CSF-1R (IC50 = 0.016 μM); FLT3 (IC50 = 0.39 μM); KIT (IC50 = 0.86 μM); AURKC (IC50 = 1 μM); KDR (IC50 = 1.1 μM)

PLX5622 (1–20 μM; 3 days) efficiently reduces microglia in cerebellar slices while having no effect on oligodendrocytes or astrocytes. After three days at 4 μM, NG2+ or PDGFRα+ cell counts are reduced by 30–40%, and at 20 μM, this number rises to 90–95%. Despite a robust (~95%) depletion of the microglial cells, slices exposed to 1 μM or 2 μM PLX5622 show no reduction of NG2+ or PDGFRα+ OPCs[3].

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Cell free kinase inhibitor profiling revealed that PLX5622 is highly specific for the CSF1R, showing > 20-fold selectivity over KIT and FLT3, the two most homologous receptors (Supplementary Table 2, 3). Two key structural differences between PLX5622 and PLX3397 contribute to the improved selectivity based on crystallographic analysis (Fig. 2b; Supplementary Table 4). First, the 2-fluoro substitution on the middle pyridine ring of PLX5622 is designed to access the CSF1R-unique space next to Gly-795 (a gate-keeper to the interior allosteric pocket); both KIT and FLT3 have a bulkier cysteine at the equivalent position. While the difference between a hydrogen atom and a fluorine atom may seem small (with slightly longer bond length and slightly larger radius, a fluorine atom extends the van der Waals edge by ~ 0.5 Å compared to a hydrogen atom), the effect is significant. Anchored on both ends by hydrogen bonds and hydrophobic packing (Fig. 2b), PLX5622 has the fluorine atom at a fixed position. As van der Waals repulsion increases in proportion to the 6th power of the distance between two atoms, a larger gate keeper residue (as seen in KIT and FLT3) will likely incur an energetic penalty for the steric hindrance. In support of this mechanism, a close analog of PLX3397 containing the same 2-fluoro substitution as PLX5622 has the same potency as PLX3397 in inhibiting CSF1R but is 10-fold more selective over KIT than PLX3397. Second, the terminal pyridine group of PLX5622 is optimized to stabilize the allosteric pocket of CSF1R vacated by the displaced juxtamembrane domain. When PLX5622 binds to KIT or FLT3, the position and orientation of the middle pyridine ring causes steric clash with the gate-keeper cysteine, compromising the optimal fit of the terminal pyridine moiety. [1]

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CSF1R kinase inhibitors PLX5622 and PLX3397 depleted microglia in cerebellar slices [3]
PLX5622 or PLX3397 were applied to cerebellar slices at increasing concentrations from 1 to 20 μM. After three days of treatment, bothPLX5622 and PLX3397 at concentrations greater than 2 μM eliminated more than 95% of microglia (Fig. 1A,B). At 1 μM, PLX5622 caused 15% more depletion than PLX 3397 (94.6±1.1% vs 79.9±2.4% Iba1+ cell loss) (Fig. 1A,B). With greater than 95% loss of microglia at 2 μM, no changes were observed in the viability or morphology of oligodendrocytes or astrocytes, as assessed by PLP-eGFP expression or GFAP staining with either PLX preparation (Fig. 1C). High concentrations of PLX5622 and PLX3397 were cytotoxic to OPCs ex vivo and in vitro [3]
Because CSF1R inhibitors may bind to multiple tyrosine kinases, we examined the effects of PLX5622 and PLX3397 on OPCs, which depend on the tyrosine kinase PDGFRα for survival. OPC cell numbers were assessed by NG2 or PDGFRα immunostaining following exposure of cerebellar explants to increasing PLX concentrations for 3 days. At 4 μM, PLX5622 caused a 30-40% reduction in NG2+ or PDGFRα+ cells; this increased to 90-95% at 20 μM. No reduction of NG2+ or PDGFRα+ OPCs was observed in slices exposed to 1 μM or 2 μM PLX5622 despite robust (~95%) depletion of the microglial cells (Fig. 2A,C). In contrast, treatment with PLX3397 significantly reduced OPC numbers at all concentrations tested. We observed a 30-35% loss of NG2+ or PDGFRα+ cells at 0.5 μM, despite incomplete (~70%) depletion of microglia. OPC loss with PLX3397 was concentration dependent with a 75-85% reduction at 1 μM and > 90% loss at 2 μM and 20 μM (Fig. 2B,D).

