Early cellular signaling responses to axonal injury

Detection of phosphoproteins in the retina after optic nerve injury

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 receptor-channel, 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.

TNFα 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 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.

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.

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.

Experimental Procedures

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 semi-quantitative, it lends itself to detection of changes in multiple phosphoproteins for each experimental time point.

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 1.5 ml Eppendorf tubes. Gel pieces were washed with 50 mM ammonium bicarbonate, 50% acetonitrile solution (3 × 15 min), and then in 100% acetonitrile. After removal of the solvent and drying in a Speed Vac (Thermo-Fisher, Pittsburgh, PA) concentrator, gels were rehydrated with 70–80 μl of 50 mM ammonium bicarbonate containing 0.01% (w/v) trypsin (Promega, Madison, WI). After incubation at 37°C (16–24 hr), the reactions were stopped by adding 1 volume of 5% (v/v) trifluoroacetic acid. The supernatants were removed and gel pieces further extracted twice with 100 μl of 0.1% trifluoroacetic acid/60% acetonitrile for 30 min. Combined extracts were then evaporated to dryness with a Speed Vac concentrator. The residues were dissolved in 20 μl of 0.1% formic acid/10% v/v acetonitrile).

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 Rhodamine-labeled 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 (HistoGene™; 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-Amp^® OA RNA amplification protocol (Arcturus). First strand cDNA was generated by reverse transcription using the total RNA. After the second strand cDNA was synthesized, a T7 RNA polymerase-driven cRNA synthesis was performed to obtain the first round of cRNA amplification. A second double strand cDNA synthesis was performed followed by a second round of cRNA amplification. A BioArray™ HighYield™ RNA transcript labeling protocol (T7) (Enzo Life Sciences, Famingdale, NY) was employed for the second round of amplification to biotinylate the cRNAs. To examine the reaction quality, an aliquot from the first stand cDNA synthesis in the first round cRNA amplification and another from the second strand cDNA synthesis in the second round amplification sample were removed for real-time PCR analysis.

Microarray Analysis

Amplified RNA (8 μg) was hybridized to Affymetrix (Santa Clara, CA) Mu430 v2.0 chips and processed as recommended by the manufacturer. Three chips each were used for the control and 6 hrs crush samples. For each dataset, invariant set normalization was performed using the PM/MM model for calculating signal intensities in dChip 2006 [51]. Thresholds for selecting significant genes were set at a relative fold-difference of > 1.2, absolute intensity difference between sample and baseline > 80, and paired t-test value of p <0.05. Genes meeting all of these criteria were considered as significantly different. This resulted in a list of 239 differentially expressed genes (160 upregulated and 79 down regulated, Additional file 2). Microarray data have been deposited in the GEO database with the series accession number GSE11862.

ELISA

ELISA assays for phosphoproteins were done with sandwich ELISA kits (Cell Signaling) following the manufacturer's instructions. These were used to detect phosphoJNK (Thr183, Tyr185) and phospho IκB (Ser32) in soluble tissue extracts. Briefly, extracts were diluted with the assay buffer to the desired total protein concentration (0.25, 0.5, 1.0 mg/ml) to 100 μl and applied in duplicate to the wells of the ELISA plate containing the capture antibody. Controls without added lysate were included in all assays. The plates were covered and incubated at 4°C for 12–16 hrs to allow binding of the target protein to the plate. Wells were then washed 4 times with the wash buffer supplied in the kit. The wells were then covered and incubated with the antiphosphoprotein antibody and incubated for 1 hr at 37°C. The plates were washed again and then incubated with horse radish peroxidase (HRP) linked detector antibody for 30 min at 37°C. The plate was washed again and then incubated with HRP substrate solution for 30 min at room temperature. Stop solution was added and the absorbance of converted substrate read at 450 nm in a plate reader.

Mouse TNFα was measured in soluble tissue extracts using an ELISA kit (Pierce). Briefly, tissue extracts were diluted to 0.25 or 0.5 mg/mL and 50 μl applied in duplicate to the ELISA plate. TNFα standards over the range of 35 to1225 pg/ml were measured in duplicate along with the samples. Then 50 μl of biotinylated antiTNFα antibody was added to all of the wells and the plate covered and incubated for 2 hr at room temperature. The plate was washed 5 times and then the wells were incubated with 100 μl of HRP-streptavidin solution for 30 min at room temperature. The reaction was stopped by adding an equal volume of acidic (2N HCl) stop solution. The absorbance was then read at 450 nm in a plate reader.