Neuroprotection of the Inner Retina Also Prevents Secondary Outer Retinal Pathology in a Mouse Model of Glaucoma.

Purpose: We examined structural and functional changes in the outer retina of a mouse model of glaucoma. We examined whether these changes are a secondary consequence of damage in the inner retina and whether neuroprotection of the inner retina also prevents outer retinal changes. Methods: We used an established microbead occlusion model of glaucoma whereby intraocular pressure (IOP) was elevated. Specific antibodies were used to label rod and cone bipolar cells (BCs), horizontal cells (HCs), and retinal ganglion cells (RGCs), as well as synaptic components in control and glaucomatous eyes, to assess structural damage and cell loss. ERG recordings were made to assess outer retina function. Results: We found structural and functional damage of BCs, including significant cell loss and dendritic/axonal remodeling of HCs, following IOP elevation. The first significant loss of both BCs occurred at 4 to 5 weeks after microbead injection. However, early changes in the dendritic structure of RGCs were observed at 3 weeks, but significant changes in the rod BC axon terminal structure were not seen until 4 weeks. We found that protection of inner retinal neurons in glaucomatous eyes by pharmacological blockade of gap junctions or genetic ablation of connexin 36 largely prevented outer retinal damage. Conclusions: Together, our results indicate that outer retinal impairments in glaucoma are a secondary sequalae of primary damage in the inner retina. The finding that neuroprotection of the inner retina can also prevent outer retinal damage has important implications with regard to the targets for effective neuroprotective therapy.

Structure and dynamics of photoreceptor sensory cilia.

The rod and cone photoreceptor cells of the vertebrate retina have highly specialized structures that enable them to carry out their function of light detection over a broad range of illumination intensities with optimized spatial and temporal resolution. Most prominent are their unusually large sensory cilia, consisting of outer segments packed with photosensitive disc membranes, a connecting cilium with many features reminiscent of the primary cilium transition zone, and a pair of centrioles forming a basal body which serves as the platform upon which the ciliary axoneme is assembled. These structures form a highway through which an enormous flux of material moves on a daily basis to sustain the continual turnover of outer segment discs and the energetic demands of phototransduction. After decades of study, the details of the fine structure and distribution of molecular components of these structures are still incompletely understood, but recent advances in cellular imaging techniques and animal models of inherited ciliary defects are yielding important new insights. This knowledge informs our understanding both of the mechanisms of trafficking and assembly and of the pathophysiological mechanisms of human blinding ciliopathies.

Prion-induced photoreceptor degeneration begins with misfolded prion protein accumulation in cones at two distinct sites: cilia and ribbon synapses.

Accumulation of misfolded host proteins is central to neuropathogenesis of numerous human brain diseases including prion and prion-like diseases. Neurons of retina are also affected by these diseases. Previously, our group and others found that prion-induced retinal damage to photoreceptor cells in mice and humans resembled pathology of human retinitis pigmentosa caused by mutations in retinal proteins. Here, using confocal, epifluorescent and electron microscopy we followed deposition of disease-associated prion protein (PrPSc) and its association with damage to critical retinal structures following intracerebral prion inoculation. The earliest time and place of retinal PrPSc deposition was 67 days post-inoculation (dpi) on the inner segment (IS) of cone photoreceptors. At 104 and 118 dpi, PrPSc was associated with the base of cilia and swollen cone inner segments, suggesting ciliopathy as a pathogenic mechanism. By 118 dpi, PrPSc was deposited in both rods and cones which showed rootlet damage in the IS, and photoreceptor cell death was indicated by thinning of the outer nuclear layer. In the outer plexiform layer (OPL) in uninfected mice, normal host PrP (PrPC) was mainly associated with cone bipolar cell processes, but in infected mice, at 118 dpi, PrPSc was detected on cone and rod bipolar cell dendrites extending into ribbon synapses. Loss of ribbon synapses in cone pedicles and rod spherules in the OPL was observed to precede destruction of most rods and cones over the next 2-3 weeks. However, bipolar cells and horizontal cells were less damaged, indicating high selectivity among neurons for injury by prions. PrPSc deposition in cone and rod inner segments and on the bipolar cell processes participating in ribbon synapses appear to be critical early events leading to damage and death of photoreceptors after prion infection. These mechanisms may also occur in human retinitis pigmentosa and prion-like diseases, such as AD.

