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CD36 expression in the brains of SAMP8
Archives of Gerontology and Geriatrics 56 (2013) 75–79
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CD36 expression in the brains of SAMP8 Bin Wu a,b, Masaki Ueno a,*, Takashi Kusaka c, Takanori Miki d, Yukiko Nagai e, Toshitaka Nakagawa e, Kenji Kanenishi f, Naohisa Hosomi g, Haruhiko Sakamoto a a
Department of Pathology and Host Defense, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan Department of General Practice, First Affiliated Hospital of China Medical University, San Hao Road 36, Shenyang 110003, China c Department of Maternal Perinatal Center, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan d Department of Anatomy and Neurobiology, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan e Department of Life Sciences Research Center, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan f Department of Perinatology and Gynecology, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan g Department of Clinical Neuroscience and Therapeutics, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 June 2012 Received in revised form 14 July 2012 Accepted 21 July 2012 Available online 9 August 2012
SAMP8, senescence accelerated mice with age-related deficits in memory and learning, are known to show age-related increases of amyloid precursor protein (APP) and immunopositivity for amyloid-b (Ab) proteins, and moreover to be under elevated oxidative stress. The elevated expression of class B scavenger receptor CD36, which is the receptor of oxidized LDL and also one of efflux transporters of Ab proteins in the cerebral vessels, is thought to mediate free radical production in cerebral ischemia and induce oxidative stress. Accordingly, by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical techniques, we examined whether the expression of CD36 was increased in the brains of 10–12-week-old SAMP8 with elevated oxidative stress. Ten to 12-week-old SAMR1 mice were used as controls without the features. The gene and protein expression of CD36 was significantly higher in the brains of SAMP8 than those of SAMR1. Confocal microscopic examination revealed that the CD36 immunoreactivity was seen in the cytoplasm of endothelial cells and F4/80-positive perivascular cells of the brains. These findings indicate that the expression of CD36 in the brains of SAMP8 is increased compared with that of SAMR1. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: CD36 SAMP8 Oxidative stress
1. Introduction The senescence-accelerated mouse (SAM) is a general term for ‘‘accelerated senescence prone’’ (P-series; SAMP) and ‘‘accelerated senescence resistant’’ (R-series; SAMR) mice (Takeda et al., 1981). Among SAMP, SAMP8 mice have a short life span like the other SAMP mice, reveal some senescence-accelerated symptoms, and show a remarkable age-related deterioration in the ability of memory and learning in passive and active avoidance responses (Miyamoto et al., 1986). Previous papers showed that the expression of amyloid precursor protein (APP) and its mRNA was increased with aging in the brains of SAMP8 (Morley et al., 2000; Takemura et al., 1993). Recently, some papers showed that amyloid-b (Ab) granules accumulated in the hippocampus of SAMP8 and that the number of the granules was increased with aging (del Valle et al., 2010, 2011; Manich et al., 2011).
* Corresponding author at: Department of Pathology and Host Defense, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan. Tel.: +81 87 891 2115; fax: +81 87 891 2116. E-mail address: [email protected] (M. Ueno). 0167-4943/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archger.2012.07.007
It has been suggested that vascular Ab receptors in endothelial cells transfer perivascular Ab proteins into circulation and thus contribute to the clearance of Ab from the brain (Zlokovic, 2004). Both the low-density lipoprotein receptor (LDLR) and the LDLRrelated protein 1 (LRP1) may act as Ab receptors (Abdulkarim & Hameed, 2006; Fryer et al., 2005; Sagare et al., 2007). LRP1 is a member of the LDLR family and functions both as a multifunctional scavenger and signaling receptor and as a transporter and metabolizer of cholesterol and apolipoprotein E (ApoE)containing lipoproteins (Herz & Marschang, 2003). LRP1 binds both ApoE/Ab complexes and Ab and regulates their clearance from brain to blood (Donahue et al., 2006; Shibata et al., 2000; Zlokovic, 2004). Besides the LDLR family, some other potential Ab-binding receptors have been identified. P-glycoprotein (Lam et al., 2001), scavenger receptor CD36 (Coraci et al., 2002), the formylpeptide receptor-like-1 (FPRL1) (Le et al., 2001), and the transmembrane APP itself (Lorenzo et al., 2000) also function as Ab receptors. We previously investigated the expression of P-glycoprotein, LDL receptor, and LRP1, all of which were efflux transporters of Ab proteins, and reported that the expression of P-glycoprotein and LDL receptor but not LRP1 was increased in the brains of SAMP8 compared with that of SAMR1 (Wu, Ueno, Kusaka, et al., 2009; Wu,
