- About this Journal ·
- Abstracting and Indexing ·
- Advance Access ·
- Aims and Scope ·
- Annual Issues ·
- Article Processing Charges ·
- Articles in Press ·
- Author Guidelines ·
- Bibliographic Information ·
- Citations to this Journal ·
- Contact Information ·
- Editorial Board ·
- Editorial Workflow ·
- Free eTOC Alerts ·
- Publication Ethics ·
- Reviewers Acknowledgment ·
- Submit a Manuscript ·
- Subscription Information ·
- Table of Contents
International Journal of Alzheimer's Disease
Volume 2012 (2012), Article ID 685739, 11 pages
Microglial Amyloid-1-40 Phagocytosis Dysfunction Is Caused by High-Mobility Group Box Protein-1: Implications for the Pathological Progression of Alzheimer’s Disease
1Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan
2Department of Molecular Cell Physiology, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan
3Department of Neurology, Mie University, Graduate School of Medicine, Tsu 514-8507, Japan
4Department of Neurology, School of Medicine, Sapporo Medical University, S1W16, Chuo-ku, Sapporo 060-8543, Japan
Received 30 November 2011; Accepted 24 February 2012
Academic Editor: Akio Suzumura
Copyright © 2012 Kazuyuki Takata et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
In Alzheimer disease (AD) patient brains, the accumulation of amyloid- (A) peptides is associated with activated microglia. A is derived from the amyloid precursor protein; two major forms of A, that is, A1-40 (A40) and A1-42 (A42), exist. We previously reported that rat microglia phagocytose A42, and high mobility group box protein 1 (HMGB1), a chromosomal protein, inhibits phagocytosis. In the present study, we investigated the effects of exogenous HMGB1 on rat microglial A40 phagocytosis. In the presence of exogenous HMGB1, A40 markedly increased in microglial cytoplasm, and the reduction of extracellular A40 was inhibited. During this period, HMGB1 was colocalized with A40 in the cytoplasm. Furthermore, exogenous HMGB1 inhibited the degradation of A40 induced by the rat microglial cytosolic fraction. Thus, extracellular HMGB1 may internalize with A40 in the microglial cytoplasm and inhibit A40 degradation by microglia. This may subsequently delay A40 clearance. We further confirmed that in AD brains, the parts of senile plaques surrounded by activated microglia are composed of A40, and extracellular HMGB1 is deposited on these plaques. Taken together, microglial A phagocytosis dysfunction may be caused by HMGB1 that accumulates extracellularly on A plaques, and it may be critically involved in the pathological progression of AD.
Alzheimer’s disease (AD) is characterized by the deposition of amyloid- (A) plaques, accumulation of neurofibrillary tangles (NFTs), and loss of synapses and neurons in particular brain areas . Experimental studies using transgenic AD mouse models have demonstrated that A accelerates NFT formation [2, 3] and is closely associated with synaptic damage . In contrast, A reduction in the brain by A immunization restores cognitive functions in transgenic AD mouse models [5–9] and also appears to slow cognitive decline in human AD patients . Thus, the accumulation of A may play a key role in the pathogenesis of AD .
A is derived from the sequential proteolysis of amyloid precursor protein (APP) by - and γ-secretases and is composed of 37–43 amino acid residues because γ-secretase, which is a protein complex including presenilin (PS), generates the C-terminal of A with different lengths . Among the variations in A, A1-40 (A40) and A1-42 (A42) are the major species found in AD brains. The most predominant species deposited in A plaques is A42 , which is prone to aggregation  and indicates increased neurotoxicity . On the other hand, A40 is the major soluble species; its secretion is 10-fold more than that of A42 in normal brains. A previous study demonstrated that the deposition of A40 in AD brains is particularly correlated with synaptic and neuronal loss . Thus, lowering the concentration of A40 and A42 in the brain may serve as a disease-modifying therapy for AD patients.
Activated microglia accumulate on A plaques in AD brains. Although microglial accumulation was initially believed to be involved in the formation of A plaques , experimental studies later demonstrated the ability of microglia to phagocytose A peptides [18, 19]. In addition, we demonstrated microglial contribution in A42 clearance using primary cultured rat microglia [20–25]. However, it has been reported that microglial dystrophy occurs in aging human brains , and the age-related disability of microglial A phagocytosis has been demonstrated experimentally . Thus, the dysfunction of microglial A phagocytosis appears to be closely involved in the progression of AD pathology.
High-mobility group box protein 1 (HMGB1) is an abundant nonhistone chromosomal protein that is released from cells undergoing necrosis [28, 29]. The released HMGB1 behaves like an inflammatory mediator by acting on receptor for advanced glycation end products (RAGE) and Toll-like receptors (TLRs) 2 and 4 [30, 31]. We have previously reported that HMGB1 is extracellularly associated with A plaques in AD brain and is involved in the pathogenesis of AD as an inhibitory factor against microglial A42 phagocytosis by interfering with uptake [32, 33]. However, the effect of extracellular HMGB1 on the microglial phagocytosis of A40, but not A42, has not been elucidated. Therefore, in the present study, we analyzed rat microglial A40 phagocytosis in the presence and absence of exogenous HMGB1.