A similar effect on OPCs was observed in purified primary murine OPC cultures. Cell type specific marker staining showed that the cultures consisted of >92% OPCs (PDGFRα), <1% astrocytes (GFAP), and 3.7±2.1% microglia (Iba1) (Supplemental Fig. S1A). Most oligodendroglial cells were OPCs but, occasionally (<0.5%) maturing OLs (04+ with a highly complex network of processes) were noted (data not shown). Low concentration (0.5 μM) of PLX5622 had no effect on viability after 24 h; however, 20 μM PLX5622 resulted in increased OPC death. In contrast, PLX3397 treatment caused significant cytotoxicity at 0.5 μM and 20μM (Supplemental Fig. S1B,C). These results indicate that the PLX CSF1R inhibitors can directly impair OPC viability in a concentration-dependent manner.

To examine the effect of long-term microglia depletion on OPC survival, 2 μM PLX5622 was applied to cerebellar slices for 8 days. Although microglia were completely eliminated, we observed no difference in NG2+ cell numbers when compared to DMSO-treated controls. In addition, long-term exposure to PLX5622 had no effect on the morphology or number of oligodendrocytes (assessed by PLP-eGFP) and astrocytes (assessed by GFAP and S100b) (Fig. 3A,B and data not shown). Myelin protein expression (measured by PLP immunofluorescence) was unchanged (Fig. 3A).

In concert, both ex vivo and in vitro data demonstrate that low concentrations (≤ 2 μM) of PLX5622 completely deplete microglia in the absence of direct effects on OPCs, oligodendrocytes or astrocytes. PLX3397 causes a significant reduction in viable OPCs even at low concentrations. At higher concentrations, both PLX compounds diminish OPC cell numbers in primary cell culture and cerebellar slices.

PLX5622 (1200 ppm; chow; for 3 weeks or 3 days; adult C57/Bl6 wild type mice) causes approximately 80% and 99% of microglia to be lost after 3 days and 3 weeks of treatment, respectively. The microglia in the cortex, striatum, cerebellum, and hippocampus are reduced in PLX5622 (adult C57/Bl6 wild type mice, 3 months old; fed for 3 weeks)[4].
In a total of 14 days, PLX5622 (50 mg/kg; intraperitoneal injection; once daily for neonatal rats, or twice daily for adult rats) reduces microglia by 80–90% in 3 days and by more than 90% in 7 days. Both adults and neonates experience a > 96% reduction in microglia after 14 days of PLX5622 treatment, while baseline astrocyte quantity is maintained. (Depleting microglia in neonates only requires a single daily injection of 0.65% PLX5622 suspended in 5% dimethyl sulfoxide and 20% Kolliphor RH40 in 0.01 M PBS; twice-daily injections are needed for adult depletion.)[5].
PLX5622 (formulated in AIN-76A standard chow at 1200 mg/kg; for 28 days) reduces microglia throughout the CNS in 14-month-old 5xfAD mice[6].

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Many risk genes for the development of Alzheimer's disease (AD) are exclusively or highly expressed in myeloid cells. Microglia are dependent on colony-stimulating factor 1 receptor (CSF1R) signaling for their survival. We designed and synthesized a highly selective brain-penetrant CSF1R inhibitor (PLX5622) allowing for extended and specific microglial elimination, preceding and during pathology development. We find that in the 5xFAD mouse model of AD, plaques fail to form in the parenchymal space following microglial depletion, except in areas containing surviving microglia. Instead, Aβ deposits in cortical blood vessels reminiscent of cerebral amyloid angiopathy. Altered gene expression in the 5xFAD hippocampus is also reversed by the absence of microglia. Transcriptional analyses of the residual plaque-forming microglia show they exhibit a disease-associated microglia profile. Collectively, we describe the structure, formulation, and efficacy of PLX5622, which allows for sustained microglial depletion and identify roles of microglia in initiating plaque pathogenesis.[1]