Syntaxin 3 is essential for photoreceptor outer segment protein trafficking and survival.

Trafficking of photoreceptor membrane proteins from their site of synthesis in the inner segment (IS) to the outer segment (OS) is critical for photoreceptor function and vision. Here we evaluate the role of syntaxin 3 (STX3), in trafficking of OS membrane proteins such as peripherin 2 (PRPH2) and rhodopsin. Photoreceptor-specific Stx3 knockouts [Stx3f/f(iCre75) and Stx3f/f(CRX-Cre) ] exhibited rapid, early-onset photoreceptor degeneration and functional decline characterized by structural defects in IS, OS, and synaptic terminals. Critically, in the absence of STX3, OS proteins such as PRPH2, the PRPH2 binding partner, rod outer segment membrane protein 1 (ROM1), and rhodopsin were mislocalized along the microtubules to the IS, cell body, and synaptic region. We find that the PRPH2 C-terminal domain interacts with STX3 as well as other photoreceptor SNAREs, and our findings indicate that STX3 is an essential part of the trafficking pathway for both disc (rhodopsin) and rim (PRPH2/ROM1) components of the OS.

Measurement of Diurnal Variation in Rod Outer Segment Length In Vivo in Mice With the OCT Optoretinogram.

Purpose: To investigate diurnal variation in the length of mouse rod outer segments in vivo. Methods: The lengths of rod inner and outer segments (RIS, ROS) of dark-adapted albino mice maintained on a 12-hour dark:12-hour light cycle with light onset 7 AM were measured at prescribed times (6:30 AM, 11 AM, 3:30 PM) during the diurnal cycle with optical coherence tomography (OCT), taking advantage of increased visibility, after a brief bleaching exposure, of the bands corresponding to RIS/ROS boundaries and ROS tips (ROST). Results: Deconvolution of OCT depth profiles resolved two backscatter bands located 7.4 ± 0.1 and 10.8 ± 0.2 µm (mean ± SEM) proximal to Bruch's membrane (BrM). These bands were identified with histology as arising from the apical surface of RPE and ROST, respectively. The average length of dark-adapted ROS at 6:30 AM was 17.7 ± 0.8 µm. By 11 AM, the average ROS length had decreased by 10% to 15.9 ± 0.7 µm. After 11 AM, the ROS length increased steadily at an average rate of 0.12 µm/h, returning to baseline length by 23.5 hours in the cycle. Conclusions: The diurnal variation in ROS length measured in these experiments is consistent with prior histological investigations showing that rodent rod discs are phagocytosed by the RPE maximally over several hours around the time of normal light onset. The rate of recovery of ROS to baseline length before normal light onset is consistent with the hypothesis that disc membrane synthesis is fairly constant over the diurnal cycle.

A non-canonical rhodopsin-mediated insulin receptor signaling pathway in retinal photoreceptor neurons.

We previously reported a ligand-independent and rhodopsin-dependent insulin receptor (IR) neuroprotective signaling pathway in both rod and cone photoreceptor cells, which is activated through protein-protein interaction. Our previous studies were performed with either retina or isolated rod or cone outer segment preparations and the expression of IR signaling proteins were examined. The isolation of outer segments with large portions of the attached inner segments is a technical challenge. Optiprep™ density gradient medium has been used to isolate the cells and subcellular organelles, Optiprep™ is a non-ionic iodixanol-based medium with a density of 1.320 g/mL. We employed this method to examine the expression of IR and its signaling proteins, and activation of one of the downstream effectors of the IR in isolated photoreceptor cells. Identification of the signaling complexes will be helpful for therapeutic targeting in disease conditions.

Acute tissue reactions, inner segment pathology, and effects of the antioxidant α1-microglobulin in an in vitro model of retinal detachment.

