The marine n-3 PUFA DHA evokes cytoprotection against oxidative stress and protein misfolding by inducing autophagy and NFE2L2 in human retinal pigment epithelial cells

Accumulation and aggregation of misfolded proteins is a hallmark of several diseases collectively known as proteinopathies. Autophagy has a cytoprotective role in diseases associated with protein aggregates. Age-related macular degeneration (AMD) is the most common neurodegenerative eye disease that evokes blindness in elderly. AMD is characterized by degeneration of retinal pigment epithelial (RPE) cells and leads to loss of photoreceptor cells and central vision. The initial phase associates with accumulation of intracellular lipofuscin and extracellular deposits called drusen. Epidemiological studies have suggested an inverse correlation between dietary intake of marine n-3 polyunsaturated fatty acids (PUFAs) and the risk of developing neurodegenerative diseases, including AMD. However, the disease-preventive mechanism(s) mobilized by n-3 PUFAs is not completely understood. In human retinal pigment epithelial cells we find that physiologically relevant doses of the n-3 PUFA docosahexaenoic acid (DHA) induce a transient increase in cellular reactive oxygen species (ROS) levels that activates the oxidative stress response regulator NFE2L2/NRF2 (nuclear factor, erythroid derived 2, like 2). Simultaneously, there is a transient increase in intracellular protein aggregates containing SQSTM1/p62 (sequestosome 1) and an increase in autophagy. Pretreatment with DHA rescues the cells from cell cycle arrest induced by misfolded proteins or oxidative stress. Cells with a downregulated oxidative stress response, or autophagy, respond with reduced cell growth and survival after DHA supplementation. These results suggest that DHA both induces endogenous antioxidants and mobilizes selective autophagy of misfolded proteins. Both mechanisms could be relevant to reduce the risk of developing aggregate-associate diseases such as AMD.     

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.     

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.     

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.     

Proteotoxic Stress Induces Phosphorylation of p62/SQSTM1 by ULK1 to Regulate Selective Autophagic Clearance of Protein Aggregates

Disruption of proteostasis, or protein homeostasis, is often associated with aberrant accumulation of misfolded proteins or protein aggregates. Autophagy offers protection to cells by removing toxic protein aggregates and injured organelles in response to proteotoxic stress. However, the exact mechanism whereby autophagy recognizes and degrades misfolded or aggregated proteins has yet to be elucidated. Mounting evidence demonstrates the selectivity of autophagy, which is mediated through autophagy receptor proteins (e.g. p62/SQSTM1) linking autophagy cargos and autophagosomes. Here we report that proteotoxic stress imposed by the proteasome inhibition or expression of polyglutamine expanded huntingtin (polyQ-Htt) induces p62 phosphorylation at its ubiquitin-association (UBA) domain that regulates its binding to ubiquitinated proteins. We find that autophagy-related kinase ULK1 phosphorylates p62 at a novel phosphorylation site S409 in UBA domain. Interestingly, phosphorylation of p62 by ULK1 does not occur upon nutrient starvation, in spite of its role in canonical autophagy signaling. ULK1 also phosphorylates S405, while S409 phosphorylation critically regulates S405 phosphorylation. We find that S409 phosphorylation destabilizes the UBA dimer interface, and increases binding affinity of p62 to ubiquitin. Furthermore, lack of S409 phosphorylation causes accumulation of p62, aberrant localization of autophagy proteins and inhibition of the clearance of ubiquitinated proteins or polyQ-Htt. Therefore, our data provide mechanistic insights into the regulation of selective autophagy by ULK1 and p62 upon proteotoxic stress. Our study suggests a potential novel drug target in developing autophagy-based therapeutics for the treatment of proteinopathies including Huntington’s disease.     

Autophagy and KRT8/keratin 8 protect degeneration of retinal pigment epithelium under oxidative stress

Contribution of autophagy and regulation of related proteins to the degeneration of retinal pigment epithelium (RPE) in age-related macular degeneration (AMD) remain unknown. We report that upregulation of KRT8 (keratin 8) as well as its phosphorylation are accompanied with autophagy and attenuated with the inhibition of autophagy in RPE cells under oxidative stress. KRT8 appears to have a dual role in RPE pathophysiology. While increased expression of KRT8 following autophagy provides a cytoprotective role in RPE, phosphorylation of KRT8 induces pathologic epithelial-mesenchymal transition (EMT) of RPE cells under oxidative stress, which is mediated by MAPK1/ERK2 (mitogen-activated protein kinase 1) and MAPK3/ERK1. Inhibition of autophagy further promotes EMT, which can be reversed by inhibition of MAPK. Thus, regulated enhancement of autophagy with concurrent increased expression of KRT8 and the inhibition of KRT8 phosphorylation serve to inhibit oxidative stress-induced EMT of RPE cells as well as to prevent cell death, suggesting that pharmacological manipulation of KRT8 upregulation through autophagy with combined inhibition of the MAPK1/3 pathway may be attractive therapeutic strategies for the treatment of AMD.     

