Increased receptor for advanced glycation end product expression in the human alcoholic prefrontal cortex is linked to adolescent drinking - PubMed (original) (raw)

Increased receptor for advanced glycation end product expression in the human alcoholic prefrontal cortex is linked to adolescent drinking

Ryan P Vetreno et al. Neurobiol Dis. 2013 Nov.

Abstract

Adolescence is characterized behaviorally by increased impulsivity and risk-taking that declines in parallel with maturation of the prefrontal cortex and executive function. In the brain, the receptor for advanced glycation end products (RAGE) is critically involved in neurodevelopment and neuropathology. In humans, the risk of alcoholism is greatly increased in those who begin drinking between 13 and 15years of age, and adolescents binge drink more than any other age group. We have previously found that alcoholism is associated with increased expression of neuroimmune genes. This manuscript tested the hypothesis that adolescent binge drinking upregulates RAGE and Toll-like receptor (TLR) 4 as well as their endogenous agonist, high-mobility group box 1 (HMGB1). Immunohistochemistry, Western blot, and mRNA analyses found that RAGE expression was increased in the human post-mortem alcoholic orbitofrontal cortex (OFC). Further, an earlier age of drinking onset correlated with increased expression of RAGE, TLR4, and HMGB1. To determine if alcohol contributed to these changes, we used an adolescent binge ethanol model in rats (5.0g/kg, i.g., 2-day on/2-day off from postnatal day [P] 25 to P55) and assessed neuroimmune gene expression. We found an age-associated decline of RAGE expression from late adolescence (P56) to young adulthood (P80). Adolescent intermittent ethanol exposure did not alter RAGE expression at P56, but increased RAGE in the young adult PFC (P80). Adolescent intermittent ethanol exposure also increased TLR4 and HMGB1 expression at P56 that persisted into young adulthood (P80). Assessment of young adult frontal cortex mRNA (RT-PCR) found increased expression of proinflammatory cytokines, oxidases, and neuroimmune agonists at P80, 25days after ethanol treatment. Together, these human and animal data support the hypothesis that an early age of drinking onset upregulates RAGE/TLR4-HMGB1 and other neuroimmune genes that persist into young adulthood and could contribute to risk of alcoholism or other brain diseases associated with neuroinflammation.

Keywords: +IR; AIE; Adolescence; Alcoholism; Binge drinking; Cytokine; Ethanol; HMGB1; Innate immunity; Neuroimmune; PMI; RAGE; TLR; Toll-like receptor; adolescent intermittent ethanol; high-mobility group box 1; immunoreactivity; post-mortem interval; receptor for advanced glycation end products.

© 2013.

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Figures

FIGURE 1

FIGURE 1. SAMPLE SELECTION AND EXPERIMENTAL DESIGN

(A) Representative image depicting human orbitofrontal cortex (OFC) sectioning by New South Wales Tissue Resource Centre at the University of Sydney.

Left

: Image of intact alcoholic frontal cortex tissue sample containing OFC.

Right

: Image depicting sampling of OFC from alcoholic volunteer. Scale bar = 1 cm. (B) Male Wistar rats were treated from postnatal day (P)25 to P55 with either ethanol (5.0 g/kg, 20% ethanol w/v, i.g.) or a comparable volume of water on a 2-day on/2-day off administration schedule. Blood ethanol concentrations (BECs) were measured one hr after ethanol treatment on P38 and P54. Subjects were sacrificed either 24 hr after the completion of AIE treatment (i.e., P56) or 25 days after the conclusion of AIE treatment (i.e., P80). (C) Representative micrographs of the regions of interest assessed for neuroimmune agonist and receptor expression in the prefrontal cortex. Receptor for advanced glycation end product expression was measured in subregions of the rat medial prefrontal cortex (mPFC), including the prelimbic cortex (PrL; Bregma: +4.20 mm to +2.20 mm) and infralimbic cortex (IL; Bregma: +3.20 mm to +2.20 mm) as well as the orbitofrontal cortex (OFC; Bregma: +4.20 mm to +2.20 mm) according to the atlas of Paxinos and Watson (1998). Toll-like receptor 4 and high-mobility group box 1 immunoreactivity was measured in the OFC (Bregma: +4.20 mm to +2.20 mm) according to the atlas of Paxinos and Watson (1998).

