Granulocyte Colony-Stimulating Factor-Induced TAF9 Modulates P53-TRIAP1-CASP3 Axis to Prevent Retinal Ganglion Cell Death after Optic Nerve Ischemia


 BackgroundOptic nerve head (ONH) infarct can result in progressive retinal ganglion cell (RGC) death. Some evidences indicated that the granulocyte colony-stimulating factor (GCSF) provides positive effects against ischemic damage on RGCs. However, protective mechanisms of the GCSF after ONH infarct are complex and remain unclear. MethodsTo investigate the complex mechanisms, the transcriptome profiles of the GCSF-treated retinas were examined using microarray technology. The retinal mRNA samples on days 3 and 7 post rat anterior ischemic optic neuropathy model (rAION) were analyzed by microarray and bioinformatics analyses. To evaluate the TAF9 function in RGC apoptosis, GCSF plus TAF9 siRNA-treated rats were evaluated using retrograde labeling with FluoroGold assay, TUNEL assay, and Western blotting in a rAION.ResultsGCSF treatment influenced 3101 genes and 3332 genes on days 3 and 7 post rAION, respectively. ONH infarct led to changes in 702 and 179 genes on days 3 and 7 post rAION, respectively. After cluster analysis, the TATA box-binding protein (TBP)-associated factor levels were significantly reduced after ONH infarct but significantly increased after GCSF treatment. The network analysis revealed that TBP associated factor 9 (TAF9) can bind to TP53 to induce TP53 regulated inhibitor of apoptosis 1 (TRIAP1) expression. The RGC density in the GCSF plus TAF9 siRNA-treated group was 1.95-fold (central retina) and 1.75-fold (midperipheral retina) lower than that in the GCSF-treated group (p < 0.05). The number of apoptotic RGC in the GCSF plus TAF9 siRNA-treated group is threefold higher than that in the GCSF-treated group (p < 0.05). Treatment with TAF9 siRNA significantly reduced GCSF-induced TP53 and TRIAP1 expression by 2.4-fold and 4.7-fold, respectively, in the rAION model. Overexpression of TAF9 significantly reduced apoptotic RGC and CASP3 levels and induced TP53 and TRIAP1 expression in the rAION model. ConclusionOur in vivo study is the first to report that GCSF is able to induce TAF9 expression to control RGC death and survival after ON infarct. Besides, the binding of TAF9 and TP53 is crucial to inhibit TP53 degradation and to modulate TRIAP1-CASP3 axis. Thus, we considered that TAF9 is a potential drug for patient with NAION.


Methods
To investigate the complex mechanisms, the transcriptome pro les of the GCSF-treated retinas were examined using microarray technology. The retinal mRNA samples on days 3 and 7 post rat anterior ischemic optic neuropathy model (rAION) were analyzed by microarray and bioinformatics analyses. To evaluate the TAF9 function in RGC apoptosis, GCSF plus TAF9 siRNA-treated rats were evaluated using retrograde labeling with FluoroGold assay, TUNEL assay, and Western blotting in a rAION.

Results
GCSF treatment in uenced 3101 genes and 3332 genes on days 3 and 7 post rAION, respectively. ONH infarct led to changes in 702 and 179 genes on days 3 and 7 post rAION, respectively. After cluster analysis, the TATA box-binding protein (TBP)-associated factor levels were signi cantly reduced after ONH infarct but signi cantly increased after GCSF treatment. The network analysis revealed that TBP associated factor 9 (TAF9) can bind to TP53 to induce TP53 regulated inhibitor of apoptosis 1 (TRIAP1) expression. The RGC density in the GCSF plus TAF9 siRNA-treated group was 1.95-fold (central retina) and 1.75-fold (midperipheral retina) lower than that in the GCSF-treated group (p < 0.05). The number of apoptotic RGC in the GCSF plus TAF9 siRNA-treated group is threefold higher than that in the GCSFtreated group (p < 0.05). Treatment with TAF9 siRNA signi cantly reduced GCSF-induced TP53 and TRIAP1 expression by 2.4-fold and 4.7-fold, respectively, in the rAION model. Overexpression of TAF9 signi cantly reduced apoptotic RGC and CASP3 levels and induced TP53 and TRIAP1 expression in the rAION model.

Conclusion
Our in vivo study is the rst to report that GCSF is able to induce TAF9 expression to control RGC death and survival after ON infarct. Besides, the binding of TAF9 and TP53 is crucial to inhibit TP53 degradation and to modulate TRIAP1-CASP3 axis. Thus, we considered that TAF9 is a potential drug for patient with NAION.