MOAP‐1‐mediated dissociation of p62/SQSTM1 bodies releases Keap1 and suppresses Nrf2 signaling

Nrf2 signaling is vital for protecting cells against oxidative stress. However, its hyperactivation is frequently found in liver cancer through excessive build‐up of p62/SQSTM1 bodies that sequester Keap1, an adaptor of the E3‐ubiquitin ligase complex for Nrf2. Here, we report that the Bax‐binding protein MOAP‐1 regulates p62‐Keap1‐Nrf2 signaling through disruption of p62 bodies. Upon induction of cellular stresses that stimulate formation of p62 bodies, MOAP‐1 is recruited to p62 bodies and reduces their levels independent of the autophagy pathway. MOAP‐1 interacts with the PB1‐ZZ domains of p62 and interferes with its self‐oligomerization and liquid–liquid phase separation, thereby disassembling the p62 bodies. Loss of MOAP‐1 can lead to marked upregulation of p62 bodies, enhanced sequestration of Keap1 by p62 and hyperactivation of Nrf2 antioxidant target genes. MOAP‐1‐deficient mice exhibit an elevated tumor burden with excessive levels of p62 bodies and Nrf2 signaling in a diethylnitrosamine (DEN)‐induced hepatocarcinogenesis model. Together, our data define MOAP‐1 as a negative regulator of Nrf2 signaling via dissociation of p62 bodies.     

Sorting, recognition and activation of the misfolded protein degradation pathways through macroautophagy and the proteasome

Based on a functional categorization, proteins may be grouped into three types and sorted to either the proteasome or the macroautophagy pathway for degradation. The two pathways are mechanistically connected but their capacity seems different. Macroautophagy can degrade all forms of misfolded proteins whereas proteasomal degradation is likely limited to soluble ones. Unlike the bulk protein degradation that occurs during starvation, autophagic degradation of misfolded proteins can have a degree of specificity, determined by ubiquitin modification and the interactions of p62/SQSTM1 and HDAC6. Macroautophagy is initiated in response to endoplasmic reticulum (ER) stress caused by misfolded proteins, via the ER-activated autophagy (ERAA) pathway, which activates a partial unfolded protein response involving PERK and/or IRE1, and a calcium-mediated signaling cascade. ERAA serves the function of mitigating ER stress and suppressing cell death, which may be explored for controlling protein conformational diseases. Conversely, inhibition of ERAA may be explored for sensitizing resistant tumor cells to cytotoxic agents.     

Sorting,recognition and activation of the misfolded protein degradation pathways through macroautophagy and the proteasome

Based on a functional categorization, proteins may be grouped into three types and sorted to either the proteasome or the macroautophagy pathway for degradation. The two pathways are mechanistically connected but their capacity seems different. Macroautophagy can degrade all forms of misfolded proteins whereas proteasomal degradation is likely limited to soluble ones.Unlike the bulk protein degradation that occurs during starvation, autophagic degradation of misfolded proteins can have a degree of specificity, determined by ubiquitin modification and the interactions of p62/SQSTM1 and HDAC6. Macroautophagy is initiated in response to endoplasmic reticulum (ER) stress caused by misfolded proteins, via the ER-activated autophagy (ERAA) pathway, which activates a partial unfolded protein response involving PERK and/or IRE1, and a calcium-mediated signaling cascade. ERAA serves the function of mitigating ER stress and suppressing cell death, which may be explored for controlling protein conformational diseases. Conversely, inhibition of ERAA may be explored for sensitizing resistant tumor cells to cytotoxic agents.     

Keap1 facilitates p62-mediated ubiquitin aggregate clearance via autophagy

The accumulation of ubiquitin-positive protein aggregates has been implicated in the pathogenesis of neurodegenerative diseases, heart disease and diabetes. Emerging evidence indicates that the autophagy lysosomal pathway plays a critical role in the clearance of ubiquitin aggregates, a process that is mediated by the ubiquitin binding protein p62. In addition to binding ubiquitin, p62 also interacts with LC3 and transports ubiquitin conjugates to autophagosomes for degradation. The exact regulatory mechanism of this process is still largely unknown. Here we report the identification of Keap1 as a binding partner for p62 and LC3. Keap1 inhibits Nrf2 by sequestering it in the cytosol and preventing its translocation to the nucleus and activation of genes involved in the oxidative stress response. In this study, we found that Keap1 interacts with p62 and LC3 in a stress-inducible manner, and that Keap1 colocalizes with LC3 and p62 in puromycin-induced ubiquitin aggregates. Moreover, p62 serves as a bridge between Keap1 and ubiquitin aggregates and autophagosomes. Finally, genetic ablation of Keap1 leads to the accumulation of ubiquitin aggregates, increased cytotoxicity of misfolded protein aggregates, and defective activation of autophagy. Therefore, this study assigns a novel positive role of Keap1 in upregulating p62-mediated autophagic clearance of ubiquitin aggregates.     

TXNIP在高糖诱导的小鼠视网膜Müller细胞自噬中的作用及相关机制

目的:探讨硫氧还蛋白相互作用蛋白(thioredoxin interacting protein,TXNIP)对高糖诱导的小鼠视网膜Müller细胞自噬的影响及其可能机制。方法:采用高糖诱导体外培养的小鼠视网膜Muller细胞,通过RNA干扰降低TXNIP的表达,免疫荧光、Western blot和Real-time PCR检测自噬相关蛋白及丝氨酸/苏氨酸激酶/雷帕霉素靶蛋白(serine/threonine kinase 1/mechanistic target of rapamycin kinase,AKT/m TOR)的表达。结果:高糖诱导的Muller细胞中TXNIP、微管相关蛋白1轻链3α(microtubule associated protein 1 light chain 3 alpha,LC3Ⅱ)、Sequestosome1(p62/SQSTMl)的表达均显著增加(P<0.05);而TXNIP敲降的Muller细胞中自噬相关特征性蛋白(LC3Ⅱ、P62)的表达则显著降低(P<0.05)。结论:TXNIP可能通过AKT/m TOR信号通路来抑制糖尿病性视网膜病变中Müller细胞自噬活性,并引起细胞发生凋亡。