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Age-related macular degeneration (AMD) is the leading, cause of sight loss in the elderly in the Western world. Most patients remain still without any treatment options. The targeting of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a transcription co-factor, is a putative therapy against AMD.The characteristics of AMD and their possible connection with PGC-1α as well as the transcriptional and post-transcriptional control of PGC-1α are discussed. The PGC-1α-driven control of mitochondrial functions, and its involvement in autophagy and antioxidant responses are also examined. Therapeutic possibilities via drugs and epigenetic approaches to enhance PGC-1α expression are discussed. Authors conducted a search of literature mainly from the recent decade from the PubMed database.Therapy options in AMD could include PGC-1α activation or stabilization. This could be achieved by a direct elevation of PGC-1α activity, a stabilization or modification of its upstream activators and inhibitors by chemical compounds, like 5-Aminoimidazole-4-carboxamide riboside, metformin, and resveratrol. Furthermore, manipulations with epigenetic modifiers of PGC-1α expression, including miRNAs, e.g. miR-204, are considered. A therapy aimed at PGC-1α up-regulation may be possible in other disorders besides AMD, if they are associated with disturbances in the mitochondria-antioxidant response-autophagy axis.

Age-related macular degeneration (AMD) is a complex eye disease in which decline in autophagy leads to the accumulation of sequestosome 1/p62 (SQSTM1/p62)-labeled waste material inside the retinal pigment epithelial (RPE) cells, and the condition results in activation of the inflammasome signaling and IL-1β secretion. Here, we have studied the role of SQSTM1/p62 in the production of IL-6, IL-8, and MCP-1 in the presence or absence of IL-1β. SQSTM1/p62 was either overexpressed or silenced in ARPE-19 cells, which were then exposed to IL-1β. Alternatively, bafilomycin A was used to demonstrate the functional decline of autophagy with increased SQSTM1/p62 levels. The protein concentration of SQSTM1/p62 was measured using the western blot technique, and interleukin levels were determined by ELISA. In IL-1β-loaded RPE cells, SQSTM1/p62 depletion and overexpression increased the production of MCP-1 and IL-8, respectively. Neither knock-down nor overexpression of SQSTM1/p62 induced the release of IL-6. Our data suggest that SQSTM1/p62 is a significant factor in inflammatory responses, especially following the inflammasome activation.

Age-related macular degeneration (AMD) is a progressive sight-impairing disease of the elderly. The pathogenic mechanisms of AMD are not well understood although both genetic and many environmental factors have been associated with the development of AMD. One clinical hallmark of AMD is the detrimental aggregation of damaged proteins. Recently, it has been suggested that the weakening of autophagy clearance is an important mechanism in the pathogenesis of AMD. Autophagy is important in the removal of damaged or no longer needed cellular material and its recycling. A considerable number of autophagy-targeting microRNAs (miRNAs), small RNA molecules and epigenetic regulators have been found to be either up- or down-regulated in AMD patients and experimental models. The important role of autophagy-targeting miRNAs is supported by several studies and can open the prospect of the use of these miRNAs in the therapy for AMD.

Mitochondrial dysfunction has been implicated in a wide variety of degenerative diseases, including age-related macular degeneration. Damage to mitochondria and mitochondrial DNA accumulates with age in the postmitotic retinal pigment epithelium (RPE), which could lead to RPE cell death and trigger disease. One possible mechanism for cells to avoid cell death is mitophagy, the targeted clearance of damaged mitochondria by autophagy. Here, we induced mitochondrial damage in human RPE cells (ARPE-19 and hRPE), using antimycin A, an inhibitor of complex III of the electron transport chain, and investigated cellular viability, mitochondrial structure and function, and autophagy activity. We observed that antimycin A evoked dose-dependent cell death, a rapid loss in mitochondrial membrane potential, and a collapse of oxidative phosphorylation. Mitochondria appeared swollen and there was clear damage to their cristae structure. At the same time, cells were undergoing active autophagy and were sensitive to autophagy inhibition by bafilomycin A1 or chloroquine. These results indicate that mitochondrial dysfunction can cause significant RPE damage and that autophagy is an important survival mechanism for cells suffering from mitochondrial damage.

PURPOSE. The cornea is continually exposed to highly energetic solar UV-B (280-320 nm). Our aim was to investigate whether UV-B triggers the activation of NLRP3 inflammasomes and the production of IL-1 beta and/or IL-18 in human corneal epithelial (HCE) cells. Additionally, we studied the capability of cis-urocanic acid (cis-UCA) to prevent inflammasome activation or alleviate inflammation through other signaling pathways. METHODS. HCE-2 cell line and primary HCE cells were primed using lipopolysaccharide or TNF-alpha. Thereafter, cells were exposed to UV-B before or after the addition of cis-UCA or caspase-1 inhibitor. Caspase-1 activity was measured from cell lysates by an enzymatic assay. IL-1 beta, IL-18, IL-6, IL-8, and NLRP3 levels were detected using the ELISA method from cell culture media. Additionally, intracellular NLRP3 levels were determined by the Western blot technique, and cytotoxicity was measured by the LDH assay. RESULTS. UV-B exposure significantly increased caspase-1 activity in TNF-alpha-primed HCE cells. This result was consistent with the concurrently induced IL-1 beta secretion. Both caspase-1 activity and release of IL-1 beta were reduced by cis-UCA. Additionally, UV-B stimulated the caspase-1-independent production of IL-18, an effect also reduced by cis-UCA. Cis-UCA decreased the release of IL-6, IL-8, and LDH in a time-dependent manner when administered to HCE-2 cells after UV-B exposure. CONCLUSIONS. Our findings demonstrate that UV-B activates inflammasomes in HCE cells. Cis-UCA can prevent the secretion of IL-1 beta and IL-18 and therapeutically reduces the levels of IL-6, IL-8, and LDH in UV-B-stressed HCE cells.

