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Cited by 946Neuroinflammation and Neurodegeneration MechanismsAlzheimer's disease research and treatmentsNeurological Disease Mechanisms and Treatments

Sustained microglial depletion with CSF1R inhibitor impairs parenchymal plaque development in an Alzheimer’s disease model

Open access
Spangenberg, Elizabeth, Severson, Paul L., Hohsfield, Lindsay A., Crapser, Joshua, Zhang, Jiazhong, Burton, Elizabeth A., Zhang, Ying, Spevak, Wayne +14 more·2019·Nature Communications
DOI 10.1038/s41467-019-11674-zPMC PMC6704256
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Abstract

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.

AI snapshot

Written from the paper’s full text.

TL;DR

PLX5622, a selective brain-penetrant CSF1R inhibitor, allowed sustained microglial depletion in 5xFAD mice and prevented parenchymal plaque formation, shifting Aβ deposition to cortical blood vessels. Residual plaque-forming microglia showed a disease-associated profile, and microglial repopulation restored plaque pathology.

Key findings

  • PLX5622 showed >20-fold selectivity over KIT and FLT3, ~20% brain penetrance, and 1200 ppm chow reduced microglia by 90% within 5 days; treatment achieved 97–100% microglial reduction over 24 weeks and >99% cortical elimination.
  • In 5xFAD mice, microglial depletion prevented dense-core parenchymal plaques except near surviving microglia in thalamus, retrosplenial cortex, and subiculum; cortical Aβ instead deposited in blood vessels as cerebral amyloid angiopathy. Plaque volumes were ~30–40% smaller, subicular plaque number was reduced by 33%, and plaque-core ApoE was reduced ~50–70%.
  • Aβ38/40/42 levels and APP processing were unchanged, but hippocampal RNA-seq showed 413 differentially expressed genes in 5xFAD vs wild-type, including 77 downregulated synaptic/neuronal genes that were not downregulated when microglia were absent. Residual plaque-forming microglia expressed DAM markers such as Ccl6, Clec7a, Cst7, Ctsd, Ctsz, and Itgax.
  • After PLX5622 withdrawal and microglial repopulation, plaque numbers in 5xFAD mice equaled untreated 5xFAD mice, though average plaque volumes remained smaller; vascular pathology persisted.

Methods

  • Structure-guided design and synthesis of PLX5622 from PLX3397, with crystallography, kinase profiling, PK across mouse/rat/dog/monkey, and chow formulation.
  • 5xFAD and 3xTg-AD mice; 5xFAD treatment began at 1.5 months for 10–24 weeks with groups Wild-type, PLX5622, 5xFAD, and 5xFAD + PLX5622 (n = 12/group, sex-balanced), plus behavioral, flow cytometry, histology, ELISA, immunoblot, and RNA-seq analyses (n = 4/group for RNA-seq).
  • Human postmortem tissue from non-demented, high-pathology non-demented, and Alzheimer’s disease subjects was examined from middle frontal gyrus cortex.

Limitations

  • The paper states no formal limitations section; it notes that no statistical methods were used to predetermine sample sizes and that data distribution was assumed normal but not formally tested.
  • Evident from the text: findings rely on transgenic mouse models, and surviving microglia in thalamus, retrosplenial cortex, and subiculum complicate interpretation of region-specific plaque formation.
  • Evident from the text: CAA and microbleeds appeared in microglia-depleted 5xFAD mice, and the long-term consequences of vascular Aβ deposition were not fully resolved.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41467-019-11674-z

Cites

71 of 71 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 50 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    PLX5622 administration to mice caused a 90% reduction in microglia with 1200 ppm chow within 5 days.

    “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).”
    Quote in the paperSubstantial claim
  • Findingown data

    Oral PLX5622 treatment led to almost complete microglial elimination (97-100% reduction) even with 24 weeks of treatment.

    “Oral PLX5622 treatment led to almost complete microglial elimination (97–100% reduction; Fig. 3c and quantified in e and f), even with 24 weeks of treatment, showing that PLX5622 allows for extended elimination of microglia.”
    Quote in the paperSubstantial claim
  • Findingown data

    Transcriptional analyses of residual plaque-forming microglia show they exhibit a disease-associated microglia profile.

    “Transcriptional analyses of the residual plaque-forming microglia show they exhibit a disease-associated microglia profile.”
    Quote in the paperNot a substantial claim
  • Findingown data

    PLX5622 is highly specific for CSF1R, showing >20-fold selectivity over KIT and FLT3.

