8354 • The Journal of Neuroscience, August 13, 2008 • 28(33):8354–8360

Neurobiology of Disease Microglial Dysfunction and Defective ␤-Amyloid Clearance Pathways in Aging Alzheimer’s Disease Mice

Suzanne E. Hickman,1 Elizabeth K. Allison,1 and Joseph El Khoury1,2 1Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology, and 2Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129

Early microglial accumulation in Alzheimer’s disease (AD) delays disease progression by promoting clearance of ␤-amyloid (A␤) before formation of senile plaques. However, persistent A␤ accumulation despite increasing microglial numbers suggests that the ability of microglia to clear A␤ may decrease with age and progression of AD pathology. To determine the effects of aging and A␤ deposition on microglial ability to clear A␤, we used quantitative PCR to analyze expression in freshly isolated adult microglia from 1.5-, 3-, 8-, and 14-month-old transgenic PS1-APP mice, an established mouse model of AD, and from their nontransgenic littermates. We found that microglia from old PS1-APP mice, but not from younger mice, have a twofold to fivefold decrease in expression of the A␤-binding scavenger receptors scavenger receptor A (SRA), CD36, and RAGE (receptor for advanced-glycosylation endproducts), and the A␤- degrading , , and MMP9, compared with their littermate controls. In contrast, PS1-APP microglia had a 2.5-fold increase in the proinflammatory cytokines IL-1␤ (interleukin-1␤) and tumor necrosis factor ␣ (TNF␣), suggesting that there is an inverse correlation between cytokine production and A␤ clearance. In support of this possibility, we found that incubation of cultured N9 mouse microglia with TNF␣ decreased the expression of SRA and CD36 and reduced A␤ uptake. Our data indicate that, although early microglialrecruitmentpromotesA␤clearanceandisneuroprotectiveinAD,asdiseaseprogresses,proinflammatorycytokinesproduced in response to A␤ deposition downregulate involved in A␤ clearance and promote A␤ accumulation, therefore contributing to neurodegeneration. Antiinflammatory therapy for AD should take this dichotomous microglial role into consideration. Key words: microglia; Alzheimer’s disease; transgenic mice; amyloid; phagocytosis; degradation

Introduction mation by phagocytosing A␤ (Simard et al., 2006). Indeed, our The senile plaque is a pathological hallmark of Alzheimer’s dis- own data also show that decreased early microglial accumulation ease (AD) and is composed of ␤-amyloid (A␤), activated micro- leads to increased A␤ deposition and early mortality in the trans- glia, astrocytes, and degenerating neurons (Dickson et al., 1988; genic swAPP (Tg2576) AD mouse model (El Khoury et al., 2007). Selkoe, 2000; Mott and Hulette, 2005). Microglia, the mononu- In addition to phagocytosis of A␤, microglia also secrete proteo- clear phagocytes of the brain (Perry and Gordon, 1988), accumu- lytic enzymes that degrade A␤, such as IDE (insulin-degrading late in senile plaques in AD patients and in animal models of AD ), neprilysin, matrix 9 (MMP9), and (McGeer et al., 1987; Perlmutter et al., 1990; Frautschy et al., plasminogen (Leissring et al., 2003; Yan et al., 2006), further 1998; Dickson, 1999; Stalder et al., 1999). However, their exact suggesting a neuroprotective role for these cells in AD. role in the pathogenesis of AD remains to be elucidated. A␤ can The data supporting a role for microglia in A␤ clearance are activate microglia to produce cytokines and neurotoxins, hence compelling, but these data also raise an important question. Why promoting neurodegeneration (Meda et al., 1995; El Khoury et does A␤ continue to accumulate, and why does AD pathology al., 1996, 2003; Coraci et al., 2002). In contrast, microglia also progress despite continued microglia recruitment? One possible express receptors that promote the clearance and phagocytosis of explanation for the failure of microglia to stop AD progression A␤, such as class A scavenger receptor, CD36, and receptor for would be that these cells become overwhelmed by the excess advanced-glycosylation endproducts (RAGE) (Yan et al., 1996; El amount of A␤ produced and cannot keep up with the pace of A␤ Khoury et al., 1998), and microglia can restrict senile plaque for- generation. Another possibility would be that, as AD progresses, the phenotype of accumulating microglia changes and these cells become more proinflammatory and lose their A␤-clearing capa- Received Feb. 11, 2008; revised June 11, 2008; accepted July 15, 2008. bilities, resulting in reduced A␤ uptake and degradation, and This work was supported by National Institute of Neurological Disorders and Stroke Grant NS059005 and a grant ␤ from The Dana Foundation Neuroimmunology Program (J.E.K.). We thank David Borchelt for permission to use the increased A accumulation. To investigate this hypothesis, we PS1-APP mice. developed a method to isolate fresh adult mouse microglia from Correspondence should be addressed to Dr. Joseph El Khoury, Center for Immunology and Inflammatory Dis- the bigenic APPswe/PSEN1dE9 (PS1-APP) mice (Borchelt et al., eases,MassachusettsGeneralHospital,HarvardMedicalSchool,CNY149,Room8301,14913thStreet,Charlestown, 1997; Jankowsky et al., 2001), a robust mouse model of AD, and MA 02129. