POU6F2, a risk factor for glaucoma, myopia and dyslexia, labels specific populations of retinal ganglion cells.

Pou6f2 is a genetic connection between central corneal thickness (CCT) in the mouse and a risk factor for developing primary open-angle glaucoma. POU6F2 is also a risk factor for several conditions in humans, including glaucoma, myopia, and dyslexia. Recent findings demonstrate that POU6F2-positive retinal ganglion cells (RGCs) comprise a number of RGC subtypes in the mouse, some of which also co-stain for Cdh6 and Hoxd10. These POU6F2-positive RGCs appear to be novel of ON-OFF directionally selective ganglion cells (ooDSGCs) that do not co-stain with CART or SATB2 (typical ooDSGCs markers). These POU6F2-positive cells are sensitive to damage caused by elevated intraocular pressure. In the DBA/2J mouse glaucoma model, heavily-labeled POU6F2 RGCs decrease by 73% at 8 months of age compared to only 22% loss of total RGCs (labeled with RBPMS). Additionally, Pou6f2-/- mice suffer a significant loss of acuity and spatial contrast sensitivity along with an 11.4% loss of total RGCs. In the rhesus macaque retina, POU6F2 labels the large parasol ganglion cells that form the magnocellular (M) pathway. The association of POU6F2 with the M-pathway may reveal in part its role in human glaucoma, myopia, and dyslexia.

Voluntary exercise preserves visual function and reduces inflammatory response in an adult mouse model of autosomal dominant retinitis pigmentosa.

Whole-body physical exercise has been shown to promote retinal structure and function preservation in animal models of retinal degeneration. It is currently unknown how exercise modulates retinal inflammatory responses. In this study, we investigated cytokine alterations associated with retinal neuroprotection induced by voluntary running wheel exercise in a retinal degeneration mouse model of class B1 autosomal dominant retinitis pigmentosa, I307N Rho. I307N Rho mice undergo rod photoreceptor degeneration when exposed to bright light (induced). Our data show, active induced mice exhibited significant preservation of retinal and visual function compared to inactive induced mice after 4 weeks of exercise. Retinal cytokine expression revealed significant reductions of proinflammatory chemokines, keratinocyte-derived chemokine (KC) and interferon gamma inducible protein-10 (IP-10) expression in active groups compared to inactive groups. Through immunofluorescence, we found KC and IP-10 labeling localized to retinal vasculature marker, collagen IV. These data show that whole-body exercise lowers specific retinal cytokine expression associated with retinal vasculature. Future studies should determine whether suppression of inflammatory responses is requisite for exercise-induced retinal protection.

Pentosan Polysulfate Sodium Causes Diminished Function and Subtle Morphological Changes in Retina and RPE of Mice.

Purpose: There are numerous reports of a distinctive maculopathy in adults exposed to pentosan polysulfate sodium (PPS), a drug prescribed to treat bladder discomfort associated with interstitial cystitis. We tested whether PPS treatment of mice injures RPE or retina to provide insight into the etiology of the human condition. Methods: Mice were fed PPS-supplemented chow over 14 months. RPE and retinal function was assessed by electroretinography (ERG) regularly. Following euthanasia, one eye was used for sagittal sectioning and histology, the contralateral for RPE flatmounting. ZO-1 positive RPE cell borders were imaged using confocal microscopy and cell morphology was analyzed using CellProfiler. Results: After 10 months of PPS treatment, we observed diminution of mean scotopic c-wave amplitudes. By 11 months, we additionally observed diminutions of mean scotopic a- and b-wave amplitudes. Analysis of flatmounts revealed altered RPE cell morphology and morphometrics in PPS-treated mice, including increased mean en face cell area and geometric eccentricity, decreased RPE cell solidity and extent, and cytosolic translocation of alpha-catenin, all markers of RPE cell stress. Sex and regional differences were seen in RPE flatmount measures. Shortened photoreceptor outer segments were also observed. Conclusions: PPS treatment reduced RPE and later retina function as measured by ERG, consistent with a primary RPE injury. Post-mortem analysis revealed extensive RPE pleomorphism and polymegathism and modest photoreceptor changes. We conclude that PPS treatment of mice causes slowly progressing RPE and photoreceptor damage and thus may provide a useful model for some retinal pathologies.

