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Blocking the RhoA/ROCK/cofilin/F-actin Pathway via Y27632 Protects Against MERTK-deficient Retinal Degeneration and
Lujia Feng1, Ting Zhang2, Yong Du1
1Shenzhen Eye Hospital, Shenzhen Eye Center, Southern Medical University, Shenzhen, 518040, China.
Introduction:
Retinitis pigmentosa (RP) is the most common inherited neurodegenerative retinal disease. Mer receptor tyrosine kinase (MERTK) mutations are associated with severe RP and dysfunction of the RPE. Previous studies have shown that MERTK and the Rho-associated coiled-coil-containing kinases (ROCK) pathway are involved in phagocytosis. However, the specific role of the ROCK pathway in the context of MERTK-associated RP needs to be revealed.
Methods:
We established an in vitro RP cellular model via MERTK depletion in human primary retinal pigment epithelium (HsRPE) cells by siRNAs, and RCS rats with spontaneous Mertk mutations were used as RP experimental animal models. Cell viability, apoptosis, phagocytosis, and visual function were measured by MTT, TUNEL and Annexin V/propidium iodide staining, phagocytosis assays and transmission electron microscopy, and electroretinography, respectively. The expression of RhoA, ROCK, Factin, and cofilin was determined by quantitative real-time PCR, western blotting, or immunofluorescence staining.
Results:
MERTK knockdown substantially impaired cell survival, promoted apoptosis, and suppressed phagocytosis in HsRPE cells. In RCS rats, Mertk mutations impaired phagocytosis, promoted apoptosis of the RPE, and damaged visual functions. Silencing MERTK upregulated the phosphorylation of RhoA, ROCK2, and cofilin and decreased F-actin expression in HsRPE cells. Blocking the RhoA/ROCK axis by a selective ROCK inhibitor, Y27632, rescued the MERTK depletion-induced phagocytic dysfunction and apoptosis of HsRPE cells and rat RPE.
Discussion:
Our results collectively indicate that MERTK maintains RPE survival and phagocytosis via regulating the RhoA/ROCK/cofilin/F-actin axis.
Conclusion:
This study demonstrates that MERTK deficiency impairs RPE phagocytosis and promotes cell apoptosis, leading to retinal degeneration and visual dysfunction. These findings provide new insight into RP pathogenesis and thereby offer valuable references for future drug development.
Insights
Mer receptor tyrosine kinase (MERTK) deficiency impairs retinal pigment epithelium (RPE) function and survival, leading to vision loss in retinitis pigmentosa (RP). Targeting the RhoA/ROCK pathway can restore RPE phagocytosis and cell survival.
Area of Science:
- Ophthalmology
- Genetics
- Cell Biology
Background:
- Retinitis pigmentosa (RP) is a common inherited neurodegenerative retinal disease.
- Mutations in Mer receptor tyrosine kinase (MERTK) are linked to severe RP and retinal pigment epithelium (RPE) dysfunction.
- The Rho-associated coiled-coil-containing kinases (ROCK) pathway's role in MERTK-associated RP requires elucidation.
Purpose of the Study:
- To investigate the role of the RhoA/ROCK pathway in MERTK-associated retinal pigment epithelium (RPE) dysfunction.
- To explore potential therapeutic targets for retinitis pigmentosa (RP) by understanding MERTK's regulatory mechanisms.
Main Methods:
- Established an in vitro model of RP using MERTK-depleted human primary RPE (HsRPE) cells.
- Utilized RCS rats with spontaneous Mertk mutations as an in vivo animal model.
- Assessed cell viability, apoptosis, phagocytosis, and visual function using various assays and microscopy; quantified gene and protein expression.
Main Results:
- MERTK knockdown in HsRPE cells reduced cell survival, increased apoptosis, and impaired phagocytosis.
- Mertk mutations in rats similarly affected RPE phagocytosis, apoptosis, and visual function.
- MERTK deficiency upregulated RhoA/ROCK/cofilin phosphorylation and decreased F-actin; ROCK inhibition rescued MERTK depletion-induced defects.
Conclusions:
- MERTK is crucial for maintaining RPE survival and phagocytosis through the RhoA/ROCK/cofilin/F-actin pathway.
- MERTK deficiency contributes to RP pathogenesis by impairing RPE function and promoting apoptosis.
- These findings offer insights into RP and potential avenues for future drug development.
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