An in-vivo Assay to Identify Compounds Protective Against Light Induced Apoptosis

Kanan, Yogita; Jacobi, Anne; Sawyer, Kjell; Mannel, David S.; Tink, Joyce Tombran; Al‐Ubaidi, Muayyad R.

doi:10.1007/978-0-387-74904-4_6

Cited by 3 publications

(2 citation statements)

References 9 publications

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“…Human embryonic kidney HEK-293 cells were transfected with pTarget, pTarget-RDH11-Flag, and pTarget-RDH12-Flag to generate stable cell lines expressing Flag-tagged versions of mouse RDH11 and RDH12, as described previously [20]. Stable HEK-293 clones expressing human RDH12 variants were generated using previously described expression constructs [8].…”

Section: Methodsmentioning

confidence: 99%

Retinol dehydrogenase 12 detoxifies 4-hydroxynonenal in photoreceptor cells

Marchette

Thompson

Кравцова

et al. 2010

Free Radical Biology and Medicine

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Mutations of the photoreceptor retinol dehydrogenase 12 (RDH12) gene cause the early-onset retinal dystrophy Leber congenital amaurosis (LCA) by mechanisms not completely resolved. Determining the physiological role of RDH12 in photoreceptors is the focus of this study. Previous studies showed that RDH12, and the closely related retinol dehydrogenase RDH11, can enzymatically reduce toxic lipid peroxidation products such as 4-Hydroxynonenal (4-HNE), in vitro. To explore the significance of this activity, we investigated the ability of RDH11 and RDH12 to protect stably transfected HEK-293 cells against the toxicity of 4-HNE. Both enzymes protected against 4-HNE modification of proteins and 4-HNE-induced apoptosis in HEK-293 cells. In the retina, exposure to bright light induces lipid peroxidation, 4-HNE production, and 4-HNE modification of proteins in photoreceptor inner segments, where RDH11 and RDH12 are located. In mouse retina, RDH12 -but not RDH11-protects against adduct formation, suggesting that 4-HNE is a physiological substrate of RDH12. RDH12 -but not RDH11-also protects against light-induced apoptosis of photoreceptors. We conclude that in mouse retina RDH12 reduces 4-HNE to non-toxic alcohol, protecting cellular macromolecules against oxidative modification, and protecting photoreceptors from light-induced apoptosis. This activity is of particular significance to understand the molecular mechanisms of RDH12-induced LCA.

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Section: Methodsmentioning

confidence: 99%

Retinol dehydrogenase 12 detoxifies 4-hydroxynonenal in photoreceptor cells

Marchette

Thompson

Кравцова

et al. 2010

Free Radical Biology and Medicine

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“…In this study, we used RT-PCR-based methods to explore the predicted alternative splicing of Pnpla2 . For this purpose, we used 661W cells, a mouse cone photoreceptor cell line that has been shown to respond to PEDF treatment in the event of light damage [17] and also mouse eye, heart, adipose, liver, and kidney tissues. With the tested samples, one main Pnpla2 transcript was detected.…”

Section: Introductionmentioning

confidence: 99%

Searching for Alternatively Spliced Variants of Phospholipase Domain-Containing 2 (Pnpla2), a Novel Gene in the Retina

DesJardin

Becerra

Subramanian

2013

J Clin Exp Ophthalmol

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Purpose Ensembl and other expressed sequence tag (EST) databases reveal putative alternative splice variants in mouse and rat for Pnpla2, the gene encoding pigment epithelium-derived factor-receptor (PEDF-R). The purpose of this study was to obtain experimental evidence for Pnpla2 splice variants in mouse. Materials and Methods Cultures of a mouse cell line derived from photoreceptors (661W cells) and mouse eye, heart, adipose, kidney, and liver tissues were used. Messenger RNA (mRNA) was isolated from cells and tissues, and complementary DNA (cDNA) was synthesized. Polymerase chain reaction (PCR) primer pairs were designed to flank the putative splice sites. Exon exclusion real time PCR was used to reduce amplification of the full-length Pnpla2 transcript and enhance amplification of low abundant splice variants. PCR products were resolved by agarose gel electrophoresis and detected with a UV transilluminator. Recombinant plasmids containing a human full-length PNPLA2 cDNA or a PNPLA2 cDNA lacking exon 5b (E5b) were controls to validate the techniques. Total cell lysates from 661W cells were prepared. PEDF-R protein detection was performed using western blots. Results PCR products for Pnpla2 transcripts obtained from 661W cells or various mouse tissues resolved into a single band following amplification with multiple primer pairs. Simultaneous amplification of two PNPLA2 cDNAs at various molar ratios prevented the detection of lower abundant transcripts. However, even when the cDNA for the full-length Pnpla2 transcript was significantly excluded using the exon exclusion method, no bands corresponding to Pnpla2 splice variants were detectable. Nonetheless, western blots of total 661W cell lysates with two different antibodies revealed isoforms for the PEDF-R protein. Conclusions The data provide evidence for the existence of a single, full-length Pnpla2 transcript that could give rise to a single protein product that undergoes posttranslational processing.

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Protective Effects of Flavonoids in Acute Models of Light-Induced Retinal Degeneration

Ortega¹,

Parmar²,

Golczak³

et al. 2020

Mol Pharmacol

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Degeneration of photoreceptors caused by excessive illumination, inherited mutations or aging are the principal pathology of blinding diseases. Pharmacological compounds that stabilize the visual receptor, rhodopsin, and modulate the cellular pathways triggering death of photoreceptors could avert this pathology. Interestingly, flavonoids can modulate the cellular processes such as oxidative stress, inflammatory responses, and apoptosis that are activated during retinal degeneration. As we found previously, flavonoids also bind directly to unliganded rod opsin, enhancing its folding, stability, and regeneration. In addition, flavonoids stimulate rhodopsin gene expression. Thus, we evaluated the effect of two main dietary flavonoids, quercetin and myricetin in Abca4 −/− Rdh8 −/− and WT BALB/c mice susceptible to light-induced photoreceptors' degeneration. Using in vivo imaging such as optical coherence tomography, scanning laser ophthalmoscopy and histological assessment of retinal morphology, we found that treatment with these flavonoids prior to light insult remarkably protected retina from deterioration and preserved its function. Using HPLC-MS analysis, we detected these flavonoids in the eye upon their intraperitoneal administration. The molecular events associated with the protective effect of quercetin and myricetin were related to the elevated expression of photoreceptor-specific proteins, rhodopsin and cone opsins, decreased expression of the specific inflammatory markers, and the shift of the equilibrium between BAX/BCL-2 towards an anti-apoptotic profile. These results were confirmed in photoreceptor-derived 661W cells treated with either H 2 O 2 or all-trans-retinal stressors implicated in the mechanism of retinal degeneration. Altogether, flavonoids could have significant prophylactic value for retinal degenerative diseases.

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An in-vivo Assay to Identify Compounds Protective Against Light Induced Apoptosis

Cited by 3 publications

References 9 publications

Retinol dehydrogenase 12 detoxifies 4-hydroxynonenal in photoreceptor cells

Retinol dehydrogenase 12 detoxifies 4-hydroxynonenal in photoreceptor cells

Searching for Alternatively Spliced Variants of Phospholipase Domain-Containing 2 (Pnpla2), a Novel Gene in the Retina

Protective Effects of Flavonoids in Acute Models of Light-Induced Retinal Degeneration

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