Binary Function of ARL3-GTP Revealed by Gene Knockouts.

UNC119 and PDEδ are lipid-binding proteins and are thought to form diffusible complexes with transducin-α and prenylated OS proteins, respectively, to mediate their trafficking to photoreceptor outer segments. Here, we investigate mechanisms of trafficking which are controlled by Arf-like protein 3 (Arl3), a small GTPase. The activity of ARL3 is regulated by a GEF (ARL13b) and a GAP (RP2). In a mouse germline knockout of RP2, ARL3-GTP is abundant as its intrinsic GTPase activity is extremely low. High levels of ARL3-GTP impair binding and trafficking of cargo to the outer segment. Germline knockout of ARL3 is embryonically lethal generating a syndromic ciliopathy-like phenotype. Retina- and rod-specific knockout of ARL3 allow to determine the precise mechanisms leading to photoreceptor degeneration. The knockouts reveal binary functions of ARL3-GTP as a key molecule in late-stage photoreceptor ciliogenesis and cargo displacement factor.

Molecular basis of CRX/DNA recognition and stoichiometry at the Ret4 response element.

Two retinal transcription factors, cone-rod homeobox (CRX) and neural retina leucine zipper (NRL), cooperate functionally and physically to control photoreceptor development and homeostasis. Mutations in CRX and NRL cause severe retinal diseases. Despite the roles of NRL and CRX, insight into their functions at the molecular level is lacking. Here, we have solved the crystal structure of the CRX homeodomain in complex with its cognate response element (Ret4) from the rhodopsin proximal promoter region. The structure reveals an unexpected 2:1 stoichiometry of CRX/Ret4 and unique orientation of CRX molecules on DNA, and it explains the mechanisms of pathogenic mutations in CRX. Mutations R41Q and E42K disrupt the CRX protein-protein contacts based on the structure and reduce the CRX/Ret4 binding stoichiometry, suggesting a novel disease mechanism. Furthermore, we show that NRL alters the stoichiometry and increases affinity of CRX binding at the rhodopsin promoter, which may enhance transcription of rod-specific genes and suppress transcription of cone-specific genes.

Differential Susceptibility of Retinal Neurons to the Loss of Mitochondrial Biogenesis Factor Nrf1.

The retina, the accessible part of the central nervous system, has served as a model system to study the relationship between energy utilization and metabolite supply. When the metabolite supply cannot match the energy demand, retinal neurons are at risk of death. As the powerhouse of eukaryotic cells, mitochondria play a pivotal role in generating ATP, produce precursors for macromolecules, maintain the redox homeostasis, and function as waste management centers for various types of metabolic intermediates. Mitochondrial dysfunction has been implicated in the pathologies of a number of degenerative retinal diseases. It is well known that photoreceptors are particularly vulnerable to mutations affecting mitochondrial function due to their high energy demand and susceptibility to oxidative stress. However, it is unclear how defective mitochondria affect other retinal neurons. Nuclear respiratory factor 1 (Nrf1) is the major transcriptional regulator of mitochondrial biogenesis, and loss of Nrf1 leads to defective mitochondria biogenesis and eventually cell death. Here, we investigated how different retinal neurons respond to the loss of Nrf1. We provide in vivo evidence that the disruption of Nrf1-mediated mitochondrial biogenesis results in a slow, progressive degeneration of all retinal cell types examined, although they present different sensitivity to the deletion of Nrf1, which implicates differential energy demand and utilization, as well as tolerance to mitochondria defects in different neuronal cells. Furthermore, transcriptome analysis on rod-specific Nrf1 deletion uncovered a previously unknown role of Nrf1 in maintaining genome stability.

Single-cell RNA sequencing: A new opportunity for retinal research.

Single-cell RNA sequencing (scRNA-seq) is a technology for single-cell transcriptome analysis that can be used to characterize complex dynamics of various retinal cell types. It provides deep scrutiny into the gene expression character of diverse cell types, lending insight into all the biological processes being carried out. The scRNA-seq is an alternative to regular RNA-seq, which does not achieve cellular heterogeneity. The retina, is a part of the central nervous system (CNS) and consists of six types of neurons and several types of glial cells. Studying retinal cell heterogeneity is important for understanding retinal diseases. Currently, scRNA-seq is employed to assess retina development and retinal disease pathogenesis and has improved our understanding of the relationship between the retina, its visual pathways, and the brain. Moreover, this technology provides new ideas on the sensitivity and molecular mechanisms of cell subtypes involved in retinal-related diseases. The application of scRNA-seq technology has given us a deeper understanding of the latest advancements and challenges in retinal development and diseases. We advocate scRNA-seq as one of the important tools for developing novel therapies for retinal diseases. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA in Disease and Development > RNA in Development RNA in Disease and Development > RNA in Disease.

Interplay between reactive oxygen species and autophagy in the course of age-related macular degeneration.

Pathological biomolecules such as lipofuscin, methylglyoxal-modified proteins (the major precursors of advanced glycationend products), misfolding protein deposits and dysfunctional mitochondria are source of oxidative stress and act as strong autophagic stimulators in age-related macular degeneration. Disturbed autophagy accelerates progression of the disease, since it leads to retinal cells' death and activates inflammation by the interplay with the NLRP3 inflammasome complex. Vascular dysfunction and hypoxia, as well as circulating autoantibodies against autophagy regulators (anti-S100A9, anti-ANXA5, and anti-HSPA8, A9 and B4) compromise an autophagy-mediated mechanism as well. Metformin, the autophagic stimulator, may act as a senostatic drug to inhibit the senescent phenotype in the age-related macular degeneration. PGC-1α , Sirt1 and AMPK represent new therapeutic targets for interventions in this disease.

Characterization and functional correlation of multiple imaging modalities with focal choroidal excavation.

BACKGROUND: To investigate the clinical manifestations and imaging features of near-infrared autofluorescence (NIA), infrared reflectance (IR), fundus autofluorescence (FAF), indocyanine green angiography (ICGA) and fluorescein angiography (FAG) in the detection of patients with focal choroidal excavation (FCE) identified by cross-sectional spectral-domain optical coherence tomography (SD-OCT). METHODS: This retrospective cross-sectional study included 12 eyes of 10 Taiwanese patients with FCE diagnosed by SD-OCT. The areas and depths of FCE in serial cross-sectional and en-face OCT were compared in different imaging modalities. NIA, IR, FAF, ICGA and FAG images were obtained. Best corrected visual acuity, subjective distortion area in the Amsler grid and history of maculopathies were also recorded. RESULTS: In areas where the choroid started to excavate as shown in SD-OCT, hypo-autofluorescence in NIA was noted. The area of hypo-fluorescence in NIA of all the FCE lesions showed good correlation with the size. The area of FCE was associated with complications such as choroidal neovascularization and central serous chorioretinopathy (p = 0.014, d.f = 1) and the volume (NIA area × Depth measured by SD-OCT × 1/3) was associated with subjective distortion strongly (p = 0.051, Spearman's correlation = 0.600). CONCLUSION: Among all image modalities, NIA was the most sensitive tool in area measurement of FCE and peripheral lesion detection. Also, the volume of FCE was associated with subjective distortion and the area was related to complications. Recording the area and volume of FCE could play an important role in monitoring complications.