Mutations in the PTEN-induced kinase 1 (PINK1) and Parkin RBR E3 ubiquitin-protein ligase (PARKIN) genes are associated with familial forms of Parkinson's disease (PD). PINK1, a protein kinase, and PARKIN, an E3 ubiquitin ligase, control the specific elimination of dysfunctional or superfluous mitochondria, thus fine-tuning mitochondrial network and preserving energy metabolism. PINK1 regulates PARKIN translocation in impaired mitochondria and drives their removal via selective autophagy, a process known as mitophagy. As knowledge obtained using different PINK1 and PARKIN transgenic animal models is being gathered, growing evidence supports the contribution of mitophagy impairment to several human pathologies, including PD and Alzheimer's diseases (AD). Therefore, therapeutic interventions aiming to modulate PINK1/PARKIN signalling might have the potential to treat these diseases. In this review, we will start by discussing how the interplay of PINK1 and PARKIN signalling helps mediate mitochondrial physiology. We will continue by debating the role of mitochondrial dysfunction in disorders such as amyotrophic lateral sclerosis, Alzheimer's, Huntington's and Parkinson's diseases, as well as eye diseases such as age-related macular degeneration and glaucoma, and the causative factors leading to PINK1/PARKIN-mediated neurodegeneration and neuroinflammation. Finally, we will discuss PINK1/PARKIN gene augmentation possibilities with a particular focus on AD, PD and glaucoma.

Early in vivo embryonic retinal development is a well-documented and evolutionary conserved process. The specification towards eye development is temporally controlled by consecutive activation or inhibition of multiple key signaling pathways, such as the Wnt and hedgehog signaling pathways. Recently, with the use of retinal organoids, researchers aim to manipulate these pathways to achieve better human representative models for retinal development and disease. To achieve this, a plethora of different small molecules and signaling factors have been used at various time points and concentrations in retinal organoid differentiations, with varying success. Additions differ from protocol to protocol, but their usefulness or efficiency has not yet been systematically reviewed. Interestingly, many of these small molecules affect the same and/or multiple pathways, leading to reduced reproducibility and high variability between studies. In this review, we make an inventory of the key signaling pathways involved in early retinogenesis and their effect on the development of the early retina in vitro. Further, we provide a comprehensive overview of the small molecules and signaling factors that are added to retinal organoid differentiation protocols, documenting the molecular and functional effects of these additions. Lastly, we comparatively evaluate several of these factors using our established retinal organoid methodology.

Iron accumulation causes cell death and disrupts tissue functions, which necessitates chelation therapy to reduce iron overload. However, clinical utilization of deferoxamine (DFO), an iron chelator, has been documented to give rise to systemic adverse effects, including ocular toxicity. This study provided the pathogenic and molecular basis for DFO-related retinopathy and identified retinal pigment epithelium (RPE) as the target tissue in DFO-related retinopathy. Our modeling demonstrated the susceptibility of RPE to DFO compared with the neuroretina. Intriguingly, we established upregulation of hypoxia inducible factor (HIF) 2α and mitochondrial deficit as the most prominent pathogenesis underlying the RPE atrophy. Moreover, suppressing hyperactivity of HIF2α and preserving mitochondrial dysfunction by α-ketoglutarate (AKG) protects the RPE against lesions both in vitro and in vivo. This supported our observation that AKG supplementation alleviates visual impairment in a patient undergoing DFO-chelation therapy. Overall, our study established a significant role of iron deficiency in initiating DFO-related RPE atrophy. Inhibiting HIF2α and rescuing mitochondrial function by AKG protect RPE cells and can potentially ameliorate patients' visual function.

CRB1 gene mutations can cause early- or late-onset retinitis pigmentosa, Leber congenital amaurosis, or maculopathy. Recapitulating human CRB1 phenotypes in animal models has proven challenging, necessitating the development of alternatives. We generated human induced pluripotent stem cell (iPSC)-derived retinal organoids of patients with retinitis pigmentosa caused by biallelic CRB1 mutations and evaluated them against autologous gene-corrected hiPSCs and hiPSCs from healthy individuals. Patient organoids show decreased levels of CRB1 and NOTCH1 expression at the retinal outer limiting membrane. Proximity ligation assays show that human CRB1 and NOTCH1 can interact via their extracellular domains. CRB1 patient organoids feature increased levels of WDFY1+ vesicles, fewer RAB11A+ recycling endosomes, decreased VPS35 retromer complex components, and more degradative endolysosomal compartments relative to isogenic control organoids. Taken together, our data demonstrate that patient-derived retinal organoids enable modeling of retinal degeneration and highlight the importance of CRB1 in early endosome maturation receptor recycling in the retina.

