Colony-stimulating factor 1 receptor inhibition prevents microglial plaque association and improves cognition in 3xTg-AD mice
- Nabil N. Dagher†1,
- Allison R. Najafi†1,
- Kara M. Neely Kayala1,
- Monica R. P. Elmore1,
- Terra E. White1,
- Rodrigo Medeiros1,
- Brian L. West2 and
- Kim N. Green1Email author
© Dagher et al. 2015
Received: 27 February 2015
Accepted: 21 July 2015
Published: 1 August 2015
Microglia are dependent upon colony-stimulating factor 1 receptor (CSF1R) signaling for their survival in the adult brain, with administration of the dual CSF1R/c-kit inhibitor PLX3397 leading to the near-complete elimination of all microglia brainwide. Here, we determined the dose-dependent effects of a specific CSF1R inhibitor (PLX5622) on microglia in both wild-type and the 3xTg-AD mouse model of Alzheimer’s disease.
Wild-type mice were treated with PLX5622 for up to 21 days, and the effects on microglial numbers were assessed. 3xTg-AD mice were treated with PLX5622 for 6 or 12 weeks and effects on microglial numbers and pathology subsequently assessed.
High doses of CSF1R inhibitor eliminate most microglia from the brain, but a 75 % lower-dose results in sustained elimination of ~30 % of microglia in both wild-type and 3xTg-AD mice. No behavioral or cognitive deficits were found in mice either depleted of microglia or treated with lower CSF1R inhibitor concentrations. Aged 3xTg-AD mice treated for 6 or 12 weeks with lower levels of PLX5622 resulted in improved learning and memory. Aβ levels and plaque loads were not altered, but microglia in treated mice no longer associated with plaques, revealing a role for the CSF1R in the microglial reaction to plaques, as well as in mediating cognitive deficits.
We find that inhibition of CSF1R alone is sufficient to eliminate microglia and that sustained microglial elimination is concentration-dependent. Inhibition of the CSF1R at lower levels in 3xTg-AD mice prevents microglial association with plaques and improves cognition.
Microglia are the primary immune cell of the central nervous system (CNS), comprising ~12 % of all cells found there. They are found ubiquitously throughout the CNS and function to detect pathogens and insults and respond to them through complicated physical and chemical remodeling processes. While microglia are crucial for the protection of the CNS from pathogens, as well as in clearing up cellular debris following cell death or minor injuries, they are well studied for their roles in neurodegenerative diseases and CNS injuries, such as traumatic brain injuries and stroke [1, 2]. It is generally thought that microglia mount an initial beneficial response, helping to limit damage from injury , or phagocytosing aggregating toxic peptides such as Aβ  and perhaps limiting plaque formation. However, these initial responses evolve into chronic inflammatory processes that may never resolve and can be damaging to the local brain environment, thus contributing to the disease/injury process itself . Hence, finding ways to manipulate microglial function and numbers may offer a way to treat CNS disorders. To that end, we recently discovered that treatment of adult mice with PLX3397, the dual colony-stimulating factor 1 receptor (CSF1R) and c-kit kinase inhibitor, leads to the near-complete elimination of all microglia from the CNS within 7–21 days . Furthermore, microglia remain eliminated for the duration of treatment, allowing for indefinite microglial elimination from the adult CNS. As CSF1R knockout mice are born without microglia [7, 8], and mice lacking either of its two substrates, CSF1  or IL-34 , also have reduced microglial densities; it suggests that microglia are fully dependent upon CSF1R signaling for their survival. Here, we describe the effects of a specific CSF1R inhibitor, PLX5622, on microglial homeostasis in the adult brain. PLX5622 is a potent inhibitor of CSF1R tyrosine kinase activity (KI = 5.9 nM) with at least 50-fold selectivity over 4 related kinases, and over 100-fold selectivity against a panel of 230 kinases [11–14]. As prolonged microglial elimination may not be translatable to humans for the extended duration of a neurodegenerative disease, we have explored the effects of lower, and more clinically relevant, drug exposures on microglial phenotypes and animal cognition/behavior in both adult healthy mice and a mouse model of Alzheimer’s disease.
PLX5622 was provided by Plexxikon Inc. and formulated in AIN-76A standard chow by Research Diets Inc. at the doses indicated in the text.
