Age-related dysregulation of the retinal transcriptome in African turquoise killifish.

Age-related vision loss caused by retinal neurodegenerative pathologies is becoming more prevalent in our ageing society. To understand the physiological and molecular impact of ageing on retinal homeostasis, we used the short-lived African turquoise killifish, a model known to naturally develop central nervous system (CNS) ageing hallmarks and vision loss. Bulk and single-cell RNA-sequencing (scRNAseq) of three age groups (6-, 12-, and 18-week-old) identified transcriptional ageing fingerprints in the killifish retina, unveiling pathways also identified in the aged brain, including oxidative stress, gliosis, and inflammageing. These findings were comparable to observations in the ageing mouse retina. Additionally, transcriptional changes in genes related to retinal diseases, such as glaucoma and age-related macular degeneration, were observed. The cellular heterogeneity in the killifish retina was characterized, confirming the presence of all typical vertebrate retinal cell types. Data integration from age-matched samples between the bulk and scRNAseq experiments revealed a loss of cellular specificity in gene expression upon ageing, suggesting potential disruption in transcriptional homeostasis. Differential expression analysis within the identified cell types highlighted the role of glial/immune cells as important stress regulators during ageing. Our work emphasizes the value of the fast-ageing killifish in elucidating molecular signatures in age-associated retinal disease and vision decline. This study contributes to the understanding of how age-related changes in molecular pathways may impact CNS health, providing insights that may inform future therapeutic strategies for age-related pathologies.

Age-related dysregulation of the retinal transcriptome in African turquoise killifish.

Age-related vision loss caused by retinal neurodegenerative pathologies is becoming more prevalent in our ageing society. To understand the physiological and molecular impact of ageing on retinal homeostasis, we used the short-lived African turquoise killifish, a model known to naturally develop central nervous system (CNS) ageing hallmarks and vision loss. Bulk and single-cell RNA-sequencing (scRNA-seq) of three age groups (6-, 12-, and 18-week-old) identified transcriptional ageing fingerprints in the killifish retina, unveiling pathways also identified in the aged brain, including oxidative stress, gliosis, and inflammageing. These findings were comparable to observations in ageing mouse retina. Additionally, transcriptional changes in genes related to retinal diseases, such as glaucoma and age-related macular degeneration, were observed. The cellular heterogeneity in the killifish retina was characterised, confirming the presence of all typical vertebrate retinal cell types. Data integration from age-matched samples between the bulk and scRNA-seq experiments revealed a loss of cellular specificity in gene expression upon ageing, suggesting potential disruption in transcriptional homeostasis. Differential expression analysis within the identified cell types highlighted the role of glial/immune cells as important stress regulators during ageing. Our work emphasises the value of the fast-ageing killifish in elucidating molecular signatures in age-associated retinal disease and vision decline. This study contributes to the understanding of how age-related changes in molecular pathways may impact CNS health, providing insights that may inform future therapeutic strategies for age-related pathologies.

Disrupting the Repeat Domain of Premelanosome Protein (PMEL) Produces Dysamyloidosis and Dystrophic Ocular Pigment Reflective of Pigmentary Glaucoma.

