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 9

Retinoid analyses of dissected human neural retina and RPE/choroid tissue.

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Retinoid analyses of dissected human neural retina and RPE/choroid tissu...
(A) Representative HPLC trace of RE-elution extracted from dissected neural retina from a single human eye. Representative HPLC trace of REs extracted from a single human dissected neural retina. The top right inset is a representative HPLC trace of REs from 6 pooled, 6 mm perimacular punches from human neural retina. Red dashed boxes highlight a neural retina-localized 11-cis-RE (a) and an all-trans-RE (b). (B) Representative HPLC trace of ROs extracted from a single human dissected neural retina. The top right inset is a representative HPLC trace of ROs extracted from 6 pooled, 6 mm perimacular punches from human neural retina. (C) Representative HPLC trace of REs from a single human dissected RPE/choroid. The top right inset is a representative HPLC trace of REs extracted from 6 pooled, 6 mm perimacular punches from human RPE/choroid. (D) Representative HPLC trace of ROs extracted from a single human dissected RPE/choroid. The top right inset is a representative HPLC trace of ROs extracted from 6 pooled, 6 mm perimacular punches from human RPE/choroid. Peaks corresponding to 11-cis-RE (a), all-trans-RE (b), syn-11-cis-RO (c), and all-trans-RO (d) are labeled; peaks marked with an asterisk denote nonretinoid peaks, phase-shift artifacts, or retinoid peaks that are not central to our study. (E) Spectra of individual peaks corresponding to 11-cis-RE, all-trans-RE, syn-11-cis-RO, and all-trans-RO, from A and B. λmax values are provided for each retinoid beneath each peak. (F) Immunoblots for rhodopsin (RHO) and RPE65 in 2 homogenates of whole human neural retina and RPE/choroid, taken from the right (OD) and left (OS) eyes of a single donor. RPE65 blotting was conducted on undiluted and 1:3.5-diluted (dil.) homogenates of neural retina (Ret) and RPE/choroid (RPE).