Abstract
The intrinsic mechanisms that regulate neurotoxic versus neuroprotective astrocyte phenotypes and their effects on central nervous system degeneration and repair remain poorly understood. Here we show that injured white matter astrocytes differentiate into two distinct C3-positive and C3-negative reactive populations, previously simplified as neurotoxic (A1) and neuroprotective (A2)1,2, which can be further subdivided into unique subpopulations defined by proliferation and differential gene expression signatures. We find the balance of neurotoxic versus neuroprotective astrocytes is regulated by discrete pools of compartmented cyclic adenosine monophosphate derived from soluble adenylyl cyclase and show that proliferating neuroprotective astrocytes inhibit microglial activation and downstream neurotoxic astrocyte differentiation to promote retinal ganglion cell survival. Finally, we report a new, therapeutically tractable viral vector to specifically target optic nerve head astrocytes and show that raising nuclear or depleting cytoplasmic cyclic AMP in reactive astrocytes inhibits deleterious microglial or macrophage cell activation and promotes retinal ganglion cell survival after optic nerve injury. Thus, soluble adenylyl cyclase and compartmented, nuclear- and cytoplasmic-localized cyclic adenosine monophosphate in reactive astrocytes act as a molecular switch for neuroprotective astrocyte reactivity that can be targeted to inhibit microglial activation and neurotoxic astrocyte differentiation to therapeutic effect. These data expand on and define new reactive astrocyte subtypes and represent a step towards the development of gliotherapeutics for the treatment of glaucoma and other optic neuropathies.
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Data availability
All data are available in the manuscript, Supplementary Information and online. Bulk and scRNA-seq data can be found at the NCBI Gene Expression Omnibus through accession numbers GSE245816 and GSE237675. Source data are provided with this paper.
Code availability
Cell Ranger can be found at http://10xgenomics.com/support/software/cell-ranger/downloads. All analysis packages can be found at http://bioconductor.org and original R package repository at http://cran.r-project.org.
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Acknowledgements
This work was supported in part by National Eye Institute grants R01-EY026766, R01-EY032416 (M.S.K. and J.L.G.), F32-EY025915 (E.G.C.), T32-EY027816 (A.B.T.), P30-EY026877 (J.L.G.), Medical Technology Enterprise Consortium, the Gilbert Family Foundation Vision Restoration Initiative and an unrestricted grant from Research to Prevent Blindness. We also thank the Stanford Gene Vector and Virus Core (GVVC) and the Stanford Genetics Bioinformatics Service Center for their critical contributions to this work. Illustrations created with BioRender.com.
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Contributions
E.G.C. and J.L.G. conceived the project, designed the experiments, interpreted the data and wrote the paper. E.G.C., M.N., A.B.T., L.H., B.T., W.Y. and P.N. performed RNA-seq experiments and analyses. M.N., E.G.C., S.H., C.D. and T.L.S. performed cell culture assays and associated analyses. E.G.C., M.N., A.B.T. and S.S. performed western blots, qPCR and analyses. E.G.C., A.B.T., J.G., M.N., R.D., X.X., S.H., C.K., M. Atkins, C.S. and M. Ashouri performed in vivo work and associated analyses. M.B. and K.C.C. validated and provided reagents. K.R.R. performed mouse husbandry and genotyping. M.S.K. and R.V.N. contributed to experimental oversite and provided editorial feedback. M.B.W. and B.T. assisted with figure design and illustrations. All authors discussed the results and commented on the manuscript.
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Stanford University has prepared provisional patent applications (file numbers pending) based on the ONH astrocyte-specific viral vector and the manipulation of astrocyte cAMP, on which E.G.C., A.B.T. and J.L.G. are included as inventors.
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Nature thanks Xin Duan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
Extended Data Fig. 1 Annotation and cell cycle profiles of optic nerve cell types shown in Fig. 1.
a. Dot plots of canonical cellular markers in 13 identified optic nerve cell types. Fraction of expressing cells shown as dot size and mean expression shown as dot colour. b. UMAP plots as in Fig. 1e showing all optic nerve cell clusters coloured by distribution of cells in G1, S and G2/M phase.