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.0616-08.2008 from their nontransgenic littermates at various ages and stages of Copyright © 2008 Society for Neuroscience 0270-6474/08/288354-07$15.00/0 AD-like pathology, and compared of A␤- Hickman et al. • Microglial Dysfunction in Alzheimer’s Disease J. Neurosci., August 13, 2008 • 28(33):8354–8360 • 8355 binding receptors and A␤-degrading enzymes. Our data show type 3 (Worthington Biochemicals). Brains were minced with sterile that, as PS1-APP mice age, their microglia become dysfunctional razor blades and allowed to incubate in enzymes for 45 min at 37°C, and exhibit a significant reduction in expression of their A␤- followed by addition of DNase I grade II (Roche Applied Science) at a binding receptors and A␤-degrading enzymes, but maintain their concentration of 40 U/ml and incubation for an additional 15 min. The enzymes were inactivated by addition of 20 ml of HBSS without Ca 2ϩ ability to produce proinflammatory cytokines. These cytokines 2ϩ ϭ and Mg (HBSS ) (Mediatech) containing 2 mM EDTA and 2% fetal may in turn act in an autocrine manner and further reduce ex- bovine serum (FBS) and the digested brain bits were triturated sequen- pression of A␤-binding receptors and A␤-degrading enzymes ␤ tially with a 25, 10, and 5 ml pipette (8–10 times each step). Between leading to decreased A clearance and increased accumulation. pipette sizes, the cells were allowed to settle for 1 min, and then the top 50% of the cell suspension was removed and passed over a 100 ␮m filter Materials and Methods (Thermo Fisher Scientific). This process was continued until all tissue Mice. PS1-APP transgenic mice [B6C3-Tg(APPswe, PSEN1dE9)85Dbo/J; had been triturated and passed through the filter. Cells were centrifuged stock number 004462] were purchased from The Jackson Laboratory and and resuspended in RPMI 1640/L-glutamine and mixed with physiologic subsequently bred in the animal care facilities at Massachusetts General Percoll (Sigma-Aldrich) and centrifuged at 850 ϫ g for 45 min. The cell Hospital. These mice coexpress a “humanized” Swedish amyloid precur- pellet was resuspended in RPMI 1640 and the cells were passed over a 70 ␮m filter (Thermo Fisher Scientific), washed, and then passed over a 40 sor protein mutation (APP695SWE) and a mutant exon 9-deleted variant of human presenilin 1 (PSEN1/dE9). The mutations in both genes are ␮m filter (Thermo Fisher Scientific). The cells were then incubated with associated with familial AD. The APP and PSEN1 transgenes are inte- anti-mouse Cd11b-coated microbeads (Miltenyi Biotec) for 20 min at grated into a single locus and are independently under the control of 12°C. The cell–bead mix was then washed to remove unbound beads. separate mouse prion protein promoter elements, which direct expres- The bead–cell pellet was resuspended in PBS/0.5% BSA/2 mM EDTA and sion of the transgenes predominantly to CNS neurons (Borchelt et al., passed over a magnetic MACS Cell Separation column (Miltenyi Biotec). 1997; Jankowsky et al., 2001). Mice were used in pairs of age-matched In this way, Cd11b-positive cells (i.e., cells that bound the beads, micro- transgenic or wild-type (WT) littermates at 1.5, 3, 8, and 14 months of glia/mononuclear phagocytes) were separated from Cd11b-negative cells neg age and killed according to approved institutional procedures. All pro- (CD11b ) (i.e., cells that did not bind the beads). Flowthrough was tocols were approved by the Massachusetts General Hospital Institu- collected and the column was rinsed three times with PBS/BSA/EDTA. ϩ tional Animal Care and Use Committee and met National Institutes of CD11b-positive cells (CD11b ) were eluted by removing the column Health guidelines for the humane care of animals. Brains were harvested from the magnetic holder and pushing PBS/BSA/EDTA through the col- and microglia were isolated (see below) and used fresh for flow cytometry umn with a plunger. Cells were centrifuged and the pellets were lysed in or gene expression analysis. RLT-Plus buffer from the RNeasy Plus mini kit (QIAGEN) to use for Immunohistochemistry staining for total A␤. Brains from transgenic quantitative PCR (QPCR). ϩ ϩ PS1-APP or WT littermates were harvested and fixed in 2% paraformal- Flow cytometry of CD11b cells. CD11b cells isolated by magnetic dehyde (PFA) in PBS (Mediatech), pH 7.5, overnight at 4°C. The fixed beads (above) were centrifuged and cell pellets were resuspended in brains were then placed in 30% sucrose overnight at 4°C for cryoprotec- PBS/2% FBS containing 1:100 dilution of mouse seroblock (AbD Sero- tion. Brains were embedded in Tissue-Tek OCT compound (Sakura Fi- tec) to block Fc receptors on microglia. Cells were maintained on ice for netek USA) and 10–14 ␮m frozen sections were cut. To stain for A␤, 10 min then stained with allophycocyanin (APC)-labeled anti-CD11b (2 sections were blocked in PBS/0.3% Triton X-100/2% goat serum for 30 ␮g/ml) or APC-labeled isotype control (2 ␮g/ml) (both from BD Bio- min, and then incubated overnight at 25°C with rabbit anti-pan A␤ sciences Pharmingen) for1honicefollowed by fixation with 2% PFA. antibody (BioSource International) or control antibody, at 2 ␮g/ml in Fluorescence intensity was measured using a FACSCalibur (BD Bio- PBS containing 0.3% Triton X-100 and 2% goat serum. The slides were sciences) flow cytometer as described previously (Coraci et al., 2002; El then processed using the Vectastain Elite ABC kit rabbit IgG (Vector Khoury et al., 2007). ϩ Laboratories) according to the manufacturer’s instructions. Briefly, Quantitative real-time PCR. Total