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Introduction Neuropathic pain is a debilitating condition. The importance of neuroimmune interactions in neuropathic pain has been evidenced by the involvement of different immune cells in peripheral and central sensitization of pathological pain. Macrophages and microglia are the most abundant immune cells activated in injured nerves and spinal cord, respectively. Several lines of evidence showed that macrophage/microglia survival, activation, proliferation, and differentiation require the involvement of macrophage-colony stimulating factor. In this study, we investigated whether blocking macrophage-colony stimulating factor/colony stimulating factor 1 receptor signaling can be effective in relieving neuropathic pain. Materials and methods Partial sciatic nerve ligation was performed in mice to induce neuropathic pain behavior. Mice were orally treated with a selective colony stimulating factor 1 receptor inhibitor, PLX5622, daily in both preventive (two days prior to surgery until D14 post-partial sciatic nerve ligation) and reversal paradigms (D28-D33 post-partial sciatic nerve ligation). Animal neuropathic pain behavior was monitored using von Frey hairs and acetone application. Phenotype of macrophages in injured nerves was analyzed at D3 and D33 post-injury using flow cytometry analysis. The effect of PLX5622 on microglia activation in lumbar spinal cord was further examined by immunohistochemistry using Iba-1 antibody. Results Significant alleviation of both mechanical and cold allodynia was observed in PLX5622-treated animals, both in preventive and reversal paradigms. PLX5622 treatment reduced the total number of macrophages in injured nerves, it appears colony stimulating factor 1 receptor inhibition affected more specifically CD86+ (M1 like) macrophages. Consequently, the expression of various pro-inflammatory cytokines (TNF-α, IL-1β) was reduced. Microglia activation in dorsal horn of lumbar spinal cord following partial sciatic nerve ligation was significantly inhibited with PLX5622 treatment in both preventive and reversal paradigms. Conclusion Macrophages in peripheral nerve and microglia in the spinal cord are required in the generation and maintenance of injury-associated neuropathic pain. Blocking macrophage-colony stimulating factor/colony stimulating factor 1 receptor signaling on these myeloid cells along the pain transmission pathway is an effective strategy to alleviate neuropathic pain.[2]

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Introduction Neuropathic pain is a debilitating condition. The importance of neuroimmune interactions in neuropathic pain has been evidenced by the involvement of different immune cells in peripheral and central sensitization of pathological pain. Macrophages and microglia are the most abundant immune cells activated in injured nerves and spinal cord, respectively. Several lines of evidence showed that macrophage/microglia survival, activation, proliferation, and differentiation require the involvement of macrophage-colony stimulating factor. In this study, we investigated whether blocking macrophage-colony stimulating factor/colony stimulating factor 1 receptor signaling can be effective in relieving neuropathic pain. Materials and methods Partial sciatic nerve ligation was performed in mice to induce neuropathic pain behavior. Mice were orally treated with a selective colony stimulating factor 1 receptor inhibitor, PLX5622, daily in both preventive (two days prior to surgery until D14 post-partial sciatic nerve ligation) and reversal paradigms (D28-D33 post-partial sciatic nerve ligation). Animal neuropathic pain behavior was monitored using von Frey hairs and acetone application. Phenotype of macrophages in injured nerves was analyzed at D3 and D33 post-injury using flow cytometry analysis. The effect of PLX5622 on microglia activation in lumbar spinal cord was further examined by immunohistochemistry using Iba-1 antibody. Results Significant alleviation of both mechanical and cold allodynia was observed in PLX5622-treated animals, both in preventive and reversal paradigms. PLX5622 treatment reduced the total number of macrophages in injured nerves, it appears colony stimulating factor 1 receptor inhibition affected more specifically CD86+ (M1 like) macrophages. Consequently, the expression of various pro-inflammatory cytokines (TNF-α, IL-1β) was reduced. Microglia activation in dorsal horn of lumbar spinal cord following partial sciatic nerve ligation was significantly inhibited with PLX5622 treatment in both preventive and reversal paradigms. Conclusion Macrophages in peripheral nerve and microglia in the spinal cord are required in the generation and maintenance of injury-associated neuropathic pain. Blocking macrophage-colony stimulating factor/colony stimulating factor 1 receptor signaling on these myeloid cells along the pain transmission pathway is an effective strategy to alleviate neuropathic pain.[3]