FIGURE 2

FIGURE 2. RECEPTOR FOR ADVANCED GLYCATION END PRODUCT (RAGE) EXPRESSION IS INCREASED IN THE HUMAN POST-MORTEM ORBITOFRONTAL CORTEX (OFC) AND IS COLOCALIZED ON NEURONS

(A) RAGE pixel density (×1000 Pixels/mm2) was increased in the alcoholic OFC (n=10), relative to moderate drinking controls (n=10). Representative photomicrographs of RAGE+IR in a moderate drinking control and alcoholic post-mortem OFC. Scale bar = 20 μm. (B) Western blot analyses found increased RAGE protein concentration in the human alcoholic OFC, relative to moderate drinking controls (n=9). Representative lanes from Western blot analysis of RAGE. CON is moderate drinking control and ALC alcoholic subject. Western blot analyses were run in triplicate, and the mean was reported. (C) RT-PCR assessment found increased RAGE mRNA in the human alcoholic OFC, relative to moderate drinking controls (n=8). RT-PCR analyses were run in triplicate, and the mean was reported. (D) Top and Middle: High magnification photomicrographs of RAGE (green) colocalization with the neuronal marker NeuN (red) in the post-mortem human alcoholic OFC. White arrowheads indicate a RAGE+IR cell that colocalized with NeuN. Bottom: Z-stack demonstrating colocalization of RAGE+IR cell with NeuN (yellow). Scale bar = 20 μm. * indicates p<0.05.

FIGURE 3

FIGURE 3. RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS (RAGE), TOLL-LIKE RECEPTOR 4 (TLR4), AND HIGH-MOBILITY GROUP BOX 1 (HMGB1) EXPRESSION IN THE HUMAN ALCOHOLIC POST-MORTEM ORBITOFRONTAL CORTEX IS CORRELATED WITH AGE OF DRINKING ONSET

Depicted are individual self and family reports of age of drinking onset vs. RAGE (circle; × 100000 Pixels/mm2), TLR4 (square; Cells/mm3), and HMGB1 (triangle; Cells/mm3) immunoreactivity. Across subjects, age of drinking onset negatively correlated with neuroimmune immunoreactivity (r = −0.47, p<0.001).

Note

: Each subject’s immunoreactivity data (i.e., RAGE, TLR4, and HMGB1) lie on vertical points over each subject’s reported age of drinking onset. Moderate alcohol drinking controls tended to self-report a later age of drinking onset (25±1 years of age) in comparison to individuals that met criteria for alcoholism (18±1 years of age).

FIGURE 4

FIGURE 4. THE MATURATIONAL TRAJECTORY OF RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS (RAGE) EXPRESSION IN THE YOUNG ADULT PREFRONTAL CORTEX IS DISRUPTED FOLLOWING ADOLESCENT BINGE ETHANOL EXPOSURE IN THE RAT

(A) RAGE pixel density (×1000 Pixels/mm2) was unchanged in the adolescent prelimbic cortex (PrL) following adolescent intermittent ethanol (AIE) treatment, but was increased by 280% in the young adult, relative to CONs. (B) AIE treatment did not affect RAGE+IR in the adolescent infralimbic cortex (IL), but did increase expression by 375% in the young adult, relative to CONs. (C) AIE did not affect RAGE pixel density (×1000 Pixels/mm2) in the adolescent orbitofrontal cortex, but did increase RAGE+IR by 340% in the young adult, relative to CONs.