Mutations in rhodopsin (RHO) are the most common causes of autosomal dominant retinitis pigmentosa (adRP), accounting for 20% to 30% of all cases worldwide. However, the high degree of genetic heterogeneity makes development of effective therapies cumbersome. To provide a universal solution to RHO-related adRP, we devised a CRISPR-based, mutation-independent gene ablation and replacement (AR) compound therapy carried by a dual AAV2/8 system. Moreover, we developed a novel hRHOC110R/hRHOWT humanized mouse model to assess the AR treatment in vivo. Results show that this humanized RHO mouse model exhibits progressive rod-cone degeneration that phenocopies hRHOC110R/hRHOWT patients. In vivo transduction of AR AAV8 dual vectors remarkably ablates endogenous RHO expression and overexpresses exogenous WT hRHO. Furthermore, the administration of AR during adulthood significantly hampers photoreceptor degeneration both histologically and functionally for at least 6 months compared with sole gene replacement or surgical trauma control. This study demonstrates the effectiveness of AR treatment of adRP in the human genomic context while revealing the feasibility of its application for other autosomal dominant disorders.

Achromatopsia is characterized by amblyopia, photophobia, nystagmus, and color blindness. Previous animal models of achromatopsia have shown promising results using gene augmentation to restore cone function. However, the optimal therapeutic window to elicit recovery remains unknown. Here, we attempted two rounds of gene augmentation to generate recoverable mouse models of achromatopsia including a Cnga3 model with a knock-in stop cassette in intron 5 using Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) and targeted embryonic stem (ES) cells. This model demonstrated that only 20% of CNGA3 levels in homozygotes derived from target ES cells remained, as compared to normal CNGA3 levels. Despite the low percentage of remaining protein, the knock-in mouse model continued to generate normal cone phototransduction. Our results showed that a small amount of normal CNGA3 protein is sufficient to form "functional" CNG channels and achieve physiological demand for proper cone phototransduction. Thus, it can be concluded that mutating the Cnga3 locus to disrupt the functional tetrameric CNG channels may ultimately require more potent STOP cassettes to generate a reversible achromatopsia mouse model. Our data also possess implications for future CNGA3-associated achromatopsia clinical trials, whereby restoration of only 20% functional CNGA3 protein may be sufficient to form functional CNG channels and thus rescue cone response.

Macular degeneration (MD) is characterized by the progressive deterioration of the macula and represents one of the most prevalent causes of blindness worldwide. Abnormal intracellular accumulation of lipid droplets and pericellular deposits of lipid-rich material in the retinal pigment epithelium (RPE) called drusen are clinical hallmarks of different forms of MD including Doyne honeycomb retinal dystrophy (DHRD) and age-related MD (AMD). However, the appropriate molecular therapeutic target underlying these disorder phenotypes remains elusive. Here, we address this knowledge gap by comparing the proteomic profiles of induced pluripotent stem cell (iPSC)-derived RPEs (iRPE) from individuals with DHRD and their isogenic controls. Our analysis and follow-up studies elucidated the mechanism of lipid accumulation in DHRD iRPE cells. Specifically, we detected significant downregulation of carboxylesterase 1 (CES1), an enzyme that converts cholesteryl ester to free cholesterol, an indispensable process in cholesterol export. CES1 knockdown or overexpression of EFEMP1R345W, a variant of EGF-containing fibulin extracellular matrix protein 1 that is associated with DHRD and attenuated cholesterol efflux and led to lipid droplet accumulation. In iRPE cells, we also found that EFEMP1R345W has a hyper-inhibitory effect on epidermal growth factor receptor (EGFR) signaling when compared to EFEMP1WT and may suppress CES1 expression via the downregulation of transcription factor SP1. Taken together, these results highlight the homeostatic role of cholesterol efflux in iRPE cells and identify CES1 as a mediator of cholesterol efflux in MD.

