ISSN 1990-7478, Biochemistry (Moscow) Supplement Series A: Membrane and Cell Biology, 2008, Vol. 2, No. 1, pp. 8–13. © Pleiades Publishing, Ltd., 2008. Original Russian Text © A.Yu. Abramov, V.A. Kasymov, V.P. Zinchenko, 2008, published in Biologicheskie Membrany, 2008, Vol. 25, No. 1, pp. 11–17.
b-Amyloid Activates Nitric Oxide Synthesis and Causes Neuronal Death in Hippocampal Astrocytes A. Yu. Abramov, V. A. Kasymov, and V. P. Zinchenko Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow oblast, 142290 Russia fax: (4967)33-05-09, e-mail:
[email protected] Received October 2, 2006; in final form, August 29, 2007
Abstract—Digital fluorescence imaging was used to study the effects of the amyloid-beta peptide βA (fulllength peptide, βA 1–42) and its neurotoxic fragment (βA 25–35) on nitric oxide (NO) synthesis and cell viability in mixed cultures of rat hippocampal neurons and astrocytes. It was found that both βA 1–42 and βA 25−35 stimulated NO synthesis in astrocytes, but not in neurons. L-NAME, an inhibitor of the inducible NO synthase, blocked the effect of βA on NO production almost completely, reduced βA-induced mitochondrial depolarization in astrocytes, and partly prevented neuronal death. The rate of NO synthesis was decreased in Ca2+-free medium, increased in the presence of antioxidants and the NADPH oxidase inhibitor, and decreased in the presence of the SH-reagent thimerosal. DOI: 10.1134/S1990747808010029
Alzheimer’s disease is among the most common forms of dementia associated with total personal degradation and death. Its main manifestation is the formation of senile plaques consisting predominantly of the polypeptide β-amyloid (βÄ) (39–43 amino acids). The neurotoxicity of this polypeptide is thought to be responsible for neuronal cell death in central nervous system [1].
latter can also be induced by peroxynitrite formed in the reaction of ROS with NO. Nitric oxide being a signalling molecule takes part in many physiological and pathological processes in cells [10]. However, the data on the effect of βÄ on NO production are contradictory. Thus, it was shown that βÄ can both activate [11] and inhibit [12] NO synthesis in astrocytes and microglia. In this study, we investigated the effect of βÄ on the kinetics of NO production and βÄ neurotoxicity.
Despite the great number of hypotheses concerning amyloid neurotoxicity, the pathophysiological mechanism of neuronal death in Alzheimer’s disease is still obscure. In vivo studies have demonstrated that damaging effects of βÄ on body cells are mediated by different mechanisms, such as oxidative stress, mitochondrial dysfunction, disturbances in ë‡2+ homeostasis, NO synthesis, microglia activation, enhanced secretion of cytokins and TNFα [2–4], etc. However, the order sequence of these reactions still remains to be established.
EXPERIMENTAL Mixed cultures of hippocampal neurons and glial cells were obtained as described previously [5, 7]. The cells were isolated from 2–4-day-old Sprague-Dawley rats. The hippocampi were immersed in cold (4°ë) ë‡2+-free Hanks solution containing 20 µg/ml gentamicin. Hippocampal tissue was cut into small pieces with scissors, incubated with 0.1% trypsin (10 min, 37°ë), washed from trypsin by threefold centrifugation (400 g, 3 min) and suspended in a cultural (neurobasal) medium containing B27 and 2 mM L-glutamine. Cell suspensions were added dropwise to poly-D-lysinetreated cover glasses and placed into an incubator for 6 h. After the addition of culture medium, the cells were incubated at 37°ë in a humid atmosphere (5% ëé2 + 95% air). The culture medium was replaced twice a week; experiments were carried out on cells cultured for 10–20 days. Measurement of NO and mitochondrial potentials using fluorescence imaging. Fluorescent images were obtained with the help of an inverted fluorescent microscope Nikon equipped with a ×20 fluoride objec-
Recent findings suggest that βÄ stimulates generation of calcium signals in astrocytes (but not in neurons) [5, 6] and causes mitochondrial depolarization in these cells due to enhanced production of ROS by activated NADPH oxidase [7–9]; this decreases the content of reduced glutathione and leads to neuronal death. The Abbreviations: βÄ, β-amyloid; NO, nitric oxide; TNFα, tumor necrosis factor alpha; ROS, reactive oxygen species; ∆Ψm, mitochondrial potential; Rh123, rhodamine 123; L-NIL, L-N6-(1-iminoethyl)-lysine; TEMPO, 2,2,6,6-tetramethylpiperidineoxy; DPI, diphenylene iodonium chloride; DAF-fm, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate; IL1, interleukin 1; iNOS, inducible NO synthase; GSH, glutathione (reduced); NADPH, nicotinamide dinucleotide phosphate reduced; PI, propidium iodide.