Age-Related RPE changes in Wildtype C57BL/6J Mice between 2 and 32 Months.

Purpose: This study provides a systematic evaluation of age-related changes in RPE cell structure and function using a morphometric approach. We aim to better capture nuanced predictive changes in cell heterogeneity that reflect loss of RPE integrity during normal aging. Using C57BL6/J mice ranging from P60-P730, we sought to evaluate how regional changes in RPE shape reflect incremental losses in RPE cell function with advancing age. We hypothesize that tracking global morphological changes in RPE is predictive of functional defects over time. Methods: We tested three groups of C57BL/6J mice (young: P60-180; Middle-aged: P365-729; aged: 730+) for function and structural defects using electroretinograms, immunofluorescence, and phagocytosis assays. Results: The largest changes in RPE morphology were evident between the young and aged groups, while the middle-aged group exhibited smaller but notable region-specific differences. We observed a 1.9-fold increase in cytoplasmic alpha-catenin expression specifically in the central-medial region of the eye between the young and aged group. There was an 8-fold increase in subretinal, IBA-1-positive immune cell recruitment and a significant decrease in visual function in aged mice compared to young mice. Functional defects in the RPE corroborated by changes in RPE phagocytotic capacity. Conclusions: The marked increase of cytoplasmic alpha-catenin expression and subretinal immune cell deposition, and decreased visual output coincide with regional changes in RPE cell morphometrics when stratified by age. These cumulative changes in the RPE morphology showed predictive regional patterns of stress associated with loss of RPE integrity.

Voluntary exercise modulates pathways associated with amelioration of retinal degenerative diseases.

Background: Exercise has been shown to promote a healthier and longer life and linked to a reduced risk of developing neurodegenerative diseases including retinal degenerations. However, the molecular pathways underpinning exercise-induced cellular protection are not well understood. In this work we aim to profile the molecular changes underlying exercise-induced retinal protection and investigate how exercise-induced inflammatory pathway modulation may slow the progression of retinal degenerations. Methods: Female C57Bl/6J mice at 6 weeks old were given free access to open voluntary running wheels for a period of 28 days and then subjected to 5 days of photo-oxidative damage (PD)-induced retinal degeneration. Following, retinal function (electroretinography; ERG), morphology (optical coherence tomography; OCT) and measures of cell death (TUNEL) and inflammation (IBA1) were analysed and compared to sedentary controls. To decipher global gene expression changes as a result of voluntary exercise, RNA sequencing and pathway and modular gene co-expression analyses were performed on retinal lysates of exercised and sedentary mice that were subjected to PD, as well as healthy dim-reared controls. Results: Following 5 days of PD, exercised mice had significantly preserved retinal function, integrity and reduced levels of retinal cell death and inflammation, compared to sedentary controls. In response to voluntary exercise, inflammatory and extracellular matrix integrity pathways were significantly modulated, with the gene expression profile of exercised mice more closely trending towards that of a healthy dim-reared retina. Conclusion: We suggest that voluntary exercise may mediate retinal protection by influencing key pathways involved in regulating retinal health and shifting the transcriptomic profile to a healthy phenotype.

Deletion of histone demethylase Lsd1 (Kdm1a) during retinal development leads to defects in retinal function and structure.

