Involvement of Endoplasmic Reticulum Stress in TULP1 Induced Retinal Degeneration

Inherited retinal disorders (IRDs) result in severe visual impairments in children and adults. A challenge in the field of retinal degenerations is identifying mechanisms of photoreceptor cell death related to specific genetic mutations. Mutations in the gene TULP1 have been associated with two forms of IRDs, early-onset retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). TULP1 is a cytoplasmic, membrane-associated protein shown to be involved in transportation of newly synthesized proteins destined for the outer segment compartment of photoreceptor cells; however, how mutant TULP1 causes cell death is not understood. In this study, we provide evidence that common missense mutations in TULP1 express as misfolded protein products that accumulate within the endoplasmic reticulum (ER) causing prolonged ER stress. In an effort to maintain protein homeostasis, photoreceptor cells then activate the unfolded protein response (UPR) complex. Our results indicate that the two major apoptotic arms of the UPR pathway, PERK and IRE1, are activated. Additionally, we show that retinas expressing mutant TULP1 significantly upregulate the expression of CHOP, a UPR signaling protein promoting apoptosis, and undergo photoreceptor cell death. Our study demonstrates that the ER-UPR, a known mechanism of apoptosis secondary to an overwhelming accumulation of misfolded protein, is involved in photoreceptor degeneration caused by missense mutations in TULP1. These observations suggest that modulating the UPR pathways might be a strategy for therapeutic intervention.     

BiP Inducer X: An ER Stress Inhibitor for Enhancing Recombinant Antibody Production in CHO Cell Culture

Prolonged endoplasmic reticulum (ER) stress reduces protein synthesis and induces apoptosis in mammalian cells. When dimethyl sulfoxide (DMSO), a specific monoclonal antibody productivity (q_mAb)‐enhancing reagent, is added to recombinant Chinese hamster ovary (rCHO) cell cultures (GSR cell line), it induces ER stress and apoptosis in a dose‐dependent manner. To determine an effective ER stress inhibitor, three ER stress inhibitors (BiP inducer X [BIX], tauroursodeoxycholic acid, and carbazole) are examined and BIX shows the best production performance. Coaddition of BIX (50 μm ) with DMSO extends the culture longevity and enhances q_mAb. As a result, the maximum mAb concentration is significantly increased with improved galactosylation. Coaddition of BIX significantly increases the expression level of binding immunoglobulin protein (BiP) followed by increased expression of chaperones (calnexin and GRP94) and galactosyltransferase. Furthermore, the expression levels of CHOP, a well‐known ER stress marker, and cleaved caspase‐3 are significantly reduced, suggesting that BIX addition reduces ER stress‐induced cell death by relieving ER stress. The beneficial effect of BIX on mAb production is also demonstrated with another q_mAb‐enhancing reagent (sodium butyrate) and a different rCHO cell line (CS13‐1.00). Taken together, BIX is an effective ER stress inhibitor that can be used to increase mAb production in rCHO cells.     

Mice deficient in the Vici syndrome gene Epg5 exhibit features of retinitis pigmentosa

Autophagy helps to maintain cellular homeostasis by removing misfolded proteins and damaged organelles, and generally acts as a cytoprotective mechanism for neuronal survival. Here we showed that mice deficient in the Vici syndrome gene Epg5, which is required for autophagosome maturation, show accumulation of ubiquitin-positive inclusions and SQSTM1 aggregates in various retinal cell types. In epg5^?/? retinas, photoreceptor function is greatly impaired, and degenerative features including progressively reduced numbers of photoreceptor cells and increased numbers of apoptotic cells in the outer nuclear layer are observed, while the morphology of other parts of the retina is not severely affected. Downstream targets of the unfolded protein response (UPR), including the death inducer DDIT3/CHOP, and also levels of cleaved CASP3 (caspase 3), are elevated in epg5^?/? retinas. Thus, apoptotic photoreceptor cell death in epg5^?/? retinas may result from the elevated UPR. Our results reveal that Epg5-deficient mice recapitulate key characteristics of retinitis pigmentosa and thus may provide a valuable model for investigating the molecular mechanism of photoreceptor degeneration.     