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Ueno, Onodera, et al., 2009). In addition, we reported that the expression of RAGE, an influx transporter of Ab proteins, was decreased in the brains of SAMP8 compared with that of SAMR1 brains (Wu, Ueno, Onodera, et al., 2009). CD36 belongs to the class B scavenger receptor family, which includes the receptor for selective cholesteryl ester uptake, scavenger receptor class B type I (SR-BI), and lysosomal integral membrane protein II (Febbraio, Hajjar, & Silverstein, 2001). CD36 is a surface glycoprotein and is localized in lipid rafts of plasma membrane and in mitochondria (Bonen et al., 2004; Campbell et al., 2004; Roepstorff, WulffHelge, Vistisen, & Kiens, 2004). In addition, CD 36 colocalizes with caveolin-1 in specialized plasma membrane microdomains known as caveolae (Lisanti et al., 1994). These cholesterol- and sphingolipid-enriched structures may serve to concentrate signaling molecules and facilitate the integration of signaling cascade. Multiple lines of evidence indicate that caveolae serve an integral role in the trafficking of cholesterol in cells. Oxidized LDL (oxLDL) depletes the endothelial cell caveolae of cholesterol, resulting in displacement of endothelial nitric oxide synthase and an altered response to acetylcholine (Uittenbogaard, Shaul, Yuhanna, Blair, & Smart, 2000). These disruptive effects of oxLDL are mediated by CD36 and can be blocked by interaction of SR-BI with HDL, which prevents the cholesterol depletion of caveolae. Thus, CD 36 has a role in the regulation of caveolar function. It is known that CD36 expression is broad and seen in microglia, macrophages, microvascular endothelium, adipocytes, dendritic cells, platelets, and cardiac, skeletal and smooth muscle cells (Febbraio, Guy, & Silverstein, 2004). CD36 recognizes a multitude of ligands, including oxLDL, long-chain fatty acids, thrombospondin-1, fibrillar Ab, and the membrane of cells undergoing apoptosis (Febbraio et al., 2004; Hirano et al., 2003; Medeiros et al., 2004). In addition, some findings suggest that the expression of CD36 is involved in the pathogenesis of cerebral ischemia with reactive oxygen species (ROS) production (Cho et al., 2005). The increased ROS production is suggested to contribute to the pathogenesis of the SAMP8 brain (Alvarez-Garcia et al., 2006; Sato et al., 1996; Yasui et al., 2003). Accordingly, we examined whether the expression of CD36 is increased in the brains of SAMP8 with elevated oxidative stress, and whether CD36 could be a therapeutic target to brain injury with the elevated oxidative stress.
control samples using glyceraldehydes 3-phosphate dehydrogenase (GAPDH) as a control for normalization among samples. For Western blot analysis, brain tissues were homogenized in lysis buffer containing 10 mmol/L Tris buffer containing 2 mmol/L phenylmethylsulfonyl fluoride, and 10 mmol/L N-ethylmaleimide with a protease inhibitor. The extracts were solubilized in a Tris/ glycine/sodium dodecyl sulfate (SDS) sample buffer in the presence of 5% 2-mercaptoethanol (Bio-Rad) and heated at 95 8C for 5 min. Protein extracts (40 mg) were then separated by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred from gel to nitrocellulose membranes and blocked in a blocking solution (5% dry milk in PBS) for 1 h. The membrane was incubated with a primary rabbit antibody for CD36 (1:500, ProteinTech Group, Inc., Chicago, IL, USA) at 4 8C overnight. The membranes were then incubated with HRP-labeled anti-rabbit IgG antibody (1:5000, Amersham Biosciences, Buckinghamshire, UK) for 1 h at room temperature (RT). The proteins were visualized on enhanced chemiluminescence film (Hyperfilm; Amersham Biosciences). Finally, the blots were reprobed using an antibody against actin (1:2000, Epitmics, Inc., Burlingame, CA, USA). Data are expressed as the relative differences among samples after normalization to actin expression. The immunoreactive bands were quantified by densitometric analysis (NIH image software).