    “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).”
    Quote in the paperSubstantial claim
  • Findingown data

    Significant elevations in full-length APP and C99 and C83 were found in 5xFAD mice relative to wild-type.

    “We found significant elevations in full-length (fl) APP and Carboxy-terminal fragments of APP (C99 and C83) in 5xFAD mice relative to wild-type (Fig. 5k, l), but observed no reductions in protein levels with PLX5622 treatment in 5xFAD animals.”
    Quote in the paperNot a substantial claim
  • May combine two claims. 54
  • Classified as probably not a substantial claim. 10

  • May combine two claims.

    Findingown data

    CAA occurred only in microglia-depleted 5xFAD mice, not in untreated 5xFAD mice or treated wild-type mice.

    “The appearance of CAA occurred only in microglia-depleted 5xFAD mice, as CAA was not observed in untreated 5xFAD mice or in treated wild-type mice.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Treatment of 5xFAD mice with PLX5622 reduced >99% of microglia in the cortex, but a fraction of cells remained in the thalamus.

    “Treatment of 5xFAD mice with PLX5622 again reduced > 99% of microglia in the cortex (Fig. 4d, g), but as noted in wild-type mice, a fraction of cells remained in the thalamus (Fig. 4j).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Absolute numbers of astrocytes were not highly affected by pathology or microglial depletion, except for a small increase in the RS cortex in 5xFAD mice.

    “Absolute numbers of astrocytes were not highly affected by pathology or microglial depletion, as measured by S100β+ cells (quantified in Supplementary Figure 5D), except for a small increase in the RS cortex in 5xFAD mice.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    With 24 weeks of treatment, microglia were eliminated brain wide, but Thio-S plaques persisted in the thalamus and RS cortex.

    “With an additional 14 weeks of treatment (i.e., 24 weeks treated animals), microglia were eliminated brain wide (Fig. 4e, k), but Thio-S plaques persisted in these areas (i.e., the thalamus and RS cortex).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CAA and plaques in microglia-depleted 5xFAD mice stained positively for Aβ fibrils via OC and Aβ42, but were negative for Aβ oligomers via A11.

    “In addition to Thio-S, CAA and plaques stained postiviely for Aβ fibrils, via the conformation specific antibody OC21 (Fig. 4r; Supplementary Figure 6C–D) and Aβ42 (Fig. 4s; Supplementary Figure 6E–F), but were negative for Aβ oligomers via A11 antibody22 (Fig. 4q; Supplementary Figure 6A–B).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [21] Kayed, R. et al. Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers. Mol. Neurodegener. 2, 18 (2007). source

      Not checked in the free run.

    • cites [22] Kayed, R. et al. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300, 486–489 (2003). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Pyroglutamate-3 immunoreactivity was found in areas of treated 5xFAD mice that exhibited plaque pathology (thalamus).

    “Of note, pyroglutamate-3 immunoreactivity was found in areas of treated 5xFAD mice that exhibited plaque pathology (i.e., thalamus; Supplementary Figure 6I–J).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    5xFAD animals did not show impairments in Y-Maze, Morris water maze, and Contextual fear conditioning.

    “To assess hippocampal-dependent memory, mice underwent testing using Y-Maze, Morris water maze (MWM), and Contextual fear conditioning (CFC) and 5xFAD animals did not show impairments in any of these tasks (Supplementary Figure 3E–G; Fig. 3i–k).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In wild-type mice, CSF1R inhibitor treatment for 10 weeks eliminated >99% of microglia in the cortex, but a fraction of cells remained in the thalamus.

    “In wild-type mice, CSF1R inhibitor treatment for 10 weeks eliminated > 99% of microglia in the cortex (Fig. 4a–f), but a fraction of cells remained in the thalamus (Fig. 4i; enlarged in Supplementary Figure 4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Plaques were predominantly observed in the immediate vicinity of surviving IBA1+ cells within the subiculum and associated white matter tract, while the cortex contained only vascular Thio-S+ deposits.

    “In fact, plaques were predominantly observed in the immediate vicinity of surviving IBA1+ cells – within the subiculum and associated white matter tract – while the cortex contained only vascular Thio-S+ deposits, in stark contrast to untreated 5xFAD mice (Fig. 6a vs. b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    PLX5622 has lower molecular weight, higher lipophilicity and better cell permeability than PLX3397.

    “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.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Some plaques form in specific brain areas with treatment, and these few remaining plaques are associated with a small subset of microglia initially resistant to CSF1R-inhibition.

