Depletion of Retinal Dopaminergic Activity in a Mouse Model of Rod Dysfunction Exacerbates Experimental Autoimmune Uveoretinitis: A Role for the Gateway Reflex

The gateway reflex is a mechanism by which neural inputs regulate chemokine expression at endothelial cell barriers, thereby establishing gateways for the invasion of autoreactive T cells into barrier-protected tissues. In this study, we hypothesized that rod photoreceptor dysfunction causes remodeling of retinal neural activity, which influences the blood–retinal barrier and the development of retinal inflammation. We evaluated this hypothesis using Gnat1rd17 mice, a model of night blindness with late-onset rod-cone dystrophy, and experimental autoimmune uveoretinitis (EAU). Retinal remodeling and its effect on EAU development were investigated by transcriptome profiling, target identification, and functional validation. We showed that Gnat1rd17 mice primarily underwent alterations in their retinal dopaminergic system, triggering the development of an exacerbated EAU, which was counteracted by dopamine replacement with L-DOPA administered either systemically or locally. Remarkably, dopamine acted on retinal endothelial cells to inhibit NF-κB and STAT3 activity and the expression of downstream target genes such as chemokines involved in T cell recruitment. These results suggest that rod-mediated dopamine release functions in a gateway reflex manner in the homeostatic control of immune cell entry into the retina, and the loss of retinal dopaminergic activity in conditions associated with rod dysfunction increases the susceptibility to autoimmune uveitis.

Determinants shaping the nanoscale architecture of the mouse rod outer segment

The unique membrane organization of the rod outer segment (ROS), the specialized sensory cilium of rod photoreceptor cells, provides the foundation for phototransduction, the initial step in vision. ROS architecture is characterized by a stack of identically shaped and tightly packed membrane disks loaded with the visual receptor rhodopsin. A wide range of genetic aberrations have been reported to compromise ROS ultrastructure, impairing photoreceptor viability and function. Yet, the structural basis giving rise to the remarkably precise arrangement of ROS membrane stacks and the molecular mechanisms underlying genetically inherited diseases remain elusive. Here, cryo-electron tomography (cryo-ET) performed on native ROS at molecular resolution provides insights into key structural determinants of ROS membrane architecture. Our data confirm the existence of two previously observed molecular connectors/spacers which likely contribute to the nanometer-scale precise stacking of the ROS disks. We further provide evidence that the extreme radius of curvature at the disk rims is enforced by a continuous supramolecular assembly composed of peripherin-2 (PRPH2) and rod outer segment membrane protein 1 (ROM1) oligomers. We suggest that together these molecular assemblies constitute the structural basis of the highly specialized ROS functional architecture. Our Cryo-ET data provide novel quantitative and structural information on the molecular architecture in ROS and substantiate previous results on proposed mechanisms underlying pathologies of certain PRPH2 mutations leading to blindness.

Retinoic acid signaling mediates peripheral cone photoreceptor survival in a mouse model of retina degeneration

Retinitis Pigmentosa (RP) is a wide array of progressive, debilitating visual disorders caused by mutations in a diverse set of genes. In both human patients and mouse models of RP, rod photoreceptor dysfunction leads to loss of night vision, and is followed by secondary cone photoreceptor dysfunction and degeneration, leading to loss of daylight color vision. A strategy to prevent secondary cone death could provide a generalized RP therapy to preserve daylight color vision regardless of the underlying mutation. In mouse models of RP, cones in the far peripheral retina survive long-term, despite complete rod loss. The mechanism for such peripheral cone survival had not been explored. Here, we found that active retinoic acid (RA) signaling in peripheral Muller glia is both sufficient and necessary for the extended cone survival. RA depletion by conditional knockout of RA synthesis enzymes, or overexpression of an RA degradation enzyme, abrogated peripheral cone survival. Conversely, constitutive activation of RA signaling in the central retina promoted long-term cone survival. These results indicate that RA signaling mediates the prolonged peripheral cone survival in the rd1 mouse model of retinal degeneration, and provide a basis for a generic strategy for cone survival in the many diseases that lead to loss of cone-mediated vision.

