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Chun-hong Xia, Mei Li, Audrey Kim, Ian Ferguson, Xiaohua Gong; Functional studies of sodium/proton exchanger 8 in adult mouse retina. Invest. Ophthalmol. Vis. Sci. 2016;57(12):243.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the essential role of sodium/proton exchanger 8 (NHE8) in the retina of adult mice and to investigate the mechanism of how NHE8 regulates pH homeostasis in the retinal pigment epithelium (RPE).
Using the novel clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Neisseria meningitidis (NmCas9), we studied the role of NHE8 in the retina of adult mice. Recombinant adeno-associated viruses (AAV) carrying NmCas9 and guide RNAs were used to knockdown NHE8 in the RPE of 4-week-old adult wild-type mice by subretinal injection; eye samples were collected 4 weeks post injection; immunostaining was performed to determine the distribution of NHE8 and other proteins in mouse retina; and NHE8 expression was examined by real-time PCR. Cultured RPE cells infected with AAV-NHE8-pHluorin-mcherry were used for pH measurements in intracellular compartments by monitoring the fluorescent intensity ratios between pH-sensitive ecliptic pHluorin and pH-insensitive mcherry.
Our previous studies revealed that NHE8 knockout and NHE8-M120K point mutant mice developed retinal degeneration associated with aberrant RPE with disrupted cell polarity; NHE8 proteins were predominantly colocalized with the Golgi complex and intracellular vesicles in the RPE as well as in the inner segments of rod photoreceptors. Current study showed a loss of photoreceptor cells in eyes injected with AAV-Cas9 and AAV-sgRNA target NHE8, while normal retinal morphology was observed in mice injected with AAV-Cas9 and AAV-control sgRNA. Real-time PCR analysis showed about 32% reduction of NHE8 transcripts in NHE8-sgRNA injected retinas compared to control-sgRNA injected retinas. Fluorescent intensity ratios revealed different pH values in some of intracellular compartments including protein trafficking pathways between RPE cells expressing wild-type NHE8 and mutant NHE8-M120K, infected with AAV-NHE8-pHluorin-mcherry and AAV-NHE8-M120K-pHluorin-mcherry.
Sustained function and regulation of NHE8 are essential for maintaining the polarity and function of RPE cells in the retina of adult mice. NHE8 regulates pH homeostasis in some intracellular compartments of RPE. Impaired NHE8 such as mutant NHE8-M120K would disrupt the protein trafficking or recycling to affect the polarity, phagocytosis and other functions of RPE cells, leading to photoreceptor cell death.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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