Jonathan Lin, MD, PhD

All Publications

• Evaluation of Verteporfin as a Novel Antifibrotic Agent in a Rabbit Model of Glaucoma Filtration Surgery: A Pilot Study. Ophthalmology science Sun, M. T., Cotton, R. M., Charoenkijkajorn, C., Garcia-Sanchez, J., Dalal, R., Xia, X., Lin, J. H., Singh, K., Goldberg, J. L., Liu, W. W. 2024; 4 (3): 100448

  Abstract

  Verteporfin is a benzoporphyrin derivative which is Food and Drug Administration-approved for treatment of choroidal neovascularization in conjunction with photodynamic therapy. It has been shown to prevent fibrosis and scar formation in several organs and represents a promising novel antifibrotic agent for glaucoma surgery. The goal of this study is to determine the effect of verteporfin on wound healing after glaucoma filtration surgery.Preclinical study using a rabbit model of glaucoma filtration surgery.Eight New Zealand white rabbits underwent glaucoma filtration surgery in both eyes.Eyes were randomized into 4 study groups to receive a postoperative subconjunctival injection of 1 mg/mL verteporfin (n = 4), 0.4 mg/mL mitomycin C (MMC; n = 4), 0.4 mg/mL MMC + 1 mg/mL verteporfin (n = 4), or balanced salt solution (BSS) control (n = 4). Bleb survival, vascularity, and morphology were graded using a standard scale over a 30-day period, and intraocular pressure (IOP) was monitored. At 30 days postoperative or surgical failure, histology was performed to evaluate for inflammation, local toxicity, and scarring.The primary outcome measure was bleb survival. Secondary outcome measures were IOP, bleb morphology, and bleb histology.Compared to BSS control blebs, verteporfin-treated blebs demonstrated a trend toward increased surgical survival (mean 9.8 vs. 7.3 days, log rank P = 0.08). Mitomycin C-treated blebs survived significantly longer than verteporfin-treated blebs (log rank P = 0.009), with all but 1 MMC-treated bleb still surviving at postoperative day 30. There were no significant differences in survival between blebs treated with combination verteporfin + MMC and MMC alone. Mitomycin C-treated blebs were less vascular than verteporfin-treated blebs (mean vascularity score 0.3 ± 0.5 for MMC vs. 1.0 ± 0.0 for verteporfin, P < 0.01). Bleb histology did not reveal any significant toxicity in verteporfin-treated eyes. There were no significant differences in inflammation or scarring across groups.Although verteporfin remained inferior to MMC with regard to surgical survival, there was a trend toward increased survival compared with BSS control and it had an excellent safety profile. Further studies with variations in verteporfin dosage and/or application frequency are needed to assess whether this may be a useful adjunct to glaucoma surgery.Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

  View details for DOI 10.1016/j.xops.2023.100448

  View details for PubMedID 38261964

  View details for PubMedCentralID PMC10797546

• Conjunctival blue nevus in a child - Case report and review of literature. American journal of ophthalmology case reports Yahya, Y., Nangia, P., Ahmad, H., Frauches, R. L., Lin, J. H., Mruthyunjaya, P. 2024; 36: 102151

  Abstract

  To report a rare case of a conjunctival blue nevus in a child.A 10-year-old girl underwent an excisional biopsy for an atypical growing melanocytic conjunctival lesion. The diagnosis of a conjunctival blue nevus was confirmed on histopathology. We describe the histopathology and the anterior segment optical coherence tomography features of a blue nevus in a 10-year-old child along with a review of literature.Conjunctival blue nevus is rare and has rarely been reported in a child. Multimodal imaging may help document lesion progression. This condition should remain in the differential for a growing, pigmented conjunctival lesion.

  View details for DOI 10.1016/j.ajoc.2024.102151

  View details for PubMedID 39282597

  View details for PubMedCentralID PMC11396036

• Ocular Surface Fibroma of the Bulbar Conjunctiva. Ophthalmology Frauches, R., Lin, J. 2024

  View details for DOI 10.1016/j.ophtha.2024.05.008

  View details for PubMedID 38958614

• Ethnic Variation and Structure-Function Analysis of Tauopathy-Associated <i>PERK</i> Alleles ISRAEL JOURNAL OF CHEMISTRY Park, G., Galdamez, A., Song, K., Le, M., Kim, K., Lin, J. H. 2024

  View details for DOI 10.1002/ijch.202300173

  View details for Web of Science ID 001230310000001

• The endoplasmic reticulum: Homeostasis and crosstalk in retinal health and disease. Progress in retinal and eye research Zhang, S. X., Wang, J. J., Starr, C. R., Lee, E. J., Park, S., Zhylkibayev, A., Medina, A., Lin, J. H., Gorbatyuk, M. 2023: 101231

  Abstract

  The endoplasmic reticulum (ER) is the largest intracellular organelle carrying out a broad range of important cellular functions including protein biosynthesis, folding, and trafficking, lipid and sterol biosynthesis, carbohydrate metabolism, and calcium storage and gated release. In addition, the ER makes close contact with multiple intracellular organelles such as mitochondria and the plasma membrane to actively regulate the biogenesis, remodeling, and function of these organelles. Therefore, maintaining a homeostatic and functional ER is critical for the survival and function of cells. This vital process is implemented through well-orchestrated signaling pathways of the unfolded protein response (UPR). The UPR is activated when misfolded or unfolded proteins accumulate in the ER, a condition known as ER stress, and functions to restore ER homeostasis thus promoting cell survival. However, prolonged activation or dysregulation of the UPR can lead to cell death and other detrimental events such as inflammation and oxidative stress; these processes are implicated in the pathogenesis of many human diseases including retinal disorders. In this review manuscript, we discuss the unique features of the ER and ER stress signaling in the retina and retinal neurons and describe recent advances in the research to uncover the role of ER stress signaling in neurodegenerative retinal diseases including age-related macular degeneration, inherited retinal degeneration, achromatopsia and cone diseases, and diabetic retinopathy. In some chapters, we highlight the complex interactions between the ER and other intracellular organelles focusing on mitochondria and illustrate how ER stress signaling regulates common cellular stress pathways such as autophagy. We also touch upon the integrated stress response in retinal degeneration and diabetic retinopathy. Finally, we provide an update on the current development of pharmacological agents targeting the UPR response and discuss some unresolved questions and knowledge gaps to be addressed by future research.

  View details for DOI 10.1016/j.preteyeres.2023.101231

  View details for PubMedID 38092262

• TDP43 pathology in chronic traumatic encephalopathy retinas. Acta neuropathologica communications Phansalkar, R., Goodwill, V. S., Nirschl, J. J., De Lillo, C., Choi, J., Spurlock, E., Coughlin, D. G., Pizzo, D., Sigurdson, C. J., Hiniker, A., Alvarez, V. E., Mckee, A. C., Lin, J. H. 2023; 11 (1): 152

  Abstract

  Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease associated with repetitive head trauma. Brain pathology in CTE is characterized by neuronal loss, gliosis, and a distinctive pattern of neuronal accumulation of hyper-phosphorylated tau (p-tau) and phospho-TDP43 (p-TDP43). Visual anomalies have been reported by patients with CTE, but the ocular pathology underlying these symptoms is unknown. We evaluated retinal pathology in post-mortem eyes collected from 8 contact sport athletes with brain autopsy-confirmed stage IV CTE and compared their findings to retinas from 8 control patients without CTE and with no known history of head injury. Pupil-optic nerve cross sections were prepared and stained with hematoxylin and eosin (H&E), p-tau, p-TDP43, and total TDP43 by immunohistochemistry. No significant retinal degeneration was observed in CTE eyes compared to control eyes by H&E. Strong cytoplasmic p-TDP43 and total TDP43 staining was found in 6/8 CTE eyes in a subset of inner nuclear layer interneurons (INL) of the retina, while only 1/8 control eyes showed similar p-TDP43 pathology. The morphology and location of these inner nuclear layer interneurons were most compatible with retinal horizontal cells, although other retinal cell types present in INL could not be ruled out. No p-tau pathology was observed in CTE or control retinas. These findings identify novel retinal TDP43 pathology in CTE retinas and support further investigation into the role of p-TDP43 in producing visual deficits in patients with CTE.

  View details for DOI 10.1186/s40478-023-01650-6

  View details for PubMedID 37737191

  View details for PubMedCentralID 7914059

• Lysine Ubiquitylation Drives Rhodopsin Protein Turnover. Advances in experimental medicine and biology Chen, A. P., Chea, L., Lee, E. J., Lin, J. H. 2023; 1415: 493-498

  Abstract

  Rhodopsin is a G-protein-coupled receptor that is specifically and abundantly expressed in rod photoreceptors. Over 150 rhodopsin mutations cause autosomal dominant retinitis pigmentosa (adRP). The most common mutation in the United States is the conversion of proline to histidine at position 23 (P23H) in the N-terminal domain of rhodopsin. We previously found that P23H rhodopsin was misfolded, ubiquitinylated, and rapidly degraded. Here, we investigated the role of lysine residues on P23H rhodopsin ubiquitinylation and turnover. We transfected HEK293 cells with a P23H human rhodopsin construct where all 11 lysine residues were mutated to arginine (K-null P23H). We found that the K-null P23H rhodopsin was significantly less ubiquitylated than intact P23H rhodopsin. We found that K-null P23H protein turnover was significantly slower compared to P23H rhodopsin through cycloheximide chase analysis. Finally, we also generated a wild-type rhodopsin construct where all lysines were converted to arginine and found significantly reduced ubiquitylation. Our findings identify ubiquitinylation of lysine residues as an important posttranslational modification involved in P23H rhodopsin protein degradation.

  View details for DOI 10.1007/978-3-031-27681-1_72

  View details for PubMedID 37440077

  View details for PubMedCentralID 4627472

• Interpreting Discordant Monosomy 3 FISH and Chromosomal Microarray Analysis Results in Uveal Melanoma. Diagnostics (Basel, Switzerland) Long, C. P., Coley, N., Thorson, J., Lin, J. H. 2023; 13 (5)

  Abstract

  Uveal melanoma is the most common primary ocular tumor in adults and causes morbidity through lymphovascular metastasis. The presence of monosomy 3 in uveal melanomas is one of the most important prognostic indicators for metastasis. Two major molecular pathology testing modalities used to assess monosomy 3 are fluorescence in situ hybridization (FISH) and chromosomal microarray analysis (CMA). Here, we report two cases of discordant monosomy 3 test results in uveal melanoma enucleation specimens, performed using these molecular pathology tests. The first case is of uveal melanoma from a 51-year-old male that showed no evidence of monosomy 3 when assessed by CMA, but where it was subsequently detected by FISH. The second case is of uveal melanoma from a 49-year-old male that showed monosomy 3 at the limit of detection when assessed by CMA, but where it was not detected by subsequent FISH analysis. These two cases underscore the potential benefits of each testing modality for monosomy 3. Mainly, while CMA may be more sensitive to low levels of monosomy 3, FISH may be best method for small tumors with high levels of adjacent normal ocular tissue. Our cases suggest that both testing methods should be pursued for uveal melanoma, with a single positive result for either test interpreted as indicating the presence of monosomy 3.

  View details for DOI 10.3390/diagnostics13050946

  View details for PubMedID 36900091

• Neurodegeneration Risk Factor, EIF2AK3 (PERK), Influences Tau Protein Aggregation. The Journal of biological chemistry Park, G., Xu, K., Chea, L., Kim, K., Safarta, L., Song, K., Wu, J., Park, S., Min, H., Hiramatsu, N., Han, J., Lin, J. H. 2022: 102821

  Abstract

  Tauopathies are neurodegenerative diseases caused by pathologic misfolded tau protein aggregation in the nervous system. Population studies implicate EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), better known as PERK (protein kinase R-like endoplasmic reticulum kinase), as a genetic risk factor in several tauopathies. PERK is a key regulator of intracellular proteostatic mechanisms - Unfolded Protein Response (UPR) and Integrated Stress Response (ISR). Previous studies found that tauopathy-associated PERK variants encoded functional hypomorphs with reduced signaling in vitro. But, it remained unclear how altered PERK activity led to tauopathy. Here, we chemically or genetically modulated PERK signaling in cell culture models of tau aggregation and found that PERK pathway activation prevented tau aggregation while inhibition exacerbated tau aggregation. In primary tauopathy patient brain tissues, we found that reduced PERK signaling correlated with increased tau neuropathology. We found that tauopathy-associated PERK variants targeted the ER luminal domain; and two of these variants damaged hydrogen bond formation. Our studies support that PERK activity protects against tau aggregation and pathology. This may explain why people carrying hypomorphic PERK variants have increased risk for developing tauopathies. Finally, our studies identify small molecule augmentation of PERK signaling as an attractive therapeutic strategy to treat tauopathies by preventing tau pathology.

  View details for DOI 10.1016/j.jbc.2022.102821

  View details for PubMedID 36563857

• Mitochondria and Endoplasmic Reticulum Stress in Retinal Organoids from Vision Loss Patients. The American journal of pathology Lee, E., Diaz-Aguilar, M. S., Min, H., Choi, J., Valdez Duran, D. A., Grandjean, J. M., Wiseman, R. L., Kroeger, H., Lin, J. H. 2022

  Abstract

  Activating transcription factor 6 (ATF6), a key regulator of the unfolded protein response (UPR), is required for endoplasmic reticulum (ER) function and protein homeostasis. Variants of ATF6 that abrogate transcriptional activity cause morphologic and molecular defects in cones manifesting clinically as the human vision loss disease achromatopsia (ACHM). ATF6 is expressed in all retinal cells. However, the effect of disease-associated ATF6 variants on other retinal cell types remains unclear. To investigate this question, we analyzed bulk-RNA-seq transcriptomes from retinal-organoids generated from ACHM patients carrying homozygous loss-of-function ATF6 variants. We identified marked dysregulation in mitochondrial respiratory complex gene expression and disrupted mitochondrial morphology in ACHM retinal organoids, indicating that loss of ATF6 leads to previously unappreciated mitochondrial defects in the retina. Next, we compared gene expression from control and ACHM retinal organoids with transcriptome profiles of 7 major retinal cell types generated from recent single-cell transcriptomic maps of non-diseased human retina. Our analysis revealed pronounced down-regulation of cone genes and up-regulation in Muller glia genes, with no significant effects on other retinal cells. Overall, our analysis of ACHM patient retinal organoids identifies new cellular and molecular phenotypes in addition to cone dysfunction: activation of Muller cells, increased ER stress, and disrupted mitochondrial structure and elevated respiratory chain activity gene expression.

  View details for DOI 10.1016/j.ajpath.2022.12.002

  View details for PubMedID 36535406

• Network biology analysis of P23H rhodopsin interactome identifies protein and mRNA quality control mechanisms. Scientific reports Kim, K., Safarta, L. A., Chiang, W. J., Coppinger, J. A., Lee, E. J., Lin, J. H. 2022; 12 (1): 17405

  Abstract

  Rhodopsin is essential for phototransduction, and many rhodopsin mutations cause heritable retinal degenerations. The P23H rhodopsin variant generates a misfolded rhodopsin protein that photoreceptors quickly target for degradation by mechanisms that are incompletely understood. To gain insight into how P23H rhodopsin is removed from rods, we used mass spectrometry to identify protein interaction partners of P23H rhodopsin immunopurified from RhoP23H/P23H mice and compared them with protein interaction partners of wild-type rhodopsin from Rho+/+ mice. We identified 286 proteins associated with P23H rhodopsin and 276 proteins associated with wild-type rhodopsin. 113 proteins were shared between wild-type and mutant rhodopsin protein interactomes. In the P23H rhodopsin protein interactome, we saw loss of phototransduction, retinal cycle, and rhodopsin protein trafficking proteins but gain of ubiquitin-related proteins when compared with the wild-type rhodopsin protein interactome. In the P23H rhodopsin protein interactome, we saw enrichment of gene ontology terms related to ER-associated protein degradation, ER stress, and translation. Protein-protein interaction network analysis revealed that translational and ribosomal quality control proteins were significant regulators in the P23H rhodopsin protein interactome. The protein partners identified in our study may provide new insights into how photoreceptors recognize and clear mutant rhodopsin, offering possible novel targets involved in retinal degeneration pathogenesis.

  View details for DOI 10.1038/s41598-022-22316-8

  View details for PubMedID 36258031

• Minimal change prion retinopathy: Morphometric comparison of retinal and brain prion deposits in Creutzfeldt-Jakob disease. Experimental eye research Goodwill, V. S., Dryden, I., Choi, J., De Lillo, C., Soldau, K., Llibre-Guerra, J., Sanchez, H., Sigurdson, C. J., Lin, J. H. 2022: 109172

  Abstract

  Sporadic Creutzfeldt-Jakob disease (sCJD) is the most commonly diagnosed human prion disease caused by the abnormal misfolding of the 'cellular' prion protein (PrPC) into the transmissible 'scrapie-type' prion form (PrPSc). Neuropathologic evaluation of brains with sCJD reveals abnormal PrPSc deposits in grey matter structures, often associated with micro-vacuolar spongiform changes in neuropil, neuronal loss, and gliosis. Abnormal PrPSc deposits have also been reported in the retina of patients with sCJD, but few studies have characterized the morphology of these retinal PrPSc deposits or evaluated for any retinal neurodegenerative changes. We performed histopathologic and morphometric analyses of retinal and brain prion deposits in 14 patients with sCJD. Interestingly, we discovered that the morphology of retinal PrPSc deposits generally differs from that of brain PrPSc deposits in terms of size and shape. We found that retinal PrPSc deposits consistently localize to the outer plexiform layer of the retina. Additionally, we observed that the retinal PrPSc deposits are not associated with the spongiform change, neuronal loss, and gliosis often seen in the brain. The stereotypic morphology and location of PrPSc deposits in sCJD retinas may help guide the use of ocular imaging devices in the detection of these deposits for a clinical diagnosis.

