Aging-related changes in the nigral angiotensin system enhances proinflammatory and pro-oxidative markers and 6-OHDA-induced dopaminergic degeneration

Neurobiology of Aging 33 (2012) 204.e1–204.e11 www.elsevier.com/locate/neuaging

Aging-related changes in the nigral angiotensin system enhances proinflammatory and pro-oxidative markers and 6-OHDA-induced dopaminergic degeneration Begoña Villar-Cheda, Rita Valenzuela, Ana I. Rodriguez-Perez, Maria J. Guerra, Jose L. Labandeira-Garcia* Laboratory of Neuroanatomy and Experimental Neurology, Department of Morphological Sciences, Faculty of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Networking Research Center on Neurodegenerative Diseases (CIBERNED), Santiago de Compostela, Spain Received 4 January, 2010; received in revised form 16 July 2010; accepted 9 August 2010

Abstract An age-related proinflammatory, pro-oxidant state in the nigra may increase the vulnerability of dopaminergic neurons to additional damage. Angiotensin II, via type 1 (AT1) receptors, is one of the most important known inflammation and oxidative stress inducers. However, it is not known if there are age-related changes in the nigral angiotensin system. In aged rats, we observed increased activation of the nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) complex and increased levels of the proinflammatory cytokines interleukin (IL)-1␤ and tumor necrosis factor (TNF)-␣, which indicate pro-oxidative, proinflammatory state in the nigra. We also observed enhanced 6-hydroxydopamine (6-OHDA)-induced dopaminergic cell death in aged rats. This is associated with increased expression of AT1 receptors and decreased expression of AT2 receptors in aged rats, and is reduced by treatment with the AT1 antagonist candesartan. The present results indicate that brain angiotensin is involved in changes that may increase the risk of Parkinson’s disease with aging. Furthermore, the results suggest that manipulation of the brain angiotensin system may constitute an effective neuroprotective strategy against aging-related risk of dopaminergic degeneration. © 2012 Elsevier Inc. All rights reserved. Keywords: Angiotensin; Aging; Neuroinflammation; Oxidative stress; Parkinson

1. Introduction Several studies in different tissues have shown that normal aging is associated with a proinflammatory, pro-oxidant state that may favor an exaggerated response to injury and degenerative diseases (Choi et al., 2010; Csiszar et al., 2003; Ungvari et al., 2004). Advancing age itself is 1 of the most significant risk factors for the development of neurodegenerative diseases such as Parkinson’s disease (PD; Deng et al., 2006; McCormack et al., 2004). It has been

* Corresponding author at: Department of Morphological Sciences, Faculty of Medicine, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain. Tel.: ⫹34 981563100; fax: ⫹34 981547078. E-mail address: [email protected] (J.L. Labandeira-Garcia). 0197-4580/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2010.08.006

suggested that aging may increase the vulnerability of dopaminergic (DA) neurons to additional damage and increase the risk of developing PD (Collier et al., 2007; Villar-Cheda et al., 2009). It is known that neuroinflammation, oxidative stress (OS), and particularly microglial NADPH activation play a major role in DA neuron degeneration and PD (Koprich et al., 2008; Rodriguez-Pallares et al., 2007; Wu et al., 2002, 2003). Therefore, an age-related proinflammatory, pro-oxidant state in the nigra may be particularly relevant for the development of PD. However, the changes induced in the nigra by aging and the potential mechanisms involved have not been clarified. The peptide angiotensin II (AII), via type 1 (AT1) receptors, is 1 of the most important known inflammation and oxidative stress inducers, and produces reactive oxygen

204.e2

B. Villar-Cheda et al. / Neurobiology of Aging 33 (2012) 204.e1–204.e11

species (ROS) by activation of the NADPH-oxidase complex (Cai et al., 2003; Seshiah et al., 2002; Touyz et al., 2002), which is the most important intracellular source of ROS apart from mitochondria (Babior, 1999, 2004). Several studies have involved local renin-angiotensin system (RAS) in age-related degenerative changes in several tissues (Basso et al., 2005; Heymes et al., 1998; Touyz et al., 1999), and recent evidence from AT1 receptor deficient mice indicates that disruption of AT1 promotes longevity through attenuation of OS (Benigni et al., 2009), and completely protects against the age-related progression of atherosclerosis (Umemoto, 2008). Several recent studies have suggested a role of brain RAS in PD (see Mertens et al., 2010 for review). We have previously shown that the DA cell loss induced by DA neurotoxins is amplified by AII via AT1 receptors, activation of the microglial NADPH complex and exacerbation of the glial inflammatory response (Joglar et al., 2009; Rey et al., 2007; Rodriguez-Pallares et al., 2008). However, it is not known if age-related changes in the nigral RAS may lead to a proinflammatory, pro-oxidant state that increases the vulnerability of DA neurons. In the present study, we compared the expression of AII receptors, NADPH complex activation, and levels of proinflammatory cytokines (interleukin [IL]-1␤, tumor necrosis factor [TNF]-␣) in the nigra of young adult and aged rats, and studied the effect of treatment with the neurotoxin 6-OHDA and the AT1 receptor antagonist candesartan on the observed proinflammatory changes and the DA degeneration. 2. Methods 2.1. Experimental design Male Sprague-Dawley rats were used in the present experiments. All experiments were carried out in accordance with the “Principles of laboratory animal care” (NIH publication number 86-23, revised 1985) and approved by the corresponding committee at the University of Santiago de Compostela. The rats were divided into 3 groups. Rats in group A were young rats (10 weeks of age; n ⫽ 30). Rats in group B were aged rats (24-month-old rats; n ⫽ 25). Rats in group C were young rats (n ⫽ 5) and aged rats (24month-old; n ⫽ 30) treated with the AT1 receptor antagonist candesartan (0.5 mg/kg/day in the drinking water; for 2 weeks; 3 weeks for 6-OHDA-treated rats, see below) before they were killed (Ito et al., 2001; Nishimura et al., 2000). It has been reported that candesartan is the most effective AT1 antagonist in crossing the blood-brain barrier, and that low doses have little effect on blood pressure and block brain AII effects (Gohlke et al., 2002; Unger, 2003). Rats from the different groups were injected with 6-OHDA (n ⫽ 5 per group) or saline (n ⫽ 5 per group) in the right striatum and processed for immunohistochemistry (see below). The remaining rats were killed and the area of the substantia nigra in the ventral mesencephalon carefully dissected and pro-

