Erythropoietin Modulates Autophagy Signaling in the Developing Rat Brain in an In Vivo Model of Oxygen-Toxicity

Autophagy is a self-degradative process that involves turnover and recycling of cytoplasmic components in healthy and diseased tissue. Autophagy has been shown to be protective at the early stages of programmed cell death but it can also promote apoptosis under certain conditions. Earlier we demonstrated that oxygen contributes to the pathogenesis of neonatal brain damage, which can be ameliorated by intervention with recombinant human erythropoietin (rhEpo). Extrinsic- and intrinsic apoptotic pathways are involved in oxygen induced neurotoxicity but the role of autophagy in this model is unclear. We analyzed the expression of autophagy activity markers in the immature rodent brain after exposure to elevated oxygen concentrations. We observed a hyperoxia-exposure dependent regulation of autophagy-related gene (Atg) proteins Atg3, 5, 12, Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), LC3A-II, and LC3B-II which are all key autophagy activity proteins. Interestingly, a single injection with rhEpo at the onset of hyperoxia counteracted these oxygen-mediated effects. Our results indicate that rhEpo generates its protective effect by modifying the key autophagy activity proteins.

apoptosis. In the early stages of programmed cell death [27] it is found to be protective but it can also promote apoptosis under particular circumstances [28].
We have previously described a hyperoxia-mediated increase of neuro-degeneration and modulation of apoptotic components [29][30][31][32]. A question yet to be answered is whether autophagy activity is affected by oxygen in the neonatal rat brain. Due to the neuro-protective ability of rhEpo in different neonatal brain injury models [31,[33][34][35][36], we also investigated the effect of rhEpo on key autophagy activity proteins in hyperoxia-mediated neonatal brain injury.

Erythropoietin Ameliorates Hyperoxia-Induced Changes of Beclin-1
As Beclin-1 is one of the key players in autophagy induction and can intervene at almost every step of the autophagic process [9], we investigated the oxygen and rhEpo mediated regulation of Beclin-1. Quantitative analysis of mRNA expression by real-time PCR ( Figure 1A) showed a marked up-regulation of Beclin-1 mRNA expression in brain hemispheres of rat pups after 12 h (203.1 + 18.0%) and a significant down-regulation after 24 h (62.1 + 5.4%) of hyperoxia (black bars). A single rhEpo-injection ameliorated this effect (dark grey bars) to normoxia controls (white bars). Analysis of Beclin-1 protein expression assessed by Western blot was in accordance with the real-time PCR results. Protein expression of Beclin-1 was significantly increased after 12 h (130.3 + 12.4%) and decreased after 24 h (69.7 + 12.8%); a single application of rhEpo normalized Beclin-1 protein expression to the control level ( Figure 1B). (A) Quantitative analysis of Beclin-1 mRNA expression by real-time polymerase chain reaction (PCR) after 12 or 24 h of hyperoxia with or without rhEpo treatment. (B) Beclin-1 protein expression after 12 or 24 h of hyperoxia with or without rhEpo treatment; the densitometric data represent the ratio of the pixel intensity of the Beclin-1 band to the corresponding β-actin band. Blots are representative of a series of three blots. White bar normoxic control (NOR); light grey bar normoxic control + rhEpo; black bar hyperoxia (HYP); dark grey bar hyperoxia + rhEpo. Data are normalized to levels of rat pups exposed to normoxia (control 100%; bars represent mean + SEM, n = 8 per group, *** p < 0.001, * p < 0.05, independent t-test after one-way ANOVA, bonferroni corrected compared to respective controls).

Intervention with rhEpo Modifies Oxygen Triggered Alterations of Autophagy-Related Components
Considering that autophagy is a complex multistep process involving several Atg proteins [6], we investigated the regulation of specific Atg-members known to be involved in the process of autophagosome formation.

Erythropoietin Restores Hyperoxia-Mediated Changes of LC3A-II and LC3B-II in the Developing Brain
On account of the processing of LC3 proteins involved in the formation and elongation of the autophagosome [20], we further investigated the effect of hyperoxia and concomitant rhEpo-treatment in the developing rat brain. As revealed by Western blot analysis (Figure 3), expression of the type II form of LC3A and LC3B displays a significant up-regulation after 12 h (LC3A-II: 179.2 + 15.1%; LC3B-II: 138.1 + 10.9%) and a significant down-regulation after 24 h (LC3A-II: 74.7 + 8.7%; LC3B-II: 75.6 + 9.3%) of hyperoxia. However, a single application of rhEpo restores expression of both LC3 proteins to normoxic control levels. Erythropoietin restores hyperoxia-mediated changes of LC3A-II and LC3B-II in the developing brain. (A,B) LC3A-II and LC3B-II protein expression, the densitometric data represent the ratio of the pixel intensity of the LC3A-II or LC3B-II band to the corresponding β-actin band. White bar normoxic control (NOR); light grey bar normoxic control + rhEpo; black bar hyperoxia (HYP); dark grey bar hyperoxia + rhEpo. Data are normalized to levels of rat pups exposed to normoxia (control 100%; bars represent mean + SEM, n = 8 per group, *** p < 0.001, * p < 0.05, independent t-test after one-way ANOVA, bonferroni corrected compared to respective controls).

