Inhibitory effects of angiotensin on NMDA-induced cytotoxicity in primary neuronal cultures

Brain Research Bulletin 62 (2004) 397–403

Inhibitory effects of angiotensin on NMDA-induced cytotoxicity in primary neuronal cultures Gao Jing a , Tom Grammatopoulos b , Paul Ferguson b , William Schelman c , James Weyhenmeyer b,∗ a

State Key Laboratory of Pharmaceutical Biotechnology, School of Medicine, Nanjing University, Nanjing 210093, PR China b Department of Cell and Structural Biology, University of Illinois, 346 Henry Administration Building, 506 South Wright Street, Urbana, IL 61801, USA c Department of Internal Medicine, University of Wisconsin, Madison, WI, USA Received 2 July 2002; received in revised form 1 July 2003; accepted 31 October 2003

Abstract Primary cultures from the hypothalamus/thalamus/septum/midbrain (HTSM) region of 1-day-old mice were used to investigate the effects of angiotensin on NMDA excitotoxicity. Cell viability was determined following exposure to 1–10 mM glutamate or 0.01–10 mM NMDA. Cells exposed to 1 mM glutamate or 1 mM NMDA for 24 h showed a significant increase in cell death as determined by propidium iodide staining. HTSM cultures treated with 0.1 mM NMDA revealed both DNA laddering and positive staining for TUNEL, suggesting apoptosis as the primary mechanism for the cell death. We also determined whether angiotensin II (Ang II) modulated NMDA-induced cell death in HTSM-cultured neurons. Cells pre-treated with 10 nM Ang II showed a decrease in NMDA-induced cytotoxicity, TUNEL staining and DNA laddering. NMDA-induced cell death was also reduced when cells were pre-treated with the AT1 receptor antagonist losartan. In this study, we have shown that NMDA and glutamate induce cell death through the NMDA receptor, and that Ang II, acting primarily through the AT2 receptor, can attenuate this response. © 2003 Elsevier Inc. All rights reserved. Keywords: Angiotensin; Glutamate; NMDA; Neuron; Cell death; Apoptosis

1. Introduction While angiotensin is best known for its role in cardiovascular regulation, fluid balance, and neuroendocrine function, recent studies have shown that it also influences cell growth in several tissue types [15]. These effects appear to be mediated through two angiotensin (AT) receptor subtypes, AT1 and AT2 , which were initially characterized on the basis of their affinity for losartan and PD123319, respectively. Both AT1 and AT2 receptors are differentially expressed and distributed in the brain. AT1 receptors, which are most frequently associated with cardiovascular regulation and the pathogenesis of hypertension, are widely expressed in both peripheral tissues (adipose, adrenal, heart, kidney, liver, lung, testis and vascular smooth muscle) and the central nervous system [7,23]. Functionally, AT1 receptors are considered

∗

Corresponding author. Tel.: +1-217-265-5440; fax: +1-217-265-544. E-mail address: [email protected] (J. Weyhenmeyer).

0361-9230/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2003.10.011

to play an important role in the regulation of heart rate and blood pressure, as well as fluid homeostasis and hormone secretion [11,22]. In contrast, the functional roles for the AT2 receptor are not fully understood. Both its distribution in developing tissues, including central nervous system, and its transient expression in adult tissues following injury, suggest that it may be associated with cell differentiation and tissue regeneration. Recent studies suggest that the AT2 receptor may play a role in modulating cell death [9,16,21,27]. Interestingly, the diencephalon (hypothalamus, thalamus, and septum) and midbrain regions, which have been reported to be more resistant to hypoxic damage and glutamate excitotoxicity than other brain regions, constitutively express high levels of AT2 receptors [2]. Glutamate, the major excitatory neurotransmitter in the brain, has been suggested to play an important role in the pathogenesis of several neurodegenerative disorders [3]. While glutamate ionotropic receptors have been implicated in this neurotoxicity, the NMDA receptor appears to be critical for the excitotoxic effects of glutamate on neurons