cessed for real time reverse transcription-polymerase chain reaction (RT-PCR) studies, Western blot, or enzyme-linked immunosorbent assay (ELISA) to determine expression of AT1 and AT2 receptors, levels of the proinflammatory cytokines IL-1␤ and TNF-␣, and expression of the NADPH cytosolic subunit p47, as an indicator of the level of activation of the NADPH complex. NADPH oxidase complex is composed of membrane-bound subunits and cytosolic subunits such as p47phox, which is considered a key subunit for NADPH activation (see Li and Shah, 2003). The necessary step for NADPH activation is the translocation of cytosolic subunits to the membrane, which leads to ROS generation. The level of the NADPH oxidase subunit p47phox is correlated with NADPH activity and NADPHderived superoxide formation (Rueckschloss et al., 2002; Touyz et al., 2002). 2.2. RNA extraction and real-time quantitative RT-PCR Total ribonucleic acid (RNA) from the nigral region was extracted with Trizol (Invitrogen, Paisley, UK) according to the manufacturer’s instructions. Total RNA (2.5 ␮g) was reverse-transcribed to complementary DNA (cDNA) with deoxynucleotide triphosphate (dNTP), random primers, and Moloney murine leukemia virus reverse transcriptase (MMLV; 200 U; Invitrogen). Real-time PCR was used to examine relative levels of angiotensin receptors type 1 (AT1a) and type 2 (AT2) messenger RNA (mRNA) and p47. Experiments were performed with a real-time iCycler™ PCR platform (Bio-Rad, Hercules, CA). ␤-actin was used as housekeeping gene and was amplified in parallel with the genes of interest. The comparative threshold cycle (Ct) method was used to examine the relative mRNA expression. The expression of each gene was obtained as relative to the housekeeping transcripts. Then, the data were normalized to the values of the control group of the same batch (i.e., expressed as a percentage of the young rat values; 100%) to counteract possible variability among batches. Finally, the results were expressed as mean ⫾ standard error of the mean (SEM). Primers sequences were as follows: for angiotensin receptors type 1 (AT1a), forward 5=-TTCAACCTCTACGCCAGTGTG-3=, reverse 5=-GCCAAGCCAGCCATCAGC-3=; for AT2, forward 5=-AACATCTGCTGAAGACCAATAG-3=, reverse 5=AGAAGGTCAGAACATGGAAGG-3=; for p47phox, forward 5=-CCACACCTCTTGAACTTCTTC-3=, reverse 5=-CTCGTAGTCAGCGATGGC-3=; and for ␤-actin, forward 5=-TCGTGCGTGACATTAAAGAG-3=, reverse 5=-TGCCACAGGATTCCATACC-3=. 2.3. Western blot analysis Tissue was homogenized in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail (P8340, Sigma, St Louis, MO) and phenylmethylsulfonyl fluoride (PMSF; P7626, Sigma). Homogenates were centrifuged and protein concentrations were determined with the Bradford protein assay. Equal amounts of protein were

B. Villar-Cheda et al. / Neurobiology of Aging 33 (2012) 204.e1–204.e11

separated by 5%–10% bis-Tris polyacrylamide gel, and transferred to nitrocellulose membrane. The membranes were incubated overnight with primary antibodies (1:200) against AT1 receptor (sc-31181), AT2 receptor (sc-9040), p47-phox (sc-7660), and IL-1␤ (sc-1252), all from Santa Cruz Biotechnology (Santa Cruz, CA). The horseradish peroxidise (HRP) conjugated secondary antibodies used were Protein A (NA9120V, GE Healthcare, Fairfield, USA) and Protein G (18-161, Upstate-Millipore, Jaffrey, NH). Immunoreactivity was detected with an Immun-Star HRP Chemiluminescent Kit (170-5044, Bio-Rad) and imaged with a chemiluminescence detection system (Molecular Imager ChemiDoc XRS System, Bio-Rad). Blots were stripped and reprobed for anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) (G9545, Sigma; 1:25,000) as loading control. In each animal, protein expression was measured by densitometry of the corresponding band and expressed as relative to the GAPDH band value. Then, the data were normalized to the values of the control group of the same batch (i.e., expressed as a percentage of the young rat values; 100%) to counteract possible variability among batches. Finally, the results were expressed as mean ⫾ SEM. 2.4. ELISA Tissue was homogenized in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail (P8340, Sigma) and phenylmethylsulfonyl fluoride (PMSF; P7626, Sigma). Homogenates were centrifuged at 12,000g for 20 minutes at 4 °C and protein concentrations were determined by the use of the Bradford protein assay. The levels of TNF-␣ were quantified with a rat-specific ELISA kit according to the manufacturers’ instructions (Rat TNF-␣ from Diaclone, 865.000.096; Gen-Probe Diaclone SAS, Besançon, France). The TNF-␣ contents in the brain samples were obtained in pg per milligram protein and expressed as percentage of the control group. 2.5. Intrastriatal injection of 6-OHDA and immunohistochemistry Under ketamine/xylazine anesthesia, young rats (group A rats; n ⫽ 5), aged rats (group B rats; n ⫽ 5), and aged rats treated with candesartan (group C rats; n ⫽ 5) were injected in the right striatum with 7 ␮g of 6-OHDA (in 3 ␮L of saline containing 0.2% ascorbic acid; Sigma). Stereotaxic coordinates were 0.8 mm anterior to bregma, 3.0 mm right of midline, and 5 mm ventral to the dura; tooth bar at ⫺3.3. Young and aged control animals were injected with 3 ␮L of sterile saline alone (n ⫽ 10). Group C rats were treated with candesartan for 1 week before 6-OHDA or saline injection and during the survival period. Rats were killed 2 weeks postlesion and processed for immunohistochemistry. Previous studies on the time course of 6-OHDA lesions have shown that the loss of tyrosine hydroxylase (TH)-ir neurons is complete or practically complete 2 weeks after adminis-

