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Am J Pathol. 2000 Sep; 157(3): 895–904.
PMCID: PMC1885684

Expression of Macrophage Colony-Stimulating Factor Receptor Is Increased in the AβPPV717F Transgenic Mouse Model of Alzheimer’s Disease


Inflammation is an important neuropathological change in Alzheimer’s disease (AD). However, the pathophysiological factors that initiate and maintain the inflammatory response in AD are unknown. We examined AβPPV717F transgenic mice, which show numerous brain amyloid-β (Aβ) deposits, for expression of the macrophage colony-stimulating factor (M-CSF) and its receptor (M-CSFR). M-CSF is increased in the brain in AD and dramatically augments the effects of Aβ on cultured microglia. AβPPV717F animals 12 months of age showed large numbers of microglia strongly labeled with an M-CSFR antibody near Aβ deposits. M-CSFR mRNA and protein levels were also increased in brain homogenates from AβPPV717F animals. Dystrophic neurites and astroglia showed no M-CSFR labeling in the transgenic animals. A M-CSF antibody decorated neuritic structures near hippocampal Aβ deposits in transgenic animals. M-CSF mRNA was also increased in AβPPV717F animals in comparison with wild-type controls. Simultaneous overexpression of M-CSFR and its ligand in AβPPV717F animals could result in augmentation of Aβ-induced activation of microglia. Because chronic activation of microglia is thought to result in neuronal injury, the M-CSF system may be a potential target for therapeutic intervention in AD.

Inflammation is thought to be important in neuronal injury in Alzheimer’s disease (AD). 1-3 Microglia, intrinsic immune effector cells of the brain, may be key in this process. 4 Cell-culture studies suggest that the amyloid-β peptide (Aβ) is likely to be an important stimulus for microglial activation in AD. 5-10 Further, transgenic mouse models for AD based on expression of mutant human amyloid precursor proteins (AβPP) show microgliosis in the vicinity of Aβ deposits in a pattern reminiscent of the AD brain. 11-13 However, in cell-culture systems Aβ alone has often been found to be a relatively weak activator of microglia, with pro-inflammatory augmenting agents often required for a robust response. 6,9,14 This suggests that in mutant AβPP transgenic mouse brain, factors other than the direct actions of Aβ may be important in inducing microglial activation.

We recently demonstrated that macrophage colony-stimulating factor (M-CSF) strongly augments the pro-inflammatory effects of Aβ on cultured microglial cells. 15 M-CSF, a hematopoietic cytokine also produced by neurons and glia, 16-18 has powerful activating effects on microglia and on other macrophage-like cells. 19,20 The receptor for M-CSF (M-CSFR) is encoded by the c-fms proto-oncogene, is expressed on microglia and on cells of the monocyte/macrophage lineage, and has tyrosine kinase activity. 21,22 M-CSF is elevated in cerebrospinal fluid of AD patients, 18 and there is increased M-CSFR expression on microglia surrounding Aβ deposits in the AD brain. 23 Thus, unlike other microglial activators such as lipopolysaccharide and interferon-γ that augment the effects of Aβ on microglia in vitro, 5,6,24,25 clinical and neuropathological data suggest that M-CSF and its receptor have pathophysiological relevance to AD.

To further clarify the role of M-CSF and its receptor in microglial activation in AD, we examined their expression in a mouse model of AD that shows extensive Aβ deposits, neuritic dystrophy, and astrogliosis. 12,26 Microgliosis has also been reported in these animals, 27 although little information is available on the phenotypic features of the microglia. Our results demonstrate that there is extensive microglial activation in AβPPV717F mouse brain characterized by dramatic up-regulation of M-CSFR expression.

