Induction of astrocyte reactivity promotes retinal ganglion cell degeneration in human pluripotent stem cell models through complement C3 activation

Abstract

Purpose : Astrocytes closely associate with retinal ganglion cells (RGCs) in the nerve fiber layer of the retina and optic nerve, where they provide support for RGCs but can contribute to RGC degeneration in a glaucomatous state. Complement cascade activation is a consistent feature observed in glaucoma, and neurotoxic reactive astrocytes express increased levels of complement C3. However, our understanding of how astrocytes promote neurotoxicity, and how complement activation is involved in astrocyte-mediated neurodegeneration remain incomplete.

Methods : Using human pluripotent stem cell (hPSC)-derived RGCs and astrocytes, we explored how reactive astrocytes contribute to RGC degeneration, and how complement C3 is involved in astrocyte reactivity. hPSC-astrocytes were induced to a reactive phenotype through incubation with C1q, TNFα and IL1α, and their neurotoxic effect was determined through co-cultures with RGCs. Furthermore, shRNA approaches were used to modulate C3 in astrocytes.

Results : Reactive astrocytes exhibited a hypertrophic profile, upregulation of inflammatory genes, and increased secretion of several cytokines. Reactive astrocytes promoted marked morphological changes to RGCs, including neurite retraction and reduced neurite complexity, through direct contact and via secreted factors in transwell co-cultures. Moreover, to better mimic the compartmentalized nature of RGCs and location of reactive astrocytes in the optic nerve head, microfluidic devices were developed to properly orient astrocytes along the proximal axonal compartment. Results showed a detrimental effect of reactive astrocytes over RGC axonal compartment. Importantly, complement activation contributed to the acquisition of the reactive phenotype by hPSC-derived astrocytes, as shRNA-mediated reduction of complement C3 resulted in a less pronounced hypertrophic profile and attenuated reactivity-associated features in astrocytes.

Conclusions : These results demonstrate that reactive astrocytes promote RGC neurodegeneration in a microfluidic platform that mimics reactive astrocyte orientation along the proximal axon. Results of this study demonstrate a role for the complement cascade in this neuroinflammatory response, and that the modulation of the complement cascade in reactive astrocytes could represent a novel therapeutic strategy for neuroinflammatory aspects of glaucoma.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.