Abstract
Purpose :
Photoreceptors (PRs) depend on the endolysosomal system to maintain homeostasis and vision. The unique arrangement of PRs in the retina allows for rigorous investigation of neuronal organelle trafficking. Using an autophagy reporter mouse line (CAG-RFP-EGFP-LC3B), we previously demonstrated rapid PR phagosome-lysosome fusion. Mature PR phagolysosomes are distributed along the cell body and in the F-actin-rich PR inner segment (IS)-myoid region. Here, we studied cytoskeleton-dependent endolysosome genesis, phagosome maturation, and protein aggregate targeting to lysosomes in mammalian PRs.
Methods :
C57BL/6J, C57BL/6NTac, CAG-RFP-EGFP-LC3B, and ZsGreen-expressing mice were treated with vinblastine sulphate (a microtubule-depolymerizing agent) for 6 h. Eyes were then harvested, fixed, and retinal wholemounts were prepared, which then were treated with anti-LAMP2 (a lysosome marker) and a fluor-tagged secondary IgG, and phalloidin-AF568 (to detect F-actin). Laser confocal fluorescence microscopy was used to assess phagolysosome distribution in the PR IS-myoid region.
Results :
Control C57BL/6J retinas exhibited the typical distribution of lysosomes, limited to the PR IS-myoid and cell body. Vinblastine treatment inhibited microtubule-dependent lysosome trafficking in those retinas and caused lysosome accumulation exclusively in the F-actin-rich IS-myoid region. Retinas of C57BL/6N mice (a model of Crb1-associated retinopathy), which exhibit disrupted F-actin in the IS-myoid region, exhibited normal lysosome distribution in lesion-free regions. However, lesioned areas showed markedly lower endolysosome content. Vinblastine treatment of C57BL/6N mice confirmed loss of PR endocytic capacity in the IS-myoid region of lesioned areas, indicated by complete loss of endolysosomes. Surprisingly, vinblastine treatment of CAG-RFP-EGFP-LC3B mice caused accumulation of RFP/LAMP2-positive mature phagolysosomes in the IS-myoid, without causing phagosome maturation defects. Fluorescent protein aggregates in ZsGreen-expressing mice were effectively targeted to lysosomes.
Conclusions :
These findings indicate that F-actin-dependent endocytosis occurs specifically in the PR IS-myoid, followed by microtubule-dependent cargo trafficking throughout the PR cell body. Phagosome-lysosome fusion occurs rapidly in the IS-myoid region. These data impilicate lysosomal defects in CRB1-associated retinopathy.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.