Materials and Methods

Animals and Tissue Processing

Development of animals carrying the AβPPV717F transgene and basic characterization of neuropathological changes have been described previously. 12,26 In the present study, AβPPV717F transgenic mice and wild-type animals were anesthetized with Fatal-Plus (0.1 ml/mouse, i.p.; Vortech) and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in Sorensen’s buffer. Brains were immediately removed and placed in ice-cold 4% paraformaldehyde. After 2.5 hours of fixation at 4°C, the brains were sectioned at 50 μm into ice-cold Tris-buffered saline (TBS) on a Vibroslice 752 (Campden Instruments, London, UK). Free-floating sections were used immediately for immunocytochemistry. Other transgenic and control brains were removed without perfusion, frozen immediately on dry-ice, and stored at −80°C until processed for RNA and protein. Transgenic (n = 16) and wild-type (n = 12) animals used in this study were between 4 and 23 months of age. For immunohistochemistry, 10 transgenic and six control animals were used. For reverse transcription (RT) and polymerase chain reaction (PCR), three transgenic and three wild-type animals were used, and for Western blotting, another three transgenic and three wild-type animals were used. All animal procedures were approved by institutional review boards.


Antibody reagents included a polyclonal reagent to the extracellular domain of mouse M-CSFR (diluted 1:1,000; Upstate, Lake Placid, NY), a polyclonal reagent to the intracellular domain of M-CSFR (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), a polyclonal reagent to human M-CSF (1:100; Santa Cruz Biotechnology), an antibody to phosphotyrosine (1:200; Sigma Chemical Co., St. Louis, MO), an F4/80 antibody (1:30; Harlan, Indianapolis, IN), an antibody to CD11b (5C6, 1:750; Serotec, Raleigh, NC), a polyclonal reagent to glial fibrillary acidic protein (GFAP) (1:1,000; a gift from Dr. L. Eng), an antibody to neurofilament proteins (SMI312, 1:1,000; Sternberger Monoclonals, Baltimore, MD), and a monoclonal reagent that reacts with all full-length Aβ species 28 (mAb 4.1; 1:150). For comparison, normal rabbit serum (Jackson ImmunoResearch, West Grove, PA) and normal goal serum (Zymed, South San Francisco, CA) were also used as primary antibodies. Secondary antibodies included Cy5-conjugated and Cy3-conjugated affinity-purified IgG reagents raised in goat, sheep, horse, or donkey and diluted to 1:1,000 (all from Jackson ImmunoResearch).


Free-floating sections were reacted with 10% normal blocking serum in TBS for 1 hour at room temperature, and then washed three times for 5 minutes each in TBS. The sections were then incubated with the primary antibody diluted in TBS at 4°C overnight with gentle shaking. Sections were then washed three times for 10 minutes each in TBS at room temperature. Secondary antibody (diluted in TBS) was applied to the sections and incubated at 37°C for 1 hour with gentle shaking. The sections were washed four times for 10 minutes each with PBS and twice with equilibration buffer (from SlowFade Antifade kit; Molecular Probes, Eugene, OR) for 3 minutes. Sections were then mounted on glass slides with the antifade reagent in glycerol/PBS. The coverslips were sealed with nail polish and stored in the dark at 4°C. All images were obtained within 24 hours after immunostaining.

For double-label immunocytochemistry, sections were reacted with 10% normal blocking serum (for the first secondary antibody) in TBS at room temperature for 1 hour, and then washed three times for 5 minutes each in TBS. They were then incubated overnight with the first primary antibody at 4°C with gentle shaking. After washing in TBS three times for 10 minutes each, sections were reacted with the first secondary antibody diluted in TBS at 37°C for 1 hour with gentle shaking. Sections were then washed in TBS four times for 10 minutes each, and incubated with the second normal blocking serum (for the second secondary antibody) in TBS at room temperature for 1 hour. After washing in TBS three times for 5 minutes each, the sections were reacted with the second primary antibody diluted in serum overnight at 4°C with gentle shaking. Sections were washed three times in TBS for 10 minutes each, and then reacted with the second secondary antibody at 37°C for 1 hour. Finally, sections were washed in TBS four times for 10 minutes each and then mounted as described above. Immunohistochemical results illustrated below are representative of data obtained from a minimum of three transgenic or wild-type animals. For a given primary antibody, immunohistochemistry was performed on at least three sections per animal.

