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. Author manuscript; available in PMC: 2008 Dec 1.
Published in final edited form as: Neurobiol Dis. 2007 Jul 28;28(3):286–292. doi: 10.1016/j.nbd.2007.07.019

A limited role for microglia in antibody mediated plaque clearance in APP mice

Monica Garcia-Alloza 1, Sarah A Dodwell 1, Brian J Ferrara 1, Gregory A Hickey 1, Bradley T Hyman 1, Brian J Bacskai 1
PMCID: PMC2193669  NIHMSID: NIHMS36077  PMID: 17822910

Abstract

Amyloid-β (Aβ) accumulation in senile plaques is a hallmark of Alzheimer’s disease (AD). Immunotherapy is a leading approach for amyloid clearance, despite the early termination of the Elan clinical trial with active immunization due to a few cases of meningoencephalitis. The mechanisms of immunotherapy-mediated amyloid clearance and this deleterious side effect are largely unknown. While clearance of Aβ probably results in part from microglia-mediated inflammation, it can be microglia-independent. Therefore, establishing the role of microglia in Aβ clearance is important for the treatment of AD. We analyzed the effects of direct microglia activation and inhibition on antibody-mediated Aβ clearance. Robust microglia activation with interferon-γ led to modest Aβ clearance alone but did not potentiate antibody mediated clearance. Microglia elimination/inactivation with immunotoxin or minocycline only partially limited antibody induced Aβ clearance suggesting that although there is a role for microglia in Aβ clearance, it does not account for the majority of the effect observed after anti-Aβ antibody treatment.

Keywords: Alzheimer, immunotherapy, multiphoton, imaging, neuritic dystrophy, senile plaque, microglia

INTRODUCTION

Alzheimer’s disease (AD) is a debilitating neurodegenerative disease characterized by intraneuronal neurofibrillary tangles, mostly composed of abnormally phosphorylated tau (Iqbal and Grundke-Iqbal, 2006), and extracellular senile plaques, largely composed of amyloid-β peptide (Hyman and Gomez-Isla, 1997). Several therapeutic strategies have been developed to eliminate excessive Aβ. Active immunization of transgenic mice with Aβ peptides reduced amyloid burden (Schenk et al., 1999) and partially restored behavioral deficits (Janus et al., 2000; Morgan et al., 2000). These experiments led to a clinical trial with active immunization using synthetic Aβ42. However, the clinical trial was discontinued due to meningoencephalitis in 6% of cases without a clear relation to serum anti-Aβ titers (Orgogozo et al., 2003). It seems that an excessive T cell response to the vaccine and microglia activation may have been responsible for the observed inflammation, and these effects should be considered for a safer anti-Aβ immunotherapy for AD (Orgogozo et al., 2003). Other approaches that are less likely to result in autoimmune reactions have been directed towards passive immunization (Morgan, 2006b), and anti-Aβ antibodies can reduce soluble and insoluble Aβ deposits (Bacskai et al., 2001) as well as restore behavioral deficits in transgenic mice (Kotilinek et al., 2002).

Microglia seem to play a key role in Aβ clearance although the underlying mechanisms have not been completely elucidated. Activated microglia cells are found in AD patients and transgenic mouse models, and they are strategically located in close proximity to senile plaques (Bacskai et al., 2001; Stalder et al., 1999). It has also been shown that Aβ itself provokes activation of microglia and astrocytes (Monsonego and Weiner, 2003). Antibody induced Aβ clearance in vitro may be mediated by cellular removal of the deposits through Fc receptor mediated phagocytosis (Bard et al., 2000). However, this route of removal is not necessary since antibody mediated amyloid clearance can occur in the absence of Fc receptor activation, suggesting that multiple mechanisms of amyloid clearance are likely (Bacskai et al., 2002a; Das et al., 2003; Wilcock et al., 2003).. Following this idea it is also possible that antibody-Aβ interaction leads to disaggregation of deposits, and a small fraction of the plaques could be either removed passively with other cerebrospinal fluid components or made available for phagocytosis and clearance by microglia without receptor stimulation. It also needs to be taken into account that the possible beneficial effect of microglia activation in Aβ removal may be counterbalanced by the negative effect of an increased secretion of inflammatory mediators that are potentially toxic to the nearby neurons (Rogers et al., 2002). In this sense, the therapeutic goal would be to suppress the neurodegenerative phenotypes of microglia that secrete pro-inflammatory cytokines without suppressing the beneficial phenotypes implicated in Aβ clearance. In the present work, we describe the effects of direct microglia activation and inactivation on antibody-mediated Aβ clearance in mouse models of amyloid deposition, with relevance to the inflammation observed in both AD patients and transgenic mouse models using active immunization (Rogers et al., 2002). Our results suggest that activation or inhibition of microglia has only a limited effect on antibody mediated clearance of Aβ.

