Dysregulated claudin-5 cycling in the inner retina causes retinal pigment epithelial cell atrophy
Natalie Hudson, Lucia Celkova, Alan Hopkins, Chris Greene, Federica Storti, Ema Ozaki, Erin Fahey, Sofia Theodoropoulou, Paul F. Kenna, Marian M. Humphries, Annie M. Curtis, Eleanor Demmons, Akeem Browne, Shervin Liddie, Matthew S. Lawrence, Christian Grimm, Mark T. Cahill, Pete Humphries, Sarah L. Doyle, Matthew Campbell
Natalie Hudson, Lucia Celkova, Alan Hopkins, Chris Greene, Federica Storti, Ema Ozaki, Erin Fahey, Sofia Theodoropoulou, Paul F. Kenna, Marian M. Humphries, Annie M. Curtis, Eleanor Demmons, Akeem Browne, Shervin Liddie, Matthew S. Lawrence, Christian Grimm, Mark T. Cahill, Pete Humphries, Sarah L. Doyle, Matthew Campbell
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Research Article Ophthalmology Vascular biology

Dysregulated claudin-5 cycling in the inner retina causes retinal pigment epithelial cell atrophy

Abstract

Age-related macular degeneration (AMD) is the leading cause of central retinal vision loss worldwide, with an estimated 1 in 10 people over the age of 55 showing early signs of the condition. There are currently no forms of therapy available for the end stage of dry AMD, geographic atrophy (GA). Here, we show that the inner blood-retina barrier (iBRB) is highly dynamic and may play a contributory role in GA development. We have discovered that the gene CLDN5, which encodes claudin-5, a tight junction protein abundantly expressed at the iBRB, is regulated by BMAL1 and the circadian clock. Persistent suppression of claudin-5 expression in mice exposed to a cholesterol-enriched diet induced striking retinal pigment epithelium (RPE) cell atrophy, and persistent targeted suppression of claudin-5 in the macular region of nonhuman primates induced RPE cell atrophy. Moreover, fundus fluorescein angiography in human and nonhuman primate subjects showed increased retinal vascular permeability in the evening compared with the morning. These findings implicate an inner retina–derived component in the early pathophysiological changes observed in AMD, and we suggest that restoring the integrity of the iBRB may represent a novel therapeutic target for the prevention and treatment of GA secondary to dry AMD.

Authors

Natalie Hudson, Lucia Celkova, Alan Hopkins, Chris Greene, Federica Storti, Ema Ozaki, Erin Fahey, Sofia Theodoropoulou, Paul F. Kenna, Marian M. Humphries, Annie M. Curtis, Eleanor Demmons, Akeem Browne, Shervin Liddie, Matthew S. Lawrence, Christian Grimm, Mark T. Cahill, Pete Humphries, Sarah L. Doyle, Matthew Campbell

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Figure 1

Claudin-5 cycling in the inner retina.

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Claudin-5 cycling in the inner retina. (A) Claudin-5 transcript cycling ...
(A) Claudin-5 transcript cycling in the mouse cerebellum, hypothalamus, and brainstem. (B) Claudin-5 cycling in the mouse aorta, lung, heart, white fat, liver, adrenal gland, muscle, and brown fat (*P = 0.00051), from data collated from open-source data available on http://cirgrdb.biols.ac.cn/ (C) Claudin-5 transcript cycling in the neural retina over a 24-hour period (**P < 0.01, *P < 0.05, and n = 7–11 mice). (D) Western blot analysis of claudin-5 expression at 8 am compared with 8 pm. Densitometric analysis (right, **P = 0.0064). (n = 5 mice for each time point.) (E) qPCR analysis of claudin-5 transcript at 8 am compared with 8 pm (*P = 0.042, and n = 5 mice for both time points). (F) Transcript levels at 8 am compared with 8 pm in mice dark adapted (DA) for 24 hours (*P = 0.0483, n = 5 mice for DA-AM, and n = 4 mice for DA-PM). (G) Levels of claudin-5 transcript in mice with inverted circadian rhythm (*P = 0.0235, n = 3 mice for morning, and n = 4 mice for inverted). (H) Claudin-5 (red) levels in the retinas of mice at 8 am (left) compared with 8 pm (right). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; scale bar: 100 μm. High-magnification images of claudin-5 (red) in the retinas of mice at 8 am (left) compared with 8 pm (right); scale bar: 50 μm. (I) EZ-Link-Biotin (red) extravasation in the inner and outer segments of the photoreceptors at 8 pm compared with 8 am. Retinal vasculature stained with isolectin IB4 (green); scale bar: 50 μm. (J) Significant extravasation of EZ-Link-Biotin in the outer segments, ONL, and OPL (*P = 0.0184). No significant signal observed in the INL, inner plexiform layer (IPL), or GCL. Student’s t test, with significance represented by a P value of less than or equal to 0.05. For multiple comparisons, ANOVA was used with Bonferroni’s post hoc test and significance represented by a P value of less than or equal to 0.05.