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[10]. NMDA receptors are known to induce both neuronal necrosis and apoptosis, with the latter being an important feature of several neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, and metabolic deficiencies of brain related to ischemic or epileptic injury [3]. Neurons cultured from the hypothalamus/thalamus/ septum/midbrain (HTSM) region constitutively express both the AT1 , AT2 and NMDA receptors, providing a model in vitro system for examining the effects of angiotensin on NMDA-induced excitotoxicity. Several lines of evidence suggest that angiotensin, acting through the AT2 receptor, can modulate NMDA-induced cell death. For example, Bains et al. [2] have shown that magnocellular neurons, from the paraventricular nucleus of the hypothalamus, express high levels of the AT2 receptors and are more resistant to NMDA receptor-mediated excitotoxicity than other hypothalamic neurons. Further supporting a functional relationship between the NMDA and angiotensinergic systems, we have shown that angiotensin decreases NMDA-induced nitric oxide and cGMP production in both PC12W and NG108 cells [20]. These effects can be blocked by PD123319, a specific AT2 receptor antagonist, suggesting that the AT2 receptor modulates NMDA receptor signaling in neurons. Recently, we reported that hypoxia-induced apoptosis is NMDA receptor-mediated in cortical neuronal cultures and that angiotensin, acting through the AT2 receptor, can protect cultured cortical neurons from this apoptotic cell death [9]. Further understanding of the functional interaction between AT and NMDA receptors, especially as it relates to the modulatory function of angiotensin in preventing NMDA-induced neuronal injury, should provide additional insight into potential therapeutic uses of angiotensin II analogs under conditions of pathological (apoptotic) cell death. In this study, we demonstrate that glutamate and NMDA induce cell death in HTSM-cultured neurons in a dose-dependent manner, and provide evidence to support a neuroprotective role for angiotensin under conditions of NMDA-induced excitotoxicity.

2. Material and methods 2.1. Materials Neurobasal media, B27 supplement, Dulbecco’s modified Eagles medium (DMEM) and penicillin/streptomycin were obtained from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS) was obtained from Summit (Ft. Collins, CO, USA). PD123319 was generously provided by Dr. Joan Keiser at Parke Davis (Ann Arbor, MI, USA). Losartan was obtained from DuPont Merck (Wilmington, DE, USA). TUNEL staining and cytotoxicity detection kits were obtained from Boehringer Mannheim (Indianapolis, IN, USA). All other reagents were obtained from Sigma (St. Louis, MO, USA) unless otherwise indicated.

2.2. Primary cell cultures Neonatal (1-day-old) BALB-c mice were CO2 anesthetized, brains were removed and the cortex was dissected and placed in isotonic Wilson’s buffer. Meninges and blood vessels were stripped and the tissue was minced into 1 mm3 pieces and trypsinized. DMEM containing 10% FBS and DNAse (4 kU/ml) was added to inactivate trypsin and decrease DNA agglutination, respectively. Cells were mechanically dissociated, washed with DMEM/FBS and plated on poly-l-lysine coated plates at a density of 2.5×106 cells/ml. The medium was changed from DMEM/FBS to neurobasal supplemented with B27 and cultures were maintained in a humidified CO2 incubator at 37 ◦ C. The procedure typically yields cultures containing >90% neurons and <10% support cells. This protocol was approved by the University of Illinois Laboratory Animal Care Advisory Committee. 2.3. Cell culture treatments Excitotoxicity was induced by treating cultures with 1–10 mM glutamate or 0.01–10 mM NMDA for 30 min and the cells were assayed 6–24 h later. To determine whether Ang II influenced glutamate or NMDA-induced cell death, cells were pre-treated with the peptide (1–100 nM). To determine which AT receptor subtype was involved, cells were pre-treated with either 1 ␮M losartan (AT1 receptor antagonist) or 1 ␮M PD123319 (AT2 receptor antagonist) [7,9]. 2.4. Propidium iodide (PI) staining Cells were permeabilized with 80% methanol for 15 min at 4 ◦ C, stained with propidium iodide (5 mg/ml) for 5 min and examined by fluorescence microscopy. 2.5. Cell death (MTT) assay NMDA-induced cytotoxicity was determined using the mitochondrial dehydrogenase assay. Briefly, cells were washed with Mg2+ -free Hank’s balanced salt solution (HBSS) containing 2 mM CaCl2 , 10 ␮M glycine and 10 ␮M glucose. After treatment for 16 h, the media was replaced with organic MTT dye (3-[4, 5-dimethylthiazol-2yl] 2.5-diphenyl tetrazolium bromide) (1 mg/ml) for 3 h at 37 ◦ C. In Triton-treated wells, cells were treated with 1% Triton-X in addition to the organic MTT dye. Absorbance was read using an ELISA plate reader (540 nm filter). The cytotoxicity index (CI) was calculated as follows: absorbance of medium control wells −absorbance of treatment wells absorbance of medium control wells − Triton-treated wells 2.6. DNA laddering Cells were harvested from six-well plates 24 h posttreatment with DNA lysis buffer (10 mM Tris/HCl, 25 mM

