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

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.     

Microglial cell death induced by glycated bovine serum albumin: nitric oxide involvement

Nonenzymatic glycation results in the formation of advanced glycation end products (AGEs) through a nonenzymatic multistep reaction of reducing sugars with proteins. AGEs have been suspected to be involved in the pathogenesis of several chronic clinical neurodegenerative complications including Alzheimer's disease, which is characterized with the activation of microglial cells in neuritic plaques. To find out the consequence of this activation on microglial cells, we treated the cultured microglial cells with different glycation levels of Bovine Serum Albumin (BSA) which were prepared in vitro. Extent of glycation of protein has been characterized during 16 weeks of incubation with glucose. Treatment of microglial cells with various levels of glycated albumin induced nitric oxide (NO) production and consequently cell death. We also tried to find out the mode of death in AGE-activated microglial cells. Altogether, our results suggest that AGE treatment causes microglia to undergo NO-mediated apoptotic and necrotic cell death in short term and long term, respectively. NO production is a consequence of iNOS expression in a JNK dependent RAGE signalling after activation of RAGE by AGE-BSA.     

RAGE: a potential target for Abeta-mediated cellular perturbation in Alzheimer's disease

This review focuses on the current findings regarding interaction between amyloid beta peptide (Abeta) and receptor for advanced glycation endproducts (RAGE) and its roles in the pathogenesis of Alzheimer's disease (AD). As a ubiquitously expressed cell surface receptor, RAGE mediates the effects of Abeta on microglia, blood-brain barrier (BBB) and neurons through activating different signaling pathways. Data from autopsy brain tissues, in vitro cell cultures and transgenic mouse models suggest that Abeta-RAGE interaction exaggerates neuronal stress, accumulation of Abeta, impaired learning memory, and neuroinflammation. Blockade of RAGE protects against Abeta-mediated cellular perturbation. These findings may have an important therapeutic implication for neurodegenerative disorders relevant to AD.     

A free radical-generating system induces the cholesterol biosynthesis pathway: a role in Alzheimer's disease

Oxidative stress, which plays a critical role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), is intimately linked to aging – the best established risk factor for AD. Studies in neuronal cells subjected to oxidative stress, mimicking the situation in AD brains, are therefore of great interest. This paper reports that, in human neuronal cells, oxidative stress induced by the free radical-generating xanthine/xanthine oxidase (X-XOD) system leads to apoptotic cell death. Microarray analyses showed a potent activation of the cholesterol biosynthesis pathway following reductions in the cell cholesterol synthesis caused by the X-XOD treatment; furthermore, the apoptosis was reduced by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase ( HMGCR ) expression with an interfering RNA. The potential importance of this mechanism in AD was investigated by genetic association, and it was found that HMGCR , a key gene in cholesterol metabolism and among those most strongly upregulated, was associated with AD risk. In summary, this work presents a human cell model prepared to mimic the effect of oxidative stress in neurons that might be useful in clarifying the mechanism involved in free radical-induced neurodegeneration. Gene expression analysis followed by genetic association studies indicates a possible link among oxidative stress, cholesterol metabolism and AD.     

Apoptosis in Alzheimer's disease—an update

Alzheimer's disease (AD) is the most common human neurodegenerative disorder characterized by the progressive deterioration of cognition and memory in association with the presence of senile plaques, neurofibrillary tangles, and massive loss of neurons. Most cases of AD are late-onset and sporadic, but in some cases the disease is inherited as an autosomal dominant trait. Four different genes, the amyloid precursor protein, apolipoprotein E, and presenilins 1 and 2 have been implicated in the etiology of familial AD. It is now generally accepted that massive neuronal death due to apoptosis is a commmon characteristic in the brains of patients suffering from neurodegenerative diseases, and apoptotic cell death has been found in neurons and glial cells in AD. This review summarizes the current findings regarding the evidence for apoptosis in AD and discusses the possible involvement of apoptosis-regulating factors in the pathology of AD. Modification of the apoptotic cascade could be considered as a primary therapeutic strategy for the disease.     