    “While some plaques form in specific brain areas with treatment, we find that these few remaining plaques are associated with a small subset of microglia initially resistant to CSF1R-inhibition.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    These features were common to all areas explored, including the subiculum, where some IBA1+ cells were still present.

    “These features were common to all areas explored, including in the subiculum, where some IBA1+ cells were still present, suggesting that some threshold density of microglial cells may be required for full plaque compaction/restructuring.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    These plaques displayed an irregular shape with structured filaments radiating out from a central fissure/point and lacked the homogenous staining intensity of plaques in microglia-intact 5xFAD mice.

    “These plaques displayed an irregular shape with structured filaments radiating out from a central fissure/point and lacked the homogenous staining intensity of plaques in microglia-intact 5xFAD mice.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Treatment of 1.5-month-old 5xFAD mice with PLX3397 600 ppm until 5 months of age showed minimal plaque pathology and the appearance of CAA.

    “Again, examination of the brains of these treated mice showed minimal plaque pathology and the appearance of CAA (Fig. 7c, d – zoomed image of CAA in D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Untreated 5xFAD mice exhibit cortical plaques at 4 months of age, but 5xFAD mice devoid of microglia show reduced plaque formation.

    “As demonstrated previously, untreated 5xFAD mice exhibit cortical plaques at 4 months of age, but 5xFAD mice devoid of microglia show reduced plaque formation (Fig. 4d, g, l).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    Transgene expression in 5xFAD mice (App, Psen1, and Thy1) was increased by ~2-fold over endogenous expression across brain regions and was not impacted by treatment.

    “Transgene expression in 5xFAD mice (consisting of App, Psen1, and Thy1) was increased by ~2-fold over endogenous expression across brain regions (Fig. 9b) and was not impacted by treatment, consistent with the protein expression data (Fig. 5k, l).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Plaque circularity and mean Thio-S intensities were reduced in the 5xFAD mice devoid of microglia.

    “As such, both plaque circularity (Fig. 6j) and mean Thio-S intensities (Fig. 6k) were reduced in the 5xFAD mice devoid of microglia.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Enrichment analyses of the hippocampus revealed an upregulation in immune function-associated pathways in 5xFAD vs. wild-type, and these pathways were all highly significantly downregulated with PLX5622 treatment.

    “Enrichment analyses of the hippocampus revealed an upregulation in immune function-associated pathways in 5xFAD vs. wild-type, and accordingly, these pathways were all highly significantly downregulated with PLX5622 treatment (Fig. 9f).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Microglia repopulated all areas of the brain in both wild-type and 5xFAD mice, although overall densities were lower than untreated mice in cortical regions.

    “Microglia repopulated all areas of the brain in both wild-type and 5xFAD mice (Fig. 10b), although overall densities were lower than the untreated mice in cortical regions (Fig. 10c, d; quantified in F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    ApoE immunoreactivity was apparent within dense core plaques, but also in cellular processes surrounding the cores.

    “Consistent with this, ApoE immunoreactivity was apparent within dense core plaques, but also in cellular processes surrounding the cores (Fig. 6l).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The hippocampus was most impacted by AD pathology (413 differentially expressed genes), while cortex and thalamus exhibited fewer changes (141 and 107 genes respectively).

    “We compared the number of differentially expressed genes (adjusted p-values < 0.05) induced by AD pathology in each of the three microdissected brain regions (i.e., wild-type vs. 5xFAD) and found that the hippocampus was most impacted (413 genes), while both cortex and thalamus exhibited fewer changes in gene expression (141 and 107 genes respectively; Fig. 9a).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    PLX3397 at 75 ppm achieved brain levels of ~1 μM, compared to ~10 μM with 600 ppm, and microglia numbers were unchanged relative to untreated animals.

    “This lower dose achieved brain levels of ~1 μM, compared to ~10 μM with the higher dose (Fig. 7b), and accordingly, microglia numbers were unchanged relative to untreated animals (Fig. 7e–i).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Microglia-repopulated 5xFAD brains revealed the appearance of robust plaque pathology with plaque numbers being equal to untreated 5xFAD mice, although average plaque volumes were smaller.

    “However, examination of microglia-repopulated 5xFAD brains revealed the appearance of robust plaque pathology (Fig. 10d) with plaque numbers being equal to the untreated 5xFAD mice (Fig. 10g), although average plaque volumes were smaller (Fig. 10h).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    All common genes significantly changed in all 3 brain regions in 5xFAD vs. wild-type mice were upregulated and the vast majority are microglia-associated.