All rodent experiments were performed in accordance with animal protocols approved by the Institutional Animal Care and Use Committee at the University of California, Irvine (UCI). LPS treatment: LPS (from Escherichia coli 055:B5; Sigma) was dissolved in phosphate buffered saline (PBS) at a concentration of 0.1 mg/ml and administered intraperitoneally (IP) at a dose of 0.5 mg/kg body weight. Following any treatments, mice were sacrificed and brains isolated. One-half of the brain was fixed in 4 % paraformaldehyde and the other half was snap frozen on dry ice and stored at −80 °C until analysis.
Thioflavin S staining
Brain sections were incubated in 0.5 % thioflavin S solution in 50 % ethanol for 10 min, rinsed twice in 50 % ethanol, then rinsed twice in water. Sections were visualized with a confocal microscope. Average plaque number, size, and percentage distribution of size were obtained using Bitplane Imaris 7.4 software.
Fluorescent immunolabeling followed a standard indirect technique (primary antibody followed by fluorescent secondary antibody) as described in . Cell counts and sizes were obtained by scanning regions at 10× at comparable sections in each animal, followed by automatic analyses using Bitplane Imaris 7.4. Acid pretreatments were used for 6e10 detection. The following antibodies were used: anti-IBA1 (1:1000; Wako), anti-GFAP (1:1000; Dako), anti-6e10 (1:1000; Chemicon), and anti-S100 (1:1000; Abcam). Stained tissue was mounted on slides and cover slipped with Dapi Fluoromount-G (SouthernBiotech).
Aß1–40 and Aß1–42 were measured using a sensitive sandwich ELISA system as previously described .
mRNA extraction and real-time PCR
Total mRNA was extracted from frozen half brains, cDNA was synthesized, and real-time PCR (RT-PCR) was performed with commercially available kits for TNFα (F, 5′-GGTGCCTATGTCTCAGCCTCTT; R, 5′-GCCATAGAACTGATGAGAGGGAG) and IL1β (F, 5′-TGGACCTTCCAGGATGAGGACA; R, 5′- GTTCATCTCGGAGCCTGTAGTG). CT values were normalized to GAPDH and expressed as a percent of control. Two-month-old wild-type mice were treated with PLX5622 (1200 mg/kg chow; n = 4) or vehicle (n = 4) for 14 days. On day 14, half of the mice in each dietary group were administered with either LPS (0.5 mg/kg) or an equivalent volume of PBS via IP injection (n = 4 per group). Mice were euthanized 6 h after injection, perfused with PBS, and their brains were collected and snap frozen with dry ice for RNA extraction.
Open field: Open-field testing was employed as a measure of anxiety as well as motor ability. Mice were placed in an opaque white box (33.7 cm L × 27.3 cm W × 21.6 cm H) for 5 min while their behavior was video-recorded. The amount of time spent in the center versus the perimeter of the box and motor readouts (distance moved and velocity) were obtained. Barnes maze: Mice were tested in the Barnes maze (table diameter 122 cm, 40 holes with diameter 4.8 cm, elevated 140 cm above the ground) for 5 days (acquisition, 4 days, 2 trials/day, maximum 120 s/trial, 15 min intertrial interval; 24-hr probe, 1 day, 1 trial, maximum of 120 s), as a measure of spatial learning and memory. Latency to find the target and enter the target box (i.e., escape latency) was recorded live each day of acquisition, while latency to find the target and number of errors prior to finding the target was recorded live on the probe day. Morris water maze: Hidden platform Morris water maze (MWM) training and testing were conducted as described previously . Novel object and place recognition tasks: Novel object and place recognition training and testing were conducted as described previously .
Aβ42 was oligomerized as previously described . One micromolar of this stock was added to BV2 cell cultures 24 h before the assay to create Aβ-stimulated enriched media. This media was used as a chemoattractant in the ChemoTx® Chemotaxis System. BV2 cells were treated either 15 min or 24 h before the assay with 1- or 10 μM of PLX5622 in DMSO, and 50,000 cells were added to each well on the assay plate. Cells were allowed to migrate for 3 h. Migrated cells were stained with H+E and counted.
