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Keith Zimmerman, Milton Tyler, Ningning Wang, Ronald Raab, Robert McKown, Gordon Laurie; Lacritin accelerated autophagy promotes clearance of aggregated proteins and is dependent on stress. Invest. Ophthalmol. Vis. Sci. 2013;54(15):908.
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© ARVO (1962-2015); The Authors (2016-present)
Lacritin is a natural tear protein that promotes the survival of human corneal epithelial (HCE) cells stressed with inflammatory cytokines INFG and TNF, both of which are elevated in dry eye. Survival is dependent on lacritin stimulated autophagic flux, which is likely to remove stress-aggregated and damaged proteins. We have now addressed this assumption by transduction of mCFP tagged Huntingtin mutant constructs Htt103Q and Htt25Q in HCE cells stably transduced with autophagy marker LC3B double tagged with mCherry and EGFP. Htt103Q forms toxic aggregates whereas Htt25Q remains soluble, thus only Htt103Q cells are significantly stressed.
Retroviral pBabe-puro-mCherry-EGFP-LC3B and pBabe-Hygro-Htt103Q-mCFP or pBabe-Hygro-Htt25Q-mCFP were expanded in HEK293T cells, and used to co-transduce human corneal epithelial (HCE) cells (Riken). Cells grown on glass cover slips were then treated either with 10 nM lacritin or inactive C-25 lacritin for 10, 30, 60 min. After washing, cells were fixed, rinsed, mounted and examined in a Zeiss LMS 700 confocal microscope. Colocalization of mCherry with mCFP was analyzed using Fiji.
Lacritin, but not C-25, stimulated the autophagic capture and transfer of toxic Htt103Q aggregates into autolysosomes. Capture and transfer peaked at 30 min. Transfer was monitored over time as EGFP (mCherry-EGFP-LC3B) was quenched by autolysosomal acidity , unlike pH insensitive mCherry. No lacritin stimulated autophagy was evident in Htt25Q cells in keeping with the absence of stress.
Lacritin signaling rids cells of toxic aggregated proteins that develop during stress by rapidly and transiently stimulating autophagy. Autolysosomal digestion products then feed into and restore oxidative phosphorylation.
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