PPARγ/RXRα-Induced and CD36-Mediated Microglial Amyloid-β Phagocytosis Results in Cognitive Improvement in Amyloid Precursor Protein/Presenilin 1 Mice

As AD progresses, microglia acquire a chronically activated phenotype through unremitting stimulation by cytokines and Aβ itself, which compromises their clearance function.

“However, as AD progresses, microglia acquire a chronically activated phenotype through unremitting stimulation by cytokines and A␤ itself, which compromises their clearance function (Bamberger et al., 2003;Hickman et al., 2008;Heneka et al., 2010a) .”

• cites Bamberger et al., 2003 sourcePartly supported

  The Introduction says plaque-associated microglia acquire a reactive proinflammatory phenotype after contact with fibrillar Aβ and links Aβ-stimulated signaling to sustained inflammation. The section on Aβ-stimulated signaling also says IL-1β augments the inflammatory response. Neither passage establishes that microglial activation progressively compromises their clearance function. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites Hickman et al., 2008 sourcePartly supported

  The Discussion reports that microglia in aging AD-model mice continue producing pro-inflammatory cytokines while losing Aβ-clearing capacity; it also links Aβ-induced inflammation to reduced expression of clearance receptors. The paper does not establish that unremitting stimulation by both cytokines and Aβ causes a chronically activated phenotype, and it frames some cytokine effects as possible mechanisms rather than demonstrated causes. Page numbers were not supplied for these passages. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites Heneka et al., 2010aNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Microglial expression of receptors that promote clearance and phagocytosis of Aβ, such as CD36, TLR4, and TLR6, are mandatory for restricting amyloid plaque formation.

“Microglial expression of receptors that promote the clearance and phagocytosis of A␤, such as CD36, TLR4, and TLR6 (Lee and Landreth, 2010), are mandatory for restricting amyloid plaque formation.”

• cites Lee and Landreth, 2010 sourcePartly supported

  The Fibrillar Aβ clearance section identifies CD36, TLR4 and TLR6 in the microglial receptor complex involved in Aβ uptake and phagocytosis. It does not establish that expression of all three is mandatory for restricting plaques: it calls the effects of TLR inactivation on Aβ deposition confusing and reports differing outcomes across mouse models. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Restricting microglial Aβ uptake leads to increased cerebral Aβ levels.

“Evidence for a important role of microglial phagocytosis comes from studies that show that restricting microglial A␤ uptake leads to increased cerebral A␤ levels (El Khoury et al., 2007;Lee and Landreth, 2010).”

• cites El Khoury et al., 2007 sourceNot checked — no full text of the source
• cites Lee and Landreth, 2010 sourcePartly supported

  The Fibrillar Aβ clearance section reports that mice with inactive TLR4 had greater cortical and hippocampal Aβ burden, and that their microglia were less efficient at taking up Aβ. It describes reduced microglial clearance as the study authors’ proposed explanation, not an established causal finding, and notes differing results for TLR2-deficient mice. The claim states the causal relationship more definitively than this review does. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

A subset of NSAIDs directly regulates gene expression through binding and activating PPARγ.

“Of note, a subset of NSAIDs directly regulates gene expression through binding and activating the peroxisome proliferator-activated receptor-␥ (PPAR␥) (Lehmann et al., 1997) that belongs to the nuclear receptor superfamily and forms a heterodimer with the retinoid X receptor (RXR) before binding to PPAR response elements in the promoter region of target genes.”

• cites Lehmann et al., 1997 sourceSupported

Impaired Aβ clearance is likely to account for the majority of sporadic AD cases.

“Because impaired A␤ clearance is likely to account for the majority of sporadic AD cases (Mawuenyega et al., 2010), identification of factors the regulate microglial A␤ uptake seems to be of upmost importance.”

• cites Mawuenyega et al., 2010 sourcePartly supported

  The paper reports slower Aβ clearance in patients with AD than in controls and says the common late-onset form of AD may involve impaired clearance. It does not establish that impaired clearance accounts for a majority of sporadic cases; the study also cautions that its association does not prove causality (Abstract and study limitations). (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Microglia are critical for Aβ phagocytosis.

“Because microglia are critical for A␤ phagocytosis, the microglial presence at the A␤ plaque can be interpreted as an attempt to clear this pathological deposit.”

