ライフサイエンスコーパス: A2E

1 A2E accumulation and retinal degeneration were prevented

2 A2E free cells were also irradiated and analyzed.

3 A2E has been shown to undergo light-dependent oxidation

4 A2E in PBS, with and without an oxygen-depleter or singl

5 A2E localized to lysosomes in cultured RPE as well as in

6 A2E that had accumulated in ARPE-19 cells exhibited irra

7 A2E that was not cell-associated underwent irradiation-i

8 A2E was synthesized and incubated with an adult RPE cell

9 A2E, a major lipofuscin fluorophore that accumulated dur

10 A2E, the major fluorophore of lipofuscin, and its precur

11 A2E-loaded RPE cells in culture bound and internalized i

12 A2E-loaded RPE degraded outer segment proteins efficient

13 scence spectroscopy revealed that A2PE-H(2), A2E, and N-retinylidene-N-retinyl-phosphatidylethanolami

14 with this, the lipofuscin fluorophores A2PE, A2E, and A2PE-H(2), which form from retinaldehyde, were

15 Mice lacking RmP accumulate A2E in RPE cells at a greatly increased rate over contro

16 Human RPE cells (ARPE-19) accumulated A2E from 10, 50, and 100 microM concentrations in media,

17 ARPE-19 cells accumulated A2E before exposure to blue light.

18 inal pigment epithelium that had accumulated A2E and were irradiated (430 nm).

19 overlying ARPE-19 cells that had accumulated A2E and were irradiated to induce A2E photooxidation.

20 man RPE (ARPE-19) cells that had accumulated A2E were exposed to blue light in the presence and absen

21 man RPE cells (ARPE-19) that had accumulated A2E were exposed to blue light.

22 In RPE cells that had previously accumulated A2E, caspase-3 activity was detected within 5 hours of b

23 ng to both A2E and A2E-rabbit serum albumin (A2E-RSA) conjugate but no cross-reactivity with various

24 Also, A2E levels in abcr(-/-) mice reared under cyclic light a

25 Accumulation of A2PE-H(2) and A2E in abcr+/- retina and RPE, respectively, was strongl

26 ot analysis revealed binding to both A2E and A2E-rabbit serum albumin (A2E-RSA) conjugate but no cros

27 e mice resulted in the formation of A2PE and A2E.

28 light and electron microscopic analysis, and A2E quantification.

29 epithelium, photoreceptor degeneration, and A2E accumulation.

30 correlate the distribution of lipofuscin and A2E across the human RPE.

31 tween the emission spectra of lipofuscin and A2E is fortuitous, and the collective data indicate the

32 Lipofuscin and A2E levels in the RPE increased with age and more so in

33 and rates of increase of RPE lipofuscin and A2E were not different between dark-reared and cyclic li

34 posure in the accumulation of lipofuscin and A2E, we analyzed RPEs and isolated rod photoreceptors fr

35 A2E was not found in any of these mice, and A2E oxidation was not induced by blue light and UV illum

36 ygen-depleter or singlet-oxygen quencher and A2E-laden RPE, were exposed to 430-nm light and examined

37 gy is dispensable for retinoid recycling and A2E deposition; however, autophagy plays a role in copin

38 effects of HPR on visual cycle retinoids and A2E biosynthesis, HPR was chronically administered to AB

39 NF-alpha, IL-1beta, and TLR4 transcripts and A2E were significantly lower in naloxone-treated DKO ani

40 fluorescence spectroscopy studies, antibody-A2E binding was evidenced by a fluorescence increase and

41 compounds within this lipofuscin mixture are A2E and all-trans-retinal dimer.

42 of the final product A2E and was consumed as A2E accumulated.

43 Fluorescent bisretinoids, such as A2E and all-trans-retinal dimer, form as a by-product of

44 of cytotoxic lipofuscin fluorophores such as A2E within the retinal pigment epithelium.

45 ion and to the formation of pigments such as A2E, and is believed to play a key role in the formation

46 indicate that bis-retinoid pigments, such as A2E, that accumulate as lipofuscin in retinal pigment ep

47 Using cell-associated A2E as a model of RPE bisretinoid behavior, photobleachi

48 ughter ions that were identical to authentic A2E and its daughter ions.

49 Because A2E accumulation in the RPE is associated with pathogene

50 the RPE to be characterized as yet have been A2E and its isomers.

51 rans-retinal and not, as generally believed, A2E or other retinoid condensation products.