  View details for DOI 10.1016/j.exer.2022.109172

  View details for PubMedID 35803332

• Preferentially Expressed Antigen in Melanoma Immunohistochemistry Labeling in Uveal Melanomas OCULAR ONCOLOGY AND PATHOLOGY Ahmadian, S. S., Dryden, I. J., Naranjo, A., Toland, A., Cayrol, R. A., Born, D. E., Egbert, P. S., Brown, R. A., Mruthyunjaya, P., Lin, J. H. 2022; 8 (2): 133-140

  View details for DOI 10.1159/000524051

  View details for Web of Science ID 000812351000009

• A Novel ATF6-Achromatopsia Allele Regulated by Nonsense-Mediated mRNA Decay Al Moujahed, A., Safarta, L., Vollrath, D., Lin, J. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2022

  View details for Web of Science ID 000844401301167

• Preferentially Expressed Antigen in Melanoma Immunohistochemistry Labeling in Uveal Melanomas. Ocular oncology and pathology Ahmadian, S. S., Dryden, I. J., Naranjo, A., Toland, A., Cayrol, R. A., Born, D. E., Egbert, P. S., Brown, R. A., Mruthyunjaya, P., Lin, J. H. 2022; 8 (2): 133-140

  Abstract

  Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, and despite treatment of the primary tumor, approximately 15%-50% of patients will develop metastatic disease. Based on gene expression profiling (GEPs), UM can be categorized as Class 1A (low metastatic risk), Class 1B (intermediate metastatic risk), or Class 2 (high metastatic risk). PReferentially expressed Antigen in MElanoma (PRAME) status is an independent prognostic UM biomarker and a potential target for immunotherapy in metastatic UM. PRAME expression status can be detected in tumors using reverse-transcription polymerase chain reaction (RT-PCR). More recently, immunohistochemistry (IHC) has been developed to detect PRAME protein expression. Here, we employed both techniques to evaluate PRAME expression in 18 UM enucleations.Tumor material from the 18 UM patients who underwent enucleation was collected by fine-needle aspiration before or during enucleation and sent for GEP and PRAME analysis by RT-PCR. Histologic sections from these patients were stained with an anti-PRAME monoclonal antibody. We collected patient demographics and tumor characteristics and included this with our analysis of GEP class, PRAME status by RT-PCR, and PRAME status by IHC. PRAME IHC and RT-PCR results were compared.Twelve males (12/18) and 6 females (6/18) with an average age of 60.6 years underwent enucleation for UM. TNM staging of the UM diagnosed Stage I in 2 patients (2/18), Stage II in 7 patients (7/18), Stage III in 8 patients (8/18), and Stage IV in 1 (1/18). GEP was Class 1A in 6 tumors (6/18), Class 1B in 6 tumors (6/18), and Class 2 in 6 tumors (6/18). PRAME IHC showed diffusely positive labeling of all UM cells in 2/18 enucleations; negative IHC labeling of UM cells in 9/18 enucleations; and IHC labeling of subsets of UM cells in 7/18 enucleations. Eleven of the 17 UMs tested for PRAME by both RT-PCR and IHC had consistent PRAME results. In the remaining 6/17 cases tested by both modalities, PRAME results were discordant between RT-PCR and IHC.We find that PRAME IHC distinguishes PRAME-positive and PRAME-negative UM tumor cells. Interestingly, IHC reveals focal PRAME expression in subsets of tumor cells consistent with tumor heterogeneity. PRAME RT-PCR and IHC provide concordant results in most of our cases. We suggest that discordance in PRAME results could arise from spatial or temporal variation in PRAME expression between tumor cells. Further studies are required to determine the prognostic implications of PRAME IHC in UM.

  View details for DOI 10.1159/000524051

  View details for PubMedID 35959159

  View details for PubMedCentralID PMC9218614

• Bilateral Serpiginous-Like Chorioretinitis Associated with Ciliochoroidal Melanoma: A Clinicopathologic Correlation. Retina (Philadelphia, Pa.) Al-Moujahed, A., Lin, J. H., Gagnon, M. R., Pulido, J., Mruthyunjaya, P., Jung, J. J. 2022

  Abstract

  PURPOSE: To report the clinicopathologic correlation of a case of bilateral serpiginous-like chorioretinitis (SLC) associated with unilateral ciliochoroidal melanoma.METHODS: A 71-year-old white female was diagnosed with progressive SLC in both eyes (OU) associated with ciliochoroidal melanoma in the right eye (OD). Clinical findings and imaging before and after enucleation OD were correlated to histologic and immunohistochemistry sections.RESULTS: Examination and imaging identified a peripheral bilobed amelanotic lesion with low reflectivity on B-scan ultrasound with an associated exudative detachment OD. Additionally, multiple areas of new SLC lesions in the macula and peripapillary region OD and along the inferior arcade in the left eye (OS) were observed. Oncologic evaluation confirmed a Class 2, ciliochoroidal melanoma, and the eye was enucleated. Autoimmune and infectious laboratory evaluation for the etiology of the SLC lesions were negative. Histopathology of the enucleated eye confirmed the diagnosis of uveal melanoma with lymphocytic inflammation at the edges of the tumor itself and in the areas of discrete SLC lesions. Immunohistochemistry identified similar predominantly cluster of differentiation (CD)3 and CD8 T-cells and fewer CD20 B-cells in both regions.CONCLUSION: Serpiginous-like chorioretinitis may present as a paraneoplastic, predominantly T-lymphocyte inflammation associated with intraocular tumor such as uveal melanoma.

  View details for DOI 10.1097/IAE.0000000000003435

  View details for PubMedID 35174798

• ATF6 is essential for human cone photoreceptor development. Proceedings of the National Academy of Sciences of the United States of America Kroeger, H., Grandjean, J. M., Chiang, W. J., Bindels, D. D., Mastey, R., Okalova, J., Nguyen, A., Powers, E. T., Kelly, J. W., Grimsey, N. J., Michaelides, M., Carroll, J., Wiseman, R. L., Lin, J. H. 2021; 118 (39)

  Abstract

  Endoplasmic reticulum (ER) stress and Unfolded Protein Response (UPR) signaling promote the pathology of many human diseases. Loss-of-function variants of the UPR regulator Activating Transcription Factor 6 (ATF6) cause severe congenital vision loss diseases such as achromatopsia by unclear pathomechanisms. To investigate this, we generated retinal organoids from achromatopsia patient induced pluripotent stem cells carrying ATF6 disease variants and from gene-edited ATF6 null hESCs. We found that achromatopsia patient and ATF6 null retinal organoids failed to form cone structures concomitant with loss of cone phototransduction gene expression, while rod photoreceptors developed normally. Adaptive optics retinal imaging of achromatopsia patients carrying ATF6 variants also showed absence of cone inner/outer segment structures but preserved rod structures, mirroring the defect in cone formation observed in our retinal organoids. These results establish that ATF6 is essential for human cone development. Interestingly, we find that a selective small molecule ATF6 signaling agonist restores the transcriptional activity of some ATF6 disease-causing variants and stimulates cone growth and gene expression in patient retinal organoids carrying these variants. These findings support that pharmacologic targeting of the ATF6 pathway can promote human cone development and should be further explored for blinding retinal diseases.

  View details for DOI 10.1073/pnas.2103196118

  View details for PubMedID 34561305

• ATF6 is required for efficient rhodopsin clearance and retinal homeostasis in the P23H rho retinitis pigmentosa mouse model. Scientific reports Lee, E., Chan, P., Chea, L., Kim, K., Kaufman, R. J., Lin, J. H. 2021; 11 (1): 16356

  Abstract

  Retinitis Pigmentosa (RP) is a blinding disease that arises from loss of rods and subsequently cones. The P23H rhodopsin knock-in (P23H-KI) mouse develops retinal degeneration that mirrors RP phenotype in patients carrying the orthologous variant. Previously, we found that the P23H rhodopsin protein was degraded in P23H-KI retinas, and the Unfolded Protein Response (UPR) promoted P23H rhodopsin degradation in heterologous cells in vitro. Here, we investigated the role of a UPR regulator gene, activating transcription factor 6 (Atf6), in rhodopsin protein homeostasis in heterozygous P23H rhodopsin (Rho+/P23H) mice. Significantly increased rhodopsin protein levels were found in Atf6-/-Rho+/P23H retinas compared to Atf6+/-Rho+/P23H retinas at early ages (~P12), while rhodopsin mRNA levels were not different. The IRE1 pathway of the UPR was hyper-activated in young Atf6-/-Rho+/P23H retinas, and photoreceptor layer thickness was unchanged at this early age in Rho+/P23H mice lacking Atf6. By contrast, older Atf6-/-Rho+/P23H mice developed significantly increased retinal degeneration in comparison to Atf6+/-Rho+/P23H mice in all retinal layers, accompanied by reduced rhodopsin protein levels. Our findings demonstrate that Atf6 is required for efficient clearance of rhodopsin protein in rod photoreceptors expressing P23H rhodopsin, and that loss of Atf6 ultimately accelerates retinal degeneration in P23H-KI mice.

  View details for DOI 10.1038/s41598-021-95895-7

  View details for PubMedID 34381136

• ARCAM-1 Facilitates Fluorescence Detection of Amyloid-Containing Deposits in the Retina. Translational vision science & technology Cao, K. J., Kim, J. H., Kroeger, H., Gaffney, P. M., Lin, J. H., Sigurdson, C. J., Yang, J. 2021; 10 (7): 5

  Abstract

  Purpose: To investigate the use of an amyloid-targeting fluorescent probe, ARCAM-1, to identify amyloid-containing deposits in the retina of a transgenic mouse model of Alzheimer's disease (AD) and in human postmortem AD patients.Methods: Aged APP/PS1 transgenic AD and wild-type (WT) mice were given an intraperitoneal (IP) injection of ARCAM-1 and their retinas imaged in vivo using a fluorescence ophthalmoscope. Eyes were enucleated and dissected for ex vivo inspection of retinal amyloid deposits. Additionally, formalin-fixed eyes from human AD and control patients were dissected, and the retinas were stained using ARCAM-1 or with an anti-amyloid-beta antibody. Confocal microscopy was used to image amyloid-containing deposits stained with ARCAM-1 or with immunostaining.Results: Four out of eight APP/PS1 mice showed the presence of amyloid aggregates in the retina during antemortem imaging. Retinas from three human AD patients stained with ARCAM-1 showed an apparent increased density of fluorescently labeled amyloid-containing deposits compared to the retinas from two healthy, cognitively normal (CN) patients. Immunolabeling confirmed the presence of amyloid deposits in both the retinal neuronal layers and in retinal vasculature.Conclusions: ARCAM-1 facilitates antemortem detection of amyloid aggregates in the retina of a mouse model for AD, and postmortem detection of amyloid-containing deposits in human retinal tissues from AD patients. These results support the hypothesis of AD pathology manifesting in the eye and highlight a novel area for fluorophore development for the optical detection of retinal amyloid in AD patients.Translational Relevance: This paper represents an initial examination for potential translation of an amyloid-targeting fluorescent probe to a retinal imaging agent for aiding in the diagnosis of Alzheimer's disease.

  View details for DOI 10.1167/tvst.10.7.5

  View details for PubMedID 34096989

• Genome Sequencing and Apoptotic Markers to Assess Treatment Response of Lacrimal Gland Adenoid Cystic Carcinoma to Intra-Arterial Cytoreductive Chemotherapy. Ophthalmic plastic and reconstructive surgery Yu, M. D., Men, C. J., Do, H., Colevas, A. D., Lin, J. H., Egbert, P. R., Tse, D. T., Kossler, A. L. 2021

  Abstract

  Adenoid cystic carcinoma of the lacrimal gland is an aggressive, malignant epithelial neoplasm. We report the case of a 30-year-old male with lacrimal gland adenoid cystic carcinoma treated with neoadjuvant intra-arterial chemotherapy through the internal carotid artery, followed by orbital exenteration and chemoradiation. Treatment response was evaluated using a novel combination of pre- and posttreatment genome sequencing coupled with immunohistochemical evaluation, which showed diffuse tumor apoptosis. A posttreatment decrease in variant allele frequency of the NOTCH1 mutation, and robust tumor cytoreduction on imaging, supports exploration of NOTCH1 analysis as a potential marker of cisplatin sensitivity. The use of genome sequencing and immunohistochemical evaluation could provide a more targeted therapeutic assessment of neoadjuvant intra-arterial chemotherapy in the management of lacrimal gland adenoid cystic carcinoma.

  View details for DOI 10.1097/IOP.0000000000002079

  View details for PubMedID 34798653

• Sinonasal Ewing sarcoma misdiagnosed as recurrent glomangiopericytoma: Case report and literature review Otolaryngology Case Reports Huang, A. E., Hwang, P. H., Lin, J. H., Chang, M. T. 2021; 21: 100369

  View details for DOI 10.1016/j.xocr.2021.100369

• Colorectal carcinoma presenting in the orbit: mass effect from an uncommon cause. Orbit (Amsterdam, Netherlands) Long, C. P., Lu, T., Ediriwickrema, L. S., Lin, J. H., Korn, B. S., Kikkawa, D. O., Liu, C. Y. 2020: 1–4

  Abstract

  An 84-year-old male with previously documented poor medical follow-up presented with progressive painless proptosis of the right eye. Right upper eyelid ptosis, limited motility, proptosis, and inferomedial displacement of the right globe were noted on the exam. Computed tomography (CT) imaging revealed a right retrobulbar extraconal heterogenous mass with ill-defined borders. Biopsy revealed a malignant adenocarcinoma with tumor markers suggestive of a colorectal primary. A rectal mass was identified during a systemic workup. After biopsy, the patient was diagnosed with stage IV metastatic rectal adenocarcinoma. He began palliative radiation therapy shortly following diagnosis.

  View details for DOI 10.1080/01676830.2020.1787466

  View details for PubMedID 32643502

• Incidence Rates of Benign and Malignant Eyelid Lesions at Stanford Healthcare from 2016-2019 Ahmadian, S., Lin, J., Cayrol, R., Born, D., Basham, R., Egbert, P. OXFORD UNIV PRESS INC. 2020: 681–82

  View details for Web of Science ID 000538796100116

• Ocular Surface Squamous Neoplasms in 75 HIV+ Mozambicans Kim, Y., Miu, C., Carrilho, C., Liu, Y., Schooley, R., Lin, J. H. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2020

  View details for Web of Science ID 000554528304009

• Multi-Exon Deletion Alleles of ATF6 Linked to Achromatopsia Kim, K., Lee, E., Chiang, W., Kroeger, H., Bi, C., Chao, D. L., Mastey, R., Tsang, S., Chea, L., Baumann, B., Audo, I. S., Kohl, S., Moore, A., Wiseman, L., Carroll, J., Lin, J. H. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2020

  View details for Web of Science ID 000554495705021

• Expanding the spectrum of Kabuki syndrome with novel neuropathological, ocular and genetic findings in an autopsy case Cayrol, R., Ahmadian, S., Michie, S., Lin, J., Born, D. OXFORD UNIV PRESS INC. 2020: 677

  View details for Web of Science ID 000538796100099

• Multiexon deletion alleles of ATF6 linked to achromatopsia. JCI insight Lee, E., Chiang, W. J., Kroeger, H., Bi, C. X., Chao, D. L., Skowronska-Krawczyk, D., Mastey, R. R., Tsang, S. H., Chea, L., Kim, K., Lambert, S. R., Grandjean, J. M., Baumann, B., Audo, I., Kohl, S., Moore, A. T., Wiseman, R. L., Carroll, J., Lin, J. H. 2020; 5 (7)

  Abstract

  Achromatopsia (ACHM) is an autosomal recessive disease that results in severe visual loss. Symptoms of ACHM include impaired visual acuity, nystagmus, and photoaversion starting from infancy; furthermore, ACHM is associated with bilateral foveal hypoplasia and absent or severely reduced cone photoreceptor function on electroretinography. Here, we performed genetic sequencing in 3 patients from 2 families with ACHM, identifying and functionally characterizing 2 mutations in the activating transcription factor 6 (ATF6) gene. We identified a homozygous deletion covering exons 8-14 of the ATF6 gene from 2 siblings from the same family. In another patient from a different family, we identified a heterozygous deletion covering exons 2 and 3 of the ATF6 gene found in trans with a previously identified ATF6 c.970C>T (p.Arg324Cys) ACHM disease allele. Recombinant ATF6 proteins bearing these exon deletions showed markedly impaired transcriptional activity by qPCR and RNA-Seq analysis compared with WT-ATF6. Finally, RNAscope revealed that ATF6 and the related ATF6B transcripts were expressed in cones as well as in all retinal layers in normal human retina. Overall, our data identify loss-of-function ATF6 disease alleles that cause human foveal disease.