• cites El Khoury et al., 2007 sourceNot checked — no full text of the source
• cites Lee and Landreth, 2010 sourceSupported

Persisting neuroinflammation and sustained exposure to proinflammatory stimuli compromises microglial clearance functions as AD progresses.

“However, persisting neuroinflammation and sustained exposure to proinflammatory stimuli compromises microglial clearance functions as AD progresses (Lee and Landreth, 2010).”

• cites Lee and Landreth, 2010 sourceSupported

PPARγ heterodimerizes with RXRs.

“PPAR␥ heterodimerizes with RXRs (Tugwood et al., 1992), and, during ligand activation, the PPAR/RXR heterodimer recruits coactivators and binds to sequence-specific PPAR response elements present in the promoter region of several target genes.”

• cites Tugwood et al., 1992 sourceCould not verify

  The Introduction and Discussion describe PPAR binding a response element in the rat ACO promoter. The Discussion mentions RXR only in comparing response-element sequences; it does not say that PPARγ heterodimerizes with RXRs. Searches for RXR heterodimerization and PPARγ found no passage addressing that claim. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

DSP-8658 treatment decreased cortical SDS-soluble Aβ1-42 levels by 32% in APP/PS1 mice after 3 months.

“Determination of cortex A␤ levels revealed 32 and 30% decrease of both SDS-soluble A␤ 1-42 and A␤ 1-40 levels, respectively, in response to the DSP-8658 treatment (Fig. 7A).”

DSP-8658-treated APP/PS1 mice show improved spatial memory performance.

“Furthermore, DSP-8658-treated mice showed improved spatial memory performance.”

DSP-8658 treatment reduces cortical and hippocampal Aβ levels in APP/PS1 mice.

“Evaluation of DSP-8658 in the amyloid precursor protein/presenilin 1 mouse model confirmed an increased microglial A␤ phagocytosis in vivo, which subsequently resulted in a reduction of cortical and hippocampal A␤ levels.”

Combination of DSP-8658 and pioglitazone with RXR agonists (retinoic acid and bexarotene) showed additive enhancement of Aβ uptake.

“Likewise, the combination of DSP-8658 and pioglitazone with RXR agonists (retinoic acid and bexarotene) showed an additive enhancement of A␤ uptake in the present study.”

DSP-8658-treated mice revealed a 30% increase in Aβ/CD11b colocalization in microglia compared with nontreated APP/PS1 mice.

“Moreover, these data were further supported by confocal microscopy showing that only a minority of microglia contained A␤ in nontreated APP/PS1 mice, while DSP-8658-treated mice revealed a 30% increase in colocalization.”

Two structurally different RXR agonists, bexarotene and retinoic acid, increase microglial Aβ phagocytosis.

“Here we show that two structurally different RXR agonists, bexarotene and retinoic acid, increase microglial A␤ phagocytosis.”

DSP-8658 treatment increases microglial Aβ phagocytosis in vivo in APP/PS1 mice.

“Interestingly, DSP-8658 treatment increased the percentage of phagocytosing cells in APP/PS1 mice compared with vehicle-treated mice (Fig. 6B).”

• Also stated in: § Abstract · plain

  “Evaluation of DSP-8658 in the amyloid precursor protein/presenilin 1 mouse model confirmed an increased microglial A␤ phagocytosis in vivo, which subsequently resulted in a reduction of cortical and hippocampal A␤ levels.”

DSP-8658 and pioglitazone enhanced CD36 mRNA and protein levels in primary microglia.

“DSP-8658 and pioglitazone enhanced CD36 mRNA and protein levels in primary microglia.”

RXRα, not RXRβ, is the functionally relevant isoform for Aβ phagocytosis in primary microglia.

“These results indicated that RXR␣ represents the functionally more relevant isoform for A␤ phagocytosis in primary microglia.”

• Also stated in: § Additive effects of combined activation of PPAR␥ and RXR in primary microglia · plain

  “In contrast, suppression of RXR␣ by siRNA inhibited the bexarotene-induced increase of A␤ uptake (Fig. 5 B, D).”

Aβ deposits are closely surrounded by CD11b+ microglia in 6-month-old APP/PS1 mice.

“A␤ deposits were closely surrounded by CD11b ϩ microglia (Fig. 6D).”