Background
In elderly individuals, the most common type of acute optic neuropathy is non-arteritic anterior ischemic optic neuropathy (NAION), with an estimated annual incidence of 3.72 per 100,000 individuals in Taiwan [1]. NAION is de ned clinically as painless visual loss with swelling of the optic disc leading to optic disc atrophy [2]. Currently, there is no effective treatment for NAION. Optic nerve (ON) ischemia induces a series of detrimental events, eventually resulting in retinal ganglion cell (RGC) loss [2]. RGC death and axon degeneration are major complications of ischemic damage and mainly caused by oxidative stress [3][4][5][6], pro-in ammatory factors [6,7], aberrant calcium ion homeostasis [8], and macrophage polarization [7,9]. Therefore, a comprehensive investigation on the complex molecular mechanisms of axonal degeneration and RGC death in ON ischemia may bring new horizon for treatment.
Several efforts in preventing ON injuries and RGC death have been made using different approaches, such as anti-in ammatory compounds [10,11], neurotropic factors [12,13], oxidative stress regulators [5], calcium channel blockers [14], microglial activation inhibitors [7,15], and blood-borne macrophage in ltration blockers [7,12,16]. These potential treatments provide some possible directions to elucidate how to control the fate of RGCs. In context with our previous study on neuroprotective effects of granulocyte-colony stimulating factor (GCSF), we found that GCSF exhibited the ability to rescue RGC from apoptosis, which may be involved in modulations of blood-ON barrier, macrophage in ltration, and in ammatory reaction [7]. Moreover, our previous ndings demonstrated that treatment with GCSF can activate PI3K/AKT pathway to protect RGC from apoptosis [17].
GCSF belongs to a member of the hematopoietic growth factor family. It is widely used in clinical practice for the treatment of neutropenia [18]. Recent ndings have suggested that GCSF also has a nonhematopoietic role in memory improvement in Alzheimer's disease and neuroprotective role in Parkinson's disease [19,20]. It also promotes angiogenesis and inhibits apoptosis and in ammation in rats with ischemic stroke [21][22][23]. Furthermore, the GCSF receptor (GCSFR) is expressed in various neural and glial cells, such as RGCs, microglia, astrocyte, and oligodendrocyte, which results in direct activation of GCSFR signaling pathways, including JAK/STAT, PI3K/AKT, and MAPK/ERK pathways [24]. These signaling pathways are involved in cell growth and differentiation. Thus, the mechanisms by which GCSF promotes RGC survival are likely complex.
To reveal these complex mechanisms, transcriptome analysis of the rat retina is a possible approach that can be used to investigate the transcriptome changes after ON infarct with or without GCSF treatment. The microarray technique is a high throughput analysis to determine the expression level of large numbers of genes simultaneously. This technique has also been used to evaluate changes after axonal injury in both the retina and isolated RGCs [25,26]. Therefore, we considered that microarray analysis is an adequate tool to explore any possible mechanism involved in RGC apoptosis and survival. Different animal models have been used to investigate RGC pathology. Herein, we used rat anterior ischemic optic neuropathy (rAION) model as it represents similar features and pathology with human and primate AION [27]. The rAION model is achieved by photodynamic therapy, which generates superoxide radicals in the ON capillaries, causing capillary thrombosis, in ammation, and oxidative stress [27].
These pathological changes are important events to induce RGC apoptosis. Thus, it is an appropriate model to use in investigating the mechanisms of RGC apoptosis.
In present study, a comparative microarray analysis was adopted to explore the dynamic transcriptome changes in the rat retina under ON infarct and GCSF treatment. We identi ed that the mRNA levels of several TATA-box binding protein (TBP)-associated factors (TAFs) were signi cantly reduced after ONH infarct but signi cantly increased after GCSF treatment. Among these genes, we targeted one gene, transcription initiation factor TFIID subunit 9 (taf9), which encodes for one of the smaller subunits of transcription factor IID (TFIID) that binds to the general transcription factor transcription initiation factor IIB (Gtf2b) and several transcriptional activators, such as p53 and Vp16 [28,29]. Taf9 physically interacts with p53 at its N-terminus, where p53 also interacts with its negative regulator, Mdm2, thereby inhibiting Mdm2 degradation of p53 [30]. Functionally, this interaction translates to an increase in p53-induced angiogenesis, DNA repair, cell arrest, cell survival, or apoptosis [30]. However, it is questionable whether TAF9 drives P53 toward cell survival or apoptosis. Thus, this study aimed to reveal the underlying mechanisms of TAF9 in RGC apoptosis and cell survival.

