in animal models or human beings affected by AD or at risk for AD was linked to reduced AD incidence, attenuated neurotoxic pathology or rescued cognitive impairment. In our laboratory, central Ang II stimulation promoted brain Ab production in normal rodents, and this was canceled by AT1R blockers [13]. However, the levels of p-tau were not assayed, despite being of crucial importance in Ab neurotoxicity and subsequent AD-related neurodegeneration. In the present study, graded doses of Ang II were delivered continuously into rat brain ventricles via osmotic pumps. Although Ang II acts as a pressor peptide, ICV administration of low- or medium-dose Ang II caused no remarkable increases in SBP, whereas high-dose Ang II did (Fig. 1A). This suggested that central Ang II-induced pressor effects are dose-dependent. The increases in SBP by high-dose Ang II were then canceled by losartan. Moreover, no statistical significance was shown between combined and single losartan, inferring that anti-hypertensive effects of losartan are not dependent on Ang II. We then assayed the cognitive performance of rats using Morris water maze. Similar to alterations in SBP, cognitive impairment was induced only by high-dose Ang II and prevented by losartan (Fig. 2A and B). However, when combined with SB, a specific GSK 3b inhibitor, high-dose Ang II-induced cognitive impairment was also prevented without decline in SBP (Figs. 1B, 2D and E).
Therefore, these findings suggested that the impairment by Ang II was not dependent on blood pressure. As noted herein, no movement dysfunction was induced by Ang II (Fig. 2C and F). Hippocampus is crucial to cognition. In the present study, hippocampal levels of Ang II after one-week infusion were assayed by EIA. As expected, hippocampal Ang II contents were remarkably elevated by doses of exogenous Ang II. It may be due to simple diffusion and enhanced synthesis by itself. Interestingly, losartan, the blocker of Ang II signaling, caused no alteration in Ang II distribution, although abundant AT1R was localized in hippocampus [29]. Similar results were shown in Ang II + SB animals, suggesting that the GSK3b signaling was not involved in Ang II levels. Although tau phosphorylation was also crucial to many physiological processes including axonal growth [30], phosphorylation at certain sites was involved in AD pathogenesis. Among the 85 putative phosphorylation sites in tau, tau phosphorylated at six representative sites accumulates in AD brains [31], and these sites were selected for analysis herein. As shown in Fig. 3, the levels of p-tau at these six sites were dose-dependently elevated after one-week Ang II infusion, although the levels of total tau were not altered. Moreover, significant tau phosphorylation was induced by lowand medium-dose Ang II without detected cognitive impairment. This implied that pathological alteration preceded visible phenotypes in AD, which was in accordance with previous reports [30]. Then, Ang II-induced tau phosphorylation was significantly attenuated by blockade of AT1R, suggesting that Ang II-induced tau phosphorylation is AT1R-mediated. As noted, losartan can also block the endogenous Ang II signaling, which may contribute to the slight decrease in single losartan-treated rats herein (Fig. 3). However, due to complicated regulatory network or alternative complementary mechanisms in body, the basal p-tau levels were not significantly altered. GSK3b, a proline-directed serine-threonine kinase, has been identified as the main tau kinase [32]. Its expression and activity is induced in AD brains [33]. Interestingly, the actions of GSK3b are also phosphorylation dependent. It can be activated by
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Fig. 5. A GSK 3b inhibitor attenuated Ang II-induced tau phosphorylation. Ang II was delivered into rat brains in combination with SB, a widely-studied GSK 3 inhibitor. (A) Representative immunoblots showing tau phosphorylated at various sites. Density analysis showing the levels of tau phosphorylated at Thr205 (B), Thr231 (C), Ser235 (D), Ser356 (E), Ser396 (F), and Ser404 (G). Data in (B–G) were normalized to total tau (anti-Tau 5), whereas the intensity of Tau 5 was normalized to that of b actin (data not shown). All data are represented as mean ± S.D., n = 12 for each group, ⁄P < 0.05 or ⁄⁄P < 0.01 versus the control group. #P < 0.05 or ##P < 0.01 versus high-dose Ang II-infused group.
