Saturday, May 17, 2025
5/17/2025
Stem cell approaches to normal tension glaucoma - ABSTRACT Glaucoma is the leading cause of irreversible blindness characterize by loss of retinal ganglion cells (RGCs). High intraocular pressure (IOP) increases risk for glaucoma, but glaucoma often occurs at low IOP (i.e., normal tension glaucoma (NTG)). As all current glaucoma therapies target high IOP, there is a critical need for new therapies for NTG that occurs at already low IOPs. We have researched the causes and cures for NTG by studying single-gene forms of disease. We discovered that duplication of the TBK1 gene causes NTG (1 of 3 known single-gene causes). TBK1 encodes a kinase that phosphorylates an autophagy receptor OPTN (which is encoded by another NTG gene) as a key step in stimulating autophagy, a ubiquitous catabolic cellular process that degrades defective organelles (mitochondria) and protein aggregates. We studied fibroblast cells from our NTG patients with TBK1 gene duplications and engineered transgenic mice with an extra dose of the TBK1 gene and showed that these mutations cause glaucoma by increasing TBK1 activity in RGCs (increased transcription, protein abundance, and phosphorylation of OPTN). Moreover, we and others have shown that TBK1 and OPTN mutations that cause NTG are also associated with dysregulation of autophagy. Mitochondria are strongly implicated as a chief target of dysregulated autophagy (mitophagy) in glaucoma. Consequently, we hypothesize that TBK1 gene duplications cause RGC death and glaucoma by dysregulating autophagy and maintenance of mitochondrial function. We plan to test this hypothesis, by creating and analyzing induced pluripotent stem cell (iPSC)-derived retinal ganglion cells with TBK1 gene duplications and their isogenic controls. We propose the following 3-part aim. AIM 1: Determine the influence of TBK1 gene duplication on RGC health and function using NTG patient cell culture, stem cell biology, and genome editing. A. Use CRISPR to correct TBK1 gene duplications in 3 iPSC lines from 3 NTG patients (iPSC lines are in hand). B. Differentiate iPSC lines into RGCs using our 3D retinal organoid protocol (3 mutant and 3 isogenic controls) C. Characterize differences in transcription, autophagy, and mitochondrial function between iPSC-derived RGC lines and isogenic controls using Seahorse, scRNAseq, and autophagy assays. We have already created iPSC lines from 3 NTG patients with TBK1 duplications and have extensive experience with genome editing, iPSC biology, RGC differentiation, and autophagy/mitochondrial assays to facilitate completion of this proposal. We have also published pilot studies of a single iPSC line with TBK1 mutations. TBK1 is at the center of the pathophysiology of a proportion of NTG cases. We have proposed experiments that will gain insight into the mechanisms by which TBK1 mutations cause disease and will facilitate future research to design and test new treatments for NTG patients who already have low IOP.
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