- Email: [email protected]
PS1dE9 mice
Neuroscience 161 (2009) 53–58
SUPPRESSION OF NUCLEAR FACTOR KAPPA B AMELIORATES ASTROGLIOSIS BUT NOT AMYLOID BURDEN IN APPswe/PS1dE9 MICE X. ZHANG,1 K. J. LUHRS,1 K. A. RYFF, W. T. MALIK, M. J. DRISCOLL AND B. CULVER*
both in and around neocortical NPs in the AD brain, and reactive astrocytes have been shown to form a halo surrounding the plaque (Ho et al., 2005). Additionally, numerous proinflammatory factors including a variety of cytokines, complement proteins, chemokines and cyclooxygenase have been reported to be upregulated in brains of both AD patients and transgenic animal models (Aarli, 2003; Lucas et al., 2006). However, it is still under heated debate whether this inflammatory response exerts a neuroprotective role or promotes neuronal degeneration. On one hand, activated microglia and astrocytes may contribute to the clearing and degradation of A, and may also mediate neurite regenerative processes (Eikelenboom et al., 2006). Conversely, microglial activation, reactive astrogliosis and excessive release of proinflammatory mediators could promote synapse loss and neuronal injury. A better understanding of the role of neuroinflammation in AD will be necessary to understand the inflammation-associated targets for drug treatment and prevention of this devastating disease. The nuclear factor kappa B, NF-B, is involved in regulating immune and inflammatory responses, as well as cell survival. Its activation boosts the expression of a number of genes including cytokines, chemokines, the major histocompatibility complex (MHC) and receptors needed for neutrophil adhesion and migration (Yamamoto and Gaynor, 2001). Cyclooxygenase (COX), tissue necrosis factor ␣ (TNF␣) and interleukin-1 (IL-1) are among major proinflammatory molecules regulated by NF-B. The two hallmarks of AD, intracellular neurofibrillary tangles and extracellular aggregates of A, have been associated with NF-B activation (Yamamoto and Gaynor, 2001). For instance, components of neurofibrillary tangles were found to produce reactive oxygen species resulting in nuclear translocation of NF-B. A was also found to activate NF-B signaling (Bonaiuto et al., 1997; Du et al., 2005). In addition, enhanced immunoreactivity was observed predominantly in and around the early NP but not the mature ones suggesting an important role of NF-B pathway in the early stages of the disease (Kaltschmidt et al., 1997). The purpose of the current study is to investigate the role of NF-B in both A deposition and neuroinflammation in a transgenic AD mouse model. Pyrrolidine dithiocarbamate (PDTC) is an established NF-B inhibitor and antioxidant. It can block NF-B activation by inhibiting the IB-ubiquitin ligase activity independent of its anti-oxidative properties (Nurmi et al., 2004). Here, we report that long term PDTC administration by intraperitoneal (i.p.) injection at a dose of 50 mg/kg can suppress induction of COX-2 and TNF␣ precursor protein, as well as attenuate astrogliosis in APPswe/PS1dE9 mice.
Division of Pharmaceutical Sciences and Graduate Neuroscience Program, University of Wyoming, College of Health Sciences, Department 3375, 1000 East University Avenue, Laramie, WY 82071-3375, USA
Abstract—Neuroinflammation has been linked to the pathologies of Alzheimer’s disease (AD), however, its effects on betaamyloid (A) burden are unclear. This study investigated the role of nuclear factor kappa B (NF-B) in regulating neuroinflammation and A deposition in a transgenic mouse model of AD. The APPswe/PS1dE9 mice and their wild-type controls received either the NF-B inhibitor pyrrolidine dithiocarbamate (PDTC, i.p. 50 mg/kg daily) or saline starting at 7 months of age for 5 months. Expression of cyclooxygenase-2 (COX-2), tissue necrosis factor alpha (TNF␣) precursor protein and microtubule-associated protein 2 was determined, and astrogliosis was assessed. Hippocampal and cortical levels of A1-40 and A1-42 were measured using ELISA. PDTC treatment effectively suppressed NF-B signaling in APPswe/PS1dE9 mice as evidenced by the abolishment of COX-2 and TNF␣ induction. Inhibition of NF-B further attenuated astrogliosis in the transgenic AD mice, yet markedly increased cerebral A1-42 burden. Our findings suggest that NF-B can mediate induction of COX-2, TNF␣ and astrogliosis in APPswe/PS1dE9 mice. Additionally, these results support the idea that neuroinflammation contributes to the clearance of A. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: Alzheimer’s disease, neuroinflammation, NF-B, beta-amyloid, astrogliosis, APPswe/PS1dE9 mice.
Alzheimer’s disease (AD) is a major cause of dementia and currently there is no cure or effective treatment to ameliorate this devastating neurodegenerative disorder. Extracellular neuritic plaques (NP) consisting of deposited beta-amyloid (A) fibrillary aggregates are one of the hallmark neuropathological lesions of the AD brain. In recent years, it has become a foremost therapeutic goal to reduce cerebral A burden in treating and preventing AD (Qu et al., 2006). Increasing evidence has shown that neuroinflammation, the inflammatory response that occurs in the central nervous system (CNS), is closely related with A pathologies. Robust microglial activation has been found 1 Denotes equal first authorship. *Corresponding author. Tel: ⫹1-307-766-6481; fax: ⫹1-307-766-2953. E-mail address: [email protected] (B. Culver). Abbreviations: AD, Alzheimer’s disease; A, beta-amyloid; CNS, central nervous system; COX, cyclooxygenase; GFAP, glial fibrillary acidic protein; i.p., intraperitoneal; MAP2, microtubule-associated protein 2; NF-B, nuclear factor kappa B; NP, neuritic plaques; PDTC, pyrrolidine dithiocarbamate; TNF␣, tissue necrosis factor ␣.
