Retinal cell death has been widely held as a central event that leads to retinal neurodegeneration, vascular dysfunction, and eventually irreversible blindness in ocular diseases, such as glaucoma, retinal degeneration, diabetic retinopathy, and uveitis. Loss of retinal ganglion cells is considered to be a direct cause of vision loss in experimental glaucoma [¹], correlating with elevated intraocular pressure (IOP) [²]. Injury of retinal pigment epithelial (RPE) cells and photoreceptors leads to photoreceptor dysfunction and retinal degeneration [³], as seen in both inherited and acquired degenerative retinal diseases such as Stargardt's Disease [⁴], retinitis pigmentosa [⁵], and age-related macular degeneration [⁶]. In diabetic retinopathy, high glucose and other diabetic insults, such as oxidants, advanced glycation end products (AGEs), and inflammatory cytokines, result in neural and vascular cell death [⁷, ⁸]. In streptozotocin-induced diabetic rats, both retinal neurons and vascular cells become apoptotic soon after the onset of diabetes [⁹]. Inflammation-driven neural and vascular cell death is also a hallmark characteristic of uveitis, a chronic eye disease that cause vision loss [¹⁰–¹³]. Together, these findings support a pivotal role of cell death in the pathogenesis of retinal diseases. Apoptosis, that is, programmed cell death, is the most common form of cell death in various cell types, including retinal cells. Apoptosis is tightly controlled by a variety of signaling pathways that either promote or inhibit the apoptotic cascades. Among the most extensively studied proapoptotic factors in retinal cells are oxidative stress, mitochondrial dysfunction, inflammation, ischemia, hyperglycemia, and excitotoxicity [¹⁴–¹⁸]. Intriguingly, recent evidence suggests that disturbed protein homeostasis and endoplasmic reticulum (ER) stress also contribute to apoptosis of retinal cells [¹⁹]. Moreover, ER stress activates a large number of genes involved in the control of cell fate, including antiapoptotic and proapoptotic molecules such as Bax and Bcl-2 [²⁰, ²¹]. Therefore, elucidating the role and mechanisms of ER stress in retinal cell apoptosis may provide important insight into the pathogenesis of retinal diseases and help in developing new drugs to protect retinal cells and to prevent vision loss. In the present review, we discuss the potential implication of ER stress in retinal cell apoptosis, with a primary focus on the signaling transduction pathways that link ER stress with apoptosis in general as well as specific for retinal cells.

The endoplasmic reticulum (ER) is the primary intracellular organelle responsible for protein folding, maturation, and trafficking [²², ²³]. The ER consists of a network of folded membranes in which secretory and most membrane proteins are synthesized, posttranslationally modified, and folded into their correct three-dimensional conformations. Only properly folded (mature) proteins can be transported to the Golgi apparatus for further processing. In addition, the ER also serves as a dynamic pool of calcium, governing the intracellular calcium homeostasis [²⁴]. Other major functions of the ER include lipid and steroid hormone synthesis, carbohydrate metabolism, and drug detoxification. Importantly, compelling evidence indicates that the ER is one of the major machinery that senses subtle environmental changes and cellular stresses, coordinates signaling pathways, and modulates cell function and cell survival. Various physiological and pathological circumstances, such as excessive mutant proteins, viral infection, energy or nutrient deprivation, as well as alteration in the redox status, can compromise the ER capacity in protein folding, resulting in the accumulation of unfolded or misfolded proteins in the ER lumen, or ER stress. In turn, misfolded proteins aggregate to form insoluble intracellular or extracellular deposit, which is toxic to the cell. It has been demonstrated that a number of age-related diseases, such as Alzheimer's diseases; inflammatory disorders, such as diabetes; and neurodegenerative diseases, such as Parkinson's disease, are associated with the build-up of misfolded or unfolded protein aggregates [²⁵–²⁸]. To eliminate the toxic protein components, cells activate an adaptive mechanism that consists of a number of intracellular signaling pathways, collectively known as unfolded protein response (UPR). The UPR relieves ER stress and restores the protein homeostasis through three complementary strategies: (1) halt the generation of more unfolded proteins by suppression of protein translation; (2) induce ER-related molecular chaperones to promote refolding of the unfolded proteins, and (3) activate the ER-associated protein degradation (ERAD) system to remove the unfolded proteins.