    “All common genes were upregulated and the vast majority are microglia-associated, reflecting the microgliosis occurring in 5xFAD mice.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The only significant changes in gene expression in 5xFAD mice compared to wild-type were the upregulation of the transgenes (App and Psen1) and Apoe, all of which were not altered by the absence of microglia, and the downregulation of myeloid-expressed Trem2, Ctsb, Ctsc, and Ctsd in the absence of microglia.

    “The only significant changes in gene expression in 5xFAD mice compared to wild-type were the upregulation of the transgenes (App and Psen1; as expected due to overexpression), and Apoe, all of which were not altered by the absence of microglia, and the downregulation of myeloid-expressed Trem2, Ctsb, Ctsc, and Ctsd in the absence of microglia, as expected.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Repopulating microglia associate with the new plaques but do not appear to react to the vascular deposits.

    “Repopulating microglia associate with the new plaques but do not appear to react to the vascular deposits (Fig. 10e).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Nanostring Neuropathology panel confirmed that overlapping microglia genes were increased and neuronal genes decreased in 5xFAD mice compared to wild-type mice, which did not occur with PLX5622 treatment.

    “Accordingly, overlapping microglia genes were increased and neuronal genes decreased in 5xFAD mice compared to wild-type mice, which did not occur with PLX5622 treatment (Supplementary Figure 9).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Expression of many of these genes (i.e., Ccl6, Clec7a, and Cst7) was detected in PLX5622-treated 5xFAD hippocampi but not cortex or thalamus, reflecting the population of surviving microglia in this region and providing a gene expression signature of plaque-forming/associated microglia.

    “Consistent with our expectations, we detected expression of many of these genes (i.e., Ccl6, Clec7a, and Cst7) in PLX5622-treated 5xFAD hippocampi (but not cortex or thalamus), reflecting the population of surviving microglia in this region (see images of subicula in Fig. 6c–d, which are included in microdissected hippocampi) and providing a gene expression signature of plaque-forming/associated”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    GFAP+ astrocytes associated with plaques in both control and repopulated 5xFAD brains, but were absent in 5xFAD brains devoid of microglia.

    “Moreover, GFAP+ astrocytes associated with plaques in both control and repopulated 5xFAD brains (Fig. 10i, j), but were absent in 5xFAD brains devoid of microglia.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PLX5622 shows high selectivity for the CSF1R, desirable PK characteristics across species including oral bioavailability, a > 20% brain penetrance, and rapid/sustained elimination of microglia.

    “To that end, we designed and created PLX5622, which shows high selectivity for the CSF1R (Supplementary Table 2), desirable PK characteristics across species (mouse, rat, dog, monkey; Supplementary Table 5) including oral bioavailability, a > 20% brain penetrance (Supplementary Table 6), and rapid/sustained elimination of microglia (Figs 2, 3).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Supplementary Table 2

      Not checked in the free run.

    • cites Supplementary Table 5

      Not checked in the free run.

    • cites Supplementary Table 6

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    The hippocampus displayed a unique gene expression profile relative to the cortex and thalamus and included a unique subset of downregulated genes.

    “The hippocampus displayed a unique gene expression profile relative to the cortex and thalamus and included a unique subset of downregulated genes.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Comparisons of changes in expression between brain regions showed regional differences in Ache, Apoe, Ptk2b, Sorl1, and Sort1.

    “Comparisons of changes in expression between brain regions showed regional differences in Ache, Apoe, Ptk2b, Sorl1, and Sort1.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In microglia-devoid 5xFAD mice, ApoE+ microglial processes surrounding dense core plaques were absent, and plaque cores lacked ApoE staining.

    “Examination of ApoE immunoreactivity in microglia-devoid 5xFAD mice showed that the ApoE+ microglial processes surrounding dense core plaques were absent, as expected, but also the plaque cores themselves lacked ApoE staining (Fig. 6m).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    With 75 ppm PLX3397, no reductions in plaque load or appearance of CAA were detected in treated 5xFAD animals compared to controls.

    “With 75 ppm PLX3397, no reductions in plaque load or appearance of CAA were detected in treated 5xFAD animals compared to controls (Fig. 7f, h, j–k).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Only 1 downregulated gene was identified in the cortex and none were detected in the thalamus.

    “Of note, only 1 downregulated gene was identified in the cortex and none were detected in the thalamus.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Of the 413 identified genes, 77 were downregulated in the 5xFAD hippocampus, and these transcripts were primarily synaptic and neuronal in nature.

    “Of the 413 identified genes, 77 were downregulated in the 5xFAD hippocampus (Fig. 9c, and individual genes plotted in D). Notably, these transcripts were primarily synaptic and neuronal in nature, including genes such as Dlk2, Dync1l1, Gls, Kcnq3, Nrg3, and Scn1b.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    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.

