Early cellular signaling responses to axonal injury

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 TNFα 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.


Introduction
Axonal damage of long projecting neurons leads to retrograde degeneration and loss of the soma of the neuron by programmed cell death over a period of several days. However, little is known about the timing of events in the soma of the neuron immediately following damage to the axon and how soon the affected neuron with axonal damage signals surrounding glia and other neurons of the catastrophic event.
Retinal ganglion cells (RGCs) are long projecting neurons whose axons make up the optic nerve. To determine the temporal sequence of cellular signals and interactions following axonal injury, we have used optic nerve crush. The somas of the RGCs are in a single layer in the retina and are easily sampled and visualized. Similarly, the glia and other retinal neurons that are associated with the RGCs are also in specific layers and can be readily observed by immunohistochemistry.
Most studies of optic nerve damage leading to RGC loss make observations of the changes in gene expression, the level of a particular molecule or activation of a pathway starting at 24-48 hrs following the injury [1][2][3]. For example, inactivation of phospho-AKT and phospho-BAD have been reported as early as 48 hrs after injury [1]. Upregulation of proapoptotic proteins such as BAX and BIM has been reported at 24 hr post injury [4][5][6]. Seven days after optic nerve crush, there is increased caspase-3 activity and significant loss of RGCs [6][7][8].
In the work presented here, we have looked for changes in the retina within 6 hrs following optic nerve crush. Our interest was not biased towards any one pathway that had changed, but to use any changes that we found to determine the timing of cellular events and cell to cell signaling, particularly those that may precede degeneration. Thus, the work presented here attempts to answer the following questions: 1. When does the soma of the RGC "sense" that its axon has been injured? 2. Which cells in the retina are signaled by the RGCs that a catastrophic event has occurred? 3. Are cell death signals apparent in the first 6 hrs following injury to the axon? 4. Are there nuclear events in the first 6 hrs following injury to the axon?
We used phosphoproteomics and microarrays to find early protein and gene expression changes following axonal damage that could be followed up with immunoblots, immunohistochemistry and RT-PCR. We then used our results to deduce a temporal sequence of cell to cell interactions and representative signaling events in the retina following optic nerve injury. We interpret our results to indicate that within 30 min of the axonal injury, the RGC soma has detected the axonal damage and has already signaled other cells throughout the retina. In addition, cell death pathways have been activated within 6 hrs of the axonal injury and changes in gene expression have been initiated by 6 hrs.

Detection of phosphoproteins in the retina after optic nerve injury
We reasoned that phosphorylation of proteins would participate in initial responses; therefore, a proteomics approach was used to establish that there are phosphorylation events within the first 6 hrs following axonal injury. Database searching of the mass spectrometry data was used to identify the captured phosphoproteins (Additional file 1) and to obtain a differential, temporal spec-trum of phosphoproteins. The detected phosphoproteins were further analyzed using Gene Ontology (GO) programs, GoMiner [9] and ProfCom [10] to identify "enrichment" of phosphoproteins within the context of categories of cellular pathways. Enrichment of phosphoproteins refers to detection of changes beyond expected from a random distribution. The "changed" p-values are based upon Fisher's test for significance [9]. Table 1 contains the GO categories of biological processes with respect to phosphoproteins that were significantly (p < 0.05) enriched, compared to control tissue, after axonal injury. Note that enrichment of phosphoproteins occurred in intracellular signaling, protein kinase, modulation of transcription and ion channel categories, among others. Table 2 presents a selected list of phosphoproteins that we culled from the enriched GO categories. These results demonstrate that within the first 6 hrs following axonal injury, there were changes in the phosphorylation of proteins associated with pathways involving intracellular signal transduction via MAPK-ERK-1, glutamate/Ca 2+ signaling, TNFα activity and transcription factors. The variety of the detected phosphoproteins indicated that signaling pathways from the plasma membrane to the nucleus become activated through phosphorylation events. We further investigated these identified pathways because of their relevance to initiation of cell death and/ or regulation of cell survival.

