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Chun-hong Xia, Mei Li, Ian Ferguson, Xiaohua Gong; Study the essential roles of sodium/proton exchanger 8 (NHE8) in RPE. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4010.
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It remains unclear for how NHE8 mutations cause slow retinal degeneration in mice. Our previous work revealed that NHE8 knockout and NHE8-M120K point mutant mice developed aberrant retinal pigment epithelium (RPE) with disrupted cell polarity. This work aims to determine whether NHE8 is essential for the maintenance of adult RPE and to investigate the underlying mechanism of NHE8 in protein trafficking and homeostasis of mouse retina.
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Neisseria meningitidis (NmCas9) and guide RNAs carried by recombinant adeno-associated viruses (AAVs) were used to knockdown NHE8 in adult mouse retina by subretinal injection; eyes were collected 4 weeks post-injection for morphological and biochemical examination. NHE8 protein distribution and pH changes were examined in cultured human RPE cell line and mouse primary RPE cells infected with AAV-NHE8-pHluorin/mCherry or transfected with NHE8 plasmids.
A loss of photoreceptor cells appeared only in the eyes injected with AAV-Cas9 and AAV- NHE8-sgRNA that were designed to knockdown NHE8 expression. Real-time PCR analysis showed a reduction of NHE8 transcripts in NHE8-sgRNA injected retinas when compared to control eyes, injected with AAV-Cas9 and AAV-control sgRNA, which displayed normal retinal morphology. Primary RPE cells infected with AAV-NHE8-pHluorin-mCherry and AAV-NHE8-M120K-pHluorin-mCherry revealed obvious differences in pH response. NHE8 proteins were predominantly colocalized with the Golgi complex and intracellular vesicles in vivo. GPF-tagged NHE8 proteins expressed from transfected plasmids were observed in Golgi-derived vesicles in cultured human and mouse primary RPE cells.
Sustained function and regulation of NHE8 protein are essential for RPE homeostasis and the survival of photoreceptor cells in adult mice. Our data suggest that NHE8 may play an important role in pH homeostasis of RPE that is necessary for cell polarity and function. Mutant NHE8-M120K probably disrupts protein trafficking or homeostasis to impair RPE cell polarity, phagocytosis, and other functions thus leading to slow retinal degeneration.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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