PGC-1α regulates the interplay between oxidative stress, senescence and autophagy in the ageing retina important in age-related macular degeneration.

We previously showed that mice with knockout in the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) gene encoding the PGC-1α protein, and nuclear factor erythroid 2 like 2 (NFE2L2) gene, exhibited some features of the age-related macular degeneration (AMD) phenotype. To further explore the mechanism behind the involvement of PGC-1α in AMD pathogenesis we used young (3-month) and old (12-month) mice with knockout in the PPARGC1A gene and age-matched wild-type (WT) animals. An immunohistochemical analysis showed age-dependent different expression of markers of oxidative stress defence, senescence and autophagy in the retinal pigment epithelium of KO animals as compared with their WT counterparts. Multivariate inference testing showed that senescence and autophagy proteins had the greatest impact on the discrimination between KO and WT 3-month animals, but proteins of antioxidant defence also contributed to that discrimination. A bioinformatic analysis showed that PGC-1α might coordinate the interplay between genes encoding proteins involved in antioxidant defence, senescence and autophagy in the ageing retina. These data support importance of PGC-1α in AMD pathogenesis and confirm the utility of mice with PGC-1α knockout as an animal model to study AMD pathogenesis.

Mitochondrial damage and clearance in retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is a devastating eye disease that causes permanent vision loss in the central part of the retina, known as the macula. Patients with such severe visual loss face a reduced quality of life and are at a 1.5 times greater risk of death compared to the general population. Currently, there is no cure for or effective treatment for dry AMD. There are several mechanisms thought to underlie the disease, for example, ageing-associated chronic oxidative stress, mitochondrial damage, harmful protein aggregation and inflammation. As a way of gaining a better understanding of the molecular mechanisms behind AMD and thus developing new therapies, we have created a peroxisome proliferator-activated receptor gamma coactivator 1-alpha and nuclear factor erythroid 2-related factor 2 (PGC1α/NFE2L2) double-knockout (dKO) mouse model that mimics many of the clinical features of dry AMD, including elevated levels of oxidative stress markers, damaged mitochondria, accumulating lysosomal lipofuscin and extracellular drusen-like structures in retinal pigment epithelial cells (RPE). In addition, a human RPE cell-based model was established to examine the impact of non-functional intracellular clearance systems on inflammasome activation. In this study, we found that there was a disturbance in the autolysosomal machinery responsible for clearing mitochondria in the RPE cells of one-year-old PGC1α/NFE2L2-deficient mice. The confocal immunohistochemical analysis revealed an increase in autophagosome marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) as well as multiple mitophagy markers such as PTE-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase (PARKIN), along with signs of damaged mitochondria. However, no increase in autolysosome formation was detected, nor was there a colocalization of the lysosomal marker LAMP2 or the mitochondrial marker, ATP synthase ß. There was an upregulation of late autolysosomal fusion Ras-related protein (Rab7) in the perinuclear space of RPE cells, together with autofluorescent aggregates. Additionally, we observed an increase in the numbers of Toll-like receptors 3 and 9, while those of NOD-like receptor 3 were decreased in PGC1α/NFE2L2 dKO retinal specimens compared to wild-type animals. There was a trend towards increased complement component C5a and increased involvement of the serine protease enzyme, thrombin, in enhancing the terminal pathway producing C5a, independent of C3. The levels of primary acute phase C-reactive protein and receptor for advanced glycation end products were also increased in the PGC1α/NFE2L2 dKO retina. Furthermore, selective proteasome inhibition with epoxomicin promoted both nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondrial-mediated oxidative stress, leading to the release of mitochondrial DNA to the cytosol, resulting in potassium efflux-dependent activation of the absent in melanoma 2 (AIM2) inflammasome and the subsequent secretion of interleukin-1ß in ARPE-19 cells. In conclusion, the data suggest that there is at least a relative decrease in mitophagy, increases in the amounts of C5 and thrombin and decreased C3 levels in this dry AMD-like model. Moreover, selective proteasome inhibition evoked mitochondrial damage and AIM2 inflammasome activation in ARPE-19 cells.