The purpose of this study was to explore acute tissue reactions, ultrastructural photoreceptor morphology with emphasis on inner segments, and the effect of antioxidant treatment in an in vitro model of rhegmatogenous retinal detachment (RRD). A previously described method of RRD simulation was used with adult retinal porcine explants kept free-floating in culture medium with or without treatment with the radical scavenger α1-microglobulin (A1M). Explants were examined at 5 time points from 1 to 24 h using transmission electron microscopy as well as quantitative real-time PCR (RT-PCR) to quantify gene expression of the cell stress marker heat shock protein 70 (Hsp70) and oxidative stress marker heme oxygenase (HO-1). The culture medium level of the cell damage marker lactate dehydrogenase (LDH) and oxidative stress DNA damage marker 8-Oxo-2'-deoxyguanosine (8-OHdG) was also assessed at each time point. We found that the levels of Hsp70 and LDH rapidly increased in both groups, and at 3 and 6 h, Hsp70 was significantly higher in A1M treated retinas. At 24 h, Hsp70 and LDH, as well as 8-OHdG were significantly lower compared with controls, whereas the tissue level of HO-1 was significantly higher. Progressive ultrastructural photoreceptor changes were seen in untreated control explants from 1 h and onwards including outer segment shortening and loss, disruption of organelles within the inner segments and loss of perikarya in the outer nuclear layer. Inner segment pathology was more rapid and extensive in rods compared with in cones. In A1M treated counterparts, damage to rod inner segment mitochondria was significantly higher after 1 h of culture, but after this time, no statistical difference was found. At 24 h, cone inner segment mitochondrial disruption was significantly higher in control retinas and the number of surviving perikarya lower. From our results, we conclude that retinal explants elicit acute cell stress reactions when placed in culture without physical support simulating a detached retina floating in the vitreous space. Photoreceptors rapidly display degenerative changes including extensive damage to inner segment mitochondria indicating loss of energy transduction as an early key event. A1M increases initial mitochondrial stress in the rods, however, subsequent pathology is attenuated by the treatment, highlighting the dynamics of protective as well as disruptive oxidative stress reactions in the detached retina.

New Developments in Murine Imaging for Assessing Photoreceptor Degeneration In Vivo.

Optical Coherence Tomography (OCT) is a powerful clinical tool that measures near infrared light backscattered from the eye and other tissues. OCT is used for assessing changes in retinal structure, including layer thicknesses, detachments and the presence of drusen in patient populations. Our custom-built OCT system for the mouse eye quantitatively images all layers of the neural retinal, the RPE, Bruchs' membrane and the choroid. Longitudinal assessment of the same retinal region reveals that the relative intensities of retinal layers are highly stable in healthy tissue, but show progressive increases in intensity in a model of retinal degeneration. The observed changes in OCT signal have been correlated with ultrastructural disruptions that were most dramatic in the inner segments and nuclei of the rods. These early changes in photoreceptor structure coincided with activation of retinal microglia, which migrated vertically from the inner to the outer retina to phagocytose photoreceptor cell bodies (Levine et al., Vis Res 102:71-79, 2014). We conclude that quantitative analysis of OCT light scattering signals may be a useful tool for early detection and subcellular localization of cell stress prior to cell death, and for assessing the progression of degenerative disease over time. Future efforts to develop sensitive approaches for monitoring microglial dynamics in vivo may likewise elucidate earlier signs of cellular stress during retinal degeneration.

Rod disc renewal occurs by evagination of the ciliary plasma membrane that makes cadherin-based contacts with the inner segment.

The outer segments of vertebrate rod photoreceptors are renewed every 10 d. Outer segment components are transported from the site of synthesis in the inner segment through the connecting cilium, followed by assembly of the highly ordered discs. Two models of assembly of discrete discs involving either successive fusion events between intracellular rhodopsin-bearing vesicles or the evagination of the plasma membrane followed by fusion of adjacent evaginations have been proposed. Here we use immuno-electron microscopy and electron tomography to show that rhodopsin is transported from the inner to the outer segment via the ciliary plasma membrane, subsequently forming successive evaginations that "zipper" up proximally, but at their leading edges are free to make junctions containing the protocadherin, PCDH21, with the inner segment plasma membrane. Given the physical dimensions of the evaginations, coupled with likely instability of the membrane cortex at the distal end of the connecting cilium, we propose that the evagination occurs via a process akin to blebbing and is not driven by actin polymerization. Disassembly of these junctions is accompanied by fusion of the leading edges of successive evaginations to form discrete discs. This fusion is topologically different to that mediated by the membrane fusion proteins, SNAREs, as initial fusion is between exoplasmic leaflets, and is accompanied by gain of the tetraspanin rim protein, peripherin.