Note

: There is a maturational reduction of RAGE+IR in the CON PrL, IL, and OFC from adolescence to young adulthood. (D) Representative photomicrographs of RAGE+IR in the OFC of adolescent (P56) and young adult (P80) CON- and AIE-treated rats. Black arrowheads indicate RAGE+IR cells. Data are presented as mean ± SEM. ** indicates p<0.01, relative to CON rats. Scale bar = 50 μm.

FIGURE 5

FIGURE 5. EXPRESSION OF RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS (RAGE) IS HIGHLY COLOCALIZED WITH NEURONS IN THE RODENT YOUNG ADULT PREFRONTAL CORTEX

(A) Quantification of RAGE colocalization with neuronal (NeuN), microglial (Iba-1), and astrocytic (GFAP) markers. Data are presented as mean ± SEM. * indicates p<0.05, relative to CON rats. (B) High magnification photomicrographs of RAGE (green) colocalization with the cellular marker NeuN, Iba-1, or GFAP (red) in the prefrontal cortex of young adult CON- and AIE-treated rats. White arrowheads indicate RAGE+IR cells that colocalized with a cellular marker (yellow). White arrows emphasize cell makers that did not colocalize with RAGE. All scale bars = 20 μm.

FIGURE 6

FIGURE 6. ADOLESCENT INTERMITTENT ETHANOL (AIE) TREATMENT PERSISTENTLY INCREASED EXPRESSION OF TOLL-LIKE RECEPTOR 4 (TLR4) IN THE ADOLESCENT (P56) AND ADULT (P80) ORBITOFRONTAL CORTEX

(A) Profile counts (Cells/mm2) revealed that AIE exposure increased TLR4+IR cells by 32% in the adolescent and 141% in the young adult orbitofrontal cortex (OFC), relative to CONs.

Note

: There is a maturational reduction of TLR4+IR in the CON OFC from adolescence to young adulthood. ** indicates p<0.01, relative to CON rats. Scale bar = 50 μm. (B) Representative photomicrographs of TLR4+IR in OFC of adolescent (P56) and young adult (P80) CON- and AIE-treated rats. Black arrowheads indicate TLR4+IR cells. Data are presented as mean ± SEM. ** indicates p<0.01, relative to CON rats. Scale bar = 50 μm.

FIGURE 7

FIGURE 7. ADOLESCENT INTERMITTENT ETHANOL (AIE) EXPOSURE INCREASES HIGH-MOBILITY GROUP BOX 1 (HMGB1) EXPRESSION IN THE ADOLESCENT (P56) ORBITOFRONTAL CORTEX THAT PERSISTS INTO YOUNG ADULTHOOD (P80)

(A) Profile counts (Cells/mm2) revealed that AIE treatment increased HMGB1+IR by 41% in the adolescent and 61% in the young adult orbitofrontal cortex (OFC), relative to CONs.

Note

: There is a maturational increase of HMGB1+IR in the CON OFC from adolescence to young adulthood. ** indicates p<0.01, relative to CON rats. Scale bar = 50 μm. (B) Representative photomicrographs of HMGB1+IR in OFC of adolescent (P56) and young adult (P80) CON- and AIE-treated rats. Black arrowheads indicate HMGB1+IR cells. Data are presented as mean ± SEM. ** indicates p<0.01, relative to CON rats. Scale bar = 50 μm.

FIGURE 8

FIGURE 8. ADOLESCENT INTERMITTENT ETHANOL (AIE) EXPOSURE INCREASES MRNA LEVELS OF PROINFLAMMATORY CYTOKINES, OXIDASES, AND NEUROIMMUNE AGONIST MARKERS IN THE YOUNG ADULT FRONTAL CORTEX