2. MATERIAL AND METHODS

2.1. Animals

APPswe/PS1dE9 mice aged 7–8.5 months were obtained from Jackson Lab (Bar Harbor, Maine) (Garcia-Alloza et al., 2006). All studies were conducted with approved protocol from the Massachusetts General Hospital Animal Care and Use Committee and in compliance with NIH guidelines for the use of experimental animals.

2.2. Reagents

Texas Red dextran 70,000 D, anti Iba-1 antibody (Wako Chemicals, Richmond, VA), anti-rabbit Alexa Fluor 568 (Molecular probes, Eugene, OR), methoxy-XO4 (gift from Dr. Klunk, U. Pittsburgh), 10D5 anti-Aβ antibody (gift from ELAN Pharmaceuticals), anti-Mac-1-saporin (Advanced Targeting Systems, San Diego, CA). Minocycline, interferon-γ (IFN-γ), thioflavin S and common chemical reagents where obtained from Sigma (St. Louis, MO),

2.3 Cranial windows and treatments

Cranial window surgeries were performed as previously described (Skoch et al., 2004). Briefly, animals were anesthetized using isoflurane or avertin, the skin and periosteum were removed and a 6 mm in diameter craniotomy was performed, making the anterior end immediately anterior to Bregma and the posterior end just anterior to Lambda. After the skull was removed, the dura was reflected to the midline to apply the treatments. 8 mm glass windows were installed and secured with dental cement. All animals received an i.p injection of methoxy-XO4 (5 mg/Kg), a fluorescent compound that crosses the blood–brain barrier and binds amyloid plaques, the day before the surgery. To facilitate finding the same sites in the brain between sessions Texas Red dextran (70.000 Da, 62.5 mg/Kg in sterile PBS) was injected into a lateral tail vein to provide a fluorescent angiogram before every imaging session.

Antibody treated animals received 10D5 (25 μl, 1.2 mg/ml) locally applied in the brain surface for 20 minutes as previously described (Bacskai et al., 2001). IFN-γ treated animals received 15 μl (50 ng/ml) in PBS (5% human serum albumin, HSA) locally in the brain surface for 20 minutes. A third group of animals received IFN-γ for 20 minutes followed by a 20 minute incubation with 10D5. For microglia inhibition studies we used anti-Mac-1-saporin or minocycline. Animals treated with anti-Mac-1-saporin received 30 μl (0.5 mg/ml) of the immunotoxin locally applied in the brain surface for 20 minutes followed by antibody treatment with 10D5 for 20 more minutes. A first set of minocycline treated animals received minocycline daily (90 mg/Kg i.p.) for 3 days prior to surgery, and 10D5 was locally applied for 20 minutes on the day of surgery. A second set of animals received minocycline (90 mg/Kg i.p.) daily i.p for 3 days, 10D5 was locally applied for 20 minutes during the cranial window implantation, and animals continued receiving minocyline treatment for 3 more days. All animals were imaged immediately after the cranial window surgery and reimaged 3 and 7 days later.