Appropriate statistical analyses were carried out to determine significance between groups using unpaired Student’s t test for comparisons between two groups, one-way ANOVA for multiple comparisons, with Newman-Keuls post hoc multiple comparison test. Multiple-day behavioral data and MSD® Multi-Spot Assay data were analyzed using a two-way ANOVA (treatment x day of testing and diet x injection, respectively) using the MIXED procedure of the Statistical Analysis Systems software (SAS Institute Inc.). For behavioral data, “mouse” was a random effect and “day of testing” was a repeated measure. Post hoc paired contrasts were used to examine biologically relevant interactions from the two-way ANOVA.
The selective CSF1R inhibitor PLX5622 reduces microglial numbers in the adult brain
We next explored the response of the microglia-depleted brain to systemically administered LPS. Two-month-old wild-type mice were treated with PLX5622 (300 or 1200 mg/kg chow) or vehicle for 14 days, and 0.5 mg/kg LPS was then administered (IP; n = 4 per group). Mice were sacrificed 6 h later, and mRNA and protein levels of inflammatory markers were measured from brain tissue via real-time PCR (normalized to the housekeeping gene GAPDH) and MSD® Multi-Spot Assay, respectively (Fig. 1c–f). As expected, LPS treatment robustly increased RNA levels of both TNFα and IL-1β message in microglia-intact animals and increased protein levels of nearly all inflammatory markers examined, with the exception of IL-4. Levels of TNFα and IL-1β mRNA in response to LPS in the 1200 mg/kg PLX5662-treated mice were significantly diminished (Fig. 1d), and protein levels of IFN-γ, IL-10, and IL-1β in response to LPS were also decreased with 1200 mg/kg PLX5622 treatment; however, no changes in inflammatory markers were seen with 300 mg/kg PLX5622 treatment (Fig. 1e, f). Interestingly, although the mRNA levels for TNFα decreased with 1200 mg/kg PLX5622 treatment in response to LPS, TNFα protein levels were not reduced. Although apparently contradictory, these data likely reflect the ability of TNFα to cross the BBB from the periphery . Indeed, plasma TNFα levels in these samples were highly and significantly elevated in all LPS-injected groups (data not shown).
Microglial depletion does not affect behavior or learning and memory
Microglial elimination with PLX5622 followed by drug removal results in rapid repopulation of the CNS
PLX5622 improves cognition in aged 3xTg-AD mice
Lower-dose PLX5622 treatment partially reduces microglial numbers
Lower-dose PLX5622 treatment prevents microglial association with plaques
3xTg-AD mice also show progressive tau pathology as they age, including somatodendritic accumulation of human tau and hyperphosphorylation . Quantification of total human tau accumulation within somatodendritic compartments of CA1 neurons showed no differences with treatment, nor tau phosphorylated at S202/T205 (Fig. 6h–k).
PLX5622 prevents chemotaxis of BV2 cells in response to Aβ-stimulated enriched media
We previously discovered that administration of the dual CSF1R/c-kit kinase inhibitor PLX3397 led to the rapid elimination of >99 % of all microglia from the CNS within 7–21 days . CSF1R knockout mice are born without microglia [7, 8], suggesting that signaling through this receptor is crucial for the development of microglia. These mice are born with developmental defects and die a few weeks after birth, making them an unsuitable model for studying microglial function. The CSF1R has two endogenous ligands—CSF1 and IL-34 . Mice lacking either one of these ligands are also born with lower densities of microglia throughout the CNS , and diminished numbers of microglia are maintained throughout life. Thus, the CSF1R seems crucial for microglial development and also population maintenance, as well as microglial proliferation during responses to neurodegeneration . As our previous study inhibited both CSF1R and c-kit, we set out to determine if inhibition of CSF1R alone was sufficient to eliminate microglia from the adult brain. Our results with the specific CSF1R inhibitor PLX5622 clearly show that inhibition of CSF1R alone is sufficient to eliminate microglia, and therefore, microglia require CSF1R signaling for their survival. Crucially, we found that lower doses of CSF1R inhibitor could lead to sustained elimination of a percentage of microglia, thus allowing us to tightly control the number of surviving microglia through different concentrations of CSF1R inhibitors. This approach may be more practical for clinical applications, where complete elimination of microglia for extended periods of time may be undesirable. Thus, we further sought to establish the effects and benefits of this paradigm in both healthy and diseased mice.