In this review we will discuss the links between autophagy, a mechanism involved in the maintenance of cellular homeostasis and controlling cellular waste management, and the DNA damage response (DDR), comprising various mechanisms preserving the integrity and stability of the genome. A reduced autophagy capacity in retinal pigment epithelium has been shown to be connected in the pathogenesis of age-related macular degeneration (AMD), an eye disease. This degenerative disease is a major and increasing cause of vision loss in the elderly in developed countries, primarily due to the profound accumulation of intra- and extracellular waste: lipofuscin and drusen. An abundance of reactive oxygen species is produced in the retina since this tissue has a high oxygen demand and contains mitochondria-rich cells. The retina is exposed to light and it also houses many photoactive molecules. These factors are clearly reflected in both the autophagy and DNA damage rates, and in both nuclear and mitochondrial genomes. It remains to be revealed whether DNA damage and DDR capacity have a more direct role in the development of AMD.

Age-related macular degeneration (AMD) is a mounting cause of loss of sight in the elderly in the developed countries, a trend enhanced by the continual ageing of the population. AMD is a multifactorial and only partly understood, malady. Unfortunately, there is no effective treatment for most AMD patients. It is known that oxidative stress (OS) damages the retinal pigment epithelium (RPE) and contributes to the progression of AMD. We review here the potential importance of two OS-related cellular systems in relation to AMD. First, the nuclear factor erythroid 2-related factor 2 (NFE2L2; NRF2)-mediated OS response signalling pathway is important in the prevention of oxidative damage and a failure of this system could be critical in the development of AMD. Second, epithelial-to-mesenchymal transition (EMT) represents a change in the cellular phenotype, which ultimately leads to the fibrosis encountered in RPE, a characteristic of AMD. Many of the pathways triggering EMT are promoted by OS. The possible interconnections between these two signalling routes are discussed here. From a broader perspective, the control of NFE2L2 and EMT as ways of preventing OS-derived cellular damage could be potentially valuable in the therapy of AMD.

DNA damage accumulates in aged postmitotic retinal pigment epithelium (RPE) cells, a phenomenon associated with the development of age-related macular degeneration. In this study, we have experimentally induced DNA damage by ultraviolet B (UVB) irradiation in interleukin-1α (IL-1α)-primed ARPE-19 cells and examined inflammasome-mediated signaling. To reveal the mechanisms of inflammasome activation, cells were additionally exposed to high levels of extracellular potassium chloride, n-acetyl-cysteine, or mitochondria-targeted antioxidant MitoTEMPO, prior to UVB irradiation. Levels of interleukin-18 (IL-18) and IL-1? mRNAs were detected with qRT-PCR and secreted amounts of IL-1?, IL-18, and caspase-1 were measured with ELISA. The role of nucleotide-binding domain and leucine-rich repeat pyrin containing protein 3 (NLRP3) in UVB-induced inflammasome activation was verified by using the NLRP3-specific siRNA. Reactive oxygen species (ROS) levels were measured immediately after UVB exposure using the cell-permeant 2?,7?-dichlorodihydrofluorescein diacetate (H2DCFDA) indicator, the levels of cyclobutane pyrimidine dimers were assayed by cell-based ELISA, and the extracellular levels of adenosine triphosphate (ATP) determined using a commercial bioluminescence assay. We found that pro-IL-18 was constitutively expressed by ARPE-19 cells, whereas the expression of pro-IL-1? was inducible by IL-1α priming. UVB induced the release of mature IL-18 and IL-1? but NLRP3 contributed only to the secretion of IL-1?. At the mechanistic level, the release of IL-1? was regulated by K+ efflux, whereas the secretion of IL-18 was dependent on ROS production. As well as K+ efflux, the cells released ATP following UVB exposure. Collectively, our data suggest that UVB clearly stimulates the secretion of mature IL-18 as a result of ROS induction, and this response is associated with DNA damage. Moreover, in human RPE cells, K+ efflux mediates the UVB-activated NLRP3 inflammasome signaling, leading to the processing of IL-1?.

The impairment of autophagic and proteasomal cleansing together with changes in pigmentation has been documented in retinal pigment epithelial (RPE) cell degeneration. However, the function and co-operation of these mechanisms in melanosome-containing RPE cells is still unclear. We show that inhibition of proteasomal degradation with MG-132 or autophagy with bafilomycin A1 increased the accumulation of premelanosomes and autophagic structures in human embryonic stem cell (hESC)-derived RPE cells. Consequently, upregulation of the autophagy marker p62 (also known as sequestosome-1, SQSTM1) was confirmed in Western blot and perinuclear staining. Interestingly, cells treated with the adenosine monophosphatedependent protein kinase activator, AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), decreased the proteasome inhibitor-induced accumulation of premelanosomes, increased the amount of autophagosomes and eradicated the protein expression of p62 and LC3 (microtubule-associated protein 1A/1B-light chain 3). These results revealed that autophagic machinery is functional in hESC-RPE cells and may regulate cellular pigmentation with proteasomes., published version, peerReviewed

CD44 is a ubiquitous cell surface glycoprotein, involved in important cellular functions including cell adhesion, migration, and modulation of signals from cell surface receptors. While most of these CD44 functions are supposed to involve hyaluronan, relatively little is known about the contribution of CD44 to hyaluronan maintenance and organization on cell surface, and the role of CD44 in hyaluronan synthesis and catabolism. Blocking hyaluronan binding either by CD44 antibodies, CD44-siRNA or hyaluronan decasaccharides (but not hexasaccharides) removed most of the hyaluronan from the surfaces of both human (HaCaT) and mouse keratinocytes, resembling results on cells from CD44⁻/⁻ animals. In vitro, compromising CD44 function led to reduced and increased amounts, respectively, of intracellular and culture medium hyaluronan, and specific accumulation below the cells. In vivo, CD44-deficiency caused no marked differences in hyaluronan staining intensity or localization in the fetal skin or in adult ear skin, while tail epidermis showed a slight reduction in epidermal hyaluronan staining intensity. However, CD44-deficient tail skin challenged with retinoic acid or tape stripping revealed diffuse accumulation of hyaluronan in the superficial epidermal layers, normally negative for hyaluronan. Our data indicate that CD44 retains hyaluronan in the keratinocyte pericellular matrix, a fact that has not been shown unambiguously before, and that hyaluronan abundance in the absence of CD44 can result in hyaluronan trapping in abnormal locations possibly interfering there with normal differentiation and epidermal barrier function.