2. Materials and Methods
2.1. Primary Culture of Rat Microglia and Drug Treatment
The primary culture experimental procedure was reviewed and approved by the Committee for Animal Research at Kyoto Pharmaceutical University. Primary cultured microglia (over 97% pure) were prepared, as described previously . Briefly, brain tissues were isolated from newborn Wistar rats, minced, and gently dissociated by trituration in Dulbecco’s modified Eagle medium (DMEM). The tissue suspension was filtered through a 50 μm diameter nylon mesh into 50 mL tubes, and cells were collected by centrifugation at 200 ×g for 10 min. Cells were resuspended in DMEM with 10% fetal calf serum, 100 units/mL penicillin, and 100 μg/mL streptomycin; they were then plated onto 100 mm diameter dishes at 37°C in humidified 5% CO2/95% air. We then harvested the floating microglia from mixed glial cultures. Microglia were transferred to 24-well plates (3.0 × 105 cells/well) and were allowed to adhere at 37°C overnight; they were then treated with sterilized phosphate-buffered saline (PBS) as the vehicle or synthetic human As (A40 or A42; Anaspec, San Jose, CA) in the presence or absence of calf thymus-purified HMGB1 (WAKO Chemicals, Osaka, Japan). We previously demonstrated that 1 μM A42 for 12 h markedly phagocytosed by rat microglia , and 0.3 μM HMGB1 inhibits the phagocytosis [32, 33]. When A40 at 1–3 μM were added into the culture, we could detect A40 phagocytosed by rat microglia by Western blot analysis . Therefore, in the present study, we adopted the concentrations at 1 μM and 0.3 μM for the treatments with As and HMGB1, respectively. To make the experimental conditions more accurate, we dissolved the lyophilized human A peptides in distilled and sterilized water at a high concentration, and small aliquots were kept at −80°C until use. Subsequently, A stock solutions were diluted using sterilized PBS, and once A was thawed, no A was refrozen to eliminate variance due to repeated freezing and thawing.
Twelve hours after A treatment, microglia were gently rinsed three times with PBS and then fixed with 4% paraformaldehyde in 100 mM phosphate buffer (PB) for 30 min. Cells were then incubated with a mouse monoclonal antibody against A (clone 6E10, 1 : 1000; Chemicon, Temecula, CA) and a rabbit polyclonal antibody against HMGB1 (1 : 1000; BD Pharmingen, San Diego, CA). The primary antibodies were followed by application of a rhodamine-labeled anti-mouse immunoglobulin (Ig)G antibody and fluorescein isothiocyanate-labeled anti-rabbit IgG antibody (each diluted 1 : 500; Molecular Probes, Eugene, OR). Furthermore, cells were incubated with Hoechst 33258 (1 : 5000; Molecular Probes) to visualize microglial nuclei. Labeled fluorescence was detected using a laser scanning confocal microscope LSM 510 (Carl Zeiss, Jena, Germany).
2.3. A Phagocytosis and Clearance Assay by Western Blot Analysis
Twelve hours after A treatment, microglia and culture media were collected and lysed with Laemmli’s sample buffer and then analyzed by Western blot analysis. Briefly, samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 20% polyacrylamide gels in Tricine buffer). Proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA) by electroelution and then incubated with a mouse monoclonal antibody against A (clone 6E10, 1 : 2000; Chemicon), followed by a horseradish peroxidase-linked secondary antibody against mouse IgG (1 : 1,000; Amersham, Buckinghamshire, UK). Subsequently, protein bands were detected on radiographic films (Kodak, Rochester, NY) using a chemiluminescence kit (ECL kit; Amersham). For semiquantitative analysis, radiographic films were scanned with a CCD color scanner (DuoScan, AGFA, Mortsel, Belgium) and then analyzed densitometrically using the public domain US National Institutes of Health image 1.56 program.
2.4. A Degradation Assay
Microglia were harvested and resuspended in 100 mM Tris-HCl buffer (pH 7.5) containing 10 mM KCl, 1.5 mM MgCl2, and 1 mM DTT and then homogenized. After centrifugation (50,000 ×g) for 20 min at 4°C, the protein concentration of the supernatant was measured and used as the microglial cytosolic fraction. The A peptide (3 μM A40 or A42) was incubated with the microglial cytosolic fraction (final concentration of 1 mg/mL) in the presence or absence of 0.3 μM HMGB1. At the time points of 0, 6, and 12 h after incubation, Laemmli’s sample buffer was added, and samples were boiled at 100°C for 5 min to stop A degradation. Subsequently, samples were analyzed using the antibody against A (clone 6E10, 1 : 2000; Chemicon) by Western blot analysis, as described previously.
HMGB1 (1.5 μg, 2.6 μM) was mixed with 3 μg of synthetic A40 (37.5 μM) in PBS. Twenty-four hours after incubation, the antibody (10 μg of IgG) against HMGB1 (BD Pharmingen) or A (clone 6E10; Chemicon) was added to the mixture and further incubated for 2 h at 4°C. Protein A-Sepharose (50 μL of a 50% slurry) was then added, and the mixture was incubated overnight at 4°C. After centrifugation, immunoprecipitates were resuspended in Laemmli’s sample buffer. Subsequently, samples were analyzed using the antibody against HMGB1 (1 : 1000; BD Pharmingen) or A (clone 6E10, 1 : 2000; Chemicon) by Western blot analysis, as described previously.