Extended Data Fig. 2 Characterization of sAC expression and function in primary cortical astrocytes.
a-b. Astrocyte marker immunostaining and characterization of primary cortical astrocyte culture purity (n = 4 cultures). Scale bars are 50 µm. c. Western blots showing sACfull (180 kDa) and sACt (50 kDa) protein expression in sACfl/fl mouse astrocytes after 15 days in vitro (DIV), 30DIV and following AAV2-GFP (Ctrl) or AAV2-Cre-GFP (KO) transduction. d. Quantification of sAC KO determined by the relative difference of sACt band intensity between CTRL and sAC KO samples with protein expression normalized to ponceau or GAPDH (n = 3 cultures). Two-tailed paired t-test. e. Illustration of cell cycle phase progression and canonical checkpoints located at the G1, G2/M and metaphase-to-anaphase transitions (spindle checkpoint). f. Experimental design to assess the role of sAC in cell cycle progression in astrocyte cultures. g. Synchronization of astrocyte proliferation with aphidicolin. Relative frequency histogram of genomic DNA content after 20 hr aphidicolin treatment and removal with hours after release indicated. h. Fraction of cells in S phase (cells between dotted lines in panel g) following the indicated hours after removal of aphidicolin (n = 3 cultures). i. Relative frequency histograms of effects of 2HE treatment on cell cycle arrest in asynchronous (A) and synchronized (S) human astrocyte cultures. j. Quantification of panel i (n = 4 cultures). Two-way ANOVA with Tukey test. p > 0.05 is non-significant, n.s. All data are shown as mean values ± s.e.m. e,f, Created with BioRender.com.
Extended Data Fig. 3 Soluble adenylyl cyclase inhibits stress-associated p21 expression in proliferating astrocytes.
Bulk RNA-seq for human astrocytes shown in Fig. 2j–k and studied using the experimental paradigm shown Extended Data Fig. 2f. a. Inhibiting sAC induces p53, HIF-1α and NF-Kβ stress signalling pathways (red) and downregulation of canonical cell cycle signalling pathways (green) in proliferating astrocytes. Gene set enrichment analysis (GSEA) of KH7-, S-phase inhibitor aphidocolin- and G2/M inhibitor nocodazole-treated human astrocyte cultures. Aphidicolin and nocodazole differentially affect stress and cell cycle signalling consistent with their unique mechanisms of action. b. Volcano plots highlighting an upregulation of p53-, HIF-1α- and NF-κB-associated gene expression (red) and downregulation of canonical cell cycle genes (green). c. Gene expression analysis of p53-, NF-κB- and Hif-1α-associated genes demonstrating p21 (CDKN1A) is the most significantly upregulated stress-associated gene induced by KH7. d. Gene expression analysis of cell cycle-associated genes showing KH7 treatment induces a significant downregulation of known p21 targets including CCNB1, CDC25A, CDC25B and CDC25C. Moderated t-statistics test; b-d .
Extended Data Fig. 4 Effects of sAC KO on astrocyte C3 and p21 expression.
a. Loss of sAC in reactive optic nerve astrocytes induces C3 expression. Representative images of C3 immunostaining in CTRL and sAC KO optic nerve astrocytes 500 µm from the crush site. Scale bar is 20 µm. b. sAC KO leads to significantly increased C3 intensity (n = 91 cells from 5 optic nerves). Dotted lines represent 25th and 75th percentiles. Dashed line represents median. Two-sided Kolmogorov-Smirnov test. c. sAC KO leads to a significant increase in neurotoxic C3-positive reactive astrocytes using CTRL median as a threshold (n = 5 optic nerves). Two-tailed unpaired t-test. d. Representative images of p21 immunostaining in CTRL and sAC KO optic nerve astrocytes 500 µm from the crush site. Scale bar is 20 µm. i. Quantification of p21+ astrocytes demonstrating a significant increase in p21 expression in GFP-positive sAC KO astrocytes relative to controls (n = 5 optic nerves). Two-tailed unpaired t-test. All data are shown as mean values ± s.e.m.
