Histone methyltransferase Ezh2 coordinates mammalian axon regeneration via regulation of key regenerative pathways

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Abstract

Current treatments for neurodegenerative diseases and neural injuries face major challenges, primarily due to the diminished regenerative capacity of neurons in the mammalian central nervous system (CNS) as they mature. Here, we investigated the role of Ezh2, a histone methyltransferase, in regulating mammalian axon regeneration. We found that Ezh2 declined in the mouse nervous system during maturation but was upregulated in adult dorsal root ganglion neurons following peripheral nerve injury to facilitate spontaneous axon regeneration. In addition, overexpression of Ezh2 in retinal ganglion cells in the CNS promoted optic nerve regeneration via both histone methylation-dependent and -independent mechanisms. Further investigation revealed that Ezh2 fostered axon regeneration by orchestrating the transcriptional silencing of genes governing synaptic function and those inhibiting axon regeneration, while concurrently activating various factors that support axon regeneration. Notably, we demonstrated that GABA transporter 2 encoded by Slc6a13 acted downstream of Ezh2 to control axon regeneration. Overall, our study underscores the potential of modulating chromatin accessibility as a promising strategy for promoting CNS axon regeneration.

Authors

Xue-Wei Wang, Shu-Guang Yang, Ming-Wen Hu, Rui-Ying Wang, Chi Zhang, Anish R. Kosanam, Arinze J. Ochuba, Jing-Jing Jiang, Ximei Luo, Yun Guan, Jiang Qian, Chang-Mei Liu, Feng-Quan Zhou

Perilipin 2–positive mononuclear phagocytes accumulate in the diabetic retina and promote PPARγ-dependent vasodegeneration

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Abstract

Type 2 diabetes mellitus (T2DM), characterized by hyperglycemia and dyslipidemia, leads to nonproliferative diabetic retinopathy (NPDR). NPDR is associated with blood-retina barrier disruption, plasma exudates, microvascular degeneration, elevated inflammatory cytokine levels, and monocyte (Mo) infiltration. Whether and how the diabetes-associated changes in plasma lipid and carbohydrate levels modify Mo differentiation remains unknown. Here, we show that mononuclear phagocytes (MPs) in areas of vascular leakage in DR donor retinas expressed perilipin 2 (PLIN2), a marker of intracellular lipid load. Strong upregulation of PLIN2 was also observed when healthy donor Mos were treated with plasma from patients with T2DM or with palmitate concentrations typical of those found in T2DM plasma, but not under high-glucose conditions. PLIN2 expression correlated with the expression of other key genes involved in lipid metabolism (ACADVL, PDK4) and the DR biomarkers ANGPTL4 and CXCL8. Mechanistically, we show that lipid-exposed MPs induced capillary degeneration in ex vivo explants that was inhibited by pharmaceutical inhibition of PPARγ signaling. Our study reveals a mechanism linking dyslipidemia-induced MP polarization to the increased inflammatory cytokine levels and microvascular degeneration that characterize NPDR. This study provides comprehensive insights into the glycemia-independent activation of Mos in T2DM and identifies MP PPARγ as a target for inhibition of lipid-activated MPs in DR.

Authors

Guillaume Blot, Rémi Karadayi, Lauriane Przegralek, Thérèse-Marie Sartoris, Hugo Charles-Messance, Sébastien Augustin, Pierre Negrier, Frédéric Blond, Frida Paulina Muñiz-Ruvalcaba, David Rivera-de la Parra, Lucile Vignaud, Aude Couturier, José-Alain Sahel, Niyazi Acar, Aida Jimenez-Corona, Cécile Delarasse, Yonathan Garfias, Florian Sennlaub, Xavier Guillonneau

Nonviral base editing of KCNJ13 mutation preserves vision in a model of inherited retinal channelopathy

