- Email: [email protected]
NADPH oxidase immunoreactivity in the mouse brain
Brain Research 988 (2003) 193–198 www.elsevier.com / locate / brainres
Short communication
NADPH oxidase immunoreactivity in the mouse brain Faridis Serrano a , Nutan S. Kolluri c , Frans B. Wientjes e , J. Patrick Card c , Eric Klann a,b,c,d , * a
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA b Division of Neuroscience, Baylor College of Medicine, Houston, TX, USA c Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA, USA d Center for the Neural Basis of Cognition, University of Pittsburgh, Pittsburgh, PA, USA e Department of Medicine, University College London, London, UK Accepted 21 July 2003
Abstract Superoxide production via NADPH oxidase has been shown to play a role in neurotoxicity, ischemic stroke, and possibly Parkinson’s and Alzheimer’s diseases. In addition, NADPH oxidase-dependent production of superoxide may be necessary for normal brain functions, including neuronal differentiation and neuronal plasticity. To improve our understanding of NADPH oxidase in the brain, we studied the localization of the various protein components of NADPH oxidase in the central nervous system of the adult mouse using immunohistochemistry. We detected staining for the cytoplasmic NADPH proteins, p40 phox , p47 phox , and p67 phox , as well as the membrane-associated NADPH oxidase proteins, p22 phox and gp91 phox in neurons throughout the mouse brain. Staining of each of the NADPH oxidase proteins was observed in neurons in all regions of the neuraxis, with particularly prominent localizations in the hippocampus, cortex, amygdala, striatum, and thalamus. The expression of NADPH oxidase proteins in neurons suggests the possibility that enzymatic production of superoxide by a NADPH oxidase may play a role in both normal neuronal function as well as neurodegeneration in the brain. 2003 Elsevier B.V. All rights reserved. Theme: Other systems of the CNS Topic: Comparative neuroanatomy Keywords: Superoxide; Hippocampus; Rac; Neurotoxicity; Neurodegeneration; Long-term potentiation
1. Introduction NADPH oxidase is a multicomponent enzyme complex that consists of the membrane-bound cytochrome b 558 phox phox phox (p22 and gp91 ) and cytoplasmic proteins (p40 , phox phox p47 , p67 ) that translocate, along with the small G-protein Rac, to the membrane during cellular stimulation to produce superoxide [5,16]. NADPH oxidase has been best characterized in phagocytic cells. However, a number of studies suggest that various NADPH oxidase proteins are expressed in non-phagocytic cells, including vascular smooth muscle cells [9], fibroblasts [21], and endothelial cells [12]. In addition, recent studies have described the isolation of NADPH oxidase homologs that are present in *Corresponding author. Tel.: 11-713-798-5630; fax: 11-713-7983475. E-mail address: [email protected] (E. Klann). 0006-8993 / 03 / $ – see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)03364-X
diverse tissues including the colon [2,18], kidney [10] and thyroid [4]. NADPH oxidase proteins also have been localized in PC12 cells [19], rat peripheral neurons [8,20], as well as cerebral cortical neurons [15] (but see Ref. [17]), hippocampal pyramidal neurons [14], and cerebellar Purkinje neurons [14]. These findings are intriguing because superoxide production via NADPH oxidase has been shown to play a role in neurotoxicity in neuronal cultures [15,20], ischemic stroke in the mouse [23], and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease [27]. NADPH oxidase-dependent production of superoxide also has been speculated to be involved in the pathogenesis underlying Alzheimer’s disease [6,11,17]. In addition to neurotoxicity and neurodegenerative diseases, superoxide production via NADPH oxidase may play a role in normal neuronal function. For example, nerve growth factor (NGF)-induced
194
F. Serrano et al. / Brain Research 988 (2003) 193–198
neuronal differentiation has been shown to be dependent on Rac1-regulated production of reactive oxygen species (ROS; [19]). In addition, NADPH oxidase has been postulated to be the source of superoxide that is required for hippocampal long-term potentiation (LTP) and hippocampus-dependent memory [13,22]. In this study we observed widespread distribution of NADPH oxidase proteins in neurons throughout the mouse brain using immunohistochemical techniques. Herein we show examples of this distribution in brain regions impacted by Parkinson’s and Alzheimer’s diseases, consistent with the idea that NADPH oxidase plays a role in the neurodegeneration associated with these diseases. In addition, we show examples of NADPH oxidase staining in the hippocampus, consistent with the possibility that NADPH oxidase plays a role in either LTP and / or hippocampusdependent memory.
mm per section throughout the rostrocaudal extent of the brain using a sliding microtome equipped with a freezing stage (Leica 200). Sections were collected sequentially in five bins of phosphate buffer and stored in cryoprotectant prior to immunohistochemical processing. Cryoprotectant was washed from sections with multiple changes of buffer and the sections were then transferred to the primary antibody diluted in 10 mM phosphate buffer containing 0.3% Triton X-100 and 1% normal donkey serum (1:500 dilution for p22 phox , p40 phox , p47 phox , gp91 phox , and 1:1000 dilution for p67 phox). Following an 48-h incubation at 4 8C, the sections were washed in three changes of 10 mM buffer over a 45-min period prior to and following a 1-h incubation in affinity purified biotinylated donkey
2. Materials and methods
2.1. Animals Six adult male mice (C57BL / 6 X C3H)F1 weighing 24–26 g were used for these studies. They were maintained in constant conditions (12-h light; light on at 07:00 h) and had free access to food and water. Experimental procedures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee.