Confocal Microscopy

Images were collected on a Molecular Dynamics (Sunnyvale, CA) MultiProbe 2010 laser-scanning confocal microscope with an argon/krypton laser and a Nikon Diaphot 200 inverted microscope. The microscope was interfaced with a Silicon Graphics Indigo2 system (Mountain View, CA) running Molecular Dynamics ImageSpace software. The instrument was configured for excitation at 568 nm (Cy3 secondary antibody), or 647 and 568 nm (Cy5 and Cy3). For all images a 50-μm pinhole aperture was used to produce true confocal images with maximum z axis resolution. Images were collected using ×10 and ×20 objectives or ×40 and ×60 oil immersion objectives. Contrast and hue were adjusted on some images using the ImageSpace software or Adobe Photoshop to provide optimal resolution of structures. However, in all cases adjustments were applied to the entire image and in no case was selective editing performed on a subregion of the image. Between two and 10 confocal images were collected from each section. A total of 603 confocal images of immunostained transgenic and wild-type sections were acquired during the course of the study.

Protein Extraction and Western Blotting

Brains were removed rapidly, the cortex and cerebellum separated from the rest of the brain by dissection, and then samples were frozen immediately in liquid nitrogen. Membrane protein extracts were prepared by chopping samples with a razor blade, suspending in lysis buffer (50 mmol/L mannitol, 5 mmol/L Hepes, pH 7.4, 1 mmol/L phenylmethyl sulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A), followed by 10 passages through a 25-gauge needle, after which CaCl2 was added with vortexing to a final concentration of 10 mmol/L. The resulting homogenate was centrifuged in a 1.5-ml tube at 15,600 × g for 1 minute. The supernatant was then centrifuged at 430,000 × g for 6 minutes in a TL-100 ultracentrifuge (Beckman, Palo Alto, CA). The resulting pellet was resuspended in lysis buffer. All protein extraction procedures were performed at 4°C using ice-cold reagents. Protein concentrations were determined using the bicinchoninic acid protein assay (Pierce, Rockford IL). For protein electrophoresis, the stacking gel was 5% sodium dodecyl sulfate-polyacrylamide, whereas the resolving gel was 8% sodium dodecyl sulfate-polyacrylamide. Five hundred μg of membrane protein extract was loaded per lane. Proteins were transferred from gels to polyvinylidene difluoride membranes (Millipore, Bedford, MA), and immunodetection was performed by incubating membranes with 10% normal goat serum in 3% nonfat milk (w/v) in PBS (PBS-milk) at room temperature for 1 hour. After two washes with PBS, the membranes were incubated overnight with agitation at 4°C with the antiserum against the extracellular domain of mouse M-CSFR in 3% PBS-milk (1:1,000; Upstate). Then the membranes were washed three times with PBS and incubated with goat anti-rabbit IgG linked to horseradish peroxidase in 3% PBS-milk (1:500; Santa Cruz Biotechnology) for 1.5 hours at room temperature with agitation. After three washes with PBS, bound antibody was detected with diaminobenzidine and hydrogen peroxide.

RNA Extraction

Frozen hemibrains were pulverized in liquid nitrogen and transferred to a 50-ml centrifuge tube along with Trizol reagent (1 ml per 100 mg tissue; Life Technologies, Inc., Gaithersburg, MD). Tissue was homogenized using a Polytron (Brinkman, Westbury, NY), and total RNA extraction was performed according to the manufacturer’s instructions. The concentration of total RNA samples dissolved in RNase-free water was determined using a spectrophotometer.