204.e3

tration of intrastriatal injections (Bjorklund et al., 1997; Sauer and Oertel, 1994). The animals used for immunohistochemistry were first perfused with 0.9% saline and then with cold 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brains were removed and subsequently washed and cryoprotected in the same buffer containing 20% sucrose, and finally cut into 40 ␮m sections on a freezing microtome. The sections were incubated for 1 hour in 10% normal swine serum with 0.25% Triton X-100 in 20 mM potassium phosphate-buffered saline containing 1% bovine serum albumin (KPBSBSA) and then incubated overnight at 4 °C with a mouse monoclonal anti-TH (Sigma; 1:10,000), as DA marker, a mouse monoclonal anti-glial fibrillary acidic protein (GFAP; Chemicon, Temecula, CA; 1:1000) as an astrocyte marker, OX6 (1:500; Serotec, Kidlington, Oxford, UK; as a marker of reactive microglia/macrophages; OX6 is a monoclonal antibody directed against a monomorphic determinant of the rat major histocompatibility complex class II antigens, expressed by activated microglia but not by resting cells), and a goat polyclonal antiserum against IL-1␤ (1:200; Santa Cruz Biotechnology). The sections were subsequently incubated, first for 60 minutes with the corresponding biotinylated secondary antibody, and then for 90 minutes with avidin-biotin-peroxidase complex (ABC, 1:100, Vector, Burlingame, CA). Finally the labeling was revealed by treatment with 0.04% hydrogen peroxide and 0.05% 3-3=diaminobenzidine (DAB, Sigma). In all experiments the control sections, in which the primary antibody was omitted, were immunonegative for these markers. The total number of TH-immunoreactive (TH-ir) neurons in the substantia nigra compacta was estimated by an unbiased stereological method (i.e., the optical fractionator). Uniform randomly chosen 40-␮m sections through the entire substantia nigra (i.e., every fourth section from the rostral tip to the caudal end) were analyzed for the total number of TH-ir cells by means of a stereological grid (fractionator). Sampling was carried out using the CASTGrid system (Computer Assisted Stereological Toolbox; Olympus, Ballerup, Denmark), which comprised an Olympus IX51 microscope, a ProScan II X-Y motorized stage (Prior Scientific, Cambridge, UK) run by a PC computer, a microcator (MT1201, Heidenhain, Germany) connected to the stage and that feeds the computer with the distance information in the Z axis, and a JVC color video camera (Yokohama, Japan). The CAST-Grid software (version 2.1.5.9) was used to delineate the substantia nigra compacta (SNc) as observed with a 4⫻ objective, and to generate counting areas. A counting frame (1800 ␮m2) was placed at random on the first counting area and systematically moved through all counting areas until the entire delineated area was sampled. The sampling frequency was chosen so that a minimum of 150 TH-positive neurons were counted in each nigra. Counting was done using a 100⫻ oil objective (numerical aperture 1.4). Guard volumes (5 ␮m from the top

204.e4

B. Villar-Cheda et al. / Neurobiology of Aging 33 (2012) 204.e1–204.e11

and the bottom of the section) were excluded from both surfaces to avoid the problem of lost caps. The penetration of the antibody was determined by registration of the depth of each counted cell that appeared in focus within the counting frame. This analysis revealed an incomplete penetration of antibody leaving 8 –10 ␮m in the center poorly stained. The total number of cells was therefore calculated excluding the volume corresponding to this portion of the sections (Torres et al., 2006). The total number of neurons was calculated according to the optical fractionator formula (Gundersen et al., 1988). The coefficient of error calculated according to this procedure was less than 0.10. The nigral volume was estimated according to Cavalieri’s method (Gundersen et al., 1988). To confirm that 6-OHDA induces cell death, series of sections through the entire substantia nigra of control rats and rats treated with 6-OHDA were counterstained with cresyl violet, and the total number of neurons in the substantia nigra was estimated by the unbiased stereology method described above for TH-ir cells. Neurons were distinguished from glial cells on morphological grounds, and neurons with visible nuclei were counted as above (see Rey et al., 2007 for details). The number of IL-1␤-ir microglial cells was estimated using the Olympus CAST-Grid system and the unbiased stereological method described above for counting TH-ir neurons. At least 4 sections through the central substantia nigra compacta of each animal were measured. The density of IL-1␤-ir microglial cells (cells/mm3) was determined by dividing the number of labeled cells by the volume that they occupied. In addition, selected sections were processed for double immunofluorescence against IL-1␤ and OX6 (as a marker of reactive microglia/macrophages) or GFAP (as a marker of astrocytes) or TH (as DA marker) to study the possible colocalization of these markers. Sections were incubated overnight at 4 °C with primary antibodies against OX6 antibody (a mouse monoclonal antibody that recognizes major histocompatibility complex (MHC) class II antigens; Serotec; 1:50), or TH (a mouse monoclonal antibody; Sigma; 1:30,000) or GFAP (a mouse monoclonal antibody; Chemicon; 1:200). After rinsing with potassium phosphatebuffered saline (KPBS), the sections were incubated for 150 minutes with the secondary antibody (rabbit anti-mouse; Sigma; 1:100) conjugated with fluorescein isothiocyanate (FITC) for TH and GFAP, or biotinylated horse anti-mouse for OX6 (Vector; 1:500). OX6 labeling was visualized by incubation of the cultures with streptavidin Alexa-fluor 488 conjugate (Molecular Probes, Invitrogen; 1:2500) for 90 minutes. For the second labeling, sections were incubated for 48 hours with specific antibodies against IL-1␤ (1:200; Santa Cruz Biotechnology). Sections were washed and incubated with the secondary antibody (rabbit anti-goat; Chemicon; 1:500) conjugated with cyanine 3.18 (Cy3). Colocalization of markers was confirmed by confocal laser microscopy (TCS-SP2; Leica, Heidelberg, Germany) and use of a sequential scan method to avoid any possible