  View details for DOI 10.1172/jci.insight.136041

  View details for PubMedID 32271167

• PREVALENCE OF MISMATCH REPAIR GENE MUTATIONS IN UVEAL MELANOMA. Retina (Philadelphia, Pa.) Toomey, C. B., Protopsaltis, N. J., Phou, S. n., Bakhoum, M. F., Thorson, J. A., Ediriwickrema, L. S., Korn, B. S., Kikkawa, D. O., Goldbaum, M. H., Lin, J. H. 2020

  Abstract

  Uveal melanomas are associated with characteristic genetic changes. Germline mutations in mismatch repair (MMR) genes and microsatellite instability have been implicated in the development of numerous malignant neoplasms such as colon and ovarian cancers. The frequency of MMR defects in uveal melanomas has yet to be determined.Here, we analyzed the frequency of MMR gene mutations in uveal melanoma specimens from the University of California, San Diego (UCSD), The Cancer Genome Atlas (TGCA), and the Catalogue of Somatic Mutations in Cancer (COSMIC).We identified only two mutations in a MMR gene: one premature stop codon in the PMS gene within the UCSD cohort (0.5% frequency) and one in-frame deletion in MSH3 within the COSMIC database (0.8% frequency). We report copy number variation of MLH1 in monosomy 3 and show decreased mRNA expression of MLH1 in uveal melanoma specimens with monosomy 3. Expression levels of MLH1 were not found to correlate with the observed number of total mutations.Overall, we show that mutations in MMR genes in uveal melanoma specimens are exceedingly rare, and although one copy of MLH1 is lost in monosomy 3, it does not seem to have pathologic consequences in uveal melanoma pathogenesis.

  View details for DOI 10.1097/IAE.0000000000002732

  View details for PubMedID 32032254

• Neuroprotective Role of Akt in Hypoxia Adaptation in Andeans. Frontiers in neuroscience Zhao, H., Lin, J., Sieck, G., Haddad, G. G. 2020; 14: 607711

  Abstract

  Chronic mountain sickness (CMS) is a disease that potentially threatens a large segment of high-altitude populations during extended living at altitudes above 2,500 m. Patients with CMS suffer from severe hypoxemia, excessive erythrocytosis and neurologic deficits. The cellular mechanisms underlying CMS neuropathology remain unknown. We previously showed that iPSC-derived CMS neurons have altered mitochondrial dynamics and increased susceptibility to hypoxia-induced cell death. Genome analysis from the same population identified many ER stress-related genes that play an important role in hypoxia adaptation or lack thereof. In the current study, we showed that iPSC-derived CMS neurons have increased expression of ER stress markers Grp78 and XBP1s under normoxia and hyperphosphorylation of PERK under hypoxia, alleviating ER stress does not rescue the hypoxia-induced CMS neuronal cell death. Akt is a cytosolic regulator of ER stress with PERK as a direct target of Akt. CMS neurons exhibited lack of Akt activation and lack of increased Parkin expression as compared to non-CMS neurons under hypoxia. By enhancing Akt activation and Parkin overexpression, hypoxia-induced CMS neuronal cell death was reduced. Taken together, we propose that increased Akt activation protects non-CMS from hypoxia-induced cell death. In contrast, impaired adaptive mechanisms including failure to activate Akt and increase Parkin expression render CMS neurons more susceptible to hypoxia-induced cell death.

  View details for DOI 10.3389/fnins.2020.607711

  View details for PubMedID 33519361

• IRE1α regulates macrophage polarization, PD-L1 expression, and tumor survival. PLoS biology Batista, A. n., Rodvold, J. J., Xian, S. n., Searles, S. C., Lew, A. n., Iwawaki, T. n., Almanza, G. n., Waller, T. C., Lin, J. n., Jepsen, K. n., Carter, H. n., Zanetti, M. n. 2020; 18 (6): e3000687

  Abstract

  In the tumor microenvironment, local immune dysregulation is driven in part by macrophages and dendritic cells that are polarized to a mixed proinflammatory/immune-suppressive phenotype. The unfolded protein response (UPR) is emerging as the possible origin of these events. Here we report that the inositol-requiring enzyme 1 (IRE1α) branch of the UPR is directly involved in the polarization of macrophages in vitro and in vivo, including the up-regulation of interleukin 6 (IL-6), IL-23, Arginase1, as well as surface expression of CD86 and programmed death ligand 1 (PD-L1). Macrophages in which the IRE1α/X-box binding protein 1 (Xbp1) axis is blocked pharmacologically or deleted genetically have significantly reduced polarization and CD86 and PD-L1 expression, which was induced independent of IFNγ signaling, suggesting a novel mechanism in PD-L1 regulation in macrophages. Mice with IRE1α- but not Xbp1-deficient macrophages showed greater survival than controls when implanted with B16.F10 melanoma cells. Remarkably, we found a significant association between the IRE1α gene signature and CD274 gene expression in tumor-infiltrating macrophages in humans. RNA sequencing (RNASeq) analysis showed that bone marrow-derived macrophages with IRE1α deletion lose the integrity of the gene connectivity characteristic of regulated IRE1α-dependent decay (RIDD) and the ability to activate CD274 gene expression. Thus, the IRE1α/Xbp1 axis drives the polarization of macrophages in the tumor microenvironment initiating a complex immune dysregulation leading to failure of local immune surveillance.

  View details for DOI 10.1371/journal.pbio.3000687

  View details for PubMedID 32520957

• IRE1α and IGF signaling predict resistance to an endoplasmic reticulum stress-inducing drug in glioblastoma cells. Scientific reports Rodvold, J. J., Xian, S. n., Nussbacher, J. n., Tsui, B. n., Cameron Waller, T. n., Searles, S. C., Lew, A. n., Jiang, P. n., Babic, I. n., Nomura, N. n., Lin, J. H., Kesari, S. n., Carter, H. n., Zanetti, M. n. 2020; 10 (1): 8348

  Abstract

  To date current therapies of glioblastoma multiforme (GBM) are largely ineffective. The induction of apoptosis by an unresolvable unfolded protein response (UPR) represents a potential new therapeutic strategy. Here we tested 12ADT, a sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor, on a panel of unselected patient-derived neurosphere-forming cells and found that GBM cells can be distinguished into "responder" and "non-responder". By RNASeq analysis we found that the non-responder phenotype is significantly linked with the expression of UPR genes, and in particular ERN1 (IRE1) and ATF4. We also identified two additional genes selectively overexpressed among non-responders, IGFBP3 and IGFBP5. CRISPR-mediated deletion of the ERN1, IGFBP3, IGFBP5 signature genes in the U251 human GBM cell line increased responsiveness to 12ADT. Remarkably, >65% of GBM cases in The Cancer Genome Atlas express the non-responder (ERN1, IGFBP3, IGFBP5) gene signature. Thus, elevated levels of IRE1α and IGFBPs predict a poor response to drugs inducing unresolvable UPR and possibly other forms of chemotherapy helping in a better stratification GBM patients.

  View details for DOI 10.1038/s41598-020-65320-6

  View details for PubMedID 32433555

• Radiation-Induced Hyalinizing Clear Cell Carcinoma of the Orbit. Ophthalmic plastic and reconstructive surgery Topping, K. L., Ortiz, A. n., Lin, J. H., Kossler, A. L. 2020

  Abstract

  Radiation-induced malignancy is rare, occurring in approximately 0.4%-1.0% of patients receiving external beam radiation therapy. Sarcomas and squamous cell carcinomas are among the most common types of cancers to occur. A 74-year-old woman presented with redness and swelling in the right periorbital region. She had history of multiple recurrent ameloblastoma of the right maxilla, invading the right orbital floor status post 4 surgical resections and 66 Gray external beam radiotherapy 5 years prior. MRI showed a poorly circumscribed mass involving the inferior and lateral orbit. Orbital biopsy revealed clear cell carcinoma with hyalinizing sclerosis and Ewing sarcoma breakpoint region 1 gene arrangement. Due to the extent of orbital disease and presence of perineural invasion, she underwent orbital exenteration. Hyalinizing clear cell carcinoma, a rare cancer, has not been reported to occur in the orbit following radiation. This case highlights the importance of lifetime monitoring in patients who have undergone radiation therapy.

  View details for DOI 10.1097/IOP.0000000000001708

  View details for PubMedID 32427738

• Reticular pseudodrusen in late-onset retinal degeneration. Ophthalmology. Retina Borooah, S. n., Papastavrou, V. n., Lando, L. n., Han, J. n., Lin, J. H., Ayyagari, R. n., Dhillon, B. n., Browning, A. C. 2020

  Abstract

  To characterize the association of reticular pseudodrusen (RPD) with late-onset retinal degeneration (L-ORD) using multimodal imaging.Prospective, two-center, longitudinal case series SUBJECTS: Twenty-nine cases with L-ORD.All subjects were evaluated within a three-year interval with near-infrared reflectance, fundus autofluorescence, and spectral-domain optical coherence tomography. In addition, a subset of patients also underwent indocyanine green angiography, fundus fluorescein angiography, mesopic microperimetry, and multifocal electroretinography.Prevalence, topographic distribution, and temporal phenotypic changes of RPD in L-ORD.A total of 29 molecularly confirmed L-ORD cases were included in this prospective study. RPD was detected in 18 cases (62%) at baseline, of which 10 were male. The prevalence of RPD varied with age. The mean age of RPD patients was 57.3±7.2 years. RPD was not seen in cases below the fifth decade (n=3 patients) or in the eighth decade (n=5 patients). RPD were found commonly in the macula with relative sparing of the fovea and were also identified in the peripheral retina. The morphology of RPD changed with follow-up. Two cases (3 eyes) demonstrated RPD regression.RPD is found frequently in cases with L-ORD and at a younger age than in individuals with AMD. RPD exhibits quick formation and collapse, change in type and morphology with time, relative foveal-sparing, and also has a peripheral retinal location in L-ORD.

  View details for DOI 10.1016/j.oret.2020.12.012

  View details for PubMedID 33352318

• Metastasis of Lung Adenocarcinoma to the Lacrimal Sac. Ophthalmic plastic and reconstructive surgery Eng, V. A., Lin, J. H., Mruthyunjaya, P. n., Erickson, B. P. 2020

  Abstract

  The authors report an unusual case of lung adenocarcinoma metastasis to the lacrimal sac. A 61-year-old woman with stage IV non-small cell lung cancer presented with left facial pain and epiphora. She was found to have an elevated tear meniscus associated with a firm, fixed medial canthal mass. Orbital imaging demonstrated nodular enlargement of the lacrimal drainage apparatus. Biopsy of the lacrimal sac was performed, and it revealed a metastatic lung adenocarcinoma. The patient received targeted radiation therapy to the lacrimal sac, and her dose of maintenance chemotherapy was increased. The patient's symptoms have since improved. This case of lung cancer involving the lacrimal sac highlights the importance of thorough oncologic surveillance, even with respect to locations atypical for metastatic spread.

  View details for DOI 10.1097/IOP.0000000000001827

  View details for PubMedID 32976332

• p16 Expression Correlates with Invasive Ocular Surface Squamous Neoplasms in HIV-Infected Mozambicans. Ocular oncology and pathology Carrilho, C. n., Miu, C. n., Kim, Y. n., Karki, S. n., Balmaceda, A. n., Challa, B. n., Diamond, S. n., Monteiro, E. n., Marole, E. n., Lorenzoni, C. n., Zambujo, Y. n., Liu, Y. T., Schooley, R. T., Lin, J. H. 2020; 6 (2): 123–28

  Abstract

  p16 immunohistochemistry is widely used to diagnose human papillomavirus (HPV)-related squamous neoplasms of cervix, anogenital, head, and neck tissues. The incidence of these HPV-related squamous neoplasms is markedly increased in the HIV-infected population. Ocular surface squamous neoplasia (OSSN) is also more common in HIV-infected patients. However, the expression pattern of p16 in OSSN among HIV-infected patients is unclear. Here, we examined the expression of p16 in OSSN surgical excisions collected from a large HIV-infected cohort from -Mozambique.OSSN surgical tissue specimens were collected from 75 Mozambican patients. Formalin-fixed, paraffin-embedded tissue blocks from these OSSNs were sectioned, stained with hematoxylin and eosin (H&E), and p16 expression by immunohistochemistry. H&E slides were reviewed to determine if OSSNs were noninvasive conjunctival intraepithelial neoplasms or invasive squamous cell carcinomas (SCC). Cases were classified as p16 positive or negative based on diffuse nuclear and cytoplasmic expression of p16 in neoplastic cells.p16 positivity was found in a minority of OSSN cases (14/75). p16 positivity was significantly associated with the invasive SCC type of OSSN in HIV-infected patients (p value of 0.026).The majority of OSSNs in our HIV-infected cohort do not express p16. However, those cases that are p16-positive are significantly more likely to be the invasive SCC form of OSSN. We propose that p16 expression may identify more aggressive OSSNs in HIV-infected populations.

  View details for DOI 10.1159/000502096

  View details for PubMedID 32258020

  View details for PubMedCentralID PMC7109427

• PERK-mediated induction of microRNA-483 disrupts cellular ATP homeostasis during the unfolded protein response. The Journal of biological chemistry Hiramatsu, N., Chiang, K., Aivati, C., Rodvold, J. J., Lee, J., Han, J., Chea, L., Zanetti, M., Koo, E. H., Lin, J. H. 2019

  Abstract

  Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), which reduces levels of misfolded proteins. However, if ER homeostasis is not restored and the UPR remains chronically activated, cells undergo apoptosis. The UPR regulator, PKR-like endoplasmic reticulum kinase (PERK), plays an important role in promoting cell death when persistently activated; however, the underlying mechanisms are poorly understood. Here, we profiled the microRNA (miRNA) transcriptome in human cells exposed to ER stress and identified miRNAs that are selectively induced by PERK signaling. We found that expression of a PERK-induced miRNA, miR-483, promotes apoptosis in human cells. miR-483 induction was mediated by a transcription factor downstream of PERK, activating transcription factor 4 (ATF4) but not by the CHOP transcription factor. We identified the creatine kinase, brain-type (CKB) gene, encoding an enzyme that maintains cellular ATP reserves through phosphocreatine production, as being repressed during the UPR and targeted by miR-483. We found that ER stress, selective PERK activation, and CKB knockdown all decrease cellular ATP levels, leading to increased vulnerability to ER stress-induced cell death. Our findings identify miR-483 as a downstream target of the PERK branch of the UPR. We propose that disruption of cellular ATP homeostasis through miR-483-mediated CKB silencing promotes ER stress-induced apoptosis.

  View details for DOI 10.1074/jbc.RA119.008336

  View details for PubMedID 31792031

• Pathomechanisms of ATF6-Associated Cone Photoreceptor Diseases. Advances in experimental medicine and biology Chiang, W. J., Kroeger, H. n., Chea, L. n., Lin, J. H. 2019; 1185: 305–10

  Abstract

  Activating transcription factor 6 (ATF6) is a key regulator of the unfolded protein response (UPR). In response to endoplasmic reticulum (ER) stress, ATF6 is transported from the ER to the Golgi apparatus where it is cleaved by intramembrane proteolysis, releasing its cytosolic fragment. The cleaved ATF6 fragment, which is a basic leucine zipper (bZip) transcription factor, translocates to the nucleus and upregulates the expression of ER protein-folding chaperones and enzymes. Mutations in ATF6 cause heritable forms of cone photoreceptor dysfunction diseases. These mutations include missense, nonsense, splice site, and deletion or duplication changes found across the entire ATF6. To date, there are 11 ATF6 mutations reported, and we classified them into three classes based on their functional defects that interrupt distinct steps in the ATF6 signaling pathway.

  View details for DOI 10.1007/978-3-030-27378-1_50

  View details for PubMedID 31884629

• Beware of the sneeze. Survey of ophthalmology Yang, P. n., Toomey, C. n., Lin, J. n., Kikkawa, D. O., Korn, B. S., Harrison, A. n. 2019

  Abstract

  Switch/sucrose non-fermentable-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1), also known as integrase interactor 1-deficient sinonasal carcinoma, is a rare entity that was first described in 2014. Since then, there have been 39 cases published in the literature, with basaloid or plasmacytoid/rhabdoid morphology being the most common pathological subtype. We report a patient with switch/sucrose non-fermentable-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (integrase interactor 1)-deficient sinonasal carcinoma who had permanent vision loss after valsalva-induced acute hemorrhage and resultant orbital compartment syndrome.