0306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.03.010
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It is important to note, however, that PDTC treatment increases cerebral A1-42 burden.
EXPERIMENTAL PROCEDURES Animals APPswe/PS1dE9 transgenic mice (stock number 004462) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). These mice carry the human APP-Swedish mutation and the DeltaE9 mutation of the human presenilin 1 gene and develop abundant amyloid deposits in the cerebral cortex and hippocampus by 10 months of age. The wild-types from the colony were used as controls. The experimental procedures were approved by the Animal Care and Use Committee at the University of Wyoming and were in accordance with US National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Every effort was made to minimize the number of animals used and their suffering. Animals were housed two mice per cage on a 12-h light/dark cycle with food and water available ad libitum.
PDTC treatment The APP/PS1 transgenic and wild-type mice were randomly divided into PDTC-treated or untreated groups with 10 animals in each group. The animals in the PDTC treatment group were injected i.p. with PDTC (50 mg/kg/day, Sigma) for 5 months, starting at the age of 7 months. The animals in the untreated groups received saline. The mice were sacrificed at 12 months of age. Brains were quickly removed, and the cortex (from the frontal lobe and parietal lobe) and hippocampus were carefully dissected and sampled according to different assay protocols.
Western blot analysis of COX-2, TNF␣, glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP2) Cortical and hippocampal samples were homogenized in a lysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 0.1% SDS and protease inhibitor cocktail. Samples were then sonicated for 15 s and centrifuged at 12,000⫻g for 20 min at 4 °C. The protein concentration was evaluated using Protein Assay Reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of protein and prestained molecular weight markers (GIBCO-BRL, Gaithersburg, MD, USA) were separated on SDS–polyacrylamide gels in a minigel apparatus (Mini-protean II, Bio-Rad) and transferred electrophoretically to polyvinylidene difluoride membranes. The membranes were incubated for 2 h in a blocking solution containing 5% skim milk in TBS, and then washed briefly in TBS and incubated overnight at 4 °C in the appropriately diluted primary antibody solution. The antibodies and dilution are as follows: anti-COX-2 (1:500), anti-TNF␣ (1: 1000) and anti-GFAP (1:1000). COX-2 polyclonal antibody was purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA. Anti-GFAP and anti-MAP2 monoclonal antibody was obtained from Sigma. Anti-TNF␣ and anti--actin antibodies were from Cell Signaling Technology, Beverly, MA, USA. After washing, blots were incubated for 1 h with the appropriate horseradish peroxidase– conjugated secondary antibody (1: 5000). Antibody binding was detected using enhanced chemiluminescence (Pierce, Rockford, IL, USA); the films were scanned and the intensity of immunoblot bands were detected with a BioRad Calibrated Densitometer (model: GS-800) (Zhang et al., 2005). -Actin was used as an internal loading control.
Immunohistochemical detection of astrogliosis Immunofluorescence was performed on zinc-fixed paraffin-embedded sections. Sections were first de-waxed and then subjected
to antigen retrieval procedures. Sections were treated for 10 min in 3% hydrogen peroxide (Sigma, St. Louis, MO, USA) and blocked in 5% normal goat serum for 2 h prior to overnight incubation with the primary antibody. The primary antibody utilized was anti-GFAP (1:400; Sigma). Sections were then washed with PBS followed by incubation with 594-conjugated secondary antibody (Molecular Probes, Eugene, OR, USA). Fluorescent images were captured at 400⫻ magnification using an Olympus BX51 fluorescent microscope equipped with a digital camera (Olympus Corp., Tokyo, Japan).
Enzyme-linked immunosorbent assay for detection of A1-40 and A1-42 peptide in brain tissues The levels of A1-40 and A1-42 were measured from freshly frozen hippocampal and cortical tissues. The samples were homogenized in guanidine–Tris buffer (5.0 M guanidine HCl/50 mM Tris–HCl, pH 8.0), then the homogenates were incubated at room temperature for 4 h before they were assayed. The levels of A1-40 and A1-42 were quantified using commercial ELISA kits (Signet Laboratories, Dedham, MA, USA) following manufacturer’s protocol. The absorbance of the plates was read at 450 nm with a microplate reader (SpectraMax). The standard curves were established using a variety of concentrations (1–2000 ng/ml) of synthetic A1-40 and A1-42 peptide. Data are expressed as nanograms of A per milligram of tissue (Qu et al., 2006).
Statistical analysis All data are expressed as mean⫾SE. Two group comparisons were evaluated by t-tests, and statistical differences among more than three groups were determined using a one-way ANOVA. A P-value less than 0.05 was considered statistically significant.
RESULTS Induction of COX-2 in APPswe/PS1dE9 mice was suppressed by PDTC treatment COX-2 is a major downstream target regulated by NF-B and is constitutively expressed in both hippocampal and cortical tissues. COX-2 levels were significantly elevated in the untreated APPswe/PS1dE9 mice compared to the wild-type animals. PDTC treatment abolished the inflated COX-2 induction in the APPswe/PS1dE9 mice without altering the normal COX-2 levels in the wild-type animals (Fig. 1). Upregulation of TNF␣ precursor protein in APPswe/ PS1dE9 mice was eliminated by PDTC treatment Another important downstream gene that is controlled by NF-B is TNF␣. The expression of TNF␣ precursor protein was elevated markedly in the untreated APPswe/PS1dE9 mice. The upregulation of TNF␣ was considerably suppressed by PDTC treatment (Fig. 2). The TNF␣ levels in the wild-type animals were not affected by PDTC. Effect of PDTC treatment on astrogliosis To determine whether PDTC treatment has anti-inflammatory effects, we assessed its ability to attenuate astrogliosis in APPswe/PS1dE9 mice. As shown in Fig. 3, the
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in Fig. 5, the MAP2 protein levels were similar in the APPswe/PS1dE9 mice to those in the wild-type animals. MAP2 protein did not appear to be affected by PDTC treatment.