Age-related decline in function of ON and OFF visual pathways

PurposeSimple psychophysical paradigm is available as a digital application in iOS devices such as iPad to measure the function of ON and OFF visual pathways. However, an age-matched normative database is not readily available. The purpose of the study is to evaluate the response of ON and OFF visual pathways as a function of age.Methods158 normal healthy adults (84 males and 74 females) whose age ranged 18-80 years participated in the study. None of them had any ocular disease (except cataract of grade II or less) and visual acuity of ≤ 20/25. Monocular testing (only one eye) was performed on the ‘EyeSpeed’ application on an iPad at 40cm distance. The targets ranged between 1 to 3 light or dark squares presented randomly in a noise background and participants responded by indicating the number of squares by touching the screen as fast as possible. The main outcome variables are reaction time, accuracy and performance index (1 / speed * accuracy).ResultsThe median reaction time was shorter (Median (IQR): 1.53s (0.49) [dark] Vs 1.76s (0.58) [light], p < 0.001) and accuracy was higher (97.21% (3.30) [dark] Vs 95.15% (5.10) [light], p < 0.001) for dark targets than the light targets. Performance index and reaction time for both target types significantly correlated with age (ρ = −0.41 to −0.43; p < 0.001).ConclusionsThis normative database will be useful to quantify disease-specific defects. More importantly, the ON pathway function can potentially serve as a surrogate for rod photoreceptor function.

Disturbed retinoid metabolism upon loss of rlbp1a impairs cone function and leads to subretinal lipid deposits and photoreceptor degeneration in the zebrafish retina

The RLBP1 gene encodes the 36 kDa cellular retinaldehyde binding protein, CRALBP, a soluble retinoid carrier, in the visual cycle of the eyes. Mutations in RLBP1 are associated with recessively inherited clinical phenotypes, including Bothnia dystrophy, retinitis pigmentosa, retinitis punctata albescens, fundus albipunctatus, and Newfoundland rod-cone dystrophy. However, the etiology of these retinal disorders is not well understood. Here, we generated homologous zebrafish models to bridge this knowledge gap. Duplication of the rlbp1 gene in zebrafish and cell-specific expression of the paralogs rlbp1a in the retinal pigment epithelium and rlbp1b in Müller glial cells allowed us to create intrinsically cell type-specific knockout fish lines. Using rlbp1a and rlbp1b single and double mutants, we investigated the pathological effects on visual function. Our analyses revealed that rlbp1a was essential for cone photoreceptor function and chromophore metabolism in the fish eyes. rlbp1a mutant fish displayed reduced chromophore levels and attenuated cone photoreceptor responses to light stimuli. They accumulated 11-cis and all-trans-retinyl esters which displayed as enlarged lipid droplets in the RPE reminiscent of the subretinal yellow-white lesions in patients with RLBP1 mutations. During aging, these fish developed retinal thinning and cone and rod photoreceptor dystrophy. In contrast, rlbp1b mutants did not display impaired vision. The double mutant essentially replicated the phenotype of the rlbp1a single mutant. Together, our study showed that the rlbp1a zebrafish mutant recapitulated many features of human blinding diseases caused by RLBP1 mutations and provided novel insights into the pathways for chromophore regeneration of cone photoreceptors.