  View details for DOI 10.1016/j.survophthal.2019.04.001

  View details for PubMedID 30978335

• ER stress and unfolded protein response in ocular health and disease. The FEBS journal Kroeger, H. n., Chiang, W. C., Felden, J. n., Nguyen, A. n., Lin, J. H. 2019; 286 (2): 399–412

  Abstract

  The human eye is the organ that is able to react to light in order to provide sharp three-dimensional and colored images. Unfortunately, the health of the eye can be impacted by various stimuli that can lead to vision loss, such as environmental changes, genetic mutations, or aging. Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) signaling have been detected in many diverse ocular diseases, and chemical and genetic approaches to modulate ER stress and specific UPR regulatory molecules have shown beneficial effects in animal models of eye disease. This review highlights specific eye diseases associated with ER stress and UPR activity, based on a recent symposia exploring this theme.

  View details for DOI 10.1111/febs.14522

  View details for PubMedID 29802807

  View details for PubMedCentralID PMC6583901

• Odontogenic choristoma presenting as dermolipoma. Orbit (Amsterdam, Netherlands) Men, C. J., Wu, F. n., Lee, B. W., Lin, J. H., Korn, B. S., Kikkawa, D. O. 2019; 38 (6): 492–94

  Abstract

  A 5-year-old otherwise healthy girl presented to the oculoplastic service with a painless superotemporal subconjunctival mass in the left eye. Visual acuity was within normal limits, and there was no evidence of proptosis or orbital enlargement. Excision was performed to remove the anterior portion of the mass for alleviation of symptoms. On histopathological analysis, the mass was comprised of fibroadipose tissue consistent with dermolipoma and contained a hard nodule found to be a calcified tooth. In the periocular region, odontogenic choristoma (tooth) is a rare lesion, and has been reported to occur within teratomas, dermoid cysts, and displaced oral embryonic epithelium. We describe an unusual case of a tooth occurring within a sporadic dermolipoma. The clinical presentation, examination, management, and histopathology are reviewed.

  View details for DOI 10.1080/01676830.2018.1558266

  View details for PubMedID 30628515

• Characterization of Retinal Structure in ATF6-Associated Achromatopsia. Investigative ophthalmology & visual science Mastey, R. R., Georgiou, M. n., Langlo, C. S., Kalitzeos, A. n., Patterson, E. J., Kane, T. n., Singh, N. n., Vincent, A. n., Moore, A. T., Tsang, S. H., Lin, J. H., Young, M. P., Hartnett, M. E., Héon, E. n., Kohl, S. n., Michaelides, M. n., Carroll, J. n. 2019; 60 (7): 2631–40

  Abstract

  Mutations in six genes have been associated with achromatopsia (ACHM): CNGA3, CNGB3, PDE6H, PDE6C, GNAT2, and ATF6. ATF6 is the most recent gene to be identified, though thorough phenotyping of this genetic subtype is lacking. Here, we sought to test the hypothesis that ATF6-associated ACHM is a structurally distinct form of congenital ACHM.Seven genetically confirmed subjects from five nonconsanguineous families were recruited. Foveal hypoplasia and the integrity of the ellipsoid zone (EZ) band (a.k.a., IS/OS) were graded from optical coherence tomography (OCT) images. Images of the photoreceptor mosaic were acquired using confocal and nonconfocal split-detection adaptive optics scanning light ophthalmoscopy (AOSLO). Parafoveal cone and rod density values were calculated and compared to published normative data as well as data from two subjects harboring CNGA3 or CNGB3 mutations who were recruited for comparative purposes. Additionally, nonconfocal dark-field AOSLO images of the retinal pigment epithelium were obtained, with quantitative analysis performed in one subject with ATF6-ACHM.Foveal hypoplasia was observed in all subjects with ATF6 mutations. Absence of the EZ band within the foveal region (grade 3) or appearance of a hyporeflective zone (grade 4) was seen in all subjects with ATF6 using OCT. There was no evidence of remnant foveal cone structure using confocal AOSLO, although sporadic cone-like structures were seen in nonconfocal split-detection AOSLO. There was a lack of cone structure in the parafovea, in direct contrast to previous reports.Our data demonstrate a near absence of cone structure in subjects harboring ATF6 mutations. This implicates ATF6 as having a major role in cone development and suggests that at least a subset of subjects with ATF6-ACHM have markedly fewer cellular targets for cone-directed gene therapies than do subjects with CNGA3- or CNGB3-ACHM.

  View details for DOI 10.1167/iovs.19-27047

  View details for PubMedID 31237654

  View details for PubMedCentralID PMC6594318

• GNAQ and PMS1 Mutations Associated with Uveal Melanoma, Ocular Surface Melanosis, and Nevus of Ota. Ocular oncology and pathology Toomey, C. B., Fraser, K. n., Thorson, J. A., Goldbaum, M. H., Lin, J. H. 2019; 5 (4): 267–72

  Abstract

  G protein mutations are common in uveal melanomas, and the vast majority target amino acid residue Q209 in either GNAQ or GNA11. The GNAQ R183Q mutation is found in a small fraction of uveal melanomas. We report a patient with an unusual presentation of uveal melanoma arising at an early age in the setting of congenital skin and ocular surface melanosis. A 34-year-old Hispanic female with congenital bilateral nevus of Ota and ocular surface melanosis presented with progressive loss of visual acuity and was found to have a juxtapapillary uveal melanoma. She was treated with brachytherapy, but the tumor relapsed. She underwent enucleation that revealed mixed spindle and epithelioid uveal melanoma cells with no extraocular or lymphovascular spread. Next-generation sequencing performed on DNA isolated from the enucleation specimen identified a GNAQ R183Q mutation and a PMS1 truncation mutation. Cytogenetic profiling revealed no monosomy 3. These findings raise the possibility that uveal melanomas bearing G protein R183 mutations may have distinct clinicopathologic profiles compared to those with Q209 mutations. Furthermore, this is the first reported case of a mutation in the mismatch repair gene PMS1 associated with uveal melanoma.

  View details for DOI 10.1159/000495508

  View details for PubMedID 31367589

  View details for PubMedCentralID PMC6615330

• Prion Seeds Distribute throughout the Eyes of Sporadic Creutzfeldt-Jakob Disease Patients. mBio Orrù, C. D., Soldau, K. n., Cordano, C. n., Llibre-Guerra, J. n., Green, A. J., Sanchez, H. n., Groveman, B. R., Edland, S. D., Safar, J. G., Lin, J. H., Caughey, B. n., Geschwind, M. D., Sigurdson, C. J. 2018; 9 (6)

  Abstract

  Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common prion disease in humans and has been iatrogenically transmitted through corneal graft transplantation. Approximately 40% of sCJD patients develop visual or oculomotor symptoms and may seek ophthalmological consultation. Here we used the highly sensitive real-time quaking-induced conversion (RT-QuIC) assay to measure postmortem prion seeding activities in cornea, lens, ocular fluid, retina, choroid, sclera, optic nerve, and extraocular muscle in the largest series of sCJD patient eyes studied by any assay to date. We detected prion seeding activity in 100% of sCJD eyes, representing three common sCJD subtypes, with levels varying by up to 4 log-fold among individuals. The retina consistently showed the highest seed levels, which in some cases were only slightly lower than brain. Within the retina, prion deposits were detected by immunohistochemistry (IHC) in the retinal outer plexiform layer in most sCJD cases, and in some eyes the inner plexiform layer, consistent with synaptic prion deposition. Prions were not detected by IHC in any other eye region. With RT-QuIC, prion seed levels generally declined in eye tissues with increased distance from the brain, and yet all corneas had prion seeds detectable. Prion seeds were also present in the optic nerve, extraocular muscle, choroid, lens, vitreous, and sclera. Collectively, these results reveal that sCJD patients accumulate prion seeds throughout the eye, indicating the potential diagnostic utility as well as a possible biohazard.IMPORTANCE Cases of iatrogenic prion disease have been reported from corneal transplants, yet the distribution and levels of prions throughout the eye remain unknown. This study probes the occurrence, level, and distribution of prions in the eyes of patients with sporadic Creutzfeldt-Jakob disease (sCJD). We tested the largest series of prion-infected eyes reported to date using an ultrasensitive technique to establish the prion seed levels in eight regions of the eye. All 11 cases had detectable prion seeds in the eye, and in some cases, the seed levels in the retina approached those in brain. In most cases, prion deposits could also be seen by immunohistochemical staining of retinal tissue; other ocular tissues were negative. Our results have implications for estimating the risk for iatrogenic transmission of sCJD as well as for the development of antemortem diagnostic tests for prion diseases.

  View details for DOI 10.1128/mBio.02095-18

  View details for PubMedID 30459197

  View details for PubMedCentralID PMC6247090

• Orbital fat regeneration following hormonal treatment of metastatic breast carcinoma. Orbit (Amsterdam, Netherlands) Alameddine, R. M., Ko, A. C., Mimura, M. n., Parker, B. A., Lin, J. H., Korn, B. S., Kikkawa, D. O. 2018; 37 (3): 187–90

  Abstract

  Enophthalmos in the setting of breast cancer metastatic to the orbit results primarily from the disease pathogenesis, or secondary to treatment effects. Orbital volume restoration and fat regeneration following endocrine treatment monotherapy has not been previously reported. A 76- year-old previously healthy female presented with progressive right enophthalmos secondary to metastatic lobular breast carcinoma. Treatment with an aromatase inhibitor (letrozole) resulted in tumor regression and orbital fat restoration with a corresponding improvement in orbital volume and enophthalmos on clinical exam. The patient is alive on continued letrozole with no progressive disease ten years after diagnosis. This case illustrates the resilience of orbital soft tissues and ability of orbital fat to regenerate in face of breast cancer metastasis. We hypothesize that endocrine monotherapy, and avoidance of radiation therapy, allowed for differentiation of remaining orbital stem cells, and facilitated the fat regenerative process.

  View details for DOI 10.1080/01676830.2017.1383468

  View details for PubMedID 29072521

• The unfolded protein response regulator ATF6 promotes mesodermal differentiation. Science signaling Kroeger, H. n., Grimsey, N. n., Paxman, R. n., Chiang, W. C., Plate, L. n., Jones, Y. n., Shaw, P. X., Trejo, J. n., Tsang, S. H., Powers, E. n., Kelly, J. W., Wiseman, R. L., Lin, J. H. 2018; 11 (517)

  Abstract

  ATF6 encodes a transcription factor that is anchored in the endoplasmic reticulum (ER) and activated during the unfolded protein response (UPR) to protect cells from ER stress. Deletion of the isoform activating transcription factor 6α (ATF6α) and its paralog ATF6β results in embryonic lethality and notochord dysgenesis in nonhuman vertebrates, and loss-of-function mutations in ATF6α are associated with malformed neuroretina and congenital vision loss in humans. These phenotypes implicate an essential role for ATF6 during vertebrate development. We investigated this hypothesis using human stem cells undergoing differentiation into multipotent germ layers, nascent tissues, and organs. We artificially activated ATF6 in stem cells with a small-molecule ATF6 agonist and, conversely, inhibited ATF6 using induced pluripotent stem cells from patients with ATF6 mutations. We found that ATF6 suppressed pluripotency, enhanced differentiation, and unexpectedly directed mesodermal cell fate. Our findings reveal a role for ATF6 during differentiation and identify a new strategy to generate mesodermal tissues through the modulation of the ATF6 arm of the UPR.

  View details for DOI 10.1126/scisignal.aan5785

  View details for PubMedID 29440509

  View details for PubMedCentralID PMC5957084

• JAK2 V617F mutation in plasma cell-free DNA preceding clinically overt myelofibrosis: Implications for early diagnosis. Cancer biology & therapy Choi, M. Y., Kato, S. n., Wang, H. Y., Lin, J. H., Lanman, R. B., Kurzrock, R. n. 2018; 19 (8): 664–68

  Abstract

  A 52 year-old man with Erdheim-Chester Disease (ECD) (a non-Langerhans polyostotic sclerosing histiocytosis) had next-generation sequencing (NGS) performed as part of his diagnostic workup. In addition to the tissue BRAF V600E mutation that is found in over 50% of ECD cases, he was also found to have a JAK2 V617F alteration in cell-free circulating tumor DNA (ctDNA) (liquid biopsy). The latter was thought to be an "incidental" finding, perhaps due to clonal hematopoiesis (though this usually occurs in older individuals), as his blood counts were normal and he had no splenomegaly. Approximately 13 months after the ctDNA test showing JAK2 V617F, he developed anemia, thrombocytopenia, and splenomegaly. Marrow biopsy then showed megakaryocytic atypia and markedly increased marrow fibrosis, consistent with WHO grade 2 of 3 myelofibrosis. Therefore, the patient was determined to have ECD with a typical BRAF V600E mutation, as well as primary myelofibrosis, with the latter diagnosis manifesting clinically over one year after the JAK2 V617F was first detected in ctDNA. He recently was started on the JAK2 inhibitor ruxolitinib. This case demonstrates that genomic alterations detected by liquid biopsy for evaluation of specific malignancies already present may serve as an early harbinger of hematological disease.

  View details for DOI 10.1080/15384047.2018.1450120

  View details for PubMedID 29565699

  View details for PubMedCentralID PMC6067874

• Tauopathy-associated PERK alleles are functional hypomorphs that increase neuronal vulnerability to ER stress. Human molecular genetics Yuan, S. H., Hiramatsu, N. n., Liu, Q. n., Sun, X. V., Lenh, D. n., Chan, P. n., Chiang, K. n., Koo, E. H., Kao, A. W., Litvan, I. n., Lin, J. H. 2018; 27 (22): 3951–63

  Abstract

  Tauopathies are neurodegenerative diseases characterized by tau protein pathology in the nervous system. EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), also known as PERK (protein kinase R-like endoplasmic reticulum kinase), was identified by genome-wide association study as a genetic risk factor in several tauopathies. PERK is a key regulator of the Unfolded Protein Response (UPR), an intracellular signal transduction mechanism that protects cells from endoplasmic reticulum (ER) stress. PERK variants had previously been identified in Wolcott-Rallison Syndrome, a rare autosomal recessive metabolic disorder, and these variants completely abrogated the function of PERK's kinase domain or prevented PERK expression. In contrast, the PERK tauopathy risk variants were distinct from the Wolcott-Rallison variants and introduced missense alterations throughout the PERK protein. The function of PERK tauopathy variants and their effects on neurodegeneration are unknown. Here, we discovered that tauopathy-associated PERK alleles showed reduced signaling activity and increased PERK protein turnover compared to protective PERK alleles. We found that iPSC-derived neurons carrying PERK risk alleles were highly vulnerable to ER stress-induced injury with increased tau pathology. We found that chemical inhibition of PERK in human iPSC-derived neurons also increased neuronal cell death in response to ER stress. Our results indicate that tauopathy-associated PERK alleles are functional hypomorphs during the UPR. We propose that reduced PERK function leads to neurodegeneration by increasing neuronal vulnerability to ER stress-associated damage. In this view, therapies to enhance PERK signaling would benefit at-risk carriers of hypomorphic alleles.

  View details for DOI 10.1093/hmg/ddy297

  View details for PubMedID 30137327

  View details for PubMedCentralID PMC6216228

• Autosomal recessive cone-rod dystrophy can be caused by mutations in the ATF6 gene. European journal of human genetics : EJHG Skorczyk-Werner, A. n., Chiang, W. C., Wawrocka, A. n., Wicher, K. n., Jarmuż-Szymczak, M. n., Kostrzewska-Poczekaj, M. n., Jamsheer, A. n., Płoski, R. n., Rydzanicz, M. n., Pojda-Wilczek, D. n., Weisschuh, N. n., Wissinger, B. n., Kohl, S. n., Lin, J. H., Krawczyński, M. R. 2017; 25 (11): 1210–16

  Abstract

  Inherited retinal dystrophies (IRDs) are clinically and genetically highly heterogeneous, making clinical diagnosis difficult. The advances in high-throughput sequencing (ie, panel, exome and genome sequencing) have proven highly effective on defining the molecular basis of these disorders by identifying the underlying variants in the respective gene. Here we report two siblings affected by an IRD phenotype and a novel homozygous c.1691A>G (p.(Asp564Gly)) ATF6 (activating transcription factor 6A) missense substitution identified by whole exome sequencing analysis. The pathogenicity of the variant was confirmed by functional analyses done on patients' fibroblasts and on recombinant p.(Asp564Gly) protein. The ATF6Asp564Gly/Asp564Gly variant shows impaired production of the ATF6 cleaved transcriptional activator domain in response to endoplasmic reticulum stress. Detailed phenotypic examination revealed extinguished cone responses but also decreased rod responses together with the ability to discriminate some colours suggestive rather for cone-rod dystrophy than achromatopsia.

