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 neurodegeneration in glaucoma. However, the mechanisms by which astrocytes contribute to neurodegeneration remain unclear. To address this, we have developed human pluripotent stem cell (hPSC)-based models to serve as powerful tools for the in vitro analysis of neurodegenerative diseases, including neuron-glia interactions.
Methods :
Using hPSC-derived RGCs and astrocytes, we explored how reactive astrocytes contribute to RGC neurodegeneration. hPSCs were differentiated into 3D retinal organoids or forebrain organoids for the isolation of RGCs and astrocytes, respectively. The induction of a reactive astrocyte phenotype (“A1”) was promoted through incubation with C1q, TNFα and IL1α.
Results :
Reactive astrocytes exhibited a hypertrophic profile and increased expression of A1-reactive markers such as complement C3, as well as upregulation of genes associated with the inflammatory pathway and cytokine signaling. Functionally, reactive astrocytes secreted increased levels of several cytokines and affected blood-brain barrier integrity by increasing barrier permeability. Subsequently, the neurotoxic potential of reactive astrocytes was determined through direct contact and transwell co-cultures with hPSC-derived RGCs. Reactive astrocytes promoted profound morphological and functional alterations including neurite retraction and reduced neurite complexity in both co-culture systems, suggesting that reactive astrocytes rely on both contact-dependent and soluble factors to induce neurodegeneration. Moreover, to better mimic the compartmentalized nature of RGCs and location of reactive astrocytes in the optic nerve head, microfluidic chips were developed to properly orient reactive astrocytes along the proximal axonal compartment, demonstrating that reactive astrocytes adversely affect RGCs in a compartmentalized manner.
Conclusions :
Taken together, these results demonstrate that hPSC-derived astrocytes can be induced to a reactive and dysfunctional state, with a predominant inflammatory and neurotoxic phenotype, contributing to RGC neurodegeneration. Thus, the modulation of reactive astrocytes could be a novel therapeutic strategy for glaucoma by rescuing RGCs from neuroinflammatory insult.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.