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tive. The excitation light from a Xenone lamp passed through a computer-controlled filter wheel (Cairn Research, UK). Fluorescent signal was recorded with a digital camera Hamamatsu Orca ER (Japan). Video records were acquired and analyzed with the help of Lucida-6 software (Kinetic Imaging, UK). Statistical and graphical analyses were performed using Origin 7 (Microcal Software Inc.). The statistic difference was significant at p < 0.05. Staining with the fluorescent probe (DAF-fm) was performed by 30-min incubation of cells in Hanks solution in the presence of 20 µM DAF-fm with subsequent washing. Hanks solution contained (in mM): NaCl, 156; KCl, 3; MgSO4, 2; KH2PO4, 1.25; CaCl2, 2; glucose, 10; HEPES, 10 (pH 7.35). Short-term irradiation of cells at 490 nm was carried out with 5–10 s intervals; fluorescence was measured at >515 nm. DAF-fm is not fluorescent but upon interaction with NO transforms irreversibly into a fluorescent form; thus the probe fluorescence does not change with a decrease of NO. This makes DAF-fm a useful tool for measuring the NO production rate per time unit. However, the probe cannot be used for determining the concentration or even relative content of nitric oxide in a cell. Prior to registration of the mitochondrial potential (∆Ψm), the cells were stained with rhodamine 123 (Rh123) (1 µg/ml, 15 min), washed with Hanks solution and mounted onto a stage of an inverted-stage fluorescent microscope equipped with ×20 objective. Mitochondrial potential ∆Ψm was measured in single cells at λexcit = 490 nm and λem = 515 nm. Due to accumulation of Rh123 in mitochondria, fluorescence of the dye is quenched. As mitochondria are depolarized, the concentration of R123 in mitochondria decreases and the dye fluorescence increases. The data were obtained on at least five cover glasses containing 2–3 different cell cultures. Differential calculation of cells. Differential calculation of the number of normal, apoptotic and necrotic cells was carried out by staining of cell nuclei with Hoechst 33342 and propidium iodide (PI) [5, 7]. The cells were transferred onto round cover glasses and incubated in the dark with 4.5 µM Hoechst 33342 and 10 µM PI in Hanks solution for 30 min. The number of cells was determined with the help of a fluorescent microscope equipped with a ×20 objective. It is known that Hoechst 33342 and PI differ in their spectral characteristics and ability to penetrate into a cell. The blue fluorescent dye Hoechst 33342 is known to penetrate freely through the plasma membrane and to stain cell chromatin. The red fluorescent dye PI penetrates exclusively into dead (predominantly necrotic) cells with impaired outer membranes. In mixed cultures, the number of neurons and astrocytes was determined morphologically (by phase contrast imaging). Cell calculation was carried out in 15 randomly selected fields on each glass; the total number of cells was summated and the number of normal, necrotic and apoptotic cells was