2.2. Antibodies Rabbit polyclonal antibodies against p22 phox , p40 phox , p47 phox and p67 phox were raised against recombinant proteins made in Escherichia Coli as previously described [1,25]. The antibody against gp91 phox was raised against a C-terminal peptide (last 30 amino acids) coupled to hemocyanine [26]. Previous studies have characterized and established the specificity of these antibodies [3,7,24,26]. To establish the specificity of staining for p40 phox , p47 phox , and p67 phox in the present study, sections from the same brain were simultaneously processed for immunohistochemistry after incubation in primary antibody or preimmune serum. All staining was absent from sections incubated in the pre-immune serum.
2.3. Tissue preparation and processing Each animal was anesthetized deeply by intraperitoneal injection of ketamine (60 mg / kg) and xylazine (7 mg / kg) prior to transcardiac perfusion of physiological saline and paraformaldehyde fixative. After the perfusion, the brain was removed, postfixed for 1 h, and cryoprotected overnight in phosphate-buffered sucrose solution at 4 8C. The brain then was serially sectioned in the coronal plane at 35
Fig. 1. Distribution of neurons immunopositive for NADPH oxidase proteins in the mouse hippocampus. Panels B, D, and F show immunopositive staining of the hippocampus with antibodies for the cytoplasmic NADPH oxidase proteins p40 phox , p47 phox , and p67 phox , respectively. Panels A, C and E show the sections treated with preimmune serum for p40 phox , p47 phox , and p67 phox , respectively. Panels G and H show immunopositive staining for the membrane-associated NADPH oxidase proteins p22 phox and gp91 phox , respectively. Scale bar: 100 mm.
F. Serrano et al. / Brain Research 988 (2003) 193–198
anti-rabbit IgG (Jackson ImmunoResearch Laboratories). The sections then were incubated in ABC complex using reagents from the Vector Elite Kit (Vector Laboratories) for 90 min at room temperature, washed in 10 mM phosphate buffer, and then incubated in 0.1 M Tris–saline buffer containing diaminobenzidine (DAB) for 10 min. The immunoperoxidase reaction product was visualized by adding 35 ml of 30% H 2 O 2 / 100 ml of DAB solution. The H 2 O 2 -catalyzed DAB reaction was monitored visually and terminated by repeated washes in 10 mM phosphate buffer. Sections then were mounted on gelatin coated slides, dried at room temperature, dehydrated in a graded ethanol series, cleared in xylene, and coverslipped with Cytoseal 60.
2.4. Tissue analysis All sections were analyzed with Zeiss Axioplan 2 photomicroscope. Digitized images (Zeiss Axiocam with Zeiss Axiovision 3.0 software) of immunoreactive neurons were acquired and Adobe Photoshop was used to assemble the plates of photomicrographs included in this report. Images presented were taken with either a 2.53 or a 103 objective, with the exception of inserts, which were taken with a 403 objective. The software was used to adjust contrast and brightness of individual photomicrographs in each plate so that they were of uniform density, but the intensity of reaction product was not altered.
195
3. Results
3.1. Specificity of antisera and cellular localization of immunoreactivity Fig. 1A–F demonstrates the specificity of the p40 phox , p47 phox , and p67 phox antibodies using one of the brain regions that stained most heavily for NADPH oxidase, the hippocampus. Staining of these populations of neurons was not observed in tissue processed with preimmune serum for either p40 phox (Fig. 1A), p47 phox (Fig. 1C), or p67 phox (Fig. 1E). The preimmune sera also failed to stain neurons in other portions of the neuraxis. Densely stained populations of neurons were observed in the pyramidal cell layer of areas CA1 and CA3 with antibodies to p40 phox (Fig. 1B), p47 phox (Fig. 1D), and p67 phox (Fig. 1F). Staining also was observed to varying degrees in stratum radiatum (Fig. 1B,D,F). Fig. 2A shows cytoplasmic localization of p47 phox in CA1 pyramidal neurons. The staining pattern in these cells was homogeneous and concentrated in the cell bodies (insert Fig. 2A). Staining also was observed in CA1 pyramidal neurons with antibodies raised against both membrane proteins, p22 phox (Fig. 1G), and gp91 phox (Fig. 1H). Interestingly, more diffuse p22 phox and gp91 phox staining was observed in CA3 pyramidal neurons (Fig. 1G,H). Fig. 2B shows higher magnifications of area CA1 stained with an antibody for one of the membrane proteins, gp91 phox . Punctate staining is present in perikarya of
Fig. 2. Intracellular staining of p47 phox and gp91 phox . Panels A and B show neurons in areas CA1 of the hippocampus stained with antibodies against p47 phox and gp91 phox , respectively. Panels C and D show neurons in the reticular nucleus stained with antibodies against p47 phox and gp91 phox , respectively. Scale bars: 100 mm, insert 50 mm.
196
F. Serrano et al. / Brain Research 988 (2003) 193–198
pyramidal neurons as well as in the proximal portions of their dendritic tree in stratum radiatum (insert Fig. 2B). Intracellular staining of p47 phox and gp91 phox in neurons in the reticular nucleus was similar to the hippocampus, with p47 phox exhibiting homogeneous intracellular staining (Fig. 2C) and gp91 phox exhibiting a punctate staining pattern (Fig. 2D).