Peroxisome proliferator-activated receptor-α activation protects against endoplasmic reticulum stress-induced HepG2 cell apoptosis

Live ischemia–reperfusion injury is associated with endoplasmic reticulum (ER) stress-induced apoptosis. Activation of peroxisome proliferator-activated receptor-α (PPARα) may inhibit hepatocyte apoptosis induced by oxidative stress and protect against liver injury. This study aimed to investigate the effects of PPARα activation, through a specific agonist, on ER stress-induced apoptosis in human liver hepatocellular carcinoma (HepG2) cells. HepG2 cells were challenged with H₂O₂ and treated with WY14643, a selective PPARα agonist, in the presence or absence of the PPARα antagonist of MK886. Cell viable assay (MTT) and immunostaining were used to evaluate cell viability. The level of apoptotic cell death was quantified through Annexin V/PI staining. Alanine aminotransferase, asparatate aminotransferase, and malondialdehyde levels were measured to determine the presence of cellular injury and oxidative stress. RT-PCR and Western blot analysis were used to detect mRNA and protein expression of PPARα, BiP, and CHOP. Immunofluorescence was utilized to determine the intracellular localization of CHOP. H₂O₂ and MK886 both reduced the viability of HepG2 cells, increased oxidative stress and apoptosis, up-regulated the BiP and CHOP expression, and induced CHOP translocation from the cytoplasm to the nucleus. Compared with cells treated with H₂O₂ alone, pre-administration of WY14643 increased cell viability, attenuated apoptosis, improved cell function, down-regulated BiP and CHOP expression and inhibited CHOP translocation. The effects of WY14643 were completely abolished using the MK886 antagonist. PPARα activation protects against H₂O₂-induced HepG2 cell apoptosis. The underlying mechanisms may be associated with its activation to suppress excessive ER stress.     

The role of endoplasmic reticulum‐related BiP/GRP78 in interferon gamma‐induced persistent Chlamydia pneumoniae infection

Direct interaction of Chlamydiae with the endoplasmic reticulum (ER) is essential in intracellular productive infection. However, little is known about the interplay between Chlamydiae and the ER under cellular stress conditions that are observed in interferon gamma (IFN‐γ) induced chlamydial persistent infection. ER stress responses are centrally regulated by the unfolded protein response (UPR) under the control of the ER chaperone BiP/GRP78 to maintain cellular homeostasis. In this study, we could show that the ER directly contacted with productive and IFN‐γ‐induced persistent inclusions of Chlamydia pneumoniae (Cpn). BiP/GRP78 induction was observed in the early phase but not in the late phase of IFN‐γ‐induced persistent infection. Enhanced BiP/GRP78 expression in the early phase of IFN‐γ‐induced persistent Cpn infection was accompanied by phosphorylation of the eukaryotic initiation factor‐2α (eIF2α) and down‐regulation of the vesicle‐associated membrane protein‐associated protein B. Loss of BiP/GRP78 function resulted in enhanced phosphorylation of eIF2α and increased host cell apoptosis. In contrast, enhanced BiP/GRP78 expression in IFN‐γ‐induced persistent Cpn infection attenuated phosphorylation of eIF2α upon an exogenous ER stress inducer. In conclusion, ER‐related BiP/GRP78 plays a key role to restore cells from stress conditions that are observed in the early phase of IFN‐γ‐induced persistent infection.     