The pathogenesis of age-related macular degeneration (AMD) essentially involves chronic oxidative stress, increased accumulation of lipofuscin in retinal pigment epithelial (RPE) cells and extracellular drusen formation, as well as the presence of chronic inflammation. The capacity to prevent the accumulation of cellular cytotoxic protein aggregates is decreased in senescent cells which may evoke lipofuscin accumulation into lysosomes in postmitotic RPE cells. This presence of lipofuscin decreases lysosomal enzyme activity and impairs autophagic clearance of damaged proteins which should be removed from cells. Proteasomes are another crucial proteolytic machine which degrade especially cellular proteins damaged by oxidative stress. This review examines the cross-talk between lysosomes, autophagy and proteasomes in RPE cell protein aggregation, their role as a possible therapeutic target and their involvement in the pathogenesis of AMD.

Estrogen has a variety of neuroprotective effects but the molecular basis of its function is still mainly unclear. Estrogen receptor (ER) signaling is highly dependent on posttranslational modifications and the assembly of coactivator and corepressor complexes. Several proteins involved in ER alpha signaling have recently been found to be acetylated, including ER alpha itself and Hsp90, a key chaperone in the functional regulation of ER alpha. ER alpha complexes also contain histone deacetylases (HDAC) which repress transactivation. Our purpose was to clarify the role of protein acetylation and Hsp90 function in the ERE-mediated ER alpha signaling in neuronal HN10 cells. We observed that increasing protein/histone acetylation status with trichostatin A, a potent HDAC inhibitor, increased the 17beta-estradiol (E2)-induced transactivation of ERE-driven luciferase in non-transfected cells, and even more extensively in pER alpha-transfected cells. E2-induced ERE-driven transactivation was blocked by ICI 182.780. Several ER antagonists, such as raloxifene and tamoxifen, were unresponsive. Valproate, an antiepileptic drug which is recently characterized as a HDAC inhibitor, was also able to potentiate the E2-induced ERE-transactivation. Inhibition of the function of Hsp90 chaperone with geldanamycin strongly inhibited the E2-induced ERE-transactivation. Overexpression of SIRT2 protein deacetylase did not inhibit the acetylation-potentiated ERE-driven transactivation indicating that SIRT2 deacetylase is not involved in ER alpha signaling. Our results reveal that ER alpha signaling is dependent on protein acetylation and epigenetic regulation.

Purpose SQSTM1/p62 is a central player in the selective autophagy directing ubiquitinated protein aggregates, mitochondria, and bacterial pathogens to autophagosomes. Autophagy culminates into the fusion of matured autophagosomes with lysosomes that contain degradative enzymes. One regulator of the fusion is Rab7, which also has a role in the movement of autophagosomes along microtubules. In this study, we have investigated the significance of SQSTM1/p62 in the functionality of human retinal pigment epithelial (RPE) cells and its possible role in the regulation of Rab7. Methods SQSTM1/p62 was blocked in ARPE-19 cells using spesific siRNA. Proteasomes were inhibited with MG-132, which has been shown to induce autophagy flux by providing more substrate to the autophagic machinery. Additionally, AMPK-activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) was used to induce autophagy. Rab7 protein levels were determined with western blot and the secretion of inflammatory cytokines using the ELISA method. Results According to our results, interference of autophagy by SQSTM1/p62 depletion, especially in the presence of other stimuli, such as MG-132 or AICAR, leads to the accumulation of Rab7. Along with higher amounts of Rab7, increased inflammatory response was observed. Conclusions Our results emphasize the importance of SQSTM1/p62 in the regulation of autophagy and inflammation in RPE cells that play the principal role in the pathogenesis of age-related macular degeneration (AMD).

Catechol-O-methyltransferase (COMT) catalyses the O-methylation of compounds having a catechol structure and its main function involves the elimination of biologically active or toxic catechols and their metabolites. By means of homologous recombination in embryonic stem cells, a strain of mice has been produced in which the gene encoding the COMT enzyme is disrupted. We report here the levels of catecholamines and their metabolites in striatal extracellular fluid in these mice as well as in homogenates from different parts of the brain, under normal conditions and after acute levodopa administration. In immunoblotting studies, COMT-knockout mice had no COMT protein in brain or kidney tissues but the amounts of catecholamine synthesizing and other metabolizing enzyme proteins were normal. Under normal conditions, COMT deficiency does not appear to affect significantly brain dopamine and noradrenaline levels in spite of relevant changes in their metabolites. This finding is consistent with previous pharmacological studies with COMT inhibitors and confirms the pivotal role of synaptic reuptake processes and monoamine oxidase-dependent metabolism in terminating the actions of catecholamines at nerve terminals. In contrast, when COMT-deficient mice are challenged with l-dihydroxyphenylalanine, they show an extensive accumulation of 3,4-dihydroxyphenylacetic acid and dihydroxyphenylglycol and even dopamine, revealing an important role for COMT under such situations. Notably, in some cases these changes appear to be Comt gene dosage-dependent, brain-region specific and sexually dimorphic. Our results may have implications for improving the treatment of Parkinson's disease and for understanding the contribution of the natural variation in COMT activity to psychiatric phenotypes.