3.2. Distribution of immunoreactive neurons in the mouse brain Each of the five NADPH oxidase proteins exhibited an
extensive distribution in all regions of the forebrain, midbrain and hindbrain. Additionally, the distribution of immunopositive neurons identified with each of the antibodies was co-extensive within a region. We chose one cytoplasmic protein, p47 phox and one membrane-associated protein, gp91 phox to exemplify some of the brain areas immunopositive for the NADPH oxidase components. Neurons immunopositive for all five proteins were observed in all regions of the neocortex. Staining for p47 phox in the neocortex (including part of the motor cortex and somatosensory cortex) is shown in Fig. 3A and staining for gp91 phox is shown in Fig. 3B. Staining was
Fig. 3. Illustration of the distribution of cells immunopositive for p47 phox and gp91 phox in selective regions of the mouse brain. The distribution of the cytoplasmic protein, p47 phox is shown in cortex (A), the habenula (Hab) and the paraventricular thalamic nucleus (PVP) of the thalamus (C), the anterior basolateral nucleus (BLA), the posterior basolateral nucleus (BLP), and the posterior basomedial nucleus (BMP) of the amygdala (E), striatum (G). Similar distribution of the membrane-associated protein, gp91 phox is shown in cortex (B), thalamus (D), amygdala (F), and striatum (H). Scale bar: 100 mm.
F. Serrano et al. / Brain Research 988 (2003) 193–198
observed in pyramidal neurons as well as smaller interneurons. The thalamus contained large numbers of immunopositive neurons that were distributed throughout all nuclear groups. Staining for p47 phox in the thalamus is shown in Fig. 3C and staining for gp91 phox is shown in Fig. 3D. All five nuclei of the amygdala (central, lateral, basolateral, basomedial, and medial nuclei) showed a homogeneous distribution of both p47 phox (Fig. 3E) and gp91 phox (Fig. 3F). Fig. 3G,H shows staining in the striatum for p47 phox and gp91 phox , respectively. Medium spiny neurons immunopositive for all five proteins were prevalent throughout the striatum. Often a subset of these cells exhibited higher staining intensity and these neurons were scattered throughout the striatum with no regional concentration. Immunopositive neurons also were prevalent throughout the globus pallidus (data not shown).
197
The expression of NADPH oxidase proteins in neurons suggests the possibility that enzymatic production of superoxide might play a role in normal neuronal function. Consistent with this idea, NGF-induced neuronal differentiation has been shown to be dependent on ROS production via a NADPH oxidase-like protein [19]. Finally, we observed NADPH oxidase in the hippocampus, consistent with the possibility that NADPH oxidase is the source of superoxide that is required for hippocampal long-term potentiation and hippocampus-dependent memory [13,22]. Therefore, the demonstration of the presence of the NADPH oxidase proteins in the normal mouse brain presented in this study could improve our understanding of the role of this enzyme in pathophysiological and physiological conditions.
Acknowledgements 4. Discussion In this manuscript, we have used immunohistochemical methods to examine the distribution of NADPH oxidase proteins in the mouse brain. We observed a wide distribution of NADPH oxidase immunoreactivity throughout the brain with coextensive distributions of immunopositive cells present in all regions of the CNS. The polyclonal antibodies for the cytoplasmic NADPH oxidase proteins, p40 phox , p47 phos and p67 phox , and the membrane-associated NADPH oxidase proteins p22 phox and gp91 phox , selectively identified neurons in all cases. This included immunoreactivity in the hippocampus, neocortex, thalamus, amygdala, reticular nucleus and striatum. In most cases, intracellular staining of the cytoplasmic NADPH oxidase proteins was homogeneous whereas the intracellular staining of the membrane-associated NADPH oxidase proteins was more punctate, consistent with differing subcellular localization of the two groups of proteins. No staining of axons, terminals, or microglia cells was observed. It has been shown that NADPH oxidase proteins are expressed in microglia under disease conditions [6,17]. Thus, it is possible that positive NADPH oxidase immunoreactivity in microglia in the mouse brain would be observed if the resting microglia were stimulated. However, this possibility remains to be determined. Furthermore, whether the NADPH oxidase immunoreactivity we have observed is either brain- and / or neuron-specific or identical to the phagocyte NADPH oxidase remains to be established. NADPH oxidase has been shown to play a role in animal models of disease [23,27]. In addition, NADPH oxidase-dependent production of superoxide also has been speculated to be involved in Alzheimer’s disease [6,11,17]. We observed NADPH oxidase immunoreactivity in the striatum and cortex, consistent with the possibility that NADPH oxidase plays a role in the neurodegeneration observed in Parkinson’s disease and Alzheimer’s disease.
This work was supported NIH grant NS34007 (E.K.).