Inner Segment Remodeling and Mitochondrial Translocation in Cone Photoreceptors in Age-Related Macular Degeneration With Outer Retinal Tubulation.

PURPOSE: To quantify impressions of mitochondrial translocation in degenerating cones and to determine the nature of accumulated material in the subretinal space with apparent inner segment (IS)-like features by examining cone IS ultrastructure. METHODS: Human donor eyes with advanced age-related macular degeneration (AMD) were screened for outer retinal tubulation (ORT) in macula-wide, high-resolution digital sections. Degenerating cones inside ORT (ORT cones) and outside ORT (non-ORT cones) from AMD eyes and unaffected cones in age-matched control eyes were imaged using transmission electron microscopy. The distances of mitochondria to the external limiting membrane (ELM), cone IS length, and cone IS width at the ELM were measured. RESULTS: Outer retinal tubulation and non-ORT cones lose outer segments (OS), followed by shortening of IS and mitochondria. In non-ORT cones, IS broaden. Outer retinal tubulation and non-ORT cone IS myoids become undetectable due to mitochondria redistribution toward the nucleus. Some ORT cones were found lacking IS and containing mitochondria in the outer fiber (between soma and ELM). Unlike long, thin IS mitochondria in control cones, ORT and non-ORT IS mitochondria are ovoid or reniform. Shed IS, some containing mitochondria, were found in the subretinal space. CONCLUSIONS: In AMD, macula cones exhibit loss of detectable myoid due to IS shortening in addition to OS loss, as described. Mitochondria shrink and translocate toward the nucleus. As reflectivity sources, translocating mitochondria may be detectable using in vivo imaging to monitor photoreceptor degeneration in retinal disorders. These results improve the knowledge basis for interpreting high-resolution clinical retinal imaging.

Light Induces Ultrastructural Changes in Rod Outer and Inner Segments, Including Autophagy, in a Transgenic Xenopus laevis P23H Rhodopsin Model of Retinitis Pigmentosa.

PURPOSE: We previously reported a transgenic Xenopus laevis model of retinitis pigmentosa in which tadpoles express the bovine form of P23H rhodopsin (bP23H) in rod photoreceptors. In this model, retinal degeneration was dependent on light exposure. Here, we investigated ultrastructural changes that occurred in the rod photoreceptors of these retinas when exposed to light. METHODS: Tadpoles expressing bP23H in rods were transferred from constant darkness to a 12-hour light:12-hour dark (12L:12D) regimen. For comparison, transgenic tadpoles expressing an inducible form of caspase 9 (iCasp9) were reared in a 12L:12D regimen, and retinal degeneration was induced by administration of the drug AP20187. Tadpoles were euthanized at various time points, and eyes were processed for confocal light and transmission electron microscopy. RESULTS: We observed defects in outer and inner segments of rods expressing bP23H that were aggravated by light exposure. Rod outer segments exhibited vesiculations throughout and were rapidly phagocytosed by the retinal pigment epithelium. In rod inner segments, we observed autophagic compartments adjacent to the endoplasmic reticulum and extensive vesiculation at later time points. These defects were not found in rods expressing iCasp9, which completely degenerated within 36 hours after drug administration. CONCLUSIONS: Our results indicate that ultrastructural defects in outer and inner segment membranes of bP23H expressing rods differ from those observed in drug-induced apoptosis. We suggest that light-induced retinal degeneration caused by P23H rhodopsin occurs via cell death with autophagy, which may represent an attempt to eliminate the mutant rhodopsin and/or damaged cellular compartments from the secretory pathway.

Robust Endoplasmic Reticulum-Associated Degradation of Rhodopsin Precedes Retinal Degeneration.