Proteinase-activated receptor-2 exerts protective and pathogenic cell type-specific effects in Alzheimer's disease

The proteinase-activated receptors (PARs) are a novel family of G protein-coupled receptors, and their effects in neurodegenerative diseases remain uncertain. Alzheimer's disease (AD) is a neurodegenerative disorder defined by misfolded protein accumulation with concurrent neuroinflammation and neuronal death. We report suppression of proteinase-activated receptor-2 (PAR2) expression in neurons of brains from AD patients, whereas PAR2 expression was increased in proximate glial cells, together with up-regulation of proinflammatory cytokines and chemokines and reduced IL-4 expression (p < 0.05). Glial PAR2 activation increased expression of formyl peptide receptor-2 (p < 0.01), a cognate receptor for a fibrillar 42-aa form of beta-amyloid (Abeta(1-42)), enhanced microglia-mediated proinflammatory responses, and suppressed astrocytic IL-4 expression, resulting in neuronal death (p < 0.05). Conversely, neuronal PAR2 activation protected human neurons against the toxic effects of Abeta(1-42) (p < 0.05), a key component of AD neuropathogenesis. Amyloid precursor protein-transgenic mice, displayed glial fibrillary acidic protein and IL-4 induction (p < 0.05) in the absence of proinflammatory gene up-regulation and neuronal injury, whereas PAR2 was up-regulated at this early stage of disease progression. PAR2-deficient mice, after hippocampal Abeta(1-42) implantation, exhibited enhanced IL-4 induction and less neuroinflammation (p < 0.05), together with improved neurobehavioral outcomes (p < 0.05). Thus, PAR2 exerted protective properties in neurons, but its activation in glia was pathogenic with secretion of neurotoxic factors and suppression of astrocytic anti-inflammatory mechanisms contributing to Abeta(1-42)-mediated neurodegeneration.     

Advanced glycation end products impair function of late endothelial progenitor cells through effects on protein kinase Akt and cyclooxygenase-2

Endothelial progenitor cells (EPCs) exhibit impaired function in the context of diabetes, and advanced glycation end products (AGEs), which accumulate in diabetes, may contribute to this. In the present study, we investigated the mechanism by which AGEs impair late EPC function. EPCs from human umbilical cord blood were isolated, and incubated with AGE-modified albumin (AGE-albumin) at different concentrations found physiologically in plasma. Apoptosis, migration, and tube formation assays were used to evaluate EPC function including capacity for vasculogenesis, and expression of the receptor for AGEs (RAGE), Akt, endothelial nitric oxide synthase (eNOS), and cycloxygenase-2 (COX-2) were determined. Anti-RAGE antibody was used to block RAGE function. AGE-albumin concentration-dependently enhanced apoptosis and depressed migration and tube formation, but did not affect proliferation, of late EPCs. High AGE-albumin increased RAGE mRNA and protein expression, and decreased Akt and COX-2 protein expression, whilst having no effect on eNOS mRNA or protein in these cells. These effects were inhibited by co-incubation with anti-RAGE antibody. These results suggest that RAGE mediates the AGE-induced impairment of late EPC function, through down-regulation of Akt and COX-2 in these cells.     

Neuronal localization of the TNFalpha converting enzyme (TACE) in brain tissue and its correlation to amyloid plaques