(A) RT-PCR assessment of proinflammatory cytokine mRNA in frontal cortex tissue samples found an approximate three-fold increase of tumor necrosis factor α (TNFα), no change in Interleukin-1β (IL-1β), and an approximate three-fold increase of monocyte chemotactic protein-1 (MCP-1), relative to CONS. (B) RT-PCR assessment of proinflammatory oxidases in frontal cortex tissue samples found an approximate three-fold increase of NADPH oxidase (NOX2) mRNA, an approximate 80% increase of cyclooxygenase-2 (COX2) mRNA, and no change in inducible nitric oxide synthase (iNOS) mRNA, relative to CONS. (C) RT-PCR assessment of neuroimmune agonist mRNA in frontal cortex tissue samples found an approximate 180% increase of S100 calcium binding protein B (S100β), a two-fold increase of fibronectin (Fbn), and a 44% increase of myeloid differentiation primary response gene 88 (MyD88), relative to CONS. RT-PCR analyses were run in triplicate. * indicates p<0.05, ** indicates p<0.01, relative to CON rats.

FIGURE 9

FIGURE 9. ADOLESCENT INTERMITTENT ETHANOL (AIE) EXPOSURE ALTERS THE DEVELOPMENTAL TRAJECTORY OF THE NEUROIMMUNE SIGNALING SYSTEM, LEADING TO UPREGULATION THAT PERSISTS INTO YOUNG ADULTHOOD

(A) A simplified schematic of AIE-induced cyclic activation of the innate immune signaling system in the prefrontal cortex that persists into young adulthood. Ethanol (EtOH) exposure causes nuclear release of high-mobility group box 1 (HMGB1 [Crews et al. 2013]) , which becomes a proinflammatory cytokine (Yang et al. 2005). Ethanol also activates glial cells (Qin and Crews 2012) to release proinflammatory cytokines (i.e., TNFα, MCP-1) and oxidases (i.e., NOX2, COX2) that increase innate immune signal agonist expression (i.e., fibronectin, S100β). These proinflammatory cytokines, oxidases, and neuroimmune signaling agonists increase the expression of Toll-like receptor 4 (TLR4), which facilitate further release of HMGB1 and other proinflammatory signals establishing the cycle of TLR4/HMGB1 innate immunity signaling (Hohne et al. 2013). Once the inflammagen (i.e., EtOH) is removed from this system, the activated innate immune signaling system continues to release proinflammatory signals leading to upregulation of the receptor for advanced glycation end products (RAGE) on neurons. (B) Schematic of the correlation of age of drinking onset with neuroimmune signaling in the human post-mortem alcoholic orbitofrontal cortex, relative to moderate drinking controls. (C) Schematic of the adolescent maturational trajectory of neuroimmune signaling. (Top) Depicted is the maturational trajectory of neuroimmune signaling from adolescence to young adulthood. (Bottom) Depicted are AIE-induced changes to the maturational trajectory of neuroimmune signaling.

References

    1. Alaux-Cantin S, Warnault V, Legastelois R, Botia B, Pierrefiche O, Vilpoux C, et al. Alcohol intoxications during adolescence increase motivation for alcohol in adult rats and induce neuroadaptations in the nucleus accumbens. Neuropharmacology. 2013;67:521–31. - PubMed
    1. Blednov YA, Benavidez JM, Geil C, Perra S, Morikawa H, Harris RA. Activation of inflammatory signaling by lipopolysaccharide produces a prolonged increase of voluntary alcohol intake in mice. Brain Behav Immun. 2011;25(Suppl 1):S92–S105. - PMC - PubMed
    1. Blednov YA, Bergeson SE, Walker D, Ferreira VM, Kuziel WA, Harris RA. Perturbation of chemokine networks by gene deletion alters the reinforcing actions of ethanol. Behav Brain Res. 2005;165:110–25. - PMC - PubMed
    1. Blednov YA, Ponomarev I, Geil C, Bergeson S, Koob GF, Harris RA. Neuroimmune regulation of alcohol consumption: behavioral validation of genes obtained from genomic studies. Addict Biol. 2012;17:108–20. - PMC - PubMed
    1. Bonaldi T, Talamo F, Scaffidi P, Ferrera D, Porto A, Bachi A, et al. Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion. Embo J. 2003;22:5551–60. - PMC - PubMed

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