CX3CR1 Deficiency Alters Microglial Activation and Reduces Beta-Amyloid Deposition in Two Alzheimer's Disease Mouse Models

The chemokine fractalkine (CX3CL1) and its cognate receptor, CX3CR1, play an important role in neuroinflammation via paracrine signaling between neurons and microglia.

“The chemokine fractalkine (CX3CL1) and its cognate receptor, CX3CR1, play an important role in neuroinflammation via paracrine signaling between neurons and microglia.[12][13][14]”

• cites [12] Limatola, 2005, Chemokine CX3CL1 protects rat hippocampal neurons against glutamate-mediated excitotoxicity, J Neuroimmunol sourceNot checked — no full text of the source
• cites [13] Cardona, 2006, Control of microglial neurotoxicity by the fractalkine receptor, Nat Neurosci sourceNot checked — no full text of the source
• cites [14] Harrison, 1998, Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia, Proc Natl Acad Sci USA sourcePartly supported

  The Discussion (page not provided) identifies neurons as the primary source of fractalkine and microglia as the principal responding cells; it also links fractalkine–CX3CR1 signaling to the response to facial motor nerve injury. This supports neuron-to-microglia signaling in a pathological setting, but the paper does not establish a broad, important role in neuroinflammation specifically, and its evidence includes membrane-anchored as well as soluble fractalkine rather than solely paracrine signaling. (LLM, conf=0.82)

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The APPPS1 model exhibits rapid deposition of primarily Aβ42.

“we mated Cx3cr1 knockout mice to two different mouse models of AD that exhibit either rapid deposition of primarily A␤42 (the APPPS1 model) 21 or gradual deposition of primarily A␤40 (the R1.40 model).[22][23][24]”

• cites [21] Radde, 2006, Abeta42-driven cerebral amyloidosis in transgenic mice reveals early and robust pathology, EMBO Rep sourceSupported

APPPS1 mice exhibit early and robust Aβ deposition beginning at 6-8 weeks of age in the cortex and 3-4 months of age in the hippocampus.

“APPPS1 mouse line coexpresses APP K670M/N671L and PS1 L166P mutations under the control of the neuronspecific Thy1 promoter and exhibits early and robust A␤ deposition beginning at 6 -8 weeks of age in the cortex and 3-4 months of age in the hippocampus.21”

• cites [21] Radde, 2006, Abeta42-driven cerebral amyloidosis in transgenic mice reveals early and robust pathology, EMBO Rep sourcePartly supported

  The Introduction identifies the APP and PS1 mutations and neuron-specific Thy1 promoter, and the Cerebral amyloidosis section reports the first neocortical plaques at 6 weeks. The hippocampal timing is less accurate: that section says deposition began in the dentate gyrus at 2–3 months and in CA1 at 4–5 months, rather than beginning throughout the hippocampus at 3–4 months. (LLM, conf=0.88)

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APPPS1 mouse line coexpresses human amyloid precursor protein harboring the K670M/N671L familial AD mutation and presenilin 1 harboring the L166P familial AD mutation, under the control of the neuron-specific Thy1 promoter.

“The APPPS1-21 (APPPS1) mouse line coexpresses human amyloid precursor protein harboring the K670M/ N671L familial AD mutation and presenilin 1 harboring the L166P familial AD mutation, under the control of the neuron-specific Thy1 promoter.21”

• cites [21] Radde, 2006, Abeta42-driven cerebral amyloidosis in transgenic mice reveals early and robust pathology, EMBO Rep sourcePartly supported

  The Introduction and mouse-generation methods identify line 21 as coexpressing APP and PS1-L166P under Thy1 promoter control. They specify the APP mutation as KM670/671NL, not the citing sentence’s K670M/N671L; that mutation designation is not supported. Page numbers are unavailable in our copy. (LLM, conf=0.91)

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The R1.40 transgene is a full genomic copy of human APP carrying the K670M/N671L mutation.

“The R1.40 transgene is a full genomic copy of human APP (carried on a 650-kb yeast artificial chromosome clone) carrying the K670M/N671L familial AD mutation associated with early-onset familial AD.”

• cites [22] Lamb, 1993, Introduction and expression of the 400 kilobase amyloid precursor protein gene in transgenic mice [corrected], Nat Genet sourceNot checked — no full text of the source
• cites [23] Lamb, 1997, Altered metabolism of familial Alzheimer's disease-linked amyloid precursor protein variants in yeast artificial chromosome transgenic mice, Hum Mol Genet sourceNot checked — no full text of the source
• cites [25] Lehman, 2003, Genetic background regulates β-amyloid precursor protein processing and β-amyloid deposition in the mouse, Hum Mol Genet sourcePartly supported

  The “Generation of R1.40 congenic strains” section says R1.40 contains the entire human APP gene on a YAC, but gives the mutation as K670N/M671L, not K670M/N671L. The Introduction identifies it as the Swedish familial AD mutation. The passages found do not specify a 650-kb YAC. (LLM, conf=0.90)

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In vivo Aβ removal is critically dependent on microglial activation status.

“However, considerable evidence suggests that in vivo A␤ removal is critically dependent on microglial activation status.33,44 -47”

• cites [33] Koenigsknecht-Talboo, 2005, Microglial phagocytosis induced by fibrillar beta-amyloid and IgGs are differentially regulated by proinflammatory cytokines, J Neurosci sourcePartly supported

  The Discussion reports that proinflammatory cytokines inhibit microglial phagocytosis of fibrillar Aβ, supporting a role for microglial state in one clearance pathway. But it says these effects were demonstrated in vitro, cautions that the chronic in vivo situation may differ, and describes other clearance mechanisms operating in parallel. It does not establish that in vivo Aβ removal is critically dependent on microglial activation status. (LLM, conf=0.87)

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• cites [44] Townsend, 2005, CD40 signaling regulates innate and adaptive activation of microglia in response to amyloid beta-peptide, Eur J Immunol sourceSupported
• cites [45] Zelcer, 2007, Attenuation of neuroinflammation and Alzheimer's disease pathology by liver x receptors, Proc Natl Acad Sci USA sourcePartly supported

  The Results (page not available) show that inflammatory signals suppress microglial phagocytosis of Aβ and that LXR treatment reverses this effect in cultured cells. The Discussion (page not available) proposes that this mechanism may affect amyloid clearance in the brain, while the in vivo finding is increased plaque burden in LXR-deficient mice. The paper does not establish that in vivo Aβ removal is critically dependent on microglial activation status. (LLM, conf=0.85)

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• cites [46] Boissonneault, 2009, Powerful beneficial effects of macrophage colony-stimulating factor on beta-amyloid deposition and cognitive impairment in Alzheimer's disease, Brain sourceNot checked — no full text of the source
• cites [47] Shie, 2005, Microglial EP2 is critical to neurotoxicity from activated cerebral innate immunity, Glia sourceNot checked — no full text of the source

CX3CR1 is exclusively expressed by microglia within the brain.

“CX3CR1 is exclusively expressed by microglia within the brain”

• Also stated in: § Discussion · plain

  “CX3CR1 is exclusively expressed by microglia in the murine CNS, 13,15”

• cites [13] Cardona, 2006, Control of microglial neurotoxicity by the fractalkine receptor, Nat Neurosci sourceNot checked — no full text of the source
• cites [15] Jung, 2000, Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion, Mol Cell Biol sourcePartly supported

  The Results and Discussion report CX3CR1 expression in mouse brain microglia and no staining in astrocytes or oligodendrocytes. They do not establish that microglia are the only CX3CR1-expressing cells anywhere in the brain, so “exclusively” is stronger than the reported finding. (LLM, conf=0.82)

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TNFα has been shown to directly reduce microglial expression of Aβ-degrading enzymes such as insulin-degrading enzyme and neprilysin.

