Abstract: Optical coherence tomography (OCT) is an ocular imaging technique that can complement the neuro-ophthalmic assessment, and inform our understanding regarding functional consequences of neuroaxonal injury in the afferent visual pathway. Indeed, OCT has emerged as a surrogate end-point in the diagnosis and follow up of several demyelinating syndromes of the central nervous system (CNS), including optic neuritis (ON) associated with: multiple sclerosis (MS), neuromyelitis optica spectrum disorder (NMOSD), and anti-myelin oligodendrocyte glycoprotein (MOG) antibodies. Recent advancements in enhanced depth imaging (EDI) OCT have distinguished this technique as a new gold standard in the diagnosis of optic disc drusen (ODD). Moreover, OCT may enhance our ability to distinguish cases of papilledema from pseudopapilledema caused by ODD. In the setting of idiopathic intracranial hypertension (IIH), OCT has shown benefit in tracking responses to treatment, with respect to reduced retinal nerve fiber layer (RNFL) measures and morphological changes in the angling of Bruch’s membrane. Longitudinal follow up of OCT measured ganglion cell-inner plexiform layer thickness may be of particular value in managing IIH patients who have secondary optic atrophy. Causes of compressive optic neuropathies may be readily diagnosed with OCT, even in the absence of overt visual field defects. Furthermore, OCT values may offer some prognostic value in predicting post-operative outcomes in these patients. Finally, OCT can be indispensable in differentiating optic neuropathies from retinal diseases in patients presenting with vision loss, and an unrevealing fundus examination. In this review, our over-arching goal is to highlight the potential role of OCT, as an ancillary investigation, in the diagnosis and management of various optic nerve disorders.
Background: Exposure to ethanol in utero leads to several brain development disorders including retinal abnormalities whose underlying cellular pathogenesis remains elusive. We have previously reported changes in electroretinogram recordings in moderate fetal alcohol exposure (MFAE) vervet monkeys. The goal of this study is to characterize the anatomical effects of moderate MFAE during the third trimester in the vervet monkey retina.
Methods: Using immunohistochemistry and Western blots, we analyzed changes in the expression of cell-type specific proteins that may occur in the MFAE retina compared to the normal retina. We also compared the basic retinal anatomy across groups by examining retinal layering and thickness.
Results: Our main result indicates that GFAP (a potent marker of astrocytes) immunoreactivity was increased in the MFAE retina indicating strong astrogliosis. There was no obvious change in the overall anatomy in the MFAE retina and no significant differences in the mean thickness of each retinal layer. Furthermore, no significant changes in the morphology of the photoreceptors, horizontal cells, bipolar cells, and amacrines cells was observed.
Conclusions: These data indicate that astrogliosis is a consequence of prenatal alcohol exposure and might explain the reported changes in the electroretinographic responses.
Background: Rods and cones are critical for light detection. Although there has been considerable work done in elucidating the molecular mechanisms involved in rod development, not much is known about how the cone cell fate decision is made by the multipotent retinal progenitor cells during development. Analysis of the promoter regions of Nrl and trβ2, rod and cone differentiation factors respectively, revealed DNA binding motifs of two POU-domain containing transcription factors, Pou2f1 and Pou2f2. Preliminary experiments showed that Pou2f1/2 are expressed during the peak of cone genesis in the embryonic retina. Therefore, we hypothesize that Pou2f1/2 specify cone cell fate in the developing retina.
Methods: We used immunofluorescence and in situ hybridization to establish the spatiotemporal expression of Pou2f1/2 during retinogenesis. We performed in vivo electroporation in post-natal mice to misexpress Pou2f1/2 and used antibodies specific to proteins expressed in cones such as Rxrγ and S-opsin to count cones. Using ex vivo electroporation of embryonic retinal explants, we knocked out Pou2f1 and Pou2f2 using CRISPR/Cas9 gRNAs at the peak of cone production window. Finally, we transfected post-natal retinal explants with a combination of regulatory elements of Nrl or thrb with control backbone vector, Pou2f1 or Pou2f2 using electroporation.
Results: We found that Pou2f1/2 are expressed in retinal progenitor cells in the developing retina and subsequently in the differentiated cones. Pou2f1/2 misexpression outside the cone genesis window led to an increase in cones at the expense of rods. Pou2f1/2 indel knockouts generated by CRISPR/Cas9 gRNAs led to a decrease in cones and a converse increase in rods. Finally, we found that Pou2f1/2 activate the cis-regulatory module (CRM) of the thrb gene and repress the activity of the CRM of Nrl.
Conclusions: These results uncover novel players that establish the complex gene regulatory network for cone photoreceptor fate specification in the retinal progenitor cells. We anticipate that this work should help us devise improved replacement therapies in the future utilizing stem cells for retinal degenerative diseases such as aged-related macular degeneration (AMD) and Stargardt’s disease.