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Elimination of microglia in aged 5xfAD mice with the specific CSF1R inhibitor PLX5622 PLX5622 is a brain-penetrant inhibitor of CSF1R that quickly eliminates microglia, but does not inhibit c-kit (Valdearcos et al., 2014; Dagher et al., 2015). To confirm the effects of PLX3397 and to rule out other off-target effects, we treated a second cohort of 5xfAD mice for 28 days with 1200 mg/kg PLX5622 in chow. We again stained tissue for dense core plaques using Thioflavin-S and immunolabelled microglia with IBA1 (Fig. 5A). Microglia were dramatically depleted with treatment, with most of the remaining cells being associated with dense core plaques, as with PLX3397 treatment (Fig. 5B and E). Quantification revealed on average about one cell per plaque remaining (Fig. 5C); however, many plaques had no IBA1+ cells associated with them (Fig. 5D). Further analyses revealed that all remaining IBA1+ cells had cell bodies containing amyloid-β (Fig. 5K); in contrast, most IBA1+ cells around plaques in untreated 5xfAD brains did not contain any amyloid-β (Fig. 5J). Indeed, some of these IBA1+ cells in the treated mice were Thioflavin-S positive (Fig. 5E). However, as with the PLX3397-treated mice, we observed no change in the plaque area or total number (Fig. 5F and G), nor any significant changes in amyloid-β protein levels (Fig. 5H and I), with this cohort of microglia-eliminated 5xfAD mice.[6]

Cerebellar Slice Culture [3]
Sagittal cerebellar slices (300μm) were prepared from PLP-eGFP mice (Mallon et al., 2002) at P10-12 and cultured on MilliCell 0.4μm membrane inserts in slice media: 25% Hank’s balanced salt solution (HBSS), 25% heat-inactivated horse serum, 50% minimum essential media (MEM), 125mM HEPES, 28mM D-Glucose, 2mM L-Glutamine, 10U/ml penicillin/streptomycin) at 37°C (Sheridan and Dev, 2012). Media was changed within the first 24 h of plating, and then every 2-3 days. Slices were cultured for 7-10 days prior to treatment. DMSO, PLX5622 and PLX3397 were applied at indicated concentrations in slice media for 3 or 8 days. Media was changed every 2-3 days. Following treatment, slices were rinsed once in PBS, fixed in 4% PFA in PBS for 30 minutes, and subjected to immunostaining.

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Purified Primary OPC Cell Culture [3]
Primary murine OPC culture was prepared from PLP-eGFP pups as described (Liu et al., 2016). Briefly, mixed glial cultures were prepared from P0-1 dissociated cortices and plated on poly-D-lysine coated T75 flask. Purified OPC monocultures were prepared by shaking flasks of confluent mixed glial cultures overnight at 200 rpm to detach the OPCs. OPCs were plated on poly-D-lysine/laminin coated dishes and maintained in media containing 10 ng/ml PDGF (platelet-derived growth factor)/FGF (fibroblast growth factor) for 24-48 h prior to experiments.

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IncuCyte Live Cell Imaging [3]
Live cell imaging was performed as described (Liu et al., 2016). Cells were grown and scanned on 24-well plates in the cell culture incubator. Each well was scanned with a 10× objective lens in 9 randomly selected positions at 15 min intervals with high definition phase contrast and epifluorescence microscopy using the 585/635 nm filter (red fluorescence, to detect the DRAQ7 dead cell nuclei). Image processing and cell counting were performed using IncuCyte and ImageJ software.

Drug preparation and treatment [2]
PLX5622 is a potent inhibitor of CSF1R tyrosine kinase activity (KI = 5.9 nM) with at least 50-fold selectivity over four related kinases and over 100-fold selectivity against a panel of 230 kinases. The molecule has been used to investigate the critical role of microglia/macrophages in many different circumstances. The dose in the current study was suggested by the company and in accordance with these previous reports where 1200 ppm in chow daily is sufficient to eliminate microglia fully or 300 ppm in chow daily can reduce partially microglia. Our 65 mg/kg dose nominally is close to the 300 ppm chow daily dose which allows us to lower macrophages in nerves. The drug was prepared following manufacturer’s protocol. Briefly, PLX5622 stock was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 130 mg/ml; 2% hydroxypropyl methyl cellulose and 25% polysorbate 80 were prepared to make a diluent. On each dosing day, PLX5622 stock was diluted 20-fold by adding 1 volume of drug stock (130 mg/ml) in 19 volumes of diluent, making a working solution at 6.5 mg/ml. Vehicle solution was prepared with a mix of diluent and DMSO. Mice were treated daily by oral gavage with 100 μl solution (6.5 mg/ml) per 10g body weight (final dose at 65 mg/kg body weight).
To study preventive effect of PLX5622, mice were treated with either a drug or vehicle two days prior to surgery, then daily until D14 post-PSNL. For the reversal effect of PLX5622, treatment started at D28, daily until D33 post-PSNL.