52 When the pyridinium bisretinoid A2E, an age-related fluorophore in the retinal pigment e

53 f RPE lipofuscin, the pyridinium bisretinoid A2E, is a diretinal conjugate that forms in photorecepto

54 The bisretinoid A2E was measured by quantitative high performance liquid

55 were allowed to accumulate the bisretinoid, A2E, and were irradiated at 430 nm.

56 light and reduced levels of the bisretinoids A2E and A2-glycero-phosphoethanolamine (A2-GPE).

57 Characterization of the bisretinoids A2E and the all-trans-retinal dimer series has shown tha

58 Analysis of the bisretinoids A2E, A2-GPE, A2-dihydropyridine-phosphatidylethanolamine

59 s dot-blot analysis revealed binding to both A2E and A2E-rabbit serum albumin (A2E-RSA) conjugate but

60 plays a role in coping with stress caused by A2E accumulation.

61 The membrane damage exhibited by A2E-accumulating RPE is dependent on the concentration o

62 onstrate, acts against apoptosis mediated by A2E, a byproduct of phototransduction that becomes toxic

63 vidence that one of the products released by A2E photodegradation is methylglyoxal, a low molecular w

64 Comparable A2E concentrations were detected in the eyes of 6-month-

65 loaded or not with the lipofuscin component A2E and inhibiting or not mitochondrial ATP synthesis ei

66 l cells accumulate the lipofuscin component, A2E.

67 and analyzed for the bis-retinoid compounds A2E and iso-A2E.

68 One of these compounds, A2E, is generated by phosphate hydrolysis of a phosphati

69 ight- exposed RPE cells that did not contain A2E remained viable.

70 lculation, based on the structure of dihydro-A2E, produced a simulated UV-visible absorbance spectrum

71 proximately fivefold for the vitamin A dimer A2E--and subsequent lipofuscinogenesis and normalizes th

72 to investigate the role of autophagy during A2E accumulation.

73 compound diretinoid-pyridinium-ethanolamine (A2E) were increased in Rdh12-deficient mice of various g

74 ation of diretinoid-pyridinium-ethanolamine (A2E), a condensation product of all-trans-retinal and a

75 cluding di-retinoid-pyridinium-ethanolamine (A2E), are thought to be transferred to RPE cells primari

76 ARPE19 cells were examined to evaluate A2E and RALdi oxidation and toxicity induced by exposure

77 on and intersystem crossing of photo-excited A2E.

78 Light-exposed A2E-laden retinal pigment epithelium exhibits a propensi

79 C measurements of the lipofuscin fluorophore A2E also revealed age-associated increases, and the fold

80 The lipofuscin fluorophore A2E has been shown to mediate blue light-induced damage

81 The lipofuscin fluorophore A2E is known to be an initiator of blue-light-induced ap

82 The major constituent is the fluorophore A2E, a pyridinium bisretinoid.

83 RPE lipofuscin, including the fluorophore A2E, forms in large part as a byproduct of the visual cy

84 A toxic vitamin A-based fluorophore (A2E) present within lipofuscin has been implicated in th

85 The major lipofuscin fluorophore, A2E, is a pyridinium bisretinoid.

86 tigated the role of lipofuscin fluorophores (A2E-lipofuscin) on oxidative stress and complement activ

87 The binding constant for A2E and purified RPE65 was calculated to be 250 nM.

88 vitro, suggesting a potential mechanism for A2E cytotoxicity.

89 lyzed ocular tissues from abcr(-/-) mice for A2E oxiranes by mass spectrometry.

90 iously hypothesized biosynthetic pathway for A2E and implicate A2PE-H(2) as a precursor in this pathw

91 intracellular lipofuscin resembles that for A2E.

92 Also, once formed, A2E was not eliminated by the RPE.

93 st in part, for cellular damage ensuing from A2E photooxidation.

94 cies of oxidized A2E, peroxy-A2E, and furano-A2E, followed by incubation with serum, also activated c

95 that the yellow-emitting fluorophores [e.g., A2E (2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-y

96 epithelium that were irradiated to generate A2E photooxidation products.

97 A2-PE; (iii) hydrolysis of A2-PE to generate A2E.

98 ction mixture with phospholipase D generated A2E, as detected by high performance liquid chromatograp

99 l illuminance was not correlated with higher A2E.

