Interplay of endoplasmic reticulum stress and autophagy in neurodegenerative disorders

The common underlying feature of most neurodegenerative diseases such as Alzheimer disease (AD), prion diseases, Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS) involves accumulation of misfolded proteins leading to initiation of endoplasmic reticulum (ER) stress and stimulation of the unfolded protein response (UPR). Additionally, ER stress more recently has been implicated in the pathogenesis of HIV-associated neurocognitive disorders (HAND). Autophagy plays an essential role in the clearance of aggregated toxic proteins and degradation of the damaged organelles. There is evidence that autophagy ameliorates ER stress by eliminating accumulated misfolded proteins. Both abnormal UPR and impaired autophagy have been implicated as a causative mechanism in the development of various neurodegenerative diseases. This review highlights recent advances in the field on the role of ER stress and autophagy in AD, prion diseases, PD, ALS and HAND with the involvement of key signaling pathways in these processes and implications for future development of therapeutic strategies.     

Stress tolerance of misfolded carboxypeptidase Y requires maintenance of protein trafficking and degradative pathways

The accumulation of aberrantly folded proteins can lead to cell dysfunction and death. Currently, the mechanisms of toxicity and cellular defenses against their effects remain incompletely understood. In the endoplasmic reticulum (ER), stress caused by misfolded proteins activates the unfolded protein response (UPR). The UPR is an ER-to-nucleus signal transduction pathway that regulates a wide variety of target genes to maintain cellular homeostasis. We studied the effects of ER stress in budding yeast through expression of the well-characterized misfolded protein, CPY*. By challenging cells within their physiological limits to resist stress, we show that the UPR is required to maintain essential functions including protein translocation, glycosylation, degradation, and transport. Under stress, the ER-associated degradation (ERAD) pathway for misfolded proteins is saturable. To maintain homeostasis, an "overflow" pathway dependent on the UPR transports excess substrate to the vacuole for turnover. The importance of this pathway was revealed through mutant strains compromised in the vesicular trafficking of excess CPY*. Expression of CPY* at levels tolerated by wild-type cells was toxic to these strains despite retaining the ability to activate the UPR.     

Molecular genetics of retinal degeneration: A Drosophila perspective

Inherited retinal degeneration in Drosophila has been explored for insights into similar processes in humans. Based on the mechanisms, I divide these mutations in Drosophila into three classes. The first consists of genes that control the specialization of photoreceptor cells including the morphogenesis of visual organelles (rhabdomeres) that house the visual signaling proteins. The second class contains genes that regulate the activity or level of the major rhodopsin, Rh1, which is the light sensor and also provides a structural role for the maintenance of rhabdomeres. Some mutations in Rh1 (NinaE) are dominant due to constitutive activity or folding defects, like autosomal dominant retinitis pigmentosa (ADRP) in humans. The third class consists of genes that control the Ca²⁺ influx directly or indirectly by promoting the turnover of the second messenger and regeneration of PIP₂, or mediate the Ca²⁺-dependent regulation of the visual response. These gene products are critical for the increase in cytosolic Ca²⁺ following light stimulation to initiate negative regulatory events. Here I will focus on the signaling mechanisms underlying the degeneration in norpA, and in ADRP-type NinaE mutants that produce misfolded Rh1. Accumulation of misfolded Rh1 in the ER triggers the unfolded protein response (UPR), while endosomal accumulation of activated Rh1 may initiate autophagy in norpA. Both autophagy and the UPR are beneficial for relieving defective endosomal trafficking and the ER stress, respectively. However, when photoreceptors fail to cope with the persistence of these stresses, a cell death program is activated leading to retinal degeneration.     

Autophagy: an ER protein quality control process

Protein quality control processes active in the endoplasmic reticulum (ER), including ER-associated protein degradation (ERAD) and the unfolded protein response (UPR), prevent the cytotoxic effects that can result from the accumulation of misfolded proteins. Characterization of a yeast mutant deficient in ERAD, a proteasome-dependent degradation pathway, revealed the employment of two overflow pathways from the ER to the vacuole when ERAD was compromised. One removes the soluble misfolded protein via the biosynthetic pathway and the second clears aggregated proteins via autophagy. Previously, autophagy had been implicated in the clearance of cytoplasmic aggresomes, but was not known to play a direct role in ER protein quality control. These findings provide insight into the molecular mechanisms that result in the gain-of-function liver disease associated with both alpha1-deficiency and hypofibrinogenemia (abnormally low levels of plasma fibrinogen, which is required for blood clotting), and emphasize the need for a more complete understanding of the molecular mechanisms of autophagy and its relationship to protein quality control.     

