Genome-wide association studies (GWAS) have identified genetic variants at 34 loci contributing to age-related macular degeneration (AMD) 1 – 3 . We generated transcriptional profiles of postmortem retina from 453 controls and cases at distinct stages of AMD and integrated retinal transcriptomes, covering 13,662 protein-coding and 1,462 non-coding genes, with genotypes at over 9 million common single nucleotide polymorphisms (SNPs) for expression quantitative trait loci (eQTL) analysis of a tissue not included in Genotype-Tissue Expression (GTEx) and other large datasets 4 , 5 . Cis -eQTL analysis identified 10,474 genes under genetic regulation, including 4,541 eQTLs detected only in the retina. Integrated analysis of AMD-GWAS with eQTLs ascertained likely target genes at six reported loci. Using transcriptome-wide association analysis (TWAS), we identified three additional genes, RLBP1 , HIC1 and PARP12 , after Bonferroni correction. Our studies expand the genetic landscape of AMD and establish the Eye Genotype Expression (EyeGEx) database as a resource for post-GWAS interpretation of multifactorial ocular traits.

Oxidative stress-induced damage to the retinal pigment epithelium (RPE) is considered to be a key factor in age-related macular degeneration (AMD) pathology. RPE cells are constantly exposed to oxidative stress that may lead to the accumulation of damaged cellular proteins, lipids, nucleic acids, and cellular organelles, including mitochondria. The ubiquitin-proteasome and the lysosomal/autophagy pathways are the two major proteolytic systems to remove damaged proteins and organelles. There is increasing evidence that proteostasis is disturbed in RPE as evidenced by lysosomal lipofuscin and extracellular drusen accumulation in AMD. Nuclear factor-erythroid 2-related factor-2 (NFE2L2) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) are master transcription factors in the regulation of antioxidant enzymes, clearance systems, and biogenesis of mitochondria. The precise cause of RPE degeneration and the onset and progression of AMD are not fully understood. However, mitochondria dysfunction, increased reactive oxygen species (ROS) production, and mitochondrial DNA (mtDNA) damage are observed together with increased protein aggregation and inflammation in AMD. In contrast, functional mitochondria prevent RPE cells damage and suppress inflammation. Here, we will discuss the role of mitochondria in RPE degeneration and AMD pathology focused on mtDNA damage and repair, autophagy/mitophagy signaling, and regulation of inflammation. Mitochondria are putative therapeutic targets to prevent or treat AMD.

Maintenance of protein homeostasis, also referred to as “Proteostasis”, integrates multiple pathways that regulate protein synthesis, folding, translocation, and degradation. Failure in proteostasis may be one of the underlying mechanisms responsible for the cascade of events leading to age-related macular degeneration (AMD). This review covers the major degradative pathways (ubiquitin-proteasome and lysosomal involvement in phagocytosis and autophagy) in the retinal pigment epithelium (RPE) and summarizes evidence of their involvement in AMD. Degradation of damaged and misfolded proteins via the proteasome occurs in coordination with heat shock proteins. Evidence of increased content of proteasome and heat shock proteins in retinas from human donors with AMD is consistent with increased oxidative stress and extensive protein damage with AMD. Phagocytosis and autophagy share key molecules in phagosome maturation as well as degradation of their cargo following fusion with lysosomes. Phagocytosis and degradation of photoreceptor outer segments ensures functional integrity of the neural retina. Autophagy rids the cell of toxic protein aggregates and defective mitochondria. Evidence suggesting a decline in autophagic flux includes the accumulation of autophagic substrates and damaged mitochondria in RPE from AMD donors. An age-related decrease in lysosomal enzymatic activity inhibits autophagic clearance of outer segments, mitochondria, and protein aggregates, thereby accelerating the accumulation of lipofuscin. This cumulative damage over a person’s lifetime tips the balance in RPE from a state of para-inflammation, which strives to restore cell homeostasis, to the chronic inflammation associated with AMD.

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.