Discussion
Our study demonstrates that high oxygen conditions lead to a modification of markers for autophagic cell death in the developing brain. Moreover, we showed that a single rhEpo-treatment neutralizes these modifications. In previous studies, our group demonstrated a marked reduction of inflammatory mediators, oxidative stress, apoptotic cell death and pro-apoptotic factors in immature rodent brains exposed to hyperoxia after rhEpo treatment [31,37,38].
As demonstrated by several groups, oxidative stress culminating in ROS generation is believed to be essential to trigger autophagy by various mechanisms [19,39]. Once autophagy is activated the formation of phagophores (precursors of autophagosomes) is initiated by Beclin-1 [40]. Here, we show that hyperoxia modulates Beclin-1 expression in the immature brain resulting in an up-regulation after 12 h of hyperoxia. In line with this finding, previous reports revealed that oxidative stress-induced autophagy coincides with an increase in the level of Beclin-1 in cortical neurons, glioma U251 and neuronal PC12 cells [41][42][43]. The elongation of phagophores requires two ubiquitin-like conjugation systems; the Atg12-Atg5-Atg16L and the phosphatidylethanolamine-LC3 system [4,18]. Some Atg genes, including LC3, were found to be up-regulated during oxidative stress-induced autophagy induction, suggesting a possible function for ROS in regulation of Atg protein expression [44]. Noteworthy, in our model of oxygen toxicity we demonstrated an increased expression of Atg3, Atg5, Atg12, LC3A-II and LC3B-II after 12 h of oxygen exposure suggesting that hyperoxia induced oxidative stress in the immature brain might trigger these acute changes in autophagy activity.
Studies using mutant mice with targeted deletion of Atg5 or Atg7 specifically in the brain suggested that autophagy is constitutively active and essential for neuronal survival [13][14][15]. However, these studies analyzed the function of autophagy activity proteins in the healthy adult brain and used a constitutive knock out model neglecting the dynamic changes of autophagic processes and their relevance in different pathological conditions. In contrast, we were interested in the regulation of autophagy activity in the immature brain and applied different levels of oxidative stress by varying the duration of hyperoxia. This might have led to an altered tissue environment with modified inflammatory mediators [38]. These different experimental setups might explain the discrepancy between hyperoxia induced brain damage and enhanced autophagy activity. Interestingly the autophagic response seems to depend on the duration of oxygen exposure, since we observed a marked down-regulation of all autophagy related proteins analyzed after 24 h.
In previous studies we demonstrated an increase in apoptotic cell death present after 6 h of hyperoxia and evident up to 72 h [29,30]. The dynamic changes of apoptotic processes combined with the regulation of autophagic activity shown here support the hypothesis of an interaction between these two mechanisms. This indicates an induction of autophagy by hyperoxia-triggered acute ROS formation with enhanced activation of apoptosis as previously shown [28]. However, with sustained oxygen exposure, triggering both extrinsic and intrinsic apoptotic cascades [29,32], autophagy might be inhibited by apoptosis [28]. In this regard it has been shown that caspase-2 deficiency leads to increased autophagy and prolonged neuronal survival whereas mitochondrial oxidative stress induced apoptosis is inhibited [45]. Of note, we have recently demonstrated that the caspase-2 initiated intrinsic apoptotic pathway plays a major role in hyperoxia-induced cell death and might be a potential explanation for the observed increased apoptosis [32] as well as reduced autophagy after longer durations of hyperoxia. This might be highly relevant for the resulting damage to the developing brain and long term functional outcome [46], since the injury cascade is in full progress within this critical time window and autophagy is necessary to degrade and recycle damaged cellular material [6,19,20].
Interestingly rhEpo reverses these hyperoxia mediated effects. However, whether these rhEpo triggered modulations are primary or secondary effects remains to be clarified. One possible mechanism would be amelioration of ROS-formation by rhEpo [36,37] indirectly inhibiting oxygen induced autophagy within the early stage (12 h). When apoptosis is predominant at 24 h it might induce anti-apoptotic signaling thereby enabling autophagy. Another explanation could be a direct modulating process on autophagy by stabilization of the outer mitochondrial membrane mediated by Bcl-2 stabilization. This would lead to impaired permeabilization of the outer mitochondrial membrane or directly via an interaction of Bcl-2 with Beclin-1 [36,47].
Although we indicate that autophagy is involved in hyperoxia mediated brain damage, we cannot exclude that our results are partly associated with apoptosis, since it has been reported that e.g. Beclin-1 may have dual roles, in apoptosis and autophagy [41,48]. Knockout mice deficient in Beclin-1 show both, neuro-degeneration and lysosomal abnormalities [49]. Since the autophagic process is dependent on the interaction of Beclin-1 and the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2), the Beclin-1-Bcl-2 complex functions as a regulator of autophagy depending on the tissue environment and the pathological conditions [50]. Accordingly, we recently demonstrated that hyperoxia triggers a decrease in Bcl-2 expression as a part of the intrinsic apoptotic pathway in the developing brain [32]. This finding might explain the observed modulation of Beclin-1 expression counteracted by rhEpo, giving similar results in a model of traumatic brain injury [36]. Furthermore, it has been shown that Beclin-1 is a substrate of caspase-3, which is a key player in the intrinsic apoptotic pathway. The cleavage products of Beclin-1 reduce autophagy and promoted apoptosis [51].
In order to distinguish to what extent apoptosis and autophagy contribute to hyperoxia mediated cell death further studies are required. Nevertheless, our data suggest that the simultaneous regulation of a broad range of autophagy activity proteins after oxygen exposure play an important role in the pathology of neonatal brain damage.
All procedures were approved by the local state authorities for animal welfare and followed institutional guidelines.