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Behavioral analysis [2]
Mice were habituated to the testing environment 1 to 2 h daily for at least two days before baseline testing. Mice were treated with either vehicle or PLX5622 daily in the morning. The investigator was blinded to the treatment conditions.

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Animal Chow Treatment [3]
Male and female PLP-eGFP animals (4-5 per group) aged 8-12 weeks were placed on formulated PLX Chow (1200 mg/kg for PLX5622 and 275 mg/kg for PLX3397) or standard diet for 7 or 21 days. Following treatment, animals were perfused with ice cold PBS followed by 4% paraformaldehyde in PBS. Brains and spinal cords were dissected, post-fixed overnight and then cryoprotected. Tissues were sectioned at 30um on a cryostat and collected in PBS for free-floating immunostaining.

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For brain-wide microglia ablation, adult C57/Bl6 wild type mice between 8-16 weeks of age were treated with CSF1R inhibitor, PLX5622, or control chow (same formula lacking only the inhibitor) for 3 weeks or 3 days as indicated. While a 3-week long PLX5622 treatment leads to a 99% microglia loss, around 80% of microglia are already lost after 3 days [4].

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Habituation to head-fixation and intraperitoneal (i.p.) injection. [4]
After a 1-2 week recovery from the surgery, the mice were randomly divided into 2 groups, one group received PLX5622 and one group received control chow (lacking inhibitor).
The mice were later put under water restriction (1.5 ml/day) and were handled and habituated daily to head-fixation and immobilization for ~2 weeks. They were immobilized in a polyethylene tube and head-fixed in the future recording environment under 2-photon microscope. During this habituation period, we increased the head-fixation period from 3 minutes to 40 minutes gradually. After they showed no signs of stress and drank water provided randomly during the 40-minute session, we switched to no-water-provided head fixation habituation for future recording.
After habituation to head-fixation, we also performed daily habituation to i.p. injection before the head-fixation session. Mice were injected i.p. with a microlitre volume equivalent to 10× the body weight in grams (10 × BW μl) of saline, matching the volume to be injected in future imaging sessions, and then performed head-fixation. This habituation was performed for ~1 week until the mice showed a reduction in clear signs of stress upon handling and i.p. injection.

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Microdialysis [4]
C57/Bl6 male mice were put on PLX5622 or control chow (n=5 PLX5622 diet, n=5 control diet). for one week. Dialysis probes were implanted and mice recovered for one week before commencing microdialysis collection. Microdialysis guide cannulae were stereotaxically implanted in the striatum (A/P: +1.4 mm; M/L: −1.0 mm; D/V: −3.8 mm from skull). Microdialysis experiments were conducted following a one-week recovery period following guide cannula implantation. Dialysis tubing was flushed prior to initial use with 70% EtOH for 5 minutes, followed by dH2O syringe pump at a flow rate of 1 μL/min. The tubing was then attached to the microdialysis probe (Cuprophane (6kD), membrane length 1mm), which was primed by placing the probe in artificial cerebrospinal fluid (aCSF; pH 7.4: 148 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl2, 0.85 mM MgCl2) and running aCSF through the microdialysis tubing and probe at a 0.8 μL/min rate. Microdialysis experiments were done in anesthetized animals. Briefly, mice were placed into a stereotaxic frame under isoflurane anesthesia (4% induction,1.75% sustained). The probe was inserted via the guide cannula and allowed to equilibrate. Dialysate was collected after 20 minutes. All collections were frozen at −80 degrees Celsius immediately following collection completion.

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Microglia inhibition via intraperitoneal injection of PLX5622 [5]
Prior CSF1R inhibitor studies using PLX3397/5622-integrated chow treated animals for at least 1 week prior to their experimental manipulation (e.g. Elmore et al., 2014; Okunukia et al., 2019). To ensure adequate microglia depletion for neonatal surgeries, we began our treatments at P1, with an equivalent pre-surgery treatment duration (9 days) for adults. Pilot testing indicated that while a single daily injection of 0.65% PLX5622 suspended in 5% dimethyl sulfoxide and 20% Kolliphor RH40 in 0.01 M PBS was sufficient for neonatal microglia depletion, adult depletion required injections twice daily (10–12 h apart). Starting at P1 (for P10 CTX) or P41 (for P50 CTX), additional rats were injected with either PLX5622 (50 mg/kg; n = 4–5/condition) or a vehicle solution (n = 3–4/condition) once (neonates) or twice (adult) a day until being sacrificed 4 days after surgery.