RNA from each sample of CD11b slides were incubated with biotinylated anti-rabbit IgG for 30 min fol- or CD11b neg cells (7.5–15 ϫ 10 5 cells) or N9 cells was isolated using the lowed by incubation with the kit ABC reagents (avidin and biotinylated RNeasy Plus mini kit for RNA isolation (QIAGEN) according to the peroxidase) for 30 min. Vector NovaRed substrate kit for peroxidase manufacturer’s instructions and all of each sample was reverse tran- (Vector Laboratories) was used to develop target-bound peroxidase for scribed using Multiscribe reverse transcriptase (Applied Biosystems). Di- detection of A␤ in brain sections. Slides were counterstained with hema- lutions of each cDNA prep were used to assess ␤2-microglobulin RNA toxylin, mounted with VectaMount (Vector Laboratories), visualized by levels, and samples were then adjusted to give equivalent levels of ␤2- bright-field microscopy, and digitally photographed. microglobulin per well in subsequent QPCRs for other genes. The QPCR Immunohistochemistry/fluorescence costaining of brains for CD11b and was performed with the MX4000 unit (Agilent Technologies) using A␤ plaques. Frozen sections from PS1-APP or WT littermate mice were SYBR Green to detect the amplification products as described previously fixed in acetone for 10 min, washed in PBS, and then treated with 0.25% (El Khoury et al., 2003, 2007). The following cycles were performed: trypsin for antigen retrieval. Endogenous peroxidase activity was initial denaturation cycle of 95°C for 10 min, followed by 40 amplifica- quenched with 0.3% H2O2 followed by blocking with 1.5% donkey se- tion cycles of 95°C for 15 s and 60°C for 1 min and ending with one cycle rum in PBS. Sections were incubated overnight at 25°C with rat anti- at 25°C for 15 s. Analysis was performed on the data output from the CD11b (clone 5C6) (AbD Serotec) or rat IgG2b negative control (AbD MX4000 software (Agilent Technologies) using Microsoft Excel XP. Rel- Serotec) each at 1:100 dilution in PBS with 1.5% donkey serum. The ative quantification of mRNA expression was calculated by the compar- slides were then processed using the Vectastain Elite ABC reagent (Vector ative cycle method described by the manufacturer (Agilent Technolo- Laboratories) according to the manufacturer’s instructions followed by gies). Primer sequences for the following murine genes can be found in development with the NovaRed Peroxidase Substrate kit. A␤-containing supplemental Table 1 (available at www.jneurosci.org as supplemental plaques were stained with 1% thioflavin-S (Sigma-Aldrich) for 5 min in material): ␤2-microglobulin, GAPDH, CD11b, CD36, scavenger recep- the dark. Finally, the sections were counterstained with hematoxylin, tor A (SRA), SRB1, macrophage receptor with collagenous domain mounted with VectaMount, and digitally photographed via bright-field (MARCO), RAGE, interleukin 1␤ (IL-1␤), tumor necrosis factor ␣ microscopy to detect CD11b, and via fluorescence to visualize (TNF␣), insulysin, neprilysin, and MMP9. thioflavin-S. Staining of N9 microglia. N9 mouse microglia (a gift from Dr. P. Isolation of CD11bϩ cells. Transgenic PS1-APP mice or their WT lit- Riciarrdi-Castagnoli, University of Milan, Bicocca, Italy) (El Khoury et termates were killed and perfused with 30 cc of PBS without Ca 2ϩ and al., 1996) were plated on 24-well plates coated with 1 ␮g of fibronectin/ Mg 2ϩ (PBS ϭ). Brains were then removed, rinsed in PBS ϭ and placed cm 2 (Sigma-Aldrich), and grown overnight in RPMI 1640 supplemented separately into a 60 mm tissue culture dish with RPMI 1640 (no phenol with10% FBS, L-glutamine (2 mM), penicillin (10 IU/ml), and strepto- red) containing 2 mML-glutamine (Mediatech), dispase and mycin (10 ␮g/ml). The following day, TNF␣ (PeproTech) was added to a 8356 • J. Neurosci., August 13, 2008 • 28(33):8354–8360 Hickman et al. • Microglial Dysfunction in Alzheimer’s Disease concentration of 50 ng/ml and cells were incu- bated overnight. For staining, cells were lifted from the plate with CellStripper (Mediatech) and resuspended in PBS/1% BSA/2% FBS con- taining 10 ␮g/ml Fc block (AbD Serotec) and allowed to sit 10 min before addition of primary antibodies. APC-labeled anti-mouse CD11b (1 ␮g/ml) (BD Biosciences Pharmingen) or APC- labeled hamster anti-mouse CD36 clone HM36 (1 ␮g/ml) (BioLegend) or Alexa647-labeled anti-CD204 (SRA) clone 2F8 (2.5 ␮g/ml) (AbD Serotec) or isotype-matched control antibodies (same concentrations as primary antibodies) were added and incubated on ice for 1 h. Cells were then fixed by the addition of 2% PFA. Flu- orescence intensity was measured using a FAC- SCalibur (BD Biosciences) flow cytometer. A␤ uptake. For uptake of A␤, N9 microglia were grown on fibronectin-coated 24-well plates and treated overnight with TNF␣ or me- dium alone. Adherent N9 microglia were rinsed once in PBS/1% BSA, followed by the addition of A␤(1–42) labeled with the fluorescent dye Figure 1. Increased number of senile-like plaques and plaque-associated microglia with aging in transgenic PS1-APP mice. ␣ ␤ HiLyte Fluor488 (AnaSpec) or with FITC as de- Top panels, Frozen sections from transgenic PS1-APP brains stained with pan- -A antibody (which recognizes all forms of ␤ ϫ ␤ scribed previously (El Khoury et al., 2007) at a -amyloid) show (original magnification, 40 ) sparse amounts of A in a transgenic mouse at 3 months of age (A) and large ␤ concentration of 5 ␮g/ml in PBS/1% BSA or numbers of A