Investigating endoplasmic reticulum (ER) stress and oxidative stress in the retina




Jade Vargas

Introduction

Vision is maintained by daily renewal of photoreceptors in the retina. Spent photoreceptor outer segments (POS) are engulfed and digested by adjacent retinal pigment epithelial (RPE) cells (Boulton 2001; Young 1967). Dysfunction or impairment with shedding, engulfment or digestion of POS is associated with disease (Mullen 1976). Oxidative stress is known to be involved with retinal dysfunction and disease (Beatty 2000; Williams 2008). A protein involved in protection from oxidative stress is the enzyme methionine-sulfoxide reductase A (MSRA). Silencing MSRA in vitro lead to decreased POS binding and engulfment by RPE and decreased RPE cell viability in response to oxidative stress (Dun 2013). In vivo, MSRA knockout leads to increased oxidative stress and impaired vision. The Finnemann Lab recently found that when MSRA knockout mice are fed an antioxidant-rich diet for four weeks, the effect of the lack of MSRA is mitigated and vision is restored. It is unclear how increased oxidative stress due to lack of MSRA leads to cellular dysfunction.

ROS are capable of inducing an endoplasmic reticulum (ER) stress response (Banerjee 2017). Additionally, ER stress is known to be involved in models of retinal degeneration (Kroeger 2012, Zhang 2014). During an ER stress response, there is upregulation in expression of many genes including transcription factors and protein folding chaperones to assist in protein folding (Walter 2011). The transcription factors include CCAAT/enhancer-binding protein homologous protein (CHOP) and X-box-binding protein 1 (XBP1). The mature XBP1 transcript is spliced during ER stress to form the XBP1 spliced (XBP1s) transcript. XBP1s encodes a highly active transcription factor protein (Yoshida 2001). The upregulated protein folding chaperones include immunoglobulin binding protein (BIP) and protein disulfide isomerase A3 (PDIA3). Thus, CHOP, BIP, PDIA3, XBP1 and XBP1s all serve as ER stress markers. In order to investigate whether ER stress is involved in the MSRA-null phenotype, the expression of ER stress markers in retinal tissue was compared between wild-type mice, MSRA knockout mice, and MSRA KO mice treated with antioxidant-rich diet.

Materials and Methods

Animals
Four month-old wild-type, MSRA knockout, and MSRA knockout mice treated with antioxidant-rich diet were sacrificed by CO2 asphyxiation. Eyeballs were enucleated and rinsed in HBSS without calcium and magnesium. The lens and vitreous humor were removed from each eye. Each animal produced one whole eye, one neural retina, and one eyecup. Each sample was placed into ice-cold RLT lysis buffer with beta-mercaptoethanol (Qiagen). Samples were then homogenized using TissueRuptor (Qiagen) and frozen at -80C until further RNA extraction.

RNA Extraction
Tissue lysates were purified from genomic DNA using Qiashredder columns (Qiagen). RNA was extracted using the Qiagen RNeasy Plus Mini Kit, following the manufacturer’s protocol. A spectrophotometer was used to measure the concentration and purity of each sample. Stock solutions of 3.33 ng/uL RNA were prepared at stored at -20C.

RT-PCR
RT-PCR was performed using the Qiagen One-Step RT-PCR Kit. Ten nanograms of RNA were amplified in a 10 uL reaction. Primers were designed to span at least one intronic region to facilitate detection of contaminating genomic DNA. Products were analyzed by gel electrophoresis. Products were then sent out for Sanger sequencing to confirm product identity (Genewiz).

Results

Expression of ER stress markers was tested in different tissues from different test groups. Whole eye, neural retina, and eyecup tissue samples were each collected from three test groups: wild-type, MSRA knockout, and MSRA knockout treated with antioxidant-rich diet (Fig. 1). No detectable change in expression of CHOP, BIP, or PDIA3 was observed using RT-PCR. GAPDH was used as a housekeeping gene. Two bands were produced by the XBP1 primer pair, which was designed to span the reported spliced region (Fig. 2). The smaller product, 170 base pairs in length, was detected in whole eye and eyecup but not in neural retina. This product was gel purified and sent for sequencing. Manual alignment of the sequence of the 170 base pair product of the XBP1 primer pair confirmed that it is the product of XBP1s (Fig. 3)

Discussion

The XBP1s transcript is a product of unconventional splicing. Inositol-requiring enzyme 1α (IRE1α) is activated during ER stress and subsequently splices the XBP1 transcript excising 26 nucleotides to form XBP1 spliced (XBP1s) (Fig. 4) (Yoshida 2001). This splicing is not spliceosome dependent. The excision of 26 nucleotides from the XBP1 transcript causes a frameshift in the open reading frame during the translation of the resulting XBP1 spliced transcript. Interestingly, this frameshift leads to a longer amino acid sequence. The protein product of XBP1s is a highly active transcription factor that promotes expression of many genes required for the ER stress response (Lee 2003).