The number of CD11b+ microglia increases with Aβ deposition in APP/PS1 mice.

“Although the number of CD11b ϩ microglia increased with the deposition, confocal microscopy showed that only a minority of microglial cells actually contained A␤ in nontreated APP/PS1 mice (Fig. 6D-F ).”

In nontreated APP/PS1 mice, only a minority of microglial cells actually contain Aβ.

“Although the number of CD11b ϩ microglia increased with the deposition, confocal microscopy showed that only a minority of microglial cells actually contained A␤ in nontreated APP/PS1 mice (Fig. 6D-F ).”

PPARγ-mediated Aβ uptake requires de novo mRNA/protein synthesis.

“Interestingly, the increasing effect of DSP-8658 and pioglitazone was abolished once either actinomycin D or cycloheximide were coincubated, suggesting that de novo mRNA/protein synthesis is required (Fig. 2 A, B).”

DSP-8658 treatment increases CD36 expression in phagocytosing microglia in APP/PS1 mice.

“Although non-phagocytosing microglia did not reveal any difference in CD36 expression, DSP-8658 treatment increased CD36 expression in phagocytosing cells (Fig. 6C).”

DSP-8658 and pioglitazone upregulate CD36 mRNA and protein levels in primary microglia in a concentration-dependent manner.

“CD36 mRNA was upregulated in response to DSP-8658 or pioglitazone, followed by an increase of the respective protein levels in a concentration-dependent manner (Fig. 2C,D).”

RXRα represents the functionally important RXR in primary microglia.

“Using siRNA-based knockdown analysis for RXR␣ or RXR␤, we revealed that RXR␣ represents the functionally important RXR in primary microglia.”

RIPA-soluble cortical Aβ1-40 levels were slightly reduced by DSP-8658 treatment in APP/PS1 mice.

“RIPA-soluble cortical A␤ 1-40 levels were slightly reduced by the same treatment (Fig. 7 B, D).”

DSP-8658 and pioglitazone enhance Aβ uptake by rat primary microglia in a concentration-dependent manner.

“Both DSP-8658 and pioglitazone enhanced the uptake of A␤ in rat primary microglia in a concentration-dependent manner (Fig. 1 A, B).”

• Also stated in: § Effects of PPAR␥ agonist on A␤ phagocytosis in rat primary microglia · hedged

  “Therefore, these results indicate that DSP-8658 and pioglitazone induce the phagocytic uptake of A␤ by microglia.”

Short-term DSP-8658 treatment for 3 days enhances CD36 expression in vivo.

“In addition, short-term DSP-8658 treatment for 3 d also enhanced CD36 expression in vivo (Fig. 6G).”

The decrease in distance traveled and escape latency was more pronounced in DSP-8658-treated APP/PS1 mice than in vehicle-treated mice.

“This decrease in distance and escape latency was more pronounced in DSP-8658-treated APP/PS1 mice than observed in vehicle-treated mice (Fig. 8).”

Methoxy-X04-positive CD11b+/CD45+ cells are detected in vehicle-treated APP/PS1 mice but almost absent in wild-type mice.

“Methoxy-X04-positive CD11b ϩ /CD45 ϩ cells were detected in vehicle-treated APP/PS1 but almost absent in wild-type mice (Fig. 6A).”

CD36 mediates the PPARγ agonist-induced increase in microglial Aβ phagocytosis.

“This PPAR␥-stimulated increase of A␤ phagocytosis was mediated by the upregulation of scavenger receptor CD36 expression.”

• Also stated in: § Effects of PPAR␥ agonist on CD36 expression in primary microglia · plain

  “Restricting the action of CD36 by either an inhibitory antibody against CD36 or siRNA knockdown of CD36 resulted in the abolishment of the stimulatory effect of DSP-8658 or pioglitazone on A␤ phagocytosis prevented the increase of A␤ uptake during incubation with DSP-8658 or pioglitazone (Fig. 3).”

• Also stated in: § Effects of PPAR␥ agonist on CD36 expression in primary microglia · boosted

  “These results identify CD36 as a key mediator of PPAR␥ agonist-induced microglia A␤ phagocytosis.”

PPARγ agonist-stimulated Aβ phagocytosis was abolished by inhibitory CD36 antibody or CD36 siRNA.

