Ophthalmology
Abstract
Elevation of intraocular pressure (IOP) due to trabecular meshwork (TM) dysfunction, leading to neurodegeneration, is the pathological hallmark of primary open-angle glaucoma (POAG). Impaired axonal transport is an early and critical feature of glaucomatous neurodegeneration. However, a robust mouse model that accurately replicates these human POAG features has been lacking. We report the development and characterization of a novel Cre-inducible mouse model expressing a DsRed-tagged Y437H mutant of human myocilin (Tg.CreMYOCY437H). A single intravitreal injection of HAd5-Cre induced selective MYOC expression in the TM, causing TM dysfunction, reducing the outflow facility, and progressively elevating IOP in Tg.CreMYOCY437H mice. Sustained IOP elevation resulted in significant loss of retinal ganglion cells (RGCs) and progressive axonal degeneration in Cre-induced Tg.CreMYOCY437H mice. Notably, impaired anterograde axonal transport was observed at the optic nerve head before RGC degeneration, independent of age, indicating that impaired axonal transport contributes to RGC degeneration in Tg.CreMYOCY437H mice. In contrast, axonal transport remained intact in ocular hypertensive mice injected with microbeads, despite significant RGC loss. Our findings indicate that Cre-inducible Tg.CreMYOCY437H mice replicate all glaucoma phenotypes, providing an ideal model for studying early events of TM dysfunction and neuronal loss in POAG.
Authors
Balasankara Reddy Kaipa, Ramesh Kasetti, Yogapriya Sundaresan, Linya Li, Sam Yacoub, J. Cameron Millar, William Cho, Dorota Skowronska-Krawczyk, Prabhavathi Maddineni, Krzysztof Palczewski, Gulab S. Zode
Abstract
BACKGROUND. Current clinical sequencing methods cannot effectively detect DNA methylation and allele-specific variation to provide parent-of-origin information from the proband alone. Parent-of-origin effects can lead to differential disease and the inability to assign this in de novo cases limits prognostication in the majority of affected individuals with retinoblastoma, a hereditary cancer with suspected parent-of-origin effects. METHODS. To directly assign parent-of-origin in retinoblastoma patients, genomic DNA was extracted from blood samples for sequencing using a programmable, targeted single-molecule long-read DNA genomic and epigenomic approach. This allowed germline variant calling and simultaneous haplotype-resolved CpG methylation in subjects with familial (n=7) and de novo (n=9) retinoblastoma. RESULTS. Targeted long-read sequencing allowed phasing genomic variation with a differentially methylated region in intron 2 of the RB1 gene to confirm parent-of-origin in known familial samples. Leveraging this approach allowed us to directly assign parent-of-origin rapidly in simple and complex de novo cases from the proband alone. The ability to assign parent-of-origin in all cases of retinoblastoma showed that harboring disease-causing variants on the paternally inherited allele, whether arising familial or de novo, is associated with more advanced cancer staging at presentation and significantly greater risk of chemotherapy failure (P=0.002). CONCLUSION. This study demonstrates the diagnostic potential of multi-omic long-read profiling to unveil the parent-of-origin effect in hereditary cancer. The approach in this work will be instrumental in assigning parent-of-origin to other genetic diseases using local and distant imprinting signals in the genome. FUNDING. National Eye Institute, NIH (K08EY033789); Gerber Foundation; Research to Prevent Blindness
Authors
Andrew W. Stacey, Kenji Nakamichi, Jennifer Huey, Jeffrey Stevens, Natalie Waligorski, Erin E. Crotty, Russell N. Van Gelder, Debarshi Mustafi
Abstract
Despite recent advances in the treatment of thyroid eye disease thyroid-related eye disease (TED), marked gaps remain in our understanding of the underlying molecular mechanisms, particularly concerning the insulin-like growth factor-1 receptor (IGF-1R) pathway. To dissect the pathophysiology of TED, we used single-nucleus RNA-Seq to analyze orbital fat specimens from both patients with TED and matched individuals acting as controls. The analysis demonstrated a marked increase in the proportion of fibroblasts transitioning to adipogenesis in the orbital fat of patients with TED compared with that in control patients. This was associated with diverse alterations in immune cell composition. Significant alterations in the IGF-1R signaling pathway were noted between TED specimens and those from control patients, indicating a potential pathological mechanism driven by IGF-1R signaling abnormalities. Additionally, our data showed that linsitinib, a small-molecule inhibitor of IGF-1R, effectively reduced adipogenesis in TED orbital fibroblasts in vitro, suggesting its potential utility as a therapeutic agent. Our findings reveal that, beyond immune dysfunction, abnormal IGF-1R signaling leading to enhanced adipogenesis is a crucial pathogenic mechanism in TED.