2. Materials and methods The experimental protocols for animal care were in compliance with institutional guidelines of Kagawa University. Ten to 12-week-old male SAMP8 (n = 10) and SAMR1 (n = 10) mice (purchased from Japan SLC, Inc., Japan) weighing 25–35 g were used. All efforts were made to minimize the number of animals used and their suffering. The animals were anesthetized with diethyl ether and then used for several experiments. For real-time RT-PCR and Western blot analyses, mice were perfused transcardially with phosphate-buffered saline (PBS). Their brains were removed and stored at 80 8C. Extraction of total RNA and reverse transcription of the total RNA were performed as reported previously (Huang et al., 2004). To quantify gene expression, TaqMan real-time quantitative PCR was performed with the ABI PRISM 7700 Sequence Detection System using Assays on-Demand Gene Expression probes of CD36 (Applied Biosystems, Foster, CA, USA). The PCR cycling conditions for all samples were as follows: 50 8C, 2 min for AmpErase UNG activation; 95 8C, 10 min for AmpliTaq Gold activation; and 50 cycles for melting (95 8C, 15 s) and annealing/extension (60 8C, 1 min) steps. Each sample was run in duplicate. The comparative threshold cycle method (Applied Biosystems) was used to calculate gene expression in each sample relative to the value observed in
Fig. 1. (a) mRNA expressions of CD36 after normalization to GAPDH expression in the brains of SAMP8 (bar indicated by oblique line) (n = 6) and SAMR1 (bar indicated by horizontal line) (n = 6) mice are shown. The gene expression of CD36 is significantly higher in the SAMP8 brains than that of SAMP8 (*, p < 0.05). (b) Western blotting analyses shows representative bands of CD36 and actin of SAMP8 and SAMR1 brains. The protein expression of CD36 is significantly higher in the SAMP8 brains than that of SAMR1 (*, p < 0.05).
B. Wu et al. / Archives of Gerontology and Geriatrics 56 (2013) 75–79
For double immunofluorescence staining, mice were perfused transcardially with PBS and then with a fixative containing 10% formalin. The brains were removed and immersed in the same fixative. The brains were immersed in 20% sucrose buffer solution for 2 days, embedded in optimal cutting temperature compound, and cut on a cryostat into sections 16 mm thick. The frozen sections were incubated with the rabbit antibody for CD36 (1:100, ProteinTech Group, Inc.) at 4 8C overnight, followed by incubation in Alexa Fluor 594-conjugated anti-rabbit IgG antibody (1:200, Molecular Probes) at RT for 1 h. After washing in PBS, the sections were incubated with a primary goat antibody for PECAM-1 (1:100, Santa Cruz Biotech, Inc., CA, USA) at 4 8C overnight, followed by incubation in Alexa Fluor 644-conjugated anti-goat IgG antibody (1:200, Molecular Probes) at RT for 1 h. In addition, the frozen sections were incubated with a mouse antibody for CD36 (1:100, abcam) at 4 8C overnight, followed by incubation in Alexa Fluor 488-conjugated anti-mouse IgG antibody (1:200, Molecular Probes) at RT for 1 h. After washing in PBS, the sections were incubated with a primary rabbit antibody for F4/80 (1:100, Santa Cruz Biotech, Inc., CA, USA) at 4 8C overnight, followed by incubation in Alexa Fluor 594-conjugated anti-rabbit IgG antibody (1:200, Molecular Probes) at RT for 1 h. Cross reactions were not detected after double staining. For nuclear staining, the sections were incubated for 15 min at RT in 40 -6-diamidino-2-phenylindole
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(DAPI, Molecular Probes, Eugene, OR, USA) or in TO-PRO-3 iodide (Molecular Probes) solution. The fluorescent signals were viewed under a confocal microscope (Bio-Rad Radiance 2100 or Carl Zeiss LSM700). As a control experiment, we performed an identical immunohistochemical procedure, but with omission of the primary antibody or using a normal serum instead of the primary antibody. Values are shown as means SD. Statistical analysis was performed by using Mann–Whitney. Differences were considered to be statistically significant when p < 0.05. 3. Results Real-time quantitative RT-PCR analysis revealed that the gene expression of CD36 was significantly higher in the brain samples of SAMP8 than those of SAMR1 (Fig. 1a). Western blot analysis using the primary antibodies for CD36 and actin revealed that relative protein expression of CD36 to that of actin was significantly higher in the brain samples of SAMP8 than those of SAMR1 (Fig. 1b). Double immunohistochemical analyses showed that the immunosignals of CD36 in the vessel wall were colocalized with those of PECAM-1, indicating that parts of the immunosignals of CD36 were present in PECAM-1-positive endothelial cells of cerebral vessels in SAMP8 (Fig. 2a) and also in SAMR1 mice