100 all-trans-retinal clearance, and the highest A2E amounts were found in Rdh8(-/-)Rdh12(-/-)Abca4(-/-)

101 The death of blue-light-illuminated A2E-laden RPE was blocked in oxygen-depleted media.

102 These studies implicate A2E as an initiator of blue light-induced apoptosis of R

103 n the presence of caspase-3 inhibitor and in A2E-loaded RPE cells that had been stably transfected wi

104 emission maximum consistent with a change in A2E milieu upon antibody binding.

105 es oxygen-dependent photochemical changes in A2E, indicates that the effects of singlet oxygen may be

106 The decline in A2E levels in the Abca4(-/-) mice corresponded to reduce

107 dant, produced a more pronounced decrease in A2E-epoxidation than vitamin C, and treatment with both

108 DNA damage was induced in A2E-containing RPE exposed to 430-nm illumination.

109 the cell death program that is initiated in A2E-containing cells in response to blue light.

110 ther autophagy-related molecules involved in A2E accumulation, we performed gene expression array ana

111 yde, which constitutes the first reactant in A2E biosynthesis.

112 l mechanism for cholesterol sequestration in A2E-loaded cells.

113 phane and N-acetylcysteine; samples included A2E-free cells.

114 ccumulated A2E and were irradiated to induce A2E photooxidation.

115 ell damage mediated by aging/disease-induced A2E accumulation.

116 jugate indicated the presence of five intact A2E molecules covalently linked to BSA, thus deeming it

117 rations in media, the levels of internalized A2E ranging from less than 5 to 64 ng/10(5) cells, as as

118 The emission spectrum of internalized A2E was also determined.

119 ation of RPE in the setting of intracellular A2E initiates a cell death program that is executed by a

120 e oxo-aldehydes are released from irradiated A2E-containing RPE cells.

121 best characterized of these fluorophores is A2E, a compound consisting of two retinoid-derived side

122 est characterized component of lipofuscin is A2E, a bis-retinoid byproduct of the normal retinoid vis

123 orescence and elevated levels of A2E and iso-A2E, major bis-retinoid pigments of lipofuscin.

124 d for the bis-retinoid compounds A2E and iso-A2E.

125 Likewise, A2E also inhibited isomerohydrolase activity in cells co

126 ts of retinal pigment epithelium lipofuscin, A2E isomers with cis olefins at positions other than the

127 However, sub-toxic levels of lysosomal A2E are sufficient to reduce phagocytic activity of RPE

128 found that physiological levels of lysosomal A2E reduced mitochondrial membrane potential and inhibit

129 measured after exposure to 50 and 100 microM A2E were attributable to membrane damage in a subpopulat

130 ting in cells incubated with 10 to 25 microM A2E were comparable to the amounts of A2E present in equ

131 ometry, we have also detected both monofuran-A2E and monoperoxy-A2E in aged human RPE and in eye cups

132 o detected both monofuran-A2E and monoperoxy-A2E in aged human RPE and in eye cups of Abca4/Abcr-/- m

133 Moreover, A2E levels declined after 8 months of age, a change not

134 e revealed that during irradiation (430 nm), A2E self-generates singlet oxygen with the singlet oxyge

135 Ocular A2E was measured using HPLC.

136 cycle retinoids and arrested accumulation of A2E and lipofuscin autofluorescence in the RPE.

137 se data demonstrate that the accumulation of A2E is not responsible for the increase in lipofuscin fl

138 No significant ocular accumulation of A2E occurred under these conditions.

139 hy, which is associated with accumulation of A2E, a diretinoid adduct within the retinal pigment epit

140 The amount of A2E, a major lipofuscin component, increased 10- to 12-f

141 Amounts of A2E in the RPE correlated with diminished all-trans-reti

142 microM A2E were comparable to the amounts of A2E present in equal numbers of RPE cells harvested from

143 sites can be assigned to the shorter arm of A2E, to the longer arm, or to both arms by analyzing cha

144 iments suggest a direct, specific binding of A2E to RPE65.

145 The role of A2PE-H(2) in the biogenesis of A2E and its relationship to other retinal fluorophores h

146 e previously proposed that the biogenesis of A2E involves the following: (i) formation of the Schiff

147 ations suggest that both the biosynthesis of A2E and its conversion to oxiranes are accelerated by li

148 ned questions related to the biosynthesis of A2E, a fluorophore that accumulates in retinal pigment e

149 Dillon & J. Sparrow, for the biosynthesis of A2E: (i) condensation of all-trans-retinaldehyde (all-tr

150 Colocalization of A2E and the Lysotracker probe revealed a preferential ac

151 stigate the cellular compartmentalization of A2E, cells were incubated simultaneously with A2E and a

152 ing RPE is dependent on the concentration of A2E and reflects the ability of this amphiphilic compoun

153 on and as a function of the concentration of A2E used to load the cells before illumination.