Methods for semiquantitative measurement of mRNA using RT-PCR have been previously described. 29 Briefly, total RNA was reverse-transcribed using Superscript II (Life Technologies, Inc.), primed by random hexamers. To equalize samples for RNA loading, the level of hypoxanthine phosphoribosyl transferase (HPRT) mRNA was determined in each sample as previously described, 29 and cDNA concentrations for subsequent PCR reactions were adjusted accordingly. For quantification of M-CSFR and M-CSF mRNA, the primers described by Raivich et al 30 were used. For M-CSFR mRNA, after a 5-minute denaturing incubation at 95°C, 35 cycles of PCR were performed in a Perkin-Elmer 9600 thermal cycler (Perkin-Elmer-Cetus, Emeryville, CA) consisting of 95°C for 45 seconds, 59°C for 45 seconds, and 72°C for 90 seconds, followed by an extension phase of 72°C for 5 minutes. For M-CSF mRNA, RT-PCR included a denaturing phase at 95°C for 5 minutes, then 30 cycles of 95°C for 45 seconds, 65°C for 45 seconds, 72°C for 1 minute and 30 seconds, and then a 5-minute 72°C extension phase. For HPRT mRNA, PCR was performed by denaturing at 96°C for 2 minutes, followed by 30 cycles of 94°C for 20 seconds, 60°C for 60 seconds, and 72°C for 2 minutes and 20 seconds.

To quantify PCR products, amplified DNA samples were transferred to a nylon membrane in a slot-blot apparatus and visualized using radiolabeled oligonucleotide probes as previously described. 29 For M-CSF mRNA detection, the probe was 5′-TCGGTGGCGTTAGCATTGGGGGTGT-3′ (bases 464 to 488, GenBank MUSCSFA, antisense orientation). For M-CSFR mRNA detection, the probe was 5′-TTTTATCTGTGGGGGCTCTGGGTGG-3′ (bases 669 to 693, GenBank MMFMSCR, antisense orientation). The HPRT probe has been previously described. 29 Autoradiograms were quantified using a densitometer. To assure that PCR amplifications had occurred in the exponential phase of the reaction, serial dilutions of cDNA were performed and quantified for each PCR reaction as described. 29



Antibodies to M-CSFR strongly labeled cells associated with Aβ deposits in hippocampus and cerebral cortex in sections from AβPPV717F transgenic mice. Labeling with the antibody to the extracellular domain of mouse M-CSFR is shown in Figure 1A . Cells decorated with the extracellular M-CSFR reagent had the morphology of activated microglia (Figure 1C) . In control brain this antibody reacted weakly with cells having the morphology of resting microglia (Figure 1B) . The reagent to the intracellular domain of M-CSFR also labeled cells with the morphology of activated microglia in transgenic mouse brain (Figure 1D) . No labeling was observed with the nonimmune sera. Likewise, the secondary antibodies showed no reactivity when the primary antibodies were omitted.

Figure 1.
Overexpression of M-CSFR in AβPPV717F transgenic mice. A: Low-power confocal image of occipital cortex in a 13-month-old AβPPV717F transgenic mouse reacted with antibody to M-CSFR, showing aggregates of activated microglia. Scale bars ...

Double-labeling experiments with M-CSFR reagents and antibodies to established microglial markers 31,32 demonstrated that the M-CSFR-reactive cells were indeed microglia. Thus, in transgenic mouse brain microglia labeled with M-CSFR reagents surrounding plaques were also labeled with CD11b, phosphotyrosine, and F4/80 antibodies (Figure 2, A–C) . In contrast, the antibody to GFAP-labeled cells with the morphology of reactive astrocytes, but never cells decorated by the M-CSFR reagents (Figure 2, D and E) . Similarly, in transgenic brain the antibody to neurofilament proteins reacted with dystrophic neurites, many of which were adjacent to M-CSFR-labeled cells. However, co-localization of neurofilament and M-CSFR labeling was not observed (Figure 3, A and B) .

Figure 2.
M-CSFR is expressed by microglia but not by astrocytes. A: Confocal image of hippocampal section from a 21-month-old AβPPV717F mouse showing microglia co-labeled with antibodies to M-CSFR (red) and CD11b (green). B: Confocal image of microglia ...
Figure 3.
No M-CSFR on dystrophic neurites; microglia overexpressing M-CSFR co-localize with Aβ. A: Low-power confocal image of posterior cerebral cortex in 19-month-old AβPPV717F mouse showing microglia labeled with antibody to M-CSFR (red) and ...

