Unfolded Protein Response in Fuchs Endothelial Corneal Dystrophy: A Unifying Pathogenic Pathway?


To assess for activation of the unfolded protein response in corneal endothelium of Fuchs endothelial corneal dystrophy patients.


Retrospective, comparative case series of laboratory specimens.


Corneal specimens of patients with Fuchs dystrophy and controls with corneal pathologic features other than Fuchs dystrophy were evaluated by transmission electron microscopy (TEM) to evaluate for structural changes of the rough endoplasmic reticulum in corneal endothelium. TEM images were evaluated for alterations of rough endoplasmic reticulum as a sign of unfolded protein response. Normal autopsy eyes, Fuchs dystrophy corneas, and keratoconus corneas were used for immunohistochemistry. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections of patient corneas for 3 unfolded protein response markers (GRP78, the α subunit of eukaryotic initiation factor 2, C/EBP homologous protein) and 2 apoptosis markers (caspase 3 and 9). Immunohistochemistry signal quantitation of corneal endothelium for evaluation of marker expression was performed using automated software. Corneal sections were assessed quantitatively for levels of immunohistochemistry marker expression.


TEM showed enlargement of rough endoplasmic reticulum in corneal endothelium of all Fuchs dystrophy specimens. Immunohistochemistry quantitation demonstrated a significant increase in mean signal in corneal endothelium from Fuchs dystrophy patients for markers GRP78, the α subunit of eukaryotic initiation factor 2, C/EBP homologous protein, and caspase 9 compared with non-Fuchs dystrophy corneas ( P < .05).


Results of both TEM and immunohistochemistry indicate activation of unfolded protein response in Fuchs dystrophy. Unfolded protein response activation leads to endothelial cell apoptosis in Fuchs dystrophy and may play a central pathogenic role in this disease.

Fuchs endothelial corneal dystrophy (Fuchs dystrophy) is a primary disease of the corneal endothelium characterized by loss of endothelial cells and abnormalities of Descemet membrane. These pathologic changes lead progressively to corneal edema and loss of vision, occurring approximately 20 years after disease onset. Fuchs dystrophy recently has been characterized as having an early-onset form, with diagnosis as early as the first decade of life, and a late-onset form with diagnosis typically between the third and fourth decades of life. Previous studies of Fuchs dystrophy pathogenesis indicate increased endothelial cell apoptosis. However, early disease stages are asymptomatic, and studies of Fuchs dystrophy tissues are limited to failed patient corneas undergoing corneal transplant surgery. Thus, the cellular pathophysiologic features of Fuchs dystrophy remain poorly understood.

Protein folding is critical for cellular functioning, and all cells have mechanisms to ensure proper protein folding and disposal of irreversibly misfolded proteins. Accumulation of misfolded proteins can result in endoplasmic reticulum stress, a condition that is toxic to cells. To counteract this stress, cells initiate the unfolded protein response, which is a comprehensive program to reduce the accumulation of toxic unfolded proteins. Endoplasmic reticulum stress and unfolded protein response have been shown to play important roles in the pathogenesis of multiple human diseases, including diabetes, Alzheimer and Parkinson diseases, and atherosclerosis. In addition, endoplasmic reticulum stress and unfolded protein response have been implicated in a variety of ophthalmologic diseases, including cataract, retinitis pigmentosa, and glaucoma.

The unfolded protein response consists of 3 effector arms mediated by 3 endoplasmic reticulum transmembrane receptors: pancreatic endoplasmic reticulum kinase–like endoplasmic reticulum kinase, activating transcription factor 6, and inositol requiring enzyme 1. These effectors are maintained in an inactive state by binding to the intraluminal endoplasmic reticulum chaperone protein GRP78. Additional important markers of unfolded protein response activation include the α subunit of eukaryotic initiation factor 2 (phospho-eIF2α), which is phosphorylated by pancreatic endoplasmic reticulum kinase–like endoplasmic reticulum kinase under endoplasmic reticulum stress conditions and C/EBP homologous protein (CHOP). Levels of GRP78, phospho-eIF2α, and CHOP are increased in cells undergoing unfolded protein response. Failure to alleviate endoplasmic reticulum stress by unfolded protein response can lead to cellular apoptosis. The complex molecular mechanisms involved in the apoptotic death of endoplasmic reticulum–stressed cells have not been elucidated fully. However, downstream effectors of unfolded protein response–mediated apoptosis in humans may include caspase 4, caspase 3, and caspase 9 ( Figure 1 ).


Simplified diagram of the unfolded protein response. Three major effectors of unfolded protein response, IRE1, ATF6, and PERK, are maintained in an inactive, membrane bound form by binding with GRP78. Binding of unfolded proteins to GRP78 causes release of GRP78 from the 3 unfolded protein response effector proteins and results in their activation. Activated ATF6 and IRE1 lead to increased activity of CHOP protein, which stimulates unfolded protein response. Activated PERK also activates CHOP via phosphorylation of eIF2α. Depending on the magnitude and duration of unfolded protein stress, unfolded protein response results in either cell recovery or cell death via apoptosis mediated by caspase 3 and 9.