Fig. 2. Confocal microscopic images of double immunostaining using antibodies for PECAM-1 (a-1, b-1) and CD36 (a-2, b-2) and nuclear staining by DAPI (a-3, b-3) and the merged images (a-4, b-4) in the SAMP8 (a) and SAMR1 (b) brains are shown. The immunosignals for CD36 are partially colocalized with those of PECAM-1 in the brains of both strains. Confocal microscopic images of double immunostaining using antibodies for CD36 (c-1, d-1) and F4/80 (c-2, d-2) and nuclear staining by TO-PRO-3 (c-3, d-3) and the merged images (c-4, d-4) in the SAMP8 (c) and SAMR1 (d) brains are shown. The immunosignals for CD36 are partially colocalized with those of F4/80 in the brains of both strains. The coincidence of two antibodies is indicated by the conversion of green (a–d-1) and red (a–d-2) to yellow (a–d-4). Scale bars indicate 20 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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(Fig. 2b). In addition, the immunosignals of CD36 were partially colocalized with those of F4/80 in vessels larger than capillaries in SAMP8 (Fig. 2c) and also in SAMR1 mice (Fig. 2d), indicating the localization of CD36 in the perivascular cells under lineage of macrophage. Sometimes, the immunosignals of CD36 showed double lines, indicating the localization of CD36 both in the endothelial cells and the perivascular cells (Fig. 2d-1). 4. Discussion This present study demonstrated that the gene and protein expressions of CD36 were higher in the brain samples of SAMP8 than in those of SAMR1 and that the immunoreactivity of CD36 was localized in the perivascular cells as well as endothelial cells. The previous study (Ueno et al., 2011) showed that the expression of CD36 was higher in the brain samples of hypertensive rats than in those of control rats and that the immunoractivity for CD36 was mainly located in the cytoplasmic membranes of the ED1-positive perivascular cells. Recently, Cho et al. (2005) found that CD36 can mediate ROS production in microglia/macrophages and induce brain injury in cerebral ischemia. The ROS production is suggested to contribute to the pathogenesis of the SAMP8 brains (AlvarezGarcia et al., 2006; Sato et al., 1996; Yasui et al., 2003) and also to the remodeling of the cerebral vasculature of the hypertensive rats (Pires, Deutsch, McClain, Rogers, & Dorrance, 2010). CD36 has been implicated in a wide variety of normal and abnormal biological functions including angiogenesis, atherosclerosis (foam cell formation), phagocytosis, inflammation, lipid metabolism, and removal of apoptotic cells (Febbraio et al., 2001; Hirano et al., 2003). CD36 may be an essential mediator of the production of ROS induced by fibrillar Ab in microglial cell cultures (Coraci et al., 2002; Moore et al., 2002). CD36 is upregulated during cerebral ischemia-reperfusion (Cho et al., 2005). Furthermore, mice lacking the CD36 receptor have a profound reduction in ROS production after ischemia and are relatively protected from ischemic injury. These findings indicate CD36-mediated ROS production as a mechanism of ischemic brain damage. Loss of CD36 confers substantial protection against atherosclerosis in the apoe-null model, suggesting that CD36 is the most relevant scavenger receptor in atherosclerosis (Febbraio et al., 2000, 2004). Antioxidants, such as a-tocopherol, diminish CD36 expression and reduce the uptake of oxLDL into macrophages (Ricciarelli, Zingg, & Azzi, 2000; Fuhrman, Volkova, & Aviram, 2002). In addition, a family of small, cell-permeable antioxidant peptides (SS peptides) that readily penetrate the BBB was recently reported (Cho et al., 2007). The SS peptides scavenge ROS and inhibit oxidation of LDL, thereby reducing the generation of oxLDL (Zhao et al., 2004). Unlike other antioxidants, the SS peptides target mitochondria and protect mitochondria against mitochondrial permeability transition, swelling, and cytochrome c release (Zhao et al., 2004). SS31, one of the SS peptides, attenuates ischemic brain injury by downregulating CD36 (Cho et al., 2007). It is likely that antioxidant substances such as SS31 can also attenuate the cerebrovascular disorders. Antisense directed at the Ab region of APP could decrease brain oxidative markers in aged senescence accelerated mice (Poon et al., 2004). In addition, we reported previously that the expression of P-glycoprotein and LDL receptor, which were also efflux transporters, was increased in the brains of SAMP8 compared with that of SAMR1 (Wu, Ueno, Kusaka, et al., 2009; Wu, Ueno, Onodera, et al., 2009). On the contrary, the expression of RAGE, which is known to be an influx transporter of Ab proteins, was decreased in the brains of SAMP8 compared with that of SAMR1 (Wu, Ueno, Onodera, et al., 2009). The lower expression of RAGE in the brains of SAMP8 may be a response to elevated oxidative stress in the brains of SAMP8, as previously reported (Wu, Ueno, Onodera, et al., 2009). Accordingly, SAMP8 may be a
