Ali Koskela

Polyphenol Stilbenes: Molecular Mechanisms of Defence against Oxidative Stress and Aging-Related Diseases

Numerous studies have highlighted the key roles of oxidative stress and inflammation in aging-related diseases such as obesity, type 2 diabetes, age-related macular degeneration (AMD), and Alzheimers disease (AD). In aging cells, the natural antioxidant capacity decreases and the overall efficiency of reparative systems against cell damage becomes impaired. There is convincing data that stilbene compounds, a diverse group of natural defence phenolics, abundant in grapes, berries, and conifer bark waste, may confer a protective effect against aging-related diseases. This review highlights recent data helping to clarify the molecular mechanisms involved in the stilbene-mediated protection against oxidative stress. The impact of stilbenes on the nuclear factor-erythroid-2-related factor-2 (Nrf2) mediated cellular defence against oxidative stress as well as the potential roles of SQSTM1/p62 protein in Nrf2/Keap1 signaling and autophagy will be summarized. The therapeutic potential of stilbene compounds against the most common aging-related diseases is discussed.

Autophagy regulates death of retinal pigment epithelium cells in age-related macular degeneration

Age-related macular degeneration (AMD) is an eye disease underlined by the degradation of retinal pigment epithelium (RPE) cells, photoreceptors, and choriocapillares, but the exact mechanism of cell death in AMD is not completely clear. This mechanism is important for prevention of and therapeutic intervention in AMD, which is a hardly curable disease. Present reports suggest that both apoptosis and pyroptosis (cell death dependent on caspase-1) as well as necroptosis (regulated necrosis dependent on the proteins RIPK3 and MLKL, caspase-independent) can be involved in the AMD-related death of RPE cells. Autophagy, a cellular clearing system, plays an important role in AMD pathogenesis, and this role is closely associated with the activation of the NLRP3 inflammasome, a central event for advanced AMD. Autophagy can play a role in apoptosis, pyroptosis, and necroptosis, but its contribution to AMD-specific cell death is not completely clear. Autophagy can be involved in the regulation of proteins important for cellular antioxidative defense, including Nrf2, which can interact with p62/SQSTM, a protein essential for autophagy. As oxidative stress is implicated in AMD pathogenesis, autophagy can contribute to this disease by deregulation of cellular defense against the stress. However, these and other interactions do not explain the mechanisms of RPE cell death in AMD. In this review, we present basic mechanisms of autophagy and its involvement in AMD pathogenesis and try to show a regulatory role of autophagy in RPE cell death. This can result in considering the genes and proteins of autophagy as molecular targets in AMD prevention and therapy.

Variation in the anthocyanin concentration of wild populations of crowberries (Empetrum nigrum L subsp. hermaphroditum).

Crowberry (Empetrum nigrum L.) is a relatively under-utilized wild berry that occurs widely throughout the northern hemisphere such as in Canada, Eurasia, and northern Europe. In this work, the anthocyanins of crowberries were analyzed from four geographically distinct crowberry populations in Finland using HPLC-DAD and HPLC-ESI/MS/MS. A total number of 15 anthocyanins were detected; 15 (11 structure elucidated) in all samples in order to profile-specific anthocyanin compositions throughout Finland. The major anthocyanin found in the samples collected from central and eastern Finland was delphinidin 3-galactoside accounting for more than 24% of the total anthocyanin content, while the cyanidin 3-galactoside was the major anthocyanin in the northernmost and in the western samples. Significant variation in the concentrations of different anthocyanins between and within crowberry populations were found suggesting that the synthesis of anthocyanins is modified by site-specific environmental conditions. The suitability of the crowberries as a potential source of health-promoting ingredients for incorporation into pharmaceutical and food industrial products is highlighted in this work due to the diverse anthocyanin profile.

Nutraceutical with Resveratrol and Omega-3 Fatty Acids Induces Autophagy in ARPE-19 Cells

Impaired autophagic and proteasomal cleansing have been documented in aged retinal pigment epithelial (RPE) cells and age-related macular degeneration (AMD). Omega-3 fatty acids and resveratrol have many positive homeostatic effects in RPE cells. In this work, ARPE-19 cells were treated with 288 ng of Resvega, containing 30 mg of trans resveratrol and 665 mg of omega-3 fatty acids, among other nutrients, with proteasome inhibitor MG-132 or autophagy inhibitor bafilomycin A1 up to 48 h. Autophagy markers p62/SQSTM1 (p62) and LC3 (microtubule-associated protein 1A/1B-light chain 3) were analyzed by Western blotting. Fluorescence microscopy with mCherry-GFP-LC3 plasmid was applied to study the autophagy flux, and cytoprotective effects were investigated with colorimetric MTT and LDH assays. Resvega induced autophagy by showing increased autolysosome formation and autophagy flux, and the change in the p62 and LC3 protein levels further confirmed the fluorescent microscopy results. Moreover, Resvega provided a clear cytoprotection under proteasome inhibition. These findings highlight the potential of the nutraceuticals containing resveratrol, omega-3 fatty acids and other nutrients in the prevention of ARPE-19 cell damage.

Autophagy regulates proteasome inhibitor-induced pigmentation in human embryonic stem cell-derived retinal pigment epithelial cells

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.

