Joanne Aiko Matsubara
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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2019)
Purpose:Age-related macular degeneration (AMD) is a devastating eye disease causing irreversible vision loss in the elderly. Retinal pigment epithelium (RPE), an important cell type afflicted in AMD, undergoes cell death in the late stages of the disease. Salient factors underlying AMD pathogenesis are aging, drusen components and NLRP3 inflammasome activity. The purpose of this dissertation is to elucidate the molecular interactions among these factors and how they contribute to RPE damage.Methods:The effects of aging on drusen components, in particular the membrane attack complex (MAC) and amyloid beta (Aβ) were examined in rats at different age. To determine the role of MAC in inflammasome activation in RPE, aurin tricarboxylic acid complex (ATAC), was administrated to naïve rats. To understand Aβ’s role in inflammasome activation, Aβ intravitreal injections were made into rat eyes in vivo and Vinpocetine was used to ameliorate the inflammatory responses. An in vitro RPE cell culture model was established to further investigate the relationship between inflammasome and X-chromosome linked inhibitor of apoptosis (XIAP). Statistical significance was set at p ≤ 0.05.Results:An age-dependent increase in MAC, Aβ, and NF-κB activation was observed in the RPE-choroid in vivo. Blocking MAC formation with ATAC led to a prominent reduction in inflammasome activity (caspase-1 cleavage and cytokine secretion), but not in NF-κB activity. Aβ intravitreal injections triggered inflammasome activation evidenced by enhanced caspase-1 cleavage and IL-1β/IL-18 release, which was suppressed by Vinpocetine mediated NF-κB inhibition. The robust inflammasome activity further led to gasdermin D-mediated activation of the pyroptotic pathway and a significant reduction in XIAP, which in turn enhanced IL-18 secretion.Conclusion:Aging is a strong risk factor for AMD, which increases the deposition of MAC and Aβ in the outer retina. The elevated levels of MAC and Aβ are triggers for inflammasome activation. By demonstrating a causal relationship between inflammasome activation and XIAP reduction, this dissertation suggests the precise regulation of XIAP, together with the suppression of MAC and NF-κB, may be crucial for controlling inflammasome activity and hence provides new avenues to prevent AMD.
The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
Purpose:Age-related macular degeneration (AMD) is a multifactorial degenerative disease that occurs in the central part of the retina − macula. The disease affects approximately one million Canadians and constitutes the number one cause of vision loss after cataract in the elderly. The Y402H polymorphism in the complement factor H (CFH) gene and drusen load are two salient risk factors for AMD. Little is known of the detailed cellular pathway(s) shared by these two factors. Here we investigate their interactions in in vitro models of AMD and in AMD samples. Methods:The biological samples came from 44 dry AMD patients, 23 postmortem eyes and retinal pigment epithelial (RPE) cell models. Drusen load and choroidal thickness in patients were measured using spectral-domain optical coherence tomography. We used analytical techniques including genotyping, Bio-Plex suspension assays, immunohistochemistry, immunofluorescence, reverse transcription PCR, Western blot, flow cytometry and lactate dehydrogenase assay Results:In dry AMD patients, higher systemic levels of interleukin (IL)-6, IL-18, and tumor necrosis factor (TNF)-α were associated with the at-risk CC variant of CFH Y402H polymorphism. IL-1β while not significant, demonstrated a similar trend. Drusen load was inversely correlated with choroidal thickness and visual acuity. Postmortem eyes genotyped with the Y402H risk variant showed significantly increased levels of GM-CSF in vitreous and immunoreactivity for CD68, C5a, IL-18 and TNF-α in Bruch’s membrane and/or choroid. Exposure to complement activation product C5a in RPE cells promoted NF-κB activation and upregulated inflammatory cytokines and growth factors. The drusen component, Aβ, induced complement activation and downregulated the membrane bound complement inhibitor, CD55, leading to sublytic MAC formation in RPE cells, which was inhibited by aurin tricarboxylic acid complex. Conclusion: Two important risk factors for AMD, CFH Y402H polymorphism and drusen load, both promote complement activation. Complement activation can mediate downstream events associated with sublytic changes in RPE, such as proinflammatory cytokine release. These results suggest that complement activation might be the central response to multiple risk factors and the complement activation products may further inflict injury on RPE cells. Complement activation products could be potential therapeutic targets to stop chronic inflammation in AMD.