Tissue Sampling
Twelve or 24 h after hyperoxia initiation on P6, Wistar rat pups (n = 8 per group) were sacrificed with chloral hydrate (1 mg/kg, IP.) and were transcardially perfused with normal saline solution. After decapitation the olfactory bulb and cerebellum were removed, brain hemispheres were snap-frozen in liquid nitrogen and stored at −80 °C until further analysis (quantitative real-time polymerase chain reaction (PCR) and Western blotting).

Semiquantitative Real-Time PCR
Total cellular RNA was isolated from snap-frozen tissue by acidic phenol/chloroform extraction and DNase I treated (Roche Diagnostics, Mannheim, Germany); 2 µg of RNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA) in 25 µL of reaction mixture. The resulting cDNA (1 µL) was amplified by real-time PCR. The PCR product of Atg3, Atg5, Atg12 and Beclin-1 was quantified in real-time, using a dye-labeled fluorogenic reporter oligonucleotide probe and primers (Table 1). All probes were labeled at their 5' ends with the reporter dye 6-carboxy-fluoresceine (FAM), at their 3' ends with the quencher dye 6-carboxytetramethylrhodamine (TAMRA) and were purchased from BioTez (Berlin, Germany). Hypoxanthineguanine phosphoribosyl-transferase (HPRT) was used as internal standard. Real-time PCR and detection were performed in triplicate and repeated four times for each sample using a total reactive volume of 13 µL which contained 6.5 µL of 2× TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 2.5 µL of 1.25 µM oligonucleotide mix and 0.5 µL (0.5 µM) of probe. The PCR amplification was performed in 96-well optical reaction plates for 40 cycles with each cycle at 94 °C for 15 s and 60 °C for 1 min. Each plate included at least three "no template controls". The expressions of Atg3, Atg5, Atg12, Beclin-1 and HPRT were analyzed with the real-time PCR ABI Prism 7500 sequence detection system (Applied Biosystems) according to the 2 −ΔΔC T method [52].

Statistical Analysis
Data were analyzed using IBM SPSS Statistics (IBM, Frankfurt, Germany) and presented as mean + standard error (SEM). Group effects were assessed by analysis of variance (ANOVA) followed by post-hoc independent sample t-test multiple comparison. p-values are presented after Bonferroni correction. Adjusted p-values of <0.05 were considered statistically significant.

Conclusions
In the present study, we report that the exposure of neonatal rats to high concentrations of oxygen causes modifications in autophagic activity in the immature rat brain. Our results show that a single rhEpo application restores hyperoxia-mediated changes in the levels of the key autophagy proteins Beclin-1, Atg3, 5, 12, LC3A-II and LC3B-II. Our findings indicate an additional mechanism might be responsible for the protective effects of rhEpo in the context of hyperoxia to the developing brain.