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expressed relative to the total number (on a per cent basis). RESULTS The addition of βÄ 1–42 (1–5 µM) or the shortchain peptide βÄ 25–35 (10–50 µM) to primary cultures of hippocampal neurons and astrocytes resulted, after a 3–5 min delay, in a significant increase in the rate of NO synthesis in astrocytes; the increment in DAF-fm fluorescence was 5.7 ± 0.3-fold (n = 97, p < 0.001) (Fig. 1). These changes were characteristic of both types of βÄ and were dose-independent within the concentration range under study. No such changes were observed when the control peptide βÄ (35–25) (50 µM) was used. The βÄ-induced increase in DAF-fm fluorescence was attributed to NO production, since preincubation of cells with the iNOS inhibitor L-NIL (50 µM) (n = 67, Fig. 1) suppressed the fluorescence growth induced by βÄ. It is of note that the initial rate of NO synthesis did not change in the presence of the inhibitor, which testifies to the lack of NO production in resting astrocytes. The neurons present in the culture did not virtually increase NO production in response to both peptides. The lack of neuronal response to βÄ and the 3–5-min delay in activation of NO synthesis in astrocytes are consistent with previously observed effects of this compound on calcium homeostasis and membrane potential of mitochondria [5–7]. It seemed therefore tempting to investigate the relationship between these processes in the mechanism of βÄ neurotoxicity. The βÄ-induced ë‡2+ signal was generated during incorporation of this peptide into the plasmalemma and channel formation, since the ability of this peptide to form channels was demonstrated in previous studies with model membranes. In experiments with astrocytes, the calcium signal showed a direct dependence on extracellular calcium [13, 14]. The addition of βÄ 1–42 (n = 56) or βÄ 25–35 (n = 48) (5 and 50 µM, respectively) to a ë‡2+-free solution increased the rate of NO production in astrocytes. However, in this case the signal was much weaker than in the ë‡2+-containing medium, viz., the intensity of DAF-fm fluorescence increased 2.8 ± 0.2-fold (cf. 5.7 ± 0.3-fold in control samples, p < 0.001, Figs. 2a and 2b). This suggests that the rate of βÄ-stimulated synthesis of NO in astrocytes partly depends on the amount of ë‡2+ entering the cell. In addition, it was demonstrated that βÄ stimulates NADPH oxidase-dependent production of reactive oxygen species (ROS) in astrocytes [7, 8]. Antioxidants and NADPH oxidase inhibitors hinder ROS formation in astrocytes and prevent neuronal death [6, 7]. As can be seen (Fig. 2b), the antioxidant mixture (TEMPO + catalase) (n = 55) not only failed to decrease the rate of NO synthesis, but even slightly increased it (the intensity of DAF-fm fluorescence increased from 570 ± 34% in control samples to 630 ± 44% in the presence of anti-
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Fig. 1. Effect of βÄ on the rate of NO production in hippocampal astrocytes. (a) Mean values of fluorescence intensity of DAF-fm in single astrocytes (data from 4 independent measurements, n = 97) in control and L-NIL (50 µM) treated (10 min) astrocytes in the presence of 50 µM βÄ 25–35. (b) The rate of NO production (%) in the presence of 5 µM βÄ 1–42. The initial rate of control astrocytes was taken for 100%. (c) Three images of cultured cells stained with DAF-fm obtained 1, 9, and 13 min after the addition of 50 µM βÄ 25–35.