DISCUSSION
Fig. 1. Long-term PDTC treatment abolished COX-2 induction in APPswe/PS1dE9 mice. APPswe/PS1dE9 mice and wild-type controls were treated with either PDTC (i.p. 50 mg/kg daily) or saline starting at age 7 months and were sacrificed at age 12 months. Hippocampal and cortical COX-2 protein expression was measured by Western blot. The levels of COX-2 protein were significantly increased in untreated APPswe/PS1dE9 mice as compared to the wild-type animals (** P⬍ 0.01). PDTC treatment abolished COX-2 induction in the transgenic mice (## P⬍0.01 compared to untreated APPswe/PS1dE9 mice). Two representative blots of each group are shown in the upper panel, and the bottom panel is a summary of the results. Data are mean⫾SE for four animals per group.
Our results demonstrated that the NF-B inhibitor, PDTC, effectively abolished COX-2 induction and upregulation of TNF␣ precursor protein and also attenuated astrogliosis in APPswe/PS1dE9 mice. However, PDTC treatment did not reduce cerebral A burden, but instead exacerbated A1-42 deposition in both the hippocampus and cortex. Therefore, our findings support the notion that neuroinflammation contributes to the clearance of A in the brain. Previous studies have shown both neuroprotective and unfavorable effects of NF-B inhibition on different CNS disease models by applying PDTC treatment (La et al., 2004; Nurmi et al., 2004; Cheng et al., 2006; Ahtoniemi et al., 2007). In the present study we first tested the efficacy of our PDTC treatment in suppressing NF-B activity in APPswe/PS1dE9 mice. The efficiency of PDTC treatment was assessed by measuring expression of two NF-Bdriven genes: COX-2 and TNF␣. Consistent with early studies (O’Banion, 1999; Cacquevel et al., 2004; Patel et al., 2005; Hoozemans et al., 2008), both pro-inflammatory mediators, COX-2 and TNF-␣, were upregulated in APPswe/ PS1dE9 mice, and PDTC administration abolished their
micrographs from the immunohistochemistry study revealed an increase in the number of GFAP positive cells (mainly astrocytes). The Western blot results showed significantly elevated protein levels of GFAP in untreated APPswe/PS1dE9 mice as compared to the wild-type animals. After PDTC treatment, the GFAP levels were markedly diminished in APPswe/PS1dE9 mice. In contrast, PDTC treatment had no effect on the GFAP expression or the number of astrocytes in wild-type animals. These data suggest that PDTC treatment attenuates reactive astrogliosis in transgenic AD mice. Effect of PDTC treatment on the A deposition and the expression of a dendrite marker-MAP2 As shown in Fig. 4, accumulation of both A1-40 and A1-42 was found in the hippocampus and cerebral cortex of APPswe/PS1dE9 mice. A1-42 was more abundant than A1-40 in both brain regions. Although PDTC had no effect on A burden in the wild-type animals, it significantly exacerbated A1-42 deposition in both hippocampus and cortex of the transgenic AD mice. However, the A1-40 burden was not notably altered by PDTC treatment (Fig. 4). MAP2 is an important cytoskeletal protein mainly detected in the dendrites of neurons. Disturbed MAP2 expression has been associated with impaired dendrites and synaptic signal transduction (Iwata et al., 2005) As shown
Fig. 2. PDTC administration suppressed TNF␣ upregulation in APPswe/PS1dE9 mice. APPswe/PS1dE9 mice and wild-type controls were treated with either PDTC (i.p. 50 mg/kg daily) or saline starting at age 7 months and were sacrificed at 12 months. Expression of TNF␣ precursor protein was measured using Western blot from both hippocampal and cortical tissue. The levels of TNF␣ precursor protein were markedly elevated in untreated APPswe/PS1dE9 mice compared to the wild-type animals (** P⬍0.01). PDTC treatment suppressed TNF␣ upregulation in the transgenic mice (## P⬍0.01 compared to untreated APPswe/PS1dE9 mice). Two representative blots of each group are shown in the upper panel, and the bottom panel is a summary of the results. Data are mean⫾SE for four animals per group.
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Currently two distinct treatment strategies are under development: treatment of AD patients with anti-inflammatory drugs and immunization with the A peptide. The former treatment approach is based on halting the inflammatory process. Conversely, the latter tactic aims to stimulate immune and inflammatory responses to promote A removal. Findings from these studies will contribute to our collective understanding in the role of inflammation on development of A accumulation and the associated progression to AD.
Fig. 3. PDTC treatment attenuated reactive astrogliosis in APPswe/ PS1dE9 mice. APPswe/PS1dE9 mice and wild-type controls were treated with either PDTC (i.p. 50 mg/kg daily) or saline starting at age 7 months and were sacrificed at age 12 months. Astrocytes and other glial cells were stained using anti-GFAP antibody. Robust astrogliosis was observed in untreated transgenic animals (** P⬍0.01 compared to wild-type animals). PDTC administration significantly attenuated astrogliosis as compared to the untreated APPswe/PS1dE9 mice (## P⬍ 0.01). Representative micrographs are shown in A. Western blot analysis of GFAP is shown in B (two representative blots from each group) and C (a summary of the results). Data are means⫾SE of four animals per group. Scale bar⫽100 m.
induction. This suggests that the PDTC treatment produces an effective suppression of NF-B signaling in this transgenic AD model. Furthermore, the robust astrogliosis found in APPswe/PS1dE9 mice was significantly reduced by PDTC treatment indicating that NF-B inhibitors exert anti-inflammatory actions in these transgenic AD mice. This finding is in agreement with the observation that PDTC is capable of attenuating the development of acute and chronic inflammation in the periphery (Cuzzocrea et al., 2002). However, it differs from the only existing report on PDTC treatment in APP/PS1 mice, which showed that giving transgenic AD mice PDTC (20 mg/kg/day) in drinking water failed to diminish inflammatory response in the brain (Malm et al., 2007). This discrepancy may be due to the variation in drug dosage and the routes of administration. Taken together, our findings suggest that NF-B induces upregulation of some inflammatory mediators including COX-2 and TNF␣, and contributes to the reactive astrogliosis in the transgenic AD mice. It is not clear, at this point, whether the individual or collective roles of activated microglia, astrocytes and each proinflammatory mediator are detrimental or beneficial.
