ADIPOR1 is essential for vision and its RPE expression is lost in the Mfrp

The knockout (KO) of the adiponectin receptor 1 (AdipoR1) gene causes retinal degeneration. Here we report that ADIPOR1 protein is primarily found in the eye and brain with little expression in other tissues. Further analysis of AdipoR1 KO mice revealed that these animals exhibit early visual system abnormalities and are depleted of RHODOPSIN prior to pronounced photoreceptor death. A KO of AdipoR1 post-development either in photoreceptors or the retinal pigment epithelium (RPE) resulted in decreased expression of retinal proteins, establishing a role for ADIPOR1 in supporting vision in adulthood. Subsequent analysis of the Mfr

A proteogenomic signature of age-related macular degeneration in blood

© 2022. The Author(s). Funding Information: The authors acknowledge the contribution of the Icelandic Heart Association (IHA) staff to the AGES-RS, as well as the involvement of all study participants. We thank the IAMDGC consortium for supplying us with their GWAS summary statistics data. National Institute on Aging (NIA) contracts N01-AG-12100 and HHSN271201200022C for V.G. financed the AGES study; retinal image collection and AMD readings were funded by the NIH Intramural Research Program (ZIAEY000401). V.G. received a funding from the NIA (1R01AG065596), and IHA received a support from Althingi (the Icelandic Parliament). The Icelandic Research Fund (IRF) funded V.E. and Va.G. with grants 195761-051, 184845-053, and 206692-051, while Va.G. received a postdoctoral research grant from the University of Iceland Research Fund Funding Information: The study was supported by the Novartis Institute for Biomedical Research. M.T., N.F., S.P., X.L., R.E., Y.Z., S.J., C.L.H., S.M.L., J.L., C.L.G., A.A.N., B.L., R.P., Z.L., L.L.J., T.E.W., Q.Z., Q.H., and J.R.L. are employees and stockholders of Novartis. All other authors have no conflict of interests to declare. Publisher Copyright: © 2022, The Author(s).Age-related macular degeneration (AMD) is one of the most common causes of visual impairment in the elderly, with a complex and still poorly understood etiology. Whole-genome association studies have discovered 34 genomic regions associated with AMD. However, the genes and cognate proteins that mediate the risk, are largely unknown. In the current study, we integrate levels of 4782 human serum proteins with all genetic risk loci for AMD in a large population-based study of the elderly, revealing many proteins and pathways linked to the disease. Serum proteins are also found to reflect AMD severity independent of genetics and predict progression from early to advanced AMD after five years in this population. A two-sample Mendelian randomization study identifies several proteins that are causally related to the disease and are directionally consistent with the observational estimates. In this work, we present a robust and unique framework for elucidating the pathobiology of AMD.Peer reviewe

Genetic Variations Strongly Influence Phenotypic Outcome in the Mouse Retina

Variation in genetic background can significantly influence the phenotypic outcome of both disease and non-disease associated traits. Additionally, differences in temporal and strain specific gene expression can also contribute to phenotypes in the mammalian retina. This is the first report of microarray based cross-strain analysis of gene expression in the retina investigating genetic background effects. Microarray analyses were performed on retinas from the following mouse strains: C57BL6/J, AKR/J, CAST/EiJ, and NOD.NON-H2-nb1 at embryonic day 18.5 (E18.5) and postnatal day 30.5 (P30.5). Over 3000 differentially expressed genes were identified between strains and developmental stages. Differential gene expression was confirmed by qRT-PCR, Western blot, and immunohistochemistry. Three major gene networks were identified that function to regulate retinal or photoreceptor development, visual perception, cellular transport, and signal transduction. Many of the genes in these networks are implicated in retinal diseases such as bradyopsia, night-blindness, and cone-rod dystrophy. Our analysis revealed strain specific variations in cone photoreceptor cell patterning and retinal function. This study highlights the substantial impact of genetic background on both development and function of the retina and the level of gene expression differences tolerated for normal retinal function. These strain specific genetic variations may also be present in other tissues. In addition, this study will provide valuable insight for the development of more accurate models for human retinal diseases