2.4. Multiphoton imaging and processing

As previously described (McLellan et al., 2003) two-photon fluorescence was generated with 800 nm excitation from a mode-locked Ti:Sapphire laser (MaiTai, Spectra-Physics, Mountain View, CA) mounted on a multiphoton imaging system (Bio-Rad 1024ES, Bio-Rad, Hercules, CA). A custom-built external detector containing three photomultiplier tubes (Hamamatsu Photonics, Bridgewater, NJ) collected emitted light in the range 380–480, 500–540 and 560–650 nm. In vivo imaging was performed using the normal scan speed and multiple z-series were collected using a 20X water immersion objective (Olympus, NA=0.95, field of view =615×615 μm; z-step, 5 μm, depth, 200 μm approximately). Images were analyzed with Image-J software (NIH, freeware) and Adobe Photoshop 7.0 software packages as previously described to determine plaque size after every session. Changes in plaque size were determined that included both reduced and cleared deposits, and data were expressed as a percentage of the size from the first imaging session.

2.5. Microglia immunohistochemistry and counting

In order to establish the effect of the selected compounds on microglia activation 1 of every 15 paraformaldehyde fixed coronal sections (30 μm, 10–12 sections per animal) located under the cranial window, were stained with anti Iba-1 antibody. Briefly, sections were incubated in 0.5 % Triton X-100 for 20 minutes. Sections were washed in tris-buffer saline (TBS) and incubated in 5 % normal goat serum (NGS) for 1h. Sections were incubated in anti-Iba1 antibody (1:2000) overnight at 4 C in 1.5% NGS. After washing in TBS, sections were incubated with anti-rabbit Alexa Fluor 568 (1:200) for 1h. Senile plaques were stained with thioflavin S 0.001 % for 15 minutes. Unbiased stereological counting was used to determine microglia number in the cortex. An image analysis system (CAST, Olympus) mounted on an upright BX51 Olympus microscope with an integrated motorized stage (Prior Scientific, Rockland, MA) was used to outline the regions, sample, and count microglia cells using a meander sampling paradigm. Region areas were determined according to Cavalieri’s principle and the total number of microglia cells was calculated.

2.5. Statistical analysis

The effects of the selected treatments on senile plaque size were determined by two-way ANOVA (treatmentXsession) with session as repeated measure, followed by Tamhane test. The effect of the treatments on microglia was determined by one-way ANOVA followed by Tamhane test.

3. RESULTS

3.1. Treatment effect on Aβ clearance

Passive immunotherapy leads to prevention and clearance of amyloid deposits (Bard et al., 2000). Our experimental protocol uses direct imaging of amyloid deposits with multiphoton microscopy before and after local treatment to unequivocally evaluate clearance of plaques (Bacskai et al., 2001; Bacskai et al., 2002b). Topical application of anti-Aβ antibody (10D5) to the brain showed a statistically significant effect on Aβ clearance (~40–50% reduction) that was evident within 3 days after treatment and at 7 days after treatment (figures 1 and 2), consistent with previous work (Bacskai et al., 2001; Bacskai et al., 2002b).

Figure 1.

Figure 1

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Effect of direct microglia activation and inhibition on clearance of senile plaques in vivo. Mice were treated with locally applied IFN-γ (15 μl, 50 ng/ml) and/or anti-Aβ antibody 10D5 (25 μl, 1.2 mg/ml). Senile plaque clearance was determined with longitudinal multiphoton imaging 3 and 7 days after treatment. Data are representative of 3–5 mice. Two-way ANOVA was used to detect statistically significant treatmentXsession effect ([F(3,2)]=4.832; p<0.001] and treatment differences were detected by one-way ANOVA followed by Tamhane test. A) Measurements on day 3 (n= 77–232) F[3,643]=19.061; *p<0.001 vs. Control group; †p<0.001 vs. 10D5 group. B) Measurements on day 7 (n= 42–160) F[3,465]=9.878; *p<0.001 vs. Control group; †p<0.001 vs. 10D5 group. The same approach was used to determine the effect of microglia inhibition on 10D5 induced senile plaque clearance. Anti-Mac-1 saporin was locally applied (30 μl, 0.5 mg/ml) prior to 10D5 application. Minocycline was administered i.p. daily (90 mg/Kg) for 3 days prior 10D5 local application, and second group received minocycline 3 days prior to antibody application followed by 3 more days after 10D5 treatment. Data are representative of 3–5 mice. Two way ANOVA revealed a statistically significant treatmentXsession effect [F(4,2)]=4.839; p<0.001]. Treatment differences were detected by one-way ANOVA followed by Tamhane test. C)Measurements on day 3 (n= 149–328) F[4,1085]=10.922; *p<0.001 vs. Control group; †p<0.001 vs. 10D5 group. D) Measurements on day 7 (n= 122–350) F[4,930]=7.928; *p<0.001 vs. Control group; †p<0.001 vs. 10D5 group.

