AßPP-induced UPR Transcriptomic Signature of Glial Cells to Oxidative Stress as an Adaptive Mechanism to Preserve Cell Function and Survival.

BACKGROUND: Alzheimer's disease (AD) and age-related macular degeneration (AMD) present similarities, particularly with respect to oxidative stress, including production of 4-Hydroxy-2- nonenal (HNE). AMD has been named the AD in the eye. The Müller cells (MC) function as a principal glia of the retina and maintain water/potassium, glutamate homeostasis and redox status. Any MC dysfunction results in retinal neurodegeneration. OBJECTIVES: We investigated the effects of HNE in human MC. RESULTS: HNE induced an increase of the reactive oxygen species associated with mitochondrial dysfunction and apoptosis. HNE induced endoplasmic reticulum (ER) stress (upregulation of GRP78/Bip, and the proapoptotic factor, CHOP). HNE also impaired expression of genes controlling potassium homeostasis (KCNJ10), glutamate detoxification (GS), and the visual cycle (RLBP1). MC adaptive response to HNE included upregulation of amyloid-ß protein precursor (AßPP). To determine the role of AßPP, we overexpressed AßPP in MC. Overexpression of AßPP induced strong antioxidant and anti-ER stress (PERK downregulation and GADD34 upregulation) responses accompanied by activation of the prosurvival branch of the unfolded protein response. It was also associated with upregulation of major genes involved in MC-controlled retinal homeostasis (KCNJ10, GS, and RLBP1) and protection against HNE-induced apoptosis. Therefore, AßPP is an ER and oxidative stress responsive molecule, and is able to stimulate the transcription of major genes involved in MC functions impaired by HNE. CONCLUSION: Our study suggests that targeting oxidative and ER stress might be a potential therapeutic strategy against glia impairment in AMD and AD, in light of the common features between the two pathologies.

Technological approaches to minimize industrial trans fatty acids in foods.

Trans fatty acids (TFAs) mainly arise from 2 major sources: natural ruminal hydrogenation and industrial partial catalytic hydrogenation. Increasing evidence suggests that most TFAs and their isomers cause harmful health effects (that is, increased risk of cardiovascular diseases). Nevertheless, in spite of the existence of an international policy consensus regarding the need for public health action, several countries (for example, France) do not adopt sufficient voluntary approaches (for example, governmental regulations and systematic consumer rejections) nor sufficient industrial strategies (for example, development of healthier manufacturing practices and innovative processes such as fat interesterifications) to eliminate deleterious TFAs from processed foods while ensuring the overall quality of the final product (for example, nutritional value and stability). In this manuscript, we first review the physical-chemical properties of TFAs, their occurrence in processed foods, their main effects on health, and the routine analytical methods to characterize TFAs, before emphasizing on the major industrial methods (that is, fat food reformulation, fat interesterification, genetically modified FAs composition) that can be used worldwide to reduce TFAs in foods.

Trans-fatty acids, dangerous bonds for health? A background review paper of their use, consumption, health implications and regulation in France.

INTRODUCTION: Trans-fatty acids (TFAs) can be produced either from bio-hydrogenation in the rumen of ruminants or by industrial hydrogenation. While most of TFAs' effects from ruminants are poorly established, there is increasing evidence that high content of industrial TFAs may cause deleterious effects on human health and life span. MATERIAL AND METHODS: Indeed, several epidemiological and experimental studies strongly suggest that high content of most TFA isomers could represent a higher risk of developing cardiovascular diseases by a mechanism that lowers the "good HDL cholesterol" and raises the "bad LDL cholesterol." RESULTS: With respect to the general precautionary principle and considering the existence of an international policy consensus regarding the need for public health action, some industrialized countries, such as France, are still not sufficiently involved in preventive strategies that aim to efficiently reduce TFAs content and TFAs consumption and produce alternative healthier fat sources. CONCLUSION: In this manuscript, we provide an overview about TFAs origins, their use and consumption among French population. We also discuss their potential human health implications as well as the preventive and regulatory measures undertaken in France.

Distinct effects of inflammation on gliosis, osmohomeostasis, and vascular integrity during amyloid beta-induced retinal degeneration.

