Monitoring basal autophagy in the retina utilizing CAG-mRFP-EGFP-MAP1LC3B reporter mouse: technical and biological considerations.

ABSTRACT

We describe the utility of a tandem-tagged autophagy reporter mouse model (CAG-RFP-EGFP-MAP1LC3B) in investigating basal macroautophagic/autophagic flux in the neural retina. Western blot, in situ hybridization, immunohistochemistry, and confocal microscopy showed that CAG promoter-driven expression of RFP-EGFP-MAP1LC3B increased "cytosolic" RFP-EGFP-LC3B-I levels, whereas RFP-EGFP-LC3B-II decorates true phagosomes. We verified that the electroretinographic (ERG) responses of tandem-tagged LC3B mice were comparable to those of age-matched controls. Optimized microscope settings detected lipofuscin autofluorescence in retinas of abca4-/- mice. The majority of retinal phagosomes in the reporter mice exhibited only RFP (not EGFP) fluorescence, suggesting rapid maturation of phagosomes. Only ~1.5% of the total phagosome population was EGFP-labeled; RFP-labeled (mature) phagosomes colocalized with lysosomal markers LAMP2 and CTSD. In the outer retina, phagosome sizes were as follows (in µm2, ave ± SEM) RPE, 0.309 ± 0.015; photoreceptor inner segment-myoid, 0.544 ± 0.031; and outer nuclear layer, 0.429 ± 0.011. Detection of RPE phagosomes by fluorescence microscopy is challenging, due to the presence of melanin. Increased lipofuscin autofluorescence, such as observed in the abca4-/- mouse model of Stargardt disease, is a strong confounding factor when attempting to study autophagy in the RPE. In addition to RPE and photoreceptor cells, phagosomes were detected in inner retinal cell types, microglia, astrocytes, and endothelial cells. We conclude that the tandem-tagged LC3B mouse model serves as a useful system for studying autophagy in the retina. This utility, however, is dependent upon several technical and biological factors, including microscope settings, transgene expression, choice of fluorophores, and lipofuscin autofluorescence.Abbreviations ACTB actin, beta; AIF1 allograft inflammatory factor 1; ATG autophagy related; CTSD cathepsin D; DAPI (4',6-diamido-2-phenylindole); DIC differential interference contrast; EGFP enhanced green fluorescent protein; ELM external limiting membrane; ERG electroretinography; GCL ganglion cell layer; GLUL glutamine-ammonia ligase (glutamine synthetase); INL inner nuclear layer; IS-E/M inner segment - ellipsoid/myoid; ISH in situ hybridization; LAMP2 lysosomal-associated membrane protein 2; L.I. laser Intensity; MAP1LC3B microtubule-associated protein 1 light chain 3 beta; MTOR mechanistic target of rapamycin kinase; O.C.T. optimal cutting temperature; OS outer segment; ONL outer nuclear layer; PE phosphatidylethanolamine; RFP red fluorescent protein; R.O.I. region of interest; RPE retinal pigment epithelium.