Figure 2.

Figure 2

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Representative in vivo images acquired with multiphoton microscopy illustrating the effect of the selected treatments on Aβ clearance induced by 10D5 in APPswe/PS1dE9 mice. In control animals, existing plaques remain, and several new plaques appear within 1 week. In contrast, many plaques disappear in anti-Aβ-antibody treated animals. IFN- γ treated mice show that some senile plaques disappear after 7 days. When animals were treated with 10D5+IFN-γ the effect on plaque clearance was similar to that observed with 10D5 alone. Anti-Mac-1-saporin limits Aβ clearance induced by 10D5. A similar reduction in the effectiveness of 10D5 was observed after inhibiting microglia with minocycline administered for 3 or 6 days. White arrows point at plaques reduced or cleared on day 7. Blood vessels (blue) are imaged with fluorescence angiography (texas red dextran iv), while both vascular and parenchymal amyloid deposits are labeled with methoxy-XO4 (red). This image is a maximum intensity projection of a 3-dimensional volume that is 615×615 microns square and 200 microns deep. Scale bar=100 μm.

We sought to evaluate the effects of direct microglia activation alone and in combination with passive immunotherapy using an anti-Aβ antibody topically applied to the brain surface. We used longitudinal imaging with multiphoton microscopy to observe plaques, and applied interferon-gamma (50 ng/ml) as a microglia activator to study its effects on clearance of senile plaques alone or in the presence of the anti-Aβ antibody 10D5. 3 days after treatment we observed a significant reduction in plaque size in IFN-γ treated animals (~20%), although this reduction did not reach the levels observed in 10D5 treated animals. Similarly, IFN-γ+10D5 treated animals showed reduced senile plaque size in the range observed for 10D5 alone (~40%), (figure 1A). A similar profile was observed 7 days after the treatment as shown in figures 1B and 2. These results demonstrate that direct activation of microglia can lead to plaque clearance but not to the extent of antibody-mediated clearance and that there is no synergistic effect of a combined approach with direct microglia activation and locally applied immunotherapy.

We next sought to test the effects of direct inhibition of microglia in combination with locally applied antibody. We used 2 strategies to inhibit microglia. The first was the immunotoxin anti-Mac1-saporin applied directly to the surface of the brain. This approach targets microglia specifically with the anti-Mac1 antibody and delivers a toxic payload (saporin) to selectively kill microglia cells. Saporin is a ribosome inactivating protein that is only effective once internalized. Saporin alone or anti-Mac1 antibody alone did not inhibit microglia activation (data not shown). The second approach to inhibit microglia used minocycline, a systemically active antibiotic that inhibits neuroinflammation and may have independent neuroprotective effects (Ryu et al., 2004; Seabrook et al., 2006). Minocycline was administered via i.p. injection for 3 days before topical 10D5 application or for 6 days (3 days before and 3 days after 10D5 treatment). When we quantitatively assessed the effect of the treatments on 10D5 induced Aβ clearance 3 days after the antibody treatment we observed a reduction in plaque size when compared to control values of about 25–35 % (Figure 1C). When we compared plaque size 7 days after the local antibody treatment we observed a similar pattern to that observed 3 days after treatment (figure 1D). However the reduction in plaque size did not reach the levels obtained with 10D5 treatment alone at 3 days, as shown in figure 1C. At 7 days, the difference between minocycline + 10D5 treated animals and 10D5 alone treated animals was not statistically significant, possibly due to the fact that some washout of minocycline occurred at this time. These results, however, demonstrate that each of these microglia inhibition approaches resulted in a partial reduction of the effectiveness of plaque clearance mediated by 10D5 antibody.

