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. 2018 Jan 5;293(1):226-244.
doi: 10.1074/jbc.M117.786756. Epub 2017 Nov 10.

Receptor for Advanced Glycation End Products Mediates Sepsis-Triggered Amyloid-β Accumulation, Tau Phosphorylation, and Cognitive Impairment

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Receptor for Advanced Glycation End Products Mediates Sepsis-Triggered Amyloid-β Accumulation, Tau Phosphorylation, and Cognitive Impairment

Juciano Gasparotto et al. J Biol Chem. .
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Abstract

Patients recovering from sepsis have higher rates of CNS morbidities associated with long-lasting impairment of cognitive functions, including neurodegenerative diseases. However, the molecular etiology of these sepsis-induced impairments is unclear. Here, we investigated the role of the receptor for advanced glycation end products (RAGE) in neuroinflammation, neurodegeneration-associated changes, and cognitive dysfunction arising after sepsis recovery. Adult Wistar rats underwent cecal ligation and perforation (CLP), and serum and brain (hippocampus and prefrontal cortex) samples were obtained at days 1, 15, and 30 after the CLP. We examined these samples for systemic and brain inflammation; amyloid-β peptide (Aβ) and Ser-202-phosphorylated Tau (p-TauSer-202) levels; and RAGE, RAGE ligands, and RAGE intracellular signaling. Serum markers associated with the acute proinflammatory phase of sepsis (TNFα, IL-1β, and IL-6) rapidly increased and then progressively decreased during the 30-day period post-CLP, concomitant with a progressive increase in RAGE ligands (S100B, Nϵ-[carboxymethyl]lysine, HSP70, and HMGB1). In the brain, levels of RAGE and Toll-like receptor 4, glial fibrillary acidic protein and neuronal nitric-oxide synthase, and Aβ and p-TauSer-202 also increased during that time. Of note, intracerebral injection of RAGE antibody into the hippocampus at days 15, 17, and 19 post-CLP reduced Aβ and p-TauSer-202 accumulation, Akt/mechanistic target of rapamycin signaling, levels of ionized calcium-binding adapter molecule 1 and glial fibrillary acidic protein, and behavioral deficits associated with cognitive decline. These results indicate that brain RAGE is an essential factor in the pathogenesis of neurological disorders following acute systemic inflammation.