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EDTA, 100 mM NaCl, 1% SDS and 200 g/ml proteinase K) and incubated overnight at 50 ◦ C. The lysate was incubated for 1 h with 0.2 mg/ml DNAase-free RNase (Ribonuclease A). DNA was precipitated with 3 mM sodium acetate and 99% ethanol after extraction with phenol/chloroform/isoamyl alcohol (2×). Pellets were resuspended in TE buffer and DNA was size-fractionated on 1.2% agarose gels. A 100-bp ladder (Invitrogen) was used as the standard. 2.7. TUNEL staining Terminal transferase biotinylated dUTP nick end labeling of DNA strand break analysis was carried out using the TUNEL kit (Boehringer Mannheim). Briefly, cells were fixed with fresh paraformaldehyde in PBS (40 g/l), washed and incubated with blocking solution (0.3% H2 O2 in methanol) and treated with permeabilization solution (0.1% Triton in 0.1% sodium citrate) at 4 ◦ C for 2 min, followed by TUNEL reaction mixture (terminal deoxynucleotidyl transferase and modified nucleotides) for 60 min at 37 ◦ C. Converter-POD was added for 30 min at 37 ◦ C and developed with metal enhanced DAB-substrate solution. Negative controls included cells incubated with TUNEL reaction mixture without terminal transferase. Positive controls included cells treated with DNAse I (4 kU/ml) following permeabilization. Cells (positive versus total) were counted from five fields or 500 cells per well at 200× using Hoffman optics.

Fig. 2. Excitotoxicity of NMDA in HTSM cultures. Cultures were treated with 1 and 10 mM NMDA for 30 min in Mg2+ -free solution and stained with PI 18–24 h later. Cells were viewed under fluorescence microscope at 200× magnification.

2.8. Statistical analyses Factorial one-factor ANOVA followed by Fisher’s PLSD and student’s t-test were used to determine significance. Val-

Fig. 1. Excitotoxicity of glutamate in HTSM cultures. Cultures were treated with glutamate (1 and 10 mM) for 30 min in Mg2+ -free solution and stained with PI 6–24 h later. Cells were viewed under fluorescence microscope at 200× magnification.

Fig. 3. NMDA dose response in HTSM cultures. Cultures were treated with 0.01–10 mM NMDA for 30 min in Mg2+ -free solution and assayed by MTT 24 h later. The results show a dose-dependent increase in cytotoxicity index. Date are reported as ±S.E.M. (n = 8) with (a) showing significance over control with P < 0.05.

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ues are presented as means ± S.E.M. from at least three independent experiments using different culture preparations.

3. Results HTSM cultures treated with 1–10 mM glutamate or NMDA for 6–24 h and stained with propidium iodide (PI) showed an increase in PI-stained cells at 18 h post-treatment with 1 mM glutamate (Fig. 1) and 1 mM NMDA (Fig. 2). An increase in cell death was seen in cells treated with 0.1–10 mM NMDA for 30 min and quantitated by MTT analysis 16 h post-treatment (Fig. 3). Based on these findings, 100 ␮M NMDA, which showed a cytotoxicity index of 0.12 ± 0.02, was chosen as the optimal concentration for all subsequent experiments. To confirm that the cell death was apoptotic, cultured cells were treated with 100 ␮M NMDA for 30 min and assayed for changes in cell morphology, TUNEL staining and DNA laddering 24 h post-treatment. Exposure to NMDA resulted in an increase in cell shrinkage and neurite retraction (Fig. 4), TUNEL positive cells (Fig. 5) and DNA laddering (Fig. 6). To determine whether angiotensin influences NMDAinduced cell death, cells were pre-treated with 1–100 nM Ang II for 5 min followed by a 30 min exposure to 100 ␮M NMDA and assayed 24 h post-treatment. Fig. 7A shows that pre-treatment with 10 nM Ang II resulted in the most significant decrease 36% ±1.9 in cell death. Morphological analyses further confirmed the protective effect of Ang II. While cells treated with 100 ␮M NMDA showed cell shrinkage and retracted neurites, which is consistent with the excitotoxic effects of NMDA, cells pre-treated with 10 nM Ang II revealed a reduction in cell shrinkage and an increase in the

number of cells with centric nuclei and branched neurites (Fig. 4). Pre-treatment with Ang II (10 nM) decreased the number of TUNEL-stained cells (Fig. 5) and DNA laddering (Fig. 6) following exposure to NMDA. To determine whether the angiotensin-mediated neuroprotection was AT receptor subtype specific, cells were pre-treated with 1 ␮M losartan (AT1 antagonist) or 1 ␮M PD123319 (AT2 antagonist) for 10 min prior to treatment with Ang II and assayed by MTT analysis. In cells pre-treated with the losartan followed by 100 ␮M NMDA, Ang II reduced the cytotoxic index by 13.8 ± 5.3% (Fig. 7B). However, when cells were pre-treated with PD123319 followed by 100 ␮M NMDA and 10 nM Ang II, the cytotoxicity index was increased by 13.7 ± 2.8% (Fig. 7B). Together, these findings suggest that Ang II acts primarily through the AT2 receptor to facilitate its neuroprotective effects.