Purpose: The purpose of this study was to investigate the role of Lysine specific demethylase 1 (Lsd1) in murine retinal development. LSD1 is a histone demethylase that can demethylate mono- and di-methyl groups on H3K4 and H3K9. Using Chx10-Cre and Rho-iCre75 driver lines, we generated novel transgenic mouse lines to delete Lsd1 in most retinal progenitor cells or specifically in rod photoreceptors. We hypothesize that Lsd1 deletion will cause global morphological and functional defects due to its importance in neuronal development. Methods: We tested the retinal function of young adult mice by electroretinogram (ERG) and assessed retinal morphology by in vivo imaging by fundus photography and SD-OCT. Afterward, eyes were enucleated, fixed, and sectioned for subsequent hematoxylin and eosin (H&E) or immunofluorescence staining. Other eyes were plastic fixed and sectioned for electron microscopy. Results: In adult Chx10-Cre Lsd1fl/fl mice, we observed a marked reduction in a-, b-, and c-wave amplitudes in scotopic conditions compared to age-matched control mice. Photopic and flicker ERG waveforms were even more sharply reduced. Modest reductions in total retinal thickness and outer nuclear layer (ONL) thickness were observed in SD-OCT and H&E images. Lastly, electron microscopy revealed significantly shorter inner and outer segments and immunofluorescence showed modest reductions in specific cell type populations. We did not observe any obvious functional or morphological defects in the adult Rho-iCre75 Lsd1fl/fl animals. Conclusion: Lsd1 is necessary for neuronal development in the retina. Adult Chx10-Cre Lsd1fl/fl mice show impaired retinal function and morphology. These effects were fully manifested in young adults (P30), suggesting that Lsd1 affects early retinal development in mice.

Treadmill exercise promotes retinal astrocyte plasticity and protects against retinal degeneration in a mouse model of light-induced retinal degeneration.

Exercise is an effective neuroprotective intervention that preserves retinal function and structure in several animal models of retinal degeneration. However, the retinal cell types governing exercise-induced neuroprotection remain elusive. Previously, we found exercise-induced retinal neuroprotection was associated with increased levels of retinal brain-derived neurotrophic factor (BDNF) and required intact signal transduction with its high-affinity receptor, tropomyosin kinase B (TrkB). Brain studies have shown astrocytes express BDNF and TrkB and that decreased BDNF-TrkB signaling in astrocytes contributes to neurodegeneration. Additionally, exercise has been shown to alter astrocyte morphology. Using a light-induced retinal degeneration (LIRD) model, we investigated how exercise influences retinal astrocytes in adult male BALB/c mice. Treadmill exercise in dim control and LIRD groups had increased astrocyte density, GFAP labeling, branching, dendritic endpoints, and arborization. Meanwhile, inactive LIRD animals had significant reductions in all measured parameters. Additionally, exercised groups had increased astrocytic BDNF expression that was visualized using proximity ligase assay. Isolated retinal astrocytes from exercised LIRD groups had significantly increased expression of a specific isoform of TrkB associated with cell survival, TrkB.FL. Conversely, inactive LIRD isolated retinal astrocytes had significantly increased expression of TrkB.T1, which has been implicated in neuronal cell death. Our data indicate exercise not only alters retinal astrocyte morphology but also promotes specific BDNF-TrkB signaling associated with cell survival and protection during retinal degeneration. These findings provide novel insights into the effects of treadmill exercise on retinal astrocyte morphology and cellular expression, highlighting retinal astrocytes as a potential cell type involved in BDNF-TrkB signaling.

Comparative Analysis of Urso- and Tauroursodeoxycholic Acid Neuroprotective Effects on Retinal Degeneration Models.

Ursodeoxycholic (UDCA) and tauroursodeoxycholic (TUDCA) acids have shown neuroprotective properties in neurodegenerative diseases, but differential effects of the two bile acids have been poorly explored. The aim of this study was to evaluate the neuroprotective effects of UDCA versus TUDCA in a neuroretinal degeneration model and to compare transcriptionally regulated pathways. The WERI-Rb-1 human cone-like cell line and retinal explants were exposed to albumin and TUDCA or UDCA. Viability, cell death, and microglial activation were quantified. Transcriptionally regulated pathways were analyzed after RNA sequencing using the edgeR bioconductor package. Pre-treatment of cone-like cells with UDCA or TUDCA significantly protected cells from albumin toxicity. On retinal explants, either bile acid reduced apoptosis, necroptosis, and microglia activation at 6 h. TUDCA induced the regulation of 463 genes, whilst 31 genes were regulated by UDCA. Only nineteen common genes were regulated by both bile acids, mainly involved in iron control, cell death, oxidative stress, and cell metabolism. As compared to UDCA, TUDCA up-regulated genes involved in endoplasmic reticulum stress pathways and down-regulated genes involved in axonal and neuronal development. Either bile acid protected against albumin-induced cell loss. However, TUDCA regulated substantially more neuroprotective genes than UDCA.