154 oducts was accompanied by the consumption of A2E, the latter being diminished, however, when illumina

155 ed in the mechanisms leading to the death of A2E-containing RPE cells after blue light illumination.

156 ries of higher molecular mass derivatives of A2E.

157 s no correlation between the distribution of A2E and lipofuscin, as the levels of A2E were highest in

158 t description of the spatial distribution of A2E in the human RPE by imaging mass spectrometry.

159 The spatial distributions of A2E and its oxides were determined using matrix-assisted

160 rum may contribute to the adverse effects of A2E accumulation, with the A2E photooxidation products b

161 Antioxidants reversed these effects of A2E, suggesting that A2E damage is mediated by oxidative

162 Given the adverse effects of A2E, there is considerable interest in combating its dep

163 Similarly, exposure of A2E-loaded ARPE-19 cells to blue light resulted in a 40%

164 Isotretinoin blocked the formation of A2E biochemically and the accumulation of lipofuscin pig

165 Finally, we showed that the formation of A2E oxiranes is strongly suppressed by treating the abcr

166 The current model for the formation of A2E requires photoactivation of rhodopsin and subsequent

167 As predicted by this model, formation of A2E was completely inhibited when abcr-/- mice were rais

168 RPE65 antagonists abolishes the formation of A2E.

169 Whereas the generation of A2E from A2-PE by acid hydrolysis was found to occur ver

170 The identity of A2E was confirmed by tandem mass spectrometry.

171 induced by blue light and UV illumination of A2E-loaded ARPE19 cells.

172 Subretinal injection of A2E significantly inhibited retinoid isomerohydrolase ac

173 ts that can be damaged by the interaction of A2E and blue light.

174 To determine the direct interaction of A2E with RPE65 protein, fluorescence binding assays were

175 Internalization of A2E by the RPE cells was evidenced by the acquisition of

176 metry detection revealed that irradiation of A2E was associated with A2E photoisomerization, photooxi

177 The levels of A2E accumulated by the cultured cells were quantified by

178 The levels of A2E accumulating in cells incubated with 10 to 25 microM

179 ased autofluorescence and elevated levels of A2E and iso-A2E, major bis-retinoid pigments of lipofusc

180 tudied the effect of physiological levels of A2E in RPE cultures on their ability to phagocytose oute

181 tion of A2E and lipofuscin, as the levels of A2E were highest in the far periphery and decreased towa

182 Loading of A2E or exposure to blue light alone had little effect on

183 Loading of A2E, with or without exposure to blue light, upregulated

184 esults corroborate the proposed mechanism of A2E biogenesis.

185 This study elucidates the mechanism of A2E biosynthesis and suggests that administration of C20

186 emonstrate the vitamin A-dependent nature of A2E biosynthesis and validate a novel therapeutic approa

187 retinal resulted in slight overproduction of A2E, a condensation product of all-trans-retinal and pho

188 -MS) we demonstrate that photodegradation of A2E and all-trans-retinal dimer generates the dicarbonyl

189 inoids have revealed that photoexcitation of A2E by wavelengths in the visible spectrum leads to sing

190 of products resulting from photooxidation of A2E might include a range of fragments that could be rec

191 glet oxygen production and photooxidation of A2E.

192 dylethanolamine (A2PE), a known precursor of A2E, share common electronic and resonant structures.

193 ations and was proposed to be a precursor of A2E.

194 The precursors of A2E identified in this study may represent pharmacologic

195 Here, we identify three precursors of A2E in ocular tissues from abcr-/- mice and humans with

196 eptides were cross-linked in the presence of A2E (adduct of two vitamin A aldehyde and ethanolamine)

197 generated by endoperoxide in the presence of A2E revealed that vitamin E, butylated hydroxytoluene, r

198 sly reported that photooxidation products of A2E can activate complement.

199 ay, reaction of collagen IV with products of A2E photodegradation resulted in reduced cleavage by the

200 the generation of reactive photo-products of A2E.

201 products in vitro Finally, quantification of A2E demonstrated the acquisition of retinal condensation

202 However, the precise role of A2E in vision loss is unclear.

203 An unbiased proteome screen of A2E-aged patient-specific iPS-derived RPE cell lines ide

204 ght-dependent oxidation to yield a series of A2E epoxides or oxiranes.