“TNF␣ has been shown to directly reduce microglial expression of A␤-degrading enzymes such as insulin-degrading enzyme and neprilysin.51,52”

• cites [51] Yamamoto, 2008, Cytokine-mediated inhibition of fibrillar amyloid-beta peptide degradation by human mononuclear phagocytes, J Immunol sourcePartly supported

  The Results section on IDE expression reports that TNF-α combined with IFN-γ reduced IDE expression in monocyte-derived macrophages, but TNF-α alone had no effect. The microglia Results section measures Aβ degradation, not enzyme expression; the copy does not report reduced microglial expression of IDE or neprilysin. (LLM, conf=0.90)

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• cites [52] Shimizu, 2008, IL-4-induced selective clearance of oligomeric beta-amyloid peptide(1-42) by rat primary type 2 microglia, J Immunol sourcePartly supported

  In “Effects of cytokines on expression of CD36, NEP, and IDE in type 2 MG” (page unavailable), the paper reports that TNF-α suppressed neprilysin expression in rat type 2 microglia. It says TNF-α had the lowest potency for inducing insulin-degrading enzyme expression, not that it reduced expression of that enzyme. (LLM, conf=0.87)

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In the AD brain, chronic activation of microglia is associated with inflammatory cytokines including TNFα that can substantially block the ability of microglia to remove or degrade Aβ.

“Within the AD brain, there is a chronic activation of microglia associated with inflammatory cytokines including TNF␣ that can substantially block of ability of the microglia to remove 47,49,50 or degrade A␤. 51,52”

• cites [47] Shie, 2005, Microglial EP2 is critical to neurotoxicity from activated cerebral innate immunity, Glia sourceNot checked — no full text of the source
• cites [49] Jimenez, 2008, Inflammatory response in the hippocampus of PS1M146L/APP751SL mouse model of Alzheimer's disease: age-dependent switch in the microglial phenotype from alternative to classic, J Neurosci sourcePartly supported

  The Introduction (page unavailable) describes chronic inflammation in AD involving activated microglia, and the Discussion (page unavailable) reports TNF-α expression by activated microglia in older AD-model mice. The paper does not show that TNF-α or other inflammatory cytokines block microglial removal or degradation of Aβ; its Discussion instead reports Aβ phagocytosis by plaque-associated microglia. (LLM, conf=0.87)

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• cites [50] Monsonego, 2003, Increased T cell reactivity to amyloid beta protein in older humans and patients with Alzheimer disease, J Clin Invest sourcePartly supported

  The Introduction associates AD plaques with activated microglia and describes microgliosis and elevated cytokines as inflammatory changes in AD (page not available). It does not identify TNFα or say that inflammatory cytokines block microglia from removing or degrading Aβ. The discussion instead describes TGF-β promoting microglial clearance in transgenic mice. (LLM, conf=0.84)

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• cites [51] Yamamoto, 2008, Cytokine-mediated inhibition of fibrillar amyloid-beta peptide degradation by human mononuclear phagocytes, J Immunol sourcePartly supported

  The Results report that TNF-α reduced a measure of fibrillar Aβ42 degradation in cultured human microglia. The Discussion says pro-inflammatory cytokines suppressed Aβ degradation in microglia, mainly through IFN-γ, but that the microglial response was small. The study does not establish substantial inhibition in chronically activated microglia within the AD brain. (LLM, conf=0.88)

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• cites [52] Shimizu, 2008, IL-4-induced selective clearance of oligomeric beta-amyloid peptide(1-42) by rat primary type 2 microglia, J Immunol sourcePartly supported

  The paper describes activated microglia around amyloid plaques and an inflammatory response involving TNFα (Abstract; page unavailable). In “Effects of cytokines on expression of CD36, NEP, and IDE in type 2 MG” (page unavailable), it reports that TNFα suppressed expression of an Aβ-degrading enzyme in cultured rat microglia. It does not show that TNFα substantially blocks microglial removal or degradation of Aβ in the AD brain. (LLM, conf=0.88)

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CCL2 and TNFα mRNA levels were elevated in APPPS1;Cx3cr1+/+ mouse brain.

“Similar to previous findings in AD brains and mouse models, 39 -42 the levels of CCL2 and TNF␣ mRNAs were elevated in the APPPS1;Cx3cr1 ϩ/ϩ mouse brain.”

• cites [39] Ishizuka, 1997, Identification of monocyte chemoattractant protein-1 in senile plaques and reactive microglia of Alzheimer's disease, Psychiatry Clin Neurosci sourceNot checked — no full text of the source
• cites [40] Simard, 2006, Bone marrow-derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease, Neuron sourceNot checked — no full text of the source
• cites [41] Heneka, 2005, Focal glial activation coincides with increased BACE1 activation and precedes amyloid plaque deposition in APP[V717I] transgenic mice, J Neuroinflammation sourcePartly supported

  The citation supports the sentence’s reference to previous inflammatory findings in AD mouse models: the Discussion reports TNFα mRNA in an earlier APP-transgenic mouse study. It does not support elevated CCL2 mRNA, and its own Results say TNFα was undetectable in APP[V717I] mice. The APPPS1;Cx3cr1+/+ result is the citing authors’ finding, not one attributed to this source. (LLM, conf=0.84)

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• cites [42] Janelsins, 2005, Early correlation of microglial activation with enhanced tumor necrosis factor-alpha and monocyte chemoattractant protein-1 expression specifically within the entorhinal cortex of triple transgenic Alzheimer's disease mice, J Neuroinflammation sourceSupported

Microglia surrounding Aβ deposits undergo extensive turnover in a mouse model of AD, exhibiting both apoptosis and proliferation.

“Microglia surrounding A␤ deposits undergo extensive turnover in a mouse model of AD, exhibiting both apoptosis and proliferation.60”

• cites [60] Bondolfi, 2002, Amyloid-associated neuron loss and gliogenesis in the neocortex of amyloid precursor protein transgenic mice, J Neurosci sourcePartly supported

  The Discussion reports many newly produced microglia near amyloid plaques in aged APP23 mice and says the findings suggest apoptotic microglial death there. It explicitly says it is unclear whether the new cells arose through microglial division or recruitment, so proliferation of the surrounding microglia is not established. Apoptosis is also presented as a suggestion rather than a definitive finding. (LLM, conf=0.88)

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CX3CL1/CX3CR1 signaling alters the phagocytic capacity of microglia, including the uptake of Aβ fibrils.

“Finally , to account for these seemingly disparate results , both in vitro and in vivo studies provided evidence that CX3CL1/CX3CR1 signaling alters the phagocytic capacity of microglia , including the uptake of A␤ fibrils.”

Blocking CX3CL1-CX3CR1 signaling increases uptake of Aβ.

“Finally, we demonstrate that CX3CL1-CX3CR1 signaling reduces microglial phagocytic capabilities and that blocking CX3CL1-CX3CR1 signaling increases uptake of A␤, suggesting that CX3CR1 deficiency leads to enhanced A␤ clearance.”

CX3CL1-CX3CR1 signaling reduces microglial phagocytic capabilities.

“Finally, we demonstrate that CX3CL1-CX3CR1 signaling reduces microglial phagocytic capabilities and that blocking CX3CL1-CX3CR1 signaling increases uptake of A␤, suggesting that CX3CR1 deficiency leads to enhanced A␤ clearance.”

CX3CR1 deficiency results in a gene dose-dependent reduction in beta-amyloid deposition in both APPPS1 and R1.40 mouse models of AD.

“Surprisingly, CX3CR1 deficiency resulted in a gene dose-dependent reduction in ␤-amyloid deposition in both the APPPS1 and R1.40 mouse models of AD.”