Sortilin participates in light-dependent photoreceptor degeneration in vivo

Both proNGF and the neurotrophin receptor p75 (p75(NTR)) are known to regulate photoreceptor cell death caused by exposure of albino mice to intense illumination. ProNGF-induced apoptosis requires the participation of sortilin as a necessary p75(NTR) co-receptor, suggesting that sortilin may participate in the photoreceptor degeneration triggered by intense lighting. We report here that light-exposed albino mice showed sortilin, p75(NTR), and proNGF expression in the outer nuclear layer, the retinal layer where photoreceptor cell bodies are located. In addition, cone progenitor-derived 661W cells subjected to intense illumination expressed sortilin and p75(NTR) and released proNGF into the culture medium. Pharmacological blockade of sortilin with either neurotensin or the "pro" domain of proNGF (pro-peptide) favored the survival of 661W cells subjected to intense light. In vivo, the pro-peptide attenuated retinal cell death in light-exposed albino mice. We propose that an auto/paracrine proapoptotic mechanism based on the interaction of proNGF with the receptor complex p75(NTR)/sortilin participates in intense light-dependent photoreceptor cell death. We therefore propose sortilin as a putative target for intervention in hereditary retinal dystrophies.     

The activation of the atypical PKC zeta in light‐induced retinal degeneration and its involvement in L‐DNase II control

Light‐induced retinal degeneration is characterized by photoreceptor cell death. Many studies showed that photoreceptor demise is caspase‐independent. In our laboratory we showed that leucocyte elastase inhibitor/LEI‐derived DNase II (LEI/L‐DNase II), a caspase‐independent apoptotic pathway, is responsible for photoreceptor death. In this work, we investigated the activation of a pro‐survival kinase, the protein kinase C (PKC) zeta. We show that light exposure induced PKC zeta activation. PKC zeta interacts with LEI/L‐DNase II and controls its DNase activity by impairing its nuclear translocation. These results highlight the role of PKC zeta in retinal physiology and show that this kinase can control caspase‐independent pathways.     

Defective trafficking of cone photoreceptor CNG channels induces the unfolded protein response and ER-stress-associated cell death

Mutations that perturb the function of photoreceptor CNG (cyclic nucleotide-gated) channels are associated with several human retinal disorders, but the molecular and cellular mechanisms leading to photoreceptor dysfunction and degeneration remain unclear. Many loss-of-function mutations result in intracellular accumulation of CNG channel subunits. Accumulation of proteins in the ER (endoplasmic reticulum) is known to cause ER stress and trigger the UPR (unfolded protein response), an evolutionarily conserved cellular programme that results in either adaptation via increased protein processing capacity or apoptotic cell death. We hypothesize that defective trafficking of cone photoreceptor CNG channels can induce UPR-mediated cell death. To test this idea, CNGA3 subunits bearing the R563H and Q655X mutations were expressed in photoreceptor-derived 661W cells with CNGB3 subunits. Compared with wild-type, R563H and Q655X subunits displayed altered degradation rates and/or were retained in the ER. ER retention was associated with increased expression of UPR-related markers of ER stress and with decreased cell viability. Chemical and pharmacological chaperones {TUDCA (tauroursodeoxycholate sodium salt), 4-PBA (sodium 4-phenylbutyrate) and the cGMP analogue CPT-cGMP [8-(4-chlorophenylthio)-cGMP]} differentially reduced degradation and/or promoted plasma-membrane localization of defective subunits. Improved subunit maturation was concordant with reduced expression of ER-stress markers and improved viability of cells expressing localization-defective channels. These results indicate that ER stress can arise from expression of localization-defective CNG channels, and may represent a contributing factor for photoreceptor degeneration.     