In normal retinas, amyloid-ß (Aß) accumulates in the subretinal space, at the interface of the retinal pigment epithelium, and the photoreceptor outer segments. However, the molecular and cellular effects of subretinal Aß remain inadequately elucidated. We previously showed that subretinal injection of Aß(1-42) induces retinal inflammation, followed by photoreceptor cell death. The retinal Müller glial (RMG) cells, which are the principal retinal glial cells, are metabolically coupled to photoreceptors. Their role in the maintenance of retinal water/potassium and glutamate homeostasis makes them important players in photoreceptor survival. This study investigated the effects of subretinal Aß(1-42) on RMG cells and of Aß(1-42)-induced inflammation on retinal homeostasis. RMG cell gliosis (upregulation of GFAP, vimentin, and nestin) on day 1 postinjection and a proinflammatory phenotype were the first signs of retinal alteration induced by Aß(1-42). On day 3, we detected modifications in the protein expression patterns of cyclooxygenase 2 (COX-2), glutamine synthetase (GS), Kir4.1 [the inwardly rectifying potassium (Kir) channel], and aquaporin (AQP)-4 water channels in RMG cells and of the photoreceptor-associated AQP-1. The integrity of the blood-retina barrier was compromised and retinal edema developed. Aß(1-42) induced endoplasmic reticulum stress associated with sustained upregulation of the proapoptotic factors of the unfolded protein response and persistent photoreceptor apoptosis. Indomethacin treatment decreased inflammation and reversed the Aß(1-42)-induced gliosis and modifications in the expression patterns of COX-2, Kir4.1, and AQP-1, but not of AQP-4 or GS. Nor did it improve edema. Our study pinpoints the adaptive response to Aß of specific RMG cell functions.

CCR2/CCL2-mediated inflammation protects photoreceptor cells from amyloid-ß-induced apoptosis.

Age-related macular degeneration is characterized by the formation of drusen containing amyloid-ß (Aß) and the degeneration of photoreceptors. To explore the largely unknown role of Aß in the retina, we investigated the effects on photoreceptors of the oligomeric form of Aß(1-42). Subretinal injection of the Aß peptide induced misplaced expression of recoverin and synaptophysin in the photoreceptors, oxidative stress in their inner and outer segments, and finally apoptosis. Aß did not induce cell death in purified photoreceptor cell cultures, but did so in retinal cell cultures, thereby suggesting that the cellular environment plays a role in Aß-induced photoreceptor apoptosis. Subretinal injection of Aß was followed by activation and migration of microglial cells and then by photoreceptor apoptosis. Microglial cells phagocytosed rhodopsin-containing debris and Aß in the subretinal space. Quantitative RT-PCR allowed us to identify a specific gene expression profile associated with the Aß-induced progression of retinal degeneration and consistent with oxidative stress, inflammation, and an apoptotic program. The gene most highly upregulated in Aß-injected retinas was that for the chemokine CCL2, and its absence or that of its cognate receptor CCR2 greatly reduced migration of activated microglial cells to the site of retinal injury and profoundly worsened photoreceptor degeneration and disorganization of the retinal pigment epithelium in Aß-injected retinas. Our study pinpoints the roles of Aß and of CCL2/CCR2 axis-dependent inflammation in photoreceptor apoptosis.

Prematurely senescent ARPE-19 cells display features of age-related macular degeneration.

The etiology of age-related macular degeneration (AMD), the leading cause of blindness in the developed world, remains poorly understood, but may be related to cumulative oxidative stress. The prime target of the disease is the retinal pigmented epithelium (RPE). To study the molecular mechanisms underlying RPE degeneration, we investigated whether repetitive oxidative stress induced premature senescence in RPE cells from the human ARPE-19 cell line. After exposure to 8 mM tert-butylhydroperoxide (tert-BHP) for 1 h daily for 5 days, the cells showed four well-known senescence biomarkers: hypertrophy, senescence-associated beta-galactosidase activity, growth arrest, and cell cycle arrest in G1. A specific low-density array followed by qRT-PCR validation allowed us to identify 36 senescence-associated genes differentially expressed in the prematurely senescent cells. Functional analysis demonstrated that premature senescence induced amyloid beta secretion, resistance to acute stress by tert-BHP and amyloid beta, and defects in adhesion and transepithelial permeability. Coculture assays with choroidal endothelial cells showed the proangiogenic properties of the senescent RPE cells. These results demonstrate that chronic oxidative stress induces premature senescence in RPE cells that modifies the transcriptome and substantially alters cell processes involved in the pathophysiology of AMD. Oxidative stress-induced premature senescence may represent an in vitro model for screening therapeutics against AMD and other retinal degeneration disorders.

Sustained versus transient ERK1/2 signaling underlies the anti- and proapoptotic effects of oxidative stress in human RPE cells.