Study design
In examining the transcriptome pro les in the retina, the rAION-inducted rats were treated by PBS or GCSF. At day 3 post rAION, the retina samples were collected in the PBS-treated group (n = 3) and GCSF-treated group (n = 3). At day 7 post rAION, the retina samples were again collected in the PBS-treated group (n = 3) and GCSF-treated group (n = 3). The retina samples in the sham-operated group (n = 3) were also collected. All retina samples were used to extract the mRNA. The mRNA samples were analyzed by RNA microarray to pro le the transcriptome in each group. The differentially expressed genes were classi ed by GO analysis. Among the differentially expressed genes, the TBP-associated proteins were classi ed into the function of cell growth. Therefore, the TBP-associated proteins were selected to predict the protein-to-protein interaction by network analysis (STRING 9.0). The TP53 was predicted to interact with many TBP-associated proteins in the network analysis. One of the TBP-associated proteins, TAF9, was selected to be the target protein to verify the function in the regulation of cell death and survival. TAF9 knockdown and overexpression experiments were performed in the rAION model. The rAION-inducted rats were treated with scramble siRNA, GCSF plus scramble siRNA, and GCSF plus TAF9 siRNA to evaluate the visual function (n = 12 in each group), RGC density (n = 12 in each group), and apoptotic RGCs (n = 6 in each group). The TAF9, TP53, and TRIAP1 levels were evaluated in each group (n = 6) using Western blot analysis. Moreover, the AAV2-mediated overexpression of TAF9 was intravitreally administered before rAION induction to examine the anti-apoptotic ability of TAF9 in the rAION model. The number of apoptotic RGCs was evaluated in the AAV2-r-TAF9-treated group (n = 6) and PBS-treated group (n = 6).

Animals
Male Wistar rats were used in the study. The rats were aged 6-8 weeks with body weight of 150-180 g. Animal care and experimental procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and the Institutional Animal Care and Use Committee (IACUC) at the Tzu Chi Medical Center approved all animal experiments.

rAION induction
The procedure of rAION induction was described in our previous study [31]. Before general anesthesia, all rats were administered Mydrin-P (Santan, Shiga, Japan) and Alcaine (Alcon, Texas, USA) eye drops for pupil dilation and topical anesthesia, respectively. Subsequently, the rats were injected intramuscularly by a mixture of ketamine (40 mg/kg body weight, P zer, UK) and xylazine (4 mg/kg body weight; Sigma, St. Louis, MO, USA) for general anesthesia. For photosensitization, 2.5 mM Rose Bengal diluted in PBS (1 ml/kg of body weight) was administered intravenously before laser application. After rose bengal injection, the optic disc was immediately exposed to an argon green laser system (MC-500 Multi-color laser, Nidek Co., Ltd, Tokyo, Japan, setting: 532 nm wavelength, 500 μm size and 80 mW power) for 12 1-s pulses. A laser fundus lens (Ocular Instruments, Inc.) was used to target the optic disc. At the end of experiment, TobraDex eye ointment (Alcon-Couvreur, Puurs-Sint-Amands, Belgium) was applied to the eyes of all experimented rats.

RNA microarray analysis (quality check, annotation, and ontology)
The retina samples were collected at days 3 and 7 post rAION. Total RNA was isolated from retina homogenate using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Complementary DNAs were synthesized using reverse transcriptase kit. RNA microarray analysis was performed using Rat OneArray kit according to the manufacturer's protocol. Clustering and principal component analysis were performed to determine the differences among biological sample replicates and their treatment conditions. Raw intensities were normalized with median scaling normalizing method, and covariance was determined by error model of Rosetta Resolver system. Normalized intensity was transformed to the log 2 ratio (fold change). Gene annotation was performed with reference to NCBI RefSeq Release 57. EnsEMBL released 70 cDNA sequences and rattus_norvegicus_core_70_5b annotations. Differentially expressed genes that showed both a log2 ratio (fold change) >1 and p < 0.05 were considered candidate genes.
FVEP recordings FVEP measurements were performed as described in our previous study [31]. After general anesthesia, the sagittal region of the skull was opened in the rats. The 4-mm screw implants were passed through the skull approximately 1.5 mm and placed at the frontal cortex and primary visual cortex region of both hemispheres using stereotaxic coordinates. A visual electrodiagnostic system (Diagnosys LLC, Lowell, MA, USA) was used to measure the FVEP. The number of sweeps per average was 64 for each rat. A comparison of the average amplitude of the P1-N2 wave in each group was made to evaluate visual function.
Retrograde labeling of RGCs and measurement of RGC density RGCs were labeled as described in our previous study [31]. Brie y, retrograde tracer dye uorogold was injected into the superior colliculus one week before the rats were euthanized. One week after labeling, the rats were euthanized, and retinas were carefully at mounted. The central and midperipheral regions in the retina were examined under a uorescence microscope with a built-in lter set (excitation lter, 350-400 nm; barrier lter, 515 nm) and connected digital imaging system. RGC density was calculated using ImageMaster 2D Platinum Software V 7.0 (GE Healthcare, Illinois, USA).