Fig. 6. Ang II-induced Ab production was not altered by the GSK3b inhibitor, SB. Hippocampal Ab levels were measured in the high-dose Ang II group with or without SB, using sandwich ELISA. Assays in Ab40 (A) and Ab42 (B) were performed in duplicate. All data are represented as mean ± S.D., n = 12 for each group, ⁄P < 0.05 or ⁄⁄P < 0.01 versus the control group.
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phosphorylation at Tyr 216 (p-Tyr 216-GSK3b) [34]. As shown in Fig. 4, despite no change in the levels of total GSK3b after continuous Ang II stimulation, the levels of p-Tyr 216-GSK3b were significantly elevated. However, these alterations were canceled by losartan, suggesting that the expression of GSK3b and its activities can be upregulated by Ang II binding to AT1R. As noted, brain GSK 3b phosphorylation can be also induced by Ab [35]. Therefore, the elevated production of Ab-induced by Ang II, as confirmed in our previous studies, could be an important mediator of Ang II-induced tau phosphorylation, which is now being investigated using Ab immunization in our laboratory. As noted herein, the activities of GSK 3b were increased by Ang II but reduced by losartan, accompanied with alterations in SBP. We then investigated the roles of blood pressure in GSK 3b activation using a genetic hypertensive model, SHR. Since Ang II exerts many effects unrelated to its pressor action, doses of a non-RAS inhibitor anti-hypertensive drug, Propranolol, were orally administrated to SHR. Elevated SBPs in SHR were confirmed by the tail cuff method (Fig. S1), while unregulated p-Tyr 216-GSK3b levels were also shown in SHR hippocampus. The SBPs in SHR were reduced by daily Propranolol, accompanied by attenuated GSK 3b activation. However, more significant decrease in GSK 3b activities was achieved in 50 mg/kg although lower SBP was shown in 100 mg/kg. These findings suggested that hypertension is an important but not the only signaling in Ang II-induced GSK 3b activation. Some other kinases, including cyclin-dependent kinase 5, casein kinase 1, protein kinase A and mitogen-activated protein kinases (MAPKs) have been implicated in tau phosphorylation [36] and in neuronal Ang II signaling [37]. The involvement of GSK3b in the Ang II-induced tau phosphorylation observed herein was confirmed using SB, a widely-studied inhibitor specific for GSK3b. As expected, the phosphorylation of all sites induced by central Ang II was significantly attenuated by inhibition of GSK3b. P-tau at Ser356 was normalized to the basal level by SB, implying that the dose of SB used herein was sufficient. However, phosphorylation at other sites was not blocked to the same extent. This inconsistency suggested that GSK3b might phosphorylate tau at specific sites only, and that it may not be the only kinase mediating Ang IIinduced tau phosphorylation. Further studies are thus warranted to elucidate the roles of other kinases, for example, MAPKs, in Ang II-induced tau phosphorylation. Ab production was also stimulated by Ang II in our previous studies [13]. The role of the GSK3b signaling in Ang II-induced Ab production was thus investigated herein. As shown in Fig. 6, inhibition of GSK3b by SB didn’t alter the levels of Ab-induced by highdose Ang II. This is in accordance with a recent study, in which in vivo Ab production was not controlled by GSK 3b [38]. Taken together, these findings suggested that Ang II, the crucial mediator of cardiovascular pathology, induced significant cognitive impairment and significant tau phosphorylation via activating GSK3b and other kinases. Attenuated tau phosphorylation protected against cognitive impairment, confirmed the crucial role of p-tau in AD pathogenesis. Given the roles of Ang II in cardiovascular events, these findings combined with our previous report will promote the establishment of the Ang II-Ab-GSK3b-tau cascade, and provide evidences for the vascular origin of AD pathogenesis. Since the blood–brain carrier is permeable to most AT1R blockers including losartan, blockade of brain Ang II signaling represents a promising strategy for anti-AD therapies. Acknowledgment This work was supported by the Medical Science and Technology Development Foundation, Nanjing Department of Health (No. ZKX 08035, No. YKK11031).
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