Angiotensin type-1-receptor antagonists reduce 6-hydroxydopamine toxicity for dopaminergic neurons

1 Angiotensin type-1-receptor antagonists reduce 6hydroxydopamine toxicity for dopaminergic neurons P. Rey,a A. Lopez-Real,a S. Sanchez-Iglesias,b A. Muñoza, R. SotoOtero,b and J. L. Labandeira-Garciaa Laboratory of Neuroanatomy and Experimental Neurology, Dept. of Morphological Sciences (a) and Laboratory of Neurochemistry, Dept. of Biochemistry and Molecular Biology (b), Faculty of Medicine, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain. Correspondence: Jose L. Labandeira-Garcia M.D., Ph. D. Dept. of Morphological Sciences, Faculty of Medicine, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain. Tel. +34-981563100; Fax: +34-981547078 E. mail: [email protected] 2 Abstract Angiotensin II activates (via type 1 receptors) NAD(P)H-dependent oxidases, which are a major source of superoxide, and is relevant in the pathogenesis of several cardiovascular diseases and certain degenerative changes associated with ageing. Given that there is a brain renin-angiotensin system and that oxidative stress is a key contributor to Parkinson’s disease, we investigated the effects of angiotensin II and angiotensin type 1 (AT1) receptor antagonists in the 6-hydroxydopamine model of Parkinson’s disease. Rats subjected to intraventricular injection of 6-hydroxydopamine showed bilateral reduction in the number of dopaminergic neurons and terminals. Injection of angiotensin alone did not induce any significant effect. However, angiotensin increased the toxic effect of 6-hydroxydopamine. Rats treated with the AT1 receptor antagonist ZD 7155 and then 6-hydroxydopamine (with or without exogenous administration of angiotensin) showed a significant reduction in 6-hydroxydopamineinduced oxidative stress (lipid peroxidation and protein oxidation) and dopaminergic degeneration. Dopaminergic degeneration was also reduced by the NAD(P)H inhibitor Apocynin. Angiotensin may play a pivotal role, via AT1 receptors, in increasing the oxidative damage of dopaminergic cells, and treatment with AT1 antagonists may reduce the progression of Parkinson’s disease. Keywords: angiotensin; basal ganglia; dopamine; neuroprotection; NAD(P)H-oxidase; Parkinson’s disease; oxidative stress, 6-hydroxydopamine 3 1. Introduction The renin-angiotensin system (RAS) was originally described as a circulating humoral system that regulates blood pressure, aldosterone release and sodium reabsorption. Angiotensin II (AII) is the most important effector peptide, and is formed by the sequential action of two enzymes, renin and angiotensin converting enzyme (ACE), on the precursor glycoprotein angiotensinogen. The actions of AII are mediated by two main cell surface receptors: AII type 1 and 2 (AT1 and AT2) receptors [2,63]. However, numerous clinical and laboratory data provide evidence that AII is relevant in the pathogenesis of several diseases, particularly of the cardiovascular system, and certain degenerative changes associated with ageing [3,11,56]. It has been also reported that AT1 antagonists reduce age-related mitochondrial dysfunction [10]. It is now generally accepted that in addition to the “classical” humoral RAS there exist local RAS in many tissues, including brain tissue [2, 46], and that locally formed AII regulates many substances such as growth factors and cytokines that are involved in processes such as cell growth/apoptosis or inflammation [56]. Interestingly, it has recently been shown that in several cell types reactive oxygen species (ROS) play a crucial role in the signaling of AII via AT1 receptors, and that AII activates NAD(P)H-dependent oxidases, which are a major source of superoxide (O2-) and are upregulated in major ageing-related diseases such as hypertension, diabetes and atherosclerosis [23,48]. Accordingly, AT1 receptor antagonists induce inhibition of oxidase, reduction of oxidative stress and improvement in endothelial dysfunction [55,67]. It is now established that the brain possesses a local angiotensin system, and that the local AII exerts multiple actions [2,46]. All components of the RAS have been observed in the striatum [1,2,8,52], and AII modulates dopamine (DA) release from the striatal DA terminals via their AT1 receptors [7,47]. Furthermore, it has recently been 4 shown that NAD(P)H-oxidase has a wide distribution throughout the