retinaldehyde and Stargardt-Disease

Retinaldehyde adducts (bisretinoids) accumulate in retinal pigment epithelial (RPE) cells as lipofuscin. Bisretinoids are implicated in some inherited and age-related forms of macular degeneration that lead to the death of RPE cells and diminished vision. By comparing albino and black-eyed mice and by rearing mice in darkness and in cyclic light, evidence indicates that bisretinoid fluorophores undergo photodegradation in the eye (Ueda et al. Proc Natl Acad Sci 113:6904-6909, 2016). Given that the photodegradation products modify and impair cellular and extracellular molecules, these processes likely impart cumulative damage to retina.

Topics: Albinism; Amines; Animals; Antioxidants; ATP-Binding Cassette Transporters; Darkness; Eye Color; Free Radical Scavengers; Light; Lipofuscin; Macular Degeneration; Melanosis; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Photochemistry; Retinal Pigment Epithelium; Retinaldehyde; Rod Cell Outer Segment; Stargardt Disease; Vitamin E

Dry age-related macular degeneration (AMD) and recessive Stargardt's disease (STGD1) lead to irreversible blindness in humans. The accumulation of all-trans-retinal (atRAL) induced by chaos in visual cycle is closely associated with retinal atrophy in dry AMD and STGD1 but its critical downstream signaling molecules remain ambiguous. Here, we reported that activation of eukaryotic translation initiation factor 2α (eIF2α) by atRAL promoted retinal degeneration and photoreceptor loss through activating c-Jun N-terminal kinase (JNK) signaling-dependent apoptosis and gasdermin E (GSDME)-mediated pyroptosis. We determined that eIF2α activation by atRAL in photoreceptor cells resulted from endoplasmic reticulum homeostasis disruption caused at least in part by reactive oxygen species production, and it activated JNK signaling independent of and dependent on activating transcription factor 4 and the activating transcription factor 4/transcription factor C/EBP homologous protein (CHOP) axis. CHOP overexpression induced apoptosis of atRAL-loaded photoreceptor cells through activating JNK signaling rather than inhibiting the expression of antiapoptotic gene Bcl2. JNK activation by eIF2α facilitated photoreceptor cell apoptosis caused by atRAL via caspase-3 activation and DNA damage. Additionally, we demonstrated that eIF2α was activated in neural retina of light-exposed Abca4

Topics: Activating Transcription Factor 4; Animals; Apoptosis; ATP-Binding Cassette Transporters; Eukaryotic Initiation Factor-2; Humans; Mice; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Retinal Pigment Epithelium; Retinaldehyde; Stargardt Disease

ABCA4, the gene implicated in Stargardt disease (STGD1), contains 50 exons, of which 17 contain multiples of three nucleotides. The impact of in-frame exon skipping is yet to be determined. Antisense oligonucleotides (AONs) have been investigated in Usher syndrome-associated genes to induce skipping of in-frame exons carrying severe variants and mitigate their disease-linked effect. Upon the identification of a STGD1 proband carrying a novel exon 17 canonical splice site variant, the activity of ABCA4 lacking 22 amino acids encoded by exon 17 was examined, followed by design of AONs able to induce exon 17 skipping.. A STGD1 proband was compound heterozygous for the splice variant c.2653+1G>A, that was predicted to result in in-frame skipping of exon 17, and a null variant [c.735T>G, p.(Tyr245*)]. Clinical characteristics of this proband were studied using multi-modal imaging and complete ophthalmological examination. The aberrant splicing of c.2653+1G>A was investigated in vitro in HEK293T cells with wild-type and mutant midigenes. The residual activity of the mutant ABCA4 protein lacking Asp864-Gly885 encoded by exon 17 was analyzed with all-trans-retinal-activated ATPase activity assay, along with its subcellular localization. To induce exon 17 skipping, the effect of 40 AONs was examined in vitro in WT WERI-Rb-1 cells and 3D human retinal organoids.. Late onset STGD1 in the proband suggests that c.2653+1G>A does not have a fully deleterious effect. The in vitro splice assay confirmed that this variant leads to ABCA4 transcripts without exon 17. ABCA4 Asp864_Gly863del was stable and retained 58% all-trans-retinal-activated ATPase activity compared to WT ABCA4. This sequence is located in an unstructured linker region between transmembrane domain 6 and nucleotide-binding domain-1 of ABCA4. AONs were designed to possibly reduce pathogenicity of severe variants harbored in exon 17. The best AON achieved 59% of exon 17 skipping in retinal organoids.. Exon 17 deletion in ABCA4 does not result in the absence of protein activity and does not cause a severe STGD1 phenotype when in trans with a null allele. By applying AONs, the effect of severe variants in exon 17 can potentially be ameliorated by exon skipping, thus generating partial ABCA4 activity in STGD1 patients.