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Compounds [6]
PLX3397 was formulated in AIN-76A standard chow by Research Diets Inc. at 290 mg/kg or 600 mg/kg, as previously described (Elmore et al., 2014). PLX5622 was provided by Plexxikon Inc. and formulated in AIN-76A standard chow at 1200 mg/kg.

Animal treatments [6]
Crossing these mice yielded CSF1R-iCRE/Rosa26YFP progeny that express yellow fluorescent protein (YFP) in all cells that either transiently or constitutively express CSF1R, which in the brain predominantly labels microglia. Two-month-old mice were treated for 7 days with PLX3397 (600 mg/kg in chow) to eliminate microglia. The 5xfAD mouse model has been previously described in detail (Oakley et al., 2006). Using a 2 × 2 factorial design, forty male and female 10-month-old wild-type (C57BL/6 background) or 5xfAD mice were treated with either PLX3397 for 28 days to eliminate microglia or control chow, creating four treatment groups (n = 10/group): Control (six males and four females), PLX3397 (six males and four females), 5xfAD (four males and six females), and 5xfAD + PLX3397 (four males and six females). At this age, 5xfAD mice display extensive pathology, synaptic loss, and neuronal loss (Oakley et al., 2006; Buskila et al., 2013; Eimer and Vassar, 2013). After 28 days of treatment, behavioural testing commenced while animals remained on their respective diets. A second cohort of 14-month-old 5xfAD mice (n = 4/group; 5xfAD = four males, and 5xfAD + PLX5622 = three males and one female) was treated with either PLX5622 for 28 days to deplete microglia or control chow (5xfAD versus 5xfAD + PLX5622). A third cohort of 1.5-month-old 5xfAD mice (n = 4/group; two males and two females) was treated with PLX3397 or control for 28 days (5xfAD versus 5xfAD + PLX3397). Following behavioural testing in the 10-month-old cohort, and following inhibitor treatment in the 14-month-old and 1.5-month-old cohorts, mice were euthanized via CO2 inhalation and transcardially perfused with phosphate-buffered saline. For both studies, brains were removed and hemispheres separated along the midline. Brain halves were either flash frozen for subsequent biochemical analysis, drop-fixed in 4% paraformaldehyde for subsequent immunohistochemical analysis, or placed in Golgi impregnation solution for subsequent dendritic spine analysis. Fixed half brains were sliced at 40 µm using a Leica SM2000 R freezing microtome. The flash-frozen hemispheres were ground with a mortar and pestle to yield a fine powder. One-half of the powder was homogenized in Tissue Protein Extraction Reagent, T-PER with protease and phosphatase inhibitors. The second half was processed with an RNA Plus Universal Mini Kit for RNA analysis.

In vivo PLX5622 demonstrated desirable PK properties in mice, rats, dogs, and monkeys (Supplementary Table 5), with a brain penetrance of ~20% (compared to ~5% for PLX339717; Supplementary Table 6). The improved blood-brain barrier (BBB) penetrance of PLX5622 over PLX3397 is consistent with the physicochemical properties of the two compounds (Supplementary Table 7). PLX5622 has lower molecular weight, higher lipophilicity and better cell permeability, all factors known to influence the ability of a compound to cross the BBB. PLX5622 was formulated in rodent chow, and administration to mice showed highly effective, causing a 90% reduction with 1200 ppm chow within 5 days of treatment (Fig. 2d, e). Instructions for the synthesis and formulation of PLX5622 are provided (Supplementary Methods). [1]

[1]. Sustained microglial depletion with CSF1R inhibitor impairs parenchymal plaque development in an Alzheimer's disease model. Nat Commun. 2019 Aug 21;10(1):3758.

[2]. Targeting macrophage and microglia activation with colony stimulating factor 1 receptor inhibitor is an effective strategy to treat injury-triggered neuropathic pain. Mol Pain. 2018 Jan-Dec;14:1744806918764979.

[3]. Concentration-dependent effects of CSF1R inhibitors on oligodendrocyte progenitor cells ex vivo and in vivo. Exp Neurol. 2019;318:32-41.

[4]. Negative feedback control of neuronal activity by microglia. Nature. 2020;586(7829):417-423.