overlap. In all experiments the control cultures, in which the primary antibody was omitted, were immunonegative for these markers. 2.6. Statistical analysis All data were obtained from at least 3 independent experiments and were expressed as means ⫾ SEM. Twogroup comparisons were analyzed by the Student t test and multiple comparisons were analyzed by 1-way analysis of variance (ANOVA) followed by a posthoc Bonferroni test. The normality of populations and homogeneity of variances were tested before each ANOVA. Differences were considered statistically significant at p ⬍ 0.05. Statistical analyses were carried out with SigmaStat 3.0 from Jandel Scientific (San Rafael, CA). 3. Results Real time RT-PCR analysis revealed significantly higher expression of AT1 receptor mRNA and much lower levels of AT2 mRNA (about 50% reduction) in aged rats than in young rats (Fig. 1A). Similarly, Western blot (WB) studies revealed a significantly higher expression of AT1 receptors (around 200%) in aged rats than in young controls, and the expression of AT2 receptors was significantly lower (about 50% reduction) in aged rats (Fig. 1B). In previous studies, we observed AT1 and AT2 receptors in DA neurons and glial cells (astrocytes and microglia), and that AII induces microglial activation and DA cell death, via AT1 receptors and activation of the NADPH complex (see Joglar et al., 2009 and Rodriguez-Pallares et al., 2008 for details). Aged rats showed significantly increased NADPH complex activation in comparison with young rats, as determined by p47 subunit expression. Real time RT-PCR analysis revealed much higher mRNA levels of p47phox in aged rats (about 250%) than in young rats (Fig. 2A and C), and WB also revealed a significant increase in the expression of p47phox in aged rats (about 300%). The presence of several NADPH complex subunits, including p47phox, was observed in DA neurons and glial cells (microglia and astrocytes) in our previous studies (see Joglar et al., 2009 and RodriguezPallares et al., 2008 for details). In addition, levels of the proinflammatory cytokines IL-1␤ and TNF-␣ were determined by WB and ELISA, respectively, and were significantly higher in aged rats than in young rats (Fig. 2B and C). Treatment of aged rats with the AT1 receptor antagonist candesartan induced a significant increase in levels of AT2 receptor mRNA and protein, although they were still significantly lower than in the young rats (Fig. 3A and B). Similarly, treatment with candesartan induced a significant decrease in levels of p47, IL-1␤, and TNF-␣. The expression of p47 in aged rats treated with candesartan was still significantly higher than in young rats. Proinflammatory cytokines, however, decreased to levels not significantly different from those of the young rats. Treatment of young

B. Villar-Cheda et al. / Neurobiology of Aging 33 (2012) 204.e1–204.e11

204.e5

rats. However, the difference in 6-OHDA-induced loss of TH-ir neurons between aged rats and young was significantly reduced by treatment of aged rats with the AT1 receptor antagonist candesartan (Fig. 5A–E). In addition,

Fig. 1. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) (A) and Western blot (B) analysis of angiotensin type 1 (AT1) and 2 (AT2) receptors in the nigral region that revealed a significant increase in expression of AT1 receptors and decrease in expression of AT2 receptors in aged rats in comparison with young rats. Protein expression was obtained as relative to the GAPDH band value and the expression of each gene was obtained as relative to the housekeeping transcripts (␤actin). Then, the results were normalized to the young rat values (100%). Data are mean ⫾ standard error of the mean (SEM). *p ⬍ 0.05 (Student t test).

rats with candesartan did not induce significant changes in levels of p47 or TNF-␣. We observed a significant decrease in levels of IL-1␤, although the decrease induced by candesartan was much lower than in aged rats (Fig. 4A and B). The effect of inhibition of AT1 receptors on increased vulnerability of aged rats to DA neurotoxins was studied by intrastriatal injection of 6-OHDA in young and aged rats. The loss of TH-ir neurons induced by administration of 6-OHDA was significantly higher in aged rats than in young

Fig. 2. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) (A) and Western blot (WB; A and C) analysis revealed a significant increase in the expression of the key NADPH oxidase subunit p47phox in aged rats. Levels of the proinflammatory cytokines interleukin (IL)-1␤ (WB) and tumor necrosis factor (TNF)-␣ enzyme-linked immunosorbent assay (ELISA) were significantly higher in aged rats than in young rats (B and C). Protein expression was obtained as relative to the GAPDH band value and the expression of each gene was obtained as relative to the housekeeping transcripts (␤-actin). Then, the results were normalized to the young rat values (100%). Data are mean ⫾ standard error of the mean (SEM). *p ⬍ 0.05 (Student t test).

204.e6

B. Villar-Cheda et al. / Neurobiology of Aging 33 (2012) 204.e1–204.e11

2010; Collier et al., 2007; McCormack et al., 2004; Sugama et al., 2003). However, the mechanisms underlying the above-mentioned differences between young and aged animals are largely unknown. The present study reveals increased expression of the NADPH oxidase complex subunit p47 and the cytokines IL-1␤ and TNF-␣, which indicate a pro-oxidative, proinflammatory state in the nigra of aged rats. It has been shown that neuroinflammation mediated by IL-1␤ increases susceptibility of DA neurons to degeneration (Koprich et al., 2008). In accordance with this, we have observed that intrastriatal injection of 6-OHDA induced significantly higher loss of nigral DA neurons in aged rats than in young rats, and significantly higher number of IL1␤-labeled microglial cells in the substantia nigra than in young rats. Laser confocal microscopy revealed that most IL-1␤-labeled cells colocalized with activated microglia (i.e., OX6-ir cells). We also observed some IL-1␤-labeled