2.6. Immunohistochemical Study Using Human AD Brain Sections
All experiments using human samples were performed in accordance with the guidelines of the ethical committees of Kyoto Pharmaceutical University. Informed consent was obtained from all subjects. For histological examination, frontal cortex tissue from a patient who was clinically and histopathologically diagnosed as human AD (age, 67 years) was used. Neuropathological assessment of AD was conducted in accordance with the criteria of the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD). Dissected tissue blocks were fixed in 10% formalin and transferred to a 15% sucrose solution in 100 mM PB containing 0.1% sodium azide at 4°C. The cryoprotected brain blocks were cut into 20 μm sections on a cryostat, and the collected sections were stored in PBS containing 0.3% Triton X-100 (PBS-T) and 0.1% sodium azide at 4°C until use.
Immunohistochemical study was essentially performed as described previously . Free-floating human brain sections were treated with 0.1% hydrogen peroxide for 30 min to quench endogenous peroxidase activity; they were then incubated with 1% goat serum to block nonspecific binding in PBS. Sections were then incubated with a mouse monoclonal antibody against A40 (1 : 1000; nanoTools, Teningen, Germany) and rabbit polyclonal antibody against A42 (1 : 1000; IBL, Gunma, Japan), a rabbit polyclonal antibody against A40 (1 : 1000; IBL) and mouse monoclonal antibody against human leukemia antigen (HLA)-DR (1 : 50; Dako, Glostrup, Denmark), or a rabbit polyclonal antibody against A42 (1 : 1000; IBL) and mouse monoclonal antibody against HLA-DR (1 : 50; Dako) in PBS-T with 0.1% sodium azide for 4 days at 4°C. After washing with PBS-T, the sections were incubated with biotinylated anti-rabbit IgG antibody (1 : 2000; Vector Laboratories, Burlingame, CA) for 2 h at room temperature. The sections were then incubated with avidin peroxidase (1 : 4000; ABC Elite Kit; Vector Laboratories) for 1 h at room temperature. Subsequently, labeling was visualized by incubation with 50 mM Tris-HCl buffer (pH 7.6) containing 0.02% 3,3′-diaminobenzidine (DAB) and 0.0045% hydrogen peroxide with nickel enhancement using 0.6% nickel ammonium sulfate, which yielded a dark blue color. In the second cycle, sections were incubated with a biotinylated anti-mouse IgG antibody (1 : 2000; Vector Laboratories) for 2 h at room temperature. The sections were then incubated with avidin peroxidase (1 : 4000; ABC Elite Kit; Vector Laboratories) for 1 h at room temperature. Subsequently, the DAB reaction was performed without nickel ammonium sulfate, which yielded a brown color.
In laser confocal microscopic analysis, human AD brain sections were treated with 1% goat serum to block nonspecific binding in PBS. Sections were then coincubated with a rabbit polyclonal antibody against A40 (1 : 1000; IBL) and a mouse monoclonal antibody against HLA-DR (1 : 50; Dako) or a mouse monoclonal antibody against A40 (1 : 1000; nanoTools) and a rabbit polyclonal antibody against HMGB1 (1 : 1000; BD Pharmingen) in PBS-T with 0.1% sodium azide for 4 days at 4°C. The primary antibodies were probed with Alexa Fluor 546-labeled anti-rabbit IgG antibody and Alexia Fluor 488-labeled anti-mouse IgG antibody or Alexa Fluor 546-labeled anti-mouse IgG antibody and Alexia Fluor 488-labeled anti-rabbit IgG antibody (each diluted 1 : 500; Molecular Probes). Subsequently, fluorescence was observed using a laser scanning confocal microscope LSM 510 (Carl Zeiss).
2.7. Statistical Evaluation
Results of the densitometric analysis are given as the mean ± standard error of mean. The statistical significance of differences was determined by analysis of variance. Further statistical analysis for post hoc comparisons was conducted using the Bonferroni/Dunn test (StatView, Abacus Concepts, Berkeley, CA).
3.1. Binding of HMGB1 with A40
In our previous study, we found that HMGB1 is extracellularly accumulated on A plaques in AD brains and further demonstrated that HMGB1 binds to A42 in in vitro cell-free study . In the present cell-free study, we first examined the binding affinity of HMGB1 for A40. Following incubation of the HMGB1 peptide alone, a 29 kDa band of HMGB1 and its high-molecular-weight aggregates was detected, while an approximately 33 kDa band (arrow in Figure 1()), which is believed to be a complex of HMGB1 and A40, appeared as an upper band 6 h after incubation of HMGB1 and A40 peptides (Figure 1()). Following incubation with A40 (Figure 1()), monomers and oligomers of A40 were the major components present in the absence of HMGB1 at each time point. Predictably, the 33 kDa band, which seemed to be a complex of HMGB1 and A40, was detected by the addition of the HMGB1 peptide (arrow in Figure 1()).
To confirm the binding affinity between HMGB1 and A40, we further examined immunoprecipitation using the anti-HMGB1 antibody (Figure 1()) or anti-A antibody (Figure 1()). As a result, in the mixture of HMGB1 and A, the complex of HMGB1 and A40 (approximately 33 kDa) was immunoprecipitated with A-Sepharose-linked antibodies against HMGB1 (Figure 1()) or A (Figure 1()). These results demonstrated that HMGB1 had a binding affinity for A40.