N‐methyl‐N‐nitrosourea induces retinal degeneration in the rat via the inhibition of NF‐κB activation

Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases characterized by the loss of photoreceptor cells through apoptosis. N‐methyl‐N‐nitrosourea (MNU) is an alkylating toxicant that induces photoreceptor cell death resembling hereditary RP. This study aimed to investigate the role of nuclear factor κB (NF‐κB) in MNU‐induced photoreceptor degeneration. Adult rats received a single intraperitoneal injection of MNU (60 mg/kg bodyweight). Hematoxylin and eosin staining demonstrated progressive outer nuclear layer (ONL) loss after MNU treatment. Transmission electron microscopy revealed nuclear pyknosis, chromatin margination in the photoreceptors, increased secondary lysosomes, and lobulated retinal‐pigmented epithelial cells in MNU‐treated rats. Numerous photoreceptor cells in the ONL showed positive TUNEL staining and apoptosis rate peaked at 24 hours. Enhanced depth imaging spectral‐domain optical coherence tomography showed ONL thinning and decreased choroid thickness. Electroretinograms showed decreased A wave amplitude that predominated in scotopic conditions. Western blot analysis showed that nuclear IκBα level increased, whereas nuclear NF‐κB p65 decreased significantly in the retinas of MNU‐treated rats. These findings indicate that MNU leads to selective photoreceptor degradation, and this is associated with the inhibition of NF‐κB activation.     

ER stress protects from retinal degeneration

The unfolded protein response (UPR) is a specific cellular process that allows the cell to cope with the overload of unfolded/misfolded proteins in the endoplasmic reticulum (ER). ER stress is commonly associated with degenerative pathologies, but its role in disease progression is still a matter for debate. Here, we found that mutations in the ER‐resident chaperone, neither inactivation nor afterpotential A (NinaA), lead to mild ER stress, protecting photoreceptor neurons from various death stimuli in adult Drosophila. In addition, Drosophila S2 cultured cells, when pre‐exposed to mild ER stress, are protected from H₂O₂, cycloheximide‐ or ultraviolet‐induced cell death. We show that a specific ER‐mediated signal promotes antioxidant defences and inhibits caspase‐dependent cell death. We propose that an immediate consequence of the UPR not only limits the accumulation of misfolded proteins but also protects tissues from harmful exogenous stresses.     

A Highlights from MBoC Selection: Translational and posttranslational regulation of XIAP by eIF2α and ATF4 promotes ER stress–induced cell death during the unfolded protein response

Endoplasmic reticulum (ER) protein misfolding activates the unfolded protein response (UPR) to help cells cope with ER stress. If ER homeostasis is not restored, UPR promotes cell death. The mechanisms of UPR-mediated cell death are poorly understood. The PKR-like endoplasmic reticulum kinase (PERK) arm of the UPR is implicated in ER stress–induced cell death, in part through up-regulation of proapoptotic CCAAT/enhancer binding protein homologous protein (CHOP). Chop^−/− cells are partially resistant to ER stress–induced cell death, and CHOP overexpression alone does not induce cell death. These findings suggest that additional mechanisms regulate cell death downstream of PERK. Here we find dramatic suppression of antiapoptosis XIAP proteins in response to chronic ER stress. We find that PERK down-regulates XIAP synthesis through eIF2α and promotes XIAP degradation through ATF4. Of interest, PERK''s down-regulation of XIAP occurs independently of CHOP activity. Loss of XIAP leads to increased cell death, whereas XIAP overexpression significantly enhances resistance to ER stress–induced cell death, even in the absence of CHOP. Our findings define a novel signaling circuit between PERK and XIAP that operates in parallel with PERK to CHOP induction to influence cell survival during ER stress. We propose a “two-hit” model of ER stress–induced cell death involving concomitant CHOP up-regulation and XIAP down-regulation both induced by PERK.     