Endoplasmic reticulum stress: a new pathway to induce autophagy

Autophagy is a response to the stress of nutrient limitation in yeast, whereby cytosolic long-lived proteins and organelles are nonselectively degraded, and the resulting macromolecules are recycled to allow new protein synthesis that is essential for survival. We recently revealed that endoplasmic reticulum (ER) stress induces autophagy. When misfolded proteins accumulate in the ER the resulting stress activates the unfolded protein response (UPR) to induce the expression of chaperones and proteins involved in the recovery process. Under conditions of ER stress, the preautophagosomal structure is assembled, and transport of autophagosomes to the vacuole is stimulated in an Atg protein-dependent manner. Interestingly, Atg1 has high kinase activity during ER stress-induced autophagy similar to the situation in starvation-induced autophagy.     

内质网应激诱导自噬的分子机制

在真核细胞中,内质网对蛋白质的折叠和运输至关重要,多种病理因素对内质网稳态的扰乱,可导致内质网腔中未折叠或错误折叠蛋白蓄积,即内质网应激(ERS)。细胞为此通过激活一种叫做未折叠蛋白反应(UPR)的防御反应来恢复内质网稳态。自噬是一种被描述为"自我吞食"的细胞代谢过程,其通过批量清除和降解未折叠蛋白以及破损细胞器在ERS时作为一种重要的保护机制。近年的研究显示这两个系统动态互联,且ERS可以通过多种方式诱导自噬的发生。在本文中,我们将总结目前关于ERS尤其是UPR诱导自噬的分子机制的相关知识,以进一步指导关于ERS与自噬关系的的研究。     

Inhibition of retrograde transport modulates misfolded protein accumulation and clearance in motoneuron diseases

Motoneuron diseases, like spinal bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS), are associated with proteins that because of gene mutation or peculiar structures, acquire aberrant (misfolded) conformations toxic to cells. To prevent misfolded protein toxicity, cells activate a protein quality control (PQC) system composed of chaperones and degradative pathways (proteasome and autophagy). Inefficient activation of the PQC system results in misfolded protein accumulation that ultimately leads to neuronal cell death, while efficient macroautophagy/autophagy-mediated degradation of aggregating proteins is beneficial. The latter relies on an active retrograde transport, mediated by dynein and specific chaperones, such as the HSPB8-BAG3-HSPA8 complex. Here, using cellular models expressing aggregate-prone proteins involved in SBMA and ALS, we demonstrate that inhibition of dynein-mediated retrograde transport, which impairs the targeting to autophagy of misfolded species, does not increase their aggregation. Rather, dynein inhibition correlates with a reduced accumulation and an increased clearance of mutant ARpolyQ, SOD1, truncated TARDBP/TDP-43 and expanded polyGP C9ORF72 products. The enhanced misfolded protein clearance is mediated by the proteasome, rather than by autophagy and correlates with the upregulation of the HSPA8 cochaperone BAG1. In line, overexpression of BAG1 increases the proteasome-mediated clearance of these misfolded proteins. Our data suggest that when the misfolded proteins cannot be efficiently transported toward the perinuclear region of the cells, where they are either degraded by autophagy or stored into the aggresome, the cells activate a compensatory mechanism that relies on the induction of BAG1 to target the HSPA8-bound cargo to the proteasome in a dynein-independent manner.     

Aerobic exercise training rescues cardiac protein quality control and blunts endoplasmic reticulum stress in heart failure rats