3.2. Microglial A Phagocytosis and Effect of Exogenous HMGB1
We previously demonstrated that microglia markedly phagocytose A42 , and extracellular HMGB1 inhibits phagocytosis on the cell surface [32, 33]. In the present study, we analyzed the microglial A40 phagocytosis and the effects of extracellular HMGB1 on phagocytosis using laser confocal microscopy (Figure 2). Endogenous HMGB1 was detected in the nuclei of primary cultured rat microglia (Figures 2()–2(), cyan). When treated with the vehicle (Figure 2()) or HMGB1 alone (Figure 2()), no A immunoreactivity was detected. Consistent with previous studies, in the presence of A42, microglia phagocytosed A42 (Figure 2(), red), exogenous HMGB1 was colocalized with A42 on the microglial cell surface, and A internalization was inhibited (Figure 2(), yellow). When treated with A40, the immunoreactivity of A40 was barely detected in the microglial cytoplasm (Figure 2(), red). Interestingly, in the presence of exogenous HMGB1, small vesicle-like immunoreactivities of A40 (Figure 2(), red) and HMGB1 (Figure 2(), green) were markedly increased in the microglial cytoplasm, and parts of them were colocalized with each other (Figure 2(), yellow).
3.3. Amounts of A40 inside and outside Microglia and Effect of Exogenous HMGB1
Twelve hours after A40 treatment, microglial cell lysate and conditioned medium were collected and subjected to Western blot analysis; semiquantitative analysis was then examined to measure the concentration of A40 inside (Figure 3(a)) and outside microglia (Figure 3(b)). When microglia were treated with the vehicle or exogenous HMGB1 alone, no A40 immunoreactivity was detected inside them (Figure 3(a)). After treatment with A40, a small amount of A40 was detected inside microglia (A40 phagocytosed by microglia), and this amount increased dramatically by simultaneous treatment with exogenous HMGB1 (Figure 3(a)). This result raises two possibilities: (i) extracellular HMGB1 increases microglial A40 uptake, and (ii) HMGB1 inhibits the degradation of A40 in the microglial cytoplasm after uptake. To address these possibilities, we measured the amount of A40 in the culture medium (A40 remaining outside microglia) (Figure 3(b)). After treatment with the vehicle or exogenous HMGB1 alone, no A was detected in the culture medium. Twelve hours after A40 treatment, the amount of A40 in the culture medium significantly increased by simultaneous treatment with exogenous HMGB1. Thus, in the presence of exogenous HMGB1, the amount of A40 both inside and outside microglia was much higher than that when treated with A40 alone. These results suggest that exogenous HMGB1 phagocytosed by microglia inhibits the degradation of A40 in the microglial cytoplasm and subsequently delays A clearance by microglia.
3.4. A Degradation with the Microglial Cytosol Fraction and Effect of Exogenous HMGB1
To confirm whether exogenous HMGB1 inhibits A40 degradation in microglial cytoplasm, we prepared cytosolic fractions from rat microglia and mixed them with A. Degradation of A40 and A42 by microglial cytosol fractions was compared (Figure 4(a)). A40 and A42 were gradually degraded by the addition of the microglial cytosolic fraction in a time-dependent manner. A40 was degraded earlier than A42 (Figure 4(a)). We next examined the effect of exogenous HMGB1 on the A40 degradation induced by the microglial cytosolic fraction (Figure 4(b)). At 6 and 24 h after incubation, the degradation of A40 was significantly delayed by the addition of exogenous HMGB1. Thus, this result suggests that exogenous HMGB1 phagocytosed by microglia inhibits the degradation of A40 in the microglial cytoplasm.
3.5. Accumulation of A40, A42, and Microglia in AD Brains
We further investigated the localization of A40 and A42 in AD brains using specific antibodies (Figures 5(a) and 5(b)) and microglial accumulation on the plaques composed of A40 (Figures 5(c) and 5(d)) and A42 (Figures 5(e) and 5(f)). The number of A40 plaques (dark blue deposits in Figure 5(a)) was lesser than that of A42 plaques (brown deposits in Figure 5(a)). High-magnification photographs revealed that A40 accumulated on A42 plaques (Figure 5(b)). Regarding microglial accumulations (Figures 5(c)–5(f)), almost all A40 plaques were markedly surrounded by activated microglia (Figure 5(c) and arrows in Figure 5(d)). Although some A42 plaques were markedly accumulated by microglia (Figure 5(e) and arrow in Figure 5(f)), others were moderately or poorly surrounded by microglia (arrowheads in Figure 5(f)).
3.6. Accumulation of HMGB1 and Microglia on A40 Plaques in AD Brains
We previously demonstrated that extracellular HMGB1 accumulates on A plaques, as detected using an anti-A antibody that reacts with a broad spectrum of A species . Therefore, in the present study, we investigated the colocalization of extracellular HMGB1 on A40 plaques in AD brains using a specific anti-A40 antibody. Consistent with the immunohistochemical study (Figures 5(c) and 5(d)), microglia (Figure 6(b)) markedly accumulated on A40 plaques (Figure 6(a)) in AD brains (Figure 6(c)). We further demonstrated that extracellular HMGB1 (Figure 6(e)) colocalized with A40 plaques (Figure 6(d)) in AD brains (Figure 6(f)).