To confirm the effectiveness of the treatments, we performed stereological counting of microglia on postmortem tissue from treated mice. As expected, the anti-Aβ antibody (10D5) showed a significant increase in number of activated microglia (2-fold) compared to control values (figures 3 and 4). In the case of microglia activation with IFN-γ, we observed a very significant increase in number of activated microglia (>7-fold) both when IFN-γ was administered alone or in combination with 10D5 (figures 3 and 4). When animals were treated with microglia inhibitors, we observed a modest reduction in microglia activation compared with control animals, and a significant reduction when compared with 10D5 alone treated animals (Figures 3 and 4). Together, these results demonstrate that direct manipulation of microglia activation has a limited role in antibody-mediated clearance of amyloid-β.

Figure 3.

Figure 3

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Effect of selected treatments on microglia numbers measured by stereological counting after immunostaining with anti Iba-1 antibody. Anti-Aβ 10D5 antibody (25 μl, 1.2 mg/ml locally applied) significantly increased microglia activation. Anti-Mac-1-saporin (30 μl, 0.5 mg/ml) locally applied before 10D5 application and minocycline (90 mg/Kg i.p.) daily administered for 6 days (days before and 3 days after local application of 10D5) reduced microglia activation. Although a similar profile was observed when minocycline was administered only for 3 days before 10D5 application, it did not reach statistical significance. IFN-γ (15 μl, 50ng/ml) alone or in combination with 10D5 showed a significant increase of microglia activation when compared to control values and 10D5 alone. Data are representative of 3–4 animals and differences are detected by one-way ANOVA followed by Tamhane test: microglia activation (F[3,8]=22.548; *p<0.001 vs. 10D5 group; †p<0.001 vs. control group),microglia inactivation (F[4,11]=6.557; *p=0.006 vs. 10D5 group).

Figure 4.

Figure 4

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Illustrative example of the effect of the selected treatments on microglia activation. In untreated APPswe/PS1dE9 transgenic animals, limited microglia activation was observed. Topical application of anti-Aβ antibody (10D5, 25 μl, 1.2 mg/ml) resulted in an increase in microglia activation. A remarkable increase of microglia activation is observed after local IFN-γ (15 μl, 50 ng/ml) alone or in combination with 10D5. A reduction in microglia activation was observed after topical application of anti-Mac-1-saporin (30 μl, 0.5 mg/ml) followed by 10D5 local application. Systemic administration of minocycline (90 mg/Kg i.p.) for 3 or 6 days in combination with 10D5 also reduced microglia activation. Microglia were immunostained using anti-Iba 1 (red) and senile plaques were stained with thioflavin S (blue). Scale bar=50 μm.

DISCUSSION

Recent efforts have been directed towards targeting and clearing Aβ as a treatment for AD. While there are endogenous mechanism for reducing Aβ in AD patients that are antibody independent (for review see (Wang et al., 2006), immunotherapy is a very effective approach. Unfortunately, side effects related to the Elan clinical trial with active immunotherapy led to the cancellation of the trial in AD patients (Orgogozo et al., 2003), highlighting the need to better understand the mechanisms of antibody-mediated clearance of Aβ.