Fig. 4. Effect of long-term PDTC treatment on cerebral A burden. APPswe/PS1dE9 mice and wild-type controls were treated with either PDTC (i.p. 50 mg/kg daily) or saline starting at age 7 months and were sacrificed at age 12 months. Total A1-40 and A1-42 in hippocampal and cortical tissue were measured using ELISA assays. PDTC has no effect on A1-40, but significantly increased A1-42 deposition in both hippocampus and cortex when compared to the untreated transgenic mice (** P⬍0.01). Results are the mean⫾SE of four to six animals per group.
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Fig. 5. Effect of PDTC on dendrite marker MAP2 expression. APPswe/PS1dE9 mice and wild-type controls were treated with either PDTC (i.p. 50 mg/kg daily) or saline starting at age 7 months and were sacrificed at age 12 months. No differences in MAP2 expression were noted among these four groups (P⬎0.05). Two representative blots of each group are shown in the upper panel, and the bottom panel is a summary of the results. Data are mean⫾SE for four animals per group.
Many anti-inflammatory drugs such as prednisone, hydroxychloroquine, naproxen (a traditional nonselective NSAID), celecoxib and rofecoxib (both selective COX-2 inhibitors) were found to lack beneficial effects on A clearance in patients with AD (Van Gool et al., 2003; Eikelenboom et al., 2006). It seems that at this time, the clinical data do not favor anti-inflammatory treatment, and our findings are in harmony with this notion. A deposition results from an imbalance between its production and clearance. In animal models of AD, diverse effects of neuroinflammation have been reported on both sides of this balance. On one hand, astrocytes and microglia were thought to function in the uptake and intracellular degradation of A. Some inflammatory mediators were also considered to promote A removal. For example, studies on hAPP mice crossed with sCrry (overexpressing an inhibitor of complement 3 activation) showed that the double transgenic mice had two to three times more A than the hAPP mice suggesting a significant role of complement in A clearance (Wyss-Coray et al., 2002). However, inflammation may be a cause of defective handling of A and the amyloid precursor protein (APP) leading to overproduction of A. Certain infections and lipopolysaccharide (LPS)-induced inflammation have been reported to increase A accumulation (von Bernhardi, 2007). In addition, some cytokines exhibit the ability to stimulate -secretase activity and increase generation of A (Blasko et al., 1999). Our results show that NF-B inhibition markedly increased A1-42 deposition in both hippocampus and cortex. This indicates that NF-B may be more important in facilitating removal of A than causing defects in A han-
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dling, at least in the early stage of AD. Furthermore, two major forms of A exist in the AD brain, known as A1-40 and A1-42. A1-42 differs from A1-40 only in two additional C-terminal residues, the Ile 41 and the Ala 42, however A1-42 aggregates more rapidly than A1-40 and it is the primary constituent of the senile plaque in diseased brains (Kim and Hecht, 2005). The present study revealed that NF-B inhibitor accelerated A1-42 aggregation, whereas, the A1-40 level was unaffected by PDTC treatment. Two possible explanations may account for the differential effects of PDTC treatment on A1-40 and A1-42. First, the dominant type of A in APPswe/PS1dE9 mice is A1-42 with a ratio of A peptide 40:42 of approximately 1:2. Mutations in the presenilin-1 (PS-1) gene (on chromosome 14) can result in an increase in the production of A1-42 (Findeis, 2007). APPswe/PS1dE9 mice express a delta E9 mutant human presenilin 1 carrying the exon-9 – deleted variant associated with familial Alzheimer’s disease. Second, the deposition of the long form A is an early step in the pathogenesis of NP in the AD brain. This is observed before a diagnosis of AD is possible, prior to the deposition of A1-40 (Parvathy et al., 2001). In the early deposition, when most deposited protein is in the form of amorphous or diffuse plaques, almost all of the A is the long form peptide. These initial A1-42 deposits may induce the further deposition of both A1-40 and A1-42 (Findeis, 2007). In the current study, the PDTC treatment started during the early stage of A aggregation; therefore it may mainly affect A1-42.
CONCLUSION In summary, we report that i.p. injection of PDTC at the dose of 50 mg/kg effectively suppressed NF-B signaling as evidenced by the abolishment of COX-2 and TNF␣ induction in APPswe/PS1dE9 mice. Inhibition of NF-B further attenuated astrogliosis in both hippocampus and cortex of the transgenic AD mice, although an increase in the cerebral A1-42 burden was strongly evident. These findings suggest that NF-B mediates neuroinflammation in APPswe/PS1dE9 mice. The present study favors the notion that neuroinflammation contributes to the clearance of A in this transgenic AD model. Acknowledgments—This research was supported in part by NIH grants NCRR BRIN P20 RR15640 (R. O. Kelley, P.I.) and NCRR COBRE P20 RR15640 (F. W. Flynn, P.I.) awarded to the University of Wyoming.