“Importantly, PPAR␥ agonist-stimulated A␤ phagocytosis was abolished by coincubation of inhibitory CD36 antibody or likewise by transfection of cells with CD36 siRNA.”

DSP-8658 treatment decreased cortical SDS-soluble Aβ1-40 levels by 30% in APP/PS1 mice after 3 months.

“Determination of cortex A␤ levels revealed 32 and 30% decrease of both SDS-soluble A␤ 1-42 and A␤ 1-40 levels, respectively, in response to the DSP-8658 treatment (Fig. 7A).”

The PPARγ agonist-induced increase in Aβ phagocytosis was abolished by transcriptional or translational inhibitors.

“Of note, this effect was abolished by coincubation with transcriptional or translational inhibitors, suggesting that the observed effect requires de novo protein synthesis.”

DSP-8658 treatment increased the percentage of phagocytosing cells in APP/PS1 mice.

“Using the amyloid dye methoxy-X04 in combination with flow cytometrical measurement of microglial isolated from brains of adult DSP-8658-treated and untreated mice, we found that DSP-8658 treatment increased the percentage of phagocytosing cells in APP/PS1 mice, confirming our in vitro observations.”

The enhancement of Aβ phagocytosis by DSP-8658 and pioglitazone is PPARγ-dependent.

“Although DSP-8658 and pioglitazone enhanced A␤ uptake at 3 M in nontarget control siRNA-transfected primary microglia, the same treatments failed to increase A␤ phagocytosis in PPAR␥ siRNA-transfected primary microglia (Fig. 1 H, I ).”

• Also stated in: § Abstract · plain

  “The PPAR␥ agonist pioglitazone and a novel selective PPAR␣/␥ modulator, DSP-8658, currently in clinical development for the treatment of type 2 diabetes, enhanced the microglial uptake of A␤ in a PPAR␥-dependent manner.”

Coincubation of PPARγ agonists (pioglitazone or DSP-8658) with RXR agonists (retinoic acid or bexarotene) produces additive enhancement of Aβ uptake in primary microglia.

“Interestingly, the combination of pioglitazone or DSP-8658 with RXR agonists revealed an additive effect on A␤ uptake (Fig. 4C-F ).”

• Also stated in: § Abstract · plain

  “In addition, combined treatment with agonists for the heterodimeric binding partners of PPAR␥, the retinoid X receptors (RXRs), showed additive enhancement of the A␤ uptake that was mediated by RXR␣ activation.”

RXR agonists retinoic acid and bexarotene enhance Aβ phagocytosis in rat primary microglia in a concentration-dependent manner.

“Both RXR agonists enhanced A␤ phagocytosis in primary microglia in a concentration-dependent manner (Fig. 4 A, B).”

In DSP-8658-treated APP/PS1 mice, distance traveled and escape latency decreased gradually during Morris water maze training.

“In DSP-8658treated APP/PS1 mice, distance traveled and escape latency decreased gradually during training.”

AUC for distance and latency verified a substantial improvement in response to DSP-8658 treatment.

“A quantitative analysis by calculating the area under the curve (AUC) for distance and latency verified a substantial improvement in response to DSP-8658 treatment.”

Aβ plaque load in the cortex decreased in DSP-8658-treated APP/PS1 mice.

“A␤ plaque load determined by thioflavin S staining of sagittal brain sections revealed a decrease in the cortex of DSP-8658treated APP/PS1 mice (Fig. 7E).”

Suppression of RXRα by siRNA inhibited the bexarotene-induced increase of Aβ uptake.

“In contrast, suppression of RXR␣ by siRNA inhibited the bexarotene-induced increase of A␤ uptake (Fig. 5 B, D).”

DSP-8658 treatment decreased hippocampal SDS-soluble Aβ1-42 levels in APP/PS1 mice.

“DSP-8658 treatment also decreased hippocampal SDS-soluble A␤ 1-42 levels (Fig. 7C).”

In the brain, PPARγ agonists reduce Aβ- and cytokine-mediated neuroinflammation and neurotoxicity in vitro and in vivo.

“In the brain, PPAR␥ agonists reduce A␤-and cytokinemediated neuroinflammation and neurotoxicity in vitro (Heneka et al., 1999;Combs et al., 2000) and in vivo (for review, see Heneka and Landreth, 2007).”