Quantitative immunohistochemical analysis revealed reduced numbers of microglia surrounding beta-amyloid deposits in CX3CR1-deficient APPPS1 animals.

“Furthermore , quantitative immunohistochemical analysis revealed reduced numbers of microglia surrounding ␤-amyloid deposits in the CX3CR1-deficient APPPS1 animals.”

There is a gene dose-dependent reduction in the number of plaque-associated microglia in the cortex of APPPS1;Cx3cr1 +/- and APPPS1;Cx3cr1 -/- animals compared to APPPS1;Cx3cr1 +/+ controls.

“We observed a gene dose-dependent reduction in the number of plaque-associated microglia in the cortex of APPPS1;Cx3cr1 ϩ/Ϫ (Figure 4, D-F) and APPPS1;Cx3cr1 Ϫ/Ϫ (Figure 4, G-I) animals when compared to APPPS1;Cx3cr1 ϩ/ϩ controls (Figure 4, A-C).”

CD45 immunoreactivity between APPPS1;Cx3cr1 +/+, APPPS1;Cx3cr1 +/-, and APPPS1;Cx3cr1 -/- genotypes appeared relatively unchanged.

“By contrast, CD45 immunoreactivity between the APPPS1; Cx3cr1 ϩ/ϩ (Figure 3D), APPPS1;Cx3cr1 ϩ/Ϫ (Figure 3F) and APPPS1;Cx3cr1 Ϫ/Ϫ (Figure 3H) genotypes appeared relatively unchanged.”

• Also stated in: § CX3CR1 Deficiency Leads to Reductions in Plaque-Associated Microglia · plain

  “CD45 immunoreactivity is relatively unchanged in APPPS1;Cx3cr1 ϩ/Ϫ and APPPS1;Cx3cr1 Ϫ/Ϫ animals compared with APPPS1;Cx3cr1 ϩ/ϩ controls (F and H, respectively).”

Expression of both TNFα and CCL2 mRNAs was increased in APPPS1;Cx3cr1 +/+ mice at four months of age relative to B6 control mice.

“Expression of both TNF␣ and CCL2 mRNAs were increased in APPPS1;Cx3cr1 ϩ/ϩ mice at four months of age relative to B6 control mice (Figure 5, A and C, respectively).”

R1.40;Cx3cr1+/− and R1.40;Cx3cr1−/− animals demonstrated a gene dose-dependent reduction in Thioflavine S-positive Aβ deposits in the cortex.

“Similar to the results in CX3CR1-deficient APPPS1 mice, R1.40; Cx3cr1 ϩ/Ϫ and R1.40;Cx3cr1 Ϫ/Ϫ animals also demonstrated a gene dose-dependent reduction in Thioflavine S-positive A␤ deposits in the cortex.”

There were no alterations in steady-state levels of brain Aβ in pre-depositing APPPS1 and R1.40 mice.

“In addition, there were no alterations in the steady-state levels of brain A␤ in pre-depositing APPPS1 and R1.40 mice (data not shown).”

R1.40;Cx3cr1+/+ controls exhibited abundant Thioflavine S positive fibrillar Aβ deposits in the cortex.

“R1.40;Cx3cr1 ϩ/ϩ controls exhibited abundant Thioflavine S positive fibrillar A␤ deposits in the cortex (see Supplemental Figure S2 at http://ajp.amjpathol.org).”

APPPS1;Cx3cr1−/− brains exhibited drastically reduced levels of biochemically extracted Aβ when compared to APPPS1;Cx3cr1+/+ brains.

“Confirming results obtained with Thioflavine S, APPPS1;Cx3cr1 Ϫ/Ϫ brains exhibited drastically reduced levels of biochemically extracted A␤ when compared to APPPS1; Cx3cr1 ϩ/ϩ brains.”

Steady-state levels of APP CTFs, including CTFβ, the direct precursor of Aβ, were unaltered in APPPS1;Cx3cr1 +/+, APPPS1;Cx3cr1 +/-, and APPPS1;Cx3cr1 -/- mice.

“The steady-state levels of APP CTFs, including CTF␤, the direct precursor of A␤, were unaltered (Figure 2, A [middle] and C).”

ELISA analysis revealed reduced Aβ42 levels in APPPS1;Cx3cr1−/− brains (1375 ± 803 pmol/g wet brain) when compared to APPPS1;Cx3cr1+/+ brains (4608 ± 861 pmol/g wet brain).

“In support of the Thioflavine S straining, ELISA analysis revealed reduced A␤42 levels in APPPS1;Cx3cr1 Ϫ/Ϫ brains (1375 Ϯ 803 pmol/g wet brain; Figure 1G) when compared to APPPS1;Cx3cr1 ϩ/ϩ brains (4608 Ϯ 861 pmol/g wet brain; Figure 1G).”

CD68 immunoreactivity was qualitatively reduced in APPPS1;Cx3cr1 +/- and APPPS1;Cx3cr1 -/- animals compared to APPPS1;Cx3cr1 +/+ controls.

“CD68 immunoreactivity was qualitatively reduced in APPPS1;Cx3cr1 ϩ/Ϫ (Figure 3E) and APPPS1;Cx3cr1 Ϫ/Ϫ animals (Figure 3G) compared to APPPS1;Cx3cr1 ϩ/ϩ controls (Figure 3C).”

• Also stated in: § CX3CR1 Deficiency Leads to Reductions in Plaque-Associated Microglia · plain

  “While age-matched APPPS1;Cx3cr1 ϩ/Ϫ and APPPS1;Cx3cr1 Ϫ/Ϫ animals exhibit gene dose-dependent reduction in CD68 immunoreactivity compared to APPPS1;Cx3cr1 ϩ/ϩ controls (E and G, respectively)”

CD68- and CD45-positive microglia are closely associated with Congo Red-positive deposits in all APPPS1 genotypes.

“CD68-and CD45-postive microglia are closely associated with Congo Red-positive deposits in all APPPS1 genotypes (Figure 3, insets).”

Staining with antibodies for dystrophic neurites (CT15 and AT8) revealed a reduction in staining in both APPPS1;Cx3cr1−/− and R1.40;Cx3cr1−/− animals.

“staining with antibodies for dystrophic neurites (CT15 and AT8) revealed a reduction in staining in both the APPPS1;Cx3cr1 Ϫ/Ϫ (see Supplemental Figure S3 at http://ajp.amjpathol.org) and R1.40;Cx3cr1 Ϫ/Ϫ animals (data not shown)”

APPPS1;Cx3cr1 +/+ mice exhibited abundant Congo Red-positive Aβ deposits that were ringed by numerous GFAP-positive astrocytes.

“By contrast, APPPS1; Cx3cr1 ϩ/ϩ mice exhibited abundant Congo Red-positive A␤ deposits that were ringed by numerous GFAP-positive astrocytes.”

There is significantly reduced GFAP immunoreactivity in APPPS1;Cx3cr1 -/- animals.

“In addition to a substantial reduction in the amount of A␤ deposition in the APPPS1;Cx3cr1 Ϫ/Ϫ animals (Figure 1), there was also significantly reduced GFAP immunoreactivity.”

In vivo, Cx3cr1−/− microglia have enhanced uptake of fibrillar Aβ compared to Cx3cr1+/+ microglia (66% vs 46% within 50 μm of injection site).

“On average, 46% of Cx3cr1 ϩ/ϩ microglia within 50 m of the needle track contained fibrillar A␤, whereas 66% of Cx3cr1 Ϫ/Ϫ microglia contained A␤ (Figure 7D; P Ͻ 0.01).”

Similar results of unaltered APP levels or processing were obtained in the R1.40 mouse model of Alzheimer's disease with CX3CR1 deficiency.