Retinal and ON sample preparation
After euthanized, there rat eyeballs were xed in 4% paraformaldehyde overnight. The eyecups and ONs were separated and transferred to 30% sucrose solution; the samples were incubated at 4°C until they settled at the bottom of the tubes. The retina cross-section and ON longitudinal sections of 15 μm were obtained using a cryostat.

TUNEL assay
To ensure the use of equivalent elds for comparison, all frozen retinal sections were prepared with a 1-2 mm distance from the ON head. We counted apoptotic cells using the TUNEL assay kit (Click-iT™ Plus TUNEL Assay, Invitrogen, Waltham, USA). Nuclei were stained with 4t,6-diamidino-2-phenylindole (DAPI). The TUNEL-positive cells in the RGC layer of each sample were counted in 10 high power eld (HPF) (400×), and an average of three sections per retina was used for further analysis (n = 6 rats per group).

Western blotting
After euthanized, and the rats' eyes were enucleated. The retinas were homogenized in lysis buffer. The protein sample was separated on 10% bis-acrylamide gel. The proteins were transferred to polyvinylidene di uoride (PVDF) membranes. The membranes were blocked with 5% non-fat dry milk for 1 h. The membranes were incubated with Taf9, TP53, TRIAP1, CASP3, ACTIN antibody at 4°C for 12-16 h, followed by incubation with a secondary antibody conjugated to HRP against the appropriate host species for 1 h at room temperature. Then, the membranes were developed using enhanced chemiluminescent (ECL) substrate. Membranes were exposed to a Western blot analyzer, and the relative density was calculated using image master platinum software V 7.0 (GE Healthcare, Illinois, USA).

Statistical analysis
All statistical analyses were performed using IBM SPSS software. The data are presented as mean ± standard deviation. A Mann-Whitney U test was used for comparisons between groups. P-values < 0.05 were considered statistically signi cant, with * representing p≤0.05.

Identi cation of differentially expressed genes by microarray
To investigate RGC death and survival in the transcriptional level, the rAION-inducted rats were treated with phosphate buffered saline (PBS) or GCSF. The transcriptome pro les were analyzed using oligonucleotide microarrays. Microarray data were analyzed using the Gene Expression Pattern Analysis Suite to identify the differentially expressed genes. In a total of 24,358 analyzed genes, 3101 and 3332 transcripts were regulated by GCSF treatment on days 3 and 7 post rAION, respectively. In addition, 702 and 179 transcripts were regulated by PBS treatment on days 3 and 7 post rAION, respectively ( Figure  1A-D). Unsupervised hierarchical clustering analysis of differentially expressed genes from all groups was conducted to investigate the similarity of the whole gene expression between the experimental samples. The result indicated that the pro le of gene expression in the GCSF-treated group was similar ( Figure 1E). Additionally, the PBS-treated rats on days 3 and 7 post rAION also exhibited similar gene expression. As stated above, the trend of gene expression is consistent between the PBS-and GCSFtreated groups.