A Tropomycin-Related Kinase B Receptor Activator for the Management of Ocular Blast-Induced Vision Loss.

Pressure waves from explosions or other traumatic events can damage the neurons of the eye and visual centers of the brain, leading to functional loss of vision. There are currently few treatments for such injuries that can be deployed rapidly to mitigate damage. Brain-derived neurotrophic factor (BDNF) and activation of its receptor tropomycin-related kinase B (TrkB) have neuroprotective effects in a number of degeneration models. Small molecule activators of TrkB, such as N-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-2-oxopiperidine-3-carboxamide (HIOC), cross the blood-brain and blood-retina barriers after systemic administration. We characterize the effects of blast-induced ocular trauma on retinal and visual function. We show that systemic administration of HIOC, a potent small molecule activator of the BDNF/TrkB receptor, preserves visual function in mice exposed to ocular blast injury. The HIOC treatment for one week preserves visual function for at least four months. The HIOC treatment effectively protected vision when the initial dose was administered up to 3 h after blast, but not if the initial treatment was delayed for 24 h. We provide evidence that the therapeutic effect of HIOC is mediated by activation of BDNF/TrkB receptors. The results indicate that HIOC may be useful for managing ocular blast injury and other forms of traumatic optic neuropathy.

Tauroursodeoxycholic Acid Protects Retinal and Visual Function in a Mouse Model of Type 1 Diabetes.

PURPOSE: Previous studies demonstrated that systemic treatment with tauroursodeoxycholic acid (TUDCA) is protective in in vivo mouse models of retinal degeneration and in culture models of hyperglycemia. This study tested the hypothesis that TUDCA will preserve visual and retinal function in a mouse model of early diabetic retinopathy (DR). METHODS: Adult C57BL/6J mice were treated with streptozotocin (STZ) and made diabetic at 8-10 weeks of age. Control and diabetic mice were treated with vehicle or TUDCA starting 1 or 3 weeks after induction of diabetes, and were assessed bimonthly for visual function via an optomotor response and monthly for retinal function via scotopic electroretinograms. RESULTS: Diabetic mice showed significantly reduced spatial frequency and contrast sensitivity thresholds compared to control mice, while diabetic mice treated early with TUDCA showed preservation at all timepoints. A-wave, b-wave, and oscillatory potential 2 (OP2) amplitudes decreased in diabetic mice. Diabetic mice also exhibited delays in a-wave and OP2-implicit times. Early TUDCA treatment ameliorated a-wave, b-wave, and OP2 deficits. Late TUDCA treatment showed reduced preservation effects compared to early treatment. CONCLUSIONS: Early TUDCA treatment preserved visual function in an STZ-mouse model of Type I diabetes. These data add to a growing body of preclinical research that may support testing whether TUDCA may be an effective early clinical intervention against declining visual function caused by diabetic retinopathy.

Systemic Treatment with Nicotinamide Riboside Is Protective in Two Mouse Models of Retinal Ganglion Cell Damage.