In the posterior hippocampal formation of transgenic animals, plaques tended to be large with numerous M-CSFR-positive microglia clustered around the central Aβ core (Figure 3C) . Dystrophic neurites labeled with the neurofilament antibody were found at approximately the same distance from the plaque core as the M-CSFR-labeled microglia (Figure 3B) . GFAP-positive astrocytes were generally found outside of the ring of M-CSFR-positive microglia (Figure 2, D and E) . In the occipital cortex, Aβ plaques tended to be small and compact. Here microglial cells intensely labeled with the M-CSFR antibody were often located at the center of the plaque, with multiple cells forming a cluster one on top of the other (Figure 3D) . These aggregates of microglial cells resembled the cap-like microglial cells described in AD brain. 33 In 4-month-old animals, cells with the morphology of activated microglia were occasionally detected with the M-CSFR reagents (Figure 3E) . These cells colocalized with Aβ deposits (Figure 3F) .

Structures with the morphology of dystrophic neurites were labeled by the M-CSF reagent in large plaques in the hippocampi of AβPPV717F transgenic mice (Figure 4A) , whereas no neuritic labeling was observed in the wild-type animals. No labeling of plaque structures with a nonimmune serum was observed. The antibody to M-CSF also labeled cells with neuronal morphology in both AβPPV717F transgenic mice and control animals. Figure 4, B and C , shows representative sections from transgenic and wild-type brains. There was no obvious difference in the intensity of immunostaining between the transgenic and control brains. Figure 4D shows the morphology of M-CSF-labeled cortical cells at higher power.

Figure 4.
M-CSF immunoreactivity in AβPPV717F mice. A: Confocal image of plaque in posterior hippocampal formation of 14-month-old AβPPV717F mouse reacted with reagent to M-CSF. Structures with the morphology of dystrophic neurites are strongly ...


Immunoblots from 4- and 12-month-old AβPPV717F transgenic mice showed that M-CSFR protein was increased in membrane protein extracts from cerebral cortices in comparison with levels in wild-type samples. In contrast, there was no increase in M-CSFR expression in cerebellum in transgenic animals in comparison with wild-type cerebellar M-CSFR levels. The protein detected by the intracellular M-CSFR antibody corresponded to the molecular weight of the mature posttranslationally modified form (∼165 kd) of M-CSFR. Results from 12-month-old wild-type and transgenic animals are shown in Figure 5A . Similar results were obtained with a second pair of 12-month-old animals.

Figure 5.
Western blot and RT-PCR for M-CSFR and M-CSF. A: Western blot of membrane extracts from cerebral cortex and cerebellum from a 12-month-old wild-type animal (WT) and from a 12-month-old AβPPV717F mouse (Tg) reacted with antibody to M-CSFR. The ...


Semiquantitative RT-PCR using total RNA extracts from hemibrains showed an increase in the mRNA encoding M-CSFR in AβPPV717F animals at both 4 months (Figure 5B ; 2.5-fold increase) and 12 months (Figure 5C ; twofold increase) of age in comparison to wild-type animals. RT-PCR also showed an increase in M-CSF mRNA in total RNA extracts from 4- and 12-month-old AβPPV717F mice compared to wild-type animals (Figure 5, B and C ; twofold increase at both ages). Similar results were obtained with a second pair of 12-month-old animals.


These results demonstrate increased expression of the M-CSFR on microglia in transgenic AβPPV717F mice. M-CSF was recently shown to augment the activating effects of Aβ on cultured microglia. 15 Specifically, combined treatment with M-CSF and Aβ induced much larger increases in the production of interleukin-1, interleukin-6, and nitric oxide than did either agent alone. Increased expression of M-CSFR on microglia in transgenic AβPPV717F mice could act synergistically with increased brain levels of M-CSF to powerfully augment Aβ-induced microglial activation. M-CSFR expression is increased in the AD brain, too. 23 Microglial activation in disease is thought to lead to production of potentially neurotoxic agents such as cytokines, nitric oxide, reactive oxygen species, and prostaglandins. 34

The close proximity of the M-CSFR-positive microglia to plaques suggests that aggregated Aβ could be the stimulus for increased M-CSFR expression. However, in this animal model Aβ deposits are but one change induced by the AβPPV717F transgene. Other changes include increased expression of mutant AβPP, neuritic dystrophy, and astrogliosis. 12 The proximity of M-CSFR-reactive microglia to dystrophic neurites and reactive astrocytes in AβPPV717F transgenic mice might also indicate that neurons and astrocytes play a role in inducing microglial M-CSFR expression.