Authors
Dong Won Kim, Soohyun Kim, Jeong Han, Karan Belday, Emily Li, Nicholas Mahoney, Seth Blackshaw, Fatemeh Rajaii
Abstract
Ocular hypertension, believed to result partly from increased contractile activity, cell adhesive interactions, and stiffness within the trabecular meshwork (TM), is a major risk factor for glaucoma, a leading cause of blindness. However, the identity of molecular mechanisms governing organization of actomyosin and cell adhesive interactions in the TM remains limited. Based on our previous findings, in which proteomics analyses revealed elevated levels of septins, including septin-9 in human TM cells treated with the ocular hypertensive agent dexamethasone, here, we evaluated the effects of septin-9 overexpression, deficiency, and pharmacological targeting in TM cells. These studies demonstrated a profound impact on actomyosin organization, cell adhesion, contraction, and phagocytosis. Overexpression raised intraocular pressure (IOP) in mice, while inhibition increased cell permeability. In addition, we replicated a significant association between a common variant (rs9038) in SEPT9 with IOP in the Genetic Epidemiology Research on Adult Healthy and Aging (GERA) cohort. Collectively, these data reveal a link between dysregulated septin cytoskeletal organization in the TM and increased IOP, likely due to enhanced cell contraction, adhesive interactions, and fibrotic activity. This suggests that targeting the septin cytoskeleton could offer a novel approach for lowering IOP in patients with glaucoma.
Authors
Rupalatha Maddala, Pallavi Gorijavolu, Levi K. Lankford, Nikolai P. Skiba, Pratap Challa, Rakesh K. Singh, K. Saidas Nair, Hélène Choquet, Ponugoti V. Rao
Abstract
Aniridia is a rare congenital condition of abnormal eye development arising principally from heterozygous mutation of the PAX6 gene. Among the multiple complications arising in the eye, aniridia-associated keratopathy (AAK) is a severe vision-impairing condition of the cornea associated with a progressive limbal stem cell deficiency that lacks suitable treatment options. Current mouse models of aniridia do not accurately represent the onset and progression dynamics of human AAK, hindering therapy development. Here, we performed deep phenotyping of a haploinsufficient Pax6+/– small-eye (Sey) mouse model on the129Sey/SvImJ background, that exhibits key features of mild presentation at birth and progressive AAK with aging, mimicking human disease. The model exhibits a slowly progressing AAK phenotype and provides new insights into the disease including disturbed basal epithelial cell organization, function and marker expression, persistent postnatal lymphangiogenesis, disrupted corneal innervation patterns, and persisting yet altered limbal stem cell marker expression with age. The model recapitulates many of the known features of human disease, enabling investigation of underlying disease mechanisms and importantly, to access a well-defined temporal window for evaluating future therapeutics.