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Archives of Gerontology and Geriatrics journal homepage: www.elsevier.com/locate/archger
CD36 expression in the brains of SAMP8 Bin Wu a,b, Masaki Ueno a,*, Takashi Kusaka c, Takanori Miki d, Yukiko Nagai e, Toshitaka Nakagawa e, Kenji Kanenishi f, Naohisa Hosomi g, Haruhiko Sakamoto a a
Department of Pathology and Host Defense, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan Department of General Practice, First Affiliated Hospital of China Medical University, San Hao Road 36, Shenyang 110003, China c Department of Maternal Perinatal Center, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan d Department of Anatomy and Neurobiology, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan e Department of Life Sciences Research Center, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan f Department of Perinatology and Gynecology, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan g Department of Clinical Neuroscience and Therapeutics, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 June 2012 Received in revised form 14 July 2012 Accepted 21 July 2012 Available online 9 August 2012
SAMP8, senescence accelerated mice with age-related deficits in memory and learning, are known to show age-related increases of amyloid precursor protein (APP) and immunopositivity for amyloid-b (Ab) proteins, and moreover to be under elevated oxidative stress. The elevated expression of class B scavenger receptor CD36, which is the receptor of oxidized LDL and also one of efflux transporters of Ab proteins in the cerebral vessels, is thought to mediate free radical production in cerebral ischemia and induce oxidative stress. Accordingly, by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical techniques, we examined whether the expression of CD36 was increased in the brains of 10–12-week-old SAMP8 with elevated oxidative stress. Ten to 12-week-old SAMR1 mice were used as controls without the features. The gene and protein expression of CD36 was significantly higher in the brains of SAMP8 than those of SAMR1. Confocal microscopic examination revealed that the CD36 immunoreactivity was seen in the cytoplasm of endothelial cells and F4/80-positive perivascular cells of the brains. These findings indicate that the expression of CD36 in the brains of SAMP8 is increased compared with that of SAMR1. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: CD36 SAMP8 Oxidative stress
1. Introduction The senescence-accelerated mouse (SAM) is a general term for ‘‘accelerated senescence prone’’ (P-series; SAMP) and ‘‘accelerated senescence resistant’’ (R-series; SAMR) mice (Takeda et al., 1981). Among SAMP, SAMP8 mice have a short life span like the other SAMP mice, reveal some senescence-accelerated symptoms, and show a remarkable age-related deterioration in the ability of memory and learning in passive and active avoidance responses (Miyamoto et al., 1986). Previous papers showed that the expression of amyloid precursor protein (APP) and its mRNA was increased with aging in the brains of SAMP8 (Morley et al., 2000; Takemura et al., 1993). Recently, some papers showed that amyloid-b (Ab) granules accumulated in the hippocampus of SAMP8 and that the number of the granules was increased with aging (del Valle et al., 2010, 2011; Manich et al., 2011).
* Corresponding author at: Department of Pathology and Host Defense, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan. Tel.: +81 87 891 2115; fax: +81 87 891 2116. E-mail address: [email protected] (M. Ueno). 0167-4943/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archger.2012.07.007
It has been suggested that vascular Ab receptors in endothelial cells transfer perivascular Ab proteins into circulation and thus contribute to the clearance of Ab from the brain (Zlokovic, 2004). Both the low-density lipoprotein receptor (LDLR) and the LDLRrelated protein 1 (LRP1) may act as Ab receptors (Abdulkarim & Hameed, 2006; Fryer et al., 2005; Sagare et al., 2007). LRP1 is a member of the LDLR family and functions both as a multifunctional scavenger and signaling receptor and as a transporter and metabolizer of cholesterol and apolipoprotein E (ApoE)containing lipoproteins (Herz & Marschang, 2003). LRP1 binds both ApoE/Ab complexes and Ab and regulates their clearance from brain to blood (Donahue et al., 2006; Shibata et al., 2000; Zlokovic, 2004). Besides the LDLR family, some other potential Ab-binding receptors have been identified. P-glycoprotein (Lam et al., 2001), scavenger receptor CD36 (Coraci et al., 2002), the formylpeptide receptor-like-1 (FPRL1) (Le et al., 2001), and the transmembrane APP itself (Lorenzo et al., 2000) also function as Ab receptors. We previously investigated the expression of P-glycoprotein, LDL receptor, and LRP1, all of which were efflux transporters of Ab proteins, and reported that the expression of P-glycoprotein and LDL receptor but not LRP1 was increased in the brains of SAMP8 compared with that of SAMR1 (Wu, Ueno, Kusaka, et al., 2009; Wu,