    “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.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The long-term absence of microglia is not detrimental to murine cognitive function and may be beneficial.

    “indicating that the long-term absence of microglia is not detrimental to murine cognitive function and may be beneficial, at least in this assay, which is consistent with our prior findings17,18.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [17] Elmore, M. R. et al. Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. Neuron 82, 380–397 (2014). source

      Not checked in the free run.

    • cites [18] Rice, R. A. et al. Elimination of Microglia Improves Functional Outcomes Following Extensive Neuronal Loss in the Hippocampus. J. Neurosci. 35, 9977–9989 (2015). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Microglia/myeloid cells facilitate plaque formation, and few cells are required for this process.

    “Importantly, these results highlight a clear relationship between microglia/myeloid cells and the appearance of plaques, suggesting a role of microglia/myeloid cells in facilitating plaque formation, and emphasizes that few cells are required for this process to occur.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The absence of microglia modulates the location of Aβ accumulation (parenchyma vs. vasculature) but does not alter the net amounts present.

    “Thus, the absence of microglia modulates the location of Aβ accumulation (i.e., parenchyma vs. vasculature) but does not alter the net amounts present.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Microglia may be critical regulators of plaque formation, and few surviving microglial cells are sufficient to facilitate some degree of plaque formation.

    “These results suggest that microglia may be critical regulators of plaque formation and that few surviving microglial cells are sufficient to facilitate some degree of plaque formation (i.e., as seen in the thalamus).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Microglia are critical and causative in the development and progression of multiple facets of AD pathology.

    “These results implicate microglia as critical and causative in the development and progression of multiple facets of AD pathology.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Removal of microglia at advanced stages of pathology protects against synaptic and neuronal loss.

    “yet the removal of the microglia at advanced stages of pathology protects against synaptic and neuronal loss10.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [10] Spangenberg, E. E. et al. Eliminating microglia in Alzheimer’s mice prevents neuronal loss without modulating amyloid-beta pathology. Brain 139, 1265–1281 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Aβ aggregation requires an acidic pH and micromolar concentrations of Aβ monomers.

    “The aggregation of Aβ is an initial step in the formation of plaques, requiring an acidic pH13 and micromolar concentrations of Aβ monomers14.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [13] Su, Y. & Chang, P. T. Acidic pH promotes the formation of toxic fibrils from beta-amyloid peptide. Brain Res 893, 287–291 (2001). source

      Not checked in the free run.

    • cites [14] Tseng, B. P. et al. Deposition of monomeric, not oligomeric, Abeta mediates growth of Alzheimer’s disease amyloid plaques in human brain preparations. Biochemistry 38, 10424–10431 (1999). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The extracellular space does not meet the conditions required for Aβ aggregation, suggesting Aβ aggregation originates elsewhere.

    “The extracellular space does not meet these requisite conditions, suggesting that Aβ aggregation originates elsewhere.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The nearly undetectable expression of homeostatic microglial genes in PLX5622-treated mice highlights their specific expression by microglia and the specificity of PLX5622 on microglial survival.

    “The expression of these genes was nearly undetectable from PLX5622-treated mice, highlighting their specific expression by microglia and the specificity of PLX5622 on microglial survival.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Long-term PLX5622-mediated microglial depletion is highly robust, sustainable, and specific to the microglial compartment.

    “Data presented here show that long-term PLX5622-mediated microglial depletion is highly robust, sustainable, and specific to the microglial compartment.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Dystrophic neurites are found in a halo around fibrillar plaques and are characterized by the accumulation of LAMP1 and APP.

    “Dystrophic neurites are found in a halo around fibrillar plaques and are characterized by the accumulation of LAMP1 and APP32–34.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [32] Andrew, R. J. et al. Lack of BACE1 S-palmitoylation reduces amyloid burden and mitigates memory deficits in transgenic mouse models of Alzheimer’s disease. Proc. Natl. Acad. Sci. USA 114, E9665–e9674 (2017). source

      Not checked in the free run.

    • cites [33] Cummings, B. J. et al. Aggregation of the amyloid precursor protein within degenerating neurons and dystrophic neurites in Alzheimer’s disease. Neuroscience 48, 763–777 (1992). source

      Not checked in the free run.

    • cites [34] Gowrishankar, S. et al. Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer’s disease amyloid plaques. Proc. Natl. Acad. Sci. USA 112, E3699–E3708 (2015). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Microglia can be indefinitely depleted via the continued administration of CSF1R inhibitors, and the microglial compartment can also be repopulated upon CSF1R inhibitor withdrawal.