Fig. 3. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR; A) and Western blot (WB; A, B) analysis of angiotensin type 2 (AT2) receptor expression in the nigral region of aged rats, and the effect of treatment with the AT1 receptor antagonist candesartan. Candesartan induced a significant increase in levels of AT2 receptor mRNA and protein, although they were still significantly lower than in the young rats. Protein expression was obtained as relative to the GAPDH band value and mRNA expression was obtained as relative to the housekeeping transcripts (␤-actin). Then, the results were normalized to the young rat values (100%). Data are mean ⫾ standard error of the mean (SEM). *p ⬍ 0.05 compared with young rats; #p ⬍ 0.05 compared with nontreated aged rats (1-way analysis of variance [ANOVA] and Bonferroni posthoc test).

aged rats lesioned with 6-OHDA showed significantly higher number of IL-1␤-ir microglial cells in the substantia nigra than young rats lesioned with 6-OHDA. However, this difference was significantly reduced by treatment with candesartan (Fig. 6). Double immunofluorescence and confocal microscopy confirmed that most IL-1␤-ir cells colocalized with the marker for activated microglia OX6 (Fig. 7A–C). However, we also observed some IL-1␤-ir cells that colocalized with the astroglial marker GFAP (Fig. 7D–F). No significant labeling for IL-1␤ was observed in TH-ir neurons (Fig. 7G–I). 4. Discussion Aging has been observed to enhance the neuroinflammatory and microglial response to DA neurotoxins (Choi et al.,

Fig. 4. Western blot (WB; A, B) and enzyme-linked immunosorbent assay (ELISA; A) analysis of protein levels of p47 (WB), interleukin (IL)-1␤ (WB) and tumor necrosis factor (TNF)-␣ (ELISA) in young rats and aged rats, and in young and aged rats treated with the AT1 receptor antagonist candesartan. Treatment with candesartan induced a significant decrease in levels of p47 (WB), IL-1␤ (WB), and TNF-␣ (ELISA) in aged rats. Treatment of young rats with candesartan did not induce significant changes in levels of p47 or TNF-␣, and the decrease in levels of IL-1␤ was much lower than in aged rats. The results were normalized to the young rat values (100%). Data are mean ⫾ standard error of the mean (SEM). *p ⬍ 0.05 compared with young rats, #p ⬍ 0.05 compared with nontreated aged rats (1-way analysis of variance [ANOVA] and Bonferroni posthoc test).

B. Villar-Cheda et al. / Neurobiology of Aging 33 (2012) 204.e1–204.e11

204.e7

Fig. 5. Dopaminergic (tyrosine hydroxylase immunoreactive; TH-ir) neurons (A–F) in the substantia nigra compacta (SNc) 2 weeks after intrastriatal injection of saline or 6-OHDA in young rats, young rats treated with 6-OHDA, aged rats, aged rats treated with 6-OHDA, and aged rats treated with 6-OHDA and the angiotensin II type 1 receptor (AT1) antagonist candesartan (aged ⫹ 6-OHDA ⫹ candesartan). The loss of TH-ir neurons induced by administration of 6-OHDA was significantly higher in aged rats than in young rats. However, the difference was significantly reduced by treatment with candesartan. The dopamine neurons were quantified as the total number of TH-ir neurons in the SNc. Representative photomicrographs and high magnification insets of the SNc of different groups of rats are shown in B–F. Data are mean ⫾ standard error of the mean (SEM). *p ⬍ 0.05 compared with the saline-treated group, #p ⬍ 0.05 compared with aged rats treated with 6-OHDA alone (1-way analysis of variance [ANOVA] and Bonferroni posthoc test). Scale bars: 500 and 100 ␮m.

astrocytes, which may be related to regenerative or protective responses (Suzumura et al., 2006). Increased expression of p47 and the cytokines IL-1␤ and TNF-␣ were associated with increased expression of

AT1 receptors and decreased expression of AT2 receptors. Furthermore, the differences between young and aged rats were significantly reduced by treatment with the AT1 receptor antagonist candesartan, which indicates

204.e8

B. Villar-Cheda et al. / Neurobiology of Aging 33 (2012) 204.e1–204.e11

Fig. 6. Interleukin (IL)-1␤-ir cells per mm3 in the substantia nigra compacta of young rats treated with 6-OHDA, aged rats treated with 6-OHDA, and aged rats treated with 6-OHDA and the AT1 antagonist candesartan. Aged rats showed significantly higher number of number of IL-1␤-ir cells in the substantia nigra than young rats. However, the difference was significantly reduced by candesartan. Data represent mean ⫾ standard error of the mean (SEM). *p ⬍ 0.05 vs. young ⫹ 6-OHDA group, #p ⬍ 0.05 vs. aged ⫹ 6-OHDA group (1-way analysis of variance [ANOVA] and Bonferroni posthoc test).

that the local RAS plays a major role in proinflammatory and pro-oxidative changes in aged substantia nigra. Increased activity of local RAS via AT1 receptors is thought to be involved in age related degenerative changes in several tissues (Basso et al., 2005; Heymes et al., 1998; Mukai et al., 2002; Touyz et al., 1999). In accordance with this, recent studies with AT1 receptor deficient mice indicate that disruption of AT1 promotes longevity through attenuation of OS and additional mechanisms (Benigni et al., 2009), and completely protects against the age-related progression of atherosclerosis (Umemoto, 2008). It is well known that AII acts via AT1 receptors to induce inflammatory responses and to release high levels of ROS mainly by activation of the NADPH complex in vascular degenerative disease and other diseases mediated by oxidative stress and chronic inflammation (Qin et al., 2004; Touyz et al., 2002). In the nigrostriatal system, we have previously shown that brain AII induces activation of the NADPH complex via AT1 receptors, leading to increased neuroinflammation, OS and DA cell death in animal models of PD (Joglar et al., 2009; Rey et al., 2007; Rodriguez-Pallares et al., 2008). We have shown NADPH expression in dopaminergic neurons and microglial cells. However, it is known that in noninflammatory cells, such as neurons, the NADPH complex produces only low rates of ROS for signaling function. In inflammatory cells such as microglia, NADPH activation produces high concentrations of ROS