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Ueno, Onodera, et al., 2009). In addition, we reported that the expression of RAGE, an influx transporter of Ab proteins, was decreased in the brains of SAMP8 compared with that of SAMR1 brains (Wu, Ueno, Onodera, et al., 2009). CD36 belongs to the class B scavenger receptor family, which includes the receptor for selective cholesteryl ester uptake, scavenger receptor class B type I (SR-BI), and lysosomal integral membrane protein II (Febbraio, Hajjar, & Silverstein, 2001). CD36 is a surface glycoprotein and is localized in lipid rafts of plasma membrane and in mitochondria (Bonen et al., 2004; Campbell et al., 2004; Roepstorff, WulffHelge, Vistisen, & Kiens, 2004). In addition, CD 36 colocalizes with caveolin-1 in specialized plasma membrane microdomains known as caveolae (Lisanti et al., 1994). These cholesterol- and sphingolipid-enriched structures may serve to concentrate signaling molecules and facilitate the integration of signaling cascade. Multiple lines of evidence indicate that caveolae serve an integral role in the trafficking of cholesterol in cells. Oxidized LDL (oxLDL) depletes the endothelial cell caveolae of cholesterol, resulting in displacement of endothelial nitric oxide synthase and an altered response to acetylcholine (Uittenbogaard, Shaul, Yuhanna, Blair, & Smart, 2000). These disruptive effects of oxLDL are mediated by CD36 and can be blocked by interaction of SR-BI with HDL, which prevents the cholesterol depletion of caveolae. Thus, CD 36 has a role in the regulation of caveolar function. It is known that CD36 expression is broad and seen in microglia, macrophages, microvascular endothelium, adipocytes, dendritic cells, platelets, and cardiac, skeletal and smooth muscle cells (Febbraio, Guy, & Silverstein, 2004). CD36 recognizes a multitude of ligands, including oxLDL, long-chain fatty acids, thrombospondin-1, fibrillar Ab, and the membrane of cells undergoing apoptosis (Febbraio et al., 2004; Hirano et al., 2003; Medeiros et al., 2004). In addition, some findings suggest that the expression of CD36 is involved in the pathogenesis of cerebral ischemia with reactive oxygen species (ROS) production (Cho et al., 2005). The increased ROS production is suggested to contribute to the pathogenesis of the SAMP8 brain (Alvarez-Garcia et al., 2006; Sato et al., 1996; Yasui et al., 2003). Accordingly, we examined whether the expression of CD36 is increased in the brains of SAMP8 with elevated oxidative stress, and whether CD36 could be a therapeutic target to brain injury with the elevated oxidative stress.
control samples using glyceraldehydes 3-phosphate dehydrogenase (GAPDH) as a control for normalization among samples. For Western blot analysis, brain tissues were homogenized in lysis buffer containing 10 mmol/L Tris buffer containing 2 mmol/L phenylmethylsulfonyl fluoride, and 10 mmol/L N-ethylmaleimide with a protease inhibitor. The extracts were solubilized in a Tris/ glycine/sodium dodecyl sulfate (SDS) sample buffer in the presence of 5% 2-mercaptoethanol (Bio-Rad) and heated at 95 8C for 5 min. Protein extracts (40 mg) were then separated by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred from gel to nitrocellulose membranes and blocked in a blocking solution (5% dry milk in PBS) for 1 h. The membrane was incubated with a primary rabbit antibody for CD36 (1:500, ProteinTech Group, Inc., Chicago, IL, USA) at 4 8C overnight. The membranes were then incubated with HRP-labeled anti-rabbit IgG antibody (1:5000, Amersham Biosciences, Buckinghamshire, UK) for 1 h at room temperature (RT). The proteins were visualized on enhanced chemiluminescence film (Hyperfilm; Amersham Biosciences). Finally, the blots were reprobed using an antibody against actin (1:2000, Epitmics, Inc., Burlingame, CA, USA). Data are expressed as the relative differences among samples after normalization to actin expression. The immunoreactive bands were quantified by densitometric analysis (NIH image software).