Purpose The constant light exposure, high metabolic rate and high lipid concentration expose retinal pigment epithelial (RPE) cells to chronic oxidative stress, which may lead to RPE cell degeneration and evokes secondarily photoreceptor damage and visual loss. In this work, we assessed the protective role of wood derived polyphenol, pinosylvin (PS), in the prevention of oxidative stress and the molecular mechanisms behind these effects Methods ARPE-19 cells were exposed either to PS or hydroquinone (HQ) or both simultaneoulsy. Toxicity and protective effects of PS against HQ-induced oxidative stress were determined by colorimetric cell viability test (MTT-test). Signalling mechanism of PS was studied by analyzing mRNA levels of Nrf2 (nuclear factor-erythroid 2-related factor-2) and its target genes heme oxygenase-1 (HO-1), glutathione S-transferase pi 1 (GSTP1) and sequestosome 1 (p62) by PCR. Moreover, Nrf2 and p62 were silenced by using siRNA technology. Results PS treatment protected ARPE-19 cells from HQ, when analyzed by MTT assay. PS treatment significantly increased the expression of HO-1 that was abolished by Nrf2 siRNA . PS did not protect p62 siRNA-treated cells from HQ induced oxidative stress. Conclusion Our results suggest that PS treatment conferred protection against HQ-induced oxidative stress through the induction of HO-1 in ARPE-19 cells. Consequently, PS-stilbene compounds may possess health-promoting properties against oxidative stress-related diseases such as AMD.

Processing of misfolded proteins is important in order for the cell to maintain its normal functioning and homeostasis. Three systems control the quality of proteins: chaperone-mediated refolding, proteasomal degradation of ubiquitinated proteins, and finally, when the two others fail, aggrephagy, as selective form of autophagy, degrades ubiquitin-labelled aggregated cargos. In this route misfolded proteins gradually form larger aggregates, aggresomes and they eventually become double membrane-wrapped organelles called autophagosomes, which become degraded when they fuse to lysosomes, for reuse by the cell. The stages, the main molecules participating in the process, and the regulation of aggrephagy are discussed here, as is the role of protein aggregation in protein accumulation diseases. In particular, we emphasize that both Alzheimer's disease and age-related macular degeneration, two of the most common pathologies in the aged, are characterized by altered protein clearance and deposits. Based on the hypothesis that manipulations of autophagy may be potentially useful in these and other aggregation-related diseases, we will discuss some promising therapeutic strategies to counteract protein aggregates-induced cellular toxicity.

Hyaluronan is a ubiquitous glycosaminoglycan involved in embryonic development, inflammation and cancer. In mammals, three hyaluronan synthase isoenzymes (HAS1-3) inserted in the plasma membrane produce hyaluronan directly on cell surface. The mRNA level and enzymatic activity of HAS1 are lower than those of HAS2 and HAS3 in many cells, obscuring the importance of HAS1. Here we demonstrate using immunocytochemistry and transfection of fluorescently tagged HAS1 that its enzymatic activity depends on the ER-Golgi-plasma membrane traffic, like reported for HAS2 and HAS3. When cultured in 5 mM glucose, HAS1-transfected MCF-7 cells show very little cell surface hyaluronan, detected with a fluorescent hyaluronan binding probe. However, a large hyaluronan coat was seen in cells grown in 20 mM glucose and 1 mM glucosamine, or treated with IL-1β, TNF-α, or TGF-β. The coats were mostly removed by the presence of hyaluronan hexasaccharides, or Hermes1 antibody, indicating that they depended on the CD44 receptor, which is in a contrast to the coat produced by HAS3, remaining attached to HAS3 itself. The findings suggest that HAS1-dependent coat is induced by inflammatory agents and glycemic stress, mediated by altered presentation of either CD44 or hyaluronan, and can offer a rapid cellular response to injury and inflammation.

Mammals have three homologous genes encoding proteins with hyaluronan synthase activity (Has1-3), all producing an identical polymer from UDP-N-acetylglucosamine and UDP-glucuronic acid. To compare the properties of these isoenzymes, COS-1 cells, with minor endogenous hyaluronan synthesis, were transfected with human Has1-3 isoenzymes. HAS1 was almost unable to secrete hyaluronan or form a hyaluronan coat, in contrast to HAS2 and HAS3. This failure of HAS1 to synthesize hyaluronan was compensated by increasing the cellular content of UDP-N-acetyl glucosamine by ∼10-fold with 1 mm glucosamine in the growth medium. Hyaluronan synthesis driven by HAS2 was less affected by glucosamine addition, and HAS3 was not affected at all. Glucose-free medium, leading to depletion of the UDP-sugars, markedly reduced hyaluronan synthesis by all HAS isoenzymes while raising its concentration from 5 to 25 mm had a moderate stimulatory effect. The results indicate that HAS1 is almost inactive in cells with low UDP-sugar supply, HAS2 activity increases with UDP-sugars, and HAS3 produces hyaluronan at high speed even with minimum substrate content. Transfected Has2 and particularly Has3 consumed enough UDP-sugars to reduce their content in COS-1 cells. Comparison of different human cell types revealed ∼50-fold differences in the content of UDP-N-acetylhexosamines and UDP-glucuronic acid, correlating with the expression level of Has1, suggesting cellular coordination between Has1 expression and the content of UDP-sugars.

3-Chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF), a chlorine disinfection by-product in drinking water, was mutagenic in the Salmonella/his (Ames) assay for both base-pair substitution strains (TA1535, TA100, TA102) and frameshift strains (TA97, TA98) with the highest mutagenic response observed in strain TA100 (1292 revertants/microgram). The presence in TA100 of pKM101 plasmid, which enhances error-phone DNA repair, greatly increased susceptibility to CMCF mutagenicity relative to the isogenic strain TA1535 lacking pKM101. In the Chinese hamster ovary (CHO) cell hprt (6-thioguanine resistance) locus assay, the mutagenicity of CMCF (1.04 mutants/10(6) clonable cells per microgram/ml) was barely detectable because of the low mutagenicity/cytotoxicity ratio. From the present experiments it appears that CMCF acts in a manner similar to that of another drinking water mutagen, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). However, CMFC appears to be a less potent mutagen in vitro than MX.

