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

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

Ciliogenesis is reciprocally regulated by PPARA and NR1H4/FXR through controlling autophagy in vitro and in vivo

<p>The primary cilia are evolutionarily conserved microtubule-based cellular organelles that perceive metabolic status and thus link the sensory system to cellular signaling pathways. Therefore, ciliogenesis is thought to be tightly linked to autophagy, which is also regulated by nutrient-sensing transcription factors, such as PPARA (peroxisome proliferator activated receptor alpha) and NR1H4/FXR (nuclear receptor subfamily 1, group H, member 4). However, the relationship between these factors and ciliogenesis has not been clearly demonstrated. Here, we present direct evidence for the involvement of macroautophagic/autophagic regulators in controlling ciliogenesis. We showed that activation of PPARA facilitated ciliogenesis independently of cellular nutritional states. Importantly, PPARA-induced ciliogenesis was mediated by controlling autophagy, since either pharmacological or genetic inactivation of autophagy significantly repressed ciliogenesis. Moreover, we showed that pharmacological activator of autophagy, rapamycin, recovered repressed ciliogenesis in <i>ppara⁻^/− </i> cells. Conversely, activation of NR1H4 repressed cilia formation, while knockdown of NR1H4 enhanced ciliogenesis by inducing autophagy. The reciprocal activities of PPARA and NR1H4 in regulating ciliogenesis were highlighted in a condition where de-repressed ciliogenesis by NR1H4 knockdown was further enhanced by PPARA activation. The in vivo roles of PPARA and NR1H4 in regulating ciliogenesis were examined in greater detail in <i>ppara⁻^/⁻ </i> mice. In response to starvation, ciliogenesis was facilitated in wild-type mice via enhanced autophagy in kidney, while <i>ppara⁻^/⁻ </i> mice displayed impaired autophagy and kidney damage resembling ciliopathy. Furthermore, an NR1H4 agonist exacerbated kidney damage associated with starvation in <i>ppara⁻^/⁻ </i> mice. These findings indicate a previously unknown role for PPARA and NR1H4 in regulating the autophagy-ciliogenesis axis in vivo.</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

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

List of antibodies used in this study.

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

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

Kaplan-Meier survival curves for MACCE during a year following primary PCI showed high sST2 level was associated with a poorer prognosis (A) and that high sST2 and high NT-proBNP levels in combination were associated with a worst prognosis than any other levels of combination (B).

<p>Kaplan-Meier survival curves for MACCE during a year following primary PCI showed high sST2 level was associated with a poorer prognosis (A) and that high sST2 and high NT-proBNP levels in combination were associated with a worst prognosis than any other levels of combination (B).</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