Zebrafish and mouse models for studying deubiquitinating enzyme genes as candidates for retinal dystrophies

[eng] The retina consists of several structured layers of highly specialized neurons that capture and process light stimuli enabling vision. Such a fine architecture turns retinal differentiation into an extremely complex event that must be accurately regulated. The ubiquitin-proteasome system (UPS) is considered one of the most dynamic and versatile mechanisms of protein regulation in eukaryotic cells. As ubiquitination is reversible, deubiquitinating enzymes (DUBs) play a major regulatory role in the UPS. Despite the importance of proteostasis and the UPS in health and disease, a more comprehensive in-depth analysis of DUB expression and function on particular tissues or organs, such as the retina, is still missing. Combining expression quantification, mRNA localization assays and functional analyses in animal and cellular models, we analyzed the function of several DUB genes in the retina to identify DUBs that regulate important retinal cell mechanisms, explore their relevance in retinal function in health and disease, and finally, posit them as new potential candidate genes for retinal dystrophies. Taking into consideration our results in the expression levels and pattern of DUBs in the retina, we first selected USP45 to perform functional assays in animal models in order to define its role and function in the retina. By morpholino-knockdown of usp45 in zebrafish embryos, our results showed moderate to severe eye morphological defects, eye size reduction, small body size with small tail or without tail, and disruption in notochord formation. There is also defective lamination and formation of the retinal structures, with no distinguishable layers and smaller retinas. Overall, our results supported the relevance of USP45 in the normal development and formation of the vertebrate retina, and we proposed this gene as a good candidate for causing hereditary retinal dystrophies, as later confirmed by other authors in several families. We also selected ATXN3, a DUB gene that causes the dominant polyQ disease Spinocerebellar ataxia type 3 (SCA3), and we aimed to analyze its function in the retina. We showed that depletion of Atxn3 in zebrafish and mice caused retinal morphological and functional alterations with photoreceptor outer segment elongation, cone opsin mislocalization, and cone hyperexcitation upon light stimuli. A pool of ATXN3 resides at the basal body and axoneme of the photoreceptor cilium, where it controls the levels and recruitment of the regulatory proteins KEAP1 and HDAC6. Abrogation of Atxn3 expression causes delayed phagosome maturation in the retinal pigment epithelium. We propose that ATXN3 regulates two relevant biological processes in the retina, ciliogenesis and phagocytosis, by modulating microtubule polymerization and microtubule-dependent retrograde transport, and propose ATXN3 as a causative or modifier gene in retinal/macular dystrophies. We further aimed to explore whether the SCA3 humanized mouse model showed specific retinal phenotype traits. We showed that polyQ-expanded ATXN3 protein formed a high number of progressive pathogenic aggregates in the retinal layers of transgenic Atxn3Q84 mice, and caused a decrease in the number of cone photoreceptors. Optical coherence tomography revealed a general decrease in the thickness of the retinal layers whereas retinal electrophysiological analyses showed a strong decrease in photoreceptor response to light, thus supporting severe retinal dysfunction in Atxn3Q84 mice. Similar analyses in human patients detected a correlation of retinal alterations with the number of CAG repeats and the age of onset of SCA3 symptoms. We propose that retinal alterations detected by non-invasive eye examination and electroretinography tests in SCA3 patients could serve as a valuable early-onset symptom and a biological marker of disease progression. As a conclusion, our work posits several DUB genes as candidates for inherited retinal dystrophies, but further investigation is needed to dissect the function of DUBs in retinal cell differentiation, photoreceptor function, and retinal homeostasis

By the Tips of Your Cilia: Ciliogenesis in the Retina and the Ubiquitin-Proteasome System.

Primary cilia are microtubule-based sensory organelles that are involved in the organization of numerous key signals during development and in differentiated tissue homeostasis. In fact, the formation and resorption of cilia highly depends on the cell cycle phase in replicative cells, and the ubiquitin proteasome pathway (UPS) proteins, such as E3 ligases and deubiquitinating enzymes, promote microtubule assembly and disassembly by regulating the degradation/availability of ciliary regulatory proteins. Also, many differentiated tissues display cilia, and mutations in genes encoding ciliary proteins are associated with several human pathologies, named ciliopathies, which are multi-organ rare diseases. The retina is one of the organs most affected by ciliary gene mutations because photoreceptors are ciliated cells. Photoreception and phototransduction occur in the outer segment, a highly specialized neurosensory cilium. In this review, we focus on the function of UPS proteins in ciliogenesis and cilia length control in replicative cells and compare it with the scanty data on the identified UPS genes that cause syndromic and non-syndromic inherited retinal disorders. Clearly, further work using animal models and gene-edited mutants of ciliary genes in cells and organoids will widen the landscape of UPS involvement in ciliogenesis and cilia homeostasis

On the Wrong Track: Alterations of Ciliary Transport in Inherited Retinal Dystrophies