4. Discussion In the present study, we have used primary neuronal cultures derived from the mouse hypothalamus/thalamus/ septum/midbrain (HTSM) to investigate a proposed role for angiotensin in attenuating cell death by glutamate/ NMDA-induced over-stimulation of NMDA receptors. HTSM cultures provide an ideal in vitro model system for examining glutamate excitotoxicity in neurons since these cells are sensitive to glutamate and NMDA, as determined by propidium iodide staining, mitochondrial dehydrogenase activity, cell morphology, TUNEL staining and DNA laddering. In vitro and in vivo CNS studies provide evidence for both apoptotic and necrotic neuronal death following over-stimulation of glutamate/NMDA receptors

Fig. 4. Effects of angiotensin on NMDA-induced morphology changes in HTSM-cultured neurons. Cultures (200×, Hoffman optics) showing neuronal morphology following treatment with 100 ␮M NMDA and the neuroprotective effect of 10 nM angiotensin. (A) Control showing normal morphology with extended neurites. (B) Cells treated with 100 ␮M NMDA showing retracted neurites and condensed cell bodies. (C) Pre-treatment with 10 nM angiotensin followed by 100 ␮M NMDA, showing partial normal neuronal morphology with some condensed cell bodies with retracted neurite branching. (D) Cells pre-treated with 1 ␮M losartan followed by 10 nM angiotensin and 100 ␮M NMDA showing normal morphology. (E) Cells pre-treated with PD123319 followed by 10 nM angiotensin and 100 ␮M NMDA, showing mostly condensed cell bodies with retracted neurites. (F) Cells pre-treated with 1 ␮M MK801 followed by 100 ␮M NMDA, showing the protective effect of the NMDA receptor specific antagonist.

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Fig. 6. Effects of angiotensin on NMDA-induced DNA laddering. DNA laddering induced by 100 ␮M NMDA and attenuated by pre-treatment with 10 nM angiotensin with or without the 1 ␮M of the angiotensin receptor specific antagonists losartan and PD123319. Lane 1, control; lane 2, DNA fragmentation induced by treatment with 100 ␮M NMDA (N); lane 3, pre-treatment with 10 nM angiotensin (A) attenuates NMDA-induced DNA fragmentation; lane 4–6, treatment with 1 ␮M losartan (L), 1 ␮M PD123319 (P) or both, respectively, prior to angiotensin and NMDA stimulation, respectively. Experiments were repeated in three separate experiments.

Fig. 5. Effects of angiotensin on NMDA-induced apoptosis in HTSM-cultured neurons. (A) Cultures (200×, Hoffman optics) showing apoptosis by TUNEL staining. (B) Number of apoptotic cells: (1) control; (2) cultures treated with 100 ␮M NMDA (N) for 30 min resulting in increase of TUNEL-positive cells; (3) pre-treatment of 10 nM angiotensin (A), showing a reduction in NMDA-induced apoptotic cells; (4) cells pre-treated with 1 ␮M losartan (L) and 1 ␮M PD123319 (P) followed by 10 nM angiotensin and 100 ␮M NMDA show inhibition of angiotensin protection.

[1]. Whether the cell death is necrotic or apoptotic appears to be dependent on the levels of glutamate, with “low” levels having no toxic effect, “moderate” levels inducing apoptosis, and “high” levels inducing necrosis. Excessive glutamate levels have been implicated as a putative underlying mechanism for ischemic injury (stroke) as well as other neurodegenerative disorders [3]. Several studies have identified the NMDA receptor as an important mediator of glutamate excitotoxicity under conditions of hypoxic injury [4]. Although elevated levels of glutamate have been shown to induce neuronal cell death,