[5]. Astrocytic response to neural injury is larger during development than in adulthood and is not predicated upon the presence of microglia, Brain, Behavior, & Immunity-Health, Volume 1, 2020, 100010, ISSN 2666-3546.

[6]. Eliminating microglia in Alzheimer's mice prevents neuronal loss without modulating amyloid-β pathology. Brain. 2016;139(Pt 4):1265-1281.

PLX5622 is a highly selective brain penetrant and orally active CSF1R inhibitor. PLX5622 allows for extended and specific microglial cells elimination, preceding and during pathology development. PLX5622 demonstrates desirable PK properties in varies animals. PLX5622 is mostly used in the way of feed free diet.

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In conclusion, we have designed and created a specific CSF1R inhibitor, PLX5622, that allows for the sustained and specific elimination of microglia. This novel method of microglial depletion provided us with the means to eliminate microglia for the duration of AD pathogenesis. Ultimately, these data demonstrate that microglial elimination is associated with the prevention of plaque formation and the downregulation of hippocampal neuronal genes that occur in a preclinical model of AD progression. These results indicate that microglia appear to contribute to multiple facets of AD etiology – microglia appear crucial to the initial appearance and structure of plaques, and following plaque formation, promote a chronic inflammatory state modulating neuronal gene expression changes in response to Aβ/AD pathology. [1]

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Synergistic blocking spinal microglia and nerve macrophages activation could result in significant alleviation of neuropathic pain behaviors, which was evidenced by the effect of CSF1R inhibitor in our study. Peripheral macrophage activation, more specifically inflammatory macrophages expressing CD86, TNF-α, or IL-1β in injured peripheral nerves, is prominently affected by CSF1R inhibition while CD206 expressing macrophages were not significantly altered. Moreover, PLX5622 treatment effectively reduced microglia activation in spinal cord following nerve injury. Although our results provided a proof of concept on the critical role of microglia/macrophages in neuropathic pain, and that CSF1R could be an interesting target for neuropathic pain alleviation, it is however important to bear in mind that blocking M-CSF/CSF1R signaling, especially when the drug is given systemically, it affects not only microglia/macrophage in the nervous system, myeloid cells in other tissues could be most likely affected. Thus, to promote the translation of the current finding, not only more evidence from other animal models of chronic pain are awaited but also thorough analysis of the properties/safety of the compound in other organs is required.[2]

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In summary, by comparing the effect of PLX5622 and PLX3397, we confirm that CSF1R inhibitors can efficiently deplete microglia ex vivo and in vivo without affecting mature oligodendrocytes or myelin protein expression. In addition, we speculate that CSF1R inhibitors may directly impact OPC viability through off-target binding to PDGFRα, a member of the type III tyrosine kinase receptor family. Therefore, inhibitor concentration and duration of treatment must be carefully adjusted for the relevant experimental system. These data also question whether microglia are essential for OPC viability in ex vivo slice cultures and adult brain.[3]

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In summary, we demonstrated glial responses to CTX that varied based on developmental stage. Adult CTX induces large microglia and small astrocyte responses, but CTX at P10 results in small microglia and large astrocyte responses, which are associated with differences in CTX recovery (Kopka et al., 2000; Martin et al., 2019; Reddaway et al., 2012; Sollars, 2005; St. John et al., 1995). We are the first to demonstrate that i.p. administration of PLX5622 to both early postnatal and adult rats depletes microglia without impacting animal health (as assessed by weight) or astrocyte quantity. While such depletion eliminated the adult astrocyte response, the neonatal response appeared unaffected, showing that microglia are likely not required for neonatal CTX-induced astrogliosis. Our findings suggest the presence of pathway-specific developmental differences which may contribute to differences in recovery across development. Our results add to existing knowledge regarding differences in glia function across development and factors that may contribute to why the developing gustatory system has difficulty recovering from injury. Our findings highlight the potential importance of central glia to both the developing and injured gustatory system and suggest differences in reciprocal interactions between microglia and astrocytes across maturational stages.[5]