Cardiac endoplasmic reticulum (ER) stress through accumulation of misfolded proteins plays a pivotal role in cardiovascular diseases. In an attempt to reestablish ER homoeostasis, the unfolded protein response (UPR) is activated. However, if ER stress persists, sustained UPR activation leads to apoptosis. There is no available therapy for ER stress relief. Considering that aerobic exercise training (AET) attenuates oxidative stress, mitochondrial dysfunction and calcium imbalance, it may be a potential strategy to reestablish cardiac ER homoeostasis. We test the hypothesis that AET would attenuate impaired cardiac ER stress after myocardial infarction (MI). Wistar rats underwent to either MI or sham surgeries. Four weeks later, rats underwent to 8 weeks of moderate‐intensity AET. Myocardial infarction rats displayed cardiac dysfunction and lung oedema, suggesting heart failure. Cardiac dysfunction in MI rats was paralleled by increased protein levels of UPR markers (GRP78, DERLIN‐1 and CHOP), accumulation of misfolded and polyubiquitinated proteins, and reduced chymotrypsin‐like proteasome activity. These results suggest an impaired cardiac protein quality control. Aerobic exercise training improved exercise capacity and cardiac function of MI animals. Interestingly, AET blunted MI‐induced ER stress by reducing protein levels of UPR markers, and accumulation of both misfolded and polyubiquinated proteins, which was associated with restored proteasome activity. Taken together, our study provide evidence for AET attenuation of ER stress through the reestablishment of cardiac protein quality control, which contributes to better cardiac function in post‐MI heart failure rats. These results reinforce the importance of AET as primary non‐pharmacological therapy to cardiovascular disease.     

A Role for Macro-ER-Phagy in ER Quality Control

The endoplasmic-reticulum quality-control (ERQC) system shuttles misfolded proteins for degradation by the proteasome through the well-defined ER-associated degradation (ERAD) pathway. In contrast, very little is known about the role of autophagy in ERQC. Macro-autophagy, a collection of pathways that deliver proteins through autophagosomes (APs) for degradation in the lysosome (vacuole in yeast), is mediated by autophagy-specific proteins, Atgs, and regulated by Ypt/Rab GTPases. Until recently, the term ER-phagy was used to describe degradation of ER membrane and proteins in the lysosome under stress: either ER stress induced by drugs or whole-cell stress induced by starvation. These two types of stresses induce micro-ER-phagy, which does not use autophagic organelles and machinery, and non-selective autophagy. Here, we characterize the macro-ER-phagy pathway and uncover its role in ERQC. This pathway delivers 20–50% of certain ER-resident membrane proteins to the vacuole and is further induced to >90% by overexpression of a single integral-membrane protein. Even though such overexpression in cells defective in macro-ER-phagy induces the unfolded-protein response (UPR), UPR is not needed for macro-ER-phagy. We show that macro-ER-phagy is dependent on Atgs and Ypt GTPases and its cargo passes through APs. Moreover, for the first time the role of Atg9, the only integral-membrane core Atg, is uncoupled from that of other core Atgs. Finally, three sequential steps of this pathway are delineated: Atg9-dependent exit from the ER en route to autophagy, Ypt1- and core Atgs-mediated pre-autophagsomal-structure organization, and Ypt51-mediated delivery of APs to the vacuole.     

The unfolded protein response in melanocytes: activation in response to chemical stressors of the endoplasmic reticulum and tyrosinase misfolding

Accumulation of proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), comprising three signaling pathways initiated by Ire1, Perk and Atf6 respectively. Unfolded protein response activation was compared in chemically stressed murine wildtype melanocytes and mutant melanocytes that retain tyrosinase in the ER. Thapsigargin, an ER stressor, activated all pathways in wildtype melanocytes, triggering Caspase 12-mediated apoptosis at toxic doses. Albino melanocytes expressing mutant tyrosinase showed evidence of ER stress with increased Ire1 expression, but the downstream effector, Xbp1, was not activated even following thapsigargin treatment. Attenuation of Ire1 signaling was recapitulated in wildtype melanocytes treated with thapsigargin for 8 days, with diminished Xbp1 activation observed after 4 days. Atf6 was also activated in albino melanocytes, with no response to thapsigargin, while the Perk pathway was not activated and thapsigargin treatment elicited robust expression of the downstream effector CCAAT-enhancer-binding protein homologous protein. Thus, melanocytes adapt to ER stress by attenuating two UPR pathways.     