  View details for DOI 10.1038/ejhg.2017.131

  View details for PubMedID 28812650

  View details for PubMedCentralID PMC5643965

• Achromatopsia mutations target sequential steps of ATF6 activation. Proceedings of the National Academy of Sciences of the United States of America Chiang, W. C., Chan, P. n., Wissinger, B. n., Vincent, A. n., Skorczyk-Werner, A. n., Krawczyński, M. R., Kaufman, R. J., Tsang, S. H., Héon, E. n., Kohl, S. n., Lin, J. H. 2017; 114 (2): 400–405

  Abstract

  Achromatopsia is an autosomal recessive disorder characterized by cone photoreceptor dysfunction. We recently identified activating transcription factor 6 (ATF6) as a genetic cause of achromatopsia. ATF6 is a key regulator of the unfolded protein response. In response to endoplasmic reticulum (ER) stress, ATF6 migrates from the ER to Golgi to undergo regulated intramembrane proteolysis to release a cytosolic domain containing a basic leucine zipper (bZIP) transcriptional activator. The cleaved ATF6 fragment migrates to the nucleus to transcriptionally up-regulate protein-folding enzymes and chaperones. ATF6 mutations in patients with achromatopsia include missense, nonsense, splice site, and single-nucleotide deletion or duplication changes found across the entire gene. Here, we comprehensively tested the function of achromatopsia-associated ATF6 mutations and found that they group into three distinct molecular pathomechanisms: class 1 ATF6 mutants show impaired ER-to-Golgi trafficking and diminished regulated intramembrane proteolysis and transcriptional activity; class 2 ATF6 mutants bear the entire ATF6 cytosolic domain with fully intact transcriptional activity and constitutive induction of downstream target genes, even in the absence of ER stress; and class 3 ATF6 mutants have complete loss of transcriptional activity because of absent or defective bZIP domains. Primary fibroblasts from patients with class 1 or class 3 ATF6 mutations show increased cell death in response to ER stress. Our findings reveal that human ATF6 mutations interrupt distinct sequential steps of the ATF6 activation mechanism. We suggest that increased susceptibility to ER stress-induced damage during retinal development underlies the pathology of achromatopsia in patients with ATF6 mutations.

  View details for DOI 10.1073/pnas.1606387114

  View details for PubMedID 28028229

  View details for PubMedCentralID PMC5240680

• Genetic analysis of 10 pedigrees with inherited retinal degeneration by exome sequencing and phenotype-genotype association. Physiological genomics Biswas, P. n., Duncan, J. L., Maranhao, B. n., Kozak, I. n., Branham, K. n., Gabriel, L. n., Lin, J. H., Barteselli, G. n., Navani, M. n., Suk, J. n., Parke, M. n., Schlechter, C. n., Weleber, R. G., Heckenlively, J. R., Dagnelie, G. n., Lee, P. n., Riazuddin, S. A., Ayyagari, R. n. 2017; 49 (4): 216–29

  Abstract

  Our purpose was to identify causative mutations and characterize the phenotype associated with the genotype in 10 unrelated families with autosomal recessive retinal degeneration. Ophthalmic evaluation and DNA isolation were carried out in 10 pedigrees with inherited retinal degenerations (IRD). Exomes of probands from eight pedigrees were captured using Nimblegen V2/V3 or Agilent V5+UTR kits, and sequencing was performed on Illumina HiSeq. The DHDDS gene was screened for mutations in the remaining two pedigrees with Ashkenazi Jewish ancestry. Exome variants were filtered to detect candidate causal variants using exomeSuite software. Segregation and ethnicity-matched control sample analysis were performed by dideoxy sequencing. Retinal histology of a patient with DHDDS mutation was studied by microscopy. Genetic analysis identified six known mutations in ABCA4 (p.Gly1961Glu, p.Ala1773Val, c.5461-10T>C), RPE65 (p.Tyr249Cys, p.Gly484Asp), PDE6B (p.Lys706Ter) and DHDDS (p.Lys42Glu) and ten novel potentially pathogenic variants in CERKL (p.Met323Val fsX20), RPE65 (p.Phe252Ser, Thr454Leu fsX31), ARL6 (p.Arg121His), USH2A (p.Gly3142Ter, p.Cys3294Trp), PDE6B (p.Gln652Ter), and DHDDS (p.Thr206Ala) genes. Among these, variants/mutations in two separate genes were observed to segregate with IRD in two pedigrees. Retinal histopathology of a patient with a DHDDS mutation showed severe degeneration of retinal layers with relative preservation of the retinal pigment epithelium. Analysis of exome variants in ten pedigrees revealed nine novel potential disease-causing variants and nine previously reported homozygous or compound heterozygous mutations in the CERKL, ABCA4, RPE65, ARL6, USH2A, PDE6B, and DHDDS genes. Mutations that could be sufficient to cause pathology were observed in more than one gene in one pedigree.

  View details for DOI 10.1152/physiolgenomics.00096.2016

  View details for PubMedID 28130426

  View details for PubMedCentralID PMC5407181

• Intercellular transmission of the unfolded protein response promotes survival and drug resistance in cancer cells. Science signaling Rodvold, J. J., Chiu, K. T., Hiramatsu, N. n., Nussbacher, J. K., Galimberti, V. n., Mahadevan, N. R., Willert, K. n., Lin, J. H., Zanetti, M. n. 2017; 10 (482)

  Abstract

  Increased protein translation in cells and various factors in the tumor microenvironment can induce endoplasmic reticulum (ER) stress, which initiates the unfolded protein response (UPR). We have previously reported that factors released from cancer cells mounting a UPR induce a de novo UPR in bone marrow-derived myeloid cells, macrophages, and dendritic cells that facilitates protumorigenic characteristics in culture and tumor growth in vivo. We investigated whether this intercellular signaling, which we have termed transmissible ER stress (TERS), also operates between cancer cells and what its functional consequences were within the tumor. We found that TERS signaling induced a UPR in recipient human prostate cancer cells that included the cell surface expression of the chaperone GRP78. TERS also activated Wnt signaling in recipient cancer cells and enhanced resistance to nutrient starvation and common chemotherapies such as the proteasome inhibitor bortezomib and the microtubule inhibitor paclitaxel. TERS-induced activation of Wnt signaling required the UPR kinase and endonuclease IRE1. However, TERS-induced enhancement of cell survival was predominantly mediated by the UPR kinase PERK and a reduction in the abundance of the transcription factor ATF4, which prevented the activation of the transcription factor CHOP and, consequently, the induction of apoptosis. When implanted in mice, TERS-primed cancer cells gave rise to faster growing tumors than did vehicle-primed cancer cells. Collectively, our data demonstrate that TERS is a mechanism of intercellular communication through which tumor cells can adapt to stressful environments.

  View details for DOI 10.1126/scisignal.aah7177

  View details for PubMedID 28588081

  View details for PubMedCentralID PMC5962022

• Masquerading Orbital Sarcoidosis with Isolated Extraocular Muscle Involvement. The open ophthalmology journal Kim, J. S., Scawn, R. L., Lee, B. W., Lin, J. H., Korn, B. S., Kikkawa, D. O. 2016; 10: 140–45

  Abstract

  Two patients, previously diagnosed and treated for euthyroid, autoantibody-negative thyroid eye disease, presented with active orbitopathy. An atypical disease course and presentation prompted orbital biopsy. Extraocular muscle histopathology demonstrated noncaseating granulomatous inflammation consistent with presumed orbital sarcoidosis involving multiple extraocular muscles, including the inferior oblique in one of the cases. These two cases emphasize the importance of a broad differential diagnosis and the utility of an orbital biopsy in the context of an unusual disease presentation or poor treatment response. The patients' clinical course is discussed alongside important clinical signs, imaging findings, and biopsy results that support a diagnosis of isolated orbital sarcoidosis.

  View details for DOI 10.2174/1874364101610010140

  View details for PubMedID 28484581

  View details for PubMedCentralID PMC5396127

• Benign and Premalignant Tumors of the Eyelid Diseases and Disorders of the Orbit and Ocular Adnexa Wu, F., Lin, J. H., Korn, B. S., Kikkawa, D. O. Elsevier. 2016
• Pathology and mechanism of eye diseases Ophthalmic disease mechanisms and drug discovery Wert, K. J., Kroeger, H., Wu, F., Tsang, S. H., Lin, J. H. 2016

  View details for DOI 10.1142/9789814663076_0002

• Large benign de novo conjunctival hemangioma in an 11-year-old boy: case report and literature review. Journal of AAPOS : the official publication of the American Association for Pediatric Ophthalmology and Strabismus Godfrey, K. J., Kinori, M. n., Lin, J. H., Snyder, V. S., Granet, D. B., Heichel, C. W., Robbins, S. L. 2016; 20 (5): 462–64

  Abstract

  Benign hemangiomas are rare vascular tumors of the conjunctiva that typically present clinically in the first few weeks of life and resolve spontaneously. De novo presentation later in childhood has not been well documented. We present the unusual case of an 11-year-old boy with a rapidly growing benign de novo conjunctival hemangioma that was treated with surgical excision.

  View details for DOI 10.1016/j.jaapos.2016.06.002

  View details for PubMedID 27647119

• Ablation of Chop Transiently Enhances Photoreceptor Survival but Does Not Prevent Retinal Degeneration in Transgenic Mice Expressing Human P23H Rhodopsin. Advances in experimental medicine and biology Chiang, W. C., Joseph, V. n., Yasumura, D. n., Matthes, M. T., Lewin, A. S., Gorbatyuk, M. S., Ahern, K. n., LaVail, M. M., Lin, J. H. 2016; 854: 185–91

  Abstract

  RHO (Rod opsin) encodes a G-protein coupled receptor that is expressed exclusively by rod photoreceptors of the retina and forms the essential photopigment, rhodopsin, when coupled with 11-cis-retinal. Many rod opsin disease -mutations cause rod opsin protein misfolding and trigger endoplasmic reticulum (ER) stress, leading to activation of the Unfolded Protein Response (UPR) signal transduction network. Chop is a transcriptional activator that is induced by ER stress and promotes cell death in response to chronic ER stress. Here, we examined the role of Chop in transgenic mice expressing human P23H rhodopsin (hP23H Rho Tg) that undergo retinal degeneration. With the exception of one time point, we found no significant induction of Chop in these animals and no significant change in retinal degeneration by histology and electrophysiology when hP23H Rho Tg animals were bred into a Chop (-/-) background. Our results indicate that Chop does not play a significant causal role during retinal degeneration in these animals. We suggest that other modules of the ER stress-induced UPR signaling network may be involved photoreceptor disease induced by P23H rhodopsin.

  View details for DOI 10.1007/978-3-319-17121-0_25

  View details for PubMedID 26427410

  View details for PubMedCentralID PMC4731204

• iPSC-Derived Retinal Pigment Epithelium Allografts Do Not Elicit Detrimental Effects in Rats: A Follow-Up Study. Stem cells international Westenskow, P. D., Bucher, F. n., Bravo, S. n., Kurihara, T. n., Feitelberg, D. n., Paris, L. P., Aguilar, E. n., Lin, J. H., Friedlander, M. n. 2016; 2016: 8470263

  Abstract

  Phototransduction is accomplished in the retina by photoreceptor neurons and retinal pigment epithelium (RPE) cells. Photoreceptors rely heavily on the RPE, and death or dysfunction of RPE is characteristic of age-related macular degeneration (AMD), a very common neurodegenerative disease for which no cure exists. RPE replacement is a promising therapeutic intervention for AMD, and large numbers of RPE cells can be generated from pluripotent stem cells. However, questions persist regarding iPSC-derived RPE (iPS-RPE) viability, immunogenicity, and tumorigenesis potential. We showed previously that iPS-RPE prevent photoreceptor atrophy in dystrophic rats up until 24 weeks after implantation. In this follow-up study, we longitudinally monitored the same implanted iPS-RPE, in the same animals. We observed no gross abnormalities in the eyes, livers, spleens, brains, and blood in aging rats with iPSC-RPE grafts. iPS-RPE cells that integrated into the subretinal space outlived the photoreceptors and survived for as long as 2 1/2 years while nonintegrating RPE cells were ingested by host macrophages. Both populations could be distinguished using immunohistochemistry and electron microscopy. iPSC-RPE could be isolated from the grafts and maintained in culture; these cells also phagocytosed isolated photoreceptor outer segments. We conclude that iPS-RPE grafts remain viable and do not induce any obvious associated pathological changes.

  View details for DOI 10.1155/2016/8470263

  View details for PubMedID 26880994

  View details for PubMedCentralID PMC4736415

• Endoplasmic reticulum stress in human photoreceptor diseases. Brain research Chan, P. n., Stolz, J. n., Kohl, S. n., Chiang, W. C., Lin, J. H. 2016; 1648 (Pt B): 538–41

  Abstract

  Photoreceptors are specialized sensory neurons essential for light detection in the human eye. Photoreceptor cell dysfunction and death cause vision loss in many eye diseases such as retinitis pigmentosa and achromatopsia. Endoplasmic reticulum (ER) stress and Unfolded Protein Response (UPR) signaling have been implicated in the development and pathology of heritable forms of retinitis pigmentosa and achromatopsia. We review the role of ER stress and UPR in retinitis pigmentosa arising from misfolded rhodopsins (RHO) and in achromatopsia arising from genetic mutations in Activating Transcription Factor 6 (ATF6). This article is part of a Special Issue entitled SI:ER stress.

  View details for DOI 10.1016/j.brainres.2016.04.021

  View details for PubMedID 27117871

  View details for PubMedCentralID PMC5036988

• Extraocular Muscle Fibrosis in Idiopathic Orbital Inflammation. Journal of pediatric ophthalmology and strabismus Kinori, M. n., Ko, A. C., Alameddine, R. M., Snyder, V. n., Lin, J. H., Korn, B. S., Granet, D. B. 2016; 53 (4): 256

  View details for DOI 10.3928/01913913-20160506-02

  View details for PubMedID 27428626

  View details for PubMedCentralID PMC5117192

• In Vivo Visualization of Endoplasmic Reticulum Stress in the Retina Using the ERAI Reporter Mouse. Investigative ophthalmology & visual science Alavi, M. V., Chiang, W. C., Kroeger, H. n., Yasumura, D. n., Matthes, M. T., Iwawaki, T. n., LaVail, M. M., Gould, D. B., Lin, J. H. 2015; 56 (11): 6961–70

  Abstract

  Endoplasmic reticulum (ER) stress activates inositol requiring enzyme 1 (IRE1), a key regulator of the unfolded protein response. The ER stress activated indicator (ERAI) transgenic mouse expresses a yellow fluorescent GFP variant (Venus) when IRE1 is activated by ER stress. We tested whether ERAI mice would allow for real-time longitudinal studies of ER stress in living mouse eyes.We chemically and genetically induced ER stress, and qualitatively and quantitatively studied the Venus signal by fluorescence ophthalmoscopy. We determined retinal cell types that contribute to the signal by immunohistology, and we performed molecular and biochemical assays using whole retinal lysates to assess activity of the IRE1 pathway.We found qualitative increase in vivo in fluorescence signal at sites of intravitreal tunicamycin injection in ERAI eyes, and quantitative increase in ERAI mice mated to RhoP23H mice expressing ER stress-inducing misfolded rhodopsin protein. As expected, we found that increased Venus signal arose primarily from photoreceptors in RhoP23H/+;ERAI mice. We found increased Xbp1S and XBP1s transcriptional target mRNA levels in RhoP23H/+;ERAI retinas compared to Rho+/+;ERAI retinas, and that Venus signal increased in ERAI retinas as a function of age.Fluorescence ophthalmoscopy of ERAI mice enables in vivo visualization of retinas undergoing ER stress. ER stress activated indicator mice enable identification of individual retinal cells undergoing ER stress by immunohistochemistry. ER stress activated indicator mice show higher Venus signal at older ages, likely arising from amplification of basal retinal ER stress levels by GFP's inherent stability.

  View details for DOI 10.1167/iovs.15-16969

  View details for PubMedID 26513501

  View details for PubMedCentralID PMC4627472

• The loss of glucose-regulated protein 78 (GRP78) during normal aging or from siRNA knockdown augments human alpha-synuclein (α-syn) toxicity to rat nigral neurons. Neurobiology of aging Salganik, M. n., Sergeyev, V. G., Shinde, V. n., Meyers, C. A., Gorbatyuk, M. S., Lin, J. H., Zolotukhin, S. n., Gorbatyuk, O. S. 2015; 36 (6): 2213–23

  Abstract

  Age-related structural changes and gradual loss of key enzymes significantly affect the ability of the endoplasmic reticulum (ER) to facilitate proper protein folding and maintain homeostasis. In this work, we present several lines of evidence supporting the hypothesis that the age-related decline in expression of the ER chaperone glucose-regulated protein 78 (GRP78) could be related to the development of Parkinson's disease. We first determined that old (24 months) rats exhibit significantly lower levels of GRP78 protein in the nigrostriatal system as compared with young (2 months) animals. Then using recombinant adeno-associate virus-mediated gene transfer, we found that GRP78 downregulation by specific small interfering RNAs (siRNAs) aggravates alpha-synuclein (α-syn) neurotoxicity in nigral dopamine (DA) neurons. Moreover, the degree of chaperone decline corresponds with the severity of neurodegeneration. Additionally, comparative analysis of nigral tissues obtained from old and young rats revealed that aging affects the capacity of nigral DA cells to upregulate endogenous GRP78 protein in response to human α-syn neurotoxicity. Finally, we demonstrated that a sustained increase of GRP78 protein over the course of 9 months protected aging nigral DA neurons in the α-syn-induced rat model of Parkinson's-like neurodegeneration. Our data indicate that the ER chaperone GRP78 may have therapeutic potential for preventing and/or slowing age-related neurodegeneration.

