Upregulation of retinal VEGF and connexin 43 in murine nonarteritic anterior ischemic optic neuropathy induced with 577 nm laser.

Nonarteritic anterior ischemic optic neuropathy (NAION) is a common acute optic neuropathy and cause of irreversible vision loss in those older than 50 years of age. There is currently no effective treatment for NAION and the biological mechanisms leading to neuronal loss are not fully understood. Promising novel targets include glial cells activation and intercellular communication mediated by molecules such as gap junction protein Connexin 43 (Cx43), which modulate neuronal fate in central nervous system disorders. In this study, we investigated retinal glial changes and neuronal loss following a novel NAION animal model using a 577 nm yellow laser. We induced unilateral photochemical thrombosis using rose bengal at the optic nerve head vasculature in adult C57BL/6 mice using a 577 nm laser and performed morphometric analysis of the retinal structure using serial in vivo optical coherence tomography (OCT) and histology for glial and neuronal markers. One day after experimental NAION, in acute phase, OCT imaging revealed peripapillary thickening of the retinal ganglion cell complex (GCC, baseline: 79.5 ±â€¯1.0 µm, n = 8; NAION: 93.0 ±â€¯2.5 µm, n = 8, P < 0.01) and total retina (baseline: 202.9 ±â€¯2.4 µm, n = 8; NAION: 228.1 ±â€¯6.8 µm, n = 8, P < 0.01). Twenty-one days after ischemia, at a chronic phase, there was significant GCC thinning (baseline 78.3 ±â€¯2.1 µm, n = 6; NAION: 72.2 ±â€¯1.9 µm, n = 5, P < 0.05), mimicking human disease. Examination of molecular changes in the retina one day after ischemia revealed that NAION induced a significant increase in retinal VEGF levels (control: 2319 ±â€¯195, n = 5; NAION: 4549 ±â€¯683 gray mean value, n = 5, P < 0.05), which highly correlated with retinal thickness (r = 0.89, P < 0.05). NAION also led to significant increase in mRNA level for Cx43 (Gj1a) at day 1 (control: 1.291 ±â€¯0.38; NAION: 3.360 ±â€¯0.58 puncta/mm2, n = 5, P < 0.05), but not of glial fibrillary acidic protein (Gfap) at the same time (control: 2,800 ±â€¯0.59; NAION: 4,690 ±â€¯0.90 puncta/mm2 n = 5, P = 0.19). Retinal ganglion cell loss at day 21 was confirmed by a 30% decrease in Brn3a+ cells (control: 2,844 ±â€¯235; NAION: 2,001 ±â€¯264 cells/mm2, n = 4, P < 0.05). We described a novel protocol of NAION induction by photochemical thrombosis using a 577 nm laser, leading to retinal edema and VEGF increase at day 1 and RGCs loss at day 21 after injury, consistent with the pathophysiology of human NAION. Early changes in glial cells intercommunication revealed by increased Cx43+ gap junctions are consistent with a retinal glial role in mediating cell-to-cell signaling after an ischemic insult. Our study demonstrates an early glial response in a novel NAION animal model and reveals glial intercommunication molecules such as Cx43 as a promising therapeutic target in acute NAION.

Human CRB1 and CRB2 form homo- and heteromeric protein complexes in the retina.

Crumbs homolog 1 (CRB1) is one of the key genes linked to retinitis pigmentosa and Leber congenital amaurosis, which are characterized by a high clinical heterogeneity. The Crumbs family member CRB2 has a similar protein structure to CRB1, and in zebrafish, Crb2 has been shown to interact through the extracellular domain. Here, we show that CRB1 and CRB2 co-localize in the human retina and human iPSC-derived retinal organoids. In retina-specific pull-downs, CRB1 was enriched in CRB2 samples, supporting a CRB1-CRB2 interaction. Furthermore, novel interactors of the crumbs complex were identified, representing a retina-derived protein interaction network. Using co-immunoprecipitation, we further demonstrate that human canonical CRB1 interacts with CRB1 and CRB2, but not with CRB3, which lacks an extracellular domain. Next, we explored how missense mutations in the extracellular domain affect CRB1-CRB2 interactions. We observed no or a mild loss of CRB1-CRB2 interaction, when interrogating various CRB1 or CRB2 missense mutants in vitro. Taken together, our results show a stable interaction of human canonical CRB2 and CRB1 in the retina.