Primary cilia and autophagic dysfunction in Huntington's disease

Huntington''s disease (HD) is an inherited, neurodegenerative disorder caused by a single-gene mutation: a CAG expansion in the huntingtin (HTT) gene that results in production of a mutated protein, mutant HTT, with a polyglutamine tail (polyQ-HTT). Although the molecular pathways of polyQ-HTT toxicity are not fully understood, because protein misfolding and aggregation are central features of HD, it has long been suspected that cellular housekeeping processes such as autophagy might be important to disease pathology. Indeed, multiple lines of research have identified abnormal autophagy in HD, characterized generally by increased autophagic induction and inefficient clearance of substrates. To date, the origin of autophagic dysfunction in HD remains unclear and the search for actors involved continues. To that end, recent studies have suggested a bidirectional relationship between autophagy and primary cilia, signaling organelles of most mammalian cells. Interestingly, primary cilia structure is defective in HD, suggesting a potential link between autophagic dysfunction, primary cilia and HD pathogenesis. In addition, because polyQ-HTT also accumulates in primary cilia, the possibility exists that primary cilia might play additional roles in HD: perhaps by disrupting signaling pathways or acting as a reservoir for secretion and propagation of toxic, misfolded polyQ-HTT fragments. Here, we review recent research suggesting potential links between autophagy, primary cilia and HD and speculate on possible pathogenic mechanisms and future directions for the field.     

Maintenance of immune hyporesponsiveness to melanosomal proteins by DHICA-mediated antioxidation: Possible implications for autoimmune vitiligo

Melanocyte destruction in the skin of vitiligo patients has been considered to be a consequence of an autoimmune response against melanosomal proteins. However, little is known about the molecular mechanisms by which the immune system recognizes these sequestered intracellular self-proteins, which are confined in specialized organelles termed melanosomes, and is provoked into an autoimmune response to melanocytes. Here, we utilize a sucrose density-gradient ultracentrifugation protocol to enrich melanosomal components from dopachrome tautomerase (Dct)-mutant or wild-type melanocytes exposed to a pulse of hydrogen peroxide at a noncytotoxic concentration to evaluate their immunogenicity in mice challenged with the corresponding melanosomal proteins. The results demonstrate that enhanced humoral and cellular immune responses to a challenge with late-stage melanosomal proteins, especially with those derived from Dct-mutant melanocytes, are found in the immunized mice. To elucidate whether a reduced 5,6-dihydroxyindole-2-carboxylic acid (DHICA) content in melanin might cause a loss in antioxidative protection to the proteins, we incubated these melanosomal proteins in vitro with synthetic 5,6-dihydroindole (DHI)-melanin or DHI/DHICA (1:1)-melanin and then used them to immunize mice. T cell proliferation and IgG antibody responsiveness to the challenges were significantly induced by melanosomal proteins treated with DHI-melanin, but not by those treated with DHI/DHICA (1:1)-melanin. Moreover, we observed that melanosomal proteins derived from Dct-mutant melanocytes are subject to oxidative modifications that alter their antigenic configurations to attain an enhanced immunogenicity compared with those derived from wild-type melanocytes. From these results, we conclude that DHICA-mediated antioxidation plays a critical role in the maintenance of immune hyporesponsiveness to melanosomal proteins.     

Protein kinase RNA-like ER kinase/eukaryotic translation initiation factor 2α pathway attenuates tumor necrosis factor alpha-induced apoptosis in nucleus pulposus cells by activating autophagy

Cellular loss induced by tumor necrosis factor alpha (TNF-α) contributes to the pathogenesis of intervertebral disc (IVD) degeneration. Cellular stress induced by TNF-α activates several processes to restore cell homeostasis. These processes include autophagy, endoplasmic reticulum stress, and related unfolded protein response (UPR). However, the effect and mechanism of UPR and autophagy regulated by TNF-α in IVD degeneration (IDD) remain unclear. The effect of autophagy on biological changes in nucleus pulposus cells (NPCs) also remains elusive. In this study, rat NPCs were cultured with TNF-α in the presence or absence of the UPR or autophagy pathway small-interfering RNAs. The associated genes and proteins were evaluated through immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analyses to monitor UPR and autophagy signaling and identify the regulatory mechanism of autophagy by the UPR pathway. Trypan blue exclusion assay, cell flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, qRT-PCR, and western blot analyses were performed to examine the apoptosis of NPCs. The results showed that the acute exposure of TNF-α induced the apoptosis of rat NPCs and activated the protein kinase RNA-like ER kinase/eukaryotic translation initiation factor 2α (PERK/eIF2α) pathway of UPR and initiated autophagy. Silencing the PERK/eIF2α pathway or inhibiting autophagy enhanced the apoptosis of NPCs. Interference of the PERK/eIF2α pathway suppressed the autophagy of rat NPCs under TNF-α stimulation. Taken together, the PERK/eIF2α pathway reinforces the survival of NPCs under TNF-α stimulation by activating autophagy. Therefore, PERK/eIF2α-dependent autophagy could be a novel biological therapeutic target for IDD.     

