Molecular mechanisms of neuronal death in glaucoma: development of a gene therapy approach for neuroprotection

In glaucoma, loss of retinal ganglion cells (RGCs) and axons leads to blindness; preservation of RGC neurons is therefore a major therapeutic goal. Increasing pressure (intraocular pressure, IOP) is a major risk factor in primary open angle glaucoma (POAG). Therapeutic lowering of IOP has been shown to be protective against glaucoma. However, despite IOP lowering many patients continue to demonstrate progressive glaucomatous neuropathy thus warranting additional intervention strategies. Brain-derived neurotrophic factor (BDNF) and its high affinity receptor tropomyosin receptor kinase B (TrkB) are reported to play important role in preservation of RGCs. In this thesis, I have investigated BDNF-TrkB signaling in neuronal cells and have explored the binding pattern of agonist and antagonist to the neurotrophin receptors. I have also investigated TrkB activity by modulating Shp2 phosphatase (PTPN11) in SH-SY5Y cells, in both healthy rodents and an experimental glaucoma animal model, using viral vector gene therapy. The results show that BDNF regulates the GSK3β activity in RGC-5, PC-12 and animal RGCs. Shp2 modulation regulates TrkB phosphorylation and endoplasmic stress response in SH-SY5Y and in the RGCs of both healthy and experimental glaucoma animal models. Shp2 overexpression in SH-SY5Y cells and animal retina, was associated with TrkB antagonism, reduced neuritogenesis, loss of retinal structural & functional integrity and enhanced ER stress response leading to apoptotic changes. Conversely, Shp2 knock down in an animal model of elevated IOP resulted in protection of the retinal structural and functional integrity. These observations correlated with enhanced TrkB phosphorylation in RGCs in response to genetic knockdown of Shp2 expression in experimental animal glaucoma model. The current findings reinforce the role played by Shp2 in regulating signaling in neuronal cells and highlight that Shp2 dysregulation is detrimental for the inner retina. Based on these observations we propose that selective targeting of Shp2 in RGCs may be a promising therapeutic strategy in glaucoma.