Elaboration of signaling pathways detected by phosphorylation screening after optic nerve injury ERK-1 signaling
Although the phosphoproteomics method did not detect phosphorylated ERK-1 (Map kinase 1), we detected the phosphorylation of regulators of ERK-1 activation ( Table  2). For example, OSBP (oxysterol binding protein) regulates the activation of ERK-1 by way of binding to phosphatases that act on phosphorylated ERK-1 [11], MAGI-3 regulates activation of ERK-1 by lysophosphatidic acid (LPA) [12] and USP8, also known as UBPY, is a deubiquination enzyme that promotes degradation of the epiderminal growth factor receptor [13], which is a known activator of the ERK-1 pathway. Therefore, we tested for the activation of ERK-1. By immunoblot, activated ERK-1 (pERK1) appeared to decrease at 30 and 60 min after optic nerve injury, but increased significantly at 6 hrs ( Figure 1A-B). Immunohistochemistry showed activated ERK-1 primarily in the Muller cells of control retinas ( Figure 1C). However, after optic nerve injury, the distribution of pERK-1 changed dramatically. At 30 min, pERK-1 was no longer detected in Muller cells but now appeared in the OPL and the inner layer of the IPL ( Figure 1D). Immunolabeling for pERK-1 in the OPL, which contains photoreceptor synapses, was present at 30 min and persisted for at least 6 hrs after optic nerve crush ( Figure 1D-F). The increased labeling in the outer plexiform layer suggests that there were signals to the photoreceptors within 30 min following optic nerve crush. There was also immunolabeling for pERK-1 in the inner stratum of the inner plexiform layer at 30 min and 6 hrs and labeling in the ganglion cell layer at 60 min. This dynamic, cellular redistribution of activated ERK-1 following optic nerve injury is striking and suggests that other cells in the retina are rapidly responding to the axonal injury of the RGCs.
Glutamate/calcium signaling After optic nerve crush, we detected phosphorylation of calmodulin (CALM1), the ionotropic glutamate receptorchannel, GluR1 (GRIA), and other ion transport-related proteins ( Table 2). These results indicated altered calcium signaling and increased activation of glutamate receptors. Phosphorylation of the GluR1 occurs on tyrosine and serine residues [14]. Using lysates and membrane fractions from whole retinas, we performed immunoblots with site specific phosphoserine antibodies to GluR1. The Ser-831 and Ser-845 sites are adjacent to a putative tyrosine phosphorylation site (Tyr-812) in the carboxyl-terminal domain of the receptor [14]. Increased phosphorylation of Ser-831 was evident at 6 hrs ( Figure 2A). Phosphorylation of Ser-845 was detected in the membrane pellet fraction ( Figure 2B) and was also increased at 6 hrs.
Immunohistochemistry was used to localize where in the retina was phosphorylation of GluR1 occurring following optic nerve crush. As shown in Figure 2C-F, there was increased labeling of GluR1 in the ganglion cell layer by 6 hrs after axonal injury; whereas the labeling in the outer plexiform layer remained relatively constant. The increased phosphorylation of GluR1 by 6 hrs was consistent using proteomics and immunohistochemistry.

TNF signaling
In our phosphoprotein screen, several proteins related to the TNFα pathway were detected following optic nerve crush (Table 2). Therefore, we determined in the retina the presence of the ligand and downstream protein kinases that can be activated through the TNFα pathway. As shown in Figure 3A, TNFα was detected in control retinas but TNFα levels increased markedly by 6 hrs after optic nerve injury.
If the TNFα pathway was activated, two major intracellular signaling pathways may also be activated: SAPK/JNK and NFκB. The activation of SAPK/JNK over the 6 hrs time course is shown in Figure 3B-C. Consistent with the increased levels of TNFα by 6 hrs, there was a significant increase in activated SAPK/JNK by 6 hrs after optic nerve injury. By immunohistochemistry, pJNK was found throughout the inner retina under control conditions and at 30 and 60 min post optic nerve crush ( Figure 3D, E and 3F). However, by 6 hrs after optic nerve injury ( Figure  3G), increased activation of JNK was present within the ganglion cell layer, which includes the RGCs. Thus, the proteomics, immunoblots, ELISA and immunohistochemistry all identify activation of the TNFα pathway, most likely in the RGCs, by 6 hrs following optic nerve injury.
We assayed for the activation of NFκB by means of a specific ELISA for phospho-Ser32 on the IκB subunit of NFκB, immunoblot for phosphorylated protein and phosphorylation of the p65 subunit of NFκB. None of these assays demonstrated activation of the NFκB pathway by 6 hrs following optic nerve crush (data not shown). Other phosphoproteins related to TNFα signaling (Table 2) may be responsible for the down-regulation of NFκB (See Discussion).

