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

Abstract

Duplication of theTBK1 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 in vitro. iPSCs were generated from dermal fibroblasts obtained from a patient with TBK1-associated NTG, via viral transduction of the transcription factors OCT4, SOX2, KLF4, and c-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 with TBK1 gene duplication have increased levels of LC3-II protein (a key marker of autophagy). Duplication of TBK1 has been previously shown to increase expression of TBK1 and here we demonstrate that the same duplication leads to activation of LC3-II. This suggests that TBK1-associated glaucoma may be caused by dysregulation (over-activation) of this catabolic pathway.

Keywords: Autophagy; Glaucoma; Retinal ganglion cells; Stem cells; TBK1; iPSC.

Figure 1

NTG pedigree 441. Members of this African American family with NTG are indicated with black symbols, while those that did not meet diagnostic criteria for glaucoma but were considered to have unknown glaucoma status because of their age are indicated with grey symbols. Family members with unknown glaucoma status because they were unavailable for examination are indicated with grey symbols and asterisks.

Figure 2

Derivation of iPSCs from a patient affected with TBK1-associated Glaucoma. (A) Phase micrograph of a TBK1-iPSC colony demonstrating a pluripotent morphology. (B) rt-PCR analysis of RNA isolated from WT-iPSCs and TBK1-iPSCs targeted against pluripotency marker expression. (C–F) H&E staining of TBK1-iPSC derived teratomas that show each of the three embryonic germ layers ((C) ectoderm, (D) mesoderm, and (E and F) endoderm). Scale bar=100 microns.

Figure 3

Differentiation of human TBK1-associated iPSCs into retinal ganglion cell-like neurons. (A) Schematic of methods used to produce retinal ganglion cell-like neurons. (B) rt-PCR analysis of RNA isolated from TBK1-iPSC or WT-iPSC derived retinal ganglion cell-like neurons shows expression of markers expressed by RGCs. (C and D) Immunocytochemical analysis of TBK1-iPSC derived retinal ganglion cell-like neurons targeted against the neural/retinal ganglion cell markers NF200 (C) and ATOH7 (D). (E) Schematic diagram illustrating the paradigm utilized to isolate/purify TBK1-iPSC derived retinal ganglion cell like neurons from a heterogeneous culture of differentiated cells. (F) Microscopic/morphological analysis of TBK1-associated iPSC-derived retinal ganglion cell-like neurons post-magnetic bead isolation. Scale bar=200 microns for panel C and D, and 400 microns for panel F.

Figure 4

Quantitative PCR assessment of TBK1 gene dose. The number of copies of the TBK1 gene was assessed in genomic DNA collected from white blood cells (A) and from iPSC-derived retinal ganglion cells (B) from an NTG patient from family 441 (Figure 1, III-1) and from control subject lymphocytes. This experiment confirms that the iPSC-derived cells carry theTBK1-gene duplication originally detected in lymphocytes.

Figure 5

Western blot analysis of fibroblast cells (A) and iPSC-derived retinal ganglion cells (B) with LC3B antibody. LC3-II, the lipidated isoform of LC3, is more abundant in both cell types that carry a TBK1 gene duplication than in cells with no duplication (NL), but is especially increased in iPSC-derived retinal ganglion cell-like neurons (B). Increased LC3-II suggests that autophagy is activated in iPSC-derived neurons that carry a TBK1 gene duplication.