NMDA receptor blockade in the absence of stress can also induce apoptosis in cultured cortical neurons [12,13]. While glutamate appears to play an important role in the neuronal cell death seen in several neurodegenerative disorders, its role as the primary excitatory neurotransmitter in the brain argues against the use of general NMDA receptor antagonists to treat these disorders. Therefore, understanding how glutamate/NMDA receptors interact with other cellular pathways during the neurodegenerative process may provide additional insight into potential therapeutic strategies for certain neurodegenerative disorders, such as stroke. Interestingly, the brain has regional sensitivity to excitotoxic and ischemic injury. For example, the cerebellum and cortex are substantially more susceptible to ischemic and excitotoxic injury than the diencephalon and midbrain, with the latter being more resistant to glutamate-induced cell death. Bains et al. [2] have suggested that a possible explanation for the increased resistance to glutamate excitotoxicity in the diencephalon and midbrain may be the neuroprotective effect of Ang II mediated through its specific receptors. To determine whether AT receptors can influence glutamate-mediated excitotoxicity, we pre-treated HTSMcultured cells with Ang II prior to exposing them to excitotoxic levels of NMDA. Our findings not only suggest that Ang II attenuates NMDA-induced cell death, but the neuroprotection is primarily mediated through the AT2 receptor.

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Fig. 7. Effects of angiotensin on NMDA-induced cytotoxicity index. HTSM neuronal cultures were treated with 100 ␮M NMDA (N) for 30 min and assayed by MTT 24 h later. (A) Cultures were pre-treated with 1–100 nM angiotensin (A) prior to the addition of 100 ␮M NMDA (N). (B) Pre-treatment with 1 ␮M of losartan (L) (AT1 receptor antagonist) and/or 1 ␮M PD123319 (P) (AT2 receptor antagonist), were added prior to 10 nM angiotensin (A) and then stimulated with 100 ␮M NMDA (N). The data are reported as the ±S.E.M. (n = 8) with (a) showing significance over control, (b) showing significance over N and (c) showing significance over N + A 10 nM, with P < 0.05.

Both TUNEL and DNA laddering results indicate that the NMDA-induced cell death is mostly apoptotic and that angiotensin attenuates the process through the AT2 receptor. This is further supported by recent studies showing a putative role for angiotensin in ischemic injury [27]. Interestingly, AT2 receptor mRNA is up-regulated in the cortex and hippocampus following global ischemia [16], while AT2 receptor mRNA levels and receptor binding are markedly increased by glutamate stimulation in cortical cells [17]. Grammatopoulos et al. [9] have shown that mouse cortical neurons exposed to chemical hypoxia undergo apoptosis that is primarily mediated through the NMDA receptor. They also found that Ang II, acting through the AT2 receptor, protects cortical neurons from hypoxic-induced apoptosis. Given that NMDA excitotoxicity has been implicated in the pathogenesis of several neurodegenerative disorders, including stroke, findings from our lab and others would suggest that angiotensinergic system, and more specific the AT2 receptor, may be a therapeutic target for these disorders. In addition to the actions of the AT2 receptor, antagonists for the AT1 receptor have been shown to have possible therapeutic value in neurodegenerative disorders [5]. Blockade of AT1 receptors has been shown to improve neurological outcome and reduce expression of AP-1 transcription factors after focal brain ischemia in rats [6]. While further studies will be necessary to determine the mechanism(s) underlying angiotensin’s ability to modulate NMDA excitotoxicity, one possible explanation is the involvement of Ang II in the cell’s ionic flux mechanisms.

The role of ion channels is slowly emerging as a significant contributor to both the induction and protection of neurons from ischemic and excitotoxic cell death. Blockade of Na+ voltage-gated channels have been shown to prevent hypoxic-induced injury by maintaining ionic homeostasis and preventing plasma membrane depolarization [8,18]. Calcium channel blockers, such as LY393615, have a neuroprotective effect during in vitro and in vivo cerebral ischemia [19]. Recently, potassium channels have been implicated in ischemic injury, and activation of these channels may have a protective role in NMDA receptor-mediated cell death [24]. The NMDA receptor is responsible for both Ca+2 and Na+ influx. In addition, high external levels of K+ can inhibit NMDA receptor activation [14]. Zhu et al. [25,26] have reported that AT1 receptors can inhibit K+ channel currents through a calmodulin/cam kinase pathway and that AT2 receptors activate K+ channel currents through the activation of phospholipase A and PP2A. Preliminary findings from our lab suggest that angiotensin protection against NMDA-mediated excitotoxicity may result from ion regulation in the cell and/or direct modulation of NMDA receptor subunit phosphorylation and is currently being examined. In this study, we have shown that NMDA induces cell death in HTSM primary neuronal cultures and that angiotensin, acting through the AT2 receptor, can attenuate this response. This study provides further evidence to suggest a possible role for angiotensin in glutamate mediated neurodegenative diseases.

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