Neuronal injury external to the retina rapidly activates retinal glia, followed by elevation of markers for cell cycle re-entry and death in retinal ganglion cells.

Retinal ganglion cells (RGCs) are neurons that relay visual signals from the retina to the brain. The RGC cell bodies reside in the retina and their fibers form the optic nerve. Full transection (axotomy) of the optic nerve is an extra-retinal injury model of RGC degeneration. Optic nerve transection permits time-kinetic studies of neurodegenerative mechanisms in neurons and resident glia of the retina, the early events of which are reported here. One day after injury, and before atrophy of RGC cell bodies was apparent, glia had increased levels of phospho-Akt, phospho-S6, and phospho-ERK1/2; however, these signals were not detected in injured RGCs. Three days after injury there were increased levels of phospho-Rb and cyclin A proteins detected in RGCs, whereas these signals were not detected in glia. DNA hyperploidy was also detected in RGCs, indicative of cell cycle re-entry by these post-mitotic neurons. These events culminated in RGC death, which is delayed by pharmacological inhibition of the MAPK/ERK pathway. Our data show that a remote injury to RGC axons rapidly conveys a signal that activates retinal glia, followed by RGC cell cycle re-entry, DNA hyperploidy, and neuronal death that is delayed by preventing glial MAPK/ERK activation. These results demonstrate that complex and variable neuro-glia interactions regulate healthy and injured states in the adult mammalian retina

RGCs re-express Cyclin A after axotomy: marker of S-phase cell cycle re-entry.

<p>(<b>a</b>) Cyclin D3 immunostaining in FG-labeled RGCs. Control and injured retinas had similar staining. (<b>c</b>) E2F1 transcription factor immunostaining in FG-labeled RGCs. E2F1 was found in RGCs. Control and injured retinas had similar staining. (<b>b</b>) Cyclin A immunostaining in FG–labeled RGCs. Low expression of cyclin A was found in RGCs of control uninjured eyes. At day 5 post-axotomy, stronger cyclin A immunoreactivity was detected in some RGCs. For each picture, an RGC indicated with a small white rectangle is shown enlarged (lower right corner). (<b>d</b>) Quantification of FG⁺ Cyclin A⁺ RGCs in uninjured and axotomized eyes, 5 days after axotomy, total of >70 images taken from n = 7 retinas per group, *p<0.001. FG: fluorogold, GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer. Scale bar 30 µm.</p

After axotomy phospho-Akt is increased in Müller cells.

<p>(<b>a–c</b>) In control and in experimental retinas p-Akt immunostaining was exclusively detected in Müller cells, based on their typical morphology and co-localization with CRALBP. One day post-axotomy, p-Akt staining increased in the processes of Müller cells. Three days post-axotomy, Müller cells showed signs of gliosis and strong p-Akt immunoreactivity. (<b>b</b>) Micrographs of the full thickness of the retinas, showing p-Akt immunostaining in Müller cell soma, and exclusively in the Müller cell fibers oriented towards the injured RGCs (top section, GCL). Arrowheads indicate that Müller cell fibers with p-Akt immunostaining are not oriented towards the photoreceptors (bottom section, PhR and RPE). (<b>d</b>) p-Akt is not expressed in astrocytes. P-Akt immunoreactivity was not detected in GFAP⁺ cells in both control and experimental retinas. GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; PhR: photoreceptor layer; RPE: retinal pigmented epithelium. Scale bar, 30 µm.</p

Retinal glia rapidly phosphorylate ERK1/2 after RGC axotomy.