that are released extracellularly to kill invading microorganisms or cells (Babior, 1999, 2004). In accordance with this, we observed that AII was not able to increase DA neuron death in the absence of microglial cells (Joglar et al., 2009; Rodriguez-Pallares et al., 2008). The present results support that age-related changes in RAS activity may be involved in the greater vulnerability of DA neurons with aging. The observed upregulation of AT1 receptors in aged rats may contribute to increased DA cell vulnerability. This is supported by the present experiments, in which we have observed that the enhanced susceptibility of DA neurons and the increased microglial response to 6-OHDA in aged rats was significantly decreased by AT1 receptor inhibition with candesartan. It is also interesting to note that we observed decreased expression of AT2 receptors in aged rats. It is known that AT2 receptors counterbalance the deleterious effect of AT1 receptor stimulation, and functional interactions between the 2 receptor subtypes may determine the AII-induced effects (Sohn et al., 2000). Thus, increased function of AT2 receptors limits AT1mediated cardiovascular pathologies (Jones et al., 2008; Touyz et al., 1999). In aged rats, we observed the absence of a counter-regulatory increase in AT2 expression, and a decreased expression of AT2 mRNA and protein, despite increased expression of AT1 receptors and p47 (i.e., NADPH activation). This may contribute to further enhancement of a pro-oxidative, proinflammatory state and DA cell vulnerability. Although increased activity of local RAS via AT1 receptors has been involved in age-related degenerative changes in several tissues (Heymes et al., 1998; Mukai et al., 2002; Touyz et al., 1999), this may be of particular interest in the dopaminergic nigrostriatal system. Several recent studies have shown an important interaction between DA and AII receptors in peripheral tissues, particularly as regards regulating renal sodium excretion and cardiovascular function (Gildea, 2009; Khan et al., 2008; Zeng et al., 2006). Recent evidence suggests that DA and AII systems directly counterregulate each other in renal cells (Gildea, 2009), that there is a negative reciprocity between DA and AT1 receptors (Khan et al., 2008), and that abnormal counterregulation between DA and AII plays an important role in degenerative changes and hypertension (Li et al., 2008). In accordance with this, abnormal counterregulation in the basal ganglia may also lead to increased RAS activity and progression of DA cell vulnerability or death. Interestingly, several studies have shown that there is an aging-related decrease in DA release, which cannot be totally counteracted by functional compensatory changes and results in a progressive decrease in motor activity (Collier et al., 2007; Gerhardt et al., 2002). The upregulation of AT1 receptors observed in aged rats in the present study may be part of the compensatory changes to increase DA levels. However, increased RAS activity via AT1 receptors may also induce the above-mentioned proinflammatory, pro-oxidative state,

B. Villar-Cheda et al. / Neurobiology of Aging 33 (2012) 204.e1–204.e11

204.e9

Fig. 7. Double immunofluorescence and laser confocal microscopy for OX6, glial fibrillary acidic protein (GFAP), or tyrosine hydroxylase (TH; green) and interleukin (IL)-1␤ (red) in the substantia nigra of aged rats treated with 6-OHDA. Most IL-1␤-ir cells showed colocalization (yellow) with the marker of activated microglia OX6 (A–C). However, some IL-1␤-ir cells showed colocalization (yellow) with the marker of astroglia GFAP (D–F). We did not observe colocalization with the dopaminergic marker TH (G–I). Scale bars: 50 ␮m (A–F), and 100 ␮m (G–I).

which may be further enhanced by a lack of compensatory upregulation of AT2 receptors in aged rats. In the present study we have observed that inhibition of AT1 receptors with the selective antagonist candesartan induced a decrease in the higher susceptibility of aged rats to DA neurotoxins, and a decrease in the expression of nigral proinflammatory cytokines. Candesartan also induced a significant reduction in p47 expression (i.e., NADPH activation) in aged rats, although it was still significantly higher than in young rats, which suggests that additional factors may be inducing NADPH activation in aged rats. Interestingly, we observed a significant increase in AT2 expression in aged rats treated with candesartan, although levels of AT2 receptor expression were still lower than those of young rats. In conclusion, the present results indicate that agingrelated increase in RAS activity is involved in proinflammatory and pro-oxidative changes in the nigra, and in the increased

susceptibility to DA neurotoxins with aging, and suggest that increased RAS activity may constitute a major factor in the increased risk of PD with aging. Furthermore, the present results suggest that manipulation of the brain RAS may constitute an effective neuroprotective strategy against aging-related risk of dopaminergic degeneration.

Disclosure statement The authors certify that no conflict of interest exists.

Acknowledgements Funding: Spanish Ministry of Education (MEC), Spanish Ministry of Health (RD06/0010/0013 and Ciberned) and Galician Government (XUGA).

204.e10

B. Villar-Cheda et al. / Neurobiology of Aging 33 (2012) 204.e1–204.e11

The authors thank Pilar Aldrey and Iria Novoa for their excellent technical assistance.