There are three branches of UPR that are initiated by distinct ER stress transducers located on the ER membrane: PKR-like endoplasmic reticulum kinase (PERK) [²⁹], inositol-requiring enzyme 1 (IRE1) [³⁰, ³¹], and activating transcription factor 6 (ATF6) [²²]. In nonstressed cells, all three ER stress transducers are kept in an inactive state through binding to the ER chaperon glucose-regulated protein 78 (Bip), which is also known as immunoglobulin binding protein (Bip) [³², ³³]. Upon ER stress, excessive unfolded proteins accumulate in the ER lumen, resulting in the dissociation of GPR78 from the ER stress transducers [³⁴], which triggers activation of the UPR branches. In eukaryotic cells, UPR is an adaptive cellular response to the disturbance of normal ER functions, which attenuate the aggregation of unfolded or misfolded proteins and promote cell survival [³⁵]. However, during prolonged or overwhelming ER stress, UPR fails to restore the normal function of the ER, and apoptotic cascade will be activated [³⁶, ³⁷] (Figure 1). The exact mechanism underlying the switch of the UPR from a prosurvival mechanism to a proapoptotic response is not clear.

Previous studies suggest that the cell fate is dependent on the balance between the extent/severity of ER stress and the capacity of the ER to restore ER homeostasis through the UPR [⁵⁷–⁵⁹]. Temporal and mild ER stress can be overcome by the adaptive UPR, cell function maintained, and cells survive. However, if the stress condition is prolonged and the UPR fails to restore the ER homeostasis, the apoptotic signaling pathways will be initiated to remove the unhealthy cells. Recently, several independent studies have provided ample evidence that ER stress is a potential cause of retinal vascular and neuronal cell death in diseases such as glaucoma, diabetic retinopathy and age-related macular degeneration [¹⁹, ⁶⁰–⁶²]. ER stress has been observed in both cultured retinal cells (vascular endothelial cells, pericytes, ganglion cells, Muller cells, as well as RPE cells) and in the retina from animal models of various diseases. Not surprisingly, the role of ER stress has been extensively studied in the pathogenesis of retinitis pigmentosa (RP) with mutations of various retinal genes. In 2004, Rebello and associates reported that expression of a mutant (R14W) of carbonic anhydrase IV, a glycosylphosphatidylinositol-anchored protein that is highly expressed in the choriocapillaris of the human eye, induced upregulation of Bip, PERK, and CHOP, markers of ER stress and the unfolded protein response, accompanied by apoptosis [⁶³]. Similarly, enhanced ER stress was reported in RP induced by the rhodopsin mutation P23H in Xenopus laevis [⁶⁴] and in rats [⁶⁵, ⁶⁶]. Further, stimulation of the UPR in the retina or cultured retinal cells by preexposure to mild ER stress protected photoreceptor neurons from oxidative damage and cell death [⁶⁷]. Moreover, overexpression of Bip, an ER chaperone that facilitates protein folding and reduces ER stress, attenuated retinal expression of CHOP and the activation of apoptotic cascade and restored retinal photoreceptor function in P23H rats [⁶¹]. In addition to the genetic models of RP, ER stress was found remarkably enhanced in retinal photoreceptors, coincident with photoreceptor cell apoptosis, in a rodent model of light damage-induced retinal degeneration [⁶²]. These findings collectively support a causal role of ER stress in photoreceptor cell death and retinal degeneration.

Another well-studied area for ER stress-related retinal cell death is glaucoma. Increased ER stress markers were observed in retinal ganglion cells in animal models of ischemia-reperfusion and chronic glaucoma [⁶⁸]. Cultured retinal ganglion cells (RGC-5, a transformed rat ganglion cell line) treated with tunicamycin, a common ER stress inducer, undergo apoptosis, accompanied by increased production of ER stress-related proteins [¹⁹, ⁶⁹]. In vivo, intravitreal injection of tunicamycin resulted in loss of retinal ganglion cells and reduced thickness of the inner retina. Moreover, raising IOP or intravitreal injection of N-methyl-D-aspartate (NMDA), an excitotoxin that binds to the NMDA receptor and induces neuron cell death, also increased the expression of ER stress markers in retinal ganglion cells, amacrine cells, and microglial cells [¹⁹]. Pharmaceutical induction of Bip significantly attenuated tunicamycin- or NMDA-induced apoptosis in retinal ganglion cells, suggesting a pivotal role of ER stress in retinal neuron cell death [⁶⁹].