Pigmentary glaucoma has recently been associated with missense mutations in PMEL that are dominantly inherited and enriched in the protein's fascinating repeat domain. PMEL pathobiology is intriguing because PMEL forms functional amyloid in healthy eyes, and this PMEL amyloid acts to scaffold melanin deposition. This is an informative contradistinction to prominent neurodegenerative diseases where amyloid formation is neurotoxic and mutations cause a toxic gain of function called "amyloidosis". Preclinical animal models have failed to model this PMEL "dysamyloidosis" pathomechanism and instead cause recessively inherited ocular pigment defects via PMEL loss of function; they have not addressed the consequences of disrupting PMEL's repetitive region. Here, we use CRISPR to engineer a small in-frame mutation in the zebrafish homolog of PMEL that is predicted to subtly disrupt the protein's repetitive region. Homozygous mutant larvae displayed pigmentation phenotypes and altered eye morphogenesis similar to presumptive null larvae. Heterozygous mutants had disrupted eye morphogenesis and disrupted pigment deposition in their retinal melanosomes. The deficits in the pigment deposition of these young adult fish were not accompanied by any detectable glaucomatous changes in intraocular pressure or retinal morphology. Overall, the data provide important in vivo validation that subtle PMEL mutations can cause a dominantly inherited pigment pathology that aligns with the inheritance of pigmentary glaucoma patient pedigrees. These in vivo observations help to resolve controversy regarding the necessity of PMEL's repeat domain in pigmentation. The data foster an ongoing interest in an antithetical dysamyloidosis mechanism that, akin to the amyloidosis of devastating dementias, manifests as a slow progressive neurodegenerative disease.

The inner junction protein CFAP20 functions in motile and non-motile cilia and is critical for vision.

Motile and non-motile cilia are associated with mutually-exclusive genetic disorders. Motile cilia propel sperm or extracellular fluids, and their dysfunction causes primary ciliary dyskinesia. Non-motile cilia serve as sensory/signalling antennae on most cell types, and their disruption causes single-organ ciliopathies such as retinopathies or multi-system syndromes. CFAP20 is a ciliopathy candidate known to modulate motile cilia in unicellular eukaryotes. We demonstrate that in zebrafish, cfap20 is required for motile cilia function, and in C. elegans, CFAP-20 maintains the structural integrity of non-motile cilia inner junctions, influencing sensory-dependent signalling and development. Human patients and zebrafish with CFAP20 mutations both exhibit retinal dystrophy. Hence, CFAP20 functions within a structural/functional hub centered on the inner junction that is shared between motile and non-motile cilia, and is distinct from other ciliopathy-associated domains or macromolecular complexes. Our findings suggest an uncharacterised pathomechanism for retinal dystrophy, and potentially for motile and non-motile ciliopathies in general.

Zebrafish and inherited photoreceptor disease: Models and insights.

Photoreceptor dysfunctions and degenerative diseases are significant causes of vision loss in patients, with few effective treatments available. Targeted interventions to prevent or reverse photoreceptor-related vision loss are not possible without a thorough understanding of the underlying mechanism leading to disease, which is exceedingly difficult to accomplish in the human system. Cone diseases are particularly challenging to model, as some popular genetically modifiable model animals are nocturnal with a rod-dominant visual system and cones that have dissimilarities to human cones. As a result, cone diseases, which affect visual acuity, colour perception, and central vision in patients, are generally poorly understood in terms of pathology and mechanism. Zebrafish (Danio rerio) provide the opportunity to model photoreceptor diseases in a diurnal vertebrate with a cone-rich retina which develops many macular degeneration-like pathologies. Zebrafish undergo external development, allowing early-onset retinal diseases to be detected and studied, and many ophthalmic tools are available for zebrafish visual assessment during development and adulthood. There are numerous zebrafish models of photoreceptor disease, spanning the various types of photoreceptor disease (developmental, rod, cone, and mixed photoreceptor diseases) and genetic/molecular cause. In this review, we explore the features of zebrafish that make them uniquely poised to model cone diseases, summarize the established zebrafish models of inherited photoreceptor disease, and discuss how disease in these models compares to the human presentation, where applicable. Further, we highlight the contributions of these zebrafish models to our understanding of photoreceptor biology and disease, and discuss future directions for utilising and investigating these diverse models.

Whole exome sequencing reveals putatively novel associations in retinopathies and drusen formation.