References [1] A. Abo, A. Boyhan, I. West, A.J. Thrasher, A.W. Segal, Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1, and cytochrome b-245, J. Biol. Chem. 267 (1992) 6767–6770. [2] B. Banfi, A. Maturana, S. Jaconi, S. Arnaudeau, T. Laforge, B. Sinha, E. Ligeti, N. Demaurex, K.-H. Krause, A mammalian H 1 channel generated through alternative splicing of he NADPH oxidase homolog NOH-1, Science 287 (2000) 138–142. [3] G. Batot, C. Martel, N. Capdeville, F. Wientjes, F. Morel, Characterization of neutrophil NADPH oxidase activity reconstituted in a cell-free assay using specific monoclonal antibodies raised against cytochrome b 558 , Eur. J. Biochem. 234 (1995) 208–215. [4] X. De Deken, D. Wang, M.-C. Many, S. Costagliola, F. Libert, G. Vassart, J.E. Dumont, F. Miot, Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family, J. Biol. Chem. 275 (2000) 23227–23233. [5] F.R. DeLeo, M.T. Quinn, Assembly of the phagocyte NADPH oxidase: molecular interaction of oxidase proteins, J. Leukoc. Biol. 60 (1996) 677–691. [6] V. Della Bianca, S. Dusi, E. Bianchini, I. Dal Pra, F. Rossi, b-Amyloid activates the O ?2 2 forming NADPH oxidase in microglia, monocytes, and neutrophils, J. Biol. Chem. 274 (1999) 15493– 15499. [7] S. Dusi, K.A. Nadalini, M. Donini, L. Zentilin, F.B. Wientjis, D. Roos, M. Giacca, F. Rossi, Nicotinamide-adenine dinucleotide phosphate oxidase assembly and activation in EBV-transformed B lymphoblastoid cell lines of normal and chronic granulomatous disease patients, J. Immunol. 161 (1998) 4968–4974. ¨ [8] M. Dvorakova, B. Hohler, E. Richter, J.B. Burritt, W. Kummer, Rat sensory neurons contain cytochrome b 558 large subunit immunoreactivity, NeuroReport 10 (1999) 2615–2617. [9] T. Fukui, B. Lassegue, H. Kai, R.W. Alexander, K.K. Griendling, Cytochrome b-558 alpha-subunit cloning and expression in rat aortic smooth muscle cells, Biochim. Biophys. Acta 1231 (1995) 215–219. [10] M. Geiszt, J.B. Kopp, P. Varnai, T.L. Leto, Identification of Renox, an NAD(P)H oxidase in kidney, Proc. Natl. Acad. Sci. USA 97 (2000) 8010–8014. [11] Y. Hashimoto, T. Niikura, Y. Ito, K. Terashita, I. Nishimoto,
198
[12]
[13]
[14]
[15]
[16] [17]
[18]
[19]
F. Serrano et al. / Brain Research 988 (2003) 193–198 Neurotoxic mechanisms by Alzheimer’s disease-linked N141I mutant presenilin 2, J. Pharmacol. Exp. Ther. 300 (2002) 736–745. S.A. Jones, V.B. O’Donnell, J.D. Wood, J.P. Broughton, E.J. Hughes, O.T. Jones, Expression of phagocytic NADPH oxidase components in human endothelial cells, Am. J. Physiol. 271 (1996) H1626– H1634. L.T. Knapp, E. Klann, The role of reactive oxygen species in hippocampal long-term potentiation: contributory or inhibitory?, J. Neurosci. Res. 70 (2002) 1–7. K. Mizuki, K. Kadomatsu, K. Hata, T. Ito, Q.-W. Fan, Y. Kage, Y. Fukumaki, Y. Sakaki, K. Takeshige, H. Sumimoto, Functional modules and expression of mouse p40 phox and p67 phox , SH3domain-containing proteins involved in the phagocyte NADPH oxidase complex, Eur. J. Biochem. 251 (1998) 573–582. K.-M. Noh, J.-Y. Koh, Induction and activation by zinc of NADPH oxidase in cultured cortical neurons and astrocytes, J. Neurosci. 20 (2000) RC111. A.W. Segal, A. Abo, The biochemical basis of the NADPH oxidase of phagocytes, Trends Biochem. Sci. 18 (1993) 43–47. S. Shimohama, H. Tanino, N. Kawakami, N. Okamura, H. Kodama, T. Yamaguchi, T. Hayakawa, A. Nunomura, S. Chiba, G. Perry, M.A. Smith, S. Fujimoto, Activation of NADPH oxidase in Alzheimer’s disease brains, Biochem. Biophys. Res. Commun. 273 (2000) 5–9. Y.-A. Suh, R.S. Arnold, B. Lassegue, J. Shi, X. Xu, D. Sorescu, A.B. Chung, K.K. Griendling, J.D. Lambeth, Cell transformation by the superoxide-generating Mox1, Nature 401 (1999) 79–82. K. Suzukawa, K. Miura, J. Mitsushita, J. Resau, K. Hirose, R. Crystal, T. Kamata, Nerve growth factor-induced neuronal differentiation requires generation of Rac1-regulated reactive oxygen species, J. Biol. Chem. 275 (2000) 13175–13178.
[20] S.P. Tammariello, M.T. Quinn, S. Estus, NADPH oxidase contributes to oxidative stress and apoptosis in nerve growth factordeprived sympathetic neurons, J. Neurosci. 20 (2000) RC53. [21] V.J. Thannickal, B.L. Fanburg, Activation of an H 2 O 2 -generating NADH oxidase in human lung fibroblasts by transforming growth factor beta 1, J. Biol. Chem. 270 (1995) 30334–30338. [22] E. Thiels, N.N. Urban, G.R. Gonzalez-Burgos, B.I. Kanterewicz, G. Barrionuevo, C.T. Chu, T.D. Oury, E. Klann, Impairment of longterm potentiation and associative memory in mice that overexpress extracellular superoxide dismutase, J. Neurosci. 20 (2000) 7631– 7639. [23] C.E. Walder, S.P. Green, W.C. Darbonne, J. Mathias, J. Rae, M.C. Dinauer, J.T. Curnutte, G.R. Thomas, Ischemic stroke injury is reduced in mice lacking a functional NADPH oxidase, Stroke 28 (1997) 2252–2258. [24] F.B. Wientjes, J.J. Hsuan, N.F. Totti, A.W. Segal, p40 phox , a third component of the activation complex of the NADPH oxidase to contain Src homology 3 domains, Biochem. J. 296 (1993) 557–561. [25] F.B. Wientjes, G. Panayotou, E. Reeves, A.W. Segal, Interactions between cytosolic components of the NADPH oxidase: p40phox interacts with both p67phox and p47phox, Biochem. J. 317 (1996) 919–924. [26] F.B. Wientjes, A.W. Segal, J.H. Hartwig, Immunoelectron microscopy shows a clustered distribution of NADPH oxidase components in the human neutrophil plasma membrane, J. Leukoc. Biol. 61 (1997) 303–312. [27] D.C. Wu, V. Jackson-Lewis, M. Vila, K. Tieu, P. Teismann, C. Vadseth, D.K. Choi, H. Ischiropoulos, S. Przedborski, Blockade of microglial activation is neuroprotective in the 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine mouse model of Parkinson disease, J. Neurosci. 22 (2002) 1763–1771.