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Abstract

Clinical genome editing is emerging for rare disease treatment, but one of the major limitations is the targeting of CRISPR editors delivery. We delivered base editors to the retinal pigmented epithelium (RPE) in the mouse eye using silica nanocapsules (SNC) as a treatment for retinal degeneration. Leber Congenital Amaurosis (LCA16) is a rare pediatric blindness caused by point mutations in the KCNJ13 gene, a loss-of-function inwardly rectifying potassium channel (Kir7.1) in the RPE. SNC carrying adenine base editor (ABE8e) mRNA and single-guide RNA precisely and efficiently corrected KCNJ13W53X/W53X mutation. Editing in both patient fibroblasts (47%) and human-induced pluripotent stem cell-derived RPE (LCA16-iPSC-RPE) (17%) had a negligible off-target response. We detected functional Kir7.1 channels in the edited LCA16-iPSC-RPE. In the LCA16 mouse model (Kcnj13W53X/+∆R), RPE cells targeted SNC delivery of ABE8e mRNA preserved normal visual function measured by full-field electroretinogram (ERG). Moreover, multifocal ERG confirmed the topographic measure of electrical activity primarily originating from the edited retinal area at the injection site. Preserved retina structure, post-treatment, was established by Optical Coherence Tomography (OCT). This preclinical validation of targeted ion channel functional rescue, a challenge for pharmacological and genomic interventions, reinforced the effectiveness of nonviral genome editing therapy for rare inherited disorders.

Authors

Meha Kabra, Pawan K. Shahi, Yuyuan Wang, Divya Sinha, Allison Spillane, Gregory A. Newby, Shivani Saxena, Yao Tong, Yu Chang, Amr A Abdeen, Kimberly L. Edwards, Cole O. Theisen, David R. Liu, David M. Gamm, Shaoqin Gong, Krishanu Saha, Bikash R. Pattnaik

Targeting hypoxia-inducible factors with 32-134D safely and effectively treats diabetic eye disease in mice

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Abstract

Many patients with diabetic eye disease respond inadequately to anti-VEGF therapies, implicating additional vasoactive mediators in its pathogenesis. We demonstrate that levels of angiogenic proteins regulated by hypoxia-inducible factor (HIF)-1 and -2 (HIFs) remain elevated in diabetic eyes despite treatment with anti-VEGF therapy. Conversely, by inhibiting HIFs we normalized the expression of multiple vasoactive mediators in mouse models of diabetic eye disease. Accumulation of HIFs and HIF-regulated vasoactive mediators in hyperglycemic animals was observed in the absence of tissue hypoxia, suggesting that targeting HIFs may be an effective early treatment for diabetic retinopathy. However, while the HIF-inhibitor acriflavine prevented retinal vascular hyperpermeability in diabetic mice for several months following a single intraocular injection, accumulation of acriflavine in the retina resulted in retinal toxicity over time, raising concerns for its use in patients. Conversely, 32-134D, a recently developed HIF inhibitor structurally unrelated to acriflavine, was not toxic to the retina, yet effectively inhibited HIF accumulation and normalized HIF-regulated gene expression in mice and in human retinal organoids. Intraocular administration of 32-134D prevented retinal neovascularization and vascular hyperpermeability in mice. These results provide the foundation for clinical studies assessing 32-134D for the treatment of patients with diabetic eye disease.

Authors

Jing Zhang, Deepti Sharma, Aumreetam Dinabandhu, Jaron Sanchez, Brooks Applewhite, Kathleen Jee, Monika Deshpande, Miguel Flores-Bellver, Ming-Wen Hu, Chuanyu Guo, Shaima Salman, Yousang Hwang, Nicole M. Anders, Michelle A. Rudek, Jiang Qian, Valeria Canto-Soler, Gregg L. Semenza, Silvia Montaner, Akrit Sodhi

iPSC–derived retinal pigmented epithelial cells from patients with macular telangiectasia show decreased mitochondrial function

