Retinol tracing within murine neural retina reveals cell type–specific retinol transport and distribution
Zachary J. Engfer, Grazyna Palczewska, Samuel W. Du, Jianye Zhang, Zhiqian Dong, Carolline Rodrigues Menezes, Jun Wang, Jianming Shao, Budd A. Tucker, Robert F. Mullins, Rui Chen, Philip D. Kiser, Krzysztof Palczewski
Zachary J. Engfer, Grazyna Palczewska, Samuel W. Du, Jianye Zhang, Zhiqian Dong, Carolline Rodrigues Menezes, Jun Wang, Jianming Shao, Budd A. Tucker, Robert F. Mullins, Rui Chen, Philip D. Kiser, Krzysztof Palczewski
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Research Article Cell biology Ophthalmology

Retinol tracing within murine neural retina reveals cell type–specific retinol transport and distribution

Abstract

11-cis-Retinal is essential for light perception in mammalian photoreceptors (PRs), and aberrations in retinoid transformations cause severe retinal diseases. Understanding these processes is crucial for combating blinding diseases. The visual cycle, operating within PRs and the retinal pigment epithelium (RPE), regenerates 11-cis-retinal to sustain light sensitivity. Retinoids are also present in Müller glia (MG), hypothesized to supply 11-cis-retinol to cone PRs and retinal ganglion cells (RGCs). To trace retinoid movement through retinal cell types, we used cell-specific knockin of lecithin:retinol acyltransferase (LRAT), which converts retinols into stable retinyl esters (REs). Ectopic LRAT expression in murine PRs, MG, and RGCs resulted in RE synthesis, with REs differing in abundance and isomeric composition across cell types under genetic and light-based perturbations. PR inner segments showed high 11-cis-RE content, suggesting a constant 11-cis-retinoid supply for pigment regeneration. In MG expressing LRAT, all-trans-REs were detected, contrasting with 11-cis-REs in PRs. The MG-specific LRAT phenotype mirrored the RE-rich human neural retina, suggesting human MG may utilize LRAT to maintain retinoid reservoirs. Our findings reveal tightly controlled retinoid flux throughout the mammalian retina that supports sustained vision, expanding understanding of the visual cycle to combat retinal diseases.

Authors

Zachary J. Engfer, Grazyna Palczewska, Samuel W. Du, Jianye Zhang, Zhiqian Dong, Carolline Rodrigues Menezes, Jun Wang, Jianming Shao, Budd A. Tucker, Robert F. Mullins, Rui Chen, Philip D. Kiser, Krzysztof Palczewski

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Figure 7

Retinosome-like structures in the mouse GCL after intravitreal injection with AAV2-hSyn-Lrat.

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Retinosome-like structures in the mouse GCL after intravitreal injection...
(A and B) Data from TP-excitation imaging through the pupil in a live mouse. Excitation wavelengths are indicated in images. (A) Fluorescence intensity–based images (top row) and phasor plots (bottom row), including an uninjected (uninj.) WT eye on the RPE layer; an uninj. WT eye on the GCL; a WT eye 6 weeks after IVt injection with AAV2-hSyn-Lrat on the GCL; a WT eye 6 weeks after IVt injection with AAV2-hSyn-Lrat, supplemented with rhodamine-conjugated anti-Thy1.2 antibody. Phasor lifetimes indicated in phasor plots. Red arrows indicate bright granules. Scale bar: 50 μm. (B) Immunohistochemistry. Top (740 nm laser) and bottom (800 nm laser) images show the GCL layer 6 weeks after IVt injection with AAV2-hSyn-Lrat and 48 hours after IVt injection with αThy1-rhodamine. Detection pass bands: 430–530 nm for retinosome emission, and 600–720 nm for rhodamine emission. Scale bar: 50 µm. (C) 3D-volume visualizations assembled from en face fluorescence intensity–based images acquired every 2 μm along the retinal thickness in the ex vivo mouse eyes. Scales are given in μm; images are pseudo-colored in green. (D) Ex vivo en face FLIM of GCL in uninj. and injected mouse eyes. Phasor scales are provided in white. Scale bar: 50 μm. (E) TP fluorescence emission spectra from WT RPE, PR-Lrat+/–Lrat+/– PRs, and the GCL from AAV2-hSyn-Lrat–injected WT mice. (F) HPLC traces of retinoid extracts from the neural retinas of B6(Cg)-Tyrc-2J/J (WT) mice 6 weeks after IVt injection with AAV2-hSyn-Lrat and TP imaging (light-adapted, n = 4). (G) HPLC traces of retinoid extracts from the RPE/choroid of B6(Cg)-Tyrc-2J/J (WT) mice 6 weeks after IVt injection with AAV2-hSyn-Lrat and TP imaging (light-adapted, n = 4). For F and G, all-trans-RE peaks are labeled a and b. Left traces are focused on the RE-elution and the right traces on the RO-elution. Full sets of retinoid traces in F and G are featured in Supplemental Figure 13, A and B.