Ophthalmology
Abstract
Leber’s hereditary optic neuropathy (LHON) is a maternally inherited eye disease. X-linked nuclear modifiers were proposed to modify the phenotypic manifestation of LHON-associated mitochondrial DNA (mtDNA) mutations. By whole exome sequencing, we identified the X-linked LHON modifier (c.157C>T, p. Arg53Trp) in the PRICKLE3 encoding a mitochondrial protein linked to biogenesis of ATPase in three Chinese families. All affected individuals carried both ND4 11778G>A and p.Arg53Trp mutations, while subjects bearing only single mutation exhibited normal vision. The cells carrying the p.Arg53Trp mutation exhibited the defective assembly, stability and function of ATP synthase, verified by PRICKLE3 knock-down cells. Co-immunoprecipitation indicated the direct interaction of PRICKLE3 with ATP synthase via ATP8. Strikingly, mutant cells bearing both p.Arg53Trp and m.11778G>A mutations displayed greater mitochondrial dysfunctions than those carrying only single mutation. These indicated that the p.Arg53Trp mutation acted in synergy with m.11778G>A mutation and deteriorated mitochondrial dysfunctions necessary for the expression of LHON. Furthermore, we demonstrated that Prickle3 deficient mice exhibited the pronounced ATPase deficiencies. Prickle3 knock-out mice recapitulated LHON phenotypes with retina deficiencies including degeneration of retinal ganglion cells and abnormal vasculature. Our findings provided new insights into pathophysiology of LHON that were manifested by interaction between mtDNA mutation and X-linked nuclear modifier.
Authors
Jialing Yu, Xiaoyang Liang, Yanchun Ji, Cheng Ai, Junxia Liu, Ling Zhu, Zhipeng Nie, Xiaofen Jin, Chenghui Wang, Juanjuan Zhang, Fuxin Zhao, Shuang Mei, Xiaoxu Zhao, Xiangtian Zhou, Minglian Zhang, Meng Wang, Taosheng Huang, Pingping Jiang, Min-Xin Guan
Abstract
Retinitis pigmentosa (RP) is a genetically heterogenous group of eye diseases in which initial degeneration of rods triggers secondary degeneration of cones, leading to significant loss of daylight, color, and high-acuity vision. Gene complementation with adeno-associated viral (AAV) vectors is one strategy to treat RP. Its implementation faces substantial challenges, however — e.g., the tremendous number of loci with causal mutations. Gene therapy targeting secondary cone degeneration is an alternative approach that could provide a much-needed generic treatment for many RP patients. Here, we show that microglia are required for the upregulation of potentially neurotoxic inflammatory factors during cone degeneration in RP, creating conditions that might contribute to cone dysfunction and death. To ameliorate the effects of such factors, we used AAV vectors to express isoforms of the anti-inflammatory cytokine transforming growth factor-beta (TGF-β). AAV-mediated delivery of TGF-β1 rescued degenerating cones in three mouse models of RP carrying different pathogenic mutations. Treatment with TGF-β1 protected vision, as measured by two behavioral assays, and could be pharmacologically disrupted by either depleting microglia or blocking the TGF-β receptors. Our results suggest that TGF-β1 may be broadly beneficial for patients with cone degeneration, and potentially other forms of neurodegeneration, through a pathway dependent upon microglia.
Authors
Sean K. Wang, Yunlu Xue, Constance L. Cepko
Abstract
Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults. Vascular pericyte degeneration is the predominant clinical manifestation of DR, yet the mechanism governing pericyte degeneration is poorly understood. Circular RNAs (circRNAs) play important roles in multiple biological processes and disease progression. Here, we investigated the role of circRNA in pericyte biology and diabetes-induced retinal vascular dysfunction. cZNF532 expression was upregulated in pericytes under diabetic stress, in the retinal vessels of a diabetic murine model, and in the vitreous humor of diabetic patients. cZNF532 silencing reduced the viability, proliferation, and differentiation of pericytes and suppressed the recruitment of pericytes toward endothelial cells in vitro. cZNF532 regulated pericyte biology by acting as a miR-29a-3p sponge and inducing increased expression of NG2, LOXL2, and CDK2. Knockdown of cZNF532 or overexpression of miR-29a-3p aggravated streptozotocin-induced retinal pericyte degeneration and vascular dysfunction. By contrast, overexpression of cZNF532 or inhibition of miR-29a-3p ameliorated human diabetic vitreous-induced retinal pericyte degeneration and vascular dysfunction. Collectively, these data identify a circRNA-mediated mechanism that coordinates pericyte biology and vascular homeostasis in DR. Induction of cZNF532 or antagonism of miR-29a-3p is an exploitable therapeutic approach for the treatment of DR.