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Abstract

Patient-derived induced pluripotent stem cells (iPSCs) provide a powerful tool for identifying cellular and molecular mechanisms of disease. Macular telangiectasia type 2 (MacTel) is a rare, late-onset degenerative retinal disease with an extremely heterogeneous genetic architecture, lending itself to the use of iPSCs. Whole-exome sequencing screens and pedigree analyses have identified rare causative mutations that account for less than 5% of cases. Metabolomic surveys of patient populations and GWAS have linked MacTel to decreased circulating levels of serine and elevated levels of neurotoxic 1-deoxysphingolipids (1-dSLs). However, retina-specific, disease-contributing factors have yet to be identified. Here, we used iPSC-differentiated retinal pigmented epithelial (iRPE) cells derived from donors with or without MacTel to screen for novel cell-intrinsic pathological mechanisms. We show that MacTel iRPE cells mimicked the low serine levels observed in serum from patients with MacTel. Through RNA-Seq and gene set enrichment pathway analysis, we determined that MacTel iRPE cells are enriched in cellular stress pathways and dysregulation of central carbon metabolism. Using respirometry and mitochondrial stress testing, we functionally validated that MacTel iRPE cells had a reduction in mitochondrial function that was independent of defects in serine biosynthesis and 1-dSL accumulation. Thus, we identified phenotypes that may constitute alternative disease mechanisms beyond the known serine/sphingolipid pathway.

Authors

Kevin T. Eade, Brendan Robert E. Ansell, Sarah Giles, Regis Fallon, Sarah Harkins-Perry, Takayuki Nagasaki, Simone Tzaridis, Martina Wallace, Elizabeth A. Mills, Samaneh Farashi, Alec Johnson, Lydia Sauer, Barbara Hart, Elena D. Rubio, Melanie Bahlo, Christian Metallo, Rando Allikmets, Marin L. Gantner, Paul S. Bernstein, Martin Friedlander

Early-life peripheral infections reprogram retinal microglia and aggravate neovascular age-related macular degeneration in later life

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Abstract

Pathological neovascularization in age-related macular degeneration (nvAMD) drives the principal cause of blindness in the elderly. While there is a robust genetic association between genes of innate immunity and AMD, genome-to-phenome relationships are low, suggesting a critical contribution of environmental triggers of disease. Possible insight comes from the observation that a past history of infection with pathogens such as Chlamydia pneumoniae, or other systemic inflammation, can predispose to nvAMD in later life. Using a mouse model of nvAMD with prior C. pneumoniae infection, endotoxin exposure, and genetic ablation of distinct immune cell populations, we demonstrated that peripheral infections elicited epigenetic reprogramming that led to a persistent memory state in retinal CX3CR1+ mononuclear phagocytes (MNPs). The immune imprinting persisted long after the initial inflammation had subsided and ultimately exacerbated choroidal neovascularization in a model of nvAMD. Single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) identified activating transcription factor 3 (ATF3) as a central mediator of retina-resident MNP reprogramming following peripheral inflammation. ATF3 polarized MNPs toward a reparative phenotype biased toward production of proangiogenic factors in response to subsequent injury. Therefore, a past history of bacterial endotoxin–induced inflammation can lead to immunological reprograming within CNS-resident MNPs and aggravate pathological angiogenesis in the aging retina.

Authors

Masayuki Hata, Maki Hata, Elisabeth M.M.A. Andriessen, Rachel Juneau, Frédérique Pilon, Sergio Crespo-Garcia, Roberto Diaz-Marin, Vera Guber, Francois Binet, Frédérik Fournier, Manuel Buscarlet, Caroline Grou, Virginie Calderon, Emilie Heckel, Heather J. Melichar, Jean-Sebastien Joyal, Ariel M. Wilson, Przemyslaw Sapieha

Thrombospondin 1 missense alleles induce extracellular matrix protein aggregation and TM dysfunction in congenital glaucoma