References Babior, B., 1999. NADPH oxidase: An update. Blood 93, 1464 –1476. Babior, B.M., 2004. NADPH oxidase. Curr. Opin. Immunol. 16, 42– 47. Basso, N., Paglia, N., Stella, I., de Cavanagh, E.M., Ferder, L., del Rosario, L., Arnaiz, M., Inserta, F., 2005. Protective effect of the inhibition of the renin-angiotensin system on aging. Regul. Pept. 128, 247–252. Benigni, A., Corna, D., Zoja, C., Sonzogni, A., Latini, R., Salio, M., Conti, S., Rottoli, D., Longaretti, L., Cassis, P., Morigi, M., Coffman, T.M., Remuzzi, G., 2009. Disruption of the Ang II type 1 receptor promotes longevity in mice. J. Clin. Invest. 119, 524 –530. Bjorklund, A., Rosenblad, C., Winkler, C., Kirik, D., 1997. Studies on neuroprotective and regenerative effects of GDNF in a partial lesion model of Parkinson’s disease. Neurobiol. Dis. 4, 186 –200. Cai, H., Griendling, K.K., Harrison, D.G., 2003. The vascular NAD(P)H oxidases as therapeutic targets in cardiovascular diseases. Trends Pharmacol. Sci. 24, 471– 478. Choi, D.Y., Zhang, J., Bing, G., 2010. Aging enhances the neuroinflammatory response and alpha-synuclein nitration in rats. Neurobiol. Aging 31, 1649 –1653. Collier, T.J., Lipton, J., Daley, B.F., Palfi, S., Chu, Y., Sortwell, C., Collier, T.J., Carvey, P.M., 2007. Aging-related changes in the nigrostriatal dopamine system and the response to MPTP in nonhuman primates: diminished compensatory mechanisms as a prelude to parkinsonism. Neurobiol. Dis. 26, 56 – 65. Csiszar, A., Ungvari, Z., Koller, A., Edwards, J.G., Kaley, G., 2003. Aging-induced proinflammatory shift in cytokine expression profile in coronary arteries. FASEB J. 17, 1183–1185. Deng, X.H., Bertini, G., Xu, Y.Z., Yan, Z., Bentivoglio, M., 2006. Cytokine-induced activation of glial cells in the mouse brain is enhanced at an advanced age. Neuroscience 141, 645– 661. Gerhardt, G.A., Cass, W.A., Yi, A., Zhang, Z., Gash, D.M., 2002. Changes in somatodendritic but not terminal dopamine regulation in aged rhesus monkeys. J. Neurochem. 80, 168 –177. Gildea, J.J., 2009. Dopamine and angiotensin as renal counterregulatory systems controlling sodium balance. Curr. Opin. Nephrol. Hypertens. 18, 28 –32. Gohlke, P., Von Kugelgen, S., Jurgensen, T., Kox, T., Rascher, W., Culman, J., Unger, T., 2002. Effects of orally applied candesartan cilexetil on central responses to angiotensin II in conscious rats. J. Hypertens. 20, 909 –918. Gundersen, H.J.G., Bendsen, T.F., Korbo, L., Marcussen, N., Moller, A., Nielsen, K., 1988. Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 96, 379 –394. Heymes, C., Silvestre, J.S., Llorens-Cortes, C., Chevalier, B., Marotte, F., Levy, B.I., Swynghedauw, B., Samuel, J.L., 1998. Cardiac senescence is associated with enhanced expression of angiotensin II receptor subtypes. Endocrinology 139, 2579 –2587. Ito T., Nishimura, Y., Saavedra, J., 2001. Pre-treatment with candesartan protects from cerebral ischaemia. J. Renin. Angiotensin Aldosterone Syst. 2,174 –179. Joglar, B., Rodriguez-Pallares, J., Rodríguez-Perez, A.I., Rey, P., Guerra, M.J., Labandeira-Garcia, J.L., 2009. The inflammatory response in the MPTP model of Parkinson’s disease is mediated by brain angiotensin: relevance to progression of the disease. J. Neurochem. 109, 656 – 669. Jones, E.S., Vinh, A., McCarthy, C.A., Gaspari, T.A., Widdop, R.E., 2008. AT2 receptors: functional relevance in cardiovascular disease. Pharmacol. Ther. 120, 292–316. Khan, F., Spicarová, Z., Zelenin, S., Holtbäck, U., Scott, L., Aperia, A., 2008. Negative reciprocity between angiotensin II type 1 and dopamine