PURPOSE: Oxidative stress is thought to contribute to the pathogenesis of age-related macular degeneration (AMD), which involves retinal pigmented epithelial (RPE) cell death. However, signaling pathways involved in the oxidative-stress-induced RPE cell death are poorly understood. This study was conducted to investigate the involvement of the MAP kinase pathways during the induction of RPE cell death by oxidative stress. METHODS: ARPE-19 cells were exposed to the oxidant tert-butyl hydroperoxide (t-BHP). Cell viability was assessed by cell counting and MTT-staining, and apoptosis was quantified by TUNEL and flow cytometry. Activation of JNK1/3, p38 alphabeta MAPKs and ERK1/2 and their potential targets was detected by Western blot analysis and immunochemistry with specific anti-phospho protein antibodies. Specific pharmacologic inhibitors directed against the MAPKs were used to analyze the signaling involved in cell death of RPE cells exposed to t-BHP. RESULTS: Exposure of RPE cells to t-BHP, associated with increase in reactive oxygen species and intracellular glutathione depletion, induced time- and concentration-dependent apoptosis, which was associated with the accumulation of inactive ERK1/2 in cell nuclei and a transient and weak ERK1/2 activation. This activation was accompanied by a deactivation of P90(RSK), the major target of ERK1/2 and consequently by the delayed activation of its transcription factor CREB. MEK1/2 inhibition completely suppressed the transient activation of ERK1/2 and completely blocked apoptosis, demonstrating the role of the MEK-ERK module in mediating oxidative-stress-induced RPE cell death. In contrast, neither JNKs nor p38 alphabeta MAPKs were involved in mediating t-BHP-induced apoptotic signaling in RPE cells. CONCLUSIONS: The results suggest that inhibiting the MEK-ERK module may allow the development of selective methods for treating oxidative-stress-induced RPE degeneration, such as AMD.

[The teaching of the biology of ageing in France 1990-2005: DEA with the course of speciality of Master Seeks]. / L'enseignement de la biologie du vieillissement en France 1990-2005: du DEA au parcours de spécialité de Master Recherche.

In 1990, following an idea arising from an Inserm study section on aging, the Diplôme d'études approfondies (DEA) de Biologie du vieillissement was created. Since then, more than 300 students have followed these courses which cover the cellular mechanisms of aging and associated diseases, from basic causes of aging to CNS and sensory organs aging, as well as nutritional aspects, sarcopenia and osteoporosis, vascular and neuroendocrine aging. More than 150 thesis have been defended and more than a quarter of students has been recruited on permanent positions in French universities and research institutions (10 %) and hospitals (16 %). Since its creation, one of the particularities of the DEA was the formal links between academia and industry since teaching takes place on private laboratory settings.

L-DNase II associated with active process during ethanol induced cell death in ARPE-19.

PURPOSE: To analyse the mechanism of ethanol-induced cell death, and particularly, the activation of the leucocyte elastase inhibitor (LEI) pathway. METHODS: Cultured ARPE-19 cells were exposed to 0-13% ethanol for 24 h. Cytotoxicity was estimated by morphologic changes within the nucleus and breakdown of DNA, assessed by agarose gel electrophoresis or flow cytometry cell sorter. Poly(ADP-ribose)polymerase cleavage (PARP) was determined by western blot analysis. Changes in transcription and translation of LEI were assessed by analysis of mRNA levels and expression of protein product (immunohistochemistry), respectively. RESULTS: We established the ability of ethanol to induce cell death in ARPE-19 cells. After a 24 h incubation with 4% ethanol, 50% of the cells died; all the cells died in the presence of 10% ethanol. After ethanol incubation, we observed nuclear condensation and DNA fragmentation; the amount of fragmentation was proportional to the ethanol level. By flow cytometry analysis and agarose gel electrophoresis, the pattern of DNA cleavage exhibited a sub-G1 peak, suggesting necrotic cell death. However, other observations, i.e. nuclei shrinkage, PARP cleavage and inhibition of cell death by cycloheximide, and activation of a caspase independent LEI/DNase II pathway were observed and are features associated with apoptotic cell death. During ethanol stress, an LEI/L-DNase II intermediate was lost, leading to complete activation L-DNase II (24 kDa). RT-PCR analysis showed an early and specific increase of the LEI mRNA. Cycloheximide inhibited LEI synthesis and protected cells against apoptosis. CONCLUSIONS: Our data indicate that ethanol stress on ARPE-19 cells can induce a pathway which is a form of programmed cell death with characteristics of both apoptosis and necrosis, possibly by triggering conversion of LEI to L-DNase II.