brain, including striatum and substantia nigra [30,57], and that NAD(P)H-oxidase-derived ROS play a major role in AII signaling in neurons [49,65]. There is growing evidence indicating that the oxidative stress is a key contributor to the pathogenesis and progression of Parkinson’s disease (PD) and ageing-related loss of DA neurons [29,51]. In addition, increased ACE activity in the cerebrospinal fluid [32], association between genetic polymorphism of the ACE gene and the disease [39], and increased expression of NAD(P)H:quinone oxidoreductase in neurons [64] have been observed in PD patients. Altogether this suggests that, as previously observed in cardiovascular diseases, the brain RAS may play a major role in the pathogenesis and progression of PD and ageing-related loss of DA neurons, and that manipulation of RAS components may be useful for neuroprotection in PD patients. In the present study, we treated rats with intraventricular injections of the DA neurotoxin 6-hydroxydopamine (6-OHDA), AII, the AT1 receptor antagonist ZD 7155 or the NAD(P)H inhibitor Apocynin, to study the effects of the RAS on the 6-OHDA-induced degeneration of DA neurons and the level of 6-OHDA-induced oxidative stress in the ventral midbrain and striatum. 2. Materials and methods Experimental design Male adult Sprague-Dawley rats (weighing about 200 g) were used in the present experiments. All experiments were carried out in accordance with the “Principles of laboratory animal care” (NIH publication No. 86-23, revised 1985) and approved by the corresponding committee at the University of Santiago de Compostela. The rats were divided into 10 groups (A-J; see Table 1). Rats in group A were used as normal (i.e. non-lesioned) controls, and received the corresponding intraventricular injections of 5 vehicle (saline containing 0.2% ascorbic acid, 3 l; see below). Rats in group B were injected in the third ventricle with Angiotensin II (AII; 5 g; Sigma) in 3 l of sterile ascorbate saline 24 h before, 25 min before and 24 h after ascorbate saline injection. Rats in group C were injected in the third ventricle with 3 l of sterile ascorbate saline 24 h before, 25 min before and 24 h after 6-OHDA injection (200 g of 6-OHDA in 3 l of sterile ascorbate saline). Rats in group D were injected intraventricularly with AII, as in group-B rats, and 3 l ascorbate saline containing 6-OHDA. Rats in groups E and H were injected as those in group D but were pretreated with the AT1 receptor antagonist ZD 7155 hydrochloride (Tocris; 50g/3 l, intraventricularly; group E) or Apocynin (Fluka; 750g /5 l, intraventricularly; group H) 20 min before each AII injection. Rats in group F and I were injected intraventricularly as those in group D but were pretreated with ZD 7155 (group F) or Apocynin (group I) rather than AII (i.e. without exogenous administration of AII). Finally, rats in groups G and J were injected with ZD 7155 (group G) or Apocynin (group J) alone (i.e. without administration of 6OHDA or AII). The injections were performed using a single cannula placed in the third ventricle during the whole injection period (stereotaxic coordinates: 0.8 mm posterior to bregma, midline, 6.5 mm ventral to the dura, and tooth bar at 0). The solution was injected with a 10 l Hamilton syringe coupled to a motorized injector (Stoelting), at 0.5 l/min. All surgery was performed under ketamine/xylazine anesthesia, and thirty minutes prior to injection of 6-OHDA, rats received desipramine (Sigma, 25 mg/kg i.p.) to prevent uptake of 6-OHDA by noradrenergic terminals. The accuracy of the injections and cannula placement were confirmed by post-mortem analysis. Five rats of each group were used to study the level of 6-OHDA-induced oxidative stress, which was estimated by determination of lipid peroxidation (TBARS formation) and protein oxidation (carbonyl content) in the ventral midbrain (i.e. from 6 bregma -4.5 to bregma -6.5, and ventral +6.5 to ventral +9.5; see ref. 50) and the striatum (from bregma +2.5 to bregma -1). For biochemical studies on oxidative stress rats were killed 48 h after lesion, because we observed the highest levels of 6-OHDAinduced oxidative stress indicators at this time in preliminary experiments (unpublished data). Six rats of each group were used for immunohistochemical studies. Given that previous studies on the time course of the DA degeneration in intraventricular 