Keywords: Tau protein (Tau); amyloid-β (Aβ); neurodegeneration; neuroinflammation; receptor for advanced glycation end products (RAGE); sepsis.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Content of pro-inflammatory cytokines in serum, hippocampus, and prefrontal cortex at 1, 15, and 30 days after CLP. The content of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α was assessed by ELISA in serum (A), hippocampus (B), and prefrontal cortex (C). Values represent relative quantification considering control (sham group) as 100%. Scattered individual data points (n = 6) and standard deviations are represented. Differences between sham and CLP groups on each day were considered significant when p < 0.05 according Student's t test (two-tailed) analysis (*, p < 0.05, and **, p < 0.001).
Figure 2.
Figure 2.
Content of pro-inflammatory markers in hippocampus and prefrontal cortex at 1, 15, and 30 days after CLP. TLR4, GFAP, and nNOS protein levels in hippocampus (A–C, respectively) and prefrontal cortex (D–F, respectively) were evaluated by Western blotting. Scattered individual data points (n = 6) and standard deviation are represented. Representative Western blots are demonstrated with graphs. Differences between sham and CLP groups in each day were considered significant when p < 0.05 according to Student's t test (two-tailed) analysis. Individual p values are depicted when differences were detected.
Figure 3.
Figure 3.
Content of Aβ and phosphorylated Tau in hippocampus and prefrontal cortex of animals at 1, 15, and 30 days after CLP. The levels of Aβ and Tau phosphorylated at Ser-202 (p-TauSer-202) in hippocampus (A and B, respectively) and prefrontal cortex (C and D, respectively) were evaluated by Western blotting. Scattered individual data points (n = 6) and standard deviation are represented. Representative Western blots are demonstrated. Differences between sham and CLP groups in each day were considered significant when p < 0.05 according to Student's t test (two-tailed) analysis; individual p values are depicted. Immunofluorescence-based visualization of Aβ and p-TauSer-202 was performed in hippocampus (E) and prefrontal cortex (F) samples of animals 30 days after CLP. DAPI was used for nuclear staining. Magnification bar length is 100 μm. Insets show staining details.
Figure 4.
Figure 4.
RAGE ligands, sRAGE, and RAGE in serum and brain. The circulating content of the RAGE ligands S100B, CML, HSP70, and HMGB1 and of the sRAGE isoform in serum was assessed by ELISA (A). Values represent relative quantification considering control (sham group) as 100%. The immunocontent of CML, HMGB1, HSP70, and RAGE in hippocampus (B) and prefrontal cortex C) was assessed by Western blotting. Representative Western blots are demonstrated. Scattered individual data points (n = 6) and standard deviation are represented for all data. Differences between sham and CLP groups were considered significant when p < 0.05 according to Student's t test (two-tailed) analysis (*, p < 0.05, and **, p < 0.001).
Figure 5.
Figure 5.
Effects of hippocampal RAGEab injection over RAGE and markers of neuroinflammation and neurodegeneration in hippocampus of animals submitted to CLP. RAGEab was administered bilaterally into the hippocampus at 100 μg/kg at days 15, 17, and 19 after CLP. Control animals received 100 μg/kg of isotype IgG. At day 30 after CLP, the hippocampus was prepared for immunofluorescence detection of RAGE (A), Iba-1 (B), GFAP (C), Aβ (D), and phospho-Tau (E). DAPI was used for nuclear staining. Magnification bar length is 400 μm in the bigger panel and 100 μm in all other panels.
Figure 6.
Figure 6.
Effects of hippocampal RAGEab injection in prefrontal cortex RAGE and markers of neuroinflammation and neurodegeneration in animals submitted to CLP. RAGEab was administered bilaterally into the hippocampus at 100 μg/kg at days 15, 17, and 19 after CLP. Control animals received 100 μg/kg of isotype IgG. At day 30 after CLP, the prefrontal cortex was prepared for immunofluorescence detection of RAGE (A), Iba-1 (B), GFAP (C), Aβ (D), and phospho-Tau (E). DAPI was used for nuclear staining. Magnification bar length is 100 μm.
Figure 7.
Figure 7.
Effects of hippocampal RAGEab injection over phospho-Tau and NeuN staining in hippocampus and prefrontal cortex of animals submitted to CLP. RAGEab was administered bilaterally into the hippocampus at 100 μg/kg at days 15, 17, and 19 after CLP. Control animals received 100 μg/kg isotype IgG. At day 30 after CLP, the hippocampus (A) and the prefrontal cortex (B) were prepared for immunofluorescence detection of phospho-Tau (red) and the neuron nuclear marker NeuN (green). DAPI was used for nuclear staining. Magnification bar length in hippocampus: left panels, 1000 μm; central panels, 200 μm; and right panels, 100 μm. In prefrontal cortex panels, the magnification bar length is 100 μm.
Figure 8.
Figure 8.
Morphological observation of Aβ immunofluorescence staining in hippocampus and prefrontal cortex 30 days after CLP. Sections of tissues from CLP and CLP + RAGEab groups are shown at left panels (magnification bar length is 100 μm). White arrows indicate isolated cells detailed in augmented visualization. DAPI-merged and isolated Aβ stainings are compared. Morphology is indicative of intraneuronal localization of Aβ. Letters and numbers in upper left of images identify panels that originated the detailed images of isolated cells.
Figure 9.
Figure 9.
Morphological observation of phospho-Tau and NeuN immunofluorescence double staining in hippocampus and prefrontal cortex 30 days after CLP. Sections of tissues from CLP group are shown at upper left panels (magnification bar length is 100 μm). Costaining with phospho-Tau (red) and the neuronal nuclear marker NeuN (green) are used to confirm neuronal localization of phospho-Tau. Details of highlighted areas are compared for each type of staining, including DAPI and merged images. Morphology is indicative of intraneuronal localization of filamentous phospho-Tau deposition in hippocampus. Prefrontal cortex did not show phospho-Tau staining, confirming Western blotting and immunofluorescence data for 30 days post-CLP animals. Letters and numbers in upper left of images identify panels that originated the detailed images of isolated cells.
Figure 10.
Figure 10.
ERK1/2, Akt, and mTOR phosphorylation in hippocampus and prefrontal cortex of animals at 1, 15, and 30 days after CLP. The phosphorylated levels of hippocampal ERK1/2 (A), Akt (B), and mTOR (C), and the phosphorylated levels of prefrontal cortex ERK1/2 (D), Akt (E), and mTOR (F) relative to their total respective protein levels were assessed by Western blotting. Scattered individual data points (n = 6) and standard deviation are represented for all data. Representative Western blots are demonstrated. Differences between sham and CLP groups in each day were considered significant when p < 0.05 according student's t test (two-tailed) analysis; individual p values are depicted.
Figure 11.
Figure 11.
Akt and mTOR phosphorylation are associated with RAGE and markers of neurodegeneration in brain. RAGEab was administered bilaterally into the hippocampus at 100 μg/kg at days 15, 17, and 19 after CLP. Hippocampus was isolated, and the levels of phosphorylated Akt (A) and mTOR (B), RAGE (C), Aβ (D), and phosphorylated Tau (E) 30 days after CLP were analyzed by Western blotting. Similarly, in isolated prefrontal cortex, phosphorylated Akt (F) and mTOR (G), RAGE (H), Aβ (I), and phosphorylated Tau (J) were assessed. Scattered individual data points (n = 6) and standard deviation are represented for all data. Representative Western blottings are demonstrated. Differences between groups were considered significant when p < 0.05 according to one-way ANOVA with Tukey's post hoc test; individual p values are depicted.
Figure 12.
Figure 12.
Effect of RAGEab injection into hippocampus over cognitive tests in animals subjected to CLP. RAGEab was administered bilaterally into the hippocampus at 100 μg/kg at days 15, 17, and 19 after CLP. For inhibitory avoidance tasks (A), training sessions were performed at day 30 after surgery. Test sessions were carried out 24 h after training, and the step-down latency was used as a measure of retention. For object recognition task (B), training at day 30 after CLP was conducted by placing rats in the field with two identical objects (objects A1 and A2). Twenty four hours later, animals were allowed to explore the field in the presence of the familiar object A and a novel object C. A recognition index was calculated as the ratio TB/(TA + TB), with TA = time spent exploring the familiar object A; TB = time spent exploring the novel object B. Scattered individual data points with mean and standard deviation from animals from two different experiments are represented; comparisons among groups were performed using the Mann-Whitney U test. For behavioral analyses, individual groups were compared by the Wilcoxon tests. Animals that presented unusual locomotor activity or any other sign of altered behavior during training sessions were excluded from tests. Differences were considered significant when p < 0.05 (*).

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