Abstract
Purpose :
A significant challenge for phagocytic tissues is balancing the competing demands of phagolysosomal degradation and autophagy. In the retina, circadian phagocytosis and degradation of photoreceptor outer segments by the postmitotic retinal pigment epithelium (RPE) is fundamental for vision. Disrupted autophagy due to abnormal mTOR activation in the RPE is associated with blinding macular degenerations; however, outer segment degradation is unaffected in these diseases, indicating that these clearance mechanisms are regulated by distinct mechanisms. Here, we investigated how this plasticity is achieved in healthy and disease states to maintain efficient phagosome clearance.
Methods :
Polarized primary porcine RPE were fed with purified porcine outer segments for 30 min followed by specific chase periods. For in vivo studies, outer segment phagocytosis was monitored in pigmented wild-type and Abca4−/− (Stargardt disease) mice at specific times after light onset. Molecular mechanisms regulating phagocytosis and phagosome clearance were examined using high-speed, high-resolution live-cell imaging, genetic strategies, immunostaining, immunoblotting, and transcriptomics.
Results :
Our data implicate optineurin in scissioning outer segment tips prior to engulfment, akin to microglial trogocytosis of neuronal processes. Optineurin is essential for recruiting LC3, which anchors outer segment phagosomes to microtubules and facilitates phagosome maturation. This dynamically activates the transcription factor EB (TFEB) to induce lysosome biogenesis in an mTOR-independent, TRPML1-dependent manner. RNAseq analyses show that expression of TFEB target genes temporally tracks with optineurin recruitment and that lysosomal and autophagy genes are controlled by distinct transcriptional programs in the RPE.
Conclusions :
We identify optineurin as a key regulator that tunes phagocytosis and lysosomal capacity to meet circadian demands and prioritize outer segment clearance by the RPE. The unconventional plasma membrane-to-nucleus signaling ensures outer segment degradation under conditions of impaired autophagy in macular degeneration models. Independent regulation of these critical clearance mechanisms could safeguard metabolic fitness of the RPE and provide the basis for selective therapeutic targeting in disease.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.