Authors
Dina Javidjam, Petros Moustardas, Mojdeh Abbasi, Ava Dashti, Yedizza Rautavaara, Neil Lagali
Abstract
The degeneration of retinal ganglion cells (RGC) due to mitochondrial dysfunctions manifests optic neuropathy. However, the molecular components of RGC linked to optic neuropathy manifestations remain largely unknown. Here, we identified a novel optic atrophy-causative CRYAB gene encoding a highly conserved major lens protein acting as mitochondrial chaperone and possessing anti-apoptotic activities. The heterozygous CRYAB mutation (c.313G>A, p. Glu105Lys) was cosegregated with autosomal dominant inheritance of optic atrophy in 3 Chinese families. The p.E105K mutation altered the structure and function of CRYAB, including decreased stability, reduced formation of oligomers and decreasing chaperone activity. Coimmunoprecipitation indicated that the p.E105K mutation reduced the interaction of CRYAB with apoptosis-associated cytochrome c and VDAC. The cell lines carrying the p.E105K mutation displayed promoting apoptosis, defective assembly, stability and activities of oxidative phosphorylation system and imbalance of mitochondrial dynamics. Involvement of CRYAB in optic atrophy was confirmed by phenotypic evaluations of Cryabp.E105K knock-in mice. These mutant mice exhibited ocular lesions including changing intra-retina layers, degeneration of RGCs, photoreceptor deficits and abnormal retinal vasculature. Furthermore, Cryab-deficient mice displayed elevated apoptosis and mitochondrial dysfunctions. Our findings provide new insight of pathophysiology of optic atrophy arising from RGC degeneration caused by CRYAB deficiency-induced elevated apoptosis and mitochondrial dysfunctions.
Authors
Chenghui Wang, Liyao Zhang, Zhipeng Nie, Min Liang, Hanqing Liu, Qiuzi Yi, Chunyan Wang, Cheng Ai, Juanjuan Zhang, Yinglong Gao, Yanchun Ji, Min-Xin Guan
Abstract
Retinitis pigmentosa (RP) is a complex group of inherited retinal diseases characterized by progressive death of photoreceptor cells and eventual blindness. Pde6a, which encodes a cGMP-specific phosphodiesterase, is a crucial pathogenic gene for autosomal recessive RP (RP43); there is no effective therapy for this form of RP. The compact CRISPR/SaCas9 system, which can be packaged into a single adeno-associated virus, holds promise for simplifying effective gene therapy. Here, we demonstrated that all-in-one AAV-SaCas9-mediated Nrl gene inactivation can efficiently prevent retinal degeneration in a RP mouse model with Pde6anmf363/nmf363 mutation. We screened single guide RNAs (sgRNAs) capable of efficiently editing mouse Nrl gene in N2a cells and then achieved effective gene editing by using a single AAV to co-deliver SaCas9 and an optimal Nrl-sg2 into the mouse retina. Excitingly, in vivo inactivation of Nrl improved photoreceptor cell survival and rescued retinal function in treated Pde6a deficient mice. Thus, we showed that a practical, gene-independent method, AAV-SaCas9-mediated Nrl inactivation, holds promise for future therapeutic applications in patients with RP.
Authors
Zhiquan Liu, Siyu Chen, Chien-Hui Lo, Qing Wang, Yang Sun
Abstract
Hereditary Macular Dystrophies (HMDs) are a genetically diverse group of disorders that cause central vision loss due to photoreceptor and retinal pigment epithelium (RPE) damage. We investigated a family with a presumed novel autosomal dominant HMD characterized by faint, hypopigmented RPE changes involving the central retina. Genome and RNA sequencing identified the disease-causing variant to be a 560 kilobase tandem duplication on chromosome 17 [NC_000017.10 (hg19): g.4012590_4573014dup], which led to the formation of a novel ZZEF1-ALOX15 fusion gene, that upregulates ALOX15. ALOX15 encodes a lipoxygenase involved in polyunsaturated fatty acid metabolism. Functional studies showed retinal disorganization, and photoreceptor and RPE damage following electroporation of the chimera transcript in mouse retina. Photoreceptor damage also occurred following electroporation with a native ALOX15 transcript but not with a near-null ALOX15 transcript. Affected patients’ lymphoblasts demonstrated lower levels of ALOX15 substrates and an accumulation of neutral lipids. We implicated the fusion gene as the cause of this family’s HMD, due to mis-localization and overexpression of ALOX15, driven by the ZZEF1 promoter. To our knowledge, this is the first reported instance of a fusion gene leading to HMD or inherited retinal dystrophy, highlighting the need to prioritize duplication analysis in unsolved retinal dystrophies.