ATF6 = activating transcription factor 6; CHOP = C/EBP homologous protein; GRP78 = glucose response protein 78; IRE1 = inositol requiring enzyme 1; PERK = pancreatic ER kinase (PKR)-like ER kinase; phospho-eIF2α = phosphorylated eukaryotic initiation factor 2α.

We hypothesized that unfolded protein response is activated in Fuchs dystrophy by the abundant production of misfolded protein, as indicated by thickening of Descemet membrane and the accumulation of wide-spaced collagen aggregates, which are hallmark findings in Fuchs dystrophy. We tested this hypothesis by electron microscopic assessment of endoplasmic reticulum in Fuchs dystrophy patients to evaluate for structural evidence of unfolded protein response activation. In addition, we performed immunohistochemistry of Fuchs dystrophy corneal endothelium for 3 unfolded protein response markers (GRP78, phospho-eIF2α, and CHOP) and 2 apoptosis markers (caspase 3 and 9).


Patients and Controls

The diagnosis of Fuchs dystrophy and keratoconus was made by fellowship-trained corneal specialists. For Fuchs dystrophy specimens, diagnosis was based on the presence of confluent endothelial guttae in both eyes and presence of corneal edema (stromal, epithelial, or both) in the operative eye. Diagnosis of keratoconus was based on corneal topography along with the presence of standard clinical signs. Corneas from autopsy cases with no history of corneal disease or pathologic corneas with diagnoses other than Fuchs dystrophy served as controls. Patient and control characteristics are summarized in Table 1 (TEM experiments) and Table 2 (immunohistochemistry).


Presence of Prominent Rough Endoplasmic Reticulum in the Corneal Endothelium of Fuchs Dystrophy and Control Corneas Assessed by Transmission Electron Microscopy

Prominent RER +/− Diagnosis
1 Granular dystrophy
2 Lattice dystrophy
3 + Macular dystrophy
4 +/− Lattice dystrophy
5 Macular dystrophy
6 Lattice dystrophy
7 Macular dystrophy
8 +/− Granular dystrophy
9 Granular dystrophy
Fuchs dystrophy
1 + Fuchs dystrophy
2 + Fuchs dystrophy
3 + Fuchs dystrophy
4 + Fuchs dystrophy
5 + Fuchs dystrophy
6 + Fuchs dystrophy
7 + Fuchs dystrophy
8 + Fuchs dystrophy
9 + Fuchs dystrophy
10 + Fuchs dystrophy

RER = rough endoplasmic reticulum.


Patient Information for Corneas Used for Immunohistochemistry

Autopsy Corneas
Age (yrs) Cause of Death Corneal Findings Gender Death to Preservation Time (hours)
19 Cystic fibrosis Normal Male 16
54 Larynx carcinoma Normal Female 11
70 Liver cirrhosis Normal Male 44
92 Aspiration pneumonia Normal Male 49
58 Human immunodeficiency virus Normal Male 13
65 Pneumonia Normal Female 58
59 Adenocarcinoma gall bladder Normal Female 15
53 Multisystem organ failure Normal Male 21
6 Cerebral infarctions Normal Male 23
63 Metastatic carcinoma Normal Male 52
75 Aneurysm rupture Normal Female 53
80 Aspiration pneumonia Normal Male 46
47 Lung cancer Normal Male 59
60 Necrotizing pancreatitis Normal Male 7
45 Pulmonary failure, liver transplantation rejection Normal Male 63
51 Metastatic melanoma Normal Male 24
71 Adenocarcinoma colon Normal Male 30
67 Gastrointestinal hemorrhage Normal Male 43
Mean 58 35
SD 20 19

Fuchs Dystrophy Corneas
Age (yrs) Clinical Diagnosis Pathologic Diagnosis Gender
38 Fuchs dystrophy Fuchs dystrophy Female
60 Fuchs dystrophy Fuchs dystrophy Male
89 Fuchs dystrophy Fuchs dystrophy Female
68 Fuchs dystrophy Fuchs dystrophy Male
34 Pseudophakic bullous keratopathy Fuchs dystrophy Male
68 Fuchs dystrophy Fuchs dystrophy Male
74 Fuchs dystrophy Fuchs dystrophy Female
67 Fuchs dystrophy Fuchs dystrophy Male
Mean 62
SD 18

Jan 17, 2017 | Posted by in OPHTHALMOLOGY | Comments Off on Unfolded Protein Response in Fuchs Endothelial Corneal Dystrophy: A Unifying Pathogenic Pathway?
Premium Wordpress Themes by UFO Themes