deposits in a 14-month-old transgenic mouse distributed throughout cortex and hippocampus (B). The inset in B ϫ ϫ diluent alone and incubated at 37°C for 2 h. showstheplaquesignifiedbyanarrowmagnifiedto400 originalmagnification.Bottompanels,400 originalmagnification. ␣ ␤ Cells were then rinsed gently three times in PBS Microglia are stained using -CD11b antibody (reddish brown Nova red development) and A -containing plaques are stained with divalent cations and then lifted from wells with thioflavin-S (green). In a 3-month-old mouse, plaques are rarely seen and microglia are evenly distributed throughout the using CellStripper. Cells were fixed in 2% PFA hippocampus region shown in C. D, In contrast, florid plaques are seen in a transgenic mouse at 14 months of age, and intensely and cell-associated fluorescence was analyzed stained microglia are clustered around the plaques. The staining was repeated in six mice in each age group with similar staining. by flow cytometry. To determine whether cell-associated fluorescence was attributable to Freshly isolated microglia express CD11b and scavenger binding or endocytosis of labeled A␤, N9 cells grown overnight on four- receptors, but not astrocyte or endothelial markers ϩ well glass slides were incubated with RPMI 1640/1% BSA alone or FITC- We developed a method to isolate microglia (CD11b cells) from labeled A␤(1–42) at 4 or 37°C for 4 h. Wells were then rinsed three times adult mouse brains and used freshly isolated cells to examine with ice-cold PBS to remove unbound A␤ and unfixed cells were ana- expression of A␤ receptors, A␤-degrading enzymes, and proin- lyzed by flow cytometry. To differentiate between bound versus endocy- flammatory cytokines. Figure 2A shows a diagram of our proto- tosed A␤, trypan blue was added to some samples to quench fluorescence col for isolation of adult microglia, which is completed in Ͻ4h. of FITC-A␤ on the outside of the cell as described previously (Thomas et Immediately after brain removal, the brain is digested with dis- al., 2000). Because cells are not fixed, the live cells are not permeable to pase and collagenase, followed by trituration and filtration of trypan blue and any fluorescence detected would be intracellular. large debris. Intact cells are enriched by centrifugation through Statistical analysis. Statistical analysis was performed using Student’s t Percoll. Microglia are then purified by binding to anti-CD11b- test and one-way ANOVA provided in the OriginLab Origin 8 graphics Ͻ coated magnetic microbeads followed by separation from and statistics software. Values of p 0.05 were considered significant. CD11b neg cells by passing through a magnetic column. Column- bound CD11b ϩ cells are eluted and centrifuged, and the cell Results pellet is immediately lysed and the total RNA isolated for use in Increased number of senile-like plaques and plaque- QPCR. Because gene expression patterns of cultured microglia associated microglia with aging in transgenic PS1-APP mice may differ from microglia in situ, the use of freshly isolated cells Frozen sections prepared from the brains of young (3 months) gives a “snapshot” of genes that are expressed in the environment and old (14 months) PS1-APP mice were stained with an anti- in which the cells were residing just before isolation. To control pan-A␤ antibody. At 3 months of age, transgenic PS1-APP mice for any changes in gene expression that may occur as a result of have sparse, but detectable A␤ deposits (Fig. 1A). By 14 months the isolation procedure itself, all mouse brains were isolated in pairs of transgenic and nontransgenic WT littermates harvested of age, A␤ deposits are distributed throughout the cortex and at the same time, under the same condition, using the same hippocampus (Fig. 1B). To visualize microglial distribution in reagents. young and old mice, sections were first stained with anti-CD11b Figure 2, B and C, shows a typical RNA expression profile of antibody followed by costaining with thioflavin-S to detect fibril- ϩ neg ␤ CD11b microglia and CD11b cells (mostly endothelial cells lar A plaques. In 3-month-old mice, plaques are rarely seen and and astrocytes) prepared using our method. Analysis by flow microglia are evenly distributed throughout the hippocampus cytometry showed that Ն96% of our CD11b ϩ populations iso- region (Fig. 1C). In contrast, florid plaques are seen in transgenic lated in this protocol stained with CD11b (supplemental Fig. 1, mice at 14 months of age and intensely stained microglia are available at www.jneurosci.org as supplemental material). RNA clustered around the plaques (Fig. 1D). These data indicate that expression is shown as the ratio of copies of target RNA compared significant microgliosis accompanies A␤ deposition in PS1-APP with ␤2-microglobulin RNA in the same sample. To determine mice and confirm that these mice are a robust model to study the the purity of our isolated cell populations, CD11b ϩ and role of microglia in AD. CD11b neg cells were compared by QPCR for expression of cell Hickman et al. • Microglial Dysfunction in Alzheimer’s Disease J. Neurosci., August 13, 2008 • 28(33):8354–8360 • 8357

isolated CD11b ϩ cells also express RNA for the three microglial scavenger recep- tors SRA, SRB1, and CD36 (El Khoury et al., 1998). Freshly isolated microglia ex- press small but detectable levels of RAGE (Yan et al., 1996) and negligible levels of MARCO (Alarco´n et al., 2005).