The mutagenicity of chlorinated humic drinking waters is accounted for mainly by a single contaminant, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), as assessed in Salmonella typhimurium strain TA100. In the present study 3,4-dichloro-5-hydroxy-2(5H)-furanone (mucochloric acid, MA), another drinking water contaminant much less potent as a mutagen in TA100 than MX, was tested in Chinese hamster ovary (CHO) cells for the induction of mutation at the hypoxanthine phosphoribosyl transferase (hprt) locus to 6-thioguanine resistance (TGr). Unexpectedly, MA induced TGr mutants in CHO cells with a potency comparable to that reported previously for MX. In subsequent experiments with S. typhimurium, the presence of pKM101 plasmid in strain TA100 increased susceptibility to the mutagenicity of MA, but much less than to that of MX, relative to the parental strain TA1535 lacking pKM101. The difference between the two compounds in TA100 thus appears to be due to a higher enhancement of the mutagenicity of MX than that of MA by pKM101 mediated error-prone DNA repair.

Macroautophagy is an important route in cellular maintenance, in the breakdown and reuse of intracellular materials. It is closely related to endocytosis, the means by which the cell can absorb extracellular material, as both macroautophagy and endocytosis have converging steps and common participating molecules. The point where autophagosomes and endosomes fuse with lysosomes to permit for the final degradation of their contents is important. One of the most substantial molecules in the maturation of autophagosomes/endosomes is Rab7, a member of small GTPases. Rab7 designates the maturation of endosomes and also autophagosomes, directing the trafficking of cargos along microtubules, and finally, participating in the fusion step with lysosomes. Rab7 is an effective multifunctional regulator of autophagy and endocytosis. Since many aggregation-based diseases, e.g. age-related macular degeneration of the eye (AMD) and Alzheimer's disease are due of malfunctioning in the autophagic process, the management of Rab7 activity might hold potential as a therapeutic target against these diseases.

Age-related macular degeneration (AMD) is the most common reason for blindness in developed countries. AMD essentially involves chronic oxidative stress, increased accumulation of lipofuscin in retinal pigment epithelial (RPE) cells, and extracellular drusen formation, as well as presence of chronic inflammation in the retina. The capacity to prevent the accumulation of cellular cytotoxic protein aggregates is decreased in senescent cells, which may evoke lipofuscin accumulation into lysosomes in postmitotic RPE cells. The formation of lipofuscin, in turn, decreases the lysosomal enzyme activity and impairs the autophagic clearance of damaged proteins destined for cellular removal. 5'-Adenosine monophosphate-activated protein kinase (AMPK) is a well-known inhibitor of mammalian target of rapamycin (mTOR) that subsequently evokes induction of autophagy. This review examines the novel potential therapeutic targets on the AMPK-mTOR axis and the ways in which autophagy clearance can suppress or prevent RPE degeneration and development of AMD.

Purpose The pathogenesis of age-related macular degeneration involves impaired protein degradation in retinal pigment epithelial (RPE) cells. The ubiquitin-proteasome pathway and the lysosomal pathway including autophagy are the major proteolytic systems in eukaryotic cells. Recently, p62/sequestosome 1 (p62) has been shown to be a key player linking the proteasomal and lysosomal clearance systems. In the present study, the effects of AICAR (AICA ribonucleotide, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) and MG-132 (proteasome inhibitor) on autophagy regulation in ARPE-19 cells were evaluated. Methods The AMP activated protein kinase (AMPK), p62 and ubiquitin protein levels were analyzed by western blotting. pDendra2-hLC3 construct was used to detect macroautophagy in confocal microscopy analysis. Transmission electron microscopy was used to detect protein aggregates and autophagosomes. Cellular permeability was measured by analyzing lactate dehydrogenase levels in culture medium. Results MG-132 (5 microM) triggered the accumulation of perinuclear aggregates that strongly colocalized with p62 and ubiquitin. AICAR (2mM) induced autophagy clearance of p62 and ubiquitin positive protein aggregates without increasing cellular permeability. Cellular energy status regulator AMPK or p-AMPK levels were not significantly changed in response to AICAR treatment. Conclusion Our findings open new avenues for understanding the mechanisms of proteolytic processes and indicate that AICAR could be useful in the acceleration of protein clearance in RPE cells.

Adenosine monophosphate-activated protein kinase (AMPK) is a crucial regulator of energy metabolic homeostasis and thus a major survival factor in a variety of metabolic stresses and also in the aging process. Metabolic syndrome is associated with a low-grade, chronic inflammation, primarily in adipose tissue. A low-level of inflammation is also present in the aging process. There are emerging results indicating that AMPK signaling can inhibit the inflammatory responses induced by the nuclear factor-κB (NF-κB) system. The NF-κB subunits are not direct phosphorylation targets of AMPK, but the inhibition of NF-κB signaling is mediated by several downstream targets of AMPK, e.g., SIRT1, PGC-1α, p53, and Forkhead box O (FoxO) factors. AMPK signaling seems to enhance energy metabolism while it can repress inflammatory responses linked to chronic stress, e.g., in nutritional overload and during the aging process. AMPK can inhibit endoplasmic reticulum and oxidative stresses which are involved in metabolic disorders and the aging process. Interestingly, many target proteins of AMPK are so-called longevity factors, e.g., SIRT1, p53, and FoxOs, which not only can increase the stress resistance and extend the lifespan of many organisms but also inhibit the inflammatory responses. The activation capacity of AMPK declines in metabolic stress and with aging which could augment the metabolic diseases and accelerate the aging process. We will review the AMPK pathways involved in the inhibition of NF-κB signaling and suppression of inflammation. We also emphasize that the capacity of AMPK to repress inflammatory responses can have a significant impact on both healthspan and lifespan.