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Age-related macular degeneration (AMD) is the leading cause of blindness among older adults in the developed world. Although the pathological mechanisms have not been definitively elucidated, evidence suggests a key role for mitochondrial (mt) dysfunction. The current study used our unique collection of human retinal samples graded for the donor's stage of AMD to address fundamental questions about mtDNA damage in the retina. To evaluate the distribution of mtDNA damage in the diseased retina, damage in the retinal pigment epithelium (RPE) and neural retina from individual donors were compared. To directly test a long-held belief that the macula is selectively damaged with AMD, RPE mtDNA damage was measured in the macula and peripheral sections from individual donors. Small segments of the entire mt genome were examined to determine whether specific regions are preferentially damaged. Our results show that mtDNA damage is limited to the RPE, equivalent mtDNA damage is found in the macular and peripheral RPE, and sites of damage are localized to regions of the mt genome that may impact mt function. These results provide a scientific basis for targeting the RPE mitochondria with therapies that protect and enhance mt function as a strategy for combating AMD.

The proteasome is the main protease for degrading oxidized proteins. We asked whether altered proteasome function contributes to the accumulation of oxidized muscle proteins with aging. Proteasome structure, function, and oxidation state were compared in young and aged F344BN rat fast-twitch skeletal muscle. In proteasome-enriched homogenates from aged muscle, we observed a two- to threefold increase in content of the 20S proteasome that was due to a corresponding increase in immunoproteasome. Content of the regulatory proteins, PA700 and PA28, relative to the 20S were reduced 75% with aging. Upon addition of exogenous PA700, there was a twofold increase in peptide hydrolysis in aged muscle, suggesting the endogenous content of PA700 is inadequate for complete activation of the 20S. Measures of catalytic activity showed a 50% reduction in specific activity for proteasome-enriched homogenates with aging. With purification of the 20S, proteasome specific activity was equivalent between ages, indicating that endogenous regulators inhibit proteasome in aged muscle. Significantly less degradation of oxidized calmodulin by the 20S from aged muscle was observed. Partial rescue of activity for aged 20S by DTT implies oxidation of functionally significant cysteines. These results demonstrate significant age-related changes in proteasome structure, function, and oxidation state that could inhibit removal of oxidized proteins.

We have investigated the mechanisms that target oxidized calmodulin for degradation by the proteasome. After methionine oxidation within calmodulin, rates of degradation by the 20 S proteasome are substantially enhanced. Mass spectrometry was used to identify the time course of the proteolytic fragments released from the proteasome. Oxidized calmodulin is initially degraded into large proteolytic fragments that are released from the proteasome and subsequently degraded into small peptides that vary in size from 6 to 12 amino acids. To investigate the molecular determinants that result in the selective degradation of oxidized calmodulin, we used circular dichroism and fluorescence spectroscopy to assess oxidant-induced structural changes. There is a linear correlation between decreases in secondary structure and the rate of degradation. Calcium binding or the repair of oxidized calmodulin by methionine sulfoxide reductase induces comparable changes in ␣-helical content and rates of degradation. In contrast, alterations in the surface hydrophobicity of oxidized calmodulin do not alter the rate of degradation by the proteasome, indicating that changes in surface hydrophobicity do not necessarily lead to enhanced proteolytic susceptibility. These results suggest that decreases in secondary structure expose proteolytically sensitive sites in oxidized calmodulin that are cleaved by the proteasome in a nonprocessive manner.Critical to the ability of a cell to reestablish cellular homeostasis after a range of different environmental stresses is the removal of oxidatively modified proteins by the proteasome (1, 2). The proteasome represents approximately 1% of the total cellular protein and is present in the cytosol and nuclei of all mammalian cells in two major forms (i.e. the 20 S and 26 S proteasomes) (3). The 20 S proteasome is a 700-kDa complex with a 10 -20-Å-diameter opening into an internal cavity that provides a sequestered environment for proteolysis. The 26 S proteasome is a 2000-kDa complex containing two 19 S regulatory complexes bound to the 20 S multicatalytic core. The 26 S proteasome is responsible for the degradation of the majority of cellular proteins through an ATP-dependent and ubiquitinmediated pathway (4 -6). In contrast, the 20 S proteasome core selectively degrades a range of different oxidized proteins in an ATP-independent manner and has been suggested to represent the primary mechanism in the rapid removal of oxidized proteins after oxidative stress (7-11). The signal for recognition and degradation of oxidized proteins by the 20 S proteasome is unknown but has been suggested to involve (i) exposure of hydrophobic surfaces after oxidative modification, (ii) recognition of molecular "markers" associated with the oxidative modification of specific amino acid side chains, and (iii) increases in the conformational flexibility of oxidized proteins (12-16).To distinguish which signals are involved in targeting an oxidized protein for degradation by the 20 S proteasome, we have investigated the mech...