The tumor necrosis factor (TNF)-alpha converting enzyme (TACE) can cleave the cell-surface ectodomain of the amyloid-beta precursor protein (APP), thus decreasing the generation of amyloid-beta (Abeta) by cultured non-neuronal cells. While the amyloidogenic processing of APP in neurons is linked to the pathogenesis of Alzheimer's disease (AD), the expression of TACE in neurons has not yet been examined. Thus, we assessed TACE expression in a series of neuronal and non-neuronal cell types by Western blots. We found that TACE was present in neurons and was only faintly detectable in lysates of astrocytes, oligodendrocytes, and microglial cells. Immunohistochemical analysis was used to determine the cellular localization of TACE in the human brain, and its expression was detected in distinct neuronal populations, including pyramidal neurons of the cerebral cortex and granular cell layer neurons in the hippocampus. Very low levels of TACE were seen in the cerebellum, with Purkinje cells at the granular-molecular boundary staining faintly. Because TACE was localized predominantly in areas of the brain that are affected by amyloid plaques in AD, we examined its expression in a series of AD brains. We found that AD and control brains showed similar levels of TACE staining, as well as similar patterns of TACE expression. By double labeling for Abeta plaques and TACE, we found that TACE-positive neurons often colocalized with amyloid plaques in AD brains. These observations support a neuronal role for TACE and suggest a mechanism for its involvement in AD pathogenesis as an antagonist of Abeta formation.     

De-N-glycosylation or G82S mutation of RAGE sensitizes its interaction with advanced glycation endproducts

Interactions between advanced glycation endproducts (AGE) and the receptor for AGE (RAGE) have been implicated in the development of diabetic vascular complications. RAGE has two N-glycosylation sites in and near the AGE-binding domain, and G82S mutation in the second N-glycosylation motif was recently reported in human. In this study, we examined whether de-N-glycosylation or G82S of RAGE affect its ability to bind AGE and cellular response to AGE. Recombinant wild-type, de-N-glycosylation and G82S RAGE proteins were produced in COS-7 cells, purified and assayed for ligand-binding abilities. De-N-glycosylation at N81 and G82S mutation decreased Kd for glycolaldehyde-derived AGE to three orders of magnitude lower levels compared with wild-type. AGE-induced upregulation of VEGF mRNA was significantly augmented in endothelial cell-derived ECV304 cells expressing de-N-glycosylated and G82S RAGE when compared with wild-type expressor. Exposure to low glucose resulted in the appearance of RAGE proteins of deglycosylated size in wild-type RAGE-expressing cells and significantly enhanced glycolaldehyde-derived AGE-induced VEGF mRNA expression. De-N-glycosylation or G82S mutation of RAGE increases affinity for AGE ligands, and may sensitize cells or conditions with it to AGE.     

Neuronal localization of the TNFα converting enzyme (TACE) in brain tissue and its correlation to amyloid plaques

The tumor necrosis factor (TNF)‐α converting enzyme (TACE) can cleave the cell‐surface ectodomain of the amyloid‐β precursor protein (APP), thus decreasing the generation of amyloid‐β (Aβ) by cultured non‐neuronal cells. While the amyloidogenic processing of APP in neurons is linked to the pathogenesis of Alzheimer's disease (AD), the expression of TACE in neurons has not yet been examined. Thus, we assessed TACE expression in a series of neuronal and non‐neuronal cell types by Western blots. We found that TACE was present in neurons and was only faintly detectable in lysates of astrocytes, oligodendrocytes, and microglial cells. Immunohistochemical analysis was used to determine the cellular localization of TACE in the human brain, and its expression was detected in distinct neuronal populations, including pyramidal neurons of the cerebral cortex and granular cell layer neurons in the hippocampus. Very low levels of TACE were seen in the cerebellum, with Purkinje cells at the granular‐molecular boundary staining faintly. Because TACE was localized predominantly in areas of the brain that are affected by amyloid plaques in AD, we examined its expression in a series of AD brains. We found that AD and control brains showed similar levels of TACE staining, as well as similar patterns of TACE expression. By double labeling for Aβ plaques and TACE, we found that TACE‐positive neurons often colocalized with amyloid plaques in AD brains. These observations support a neuronal role for TACE and suggest a mechanism for its involvement in AD pathogenesis as an antagonist of Aβ formation. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 40–46, 2001     

Effects of oxidative and nitrosative stress in brain on p53 proapoptotic protein in amnestic mild cognitive impairment and Alzheimer disease