D1 receptors in rat renal proximal tubule cells. Am. J. Physiol. Ren. Physiol. 295, F1110 –F1116. Koprich, J.B., Reske-Nielsen, C., Mithal, P., Isacson, O., 2008. Neuroinflammation mediated by IL-1beta increases susceptibility of dopamine neurons to degeneration in an animal model of Parkinson’s disease. J. Neuroinflammation 5, 8. Li, H., Armando, I., Yu, P., Escano, C., Mueller, S.C., Asico, L., Pascua, A., Lu, Q., Wang, X., Villar, V.A., Jones, J.E., Wang, Z., Periasamy, A., Lau, Y.S., Soares-da-Silva, P., Creswell, K., Guillemette, G., Sibley, D.R., Eisner, G., Gildea, J.J., Felder, R.A., Jose, P.A., 2008. Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells. J. Clin. Invest. 118, 2180 –2189. Li, J.M., Shah, A.M., 2003. Mechanism of endothelial cell NADPH oxidase activation by angiotensin II. Role of the p47phox subunit. J. Biol. Chem. 278, 12094 –12100. McCormack, A.L., Di Monte, D.A., Delfani, K., Irwin, I., DeLanney, L.E., Langston, W.J., Janson, A.M., 2004. Aging of the nigrostriatal system in the squirrel monkey. J. Comp. Neurol. 471, 387–395. Mertens, B., Vanderheyden, P., Michotte, Y., Sarre, S., 2010. The role of the central renin-angiotensin system in Parkinson’s disease. J. Renin Angiotensin Aldosterone Syst. 11, 49 –56. Mukai, Y., Shimokawa, H., Higashi, M., Morikawa, K., Matoba, T., Hiroki, J., Kunihiro, I., Talukder, H.M., Takeshita, A., 2002. Inhibition of reninangiotensin system ameliorates endothelial dysfunction associated with aging in rats. Arterioscler. Thromb. Vasc. Biol. 22, 1445–1450. Nishimura, Y., Ito, T., Hoe, K., Saavedra, J.M., 2000. Chronic peripheral administration of the angiotensin II AT(1) receptor antagonist candesartan blocks brain AT(1) receptors. Brain Res. 871, 29 –38. Qin, L., Liu, Y., Wang, T., Wei, S.J., Block, M.L., Wilson, B., Liu, B., Hong, J.S., 2004. NADPH oxidase mediates lipopolysaccharide-induced neurotoxicity and proinflammatory gene expression in activated microglia. J. Biol. Chem. 279, 1415–1421. Rey, P., Lopez-Real, A., Sanchez-Iglesias, S., Muñoz, A., Soto-Otero, R., Labandeira-Garcia, J.L., 2007. Angiotensin type-1-receptor antagonists reduce 6-hydroxydopamine toxicity for dopaminergic neurons. Neurobiol. Aging 28, 555–567. Rodriguez-Pallares, J., Parga, J.A., Muñoz, A., Rey, P., Guerra, M.J., Labandeira-Garcia, J.L., 2007. Mechanism of 6-hydroxydopamine neurotoxicity: the role of NADPH oxidase and microglial activation in 6-hydroxydopamine-induced degeneration of dopaminergic neurons. J. Neurochem. 103, 145–156. Rodriguez-Pallares, J., Rey, P., Parga, J.A., Muñoz, A., Guerra, M.J., Labandeira-Garcia, J.L., 2008. Brain angiotensin enhances dopaminergic cell death via microglial activation and NADPH-derived ROS. Neurobiol. Dis. 31, 58 –73. Rueckschloss, U., Quinn, M.T., Holtz, J., Morawietz, H., 2002. Dosedependent regulation of NAD(P)H oxidase expression by angiotensin II in human endothelial cells: protective effect of angiotensin II type 1 receptor blockade in patients with coronary artery disease. Arterioscler. Thromb. Vasc. Biol. 22, 1845–1851. Sauer, H., Oertel, W.H., 1994. Progressive degeneration of nigrostriatal dopamine neurons following intrastriatal terminal lesions with 6-hydroxydopamine: a combined retrograde tracing and immunohistochemical study in the rat. Neuroscience 59, 401– 415. Seshiah, P.N., Weber, D.S., Rocic, P., Valppu, L., Taniyama, Y., Griendling, K.K., 2002. Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators. Circ. Res. 91, 406 – 413. Sohn, H.Y., Raff, U., Hoffmann, A., Gloe, T., Heermeier, K., Galle, J., Pohl, U., 2000. Differential role of angiotensin II receptor subtypes on endothelial superoxide formation. Br. J. Pharmacol. 131, 667– 672. Sugama, S., Yang, L., Cho, B.P., DeGiorgio, L.A., Lorenzl, S., Albers, D.S., Beal, M.F., Volpe, B.T., Joh, T.H., 2003. Age-related microglial activation in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurodegeneration in C57BL/6 mice. Brain Res. 964, 288 –294.

B. Villar-Cheda et al. / Neurobiology of Aging 33 (2012) 204.e1–204.e11 Suzumura, A., Takeuchi, H., Zhang, G., Kuno, R., Mizuno, T., 2006. Roles of glia-derived cytokines on neuronal degeneration and regeneration. Ann. N Y Acad. Sci. 1088, 219 –229. Torres, E.M., Meldrum, A., Kirik, D., Dunnett, S.B., 2006. An investigation of the problem of two-layered immunohistochemical staining in paraformaldehyde fixed sections. J. Neurosci. Methods 158, 64 –74. Touyz, R.M., Chen, X., Tabet, F., Yao, G., He, G., Quinn, M.T., Pagano, P.J., Schiffrin, E.L., 2002. Expression of a functionally active gp91phox-containing neutrophil-type NAD(P)H oxidase in smooth muscle cells from human resistance arteries, regulation by angiotensin II. Circ. Res. 14, 1205–1213. Touyz, R.M., Endemann, D., He, G., Li, J.S., Schiffrin, E.L., 1999. Role of AT2 receptors in angiotensin II-stimulated contraction of small mesenteric arteries in young SHR. Hypertension 33, 366 –372. Umemoto, S., 2008. Angiotensin II type 1 (AT1) receptor deficiency halts the progression of age-related atherosclerosis in hypercholesterolemia: molecular link between the AT1 receptor and hypercholesterolemia. Hypertens. Res. 31, 1495–1497. Unger, T., 2003. Inhibiting angiotensin receptors in the brain: possible therapeutic implications. Curr. Med. Res. Opin. 19, 449 – 451.

204.e11

Ungvari, Z., Csiszar, A., Kaley, G., 2004. Vascular inflammation in aging. Herz 29, 733–740. Villar-Cheda, B., Sousa-Ribeiro, D., Rodriguez-Pallares, J., RodriguezPerez, A.I., Guerra, M.J., Labandeira-Garcia, J.L., 2009. Aging and sedentarism decrease vascularization and VEGF levels in the rat substantia nigra. Implications for Parkinson’s disease. J. Cereb. Blood Flow Metab. 29, 230 –239. Wu, D., Teisman, P., Tieu, K., Vila, M., Jackson-Lewis, V., Ischiropoulos, H., Przedborski, S., 2003. NADPH oxidase mediates oxidative stress in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson¨ s disease. Proc. Natl. Acad. Sci. U. S. A. 100, 6145– 6150. Wu, D., Teisman, P., Tieu, K., Vila, M., Jackson-Lewis, V., Ischiropoulos, H., Przedborski, S., 2003. NADPH oxidase mediates oxidative stress in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson¨ s disease. Proc. Natl. Acad. Sci. U. S. A. 100, 6145– 6150. Zeng, C., Liu, Y., Wang, Z., He, D., Huang, L., Yu, P., Zheng, S., Jones, J.E., Asico, L.D., Hopfer, U., Eisner, G.M., Felder, R.A., Jose, P.A., 2006. Activation of D3 dopamine receptor decreases angiotensin II type 1 receptor expression in rat renal proximal tubule cells. Circ. Res. 99, 494 –500.