Additional file 1: Table S1. of Age dependent accumulation patterns of advanced glycation end product receptor (RAGE) ligands and binding intensities between RAGE and its ligands differ in the liver, kidney, and skeletal muscle

Antibodies list used for Immunohistochemistry, ELISA and immunoblotting. Table S2. List of primers for Quantitative polymerase chain reaction (qRT-PCR). (DOC 46 kb

List of characteristics of human specimens including alcoholic individuals (1–5) and control (6–10).

<p>List of characteristics of human specimens including alcoholic individuals (1–5) and control (6–10).</p

Microglial AGE-Albumin Is Critical in Promoting Alcohol-Induced Neurodegeneration in Rats and Humans

<div><p>Alcohol is a neurotoxic agent, since long-term heavy ingestion of alcohol can cause various neural diseases including fetal alcohol syndrome, cerebellar degeneracy and alcoholic dementia. However, the molecular mechanisms of alcohol-induced neurotoxicity are still poorly understood despite numerous studies. Thus, we hypothesized that activated microglial cells with elevated AGE-albumin levels play an important role in promoting alcohol-induced neurodegeneration. Our results revealed that microglial activation and neuronal damage were found in the hippocampus and entorhinal cortex following alcohol treatment in a rat model. Increased AGE-albumin synthesis and secretion were also observed in activated microglial cells after alcohol exposure. The expressed levels of receptor for AGE (RAGE)-positive neurons and RAGE-dependent neuronal death were markedly elevated by AGE-albumin through the mitogen activated protein kinase pathway. Treatment with soluble RAGE or AGE inhibitors significantly diminished neuronal damage in the animal model. Furthermore, the levels of activated microglial cells, AGE-albumin and neuronal loss were significantly elevated in human brains from alcoholic indivisuals compared to normal controls. Taken together, our data suggest that increased AGE-albumin from activated microglial cells induces neuronal death, and that efficient regulation of its synthesis and secretion is a therapeutic target for preventing alcohol-induced neurodegeneration.</p></div

AGE-albumin level in rat brains.

<p>Triple-labeled confocal microscopic image analyses were used to study the distribution and relative levels of albumin (green), AGE (red), and DAPI (blue) in the hippocampus Cornu Ammonis Area1 (CA1), Cornu Ammonis Area2 (CA2), Cornu Ammonis Area3 (CA3), Dendate Gyrus (DG) of the control rats (A) and binge-alcohol exposed rats (B). Scale bar  = 50 µm. (C, D) Co-immuonoprecipitation analysis of AGE-ALB in the hippocampus (C) and cerebellum (D) in control or alcohol-exposed rats on days 9 and 11. Scale bar  = 50 µm.</p

Distribution of activated microglial cells and neuron in the hippocampus of the control or rats exposed to binge-alcohol at days 9 and 11.

<p>(A) OX-42-positive microglial cells were measured by immunohistochemical staining in control or alcohol-exposed rats brains. (B) Cresyl violet staining was used to detect decreasing neuronal cell number in the hippocampus of alcohol-exposed rats. (C) Number of OX-42 positive and cresyl violet stained cells in the hippocampus in control or alcohol-exposed rats. (D) Number of OX-42 positive and cresyl violet stained cells in the cerebellum at control or alcohol-exposed rats (white bar; OX-42 positive cell (activated microglia), black bar; cresyl violet positive cell (neuron)). Scale bar  = 200 µm. **; p<0.001, ***; p<0.0001.</p

Protection of ethanol-mediated neuronal cell death by the AGE inhibitor pyridoxamine by decreasing RAGE levels.

<p>(A) Relative levels of neurons in the hippocampus of rat brains were evaluated by cresyl violet staining after alcohol and pyridoxamine (PM) co-treatment. Scale bar  = 200 µm (B) The relative levels of RAGE positive neurons with Neun (green), RAGE (red), and DAPI (blue) in the hippocampus of the control or rats exposed to bingealcohol with or without pyridoxamine co-treatment were evaluated by triple confocal microscopic image analyses. Scale bar  = 200 µm (C–D) Immunoblot analysis was conducted to evaluate the expressed levels of RAGE, ERK1/2, pERK1/2, SAPK/JNK, pSAPK/JNK, p38, pp38, and β-actin in the hippocampus of the control or binge alcohol-exposed rats with or without pyridoxamine co-treatment (C) and neuron cell lysate (D). β-actin was used as an internal control for equal protein loading of each lane (CON; Control, ALC; Alcoholism, PM/ALC; Pyridoxamine/alcoholism, A-A; AGE-Albumin, PM/A-A; Pyridoxamine/AGE-Albumin). (E) Neuronal cell apoptosis was evaluated by triple labeling with NEUN (neuron marker, green), TUNEL (apoptotic cell marker, red) and DAPI (blue) in the hippocampus of the control or rats exposed to binge alcohol with or without pyridoxamine co-treatment. Scale bar  = 50 µm.</p

List of antibodies used in this study.

<p>List of antibodies used in this study.</p

Increased synthesis of AGE-albumin in human microglial cells and its extracellular secretion.

<p>(A) Triple-labeled confocal microscopic image analyses were used to study the distribution and relative levels of AGE (blue), albumin (green), and Iba-1 (red) in human microglial cells after alcohol treatment. (B) The dose-dependent increments of AGE-albumin in total lysates of HMO6 cells treated with 0, 25, 50 or 100 mM ethanol for 24 h were determined by co-immunoprecipitation. (C) Graph illustrating the dose-dependent changes in intracellular (cell lysates) and extracellular (culture supernatant) levels of AGE-albumin in HMO6 cells treated with 0, 25, 50 or 100 mM ethanol for 24 hr, as determined by ELISA. Scale bar  = 50 µm.</p

Distribution of AGE-albumin and activated microglial cells in the brains of human alcoholics and control people.

<p>(A) Triple-labeled confocal microscopic image analyses were used to study the distribution and relative levels of AGE (blue), albumin (green), and Iba-1 (red) in the cerebral cortex of human brains from normal people or alcoholic individuals. (B) Densitometry analyses of AGE, albumin, and Iba-1 colocalization were evaluated using Zeiss Zen software. Scale bar  = 50 µm. **p<0.001.</p

Induction of neuronal cell death by AGE-albumin through up-regulation of RAGE in the hippocampus of rats exposed to binge alcohol.

<p>(A) Relative levels of Neun (green), RAGE (red), and DAPI (blue) in the hippocampus of the control or rats exposed to binge-alcohol were evaluated by triple confocal microscopic image analyses. Scale bar  = 200 µm. (B) Immunoblot analysis was performed to determine the expressed levels of RAGE, ERK1/2, pERK1/2, SAPK/JNK, pSAPK/JNK, p38, pp38, and β-actin, used as an internal control for equal protein loading of each lane. (C) Neuronal cell apoptosis was evaluated by triple labeling with NEUN (neuron marker, green), TUNEL (apoptotic cell marker, red) and DAPI (blue). Scale bar  = 50 µm.</p