Nuclear activity
Active transcription involves the dynamic, post-translational modification of histones and other proteins associated with chromatin, as well as transcription factors that translocate to the nucleus depending upon their phosphorylation state. Table 2 contains several transcriptional *Listing the proteins in Table 2 was based upon the following criteria: 1) the underlying peptides were detected at least twice in the data, 2) the identified protein has known phosphorylation sites in the Phosphosite database [50], and 3) the phosphoprotein levels changed (up/down) by approximately two-fold after axonal injury. **Direction is the up or down change in phosphopeptide level based upon the average of the 30, 60 min and 6 hrs values divided by the control value.
Activation of ERK-1 (MAPK1) 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. Phosphorylation of GluR1 cofactors, such as TCP20, and HES6. TCP20 (also known as SPBP-streomelysin-1 platelet-derive growth factor responsive element binding protein) enhances the activity of various transcription factors, including c-Jun [15]. HES6 is a basic helix-loop-helix transcription factor that promotes neuronal differentiation [16] but inhibits astrogenic differentiation [17]. HES6 is phosphorylated by ERK-1 which is necessary for its anti-astrogenic activity [17].
In our survey for nuclear phosphoproteins based on mass spectrometry, we also found that H2A, JMJD1A (a histone demethylase) and SETD2 (a histone methylase) were phosphorylated within 6 hrs after optic nerve injury.
Using an antibody to phosphorylated H2A (pSer139), we confirmed the phosphorylation state of this histone in the neural retina following optic nerve injury. Western blotting indicated that H2A is phosphorylated and that the level of phosphorylation increased during the 6 hrs after optic nerve injury ( Figure 4A). The phosphorylation of H2A may be related to chromatin remodeling [18,19] and is also a feature of the initiation of apoptosis [20,21].
Because we detected phosphorylation of two proteins involved in histone methlation/demethylation (JMJD1A and SETD2 in Table 2), we further investigated selected histone H3 methylation sites. To detect changes in histone methylation, we used an antibody directed towards histone H3 di and tri-methylated at K4. Trimethylation at this site is associated with the active transcription of a number of genes [22]. As shown in Figure 4B, H3-K4 trimethylation is constitutive, but decreases abruptly by 30 min, and recovers slightly at 6 hrs after optic nerve crush. H3-K4 dimethylation also decreased with time after injury (data not shown). Thus, the increase in H2A phosphorylation and changes in histone H3 methylation may be linked to decreased transcription of certain genes within 6 hrs following optic nerve crush.