Topics: Adenosine Triphosphatases; ATP-Binding Cassette Transporters; Exons; HEK293 Cells; Humans; Mutant Proteins; Retinaldehyde; Stargardt Disease

The breakdown of all-trans-retinal (atRAL) clearance is closely associated with photoreceptor cell death in dry age-related macular degeneration (AMD) and autosomal recessive Stargardt's disease (STGD1), but its mechanisms remain elusive. Here, we demonstrate that activation of gasdermin E (GSDME) but not gasdermin D promotes atRAL-induced photoreceptor damage by activating pyroptosis and aggravating apoptosis through a mitochondria-mediated caspase-3-dependent signaling pathway. Activation of c-Jun N-terminal kinase was identified as one of the major causes of mitochondrial membrane rupture in atRAL-loaded photoreceptor cells, resulting in the release of cytochrome c from mitochondria to the cytosol, where it stimulated caspase-3 activation required for cleavage of GSDME. Aggregation of the N-terminal fragment of GSDME in the mitochondria revealed that GSDME was likely to penetrate mitochondrial membranes in photoreceptor cells after atRAL exposure. ABC (subfamily A, member 4) and all-trans-retinol dehydrogenase 8 are two key proteins responsible for clearing atRAL in the retina. Abca4

Topics: Animals; ATP-Binding Cassette Transporters; Caspase 3; Mice; Photoreceptor Cells; Pore Forming Cytotoxic Proteins; Retina; Retinaldehyde; Stargardt Disease

Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the

Topics: Animals; ATP-Binding Cassette Transporters; c-Mer Tyrosine Kinase; Cells, Cultured; Disease Models, Animal; Lipofuscin; Lysosomes; Macular Degeneration; Mice; Mice, Inbred BALB C; Mice, Knockout; Phagocytosis; Photoreceptor Cells; Retina; Retinal Degeneration; Retinal Pigment Epithelium; Retinaldehyde; Rhodopsin; Stargardt Disease

Gene and drug therapies are being developed to alleviate vision loss in patients with Stargardt's disease and age-related macular degeneration (AMD). To evaluate the therapeutic effects of these treatments, organic solvents are routinely used to extract and quantify bisretinoid lipofuscin constituents, such as N-retinylidene-N-retinyl-ethanolamine (A2E) and all-trans-retinal dimer (ATR-dimer). By high-performance liquid chromatography (HPLC), we found that A2E and ATR-dimer were both altered by tetrahydrofuran (THF) and chloroform, but were stable in dimethyl sulfoxide (DMSO) or methanol (MeOH). In addition, cyclohexane and ethanol (EtOH) did not alter ATR-dimer, whereas an alteration of A2E occurred in EtOH. On the basis of these findings, we designed processes II-IV, generated by modifications of process I, a routine method to measure bisretinoid compounds in vivo. Extra amounts of either ATR-dimer or A2E in mouse eyecups were released by processes II-IV versus process I. Efforts to clarify the effects of organic solvents on lipofuscin pigments are important because such studies can guide the handling of these fluorophores in related experiments.

Topics: Animals; Chromatography, High Pressure Liquid; Lipofuscin; Macular Degeneration; Mice; Mice, Inbred C57BL; Pigment Epithelium of Eye; Retinaldehyde; Solvents; Stargardt Disease

Fluorescent bisretinoid compounds accumulate in retinal pigment epithelial (RPE) cells as a consequence of two processes: random reactions of vitamin A aldehyde in photoreceptor cell outer segments, and phagocytosis of discarded photoreceptor outer segment discs by RPE. The formation of bisretinoid is accentuated in some forms of retinal degeneration. The detection of a novel bisretinoid fluorophore that is a conjugate of all-trans-retinal and glycerophosphoethanolamine is reported.. Human RPE/choroid, eyes harvested from Abca4 (ATP-binding cassette transporter 4) null mutant mice, and biosynthetic reaction mixtures were analyzed by ultra performance liquid chromatography coupled to mass spectrometry and by nuclear magnetic resonance spectra and spectrofluorometry.. A fluorescent compound in mouse eyes and in human RPE/choroid corresponded to the product of the reaction between all-trans-retinal and glycerophosphoethanolamine (A2-GPE), as determined on the basis of molecular weight (m/z 746), absorbance (approximately 338,443 nm), and retention time. Nuclear magnetic resonance spectra were consistent with a pyridinium molecule with a glycerophosphate moiety. The emission maximum of A2-GPE was approximately 610 nm. A2-GPE accumulated with age in mouse eyes and was more abundant in Abca4(-/-) mice, a model of recessive Stargardt disease.. To date, several bisretinoids of RPE lipofuscin have been isolated and characterized, and for all of these, formation involves the membrane phospholipid phosphatidylethanolamine. Conversely, the bisretinoid A2-GPE is detected as sn-glycero-3-phosphoethanolamine (GPE) derivatized by two all-trans-retinal. The pathways by which A2-GPE may form under conditions of increased availability of all-trans-retinal, for instance in the Abca4(-/-) mouse, are discussed.

Topics: Animals; ATP-Binding Cassette Transporters; Chromatography, High Pressure Liquid; Disease Models, Animal; Diterpenes; Humans; Lipofuscin; Macular Degeneration; Magnetic Resonance Spectroscopy; Mass Spectrometry; Mice; Mice, Knockout; Phosphatidylethanolamines; Pyridinium Compounds; Retina; Retinal Pigment Epithelium; Retinaldehyde; Retinoids; Spectrometry, Fluorescence; Stargardt Disease; Vitamin A