Microglia from old transgenic PS1-APP mice have reduced expression of A␤-binding receptors To determine whether aging and A␤ dep- osition affect expression of A␤-binding re- ceptors SRA, SRB1, CD36, RAGE, and MARCO, we measured expression of these receptors in CD11b ϩ cells isolated from PS1-APP mice and their age-matched wild-type littermates at 1.5, 3, 8, and 14 months of age (five to six mice per geno- type per age group). At 1.5 months of age, expression of SRA, SRB1, CD36, and RAGE RNAs were comparable between transgenic and WT mice. At 3 months of age, ex- ϩ pression of SRA, CD36, and RAGE RNAs Figure 2. Gene expression profile of adult WT CD11b microglia and CD11b neg cells (endothelial cells and astrocytes) mea- suredbyQPCR.A,ProtocolusedforisolatingCD11b ϩ cellsfromadultmousebrains.B,CD11b ϩ cells(blackbars)havehighlevels was 1.4- to 1.6-fold lower in PS1-APP ofCD11bRNAandbarelydetectablelevelsofcellmarkersforastrocytes(GFAP)andendothelialcells(CD31)confirminghighpurity mice than in their age-matched WT lit- of the CD11b ϩ cells preparations. The CD11b neg population (gray bars) expresses CD31 and GFAP, but no CD11. C, CD11b ϩ cells termates, but the differences were not isolated using our protocol expressed SRA, SRB1, and CD36 (3 known microglia receptors), some RAGE, and negligible levels of statistically significant. However, by 8 MARCO. months of age, microglia from PS1-APP transgenic mice showed significantly re- duced expression of SRA (1.9-fold de- crease; p Ͻ 0.002), CD36 (2.6-fold de- crease; p Ͻ 0.0002), and RAGE (twofold decrease; p Ͻ 0.042) compared with their age-matched WT littermates (Fig. 3A,C,D). At 14 months of age, micro- glial RNA levels for these genes had de- clined even further in PS1-APP trans- genic mice compared with their WT littermates: SRA (2.5-fold decrease; p Ͻ 0.01), CD36 (fivefold decrease; p Ͻ 0.0003), and RAGE (sixfold decrease; p Ͻ 0.003) (Fig. 3A,C,D). There are no significant differences in expression of SRB1 observed between transgenic and WT mice at any age (Fig. 3B). These data indicate that microglia in mice with ad- vanced AD-like pathology have de- creased expression of A␤ receptors and suggest that they may have decreased Figure 3. Reduced expression of A␤-binding receptors in microglia from old transgenic PS1-APP mice. Expression of A␤- capacity to bind and subsequently bindingreceptorsinCD11b ϩ cellswascomparedbetweentransgenicPS1-APPmiceandtheirage-matchedWTlittermatesat1.5, clear A␤. 3,8,and14monthsofage.DatarepresenttheaveragevaluesobtainedbyQPCRonsixsetsofmicefor3and14monthsofageand Interestingly, WT mice also exhibited onfivesetsofmiceat1.5and8monthsofage.*Valuesofpareshownonlyifdifferencesaresignificant(Ͻ0.05).A,B,D,At8and an age-related decrease in expression of 14 months of age, PS1-APP transgenic mice show significantly reduced expression of SRA (A), CD36 (B), and RAGE (D) compared SRA, CD36, and SRB1 RNAs in 8- and 14- withtheirWTlittermates.C,TherearenosignificantdifferencesinexpressionofSRB1observedbetweentransgenicandWTmice month-old mice compared with younger at any age. E, There was negligible expression of MARCO. Error bars indicate SEM. WT mice. However, only reduction in CD36 was statistically significant ( p Ͻ markers for astrocytes (GFAP), endothelial cells (CD31), and 0.03). Furthermore, the reduction in expression of SRA and microglia (CD11b). As expected, CD11b ϩ cells expressed high CD36 was significantly more pronounced between microglia levels of CD11b, and the CD11b neg cells expressed CD31 and from transgenic mice and WT littermates than between WT mice GFAP and no CD11b. Additional analysis showed that freshly of different age groups. 8358 • J. Neurosci., August 13, 2008 • 28(33):8354–8360 Hickman et al. • Microglial Dysfunction in Alzheimer’s Disease

Microglia from old PS1-APP mice have reduced expression of A␤-degrading enzymes In addition to their ability to phagocytose A␤, microglia can also clear A␤ by degra- dation via A␤-degrading enzymes (Leiss- ring et al., 2003; Yan et al., 2006). To de- termine whether aging and A␤ deposition affect microglial expression of major A␤- degrading enzymes, we quantified by QPCR the mRNA levels of insulysin, ne- ϩ prilysin, and MMP-9 in CD11b cells Figure 4. Decreased expression of A␤-degrading enzymes in microglia from old transgenic PS1-APP mice. Expression of from transgenic PS1-APP mice and their A␤-degrading enzymes in CD11b ϩ cells from transgenic PS1-APP mice was compared with their age-matched wild-type litter- age-matched WT littermates at 1.5, 3, 8, mates at 1.5, 3, 8, and 14 months of age. Data are expressed for transgenic mice as percentage expression of WT. *Values of p are and 14 months of age. Expression of RNA only shown if differences are significant. A–C, Expression of insulysin (A), neprilysin (B), and MMP9 (C), enzymes thought to levels of the A␤-degrading enzymes insu- degradeA␤,iscomparableacrossWTandtransgenicmicrogliafrommiceat1.5and3monthsofage.A,In8-month-oldPS1-APP lysin, neprilysin, and MMP9, were compa- transgenic mice, there is a statistically significant reduction only in insulysin RNA levels. However, by 14 months of age, the rable in WT and transgenic mice at 1.5 and reduction in expression of all three enzymes is statistically significant (A–C). The horizontal lines across the graphs indicate 100% 3 months of age (Fig. 4A–C). However, in WT levels. Error bars indicate SEM. 8-month-old transgenic mice, insulysin RNA levels decreased to 50% ( p Ͻ 0.001) of age-matched WT A B IL-1β p< 0.025 TNFα littermates. This level of reduction in transgenic mice is also ob- ** 350 p<0.041 served at 14 months of age (50% of WT; p Ͻ 0.035) (Fig. 4A). At 250 p<0.015 300 8 months of age, transgenic mice express reduced levels of RNA 200 ** * 250 p<0.015 for neprilysin and MMP9 compared with their WT littermates, 150 200 150 * but the differences were not statistically significant (Fig. 4B,C). 100 100 However, by 14 months of age, microglial levels of both neprily- 50 Ͻ Ͻ 50 sin ( p 0.015) and MMP9 ( p 0.05) in transgenic mice were 0 0 (% of WT littermates) reduced to only 20% of the levels seen in their age-matched WT 1.5 3 8 14 1.5 3 8 14 littermates (Fig. 4B,C). These data show that microglia in mice Age (months)