Differential gene expression in the ganglion cell layer post retinal injury
To examine the early changes in gene expression within the ganglion cell layer after optic nerve crush, we performed laser capture microdissection on retinal tissue sections to extract material from the ganglion cell layer. This layer contains RGCs as well as astrocytes, displaced amacrine cells, microglia, vascular endothelia and the processes from Muller cells. Thus, laser capture microscopy enriched our sample for RGCs compared to having used full thickness retina. mRNA was prepared from ganglion cell layer samples obtained from retina sections of eyes with optic nerve crush and compared to ganglion cell layer samples from control retina sections. Samples were subjected to linear amplification and processing, followed by hybridization to a mouse microarray (Affymetrix Mu430 V2). A list of 220 differentially expressed genes was obtained after filtering for a minimum 1.2-fold change and p < 0.05 (Additional file 2). Changes in selected genes were verified using qRT-PCR. We used gene ontology analysis (GoMiner and ProfCom) to analyze changes in gene expression with regard to cellular signaling.
In the gene ontology analysis, expression of genes involved in transcription and transcriptional regulation were one of the most significant with 31 differentially expressed genes (p = 0.01, Additional file 3). The increase in nuclear gene phosphorylation that we found with phosphoproteomics apparently leads to alterations in transcriptional activity. Another category containing significantly altered gene expression was phosphorylation with 16 differentially expressed genes (p = 0.0035, Additional file 3). Table 3 contains a curated list of genes we found in these GO categories grouped with respect to functions in signal transduction, Ca 2+ homeostasis and cell death. We also found that expression of genes related to cytokine/growth factor signaling was altered at 6 hrs post injury. Socs3 and Atf2 were upregulated and Map3K10 was downregulated (Table 3). Atf2 is a transcription activator downstream of JNK, while Map3K10 is an activating kinase upstream of JNK. Consistent with the microarray results, qRT-PCR data confirmed the upregulation of Socs3 (4.4-fold). Several differentially expressed genes at 6 hrs post injury were associated with regulation of cell death pathways: Aifm3 and Bax are directly associated with mitochrondria permeability channels that, when opened, leads to apoptosis. We confirmed the upregulation of Bax expression (1.6-fold) using qRT-PCR.

Histone phosphorylation and methylation
Using immunohistochemistry, we investigated which cell types are differentially expressing SOCS3 and BAX proteins at 6 hrs after optic nerve injury. As seen in Fig. 5A-B, SOCS3 appears to be present throughout the retina but is increased at 6 hrs, particularly in the Muller cells. Similarly, BAX increased at 6 hrs, particularly in the ganglion cell layer (Fig. 5C-D). Thus, the immunohistochemistry demonstrating proteins was consistent with the mRNA expression data. Expression of other cell death genes was also apparent at 6 hrs (Table 3). Consistent with the lack of activation of the NFkB pathway, we did not observe upregulation of the anti-apoptotic factor Bcl-2 or caspase inhibitors [23]. Thus, changes associated with the initiation of programmed cell death may already have started in RGCs within 6 hrs after optic nerve injury.

