Human Stem Cell Differentiated Retinal Ganglion Cells for Developing Glaucoma Neuroprotection and Cell Replacement Strategies

Abstract

Progressive loss of retinal ganglion cells (RGCs) leads to glaucoma. Early diagnosis offers an opportunity to protect existing RGCs. In advanced glaucoma, most RGCs are lost causing blindness and cell replacement therapy the only option. We used a human stem cell-based RGC differentiation model to develop neuroprotection by restoring mitochondrial homeostasis and enhancing RGC differentiation efficiency to increase the success of cell replacement therapy. Unmyelinated axons in RGCs require high levels of ATP, making disrupted mitochondria a risk factor in glaucoma. Our goal was to restore mitochondrial homeostasis through mitophagy (mitochondrial autophagy) and mitobiogenesis (mitochondrial biogenesis). Mutations in the mitophagy protein Optineurin (OPTNE50K) are found in patients with normal tension glaucoma and hence, we also used RGCs with the E50K mutation. We discovered that hRGCE50Ks suffer from mitobiogenesis issues, Parkin/Pink mediated mitophagy defects, and have OPTNE50K-Tank binding kinase-1 (TBK1) aggregates. hRGCE50Ks have lower mitochondrial mass and a higher mitochondrial load. We inhibited TBK1 to induce mitochondrial biogenesis and dissolve OPTNE50K-TBK1 aggregates. Our results show TBK1 inhibition triggered mitobiogenesis, dissolved aggregates, decreased mitochondrial ATP production load, and increased spare respiratory capacity, leading to neuroprotection. With complete RGC loss, enhancing differentiation to progenitor cells with lower cell division capacity can improve the success of cell replacement therapy and reduce teratoma formation and poor tissue integration. We observed that stem cells use proteasomes for mitochondrial degradation, while hRGCs use the lysosomal mitophagy pathway. Our results indicate that proteasomal activity declines during differentiation to hRGCs. Inhibition of proteasomal activity during early differentiation resulted in higher and faster RGC differentiation, with similar effects seen in motor neuron differentiation. We did not observe metabolic reprogramming in differentiating cells upon proteasomal activity inhibition but saw changes in cell cycle distribution, specifically an increase in the number of cells in the G1 phase. Proteomics analysis post-inhibitory treatment showed elevated neuronal differentiation proteins. Our results can be translated to minimize injection cell numbers and other risks of cell replacement therapy. In summary, my research identifies novel mechanisms for restoring mitochondrial homeostasis for neuroprotection in glaucomatous RGCs and develops an enhanced differentiation strategy to aid the success of cell replacement therapy.