The presence of XBP1s in eyecup samples in wild-type, MSRA KO, and MSRA KO treated with antioxidant-rich diet suggests that XBP1s is present in the tissues in the eyecup, including RPE cells, during physiological conditions. Recent studies have found that XBP1s is required for some normal RPE cell function (Ma 2016; McLaughlin 2018). Therefore, XBP1s is involved in normal cellular function as well as ER stress response in the retina. Further studies are required to determine whether ER stress is involved in the retinal cellular dysfunction observed in MSRA knockout mice.

References

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Beatty S, Koh H, Phil M, Henson D, Boulton M. The role of oxidative stress in the pathogenesis of age-related macular degeneration. Surv Ophthalmol. 2000;45: 115-134.

Boulton, M., Dayhaw-Barker, P. The role of the retinal pigment epithelium: topographical variation and ageing changes. Eye. 2001;15.3: 384-389.

D. L. Williams, “Oxidative stress and the eye,” Veterinary Clinics of North America: Small Animal Practice, vol. 38, no. 1, pp. 179–192, 2008.

Dun Y, Vargas J, Brot N, Finnemann SC. Independent roles of methionine sulfoxide reductase A in mitochondrial ATP synthesis and as antioxidant in retinal pigment epithelial cells. Free radical biology & medicine. 2013;65:10.1016.

Kroeger H, Messah C, Ahern K, et al. Induction of Endoplasmic Reticulum Stress Genes, BiP and Chop, in Genetic and Environmental Models of Retinal Degeneration. Investigative Ophthalmology & Visual Science. 2012;53(12):7590-7599.

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Margariti, A., Li, H., Chen, T., Martin, D., Vizcay-Barrena, G., Alam, S. Karamariti, E., Xiao, Q., Zampetaki, A., Zhang, Z. et al. XBP1 mRNA splicing triggers an autophagic response in endothelial cells through BECLIN-1 transcriptional activation. J. Biol. Chem. 2013;288: 859-872.

McLaughlin T, Falkowski M, Park JW, et al. Loss of XBP1 accelerates age-related decline in retinal function and neurodegeneration. Molecular Neurodegeneration. 2018;13:16.

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Figures


Figure 1-Depiction of the three mice test groups.


Figure 2-RT-PCR results for all ER stress markers and housekeeping gene in whole eye, neural retina, and eyecup from wild-type (WT), MSRA knockout (KO), and MSRA knockout mice treated with antioxidant-rich diet (KO+). A 170 base pair product was obtained from XBP1 primers.


Figure 3-RT-PCR results for XBP1 primers. Sequencing confirmed that the 170 base pair product was XBP1s.


Figure 4-Diagram depicting XBP1 splicing by IRE1α. Image adapted from Margariti 2013.


Dysfunction or impairment with daily photoreceptor renewal by retinal pigment epithelial (RPE) cells is associated with disease. RPE function and viability is compromised when methionine-sulfoxide reductase A (MSRA) is silenced or knocked out. It is unclear how increased oxidative stress due to lack of MSRA contributes to cellular dysfunction. Given that reactive oxygen species (ROS) are capable of triggering an endoplasmic reticulum (ER) stress response, ER stress may be involved. To investigate whether ER stress is involved in cellular dysfunction in MSRA knockout mice, four transcripts that are upregulated during an ER stress response were used as ER stress markers. RNA was isolated from whole eye, neural retina, and eyecup tissues from wild-type, MSRA knockout, and MSRA knockout mice treated with an antioxidant-rich diet. No changes in expression of CHOP, BIP, or PDIA3 were detected by RT-PCR. An unconventional splicing product, XBP1 spliced, was detected in the eyecup but not in the neural retina in all test groups. Further studies utilizing a more sensitive assay such as quantitative RT-PCR may be required to determine whether ER stress is involved in cellular dysfunction in MSRA knockout mice.


Full Paper

Acknowledgments

I would like to thank Dr. Finnemann for providing me with the mice used for this project and assisting me with project design. I would like to thank Faaria Fasih-Ahmad and Anthony Evans for providing constant and tireless support. Finally, I would like to thank Dr. Rubin for helpful feedback and guidance, and for making this project possible.


This document was last modified 05/15/2018.
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