    “While microglia can be indefinitely depleted via the continued administration of CSF1R inhibitors, the microglial compartment can also be repopulated upon CSF1R inhibitor withdrawal17,40,41.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [17] Elmore, M. R. et al. Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. Neuron 82, 380–397 (2014). source

      Not checked in the free run.

    • cites [40] Elmore, M. R. P. et al. Replacement of microglia in the aged brain reverses cognitive, synaptic, and neuronal deficits in mice. Aging Cell  17, e12832 (2018). source

      Not checked in the free run.

    • cites [41] Rice, R. A. et al. Microglial repopulation resolves inflammation and promotes brain recovery after injury. Glia 65, 931–944 (2017). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Several of these genes are known markers of disease-associated microglia (DAM).

    “Notably, several of these genes are known markers of disease-associated microglia39 (DAM).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] Keren-Shaul, H. et al. A unique microglia type associated with restricting development of Alzheimer’s disease. Cell 169, 1276–1290 e17 (2017). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    PLX3397 at 75 ppm provides robust peripheral CSF1R inhibition without microglial elimination.

    “We further examined the role of peripheral CSF1R inhibition on AD pathology by treating 1.5-month-old 5xFAD mice until 5 months of age with PLX3397 (75 ppm) chow (Fig. 7a) – a formulation that provides robust peripheral CSF1R inhibition without microglial elimination17.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [17] Elmore, M. R. et al. Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. Neuron 82, 380–397 (2014). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Both AD pathology and the absence of microglia have minimal effects on AD-related genes.

    “Thus, both AD pathology and the absence of microglia have minimal effects on AD-related genes, and importantly, we observe no alterations in gene expression or protein production in the absence of microglia that could account for reduced plaque formation.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Premisenone given

    CSF1R inhibition is the only currently available method capable of achieving sustained long-term microglial elimination.

    “Most importantly, CSF1R inhibition is the only currently available method capable of achieving sustained long-term microglial elimination.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Peripheral CSF1R inhibition or simply the presence of CSF1R inhibitor within the CNS do not contribute to the reductions in plaque pathology.

    “Thus, peripheral CSF1R inhibition or simply the presence of CSF1R inhibitor within the CNS do not attribute to the reductions in plaque pathology and more complete microglial elimination is necessary to perturb plaque formation processes.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Previous reports describe the coaggregation of ApoE with Aβ fibrils in both plaques and vascular pathology.

    “Focusing on plaque structure, we next evaluated the presence of ApoE within plaque cores – previous reports describe the coaggregation of ApoE with Aβ fibrils in both plaques and vascular pathology29,30.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [29] Liao, F. et al. Murine versus human apolipoprotein E4: differential facilitation of and co-localization in cerebral amyloid angiopathy and amyloid plaques in APP transgenic mouse models. Acta Neuropathol. Commun. 3, 70 (2015). source

      Not checked in the free run.

    • cites [30] Parhizkar, S. et al. Loss of TREM2 function increases amyloid seeding but reduces plaque-associated ApoE. Nat Neurosci (2019). source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Prior studies show that microglia form a physical barrier around plaques and compact Aβ into dense deposits, protecting against local neurite damage.

    “These results are consistent with prior studies showing that microglia form a physical barrier around plaques and compact Aβ into dense deposits, protecting against local neurite damage35–37.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [35] Condello, C., Yuan, P., Schain, A. & Grutzendler, J. Microglia constitute a barrier that prevents neurotoxic protofibrillar Abeta42 hotspots around plaques. Nat. Commun. 6, 6176 (2015). source

      Not checked in the free run.

    • cites [36] Wang, Y. et al. TREM2-mediated early microglial response limits diffusion and toxicity of amyloid plaques. J. Exp. Med 213, 667–675 (2016). source

      Not checked in the free run.

    • cites [37] Yuan, P. et al. TREM2 Haplodeficiency in Mice and Humans Impairs the Microglia Barrier Function Leading to Decreased Amyloid Compaction and Severe Axonal Dystrophy. Neuron 90, 724–739 (2016). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationcitation

    Microglia appear to have complex and contrasting roles within AD, namely their facilitation of plaque formation, plaque growth, plaque compaction, and neuronal damage.

    “Therefore, microglia appear to have complex and contrasting roles within AD61, namely their facilitation of plaque formation, plaque growth, plaque compaction, and neuronal damage.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [61] Moore, Z., Taylor, J. M. & Crack, P. J. The involvement of microglia in Alzheimer’s disease: a new dog in the fight. Br. J. Pharmacol. (2018). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Microglia contribute to multiple facets of AD etiology: they are 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.