We observed an increase in M-CSF mRNA and labeling of structures with the morphology of dystrophic neurites by an M-CSF antibody in the AβPPV717F transgenic brain. These findings may be consistent with a report that M-CSF is increased in cerebrospinal fluid from patients with AD. 18 Although we found that the M-CSF antibody labeled neuronal cell bodies, we did not observe an obvious increase in M-CSF immunostaining in the transgenic animals in comparison with controls. This finding is at variance with the observation of Du Yan et al 18 that neuronal M-CSF immunoreactivity in the AD brain is markedly greater than that in control brain. Future studies using quantitative image analysis of M-CSF immunoreactivity in transgenic and wild-type brains might detect an increase in staining intensity in the transgenic neurons. Some reports have localized M-CSF expression to astroglia rather to neurons. 16,17,35 We found no immunostaining of astrocytes with the M-CSF antibody. However, M-CSF exists in a variety of forms, including membrane bound, soluble, and bound to the extracellular matrix. 36 It is possible that the antibody we used did not detect the predominant form expressed by astrocytes. In situ hybridization studies of AβPPV717F transgenic mice would contribute toward clarifying this issue. Regardless of the source of M-CSF in AβPPV717F transgenic brain, increased expression of this cytokine and its receptor could act synergistically to activate microglia.

Extensive microglial activation has been reported in two other transgenic mouse models for AD characterized by Aβ deposits in the brain. 11,13 It will be interesting to determine whether expression of M-CSFR and/or M-CSF is altered in either of these two models. Increased M-CSFR expression by brain microglia has recently been observed in mouse mechanical injury and ischemia models. 30,37 Indeed, it has been proposed that M-CSFR is a highly specific indicator of activation of all macrophage-like cells. 38 It is conceivable that a variety of neurological insults lead to activation of microglia via the M-CSF/M-CSFR system. Interestingly, mechanical injury resulted in a increase in M-CSFR expression but did not change the constitutive expression of M-CSF, 30 in contrast to our findings in the AβPPV717F transgenic model. In the ischemia model, neuronal expression of M-CSF was detected near the lesion, 37 whereas in the mechanical injury model 30 and in the AβPPV717F transgenic animals no neuronal M-CSFR expression was found. These contrasting results may reflect important underlying differences in the regulation of M-CSFR and its ligand among models of neurological disease.

Little is known about the specific signals that regulate M-CSFR expression in microglia. In other cell types, M-CSFR expression has been shown to be regulated by growth factors and cytokines such as heparin-binding epidermal growth factor-like growth factor, interleukin-10, and transforming growth factor-β. 39-41 The neuritic plaque in AD is rich in a variety of growth factors and cytokines thought to be produced by neurons, astrocytes, and microglia. 34 One or more of these agents could induce M-CSFR expression by microglia, ultimately leading to an augmentation of the microglial activating effects of Aβ. Determining the signals for increased M-CSFR expression in AD and in animal models for AD may lead to new strategies for preventing the microglial-mediated neuronal injury in AD.


We thank Dr. Linda Higgins, Xiaorong Ou, and Dr. Susan Palmieri for their valuable assistance; and Lilly Neuroscience for kindly providing the AβPPV717F transgenic mice.


Address reprint requests to Greer M. Murphy, Jr., M.D., Ph.D., Neuroscience Research Laboratories, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, MSLS P-104, Stanford, CA 94305-5485. E-mail: .ude.drofnats.dnalel@yhprumg

Supported by National Institute of Mental Health grants MH01239, MH 57833, and MH40041.


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