2. Materials and methods The experimental protocols for animal care were in compliance with institutional guidelines of Kagawa University. Ten to 12-week-old male SAMP8 (n = 10) and SAMR1 (n = 10) mice (purchased from Japan SLC, Inc., Japan) weighing 25–35 g were used. All efforts were made to minimize the number of animals used and their suffering. The animals were anesthetized with diethyl ether and then used for several experiments. For real-time RT-PCR and Western blot analyses, mice were perfused transcardially with phosphate-buffered saline (PBS). Their brains were removed and stored at 80 8C. Extraction of total RNA and reverse transcription of the total RNA were performed as reported previously (Huang et al., 2004). To quantify gene expression, TaqMan real-time quantitative PCR was performed with the ABI PRISM 7700 Sequence Detection System using Assays on-Demand Gene Expression probes of CD36 (Applied Biosystems, Foster, CA, USA). The PCR cycling conditions for all samples were as follows: 50 8C, 2 min for AmpErase UNG activation; 95 8C, 10 min for AmpliTaq Gold activation; and 50 cycles for melting (95 8C, 15 s) and annealing/extension (60 8C, 1 min) steps. Each sample was run in duplicate. The comparative threshold cycle method (Applied Biosystems) was used to calculate gene expression in each sample relative to the value observed in
Fig. 1. (a) mRNA expressions of CD36 after normalization to GAPDH expression in the brains of SAMP8 (bar indicated by oblique line) (n = 6) and SAMR1 (bar indicated by horizontal line) (n = 6) mice are shown. The gene expression of CD36 is significantly higher in the SAMP8 brains than that of SAMP8 (*, p < 0.05). (b) Western blotting analyses shows representative bands of CD36 and actin of SAMP8 and SAMR1 brains. The protein expression of CD36 is significantly higher in the SAMP8 brains than that of SAMR1 (*, p < 0.05).
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For double immunofluorescence staining, mice were perfused transcardially with PBS and then with a fixative containing 10% formalin. The brains were removed and immersed in the same fixative. The brains were immersed in 20% sucrose buffer solution for 2 days, embedded in optimal cutting temperature compound, and cut on a cryostat into sections 16 mm thick. The frozen sections were incubated with the rabbit antibody for CD36 (1:100, ProteinTech Group, Inc.) at 4 8C overnight, followed by incubation in Alexa Fluor 594-conjugated anti-rabbit IgG antibody (1:200, Molecular Probes) at RT for 1 h. After washing in PBS, the sections were incubated with a primary goat antibody for PECAM-1 (1:100, Santa Cruz Biotech, Inc., CA, USA) at 4 8C overnight, followed by incubation in Alexa Fluor 644-conjugated anti-goat IgG antibody (1:200, Molecular Probes) at RT for 1 h. In addition, the frozen sections were incubated with a mouse antibody for CD36 (1:100, abcam) at 4 8C overnight, followed by incubation in Alexa Fluor 488-conjugated anti-mouse IgG antibody (1:200, Molecular Probes) at RT for 1 h. After washing in PBS, the sections were incubated with a primary rabbit antibody for F4/80 (1:100, Santa Cruz Biotech, Inc., CA, USA) at 4 8C overnight, followed by incubation in Alexa Fluor 594-conjugated anti-rabbit IgG antibody (1:200, Molecular Probes) at RT for 1 h. Cross reactions were not detected after double staining. For nuclear staining, the sections were incubated for 15 min at RT in 40 -6-diamidino-2-phenylindole
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(DAPI, Molecular Probes, Eugene, OR, USA) or in TO-PRO-3 iodide (Molecular Probes) solution. The fluorescent signals were viewed under a confocal microscope (Bio-Rad Radiance 2100 or Carl Zeiss LSM700). As a control experiment, we performed an identical immunohistochemical procedure, but with omission of the primary antibody or using a normal serum instead of the primary antibody. Values are shown as means SD. Statistical analysis was performed by using Mann–Whitney. Differences were considered to be statistically significant when p < 0.05. 3. Results Real-time quantitative RT-PCR analysis revealed that the gene expression of CD36 was significantly higher in the brain samples of SAMP8 than those of SAMR1 (Fig. 1a). Western blot analysis using the primary antibodies for CD36 and actin revealed that relative protein expression of CD36 to that of actin was significantly higher in the brain samples of SAMP8 than those of SAMR1 (Fig. 1b). Double immunohistochemical analyses showed that the immunosignals of CD36 in the vessel wall were colocalized with those of PECAM-1, indicating that parts of the immunosignals of CD36 were present in PECAM-1-positive endothelial cells of cerebral vessels in SAMP8 (Fig. 2a) and also in SAMR1 mice