Discussion
We used a multidisciplinary approach to investigate the temporal, intercellular and intracellular signaling that immediately follow optic nerve injury. Our hypothesis was that there are cellular events in different cells in the retina very early after optic nerve injury. Our intent was not to investigate all pathways or any one pathway indepth, but to find multiple signaling pathways, that would be representative of sequential changes. Our data provide a temporal, sequential framework of early events within the first 6 hrs after optic nerve injury. Previous studies have investigated changes in selected protein phosphorylation [1] or gene expression [2,3] at one day to several weeks after optic nerve injury. Thus, our data pro- vides the first observation of responses in the neural retina as early as 30 min after axonal injury.
As answers to the questions that were raised in the Introduction of this paper, we believe that: 1. The soma of the RGC "senses" that its axon has been injured within 30 min. This interpretation of our data is based on the dramatic de-activation of the phosphorylation state of ERK1 in the Muller cells within 30 min. Muller cells and astrocytes express activated ERK-1 in the retina, and these cells express higher levels in retinas from glaucomatous donors [24]. Muller cells wrap around the somas of RGCs and have many points of contact. We hypothesize that within 30 min, the somas of the RGCs signal the Muller cells, which lead to loss of the activation/ phosphorylation state of ERK1 in the Muller cells. If there are changes in Muller cell activity by 30 min post axonal injury, the likely source of the signals to affect these changes is the somas of the RGCs with damaged axons. Thus, the somas of the RGCs have presumably "sensed" that a cellular event has occurred.
2. The somas of the RGCs probably signal that a catastrophic event has occurred to many neurons and glia in the retina within 30 min. Surprisingly, 30 min after acute injury, when Muller cells have lost ERK-1 activation/phosphorylation, ERK-1 activation/phosphorylation simultaneously appears in the outer plexiform layer, the location of the photoreceptor synapses, in the inner nuclear layer and in the inner plexiform layer.
3. Cell death signals are apparent within 6 hrs following injury to the RGC axon. These death signals include: an Immunohistochemistry of differentially expressed proteins 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.
increase in TNFα production and changes in phosphorylation of related TNFα pathway proteins (CASP8AP2 (Flash), TTRAP, SDCCAG3, JNK, CARD9, and DAP2IP). DABIP2 (AIP1) [25,26] is directly involved in signaling from the TNFα receptors to activate JNK, while SDCCAG3 is involved in receptor trafficking [27]. These signals may not be sufficient to induce cell death at 6 hrs but may be part of the early events that lead to programmed cell death. The lack of activation of the NFκB survival pathway is consistent with changes in the phosphorylation of other TNFα pathway components, (eg TTRAP, CARD9, CASP8AP2) that can negatively regulate NFκB activation [28][29][30]. Finally, protein kinase MAST2, which is upregulated 6 hrs post optic nerve crush (Table 3), interacts with TRAF6 in the TNFα pathway so as to decrease activation of NFκB [31].
4. There are nuclear events causing new protein synthesis within 6 hrs following injury to the RGC axon. Two new proteins, for example, BAX and AIFM3, are associated with the initation of programmed cell death; whereas, another, RTN4, sequesters the antiapoptotic BCL-2 protein [32]. The decrease in histone methylation ( Figure 5) and the upregulation of transcription factors (Table 3) at 6 hrs after optic nerve crush are consistent with changes in transcriptional activity. Furthermore, phosphorylation of H2A at Ser-139 is associated with release of H2A from the nucleus and apoptosis [18].
The increase in expression of Socs3 can be related to JAK1 activation [33] and cytokine induced degeneration. For example, Socs3 is also upregulated in the neural retina upon light-induced injury [34]. In these studies, the activity of one or more of the IL-6 family of cytokines (IL-6, CNTF, NP, LIF, CLC) was the stimulus for Socs3 upregulation. We did not detect upregulation of any single member of the IL-6 family in our microarray data. Thus, as with light-induced injury, multiple cytokines likely lead to the increase of Socs3 expression.
The increased expression of Bax and other genes associated with programmed cell death is consistent with the beginnings of a pro-apoptotic program that eventually translocates BAX [5,6] and the related proapoptotic protein BIM [4,35] to the mitochondria to induce death in RGCs. Upregulation of BAX protein has been shown to persist after optic nerve crush [1,6]. Although a detailed promoter analysis has not been reported, BAX upregulation has been linked to JNK activation [36] that we observed within 6 hrs of optic nerve injury. Bax knockout mice are more resistant to RGC cell death after optic nerve crush, but not to degeneration induced by glutamate excitotoxicity [37]. RGC cells in Bim knockout mice are also protected from optic nerve axotomy-induced death [4]. The proapoptotic activity of Bim is negatively regulated by ERK-1 phosphorylation, while phosphorylation by JNK enhances Bim activity possibly by dissociation from intracellular sequestration [38]. Phosphorylation of BIM by ERK-1 causes its degradation by the proteosome [39] so that the regional differences we observe in ERK-1 and JNK activation (Figs 3 and 4) could affect Bim levels in various cell types.
In addition, we note that there is limited survival signaling in the retina immediately after optic nerve injury. Previous studies have shown that survival signaling by IGF-1 through the phosphoinositide-Akt pathway begins to decrease within two days after optic nerve crush [1]. The loss of IGF-1 signaling may be due to the upregulation of Socs3 (Table 3) which is known to antagonize this pathway [40] and interacts directly with the IGF-1 receptor [41]. The changes in glutamate receptor phosphorylation that we observed after optic nerve crush suggests that altered Ca 2+ signaling is part of the degenerative process. Brain-derived neurotrophic factor (BDNF) is an important trophic factor for RGC cells and has been shown to be neuroprotective in RGC injury paradigms (Reviewed in [42]). However, the upregulation of Camk2 and related Ca 2+ signaling (Tables 2 and 3) antagonizes the trophic activity of BDNF [43]. Thus, application of BDNF, IGF-1, and related factors [44] may be of only short-term benefit after optic nerve injury.
Based on our data we offer the following hypothesis: The soma of the RGC "senses" that its axon is damaged within 30 min and signals the Muller cells, which, we believe, signal the entire retina that a catastrophic event has occurred. Furthermore, within 6 hrs of damage to the optic nerve, death signals are present in the retina that will ultimately lead to RGC degeneration. The temporal rapidity with which these events occur suggest that attempting to interfere with programmed cell death at a later time may be fruitless and, perhaps, not possible

