Abstract
Purpose :
In the AMD (Age-related Macular Degeneration) RPE transmitochondrial cell lines, the protective effects of Humanin G (HNG) mitochondrial derived peptide are mediated through the extracellular trimeric CNTFR/gp130/WSX-1 receptor. Binding of Humanin to this receptor leads to the activation of downstream signaling factors, phosphorylation of JAK2 and STAT3, and transcription of genes that confer cytoprotection in AMD.
Methods :
We created a novel CRISPR-edited triple knock-out (KO) cell pool of the extracellular trimeric CNTFR/gp130/WSX-1 receptor genes in a wet AMD RPE transmitochondrial cybrid cell line by sequential knock-out of these genes and performed Sanger sequencing to confirm the knockout. HNG was exogenously added to AMD RPE transmitochondrial cybrid cells, which had identical nuclei from the mitochondria-deficient ARPE-19 cells but differed in mitochondrial DNA (mtDNA) content which was derived from a clinically characterized AMD patient. Cell viability and apoptotic cell death were compared between AMD wild-type (WT) vs. AMD KO cells using the MTT assay and Caspase- 3/7/ NucLight IncuCyte® live-cell imaging.
Results :
Sanger sequencing of the KO cell pool results: (1) CNTFR gene: KO score=88 %; Indel=94 %; (2) gp130 gene: KO score=62 %; Indel=64 %; (3) WSX-1 gene: KO score=94 %; Indel=99 %. These results demonstrate successful CRISPR editing of the trimeric receptor genes. There was decreased (P = 0.02) cellular metabolic activity that reflects cell viability in AMD KO untreated cells (0.88 ± 0.03) compared to AMD WT untreated cells (1 ± 0.04). Higher viable cell number (IncuCyte live-cell imaging) was observed in HNG-treated AMD WT cells (1.14 ± 0.02) compared with untreated AMD WT cells (1 ± 0.04, P = 0.03). We found significant reduction (P<0.01) in cell number in untreated AMD KO vs. untreated AMD WT cells. HNG-induced rescue was reduced significantly in AMD KO cells compared to that in AMD WT cells (P<0.05).
Conclusions :
The CRISPR-Cas9 knockout (CRISPR-KO) of the CNTFR/gp130/WSX-1 receptor reduces cell survival in AMD KO cybrids in vitro and blunts the protective effects of exogenous HNG, demonstrating the crucial role of this receptor in HNG-mediated cytoprotection.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.