Leber hereditary optic neuropathy (LHON) is a paradigm for mitochondrial retinopathy due to mitochondrial DNA (mtDNA) mutations. However, the mechanism underlying retinal cell-specific effects of LHON-linked mtDNA mutations remains poorly understood and there has been no effective treatment or cure for this disorder. Using a mice model bearing a LHON-linked ND6P25L mutation, we demonstrated that the mutation caused retinal cell-specific deficiencies, especially in retinal ganglion cells (RGC), rods and Müller cells. Single-cell RNA sequencing revealed cell-specific dysregulation of oxidative phosphorylation and visual signaling pathways in the mutant retina. Strikingly, ND6 mutation-induced dysfunctions caused abnormal vitamin A (VA) metabolism essential for visual function. VA supplementation remarkably alleviated retinal deficiencies, including reduced fundus lesion and retinal thickness, and increasing numbers of RGCs, photoreceptors and Müller cell neurites. The restoration of visual functions with VA treatment were further evidenced by correcting dysregulations of phototransduction cascade and neurotransmitter transmission and restoring electrophysiological properties. Interestingly, VA supplementation markedly rescued the abnormal mitochondrial morphologies and functions in the mutant retina. These findings provide new insight into retina-specific pathophysiology of mitochondrial retinopathy arising from vitamin A deficiency and mitochondrial dysfunction induced by mtDNA mutation and step toward for therapeutic intervention for LHON and other mitochondrial retinopathy.
Cheng Ai, Huiying Li, Chunyan Wang, Yanchun Ji, Douglas C. Wallace, Junbin Qian, Yimin Zhu, Min-Xin Guan
The high-temperature requirement A1 (HTRA1), a serine protease, has been demonstrated to play a pivotal role in the extracellular matrix (ECM) and has been reported to be associated with the pathogenesis of age-related macular degeneration (AMD). To delineate its role in the retina, the phenotype of homozygous Htra1-KO (Htra1–/–) mice was characterized to examine the effect of Htra1 loss on the retina and retinal pigment epithelium (RPE) with age. The ablation of Htra1 led to a significant reduction in rod and cone photoreceptor function, primary cone abnormalities followed by rods, and atrophy in the RPE compared with WT mice. Ultrastructural analysis of Htra1–/– mice revealed RPE and Bruch’s membrane (BM) abnormalities, including the presence of sub-RPE deposits at 5 months (m) that progressed with age accompanied by increased severity of pathology. Htra1–/– mice also displayed alterations in key markers for inflammation, autophagy, and lipid metabolism in the retina. These results highlight the crucial role of HTRA1 in the retina and RPE. Furthermore, this study allows for the Htra1–/– mouse model to be utilized for deciphering mechanisms that lead to sub-RPE deposit phenotypes including AMD.
Pooja Biswas, DaNae R. Woodard, T.J. Hollingsworth, Naheed W. Khan, Danielle R. Lazaro, Anne Marie Berry, Manisha Dagar, Yang Pan, Donita Garland, Peter X. Shaw, Chio Oka, Takeshi Iwata, Monica M. Jablonski, Radha Ayyagari
Autoimmune uveitis (AU) is a sight-threatening ocular autoimmune disorder that often manifests as retinal vasculitis. Increased neutrophil infiltration around retinal vessels has been reported during the progression of AU, while how they function is not fully recognized. Neutrophil extracellular traps (NETs), produced by activated neutrophils, have been suggested to be detrimental in autoimmune diseases. Here, we found that NETs were elevated in patients with active AU, and this was verified in an experimental AU (EAU) mouse model. Depletion of neutrophils or degradation of NETs with deoxyribonuclease-I (DNase I) could decrease CD4+ effector T cell (Teff) infiltration in retina and spleen to alleviate EAU. Moreover, we found that the expression of adhesion molecules, selectin, and antigen-presenting molecules was elevated in EAU retina and in retinal microvascular endothelial cells (RMECs) cocultured with NETs. The stimulated RMECs further facilitated CD4+ T cell adhesion, activation, and differentiation into Teffs. Mechanistically, NETs trigger RMEC activation by hastening cell senescence through the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway. Slowing down senescence or inhibiting the cGAS/STING pathway in RMECs reduces the activation and differentiation of CD4+ T cells. These results suggest a deleterious role of NETs in AU. Targeting NETs would offer an effective therapeutic method.
Zuoyi Li, Zhuang Li, Yunwei Hu, Yanyan Xie, Yuxun Shi, Guanyu Chen, Jun Huang, Zhiqiang Xiao, Wenjie Zhu, Haixiang Huang, Minzhen Wang, Jianping Chen, Xiaoqing Chen, Dan Liang
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.
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
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
Andrew W. Stacey, Kenji Nakamichi, Jennifer Huey, Jeffrey Stevens, Natalie Waligorski, Erin E. Crotty, Russell N. Van Gelder, Debarshi Mustafi
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.
Dong Won Kim, Soohyun Kim, Jeong Han, Karan Belday, Emily Li, Nicholas Mahoney, Seth Blackshaw, Fatemeh Rajaii
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.
Rupalatha Maddala, Pallavi Gorijavolu, Levi K. Lankford, Nikolai P. Skiba, Pratap Challa, Rakesh K. Singh, K. Saidas Nair, Hélène Choquet, Ponugoti V. Rao
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.
Dina Javidjam, Petros Moustardas, Mojdeh Abbasi, Ava Dashti, Yedizza Rautavaara, Neil Lagali
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.
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
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.
Zhiquan Liu, Siyu Chen, Chien-Hui Lo, Qing Wang, Yang Sun
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