Extended Data Fig. 5 AAV5 and AAV8 transduction in the retina with CMV, GFAP and gfaABC(1)D promoters.
a. Representative optic nerve head (ONH) cross-sections demonstrating tdTomato reporter expression for each viral vector tested. ONH region defined as the area between the glial lamina and optic disc rim ~200 µm from the centre of the retina. Scale bar is 50 µm. b. Representative cross-sections of transduced retinas at a distance (>500 µm) from the ONH. Closed arrowheads showing transduced NFL astrocytes. Open arrowheads showing transduced Muller glial cells in the inner nuclear layer (INL). NFL astrocytes defined by GFAP and Sox9 in the inner retina. Muller glia defined by Sox9 and DAPI in the INL. Photoreceptors defined by DAPI in the outer nuclear later (ONL). Scale bar is 50 µm. c. Representative retinal flat-mounts showing tdTomato reporter expression for each viral vector tested. RGCs identified by RBPMS immunostaining. Scale bar is 500 µm. d. Magnified ONH and retinal regions from retinal flat-mount (dashed in c). Open arrowheads showing transduced RGCs. Scale bar is 50 µm. e. Representative ONH cross-sections immunostained for Iba1 showing microglia and virus co-localization. Scale bar is 50 µm. f. Quantification of reporter expression in principal retinal cell types (AAV5.gfaABC(1)D, n = 5 retinas; AAV8.gfaABC(1)D, n = 3 retinas; AAV5.GFAP, n = 3 retinas; AAV8.GFAP, n = 3 retinas; AAV5.CMV, n = 2 retinas; AAV8.CMV, n = 2 retinas). One-way ANOVA with Tukey test. All data are shown as mean values ± s.e.m.
Extended Data Fig. 6 Compartmented cAMP in optic nerve head (ONH) astrocytes differentially regulates proliferation and C3 expression after optic nerve crush injury.
a. Representative images of AAV5.gfaABC(1)D.tdTomato (control), NES-sponge and NLS-sponge effects on ONH astrocyte proliferation after injury. Scale bar is 50 µm. b. Buffering cytoplasmic cAMP in ONH astrocytes promotes proliferation after injury (n = 4 retinas). One-way ANOVA with Tukey test. c. Representative images of ONH C3 immunoreactivity in AAV5.gfaABC(1)D.tdTomato (control), NES-sponge and NLS-sponge transduced retinas after injury. Scale bar is 50 µm. d. Buffering nuclear cAMP in ONH astrocytes induces significant C3 expression (n = 4 retinas). One-way ANOVA with Tukey test. e. Representative images of ONH astrocyte proliferation in AAV5.gfaABC(1)D.GFP (control) and AAV5.gfaABC(1)D.Cre-GFP (sAC KO) injected sACfl/fl mouse eyes following injury. f. sAC KO inhibits ONH astrocyte proliferation after injury (n = 2 retinas). p > 0.05 is non-significant, n.s. All data are shown as mean values ± s.e.m.
Extended Data Fig. 7 Model for sAC-dependent reactive astrocyte differentiation and regulation of glial–glial and glial-neuronal signalling in response to injury.
Nuclear and cytoplasmic sAC-derived cAMP differentially regulate neuroprotective astrocyte proliferation through the regulation of p21. Proliferating neuroprotective reactive astrocytes inhibit deleterious microglial activation and downstream neurotoxic astrocyte reactivity, thus influencing the balance between neuroprotective and neurotoxic reactive phenotypes and the extent of retinal ganglion cell (RGC) survival.
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Supplementary Fig. 1 and Tables 1–3.
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Cameron, E.G., Nahmou, M., Toth, A.B. et al. A molecular switch for neuroprotective astrocyte reactivity. Nature 626, 574–582 (2024). https://doi.org/10.1038/s41586-023-06935-3
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DOI: https://doi.org/10.1038/s41586-023-06935-3
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