6-OHDA lesion model have shown that the DA lesion is complete or practically complete 1 week after 6-OHDA injection [53], the rats processed for immunohistochemistry (see below) were killed 1 week postlesion. Furthermore, in preliminary experiments the doses of 6OHDA were reduced to the minimal levels that induced a significant loss of nigral DA neurons and high survival-rate. Immunohistochemistry and Cresyl violet staining One week post-lesion, the animals were killed by chloral hydrate overdose and perfused first 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 on a freezing microtome. Sections were processed for tyrosine hydroxylase (TH) immunohistochemistry (as follows). After incubation for 1 hour in 10% normal swine serum with 0.25% Triton-X-100 in 0.02 M potassium phosphate-buffered saline containing 1% bovine serum albumin (KPBS-BSA), sections were incubated overnight at room temperature with rabbit polyclonal antiserum to TH (Peel-Freez; 1:500 in KPBS-BSA containing 2% normal swine serum and 0.25% Triton-X-100). The sections were subsequently incubated first for 90 min with the corresponding biotinylated secondary antibody (Vector, USA; diluted 1:200) and then for 90 min with an avidin- 7 biotin-peroxidase complex (ABC, Vector, USA). Finally, the labeling was visualized with 0.04% hydrogen peroxide and 0.05% 3-3’ diaminobenzidine (DAB, Sigma). Estimation of the total TH-immunoreactive (TH-ir) neurons was made in the substantia nigra compacta (SNc; see ref. 50). We used an unbiased stereology 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 CAST-Grid system (Computer Assisted Stereological Toolbox; Olympus, Denmark), which comprised an Olympus IX51 microscope, a ProScan II X-Y motorized stage (Prior Scientific, 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 (Japan). The CAST-Grid software (version 2.1.5.9) was used to delineate the SNc as observed with a 4X objective, and to generate counting areas. A counting frame (1800 µm2) 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 100X oil objective (NA 1.4). Guard volumes (5 µm from the top and the bottom of the section) were excluded from both surfaces to avoid the problem of lost caps, and only the profiles that came into focus within the counting volume (with a depth of 12 µm) were counted. The total number of neurons was calculated according to the optical fractionator formula [24, 66]. The coefficient of error associated with the estimation was calculated according to and was less than 0.10. The nigral volume was estimated according to Cavalieri’s method [24]. 8 The density of striatal dopaminergic terminals was estimated as the optical density of the striatal TH-ir with the aid of NIH-Image 1.55 image analysis software (Wayne Rasband, MIMH) on a personal computer coupled to a videocamera (CCD-72, MTI) and a constant illumination light table (Northern Light, St. Catharines, Canada). At least four sections through the central striatum (i.e. from bregma +1 to bregma +0; see ref. 50) of each rat were measured (both the right and left striatum), and for each section optical densities were corrected by subtraction of background, as observed in the corpus callosum. In order to confirm that 6-OHDA induces cell death and not just phenotypic downregulation in TH activity, series of sections through the entire substantia nigra of control rats and rats treated with 6-OHDA (groups A and C) were counterstained with Cresyl violet, and the total number of neurons in the SNc was estimated using 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 Determination of TBARS The TBARS determination was performed spectrophotometrically. The striata or ventral midbrain tissue were homogenized with three volumes (w/v) of a Na2PO4/KH2PO4 buffer (pH 7.4) isotonized with KCl and containing butylated hydroxytoluene (200 M) and desferrioxamine (200 M). An aliquot of the resulting sample (200 l) was treated with SDS (8%, w/v) followed by acetic acid (20%) and the mixture vortexed for 1 min. Thiobarbituric acid (0.8%) was then added and the resulting mixture incubated at 95 ºC for 60 min. After cooling to room temperature, 3 ml of n-butanol were added and the mixture shaken vigorously. After centrifugation at 4000 r.p.m. for 5 min, the absorbance of the supernatant (organic layer) was measured at 532 nm using an Ultrospec III 9 spectrophotometer (Pharmacia