Ciliopathies are a group of heterogeneous inherited disorders associated with dysfunction of the cilium, a ubiquitous microtubule-based organelle involved in a broad range of cellular functions. Most ciliopathies are syndromic, since several organs whose cells produce a cilium, such as the retina, cochlea or kidney, are affected by mutations in ciliary-related genes. In the retina, photoreceptor cells present a highly specialized neurosensory cilium, the outer segment, stacked with membranous disks where photoreception and phototransduction occurs. The daily renewal of the more distal disks is a unique characteristic of photoreceptor outer segments, resulting in an elevated protein demand. All components necessary for outer segment formation, maintenance and function have to be transported from the photoreceptor inner segment, where synthesis occurs, to the cilium. Therefore, efficient transport of selected proteins is critical for photoreceptor ciliogenesis and function, and any alteration in either cargo delivery to the cilium or intraciliary trafficking compromises photoreceptor survival and leads to retinal degeneration. To date, mutations in more than 100 ciliary genes have been associated with retinal dystrophies, accounting for almost 25% of these inherited rare diseases. Interestingly, not all mutations in ciliary genes that cause retinal degeneration are also involved in pleiotropic pathologies in other ciliated organs. Depending on the mutation, the same gene can cause syndromic or non-syndromic retinopathies, thus emphasizing the highly refined specialization of the photoreceptor neurosensory cilia, and raising the possibility of photoreceptor-specific molecular mechanisms underlying common ciliary functions such as ciliary transport. In this review, we will focus on ciliary transport in photoreceptor cells and discuss the molecular complexity underpinning retinal ciliopathies, with a special emphasis on ciliary genes that, when mutated, cause either syndromic or non-syndromic retinal ciliopathies

In vitro gene delivery in retinal pigment epithelium cells by plasmid DNA-wrapped gold nanoparticles

Many rare diseases course with affectation of neurosensory organs. Among them, the neuroepithelial retina is very vulnerable due to constant light/oxidative stress, but it is also the most accessible and amenable to gene manipulation. Currently, gene addition therapies targeting retinal tissue (either photoreceptors or the retinal pigment epithelium), as a therapy for inherited retinal dystrophies, use adeno-associated virus (AAV)-based approaches. However, efficiency and safety of therapeutic strategies are relevant issues that are not always resolved in virus-based gene delivery and alternative methodologies should be explored. Based on our experience, we are currently assessing the novel physical properties at the nanoscale of inorganic gold nanoparticles for delivering genes to the retinal pigment epithelium (RPE) as a safe and efficient alternative approach. In this work, we present our preliminary results using DNA-wrapped gold nanoparticles (DNA-gold NPs) for successful in vitro gene delivery on human retinal pigment epithelium cell cultures, as a proof-of-principle to assess its feasibility for retina in vivo gene delivery. Our results show faster expression of a reporter gene in cells transfected with DNA-gold NPs compared to DNA-liposome complexes. Furthermore, we show that the DNA-gold NPs follow different uptake, internalization and intracellular vesicle trafficking routes compared to pristine NPs

Induced Pluripotent Stem Cells and Genome-Editing Tools in Determining Gene Function and Therapy for Inherited Retinal Disorders

Inherited retinal disorders (IRDs) affect millions of people worldwide and are a major cause of irreversible blindness. Therapies based on drugs, gene augmentation or transplantation approaches have been widely investigated and proposed. Among gene therapies for retinal degenerative diseases, the fast-evolving genome-editing CRISPR/Cas technology has emerged as a new potential treatment. The CRISPR/Cas system has been developed as a powerful genome-editing tool in ophthalmic studies and has been applied not only to gain proof of principle for gene therapies in vivo, but has also been extensively used in basic research to model diseases-in-a-dish. Indeed, the CRISPR/Cas technology has been exploited to genetically modify human induced pluripotent stem cells (iPSCs) to model retinal disorders in vitro, to test in vitro drugs and therapies and to provide a cell source for autologous transplantation. In this review, we will focus on the technological advances in iPSC-based cellular reprogramming and gene editing technologies to create human in vitro models that accurately recapitulate IRD mechanisms towards the development of treatments for retinal degenerative diseases

Expression of deubiquitinating enzyme genes in the developing mammal retina

Purpose: Genes involved in the development and differentiation of the mammalian retina are also associated with inherited retinal dystrophies (IRDs) and age-related macular degeneration. Transcriptional regulation of retinal cell differentiation has been addressed by genetic and transcriptomic studies. Much less is known about the posttranslational regulation of key regulatory proteins, although mutations in some genes involved in ubiquitination and proteostasis E3 ligases and deubiquitinating enzymes (DUBs) cause IRDs. This study intends to provide new data on DUB gene expression during different developmental stages of mouse and human fetal retinas. Methods: We performed a comprehensive transcriptomic analysis of all the annotated human and mouse DUBs (87) in the developing mouse retina at several embryonic and postnatal time points compared with the transcriptome of the fetal human retina. An integrated comparison of data from transcriptomics, reported chromatin immunoprecipitation sequencing (ChIP-seq) of CRX and NRL transcription factors, and the phenotypic retinal alterations in different animal models is presented. Results: Several DUB genes are differentially expressed during the development of the mouse and human retinas in relation to proliferation or differentiation stages. Some DUB genes appear to be distinctly expressed during the differentiation stages of rod and cone photoreceptor cells, and their expression is altered in mouse knockout models of relevant photoreceptor transcription factors. We complemented this RNA-sequencing (RNA-seq) analysis with other reported expression and phenotypic data to underscore the involvement of DUBs in cell fate decision and photoreceptor differentiation. Conclusions: The present results highlight a short list of potential DUB candidates for retinal disorders, which require further study