TAF9 knockdown impaired the anti-apoptotic ability of GCSF
The numbers of TUNEL + cells in the sham-operated, scramble siRNA-treated, GCSF plus scramble siRNAtreated, and GCSF plus TAF9 siRNA-treated groups were 0.2 ± 0.4/HPF, 7.4 ± 2.7/HPF, 2.1 ± 1.3/HPF, and 6.3 ± 2.2/HPF, respectively. The number of TUNEL + cell in the GCSF plus TAF9 siRNA-treated group signi cantly increased by threefold compared to that in the GCSF plus scramble siRNA-treated group (p < 0.05), but there was no signi cant difference between the scramble siRNA-treated and GCSF plus TAF9 siRNA-treated groups (Figure 6), further suggesting a survival pathway dependent on TAF9.
TAF9 knockdown suppressed GCSF-induced TP53 and TRIAP1 expression Western blotting con rmed that the GCSF plus scramble siRNA-treated group exhibited the highest protein level of TAF9 compared with other groups (Figure 7, p < 0.05). GCSF plus TAF9 siRNA treatment signi cantly repressed TAF9 protein expression by 6.9-fold compared to GCSF plus scramble siRNA treatment (p < 0.05). In the GCSF plus TAF9 siRNA-treated group, the TP53 level was reduced by 2.4-fold compared to that in the GCSF plus scramble siRNA-treated group (p < 0.05). One of TP53 regulated genes, TP53-regulated inhibitor of apoptosis gene 1 (TRIAP1), can inhibit apoptosis through interaction with APAF1 and heat shock protein 70 (HSP70) complex [33]. Our Western blotting data demonstrated that the TRIAP1 level was reduced by 4.7-fold in the GCSF plus TAF9 siRNA-treated group compared with that in the GCSF plus scramble siRNA-treated group (p < 0.05).

Overexpression of TAF9 inhibited RGC death by modulating TP53-TRIAP1-CASP3 axis
To explore the role of TAF9 in the regulation of RGC death and survival, the AAV2-rTAF9 was used to overexpress the TAF9 level in the rAION model. Four weeks after rAION, the numbers of TUNEL positive cells in the PBS-treated and AAV2-r-TAF9-treated groups were 7.4 ± 2.7/HPF and 2.4 ± 1.7/HPF, respectively ( Figure 8A). The number of TUNEL positive cell was 3.1-fold lower in the AAV2-r-TAF9-treated group than that in the PBS-treated group (p < 0.05). Western blotting con rmed that the TP53 and TRIAP1 levels in the AAV2-r-TAF9-treated group were signi cantly increased by 2.04-and 2.71-fold, respectively, compared to those in the PBS-treated group ( Figure 8B, p < 0.05). Moreover, the cleaved-caspase 3 (Cl-casp3) level was reduced by 2.33-fold in the AAV2-rTAF9-treated group compared to that in the PBStreated group ( Figure 8B, p < 0.05).