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(Accepted 5 March 2009) (Available online 13 March 2009)
SUPPRESSION OF NUCLEAR FACTOR KAPPA B AMELIORATES ASTROGLIOSIS BUT NOT AMYLOID BURDEN IN APPswe/PS1dE9 MICE X. ZHANG,1 K. J. LUHRS,1 K. A. RYFF, W. T. MALIK, M. J. DRISCOLL AND B. CULVER*
both in and around neocortical NPs in the AD brain, and reactive astrocytes have been shown to form a halo surrounding the plaque (Ho et al., 2005). Additionally, numerous proinflammatory factors including a variety of cytokines, complement proteins, chemokines and cyclooxygenase have been reported to be upregulated in brains of both AD patients and transgenic animal models (Aarli, 2003; Lucas et al., 2006). However, it is still under heated debate whether this inflammatory response exerts a neuroprotective role or promotes neuronal degeneration. On one hand, activated microglia and astrocytes may contribute to the clearing and degradation of A, and may also mediate neurite regenerative processes (Eikelenboom et al., 2006). Conversely, microglial activation, reactive astrogliosis and excessive release of proinflammatory mediators could promote synapse loss and neuronal injury. A better understanding of the role of neuroinflammation in AD will be necessary to understand the inflammation-associated targets for drug treatment and prevention of this devastating disease. The nuclear factor kappa B, NF-B, is involved in regulating immune and inflammatory responses, as well as cell survival. Its activation boosts the expression of a number of genes including cytokines, chemokines, the major histocompatibility complex (MHC) and receptors needed for neutrophil adhesion and migration (Yamamoto and Gaynor, 2001). Cyclooxygenase (COX), tissue necrosis factor ␣ (TNF␣) and interleukin-1 (IL-1) are among major proinflammatory molecules regulated by NF-B. The two hallmarks of AD, intracellular neurofibrillary tangles and extracellular aggregates of A, have been associated with NF-B activation (Yamamoto and Gaynor, 2001). For instance, components of neurofibrillary tangles were found to produce reactive oxygen species resulting in nuclear translocation of NF-B. A was also found to activate NF-B signaling (Bonaiuto et al., 1997; Du et al., 2005). In addition, enhanced immunoreactivity was observed predominantly in and around the early NP but not the mature ones suggesting an important role of NF-B pathway in the early stages of the disease (Kaltschmidt et al., 1997). The purpose of the current study is to investigate the role of NF-B in both A deposition and neuroinflammation in a transgenic AD mouse model. Pyrrolidine dithiocarbamate (PDTC) is an established NF-B inhibitor and antioxidant. It can block NF-B activation by inhibiting the IB-ubiquitin ligase activity independent of its anti-oxidative properties (Nurmi et al., 2004). Here, we report that long term PDTC administration by intraperitoneal (i.p.) injection at a dose of 50 mg/kg can suppress induction of COX-2 and TNF␣ precursor protein, as well as attenuate astrogliosis in APPswe/PS1dE9 mice.
Division of Pharmaceutical Sciences and Graduate Neuroscience Program, University of Wyoming, College of Health Sciences, Department 3375, 1000 East University Avenue, Laramie, WY 82071-3375, USA
Abstract—Neuroinflammation has been linked to the pathologies of Alzheimer’s disease (AD), however, its effects on betaamyloid (A) burden are unclear. This study investigated the role of nuclear factor kappa B (NF-B) in regulating neuroinflammation and A deposition in a transgenic mouse model of AD. The APPswe/PS1dE9 mice and their wild-type controls received either the NF-B inhibitor pyrrolidine dithiocarbamate (PDTC, i.p. 50 mg/kg daily) or saline starting at 7 months of age for 5 months. Expression of cyclooxygenase-2 (COX-2), tissue necrosis factor alpha (TNF␣) precursor protein and microtubule-associated protein 2 was determined, and astrogliosis was assessed. Hippocampal and cortical levels of A1-40 and A1-42 were measured using ELISA. PDTC treatment effectively suppressed NF-B signaling in APPswe/PS1dE9 mice as evidenced by the abolishment of COX-2 and TNF␣ induction. Inhibition of NF-B further attenuated astrogliosis in the transgenic AD mice, yet markedly increased cerebral A1-42 burden. Our findings suggest that NF-B can mediate induction of COX-2, TNF␣ and astrogliosis in APPswe/PS1dE9 mice. Additionally, these results support the idea that neuroinflammation contributes to the clearance of A. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: Alzheimer’s disease, neuroinflammation, NF-B, beta-amyloid, astrogliosis, APPswe/PS1dE9 mice.
Alzheimer’s disease (AD) is a major cause of dementia and currently there is no cure or effective treatment to ameliorate this devastating neurodegenerative disorder. Extracellular neuritic plaques (NP) consisting of deposited beta-amyloid (A) fibrillary aggregates are one of the hallmark neuropathological lesions of the AD brain. In recent years, it has become a foremost therapeutic goal to reduce cerebral A burden in treating and preventing AD (Qu et al., 2006). Increasing evidence has shown that neuroinflammation, the inflammatory response that occurs in the central nervous system (CNS), is closely related with A pathologies. Robust microglial activation has been found 1 Denotes equal first authorship. *Corresponding author. Tel: ⫹1-307-766-6481; fax: ⫹1-307-766-2953. E-mail address: [email protected] (B. Culver). Abbreviations: AD, Alzheimer’s disease; A, beta-amyloid; CNS, central nervous system; COX, cyclooxygenase; GFAP, glial fibrillary acidic protein; i.p., intraperitoneal; MAP2, microtubule-associated protein 2; NF-B, nuclear factor kappa B; NP, neuritic plaques; PDTC, pyrrolidine dithiocarbamate; TNF␣, tissue necrosis factor ␣.