oxidants) (p < 0.05). Incubation of cells with the NADPH oxidase inhibitor DPI (n = 62) (20 min) also augmented NO synthesis in astrocytes in response to βÄ addition (the signal was enhanced from 570 ± 34% in control samples to 680 ± 41% in the presence of antioxidants (p < 0.05). This effect can be attributed to the interaction of ROS with NO with the formation of peroxynitrite [15] (the latter failed to be detected with the help of DAF-fm or ROS-specific probes) rather than to direct effect of ROS on NO synthesis. In experiments designed to investigate the effect of ROS on NO production, we used thimerosal as an SH-reagent. Earlier studies have shown that thimerosal decreases the GSH content and stimulates production of ROS in astrocytes [16]. In our study, thimerosal strongly attenuated the effect of βÄ 1–42 on the rate of NO synthesis, viz., the increase in DAF-fm fluorescence diminished from 5.7 ± 0.3-fold to 3.2 ± 0.2-fold (n = 45) (p < 0.05; Fig. 2). This effect can also be attributed to the interaction of NO with the superoxide leading to peroxynitrite formation. In this way, βÄ stimulated ROS and NO production and their possible interaction in a ë‡2+-dependent manner. Considering that NO inhibits the respiratory complex IV [17], we studied the effect of NO on βÄ-induced mitochondrial depolarization with the ultimate goal to elucidate the role of NO in the mechanism of βÄ neu-
rotoxicity. βÄ induced three kinds of mutually convertible changes in the signal generated by the potential-sensitive probe Rh123: (I) slow depolarization, (II) spike-shape depolarization concomitant with the recovery, and (III) spike-shape depolarization without potential recovery (Figs. 3a, 3c). Incubation of cells with the NO synthase inhibitor L-NIL (Figs. 3b, 3d) diminished the mean values of Rh123 fluorescence in astrocytes from 64 ± 13 to 38 ± 6% (n = 65, p < 0.05). It is of note that this effect was due to reduction of the number of cells with slow mitochondrial depolarization and sharp peaks of the Rh123 signal (Fig. 3c) rather than by inhibition of signal neurotoxicity. Thus, NO is partly involved in βA-stimulated mitochondrial depolarization of hippocampal astrocytes. Presumably, the effect of NO on the mitochondrial potential observed in this study is due to its interaction with ROS rather than with respiratory chain components. Twenty-four hour incubation of primary mixed cultures of astrocytes and hippocampal neurons in the presence of 5 µM βÄ 1–42 induced the death of 51 ± 3% neurons and 23 ± 2% astrocytes (Figs. 4a, 4b) (data from 7 independent measurements). Interestingly, in NO-producing astrocytes, the NO inhibitor 50 µM L-NIL (data from 5 independent measurements) diminished the number of dead cells only insignificantly (up to 19 ± 3%), whereas incubation of neurons unable to
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Fig. 2. Effects of extracellular ë‡2+ and ROS production on βÄ-induced synthesis of NO in astrocytes. (a) Mean fluorescence intensity of DAF-fm in single astrocytes in control and in a ë‡2+-free solution (+0.5 mM EGTA) induced by 5 µM βÄ 1–42. (b) The rate of NO synthesis (%) induced by 5 µM βÄ 1–42. Thimerosal (15 µM), DPI (0.5 µM), TEMPO (0.5 mM) + 250 unit/ml catalase were added to the samples 20 min prior to the assay. The initial rates of control astrocytes were taken for 100%.
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Fig. 3. Effect of the NO synthesis inhibitor on βÄ-induced depolarization of mitochondria. Changes in the fluorescence of Rh123 in control astrocytes (a) and in astrocytes incubated for 30 min with 50 µM L-NIL (b) in the presence of 50 µM βÄ 25–35. (c, d) Effect of L-NIL on the ratio of mitochondrial depolarization types and mean values of changes in the fluorescence of Rh123, respectively. BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES A: MEMBRANE AND CELL BIOLOGY
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Fig. 4. βÄ-Induced death of cultured hippocampal neurons (a) and astrocytes (b). Effect of 24-h incubation with 50 µM βÄ 25–35 or 5 µM βÄ 1–42 on the viability of 10–20 dayold mixed cell cultures. The number of dead cells was determined by staining with propidium iodide. Live cells were stained with Hoechst 33342. L-NIL (50 µM) was added to the samples 20 min prior to βÄ addition; the resulting mixture was incubated in the medium for 24 h.