Many studies reported that oxidative and nitrosative stress might be important for the pathogenesis of Alzheimer's disease (AD) beginning with arguably the earliest stage of AD, i.e., as mild cognitive impairment (MCI). p53 is a proapoptotic protein that plays an important role in neuronal death, a process involved in many neurodegenerative disorders. Moreover, p53 plays a key role in the oxidative stress-dependent apoptosis. We demonstrated previously that p53 levels in brain were significantly higher in MCI and AD IPL (inferior parietal lobule) compared to control brains. In addition, we showed that in AD IPL, but not in MCI, HNE, a lipid peroxidation product, was significantly bound to p53 protein. In this report, we studied by means of immunoprecipitation analysis, the levels of markers of protein oxidation, 3-nitrotyrosine (3-NT) and protein carbonyls, in p53 in a specific region of the cerebral cortex, namely the inferior parietal lobule, in MCI and AD compared to control brains. The focus of these studies was to measure the oxidation and nitration status of this important proapoptotic protein, consistent with the hypothesis that oxidative modification of p53 could be involved in the neuronal loss observed in neurodegenerative conditions.     

Interleukin-6 and alpha-2-macroglobulin indicate an acute-phase state in Alzheimer's disease cortices.

Recent studies indicated that the formation of a major constituent of Alzheimer's disease (AD) senile plaques, called beta A4-peptide, does not result from normal processing of its precursor, amyloid precursor protein (APP). Since proteolytic cleavage of APP inside its beta A4 sequence was found to be part of APP processing the formation of the beta A4-peptide seems to be prevented under normal conditions. We considered whether in AD one of the endogenous proteinase inhibitors might interfere with APP processing. After we had recently found that cultured human neuronal cells synthesize the most potent of the known human proteinase inhibitors, alpha-2-macroglobulin (alpha 2M), upon stimulation with the inflammatory mediator interleukin-6 (IL-6) we now investigated whether alpha 2M and IL-6 could be detected in AD brains. Here we report that AD cortical senile plaques display strong alpha 2M and IL-6 immunoreactivity while no such immunoreactivity was found in age-matched control brains. Strong perinuclear alpha 2M immunoreactivity in hippocampal CA1 neurons of Alzheimer's disease brains indicates that neuronal cells are the site of alpha 2M synthesis in AD brains. We did not detect elevated IL-6 or alpha 2M levels in the cerebrospinal fluid of AD patients. Our data indicate that a sequence of immunological events which seem to be restricted to the local cortical environment is part of AD pathology.     

The 1.5 Å crystal structure of human receptor for advanced glycation endproducts (RAGE) ectodomains reveals unique features determining ligand binding

Interaction of the pattern recognition receptor, RAGE with key ligands such as advanced glycation end products (AGE), S100 proteins, amyloid β, and HMGB1 has been linked to diabetic complications, inflammatory and neurodegenerative disorders, and cancer. To help answer the question of how a single receptor can recognize and respond to a diverse set of ligands we have investigated the structure and binding properties of the first two extracellular domains of human RAGE, which are implicated in various ligand binding and subsequent signaling events. The 1.5-Å crystal structure reveals an elongated molecule with a large basic patch and a large hydrophobic patch, both highly conserved. Isothermal titration calorimetry (ITC) and deletion experiments indicate S100B recognition by RAGE is an entropically driven process involving hydrophobic interaction that is dependent on Ca²⁺ and on residues in the C′D loop (residues 54–67) of domain 1. In contrast, competition experiments using gel shift assays suggest that RAGE interaction with AGE is driven by the recognition of negative charges on AGE-proteins. We also demonstrate that RAGE can bind to dsDNA and dsRNA. These findings reveal versatile structural features of RAGE that help explain its ability to recognize of multiple ligands.     