with significant AD-like pathology have reduced expression of mice Levels in Transgenic mRNA three major A␤-degrading enzymes and suggest that these micro- ␤ ␣ glia may have become defective in their capacity to degrade A␤. Figure5. IncreasedexpressionofIL-1 andTNF inmicrogliafromoldtransgenicPS1-APP mice. A, B, Expression of IL-1␤ (A) and TNF␣ (B) was measured by QPCR in CD11b ϩ cells from transgenic PS1-APP mice and their age-matched, nontransgenic littermates at 1.5, 3, 8, and 14 Microglia from old PS1-APP mice have increased expression months of age. Data are displayed for transgenic mice as percentage expression of their WT ␤ of IL-1␤ and TNF␣ littermates at the same ages. In young mice (1.5 and 3 months of age), expression of IL-1 (A) ␣ Mouse microglia and macrophages stimulated with A␤ upregu- and TNF (B) is comparable across all groups, but at 8 and 14 months of age there are signifi- cant increases ( p Ͻ 0.05) in RNA expression of both IL-1␤ and TNF␣ in transgenic compared late their expression of several proinflammatory and with WT mice. Error bars indicate SEM. cytokines including IL-1␤ and TNF␣ (El Khoury et al., 2003). In addition, TNF␣ is upregulated in the cortex of triple transgenic AD mice (Janelsins et al., 2005). To determine the effects of aging microglia, but a rather selective defect in their A␤-clearing and A␤ deposition on expression of TNF␣ and IL-1␤ in micro- pathways. glia, we measured RNA levels of these two cytokines in CD11b ϩ cells from transgenic PS1-APP mice and their WT littermates at TNF␣ downregulates expression of SRA and CD36 and 1.5, 3, 8, and 14 months of age using QPCR. IL-1␤ expression was reduces uptake of A␤ in N9 microglia comparable in microglia from transgenic and WT mice at 1.5 and Because microglia from old transgenic PS1-APP mice exhibited 3 months (Fig. 5A). In contrast, at 8 months of age, microglial higher expression of proinflammatory cytokines that are potent IL-1␤ RNA in transgenic mice had increased to 150% of their WT regulators of gene expression in mononuclear phagocytes and littermates ( p Ͻ 0.015), and by 14 months, IL-1␤ RNA was in- microglia, we hypothesized that these cytokines regulate expres- creased to 250% of WT ( p Ͻ 0.025) (Fig. 5A). Similarly, levels of sion of A␤ receptors and A␤-degrading enzymes. To test this TNF␣ RNA were also comparable between transgenic and WT hypothesis, we incubated murine N9 microglia with IL-1␤, mice at 1.5 and 3 months of age (Fig. 5B). Statistically significant TNF␣, or medium alone and measured expression of SRA, CD36, increases in TNF␣ RNA were observed in transgenic microglia neprilysin, and insulysin by QPCR. We found that incubation from 8-month-old mice (150% of WT; p Ͻ 0.015) and 14- with TNF␣ for 18 h significantly downregulated expression of month-old mice (200% of WT; p Ͻ 0.041), compared with their SRA and CD36 to 65% ( p Ͻ 0.0003) and 52.8% ( p Ͻ 0.0001) of age-matched WT littermates. Together, these data show that mi- untreated N9 cells, respectively (Fig. 6A). In preliminary experi- croglia in aged AD mice retain their proinflammatory response in ments, similar results were obtained with IL-1␤, albeit to a lesser the presence of continued A␤ deposition. Because expression of extent, and differences were not statistically significant (data not these two cytokines are increased, these data also suggest that the shown). TNF␣-induced reduction in RNA levels corresponded decreased expression of A␤ receptors and A␤-degrading enzymes to a reduction of both SRA and CD36 surface expression, as observed in microglia from aging PS1-APP mice is not an indi- assessed by staining and flow cytometry. TNF␣ reduced SRA and cation of a generalized “shut down” in the functions of these CD36 surface expression to 54.7% ( p Ͻ 0.008) and 64.5% ( p Ͻ Hickman et al. • Microglial Dysfunction in Alzheimer’s Disease J. Neurosci., August 13, 2008 • 28(33):8354–8360 • 8359

Discussion Increasing evidence indicates that microglia may play a protective role in AD by mediating clearance of A␤. Indeed, early microglial accumulation appears to delay progression of AD-like pathology, and marrow-derived microglia may restrict plaque forma- tion in transgenic mice (Simard et al., 2006; El Khoury et al., 2007). As AD progresses, however, microglial accumulation ap- pears to parallel disease progression, and the presence of in- creased numbers of microglia does not appear to prevent forma- tion of plaques or AD development (Perlmutter et al., 1990; Dickson, 1999; Mott and Hulette, 2005). The data presented in this study show that, in the PS1-APP transgenic mouse model of AD, the phenotype of accumulating microglia changes as AD-like pathology progresses. Microglia continue to produce proinflam- matory cytokines, but lose their A␤-clearing capabilities. Expres- sion of microglial A␤ receptors and A␤-degrading enzymes is reduced, resulting in reduced A␤ uptake and degradation, and increased A␤ accumulation. This