205 lyzing changes in the UV-visible spectrum of A2E, and we have observed a preference for oxidation on

206 ules also resembles the emission spectrum of A2E, but the spectrum of individual granules varies sign

207 were located in blue light- exposed zones of A2E-containing RPE cells, whereas cells situated outside

208 performed gene expression array analysis on A2E-treated human RPE cells and found up-regulation of f

209 to define the effect of increasing light on A2E accumulation.

210 Exposure to H2O(2) or A2E-mediated photooxidation also resulted in a twofold t

211 exposed to continuously generated H2O(2) or A2E-mediated photooxidation.

212 osure of RPE cells to H(2)O(2), paraquat, or A2E-mediated photooxidation resulted in increased expres

213 Singly- and doubly-oxidized A2E had distributions similar to A2E, but represented <1

214 However oxidized A2E was not found in any of these mice, and A2E oxidatio

215 crotiter plates with two species of oxidized A2E, peroxy-A2E, and furano-A2E, followed by incubation

216 he production of endoperoxide-containing oxo-A2E may account, at least in part, for cellular damage e

217 By studying the oxidation products (oxo-A2E) generated using oxidizing agents that add one or tw

218 tes with two species of oxidized A2E, peroxy-A2E, and furano-A2E, followed by incubation with serum,

219 rum incubated in wells precoated with peroxy-A2E, the lipofuscin pigment all-trans-retinal dimer, and

220 on at m/z 672.8, representing the phosphoryl-A2E fragment of A2-PE.

221 different chain lengths, and the phosphoryl-A2E moiety, m/z 673.

222 ning moieties generated within photooxidized A2E include a 5,8-monofuranoid and a cyclic 5,8-monopero

223 The autofluorescent lipofuscin pigment A2E accumulates in retinal pigment epithelial cells with

224 f free all-trans-retinal, thereby preventing A2E accumulation and photoreceptor cell death.

225 Although endogenously produced A2E in the RPE has been associated with macular degenera

226 ce it formed in advance of the final product A2E and was consumed as A2E accumulated.

227 s accumulation of its condensation products, A2E, and all-trans-retinal dimer (RALdi), both associate

228 xen-1- yl)-1E,3E,5E-hexatrienyl]-pyridinium (A2E) is followed by RPE atrophy.

229 gical concentrations of glucose or pyruvate, A2E significantly inhibited phagocytosis.

230 ments in mice, and to report changes in qAF, A2E bisretinoid concentration, and outer nuclear layer (

231 autofluorescence, decreased HPLC-quantified A2E, outer nuclear layer thinning, and increased methylg

232 Rather, A2E prevents cholesterol efflux from these organelles, w

233 g, the rabbit polyclonal antibody recognized A2E that had accumulated in cultured cells, whereas dot-

234 and Abca4 all protect the retina and reduce A2E production by facilitating all-trans-retinal clearan

235 e (PNU-83836-E), and bilberry extract reduce A2E-epoxidation, whereas single cell gel electrophoresis

236 l, the trolox analogue, and bilberry reduced A2E-epoxidation by quenching singlet oxygen.

237 f 11-cis-retinal, is associated with reduced A2E accumulation.

238 Funduscopy, histopathology, retinal A2E quantification, proteomics, RT-PCR gene expression a

239 Pyridinium bis-retinoid A2E is a major component of lipofuscin which accumulates

240 cludes N-retinylidene-N-retinylethanolamine (A2E) as the major autofluorescent component.

241 uch as N-retinylidene-N-retinylethanolamine (A2E) in cells of the retinal pigment epithelium.

242 ion of N-retinylidene-N-retinylethanolamine (A2E), a toxic substance known to contribute to retinal d

243 sed to N-retinylidene-N-retinylethanolamine (A2E), tamoxifen, or chloroquine.

244 uch as N-retinylidene-N-retinylethanolamine (A2E).

245 inoid, N-retinylidene-N-retinylethanolamine (A2E).

246 inoid, N-retinylidene-N-retinylethanolamine (A2E).

247 tinoid N-retinylidene-N-retinylethanolamine (A2E).

248 bsorbing chromophores in lipofuscin and show A2E is not the dominant yellow-emitting fluorophore in m

249 etic defects in vitamin A processing, slowed A2E biosynthesis.

