Experimental approaches to the study of A2E, a bisretinoid lipofuscin chromophore of retinal pigment epithelium

Abstract

Bisretinoid lipofuscin compounds that accumulate in retinal pigment epithelial (RPE) cells are implicated in the pathogenesis of some forms of macular degeneration. In the development of approaches to the amelioration of retinal disorders characterized by enhanced RPE lipofuscin formation, attention is being given to therapies that reduce the production of these damaging pigments. An understanding of the biosynthetic pathways by which these molecules form is essential to the development of these therapies. Thus methods for studying the biosynthesis of these compounds are presented. A tissue culture model is also described whereby a human RPE cell line that is otherwise devoid of bisretinoid lipofuscin compounds is employed and synthesized A2E is delivered to the cells. This approach allows for a population of RPE cells that have accumulated the lipofuscin fluorophore A2E in addition to A2E-free cells.

Fig. 18.1

Structures and absorbance maxima (λ_max). (a) Structures and λ_max of A2E and the biosynthetic precursors dihydropyridinium-A2PE and A2PE. The structure of A2E is that of a pyridinium bisretinoid. (b) Structures and λ_max of the all-trans-retinal dimer series of compounds: all-trans-retinal dimer-PE, all-trans-retinal dimer-E, and all-trans-retinal dimer. (c) Structure and λ_max of all-trans-retinol. Absorbance maxima of bischromophores in (a) and (b) can be assigned to the shorter and longer side arms. All-trans-retinol (c) has an absorbance maximum at 325 nm that reflects an extended conjugation system consisting of a polyene chain of four double bonds with a fifth conjugated olefin in the β-ionone ring. For the bischromophore dihydropyridinium-A2PE, an absorbance maximum of ~330 nm is generated by the same conjugation system along the short arm. Similarly, absorbance maxima of A2E and A2PE originate from the shorter and longer side arms extending from the pyridinium ring. Note that the short arm of all-trans-retinal dimer-PE and all-trans-retinal dimer-E has only four double bonds (three along the polyene chain and one in the β-ionone ring); accordingly, the absorbances generated from the short arms of these compounds are blue-shifted relative to the absorbances generated from the short arm of A2E, dihydropyridinium-A2PE, and A2PE. For all-trans-retinal dimer-PE and all-trans-retinal dimer-E, the absorbance generated from the long arm exhibits a bathochromic shift (red shift) due to protonation of the imine functional group (–C=N–).

Fig. 18.2

Biosynthetic pathway of A2E. All-trans-retinal generated by light-mediated isomerization of 11-cis-retinal reacts with phosphatidylethanolamine (PE) to generate the Schiff base adduct N-retinylidene-phosphatidylethanolamine (NRPE). After [1,6]-proton tautomerization, reaction with a second molecule of all-trans-retinal and 6π-electrocyclization, a phosphatidyl-dihydropyridinium bisretinoid (dihydropyridinium-A2PE) is formed. Dihydropyridinium-A2PE would undergo a 1,3-H shift and hydrogen atom elimination to give rise to A2-DHP-PE or eliminate two hydrogens to form A2PE, a phosphatidyl-pyridinium bisretinoid. Following aromatic autooxidation, A2PE is generated. Hydrolysis of the phosphate ester of A2PE by phospholipase D yields A2PE. Under room light, A2E and isoA2E reach photoequilibrium at a ratio of 4:1.

Fig. 18.3

HPLC monitoring of biomimetic synthesis of A2PE and A2E. (a) Synthesis of A2E with all-trans-retinal and ethanolamine as starting materials. Incubation for 7 days. In this reaction mixture ethanolamine was substituted for phosphatidylethanolamine; the latter would be the reactant in vivo. Dihydropyridinium-A2E (A2E-H₂) is detected as an intermediate of this biosynthetic reaction. Oxidation of the latter intermediate yields A2E. (b) Biomimetic synthesis of lipofuscin pigments using all-trans-retinal and phosphatidylethanolamine as starting materials. Incubation for 7 days. Compounds generated within the reaction mixture include N-retinylidene-phosphatidylethanolamine (NRPE), A2-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE), and A2PE, known intermediates of the A2E biosynthetic pathway. Also generated are compounds of the all-trans-retinal dimer series: all-trans-retinal dimer (atRALdi) and all-trans-retinal dimer-PE (atRALdi-PE). (c) A2E is generated from A2PE by phospholipase D (PLD)-mediated hydrolysis. A2PE was incubated in the absence (left) and presence (right) of the lysosomal enzyme PLD for 3 h. Reverse-phase C18 (a, b) and C4 (inset in b, c) columns and monitoring at 490 (a, b) and 430 nm (c).