0306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.03.010
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It is important to note, however, that PDTC treatment increases cerebral A1-42 burden.
EXPERIMENTAL PROCEDURES Animals APPswe/PS1dE9 transgenic mice (stock number 004462) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). These mice carry the human APP-Swedish mutation and the DeltaE9 mutation of the human presenilin 1 gene and develop abundant amyloid deposits in the cerebral cortex and hippocampus by 10 months of age. The wild-types from the colony were used as controls. The experimental procedures were approved by the Animal Care and Use Committee at the University of Wyoming and were in accordance with US National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Every effort was made to minimize the number of animals used and their suffering. Animals were housed two mice per cage on a 12-h light/dark cycle with food and water available ad libitum.
PDTC treatment The APP/PS1 transgenic and wild-type mice were randomly divided into PDTC-treated or untreated groups with 10 animals in each group. The animals in the PDTC treatment group were injected i.p. with PDTC (50 mg/kg/day, Sigma) for 5 months, starting at the age of 7 months. The animals in the untreated groups received saline. The mice were sacrificed at 12 months of age. Brains were quickly removed, and the cortex (from the frontal lobe and parietal lobe) and hippocampus were carefully dissected and sampled according to different assay protocols.
Western blot analysis of COX-2, TNF␣, glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP2) Cortical and hippocampal samples were homogenized in a lysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 0.1% SDS and protease inhibitor cocktail. Samples were then sonicated for 15 s and centrifuged at 12,000⫻g for 20 min at 4 °C. The protein concentration was evaluated using Protein Assay Reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of protein and prestained molecular weight markers (GIBCO-BRL, Gaithersburg, MD, USA) were separated on SDS–polyacrylamide gels in a minigel apparatus (Mini-protean II, Bio-Rad) and transferred electrophoretically to polyvinylidene difluoride membranes. The membranes were incubated for 2 h in a blocking solution containing 5% skim milk in TBS, and then washed briefly in TBS and incubated overnight at 4 °C in the appropriately diluted primary antibody solution. The antibodies and dilution are as follows: anti-COX-2 (1:500), anti-TNF␣ (1: 1000) and anti-GFAP (1:1000). COX-2 polyclonal antibody was purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA. Anti-GFAP and anti-MAP2 monoclonal antibody was obtained from Sigma. Anti-TNF␣ and anti--actin antibodies were from Cell Signaling Technology, Beverly, MA, USA. After washing, blots were incubated for 1 h with the appropriate horseradish peroxidase– conjugated secondary antibody (1: 5000). Antibody binding was detected using enhanced chemiluminescence (Pierce, Rockford, IL, USA); the films were scanned and the intensity of immunoblot bands were detected with a BioRad Calibrated Densitometer (model: GS-800) (Zhang et al., 2005). -Actin was used as an internal loading control.
Immunohistochemical detection of astrogliosis Immunofluorescence was performed on zinc-fixed paraffin-embedded sections. Sections were first de-waxed and then subjected
to antigen retrieval procedures. Sections were treated for 10 min in 3% hydrogen peroxide (Sigma, St. Louis, MO, USA) and blocked in 5% normal goat serum for 2 h prior to overnight incubation with the primary antibody. The primary antibody utilized was anti-GFAP (1:400; Sigma). Sections were then washed with PBS followed by incubation with 594-conjugated secondary antibody (Molecular Probes, Eugene, OR, USA). Fluorescent images were captured at 400⫻ magnification using an Olympus BX51 fluorescent microscope equipped with a digital camera (Olympus Corp., Tokyo, Japan).
Enzyme-linked immunosorbent assay for detection of A1-40 and A1-42 peptide in brain tissues The levels of A1-40 and A1-42 were measured from freshly frozen hippocampal and cortical tissues. The samples were homogenized in guanidine–Tris buffer (5.0 M guanidine HCl/50 mM Tris–HCl, pH 8.0), then the homogenates were incubated at room temperature for 4 h before they were assayed. The levels of A1-40 and A1-42 were quantified using commercial ELISA kits (Signet Laboratories, Dedham, MA, USA) following manufacturer’s protocol. The absorbance of the plates was read at 450 nm with a microplate reader (SpectraMax). The standard curves were established using a variety of concentrations (1–2000 ng/ml) of synthetic A1-40 and A1-42 peptide. Data are expressed as nanograms of A per milligram of tissue (Qu et al., 2006).
Statistical analysis All data are expressed as mean⫾SE. Two group comparisons were evaluated by t-tests, and statistical differences among more than three groups were determined using a one-way ANOVA. A P-value less than 0.05 was considered statistically significant.
RESULTS Induction of COX-2 in APPswe/PS1dE9 mice was suppressed by PDTC treatment COX-2 is a major downstream target regulated by NF-B and is constitutively expressed in both hippocampal and cortical tissues. COX-2 levels were significantly elevated in the untreated APPswe/PS1dE9 mice compared to the wild-type animals. PDTC treatment abolished the inflated COX-2 induction in the APPswe/PS1dE9 mice without altering the normal COX-2 levels in the wild-type animals (Fig. 1). Upregulation of TNF␣ precursor protein in APPswe/ PS1dE9 mice was eliminated by PDTC treatment Another important downstream gene that is controlled by NF-B is TNF␣. The expression of TNF␣ precursor protein was elevated markedly in the untreated APPswe/PS1dE9 mice. The upregulation of TNF␣ was considerably suppressed by PDTC treatment (Fig. 2). The TNF␣ levels in the wild-type animals were not affected by PDTC. Effect of PDTC treatment on astrogliosis To determine whether PDTC treatment has anti-inflammatory effects, we assessed its ability to attenuate astrogliosis in APPswe/PS1dE9 mice. As shown in Fig. 3, the
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in Fig. 5, the MAP2 protein levels were similar in the APPswe/PS1dE9 mice to those in the wild-type animals. MAP2 protein did not appear to be affected by PDTC treatment.