RPGeNet v2 .0: expanding the universe of retinal disease gene interactions network

RPGeNet offers researchers a user-friendly queriable tool to visualize the interactome network of visual disorder genes, thus enabling the identification of new potential causative genes and the assignment of novel candidates to specific retinal or cellular pathways. This can be highly relevant for clinical applications as retinal dystrophies affect 1:3000 people worldwide, and the causative genes are still unknown for 30% of the patients. RPGeNet is a refined interaction network interface that limits its skeleton network to the shortest paths between each and every known causative gene of inherited syndromic and non-syndromic retinal dystrophies. RPGeNet integrates interaction information from STRING, BioGRID and PPaxe, along with retina-specific expression data and associated genetic variants, over a Cytoscape.js web interface. For the new version, RPGeNet v2.0, the database engine was migrated to Neo4j graph database manager, which speeds up the initial queries and can handle whole interactome data for new ways to query the network. Further, user facilities have been introduced as the capability of saving and restoring a researcher customized network layout or as novel features to facilitate navigation and data projection on the network explorer interface. Responsiveness has been further improved by transferring some functionality to the client side

Expression atlas of the Deubiquitinating enzymes in the adult mouse retina, their evolutionary diversification and phenotypic roles

Ubiquitination is a relevant cell regulatory mechanism to determine protein fate and function. Most data has focused on the role of ubiquitin as a tag molecule to target substrates to proteasome degradation, and on its impact in the control of cell cycle, protein homeostasis and cancer. Only recently, systematic assays have pointed to the relevance of the ubiquitin pathway in the development and differentiation of tissues and organs, and its implication in hereditary diseases. Moreover, although the activity and composition of ubiquitin ligases has been largely addressed, the role of the deubiquitinating enzymes (DUBs) in specific tissues, such as the retina, remains mainly unknown. In this work, we undertook a systematic analysis of the transcriptional levels of DUB genes in the adult mouse retina by RT-qPCR and analyzed the expression pattern by in situ hybridization and fluorescent immunohistochemistry, thus providing a unique spatial reference map of retinal DUB expression. We also performed a systematic phylogenetic analysis to understand the origin and the presence/absence of DUB genes in the genomes of diverse animal taxa that represent most of the known animal diversity. The expression landscape obtained supports the potential subfunctionalization of paralogs in those families that expanded in vertebrates. Overall, our results constitute a reference framework for further characterization of the DUB roles in the retina and suggest new candidates for inherited retinal disorders

Increasing the genetic diagnosis yield in inherited retinal dystrophies: assigning pathogenicity to novel non-canonical splice site variants

Aims: We aimed to validate the pathogenicity of genetic variants identified in inherited retinal dystrophy (IRD) patients, which were located in non-canonical splice sites (NCSS). Methods: After next generation sequencing (NGS) analysis (target gene panels or whole exome sequencing (WES)), NCSS variants were prioritized according to in silico predictions. In vivo and in vitro functional tests were used to validate their pathogenicity. Results: Four novel NCSS variants have been identified. They are located in intron 33 and 34 of ABCA4 (c.4774-9G>A and c.4849-8C>G, respectively), intron 2 of POC1B (c.101-3T>G) and intron 3 of RP2 (c.884-14G>A). Functional analysis detected different aberrant splicing events, including intron retention, exon skipping and intronic nucleotide addition, whose molecular effect was either the disruption or the elongation of the open reading frame of the corresponding gene. Conclusions: Our data increase the genetic diagnostic yield of IRD patients and expand the landscape of pathogenic variants, which will have an impact on the genotype-phenotype correlations and allow patients to opt for the emerging gene and cell therapies

The Deubiquitinating Enzyme Ataxin-3 Regulates Ciliogenesis and Phagocytosis in the Retina

Expansion of a CAG repeat in ATXN3 causes the dominant polyglutamine disease spinocerebellar ataxia type 3 (SCA3), yet the physiological role of ATXN3 remains unclear. Here, we focus on unveiling the function of Ataxin-3 (ATXN3) in the retina, a neurological organ amenable to morphological and physiological studies. Depletion of Atxn3 in zebrafish and mice causes morphological and functional retinal alterations and, more precisely, photoreceptor cilium and outer segment elongation, cone opsin mislocalization, and cone hyperexcitation. ATXN3 localizes at the basal body and axoneme of the cilium, supporting its role in regulating ciliary length. Abrogation of Atxn3 expression causes decreased levels of the regulatory protein KEAP1 in the retina and delayed phagosome maturation in the retinal pigment epithelium. We propose that ATXN3 regulates two relevant biological processes in the retina, namely, ciliogenesis and phagocytosis, by modulating microtubule polymerization and microtubule-dependent retrograde transport, thus positing ATXN3 as a causative or modifier gene in retinal/macular dystrophies