A murine glaucoma model induced by rapid <i>in vivo</i> photopolymerization of hyaluronic acid glycidyl methacrylate

<div><p>Glaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP) resulting in progressive loss of retinal ganglion cells (RGCs) and optic nerve degeneration, leading to blindness. New therapeutic approaches that better preserve the visual field by promoting survival and health of RGCs are highly needed since RGC death occurs despite good IOP control in glaucoma patients. We have developed a novel approach to reliably induce chronic IOP elevation in mouse using a photopolymerizable biomatrix, hyaluronic acid glycidyl methacrylate. This is achieved by rapid <i>in vivo</i> crosslinking of the biomatrix at the iridocorneal angle by a flash of ultraviolet A (UVA) light to impede the aqueous outflow pathway with a controllable manner. Sustained IOP elevation was induced after a single manipulation and was maintained at ~45% above baseline for >4 weeks. Significant thinning of the inner retina and ~35% reduction in RGCs and axons was noted within one month of IOP elevation. Optic nerve degeneration showed positive correlation with cumulative IOP elevation. Activation of astrocytes and microglia appeared to be an early event in response to IOP elevation preceding detectable RGC and axon loss. Attenuated glial reactivity was noted at later stage where significant RGC/axon loss had occurred suggesting astrocytes and microglia may play different roles over the course of glaucomatous degeneration. This novel murine glaucoma model is reproducible and displays cellular changes that recapitulate several pathophysiological features of glaucoma.</p></div

Astrocyte activation was observed in the hypertensive retinas with more prominent reactivity detected on Day 3 compared to Day 30.

<p>(A) Immunostaining of GFAP in retinal vertical sections. HAMA xl induced dramatic astrocyte activation with higher reactivity detected at early time point Day 3 (d) compared to Day 30 (e). In contrast, PBS+ UVA light (data not shown) or HAMA monomer (b,c) did not induce detectable astrocyte activation compared to naive controls. Images were collected from approximately 1.2–1.5mm from the optic nerve head in the retinal vertical sections. Scale bars: 50 μm. (B) Representative GFAP-immunoreactivity in retinal flatmounts. Top panel: GFAP immunostianing in retinal flatmounts from naive (f), hypertensive retina on day 3 (g) and day 30 (h); bottom panel: signals delineated by HALO software for corresponding images in f,g,h. No activation of astrocytes was noted in the retinal flatmounts from the PBS+UVA or HAMA monomer controls, data not shown. (C) Quantification of GFAP-immunoreactivity by HALO based on the total area covered by GFAP-immunofluorescence and the signal intensity. The intensity of GFAP-immunofluorescent signal was categorized as strong, moderate and weak by HALO. GFAP-immunoreactivity was significantly upregulated in the hypertensive retinas on Day 3 (P = 0.0027) and Day 30 (P = 0.0145) compared to naive controls. A significant attenuation of GFAP-immunoreactivity was also detected on Day 30 compared to Day 3 (P = 0.0036). Error bars: SEM. (D) GFAP mRNA was significantly upregulated in the hypertensive retinas on Day 3, which attenuated largely on Day 30. ** P = 0.008, multiple t-tests. Error bars: SEM.</p

Summary of existing surgically-induced rodent ocular hypertension models.

<p>Summary of existing surgically-induced rodent ocular hypertension models.</p

Ocular hypertension induced by HAMA xl led to significant loss of retinal ganglion cells (RGCs) one month post-operation.