We previously demonstrated that elimination of microglia with PLX3397 had no detrimental effects on locomotion, cognition, or behavior, despite mice being depleted of microglia for up to 2 months . This was an unexpected finding, as a role for microglia in synaptic sculpting [5, 27] and neuronal communication  is emerging. However, we confirm our prior results and show that elimination of microglia with PLX5622 leads to no discernable deficits in behavior or learning and memory in the tasks tested. Likewise, lower levels of CSF1R inhibitor treatment had no effects on behavior or learning in wild-type mice. As prolonged near-complete microglial elimination, which is achieved with the higher doses of CSF1R inhibitors, may not be translatable for the full duration of a neurodegenerative disease, lower levels of CSF1R inhibitors may offer a chronic option for the treatment of neuroinflammation. To that end, we also tested chronic treatment with the lower dose of PLX5622 in aged 3xTg-AD mice. As with wild-type mice, this lower dose resulted in sustained elimination of only ~30 % of microglia, even over a 3 month period. Strikingly, however, this treatment strongly diminished the association between microglia and plaques; untreated 3xTg-AD mice have high plaque burdens and all plaques are tightly surrounded by numerous microglia, yet treated 3xTg-AD mice have the same plaque burdens, but their microglia no longer surround them. Despite this lack of microglia associated with plaques, Aβ levels and plaque numbers and sizes were not altered, suggesting that microglia surrounding plaques are not actively restricting their growth or formation, consistent with previous findings .
Inflammatory status has been linked to cognitive deficits in AD patients , with anti-inflammatory treatments improving cognition in transgenic models of the disease . Here, we found that targeting microglia with CSF1R inhibitors also led to improvements in cognition in the 3xTg-AD mice. We find that low dose treatments reduce brain microglial number by 30 %, but this does not diminish the overall levels of inflammatory markers. In line with these results, we explored the inflammatory response to LPS in wild-type mice with the same 300 mg/kg PLX5622 treatment and also found no significant effects of treatment. Moreover, we actually found increases in the levels of the typically proinflammatory markers TNFα and CXCL1 with treatment in the 3xTg-AD mice. Though seemingly counterintuitive, these results direct our attention towards other possible, non-inflammatory, mechanisms of action of PLX5622 treatment for improved behavior, potentially by acting as a “microglial shaper.” Notably, we find that this treatment prevents the association of microglia with plaques, suggesting that CSF1R inhibition alters the microglial phenotype and results in behavioral improvements. While we cannot determine the relative contributions of modest reductions in microglial numbers vs. prevention of microglial association with plaques, treatment with 300 mg/kg PLX5622 ultimately results in improved cognition. Although average cell area was increased in surviving microglia, which we have found to be a stereotypical response to CSF1R inhibition , we found average IBA1 staining intensity to be reduced in PLX5622-treated mice. As differential levels of IBA1 expression have also been linked to migratory function , the reduction in IBA1 staining intensity supports the hypothesis that microglial function is altered with 300 mg/kg PLX5622 treatment.
Of interest, recent studies investigating the effects of the AD-associated TREM2 gene on AD pathology found that heterozygous loss of one or two TREM2 alleles altered the microglial response to Aβ plaques [32–34], paralleling the effects of lower doses of PLX5622. CSF1 signaling can be regulated by TREM2 [35, 34], which could suggest that the effects of TREM2 on microglia in the AD brain may be partly mediated by the CSF1 signaling cascade. It may be that association and chemotaxis of microglia to Aβ deposits are protective in the initial stages of the disease when the microglia can help clear the aggregates from the brain, but that preventing this association at later stages alters the chronic neuroinflammatory response and becomes beneficial, as we describe here.
We find that inhibition of CSF1R alone is sufficient to eliminate microglia but the level of elimination is both dose-dependent and chronically sustainable. Elimination of microglia does not impair behavior or cognition in wild-type mice. Of disease and translational relevance, lower dose inhibition of the CSF1R in 3xTg-AD mice prevents microglial association with plaques and improves cognition.
This work was supported by the National Institutes of Health under awards 1R01NS083801 (NINDS) and P50 AG016573 (NIA) to KNG, as well as the Whitehall foundation to KNG, the American Federation of Aging Research to KNG, and the Alzheimer’s Association to KNG. ARN and ME are supported by the NIH training fellowship AG00538. BLW is an employee of Plexxikon Inc. The Αβ peptides and anti-Αβ antibodies were provided by the University of California Alzheimer’s Disease Research Center (UCI-ADRC) NIH/NIA Grant P50 AG16573.
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