Biotech, Uppsala, Sweden). For calibration, a standard curve (5-150 nM) was generated using the malonodialdehyde (MDA) derived by acid hydrolysis (SO4H2; 1.5%, v/v) of 1,1,3,3-tetraethoxypropane and the TBARS results expressed as nmol MDA/mg protein. The protein concentration of the sample obtained was determined according to a previously published method [45], using BSA as the standard. Estimation of protein carbonyl content The protein carbonyl content was assessed spectrophotometrically. An aliquot of the sample obtained after the homogenization of brain tissue for TBARS (200 l) was submitted to precipitation of nucleic acids with 1% streptomycin sulphate (1:9, v/v) followed by centrifugation at 13000 r.p.m. The pellet was then discarded and the supernatant treated with trichloroacetic acid (1 M) followed consecutively by sonication (Branson Sonic Corp., Danbury, USA) and centrifugation in a microcentrifuge (model E, Beckman Instruments) at 13000 r.p.m. for 5 min. The resulting pellet was reconstituted in NaOH (0.5 M) with vigorous vortexing for 3 min. Ten mM 2,4dinitrophenylhydrazine in 2 M chloric acid was then added and the mixture incubated at room temperature for 1 hr, in darkness, and with continuous agitation. After the addition of trichloroacetic acid (1 M), the resulting mixture was centrifuged at 13000 r.p.m. for 5 min. The resulting pellet was washed twice with ethyl acetate:ethanol (1:1, v/v), and the washed pellet was then reconstituted with 6 M guanidine in a 20 mM KH2PO4 buffer (pH 2.3) and the absorbance of the resulting solution measured at 370 nm. The carbonyl content was calculated from the absorbance data using = 22000 M−1·cm−1 as the value of the absorption coefficient for dinitrophenylhydrazone and expressing this parameter as nmol carbonyls/mg protein. Because of the numerous washing steps, the protein 16 administration of too high concentrations of AII may be counteracted by downregulation of AT1 receptors [25]. The results suggest, therefore, that administration of exogenous AII is not sufficient by itself to induce a significant loss of DA neurons, at least after a 7 day survival period, but it is sufficient to induce a detectable amplifying effect on the 6-OHDA toxicity. More interestingly, the inhibition of the effect of the endogenous and /or exogenous AII on the AT1 receptors by ZD 7155 decreased the 6OHDA-induced loss of DA neurons. The neuroprotective effect induced by administration of the AT1 antagonist appears, however, more marked than the neurotoxic effect induced by administration of exogenous AII (see below). 6-OHDA is a selective catecholaminergic neurotoxin widely used to investigate the pathogenesis and progression of PD. In the present study, administration of 6OHDA induced a loss of TH-ir nigral neurons and TH-ir striatal terminals. Changes in TH expression do not necessarily imply neuronal death. However, the animal model used in the present study (i.e. injection of 6-OHDA in the third ventricle) has recently been described in detail [53], and it has been shown that the lesion is complete or practically complete 1 week after injection, and that the loss of TH immunoreactivity in neurons corresponds with neuronal death, which was also confirmed in the present experiments by using Cresyl-violet stained sections. The specificity of 6-OHDA neurotoxicity has been associated with its uptake and accumulation by a transport mechanism specific for catecholaminergic neurons [27]. It has been observed that 6OHDA is readily oxidized, thereby producing a variety of cytotoxic radical products [33,60], and its neurotoxicity has been shown to be related to the production of reactive oxygen species. However, it has been suggested that 6-OHDA also acts directly by inhibiting the mitochondrial respiratory chain at the level of complex I of the electron transport chain [22]. Therefore oxidative stress is a major molecular mechanism 17 underlying both the DA degeneration induced by 6-OHDA and that observed in PD patients [51,60]. The NAD(P)H oxidase complex is the most important intracellular source of ROS other than mitochondria. Furthermore, earlier studies demonstrated that extramitochondrial ROS can induce mitochondrial DNA damage, thereby destroying respiratory enzymes to produce more reactive oxygen species [37]. Thus, ROS originated by NAD(P)H oxidases favour their own production via mitochondria, intracellular