title: Duplication of TBK1 Stimulates Autophagy in iPSC-derived Retinal Cells from a Patient with Normal Tension Glaucoma.
abstract: Duplication of theTBK1gene causes normal tension glaucoma (NTG); however the mechanism by which this copy number variation leads to retinal ganglion cell death is poorly understood. The ability to use skin-derived induced pluripotent stem cells (iPSCs) to investigate the function or dysfunction of a mutant gene product in inaccessible tissues such as the retina now provides us with the ability to interrogate disease pathophysiologyin vitro. iPSCs were generated from dermal fibroblasts obtained from a patient withTBK1-associated NTG, via viral transduction of the transcription factorsOCT4,SOX2,KLF4, andc-MYC. Retinal progenitor cells and subsequent retinal ganglion cell-like neurons were derived using our previously developed stepwise differentiation protocol. Differentiation to retinal ganglion-like cells was demonstrated via rt-PCR targeted against TUJ1, MAP2, THY1, NF200, ATOH7 and BRN3B and immunohistochemistry targeted against NF200 and ATOH7. Western blot analysis demonstrated that both fibroblasts and retinal ganglion cell-like neurons derived from NTG patients withTBK1gene duplication have increased levels of LC3-II protein (a key marker of autophagy). Duplication ofTBK1has been previously shown to increase expression ofTBK1and here we demonstrate that the same duplication leads to activation of LC3-II. This suggests thatTBK1-associated glaucoma may be caused by dysregulation (over-activation) of this catabolic pathway.
authors.affiliation: Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA ; Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Budd A
authors.initials: BA
authors.lastname: Tucker
authors.affiliation: Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA ; Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Frances
authors.initials: F
authors.lastname: Solivan-Timpe
authors.affiliation: Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA ; Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Ben R
authors.initials: BR
authors.lastname: Roos
authors.affiliation: Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA ; Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Kristin R
authors.initials: KR
authors.lastname: Anfinson
authors.affiliation: Department of Ophthalmology, Johns Hopkins University, Baltimore, MD 21287, USA ; Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Alan L
authors.initials: AL
authors.lastname: Robin
authors.affiliation: Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA ; Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Luke A
authors.initials: LA
authors.lastname: Wiley
authors.affiliation: Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA ; Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Mullins
authors.affiliation: Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA ; Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: John H
authors.initials: JH
authors.lastname: Fingert
keywords: Autophagy; Glaucoma; Retinal ganglion cells; Stem cells; TBK1; iPSC