Glaucoma etiology often includes retinal ganglion cell (RGC) death associated with elevated intraocular pressure (IOP). However, even when IOP is managed well, disease can progress. It is thus important to develop therapeutic approaches that directly protect RGCs in an IOP-independent manner. Compromised nicotinamide adenine dinucleotide (NAD+) metabolism occurs in neurodegenerative diseases, including models of glaucoma. Here we report testing the protective effects of prophylactically systemically administered nicotinamide riboside (NR), a NAD+ precursor, in a mouse model of acute RGC damage (optic nerve crush (ONC)), and in a chronic model of RGC degeneration (ocular hypertension induced by intracameral injection of microbeads). For both models, treatment enhanced RGC survival, assessed by counting cells in retinal flatmounts immunostained for Brn3a+. In the ONC model, treatment preserved RGC function, as assessed by pattern electroretinogram, and suppressed retinal inflammation, as assessed by immunofluorescence staining of retinal fixed sections for glial fibrillary acidic protein (GFAP). This is the first study to demonstrate that systemic treatment with NR is protective in acute and chronic models of RGC damage. The protection is significant and, considering that NR is highly bioavailable in and well-tolerated by humans, may support the proposition of prospective human subject studies.

Electrophysiologic and Morphologic Strain Differences in a Low-Dose NaIO3-Induced Retinal Pigment Epithelium Damage Model.

Purpose: We aimed to explore differences in the NaIO3-elicited responses of retinal pigment epithelium (RPE) and other retinal cells associated with mouse strains and dosing regimens. Methods: One dose of NaIO3 at 10 or 15 mg/kg was given intravenously to adult male C57BL/6J and 129/SV-E mice. Control animals were injected with PBS. Morphologic and functional changes were characterized by spectral domain optical coherence tomography, electroretinography, histologic, and immunofluorescence techniques. Results: Injection with 10 mg/kg of NaIO3 did not cause consistent RPE or retinal changes in either strain. Administration of 15 mg/kg of NaIO3 initially induced a large transient increase in scotopic electroretinography a-, b-, and c-wave amplitudes within 12 hours of injection, followed by progressive structural and functional degradation at 3 days after injection in C57BL/6J mice and at 1 week after injection in 129/SV-E mice. RPE cell loss occurred in a large posterior-central lesion with a ring-like transition zone of abnormally shaped cells starting 12 hours after NaIO3 treatment. Conclusions: NaIO3 effects depended on the timing, dosage, and mouse strain. The RPE in the periphery was spared from damage compared with the central RPE. The large transient increase in the electroretinography was remarkable. Translational Relevance: This study is a phase T1 translational research study focusing on the development and validation of a mouse model of RPE damage. It provides a detailed foundation for future research, informing choices of mouse strain, dosage, and time points to establish NaIO3-induced RPE damage.

Age-Related Retinal Changes in Wild-Type C57BL/6J Mice Between 2 and 32 Months.

Purpose: The purpose of this study was to extend our understanding of how aging affects normal retina function and morphology in wild-type C57BL/6J mice, by analyzing electrophysiological recordings and in vivo and post mortem anatomy. Methods: Electroretinograms (ERGs), spectral domain optical coherence tomography (SD-OCT), and confocal scanning laser ophthalmoscope (cSLO) in vivo images were obtained from mice between the ages of 2 and 32 months in four groups: group 1 (<0.5 years), group 2 (1.0-1.5 years), group 3 (1.5-2.0 years), and group 4 (>2.0 years). Afterward, mouse bodies and eyes were weighed. Eyes were stained with hematoxylin and eosin (H&E) and cell nuclei were quantified. Results: With aging, mice showed a significant reduction in both a- and b-wave ERG amplitudes in scotopic and photopic conditions. Additionally, total retina and outer nuclear layer (ONL) thickness, as measured by SD-OCT images, were significantly reduced in older groups. The cSLO images showed an increase in auto-fluorescence at the photoreceptor-RPE interface as age increases. H&E cell nuclei quantification showed significant reduction in the ONL in older ages, but no differences in the inner nuclear layer (INL) or ganglion cell layer (GCL). Conclusions: By using multiple age groups and extending the upper age limit of our animals to approximately 2.65 years (P970), we found that natural aging causes negative effects on retinal function and morphology in a gradual, rather than abrupt, process. Future studies should investigate the exact mechanisms that contribute to these gradual declines in order to discover pathways that could potentially serve as therapeutic targets.