    “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.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductcitation

    No statistical methods were used to predetermine sample sizes; however, the sample sizes are similar to those reported in recently published similar studies.

    “No statistical methods were used to predetermine sample sizes; however, our sample sizes are similar to those reported in recently published similar studies10,28.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [10] Spangenberg, E. E. et al. Eliminating microglia in Alzheimer’s mice prevents neuronal loss without modulating amyloid-beta pathology. Brain 139, 1265–1281 (2016). source

      Not checked in the free run.

    • cites [28] Olmos-Alonso, A. et al. Pharmacological targeting of CSF1R inhibits microglial proliferation and prevents the progression of Alzheimer’s-like pathology. Brain 139, 891–907 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Microglia appear to have detrimental and beneficial roles in a preclinical model of AD.

    “Therefore, microglia appear to have detrimental and beneficial roles in a preclinical model of AD.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Plaque-associated microglia may protect against local neurite damage.
60 of 60

  • The paper relies on this without support.

    5xFAD mice exhibit plaque pathology from 3 months of age.

    “To that end, 5xFAD animals, which exhibit plaque pathology from 3 months of age, underwent treatment at 1.5 months of age with PLX5622-formulated chow (1200 ppm) or control diet continuously for either 10–24 weeks.”

  • The paper relies on this without support.

    The 5xFAD mouse model is a valid model of Alzheimer's disease pathology relevant to human AD.

    “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.”

  • The paper relies on this without support.

    Plaque-associated microglia may protect against local neurite damage.

    “Although plaque-associated microglia may protect against local neurite damage, the presence of microglia is also required for the reduction in expression of synaptic and neuronal genes in the hippocampus associated with AD.”

  • The authors state this as an assumption.

    Data distribution is normal.

    “Data distribution was assumed to be normal, but this was not formally tested.”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Our results are in line with studies showing that reduced microglial colocalization with β-amyloid is associated with a reduced β-amyloid burden.

    “Our results are in line with studies showing that reduced microglial colocalization with β-amyloid is associated with a reduced β-amyloid burden (D’Errico et al., 2022; Spangenberg et al., 2019).”

    From A novel molecular class that recruits HDAC/MECP2 complexes to PU.1 motifs reduces neuroinflammation (2023) · cites this paper as Spangenberg et al., 2019

    The Results section (“Microglial elimination impairs plaque formation”; page unavailable) reports fewer parenchymal Aβ plaques in microglia-depleted 5xFAD mice, supporting the association if ‘burden’ means plaque burden. The same section says soluble and insoluble Aβ levels did not significantly change: depletion shifted where Aβ accumulated rather than reducing its net amount. The source therefore does not support a reduction in overall Aβ burden. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • Previous studies have shown that microglial depletion can have beneficial effects in transgenic mice that model AD hallmarks, including reduction in brain accumulation of both amyloid-β and tau.

    “In support of this interpretation, previous studies have shown that microglial depletion can have beneficial effects in transgenic mice that model AD hallmarks, including reduction in brain accumulation of both amyloid-β and tau (Sosna et al., 2018; Spangenberg et al., 2019).”

    From A novel molecular class that recruits HDAC/MECP2 complexes to PU.1 motifs reduces neuroinflammation (2023) · cites this paper as Spangenberg et al., 2019

    The Results section ('Microglial elimination impairs plaque formation'; page unavailable) reports fewer cortical amyloid-β plaques in microglia-depleted 5xFAD mice, supporting the amyloid-plaque part of the claim. It also says total amyloid-β amounts were unchanged and accumulation shifted toward blood vessels, so it does not support reduced overall brain accumulation. The passages found do not report reduced tau accumulation. (LLM, conf=0.89)

    Experimental — accuracy not yet measured

  • PLX5622 enables sustained microglial depletion without breaching the BBB, without toxicity and without initiating an inflammatory response.

    “It enables sustained microglial depletion without breaching the BBB, without toxicity and without initiating an inflammatory response, as seen with other microglial depletion methods [295, 299, 331].”

    From Microglia and monocytes in inflammatory CNS disease: integrating phenotype and function (2022) · cites this paper as [295]

    The Discussion (page unavailable) reports sustained PLX5622-mediated microglial depletion and describes CSF1R-inhibitor depletion as lacking an inflammatory response. It also reports no behavioral or cognitive impairment, but that does not establish an absence of toxicity generally. The Results section on inhibitor development (page unavailable) describes PLX5622 crossing the BBB; it does not establish that BBB integrity was unaffected. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • Microglia depletion before or after Aβ pathology in AD mouse models improved contextual memory.