Autophagy Stimulus Promotes Early HuR Protein Activation and p62/SQSTM1 Protein Synthesis in ARPE-19 Cells by Triggering Erk1/2, p38MAPK, and JNK Kinase Pathways

RNA-binding protein dysregulation and altered expression of proteins involved in the autophagy/proteasome pathway play a role in many neurodegenerative disease onset/progression, including age-related macular degeneration (AMD). HuR/ELAVL1 is a master regulator of gene expression in human physiopathology. In ARPE-19 cells exposed to the proteasomal inhibitor MG132, HuR positively affects at posttranscriptional level p62 expression, a stress response gene involved in protein aggregate clearance with a role in AMD. Here, we studied the early effects of the proautophagy AICAR + MG132 cotreatment on the HuR-p62 pathway. We treated ARPE-19 cells with Erk1/2, AMPK, p38MAPK, PKC, and JNK kinase inhibitors in the presence of AICAR + MG132 and evaluated HuR localization/phosphorylation and p62 expression. Two-hour AICAR + MG132 induces both HuR cytoplasmic translocation and threonine phosphorylation via the Erk1/2 pathway. In these conditions, p62 mRNA is loaded on polysomes and its translation in de novo protein is favored. Additionally, for the first time, we report that JNK can phosphorylate HuR, however, without modulating its localization. Our study supports HuRs role as an upstream regulator of p62 expression in ARPE-19 cells, helps to understand better the early events in response to a proautophagy stimulus, and suggests that modulation of the autophagy-regulating kinases as potential therapeutic targets for AMD may be relevant.

Loss of NRF-2 and PGC-1α genes leads to retinal pigment epithelium damage resembling dry age-related macular degeneration

Age-related macular degeneration (AMD) is a multi-factorial disease that is the leading cause of irreversible and severe vision loss in the developed countries. It has been suggested that the pathogenesis of dry AMD involves impaired protein degradation in retinal pigment epithelial cells (RPE). RPE cells are constantly exposed to oxidative stress that may lead to the accumulation of damaged cellular proteins, DNA and lipids and evoke tissue deterioration during the aging process. The ubiquitin-proteasome pathway and the lysosomal/autophagosomal pathway are the two major proteolytic systems in eukaryotic cells. NRF-2 (nuclear factor-erythroid 2-related factor-2) and PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) are master transcription factors in the regulation of cellular detoxification. We investigated the role of NRF-2 and PGC-1α in the regulation of RPE cell structure and function by using global double knockout (dKO) mice. The NRF-2/PGC-1α dKO mice exhibited significant age-dependent RPE degeneration, accumulation of the oxidative stress marker, 4-HNE (4-hydroxynonenal), the endoplasmic reticulum stress markers GRP78 (glucose-regulated protein 78) and ATF4 (activating transcription factor 4), and damaged mitochondria. Moreover, levels of protein ubiquitination and autophagy markers p62/SQSTM1 (sequestosome 1), Beclin-1 and LC3B (microtubule associated protein 1 light chain 3 beta) were significantly increased together with the Iba-1 (ionized calcium binding adaptor molecule 1) mononuclear phagocyte marker and an enlargement of RPE size. These histopathological changes of RPE were accompanied by photoreceptor dysmorphology and vision loss as revealed by electroretinography. Consequently, these novel findings suggest that the NRF-2/PGC-1α dKO mouse is a valuable model for investigating the role of proteasomal and autophagy clearance in the RPE and in the development of dry AMD.

Fatty acids and oxidized lipoproteins contribute to autophagy and innate immunity responses upon the degeneration of retinal pigment epithelium and development of age-related macular degeneration

Retinal pigment epithelium (RPE) damage is a primary sign in the development of age-related macular degeneration (AMD) the leading cause of blindness in western countries. RPE cells are exposed to chronic oxidative stress due to constant light exposure, active fatty acid metabolism and high oxygen consumption. RPE cells phagocytosize lipid rich photoreceptor outer segment (POS) which is regulated by circadian rhytmn. Docosahexaenoic acid is present in high quantity in POS and increases oxidative stress, while its metabolites have cytoprotective effects in RPE. During RPE aging, reactive oxygen species and oxidized lipoproteins are considered to be major causes of disturbed autophagy clearance that lead to chronic innate immunity response involved in NOD-Like, Toll-Like, Advanced Glycation End product Receptors (NLRP, TLR, RAGE, respectively), pentraxins and complement systems. We discuss role of fatty acids and lipoproteins in the degeneration of RPE and development of AMD.

Loss of Nrf-2 and PGC1-alpha genes changes macromorphology of the eye and evokes microstructural and pigmentation pattern changes of the retinal pigmented epithelium

Purpose Nrf2 (NF-E2-related factor 2) and PGC1- α (peroxisome proliferator-activated receptor-gamma coactivator 1-alpha) regulate oxidative stress response in cells. Nrf-2 and PGC1-alpha double-knock-out (dKO) mice were used to monitor macro and morphological changes of eye, retina and retinal pigmented epithelium (RPE), respectively. Methods The mMRI and mCT imaging were carried out for mice aged at 6 weeks (mCT), 12 weeks and 12 months (mMRI). The retinal and RPE microanatomy and pigmentation were studied from HE-stained thin wax and toluidine blue-stained epoxy sections. Finally, cellular proliferation and pigmentation patterns were studied in the primary cell cultures. Results The dKO samples showed smaller body parameters and weight. mMRI, mCT assays indicated size differences and dysmorphic body features in the dKO mice. Moreover, dKOs exhibited reduced retinal full tickness joined with retained RPE morphology. The melanosomes of dKO RPEs were heterogeneous in shape morphology, but comparable in size to aged matched wild type melanosomes, respectively. However, there was a striking trend of increase in the density of melanosomes in RPE of dKOs that was convinced by the primary RPE cultures. Conclusions Nrf-2/PGC-1α knockout mice provide a novel model to study degenerative changes in retina and RPE.