Morphometric Analysis of Retinal Pigment Epithelial Cells From C57BL/6J Mice During Aging.

Purpose: To quantitatively evaluate the changes in orientation and morphometric features of mouse retinal pigment epithelial (RPE) cells in different regions of the eye during aging. Methods: We segmented individual RPE cells from whole RPE flatmount images of C57BL/6J mice (postnatal days 30 to 720) using a machine-learning method and evaluated changes in morphometric features, including our newly developed metric combining alignment and shape of RPE cells during aging. Results: Mainly, the anterior part of the RPE sheet grows during aging, while the posterior part remains constant. Changes in size and shape of the peripheral RPE cells are prominent with aging as cells become larger, elongated, and concave. Conversely, the central RPE cells maintain relatively constant size and numbers with aging. Cell count in the central area and the overall cell count (approximately 50,000) were relatively constant over different age groups. RPE cells also present a specific orientation concordance that matches the shape of the specific region of the eyeball. Those cells near the optic disc or equator have a circumferential orientation to cover the round shape of the eyeball, whereas those cells in the periphery have a radial orientation and corresponding radial elongation, the extent of which increases with aging and matches with axial elongation of the eyeball. Conclusions: These results suggest that the fluid RPE morphology reflects various growth rates of underlying eyeball, and RPE cells could be classified into four regional classes (near the optic disc, central, equatorial, and peripheral) according to their morphometric features.

Drug Tissue Distribution of TUDCA From a Biodegradable Suprachoroidal Implant versus Intravitreal or Systemic Delivery in the Pig Model.

Purpose: To determine local ocular tissue levels of the bile acid, tauroursodeoxycholic acid (TUDCA), in the pig model using oral, intravenous (IV), intravitreal injection (IVitI) and low- and high-dose suprachoroidal, sustained-release implants (SCI-L or SCI-H). Methods: Forty-six pigs (92 globes) were included in the study. TUDCA was delivered orally in 5 pigs, IV in 4, IVitI in 6, SCI-L in 17, and SCI-H in 14. Testing timeframes varied from the same day (within minutes) for IV; 1 to 6 days, oral; and 1 to 4 weeks, IVitI and SCI. Enucleated globes were dissected, specimens from specific tissues were separated, and TUDCA was extracted and quantified using mass spectrometry. Results: The highest TUDCA tissue levels occurred after IV delivery in the macula (252 ± 238 nM) and peripheral retina (196 ± 171 nM). Macular choroid and peripheral choroid levels were also high (1032 ± 1269 and 1219 ± 1486 nM, respectively). For IVitI delivery, macular levels at day 6 were low (0.5 ± 0.5 nM), whereas peripheral choroid was higher (15.3 ± 16.7 nM). Neither the SCI-L nor SCI-H implants delivered meaningful macular doses (≤1 nM); however, peripheral retina and choroid levels were significantly higher. Bile acid isoforms were found in the serum specimens. Conclusions: The highest TUDCA tissue levels in the pig model were obtained using IV delivery. Oral delivery was associated with reasonable tissue levels. Local delivery (IVitI and SCI) was able to achieve measurable local ocular tissue levels. Translational Relevance: Diffusional kinetics from the suprachoroidal space follow the choroidal blood flow, away from the macula and toward the periphery.

Systemic Treatment With Nicotinamide Riboside Is Protective in a Mouse Model of Light-Induced Retinal Degeneration.