Authors
Qin Jiang, Chang Liu, Chaopeng Li, Shanshan Xu, Mudi Yao, Huimin Ge, Yanan Sun, Xiumiao Li, Shujie Zhang, Kun Shan, Baihui Liu, Jin Yao, Chen Zhao, Biao Yan
Abstract
Background: In retinitis pigmentosa (RP) rod photoreceptors degenerate from one of many mutations after which cones are compromised by oxidative stress. N-acetylcysteine (NAC) reduces oxidative damage and increases cone function/survival in RP models. We tested the safety, tolerability, and visual function effects of oral NAC in RP patients. Methods: Subjects (n = 10 per cohort) received 600 mg (cohort 1), 1200 mg (cohort 2), or 1800 mg (cohort 3) NAC BID for 12 weeks and then TID for 12 weeks. Best-corrected visual acuity (BCVA), macular sensitivity, ellipsoid zone (EZ) width, and aqueous NAC were measured. Linear mixed effects models were used to estimate the rates of changes during the treatment period. Results: There were 9 drug-related gastrointestinal adverse events which resolved spontaneously or with dose reduction (MTD 1800 mg bid). During the 24 week treatment period, mean BCVA significantly improved at 0.4 (95% CI 0.2–0.6, P < 0.001), 0.5 (95% CI 0.3–0.7, P < 0.001) and 0.2 (95% CI 0.02–0.4, P = 0.03) letters/month in cohorts 1, 2 and 3, respectively. There was no significant improvement in mean sensitivity (MS) over time in cohorts 1 and 2, but there was in cohort 3 (0.15 dB/month, 95%CI 0.04–0.26). There was no significant change in mean EZ width in any cohort. Conclusion: Oral NAC is safe and well-tolerated in patients with moderately advanced RP and may improve suboptimally functioning macular cones. A randomized, placebo-controlled trial is needed to determine if oral NAC can provide long term stabilization and/or improvement in visual function in patients with RP.
Authors
Peter A. Campochiaro, Mustafa Iftikhar, Gulnar Hafiz, Anam Akhlaq, Grace Tsai, Dagmar Wehling, Lili Lu, G. Michael Wall, Mandeep S. Singh, Xiangrong Kong
Abstract
Increases in the number of cell therapies in the preclinical and clinical phases have prompted the need for reliable and non-invasive assays to validate transplant function in clinical biomanufacturing. We developed a robust characterization methodology composed of quantitative bright-field absorbance microscopy (QBAM) and deep neural networks (DNNs) to non-invasively predict tissue function and cellular donor identity. The methodology was validated using clinical-grade induced pluripotent stem cell derived retinal pigment epithelial cells (iPSC-RPE). QBAM images of iPSC-RPE were used to train DNNs that predicted iPSC-RPE monolayer transepithelial resistance, predicted polarized vascular endothelial growth factor (VEGF) secretion, and matched iPSC-RPE monolayers to the stem cell donors. DNN predictions were supplemented with traditional machine learning algorithms that identified shape and texture features of single cells that were used to predict tissue function and iPSC donor identity. These results demonstrate non-invasive cell therapy characterization can be achieved with QBAM and machine learning.
Authors
Nicholas J. Schaub, Nathan A. Hotaling, Petre Manescu, Sarala Padi, Qin Wan, Ruchi Sharma, Aman George, Joe Chalfoun, Mylene Simon, Mohamed Ouladi, Carl G. Simon, Jr., Peter Bajcsy, Kapil Bharti
Abstract
Mutations in genes encoding components of the mitochondrial DNA (mtDNA) replication machinery cause mtDNA depletion syndromes (MDS), which associate ocular features with severe neurological syndromes. Here, we identified heterozygous missense mutations in SSBP1 in five unrelated families, leading to the R38Q and R107Q amino-acid changes in the mitochondrial single-stranded DNA-binding protein, a crucial protein involved in mtDNA replication. All affected individuals presented optic atrophy, associated with foveopathy in half of the cases. To uncover the structural features underlying SSBP1 mutations, we determined a new revised SSBP1 crystal structure. Structural analysis suggests that both mutations affect dimer interactions and presumably distort the DNA binding region. Using patient fibroblasts, we validated that the R38Q variant destabilizes SSBP1 dimer/tetramer formation, affects mtDNA replication and induces mtDNA depletion. Our study, showing that mutations in SSBP1 cause a novel form of dominant optic atrophy frequently accompanied with foveopathy, brings new insights into mtDNA maintenance disorders.