Loss of retinal vascular cells and apoptosis of retinal neurons have been recognized as critical events and pathological features of diabetic retinopathy [⁷⁰–⁷²]. Although currently it remains to be investigated how ER stress signaling pathways contribute to retinal cell death induced by diabetes, recent studies by our group and others demonstrated that ER stress was induced in early stage of diabetic retinopathy and was implicated in retinal inflammation and vascular damage [⁷³–⁷⁶]. In 2009, we reported increased ER stress markers in the retina of diabetic Akita mice, in parallel with elevated expression of inflammatory genes [⁷³]. In cultured retinal endothelial cells, ER stress was induced by hypoxia, a potent stimulator of inflammation and angiogenesis, and prevented by chemical chaperones. Moreover, we showed that induction of ER stress in the retina was sufficient to trigger an upregulation of inflammatory genes. Conversely, inhibiting ER stress protected the retina and retinal endothelial cells from inflammatory damage. In addition, activation of the adaptive UPR by preconditioning with ER stress also successfully prevented inflammatory damage to retinal endothelial cells and vascular leakage induced by diabetic stimulus [⁷⁴]. These results suggest that ER stress is implicated in retinal cell damage caused by diabetes.

It is currently unclear how ER stress induces apoptosis in various retinal cells. Generally, there are two major pathways for the initiation of apoptosis: extrinsic and intrinsic pathways [⁷⁷]. The extrinsic pathway is mediated by the cell membrane death receptors. Activation of the death receptor recruits adaptor molecules and activates caspase-8 or caspase-10, which cleaves the downstream substrates, that is, other caspases including caspase-3, resulting in apoptosis [⁷⁸]. The intrinsic pathway is closely related to factors anchored on the mitochondria. The insertion of these proapoptotic proteins changes the mitochondrial membrane permeability, resulting in the release of cytochrome c from mitochondria into the cytosol. Then cytochrome c binds to Apaf-1 and activates caspase-9 and then caspase-3, leading to the execution of cell death. In addition, accumulating evidence suggests that calcium release from the ER can also initiate the cell death signals, either by directly activating death receptors or by altering the sensitivity of mitochondria. Finally, these apoptotic pathways converge on caspase-3, resulting in the cleavage of other proteases and leading to apoptosis. In addition to caspase-dependent pathways, caspase-independent pathway is also implicated in retina apoptosis [⁷⁹]. In the following sections, we discuss the potential pathways that may play a role in ER stress-associated apoptosis in various retinal diseases, such as age-related macular degeneration, glaucoma, and diabetic retinopathy (Figure 2).

Emerging evidence suggests that ER stress plays a pivotal role in retinal apoptosis and cell death. Studies in other fields have identified a number of signaling pathways that are implicated in ER stress-mediated apoptotic process. These include CHOP induction, caspase-12 activation, mitochondria dysfunction, JNK activation, Fas-FasL system and the STAT1 pathway. Blocking each of these pathways reduces or prevents ER stress-induced apoptosis to a certain extent; however, induction of an individual proapoptotic pathway may not be sufficient to induce apoptosis [¹¹⁷]. This suggests that prolonged ER stress may activate multiple subthreshold proapoptotic pathways, and these pathways interact and regulate each other to execute apoptosis. In addition, prolonged ER stress may also suppress the compensatory cell survival pathways induced by the UPR, such as the IFN-β and Akt-p38α pathways [¹¹⁷]. Recently, we demonstrated that enhancing the endogenous adaptive UPR system by preconditioning retinal cells with mild ER stress was able to reduce vascular inflammation and retinal vascular leakage [⁷⁴]. Moreover, inducing molecular chaperones, for example, heat shock protein 90 (Hsp90), by antibiotics [¹¹⁸] prevents protein aggregation and that protects photoreceptors against retinal degeneration in a murine model of autosomal dominant retinitis pigmentosa (ADRP) [¹¹⁹]. In addition, overexpressing Bip/Bip in retinal photoreceptors alleviated ER stress, reduced CHOP expression, and mitigated photoreceptor apoptosis in P23H rhodopsin transgenic rats [⁶¹]. These findings support an essential role of the adaptive URP system and the URP-activated survival pathways in protecting retinal cells against apoptosis and cell death. Therefore, identifying the key proapoptotic and antiapoptotic pathways implicated in ER stress-associated apoptosis and addressing how these pathways are involved in different pathological conditions of retinal cells may offer the opportunity for developing new drugs to treat retinal diseases.

This work was supported by the National Institutes of Health Grant EY019949 and research awards from the American Diabetes Association, Juvenile Diabetes Research Foundation, Oklahoma Center for the Advancement of Science and Technology, American Health Assistance Foundation, and Harold Hamm Diabetes Center (all to S. X. Zhang).