<p>(<b>a–d</b>) p-ERK immunoreactivity in neurons (<b>a</b>) In uninjured retinas there is expression of ERK1/2 in all cellular compartments, both in neuronal and in non-neuronal cells, detected with an antibody to total ERK1/2. Total ERK1/2 staining continued to be expressed to the same level in retinas at days 1 and 3 post-axotomy (data not shown). (<b>c</b>) In uninjured retinas ERK1/2 is phosphorylated in RGCs. (<b>b</b>) At day 1 after ON axotomy, there was a robust increase of p-ERK1/2 immunoreactivity in putative glial cells in the GCL, INL, and both plexiform layers. Note the relative reduction of p-ERK1/2 in the GCL versus control normal retinas. (<b>d</b>) At day 3 after ON axotomy both the soma and the processes of retinal glia are robustly labeled with p-ERK1/2. Note that, unlike what was shown for p-Akt, the glial fibers with p-ERK1/2 point both towards the injured RGCs and towards the photoreceptors (full arrows). Arrowheads point to the soma of glia. (<b>e,f</b>) p-ERK immunoreactivity in glia. (<b>e</b>) p-ERK is strongly expressed in the end-feet, somas and processes of Müller cells expanding throughout the PhR layer rapidly after axotomy, as shown by co-localization of p-ERK with CRALBP. (<b>f</b>) The expression of p-ERK was low in astrocytes, as revealed by the weak co-localization of p-ERK with GFAP. GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; PhR: photoreceptor layer. Scale bar, 30 µm.</p

Hypothetical model of events leading to RGC death after axotomy.

<p>(<b>a</b>) Time-dependent retinal changes for cell cycle markers in RGCs (blue) and Müller cells (orange) after optic nerve transaction (extra-retinal damage). (<b>b</b>) Neuro-glia interactions. Optic nerve transaction induces retrograde transport of “injury signals” and migration of reactive astrocytes to the retina. Injured RGCs and glial cells are in intimate contact and also communicate by releasing soluble molecules. The interaction may be bi-directional. Early after RGC injury, glial cells rapidly activate p-Akt, and p-ERK pathways. The p-Akt response in Müller cells is specifically oriented towards the injured RGCs. Early after RGC injury glia do not undergo Rb phosphorylation or cell cycle re-entry, but they do undergo hypertrophy. Shortly thereafter, and before significant RGC death, the injured RGCs decrease p-S6, phosphorylate-Rb, re-enter the cell cycle and undergo DNA synthesis. Later the RGCs undergo frank cell death and their somata are cleared from the retina, but RGC death can be delayed by inhibiting p-ERK in glial cells.</p

Up-regulation of phospho-Akt (p-Akt) after axotomy has a vectorial orientation towards the RGCs.

<p>(<b>a</b>) p-Akt immunoreactivity in control uninjured retinas, and at day 1 and day 3 after axotomy. No other antibody was used, to depict clearly the specific p-Akt reactivity. (<b>b</b>) Quantification of p-Akt area in uninjured and day 3 post-axotomy retinas. Area was significantly increased in axotomized eyes, as a sign of gliosis and hypertrophy of putative Müller cells after injury, >90 images taken from n = 9 retinas per group, *p<0.01. GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; PhR: photoreceptor layer. Scale bar, 30 µm.</p

Expression of phospho-S6 (p-S6) decreases in the GCL after axotomy.

<p>(<b>a</b>) p-S6 immunoreativity in neurons of the GCL. In control uninjured retinas, p-S6 immunoreactivity was detected in some neurons in the GCL (likely RGCs) and other cell types in the INL (likely horizontal cells and microglia). One day after axotomy, p-S6 decreased in the GCL, and three days after axotomy it was undetectable in neurons of the GCL. p-S6 did not change in neurons of the INL. (<b>b</b>) Quantification of NeuN⁺ p-S6⁺ cells in the GCL, comparing uninjured control (left) to injured (right) eye in each animal at 1 and 3 days after axotomy, total of >40 images taken from n = 4 retinas per group, *p<0.01. (<b>c, d</b>) lack of p-S6 immunoreactivity in glia. Neither astrocytes (labeled with GFAP) nor Müller cells (labeled with CRALBP) were p-S6⁺ 1 day after axotomy. GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; PhR: photoreceptor layer. Scale bar, 30 µm.</p

Cell size distribution and propidium iodide (PI) intensity in the retina 5 days after axotomy.

<p>Fluorogold⁺ PI⁺ RGCs were classified as small and large depending on their diameter in pixels. Cells with values between 100 and 400 pixels were arbitrarily considered “small” and cells with values between 400 and 1000 were consider “large”. More than 7000 cells per group were counted as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101349#s2" target="_blank">Methods</a> and all retinal areas were averaged for each group.</p