Rhodopsin is a G protein-coupled receptor essential for vision and rod photoreceptor viability. Disease-associated rhodopsin mutations, such as P23H rhodopsin, cause rhodopsin protein misfolding and trigger endoplasmic reticulum (ER) stress, activating the unfolded protein response (UPR). The pathophysiologic effects of ER stress and UPR activation on photoreceptors are unclear. Here, by examining P23H rhodopsin knock-in mice, we found that the UPR inositol-requiring enzyme 1 (IRE1) signaling pathway is strongly activated in misfolded rhodopsin-expressing photoreceptors. IRE1 significantly upregulated ER-associated protein degradation (ERAD), triggering pronounced P23H rhodopsin degradation. Rhodopsin protein loss occurred as soon as photoreceptors developed, preceding photoreceptor cell death. By contrast, IRE1 activation did not affect JNK signaling or rhodopsin mRNA levels. Interestingly, pro-apoptotic signaling from the PERK UPR pathway was also not induced. Our findings reveal that an early and significant pathophysiologic effect of ER stress in photoreceptors is the highly efficient elimination of misfolded rhodopsin protein. We propose that early disruption of rhodopsin protein homeostasis in photoreceptors could contribute to retinal degeneration.

Contrast sensitivity and foveal microstructure following vitrectomy for epiretinal membrane.

PURPOSE: To evaluate contrast sensitivity (CS) in patients with epiretinal membrane (ERM) following vitrectomy and to investigate the relationship between CS and foveal microstructures with spectral-domain optical coherence tomography (SD-OCT). METHODS: Thirty-one eyes of 31 patients with ERM were included. We examined CS with a CSV-1000E chart, a logMAR best-corrected visual acuity (BCVA), and foveal microstructure by using SD-OCT before and at 6 months after surgery. From the CS data, the area under the log contrast sensitivity function (AULCSF) was calculated. Based on the OCT images, we quantified the mean thickness of the ganglion cell layer (GCL), the inner nuclear layer (INL), and the outer retinal layer (outer nuclear layer and outer plexiform layer [ONL+OPL]). The status of the photoreceptor inner and outer segment junction (IS/OS) and external limiting membrane (ELM) was also evaluated. RESULTS: Vitrectomy significantly improved logMAR BCVA and AULCSF. Even in patients with poor improvement of visual acuity (changes in logMAR BCVA by surgery was ≤0.2), postoperative AULCSF significantly increased by treatment (P < 0.05). Postoperative AULCSF showed a significant correlation with preoperative (P < 0.05) and postoperative (P < 0.05) ONL+OPL thickness, whereas other parameters were not relevant. Postoperative logMAR BCVA significantly correlated with postoperative status of IS/OS (P < 0.05) and preoperative ONL+OPL thickness (P < 0.05). CONCLUSIONS: In patients with ERM, CS improved even though their visual acuity did not recover significantly by vitrectomy. CS was associated with the thickness of outer retinal layer.

[Endogenous content of the nitric oxide in the cell layers of the eye retina].

Nitric oxide is a universal molecule that regulates many different functions in an organism. In the eye retina nitric oxide plays both a regulatory role by modulation of the synaptic transmission between photoreceptors and bipolar cells and a toxic role in apoptosis induction in the outer nuclear layer and in the layer of ganglion cells. In this paper there has been made the first attempt to estimate the endogenous NO concentration in retina layers in vivo. The concentration of the nitric oxide was determined by two indepen- dent techniques: ESR spectrometry using spin trap for in vivo determination and NO-sensitive microelectrode for in situ determination in the survival isolated frog retina. The distinct NO con- centration was detected only in the ganglion cells layer (~0.25 µM) and in the inner segments layer of the photoreceptors (~0.6 µM). The activity and the kinetic characteristic of the NO-synthase localized in the same layers were also determined. Key words: retina cells layers, nitric oxide, ESR, NO-sensitive microelectrodes.

Real-time imaging of rabbit retina with retinal degeneration by using spectral-domain optical coherence tomography.