Usher syndrome Type 1-associated gene, pcdh15b, is required for photoreceptor structural integrity in zebrafish

Blindness associated with Usher Syndrome Type 1 (USH1) is typically characterized as rod photoreceptor degeneration, followed by secondary loss of cones. The mechanisms leading to blindness are unknown since most genetic mouse models only recapitulate auditory defects. We generated zebrafish mutants for one of the USH1 proteins, protocadherin-15b (pcdh15b), a putative cell adhesion molecule. Zebrafish pcdh15 is expressed exclusively in photoreceptors within calyceal processes (CPs), at the base of the outer segment (OS), and within the synapse. In our mutants, rod and cone photoreceptor integrity is compromised with early and progressively worsening abnormal OS disc growth and detachment, in part due to weakening CP contacts. These effects were attenuated or exacerbated by growth in dark and bright light conditions, respectively. We also describe novel evidence for structural defects in synapses of pcdh15 mutant photoreceptors. Cell death does not accompany these defects at early stages, suggesting that photoreceptor structural defects, rather than overt cell loss, may underlie vision deficits. Thus, we present the first genetic animal model of a pcdh15-associated retinopathy that can be used to understand the etiology of blindness in USH1.

Dual-Cell Patch-Clamp Recording Revealed a Mechanism for a Ribbon Synapse to Process Both Digital and Analog Inputs and Outputs

A chemical synapse is either an action potential (AP) synapse or a graded potential (GP) synapse but not both. This study investigated how signals passed the glutamatergic synapse between the rod photoreceptor and its postsynaptic hyperpolarizing bipolar cells (HBCs) and light responses of retinal neurons with dual-cell and single-cell patch-clamp recording techniques. The results showed that scotopic lights evoked GPs in rods, whose depolarizing Phase 3 associated with the light offset also evoked APs of a duration of 241.8 ms and a slope of 4.5 mV/ms. The depolarization speed of Phase 3 (Speed) was 0.0001–0.0111 mV/ms and 0.103–0.469 mV/ms for rods and cones, respectively. On pairs of recorded rods and HBCs, only the depolarizing limbs of square waves applied to rods evoked clear currents in HBCs which reversed at −6.1 mV, indicating cation currents. We further used stimuli that simulated the rod light response to stimulate rods and recorded the rod-evoked excitatory current (rdEPSC) in HBCs. The normalized amplitude (R/Rmax), delay, and rising slope of rdEPSCs were differentially exponentially correlated with the Speed (all p &lt; 0.001). For the Speed &lt; 0.1 mV/ms, R/Rmax grew while the delay and duration reduced slowly; for the Speed between 0.1 and 0.4 mV/ms, R/Rmax grew fast while the delay and duration dramatically decreased; for the Speed &gt; 0.4 mV/ms, R/Rmax reached the plateau, while the delay and duration approached the minimum, resembling digital signals. The rdEPSC peak was left-shifted and much faster than currents in rods. The scotopic-light-offset-associated major and minor cation currents in retinal ganglion cells (RGCs), the gigantic excitatory transient currents (GTECs) in HBCs, and APs and Phase 3 in rods showed comparable light-intensity-related locations. The data demonstrate that the rod-HBC synapse is a perfect synapse that can differentially decode and code analog and digital signals to process enormously varied rod and coupled-cone inputs.

Subcellular localization of mutant P23H rhodopsin in an RFP fusion knockin mouse model of retinitis pigmentosa