    “microglia depletion before or after the formation of Aβ pathology in mouse models of AD resulted in improved contextual memory”

    From Microglia and monocytes in inflammatory CNS disease: integrating phenotype and function (2022) · cites this paper as [295]

    The Discussion (page unavailable) describes microglial depletion before plaque formation in this study and after extensive pathology in a prior study. But the Results, under “Absence of abnormalities with microglial elimination” (page unavailable), report no contextual fear-conditioning impairment in 5xFAD mice and do not report improved contextual memory after depletion at either time point. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • TREM2, expressed by DAM/MGnD, exerts protective function in AD via ligation of ApoE or Aβ oligomers.

    “TREM2, a receptor expressed by DAM/MGnD, which exerts its protective function in AD via ligation of ApoE (apolipoprotein E), which binds to loose Aβ aggregates, or direct ligation of Aβ oligomers [286, 295, 338, 357, 366]”

    From Microglia and monocytes in inflammatory CNS disease: integrating phenotype and function (2022) · cites this paper as [295]

    The Discussion (page not indicated) links TREM2 signaling to plaque-associated microglia and describes a protective barrier around plaques. The Results report ApoE in plaque-associated microglia, but neither passage establishes that TREM2 is expressed specifically by DAM/MGnD or that its protective function works through ligation of ApoE or Aβ oligomers. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Cortex described as plaque-free despite persistent retrosplenial plaques

    Minor

    The statement that the treated cortex contained only vascular Thio-S+ deposits conflicts with the report that Thio-S plaques persisted in the retrosplenial cortex after 24 weeks of treatment.

  • Brain-wide elimination conflicts with surviving subicular cells

    Minor

    The text says microglia were eliminated brain-wide after 24 weeks, but later reports surviving IBA1+ cells in the subiculum of mice treated through the same age range.

2 of 2

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Authors
Spangenberg, E., Severson, P. L., Hohsfield, L. A., Crapser, J., Zhang, J., Burton, E. A., Zhang, Y., Spevak, W., Lin, J., Phan, N. Y., Habets, G., Rymar, A., Tsang, G., Walters, J., Nespi, M., Singh, P., Broome, S., Ibrahim, P., Zhang, C., Bollag, G., West, B. L., & Green, K. N.
Affiliations
  • University of California, Irvine (US)
  • Plexxikon (United States) (US)
Corresponding author
Green, Kim N.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2019-08-21
Volume
10
Issue
1
Pages
3758-3758
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-019-11674-z
PMC
PMC6704256
OpenAlex
W2969940182
Semantic Scholar
3e694fe2e99a78fdb9cd33e218acd99a93cc12a0
Cited by
939
References
71
Citations by year
2026: 114 · 2025: 122 · 2024: 201 · 2023: 139 · 2022: 137 · 2021: 132 · 2020: 82 · 2019: 10 · 2018: 1
FWCI
39.95
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • eScholarship (California Digital Library) (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion, cc-by)
Funders
  • U.S. Department of Energy (-AC02-05CH11231, 05CH11231, DE AC02 05CH11231, DE-AC02-)
  • Alzheimer's Association (AARF-16-442762)
  • National Institutes of Health (DE-AC02–05CH11231)
  • Office of Science (DEAC02–05CH11231, AC02-05CH11231, DE-AC02)
  • National Institute on Aging (R01 AG056768, T32 AG00096, P50AG016573, F31AG059367, RF1AG056768, T32AG000096)
  • National Institute of Neurological Disorders and Stroke (R01NS083801, R01AG056768)
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Physiology › Alzheimer's disease research and treatments
  • Life Sciences › Neuroscience › Neurology › Neurological Disease Mechanisms and Treatments
Keywords
Alzheimer's disease, microglia, CSF1R inhibition, PLX5622, microglial depletion, amyloid plaques, 5xFAD mice, cerebral amyloid angiopathy, amyloid-beta deposition, disease-associated microglia, hippocampus
MeSH terms
Alzheimer Disease, Animals, Behavior, Animal, Brain, Disease Models, Animal, Gene Expression Regulation, Hippocampus, Humans, Memory, Mice, Transgenic, Organic Chemicals, Receptors, Granulocyte-Macrophage Colony-Stimulating Factor, Microglia, Mice, Plaque, Amyloid, Transcriptome

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-06.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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