  View details for DOI 10.1016/j.neurobiolaging.2015.02.018

  View details for PubMedID 25863526

  View details for PubMedCentralID PMC4433578

• Transcriptome sequencing uncovers novel long noncoding and small nucleolar RNAs dysregulated in head and neck squamous cell carcinoma. RNA (New York, N.Y.) Zou, A. E., Ku, J. n., Honda, T. K., Yu, V. n., Kuo, S. Z., Zheng, H. n., Xuan, Y. n., Saad, M. A., Hinton, A. n., Brumund, K. T., Lin, J. H., Wang-Rodriguez, J. n., Ongkeko, W. M. 2015; 21 (6): 1122–34

  Abstract

  Head and neck squamous cell carcinoma persists as one of the most common and deadly malignancies, with early detection and effective treatment still posing formidable challenges. To expand our currently sparse knowledge of the noncoding alterations involved in the disease and identify potential biomarkers and therapeutic targets, we globally profiled the dysregulation of small nucleolar and long noncoding RNAs in head and neck tumors. Using next-generation RNA-sequencing data from 40 pairs of tumor and matched normal tissues, we found 2808 long noncoding RNA (lncRNA) transcripts significantly differentially expressed by a fold change magnitude ≥2. Meanwhile, RNA-sequencing analysis of 31 tumor-normal pairs yielded 33 significantly dysregulated small nucleolar RNAs (snoRNA). In particular, we identified two dramatically down-regulated lncRNAs and one down-regulated snoRNA whose expression levels correlated significantly with overall patient survival, suggesting their functional significance and clinical relevance in head and neck cancer pathogenesis. We confirmed the dysregulation of these noncoding RNAs in head and neck cancer cell lines derived from different anatomic sites, and determined that ectopic expression of the two lncRNAs inhibited key EMT and stem cell genes and reduced cellular proliferation and migration. As a whole, noncoding RNAs are pervasively dysregulated in head and squamous cell carcinoma. The precise molecular roles of the three transcripts identified warrants further characterization, but our data suggest that they are likely to play substantial roles in head and neck cancer pathogenesis and are significantly associated with patient survival.

  View details for DOI 10.1261/rna.049262.114

  View details for PubMedID 25904139

  View details for PubMedCentralID PMC4436665

• Robust Endoplasmic Reticulum-Associated Degradation of Rhodopsin Precedes Retinal Degeneration. Molecular neurobiology Chiang, W. C., Kroeger, H. n., Sakami, S. n., Messah, C. n., Yasumura, D. n., Matthes, M. T., Coppinger, J. A., Palczewski, K. n., LaVail, M. M., Lin, J. H. 2015; 52 (1): 679–95

  Abstract

  Rhodopsin is a G protein-coupled receptor essential for vision and rod photoreceptor viability. Disease-associated rhodopsin mutations, such as P23H rhodopsin, cause rhodopsin protein misfolding and trigger endoplasmic reticulum (ER) stress, activating the unfolded protein response (UPR). The pathophysiologic effects of ER stress and UPR activation on photoreceptors are unclear. Here, by examining P23H rhodopsin knock-in mice, we found that the UPR inositol-requiring enzyme 1 (IRE1) signaling pathway is strongly activated in misfolded rhodopsin-expressing photoreceptors. IRE1 significantly upregulated ER-associated protein degradation (ERAD), triggering pronounced P23H rhodopsin degradation. Rhodopsin protein loss occurred as soon as photoreceptors developed, preceding photoreceptor cell death. By contrast, IRE1 activation did not affect JNK signaling or rhodopsin mRNA levels. Interestingly, pro-apoptotic signaling from the PERK UPR pathway was also not induced. Our findings reveal that an early and significant pathophysiologic effect of ER stress in photoreceptors is the highly efficient elimination of misfolded rhodopsin protein. We propose that early disruption of rhodopsin protein homeostasis in photoreceptors could contribute to retinal degeneration.

  View details for DOI 10.1007/s12035-014-8881-8

  View details for PubMedID 25270370

  View details for PubMedCentralID PMC4383737

• The unfolded protein response is shaped by the NMD pathway. EMBO reports Karam, R. n., Lou, C. H., Kroeger, H. n., Huang, L. n., Lin, J. H., Wilkinson, M. F. 2015; 16 (5): 599–609

  Abstract

  Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), an essential adaptive intracellular pathway that relieves the stress. Although the UPR is an evolutionarily conserved and beneficial pathway, its chronic activation contributes to the pathogenesis of a wide variety of human disorders. The fidelity of UPR activation must thus be tightly regulated to prevent inappropriate signaling. The nonsense-mediated RNA decay (NMD) pathway has long been known to function in RNA quality control, rapidly degrading aberrant mRNAs, and has been suggested to regulate subsets of normal mRNAs. Here, we report that the NMD pathway regulates the UPR. NMD increases the threshold for triggering the UPR in vitro and in vivo, thereby preventing UPR activation in response to normally innocuous levels of ER stress. NMD also promotes the timely termination of the UPR. We demonstrate that NMD directly targets the mRNAs encoding several UPR components, including the highly conserved UPR sensor, IRE1α, whose NMD-dependent degradation partly underpins this process. Our work not only sheds light on UPR regulation, but demonstrates the physiological relevance of NMD's ability to regulate normal mRNAs.

  View details for DOI 10.15252/embr.201439696

  View details for PubMedID 25807986

  View details for PubMedCentralID PMC4428047

• Multiple Mechanisms of Unfolded Protein Response-Induced Cell Death. The American journal of pathology Hiramatsu, N. n., Chiang, W. C., Kurt, T. D., Sigurdson, C. J., Lin, J. H. 2015; 185 (7): 1800–1808

  Abstract

  Eukaryotic cells fold and assemble membrane and secreted proteins in the endoplasmic reticulum (ER), before delivery to other cellular compartments or the extracellular environment. Correctly folded proteins are released from the ER, and poorly folded proteins are retained until they achieve stable conformations; irreparably misfolded proteins are targeted for degradation. Diverse pathological insults, such as amino acid mutations, hypoxia, or infection, can overwhelm ER protein quality control, leading to misfolded protein buildup, causing ER stress. To cope with ER stress, eukaryotic cells activate the unfolded protein response (UPR) by increasing levels of ER protein-folding enzymes and chaperones, enhancing the degradation of misfolded proteins, and reducing protein translation. In mammalian cells, three ER transmembrane proteins, inositol-requiring enzyme-1 (IRE1; official name ERN1), PKR-like ER kinase (PERK; official name EIF2AK3), and activating transcription factor-6, control the UPR. The UPR signaling triggers a set of prodeath programs when the cells fail to successfully adapt to ER stress or restore homeostasis. ER stress and UPR signaling are implicated in the pathogenesis of diverse diseases, including neurodegeneration, cancer, diabetes, and inflammation. This review discusses the current understanding in both adaptive and apoptotic responses as well as the molecular mechanisms instigating apoptosis via IRE1 and PERK signaling. We also examine how IRE1 and PERK signaling may be differentially used during neurodegeneration arising in retinitis pigmentosa and prion infection.

  View details for DOI 10.1016/j.ajpath.2015.03.009

  View details for PubMedID 25956028

  View details for PubMedCentralID PMC4484218

• Mutations in the unfolded protein response regulator ATF6 cause the cone dysfunction disorder achromatopsia. Nature genetics Kohl, S. n., Zobor, D. n., Chiang, W. C., Weisschuh, N. n., Staller, J. n., Gonzalez Menendez, I. n., Chang, S. n., Beck, S. C., Garcia Garrido, M. n., Sothilingam, V. n., Seeliger, M. W., Stanzial, F. n., Benedicenti, F. n., Inzana, F. n., Héon, E. n., Vincent, A. n., Beis, J. n., Strom, T. M., Rudolph, G. n., Roosing, S. n., Hollander, A. I., Cremers, F. P., Lopez, I. n., Ren, H. n., Moore, A. T., Webster, A. R., Michaelides, M. n., Koenekoop, R. K., Zrenner, E. n., Kaufman, R. J., Tsang, S. H., Wissinger, B. n., Lin, J. H. 2015; 47 (7): 757–65

  Abstract

  Achromatopsia (ACHM) is an autosomal recessive disorder characterized by color blindness, photophobia, nystagmus and severely reduced visual acuity. Using homozygosity mapping and whole-exome and candidate gene sequencing, we identified ten families carrying six homozygous and two compound-heterozygous mutations in the ATF6 gene (encoding activating transcription factor 6A), a key regulator of the unfolded protein response (UPR) and cellular endoplasmic reticulum (ER) homeostasis. Patients had evidence of foveal hypoplasia and disruption of the cone photoreceptor layer. The ACHM-associated ATF6 mutations attenuate ATF6 transcriptional activity in response to ER stress. Atf6(-/-) mice have normal retinal morphology and function at a young age but develop rod and cone dysfunction with increasing age. This new ACHM-related gene suggests a crucial and unexpected role for ATF6A in human foveal development and cone function and adds to the list of genes that, despite ubiquitous expression, when mutated can result in an isolated retinal photoreceptor phenotype.

  View details for DOI 10.1038/ng.3319

  View details for PubMedID 26029869

  View details for PubMedCentralID PMC4610820

• Cysticercosis with an Orbital Tropism in Twins. The American journal of tropical medicine and hygiene Lee, B. W., Kumar, U. R., Lin, J. H., Amaro, D. E., Kikkawa, D. O., Alameddine, R. M., Lowe, M. C., Hilger, P. A., Vinetz, J. M., Korn, B. S. 2015; 93 (4): 828–30

  Abstract

  Two fraternal twin sisters developed cysticercosis localizing to the right lateral orbit over the same period after a presumed common-source exposure in China. This case demonstrates that cysticercosis can be related to travel. Similar temporal and spatial occurrences of these infections suggest a genetic tropism of the infecting organism in these twins.

  View details for DOI 10.4269/ajtmh.15-0205

  View details for PubMedID 26217041

  View details for PubMedCentralID PMC4596607

• p16INK4A expression is frequently increased in periorbital and ocular squamous lesions. Diagnostic pathology Kobalka, P. J., Abboud, J. P., Liao, X. n., Jones, K. n., Lee, B. W., Korn, B. S., Kikkawa, D. O., Lin, J. H. 2015; 10: 175

  Abstract

  p16 expression is a well established biomarker of cervical dysplasia and carcinoma arising from high risk human papilloma virus infection. Increased p16 expression is also seen in squamous neoplasms arising at other sites, including head, neck, and oropharyngeal tract. Squamous lesions are also frequently encountered at ocular surface and peri-orbital skin sites, but the prevalence of increased p16 expression in these lesions has been poorly studied.We retrospectively surveyed 13 ocular surface and 16 orbital squamous lesions biopsied at UC San Diego Healthcare System and VA San Diego Healthcare System for p16 expression by immunohistochemistry. These cases included ocular surface lesions with diagnoses of conjunctival intraepithelial neoplasm (CIN) and squamous cell carcinoma in situ. Peri-orbital eyelid biopsies included lesions with diagnoses of SCCis and invasive squamous cell carcinoma. We performed multivariate logistic regression, followed by student's T-test or Fisher's exact test to determine if there were statistically significant associations between p16 immunoreactivity and patient age, gender, diagnosis, and ethnicity. Statistical significance was defined as p < 0.05.We found an unexpectedly large prevalence of strong nuclear and cytoplasmic p16 immunoreactivity in our cases. Almost all of the ocular surface squamous lesions were diffusely positive for p16 expression (12/13). All of the periorbital lesions showed diffuse p16 immunoreactivity (16/16). Altogether, 28/29 lesions tested showed strong and diffuse p16 expression. We found no statistically significant correlation between p16 expression and patient age, gender, ethnicity, or diagnosis. In 6 of the peri-orbital biopsies, we had sufficient tissue to assess high-risk HPV expression by in situ hybridization. Interestingly, all of these cases were negative for HPV, despite strong p16 expression.Strong p16 expression was observed in virtually all of the ocular surface and peri-orbital squamous neoplasms in our study. The relationship between p16 expression and HPV infection in ocular surface and peri-orbital sites requires further investigation.

  View details for DOI 10.1186/s13000-015-0396-8

  View details for PubMedID 26400483

  View details for PubMedCentralID PMC4581440

• Orbital Granulomatosis With Polyangiitis (Wegener Granulomatosis) Clinical and Pathologic Findings ARCHIVES OF PATHOLOGY & LABORATORY MEDICINE Muller, K., Lin, J. H. 2014; 138 (8): 1110–14

  Abstract

  The pathology of granulomatosis with polyangiitis (GPA), formerly Wegener granulomatosis, typically features a granulomatous and sometimes necrotizing vasculitis targeting the respiratory tract and kidneys. However, orbital involvement occurs in up to 60% of patients and is frequently the first or only clinical presentation in patients with systemic or limited forms of GPA. Orbital GPA can cause significant morbidity and potentially lead to complete loss of vision and permanent facial deformity. Fortunately, GPA is highly responsive to medical treatment with corticosteroids combined with cyclophosphamide or, more recently, rituximab. Therefore, it is imperative for this disease to be accurately diagnosed on orbital biopsy and distinguished from other histologically similar orbital lesions. Herein, we review the clinical and pathologic findings of orbital GPA, focusing on the differentiation of this disease from other inflammatory orbital lesions.

  View details for DOI 10.5858/arpa.2013-0006-RS

  View details for Web of Science ID 000339619900021

  View details for PubMedID 25076302

  View details for PubMedCentralID PMC4140401

• Endoplasmic reticulum stress in vertebrate mutant rhodopsin models of retinal degeneration. Advances in experimental medicine and biology Kroeger, H. n., LaVail, M. M., Lin, J. H. 2014; 801: 585–92

  Abstract

  Rhodopsin mutations cause many types of heritable retinitis pigmentosa (RP). Biochemical and in vitro studies have demonstrated that many RP-linked mutant rhodopsins produce misfolded rhodopsin proteins, which are prone to aggregation and retention within the endoplasmic reticulum, where they cause endoplasmic reticulum stress and activate the Unfolded Protein Response signaling pathways. Many vertebrate models of retinal degeneration have been created through expression of RP-linked rhodopsins in photoreceptors including, but not limited to, VPP/GHL mice, P23H Rhodopsin frogs, P23H rhodopsin rats, S334ter rhodopsin rats, C185R rhodopsin mice, T17M rhodopsin mice, and P23H rhodopsin mice. These models have provided many opportunities to test therapeutic strategies to prevent retinal degeneration and also enabled in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death. Here, we examine and compare the contribution of endoplasmic reticulum stress to retinal degeneration in several vertebrate models of RP generated through expression of mutant rhodopsins.

  View details for DOI 10.1007/978-1-4614-3209-8_74

  View details for PubMedID 24664747

  View details for PubMedCentralID PMC4103620

• WNT7A and PAX6 define corneal epithelium homeostasis and pathogenesis. Nature Ouyang, H. n., Xue, Y. n., Lin, Y. n., Zhang, X. n., Xi, L. n., Patel, S. n., Cai, H. n., Luo, J. n., Zhang, M. n., Zhang, M. n., Yang, Y. n., Li, G. n., Li, H. n., Jiang, W. n., Yeh, E. n., Lin, J. n., Pei, M. n., Zhu, J. n., Cao, G. n., Zhang, L. n., Yu, B. n., Chen, S. n., Fu, X. D., Liu, Y. n., Zhang, K. n. 2014; 511 (7509): 358–61

  Abstract

  The surface of the cornea consists of a unique type of non-keratinized epithelial cells arranged in an orderly fashion, and this is essential for vision by maintaining transparency for light transmission. Cornea epithelial cells (CECs) undergo continuous renewal from limbal stem or progenitor cells (LSCs), and deficiency in LSCs or corneal epithelium--which turns cornea into a non-transparent, keratinized skin-like epithelium--causes corneal surface disease that leads to blindness in millions of people worldwide. How LSCs are maintained and differentiated into corneal epithelium in healthy individuals and which key molecular events are defective in patients have been largely unknown. Here we report establishment of an in vitro feeder-cell-free LSC expansion and three-dimensional corneal differentiation protocol in which we found that the transcription factors p63 (tumour protein 63) and PAX6 (paired box protein PAX6) act together to specify LSCs, and WNT7A controls corneal epithelium differentiation through PAX6. Loss of WNT7A or PAX6 induces LSCs into skin-like epithelium, a critical defect tightly linked to common human corneal diseases. Notably, transduction of PAX6 in skin epithelial stem cells is sufficient to convert them to LSC-like cells, and upon transplantation onto eyes in a rabbit corneal injury model, these reprogrammed cells are able to replenish CECs and repair damaged corneal surface. These findings suggest a central role of the WNT7A-PAX6 axis in corneal epithelial cell fate determination, and point to a new strategy for treating corneal surface diseases.