Authors
Camille Piro-Mégy, Emmanuelle Sarzi, Aleix Tarrés-Solé, Marie Péquignot, Fenna Hensen, Mélanie Quilès, Gaël Manes, Arka Chakraborty, Audrey Sénéchal, Béatrice Bocquet, Chantal Cazevieille, Agathe Roubertie, Agnès Müller, Majida Charif, David Goudenège, Guy Lenaers, Helmut Wilhelm, Ulrich Kellner, Nicole Weisschuh, Bernd Wissinger, Xavier Zanlonghi, Christian Hamel, Johannes N. Spelbrink, Maria Solà, Cécile Delettre
Abstract
Inherited optic neuropathies include complex phenotypes, mostly driven by mitochondrial dysfunction. We report an optic atrophy spectrum disorder, including retinal macular dystrophy and kidney insufficiency leading to transplantation, associated with mitochondrial DNA (mtDNA) depletion without accumulation of multiple deletions. By whole-exome sequencing, we identified mutations affecting the mitochondrial single strand binding protein (SSBP1) in four families with dominant and one with recessive inheritance. We show that SSBP1 mutations in patient-derived fibroblasts variably affect its amount and alter multimer formation, but not the binding to ssDNA. SSBP1 mutations impaired mtDNA, nucleoids and 7S-DNA amounts as well as mtDNA replication, impacting replisome machinery. The variable mtDNA depletion in cells reflected in severity of mitochondrial dysfunction, including respiratory efficiency, OXPHOS subunits and complexes amount and assembly. mtDNA depletion and cytochrome c oxidase-negative cells were found ex-vivo in biopsies of affected tissues, like kidney and skeletal muscle. Reduced efficiency of mtDNA replication was also reproduced in vitro, confirming the pathogenic mechanism. Furthermore, ssbp1 suppression in zebrafish induced signs of nephropathy and reduced optic nerve size, the latter phenotype complemented by wild-type mRNA but not by SSBP1 mutant transcripts. This previously unrecognized disease of mtDNA maintenance implicates SSBP1 mutations as cause of human pathology.
Authors
Valentina Del Dotto, Farid Ullah, Ivano Di Meo, Pamela Magini, Mirjana Gusic, Alessandra Maresca, Leonardo Caporali, Flavia Palombo, Francesca Tagliavini, Evan H. Baugh, Bertil Macao, Zsolt Szilagyi, Camille Péron, Margaret A. Gustafson, Kamal Khan, Chiara La Morgia, Piero Barboni, Michele Carbonelli, Maria Lucia Valentino, Rocco Liguori, Vandana Shashi, Jennifer A. Sullivan, Shashi Nagaraj, Mays El-Dairi, Alessandro Iannaccone, Ioana Cutcutache, Enrico Bertini, Rosalba Carrozzo, Francesco Emma, Francesca Diomedi-Camassei, Claudia Zanna, Martin Armstrong, Matthew J Page, Sylvia Boesch, Saskia B. Wortmann, Robert Kopajtich, Nicholas Stong, Wolfgang Sperl, Erica Davis, William C. Copeland, Marco Seri, Maria Falkenberg, Holger Prokisch, Nicholas Katsanis, Valeria Tiranti, Tommaso Pippucci, Valerio Carelli
Abstract
The majority of patients with diabetic macular edema (DME), the most common cause of vision loss in working-age Americans, do not respond adequately to current therapies targeting VEGFA. Here, we show that expression of angiopoietin-like 4 (ANGPTL4), a HIF-1–regulated gene product, is increased in the eyes of diabetic mice and patients with DME. We observed that ANGPTL4 and VEGF act synergistically to destabilize the retinal vascular barrier. Interestingly, while ANGPTL4 modestly enhanced tyrosine phosphorylation of VEGF receptor 2, promotion of vascular permeability by ANGPTL4 was independent of this receptor. Instead, we found that ANGPTL4 binds directly to neuropilin 1 (NRP1) and NRP2 on endothelial cells (ECs), leading to rapid activation of the RhoA/ROCK signaling pathway and breakdown of EC-EC junctions. Treatment with a soluble fragment of NRP1 (sNRP1) prevented ANGPTL4 from binding to NRP1 and blocked ANGPTL4-induced activation of RhoA as well as EC permeability in vitro and retinal vascular leakage in diabetic animals in vivo. In addition, sNRP1 reduced the stimulation of EC permeability by aqueous fluid from patients with DME. Collectively, these data identify the ANGPTL4/NRP/RhoA pathway as a therapeutic target for the treatment of DME.