DISCUSSION
Fig. 1. Long-term PDTC treatment abolished COX-2 induction in APPswe/PS1dE9 mice. APPswe/PS1dE9 mice and wild-type controls were treated with either PDTC (i.p. 50 mg/kg daily) or saline starting at age 7 months and were sacrificed at age 12 months. Hippocampal and cortical COX-2 protein expression was measured by Western blot. The levels of COX-2 protein were significantly increased in untreated APPswe/PS1dE9 mice as compared to the wild-type animals (** P⬍ 0.01). PDTC treatment abolished COX-2 induction in the transgenic mice (## P⬍0.01 compared to untreated APPswe/PS1dE9 mice). Two representative blots of each group are shown in the upper panel, and the bottom panel is a summary of the results. Data are mean⫾SE for four animals per group.
Our results demonstrated that the NF-B inhibitor, PDTC, effectively abolished COX-2 induction and upregulation of TNF␣ precursor protein and also attenuated astrogliosis in APPswe/PS1dE9 mice. However, PDTC treatment did not reduce cerebral A burden, but instead exacerbated A1-42 deposition in both the hippocampus and cortex. Therefore, our findings support the notion that neuroinflammation contributes to the clearance of A in the brain. Previous studies have shown both neuroprotective and unfavorable effects of NF-B inhibition on different CNS disease models by applying PDTC treatment (La et al., 2004; Nurmi et al., 2004; Cheng et al., 2006; Ahtoniemi et al., 2007). In the present study we first tested the efficacy of our PDTC treatment in suppressing NF-B activity in APPswe/PS1dE9 mice. The efficiency of PDTC treatment was assessed by measuring expression of two NF-Bdriven genes: COX-2 and TNF␣. Consistent with early studies (O’Banion, 1999; Cacquevel et al., 2004; Patel et al., 2005; Hoozemans et al., 2008), both pro-inflammatory mediators, COX-2 and TNF-␣, were upregulated in APPswe/ PS1dE9 mice, and PDTC administration abolished their
micrographs from the immunohistochemistry study revealed an increase in the number of GFAP positive cells (mainly astrocytes). The Western blot results showed significantly elevated protein levels of GFAP in untreated APPswe/PS1dE9 mice as compared to the wild-type animals. After PDTC treatment, the GFAP levels were markedly diminished in APPswe/PS1dE9 mice. In contrast, PDTC treatment had no effect on the GFAP expression or the number of astrocytes in wild-type animals. These data suggest that PDTC treatment attenuates reactive astrogliosis in transgenic AD mice. Effect of PDTC treatment on the A deposition and the expression of a dendrite marker-MAP2 As shown in Fig. 4, accumulation of both A1-40 and A1-42 was found in the hippocampus and cerebral cortex of APPswe/PS1dE9 mice. A1-42 was more abundant than A1-40 in both brain regions. Although PDTC had no effect on A burden in the wild-type animals, it significantly exacerbated A1-42 deposition in both hippocampus and cortex of the transgenic AD mice. However, the A1-40 burden was not notably altered by PDTC treatment (Fig. 4). MAP2 is an important cytoskeletal protein mainly detected in the dendrites of neurons. Disturbed MAP2 expression has been associated with impaired dendrites and synaptic signal transduction (Iwata et al., 2005) As shown
Fig. 2. PDTC administration suppressed TNF␣ upregulation in APPswe/PS1dE9 mice. APPswe/PS1dE9 mice and wild-type controls were treated with either PDTC (i.p. 50 mg/kg daily) or saline starting at age 7 months and were sacrificed at 12 months. Expression of TNF␣ precursor protein was measured using Western blot from both hippocampal and cortical tissue. The levels of TNF␣ precursor protein were markedly elevated in untreated APPswe/PS1dE9 mice compared to the wild-type animals (** P⬍0.01). PDTC treatment suppressed TNF␣ upregulation in the transgenic mice (## P⬍0.01 compared to untreated APPswe/PS1dE9 mice). Two representative blots of each group are shown in the upper panel, and the bottom panel is a summary of the results. Data are mean⫾SE for four animals per group.
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Currently two distinct treatment strategies are under development: treatment of AD patients with anti-inflammatory drugs and immunization with the A peptide. The former treatment approach is based on halting the inflammatory process. Conversely, the latter tactic aims to stimulate immune and inflammatory responses to promote A removal. Findings from these studies will contribute to our collective understanding in the role of inflammation on development of A accumulation and the associated progression to AD.
Fig. 3. PDTC treatment attenuated reactive astrogliosis in APPswe/ PS1dE9 mice. APPswe/PS1dE9 mice and wild-type controls were treated with either PDTC (i.p. 50 mg/kg daily) or saline starting at age 7 months and were sacrificed at age 12 months. Astrocytes and other glial cells were stained using anti-GFAP antibody. Robust astrogliosis was observed in untreated transgenic animals (** P⬍0.01 compared to wild-type animals). PDTC administration significantly attenuated astrogliosis as compared to the untreated APPswe/PS1dE9 mice (## P⬍ 0.01). Representative micrographs are shown in A. Western blot analysis of GFAP is shown in B (two representative blots from each group) and C (a summary of the results). Data are means⫾SE of four animals per group. Scale bar⫽100 m.
induction. This suggests that the PDTC treatment produces an effective suppression of NF-B signaling in this transgenic AD model. Furthermore, the robust astrogliosis found in APPswe/PS1dE9 mice was significantly reduced by PDTC treatment indicating that NF-B inhibitors exert anti-inflammatory actions in these transgenic AD mice. This finding is in agreement with the observation that PDTC is capable of attenuating the development of acute and chronic inflammation in the periphery (Cuzzocrea et al., 2002). However, it differs from the only existing report on PDTC treatment in APP/PS1 mice, which showed that giving transgenic AD mice PDTC (20 mg/kg/day) in drinking water failed to diminish inflammatory response in the brain (Malm et al., 2007). This discrepancy may be due to the variation in drug dosage and the routes of administration. Taken together, our findings suggest that NF-B induces upregulation of some inflammatory mediators including COX-2 and TNF␣, and contributes to the reactive astrogliosis in the transgenic AD mice. It is not clear, at this point, whether the individual or collective roles of activated microglia, astrocytes and each proinflammatory mediator are detrimental or beneficial.