Purpose: Maintaining levels of nicotinamide adenine dinucleotide (NAD+), a coenzyme critical for cellular energetics and biosynthetic pathways, may be therapeutic in retinal disease because retinal NAD+ levels decline during retinal damage and degeneration. The purpose of this study was to investigate whether systemic treatment with nicotinamide riboside (NR), a NAD+ precursor that is orally deliverable and well-tolerated by humans, is protective in a mouse model of light-induced retinal degeneration. Methods: Mice were injected intraperitoneally with vehicle or NR the day before and the morning of exposure to degeneration-inducing levels of light. Retinal function was assessed by electroretinography and in vivo retinal morphology and inflammation was assessed by optical coherence tomography. Post mortem retina sections were assessed for morphology, TUNEL, and inflammatory markers Iba1 and GFAP. Retinal NAD+ levels were enzymatically assayed. Results: Exposure to degeneration-inducing levels of light suppressed retinal NAD+ levels. Mice undergoing light-induced retinal degeneration exhibited significantly suppressed retinal function, severely disrupted photoreceptor cell layers, and increased apoptosis and inflammation in the outer retina. Treatment with NR increased levels of NAD+ in retina and prevented these deleterious outcomes. Conclusions: This study is the first to report the protective effects of NR treatment in a mouse model of retinal degeneration. The positive outcomes, coupled with human tolerance to NR dosing, suggest that maintaining retinal NAD+ via systemic NR treatment should be further explored for clinical relevance.

Initial Assessment of Lactate as Mediator of Exercise-Induced Retinal Protection.

Physical exercise is protective in rodent models of retinal injury and disease. Data suggest that this is in part mediated by brain-derived neurotrophic factor (BDNF) signal transduction. It has been hypothesized that exercised-induced neuroprotection may be mediated by increases in circulating lactate that in turn alter BDNF secretion. We therefore tested whether mice undergoing a treadmill running regimen previously shown to be protective in a mouse model of retinal degeneration (RD) have increased serum levels of lactate. Lactate levels in exercised and non-exercised mice were statistically indistinguishable. A role for circulating lactate in exercise-induced retinal protection is unsupported.

Characterization of LSD1 Expression Within the Murine Eye.

Purpose: The purpose of this study was to extend the current understanding of endogenous lysine-specific demethylase 1 (LSD1) expression spatially and temporally in the retina. Toward that end, we determined the localization and levels of LSD1 and its substrates H3K4me1 and H3K4me2 (H3K4me1/2) within the murine eye. Methods: Immunofluorescent microscopy for LSD1, H3K4me1, and H3K4me2 was conducted on murine formalin-fixed paraffin-embedded eye sections across development in addition to Western immunoblotting to assess localization and protein levels. Results: Retinal LSD1 protein levels were highest at postnatal day 7 (P7), whereas its substrates H3K4me1 and H3K4me2 had equally high levels at P2 and P14. Concentrations of all three proteins gradually decreased over developmental time until reaching a basement level of ∼60% of maximum at P36. LSD1 and H3K4me1/2 were expressed uniformly in all retinal progenitor cells. By P36, there was variability in LSD1 expression in the ganglion cell layer, uniform expression in the inner nuclear layer, and dichotomous expression between photoreceptors in the outer nuclear layer. This contrasted with H3K4me1/2 expression, which remained uniform. Additionally, LSD1 was widely expressed in the lens, cornea, and retinal pigment epithelium. Conclusions: Consistent with its known role in neuronal differentiation, LSD1 is highly and uniformly expressed throughout all retinal progenitor cells. Variability in LSD1 expression, particularly in photoreceptors, may be indicative of their unique transcriptomes and epigenetic patterns of rods and cones. Murine rod nuclei exhibit LSD1 expression in a ring or shell, rather than throughout the nucleus, consistent with their unique inverted chromatin organization. LSD1 has substantial expression throughout adulthood, especially in cone nuclei. By providing insight into endogenous LSD1 expression, our current findings could directly inform future studies to determine the exact role of Lsd1 in the development and maintenance of specific structures and cell types within the eye.

Review: The bile acids urso- and tauroursodeoxycholic acid as neuroprotective therapies in retinal disease.

