Induction of DDIT4 Impairs Autophagy Through Oxidative Stress in Dry Eye

Area of Science:

• Ophthalmology
• Cell Biology
• Molecular Biology

Background:

• Dry eye disease (DED) is a prevalent ocular surface condition characterized by inflammation and damage to the ocular surface.
• Autophagy, a cellular degradation process, plays a crucial role in maintaining cellular homeostasis and is implicated in various ocular diseases.
• The role of DNA damage-inducible transcript 4 (DDIT4) and its impact on autophagic flux in the context of DED remains largely unexplored.

Purpose of the Study:

• To investigate the alterations in DDIT4 expression and autophagic flux within the corneal epithelium during dry eye disease.
• To elucidate the underlying molecular mechanisms connecting DDIT4, oxidative stress, mitochondrial dysfunction, and impaired autophagy in DED.
• To assess the therapeutic potential of targeting DDIT4 in mitigating DED-associated cellular damage.

Main Methods:

• Dry eye disease was modeled in C57BL/6 mice using desiccating stress and in primary human corneal epithelial cells and cell lines exposed to hyperosmolarity.
• Protein and mRNA expression levels of DDIT4, LC3-II, and SQSTM1 were quantified using Western blot, immunofluorescence, RT-PCR, and quantitative real-time PCR.
• Autophagic vacuoles were visualized via electron microscopy, while cellular reactive oxygen species (ROS), mitochondrial function, cell death, and apoptosis were assessed using specific assays and flow cytometry.

Main Results:

• Corneal epithelial cells in both in vitro and in vivo dry eye models exhibited increased expression of DDIT4, LC3-II, and SQSTM1.
• Electron microscopy revealed accumulation of autophagic vacuoles with undegraded material in hyperosmolar conditions, indicative of impaired autophagic flux.
• Knockdown of DDIT4 significantly reduced LC3-II and SQSTM1 levels, attenuated ROS production, restored mitochondrial function, and inhibited apoptosis, thereby preserving cell viability.

Conclusions:

• Elevated DDIT4 expression in dry eye disease contributes to impaired autophagic flux and reduced cell viability.
• Excessive reactive oxygen species (ROS) generation, mediated by DDIT4 induction, is a key factor in the pathogenesis of dry eye.
• Targeting DDIT4 may offer a novel therapeutic strategy for managing dry eye disease by restoring autophagic function and protecting corneal epithelial cells.