In studies on familial AD, mutations in the APP, PS1, and PS2 genes have been detected, and transgenic mice models carrying these familial AD-linked mutations show enhanced A production in their brains. In particular, transgenic mice carrying the APP mutation display characteristics that closely resemble AD, such as A deposition and memory dysfunction [35, 36], and introduction of the double mutations of PS/APP exhibits the early onset of these pathologies . Thus, all mutations are involved in A generation, and the accumulation of A in the brain has been strongly suggested to be the primary event driving the pathogenesis of AD. However, familial AD accounts for less than 1% of all AD cases ; most cases develop sporadically. Although the etiology of sporadic AD remains much more elusive than that of familial cases, neurological and pathological events in sporadic AD are essentially indistinguishable from those in familial cases. In sporadic AD, a decreased A clearance rate has been reported .
One proposed mechanism of A clearance is microglial A phagocytosis [40, 41]. Reports on AD patients treated with A immunization also indicate microglial contribution to A clearance in human AD brains [42, 43]. However, it has been suggested that the ability of microglia to clear A decreases with age and progression of AD pathology [26, 27], and it may, at least in part, account for the dysregulation of A clearance in sporadic AD.
HMGB1 inhibits microglial A42 phagocytosis by interfering with A42 internalization [32, 33]. In the present study, we further showed that exogenous HMGB1 inhibits the degradation of A40 in rat microglial cytoplasm and subsequently delays A40 clearance. We demonstrated the binding affinities of HMGB1 for A40 and A42 . A contains an amino acid sequence (18VFFA21) that has been identified to be essential for aggregation and fibril formation . Interestingly, HMGB1 contains a homologous motif (16AFFV19), and this sequence is thought to be critically involved in the interactions of A with HMGB1 [33, 45]. Among the many peptidases that have been proposed as A-degrading enzymes , insulin-degrading enzyme, cathepsin D, and neprilysin are the principle enzymes involved in microglia-mediated A degradation [25, 47, 48]. Many cleavage sites that are the targets of microglial A-degrading enzymes are located on and in the vicinity of the amino acid sequence (18VFFA21) of A . Therefore, we speculate that the cleavage sites of A are masked by the binding of HMGB1; subsequently, the degradation of A40 may be inhibited in the microglial cytoplasm. In case of A42, A42 itself forms high-molecular-weight fibrils during incubation . Therefore, the binding of HMGB1 may stabilize A42 fibril formation, and high-molecular-weight complex of HMGB1 and A42 fibril may interrupt the uptake of A42 by microglia. Thus, extracellular HMGB1 may serve as a chaperone protein for A and inhibit microglial A clearance by interrupting A40 degradation and A42 internalization by microglia. On the other hand, RAGE, TLR2, and TLR4 are receptors for HMGB1 [30, 31]; they are also involved in microglial A phagocytosis [49, 50]. Therefore, there is a possibility that the interactions of HMGB1 with these receptors on microglia may be related to the inhibitory events on A.
Consistent with a previous study , plaques containing A42 predominantly existed in AD brains, and A40 accumulated on parts of A42 plaques. Despite the restricted distribution of A40, almost all plaques containing A40 were markedly surrounded by activated microglia. We previously reported that small oligomers formed by A40 strongly induce rat microglial reactions such as cytokine production . Thus, A40 may play an important role in microglial activation and/or recruitment on A plaques. However, we have found that the level of HMGB1 was significantly increased in AD brains , and extracellular HMGB1 accumulated on A plaques. Therefore, in AD brains, microglial degradation of A40 and uptake of A42 may be inhibited by extracellular HMGB1 despite the marked accumulation of reactive microglia on A plaques. Moreover, in the present study, we demonstrated that A40 is more readily degraded by the microglial cytosolic fraction than A42. However, in the presence of exogenous HMGB1, the degradation of A40 by microglia is inhibited, and a lot of A40 granules are existed in the cytoplasm of rat microglia as shown in Figure 2(). Interestingly, numerous microglia containing A40 granules, but not A42, have also been detected in AD brains . Thus, this event in AD brain is well replicated by the treatment with A40 in the presence of extracellular HMGB1 in primary-cultured rat microglia. Results suggest that our findings in the effect of HMGB1 on rat microglia may reflect on the pathological event induced in AD brain and are expected the critical implication of extracellular HMGB1 in the progression of AD pathologies. In addition, we have postulated that the origin of extracellular HMGB1 is leakage from dead neurons during the progression of AD , like ischemic neurodegeneration . Extracellular HMGB1 leaked from dead neurons may then accumulate on A plaques through its binding affinity for A in AD brains.