Experimental Procedures
Animal model Retinas were obtained from male C57BL/6J mice. All experiments were performed in accordance with the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals, the National Institutes of Health Guide for the Care and Use of Laboratory Animals, the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and institutional, federal, and state guidelines regarding animal research. Groups of 5-9 mice were subjected to optic nerve crush. Axons of the optic nerve were crushed with fine forceps for 10 sec, 2 mm posterior to the globe, under direct visualization, within an intact meningeal sheath. By performing the optic nerve crush 2 mm posterior to the globe, the injury is distal to the entry of the ophthalmic artery into the optic nerve.
Thus, care is taken to not disturb the retinal blood supply. Optic nerve crush has been widely used by many laboratories and is well documented in the literature [45,46]. At the desired times (30, 60 and 6 hrs after crush) eyes were enucleated and neural retina removed and frozen at -80°C. Controls (0 min) were contralateral eyes that had not been injured from the same animals in each group.

Strategy
The initial screening for changes in phosphorylated proteins was done using affinity capture methods coupled to mass spectrometry for protein identification. These methods included anti-phosphotyrosine beads for enrichment of tyrosine phosphorylated proteins followed by separation of the captured material using one-dimensional gel electrophoresis. We used metal-ion chelate chromatography of phosphopeptides obtained from proteins that were not captured by anti-phospho-tyrosine antibodies. We did these experiments at multiple points post injury to attempt to capture a broad spectrum of events in cellular signaling. Although the method used was only semiquantitative, it lends itself to detection of changes in multiple phosphoproteins for each experimental time point.

Isolation of phosphoproteins/peptides
Tyrosine-phosphorylated proteins were isolated by immunocapture with anti-phosphotyrosine antibody 4G10 conjugated to agarose beads (Millipore Billerica, MA). This antibody has been used previously to characterize tyrosine phosphoryated proteins in stimulated cell systems [47] and for quantitative phosphoprotein detection [48]. Lysate containing 1 mg of protein was diluted 1:10 with lysis buffer without NP-40 detergent and applied to 50 μl of a slurry of 4G10-conjugate. The antibody binding reaction was incubated at 4°C with gentle rocking for 16 to 24 hr. Beads were pelleted by centrifugation (2000 rpm, 3 min). The supernatant was removed and saved for subsequent digestion and isolation of additional phosphopeptides (see below). The beads were washed two times with 50 μl of lysis buffer without NP40 and the washings combined with the original supernatant. The beads were washed with lysis buffer (500 μl) without NP-40 and the supernatants discarded. Proteins were eluted from the beads by applying 50 μl of SDS-PAGE sample buffer (Invitrogen) and heating to 95°C for 10 min. After brief centrifugation, the supernatants were removed and applied to individual lanes of a 4-12% polyacrylamide gel (Biorad Criterion, Hercules, CA) and electrophoresed at constant voltage. Gels were stained with Simply Blue stain (Invitrogen, Carlsbad, CA) and de-stained in water.

In gel digestion of phosphotyrosine antibody-captured proteins
Each gel lane was cut into 10 bands (4 mm × 6 mm) and further chopped into ~1 mm pieces and transferred to 1.

Mass Spectrometry
Peptides from the gel bands of tyrosine phosphorylated proteins were separated on a nano-flow column (0.075 × 50 mm, C18 Agilent Santa Clara, CA). The column was eluted at 0.3 μl/min with a gradient of 20 to 65% (v/v) acetonitrile in 0.1% formic acid. Peptides from the phosphopeptide isolation cartridge were separated using automated two-dimensional chromatography on the Agilent 1100 chromatography system. In the first dimension, phosphopeptides were injected onto a sulfated-ion exchange column (PolyLC 0.3 mm × 50 mm (Columbia, MD). Peptides were then eluted with steps of 0.05, 0.1, 0.2 and 0.4 M ammonium acetate onto a nanoflow column (0.075 mm × 150 mm, Agilent). The column was eluted at 0.3 μl/min with a gradient of 5-65% acetonitrile in 0.1% formic acid. Peptides were detected using an Agilent 1100 XCT ion-trap LC-MS system as described previously [49]. Each sample from the gel band isolation was run twice.

