Duplication of TBK1 Stimulates Autophagy in iPSC-derived Retinal Cells from a Patient with Normal Tension Glaucoma.

Duplication of the gene 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 pathophysiology . iPSCs were generated from dermal fibroblasts obtained from a patient with -associated NTG, via viral transduction of the transcription factors , , , and . 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 with gene duplication have increased levels of LC3-II protein (a key marker of autophagy). Duplication of has been previously shown to increase expression of and here we demonstrate that the same duplication leads to activation of LC3-II. This suggests that -associated glaucoma may be caused by dysregulation (over-activation) of this catabolic pathway.