Non-photopic and photopic visual cycles differentially regulate immediate, early and late-phases of cone photoreceptor-mediated vision

Cone photoreceptors in the retina enable vision over a wide range of light intensities. However, the processes enabling cone vision in bright light (i.e. photopic vision) are not adequately understood. Chromophore regeneration of cone photopigments may require the retinal pigment epithelium (RPE) and/or retinal Müller glia. In the RPE, isomerization of all-trans-retinyl esters (atRE) to 11-cis-retinol (11cROL) is mediated by the retinoid isomerohydrolase Rpe65. A putative alternative retinoid isomerase, dihydroceramide desaturase-1 (DES1), is expressed in RPE and Müller cells. The retinol-isomerase activities of Rpe65 and Des1 are inhibited by emixustat and fenretinide, respectively. Here, we tested the effects of these visual cycle inhibitors on immediate, early and late phases of cone photopic vision. In zebrafish larvae raised under cyclic light conditions, fenretinide impaired late cone photopic vision, whereas emixustat-treated zebrafish unexpectedly had normal vision. In contrast, emixustat-treated larvae raised under extensive dark-adaption displayed significantly attenuated immediate photopic vision concomitant with significantly reduced 11-cis-retinaldehyde (11cRAL). Following 30 minutes of light, early photopic vision recovered, despite 11cRAL levels remaining significantly reduced. Defects in immediate cone photopic vision were rescued in emixustat- or fenretinide-treated larvae following exogenous 9-cis-retinaldehyde (9cRAL) supplementation. Genetic knockout of Des1 (degs1) or retinaldehyde-binding protein 1b (rlbp1b) did not eliminate photopic vision in zebrafish. Our findings define molecular and temporal requirements of the non-photopic or photopic visual cycles for mediating vision in bright light.European Commission Horizon 2020Irish Research CouncilNational Institutes of Health12 month embargo limited to 6 months due to H2020 - A

Mitochondrial Calcium Uniporter (MCU) defciency reveals an alternate path for ­Ca2+ uptake in photoreceptor mitochondria

Rods and cones use intracellular Ca2+ to regulate many functions, including phototransduction and neurotransmission. The Mitochondrial Calcium Uniporter (MCU) complex is thought to be the primary pathway for Ca2+ entry into mitochondria in eukaryotes. We investigate the hypothesis that mitochondrial Ca2+ uptake via MCU influences phototransduction and energy metabolism in photoreceptors using a mcu-/- zebrafish and a rod photoreceptor-specific Mcu-/- mouse. Using genetically encoded Ca2+ sensors to directly examine Ca2+ uptake in zebrafish cone mitochondria, we found that loss of MCU reduces but does not eliminate mitochondrial Ca2+ uptake. Loss of MCU does not lead to photoreceptor degeneration, mildly affects mitochondrial metabolism, and does not alter physiological responses to light, even in the absence of the Na+/Ca2+, K+ exchanger. Our results reveal that MCU is dispensable for vertebrate photoreceptor function, consistent with its low expression and the presence of an alternative pathway for Ca2+ uptake into photoreceptor mitochondria

The ciliopathy gene cc2d2a controls zebrafish photoreceptor outer segment development through a role in Rab8-dependent vesicle trafficking

Ciliopathies are a genetically and phenotypically heterogeneous group of human developmental disorders whose root cause is the absence or dysfunction of primary cilia. Joubert syndrome is characterized by a distinctive hindbrain malformation variably associated with retinal dystrophy and cystic kidney disease. Mutations in CC2D2A are found in ∼10% of patients with Joubert syndrome. Here we describe the retinal phenotype of cc2d2a mutant zebrafish consisting of disorganized rod and cone photoreceptor outer segments resulting in abnormal visual function as measured by electroretinogram. Our analysis reveals trafficking defects in mutant photoreceptors affecting transmembrane outer segment proteins (opsins) and striking accumulation of vesicles, suggesting a role for Cc2d2a in vesicle trafficking and fusion. This is further supported by mislocalization of Rab8, a key regulator of opsin carrier vesicle trafficking, in cc2d2a mutant photoreceptors and by enhancement of the cc2d2a retinal and kidney phenotypes with partial knockdown of rab8. We demonstrate that Cc2d2a localizes to the connecting cilium in photoreceptors and to the transition zone in other ciliated cell types and that cilia are present in these cells in cc2d2a mutants, arguing against a primary function for Cc2d2a in ciliogenesis. Our data support a model where Cc2d2a, localized at the photoreceptor connecting cilium/transition zone, facilitates protein transport through a role in Rab8-dependent vesicle trafficking and fusion

Biochemical adaptations of the retina and retinal pigment epithelium support a metabolic ecosystem in the vertebrate eye

Here we report multiple lines of evidence for a comprehensive model of energy metabolism in the vertebrate eye. Metabolic flux, locations of key enzymes, and our finding that glucose enters mouse and zebrafish retinas mostly through photoreceptors support a conceptually new model for retinal metabolism. In this model, glucose from the choroidal blood passes through the retinal pigment epithelium to the retina where photoreceptors convert it to lactate. Photoreceptors then export the lactate as fuel for the retinal pigment epithelium and for neighboring Mu ̈ ller glial cells. We used human retinal epithelial cells to show that lactate can suppress consumption of glucose by the retinal pigment epithelium. Suppression of glucose consumption in the retinal pigment epithelium can increase the amount of glucose that reaches the retina. This framework for understanding metabolic relationships in the vertebrate retina provides new insights into the underlying causes of retinal disease and age-related vision loss