Mutations in the Crumbs homologue 1 (CRB1) gene cause inherited retinal dystrophies, such as early-onset retinitis pigmentosa and Leber congenital amaurosis. A Brown Norway rat strain was reported with a spontaneous insertion-deletion (indel) mutation in exon 6 of Crb1. It has been reported that these Crb1 mutant rats show vascular abnormalities associated with retinal telangiectasia and possess an early-onset retinal degenerative phenotype with outer limiting membrane breaks and focal loss of retinal lamination at 2 months of age. Here, we further characterized the morphological phenotype of new-born and adult Crb1 mutant rats in comparison with age-matched Brown Norway rats without a mutation in Crb1. A significantly decreased retinal function and visual acuity was observed in Crb1 mutant rats at 1 and 3 months of age, respectively. Moreover, in control rats, the subcellular localization of canonical CRB1 was observed at the subapical region in Müller glial cells while CRB2 was observed at the subapical region in both photoreceptors and Müller glial cells by immuno-electron microscopy. CRB1 localization was lost in the Crb1 mutant rats, whereas CRB2 was still observed. In addition, we determined the tropism of subretinal or intravitreally administered AAV5-, AAV9- or AAV6-variant ShH10Y445F vectors in new-born control and Crb1 mutant rat retinas. We showed that subretinal injection of AAV5 and AAV9 at postnatal days 5 (P5) or 8 (P8) predominantly infected the retinal pigment epithelium (RPE) and photoreceptor cells; while intravitreal injection of ShH10Y445F at P5 or P8 resulted in efficient infection of mainly Müller glial cells. Using knowledge of the subcellular localization of CRB1 and the ability of ShH10Y445F to infect Müller glial cells, canonical hCRB1 and hCRB2 AAV-mediated gene therapy were explored in new-born Crb1 mutant rats. Enhanced retinal function after gene therapy delivery in the Crb1 rat was not observed. No timely rescue of the retinal phenotype was observed using retinal function and visual acuity, suggesting the need for earlier onset of expression of recombinant hCRB proteins in Müller glial cells to rescue the severe retinal phenotype in Crb1 mutant rats.

The developing vertebrate eye cup is partitioned into the neural retina (NR), the retinal pigmented epithelium (RPE), and the ciliary margin (CM). By single-cell analysis, we showed that fibroblast growth factor (FGF) signaling regulates the CM in its stem cell–like property of self-renewal, differentiation, and survival, which is balanced by an evolutionarily conserved Wnt signaling gradient. FGF promotes Wnt signaling in the CM by stabilizing β-catenin in a GSK3β-independent manner. While Wnt signaling converts the NR to either the CM or the RPE depending on FGF signaling, FGF transforms the RPE to the NR or CM dependent on Wnt activity. The default fate of the eye cup is the NR, but synergistic FGF and Wnt signaling promotes CM formation both in vivo and in human retinal organoid. Our study reveals that the vertebrate eye develops through phase transition determined by a combinatorial code of FGF and Wnt signaling.

Age-related macular degeneration (AMD) is the leading cause of severe vision loss and blindness in elderly people worldwide. The damage to the retinal pigment epithelium (RPE) triggered by oxidative stress plays a central role in the onset and progression of AMD and results from the excessive accumulation of reactive oxygen species (ROS) produced mainly by mitochondria. Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial molecular chaperone that contributes to the maintenance of mitochondrial integrity by decreasing the production and accumulation of ROS. The present study aimed to evaluate the presence and the role of TRAP1 in the RPE. Here, we report that TRAP1 is expressed in human adult retinal pigment epithelial cells and is located mainly in the mitochondria. Exposure of RPE cells to hydrogen peroxide decreases the levels of TRAP1. Furthermore, TRAP1 silencing increases intracellular ROS production and decreases mitochondrial respiratory capacity without affecting cell proliferation. Together, these findings offer novel insights into TRAP1 functions in RPE cells, opening possibilities to develop new treatment options for AMD.