Inherited retinal dystrophies (IRDs) affect 1 in 3000 individuals worldwide and are genetically heterogeneous, with over 270 identified genes and loci; however, there are still many identified disorders with no current genetic etiology. Whole exome sequencing (WES) provides a hypothesis-free first examination of IRD patients in either a clinical or research setting to identify the genetic cause of disease. We present a study of IRD in ten families from Alberta, Canada, through the lens of novel gene discovery. We identify the genetic etiology of IRDs in three of the families to be variants in known disease-associated genes, previously missed by clinical investigations. In addition, we identify two potentially novel associations: LRP1 in early-onset drusen formation and UBE2U in a multi-system condition presenting with retinoschisis, cataracts, learning disabilities, and developmental delay. We also describe interesting results in our unsolved cases to provide further information to other investigators of these blinding conditions.

A homozygous POC1B variant causes recessive cone-rod dystrophy.

Purpose: To report a case of initial cone dystrophy that advanced to a cone-rod dystrophy with homozygous variants in the POC1B gene.Methods: Retinal structure and visual function assessments were performed using fundoscopy, spectral-domain optical coherence tomography, full field electroretinography, semi-kinetic perimetry, and Ishihara plate testing. A DNA sample was collected and sent for diagnostic molecular genetic testing with a cone-rod dystrophy panel.Results: Clinical examination and electroretinography confirmed a clinical diagnosis of cone dystrophy. Molecular genetic testing revealed homozygous variants in POC1B (c.1355 G > A, p.(Arg452Gln)). Follow-up three years later showed progression to a cone-rod dystrophy.Conclusion: Our case describes an ophthalmological phenotype associated with a homozygous POC1B missense variant and provides clinical support for variant classification.

Zebrafish Models of Photoreceptor Dysfunction and Degeneration.

Zebrafish are an instrumental system for the generation of photoreceptor degeneration models, which can be utilized to determine underlying causes of photoreceptor dysfunction and death, and for the analysis of potential therapeutic compounds, as well as the characterization of regenerative responses. We review the wealth of information from existing zebrafish models of photoreceptor disease, specifically as they relate to currently accepted taxonomic classes of human rod and cone disease. We also highlight that rich, detailed information can be derived from studying photoreceptor development, structure, and function, including behavioural assessments and in vivo imaging of zebrafish. Zebrafish models are available for a diversity of photoreceptor diseases, including cone dystrophies, which are challenging to recapitulate in nocturnal mammalian systems. Newly discovered models of photoreceptor disease and drusenoid deposit formation may not only provide important insights into pathogenesis of disease, but also potential therapeutic approaches. Zebrafish have already shown their use in providing pre-clinical data prior to testing genetic therapies in clinical trials, such as antisense oligonucleotide therapy for Usher syndrome.

Progressive Photoreceptor Dysfunction and Age-Related Macular Degeneration-Like Features in rp1l1 Mutant Zebrafish.

Photoreceptor disease results in irreparable vision loss and blindness, which has a dramatic impact on quality of life. Pathogenic mutations in RP1L1 lead to photoreceptor degenerations such as occult macular dystrophy and retinitis pigmentosa. RP1L1 is a component of the photoreceptor axoneme, the backbone structure of the photoreceptor's light-sensing outer segment. We generated an rp1l1 zebrafish mutant using CRISPR/Cas9 genome editing. Mutant animals had progressive photoreceptor functional defects as determined by electrophysiological assessment. Optical coherence tomography showed gaps in the photoreceptor layer, disrupted photoreceptor mosaics, and thinner retinas. Mutant retinas had disorganized photoreceptor outer segments and lipid-rich subretinal drusenoid deposits between the photoreceptors and retinal pigment epithelium. Our mutant is a novel model of RP1L1-associated photoreceptor disease and the first zebrafish model of photoreceptor degeneration with reported subretinal drusenoid deposits, a feature of age-related macular degeneration.

RP1L1 and inherited photoreceptor disease: A review.