It has been reported that the released HMGB1 is involved in the pathologies of various inflammatory-related disease . In ischemic stroke  and intracerebral hemorrhage especially , extracellular HMGB1 is suggested to exacerbate brain insult through the disruption of the blood-brain barrier (BBB), overfacilitation of microglia, and intense production of proinflammatory molecules. These studies also demonstrated that a neutralizing anti-HMGB1 monoclonal antibody and glycyrrhizin which bind to and inhibit cytokine-like activity of HMGB1 attenuate the brain insult induced by transient ischemia and intracerebral hemorrhage in rat, respectively. Therefore, there is a possibility that the neutralizing anti-HMGB1 monoclonal antibody and glycyrrhizin may bind to the extracellular HMGB1 accumulated on the A plaques in the AD brain, cancel the inhibitory effects of HMGB1 on microglial A phagocytosis, and then may provide novel therapeutic options for the AD treatment.
In conclusion, in the present study, we found that HMGB1 extracellularly accumulates on A plaques containing A40 in AD brains. We further demonstrated that HMGB1 has a binding affinity for A40, and exogenous HMGB1 is internalized into rat microglial cytoplasm with A40 and inhibits A40 degradation. Subsequently, exogenous HMGB1 delays A40 clearance in the culture medium. Thus, these results suggest that extracellular HMGB1 attenuates microglial A clearance and is possibly involved in the progression of AD pathology.
The authors thank Toshiyuki Kawasaki for his technical assistance. This study was supported by the Frontier Research Programs of the Ministry of Education, Culture, Sports, Science and Technology of Japan; Grants-in-Aid from the Japan Society for the Promotion of Science; and Kyoto Pharmaceutical University Fund for the Promotion of Scientific Research.
- D. J. Selkoe, “Alzheimer's disease is a synaptic failure,” Science, vol. 298, no. 5594, pp. 789–791, 2002.
- J. Götz, F. Chen, J. Van Dorpe, and R. M. Nitsch, “Formation of neurofibrillary tangles in P301L tau transgenic mice induced by Aβ42 fibrils,” Science, vol. 293, no. 5534, pp. 1491–1495, 2001.
- J. Lewis, D. W. Dickson, W. L. Lin et al., “Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP,” Science, vol. 293, no. 5534, pp. 1487–1491, 2001.
- K. Takata, Y. Kitamura, Y. Nakata et al., “Involvement of WAVE accumulation in Aβ/APP pathology-dependent tangle modification in Alzheimer's disease,” American Journal of Pathology, vol. 175, no. 1, pp. 17–24, 2009.
- F. Bard, C. Cannon, R. Barbour et al., “Peripherally administered antibodies against amyloid β-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease,” Nature Medicine, vol. 6, no. 8, pp. 916–919, 2000.
- J. C. Dodart, K. R. Bales, K. S. Gannon et al., “Immunization reverses memory deficits without reducing brain Aβ burden in Alzheimer's disease model,” Nature Neuroscience, vol. 5, no. 5, pp. 452–457, 2002.
- C. Janus, J. Pearson, J. McLaurin et al., “Aβ peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer's disease,” Nature, vol. 408, no. 6815, pp. 979–982, 2000.
- D. Morgan, D. M. Diamond, P. E. Gottschall et al., “A β peptide vaccination prevents memory loss in an animal model of Alzheimer's disease,” Nature, vol. 408, no. 6815, pp. 982–985, 2000.
- D. Schenk, R. Barbour, W. Dunn et al., “Immunization with amyloid-β attenuates Alzheimer disease-like pathology in the PDAPP mouse,” Nature, vol. 400, no. 6740, pp. 173–177, 1999.
- C. Hock, U. Konietzko, J. R. Streffer et al., “Antibodies against β-amyloid slow cognitive decline in Alzheimer's disease,” Neuron, vol. 38, no. 4, pp. 547–554, 2003.
- J. Hardy and D. J. Selkoe, “The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics,” Science, vol. 297, no. 5580, pp. 353–356, 2002.
- J. Wiltfang, H. Esselmann, M. Bibl et al., “Highly conserved and disease-specific patterns of carboxyterminally truncated Aβ peptides 1-37/38/39 in addition to 1-40/42 in Alzheimer's disease and in patients with chronic neuroinflammation,” Journal of Neurochemistry, vol. 81, no. 3, pp. 481–496, 2002.
- T. Iwatsubo, A. Odaka, N. Suzuki, H. Mizusawa, N. Nukina, and Y. Ihara, “Visualization of Aβ42(43) and Aβ40 in senile plaques with end-specific Aβ monoclonals: evidence that an initially deposited species is Aβ42(43),” Neuron, vol. 13, no. 1, pp. 45–53, 1994.
- J. T. Jarrett, E. P. Berger, and P. T. Lansbury Jr., “The carboxy terminus of the β amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Alzheimer's disease,” Biochemistry, vol. 32, no. 18, pp. 4693–4697, 1993.
- K. N. Dahlgren, A. M. Manelli, W. Blaine Stine, L. K. Baker, G. A. Krafft, and M. J. Ladu, “Oligomeric and fibrillar species of amyloid-β peptides differentially affect neuronal viability,” Journal of Biological Chemistry, vol. 277, no. 35, pp. 32046–32053, 2002.
- L. F. Lue, Y. M. Kuo, A. E. Roher et al., “Soluble amyloid β peptide concentration as a predictor of synaptic change in Alzheimer's disease,” American Journal of Pathology, vol. 155, no. 3, pp. 853–862, 1999.
- L. S. Perlmutter, E. Barron, and H. C. Chui, “Morphologic association between microGlia and senile plaque amyloid in Alzheimer's disease,” Neuroscience Letters, vol. 119, no. 1, pp. 32–36, 1990.