The P23H mutation in rhodopsin (Rho), the visual pigment protein in rod photoreceptor neurons, is the most common genetic cause of autosomal dominant retinitis pigmentosa (adRP), a retinal disease that causes blindness. Despite multiple studies in animal models, the subcellular details of the fate of misfolded mutant Rho in rod photoreceptors have not been completely defined. We generated a new mouse model of adRP, in which the P23H-Rho mutant allele is fused to Tag-RFP-T (P23HhRhoRFP). In heterozygotes, outer segments formed, and WT rhodopsin was properly localized there, but mutant P23H-Rho protein was specifically mislocalized in the inner segments of rods. Despite this cellular phenotype, the P23HhRhoRFP heterozygous mice exhibited only slowly progressing retinal degeneration; in ERG recordings, scotopic a-wave amplitudes were reduced by 24% and 26% at 30 days and 90 days respectively, and the corresponding scotopic b-waves by 18% and 24%. Outer nuclear layer thickness was still 80% of WT at 90 days, but at 364 days had declined to 40% of WT. Transmission electron microscopy revealed greatly expanded membrane lamellae in the inner segment, and by fluorescence imaging, we determined that the mislocalized P23HhRhoRFP was contained in greatly expanded endoplasmic reticulum (ER) membranes. TUNEL staining revealed a slow pace of cell death involving chromosomal endonucleolytic degradation. Quantification of mRNA for markers of ER stress and the unfolded protein response revealed little or no increases in levels of messages encoding the proteins BiP, CHOP, ATF6, XBP1, PERK, Eif2α and Derlin-1, but a decreased level of total Rhodopsin (mouse + human) mRNA levels. The decline in the rate of cell death after an initial burst suggests that P23HhRhoRFP mutant rods undergo an adaptative process that prolongs survival despite gross P23HhRhoRFP protein accumulation in the ER. Because of its slowly progressing nature, and easy visualization of the mutant protein, the P23H-Rho-RFP mouse may represent a useful tool for the future study of the pathology and treatment of P23H-Rho and adRP.

Early Changes in Exo- and Endocytosis in the EAE Mouse Model of Multiple Sclerosis Correlate with Decreased Synaptic Ribbon Size and Reduced Ribbon-Associated Vesicle Pools in Rod Photoreceptor Synapses

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system that finally leads to demyelination. Demyelinating optic neuritis is a frequent symptom in MS. Recent studies also revealed synapse dysfunctions in MS patients and MS mouse models. We previously reported alterations of photoreceptor ribbon synapses in the experimental auto-immune encephalomyelitis (EAE) mouse model of MS. In the present study, we found that the previously observed decreased imunosignals of photoreceptor ribbons in early EAE resulted from a decrease in synaptic ribbon size, whereas the number/density of ribbons in photoreceptor synapses remained unchanged. Smaller photoreceptor ribbons are associated with fewer docked and ribbon-associated vesicles. At a functional level, depolarization-evoked exocytosis as monitored by optical recording was diminished even as early as on day 7 after EAE induction. Moreover compensatory, post-depolarization endocytosis was decreased. Decreased post-depolarization endocytosis in early EAE correlated with diminished synaptic enrichment of dynamin3. In contrast, basal endocytosis in photoreceptor synapses of resting non-depolarized retinal slices was increased in early EAE. Increased basal endocytosis correlated with increased de-phosphorylation of dynamin1. Thus, multiple endocytic pathways in photoreceptor synapse are differentially affected in early EAE and likely contribute to the observed synapse pathology in early EAE.

Characterization of Scotopic and Mesopic Rod Signaling Pathways in Dogs Using the On-Off Electroretinogram

Background: The On-Off, or long flash, full field electroretinogram (ERG) separates retinal responses to flash onset and offset. Depending on degree of dark-adaptation and stimulus strength the On and Off ERG can be shaped by rod and cone photoreceptors and postreceptoral cells, including ON and OFF bipolar cells. Interspecies differences have been shown, with predominantly positive Off-response in humans and other primates and a negative Off-response in rodents and dogs. However, the rod signaling pathways that contribute to these differential responses have not been characterized. In this study, we designed a long flash protocol in the dog that varied in background luminance and stimulus strength allowing for some rod components to be present to better characterize how rod pathways vary from scotopic to mesopic conditions.Results: With low background light the rod a-wave remains while the b-wave is significantly reduced resulting in a predominantly negative waveform in mesopic conditions. Through modeling and subtraction of the rod-driven response, we show that rod bipolar cells saturate with dimmer backgrounds than rod photoreceptors, resulting in rod hyperpolarization contributing to a large underlying negativity with mesopic backgrounds. Conclusions: Reduction in rod bipolar cell responses in mesopic conditions prior to suppression of rod photoreceptor responses may reflect the changes in signaling pathway of rod-driven responses needed to extend the range of lighting conditions over which the retina functions.