<p>(A) Representative graphs of the Day 30 retinal flatmounts immunostained with BRN3A, a RGC nucleus marker. Loss of RGCs was observed from the hypertensive eyes treated with HAMA xl on Day 30. Note: the mouse monoclonal BRN3A antibody used in the present study cross-reacted with the blood vessels (red arrowheads) in the retina which became more visible in the degenerative retinas. Yellow dotted lines in b, c delineate the regions with more prominent RGC loss. Scale bar: 1 mm. (B) Representative micrographs of BRN3A immunolabeling of RGCs from normotensive (a. PBS+UVA light; b. HAMA monomer) and hypertensive (c. HAMA xl) retinas. Scale bar: 50 μm. (C) Quantification of RGC density based on BRN3A+ nuclei count from retinal flatmounts. Graph was shown as interleaved box & whiskers with 95% confidence interval. n = 11–12 eyes/group for naive, HAMA monomer (Day 30) and HAMA xl (Day 3) groups; n = 20 eyes/group for PBS+UVA light (Day 30) and HAMA xl (Day 30) groups, **** P<0.0001, ns: non-significant. Two-way ANOVA followed by Tukey’s multiple comparisons test. (D) Schematic indicating the sampling of eight 563μm x 422μm rectangle area in the retinal flatmount from four quadrants at two eccentricities (central vs periphery) from the optic nerve head (ONH) for RGC quantification (refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196529#pone.0196529.s001" target="_blank">S1 Fig</a> for technical details).</p

In vivo photopolymerization of HAMA-μBeads induced sustained IOP elevation for over one month.

<p>(A) Schematic indicating the microinjection of HAMA-μBeads into the anterior chamber and photopolymerization of HAMA at the iridocorneal angle to impede the aqueous outflow. 1. An air bubble (1μl) was first injected into the central anterior chamber via an opening made at the paracentral cornea. 2. 2% HAMA-μBeads solution (2μl, indicated as blue here) was injected into the interface between the air bubble and the aqueous humor. The air bubble guided the distribution of HAMA-μBeads to the iridocorneal angle and prevented efflux of the solution upon removal of the micropipette. 3. Immediately post-injection, HAMA was photopolymerized by defined UVA light at 365nm wavelength for 10 seconds, the μBreads were immobilized within the solidified HAMA gel for long-term tracking of the morphological change of the gel inside the anterior chamber. (B) UVA lamp that was programmed to generate UVA light at 365nm for 10 seconds per action. (C) Representative anterior chamber images before and after injection. a. pre-injection, the blue dotted circle marks the limbal region where the iris joins the cornea and sclera; b. shows the HAMA-μBeads ring formed along the iridocorneal angle immediately after photopolymerization; c. shows the distribution pattern of the HAMA-μBeads inside the anterior chamber 30 days post-injection, the HAMA-μBeads gel remained in place at the angle after 1 month post-operation; d. a HAMA monomer injected eye 30 days post-injection. (D) Hematoxylin and eosin stain of ocular vertical sections at the iridocorneal angle from a naive eye (a) and a HAMA-μBeads injected eye (b), the blue matter between the iris and the cornea in (b) is polymerized HAMA. Red asterisks indicate the position of the schlemm’s canal. (E) IOP profiles from the control and HAMA xl groups. Injection of 1XPBS followed by the UVA exposure (the PBS+UVA light group), or injection of HAMA monomer without the presence of μBeads and UVA crosslink did not cause IOP elevation. In vivo photopolymerization of HAMA-μBeads induced significant and sustained IOP elevation. n = 10–12 eyes/group for naive and PBS+UVA light groups, n = 44 eyes/group for HAMA monomer and HAMA xl groups. Group comparison was performed by one-way ANOVA followed by Tukey’s multiple comparisons (F = 26.11, P<0.0001); IOP elevation in the HAMA xl group at each time point was compared with the PBS+UVA light group by Student’s T-test, * P<0.05, ** P<0.01, *** P<0.001. Error bars indicate 95% confidence interval.</p

List of primary antibodies used for this study.

<p>List of primary antibodies used for this study.</p

Ocular hypertension caused significant thinning of the inner retina.

<p>(A) Representative retina images obtained from optical coherence tomography (OCT) at baseline (left) and 1 month post-OHT induction by HAMA xl (right). Asterisk marks the position of the optic nerve head (ONH). (B) Analyses of the thicknesses of the ganglion cell complex (GCC), photoreceptor layer (PRL), and the total retina. GCC = NFL+ GCL+ IPL, as indicated by the blue solid line; PRL = ONL + IS + OS (from outer boundary of OPL to RPE), as indicated by the dotted green line; total neuroretina thickness was measured from NFL to RPE layer as indicated by the yellow dotted line. * P<0.05 by Student’s paired t-test.</p