BACKGROUND: Recently, a transgenic rabbit with rhodopsin Pro 347 Leu mutation was generated as a model of retinitis pigmentosa (RP), which is characterized by a gradual loss of vision due to photoreceptor degeneration. The purpose of the current study is to noninvasively visualize and assess time-dependent changes in the retinal structures of a rabbit model of retinal degeneration by using speckle noise-reduced spectral-domain optical coherence tomography (SD-OCT). METHODOLOGY/PRINCIPAL FINDINGS: Wild type (WT) and RP rabbits (aged 4-20 weeks) were investigated using SD-OCT. The total retinal thickness in RP rabbits decreased with age. The thickness of the outer nuclear layer (ONL) and between the external limiting membrane and Bruch's membrane (ELM-BM) were reduced in RP rabbits around the visual streak, compared to WT rabbits even at 4 weeks of age, and the differences increased with age. However, inner nuclear layer (INL) thickness in RP rabbits did not differ from that of WT during the observation period. The ganglion cell complex (GCC) thickness in RP rabbits increased near the optic nerve head but not around the visual streak in the later stages of the observation period. Hyper-reflective change was widely observed in the inner segments (IS) and outer segments (OS) of the photoreceptors in the OCT images of RP rabbits. Ultrastructural findings in RP retinas included the appearance of small rhodopsin-containing vesicles scattered in the extracellular space around the photoreceptors. CONCLUSIONS/SIGNIFICANCE: In the current study, SD-OCT provided the pattern of photoreceptor degeneration in RP rabbits and the longitudinal changes in each retinal layer through the evaluation of identical areas over time. The time-dependent changes in the retinal structure of RP rabbits showed regional and time-stage variations. In vivo imaging of RP rabbit retinas by using SD-OCT is a powerful method for characterizing disease dynamics and for assessing the therapeutic effects of experimental interventions.

Ultrastructure of the distal retina of the adult zebrafish, Danio rerio.

The organization, morphological characteristics, and synaptic structure of photoreceptors in the adult zebrafish retina were studied using light and electron microscopy. Adult photoreceptors show a typical ordered tier arrangement with rods easily distinguished from cones based on outer segment (OS) morphology. Both rods and cones contain mitochondria within the inner segments (IS), including the large, electron-dense megamitochondria previously described (Kim et al.) Four major ultrastructural differences were observed between zebrafish rods and cones: (1) the membranes of cone lamellar disks showed a wider variety of relationships to the plasma membrane than those of rods, (2) cone pedicles typically had multiple synaptic ribbons, while rod spherules had 1-2 ribbons, (3) synaptic ribbons in rod spherules were ∼2 times longer than ribbons in cone pedicles, and (4) rod spherules had a more electron-dense cytoplasm than cone pedicles. Examination of photoreceptor terminals identified four synaptic relationships at cone pedicles: (1) invaginating contacts postsynaptic to cone ribbons forming dyad, triad, and quadrad synapses, (2) presumed gap junctions connecting adjacent postsynaptic processes invaginating into cone terminals, (3) basal junctions away from synaptic ribbons, and (4) gap junctions between adjacent photoreceptor terminals. More vitread and slightly farther removed from photoreceptor terminals, extracellular microtubule-like structures were identified in association with presumed horizontal cell processes in the OPL. These findings, the first to document the ultrastructure of the distal retina in adult zebrafish, indicate that zebrafish photoreceptors have many characteristics similar to other species, further supporting the use of zebrafish as a model for the vertebrate visual system.

Facilitative glucose transporter Glut1 is actively excluded from rod outer segments.

Photoreceptors are among the most metabolically active cells in the body, relying on both oxidative phosphorylation and glycolysis to satisfy their high energy needs. Local glycolysis is thought to be particularly crucial in supporting the function of the photoreceptor's light-sensitive outer segment compartment, which is devoid of mitochondria. Accordingly, it has been commonly accepted that the facilitative glucose transporter Glut1 responsible for glucose entry into photoreceptors is localized in part to the outer segment plasma membrane. However, we now demonstrate that Glut1 is entirely absent from the rod outer segment and is actively excluded from this compartment by targeting information present in its cytosolic C-terminal tail. Our data indicate that glucose metabolized in the outer segment must first enter through other parts of the photoreceptor cell. Consequently, the entire energy supply of the outer segment is dependent on diffusion of energy-rich substrates through the thin connecting cilium that links this compartment to the rest of the cell.