“Similar results were also obtained in the R1.40 mouse model of AD with CX3CR1 deficiency (see Supplemental Figure S4 at http://ajp.amjpathol.org).”

For medium plaques (>500 μm2 and <1000 μm2), APPPS1;Cx3cr1 +/- and APPPS1;Cx3cr1 -/- mice demonstrated an average of approximately 7 and approximately 5 plaque-associated microglia, respectively, which was significantly lower than approximately 9 plaque-associated microglia in the medium plaques of APPPS1;Cx3cr1 +/+ controls.

“For medium plaques (Ͼ500 m 2 and Ͻ1000 m 2 ), APPPS1;Cx3cr1 ϩ/Ϫ and APPPS1;Cx3cr1 Ϫ/Ϫ mice demonstrated an average of ϳ7 and ϳ5 plaque associated microglia, respectively, which was significantly lower than ϳ9 plaque associated microglia in the medium plaques of APPPS1;Cx3cr1 ϩ/ϩ controls (Figure 4K).”

CX3CR1-deficient APPPS1 and R1.40 animals show no evidence for gross neurodegeneration by TUNEL and cleaved caspase 3 staining.

“TUNEL and cleaved caspase 3 staining of brain sections from CX3CR1-deficient APPPS1 and R1.40 animals revealed no evidence for any gross neurodegeneration (data not shown)”

Expression of IL1β mRNA was up-regulated in APPPS1;Cx3cr1 +/- and APPPS1;Cx3cr1 -/- animals (P < 0.05 and P < 0.01, respectively).

“By contrast, expression of IL1␤ mRNA was up-regulated in APPPS1; Cx3cr1 ϩ/Ϫ and APPPS1; Cx3cr1 Ϫ/Ϫ animals (P Ͻ 0.05 and P Ͻ 0.01, respectively; Figure 5B) as previously reported in other neuroinflammatory models.13”

• cites [13] Cardona, 2006, Control of microglial neurotoxicity by the fractalkine receptor, Nat Neurosci sourceNot checked — no full text of the source

Cx3cr1−/− microglia demonstrate equivalent phagocytic responses with or without fractalkine treatment.

“Cx3cr1 Ϫ/Ϫ microglia demonstrated equivalent phagocytic responses either with (Figure 6, C and E) or without fractalkine treatment (Figure 6, D and E)”

For small plaques (<500 μm2), differences were not statistically significant among the genotypes.

“For small plaques (Ͻ500 m 2 ), differences were not statistically significant among the genotypes (Figure 4L).”

6E10 immunohistochemistry confirmed a gene dose-dependent reduction in Aβ deposits in the cortex and hippocampus of APPPS1 mice.

“Finally, 6E10 immunohistochemistry also confirmed a gene dose-dependent reduction A␤ deposits in the cortex (see Supplemental Figure S1 at http://ajp.amjpathol.org) and hippocampus (data not shown).”

Age-matched APPPS1;Cx3cr1 +/- and APPPS1;Cx3cr1 -/- mice demonstrated reduced expression levels of both TNFα (P < 0.001) and CCL2 mRNAs (P < 0.01) relative to APPPS1;Cx3cr1 +/+ mice.

“Notably, age-matched APPPS1; Cx3cr1 ϩ/Ϫ and APPPS1;Cx3cr1 Ϫ/Ϫ mice demonstrated reduced expression levels of both TNF␣ (P Ͻ 0.001) and CCL2 mRNAs (P Ͻ 0.01) relative to APPPS1;Cx3cr1 ϩ/ϩ mice (Figure 5, A and C, respectively).”

Both Cx3cr1+/+ and Cx3cr1−/− microglia accumulated in similar numbers near the fibrillar Aβ1-42 injection sites.

“both Cx3cr1 ϩ/ϩ and Cx3cr1 Ϫ/Ϫ microglia accumulated in similar numbers near the fibrillar A␤ 1-42 injection sites (Figure 7E; P Ͻ 0.05)”

TUNEL and cleaved caspase 3 staining of brain sections from CX3CR1-deficient APPPS1 and R1.40 animals did not reveal any gross alterations in neurodegeneration.

“First, TUNEL and cleaved caspase 3 staining of brain sections from CX3CR1-deficient APPPS1 and R1.40 animals did not reveal any gross alterations in neurodegeneration (data not shown), consistent with previous studies demonstrating no overt neurodegeneration in either the APPPS1 or R1.40 animals.21,36”

The APPPS1;Cx3cr1+/− and APPPS1;Cx3cr1−/− genotypes exhibited a statistically significant difference in microglial accumulation around medium Aβ deposits.

“The APPPS1;Cx3cr1 ϩ/Ϫ and APPPS1; Cx3cr1 Ϫ/Ϫ genotypes exhibited a statistically significant difference in microglial accumulation around medium A␤ deposits (P Ͻ 0.05)”

APPPS1;Cx3cr1+/+ controls exhibited numerous fibrillar Aβ deposits in the cortex and hippocampus.

“Consistent with published reports, APPPS1;Cx3cr1 ϩ/ϩ controls exhibited numerous fibrillar A␤ deposits in the cortex and hippocampus (Figure 1A).”

Quantification revealed that R1.40;Cx3cr1+/− and R1.40;Cx3cr1−/− exhibited markedly fewer cortical Thioflavine S-positive plaques when compared to site-matched brain regions in R1.40;Cx3cr1+/+ mice (P < 0.05).

“Quantification of the total number of Thioflavine S-positive A␤ deposits in the cortex revealed that R1.40;Cx3cr1 ϩ/Ϫ and R1.40; Cx3cr1 Ϫ/Ϫ exhibited markedly fewer cortical Thioflavine S-positive plaques when compared to site-matched brain regions in R1.40;Cx3cr1 ϩ/ϩ mice (P Ͻ 0.05).”

Cx3cr1−/− microglia in Aβ1-42 injected animals exhibited a different morphology, with a more rounded appearance, than either Cx3cr1+/+ Aβ1-42 injected animals or Aβ42-1 injected controls.

“the Cx3cr1 Ϫ/Ϫ microglia in the A␤ 1-42 injected animals exhibited a different morphology, with a more rounded appearance, than either Cx3cr1 ϩ/ϩ A␤ 1-42 injected animals or A␤ 42-1 injected controls”

In APPPS1 mice, one or two copies of Cx3cr1 loss-of-function alleles resulted in a statistically significant reduction in microglia surrounding large (>1000 μm2), medium (>500 and <1000 μm2), and small (<500 μm2) Aβ deposits compared to age-matched APPPS1;Cx3cr1+/+ controls.

“with either one or two copies of Cx3cr1 loss-of-function alleles exhibited a statistically significant reduction in microglia surrounding both large (Ͼ1000 m 2 ; J), medium (Ͼ500 m 2 , Ͻ1000 m 2 ; K), and small (Ͻ500 m 2 ; L) A␤ deposits when compared to age-matched APPPS1;Cx3cr1 ϩ/ϩ controls (*P Ͻ 0.05; **P Ͻ 0.001, respectively).”

No significant differences in steady-state levels of holo-APP were observed in brain extracts from two-month-old APPPS1;Cx3cr1 +/+, APPPS1;Cx3cr1 +/-, and APPPS1;Cx3cr1 -/- mice.

“No significant differences in the steady state levels of holo-APP were observed in the samples from three different genotypes (Figure 2, A [top] and B).”

Additional staining with antibodies for dystrophic neurites, including APP (CT15) and tau phosphorylation (AT8), revealed a reduction in staining in both the APPPS1;Cx3cr1−/− and R1.40;Cx3cr1−/− animals.