Short communication
NADPH oxidase immunoreactivity in the mouse brain Faridis Serrano a , Nutan S. Kolluri c , Frans B. Wientjes e , J. Patrick Card c , Eric Klann a,b,c,d , * a
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA b Division of Neuroscience, Baylor College of Medicine, Houston, TX, USA c Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA, USA d Center for the Neural Basis of Cognition, University of Pittsburgh, Pittsburgh, PA, USA e Department of Medicine, University College London, London, UK Accepted 21 July 2003
Abstract Superoxide production via NADPH oxidase has been shown to play a role in neurotoxicity, ischemic stroke, and possibly Parkinson’s and Alzheimer’s diseases. In addition, NADPH oxidase-dependent production of superoxide may be necessary for normal brain functions, including neuronal differentiation and neuronal plasticity. To improve our understanding of NADPH oxidase in the brain, we studied the localization of the various protein components of NADPH oxidase in the central nervous system of the adult mouse using immunohistochemistry. We detected staining for the cytoplasmic NADPH proteins, p40 phox , p47 phox , and p67 phox , as well as the membrane-associated NADPH oxidase proteins, p22 phox and gp91 phox in neurons throughout the mouse brain. Staining of each of the NADPH oxidase proteins was observed in neurons in all regions of the neuraxis, with particularly prominent localizations in the hippocampus, cortex, amygdala, striatum, and thalamus. The expression of NADPH oxidase proteins in neurons suggests the possibility that enzymatic production of superoxide by a NADPH oxidase may play a role in both normal neuronal function as well as neurodegeneration in the brain. 2003 Elsevier B.V. All rights reserved. Theme: Other systems of the CNS Topic: Comparative neuroanatomy Keywords: Superoxide; Hippocampus; Rac; Neurotoxicity; Neurodegeneration; Long-term potentiation
1. Introduction NADPH oxidase is a multicomponent enzyme complex that consists of the membrane-bound cytochrome b 558 phox phox phox (p22 and gp91 ) and cytoplasmic proteins (p40 , phox phox p47 , p67 ) that translocate, along with the small G-protein Rac, to the membrane during cellular stimulation to produce superoxide [5,16]. NADPH oxidase has been best characterized in phagocytic cells. However, a number of studies suggest that various NADPH oxidase proteins are expressed in non-phagocytic cells, including vascular smooth muscle cells [9], fibroblasts [21], and endothelial cells [12]. In addition, recent studies have described the isolation of NADPH oxidase homologs that are present in *Corresponding author. Tel.: 11-713-798-5630; fax: 11-713-7983475. E-mail address: [email protected] (E. Klann). 0006-8993 / 03 / $ – see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)03364-X
diverse tissues including the colon [2,18], kidney [10] and thyroid [4]. NADPH oxidase proteins also have been localized in PC12 cells [19], rat peripheral neurons [8,20], as well as cerebral cortical neurons [15] (but see Ref. [17]), hippocampal pyramidal neurons [14], and cerebellar Purkinje neurons [14]. These findings are intriguing because superoxide production via NADPH oxidase has been shown to play a role in neurotoxicity in neuronal cultures [15,20], ischemic stroke in the mouse [23], and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease [27]. NADPH oxidase-dependent production of superoxide also has been speculated to be involved in the pathogenesis underlying Alzheimer’s disease [6,11,17]. In addition to neurotoxicity and neurodegenerative diseases, superoxide production via NADPH oxidase may play a role in normal neuronal function. For example, nerve growth factor (NGF)-induced
194
F. Serrano et al. / Brain Research 988 (2003) 193–198
neuronal differentiation has been shown to be dependent on Rac1-regulated production of reactive oxygen species (ROS; [19]). In addition, NADPH oxidase has been postulated to be the source of superoxide that is required for hippocampal long-term potentiation (LTP) and hippocampus-dependent memory [13,22]. In this study we observed widespread distribution of NADPH oxidase proteins in neurons throughout the mouse brain using immunohistochemical techniques. Herein we show examples of this distribution in brain regions impacted by Parkinson’s and Alzheimer’s diseases, consistent with the idea that NADPH oxidase plays a role in the neurodegeneration associated with these diseases. In addition, we show examples of NADPH oxidase staining in the hippocampus, consistent with the possibility that NADPH oxidase plays a role in either LTP and / or hippocampusdependent memory.
mm per section throughout the rostrocaudal extent of the brain using a sliding microtome equipped with a freezing stage (Leica 200). Sections were collected sequentially in five bins of phosphate buffer and stored in cryoprotectant prior to immunohistochemical processing. Cryoprotectant was washed from sections with multiple changes of buffer and the sections were then transferred to the primary antibody diluted in 10 mM phosphate buffer containing 0.3% Triton X-100 and 1% normal donkey serum (1:500 dilution for p22 phox , p40 phox , p47 phox , gp91 phox , and 1:1000 dilution for p67 phox). Following an 48-h incubation at 4 8C, the sections were washed in three changes of 10 mM buffer over a 45-min period prior to and following a 1-h incubation in affinity purified biotinylated donkey
2. Materials and methods
2.1. Animals Six adult male mice (C57BL / 6 X C3H)F1 weighing 24–26 g were used for these studies. They were maintained in constant conditions (12-h light; light on at 07:00 h) and had free access to food and water. Experimental procedures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee.