- D. M. Paresce, R. N. Ghosh, and F. R. Maxfield, “MicroGlial cells internalize aggregates of the Alzheimer's disease amyloid β-protein via a scavenger receptor,” Neuron, vol. 17, no. 3, pp. 553–565, 1996.
- D. M. Paresce, H. Chung, and F. R. Maxfield, “Slow degradation of aggregates of the Alzheimer's disease amyloid β- protein by microGlial cells,” Journal of Biological Chemistry, vol. 272, no. 46, pp. 29390–29397, 1997.
- K. Takata, Y. Kitamura, M. Saeki et al., “Galantamine-induced amyloid-β clearance mediated via stimulation of microGlial nicotinic acetylcholine receptors,” Journal of Biological Chemistry, vol. 285, no. 51, pp. 40180–40191, 2010.
- K. Takata, C. Hirata-Fukae, A. G. Becker et al., “Deglycosylated anti-amyloid beta antibodies reduce microGlial phagocytosis and cytokine production while retaining the capacity to induce amyloid beta sequestration,” European Journal of Neuroscience, vol. 26, no. 9, pp. 2458–2468, 2007.
- K. Takata, Y. Kitamura, D. Yanagisawa et al., “MicroGlial transplantation increases amyloid-β clearance in Alzheimer model rats,” FEBS Letters, vol. 581, no. 3, pp. 475–478, 2007.
- Y. Kitamura, K. Shibagaki, K. Takata et al., “Involvement of Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE) and Rac1 in the phagocytosis of amyloid-β(1-42) in rat microGlia,” Journal of Pharmacological Sciences, vol. 92, no. 2, pp. 115–123, 2003.
- K. Takata, Y. Kitamura, D. Tsuchiya, T. Kawasaki, T. Taniguchi, and S. Shimohama, “Heat shock protein-90-induced microGlial clearance of exogenous amyloid-β1-42 in rat hippocampus in vivo,” Neuroscience Letters, vol. 344, no. 2, pp. 87–90, 2003.
- J. I. Kakimura, Y. Kitamura, K. Takata et al., “MicroGlial activation and amyloid-β clearance induced by exogenous heat-shock proteins,” FASEB Journal, vol. 16, no. 6, pp. 601–603, 2002.
- W. J. Streit, N. W. Sammons, A. J. Kuhns, and D. L. Sparks, “Dystrophic MicroGlia in the Aging Human Brain,” Glia, vol. 45, no. 2, pp. 208–212, 2004.
- S. E. Hickman, E. K. Allison, and J. El Khoury, “MicroGlial dysfunction and defective β-amyloid clearance pathways in aging alzheimer's disease mice,” Journal of Neuroscience, vol. 28, no. 33, pp. 8354–8360, 2008.
- S. Müller, P. Scaffidi, B. Degryse et al., “The double life of HMGB1 chromatin protein: architectural factor and extracellular signal,” EMBO Journal, vol. 20, no. 16, pp. 4337–4340, 2001.
- P. Scaffidi, T. Misteli, and M. E. Bianchi, “Release of chromatin protein HMGB1 by necrotic cells triggers inflammation,” Nature, vol. 418, no. 6894, pp. 191–195, 2002.
- J. S. Park, D. Svetkauskaite, Q. He et al., “Involvement of toll-like receptors 2 and 4 in cellular activation by high mobility group box 1 protein,” Journal of Biological Chemistry, vol. 279, no. 9, pp. 7370–7377, 2004.
- R. Kokkola, A. Andersson, G. Mullins et al., “RAGE is the major receptor for the proinflammatory activity of HMGB1 in rodent macrophages,” Scandinavian Journal of Immunology, vol. 61, no. 1, pp. 1–9, 2005.
- K. Takata, Y. Kitamura, D. Tsuchiya, T. Kawasaki, T. Taniguchi, and S. Shimohama, “High mobility group box protein-1 inhibits microGlial Aβ clearance and enhances Aβ neurotoxicity,” Journal of Neuroscience Research, vol. 78, no. 6, pp. 880–891, 2004.
- K. Takata, Y. Kitamura, J. I. Kakimura et al., “Role of high mobility group protein-1 (HMG1) in amyloid-β homeostasis,” Biochemical and Biophysical Research Communications, vol. 301, no. 3, pp. 699–703, 2003.
- Y. Kitamura, S. Shimohama, W. Kamoshima et al., “Alteration of proteins regulating apoptosis, Bcl-2, Bcl-x, Bax, Bak, Bad, ICH-1 and CPP32, in Alzheimer's disease,” Brain Research, vol. 780, no. 2, pp. 260–269, 1998.
- D. Games, D. Adams, R. Alessandrini et al., “Alzheimer-type neuropathology in transgenic mice overexpressing V717F β-amyloid precursor protein,” Nature, vol. 373, no. 6514, pp. 523–527, 1995.
- K. Hsiao, P. Chapman, S. Nilsen et al., “Correlative memory deficits, Aβ elevation, and amyloid plaques in transgenic mice,” Science, vol. 274, no. 5284, pp. 99–102, 1996.
- L. Holcomb, M. N. Gordon, E. Mcgowan et al., “Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes,” Nature Medicine, vol. 4, no. 1, pp. 97–100, 1998.