Intra- and extracellular Abeta and PHF in clinically evaluated cases of Alzheimer's disease

Temporal cortical sections from postmortem brains of individuals without any dementing condition and with different degrees of severity of Alzheimer's disease (AD) evaluated by the Clinical Dementia Rating scale (CDR 0-CDR 3) were analyzed using immunohistochemical procedures. To demonstrate the amyloid-beta-peptide (Abeta) deposition and the neurofibrillary pathology, two monoclonal antibodies were used, a human CERAD Abeta (10D5) antibody raised against the N-terminal region of the Abeta-peptide, and an antibody raised against paired helical filaments (PHF-1). The neuron cell bodies and the glial cells were also recognized by two polyclonal antibodies raised, respectively, against the protein gene peptide (PGP 9.5) and glial fibrillary acidic protein (GFAP). Directly related to severity of AD, progressive deposits of Abeta-peptide were found within cortical pyramidal-like neurons and forming senile plaques. Ultrastructurally, Abeta-peptide deposits were related to neuronal intracytoplasmic organelles, such as the ER, the mitochondria, the Nissl bodies and lipofuscin. We have also found that the intracellular deposition of the Abeta peptide is a neuropathological finding prior to the appearance of PHF-immunoreactive structures. We suggest that the intracellular Abeta deposition in cortical pyramidal neurons is a first neurodegenerative event in AD development and that it is involved in cell dysfunction, neuronal death, and plaque formation.     

Oxidative insults to neurons and synapse are prevented by aged garlic extract and S-allyl-L-cysteine treatment in the neuronal culture and APP-Tg mouse model

Alzheimer's disease (AD) is one of the most common forms of dementia in the elderly. In AD patients, β-amyloid peptide (Aβ) plaques and neurofibrillary tangles are common features observed in the CNS. Aβ deposition results in the production of reactive oxygen species (ROS) leading to the hyperphosphorylation of tau that are associated with neuronal damage. Cholinesterase inhibitors and a partial NMDA receptor antagonist (memantine) have been identified as potential treatment options for AD. However, clinical studies have found that these drugs fail to prevent the disease progression. From ancient times, garlic (Allium sativum) has been used to treat several diseases. By 'aging' of garlic, some adverse reactions of garlic can be eliminated. Recent findings suggest that 'aged garlic extract' (AGE) may be a therapeutic agent for AD because of its antioxidant and Aβ lowering properties. To date, the molecular properties of AGE have been sparsely studied in vitro or in vivo. The present study tested specific biochemical and molecular effects of AGE in neuronal and AD rodent models. Furthermore, we identified S-allyl-L-cysteine (SAC) as one of the most active chemicals responsible for the AGE-mediated effect(s). We observed significant neuroprotective and neurorescue properties of AGE and one of its ingredients, SAC, from ROS (H(2)O(2))-mediated insults to neuronal cells. Treatment of AGE and SAC were found to protect neuronal cells when they were independently co-treated with ROS. Furthermore, a novel neuropreservation effect of AGE was detected in that pre-treatment with AGE alone protected ～ 80% neuronal cells from ROS-mediated damage. AGE was also found to preserve pre-synaptic protein synaptosomal associated protein of 25 kDa (SNAP25) from ROS-mediated insult. For example, treatment with 2% AGE containing diet and SAC (20 mg/kg of diet) independently increased (～70%) levels of SNAP25 and synaptophysin in Alzheimer's amyloid precursor protein-transgenic mice, of which the latter was significantly decreased in AD. Taken together, the neuroprotective, including preservation of pre-synaptic proteins by AGE and SAC can be utilized in future drug development in AD.     

Microglia Acquire Distinct Activation Profiles Depending on the Degree of α-Synuclein Neuropathology in a rAAV Based Model of Parkinson's Disease