  View details for DOI 10.1038/nature13465

  View details for PubMedID 25030175

  View details for PubMedCentralID PMC4610745

• De novo prion aggregates trigger autophagy in skeletal muscle. Journal of virology Joshi-Barr, S. n., Bett, C. n., Chiang, W. C., Trejo, M. n., Goebel, H. H., Sikorska, B. n., Liberski, P. n., Raeber, A. n., Lin, J. H., Masliah, E. n., Sigurdson, C. J. 2014; 88 (4): 2071–82

  Abstract

  In certain sporadic, familial, and infectious prion diseases, the prion protein misfolds and aggregates in skeletal muscle in addition to the brain and spinal cord. In myocytes, prion aggregates accumulate intracellularly, yet little is known about clearance pathways. Here we investigated the clearance of prion aggregates in muscle of transgenic mice that develop prion disease de novo. In addition to neurodegeneration, aged mice developed a degenerative myopathy, with scattered myocytes containing ubiquitinated, intracellular prion inclusions that were adjacent to myocytes lacking inclusions. Myocytes also showed elevated levels of the endoplasmic reticulum chaperone Grp78/BiP, suggestive of impaired protein degradation and endoplasmic reticulum stress. Additionally, autophagy was induced, as indicated by increased levels of beclin-1 and LC3-II. In C2C12 myoblasts, inhibition of autophagosome maturation or lysosomal degradation led to enhanced prion aggregation, consistent with a role for autophagy in prion aggregate clearance. Taken together, these findings suggest that the induction of autophagy may be a central strategy for prion aggregate clearance in myocytes. IMPORTANCE In prion diseases, the prion protein misfolds and aggregates in the central nervous system and sometimes in other organs, including muscle, yet the cellular pathways of prion aggregate clearance are unclear. Here we investigated the clearance of prion aggregates in the muscle of a transgenic mouse model that develops profound muscle degeneration. We found that endoplasmic reticulum stress pathways were activated and that autophagy was induced. Blocking of autophagic degradation in cell culture models led to an accumulation of aggregated prion protein. Collectively, these findings suggest that autophagy has an instrumental role in prion protein clearance.

  View details for DOI 10.1128/JVI.02279-13

  View details for PubMedID 24307586

  View details for PubMedCentralID PMC3911572

• General pathophysiology in retinal degeneration. Developments in ophthalmology Wert, K. J., Lin, J. H., Tsang, S. H. 2014; 53: 33-43

  Abstract

  Retinal degeneration, including that seen in age-related macular degeneration and retinitis pigmentosa (RP), is the most common form of neural degenerative disease in the world. There is great genetic and allelic heterogeneity of the various retinal dystrophies. Classifications of these diseases can be ambiguous, as there are similar clinical presentations in retinal degenerations arising from different genetic mechanisms. As would be expected, alterations in the activity of the phototransduction cascade, such as changes affecting the renewal and shedding of the photoreceptor OS, visual transduction, and/or retinol metabolism have a great impact on the health of the retina. Mutations within any of the molecules responsible for these visual processes cause several types of retinal and retinal pigment epithelium degenerative diseases. Apoptosis has been implicated in the rod cell loss seen in a mouse model of RP, but the precise mechanisms that connect the activation of these pathways to the loss of phosphodiesterase (PDE6β) function has yet to be defined. Additionally, the activation of apoptosis by CCAAT/-enhancer-binding protein homologous protein (CHOP), after activation of the unfolded protein response pathway, may be responsible for cell death, although the mechanism remains unknown. However, the mechanisms of cell death after loss of function of PDE6, which is a commonly studied mammalian model in research, may be generalizable to loss of function of different key proteins involved in the phototransduction cascade.

  View details for DOI 10.1159/000357294

  View details for PubMedID 24732759

  View details for PubMedCentralID PMC4405532

• Translational and posttranslational regulation of XIAP by eIF2α and ATF4 promotes ER stress-induced cell death during the unfolded protein response. Molecular biology of the cell Hiramatsu, N. n., Messah, C. n., Han, J. n., LaVail, M. M., Kaufman, R. J., Lin, J. H. 2014; 25 (9): 1411–20

  Abstract

  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.

  View details for DOI 10.1091/mbc.E13-11-0664

  View details for PubMedID 24623724

  View details for PubMedCentralID PMC4004591

• Lymphocyte infiltration in CAPN5 autosomal dominant neovascular inflammatory vitreoretinopathy. Clinical ophthalmology (Auckland, N.Z.) Mahajan, V. B., Lin, J. H. 2013; 7: 1339–45

  Abstract

  To describe immunohistopathological findings in autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV).An enucleated eye specimen from a patient with Stage V ADNIV was examined using standard histopathological methods and lymphocyte markers.A c.731T>C CAPN5 mutation resulted in a p.Leu244Pro substitution in calpain-5. The eye showed exudative retinal detachment and neovascularization, intraocular fibrosis, and features of phthisis bulbi. Chronic inflammatory CD3-positive cell infiltrates were identified throughout the uvea, vitreous and retina, consistent with chronic uveitis.Mutations in CAPN5 trigger autoimmune uveitis characterized by inflammatory T-cells and severe neovascularization.

  View details for DOI 10.2147/OPTH.S46450

  View details for PubMedID 23861576

  View details for PubMedCentralID PMC3704602

• IRE1 directs proteasomal and lysosomal degradation of misfolded rhodopsin. Molecular biology of the cell Chiang, W. C., Messah, C. n., Lin, J. H. 2012; 23 (5): 758–70

  Abstract

  Endoplasmic reticulum (ER) is responsible for folding of secreted and membrane proteins in eukaryotic cells. Disruption of ER protein folding leads to ER stress. Chronic ER stress can cause cell death and is proposed to underlie the pathogenesis of many human diseases. Inositol-requiring enzyme 1 (IRE1) directs a key unfolded protein response signaling pathway that controls the fidelity of ER protein folding. IRE1 signaling may be particularly helpful in preventing chronic ER stress and cell injury by alleviating protein misfolding in the ER. To examine this, we used a chemical-genetic approach to selectively activate IRE1 in mammalian cells and tested how artificial IRE1 signaling affected the fate of misfolded P23H rhodopsin linked to photoreceptor cell death. We found that IRE1 signaling robustly promoted the degradation of misfolded P23H rhodopsin without affecting its wild-type counterpart. We also found that IRE1 used both proteasomal and lysosomal degradation pathways to remove P23H rhodopsin. Surprisingly, when one degradation pathway was compromised, IRE1 signaling could still promote misfolded rhodopsin degradation using the remaining pathway. Last, we showed that IRE1 signaling also reduced levels of several other misfolded rhodopsins with lesser effects on misfolded cystic fibrosis transmembrane conductance regulator. Our findings reveal the diversity of proteolytic mechanisms used by IRE1 to eliminate misfolded rhodopsin.

  View details for DOI 10.1091/mbc.E11-08-0663

  View details for PubMedID 22219383

  View details for PubMedCentralID PMC3290636

• Genetic Pathways in Retinal Degenerations and Targets for Therapy Genetic Diseases of the Eye Leung, L. B., Voleti, V. B., Lin, J. H., Tsang, S. H. 2012

  View details for DOI 10.1093/med/9780195326147.003.0021

• Glucose regulated protein 78 diminishes α-synuclein neurotoxicity in a rat model of Parkinson disease. Molecular therapy : the journal of the American Society of Gene Therapy Gorbatyuk, M. S., Shabashvili, A. n., Chen, W. n., Meyers, C. n., Sullivan, L. F., Salganik, M. n., Lin, J. H., Lewin, A. S., Muzyczka, N. n., Gorbatyuk, O. S. 2012; 20 (7): 1327–37

  Abstract

  Accumulation of human wild-type (wt) α-synuclein (α-syn) induces neurodegeneration in humans and in experimental rodent models of Parkinson disease (PD). It also leads to endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). We overexpressed glucose regulated protein 78, also known as BiP (GRP78/BiP), to test the hypothesis that this ER chaperone modulates the UPR, blocks apoptosis, and promotes the survival of nigral dopamine (DA) neurons in a rat model of PD induced by elevated level of human α-syn. We determined that α-syn activates ER stress mediators associated with pancreatic ER kinase-like ER kinase (PERK) and activating transcription factor-6 (ATF6) signaling pathways as well as proaoptotic CCAAT/-enhancer-binding protein homologous protein (CHOP) in nigral DA neurons. At the same time, overexpression of GRP78/BiP diminished α-syn neurotoxicity by down regulating ER stress mediators and the level of apoptosis, promoted survival of nigral tyrosine hydroxylase (TH) positive cells and resulted in higher levels of striatal DA, while eliminating amphetamine induced behavioral asymmetry. We also detected a complex between GRP78/BiP and α-syn that may contribute to prevention of the neurotoxicity caused by α-syn. Our data suggest that the molecular chaperone GRP78/BiP plays a neuroprotective role in α-syn-induced Parkinson-like neurodegeneration.

  View details for DOI 10.1038/mt.2012.28

  View details for PubMedID 22434142

  View details for PubMedCentralID PMC3392977

• Functional rescue of P23H rhodopsin photoreceptors by gene delivery. Advances in experimental medicine and biology Gorbatyuk, M. S., Gorbatyuk, O. S., LaVail, M. M., Lin, J. H., Hauswirth, W. W., Lewin, A. S. 2012; 723: 191–97

  View details for DOI 10.1007/978-1-4614-0631-0_26

  View details for PubMedID 22183333

  View details for PubMedCentralID PMC3365920

• ER stress in retinal degeneration in S334ter Rho rats. PloS one Shinde, V. M., Sizova, O. S., Lin, J. H., LaVail, M. M., Gorbatyuk, M. S. 2012; 7 (3): e33266

  Abstract

  The S334ter rhodopsin (Rho) rat (line 4) bears the rhodopsin gene with an early termination codon at residue 334 that is a model for several such mutations found in human patients with autosomal dominant retinitis pigmentosa (ADRP). The Unfolded Protein Response (UPR) is implicated in the pathophysiology of several retinal disorders including ADRP in P23H Rho rats. The aim of this study was to examine the onset of UPR gene expression in S334ter Rho retinas to determine if UPR is activated in ADRP animal models and to investigate how the activation of UPR molecules leads to the final demise of S334ter Rho photoreceptors. RT-PCR was performed to evaluate the gene expression profiles for the P10, P12, P15, and P21 stages of the development and progression of ADRP in S334ter Rho photoreceptors. We determined that during the P12-P15 period, ER stress-related genes are strongly upregulated in transgenic retinas, resulting in the activation of the UPR that was confirmed using western blot analysis and RT-PCR. The activation of UPR was associated with the increased expression of JNK, Bik, Bim, Bid, Noxa, and Puma genes and cleavage of caspase-12 that together with activated calpains presumably compromise the integrity of the mitochondrial MPTP, leading to the release of pro-apoptotic AIF1 into the cytosol of S334ter Rho photoreceptor cells. Therefore, two major cross-talking pathways, the UPR and mitochondrial MPTP occur in S334ter-4 Rho retina concomitantly and eventually promote the death of the photoreceptor cells.

  View details for DOI 10.1371/journal.pone.0033266

  View details for PubMedID 22432009

  View details for PubMedCentralID PMC3303830

• Induction of endoplasmic reticulum stress genes, BiP and chop, in genetic and environmental models of retinal degeneration. Investigative ophthalmology & visual science Kroeger, H. n., Messah, C. n., Ahern, K. n., Gee, J. n., Joseph, V. n., Matthes, M. T., Yasumura, D. n., Gorbatyuk, M. S., Chiang, W. C., LaVail, M. M., Lin, J. H. 2012; 53 (12): 7590–99

  Abstract

  Endoplasmic reticulum (ER) stress has been observed in animal models of retinitis pigmentosa expressing P23H rhodopsin. We compared levels of tightly induced ER stress genes, Binding of immunoglobulin protein (BiP) and CCAAT/enhancer-binding protein homologous protein (Chop), in seven additional models of retinal degeneration arising from genetic or environmental causes.Retinas from transgenic S334ter rhodopsin (lines 3, 4, and 5) and Royal College of Surgeons (RCS and RCS-p+) rats from postnatal (P) days 10 to 120 were analyzed. In a constant light (CL) model of retinal degeneration, BALB/c mice were exposed to 15,000 lux of CL for 0 to 8 hours. Retinal tissues from three to eight animals per experimental condition were collected for histologic and molecular analyses.S334ter animals revealed significant increases in BiP, S334ter-3 (3.3× at P15), S334ter-4 (4× at P60), and S334ter-5 (2.2× at P90), and Chop, S334ter-3 (1.3× at P15), S334ter-4 (1.5× at P30), and S334ter-5 (no change), compared with controls. P23H-3 rats showed significant increase of BiP at P60 (2.3×) and Chop (1.6×). RCS and RCS-p+ rats showed significant increases in BiP at P60 (2.4×) and P20 (1.8×), respectively, but no statistically significant changes in Chop. BALB/c mice showed increases in BiP (1.5×) and Chop (1.3×) after 4 hours of CL. Increased levels of these ER stress markers correlated with photoreceptor cell loss.Our study reveals surprising increases in BiP and to a lesser degree Chop in retinal degenerations arising from diverse causes. We propose that manipulation of ER stress responses may be helpful in treating many environmental and heritable forms of retinal degeneration.

  View details for DOI 10.1167/iovs.12-10221

  View details for PubMedID 23074209

  View details for PubMedCentralID PMC3495601

• Selective activation of ATF6 and PERK endoplasmic reticulum stress signaling pathways prevent mutant rhodopsin accumulation. Investigative ophthalmology & visual science Chiang, W. C., Hiramatsu, N. n., Messah, C. n., Kroeger, H. n., Lin, J. H. 2012; 53 (11): 7159–66

  Abstract

  Many rhodopsin mutations that cause retinitis pigmentosa produce misfolded rhodopsin proteins that are retained within the endoplasmic reticulum (ER) and cause photoreceptor cell death. Activating transcription factor 6 (ATF6) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) control intracellular signaling pathways that maintain ER homeostasis. The aim of this study was to investigate how ATF6 and PERK signaling affected misfolded rhodopsin in cells, which could identify new molecular therapies to treat retinal diseases associated with ER protein misfolding.To examine the effect of ATF6 on rhodopsin, wild-type (WT) or mutant rhodopsins were expressed in cells expressing inducible human ATF6f, the transcriptional activator domain of ATF6. Induction of ATF6f synthesis rapidly activated downstream genes. To examine PERK's effect on rhodopsin, WT or mutant rhodopsins were expressed in cells expressing a genetically altered PERK protein, Fv2E-PERK. Addition of the dimerizing molecule (AP20187) rapidly activated Fv2E-PERK and downstream genes. By use of these strategies, it was examined how selective ATF6 or PERK signaling affected the fate of WT and mutant rhodopsins.ATF6 significantly reduced T17M, P23H, Y178C, C185R, D190G, K296E, and S334ter rhodopsin protein levels in the cells with minimal effects on monomeric WT rhodopsin protein levels. By contrast, the PERK pathway reduced both levels of WT, mutant rhodopsins, and many other proteins in the cell.This study indicates that selectively activating ATF6 or PERK prevents mutant rhodopsin from accumulating in cells. ATF6 signaling may be especially useful in treating retinal degenerative diseases arising from rhodopsin misfolding by preferentially clearing mutant rhodopsin and abnormal rhodopsin aggregates.

  View details for DOI 10.1167/iovs.12-10222

  View details for PubMedID 22956602

  View details for PubMedCentralID PMC3474590

• Endoplasmic reticulum-associated degradation (ERAD) of misfolded glycoproteins and mutant P23H rhodopsin in photoreceptor cells. Advances in experimental medicine and biology Kroeger, H. n., Chiang, W. C., Lin, J. H. 2012; 723: 559–65

  View details for DOI 10.1007/978-1-4614-0631-0_71

  View details for PubMedID 22183378

  View details for PubMedCentralID PMC3651033

• The unfolded protein response is a major mechanism by which LRP1 regulates Schwann cell survival after injury. The Journal of neuroscience : the official journal of the Society for Neuroscience Mantuano, E. n., Henry, K. n., Yamauchi, T. n., Hiramatsu, N. n., Yamauchi, K. n., Orita, S. n., Takahashi, K. n., Lin, J. H., Gonias, S. L., Campana, W. M. 2011; 31 (38): 13376–85

  Abstract

  In peripheral nerve injury, Schwann cells (SCs) must survive to exert a continuing and essential role in successful nerve regeneration. Herein, we show that peripheral nerve injury is associated with activation of endoplasmic reticulum (ER) stress and the adaptive unfolded protein response (UPR). The UPR culminates in expression of C/EBP homology protein (CHOP), a proapoptotic transcription factor in SCs, unless counteracted by LDL receptor-related protein-1 (LRP1), which serves as a major activator of phosphatidylinositol 3-kinase (PI3K). Sciatic nerve crush injury in rats induced expression of the ER chaperone GRP78/BIP, reflecting an early, corrective phase of the UPR. However, when LRP1 signaling was inhibited with receptor-associated protein, PI3K activity was decreased and CHOP protein expression increased, particularly in myelinating SCs. In cultured SCs, the PKR-like ER kinase target eIF2α was phosphorylated and CHOP was induced by (1) inhibiting PI3K, (2) treating the cells with tumor necrosis factor-α (TNF-α), or (3) genetic silencing of LRP1. CHOP gene deletion in SCs decreased cell death in response to TNF-α. Furthermore, the effects of TNF-α on phosphorylated eIF2α, CHOP, and SC death were blocked by adding LRP1 ligands that augment LRP1-dependent cell signaling to PI3K. Collectively, our results support a model in which UPR-activated signaling pathways represent a major challenge to SC survival in nerve injury. LRP1 functions as a potent activator of PI3K in SCs and, by this mechanism, limits SC apoptosis resulting from increased CHOP expression in nerve injury.

