Neurotoxic Reactive Astrocytes Drive Neuronal Death after Retinal Injury

Abstract

Glaucoma is a neurodegenerative disease that features the death of retinal ganglion cells (RGCs) in the retina, often as a result of prolonged increases in intraocular pressure. We show that preventing the formation of neuroinflammatory reactive astrocytes prevents the death of RGCs normally seen in a mouse model of glaucoma. Furthermore, we show that these spared RGCs are electrophysiologically functional and thus still have potential value for the function and regeneration of the retina. Finally, we demonstrate that the death of RGCs depends on a combination of both an injury to the neurons and the presence of reactive astrocytes, suggesting a model that may explain why reactive astrocytes are toxic only in some circumstances. Altogether, these findings highlight reactive astrocytes as drivers of RGC death in a chronic neurodegenerative disease of the eye.

Keywords: astrocytes; astrogliosis; glaucoma; glia; neurodegeneration; neuroprotection; optic nerve crush; reactive astrocyte.

FIGURE 1.. Reactive astrocytes drive death of retinal ganglion cells.

A. RBPMS (RNA-binding protein with multiple splicing, an RGC marker) immunostaining of whole-mount mouse retinas showing decreased number of retinal ganglion cells (RGCs) following optic nerve crush (ONC) at 28 days, which was prevented in Il1a^−/−Tnf^−/−C1qa^−/− (tKO) mice. B-C. Quantification of RGC survival 1,3, 7,14, and 28 days following optic nerve crush in wild-type (B) and tKO (C) mice. D. RBPMS immunostaining of whole-mount rat retinas showing decreased number of RGCs 28 days following ONC and IgG control antibody injection (E) which was prevented with Il-1α, TNFα, and C1q neutralizing antibody treatment (F). Data normalized to contralateral un-manipulated eye. * P < 0.05, one-way ANOVA followed by Tukey’s multiple comparisons test. Individual data points are plotted and represent individual animals, while bars represent mean ± s.e.m. Scale bar is 20 μm for all micrographs in A/D.

FIGURE 2.. Bead injection glaucoma model increases intraocular pressure and causes astrocyte-dependent RGC loss.

A-D. Bead injection glaucoma produced increases in intraocular pressure (IOP) in wildtype (WT, A) and triple knock-out mice (tKO, Il1a^−/−Tnf^−/−C1qa^−/−, B). Data in C/D are normalized around the day of peak (P) IOP increase. Refer to Supp. Figure S1 for individual animal plots. E. IOP maximum increase was approximately 20–30% in both wildtype (WT) and tKO mice. Similar maximal levels were reported in mice that had a sustained (S) increase in IOP (A/B), or transient (T) increase (C/D). F. Representative RBPMS+ staining of whole mount retinas from WT and tKO animals following sustained and transient IOP increase. Quantified in (G/I). G. IOP increase paired with death of RBPMS+ retinal ganglion cells in WT but not tKO mice following bead occlusion (compared to contralateral eye). There was a particularly large drop in SMI-32+ retinal ganglion cells (H). I. Mice with transient increase in IOP (C/D) had no loss of RGCs. J. Heat map of z-scores from microfluidic qPCR analysis highlights enrichment of reactive astrocyte transcripts in retina, optic nerve head, and optic nerve of bead-injected but not PBS-injected eyes of WT mice (largely absent in bead-injected eyes from tKO mice). K-N. Analysis of average fold induction for all reactive astrocyte transcripts (taken from J) highlights retinas from mice with transient increase in IOP that returned to baseline (see C) showed upregulated reactive astrocyte transcripts (same level as sustained IOP increase in the retina (K/L), optic nerve head (M), or optic nerve (N), see also Figure S2, S3). See also Supplemental Figures S1-S3. Abbreviations: tKO, triple knock out mice (Il1a^−/−Tnf^−/−C1qa^−/−, neuroinflammatory reactive astrocyte deficient); S, sustained (IOP increase); T, transient (IOP increase); WT, wildtype. * P < 0.05, one-way ANOVA followed by Tukey’s multiple comparisons test. Individual data points representative of individual animals are plotted, while bars represent mean ± s.e.m. Scale bar is 20 μm for all micrographs in F.

FIGURE 3. Retinal ganglion cells require both damage and astrocyte-derived toxins for targeted removal.

Two weeks following optic nerve crush (ONC) in wild type (WT) mice, retinal ganglion cell number was decreased approximately 75%. This death was not seen in ONC of neuroinflammatory reactive astrocyte-deficient tKO mice, or in mice that had intact optic nerves but received an injection of toxic reactive astrocyte conditioned media (neurotoxic ACM) which was otherwise toxic in in vitro assays. Triple knockout mice that would normally not show loss of RGCs following ONC had loss of cells when paired with injection of neurotoxic ACM. RGC numbers normalized to contralateral control eye. * P < 0.05, one-way ANOVA followed by Tukey’s multiple comparisons test. Individual data points are plotted and represent individual animals, while bars represent mean ± s.e.m.

FIGURE 4.. Dendrites of OFF-S RGCs in Il1a^−/−Tnf^−/−C1qa^−/− KO mice stratify more broadly in the IPL following elevated IOP.