2.2. Antibodies Rabbit polyclonal antibodies against p22 phox , p40 phox , p47 phox and p67 phox were raised against recombinant proteins made in Escherichia Coli as previously described [1,25]. The antibody against gp91 phox was raised against a C-terminal peptide (last 30 amino acids) coupled to hemocyanine [26]. Previous studies have characterized and established the specificity of these antibodies [3,7,24,26]. To establish the specificity of staining for p40 phox , p47 phox , and p67 phox in the present study, sections from the same brain were simultaneously processed for immunohistochemistry after incubation in primary antibody or preimmune serum. All staining was absent from sections incubated in the pre-immune serum.
2.3. Tissue preparation and processing Each animal was anesthetized deeply by intraperitoneal injection of ketamine (60 mg / kg) and xylazine (7 mg / kg) prior to transcardiac perfusion of physiological saline and paraformaldehyde fixative. After the perfusion, the brain was removed, postfixed for 1 h, and cryoprotected overnight in phosphate-buffered sucrose solution at 4 8C. The brain then was serially sectioned in the coronal plane at 35
Fig. 1. Distribution of neurons immunopositive for NADPH oxidase proteins in the mouse hippocampus. Panels B, D, and F show immunopositive staining of the hippocampus with antibodies for the cytoplasmic NADPH oxidase proteins p40 phox , p47 phox , and p67 phox , respectively. Panels A, C and E show the sections treated with preimmune serum for p40 phox , p47 phox , and p67 phox , respectively. Panels G and H show immunopositive staining for the membrane-associated NADPH oxidase proteins p22 phox and gp91 phox , respectively. Scale bar: 100 mm.
F. Serrano et al. / Brain Research 988 (2003) 193–198
anti-rabbit IgG (Jackson ImmunoResearch Laboratories). The sections then were incubated in ABC complex using reagents from the Vector Elite Kit (Vector Laboratories) for 90 min at room temperature, washed in 10 mM phosphate buffer, and then incubated in 0.1 M Tris–saline buffer containing diaminobenzidine (DAB) for 10 min. The immunoperoxidase reaction product was visualized by adding 35 ml of 30% H 2 O 2 / 100 ml of DAB solution. The H 2 O 2 -catalyzed DAB reaction was monitored visually and terminated by repeated washes in 10 mM phosphate buffer. Sections then were mounted on gelatin coated slides, dried at room temperature, dehydrated in a graded ethanol series, cleared in xylene, and coverslipped with Cytoseal 60.
2.4. Tissue analysis All sections were analyzed with Zeiss Axioplan 2 photomicroscope. Digitized images (Zeiss Axiocam with Zeiss Axiovision 3.0 software) of immunoreactive neurons were acquired and Adobe Photoshop was used to assemble the plates of photomicrographs included in this report. Images presented were taken with either a 2.53 or a 103 objective, with the exception of inserts, which were taken with a 403 objective. The software was used to adjust contrast and brightness of individual photomicrographs in each plate so that they were of uniform density, but the intensity of reaction product was not altered.
195
3. Results
3.1. Specificity of antisera and cellular localization of immunoreactivity Fig. 1A–F demonstrates the specificity of the p40 phox , p47 phox , and p67 phox antibodies using one of the brain regions that stained most heavily for NADPH oxidase, the hippocampus. Staining of these populations of neurons was not observed in tissue processed with preimmune serum for either p40 phox (Fig. 1A), p47 phox (Fig. 1C), or p67 phox (Fig. 1E). The preimmune sera also failed to stain neurons in other portions of the neuraxis. Densely stained populations of neurons were observed in the pyramidal cell layer of areas CA1 and CA3 with antibodies to p40 phox (Fig. 1B), p47 phox (Fig. 1D), and p67 phox (Fig. 1F). Staining also was observed to varying degrees in stratum radiatum (Fig. 1B,D,F). Fig. 2A shows cytoplasmic localization of p47 phox in CA1 pyramidal neurons. The staining pattern in these cells was homogeneous and concentrated in the cell bodies (insert Fig. 2A). Staining also was observed in CA1 pyramidal neurons with antibodies raised against both membrane proteins, p22 phox (Fig. 1G), and gp91 phox (Fig. 1H). Interestingly, more diffuse p22 phox and gp91 phox staining was observed in CA3 pyramidal neurons (Fig. 1G,H). Fig. 2B shows higher magnifications of area CA1 stained with an antibody for one of the membrane proteins, gp91 phox . Punctate staining is present in perikarya of
Fig. 2. Intracellular staining of p47 phox and gp91 phox . Panels A and B show neurons in areas CA1 of the hippocampus stained with antibodies against p47 phox and gp91 phox , respectively. Panels C and D show neurons in the reticular nucleus stained with antibodies against p47 phox and gp91 phox , respectively. Scale bars: 100 mm, insert 50 mm.
196
F. Serrano et al. / Brain Research 988 (2003) 193–198
pyramidal neurons as well as in the proximal portions of their dendritic tree in stratum radiatum (insert Fig. 2B). Intracellular staining of p47 phox and gp91 phox in neurons in the reticular nucleus was similar to the hippocampus, with p47 phox exhibiting homogeneous intracellular staining (Fig. 2C) and gp91 phox exhibiting a punctate staining pattern (Fig. 2D).
3.2. Distribution of immunoreactive neurons in the mouse brain Each of the five NADPH oxidase proteins exhibited an
extensive distribution in all regions of the forebrain, midbrain and hindbrain. Additionally, the distribution of immunopositive neurons identified with each of the antibodies was co-extensive within a region. We chose one cytoplasmic protein, p47 phox and one membrane-associated protein, gp91 phox to exemplify some of the brain areas immunopositive for the NADPH oxidase components. Neurons immunopositive for all five proteins were observed in all regions of the neocortex. Staining for p47 phox in the neocortex (including part of the motor cortex and somatosensory cortex) is shown in Fig. 3A and staining for gp91 phox is shown in Fig. 3B. Staining was
Fig. 3. Illustration of the distribution of cells immunopositive for p47 phox and gp91 phox in selective regions of the mouse brain. The distribution of the cytoplasmic protein, p47 phox is shown in cortex (A), the habenula (Hab) and the paraventricular thalamic nucleus (PVP) of the thalamus (C), the anterior basolateral nucleus (BLA), the posterior basolateral nucleus (BLP), and the posterior basomedial nucleus (BMP) of the amygdala (E), striatum (G). Similar distribution of the membrane-associated protein, gp91 phox is shown in cortex (B), thalamus (D), amygdala (F), and striatum (H). Scale bar: 100 mm.