process is observed at the age of 8 months and appears to precede or parallel the increase in A␤ levels observed in PS1-APP mice at that age (Jankowsky et al., 2001, 2004). In contrast to their protective role early in the disease process, as AD-like pathology progresses, microglial dysfunction and their failure to phagocytose and/or degrade A␤ further con- tributes to disease progression. In support of this possibility, Fiala et al. (2005) found that monocytes and macrophages from AD patients exhibited ineffective phagocytosis of A␤ when compared with monocytes and macrophages from age-matched control Figure 6. Treatment of N9 cells with TNF␣ decreases expression of SRA and CD36 and re- non-AD patients. duces uptake of A␤. A, B, N9 microglia-like cells were treated with TNF␣ overnight, and ex- Our data also suggest that failure of the microglia to perform pression of SRA and CD36 were measured by QPCR (A) and by cell surface staining and flow their A␤-clearing functions may be a direct result of the A␤- cytometry (B). Data represent the average of five separate experiments and are displayed for induced inflammatory response. TNF␣, a major cytokine pro- treated cells as percentage expression (%) of untreated controls (100%). B, Far right, Results of ␤ ␣ ␤ ␣ duced by microglia in response to A stimulation, reduced ex- TNF- treatment on binding/uptake of A -labeled with Hylite fluor-488. TNF- -treated or ␤ untreated N9 cells were incubated with fluorescent A␤ for 2 h, and then cell-associated fluo- pression of the A receptors SRA and CD36, similar to the rescence was measured by flow cytometry. Data represent an average of five experiments and reduction observed in microglia from aging PS1-APP mice. It is are displayed for TNF␣-treated cells as percentage (%) of mean fluorescence intensity of un- not clear what mechanism accounts for reduced expression of the treated cells. Error bars indicate SEM. degrading enzymes, but reactive oxygen species and a number of proinflammatory cytokines have been found to be upregulated in transgenic mice with AD-like pathology. It is possible that one or more of these cytokines is responsible for downregulation of the 0.00005) of untreated cells, respectively (Fig. 6B). Thus, treat- A␤ degrading enzymes. ment of N9 microglia with TNF␣ resulted in a significant de- Interestingly, in addition to downregulating A␤ clearance crease in expression of both SRA and CD36 RNAs and membrane pathways, proinflammatory cytokines may also contribute to A␤ protein. Interestingly, treatment with these proinflammatory cy- accumulation by another mechanism. Recently, it was shown tokines did not affect expression of neprilysin and insulysin in N9 that TNF␣ and interferon-␥ upregulate ␤-secretase (BACE1), an microglia, suggesting that the reduction in RNA levels of these enzyme involved in A␤ production (Yamamoto et al., 2007). enzymes observed in vivo may be mediated by a different TNF␣, IL-1␤, and interferon-␥ were also found to stimulate mechanism(s). ␥-secretase-mediated cleavage of APP (Liao et al., 2004). Proin- N9 microglia bind to and phagocytose fluorescently labeled flammatory cytokines therefore may also contribute to AD-like ␤ A (supplemental Fig. 2, available at www.jneurosci.org as sup- pathology by promoting A␤ generation. plemental material). To determine whether decreased expression Our data provide evidence to support the paradigm that the ␣ of SRA and CD36 on N9 microglia in response to TNF treat- inflammatory response in AD is a “double-edged sword.” Micro- ment affected the ability of these cells to bind and/or phagocytose glia are recruited to sites of A␤ deposition as part of the attempt of A␤, we incubated TNF␣-treated and untreated N9 microglia the brain to clear these neurotoxic peptides, and early microglial with fluorescently labeled A␤ for 2 h, and measured cell- accumulation in AD delays disease progression. However, as AD associated fluorescence intensity by flow cytometry. Cells treated mice age, their microglia become dysfunctional and show a sig- with TNF␣ showed a 30% decrease in binding/uptake of labeled nificant reduction in expression of their A␤-binding receptors A␤ compared with untreated cells ( p Ͻ 0.01) (Fig. 6B). These and A␤-degrading enzymes, but maintain their ability to produce data show that TNF␣ reduces expression of genes involved in proinflammatory cytokines. These cytokines may in turn act in binding and phagocytosis of A␤, resulting in decreased uptake of an autocrine manner and promote A␤ production by stimulating A␤ by N9 microglia. These data also suggest that the reduction in ␤- and ␥-secretases and/or reduce A␤ clearance by reducing ex- expression of A␤-binding receptors observed in aged PS1-APP pression of A␤-binding receptors and A␤-degrading enzymes. microglia may, in part, be mediated by TNF␣ The convergence of both of these cytokine-mediated pathways 8360 • J. Neurosci., August 13, 2008 • 28(33):8354–8360 Hickman et al. • Microglial Dysfunction in Alzheimer’s Disease leads to increased A␤ accumulation and contributes to disease DR (2001) Co-expression of multiple transgenes in mouse CNS: a com- progression. Antiinflammatory therapy that distinguishes be- parison of strategies. Biomol Eng 17:157–165. tween such dichotomous functions of microglia and promotes Jankowsky JL, Fadale DJ, Anderson J, Xu GM, Gonzales V, Jenkins NA, Cope- ␤ land NG, Lee MK, Younkin LH, Wagner SL, Younkin SG, Borchelt DR their ability to clear A , while decreasing their ability to produce (2004) Mutant presenilins specifically elevate the levels of the 42 residue