Early cellular signaling responses to axonal injury

Abstract

Background: We have used optic nerve injury as a model to study early signaling events in neuronal tissue following axonal injury. Optic nerve injury results in the selective death of retinal ganglion cells (RGCs). The time course of cell death takes place over a period of days with the earliest detection of RGC death at about 48 hr post injury. We hypothesized that in the period immediately following axonal injury, there are changes in the soma that signal surrounding glia and neurons and that start programmed cell death. In the current study, we investigated early changes in cellular signaling and gene expression that occur within the first 6 hrs post optic nerve injury.

Results: We found evidence of cell to cell signaling within 30 min of axonal injury. We detected differences in phosphoproteins and gene expression within the 6 hrs time period. Activation of TNFalpha and glutamate receptors, two pathways that can initiate cell death, begins in RGCs within 6 hrs following axonal injury. Differential gene expression at 6 hrs post injury included genes involved in cytokine, neurotrophic factor signaling (Socs3) and apoptosis (Bax).

Conclusion: We interpret our studies to indicate that both neurons and glia in the retina have been signaled within 30 min after optic nerve injury. The signals are probably initiated by the RGC soma. In addition, signals activating cellular death pathways occur within 6 hrs of injury, which likely lead to RGC degeneration.

Figure 1

Activation of ERK-1 (MAPK1). A. Western blots were probed with anti-ERK-1 (pThr202/pTyr204) antibody. There was constitutive activation prior to optic nerve crush. However, the phosphorylation ratio (pERK/ERK) increased further at 360 min although the total ERK extracted was lower. B. Graph of the data in A normalized to the total ERK-1 protein. C-F Immunohistochemistry of activated ERK-1, at 0, 30 min, 60 min, and 360 min (6 hr) after optic nerve crush, respectively. Increased labeling of cells in the OPL (down arrows) are particularly evident at 30 min as well as decreased labeling of Muller cells that extend the from the ONL to the GCL (arrowheads). Also note that there is immunolabeling for pERK-1 in the inner stratum of the inner plexiform layer at 30 min and 6 hrs (up arrows) and labeling in the ganglion cell layer at 60 min (arrowheads). The grayscale inset in C shows the staining of the Muller cell marker glutamine synthase. Red = pERK-1, Blue = DAPI.

Figure 2

Phosphorylation of GluR1. A. Phosphorylation of GluR1 at Ser-831 increased at 6 hrs after optic nerve crush. B. GluR1 protein levels remained relatively constant, while phosphorylation of Ser-845 increased by 6 hrs. C-F. Immunohistochemistry of phosphorylated GluR1 (Ser-845) at 0, 0.5, 1, and 6 hrs after optic nerve crush. Increased pGluR1 is apparent in the ganglion cell layer (arrowheads) at 6 hrs after optic nerve injury. Red, = pGluR1, Blue = DAPI. IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Figure 3

Activation of TNFα pathways. A. Detection of TNFα by ELISA in the neural retina. Cytokine levels were determined in three samples at each time point. The level at 6 hr was significantly higher. B-C. Detection of phosphorylated JNK in the neural retina. B. Western blots (upper image) were probed with an anti pJNK antibody. C. ELISA confirmed the change in activation of JNK at 6 hrs after optic nerve injury. Data are normalized to total protein. D-G. Immunohistochemistry of activated JNK in the neural retina. Antibodies to pJNK were used on sections at 0, 30 min, 60 min and 6 hr after optic nerve crush. Increased labeling is evident in the GCL (arrows) at 6 hr.

Figure 4

Histone phosphorylation and methylation. Western blots probed with A. antiphospho-H2A (Ser-139) and B. trimethyl-K4 H3 histone. Phosphorylation of H2A increased while methylation of histone H3 (at lysine-4) decreased after optic nerve injury. The demethylation of H3 persisted for several hours and started to recover at 6 hr post injury. Actin served as a loading control in these experiments.

Figure 5

Immunohistochemistry of differentially expressed proteins. A-B: Expression of SOCS3 and C-D: BAX in the control and 6 hrs after optic nerve crush. SOCS3 was expressed throughout the retina and appeared increased in Muller cells (arrowheads) after optic nerve injury. Increased BAX labeling after optic nerve injury was particularly evident in the ganglion cell layer (arrows), consistent with pending programmed cell death of RGCs. Red = target antibody, Blue = DAPI.