Background: Retinal pigment epithelium (RPE) is vital for the homeostasis of the subretina including photoreceptors and choroid. Interestingly, our previous results suggested that the recently discovered lactate receptor GPR81 is abundantly expressed in RPE. To date, only one previous study has shown that activating GPR81 could enhance DNA repair by activating HDAC1. Consequently, we investigated whether GPR81 exhibits epigenetic modification in the subretina by using GPR81?/? mice.
Methods: GPR81?/? mice and wide type littermates were generated on a background of C57BL/6J mice. The thicknesses of their choroid were evaluated by immunohistochemistry. Meanwhile, Q-PCR, western blot and choroid sprout assay were performed. In vitro, primary retinal pigment epithelium (pRPE) cells were isolated from mice, and cultured for treatments.
Results: The thickness of choroid was reduced in GPR81?/? mice compared to GPR81+/+ mice, suggesting that GPR81 is important for the integrity of choroid. In the choroid sprout assay, lactate treated RPE/choroid complex showed a significant increase in angiogenesis compared to controls while lactate treated KO RPE/choroid complex showed no difference compared to their controls. For Q-PCR, most of the genes screened elevated their expression in GPR81?/? mice compared to WT mice, suggesting epigenetic modification may exist, which were confirmed by histone acetylation and HDACs activity assay.
Conclusions: Taking together, the lactate receptor GPR81 in RPE is very important for maintaining homeostasis of the subretina. This novel discovery sheds new light on the relationship between metabolism and epigenetic modification.
Background: Retinol dehydrogenase 8 (RDH8) is a 312-amino acid (aa) protein involved in the visual cycle. Bound to the outer segment disk membranes of photoreceptors, it reduces all-trans-retinal to all-trans-retinol1 as one of the rate-limiting steps of the visual cycle2. RDH8 is a member of the short-chain dehydrogenase/reductase family. Its C-terminal segment allows its membrane-anchoring through the postulated presence of an amphipathic α-helix and of 1 to 3 acyl groups at positions 299, 302 and 3043. The secondary structure and membrane binding characteristics of RDH8 and its C-terminal segment have not yet been described.
Methods: To evaluate the membrane binding of RDH8, the full-length protein (aa 1–312), a truncated form (aa 1–296), its C-terminal segment (aa 281–312 and 297–312) as well as different additional variants of this segment were used. The truncated protein binds membranes less efficiently than the full-length form. Thus, the C-terminal segment of RDH8 is essential for the binding and has thus been further examined. The intrinsic fluorescence of tryptophan residues at positions 289 and 310 of the wild-type C-terminal segment of RDH8 and the mutants W289F, W310F and W310R have thus been used to determine their extent of binding to lipid vesicles and to monitor their local environment. Unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or a mixture of POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) were used to mimic the phospholipid content of the outer segment disk membranes of photoreceptors.
Results: An increase in fluorescence intensity and in fluorescence lifetime is observed upon increasing the concentration of lipid vesicles. These data allowed calculating values of partition coefficient of the C-terminal segment of RDH8 varying between Kp =1.1 E6 to 1.7 E6. It is noteworthy that the observation of a more intense shift to lower wavelengths upon membrane binding of the mutant W310R and W310F indicates a deeper incorporation of the remaining tryptophan residue at position 289 into the lipid bilayer. The secondary structure of the C-terminal segment of RDH8 observed by circular dichroism and infrared spectroscopy shows a superposition of α-helical, β-turn and unordered structures.
Conclusions: The peptides derived from the C-terminal segment of RDH8 show a strong binding to lipid vesicles. These strength of binding is independent of the type of lipid and the presence of a mutation.
Abstract: Mononuclear phagocytes (MP) comprise a family of cells that include microglial cells (MC), monocytes, and macrophages. The subretinal space, located between the RPE and the photoreceptor outer segments, is physiologically devoid of MPs and a zone of immune privilege mediated, among others, by immunosuppressive RPE signals. Age-related macular degeneration (AMD) is a highly heritable major cause of blindness, characterized by a breakdown of the subretinal immunosuppressive environment and an accumulation of pathogenic inflammatory MPs. Studies in mice and humans suggest that the AMD-associated APOE2 isoform promotes the breakdown of subretinal immunosuppression and increased MP survival. Of all genetic factors, variants of complement factor H (CFH) are associated with greatest linkage to AMD. Using loss of function genetics and orthologous models of AMD, we provide mechanistic evidence that CFH inhibits the elimination of subretinal MPs. Importantly, the AMD-associated CFH402H isoform markedly increased this inhibitory effect on microglial cells, indicating a causal link to disease etiology. Pharmacological acceleration of resolution of subretinal inflammation might be a powerful tool for controlling inflammation and neurodegeneration in late AMD.