It has been postulated that there is a dynamic balance between amyloid deposition and resolution (Hyman et al., 1993), and in support of this idea, it has been suggested that microglial clearance of Aβ may occur but not at a sufficiently rapid rate to prevent overall accumulation (Rogers et al., 2002). In this sense, it is likely that the brain is able to compensate for toxic effects of Aβ up to a certain threshold of damage (Baron et al., 2007). On the other hand, Aβ stimulation of microglia and astrocytes leads to increased production of NO (Monsonego and Weiner, 2003) and general microglia activation is also accompanied by increased secretion of inflammatory mediators that are toxic to nearby neurons. A recent report demonstrated that paralysis of microglia in an animal model abolished the release of microglia-derived nitrite and proinflammatory cytokines and chemokines, and inhibited the development and maintenance of inflammatory CNS lesions (Heppner et al., 2005). On the other hand, a recent study showed that CC-chemokine receptor 2 deficiency led to decreased microglia accumulation in Tg2576 mice, resulting in increased Aβ deposition (El Khoury et al., 2007). Therefore, exploring the role of microglia in Aβ clearance, as well as finding a balance of the benefits and risks of microglia activation are important to understand and optimize the mechanism of antibody mediated Aβ clearance (for review see (Morgan, 2006a; Morgan et al., 2005).

Taking these considerations into account, we explored the effect of IFN-γ, a strong microglia activator, on senile plaque clearance. This key cytokine can induce microglia differentiation into antigen presenting cells (Matsubara et al., 1999). It also facilitates microglia motility and uptake of Aβ and T cell motility (Monsonego et al., 2006). Although our study was centered on the microglial activator IFN-γ, other compounds such as transforming growth factor beta (TGF-β) led to similar results, however the significant distortion observed in brain vasculature impeded its regular use (data not shown). In our hands, IFN-γ induced significant amyloid plaque clearance but it did not reach anti-Aβ antibody (10D5) clearance levels, despite a dramatic increase in the number of activated microglia. Moreover, when we administered IFN-γ and 10D5 simultaneously, no synergistic effect was observed; amyloid clearance was the same as that observed with 10D5 alone (with limited microglia activation). It has previously been shown in vitro that Aβ in combination with IFN-γ has a synergistic effect on microglia activation as well as on neuron mortality (Meda et al., 1995). IFN-γ in vitro also seems to increase Aβ clearance by microglia as well as activate microglia to facilitate T cell motility (Monsonego et al., 2006). Amyloid precursor protein (APP)/IFN-γ transgenic mice show increased microglia activation accompanied by a reduction of Aβ staining at the sites of inflammation (Monsonego et al., 2006). These mice also show meningoencephalitis involving migration of T cells to brain areas where Aβ is accumulated. All together these data show a strong effect of IFN-γ on microglia activation, as we also observed in our experiments. However, this effect in itself does not lead to increased clearance of Aβ, and possible deleterious effects due to the non-specific neuro-inflammation cannot be excluded. It is also possible that IFN-γ is not targeting the microglia that may mediate 10D5 induced clearance. This idea is consistent with previous studies where it is suggested that a shift in the microglial phenotype, from a condition associated with inflammation that is ineffective in clearing Aβ deposits to one exhibiting reduced inflammation but is more capable of clearing deposited amyloid (Morgan, 2006b). In this sense, it is important to note that our classification of “activated” microglia relies exclusively on Iba-1 immunoreactivity.

After measuring the modest effect of microglia activation on Aβ clearance we assessed the effect of microglia inhibition on Aβ induced clearance by 10D5. We selected the immunotoxin anti-Mac-1-saporin to target and selectively kill activated microglia. Anti-Mac-1-saporin is a conjugate of a monoclonal antibody that recognizes the Mac-1 antigen (CD11) to the ribosome-inactivating protein saporin. Complete immunolesions have been shown with the immunotoxin approach as soon as 2 days after treatment (Heckers et al., 1994). We observed that local lesions with anti-Mac-1-saporin led to a limited, but detectable reduction of activated microglia 7 days after treatment. It is likely that microglia recruitment occurs within this time frame, limiting the ability to abolish microglia activity completely. Nonetheless, anti-Mac-1-saporin reduced the Aβ clearance mediated by 10D5 both 3 and 7 days after the administration, although appreciable clearance was still present. It has been suggested that down-regulation of microglia activation may represent a therapeutic strategy in neurodegenerative disorders (Heppner et al., 2005; McCarty, 2006). In our hands limiting microglia activation also reduces the effectiveness of clearance of Aβ with anti-Aβ antibody. This effect suggests that although it does not seem to be the primary mechanism, microglia play a role in eliminating Aβ. However, previous approaches to inhibit microglia activation using dexamethasone (Wilcock et al., 2004), led to a significant reduction in anti Aβ antibody effectiveness. The complexity of microglia activation may account for these differences and it is possible that the different compounds target different microglia populations.