F. Serrano et al. / Brain Research 988 (2003) 193–198
observed in pyramidal neurons as well as smaller interneurons. The thalamus contained large numbers of immunopositive neurons that were distributed throughout all nuclear groups. Staining for p47 phox in the thalamus is shown in Fig. 3C and staining for gp91 phox is shown in Fig. 3D. All five nuclei of the amygdala (central, lateral, basolateral, basomedial, and medial nuclei) showed a homogeneous distribution of both p47 phox (Fig. 3E) and gp91 phox (Fig. 3F). Fig. 3G,H shows staining in the striatum for p47 phox and gp91 phox , respectively. Medium spiny neurons immunopositive for all five proteins were prevalent throughout the striatum. Often a subset of these cells exhibited higher staining intensity and these neurons were scattered throughout the striatum with no regional concentration. Immunopositive neurons also were prevalent throughout the globus pallidus (data not shown).
197
The expression of NADPH oxidase proteins in neurons suggests the possibility that enzymatic production of superoxide might play a role in normal neuronal function. Consistent with this idea, NGF-induced neuronal differentiation has been shown to be dependent on ROS production via a NADPH oxidase-like protein [19]. Finally, we observed NADPH oxidase in the hippocampus, consistent with the possibility that NADPH oxidase is the source of superoxide that is required for hippocampal long-term potentiation and hippocampus-dependent memory [13,22]. Therefore, the demonstration of the presence of the NADPH oxidase proteins in the normal mouse brain presented in this study could improve our understanding of the role of this enzyme in pathophysiological and physiological conditions.
Acknowledgements 4. Discussion In this manuscript, we have used immunohistochemical methods to examine the distribution of NADPH oxidase proteins in the mouse brain. We observed a wide distribution of NADPH oxidase immunoreactivity throughout the brain with coextensive distributions of immunopositive cells present in all regions of the CNS. The polyclonal antibodies for the cytoplasmic NADPH oxidase proteins, p40 phox , p47 phos and p67 phox , and the membrane-associated NADPH oxidase proteins p22 phox and gp91 phox , selectively identified neurons in all cases. This included immunoreactivity in the hippocampus, neocortex, thalamus, amygdala, reticular nucleus and striatum. In most cases, intracellular staining of the cytoplasmic NADPH oxidase proteins was homogeneous whereas the intracellular staining of the membrane-associated NADPH oxidase proteins was more punctate, consistent with differing subcellular localization of the two groups of proteins. No staining of axons, terminals, or microglia cells was observed. It has been shown that NADPH oxidase proteins are expressed in microglia under disease conditions [6,17]. Thus, it is possible that positive NADPH oxidase immunoreactivity in microglia in the mouse brain would be observed if the resting microglia were stimulated. However, this possibility remains to be determined. Furthermore, whether the NADPH oxidase immunoreactivity we have observed is either brain- and / or neuron-specific or identical to the phagocyte NADPH oxidase remains to be established. NADPH oxidase has been shown to play a role in animal models of disease [23,27]. In addition, NADPH oxidase-dependent production of superoxide also has been speculated to be involved in Alzheimer’s disease [6,11,17]. We observed NADPH oxidase immunoreactivity in the striatum and cortex, consistent with the possibility that NADPH oxidase plays a role in the neurodegeneration observed in Parkinson’s disease and Alzheimer’s disease.
This work was supported NIH grant NS34007 (E.K.).