Abstract: Subretinal inflammation plays a critical role in retinal degenerative diseases. Although activated macrophages have been shown to play a key role in the progression of retinopathies and specifically in age-related macular degeneration, little is known about the mechanisms involved in the loss of photoreceptors leading to vision impairment. In our study on retinal damages induced by photo-oxidative stress, we have observed that CD36-deficient mice featured less subretinal macrophage accumulation with attenuated photoreceptor degeneration compared to wild-type (WT) mice. Treatment with CD36-selective azapeptide ligand (labelled MPE-001) as modulator of the inflammatory environment of the retina reduced subretinal macrophage/activated microglia accumulation with preservation of photoreceptor layers and function assessed by ERG in WT, in a CD36-dependent manner. The azapeptide modulated the transcriptome of subretinal macrophage/activated microglia by reducing pro-inflammatory markers. In isolated macrophages, the CD36-selective azapeptide induced dissociation of the CD36-TLR2/6 heterodimer complex (using FRET) altering the TLR2 signaling pathway, thus decreasing NF-KB activation and inflammasome activity. The azapeptide also incurred cytoprotection against photoreceptor apoptosis elicited by activated macrophages. These findings suggest that the azapeptide as ligand of co-receptor CD36 decreases the inflammatory response by modulating CD36-TLR2/6 complex signaling pathway in macrophages, and suggests its potential application in the treatment of retinal degenerative diseases.
Abstract: Autophagy recycles intracellular substrate in part to fuel mitochondria during starvation. Deregulated autophagy caused by dyslipidemia, oxidative stress, and aging is associated with early signs of age-related macular degeneration (AMD), such as lipofuscin and perhaps drusen accumulation. Intracellular nutrient sensors for glucose and amino acids regulate autophagy. The role of lipid sensors in controlling autophagy, however, remains ill-defined. Here we will show that abundant circulating lipids trigger a satiety signal through FA receptors that restrain autophagy and oxidative mitochondrial metabolism. In the presence of excess dietary lipids, fatty acid sensors might protect tissues with high metabolic rates against lipotoxicity, favoring their storage, instead, in adipose tissues. However, sustained exposure to lipid reduces retinal metabolic efficiency. In photoreceptors with high metabolic needs, it predisposes to an energy failure and triggers compensatory albeit pathological angiogenesis, leading to blinding neovascular AMD.
Abstract: The inverted retina is a basic characteristic of the vertebrate eye. This implies that vertebrates must have a common ancestor with an inverted retina. Of the two groups of chordates, cephalochordates have an inverted retina and urochordates a direct retina. Surprisingly, recent genetics studies favor urochordates as the closest ancestor to vertebrates. The evolution of increasingly complex organs such as the eye implies not only tissular but also structural modifications at the organ level. How these configurational modifications give rise to a functional eye at any step is still subject to debate and speculation. Here we propose an orderly sequence of phylogenetic events that closely follows the sequence of developmental eye formation in extant vertebrates. The progressive structural complexity has been clearly recorded during vertebrate development at the period of organogenesis. Matching the chain of increasing eye complexity in Mollusca that leads to the bicameral eye of the octopus and the developmental sequence in vertebrates, we delineate the parallel evolution of the two-chambered eye of vertebrates starting with an early ectodermal eye. This sequence allows for some interesting predictions regarding the eyes of not preserved intermediary species. The clue to understanding the inverted retina of vertebrates and the similarity between the sequence followed by Mollusca and chordates is the notion that the eye in both cases is an ectodermal structure, in contrast to an exclusively (de novo) neuroectodermal origin in the eye of vertebrates. This analysis places cephalochordates as the closest branch to vertebrates contrary to urochordates, claimed as a closer branch by some researchers that base their proposals in a genetic analysis.
Abstract: Pediatric neuro-ophthalmology is a subspecialty within neuro-ophthalmology. Pediatric neuro-ophthalmic diseases must be considered separate from their adult counterparts, due to the distinctive nature of the examination, clinical presentations, and management choices. This manuscript will highlight four common pediatric neuro-ophthalmic disorders by describing common clinical presentations, recommended management, and highlighting recent developments. Diseases discussed include pediatric idiopathic intracranial hypertension (IIH), pseudopapilledema, optic neuritis (ON) and optic pathway gliomas (OPG). The demographics, diagnosis and management of common pediatric neuro-ophthalmic disease require a working knowledge of the current research presented herein. Special attention should be placed on the differences between pediatric and adult entities such that children can be appropriately diagnosed and treated.
