MAY 02, 2022
Duloxetine-activated PAX6 transcriptional responsive elements were inserted into a TRE-tomato-HEK293 reporter construct and transduced into mutant limbal stem cells. Rescue of function compared to wild-type was investigated.
The authors generated a TRE-tomato-HEK293 reporter cell line in which multiple copies of PAX6 transcriptional responsive elements had been inserted upstream. A phenotypic screening of FDA-approved biologics was conducted with the modified HEK cell line, and the selective serotonin and norepinephrine reuptake inhibitor duloxetine was identified as a promising compound. Western blots, qRT-PCR, and cell migration tests in wild-type and mutant limbal stem cells (LSCs) with nonsense mutations on one allele of PAX6 were used to analyze the effect of duloxetine on levels of endogenous PAX6 and to assess PAX6-target gene expression.
Analysis by qRT-PCR revealed that PAX6 haploinsufficiency was rescued by duloxetine, and the migration test showed that duloxetine-treated LSCs had closure kinetics comparable to wild-type LSCs. Treatment of mut-LSC with 1 μM duloxetine for 48 hours restored endogenous PAX6 expression to levels normally found in LSC parental cells. Western blotting revealed that duloxetine also activates PAX6 partially through inhibition of the ERK pathway.
Although duloxetine reduced the phosphorylation of ERK in mutant LSCs, its effect on phosphorylated ERK in wild-type cells was much weaker, raising questions about the strength of modulation. While the authors offer a hypothesis on the link between PAX6 expression in the central nervous system and the cornea, further work will be useful in elucidating the exact mechanism of duloxetine’s action.
Aniridia, caused by a dominant heterozygous mutation in the PAX6 gene, results in abnormal development of multiple eye structures, leading to conditions like aniridia-related keratopathy (ARK), which is characterized by progressive corneal opacification. Stem cell therapies and ocular surface reconstruction are stopgap measures that offer temporary improvements in vision before the disease and symptoms return. The repurposing of duloxetine into locally administered eye drops could provide a potential treatment for ARK, via enhancement of PAX6 expression.