Fig. 2. Confocal microscopic images of double immunostaining using antibodies for PECAM-1 (a-1, b-1) and CD36 (a-2, b-2) and nuclear staining by DAPI (a-3, b-3) and the merged images (a-4, b-4) in the SAMP8 (a) and SAMR1 (b) brains are shown. The immunosignals for CD36 are partially colocalized with those of PECAM-1 in the brains of both strains. Confocal microscopic images of double immunostaining using antibodies for CD36 (c-1, d-1) and F4/80 (c-2, d-2) and nuclear staining by TO-PRO-3 (c-3, d-3) and the merged images (c-4, d-4) in the SAMP8 (c) and SAMR1 (d) brains are shown. The immunosignals for CD36 are partially colocalized with those of F4/80 in the brains of both strains. The coincidence of two antibodies is indicated by the conversion of green (a–d-1) and red (a–d-2) to yellow (a–d-4). Scale bars indicate 20 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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(Fig. 2b). In addition, the immunosignals of CD36 were partially colocalized with those of F4/80 in vessels larger than capillaries in SAMP8 (Fig. 2c) and also in SAMR1 mice (Fig. 2d), indicating the localization of CD36 in the perivascular cells under lineage of macrophage. Sometimes, the immunosignals of CD36 showed double lines, indicating the localization of CD36 both in the endothelial cells and the perivascular cells (Fig. 2d-1). 4. Discussion This present study demonstrated that the gene and protein expressions of CD36 were higher in the brain samples of SAMP8 than in those of SAMR1 and that the immunoreactivity of CD36 was localized in the perivascular cells as well as endothelial cells. The previous study (Ueno et al., 2011) showed that the expression of CD36 was higher in the brain samples of hypertensive rats than in those of control rats and that the immunoractivity for CD36 was mainly located in the cytoplasmic membranes of the ED1-positive perivascular cells. Recently, Cho et al. (2005) found that CD36 can mediate ROS production in microglia/macrophages and induce brain injury in cerebral ischemia. The ROS production is suggested to contribute to the pathogenesis of the SAMP8 brains (AlvarezGarcia et al., 2006; Sato et al., 1996; Yasui et al., 2003) and also to the remodeling of the cerebral vasculature of the hypertensive rats (Pires, Deutsch, McClain, Rogers, & Dorrance, 2010). CD36 has been implicated in a wide variety of normal and abnormal biological functions including angiogenesis, atherosclerosis (foam cell formation), phagocytosis, inflammation, lipid metabolism, and removal of apoptotic cells (Febbraio et al., 2001; Hirano et al., 2003). CD36 may be an essential mediator of the production of ROS induced by fibrillar Ab in microglial cell cultures (Coraci et al., 2002; Moore et al., 2002). CD36 is upregulated during cerebral ischemia-reperfusion (Cho et al., 2005). Furthermore, mice lacking the CD36 receptor have a profound reduction in ROS production after ischemia and are relatively protected from ischemic injury. These findings indicate CD36-mediated ROS production as a mechanism of ischemic brain damage. Loss of CD36 confers substantial protection against atherosclerosis in the apoe-null model, suggesting that CD36 is the most relevant scavenger receptor in atherosclerosis (Febbraio et al., 2000, 2004). Antioxidants, such as a-tocopherol, diminish CD36 expression and reduce the uptake of oxLDL into macrophages (Ricciarelli, Zingg, & Azzi, 2000; Fuhrman, Volkova, & Aviram, 2002). In addition, a family of small, cell-permeable antioxidant peptides (SS peptides) that readily penetrate the BBB was recently reported (Cho et al., 2007). The SS peptides scavenge ROS and inhibit oxidation of LDL, thereby reducing the generation of oxLDL (Zhao et al., 2004). Unlike other antioxidants, the SS peptides target mitochondria and protect mitochondria against mitochondrial permeability transition, swelling, and cytochrome c release (Zhao et al., 2004). SS31, one of the SS peptides, attenuates ischemic brain injury by downregulating CD36 (Cho et al., 2007). It is likely that antioxidant substances such as SS31 can also attenuate the cerebrovascular disorders. Antisense directed at the Ab region of APP could decrease brain oxidative markers in aged senescence accelerated mice (Poon et al., 2004). In addition, we reported previously that the expression of P-glycoprotein and LDL receptor, which were also efflux transporters, was increased in the brains of SAMP8 compared with that of SAMR1 (Wu, Ueno, Kusaka, et al., 2009; Wu, Ueno, Onodera, et al., 2009). On the contrary, the expression of RAGE, which is known to be an influx transporter of Ab proteins, was decreased in the brains of SAMP8 compared with that of SAMR1 (Wu, Ueno, Onodera, et al., 2009). The lower expression of RAGE in the brains of SAMP8 may be a response to elevated oxidative stress in the brains of SAMP8, as previously reported (Wu, Ueno, Onodera, et al., 2009). Accordingly, SAMP8 may be a
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