A. Example z-projection of a stack of confocal images taken from an OFF-S RGC from Il1a^−/−Tnf^−/−C1qa^−/− mice under control conditions showing the level of RGC dendrites (red) and ChAT bands (cyan). B. Same as in (A) but from OFF-S RGCs from Il1a^−/−Tnf^−/−C1qa^−/− mice under elevated IOP conditions. Dendrites stratify in approximately the correct location, but more broadly. C. The position and width of the dendrites in the IPL can be quantified. The normalized fluorescence intensity of the dendritic signal relative to that of the ChAT bands across a portion of the IPL (denoted by yellow box in (B)) is shown for a single RGC. By plotting the position of the dendrites relative to the normalized position of the ChAT bands, the position and width of the dendritic stratification can be quantified. D. Quantification of average stratification position within the IPL of OFF-S RGCs from Il1a^−/−Tnf^−/−C1qa^−/− mice under control and bead-injected retinas. E. The dendrites of RGCs from bead-injected Il1a^−/−Tnf^−/−C1qa^−/− mice stratified across a broader region of the IPL than RGCs from control retinas. F. Representative dye-filled RGC used for electrophysiological recordings. G-H. Ray-trace images of dye-fills pictured in (F) shown from the perspective of a flat retina (z-projection) to visualize the radial branching of RGCs (G) and from a side view(y-projection) to visualize dendritic position across layers of the retina (H). I. Sholl analysis of RGC process branching in control or bead-injected eyes. J-L. Quantification of number of dendritic branch points per cell (J), cell radius (K), and total neurite length (J) for RGCs in spared vs control retinas. All quantification performed on mice with sustained increases in IOP. * P < 0.05, unpaired t-test. Individual data points are plotted and represent individual cells from 4 animals (control) and 9 animals (bead-injected), while bars represent mean ± s.e.m. Scale bar is 100 μm for all micrographs in F.

FIGURE 5.. OFF-S RGCs in Il1a^−/−Tnf^−/−C1qa^−/− KO mice show subtle changes in light response properties following elevated IOP.

A. Response of an OFF-S RGC from Il1a^−/−Tnf^−/−C1qa^−/− mice to a one second long negative contrast pulse under control conditions (top) and after elevated IOP following bead injection (bottom). B. Mean normalized spike rate histogram from a population of OFF-S RGCs from Il1a^−/−Tnf^−/−C1qa^−/− mice. Inset shows components of the spiking response used to analyze response properties in (C). C. Analysis of various light response properties of OFF-S RGCs. The peak spike rate (left) was significantly reduced in OFF-S RGCs from Il1a^−/−Tnf^−/−C1qa^−/− mice that experienced elevated IOP. Other measurements of light response properties of OFF-S RGCs (decay, latency, and final/peak ratio) remained unchanged following bead-occlusion. D. Mean normalized responses of OFF-S RGCs to spots of increasing diameter in Il1a^−/−Tnf^−/−C1qa^−/− KO mice under elevated IOP (red) or control (grey) conditions. Elevated IOP significantly reduced responses to spot sizes below 600 microns. E. Mean normalized responses of a single OFF-S RGC to spots of increasing size (left). Spot size response function was fit (smooth line) and the spot size that produced a half maximal response (dashed lines) was calculated. Half maximal responses for a population of OFF-S RGCs from control and bead-injected Il1a^−/−Tnf^−/− C1qa^−/− mice (right). The spots size that produced a half-maximal response was significantly larger in bead-injected mice. All quantification performed on mice with sustained increases in IOP. * P < 0.05, unpaired t-test or one-way ANOVA as appropriate. Individual data points are plotted and represent individual cells from 4 animals (control) and 9 animals (bead-injected), while bars represent mean ± s.e.m.

FIGURE 6.. Spatial summation properties of OFF-S RGCs in Il1a^−/−Tnf^−/−C1qa^−/− mice are unchanged following elevated IOP.

A. Response of an OFF-S RGC from an Il1a^−/−Tnf^−/−C1qa^−/− mice after elevated IOP to contrast reversing gratings (CRGs) presented for four seconds at 2 Hz. B. Typical normalized post stimulus time histograms (PSTHs) from one cycle of the CRG stimulus of an OFF-S RGC from Il1a^−/−Tnf^−/−C1qa^−/− mice after elevated IOP to one cycle of CRGs at a single spatial frequency at four spatial phases. The response modulates primarily at the temporal frequency of the stimulus. C. Spatial frequency tuning curves for the first Fourier harmonic (F1; black) and the second Fourier harmonic (F2; red) for OFF-S RGCs from Il1a^−/−Tnf^−/−C1qa^−/− mice under control (left) and after elevated IOP (right). The F2 component of the response is small at all spatial frequencies tested. D. Spatial summation nonlinearities of OFF-S RGCs from Il1a^−/−Tnf^−/−C1qa^−/− mice under control and elevated IOP conditions. The nonlinearity index is defined as the maximum value of the ratio between the second and the first harmonics (F2/F1) over all spatial frequencies tested. All quantification performed on mice with sustained increases in IOP. n.s. P > 0.05, unpaired t-test. Individual data points are plotted and represent individual cells from 4 animals (control) and 9 animals (bead-injected), while bars represent mean ± s.e.m.