Post-mortem analysis of brains from Parkinson''s disease (PD) patients strongly supports microglia activation and adaptive immunity as factors contributing to disease progression. Such responses may be triggered by α-synuclein (α-syn), which is known to be the main constituent of the aggregated proteins found in Lewy bodies in the brains of PD patients. To investigate this we used a recombinant viral vector to express human α-syn in rat midbrain at levels that induced neuronal pathology either in the absence or the presence of dopaminergic cell death, thereby mimicking early or late stages of the disease. Microglia activation was assessed by stereological quantification of Mac1+ cells, as well as the expression patterns of CD68 and MCH II. In our study, when α-syn induced neuronal pathology but not cell death, a fast transient increase in microglia cell numbers resulted in the long-term induction of MHC II+ microglia, denoting antigen-presenting ability. On the other hand, when α-syn induced both neuronal pathology and cell death, there was a delayed increase in microglia cell numbers, which correlated with long-lasting CD68 expression and a morphology reminiscent of peripheral macrophages. In addition T-lymphocyte infiltration, as judged by the presence of CD4+ and CD8+ cells, showed distinct kinetics depending on the degree of neurodegeneration, and was significantly higher when cell death occurred. We have thus for the first time shown that the microglial response differs depending on whether α-syn expression results on cell death or not, suggesting that microglia may play different roles during disease progression. Furthermore, our data suggest that the microglial response is modulated by early events related to α-syn expression in substantia nigra and persists at the long term.     

AGEs Induce Cell Death via Oxidative and Endoplasmic Reticulum Stresses in Both Human SH-SY5Y Neuroblastoma Cells and Rat Cortical Neurons

Advanced glycation endproducts (AGEs) are elevated in aging and neurodegenerative diseases such as Alzheimer??s disease (AD), and they can stimulate the generation of reactive oxygen species (ROSs) via NADPH oxidase, induce oxidative stress that lead to cell death. In the current study, we investigated the molecular events underlying the process that AGEs induce cell death in SH-SY5Y cells and rat cortical neurons. We found: (1) AGEs increase intracellular ROSs; (2) AGEs cause cell death after ROSs increase; (3) oxidative stress-induced cell death is inhibited via the blockage of AGEs receptor (RAGE), the down-regulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and the increase of scavenging by anti-oxidant alpha-lipoic acid (ALA); (4) endoplasmic reticulum (ER) stress was triggered by AGE-induced oxidative stress, resulting in the activation of C/EBP homologous protein (CHOP) and caspase-12 that consequently initiates cell death, taurine-conjugated ursodeoxycholic acid (TUDCA) inhibited AGE-induced ER stress and cell death. Blocking RAGE?CNADPH oxidase, and RAGE?CNADPH oxidase?CROSs and ER stress scavenging pathways could efficiently prevent the oxidative and ER stresses, and consequently inhibited cell death. Our results suggest a new prevention and or therapeutic approach in AGE-induced cell death.     

Aminoguanidine and metformin prevent the reduced rate of HDL-mediated cell cholesterol efflux induced by formation of advanced glycation end products

OBJECTIVE: The mechanisms whereby advanced glycation end products (AGE) contribute to atherogenesis in diabetes mellitus are not fully understood. In this study we analyzed in vitro the influence of advanced glycated albumin (AGE-albumin) as well as the role of the AGE inhibitors--aminoguanidine (AMG) and metformin (MF)--on the cell cholesterol efflux. METHODS: HDL3 and albumin-mediated cholesterol efflux was measured in mouse peritoneal macrophages and in SR-BI transfected cells that had been treated along time with dicarbonyl sugars or AGE-albumin, both in the presence or in the absence of AMG and MF. 125I-HDL3 cell binding and 125I-AGE-albumin cell degradation were measured. Carboxymethyllysine (CML) formation and SR-BI expressions were determined by immunoblot. RESULTS: AGE-albumin efficiently trapped cell cholesterol but impaired the HDL-mediated cell cholesterol efflux by decreasing HDL binding to the cell surface and inducing intracellular glycoxidation, without interfering with the SR-BI expression. Cell treatment with dicarbonyl sugars also disrupted the HDL-mediated cell cholesterol efflux, but this was prevented by AMG and MF that reduced CML formation. CONCLUSIONS: By adversely impairing the HDL-mediated cell cholesterol removal rate, AGE-albumin and cell glycoxidation could facilitate the development of premature atherosclerosis in diabetes mellitus (DM) and in other diseases associated with carbonyl and oxidative stress like in chronic uremia. Thus, drugs that prevent AGE formation may be useful to correct disturbances in cell cholesterol transport.