Oxidative Stress and Cellular Protein Accumulation Are Present in Keratoconus, Macular Corneal Dystrophy, and Fuchs Endothelial Corneal Dystrophy.

The aim of the study was to investigate oxidative stress as well as cellular protein accumulation in corneal diseases including keratoconus (KC), macular corneal dystrophy (MCD), and Fuchs endothelial corneal dystrophy (FECD) at their primary affecting sites. Corneal buttons from KC, MCD, and FECD patients, as well as healthy controls, were analyzed immunohistochemically to evaluate the presence of oxidative stress and the function of the proteostasis network. 4-Fydroxynonenal (4-HNE) was used as a marker of oxidative stress, whereas the levels of catalase and heat-shock protein 70 (HSP70) were analyzed to evaluate the response of the antioxidant defense system and molecular chaperones, respectively. Sequestosome 1 (SQSTM1) levels were determined to assess protein aggregation and the functionality of autophagic degradation. Basal epithelial cells of the KC samples showed increased levels of oxidative stress marker 4-HNE and antioxidant enzyme catalase together with elevated levels of HSP70 and accumulation of SQSTM1. Corneal stromal cells and endothelial cells from MCD and FECD samples, respectively, showed similarly increased levels of these markers. All corneal diseases showed the presence of oxidative stress and activation of the molecular chaperone response to sustain protein homeostasis. However, the accumulation of protein aggregates suggests insufficient function of the protective mechanisms to limit the oxidative damage and removal of protein aggregates via autophagy. These results suggest that oxidative stress has a role in KC, MCD, and FECD at the cellular level as a secondary outcome. Thus, antioxidant- and autophagy-targeted therapies could be included as supporting care when treating KC or corneal dystrophies.

Pinosylvin Extract Retinari™ Sustains Electrophysiological Function, Prevents Thinning of Retina, and Enhances Cellular Response to Oxidative Stress in NFE2L2 Knockout Mice.

Chronic oxidative stress eventually leads to protein aggregation in combination with impaired autophagy, which has been observed in age-related macular degeneration. We have previously shown an effective age-related macular degeneration disease model in mice with nuclear factor-erythroid 2-related factor-2 (NFE2L2) knockout. We have also shown pinosylvin, a polyphenol abundant in bark waste, to increase human retinal pigment epithelium cell viability in vitro. In this work, the effects of commercial natural pinosylvin extract, Retinari™, were studied on the electroretinogram, optical coherence tomogram, autophagic activity, antioxidant capacity, and inflammation markers. Wild-type and NFE2L2 knockout mice were raised until the age of 14.8 ± 3.8 months. They were fed with either regular or Retinari™ chow (141 ± 17.0 mg/kg/day of pinosylvin) for 10 weeks before the assays. Retinari™ treatment preserved significant retinal function with significantly preserved a- and b-wave amplitudes in the electroretinogram responses. Additionally, the treatment prevented thinning of the retina in the NFE2L2 knockout mice. The NFE2L2 knockout mice showed reduced ubiquitin-tagged protein accumulation in addition to local upregulation of complement factor H and antioxidant enzymes superoxide dismutase 1 and catalase. Therefore, the treatment in the NFE2L2 KO disease model led to reduced chronic oxidative stress and sustained retinal function and morphology. Our results demonstrate that pinosylvin supplementation could potentially lower the risk of age-related macular degeneration onset and slow down its progression.

Disturbed mitochondrial function restricts glutamate uptake in the human Müller glia cell line, MIO-M1.

Using the human Müller cell line, MIO-M1, the aim was to study the impact of mitochondrial inhibition in Müller glia through antimycin A treatment. MIO-M1 cell survival, levels of released lactate, mitochondrial function, and glutamate uptake were studied in response to mitochondrial inhibition and glucose restriction. Lactate release decreased in response to glucose restriction. Combined glucose restriction and blocked mitochondrial activity decreased survival and caused collapse of the respiratory chain measured by oxygen consumption rate and extracellular acidification rate. Mitochondrial inhibition caused impaired glutamate uptake and decreased mRNA expression of the glutamate transporter, EAAT1. Over all, we show important roles of mitochondrial activity in MIO-M1 cell function and survival.