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Abstract

Glaucoma is a highly heritable disease that is a leading cause of blindness worldwide. Here, we identified heterozygous thrombospondin 1 (THBS1) missense alleles altering p.Arg1034, a highly evolutionarily conserved amino acid, in 3 unrelated and ethnically diverse families affected by congenital glaucoma, a severe form of glaucoma affecting children. Thbs1R1034C-mutant mice had elevated intraocular pressure (IOP), reduced ocular fluid outflow, and retinal ganglion cell loss. Histology revealed an abundant, abnormal extracellular accumulation of THBS1 with abnormal morphology of juxtacanalicular trabecular meshwork (TM), an ocular tissue critical for aqueous fluid outflow. Functional characterization showed that the THBS1 missense alleles found in affected individuals destabilized the THBS1 C-terminus, causing protein misfolding and extracellular aggregation. Analysis using a range of amino acid substitutions at position R1034 showed that the extent of aggregation was correlated with the change in protein-folding free energy caused by variations in amino acid structure. Extracellular matrix (ECM) proteins, especially fibronectin, which bind to THBS1, also accumulated within THBS1 deposits. These results show that missense variants altering THBS1 p.Arg1034 can cause elevated IOP through a mechanism involving impaired TM fluid outflow in association with accumulation of aggregated THBS1 in the ECM of juxtacanalicular meshwork with altered morphology.

Authors

Haojie Fu, Owen M. Siggs, Lachlan S.W. Knight, Sandra E. Staffieri, Jonathan B. Ruddle, Amy E. Birsner, Edward Ryan Collantes, Jamie E. Craig, Janey L. Wiggs, Robert J. D’Amato

Aflibercept is more effective than bevacizumab at weaning neovascular age-related macular degeneration patients off therapy

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Abstract

BACKGROUND. Studies assessing the efficacy of therapies for neovascular age-related macular degeneration (nvAMD) have demonstrated that aflibercept may have a longer treatment interval than its lesser-expensive alternative, bevacizumab. However, whether this benefit justifies the additional cost of aflibercept remains under debate. We have recently reported that a “treat-and-extend-pause/monitor” (TEP/M) approach can be used to successfully wean 31% of nvAMD patients off anti-VEGF therapy. Here we examine whether the choice of therapy influences the outcomes of this approach. METHODS. In this retrospective analysis, 122 eyes of 106 patients with nvAMD underwent 3 consecutive monthly injections with either aflibercept (n=70) or bevacizumab (n=52) followed by a treat-and-extend protocol in which the decision to extend the interval between treatments was based on visual acuity, clinical exam, and the presence or absence of fluid on optical coherence tomography (OCT). Eyes that remained stable 12 weeks from their prior treatment were given a 6-week trial of holding further treatment, followed by quarterly monitoring. Treatment was resumed for worsening vision, clinical exam, or OCT findings. RESULTS. At the end of one year, eyes receiving bevacizumab had similar vision but required more injections (8.7 ±0.3 vs. 7.2 ±0.3) compared to aflibercept. However, eyes treated with aflibercept were almost 3-times more likely to be weaned off treatment (43% vs. 15%) compared to eyes treated with bevacizumab at the end of one year. CONCLUSIONS. These observations expose a previously unappreciated advantage of aflibercept over bevacizumab and have important clinical implications for the selection of therapy for patients with nvAMD.

Authors

Xuan Cao, Jaron Castillo Sanchez, Tapan P. Patel, Zhiyong Yang, Chuanyu Guo, Danyal Malik, Anuoluwapo Sopeyin, Silvia Montaner, Akrit Sodhi