“Second, additional staining with antibodies for dystrophic neurites, including APP (antibody CT15) and tau phosphorylation (antibody AT8) revealed a reduction in staining in both the APPPS1;Cx3cr1 Ϫ/Ϫ (See Supplemental Figure S3 at http://ajp.amjpathol.org) and R1.40;Cx3cr1 Ϫ/Ϫ animals (data not shown) that likely reflects the reduction in A␤ deposition observed in these mice.”

CX3CL1 treatment reduces the phagocytic capacity of Cx3cr1+/+ microglia, as measured by microspheres per cell (from ~4 to ~2).

“On average, Cx3cr1 ϩ/ϩ microglia phagocytosed ϳ4 microspheres per cell (Figure 6, A and E) whereas CX3CL1-treated Cx3cr1 ϩ/ϩ microglia phagocytosed only ϳ2 microspheres per cell (Figure 6, B and E; P Ͻ 0.05).”

Aβ deposition was reduced in the hippocampus by 70% and 85% in the APPPS1;Cx3cr1+/− and APPPS1;Cx3cr1−/− animals (P < 0.001), when compared to control mice.

“A␤ deposition was also reduced in the hippocampus by 70% and 85% in the APPPS1;Cx3cr1 ϩ/Ϫ and APPPS1;Cx3cr1 Ϫ/Ϫ animals (P Ͻ 0.001), when compared to control mice (Figure 1E).”

There is a substantial reduction in the amount of Aβ deposition in APPPS1;Cx3cr1 -/- animals.

“In addition to a substantial reduction in the amount of A␤ deposition in the APPPS1;Cx3cr1 Ϫ/Ϫ animals (Figure 1), there was also significantly reduced GFAP immunoreactivity.”

CX3CR1 deficiency did not alter steady-state APP or APP CTF levels in APPPS1 and R1.40 mice.

“the levels of steady-state APP and APP CTFs remained unchanged in CX3CR1 deficient APPPS1 and R1.40 mice (Figure 2 and see Supplemental Figure S4 at http://ajp.amjpathol.org).”

IL1β mRNA levels were increased in a gene dose-dependent manner in CX3CR1-deficient APPPS1 mice.

“while brain levels of IL1␤ mRNA were increased”

• Also stated in: § Microglial Removal of A␤ · plain

  “Furthermore, IL1␤ mRNA levels were significantly increased in a gene dose-dependent manner in the CX3CR1-deficient APPPS1 animals”

CX3CR1 deficiency reduced CCL2 and TNFα mRNA levels in APPPS1 mouse brain to those observed in non-transgenic controls.

“Notably, CX3CR1 deficiency reduced the levels of CCL2 and TNF␣ mRNA to that observed in the non-transgenic controls, while brain levels of IL1␤ mRNA were increased.”

• Also stated in: § Microglial Removal of A␤ · plain

  “In support of this possibility, TNF␣ mRNA levels were selectively down-regulated in CX3CR1 deficient APPPS1 animals”

MCP-1 mRNA levels were reduced in CX3CR1-deficient APPPS1 animals.

“mRNA levels of MCP-1, which promotes microglia-mediated A␤ oligomerization, 54 was reduced in CX3CR1-deficient APPPS1 ani-mals”

Microglial Aβ phagocytosis was enhanced in CX3CR1-deficient APPPS1 animals.

“Finally, we confirmed that microglial A␤ phagocytosis was enhanced in CX3CR1-deficient APPPS1 animals (Figure 7).”

Cx3cr1+/+ and Cx3cr1−/− microglia accumulated in similar numbers around the injection sites.

“However, Cx3cr1 ϩ/ϩ and Cx3cr1 Ϫ/Ϫ microglia accumulated in similar numbers around the injection sites, suggesting that microglial migration was not impaired by CX3CR1 deficiency.”

No enhancement of phagocytosis was observed in Cx3cr1−/− compared to untreated Cx3cr1+/+ microglia.

“We did not observe an enhancement of phagocytosis in Cx3cr1 Ϫ/Ϫ compared to untreated Cx3cr1 ϩ/ϩ microglia.”

CX3CR1-deficient APPPS1 animals show a substantial reduction in astrocytosis.

“we also observed a substantial reduction in astrocytosis in CX3CR1-deficient APPPS1 animals (see Supplemental Figure S5 at http://ajp.amjpathol.org)”

The phagocytic capacity of Cx3cr1−/− primary microglia did not change with CX3CL1 treatment.

“Importantly, the phagocytic capacity of Cx3cr1 Ϫ/Ϫ primary microglia did not change with CX3CL1 treatment, indicating that the reduced microglial phagocytosis observed is mediated by CX3CR1 signaling.”

CX3CL1 treatment of primary microglia led to a 50% reduction in the number of phagocytosed microspheres in wild-type Cx3cr1+/+ primary microglia.

“CX3CL1 treatment of primary microglia led to a 50% reduction in the number of phagocytosed microspheres in wild-type Cx3cr1 ϩ/ϩ primary microglia.”

APPPS1 mice with either one or two copies of Cx3cr1 loss-of-function alleles exhibited a statistically significant reduction in microglia surrounding large, medium, and small A␤ deposits compared to age-matched APPPS1;Cx3cr1+/+ controls.

“APPPS1 mice with either one or two copies of Cx3cr1 loss-of-function alleles exhibited a statistically significant reduction in microglia surrounding both large (Ͼ1000 m 2 ; J), medium (Ͼ500 m 2 , Ͻ1000 m 2 ; K), and small (Ͻ500 m 2 ; L) A␤ deposits when compared to age-matched APPPS1;Cx3cr1 ϩ/ϩ controls (*P Ͻ 0.05; **P Ͻ 0.001, respectively).”

APPPS1;Cx3cr1+/+ controls exhibit extensive accumulation of Iba1-positive microglia around senile plaques.

“APPPS1;Cx3cr1 ϩ/ϩ controls exhibit extensive accumulation of Iba1-positive microglia around senile plaques (A-C).”

The reduction in A␤ deposition in CX3CR1-deficient APPPS1 and R1.40 mice led to a reduction in the formation of dystrophic neurites.

“the reduction in A␤ deposition in the CX3CR1deficient APPPS1 and R1.40 mice also led to a reduction in the formation of dystrophic neurites”

There is enhanced A␤ phagocytosis in CX3CR1-deficient mice.

“Enhanced A␤ phagocytosis in CX3CR1-deficient mice.”

CX3CR1 genotype has no effect on APP processing.

“Lack of effect of CX3CR1 genotype on APP processing.”

APPPS1;Cx3cr1+/+ animals show abundant CD68 and CD45 immunoreactivity, especially in the immediate vicinity of Congo Red-positive A␤ deposits.

“APPPS1; Cx3cr1 ϩ/ϩ animals show abundant CD68 (C) and CD45 immunoreactivity (D), especially in the immediate vicinity of Congo Red-positive A␤ deposits (insets).”

Microglial proliferation and cell death were unaltered in APPPS1 mice deficient for CX3CR1 as assessed via bromodeoxyuridine incorporation and cleaved caspase 3 labeling.

“However, our preliminary analysis suggests that microglial proliferation and cell death were unaltered in APPPS1 mice deficient for CX3CR1 as assessed via bromodeoxyuridine incorporation and cleaved caspase 3 labeling, respectively (data not shown).”

Age-matched APPPS1;Cx3cr1+/- and APPPS1;Cx3cr1-/- animals exhibited a gene dose-dependent reduction in fibrillar A␤ deposition throughout the cortex and hippocampus.

“age-matched APPPS1;Cx3cr1 ϩ/Ϫ (B) and APPPS1;Cx3cr1 Ϫ/Ϫ (C) animals exhibited a gene dose-dependent reduction in fibrillar A␤ deposition throughout the cortex and hippocampus.”