Purpose Retinal pigment epithelial (RPE) cells are continually exposed to oxidative stress that contributes to protein misfolding, aggregation and functional abnormalities during aging. The protein aggregates formed at the cell periphery are delivered along the microtubulus network by dynein dependent retrograde trafficking to a juxtanuclear location. Methods Cellular organelles were analysed by transmission electron microscopy of ARPE-19 cells exposed 5 µM MG-132, 0.25 µM geldanamycin (GA), 1 µM trichostatin A (TSA), 1 µM taxol (TAX) or 5 µM nocodazole (NOC) for 24 hours. In addition, the cells were treated simultaneously with GA or TSA or TAX or NOC and MG-132 up to 24 hours. Ubiquitin, Hsp90, Hsp70, acetylated tubulin and Hsc70 protein levels were analyzed by western blotting. Results Hsp90 inhibition by geldanamycin can effectively suppress proteasome inhibitor, MG-132 –induced protein aggregation in a way that is an independent of HDAC inhibition, or the tubulin acetylation levels in ARPE-19 cells. However, the tubulin acetylation and polymerization state affects the localization of the proteasome-inhibitor –induced aggregation. Conclusion Hsp90 inhibition is effectively related to regulation of protein aggregation that is independent of HDAC inhibition or tubulin acetylation levels in the RPE cells. Our findings open new perspectives for understanding the pathogenesis of protein aggregation in retinal cells and can be useful for the development of therapeutic treatments to prevent retinal cell deterioration.

Age-related macular degeneration (AMD) can be classified into two main categories: the atrophic, dry form and the exudative, wet form. The crucial difference between dry and wet AMD is the development of choroidal neovascularization in wet AMD. One fundamental cause of the neovascularization is the increased expression of VEGF (vascular endothelial growth factor) in retinal pigment epithelial cells. Progression of AMD is linked to augmentation of cellular stress, for example, oxidative stress, proteotoxic stress, inflammation and hypoxia. All these conditions can trigger stress in endoplasmic reticulum (ER), which maintains protein quality control in cells. ER stress induces the unfolded protein response (UPR) via IRE1 (inositol-requiring protein-1), PERK (protein kinase RNA-like ER kinase) and ATF6 (activating transcription factor-6) transducers. UPR signaling is a double-edged sword, that is, it can restore cellular homeostasis as far as possible, but ultimately may lead to chronic, overwhelming stress that can cause apoptotic cell death. Interestingly, ER stress is a well-known inducer of angiogenesis in cancer. Moreover, stress conditions associated with the progress of AMD can induce the expression of VEGF. We discuss the role of ER stress in the regulation of neovascularization and the conversion of dry AMD to its wet, detrimental counterpart.

Since estrogen and selective estrogen receptor modulators can inhibit inflammatory responses, we studied the regulatory role of several selective estrogen receptor modulators on interleukin-6 (IL-6) expression in human retinal pigment epithelial cells (ARPE-19). ARPE-19 cells were exposed to lipopolysaccharide with simultaneous exposure to different selective estrogen receptor modulators with the secretion of IL-6 cytokine being analyzed by enzyme-linked immunosorbent assay (ELISA). We demonstrate that 17beta-estradiol and HM-D, a novel selective estrogen receptor modulator compound, clearly reduced the IL-6 expression levels after lipopolysaccharide exposure in ARPE-19 cells. Molecular effects of selective estrogen receptor modulators and estrogen on the estrogen response element-mediated transcription were studied using MCF-7 and ARPE-19 cell lines carrying the estrogen response element-luciferase reporter gene. Estrogen and HM-D stimulated the activity of estrogen response element-reporter gene in MCF-7 cells but did not affect the activity in ARPE-19 cells. In addition, HM-D did not activate estrogen receptor alpha when studied by nuclear receptor peptide estrogen receptor alpha ELISA in ARPE-19 cells. These results indicate that estrogen and HM-D can suppress the lipopolysaccharide-induced inflammatory response but signalling is not mediated through estrogen response element transcription in human retinal pigment epithelial cells.

The pathogenesis of age-related macular degeneration involves chronic oxidative stress, impaired degradation of membranous discs shed from photoreceptor outer segments and accumulation of lysosomal lipofuscin in retinal pigment epithelial (RPE) cells. It has been estimated that a major part of cellular proteolysis occurs in proteasomes, but the importance of proteasomes and the other proteolytic pathways including autophagy in RPE cells is poorly understood. Prior to proteolysis, heat shock proteins (Hsps), agents that function as molecular chaperones, attempt to refold misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. In the present study, the roles of the Hsp70 molecular chaperone and proteasomal and lysosomal proteolytic pathways were evaluated in human RPE cells (ARPE-19). The Hsp70 and ubiquitin protein levels and localization were analysed by Western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. Hsp70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. The proteasome inhibitor MG-132 evoked the accumulation of perinuclear aggregates positive for Hsp70, ubiquitin-protein conjugates and the lysosomal membrane protein LAMP-2. Interestingly, the hsp70 mRNA depletion significantly increased cell death in conjunction with proteasome inhibition. We found that the accumulation of lysosomes was reversible: a cessation of proteasome inhibition led to clearance of the deposits via a mechanism believed to include autophagy. The molecular chaperone Hsp70, proteasomes and autophagy have an important regulatory role in the protein turnover of human RPE cells and may thus open new avenues for understanding degenerative processes in retinal cells.