iron uptake and other intracellular sources [9]. These feed-forward mechanisms form a vicious circle and may amplify and sustain ROS production induced by low doses of 6-OHDA or other neurotoxins, contributing to DA cell death. Furthermore, it has been recently reported that AII also stimulates mitochondrial ROS production through the opening of mitochondrial ATP-sensitive potassium channels [31] Recent studies have shown the presence of NAD(P)H oxidase in neurons and glial cells [30,57], and that in these cells, NAD(P)H oxidase and ROS are involved in signaling by AII via AT1 receptors [49,65]. In addition, it has been observed that in experimental stroke ischemic injury is modulated by AII via neuronal AT1 receptors, and that AT1 antagonists reduced the expression of NAD(P)H and lipid peroxidation, lessened cerebral infarction and improved the neurological outcome [34,43]. In agreement, in a series of interesting studies using mesencephalic cell cultures, Gao et al. [17-19] have shown that several well-known dopaminergic toxins (i.e. MPTP, rotenone and inflammogen lipopolysaccharide) induce NAD(P)H oxidase-mediated generation of ROS in microglial cells, and suggested that the release of superoxide from activated microglia greatly enhanced the neurotoxin-induced DA degeneration. Interestingly, it was observed that low and apparently non-toxic doses of two neurotoxins can act synergistically to induce DA degeneration, and that NAD(P)H oxidase-mediated 18 generation of ROS appeared to be a key contributor to synergistic DA neurotoxicity [17,19]. The present results suggest that endogenous AII increases the neurotoxic effect of 6-OHDA on DA neurons, as previously observed after simultaneous administration of low doses of two DA neurotoxins to DA cell cultures [17,19]. This effect was blocked by administration of the AT1 antagonist ZD 7155, suggesting that this increasing effect of AII on 6-OHDA neurotoxicity is exerted via AT1 receptors. Furthermore, pretreatment with the NAD(P)H inhibitor Apocynin inhibited the neurotoxic effect of 6-OHDA + AII, suggesting that NAD(P)H oxidase-mediated superoxide production plays a major role in the synergistic neurotoxicity of 6-OHDA and AII. Moreover, treatment with ZD 7155 or Apocynin not only reduced the increase in DA cell loss induced by administration of AII, but also counteracted the neurotoxic effect of the 6-OHDA in rats treated with 6-OHDA + AII, which was confirmed in rats treated with 6-OHDA and apocynin or ZD 7155 alone (i.e. without administration of exogenous AII). The present studies on the levels of major oxidative stress indicators (i.e. lipid peroxidation and protein oxidation) induced by 6-OHDA in the main areas of DA system degeneration (i.e. the striatum and the ventral midbrain) showed that inhibition of AT1 receptor activity lead to a marked reduction in 6-OHDA-induced increase in lipid peroxidation (75 and 60% reduction, respectively) and protein oxidation (50 and 60% reduction, respectively). In agreement, administration of ZD 7155 alone (i.e. without 6-OHDA) induced a significant reduction in the levels of lipid peroxidation and protein oxidation in comparison with controls. This is consistent with the neuroprotective effect of the AT1 antagonist ZD 7155 observed in the immunohistochemical studies. Forty-eight hours after treatment with 6-OHDA and AII no significant increase in lipid peroxidation or protein oxidation was found in 19 comparison with rats treated with 6-OHDA alone, despite the immunohistochemical data showing that administration of exogenous AII increased 6-OHDA-induced lesion 1 week after treatment. For biochemical studies, rats were killed 48 h after lesion because we observed the highest levels of 6-OHDA-induced oxidative stress indicators at this time point in preliminary experiments. However, 48 h after the 6-OHDA injection may be an inadequate time point for detecting the increasing effect of the AII injection on the 6-OHDA-induced ROS (or the increase in ROS induced by AII alone, group-B rats). Firstly, 48 h after the 6-OHDA injection is probably not the time point corresponding to the maximal effect of the injected AII on the ROS levels. Secondly, an increase in ROS induced by the injection of AII may be non-significant at the time when maximal levels of ROS induced by 6-OHDA occur (i.e. 48 h). In any case, the neuroprotective effect