  View details for DOI 10.1523/JNEUROSCI.2850-11.2011

  View details for PubMedID 21940431

  View details for PubMedCentralID PMC3188465

• Rescue of photoreceptor degeneration by curcumin in transgenic rats with P23H rhodopsin mutation. PloS one Vasireddy, V. n., Chavali, V. R., Joseph, V. T., Kadam, R. n., Lin, J. H., Jamison, J. A., Kompella, U. B., Reddy, G. B., Ayyagari, R. n. 2011; 6 (6): e21193

  Abstract

  The P23H mutation in the rhodopsin gene causes rhodopsin misfolding, altered trafficking and formation of insoluble aggregates leading to photoreceptor degeneration and autosomal dominant retinitis pigmentosa (RP). There are no effective therapies to treat this condition. Compounds that enhance dissociation of protein aggregates may be of value in developing new treatments for such diseases. Anti-protein aggregating activity of curcumin has been reported earlier. In this study we present that treatment of COS-7 cells expressing mutant rhodopsin with curcumin results in dissociation of mutant protein aggregates and decreases endoplasmic reticulum stress. Furthermore we demonstrate that administration of curcumin to P23H-rhodopsin transgenic rats improves retinal morphology, physiology, gene expression and localization of rhodopsin. Our findings indicate that supplementation of curcumin improves retinal structure and function in P23H-rhodopsin transgenic rats. This data also suggest that curcumin may serve as a potential therapeutic agent in treating RP due to the P23H rhodopsin mutation and perhaps other degenerative diseases caused by protein trafficking defects.

  View details for DOI 10.1371/journal.pone.0021193

  View details for PubMedID 21738619

  View details for PubMedCentralID PMC3126808

• Monitoring and manipulating mammalian unfolded protein response. Methods in enzymology Hiramatsu, N. n., Joseph, V. T., Lin, J. H. 2011; 491: 183–98

  Abstract

  The unfolded protein response (UPR) is a conserved, intracellular signaling pathway activated by endoplasmic reticulum (ER) stress. In mammalian cells, the UPR is controlled by three ER-resident transmembrane proteins: inositol-requiring enyzme-1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor-6 (ATF6), by which cytoprotective mechanisms are initiated to restore ER functions. However, if cellular homeostasis is not restored by the UPR's initial events, UPR signaling triggers apoptotic cell death, which correlates with the pathogenesis of a wide range of human diseases. The intrinsic function of the UPR in regulating cell survival and death suggests its importance as a mechanistic link between ER stress and disease pathogenesis. Understanding UPR regulatory molecules or signaling pathways involved in disease pathogenesis is critical to establishing therapeutic strategies. For this purpose, several experimental tools have been developed to evaluate individual UPR components. In this chapter, we present methods to monitor and quantify activation of individual UPR signaling pathways in mammalian cells and tissues, and we review strategies to artificially and selectively activate individual UPR signaling pathways using chemical-genetic approaches.

  View details for DOI 10.1016/B978-0-12-385928-0.00011-0

  View details for PubMedID 21329801

  View details for PubMedCentralID PMC3658618

• Restoration of visual function in P23H rhodopsin transgenic rats by gene delivery of BiP/Grp78. Proceedings of the National Academy of Sciences of the United States of America Gorbatyuk, M. S., Knox, T. n., LaVail, M. M., Gorbatyuk, O. S., Noorwez, S. M., Hauswirth, W. W., Lin, J. H., Muzyczka, N. n., Lewin, A. S. 2010; 107 (13): 5961–66

  Abstract

  The P23H mutation within the rhodopsin gene (RHO) causes rhodopsin misfolding, endoplasmic reticulum (ER) stress, and activates the unfolded protein response (UPR), leading to rod photoreceptor degeneration and autosomal dominant retinitis pigmentosa (ADRP). Grp78/BiP is an ER-localized chaperone that is induced by UPR signaling in response to ER stress. We have previously demonstrated that BiP mRNA levels are selectively reduced in animal models of ADRP arising from P23H rhodopsin expression at ages that precede photoreceptor degeneration. We have now overexpressed BiP to test the hypothesis that this chaperone promotes the trafficking of P23H rhodopsin to the cell membrane, reprograms the UPR favoring the survival of photoreceptors, blocks apoptosis, and, ultimately, preserves vision in ADRP rats. In cell culture, increasing levels of BiP had no impact on the localization of P23H rhodopsin. However, BiP overexpression alleviated ER stress by reducing levels of cleaved pATF6 protein, phosphorylated eIF2alpha and the proapoptotic protein CHOP. In P23H rats, photoreceptor levels of cleaved ATF6, pEIF2alpha, CHOP, and caspase-7 were much higher than those of wild-type rats. Subretinal delivery of AAV5 expressing BiP to transgenic rats led to reduction in CHOP and photoreceptor apoptosis and to a sustained increase in electroretinogram amplitudes. We detected complexes between BiP, caspase-12, and the BH3-only protein BiK that may contribute to the antiapoptotic activity of BiP. Thus, the preservation of photoreceptor function resulting from elevated levels of BiP is due to suppression of apoptosis rather than to a promotion of rhodopsin folding.

  View details for DOI 10.1073/pnas.0911991107

  View details for PubMedID 20231467

  View details for PubMedCentralID PMC2851865

• Misfolded proteins and retinal dystrophies. Advances in experimental medicine and biology Lin, J. H., Lavail, M. M. 2010; 664: 115–21

  Abstract

  Many mutations associated with retinal degeneration lead to the production of misfolded proteins by cells of the retina. Emerging evidence suggests that these abnormal proteins cause cell death by activating the Unfolded Protein Response, a set of conserved intracellular signaling pathways that detect protein misfolding within the endoplasmic reticulum and control protective and proapoptotic signal transduction pathways. Here, we review the misfolded proteins associated with select types of retinitis pigmentosa, Stargadt-like macular degeneration, and Doyne Honeycomb Retinal Dystrophy and discuss the role that endoplasmic reticulum stress and UPR signaling play in their pathogenesis. Last, we review new therapies for these diseases based on preventing protein misfolding in the retina.

  View details for DOI 10.1007/978-1-4419-1399-9_14

  View details for PubMedID 20238009

  View details for PubMedCentralID PMC2955894

• Divergent effects of PERK and IRE1 signaling on cell viability. PloS one Lin, J. H., Li, H. n., Zhang, Y. n., Ron, D. n., Walter, P. n. 2009; 4 (1): e4170

  Abstract

  Protein misfolding in the endoplasmic reticulum (ER) activates a set of intracellular signaling pathways, collectively termed the Unfolded Protein Response (UPR). UPR signaling promotes cell survival by reducing misfolded protein levels. If homeostasis cannot be restored, UPR signaling promotes cell death. The molecular basis for the switch between prosurvival and proapoptotic UPR function is poorly understood. The ER-resident proteins, PERK and IRE1, control two key UPR signaling pathways. Protein misfolding concomitantly activates PERK and IRE1 and has clouded insight into their contributions toward life or death cell fates. Here, we employed chemical-genetic strategies to activate individually PERK or IRE1 uncoupled from protein misfolding. We found that sustained PERK signaling impaired cell proliferation and promoted apoptosis. By contrast, equivalent durations of IRE1 signaling enhanced cell proliferation without promoting cell death. These results demonstrate that extended PERK and IRE1 signaling have opposite effects on cell viability. Differential activation of PERK and IRE1 may determine life or death decisions after ER protein misfolding.

  View details for DOI 10.1371/journal.pone.0004170

  View details for PubMedID 19137072

  View details for PubMedCentralID PMC2614882

• Regulated Ire1-dependent decay of messenger RNAs in mammalian cells. The Journal of cell biology Hollien, J. n., Lin, J. H., Li, H. n., Stevens, N. n., Walter, P. n., Weissman, J. S. 2009; 186 (3): 323–31

  Abstract

  Maintenance of endoplasmic reticulum (ER) function is achieved in part through Ire1 (inositol-requiring enzyme 1), a transmembrane protein activated by protein misfolding in the ER. The cytoplasmic nuclease domain of Ire1 cleaves the messenger RNA (mRNA) encoding XBP-1 (X-box-binding protein 1), enabling splicing and production of this active transcription factor. We recently showed that Ire1 activation independently induces the rapid turnover of mRNAs encoding membrane and secreted proteins in Drosophila melanogaster cells through a pathway we call regulated Ire1-dependent decay (RIDD). In this study, we show that mouse fibroblasts expressing wild-type Ire1 but not an Ire1 variant lacking nuclease activity also degrade mRNAs in response to ER stress. Using a second variant of Ire1 that is activated by a small adenosine triphosphate analogue, we show that although XBP-1 splicing can be artificially induced in the absence of ER stress, RIDD appears to require both Ire1 activity and ER stress. Our data suggest that cells use a multitiered mechanism by which different conditions in the ER lead to distinct outputs from Ire1.

  View details for DOI 10.1083/jcb.200903014

  View details for PubMedID 19651891

  View details for PubMedCentralID PMC2728407

• BAX inhibitor-1 is a negative regulator of the ER stress sensor IRE1alpha. Molecular cell Lisbona, F. n., Rojas-Rivera, D. n., Thielen, P. n., Zamorano, S. n., Todd, D. n., Martinon, F. n., Glavic, A. n., Kress, C. n., Lin, J. H., Walter, P. n., Reed, J. C., Glimcher, L. H., Hetz, C. n. 2009; 33 (6): 679–91

  Abstract

  Adaptation to endoplasmic reticulum (ER) stress depends on the activation of an integrated signal transduction pathway known as the unfolded protein response (UPR). Bax inhibitor-1 (BI-1) is an evolutionarily conserved ER-resident protein that suppresses cell death. Here we have investigated the role of BI-1 in the UPR. BI-1 expression suppressed IRE1alpha activity in fly and mouse models of ER stress. BI-1-deficient cells displayed hyperactivation of the ER stress sensor IRE1alpha, leading to increased levels of its downstream target X-box-binding protein-1 (XBP-1) and upregulation of UPR target genes. This phenotype was associated with the formation of a stable protein complex between BI-1 and IRE1alpha, decreasing its ribonuclease activity. Finally, BI-1 deficiency increased the secretory activity of primary B cells, a phenomenon regulated by XBP-1. Our results suggest a role for BI-1 in early adaptive responses against ER stress that contrasts with its known downstream function in apoptosis.

  View details for DOI 10.1016/j.molcel.2009.02.017

  View details for PubMedID 19328063

  View details for PubMedCentralID PMC2818874

• Endoplasmic reticulum stress in disease pathogenesis. Annual review of pathology Lin, J. H., Walter, P. n., Yen, T. S. 2008; 3: 399–425

  Abstract

  The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins, which, collectively, represent a large fraction of the total protein output of a mammalian cell. Therefore, the protein flux through the ER must be carefully monitored for abnormalities, including the buildup of misfolded proteins. Mammalian cells have evolved an intricate set of signaling pathways from the ER to the cytosol and nucleus, to allow the cell to respond to the presence of misfolded proteins within the ER. These pathways, known collectively as the unfolded protein response, are important for normal cellular homeostasis and organismal development and may also play key roles in the pathogenesis of many diseases. This review provides background information on the unfolded protein response and discusses a selection of diseases whose pathogenesis involves ER stress.

  View details for DOI 10.1146/annurev.pathmechdis.3.121806.151434

  View details for PubMedID 18039139

  View details for PubMedCentralID PMC3653419

• IRE1 signaling affects cell fate during the unfolded protein response. Science (New York, N.Y.) Lin, J. H., Li, H. n., Yasumura, D. n., Cohen, H. R., Zhang, C. n., Panning, B. n., Shokat, K. M., Lavail, M. M., Walter, P. n. 2007; 318 (5852): 944–49

  Abstract

  Endoplasmic reticulum (ER) stress activates a set of signaling pathways, collectively termed the unfolded protein response (UPR). The three UPR branches (IRE1, PERK, and ATF6) promote cell survival by reducing misfolded protein levels. UPR signaling also promotes apoptotic cell death if ER stress is not alleviated. How the UPR integrates its cytoprotective and proapoptotic outputs to select between life or death cell fates is unknown. We found that IRE1 and ATF6 activities were attenuated by persistent ER stress in human cells. By contrast, PERK signaling, including translational inhibition and proapoptotic transcription regulator Chop induction, was maintained. When IRE1 activity was sustained artificially, cell survival was enhanced, suggesting a causal link between the duration of UPR branch signaling and life or death cell fate after ER stress. Key findings from our studies in cell culture were recapitulated in photoreceptors expressing mutant rhodopsin in animal models of retinitis pigmentosa.

  View details for DOI 10.1126/science.1146361

  View details for PubMedID 17991856

  View details for PubMedCentralID PMC3670588

• ETS gene Er81 controls the formation of functional connections between group Ia sensory afferents and motor neurons. Cell Arber, S. n., Ladle, D. R., Lin, J. H., Frank, E. n., Jessell, T. M. 2000; 101 (5): 485–98

  Abstract

  The connections formed between sensory and motor neurons (MNs) play a critical role in the control of motor behavior. During development, the axons of proprioceptive sensory neurons project into the spinal cord and form both direct and indirect connections with MNs. Two ETS transcription factors, ER81 and PEA3, are expressed by developing proprioceptive neurons and MNs, raising the possibility that these genes are involved in the formation of sensory-motor connections. Er81 mutant mice exhibit a severe motor discoordination, yet the specification of MNs and induction of muscle spindles occurs normally. The motor defect in Er81 mutants results from a failure of group Ia proprioceptive afferents to form a discrete termination zone in the ventral spinal cord. As a consequence there is a dramatic reduction in the formation of direct connections between proprioceptive afferents and MNs. ER81 therefore controls a late step in the establishment of functional sensory-motor circuitry in the developing spinal cord.

  View details for DOI 10.1016/s0092-8674(00)80859-4

  View details for PubMedID 10850491

• Functionally related motor neuron pool and muscle sensory afferent subtypes defined by coordinate ETS gene expression. Cell Lin, J. H., Saito, T. n., Anderson, D. J., Lance-Jones, C. n., Jessell, T. M., Arber, S. n. 1998; 95 (3): 393–407

  Abstract

  Motor function depends on the formation of selective connections between sensory and motor neurons and their muscle targets. The molecular basis of the specificity inherent in this sensory-motor circuit remains unclear. We show that motor neuron pools and subsets of muscle sensory afferents can be defined by the expression of ETS genes, notably PEA3 and ER81. There is a matching in PEA3 and ER81 expression by functionally interconnected sensory and motor neurons. ETS gene expression by motor and sensory neurons fails to occur after limb ablation, suggesting that their expression is coordinated by signals from the periphery. ETS genes may therefore participate in the development of selective sensory-motor circuits in the spinal cord.

  View details for DOI 10.1016/s0092-8674(00)81770-5

  View details for PubMedID 9814709

• Glomuvenous Malformation: A Rare Periorbital Lesion of the Thermoregulatory Apparatus. Ophthalmic plastic and reconstructive surgery Whipple, K. M., Godfrey, K. J., Solomon, J. P., Lin, J. H., Korn, B. S., Kikkawa, D. O. ; 33 (2): e36–e37

  Abstract

  Glomuvenous malformations (GVMs), previously referred to as glomus tumors or glomangiomas, are benign, mesenchymal venous malformations arising from glomus bodies. Glomus bodies are modified smooth muscle neuromyoarterial structures involved in temperature regulation via blood shunting. These classically occur in the digits but can occur in other locations. The authors present a case of a periorbital GVM presented following blunt trauma to the area.

  View details for DOI 10.1097/IOP.0000000000000695

  View details for PubMedID 27065433

  View details for PubMedCentralID PMC5118188

• Bilateral squamous cell carcinoma of the lacrimal sac. Ophthalmic plastic and reconstructive surgery Hodgson, N. n., Whipple, K. n., Lin, J. H., Brumund, K. T., Kikkawa, D. O., Korn, B. S. ; 29 (6): e149–51

  Abstract

  A 42-year-old man presented with right-sided epiphora, a fleshy lesion emanating from the right inferior punctum and a painless mass below the medial canthal tendon. Biopsy of the lacrimal sac mass disclosed papillary squamous cell carcinoma in situ. The patient underwent wide local excision with clear surgical margins and remained disease free until 28 months later when he returned with hemorrhagic epiphora of the OS and fullness overlying the left lacrimal sac. Biopsy confirmed papillary squamous cell carcinoma in situ. He underwent similar excision and has remained disease free for 6 months. To the best of the authors' knowledge, this is the first report of bilateral squamous cell carcinoma of the lacrimal sac.

  View details for DOI 10.1097/IOP.0b013e3182831c2d

  View details for PubMedID 24217491

  View details for PubMedCentralID PMC4081470