Steady-state levels of A␤ in predepositing CX3CR1-deficient APPPS1 and R1.40 animals remained unchanged.

“the steady-state levels of A␤ in predepositing CX3CR1-deficient APPPS1 and R1.40 animals remained unchanged (data not shown)”

In CX3CR1-deficient APPPS1 animals, there was a significant reduction in Aβ deposition.

“despite the reduction in the number of microglia around the A␤ deposits in CX3CR1-deficient APPPS1 animals, there was a significant reduction in A␤ deposition”

CX3CL1-CX3CR1 signaling inhibits microglial phagocytosis.

“CX3CL1-CX3CR1 signaling inhibits microglial phagocytosis.”

ELISAs revealed reduced A␤42 levels in CX3CR1-deficient APPPS1 animals.

“ELISAs performed on APPPS1;Cx3cr1 ϩ/ϩ (n ϭ 6) and APPPS1; Cx3cr1 Ϫ/Ϫ brain homogenates (n ϭ 5) revealed reduced A␤42 levels in CX3CR1-deficient animals (G; *P Ͻ 0.05).”

CD68- and CD45-positive microglia are mostly clustered around Congo Red-positive A␤ deposits.

“higher magnification revealed that CD68-and CD45-positive microglia are mostly clustered around Congo Red-positive A␤ deposits (insets).”

CD45 immunoreactivity is relatively unchanged in APPPS1;Cx3cr1+/- and APPPS1;Cx3cr1-/- animals compared with APPPS1;Cx3cr1+/+ controls.

“CD45 immunoreactivity is relatively unchanged in APPPS1;Cx3cr1 ϩ/Ϫ and APPPS1;Cx3cr1 Ϫ/Ϫ animals compared with APPPS1;Cx3cr1 ϩ/ϩ controls (F and H, respectively).”

Age-matched APPPS1;Cx3cr1+/- and APPPS1;Cx3cr1-/- animals exhibit gene dose-dependent reduction in the number of Iba1-positive microglia surrounding senile plaques.

“age-matched APPPS1;Cx3cr1 ϩ/Ϫ (D-F) and APPPS1;Cx3cr1 Ϫ/Ϫ animals (G-I) exhibit gene dose-dependent reduction in the number of Iba1-positive microglia surrounding the senile plaques.”

There was a significant difference in A␤ deposition in the cortex between APPPS1;Cx3cr1+/- and APPPS1;Cx3cr1-/- genotypes.

“there was also a significant difference in A␤ deposition in the cortex between APPPS1;Cx3cr1 ϩ/Ϫ and APPPS1; Cx3cr1 Ϫ/Ϫ genotypes (P Ͻ 0.05).”

There is a gene dose-dependent reduction in fibrillar A␤ deposition in the APPPS1 mouse model of AD with CX3CR1 deficiency.

“Gene dose-dependent reduction in fibrillar A␤ deposition in the APPPS1 mouse model of AD with CX3CR1 deficiency.”

There is reduced plaque-associated microglia in the APPPS1 mouse model of AD with CX3CR1 deficiency.

“Reduced plaque-associated microglia in the APPPS1 mouse model of AD with CX3CR1 deficiency.”

Control A␤ 42-1 peptide injection triggered significantly reduced microglial reaction compared to fibrillar A␤ 1-42 injection.

“control A␤ 42-1 peptide injection triggered significantly reduced microglial reaction compared to fibrillar A␤ 1-42 injection (C and E; *P Ͻ 0.05).”

APPPS1;Cx3cr1-/- brains exhibited lower steady-state A␤ levels compared with age-matched APPPS1;Cx3cr1+/+ controls.

“APPPS1;Cx3cr1 Ϫ/Ϫ brains exhibited lower steady-state A␤ levels when compared with age-matched APPPS1;Cx3cr1 ϩ/ϩ controls (F).”

Age-matched APPPS1;Cx3cr1+/- and APPPS1;Cx3cr1-/- animals exhibit gene dose-dependent reduction in CD68 immunoreactivity compared to APPPS1;Cx3cr1+/+ controls.

“age-matched APPPS1;Cx3cr1 ϩ/Ϫ and APPPS1;Cx3cr1 Ϫ/Ϫ animals exhibit gene dose-dependent reduction in CD68 immunoreactivity compared to APPPS1;Cx3cr1 ϩ/ϩ controls (E and G, respectively)”

CX3CR1 deficiency reduces extracellular A␤ deposition in two different mouse models of AD (APPPS1 and R1.40).

“our studies demonstrate that CX3CR1 deficiency reduces extracellular A␤ deposition in two different mouse models of AD”

Cx3cr1-/- microglia exhibited enhanced A␤ phagocytosis when compared with control Cx3cr1+/+ microglia.

“Cx3cr1 Ϫ/Ϫ microglia exhibited enhanced A␤ phagocytosis when compared with control Cx3cr1 ϩ/ϩ microglia (*P Ͻ 0.01; D)”

In CX3CR1-deficient animals, there was a gene dose-dependent reduction in the number of microglia associated with Aβ deposition.

“In CX3CR1-deficient animals, there was a gene dose-dependent reduction in number of microglia associated with A␤ deposition.”

• Also stated in: § Microglial Proliferation/Migration · plain

  “despite the reduction in the number of microglia around the A␤ deposits in CX3CR1-deficient APPPS1 animals”

Quantification of Thioflavine S staining revealed a statistically significant decrease in A␤ deposition in APPPS1 mice with either one or two copies of Cx3cr1 loss-of-function alleles compared with age-matched APPPS1;Cx3cr1+/+ controls.

“Quantification of Thioflavine S staining in the cortex (D) and hippocampus (E) across all animals revealed a statistically significant decrease in APPPS1 mice with either one or two copies of Cx3cr1 loss of function alleles when compared with age-matched APPPS1;Cx3cr1 ϩ/ϩ controls (*P Ͻ 0.001).”

There is altered expression of microglial markers in the APPPS1 mouse model of AD with CX3CR1 deficiency.

“Altered expression of microglial markers in the APPPS1 mouse model of AD with CX3CR1 deficiency.”

The number of microglia around the injection site did not significantly differ between genotypes.

“the number of microglia around the injection site did not significantly differ between genotypes (E).”

CX3CL1-treated Cx3cr1+/+ microglia exhibited reduced numbers of phagocytosed microspheres per cell when compared with untreated Cx3cr1+/+ and CX3CL1-treated Cx3cr1-/- microglia.

“CX3CL1-treated Cx3cr1 ϩ/ϩ microglia exhibited reduced numbers of phagocytosed microspheres per cell when compared with untreated Cx3cr1 ϩ/ϩ and CX3CL1-treated Cx3cr1 Ϫ/Ϫ microglia (E; *P Ͻ 0.05).”

There is altered expression of inflammatory markers in CX3CR1-deficient APPPS1 mouse model of AD.

“Altered expression of inflammatory markers in CX3CR1-deficient APPPS1 mouse model of AD.”

APPPS1;Cx3cr1+/- and APPPS1;Cx3cr1-/- genotypes exhibited a statistically significant difference in microglial accumulation around medium A␤ deposits.

“The APPPS1;Cx3cr1 ϩ/Ϫ and APPPS1; Cx3cr1 Ϫ/Ϫ genotypes exhibited a statistically significant difference in microglial accumulation around medium A␤ deposits (P Ͻ 0.05), suggesting a gene dose-dependent effect.”

No obvious alterations in neurodegeneration were observed in APPPS1;Cx3cr1-/- and R1.40;Cx3cr1-/- animals.

“we did not observe any obvious alterations in neurodegeneration in the APPPS1;Cx3cr1 Ϫ/Ϫ and R1.40;Cx3cr1 Ϫ/Ϫ animals”

Loss of neuron-microglial fractalkine signaling leads to reduced beta-amyloid deposition in mouse models of AD.

