Rubicon regulates A2E-induced autophagy impairment in the retinal pigment epithelium implicated in the pathology of age-related macular degeneration.

Waste product deposition and light stress in the retinal pigment epithelium (RPE) are crucial factors in the pathogenesis of various retinal degenerative diseases, including age-related macular degeneration (AMD), a leading cause of vision loss in elderly individuals worldwide. Given that autophagy in the RPE suppresses waste accumulation, determining the molecular mechanism by which autophagy is compromised in degeneration is necessary. Using polarized human RPE sheets, we found that bis-retinoid N-retinyl-N-retinylidene ethanolamine (A2E), a major toxic fluorophore of lipofuscin, causes significant impairment of autophagy and the simultaneous upregulation of Rubicon, a negative regulator of autophagy. Importantly, this impairment was reversed in Rubicon-specific siRNA-treated RPE sheets. In a retinal functional analysis using electroretinograms (ERGs), mice with the RPE-specific deletion of Rubicon showed no significant differences from control cre-expressing mice but presented partially but significantly enhanced amplitudes compared with Atg7 knockout mice. We also found that an inflammatory reaction in the retina in response to chronic blue light irradiation was alleviated in mice with the RPE-specific deletion of Rubicon. In summary, we propose that upregulating basal autophagy by targeting Rubicon is beneficial for protecting the RPE from functional damage with ageing and the inflammatory reaction caused by light-induced cellular stress.

Conjunctival dysbiosis in mucosa-associated lymphoid tissue lymphoma.

To investigate the conjunctival microbiota and the association between the development of conjunctival mucosa-associated lymphoid tissue (MALT) lymphoma and dysbiosis, DNA samples were collected from 25 conjunctival MALT lymphoma patients and 25 healthy controls. To compare the microbiota, samples were collected from the following four body locations: conjunctiva, meibomian gland, periocular skin and hand. Extracted DNA was analyzed by 16S rRNA sequences, and libraries were sequenced on an Illumina MiSeq sequencer. The differences in bacteria were characterized by using principal coordinate analysis of metagenomics data, and the differences in bacterial compositions were evaluated by linear discriminant analysis effect size. The conjunctival microbiota of MALT lymphoma patients was compositionally different from that of healthy controls. For the conjunctival MALT lymphoma patients, alterations in the microbial composition were detected, and a remarkable change was detected at the conjunctiva. Detailed analysis showed that a specific population of the microbiota, the genus Delftia, was significantly more abundant in conjunctival MALT lymphoma patients, and the genera Bacteroides and Clostridium were less abundant in the MALT lymphoma patients. A specific microbiota on the ocular surface in conjunctival MALT lymphoma patients was detected, and dysbiosis may play an important role in the pathophysiology of conjunctival MALT lymphoma.

Moderate repulsive effects of E-unit-containing chondroitin sulfate (CSE) on behavior of retinal growth cones.

Chondroitin sulfate (CS), the carbohydrate chain of chondroitin sulfate proteoglycans, is involved in neuronal circuit formation during development. CS shows great structural diversity with combination of disaccharide units of different structure (A-, C-, D-, or E-unit). However, whether its structural diversity contributes to pathway formation remains unclear. We chemically coupled the reducing end of various types of CS to the amino group of phosphatidylethanolamine (lipid-derivatized CS, CS-PE) and established an in vitro time-lapse assay to observe the behaviors of growth cones of retinal ganglion cells from embryonic day 6 chick retina on exposure to beads coated with lipid-derivatized CS (CS-PE beads). Among CS-PEs with different content of the structural units, the beads coated with E-unit-containing CS-PE [E-unit: GlcAß1-3GalNAc(4,6-O-disulfate)] (CSE-PE beads) significantly caused the growth cones to retract and to turn away from the beads, but the beads coated with CSA-, CSC- or CSD-PE beads did not. Importantly, not all the growth cones retracted equally from the CSE-PE beads, but they showed continuum of the repulsive behaviors; some behaved moderately and others remarkably. The growth cones distinguished different samples of CS: CSE and the others. Moreover, the continuum of the repulsive behaviors suggests that CS might be involved with the fine regulation of growth cones' behavior through its characteristic structure.

Influences of retinal axons on the cultural substrate containing biotin-conjugated chondroitin sulfate in vitro.

Although chondroitin sulfate (CS) is known to act as an inhibitory axon guidance cue, retinal axons show substantial growth on a culture substrate containing CS. Thus, the question arises as to how retinal axons elongate on CS-containing culture substrates. To elucidate the effects of retinal axons on a substrate containing CS, we synthesized biotinylated CS (biotin-CS) and developed a culture substrate with streptavidin-conjugated biotin-CS (complex between streptavidin and biotin-CS) to culture retinal axons. The effects of retinal axons on the streptavidin-biotin-CS complex were analyzed immunocytochemically using antibodies against CS and streptavidin, which recognize the carbohydrate and protein portions of the complex, respectively. After the axons were cultured on the substrate, areas that were CS-immunonegative but streptavidin-immunopositive were observed on the surface, corresponding to areas with or without axons, respectively. Absence of CS immunostaining was considered to be caused by structural alterations in the carbohydrate chains of the CS under the influence of the axons.