Astrocytes modulate neurodegenerative phenotypes associated with glaucoma in OPTN(E50K) human stem cell-derived retinal ganglion cells

Abstract

Although the degeneration of retinal ganglion cells (RGCs) is a primary characteristic of glaucoma, astrocytes also contribute to their neurodegeneration in disease states. Although studies often explore cell-autonomous aspects of RGC neurodegeneration, a more comprehensive model of glaucoma should take into consideration interactions between astrocytes and RGCs. To explore this concept, RGCs and astrocytes were differentiated from human pluripotent stem cells (hPSCs) with a glaucoma-associated OPTN(E50K) mutation along with corresponding isogenic controls. Initial results indicated significant changes in OPTN(E50K) astrocytes, including evidence of autophagy dysfunction. Subsequently, co-culture experiments demonstrated that OPTN(E50K) astrocytes led to neurodegenerative properties in otherwise healthy RGCs, while healthy astrocytes rescued some neurodegenerative features in OPTN(E50K) RGCs. These results are the first to identify disease phenotypes in OPTN(E50K) astrocytes, including how their modulation of RGCs is affected. Moreover, these results support the concept that astrocytes could offer a promising target for therapeutic intervention in glaucoma.

Keywords: astrocyte; autophagy; glaucoma; neurodegeneration; retina; retinal ganglion cell; stem cell.

Graphical abstract

Figure 1

Astrocyte differentiation from isogenic control and OPTN(E50K) hPSCs (A–F) Astrocytes were differentiated from both cell sources in high purity, and resulting astrocytes expressed a variety of associated markers. (G–I) Compared with isogenic control astrocytes, a smaller percentage of OPTN(E50K) astrocytes expressed GFAP. (J) Western blot analyses confirmed a significant decrease in GFAP expression, as well as increased expression of S100β and SOX9. n = 4 separate differentiation experiments each using H7 and H7(E50K) hPSCs. Scale bars, 100 μm. Error bars represent SEM. ^∗p < 0.05, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

Figure 2

Disease-related features of OPTN(E50K) astrocytes (A–C) Isogenic control astrocytes demonstrated widespread expression of LC3, while OPTN(E50K) astrocytes displayed significant aggregation of LC3 within the soma. (D–F) Mitochondria identified by TOM20 immunostaining were observed to be widespread and elongated in isogenic control astrocytes, while OPTN(E50K) astrocytes displayed condensed mitochondria aggregated within the perinuclear region. (G–I) OPTN(E50K) astrocytes displayed significantly less co-localization of LC3 and LAMP1, suggesting an inability of the autophagosome to fuse with the lysosome. (J) OPTN(E50K) astrocytes also expressed significantly higher levels of the autophagy-related p62 protein, suggesting autophagy impairment. n = 3 separate differentiation experiments each using H7 and H7(E50K) hPSCs. A minimum of 5 individual cells from each of at least 3 separate differentiation experiments were used for analysis in (A)–(I). Scale bars, 25 μm (A, B, D, and E) and 10 μm (G and H). Error bars represent SEM. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗∗p < 0.0001.

Figure 3

RNA-seq analyses of OPTN(E50K) and isogenic control astrocytes (A) Heatmap representation of differentially expressed genes between OPTN(E50K) and isogenic control astrocytes. Each row represents one sample, and each column represents one gene. The up- and down-regulated genes are yellow and blue colored, respectively. (B and C) Venn diagram (B) and (C) scatterplot of differentially expressed genes. (D) Gene Ontology (GO) and pathway terms from differential gene expression analysis of OPTN(E50K) and isogenic control astrocytes. Statistical significance (−log[p] value on the x axis) was assessed using hypergeometric test. n = 4 separate differentiation experiments each using H7 and H7(E50K) hPSCs.

Figure 4

Astrocytes modulate RGC phenotypes (A–F) Compared with isogenic control RGCs grown alone, those grown in the presence of healthy isogenic control astrocytes exhibit enhanced neurite outgrowth, while those grown in the presence of OPTN(E50K) astrocytes exhibit a reduction of primary neurites as well as soma size. (G–L) OPTN(E50K) RGCs exhibit degenerative phenotypes that can be rescued in the presence of healthy isogenic control astrocytes, whereas OPTN(E50K) astrocytes did not have an observable effect. (M–O) Quantification of results from these co-culture assays. n ≥ 3 separate differentiation experiments each using H7 and H7(E50K) hPSCs. A range of 10–15 RGCs were analyzed per condition. Scale bars, 50 μm (white bars refer to A, C, E, G, I, and K) and 100 μm (black bars refer to B, D, F, H, J, and L). Error bars represent SEM. ^∗p < 0.05, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.

Figure 5

OPTN(E50K) astrocytes confer hyperexcitable profiles to RGCs (A) Ionic currents recorded from RGCs in the presence of astrocytes in each experimental condition. (B and C) OPTN(E50K) RGCs grown with OPTN(E50K) astrocytes fired significantly more action potentials upon current injection compared with RGCs in other conditions. (D–G) RGCs grown with OPTN(E50K) astrocytes resulted in increased sodium current density, increased input resistance, decreased cell capacitance, and decreased action potential threshold. (H) Following 1 ms current injections, a higher percentage of RGCs on OPTN(E50K) astrocytes fired action potentials compared with RGCs on isogenic control astrocytes. n = 4 separate differentiation experiments each using H7 and H7(E50K) hPSCs. Error bars represent SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

Figure 6

Analysis of factors secreted by OPTN(E50K) astrocytes upon RGCs (A–D) Isogenic control and OPTN(E50K) RGCs expressing BRN3b:tdTomato in the presence of isogenic control and OPTN(E50K) astrocyte-conditioned medium. (E and F) Quantification of results demonstrated significant effects of astrocyte-conditioned medium upon RGCs, with OPTN(E50K) astrocyte-conditioned medium resulting in degenerative phenotypes compared to isogenic control conditioned medium. (G) Meso Scale Discovery analysis revealed a reduced secretion of IL-6 and IL-8 from OPTN(E50K) astrocytes compared with isogenic control astrocytes. Values are fold change compared with basal medium alone. (H–L) Representative inverted fluorescent images of BRN3b:tdTomato RGCs demonstrating a reduction of neuronal complexity due to the OPTN(E50K) mutation, which could be partially rescued following treatment with exogenous IL-6. (M and N) In response to exogenous treatment with IL-6, OPTN(E50K) RGCs expressed significantly higher levels of synaptic proteins, a measure of RGC health and maturation state. n ≥ 3 separate differentiation experiments with at least 15 individual RGCs analyzed for each experiment using H7 and H7(E50K) hPSCs. Scale bars, 30 μm (A–D) and 20 μm (H–J). Error bars represent SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.