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Alexandra Rasnitsyn, Tim Footz, Lance Patrick Doucette, May Yu, Michael A Walter; Analysis of Exocytosis Regulation by Transcription Factor FOXC1 and its Role in Axenfeld-Rieger Syndrome Pathogenesis. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3802.
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Axenfeld-Rieger syndrome (ARS) is characterized by abnormal development of the eye resulting in patients having a 50% chance of developing glaucoma, and therefore offers important insight to glaucoma pathogenesis. Mutations in the transcription factor FOXC1 were found to cause ARS. Therefore, identifying the genes that FOXC1 regulates will help to develop new treatments for glaucoma. A microarray screen performed by our lab identified potential targets for FOXC1, among them RAB3GAP1, SNAP25 and STXBP6 which are involved in the Ca2+ directed exocytosis pathway responsible for synaptic vesicle release. RAB3GAP1 and RAB3GAP2 mutations cause Micro and Martsolf syndromes, as well as keratoconus. We hypothesize that from the resulting dysregulation of protein levels in the exocytotic pathway, FOXC1 mutations lead to disruption of normal neurotransmitter release thereby contributing to development of ocular and neurodegenerative disorders.
A human tubercular meshwork cell line (TM1) was transfected with FOXC1 siRNA or plasmid, to knockdown or overexpress FOXC1, respectively. Western blot analysis was used to asses target protein expression while quantitative real-time PCR was used to examine changes in transcript levels.
We found that following knockdown of FOXC1 expression of RAB3GAP1 and RAB3GAP2 decreased, to 60% and 70%, respectively in TM1 cells (n=4; p=0.004, p=0.01 ). Western blot quantification showed that expression of SNAP25 protein increased two-fold following knockdown of FOXC1 (n=4; p=0.009).
We found that knockdown of FOXC1 in TM1 cells changed levels of key proteins in the Ca2+ directed exocytosis pathway. Our results indicate a possible role for FOXC1 as a regulator of exocytosis by positively regulating RAB3GAP1 and RAB3GAP2, involved in the priming stage, and negatively regulating SNAP25, involved in the docking and fusion stages of synaptic vesicle exocytosis. We further hypothesize that mutations which affect FOXC1 expression levels could disrupt normal synaptic vesicle exocytosis, leading to interruptions in neurotransmitter release, which could contribute to pathogenesis of ARS and glaucoma.
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