- D. Campion, C. Dumanchin, D. Hannequin et al., “Early-onset autosomal dominant Alzheimer disease: prevalence, genetic heterogeneity, and mutation spectrum,” American Journal of Human Genetics, vol. 65, no. 3, pp. 664–670, 1999.
- K. G. Mawuenyega, W. Sigurdson, V. Ovod et al., “Decreased clearance of CNS β-amyloid in Alzheimer's disease,” Science, vol. 330, no. 6012, p. 1774, 2010.
- A. R. Simard, D. Soulet, G. Gowing, J. P. Julien, and S. Rivest, “Bone marrow-derived microGlia play a critical role in restricting senile plaque formation in Alzheimer's disease,” Neuron, vol. 49, no. 4, pp. 489–502, 2006.
- J. El Khoury, M. Toft, S. E. Hickman et al., “Ccr2 deficiency impairs microGlial accumulation and accelerates progression of Alzheimer-like disease,” Nature Medicine, vol. 13, no. 4, pp. 432–438, 2007.
- D. Boche, N. Denham, C. Holmes, and J. A. R. Nicoll, “Neuropathology after active Aβ42 immunotherapy: implications for Alzheimer's disease pathogenesis,” Acta Neuropathologica, vol. 120, no. 3, pp. 369–384, 2010.
- J. A. R. Nicolll, D. Wilkinson, C. Holmes, P. Steart, H. Markham, and R. O. Weller, “Neuropathology of human Alzheimer disease after immunization with amyloid-β peptide: a case report,” Nature Medicine, vol. 9, no. 4, pp. 448–452, 2003.
- L. O. Tjernberg, D. J. E. Callaway, A. Tjernberg et al., “A molecular model of Alzheimer amyloid β-peptide fibril formation,” Journal of Biological Chemistry, vol. 274, no. 18, pp. 12619–12625, 1999.
- J. Kallijärvi, M. Haltia, and M. H. Baumann, “Amphoterin includes a sequence motif which is homologous to the Alzheimer's β-amyloid peptide (Aβ), forms amyloid fibrils in vitro, and binds avidly to Aβ,” Biochemistry, vol. 40, no. 34, pp. 10032–10037, 2001.
- N. Iwata, M. Higuchi, and T. C. Saido, “Metabolism of amyloid-β peptide and Alzheimer's disease,” Pharmacology and Therapeutics, vol. 108, no. 2, pp. 129–148, 2005.
- W. Q. Qiu, D. M. Walsh, Z. Ye et al., “Insulin-degrading enzyme regulates extracellular levels of amyloid β- protein by degradation,” Journal of Biological Chemistry, vol. 273, no. 49, pp. 32730–32738, 1998.
- E. Shimizu, K. Kawahara, M. Kajizono, M. Sawada, and H. Nakayama, “IL-4-induced selective clearance of oligomeric β-amyloid peptide 1-42 by rat primary type 2 microGlia,” Journal of Immunology, vol. 181, no. 9, pp. 6503–6513, 2008.
- L. F. Lue, D. G. Walker, L. Brachova et al., “Involvement of microGlial receptor for advanced glycation endproducts (RAGE)in Alzheimer's disease: identification of a cellular activation mechanism,” Experimental Neurology, vol. 171, no. 1, pp. 29–45, 2001.
- K. Tahara, H. D. Kim, J. J. Jin, J. A. Maxwell, L. Li, and K. I. Fukuchi, “Role of toll-like receptor signalling in Aβ uptake and clearance,” Brain, vol. 129, no. 11, pp. 3006–3019, 2006.
- K. Takata, Y. Kitamura, M. Umeki et al., “Possible involvement of small oligomers of amyloid-β peptides in 15-deoxy-Δ12,14 prostaglandin J2-sensitive microGlial activation,” Journal Pharmacological Sciences, vol. 91, no. 4, pp. 330–333, 2003.
- H. Akiyama, H. Mori, T. Saido, H. Kondo, K. Ikeda, and P. L. McGeer, “Occurrence of the diffuse amyloid β-protein (Aβ) deposits with numerous Aβ-containing Glial cells in the cerebral cortex of patients with Alzheimer's disease,” Glia, vol. 25, no. 4, pp. 324–331, 1999.
- G. Faraco, S. Fossati, M. E. Bianchi et al., “High mobility group box 1 protein is released by neural cells upon different stresses and worsens ischemic neurodegeneration in vitro and in vivo,” Journal of Neurochemistry, vol. 103, no. 2, pp. 590–603, 2007.
- M. T. Lotze and K. J. Tracey, “High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal,” Nature Reviews Immunology, vol. 5, no. 4, pp. 331–342, 2005.
- K. Liu, S. Mori, H. K. Takahashi et al., “Anti-high mobility group box 1 monoclonal antibody ameliorates brain infarction induced by transient ischemia in rats,” FASEB Journal, vol. 21, no. 14, pp. 3904–3916, 2007.
- M. Ohnishi, H. Katsuki, C. Fukutomi et al., “HMGB1 inhibitor glycyrrhizin attenuates intracerebral hemorrhage-induced injury in rats,” Neuropharmacology, vol. 61, no. 5-6, pp. 975–980, 2011.