Retinitis pigmentosa 1-like 1 (RP1L1) is a component of the photoreceptor cilium. Pathogenic variants in RP1L1 lead to photoreceptor disease, suggesting an important role for RP1L1 in photoreceptor biology, though its exact function is unknown. To date, RP1L1 variants have been associated with occult macular dystrophy (a cone degeneration) and retinitis pigmentosa (a rod disease). Here, we summarize reported RP1L1-associated photoreceptor conditions and disease-causing RP1L1 variants. We also discuss novel associations between RP1L1 and additional photoreceptor conditions-besides occult macular dystrophy and retinitis pigmentosa-and fit RP1L1 into the broader scope of photoreceptor disease. RP1L1 appears to have a complex relationship with other photoreceptor proteins and may modify disease phenotype. Ultimately, further exploration of the relationship between RP1L1, other cilium components, and their impact on photoreceptor health is needed.

Connectivity of cone photoreceptor telodendria in the zebrafish retina.

The connectivity amongst photoreceptors is critical to their function, as it underpins lateral inhibition and effective translation of stimuli into neural signals. Despite much work characterizing second-order interneurons in the outer retina, the synapses directly connecting photoreceptors have often been overlooked. Telodendria are fine processes that connect photoreceptor pedicles. They have been observed in diverse vertebrate groups, yet their roles in vision remain speculative. Here, we visualize telodendria via fluorescent protein expression in photoreceptor subtypes. We characterized short wavelength cone telodendria in adult and larval zebrafish retina. Additionally, in the larval retina, we investigated rod telodendria and UV cone telodendria in mutant and transgenic retinas with altered complements of cone types. In the adult retina, telodendria are twice as abundant and branch almost twice as often on blue cones compared to UV cones. Pedicles of neighboring UV and blue cones typically converge into contiguous pairs, despite the regular spacing of their cell bodies. In contrast to adults, larval UV cone telodendria are more numerous (1.3 times) than blue cone telodendria. UV cone telodendria are not detectably affected by ablation of blue cones, and are reduced twofold in mutant larval retina with few UV cones. We thus saw no evidence that telodendria increase in number in the absence of their typical cellular neighbors. We also found that larval rod telodendria are less abundant than short wavelength cone telodendria. In summary, we describe the development and morphology of zebrafish photoreceptor synaptic connectivity toward appreciating the function of telodendria in visual signal processing.

Rapid Recovery of Visual Function Associated with Blue Cone Ablation in Zebrafish.

Hurdles in the treatment of retinal degeneration include managing the functional rewiring of surviving photoreceptors and integration of any newly added cells into the remaining second-order retinal neurons. Zebrafish are the premier genetic model for such questions, and we present two new transgenic lines allowing us to contrast vision loss and recovery following conditional ablation of specific cone types: UV or blue cones. The ablation of each cone type proved to be thorough (killing 80% of cells in each intended cone class), specific, and cell-autonomous. We assessed the loss and recovery of vision in larvae via the optomotor behavioural response (OMR). This visually mediated behaviour decreased to about 5% or 20% of control levels following ablation of UV or blue cones, respectively (P<0.05). We further assessed ocular photoreception by measuring the effects of UV light on body pigmentation, and observed that photoreceptor deficits and recovery occurred (p<0.01) with a timeline coincident to the OMR results. This corroborated and extended previous conclusions that UV cones are required photoreceptors for modulating body pigmentation, addressing assumptions that were unavoidable in previous experiments. Functional vision recovery following UV cone ablation was robust, as measured by both assays, returning to control levels within four days. In contrast, robust functional recovery following blue cone ablation was unexpectedly rapid, returning to normal levels within 24 hours after ablation. Ablation of cones led to increased proliferation in the retina, though the rapid recovery of vision following blue cone ablation was demonstrated to not be mediated by blue cone regeneration. Thus rapid visual recovery occurs following ablation of some, but not all, cone subtypes, suggesting an opportunity to contrast and dissect the sources and mechanisms of outer retinal recovery during cone photoreceptor death and regeneration.