proinflammatory cytokines, may indeed be very helpful to delay beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific or stop the progression of Alzheimer’s disease. gamma secretase. Hum Mol Genet 13:159–170. Leissring MA, Farris W, Chang AY, Walsh DM, Wu X, Sun X, Frosch MP, References Selkoe DJ (2003) Enhanced proteolysis of beta-amyloid in APP trans- Alarco´n R, Fuenzalida C, Santiba´n˜ez M, von Bernhardi R (2005) Expression genic mice prevents plaque formation, secondary pathology, and prema- of scavenger receptors in glial cells. Comparing the adhesion of astrocytes ture death. Neuron 40:1087–1093. and microglia from neonatal rats to surface-bound beta-amyloid. J Biol Liao YF, Wang BJ, Cheng HT, Kuo LH, Wolfe MS (2004) Tumor necrosis Chem 280:30406–30415. factor-alpha, interleukin-1beta, and interferon-gamma stimulate Borchelt DR, Ratovitski T, van Lare J, Lee MK, Gonzales V, Jenkins NA, gamma-secretase-mediated cleavage of amyloid precursor protein Copeland NG, Price DL, Sisodia SS (1997) Accelerated amyloid deposi- through a JNK-dependent MAPK pathway. J Biol Chem tion in the brains of transgenic mice coexpressing mutant presenilin 1 and 279:49523–49532. amyloid precursor proteins. Neuron 19:939–945. McGeer PL, Itagaki S, Tago H, McGeer EG (1987) Reactive microglia in Coraci IS, Husemann J, Berman JW, Hulette C, Dufour JH, Campanella GK, patients with senile dementia of the Alzheimer type are positive for the Luster AD, Silverstein SC, El-Khoury JB (2002) CD36, a class B scaven- histocompatibility glycoprotein HLA-DR. Neurosci Lett 79:195–200. ger receptor, is expressed on microglia in Alzheimer’s disease brains and Meda L, Cassatella MA, Szendrei GI, Otvos L Jr, Baron P, Villalba M, Ferrari can mediate production of reactive oxygen species in response to beta- D, Rossi F (1995) Activation of microglial cells by beta-amyloid protein amyloid fibrils. Am J Pathol 160:101–112. and interferon-gamma. Nature 374:647–650. Dickson DW (1999) Microglia in Alzheimer’s disease and transgenic mod- Mott RT, Hulette CM (2005) Neuropathology of Alzheimer’s disease. Neu- els. How close the fit? Am J Pathol 154:1627–1631. roimaging Clin N Am 15:755–765, ix. Dickson DW, Farlo J, Davies P, Crystal H, Fuld P, Yen SH (1988) Alzhei- Perlmutter LS, Barron E, Chui HC (1990) Morphologic association between mer’s disease. A double-labeling immunohistochemical study of senile microglia and senile plaque amyloid in Alzheimer’s disease. Neurosci Lett plaques. Am J Pathol 132:86–101. 119:32–36. El Khoury J, Hickman SE, Thomas CA, Cao L, Silverstein SC, Loike JD Perry VH, Gordon S (1988) Macrophages and microglia in the nervous sys- (1996) Scavenger receptor-mediated adhesion of microglia to beta- amyloid fibrils. Nature 382:716–719. tem. Trends Neurosci 11:273–277. El Khoury J, Hickman SE, Thomas CA, Loike JD, Silverstein SC (1998) Mi- Selkoe DJ (2000) The origins of Alzheimer disease: a is for amyloid. JAMA croglia, scavenger receptors, and the pathogenesis of Alzheimer’s disease. 283:1615–1617. Neurobiol Aging 19:S81–S84. Simard AR, Soulet D, Gowing G, Julien JP, Rivest S (2006) Bone marrow- El Khoury J, Toft M, Hickman SE, Means TK, Terada K, Geula C, Luster AD derived microglia play a critical role in restricting senile plaque formation (2007) Ccr2 deficiency impairs microglial accumulation and accelerates in Alzheimer’s disease. Neuron 49:489–502. progression of Alzheimer-like disease. Nat Med 13:432–438. Stalder M, Phinney A, Probst A, Sommer B, Staufenbiel M, Jucker M (1999) El Khoury JB, Moore KJ, Means TK, Leung J, Terada K, Toft M, Freeman Association of microglia with amyloid plaques in brains of APP23 trans- MW, Luster AD (2003) CD36 mediates the innate host response to beta- genic mice. Am J Pathol 154:1673–1684. amyloid. J Exp Med 197:1657–1666. Thomas CA, Li Y, Kodama T, Suzuki H, Silverstein SC, El Khoury J (2000) Fiala M, Lin J, Ringman J, Kermani-Arab V, Tsao G, Patel A, Lossinsky AS, Protection from lethal gram-positive infection by macrophage scavenger Graves MC, Gustavson A, Sayre J, Sofroni E, Suarez T, Chiappelli F, receptor-dependent phagocytosis. J Exp Med 191:147–156. Bernard G (2005) Ineffective phagocytosis of amyloid-beta by macro- Yamamoto M, Kiyota T, Horiba M, Buescher JL, Walsh SM, Gendelman HE, phages of Alzheimer’s disease patients. J Alzheimers Dis 7:221–232; dis- Ikezu T (2007) Interferon-gamma and tumor necrosis factor-alpha reg- cussion 255–262. ulate amyloid-beta plaque deposition and beta-secretase expression in Frautschy SA, Yang F, Irrizarry M, Hyman B, Saido TC, Hsiao K, Cole GM Swedish mutant APP transgenic mice. Am J Pathol 170:680–692. (1998) Microglial response to amyloid plaques in APPsw transgenic Yan P, Hu X, Song H, Yin K, Bateman RJ, Cirrito JR, Xiao Q, Hsu FF, Turk mice. Am J Pathol 152:307–317. JW, Xu J, Hsu CY, Holtzman DM, Lee JM (2006) Matrix Janelsins MC, Mastrangelo MA, Oddo S, LaFerla FM, Federoff HJ, Bowers WJ metalloproteinase-9 degrades amyloid-beta fibrils in vitro and compact (2005) Early correlation of microglial activation with enhanced tumor plaques in situ. J Biol Chem 281:24566–24574. necrosis factor-alpha and monocyte chemoattractant protein-1 expres- Yan SD, Chen X, Fu J, Chen M, Zhu H, Roher A, Slattery T, Zhao L, Na- sion specifically within the entorhinal cortex of triple transgenic Alzhei- gashima M, Morser J, Migheli A, Nawroth P, Stern D, Schmidt AM mer’s disease mice. J Neuroinflammation 2:23. (1996) RAGE and amyloid-beta peptide neurotoxicity in Alzheimer’s Jankowsky JL, Slunt HH, Ratovitski T, Jenkins NA, Copeland NG, Borchelt disease. Nature 382:685–691.