Authors
Rabiat Adele, Rowaida Hussein, Erika Tavares, Kashif Ahmed, Matteo Di Scipio, Jason Charish, Minggao Liang, Simon Monis, Anupreet Tumber, Xiaoyan Chen, Tara A. Paton, Nicole M. Roslin, Christabel Eileen, Evgueni Ivakine, Nishanth E. Sunny, Michael D. Wilson, Eric Campos, Raju V.S. Rajala, Jason T. Maynes, Philippe P. Monnier, Andrew D. Paterson, Elise Héon, Ajoy Vincent
Abstract
Accumulation of extracellular matrix (ECM) proteins in trabecular meshwork (TM), which leads to increased outflow resistance of aqueous humor and consequently high intraocular pressure, is a major cause of primary open-angle glaucoma (POAG). According to our preliminary research, the RapGAP protein superfamily member, signal-induced proliferation-associated 1-like 1 protein (SIPA1L1), which is involved in tissue fibrosis, may have an impact on POAG by influencing ECM metabolism of TM. This study aims to confirm these findings and identify effects and cellular mechanisms of SIPA1L1 on ECM changes and phagocytosis in human TM (HTM) cells. Our results showed that the expression of SIPA1L1 in HTM cells was significantly increased by TGFβ2 treatment in Label-free quantitative proteomics. The aqueous humor and TM cells concentration of SIPA1L1 in POAG patients was higher than that of control. In HTM cells, TGFβ2 increased expression of SIPA1L1 along with accumulation of ECM, RhoA and p-Cofilin1. The effects of TGFβ2 were reduced by si-SIPA1L1. TGFβ2 decreased HTM cell phagocytosis by polymerizing cytoskeletal actin filaments, while si-SIPA1L1 increased phagocytosis by disassembling actin filaments. Simultaneously, overexpressing SIPA1L1 alone exhibited comparable effects to that of TGFβ2. Our studies demonstrate that SIPA1L1 not only promotes the production of ECM, but also inhibits its removal by reducing phagocytosis. Targeting SIPA1L1 degradation may become a significant therapy for POAG.
Authors
Chenyu Xu, Jiahong Wei, Dan Song, Siyu Zhao, Mingmin Hou, Yuchen Fan, Li Guo, Hao Sun, Tao Guo
Abstract
Despite advances in sequencing technologies, a molecular diagnosis remains elusive in many Mendelian disease patients. Current short-read clinical sequencing approaches cannot provide chromosomal phase information or epigenetic information without further sample processing, which is not routinely done and can result in an incomplete molecular diagnosis in patients. The ability to provide phased genetic and epigenetic information from a single sequencing run would improve the diagnostic rate of Mendelian conditions. Here we describe Targeted Long-read Sequencing of Mendelian Disease genes (TaLon-SeqMD) using a real-time adaptive sequencing approach. Optimization of bioinformatic targeting enabled selective enrichment of multiple disease-causing regions of the human genome. Haplotype-resolved variant calling and simultaneous resolution of epigenetic base modification could be achieved in a single sequencing run. The TaLon-SeqMD approach was validated in a cohort of 18 subjects with previous genetic testing targeting 373 inherited retinal disease (IRD) genes, yielding the complete molecular diagnosis in each case. This approach was then applied in two IRD cases with inconclusive testing, which uncovered non-coding and structural variants that were difficult to characterize by standard short-read sequencing. Overall, these results demonstrate TaLon-SeqMD as an approach to provide rapid phased-variant calling to provide the molecular basis of Mendelian diseases.
Authors
Kenji Nakamichi, Jennifer Huey, Riccardo Sangermano, Emily M. Place, Kinga M. Bujakowska, Molly Marra, Lesley A. Everett, Paul Yang, Jennifer R. Chao, Russell N. Van Gelder, Debarshi Mustafi
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