250 ) mice, Abca4(PV/PV) mice showed substantial A2E and lipofuscin accumulation in their RPE cells but n

251 tive to wild type and was more abundant than A2E.

252 rans-retinal clearance contributes more than A2E oxidation to light-induced cellular toxicity.

253 e efficient generator of singlet oxygen than A2E, and the all-trans-retinal dimer series was more rea

254 merohydrolase activity assays confirmed that A2E inhibited enzymatic activity of recombinant RPE65 in

255 Our results demonstrate that A2E accumulation exacerbates the effects of moderate mit

256 ss spectrometry (MS/MS), to demonstrate that A2E also undergoes photooxidation-induced degradation an

257 These results demonstrate that A2E inhibits the regeneration of 11-cis retinal, the chr

258 Our data also demonstrate that A2E, which is a cone-shaped lipid, increases the chemica

259 Here we demonstrate that A2E-epoxides, independent of singlet oxygen, exhibit rea

260 Our results provide direct evidence that A2E causes aberrant cholesterol metabolism in RPE cells

261 Taken together, these findings indicate that A2E biosynthesis involves the oxidation of a dihydropyri

262 uter segments as control RPE indicating that A2E does not alter early steps of phagocytosis.

263 Here we report that A2E efficiently inhibits RPE65 isomerohydrolase, a key e

264 Here, we show that A2E, a quaternary amine and retinoid by-product of the v

265 These results suggest that A2E accumulation causes oxidative stress, complement act

266 versed these effects of A2E, suggesting that A2E damage is mediated by oxidative processes.

267 chromatography, thus confirming A2-PE as the A2E precursor.

268 -IMS of retinal cross-sections confirmed the A2E localization data obtained in RPE flat-mounts.

269 bdamax 490 and 330 nm) was identified in the A2E biomimetic reaction mixture.

270 similar to A2E, but represented <10% of the A2E levels.

271 to membrane damage in a subpopulation of the A2E-accumulating cells, determined by fluorescence nucle

272 ight mass spectrometry (MALDI-TOF MS) of the A2E-BSA conjugate indicated the presence of five intact

273 evealed that detectable levels of A2-PE, the A2E precursor, are formed within photoreceptor outer seg

274 ator-activated receptor pathway relieves the A2E-induced block on cholesterol efflux and restores cho

275 gates in which the protein was linked to the A2E molecule via its pyridinium ethanolamine moiety.

276 dverse effects of A2E accumulation, with the A2E photooxidation products being damaging intermediates

277 In addition to A2E and related cis isomers, we previously showed that c

278 ether acute exposure of healthy RPE cells to A2E-lipofuscin affects oxidative stress and expression o

279 PE65 is also on the rate-limiting pathway to A2E formation.

280 iesterase activity that can convert A2-PE to A2E may indicate that some portion of the A2-PE that for

281 ly-oxidized A2E had distributions similar to A2E, but represented <10% of the A2E levels.

282 ells that likely were recruited to transport A2E-like condensation products to the RPE and dispose of

283 LISA) revealed that the substrate underlying A2E-containing RPE was AGE-modified after irradiation.

284 Unexpectedly, A2E levels were not higher in the albino mice.

285 Unlike A2E, the oxiranes were more abundant in albino vs. pigme

286 s more reactive with singlet oxygen than was A2E.

287 When A2E, in either acellular or cellular environments, was i

288 When A2E-laden RPE were transferred to D(2)O-based media and

289 ence of apoptotic nuclei was attenuated when A2E-containing RPE cells were exposed to blue light in t

290 determined by fluorescence imaging, whereas A2E levels were quantified by HPLC and UV-visible absorp

291 which represents a unique mechanism by which A2E may impair vision in STGD.

292 Studies in which A2E was incubated with a singlet oxygen generator yielde

293 that irradiation of A2E was associated with A2E photoisomerization, photooxidation, and photodegrada

294 ontribute to the pathologies associated with A2E.

295 were elevated in NHS placed in contact with A2E-laden retinal pigment epithelium that were irradiate

296 ence of RPE65 decreased upon incubation with A2E.

297 ith the singlet oxygen in turn reacting with A2E to generate epoxides at carbon-carbon double bonds.

298 ic titration, binding of purified RPE65 with A2E enhanced the bis-retinoid fluorescence.

299 2E, cells were incubated simultaneously with A2E and a fluorescent acidotropic probe, (Lysotracker Re

300 ) hydrolysis of the phosphate ester to yield A2E.