induced by administration of the AT1 antagonist appears more marked than the neurotoxic effect induced by administration of exogenous AII. In different cell types, it has been reported that AII-mediated NAD(P)H oxidase activation occurs via AT1 receptors, whereas the AT2 receptor appears to inhibit oxidase activation and have protective effects [11,59], and it has been suggested that AT1 receptor antagonists may enable endogenous angiotensin II to stimulate cell survival via activation of AT2 receptors. However, administration of exogenous AII would act on both AT1 and AT2 receptors. We have observed both AT1 and AT2 receptors in mesencephalic DA neurons in vitro, and that AII increased differentiation of DA neurons via AT2 receptors [54]. However, it is not clear if there are AT2 receptors in DA neurons and terminals in the adult brain, since conflicting results have been reported [15, 20]. In addition, the effect of the administration of exogenous AII may be partially counteracted by downregulation of AT1 receptors [25]. Finally, possible indirect effects of AII via glial cell receptors may also be investigated in futures studies [17-19]. 20 The most interesting point is, therefore, that pre-treatment with ZD 7155 induced a significant decrease in the DA neurotoxicity and oxidative stress induced by 6-OHDA alone, and that treatment with ZD 7155 alone induced a significant reduction of oxidative stress indicators in comparison with untreated controls. As indicated above, ROS originated by NAD(P)H oxidases may amplify and sustain the effect of low doses of 6-OHDA or other neurotoxins in animal models of PD or PD patients. Since AII is a major activator of NAD(P)H oxidase complex via AT1 receptors, treatment with AT1 antagonists may reduce the amplifying effect of NAD(P)H oxidase-derived ROS on DA neurotoxins. In preliminary experiments (A. Muñoz and J.L. Labandeira-Garcia, unpublished observations), we used radioimmunoassay (RIA; Bühlmann Laboratories AG, Switzerland) to estimate AII levels in striatal homogenates from control rats and 6OHDA lesioned rats (i.e. rats treated as those in groups A and C in the present study). Rats subjected to partial DA denervation using intraventricular 6-OHDA showed a slight increase (around 20% increase) in striatal AII levels with respect to controls. It is known that AII induces DA release from striatal DA terminals via AT1 receptors [7,15,47], and increased AII levels after partial DA denervation may constitute a compensatory response induced by a decrease in striatal DA levels. However, an increase in activation of AT1 receptors may lead to a vicious circle and amplify ROS production and DA cell degeneration. It is not known if there is an increase in striatal AII levels in PD. However, increased ACE activity in the parkinsonian cerebrospinal fluid [32] and an association between genetic polymorphisms of the ACE gene [39] and PD have been observed. Additional experiments are necessary to clarify the effects of the DA denervation on the levels of AII and other components of the striatal RAS. There is growing evidence indicating that the ageing-related loss of DA neurons and development of PD may be the result of interaction of multiple factors that act on 21 the DA neurons, which are particularly vulnerable to oxidative stress because of a number of intrinsic characteristics, including a reduced antioxidant capacity [29]. This combines to initiate the slow acting yet long term neurodegenerative process of PD. Genetic predisposition, environmental toxins, neuroinflammation and DA toxicity may be potential factors. In addition, the results of the present experiments suggest that striatal AII, via AT1 receptors, may play a pivotal role in increasing the oxidative damage to DA cells, and that manipulation of the RAS components, and particularly treatment with AT1 antagonists, widely used in cardiovascular therapy, may reduce the progression of PD. Acknowledgements This work was financially supported by grants from the Spanish Ministry of Education (MEC) and Galician Government (XUGA). The authors thank Pilar Aldrey for her excellent technical assistance. Disclosure Statement We have no actual or potential conflicts of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the work submitted that could inappropriately influence (bias) our work. 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