Authors
Akrit Sodhi, Tao Ma, Deepak Menon, Monika Deshpande, Kathleen Jee, Aumreetam Dinabandhu, Jordan Vancel, Daoyuan Lu, Silvia Montaner
Abstract
Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) positively affect the outcome of retinopathy of prematurity (ROP). Given that DHA metabolism by cytochrome P450 and soluble epoxide hydrolase (sEH) enzymes affects retinal angiogenesis and vascular stability we investigated the role of sEH in a mouse model of ROP. In wild-type mice, hyperoxia elicited the tyrosine nitration and inhibition of the sEH and decreased generation of the DHA-derived diol 19,20-dihydroxydocosapentaenoic acid (DHDP). Correspondingly in a murine model of ROP, sEH–/– mice developed a larger central avascular zone and peripheral pathological vascular tuft formation than their wild-type littermates. Astrocytes were the cells most affected by sEH deletion and hyperoxia increased astrocyte apoptosis. In rescue experiments 19,20-DHDP prevented astrocyte loss by targeting the mitochondrial membrane to prevent the hyperoxia-induced dissociation of presenilin-1 (PS-1) and PS-1 associated protein (PSAP) to attenuate PARP1 activation and mitochondrial DNA damage. Therapeutic intravitreal administration of 19,20-DHDP not only suppressed astrocyte loss but also reduced pathological vascular tuft formation in sEH–/– mice. Our data indicate that sEH activity is required for mitochondrial integrity and retinal astrocyte survival in ROP. Moreover, 19,20-DHDP may be more effective than DHA as a nutritional supplement at preventing retinopathy in preterm infants.
Authors
Jiong Hu, Sofia Iris Bibli, Janina Wittig, Sven Zukunft, Jihong Lin, Hans-Peter Hammes, Rüdiger Popp, Ingrid Fleming
Abstract
There has been great progress in ocular gene therapy, but delivery of viral vectors to the retinal pigmented epithelium (RPE) and retina can be challenging. Subretinal injection, the preferred route of delivery for most applications, requires a surgical procedure that has risks. Herein we report a novel gene therapy delivery approach, suprachoroidal injection of AAV8 vectors, which is less invasive and could be done in an outpatient setting. Two weeks after suprachoroidal injection of AAV8.GFP in rats, GFP fluorescence covered 18.9% of RPE flat mounts and extended entirely around sagittal and transverse sections in RPE and photoreceptors. After 2 suprachoroidal injections of AAV8.GFP, GFP fluorescence covered 30.5% of RPE flat mounts. Similarly, widespread expression of GFP occurred in nonhuman primate and pig eyes after suprachoroidal injection of AAV8.GFP. Compared with subretinal injection in rats of RGX-314, an AAV8 vector expressing an anti-VEGF Fab, suprachoroidal injection of the same dose of RGX-314 resulted in similar expression of anti-VEGF Fab and similar suppression of VEGF-induced vascular leakage. Suprachoroidal AAV8 vector injection provides a noninvasive outpatient procedure to obtain widespread transgene expression in retina and RPE.
Authors
Kun Ding, Jikui Shen, Zibran Hafiz, Sean F. Hackett, Raquel Lima e Silva, Mahmood Khan, Valeria E. Lorenc, Daiqin Chen, Rishi Chadha, Minie Zhang, Sherri Van Everen, Nicholas Buss, Michele Fiscella, Olivier Danos, Peter A. Campochiaro
Copyright © 2020 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)