References [1] A. Abo, A. Boyhan, I. West, A.J. Thrasher, A.W. Segal, Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1, and cytochrome b-245, J. Biol. Chem. 267 (1992) 6767–6770. [2] B. Banfi, A. Maturana, S. Jaconi, S. Arnaudeau, T. Laforge, B. Sinha, E. Ligeti, N. Demaurex, K.-H. Krause, A mammalian H 1 channel generated through alternative splicing of he NADPH oxidase homolog NOH-1, Science 287 (2000) 138–142. [3] G. Batot, C. Martel, N. Capdeville, F. Wientjes, F. Morel, Characterization of neutrophil NADPH oxidase activity reconstituted in a cell-free assay using specific monoclonal antibodies raised against cytochrome b 558 , Eur. J. Biochem. 234 (1995) 208–215. [4] X. De Deken, D. Wang, M.-C. Many, S. Costagliola, F. Libert, G. Vassart, J.E. Dumont, F. Miot, Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family, J. Biol. Chem. 275 (2000) 23227–23233. [5] F.R. DeLeo, M.T. Quinn, Assembly of the phagocyte NADPH oxidase: molecular interaction of oxidase proteins, J. Leukoc. Biol. 60 (1996) 677–691. [6] V. Della Bianca, S. Dusi, E. Bianchini, I. Dal Pra, F. Rossi, b-Amyloid activates the O ?2 2 forming NADPH oxidase in microglia, monocytes, and neutrophils, J. Biol. Chem. 274 (1999) 15493– 15499. [7] S. Dusi, K.A. Nadalini, M. Donini, L. Zentilin, F.B. Wientjis, D. Roos, M. Giacca, F. Rossi, Nicotinamide-adenine dinucleotide phosphate oxidase assembly and activation in EBV-transformed B lymphoblastoid cell lines of normal and chronic granulomatous disease patients, J. Immunol. 161 (1998) 4968–4974. ¨ [8] M. Dvorakova, B. Hohler, E. Richter, J.B. Burritt, W. Kummer, Rat sensory neurons contain cytochrome b 558 large subunit immunoreactivity, NeuroReport 10 (1999) 2615–2617. [9] T. Fukui, B. Lassegue, H. Kai, R.W. Alexander, K.K. Griendling, Cytochrome b-558 alpha-subunit cloning and expression in rat aortic smooth muscle cells, Biochim. Biophys. Acta 1231 (1995) 215–219. [10] M. Geiszt, J.B. Kopp, P. Varnai, T.L. Leto, Identification of Renox, an NAD(P)H oxidase in kidney, Proc. Natl. Acad. Sci. USA 97 (2000) 8010–8014. [11] Y. Hashimoto, T. Niikura, Y. Ito, K. Terashita, I. Nishimoto,
198
[12]
[13]
[14]
[15]
[16] [17]
[18]
[19]
F. Serrano et al. / Brain Research 988 (2003) 193–198 Neurotoxic mechanisms by Alzheimer’s disease-linked N141I mutant presenilin 2, J. Pharmacol. Exp. Ther. 300 (2002) 736–745. S.A. Jones, V.B. O’Donnell, J.D. Wood, J.P. Broughton, E.J. Hughes, O.T. Jones, Expression of phagocytic NADPH oxidase components in human endothelial cells, Am. J. Physiol. 271 (1996) H1626– H1634. L.T. Knapp, E. Klann, The role of reactive oxygen species in hippocampal long-term potentiation: contributory or inhibitory?, J. Neurosci. Res. 70 (2002) 1–7. K. Mizuki, K. Kadomatsu, K. Hata, T. Ito, Q.-W. Fan, Y. Kage, Y. Fukumaki, Y. Sakaki, K. Takeshige, H. Sumimoto, Functional modules and expression of mouse p40 phox and p67 phox , SH3domain-containing proteins involved in the phagocyte NADPH oxidase complex, Eur. J. Biochem. 251 (1998) 573–582. K.-M. Noh, J.-Y. Koh, Induction and activation by zinc of NADPH oxidase in cultured cortical neurons and astrocytes, J. Neurosci. 20 (2000) RC111. A.W. Segal, A. Abo, The biochemical basis of the NADPH oxidase of phagocytes, Trends Biochem. Sci. 18 (1993) 43–47. S. Shimohama, H. Tanino, N. Kawakami, N. Okamura, H. Kodama, T. Yamaguchi, T. Hayakawa, A. Nunomura, S. Chiba, G. Perry, M.A. Smith, S. Fujimoto, Activation of NADPH oxidase in Alzheimer’s disease brains, Biochem. Biophys. Res. Commun. 273 (2000) 5–9. Y.-A. Suh, R.S. Arnold, B. Lassegue, J. Shi, X. Xu, D. Sorescu, A.B. Chung, K.K. Griendling, J.D. Lambeth, Cell transformation by the superoxide-generating Mox1, Nature 401 (1999) 79–82. K. Suzukawa, K. Miura, J. Mitsushita, J. Resau, K. Hirose, R. Crystal, T. Kamata, Nerve growth factor-induced neuronal differentiation requires generation of Rac1-regulated reactive oxygen species, J. Biol. Chem. 275 (2000) 13175–13178.
[20] S.P. Tammariello, M.T. Quinn, S. Estus, NADPH oxidase contributes to oxidative stress and apoptosis in nerve growth factordeprived sympathetic neurons, J. Neurosci. 20 (2000) RC53. [21] V.J. Thannickal, B.L. Fanburg, Activation of an H 2 O 2 -generating NADH oxidase in human lung fibroblasts by transforming growth factor beta 1, J. Biol. Chem. 270 (1995) 30334–30338. [22] E. Thiels, N.N. Urban, G.R. Gonzalez-Burgos, B.I. Kanterewicz, G. Barrionuevo, C.T. Chu, T.D. Oury, E. Klann, Impairment of longterm potentiation and associative memory in mice that overexpress extracellular superoxide dismutase, J. Neurosci. 20 (2000) 7631– 7639. [23] C.E. Walder, S.P. Green, W.C. Darbonne, J. Mathias, J. Rae, M.C. Dinauer, J.T. Curnutte, G.R. Thomas, Ischemic stroke injury is reduced in mice lacking a functional NADPH oxidase, Stroke 28 (1997) 2252–2258. [24] F.B. Wientjes, J.J. Hsuan, N.F. Totti, A.W. Segal, p40 phox , a third component of the activation complex of the NADPH oxidase to contain Src homology 3 domains, Biochem. J. 296 (1993) 557–561. [25] F.B. Wientjes, G. Panayotou, E. Reeves, A.W. Segal, Interactions between cytosolic components of the NADPH oxidase: p40phox interacts with both p67phox and p47phox, Biochem. J. 317 (1996) 919–924. [26] F.B. Wientjes, A.W. Segal, J.H. Hartwig, Immunoelectron microscopy shows a clustered distribution of NADPH oxidase components in the human neutrophil plasma membrane, J. Leukoc. Biol. 61 (1997) 303–312. [27] D.C. Wu, V. Jackson-Lewis, M. Vila, K. Tieu, P. Teismann, C. Vadseth, D.K. Choi, H. Ischiropoulos, S. Przedborski, Blockade of microglial activation is neuroprotective in the 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine mouse model of Parkinson disease, J. Neurosci. 22 (2002) 1763–1771.