Purpose: Pathogenesis of AMD involves chronic oxidative stress, impaired degradation of membranous discs and accumulation of lysosomal lipofuscin in retinal pigment epithelial (RPE) cells. It has been estimated that a major part of cellular proteolysis occurs however in proteasomes. Prior to the proteolysis heat shock proteins tend to refold misfolded proteins and thus prevent the accumulation of protein aggregates. Connection of Hsp70, lysosomes and proteasomes were evaluated in the protein aggregation process in human RPE cells (ARPE-19). Methods: Cellular localization of Hsp70 was studied by immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and morphological changes in ARPE-19 cells. Cell viability was analyzed by MTT -assay. The modulations of Hsp70 levels were done by using siRNA and overexpression vector techniques. Results: Proteasome inhibitor caused formation of juxtanuclear protein aggregates. It also caused robust accumulation of Hsp70 protein which colocalized with the protein aggregates. The granulation of aggregates was clearly increased in response to proteasome inhibition and simultaneous hsp70 mRNA interference. We found that protein aggregation is an interim, a cease of proteasome inhibition led to autophagosome-mediated cleaning of cytoplasmic protein aggregates. The Hsp70 colocalization was observed in both primary lysosomal fractions and in autophagosomes. Conclusions: This study reveals that Hsp70, lysosomes and protesomes are connected each other in macroautophagy -mediated proteolysis in human RPE cells.

Hyaluronan is a multifunctional glycosaminoglycan up to 10(7) Da molecular mass produced by the integral membrane glycosyltransferase, hyaluronan synthase (HAS). When expressed in keratinocytes, N-terminally tagged green fluorescent protein-HAS2 and -HAS3 isoenzymes were found to travel through endoplasmic reticulum (ER), Golgi, plasma membrane, and endocytic vesicles. A distinct enrichment of plasma membrane HAS was found in cell protrusions. The total turnover time of HAS3 was 4-5 h as judged by the green fluorescent protein signal decay and hyaluronan synthesis inhibition in cycloheximide-treated cells. The transfer from ER to Golgi took about 1 h, and the dwell time on the plasma membrane was less than 2 h in experiments with a relief and introduction, respectively, of brefeldin A. Constructs of HAS3 with 16- and 45-amino-acid C-terminal deletions mostly stayed within the ER, whereas a D216A missense mutant was localized within the Golgi complex but not the plasma membrane. Both types of mutations were almost or completely inactive, similar to the wild type enzyme that had its entry to the plasma membrane experimentally blocked by brefeldin A. Inhibition of hyaluronan synthesis by UDP-glucuronic acid starvation using 4-methyl-umbelliferone also prevented HAS access to the plasma membrane. The results demonstrate that 1) a latent pool of HAS exists within the ER-Golgi pathway; 2) this pool can be rapidly mobilized and activated by insertion into the plasma membrane; and 3) inhibition of HAS activity through mutation or substrate starvation results in exclusion of HAS from the plasma membrane.

The kinds of mutations induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in the protein coding region of the hprt gene of Chinese hamster ovary (CHO) cells were determined by direct sequencing of polymerase chain reaction (PCR)-amplified cDNA. Primary mutations were found in 15 of 19 of the mutants: 11 were G:C-->T:A transversions, two were A:T-->T:A transversions and two were deletions of single G:C base pairs (-1 frameshifts). The remaining four mutants had large alterations in the cDNA that were explained by mRNA splicing errors. A group of control mutants had more diverse hprt cDNA alterations than MX-induced mutants. Transversions yielding an A:T base pair were the predominant type of MX-induced mutations, in agreement with previous findings in bacteria. This specificity may be explained by the 'A rule', that DNA polymerases preferentially insert adenine nucleotides opposite non-instructional lesions.

Keratinocyte growth factor (KGF) activates keratinocyte migration and stimulates wound healing. Hyaluronan, an extracellular matrix glycosaminoglycan that accumulates in wounded epidermis, is known to promote cell migration, suggesting that increased synthesis of hyaluronan might be associated with the KGF response in keratinocytes. Treatment of monolayer cultures of rat epidermal keratinocytes led to an elongated and lifted cell shape, increased filopodial protrusions, enhanced cell migration, accumulation of intermediate size hyaluronan in the culture medium and within keratinocytes, and a rapid increase of hyaluronan synthase 2 (Has2) mRNA, suggesting a direct influence on this gene. In stratified, organotypic cultures of the same cell line, both Has2 and Has3 with the hyaluronan receptor CD44 were up-regulated and hyaluronan accumulated in the epidermis, the spinous cell layer in particular. At the same time the expression of the early differentiation marker keratin 10 was inhibited, whereas filaggrin expression and epidermal permeability were less affected. The data indicate that Has2 and Has3 belong to the targets of KGF in keratinocytes, and support the idea that enhanced hyaluronan synthesis acts an effector for the migratory response of keratinocytes in wound healing, whereas it may delay keratinocyte terminal differentiation.

Elina Penttilä,1 Juha MT Hyttinen,2 Maria Hytti,2 Anu Kauppinen,2,3 Grigori Smirnov,1,4 Henri Tuomilehto,1,4 Juha Seppä,1 Juhani Nuutinen,1 Kai Kaarniranta2,31Department of Otorhinolaryngology, Kuopio University Hospital, and Institute of Clinical Medicine, University of Eastern Finland, 2Department of Ophthalmology, Institute of Clinical Medicine, University of Eastern Finland, 3Department of Ophthalmology, Kuopio University Hospital, 4Oivauni Sleep Clinic, Kuopio, FinlandBackground: Epiphora is a common complaint of nasolacrimal duct obstruction (NLDO) in adults. The precise pathogenesis of NLDO is still unknown, but inflammatory processes are believed to be predisposing factors. Endoscopic dacryocystorhinostomy (EN-DCR) is an effective surgical technique for treating symptomatic NLDO. The purpose of the procedure is to relieve the patient's symptoms by creating an opening, ie, a rhinostoma, between the lacrimal sac and the nasal cavity. Although the success rates after EN-DCR are high, the procedure sometimes fails due to onset of a fibrotic process at the rhinostomy site. The aim of this prospective comparative study was to investigate inflammation-related gene expression in the nasal mucosa at the rhinostomy site.Methods: Ten participants were consecutively recruited from eligible adult patients who underwent primary powered EN-DCR (five patients) or septoplasty (five controls). Nasal mucosa specimens were taken from the rhinostomy site at the beginning of surgery for analysis of gene expression. Specimens were taken from the same site on the lateral nasal wall for controls. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed for the inflammatory genes interleukin (IL)-6, IL-1ß, and CCL2, and because of a clear trend of increased inflammation in the EN-DCR samples, a wider PCR array was performed to compare inflammation-related gene expression in EN-DCR subjects and corresponding controls.Results: Our qRT-PCR results revealed a clear trend of increased transcription of IL-6, IL-1ß, and CCL2 (P=0.03). The same trend was also evident in the PCR array, which additionally revealed notable differences between EN-DCR subjects and controls with regard to expression of several other inflammation-related mediators. At 6-month follow-up, the success rate after primary EN-DCR was 60%, ie, in three of five patients.Conclusion: The present study demonstrates that there is an intense inflammation gene expression response in the nasal mucosa of patients undergoing EN-DCR.Keywords: epiphora, fibrosis, dacryocystorhinostomy, gene expression, inflammation, nasolacrimal duct obstruction

