Investigating the effects of exogenous neuroserpin administration on the retina in healthy and diseased conditions

Introduction: Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide, characterised by optic neuropathy and retinal ganglion cell degeneration. While the underlying pathophysiology of POAG is poorly understood, studies have reported that inactivation of the serine protease inhibitor neuroserpin (NeuS), promotes plasmin-proteolytic excitotoxicity. The therapeutic benefits of exogenous NeuS overexpression have previously been reported in acute glaucoma models and in various neurodegenerative diseases. However, its neuroprotective effects in chronic glaucoma conditions have not been investigated.

Objective: This study investigated the neuroprotective effects of exogenous NeuS overexpression in healthy and an experimental glaucoma mouse model. Mice were examined for functional, structural and biochemical changes.

Methods: This 8-week study utilised wild-type C57BL/6J mice (n=32). Glaucoma was induced via weekly intracameral microbead injections (glaucoma, n=8). Human recombinant NeuS was intravitreally injected in healthy (NeuS, n=8) and experimental glaucoma mice (glaucoma+NeuS, n=8). PBS was used as a vehicle control (PBS, n=8). Functional changes were analysed using electroretinogram (ERG) and positive scotopic threshold response (pSTR) amplitudes. Histological staining investigated changes to the retinal ultra-structures, while immunohistochemistry investigated changes to NeuS and plasminogen expression and localisation.

Results: Exogenous NeuS administration in experimental glaucoma mice demonstrated significant preservation of pSTR amplitudes when compared to glaucoma mice (p=0.0168). Meanwhile, mice treated with NeuS showed non-significant changes to pSTR amplitudes when compared to control and PBS treated mice (p=0.1977). Immunofluorescence revealed no significant changes to NeuS immunoreactivity in healthy and experimental glaucomatous mice. While, NeuS administration in healthy and glaucoma induced mice showed a 3-fold increase in NeuS immunoreactivity (p=0.0014, p=0.0033 respectively). There was a significant increase of plasminogen immunoreactivity in glaucoma mice (p=0.0172), which was significantly attenuated in glaucoma mice retina subjected to exogenous NeuS administration (p=0.0079). Histological quantification showed no significant changes to GCL density in control, PBS and NeuS treated mice (p=0.3659). However, there was significant rescue of the GCL population in NeuS-administered glaucoma mice when compared to glaucoma mice (p=0.0089)

Conclusion: This thesis describes the first investigation of exogenous neuroserpin administration during chronic glaucomatous conditions and demonstrates proof of concept for its therapeutic benefit. These findings also provide insight into the molecular changes due to proteolytic excitotoxicity in POAG and other neurodegenerative diseases. However, the molecular mechanisms by which neuroserpin might impart neuroprotection and the consequences of its loss remain unclear. Thus, future studies will incorporate detailed proteomics and enzymatic studies to illustrate these mechanistic aspects.