• cites Heneka et al., 1999 sourceNot checked — no full text of the source
• cites Combs et al., 2000 sourceNot checked — no full text of the source
• cites Heneka and Landreth, 2007 sourceNot checked — no full text of the source

Transfection of primary rat and mouse microglia with RXRα siRNA effectively decreased RXRα expression.

“Transfection of primary rat and mouse microglia with siRNA effectively decreased RXR␣ expression (Fig. 5 A, C), whereas RXR␤ expression was not (rat) or only to a minor level (mouse) detected in primary microglia after transfection of both nontarget control or RXR␤ siRNA.”

The PPARγ agonist-induced increase in Aβ phagocytosis requires de novo protein synthesis.

“Of note, this effect was abolished by coincubation with transcriptional or translational inhibitors, suggesting that the observed effect requires de novo protein synthesis.”

The upregulated expression of CD36 may mediate the observed increase of Aβ phagocytosis in vitro and in vivo.

“In addition, the upregulated expression of CD36 may mediate the observed increase of A␤ phagocytosis in vitro and in vivo.”

PPARγ agonists can be more effective in AD.

“These results are consistent with our in vitro observations and suggest that PPAR␥ agonists can be more effective in AD.”

Stimulation of microglial clearance by simultaneous activation of the PPARγ/RXRα heterodimer may prove beneficial in prevention of AD.

“Therefore, stimulation of microglial clearance by simultaneous activation of the PPAR␥/RXR␣ heterodimer may prove beneficial in prevention of AD.”

Dual activation of both heterodimer partners shows increased efficacy compared with activation of a single heterodimer partner.

“Dual activation of both heterodimer partners has been reported previously to show increased efficacy compared with the activation of a single heterodimer partner (Papi et al., 2009).”

• cites Papi et al., 2009 sourceNot checked — no full text of the source

DSP-8658 has superior ability to cross the blood-brain barrier compared to pioglitazone (brain/plasma concentration ratio 0.13:0.08).

“Based on the in vitro data presented above and the superior ability to cross the blood-brain barrier [brain/plasma concentration ratio of 0.13:0.08 for DSP-8658/ pioglitazone (Maeshiba et al., 1997)], we chose DSP-8658 for additional in vivo experiments.”

• cites Maeshiba et al., 1997Not checked — no full text of the source

The increased microglial Aβ phagocytosis resulted in improved spatial memory performance.

“Moreover, we revealed that DSP-8658 treatment increased microglial A␤ phagocytosis in vivo, resulting in an overall reduction of A␤ levels and improved spatial memory performance.”

PPARγ activation by DSP-8658 or pioglitazone alone or, more effectively, in combination with RXRα activation, enhances microglial Aβ phagocytosis by upregulation of CD36.

“In summary, PPAR␥ activation by DSP-8658 or pioglitazone alone or, more effectively, in combination with RXR␣ activation, enhances microglial A␤ phagocytosis by upregulation of CD36.”

DSP-8658 ameliorated spatial learning in AD mice.

“These results indicated that DSP-8658 ameliorated the spatial learning in AD mice.”

Microglial cell death in response to drug or Aβ exposure was excluded by LDH assay.

“Microglial cell death in response to drug or A␤ exposure was excluded by LDH assay.”

PPARγ activation increases microglial Aβ clearance.

“Together, these results suggest that microglial A␤ clearance can be increased by PPAR␥ activation.”

PPARγ agonist-induced upregulation of CD36 promotes microglia-mediated phagocytosis.

“These results indicate clearly that PPAR␥ agonistinduced upregulation of CD36 promotes microglia-mediated phagocytosis.”

Cell death was excluded as the source of reduced phagocytic response in siRNA transfection experiments.

“Although cell death was excluded as the source of this phenomenon, siRNA transfection may have led to microglial prestimulation, resulting in an overall decreased phagocytic response.”

Suppression of inflammation can restore Aβ phagocytosis.

“In turn, suppression of inflammation can restore A␤ phagocytosis (Heneka et al., 2010b).”

• cites Heneka et al., 2010bNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

The increased microglial Aβ phagocytosis resulted in an overall reduction of Aβ levels.

“Moreover, we revealed that DSP-8658 treatment increased microglial A␤ phagocytosis in vivo, resulting in an overall reduction of A␤ levels and improved spatial memory performance.”