Data Analysis
Peptide mass and fragmentation data were filtered and database searching done using Spectrum Mill software (Agilent) as described previously [49]. We used the mouse International Protein Index (IPI) database for searching the mass spectrometry data generated from each sample. From each sample run, a curated list of hits was obtained. This list was based upon the database score and the quality of mass spectral data. For comparisons, we used only those hits that were identified at least twice in each sample. The MS-data intensity values for each peptide were then averaged over the three time points (3 0 min, 60 min, 6 hrs) and divided by averaged control values. The final list of identified proteins is summarized in Additional file 1. This spreadsheet contains the sequences of phosphopeptides identified by mass spectrometry data. Also included is the analysis for the presence of phosporylation sites using Phosphosite [50].

Immunohistochemistry
Enucleated eyes were fixed in 2% wt/vol paraformaldehyde in 0.01 M phosphate buffered saline (PBS; pH 7.4) at 4°C overnight. Six animals were used for each group in all immunohistochemistry experiments. Immunohistochemistry was performed on paraffin sagittal sections of retina for pJNK (Cell signaling, Danvers, MA) using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) and diaminobenzidine as a substrate. As a negative control, sections were treated in the same manner, except that incubation with primary antibody was omitted. The sections were treated for equal time in DAB reagent and photographed at the same time. Phosphorylated GluR1 (Ser-845, Zymed, Billerica, MA), phosphorylated ERK-1 (Cell Signaling) SOCS3 (Santa Cruz P-19, Santa Cruz, CA), BAX (Santa Cruz H103), and glutamine synthase (Chemicon-Millipore) were detected using Rhodaminelabeled secondary antibody (Vector Labs). Nuclei were stained with DAPI. Sections were washed, mounted and viewed under a fluorescence microscope (Olympus, AX70). As a negative control, sections were treated in the same manner, except that incubation with primary antibody was omitted.

Isolation of retinal ganglion cell layer by laser capture microscopy (LCM)
The LCM system that we used (Veritas Microdissection System, Molecular Devices, Sunnyvale, CA) obviated the need for tissue dehydration prior to microdissection. This enabled us to isolate quiescent retinal ganglion cells directly from 8-μM frozen sections of mouse eyes, thereby increasing the yield and quality of RNA (see below). Frozen sections, mounted on special membrane-coated slides (P.A.L.M. Microbeam), which facilitated the capture of cells, were briefly (1 min) stained with hematoxylin (His-toGene™; Arcturus, Mountain View, CA). In addition to visualizing the retinal structures, this staining procedure also removed the OCT mounting medium. Labeled sections were tracked with intergral light microscope using a 20× objective. Retinal ganglion cells to be isolated were outlined with a "light" pen or cursor on a monitor screen. Such an outline defined the area that would be cut and catapulted intact into a Capsure Macro LCM cap (Molecular Devices, Sunnyvale, CA). In this manner, approximately 6000 cells from the ganglion cell layer were isolated from each eye.

Total RNA Isolation and cRNA Amplification
Total cellular RNA from LCM-captured cells was isolated and purified (Pico-Pure™; Arcturus). Samples of the total starting RNA were analyzed by capillary electrophoresis (Agilent Technologies, Palo Alto, CA) to assess the degree of purification. Approximately 60 ng of total cellular RNA could be extracted from 6000 cells from the ganglion cell layer that were isolated by LCM. When this RNA was contrasted with commercially prepared total RNA from mouse liver using picogram chips and a Bioanalyzer (Agilent Technologies), sharp bands corresponding to the 18 S and 28 S RNA were observed for all samples RNA quality was further assessed by calculating the RNA integrity number, which is based on a proprietary Agilent Technologies algorithm. Total RNA from the isolated cells was subjected to cRNA amplification. Briefly, 1.5 rounds of cRNA amplification were accomplished using a Ribo-