Discussion
In this study, we conducted a microarray analysis of rat retinas to compare the differences between PBS and GCSF treatments after rAION induction to pro le the retinal transcriptomes in response to ON ischemic injury or RGC protective environment. Dynamic transcriptome pro ling revealed many novel differentially expressed genes involved in the regulation of cell death and proliferation. Among these transcripts, some genes involved in the regulation of cell death and proliferation are the TAFs. Besides, a subsequent in silico pathway analysis revealed signi cant interactions between TAFs and TP53. One TP53 coactivator, TAF9, was selected to prove its role in the regulation of RGC death and survival because TAF9 is an apoptosis regulator [34]. TAF9 knockdown not only effectively reduced the neuroprotective effects of GCSF but also inhibited G-CSF-induced TP53 and TRIAP1 expressions in the rAION model. In corresponding to the ndings of TAF9 knockdown, overexpression of TAF9 in the rAION model induced the levels of TP53 and TRIAP1 and suppressed the level of cleaved-CASP3, which provided an anti-apoptotic effects on RGCs. Thus, we suggested that TAF9 is a key element in modulating the TP53-TRIAP1-CASP3 pathway to control RGC death and survival. This transcriptomic analysis discovered a novel GCSF-regulated pathway, which is involved in RGC death and survival.
The differentially expressed genes found in the study are involved in the regulation of RGC death. Notably, ON ischemia in uenced 702 and 179 transcripts on days 3 and 7 post rAION, respectively. We found that the numbers of ON ischemia-in uenced genes are gradually reduced from days 3 to 7 post rAION. These data indicated that a dramatic change in transcription occurs in the acute stage, but this transcriptional change returns to normal in the subacute stage. A similar observation was found in our previous study that vascular permeability was highly increased in the acute stage and reduced in the subacute stage after ON infarct [7]. Taken together, we considered that ON ischemia may cause severe pathological changes in the acute stage and the natural course of recovery may be started in the subacute stage. Therefore, the therapeutic window should be focused on the acute stage in ON ischemia. As expected, our previous ndings also demonstrated that early treatment with GCSF or methylprednisolone provided good neuroprotective effects in the rAION model [7,35].
Comparing the GCSF-treated groups with the PBS-treated groups, GCSF treatment constantly in uenced > 3000 transcripts for 7 days, but the PBS-treated rats gradually reduced the transcriptional changes from the acute to subacute stage. It indicated that immediate treatment with GCSF can in uence several genes to trigger the rescue actions after ON ischemia. This remarkable transcriptional changes provided informative messages in discovering the key pathways involved in RGC survival. In this transcriptomic analysis, we found that many TBP-associated proteins were suppressed by ischemic insult but induced by GCSF treatment. These TAFs were involved in the regulation of RGC death and survival. At the molecular level, gene expression is regulated by many core transcriptional complexes, such as TFIID, along with different cofactors [36]. Previous studies revealed TFIID as an integral component of the core transcriptional machinery for RNA polymerase II at mRNA encoding genes [36,37] and demonstrated that it is assembled with TBP and multiple TAFs [38]. To date, many TAFs and several tissue-speci c variants are characterized [39]. Some genetic studies revealed the complex role of TFIID in controlling tissuespeci c and context-dependent transcriptional processes, proving the existence of different TFIID complexes and tissue-speci c TAFs [40][41][42][43][44]. TFIID subunits regulate many cellular processes in tissuespeci c manners, which facilitated research into TAF involvement in moderating biological functions, including proliferation, differentiation, apoptosis, metastasis, and hormone response [45].
After network analysis, we found that TAF9 was predicted to interact with TBP and TP53. In addition, TAF9 was highly upregulated by GCSF treatment at days 3 and 7 post rAION. It implied that TAF9 plays an important role in modulating RGC death and survival via P53 signaling pathway. TAF9 was reported to be a crucial P53 coactivator for stabilization and activation of P53 [28,30]. TAF9 inhibits MDM2mediated degradation of p53 by reducing MDM2 binding to p53 [30]. A previous study also demonstrated that one TFIID complex lacking TAF9 in Hela cells causes apoptosis [46]. Interruption of interactions between Hedgehog transcription factors (Gli proteins) and TAF9 reduces Gli/TAF9-dependent transcription, suppresses cancer cell proliferation, and reduces xenograft growth [34]. As mentioned above, we hypothesize that the high TAF9 level activates P53 pathway to inhibit RGC apoptosis after ON infarct. To verify our hypothesis, the TAF9-knockdown experiment was performed to discover the role of TAF9 in the regulation of RGC death and survival. As expected, TAF9 knockdown effectively reduced the protective effects of GCSF in the rAION model. Therefore, we demonstrated that TAF9 plays a key role in RGC protection after ON ischemia.
Cell apoptosis is manipulated on multiple levels by the sequence-speci c transcription factor TP53, with > 100 genes existing TP53 binding sites [47]. Moreover, the function of these genes remains unclear. One of these genes is TP53-regulated inhibitor of apoptosis gene 1 (TRIAP1), which has a p53 binding site within its coding sequence and is upregulated in many cancer cells [48]. TRIAP1 was reported to protect cancer cells from apoptosis through interaction with HSP70 or repression of cyclin-dependent kinase inhibitor 1 (p21) [49,50]. Recent ndings demonstrated that TRIAP1 contributes to the resistance of apoptosis in a mitochondria-dependent manner [51,52]. Based on these evidences, we further evaluated the relationship among TAF9, P53, TRIAP1, and CASP3 in the rAION model. Remarkably, immunoblotting data demonstrated that TAF9 overexpression prevented TP53 degradation and increased TRIAP1 expression in the rAION model. Additionally, we found that TAF9 overexpression reduced apoptotic RGCs and caspase 3 level after ON infarct. Taken together, we suggested that TAF9 plays a key role in the protection of ischemia-induced RGC apoptosis by modulation of TP53-TRIAP1-CASP3 axis.

Conclusion
Therefore, our transcriptomic analysis found a novel signaling pathway to elucidate the anti-apoptotic effects of GCSF on RGCs. In this novel signaling pathway, TAF9 is a key element in the modulation of TP53-TRIAP1-CASP3 axis for preventing RGC death. All the evidences suggest that TAF9 is a potential target in developing a new drug for NAION treatment.   Network analysis of TBP-associated factors. STRING analysis shows that the TBP-associated factors are involved in the known and predicted protein-protein interactions. Network analysis exhibited that many TBP-associated factors directly interact with TP53.    Western blot analysis of TAF9, TP53, and TRIAP1 expression. Treatment with GCSF plus TAF9 siRNA signi cantly repressed TAF9, TP53, and TRIAP1 expression by 6.9-, 2.2-, and 4.7-fold compared to treatment with GCSF plus scramble siRNA (p < 0.05).