“Taken together, these results demonstrate that loss of neuron-microglial fractalkine signaling leads to reduced ␤-amyloid deposition in mouse models of AD that is potentially mediated by altered activation and phagocytic capability of CX3CR1-deficient microglia.”

The reduction in beta-amyloid deposition caused by loss of neuron-microglial fractalkine signaling is potentially mediated by altered activation and phagocytic capability of CX3CR1-deficient microglia.

“Taken together, these results demonstrate that loss of neuron-microglial fractalkine signaling leads to reduced ␤-amyloid deposition in mouse models of AD that is potentially mediated by altered activation and phagocytic capability of CX3CR1-deficient microglia.”

In the brain, CX3CR1 is exclusively expressed by microglia.

“While CX3CR1 is expressed by many leukocyte cell types in the periphery, 15 in the brain CX3CR1 is exclusively expressed by microglia 13 whereas CX3CL1 is highly expressed within neurons.14”

• cites [13] Cardona, 2006, Control of microglial neurotoxicity by the fractalkine receptor, Nat Neurosci sourceNot checked — no full text of the source

The correlation of reduced beta-amyloid pathology with reduced TNFα and CCL2 mRNAs and elevated IL1β mRNA levels suggests an altered neuroinflammatory milieu.

“The reduced ␤-amyloid pathology correlated with reduced levels of TNF␣ and CCL2 mRNAs , but elevated IL1␤ mRNA levels , suggesting an altered neuroinflammatory milieu.”

Loss of signaling between neurons and microglia via deletion of the microglial receptor CX3CR1 worsens phenotypes in various models of neurodegenerative diseases.

“Loss of signaling between neurons and microglia via deletion of the microglial receptor, CX3CR1 , worsens phenotypes in various models of neurodegenerative diseases.”

• Also stated in: § Abstract · plain

  “disruption of CX3CL1-CX3CR1 signaling promotes neurodegeneration in mouse models of Parkinson's disease, amyotrophic lateral sclerosis, as well as in neuroinflammation induced by systemic administration of lipopolysaccharide.13”

• cites [13] Cardona, 2006, Control of microglial neurotoxicity by the fractalkine receptor, Nat Neurosci sourceNot checked — no full text of the source

CX3CR1 deficiency leads to enhanced Aβ clearance.

“Finally, we demonstrate that CX3CL1-CX3CR1 signaling reduces microglial phagocytic capabilities and that blocking CX3CL1-CX3CR1 signaling increases uptake of A␤, suggesting that CX3CR1 deficiency leads to enhanced A␤ clearance.”

CX3CR1 deficiency leads to altered expression of inflammatory cytokines/chemokines.

“CX3CR1 Deficiency Leads to Altered Expression of Inflammatory Cytokines/Chemokines”

CX3CR1 deficiency results in a gene dose-dependent reduction in Aβ deposition in mouse models of AD, regardless of the kinetics of deposition.

“Taken together, these data indicate that CX3CR1 deficiency results in a gene dose-dependent reduction in A␤ deposition in mouse models of AD, regardless of the kinetics of deposition.”

The R1.40 transgenic mouse line develops gradual AD-like Aβ pathology, exhibiting both diffuse and fibrillar Aβ deposition beginning at 14-16 months of age in the cortex and 18-20 months of age in the hippocampus.

“The R1.40 transgenic mouse line develops gradual AD-like A␤ pathology, exhibiting both diffuse and fibrillar A␤ deposition beginning at 14 -16 months of age in the cortex and 18 -20 months of age in the hippocampus.36”

• cites [36] Kulnane, 2001, Neuropathological characterization of mutant amyloid precursor protein yeast artificial chromosome transgenic mice, Neurobiol Dis sourceNot checked — no full text of the source

The reduction in dystrophic neurite staining appears to reflect the reduction in Aβ deposition observed in these mice.

“that appears to reflect the reduction in A␤ deposition observed in these mice.”

The differences in Aβ deposition observed in this study are not due to altered expression or processing of APP by neurons.

“Taken together, these results indicate that the differences in A␤ deposition observed in this study are not due to altered expression or processing of APP by neurons and instead suggest involvement of brain microglia, where CX3CR1 is exclusively expressed.”

The R1.40 model exhibits gradual deposition of primarily Aβ40.

“we mated Cx3cr1 knockout mice to two different mouse models of AD that exhibit either rapid deposition of primarily A␤42 (the APPPS1 model) 21 or gradual deposition of primarily A␤40 (the R1.40 model).[22][23][24]”

• cites [22] Lamb, 1993, Introduction and expression of the 400 kilobase amyloid precursor protein gene in transgenic mice [corrected], Nat Genet sourceNot checked — no full text of the source
• cites [23] Lamb, 1997, Altered metabolism of familial Alzheimer's disease-linked amyloid precursor protein variants in yeast artificial chromosome transgenic mice, Hum Mol Genet sourceNot checked — no full text of the source
• cites [24] Lehman, 2003, Alterations in β-amyloid production and deposition in brain regions of two transgenic models, Neurobiol Aging sourceNot checked — no full text of the source

Alterations in CX3CL1-CX3CR1 signaling can lead to altered phagocytic capabilities of microglia.

“These results suggest that alterations in CX3CL1-CX3CR1 signaling can lead to altered phagocytic capabilities of microglia”

The differences in Aβ deposition observed in this study suggest involvement of brain microglia.

“Taken together, these results indicate that the differences in A␤ deposition observed in this study are not due to altered expression or processing of APP by neurons and instead suggest involvement of brain microglia, where CX3CR1 is exclusively expressed.”

• AssumptionRests on an assumption: CX3CR1 is exclusively expressed in brain microglia.

There are alterations in the expression of the chemokines/cytokines CCL2, IL1β, and TNFα in the brains of the APPPS1 mouse model of AD with CX3CR1 deficiency.

“These results suggest that there are alterations in the expression of the chemokines/cytokines CCL2, IL1␤, and TNF␣ in the brains of the APPPS1 mouse model of AD with CX3CR1 deficiency.”

CX3CL1-CX3CR1 signaling inhibits microglial phagocytic capacity.

“Our in vitro data suggested that CX3CL1-CX3CR1 signaling inhibits microglial phagocytic capacity.”

The microglial responses were specific to fibrillar Aβ1-42.

“indicating that the microglial responses were specific to fibrillar A␤ 1-42 .”

In addition to alterations in microglial activation, there is also reduced astrocytosis in CX3CR1-deficient APPPS1 mice.

“Taken together, these results indicate that, in addition to alterations in microglial activation, there is also reduced astrocytosis in CX3CR1deficient APPPS1 mice, although both of these alterations could be due to the reduced A␤ deposition observed.”

The altered morphology of Cx3cr1−/− microglia is consistent with an altered phagocytic capacity of these cells.

“which is consistent with an altered phagocytic capacity of these cells.”

The reduction in dystrophic neurite staining in APPPS1;Cx3cr1−/− and R1.40;Cx3cr1−/− animals likely reflects the reduction in Aβ deposition observed in these mice.

“Second, additional staining with antibodies for dystrophic neurites, including APP (antibody CT15) and tau phosphorylation (antibody AT8) revealed a reduction in staining in both the APPPS1;Cx3cr1 Ϫ/Ϫ (See Supplemental Figure S3 at http://ajp.amjpathol.org) and R1.40;Cx3cr1 Ϫ/Ϫ animals (data not shown) that likely reflects the reduction in A␤ deposition observed in these mice.”

CX3CL1-mediated inhibition of microglial phagocytic capacity requires CX3CR1 signaling.