Fig. 4. Effect of long-term PDTC treatment on cerebral A burden. APPswe/PS1dE9 mice and wild-type controls were treated with either PDTC (i.p. 50 mg/kg daily) or saline starting at age 7 months and were sacrificed at age 12 months. Total A1-40 and A1-42 in hippocampal and cortical tissue were measured using ELISA assays. PDTC has no effect on A1-40, but significantly increased A1-42 deposition in both hippocampus and cortex when compared to the untreated transgenic mice (** P⬍0.01). Results are the mean⫾SE of four to six animals per group.
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Fig. 5. Effect of PDTC on dendrite marker MAP2 expression. APPswe/PS1dE9 mice and wild-type controls were treated with either PDTC (i.p. 50 mg/kg daily) or saline starting at age 7 months and were sacrificed at age 12 months. No differences in MAP2 expression were noted among these four groups (P⬎0.05). Two representative blots of each group are shown in the upper panel, and the bottom panel is a summary of the results. Data are mean⫾SE for four animals per group.
Many anti-inflammatory drugs such as prednisone, hydroxychloroquine, naproxen (a traditional nonselective NSAID), celecoxib and rofecoxib (both selective COX-2 inhibitors) were found to lack beneficial effects on A clearance in patients with AD (Van Gool et al., 2003; Eikelenboom et al., 2006). It seems that at this time, the clinical data do not favor anti-inflammatory treatment, and our findings are in harmony with this notion. A deposition results from an imbalance between its production and clearance. In animal models of AD, diverse effects of neuroinflammation have been reported on both sides of this balance. On one hand, astrocytes and microglia were thought to function in the uptake and intracellular degradation of A. Some inflammatory mediators were also considered to promote A removal. For example, studies on hAPP mice crossed with sCrry (overexpressing an inhibitor of complement 3 activation) showed that the double transgenic mice had two to three times more A than the hAPP mice suggesting a significant role of complement in A clearance (Wyss-Coray et al., 2002). However, inflammation may be a cause of defective handling of A and the amyloid precursor protein (APP) leading to overproduction of A. Certain infections and lipopolysaccharide (LPS)-induced inflammation have been reported to increase A accumulation (von Bernhardi, 2007). In addition, some cytokines exhibit the ability to stimulate -secretase activity and increase generation of A (Blasko et al., 1999). Our results show that NF-B inhibition markedly increased A1-42 deposition in both hippocampus and cortex. This indicates that NF-B may be more important in facilitating removal of A than causing defects in A han-
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dling, at least in the early stage of AD. Furthermore, two major forms of A exist in the AD brain, known as A1-40 and A1-42. A1-42 differs from A1-40 only in two additional C-terminal residues, the Ile 41 and the Ala 42, however A1-42 aggregates more rapidly than A1-40 and it is the primary constituent of the senile plaque in diseased brains (Kim and Hecht, 2005). The present study revealed that NF-B inhibitor accelerated A1-42 aggregation, whereas, the A1-40 level was unaffected by PDTC treatment. Two possible explanations may account for the differential effects of PDTC treatment on A1-40 and A1-42. First, the dominant type of A in APPswe/PS1dE9 mice is A1-42 with a ratio of A peptide 40:42 of approximately 1:2. Mutations in the presenilin-1 (PS-1) gene (on chromosome 14) can result in an increase in the production of A1-42 (Findeis, 2007). APPswe/PS1dE9 mice express a delta E9 mutant human presenilin 1 carrying the exon-9 – deleted variant associated with familial Alzheimer’s disease. Second, the deposition of the long form A is an early step in the pathogenesis of NP in the AD brain. This is observed before a diagnosis of AD is possible, prior to the deposition of A1-40 (Parvathy et al., 2001). In the early deposition, when most deposited protein is in the form of amorphous or diffuse plaques, almost all of the A is the long form peptide. These initial A1-42 deposits may induce the further deposition of both A1-40 and A1-42 (Findeis, 2007). In the current study, the PDTC treatment started during the early stage of A aggregation; therefore it may mainly affect A1-42.
CONCLUSION In summary, we report that i.p. injection of PDTC at the dose of 50 mg/kg effectively suppressed NF-B signaling as evidenced by the abolishment of COX-2 and TNF␣ induction in APPswe/PS1dE9 mice. Inhibition of NF-B further attenuated astrogliosis in both hippocampus and cortex of the transgenic AD mice, although an increase in the cerebral A1-42 burden was strongly evident. These findings suggest that NF-B mediates neuroinflammation in APPswe/PS1dE9 mice. The present study favors the notion that neuroinflammation contributes to the clearance of A in this transgenic AD model. Acknowledgments—This research was supported in part by NIH grants NCRR BRIN P20 RR15640 (R. O. Kelley, P.I.) and NCRR COBRE P20 RR15640 (F. W. Flynn, P.I.) awarded to the University of Wyoming.
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(Accepted 5 March 2009) (Available online 13 March 2009)