Investigating the role of amyloid protein in the retina and its crosstalk with neuroserpin in glaucoma

<p dir="ltr">Primary open-angle glaucoma (POAG) is a subset of glaucomatous optic neuropathies characterised by progressive neurodegeneration of inner retinal ganglion cell (RGCs) populations, and axonopathy. There are several genetic and environmental factors associated with POAG susceptibility and progression, with high intra-ocular pressure (IOP) conferring the greatest risk. However, POAG retinal neurodegeneration can persist despite IOP involvement. Populational studies have increasingly reported age as a contributing factor to glaucoma and there is accumulating evidence indicating a potential overlap between the downstream cellular signalling cascades activated in glaucoma and Alzheimer’s Disease (AD). The core tenant of AD neuropathology involves amyloid protein deposition, and this has also been reported in the retinas of AD-afflicted individuals. Notably, chronic glaucoma is also capable of increasing local amyloid production with detrimental effects on the ganglion cell layer and optic nerve head to induce impaired axonal neurotrophic support. Another protein of interest is the serine protease inhibitor neuroserpin, that canonically inhibits tPA in the central nervous system, and is known to undergo oxidative inactivation in glaucomatous retina, resulting in excessive plasmin proteolytic degradation.</p><p dir="ltr">Therefore, this study investigated the role of amyloid protein during chronic glaucoma, with the precedent that enhanced mutant APP expression exacerbates experimental chronic glaucoma, and APP genetic silencing imparts retinal neuroprotection. Theron, this study seeks to determine whether genetic silencing may confer phenotype rescue of transgenic NS^-/- mice that demonstrate inner retinal functional deficits. This was achieved using a novel modified AAV-PHP.eB vector constructed on an AAV serotype-9 backbone that have been demonstrated to drive efficient neuronal gene modulation. Opening studies of this thesis validate that AAV-PHP.eB constructs are capable of inducing significant transgene expression in cultured human SH-SY5Y neurons, and in also driving visual functional decline of healthy C57BL/6J mice retinas. Subsequent studies structurally and biochemically characterise APP^swe/lon phenotypes in experimental glaucoma, with respect to current AD literature. These findings reveal extensive degradation of the retina and optic nerve, chiefly driven by astroglial and microglial cell bodies. Subsequent chapters conversely demonstrate that AAV-PHP.eB-shRNAmir-mediated genetic silencing confers structural, but not functional rescue of retinas during both healthy and experimental glaucomatous conditions, with significant amelioration of proinflammatory and apoptotic activity. Final studies of this thesis determine that in contrast to NS^-/- phenotypic rescue, concomitant APP and neuroserpin ablation results in a detrimental loss of function role, suggesting that both proteins are essential for healthy retinal homeostasis.</p><p dir="ltr">These findings are in line with growing scientific consensus, that a greater understanding of the biological role exerted by APP is necessary to develop a refined therapeutic approach to neurodegenerative pathologies such as AD and POAG. With respect to neuroserpin deficient phenotypes, this study demonstrates that despite initial confounding evidence, AAV-PHP.eB constructs are a highly efficient vector that may assist in the development of sustained gene therapy where neuroserpin deficiency is indicated. Furthermore, these studies confirm that AAV-PHP.eB constructs demonstrate relative ease in crossing the blood-retinal-barrier and efficient transduction <i>via </i>intravenous infusion alone. This may have therapeutic benefits in targeting retinal vascular diseases, or in understanding and manipulating the retinal vascular components of AD.</p>