Bile acids are produced in the liver and excreted into the intestine, where their main function is to participate in lipid digestion. Ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA) have shown antiapoptotic, anti-inflammatory, and antioxidant effects in various models of neurodegenerative diseases. However, little is known about signaling pathways and molecular mechanisms through which these bile acids act as neuroprotectors, delaying translation to the clinical setting. We review evidence supporting a potentially therapeutic role for bile acids in retinal disorders, and the mechanisms and pathways involved in the cytoprotective effects of bile acids from the liver and the enterohepatic circulation to the central nervous system and the retina. As secondary bile acids are generated by the microbiota metabolism, bile acids might be a link between neurodegenerative retinal diseases and microbiota.

Wheel running exercise protects against retinal degeneration in the I307N rhodopsin mouse model of inducible autosomal dominant retinitis pigmentosa.

Purpose: We previously reported that modest running exercise protects photoreceptors in mice undergoing light-induced retinal degeneration and in the rd10 mouse model of autosomal recessive retinitis pigmentosa (arRP). We hypothesized that exercise would protect against other types of retinal degeneration, specifically, in autosomal dominant inherited disease. We tested whether voluntary running wheel exercise is protective in a retinal degeneration mouse model of class B1 autosomal dominant RP (adRP). Methods: C57BL/6J mice heterozygous for the mutation in I307N rhodopsin (Rho) (also known as RHOTvrm4/+, or Tvrm4) are normal until exposed to brief but bright light, whereupon rod photoreceptor degeneration ensues. I307N Rho mice were given access to free spinning (active) or locked (inactive) running wheels. Five weeks later, half of each cohort was treated with 0.2% atropine eye drops and exposed to white LED light (6,000 lux) for 5 min, then returned to maintenance housing with wheels. At 1 week or 4 weeks after induction, retinal and visual function was assessed with electroretinogram (ERG) and optomotor response (OMR). In vivo retinal morphology was assessed with optical coherence tomography (OCT), and fundus blue autofluorescence assessed using a scanning laser ophthalmoscope. The mice were then euthanized, and the eyes fixed for paraffin sectioning or flatmounting. The paraffin sections were stained with hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) to assess retina morphology and apoptosis. Half of the flatmounts were stained for ZO-1 and α-catenin to assess RPE cell structure and stress. (We previously reported that translocation of α-catenin from cell membranes into the cytosol indicates RPE cell stress.) The remaining flatmounts were stained for ZO-1 and Iba-1 to assess the RPE cell size and shape, and inflammatory responses. Results: In vivo measures revealed that induction of the I307N Rho degeneration decreased retinal and visual function, decreased the thickness of the retina and photoreceptor layers, and increased the number of blue autofluorescence spots at the level of the photoreceptor-RPE interface. Post-mortem analyses showed that induction caused loss of photoreceptors in the central retinal region, and increased TUNEL labeling in the outer nuclear layer (ONL). The RPE was disrupted 1 week after induction, with changes in cell size and shape accompanied by increased α-catenin translocation and Iba-1 staining. These outcomes were partially but statistically significantly prevented in the exercised mice. The exercised mice that underwent induced I307N Rho degeneration exhibited retinal function and visual function measures that were statistically indistinguishable from that of the uninduced mice, and compared to the unexercised induced mice, had thicker retina and photoreceptor layers, and decreased numbers of subretinal autofluorescent spots. Post-mortem, the retina sections from the exercised mice that had undergone induced I307N Rho degeneration exhibited numbers of photoreceptors that were statistically indistinguishable from those of uninduced mice. Similarly, exercise largely precluded a degeneration-induced increase in TUNEL-positive cells in the ONL. Finally, the RPE of the exercised mice appeared normal, with a regular cell shape and size, and little to no alpha-catenin translocation or Iba-1 immunosignal. Conclusions: Voluntary wheel running partially protected against retinal degeneration and inflammation, and RPE disruption in a model of inducible adRP. This is the first report of exercise protection in an adult adRP animal model. It is also the first report of an RPE phenotype in the I307N Rho mouse. These findings add to a growing literature reporting that modest whole-body exercise is protective across a wide range of models of retinal damage and disease, and further highlights the potential for this accessible and inexpensive therapeutic intervention in the ophthalmic clinic.