Minocycline is a tetracycline derivative with neuroprotective effects in several central nervous system disorders such as Parkinson’s (Thomas and Le, 2004), amyotrophic lateral sclerosis (Zhu et al., 2002), and Huntington’s disease (Bantubungi et al., 2005). The inhibitory effect of minocycline on microglial proliferation and activation is well established (Ryu et al., 2004; Tikka and Koistinaho, 2001), and in relation to AD it has been shown that tetracyclines can interfere with amyloid fibrilization in vitro (Forloni et al., 2001). However, the effect in vivo is more controversial since minocycline seems to increase amyloid deposition in young transgenic AD mice (J20 APP-tg) and improve behavioral impairment, without a significant effect on microglial activation. However, when treatments commenced once amyloid deposition was established no effect was observed on amyloid deposition or memory tasks, whereas microglia activation was suppressed (Seabrook et al., 2006). On the other hand, in a recent study in a cerebral amyloid angiopathy mouse model (Tg-SwDI, expressing human Swedish/Dutch and Iowa mutants of APP), minocycline improved spatial learning and memory deficits and reduced microglial activation (Fan et al., 2007), without a significant effect on Aβ levels. In vitro studies have shown that minocycline inhibits Aβ induced microglial production of proinflammatory cytokines such as TNF-α and IL-6, whereas microglia phagocytic capacity is unaffected, suggesting that minocycline may have a positive effect in attenuating the microglia mediated inflammatory response in AD (Familian et al., 2007). Wilcock et al. (Wilcock et al., 2004) have also shown that minocycline can reduce microglia activation after local antibody treatment without a significant effect on thioflavin S-positive deposits when assessed in postmortem tissue from Tg2576 mice. We observed that the reduction of microglia activation after minocycline treatment was accompanied by a partial reduction in 10D5-induced amyloid clearance in vivo. In our hands the differences observed between 10D5 and minocycline+10D5 treatments in senile plaque sizes reached ~20%, and it is possible that such a difference can only be detected with longitudinal in vivo imaging of identified plaques, whereas this effect can be underestimated when total senile plaque burden is assessed in postmortem tissue. It is also possible that the animal model and the experimental approach used account for the differences since Wilcock et al. (Wilcock et al., 2004) also describe a transient activation of microglia 3 days after antibody treatment that is not detectable after 7 days, whereas in our hands microglia activation is still present after 7 days of 10D5 application.

Although it is possible that not only direct activation or inhibition of microglia but modulation of the activation process is important for therapeutic clearance, our data support only a partial role of microglia in the process of Aβ clearance. The effects of either direct activation or inhibition of microglia on Aβ clearance were modest despite the dramatic effects observed on the number of activated microglia with these manipulations. Therefore, while microglia play some role in the clearance of Aβ, they are not the main mediators in the elimination of excessive Aβ in AD. These results are consistent with previous reports that demonstrated effective prevention or clearance of plaques in the absence of Fc receptor stimulation (Bacskai et al., 2002a; Das et al., 2003) indicating that microglia mediated phagocytosis is not required for effective immunotherapy in mouse models. However, it can not be excluded that systemic administration of antibodies may lead to a different effect on microglia activation. Ultimately, understanding the mechanisms of antibody-mediated clearance while minimizing the inflammatory side effects should help in the elaboration of safer immunotherapy treatments for AD.

Acknowledgments

This work was supported by NIH AG020570, EB000768 (BJB), AG08487 (BTH), and a fellowship from the Fundacion Caja Madrid (M.G-A).

Footnotes

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