synthesize NO with L-NIL significantly (p < 0.05) decreased the number of dead cells (from 51 ± 3 to 32 ± 3%). It may thus be concluded that NO synthesis in astrocytes plays a role in the mechanism of βÄ neurotoxicity through regulation of ROS production and secretion of TNFα and other cytokins. DISCUSSION Despite the increasing number of publications devoted to the mechanism of βÄ neurotoxicity, no specific model of this mechanism exists. The role of NO in this process is being actively discussed in the literature, but the kinetics of its production in the presence of βÄ has not been finally established yet. The βÄ-induced production of NO in astrocytes (but not in neurons) testifies to a specific dependence of this reaction on iNOS. At the same time, the lack of NO synthesis in neurons is not suggestive of the nonspecificity of the neuronal enzyme isoform with respect to βÄ. As a matter of fact, βÄ-induced primary signals generated in astrocytes, e.g., ë‡2+ signals [5, 6], can be absent in these cells. The 3–5 min delay in NO synthesis (Figs. 1a and 2a) is yet another interesting finding of this study. As some βÄ-induced processes precede the activation of NO synthesis (ROS [7–9], IL1 and TNF [18] production), it may be assumed that NO synthesis is a secondary process. Based on our data and the previously established ë‡2+ dependence of the enzyme [19], it may be suggested that it is the calcium signal in astrocytes that is a true activator of NO synthase. At the same time, incomplete, albeit significant, inhibition of NO production in a ë‡2+-free medium (Fig. 2) entails ë‡2+-induced
enhancement of NO synthesis rather than its primary activation. Here, it is appropriate to note that iNOS is devoid of ë‡2+-binding sites; therefore, the enzyme cannot be activated by ë‡2+ ions directly. The observed dependence of NO production on ë‡2+ can be due to stimulation of protein kinase C [20]. Our data suggest that stimulation of NO production in astrocytes accelerates neuronal death. This can be attributed to direct effect of NO on hippocampal neurons and, as a consequence, deregulation of the signaling system [21, 22] and activation of molecular mechanisms of neuronal cell death. At the same time, simultaneous production of NO and ROS leads to the formation of peroxynitrite, which plays a crucial role in βÄ-dependent pathologies [23]. βÄ-Dependent secretion of TNFα and other cytokins is yet another explanation for this effect [24]. As it is known, βÄ stimulates IL1 and TNF-TNFα synthesis in astrocytes resulting in receptor-dependent expression of iNOS and generation of neurotoxic free radicals [18]. REFERENCES 1. Hardy, J. and Selkoe, D.J., The Amyloid Hypothesis of Alzheimer’s Disease: Progress and Problems on the Road to Therapeutics, Science, 2002, vol. 297, pp. 353– 356. 2. Laferla, F.M., Calcium Dyshomeostasis and Intracellular Signalling in Alzheimer’s Disease, Nat. Rev. Neurosci., 2002, vol. 3, pp. 862–872. 3. Blass, J.P. and Gibson, G.E., The Role of Oxidative Abnormalities in the Pathophysiology of Alzheimer’s Disease, Rev. Neurol. (Paris), 1991, vol. 147, pp. 513– 525. 4. Canevari, L., Abramov, A.Y., and Duchen, M.R., Toxicity of Amyloid Beta Peptide: Tales of Calcium, Mitochondria, and Oxidative Stress, Neurochem. Res., 2004, vol. 29, pp. 637–650. 5. Abramov, A.Y., Canevari, L., and Duchen, M.R., Changes in Intracellular Calcium and Glutathione in Astrocytes as the Primary Mechanism of Amyloid Neurotoxicity, J. Neurosci., 2003, vol. 23, pp. 5088–5095. 6. Abramov, A.Y., Canevari, L., and Duchen, M.R., Calcium Signals Induced by Amyloid Beta Peptide and Their Consequences in Neurons and Astrocytes in Culture, Biochim. et Biophys. Acta, 2004, vol. 6, pp. 81–87. 7. Abramov, A.Y., Canevari, L., and Duchen, M.R., BetaAmyloid Peptides Induce Mitochondrial Dysfunction and Oxidative Stress in Astrocytes and Death of Neurons through Activation of NADPH Oxidase, J. Neurosci., 2004, vol. 24, pp. 565–575. 8. Abramov, A.Y., Jacobson, J., Wientjes, F., Hothersall, J., Canevari, L., and Duchen, M.R., Expression and Modulation of an NADPH Oxidase in Mammalian Astrocytes, J. Neurosci., 2005, vol. 25, pp. 9176–9184. 9. Abramov, A.Y. and Duchen, M.R., The Role of an Astrocytic NADPH Oxidase in the Neurotoxicity of Amyloid Beta Peptides, Philos. Trans. R. Soc. Lond. B Biol. Sci., 2005, vol. 360, pp. 2309–2314. 10. Bashkatova, V.G. and Raevsky, K.S., Nitric Oxide in Mechanisms of Brain Damage Induced by Neurotoxic
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