Getting the better of ER stress

Research over the past few years has highlighted the ability of the unfolded protein response (UPR) to minimize the deleterious effects of accumulated misfolded proteins under both physiological and pathological conditions. The endoplasmic reticulum (ER) adapts to endogenous and exogenous stressors by expanding its protein-folding capacity and by stimulating protective processes such as autophagy and antioxidant responses. Although it is clear that severe ER stress can elicit cell death, several recent studies have shown that low levels of ER stress may actually be beneficial to cells by eliciting an adaptive UPR that ‘preconditions’ the cell to a subsequent lethal insult; this process is called ER hormesis. The findings have important implications for the treatment of a wide variety of diseases associated with defective proteostasis, including neurodegenerative diseases, diabetes, and cancer. Here, we review the physiological and pathological functions of the ER, with a particular focus on the molecular mechanisms that lead to ER hormesis and cellular protection, and discuss the implications for disease treatment.     

Endoplasmic reticulum-associated degradation mediated by MoHrd1 and MoDer1 is pivotal for appressorium development and pathogenicity of Magnaporthe oryzae

Most secretory proteins are folded and modified in the endoplasmic reticulum (ER); however, protein folding is error-prone, resulting in toxic protein aggregation and cause ER stress. Irreversibly misfolded proteins are subjected to ER-associated degradation (ERAD), modified by ubiquitination, and degraded by the 26S proteasome. The yeast ERAD ubiquitin ligase Hrd1p and multispanning membrane protein Der1p are involved in ubiquitination and transportation of the folding-defective proteins. Here, we performed functional characterization of MoHrd1 and MoDer1 and revealed that both of them are localized to the ER and are pivotal for ERAD substrate degradation and the ER stress response. MoHrd1 and MoDer1 are involved in hyphal growth, asexual reproduction, infection-related morphogenesis, protein secretion and pathogenicity of M. oryzae. Importantly, MoHrd1 and MoDer1 mediated conidial autophagic cell death and subsequent septin ring assembly at the appressorium pore, leading to abnormal appressorium development and loss of pathogenicity. In addition, deletion of MoHrd1 and MoDer1 activated the basal unfolded protein response (UPR) and autophagy, suggesting that crosstalk between ERAD and two other closely related mechanisms in ER quality control system (UPR and autophagy) governs the ER stress response. Our study indicates the importance of ERAD function in fungal development and pathogenesis of M. oryzae.     

Molecular genetics of retinal degeneration

Inherited retinal degeneration in Drosophila has been explored for insights into similar processes in humans. Based on the mechanisms, I divide these mutations in Drosophila into three classes. The first consists of genes that control the specialization of photoreceptor cells including the morphogenesis of visual organelles (rhabdomeres) that house the visual signaling proteins. The second class contains genes that regulate the activity or level of the major rhodopsin, Rh1, which is the light sensor and also provides a structural role for the maintenance of rhabdomeres. Some mutations in Rh1 (NinaE) are dominant due to constitutive activity or folding defects, like autosomal dominant retinitis pigmentosa (ADRP) in humans. The third class consists of genes that control the Ca²⁺ influx directly or indirectly by promoting the turnover of the second messenger and regeneration of PIP_2, or mediate the Ca²⁺-dependent regulation of the visual response. These gene products are critical for the increase in cytosolic Ca²⁺ following light stimulation to initiate negative regulatory events. Here I will focus on the signaling mechanisms underlying the degeneration in norpA, and in ADRP-type NinaE mutants that produce misfolded Rh1. Accumulation of misfolded Rh1 in the ER triggers the unfolded protein response (UPR), while endosomal accumulation of activated Rh1 may initiate autophagy in norpA. Both autophagy and the UPR are beneficial for relieving defective endosomal trafficking and the ER stress, respectively. However, when photoreceptors fail to cope with the persistence of these stresses, a cell death program is activated leading to retinal degeneration.