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We have previously shown that microglial elimination can be achieved in the healthy adult mouse brain by treatment with small-molecule inhibitors of CSF1R (Elmore et al., 2014; Dagher et al., 2015), with many groups confirming our findings (Valdearcos et al., 2014; Asai et al., 2015; Klein et al., 2015; Schreiner et al., 2015). Here, we have extended these findings in mice constitutively expressing YFP under the Rosa26 locus in all CSF1R expressing cells to definitively show that microglia are eliminated and are not simply downregulating myeloid/microglial markers. We also found that chronically activated microglia following extensive neuronal injury can be eliminated with the same approach. Our studies also revealed that microglial elimination following neuronal insult improved functional outcomes, whereas elimination of microglia during the lesion exacerbated neuronal loss, revealing differential roles of microglia in injury response (Rice et al., 2015). It is important to note that only blood–brain barrier-permeant CSF1R inhibitors are able to eliminate microglia. These include the compounds PLX3397 and PLX5622 used in this study, as well as BLZ945 (Pyonteck et al., 2013). Furthermore, sustained brain exposure levels of CSF1R inhibitors are required for effective microglial elimination. It takes at least 3 days for microglia to succumb and die within the CNS (Elmore et al., 2014). It should be noted that CSF1R inhibitors are highly versatile; at high doses they eliminate microglia, allowing for the exploration into the roles of these cells in disease and normal brain function, while at lower doses they modulate CSF1R signalling in microglia without eliminating them. To this end, we have previously used low doses of PLX5622 (300 mg/kg chow rather than the 1200 mg/kg chow used in this study) to explore the role of the CSF1R in microglial response to Alzheimer’s disease pathology and found that it regulates the chemotactic response of these cells to plaques (Dagher et al., 2015), resulting in improvements in cognition without affecting plaque burden or inflammatory profile. Moreover, the CSF1R has also been shown to be crucial for microglial proliferation during disease (Gomez-Nicola et al., 2013), also using CSF1R inhibitor paradigms that do not result in microglial elimination. In contrast, in this study, we sought to define the roles of microglia in mediating Alzheimer’s disease pathogenesis through the administration of CSF1R inhibitors at doses sufficient to eliminate microglia, rather than at lower doses that modulate microglial function without eliminating them. We first set out to determine if microglia in the brains of aged 5xfAD mice are still dependent on CSF1R signalling for their survival. Importantly, we show that 28 days of continuous treatment with either PLX3397 or PLX5622 leads to an ∼80–90% reduction in microglia, respectively, throughout the CNS in adult 5xfAD mice. [6]

C21H19F2N5O

395.4053

395.16

C, 63.79; H, 4.84; F, 9.61; N, 17.71; O, 4.05

1303420-67-8

PLX5622 hemifumarate

52936034

White to light yellow solid powder

4.1

2

7

6

29

529

0

WPOXAFXHRJYEIC-UHFFFAOYSA-N

InChI=1S/C23H27ClN4O3/c1-28-8-6-15(7-9-28)13-31-22-12-19-17(11-21(22)30-3)23(26-14-25-19)27-20-10-16(29-2)4-5-18(20)24/h4-5,10-12,14-15H,6-9,13H2,1-3H3,(H,25,26,27)

N-(2-chloro-5-methoxyphenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine

PLX5622 free base; PLX-5622 free base; PLX5622; 1303420-67-8; 6-Fluoro-N-((5-fluoro-2-methoxypyridin-3-yl)methyl)-5-((5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)pyridin-2-amine; PLX-5622; 6-fluoro-N-[(5-fluoro-2-methoxypyridin-3-yl)methyl]-5-[(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl]pyridin-2-amine; 16A8IGR8L7; 5-fluoro-N-[6-fluoro-5-[(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl]-2-pyridinyl]-2-methoxy-3-pyridinemethanamine; UNII-16A8IGR8L7; PLX 5622 free base;PLX5622; PLX-5622; PLX 5622

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 79~100 mg/mL (199.8~252.9 mM)
Ethanol: ~3.3 mg/mL (~8.4 mM)
H2O: < 0.1 mg/mL

Solubility in Formulation 1: 6.5 mg/mL (16.44 mM) in 5% DMSO + 95% (0.5% Hypromellose 1% Tween-80) (Note: To make 100 mL diluent (0.5% Hypromellose 1% Tween-80), add 25 mL of 2% Hypromellose stock and 4 mL of 25% Tween80 + stock to 71 mL ddH2O) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication.

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Solubility in Formulation 2: 5 mg/mL (12.65 mM) in 5% DMSO + 95% (20% Ethoxylated hydrogenated castor oil in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 3.12 mg/mL (7.89 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 31.2 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (6.32 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 5: ≥ 2.5 mg/mL (6.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)