Analysis of FOXC1 Regulation of Exocytosis

• Author / Creator

  Rasnitsyn, Alexandra

• Glaucoma is a neurodegenerative disease which is among the leading causes of blindness in the world. Glaucoma is characterized by progressive visual field loss, caused by retinal ganglion cell (RGC) death. Both surgical glaucoma treatments and medications are available; however, they only halt glaucoma progression and are unable to reverse damage. Furthermore, many patients do not respond well to treatments, illustrating the importance for better understanding of glaucoma pathogenesis. Patients with Axenfeld-Rieger syndrome (ARS) offer important insight into glaucoma progression. ARS patients are at 50% risk of developing early onset glaucoma and respond poorly to treatments, even when surgical treatments are combined with medications. Mutations in the transcription factor FOXC1 were shown to cause ARS. Alterations in FOXC1 levels were previously shown to cause ocular malformation and disrupt stress response in ocular tissues, thereby contributing to glaucoma progression. In the present work I have shown that FOXC1 regulates the expression of RAB3GAP1, RAB3GAP2 and SNAP25, three genes with central roles in both exocytosis and endocytosis, responsible for extracellular trafficking. I have shown that FOXC1 positively regulates RNA and protein levels of RAB3GAP1 and RAB3GAP2 in HeLa cells, and that FOXC1 regulation of SNAP25 in HeLa cells follows a bimodular model with either increase or decrease in FOXC1 beyond its normal range resulting in SNAP25 decrease. In immortalized human trabecular meshwork (TM1) cells FOXC1 positively regulates RAB3GAP1 and RAB3GAP2 at the RNA level, but only RAB3GAP1 at the protein level, while SNAP25 is negatively regulated by FOXC1 in TM1 cells, specifically isoform SNAP25a. FOXC1 regulation of RAB3GAP1, RAB3GAP2 and SNAP25 has physiological consequences, and in HeLa cells affects secretion of exogenous Myocilin (MYOC), a protein associated with juvenile onset glaucoma and steroid induced glaucoma. Knockdown of either FOXC1 or combined knockdown of all three of its targets, RAB3GAP1, RAB3GAP2 and SNAP25, resulted in decrease in both intracellular and extracellular MYOC. In turn, knockdown of either RAB3FAP1 or SNAP25 led to decrease in intracellular MYOC and increase in extracellular MYOC indicating increased MYOC secretion while RAB3GAP2 knockdown had the same effect on MYOC as FOXC1 knockdown. FOXC1 regulation of exogenous MYOC secretion is therefore complicated and mediated through its targets, RAB3GAP1, RAB3GAP2 and SNAP25. My research reveals that FOXC1 is an important regulator of exocytosis and established a new link between FOXC1 and MYOC-associated glaucoma.

• Subjects / Keywords

  • FOXC1
  • Exocytosis
  • Glaucoma
  • RAB3GAP1
  • RAB3GAP2
  • SNAP25
  • MYOC

• Graduation date

  Fall 2016

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3B56DK11

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.