Disability Grant: a precarious lifeline for HIV/AIDS patients in South Africa

Background: In South Africa, HIV/AIDS remains a major public health problem. In a context of chronic unemployment and deepening poverty, social assistance through a Disability Grant (DG) is extended to adults with HIV/AIDS who are unable to work because of a mental or physical disability. Using a mixed methods approach, we consider 1) inequalities in access to the DG for patients on ART and 2) implications of DG access for on-going access to healthcare. Methods: Data were collected in exit interviews with 1200 ART patients in two rural and two urban health sub-districts in four different South African provinces. Additionally, 17 and 18 in-depth interviews were completed with patients on ART treatment and ART providers, respectively, in three of the four sites included in the quantitative phase. Results: Grant recipients were comparatively worse off than non-recipients in terms of employment (9.1 % vs. 29.9 %) and wealth (58.3 % in the poorest half vs. 45.8 %). After controlling for socioeconomic and demographic factors, site, treatment duration, adherence and concomitant TB treatment, the regression analyses showed that the employed were significantly less likely to receive the DG than the unemployed (

Zebrafish Class 1 Phosphatidylinositol Transfer Proteins: PITPβ and Double Cone Cell Outer Segment Integrity in Retina

Phosphatidylinositol transfer proteins (PITPs) in yeast coordinate lipid metabolism with the activities of specific membrane trafficking pathways. The structurally unrelated metazoan-specific PITPs (mPITPs), on the other hand, are an under-investigated class of proteins. It remains unclear what biological activities mPITPs discharge, and the mechanisms by which these proteins function are also not understood. The soluble class 1 mPITPs include the PITPα and PITPβ isoforms. Of these, the β-isoforms are particularly poorly characterized. Herein, we report the use of zebrafish as a model vertebrate for the study of class 1 mPITP biological function. Zebrafish express PITPα and PITPβ-isoforms (Pitpna and Pitpnb, respectively) and a novel PITPβ-like isoform (Pitpng). Pitpnb expression is particularly robust in double cone cells of the zebrafish retina. Morpholino-mediated protein knockdown experiments demonstrate Pitpnb activity is primarily required for biogenesis/maintenance of the double cone photoreceptor cell outer segments in the developing retina. By contrast, Pitpna activity is essential for successful navigation of early developmental programs. This study reports the initial description of the zebrafish class 1 mPITP family, and the first analysis of PITPβ function in a vertebrate

synaptojanin1 Is Required for Temporal Fidelity of Synaptic Transmission in Hair Cells

To faithfully encode mechanosensory information, auditory/vestibular hair cells utilize graded synaptic vesicle (SV) release at specialized ribbon synapses. The molecular basis of SV release and consequent recycling of membrane in hair cells has not been fully explored. Here, we report that comet, a gene identified in an ENU mutagenesis screen for zebrafish larvae with vestibular defects, encodes the lipid phosphatase Synaptojanin 1 (Synj1). Examination of mutant synj1 hair cells revealed basal blebbing near ribbons that was dependent on Cav1.3 calcium channel activity but not mechanotransduction. Synaptojanin has been previously implicated in SV recycling; therefore, we tested synaptic transmission at hair-cell synapses. Recordings of post-synaptic activity in synj1 mutants showed relatively normal spike rates when hair cells were mechanically stimulated for a short period of time at 20 Hz. In contrast, a sharp decline in the rate of firing occurred during prolonged stimulation at 20 Hz or stimulation at a higher frequency of 60 Hz. The decline in spike rate suggested that fewer vesicles were available for release. Consistent with this result, we observed that stimulated mutant hair cells had decreased numbers of tethered and reserve-pool vesicles in comparison to wild-type hair cells. Furthermore, stimulation at 60 Hz impaired phase locking of the postsynaptic activity to the mechanical stimulus. Following prolonged stimulation at 60 Hz, we also found that mutant synj1 hair cells displayed a striking delay in the recovery of spontaneous activity. Collectively, the data suggest that Synj1 is critical for retrieval of membrane in order to maintain the quantity, timing of fusion, and spontaneous release properties of SVs at hair-cell ribbon synapses

Genetics of photoreceptor degeneration and regeneration in zebrafish

Zebrafish are unique in that they provide a useful model system for studying two critically important problems in retinal neurobiology, the mechanisms responsible for triggering photoreceptor cell death and the innate stem cell–mediated regenerative response elicited by this death. In this review we highlight recent seminal findings in these two fields. We first focus on zebrafish as a model for studying photoreceptor degeneration. We summarize the genes currently known to cause photoreceptor degeneration, and we describe the phenotype of a few zebrafish mutants in detail, highlighting the usefulness of this model for studying this process. In the second section, we discuss the several different experimental paradigms that are available to study regeneration in the teleost retina. A model outlining the sequence of gene expression starting from the dedifferentiation of Müller glia to the formation of rod and cone precursors is presented