“indicating that CX3CL1-mediated inhibition of microglial phagocytic capacity required CX3CR1 signaling.”

CX3CR1 deficiency results in a gene dose-dependent reduction in Aβ deposition in the APPPS1 mouse model of AD.

“Taken together, these data indicate that CX3CR1 deficiency results in a gene dosedependent reduction in A␤ deposition in the APPPS1 mouse model of AD.”

Blocking CX3CL1-CX3CR1 signaling leads to enhanced uptake of fibrillar Aβ within microglia.

“These data demonstrate that blocking CX3CL1-CX3CR1 signaling leads to enhanced uptake of fibrillar A␤ within microglia”

Aβ42-1-injected Cx3cr1+/+ and Cx3cr1−/− controls showed no significant differences in microglial counts.

“Furthermore, A␤ 42-1 -injected Cx3cr1 ϩ/ϩ and Cx3cr1 Ϫ/Ϫ controls showed no significant differences in microglial counts (data not shown).”

There was no significant alteration in the ability of microglia to migrate to the Aβ injection site.

“suggesting that there was no significant alteration in the ability of microglia to migrate to the A␤ injection site.”

CX3CR1 deficiency alters the microglial activation program in reaction to dense-core Aβ deposits.

“These data suggest that CX3CR1 deficiency alters the microglial activation program in reaction to dense-core A␤ deposits.”

CX3CR1 deficiency reduces the number of microglia around Aβ deposits in a gene dose-dependent manner in the APPPS1 mouse model of Alzheimer's disease.

“In summary, these results indicate that CX3CR1 deficiency reduces the number of microglia around A␤ deposits in a gene dose-dependent manner in the APPPS1 mouse model of AD.”

There is an altered neuroinflammatory milieu within CX3CR1-deficient APPPS1 animals.

“suggesting that there is an altered neuroinflammatory milieu within these animals.”

CX3CL1-CX3CR1 signaling regulates the phagocytic capacity of microglia.

“In addition, in vitro phagocytosis assay revealed that CX3CL1-CX3CR1 signaling regulates the phagocytic capacity of microglia (Figure 6).”

• AssumptionRests on an assumption: In vitro phagocytosis of microspheres by primary microglia reflects microglial phagocytosis of Aβ.

Microglial migration was not impaired by CX3CR1 deficiency.

“However, Cx3cr1 ϩ/ϩ and Cx3cr1 Ϫ/Ϫ microglia accumulated in similar numbers around the injection sites, suggesting that microglial migration was not impaired by CX3CR1 deficiency.”

• AssumptionRests on an assumption: Similar numbers of Cx3cr1+/+ and Cx3cr1−/− microglia around injection sites indicate that microglial migration is not impaired.

Taken together, these results support a role of CX3CL1-CX3CR1 signaling to microglia in modulating neurodegenerative disease phenotypes.

“Taken together these results support a role of CX3CL1-CX3CR1 signaling to microglia in modulating neurodegenerative disease phenotypes.”

CX3CR1 deficiency leads to a cytokine environment that harnesses the beneficial effects of microglial activation in response to Aβ.

“These observations suggest that CX3CR1 deficiency leads to a cytokine environment that harnesses the beneficial effects of microglial activation in response to A␤.”

• AssumptionRests on an assumption: mRNA levels of cytokines/chemokines reflect their protein levels and functional activity in the brain.

Enhanced Aβ phagocytosis by Cx3cr1−/− microglia is the more proximal and significant contributor to the reduced Aβ deposition observed in CX3CR1-deficient animals.

“the current studies point to enhanced A␤ phagocytosis by Cx3cr1 Ϫ/Ϫ microglia as the more proximal and significant contributor.”

The reduced microglial phagocytosis observed is mediated by CX3CR1 signaling.

“Importantly, the phagocytic capacity of Cx3cr1 Ϫ/Ϫ primary microglia did not change with CX3CL1 treatment, indicating that the reduced microglial phagocytosis observed is mediated by CX3CR1 signaling.”

The reduced astrogliosis in CX3CR1-deficient animals is a secondary response to primary alterations in microglia.

“because CX3CR1 is exclusively expressed by microglia in the murine CNS, 13,15 the reduced astrogliosis is by definition a secondary response to primary alterations in microglia.”

The effects of CX3CR1 deficiency are autonomous to microglia.

“the effects of CX3CR1 deficiency are autonomous to microglia, 13”

• cites [13] Cardona, 2006, Control of microglial neurotoxicity by the fractalkine receptor, Nat Neurosci sourceNot checked — no full text of the source

The reduction in extracellular A␤ deposition by CX3CR1 deficiency is via a mechanism that likely involves enhanced phagocytic clearance of extracellular A␤ fibrils.

“via a mechanism that likely involves enhanced phagocytic clearance of extracellular A␤ fibrils”

The apparent discrepancy between the results of the two studies may be reflective of the cellular location of the A␤ aggregates.

“Thus, the apparent discrepancy in the results of the two studies may be reflective of the cellular location of the A␤ aggregates”

• AssumptionRests on an assumption: In 3xTg mice at 4-6 months, there is no extracellular A␤ deposition but abundant intracellular A␤ immunoreactivity.

CX3CL1-CX3CR1 signaling inhibits microglial phagocytosis, including fibrillar Aβ.

“In support of this hypothesis, our in vitro and in vivo data demonstrate that CX3CL1-CX3CR1 signaling inhibits microglial phagocytosis, including fibrillar A␤.”

• AssumptionRests on an assumption: In vitro phagocytosis of microspheres by primary microglia reflects microglial phagocytosis of Aβ.

The reduction in Aβ deposition in CX3CR1-deficient APPPS1 animals is consistent with an enhanced capacity of microglia to remove Aβ.

“consistent with an enhanced capacity of microglia to remove A␤”

The effect of CX3CR1 deficiency appears to depend on the nature of the CNS insult.

“Indeed, the effect of CX3CR1 deficiency appears to depend on the nature of the CNS insult as recent studies demonstrated that Cx3cr1 knockout mice exhibit smaller infarcts after transient middle cerebral artery occlusion.19,20”

• cites [19] Denes, 2008, Role of CX3CR1 (fractalkine receptor) in brain damage and inflammation induced by focal cerebral ischemia in mouse, J Cereb Blood Flow Metab sourceNot checked — no full text of the source
• cites [20] Soriano, 2002, Mice deficient in fractalkine are less susceptible to cerebral ischemia-reperfusion injury, J Neuroimmunol sourceNot checked — no full text of the source

Similar types of studies did not detect robust neuronal loss in aged 3xTg animals.

“similar types of studies also did not detect robust neuronal loss in aged 3xTg animals, 62”

• cites [62] Lopes, 2009, Activation of cell cycle proteins in transgenic mice in response to neuronal loss but not amyloid-beta and tau pathology, J Alzheimers Dis sourceNot checked — no full text of the source

CX3CL1 is found in high concentrations in both normal and diseased CNS.

“Notably, CX3CL1 is found in high concentrations in both normal and diseased CNS, 55”

• cites [55] Bajetto, 2001, Chemokines and their receptors in the central nervous system, Front Neuroendocrinol sourceNot checked — no full text of the source

Multiple redundant pathways regulate Aβ clearance and deposition in different disease states and experimental conditions.

“Taken together, these data suggest multiple redundant pathways that regulate A␤ clearance and deposition in different disease states and experimental conditions.”

CX3CL1-CX3CR1 signaling establishes a permissive environment for Aβ deposition in AD brains and transgenic mouse models by downregulating the microglial phagocytic response toward Aβ.

“our results suggest that CX3CL1-CX3CR1 signaling establishes a permissive environment for A␤ deposition in the AD brains and transgenic mouse models by downregulating the microglial phagocytic response toward A␤.”