METTL23 mutation alters histone H3R17 methylation in normal-tension glaucoma

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Abstract

Normal-tension glaucoma (NTG) is a heterogeneous disease characterized by retinal ganglion cell (RGC) death leading to cupping of the optic nerve head and visual field loss at normal intraocular pressure (IOP). The pathogenesis of NTG remains unclear. Here, we described a single nucleotide mutation in exon 2 of the methyltransferase like 23 (METTL23) gene identified in a three-generation Japanese NTG family. This mutation caused METTL23 mRNA aberrant splicing, which abolished normal protein production and altered subcellular localization. Mettl23 knock-in (Mettl23+/G & Mettl23G/G) and knockout (Mettl23+/- & Mettl23-/-) mice developed a glaucoma phenotype without elevated IOP. METTL23 is a histone arginine methyltransferase expressed in murine and macaque RGCs. However, the novel mutation reduced Mettl23 expression in RGCs of Mettl23G/G mice, which recapitulated both clinical and biological phenotypes. Moreover, our findings demonstrated that Mettl23 catalyzed the dimethylation of H3R17 in the retina, and was required for the transcription of pS2, an estrogen receptor α target gene that was critical to RGC homeostasis through the negative regulation of NF-κB-mediated TNF-α/IL-1β feedback. These findings suggest an etiologic role of METTL23 in NTG with tissue-specific pathology.

Authors

Yang Pan, Akiko Suga, Itaru Kimura, Chojiro Kimura, Yuriko Minegishi, Mao Nakayama, Kazutoshi Yoshitake, Daisuke Iejima, Naoko Minematsu, Megumi Yamamoto, Fumihiko Mabuchi, Mitsuko Takamoto, Yukihiro Shiga, Makoto Araie, Kenji Kashiwagi, Makoto Aihara, Toru Nakazawa, Takeshi Iwata

A deep-learning system predicts glaucoma incidence and progression using retinal photographs

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Abstract

Background Deep learning has been widely used for glaucoma diagnosis. However, there is no clinically validated algorithm for glaucoma incidence and progression prediction. This study aims to develop a clinically feasible deep-learning system for predicting and stratifying the risk of glaucoma onset and progression based on color fundus photographs (CFPs), with clinical validation of performance in external population cohorts.Methods We established data sets of CFPs and visual fields collected from longitudinal cohorts. The mean follow-up duration was 3 to 5 years across the data sets. Artificial intelligence (AI) models were developed to predict future glaucoma incidence and progression based on the CFPs of 17,497 eyes in 9346 patients. The area under the receiver operating characteristic (AUROC) curve, sensitivity, and specificity of the AI models were calculated with reference to the labels provided by experienced ophthalmologists. Incidence and progression of glaucoma were determined based on longitudinal CFP images or visual fields, respectively.Results The AI model to predict glaucoma incidence achieved an AUROC of 0.90 (0.81–0.99) in the validation set and demonstrated good generalizability, with AUROCs of 0.89 (0.83–0.95) and 0.88 (0.79–0.97) in external test sets 1 and 2, respectively. The AI model to predict glaucoma progression achieved an AUROC of 0.91 (0.88–0.94) in the validation set, and also demonstrated outstanding predictive performance with AUROCs of 0.87 (0.81–0.92) and 0.88 (0.83–0.94) in external test sets 1 and 2, respectively.Conclusion Our study demonstrates the feasibility of deep-learning algorithms in the early detection and prediction of glaucoma progression.FUNDING National Natural Science Foundation of China (NSFC); the High-level Hospital Construction Project, Zhongshan Ophthalmic Center, Sun Yat-sen University; the Science and Technology Program of Guangzhou, China (2021), the Science and Technology Development Fund (FDCT) of Macau, and FDCT-NSFC.

Authors

Fei Li, Yuandong Su, Fengbin Lin, Zhihuan Li, Yunhe Song, Sheng Nie, Jie Xu, Linjiang Chen, Shiyan Chen, Hao Li, Kanmin Xue, Huixin Che, Zhengui Chen, Bin Yang, Huiying Zhang, Ming Ge, Weihui Zhong, Chunman Yang, Lina Chen, Fanyin Wang, Yunqin Jia, Wanlin Li, Yuqing Wu, Yingjie Li, Yuanxu Gao, Yong Zhou, Kang Zhang, Xiulan Zhang