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is a potent direct-acting mutagen found in chlorinated drinking water. In the present study, the induction of DNA strand breaks and apurinic/apyrimidinic (AP) sites by MX in supercoiled PM2 DNA was examined using exonuclease III, which specifically cleaves the DNA at AP sites. The results showed that MX induced AP sites in great excess of direct strand breaks. In view of the known mutagenicity of AP sites, these results provide insight into the mechanism of MX-induced mutagenesis.

The mutational specificity of three chlorohydroxyfuranones found in chlorinated drinking water, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), 3-chloro-4(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF) and 3,4-dichloro-5-hydroxy-2(5H)-furanone (mucochloric acid, MCA), was examined in Salmonella typhimurium strain TA100. DNA colony-hybridization of TA100 revertants showed that MX and CMCF both induced predominantly G:C-->T:A transversions (87 and 75% of total, respectively) with a 3:1 preference for the second position of the hisG46 (CCC) target codon. By contrast, MCA produced primarily G:C-->A:T transitions (66% of the total) with a 4:1 preference for the second position of the CCC codon. The mutational specificity of MCA is consistent with the idea that chloroacetaldehyde, a degradation product of MCA, is responsible for the observed mutations. The chemical mechanism by which either MX or CMCF induces G:C-->T:A transversions remains unknown.

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a potent direct-acting Salmonella mutagen found in chlorinated drinking water, was tested in Chinese hamster ovary (CHO) cells for the induction of mutation at the hypoxanthine phosphoribosyl transferase locus to 6-thioguanine resistance (TGr). MX treatment of CHO cells for 3 h at 37 degrees C resulted in significant dose-related increases in mutant frequency. The lowest observed effective dose was 2.5 micrograms/ml, where the cloning efficiency estimated on the day after treatment was not affected. The relationship between the dose of MX and the frequency of TGr mutants was approximately linear over the range of 0-5 micrograms/ml with an estimated slope (+/- 95% confidence limits) of 7.2 +/- 2.6 mutants per 10(6) clonable cells per microgram/ml.

Hyaluronan, a major extracellular matrix molecule in the vital cell layers of skin epidermis, has been suggested to support proliferation and migration of keratinocytes, during challenges like wounding and inflammation. An organotypic keratinocyte culture originated from continuous rat epidermal keratinocyte cell line was subjected to the proliferative and antiproliferative growth factors epidermal growth factor and transforming growth factor beta, respectively, to study their influence on hyaluronan synthesis and epidermal morphology. Epidermal growth factor induced a 4-fold increase of epidermal hyaluronan concentration. This was associated with upregulation of the hyaluronan synthases Has2 and Has3, and the hyaluronan receptor CD44. 5-Bromo-2'-deoxyuridine labeling, basal cell height, and the thickness of vital epidermis were increased, reflecting the hyperplastic effects of epidermal growth factor. The expression of keratin 10 and the maturation of filaggrin were inhibited, and epidermal permeability barrier became less efficient, indicating compromised terminal differentiation by epidermal growth factor. In contrast, transforming growth factor beta reduced the content of hyaluronan and the mRNA of Has2 and Has3. At the same time, transforming growth factor beta suppressed keratinocyte proliferation and epidermal thickness, but retained intact differentiation. The results suggest that epidermal hyaluronan synthesis, controlled by epidermal growth factor and transforming growth factor beta through changes in the expression of Has2 and Has3, correlates with epidermal proliferation, thickness, and differentiation.

Several pre-clinical and clinical studies have demonstrated zoledronic acid (Zol), which regulates the mevalonate pathway, has efficient anti-cancer effects. Zol can also induce autophagy. The aim of this study is to add new understanding to the mechanism of autophagy induction by Zol. LC3B-II, the marker for autophagy was increased by Zol treatment in breast cancer cells. Autophagosomes induced by Zol were visualized and quantified in both transient (pDendra2-hLC3) and stable MCF-7-GFP-LC3 cell lines. Acidic vesicular organelles were quantified using acridine orange. Zol induced a dose and time dependent autophagy. Treatment of Zol increased oxidative stress in MCF-7 cells, which was reversed by GGOH or anti-oxidants. On the other hand, treatment with GGOH or anti-oxidants resulted in decreased levels of LC3B-II. Further, the induced autophagy was irreversible, as the washout of Zol after 2 h or 24 h resulted in similar levels of autophagy, as induced by continuous treatment after 72 h. Thus, it can be summarized that Zol can induce a dose dependent but irreversible autophagy, by its effect on the mevalonate pathway and oxidative stress. This study adds to the understanding of the mechanism of action of Zol, and that it can induce autophagy at clinically relevant shorter exposure times in cancer cells.