Inhibiting autophagy reduces retinal degeneration caused by protein misfolding

Mutations in the genes necessary for the structure and function of vertebrate photoreceptor cells are associated with multiple forms of inherited retinal degeneration. Mutations in the gene encoding RHO (rhodopsin) are a common cause of autosomal dominant retinitis pigmentosa (adRP), with the Pro23His variant of RHO resulting in a misfolded protein that activates endoplasmic reticulum stress and the unfolded protein response. Stimulating macroautophagy/autophagy has been proposed as a strategy for clearing misfolded RHO and reducing photoreceptor death. We found that retinas from mice heterozygous for the gene encoding the RHO^P23H variant (hereafter called P23H) exhibited elevated levels of autophagy flux, and that pharmacological stimulation of autophagy accelerated retinal degeneration. In contrast, reducing autophagy flux pharmacologically or by rod-specific deletion of the autophagy-activating gene Atg5, improved photoreceptor structure and function. Furthermore, proteasome levels and activity were reduced in the P23H retina, and increased when Atg5 was deleted. Our findings suggest that autophagy contributes to photoreceptor cell death in P23H mice, and that decreasing autophagy shifts the degradation of misfolded RHO protein to the proteasome and is protective. These observations suggest that modulating the flux of misfolded proteins from autophagy to the proteasome may represent an important therapeutic strategy for reducing proteotoxicity in adRP and other diseases caused by protein folding defects.     

Otx2 enhances transdifferentiation of Müller cells‐derived retinal stem cells into photoreceptor‐like cells

Retinal Müller glial cells have the potential of neurogenic retinal progenitor cells, and could reprogram into retinal‐specific cell types such as photoreceptor cells. How to promote the differentiation of Müller cells into photoreceptor cells represents a promising therapy strategy for retinal degeneration diseases. This study aimed to enhance the transdifferentiation of rat Müller cells‐derived retinal stem cells (MC‐RSCs) into photoreceptor‐like cells and explore the signalling mechanism. We dedifferentiated rat Müller cells into MC‐RSCs which were infected with Otx2 overexpression lentivirus or control. The positive rate of photoreceptor‐like cells among MC‐RSCs treated with Otx2 overexpression lentivirus was significantly higher compared to control. Furthermore, pre‐treatment with Crx siRNA, Nrl siRNA, or GSK‐3 inhibitor SB‐216763 reduced the positive rate of photoreceptor‐like cells among MC‐RSCs treated with Otx2 overexpression lentivirus. Finally, Otx2 induced photoreceptor precursor cells were injected into subretinal space of N‐methyl‐N‐nitrosourea induced rat model of retinal degeneration and partially recovered retinal degeneration in the rats. In conclusion, Otx2 enhances transdifferentiation of MC‐RSCs into photoreceptor‐like cells and this is associated with the inhibition of Wnt signalling. Otx2 is a potential target for gene therapy of retinal degenerative diseases.     

Phloroglucinol protects retinal pigment epithelium and photoreceptor against all‐trans‐retinal–induced toxicity and inhibits A2E formation

Among retinal macular diseases, the juvenile recessive Stargardt disease and the age‐related degenerative disease arise from carbonyl and oxidative stresses (COS). Both stresses originate from an accumulation of all‐trans‐retinal (atRAL) and are involved in bisretinoid formation by condensation of atRAL with phosphatidylethanolamine (carbonyl stress) in the photoreceptor and its transformation into lipofuscin bisretinoids (oxidative stress) in the retinal pigment epithelium (RPE). As atRAL and bisretinoid accumulation contribute to RPE and photoreceptor cell death, our goal is to select powerful chemical inhibitors of COS. Here, we describe that phloroglucinol, a natural phenolic compound having anti‐COS properties, protects both rat RPE and mouse photoreceptor primary cultures from atRAL‐induced cell death and reduces hydrogen peroxide (H₂O₂)‐induced damage in RPE in a dose‐dependent manner. Mechanistic analyses demonstrate that the protective effect encompasses decrease in atRAL‐induced intracellular reactive oxygen species and free atRAL levels. Moreover, we show that phloroglucinol reacts with atRAL to form a chromene adduct which prevents bisretinoid A2E synthesis in vitro. Taken together, these data show that the protective effect of phloroglucinol correlates with its ability to trap atRAL and to prevent its further transformation into deleterious bisretinoids. Phloroglucinol might be a good basis to develop efficient therapeutic derivatives in the treatment of retinal macular diseases.