Heme induces autophagic cell death via ER stress in neuron

Aims Intracerebral hemorrhage (ICH) is serious medical problem and the effective treatment is limited. Hemorrhaged blood is highly toxic to the brain, and heme mainly released from hemoglobin plays a vital role in neurotoxicity. However, the specific mechanism involved in heme mediated neurotoxicity has not been well studied. Methods In this study, we investigated the neurotoxicity of heme in neurons. Neurons were administrated with heme, and the cell death, autophagy and ER stress were analyzed. In addition, the relationship between autophagy and apoptosis in heme-induced cell death and the downstream effects were also detected. Results We showed that heme induced cell death and autophagy in neurons. The suppression of autophagy using either pharmacologic inhibitors (3-methyladenine) or RNA interference in essential autophagy genes (BECN1 and ATG5) decreased the cell death induced by heme in neurons. Moreover, ER stress activator thapsigargin increased the cell autophagy and cell death ratio following heme treatment. Autophagy promotes cell apoptosis and cell death induced by heme through BECN1/ ATG5 pathway. Conclusions Our findings suggest that heme potentiates neuron autophagy via ER stress, in turn inducing cell death via BECN1/ATG5 pathway. Targeting ER stress mediated autophagy might be a promising therapeutic strategy for ICH.


Introduction
Intracerebral hemorrhage (ICH) accounts for 10%-15% of all strokes, and causes severe disability and mortality [1,2,3]. During ICH, large numbers of erythrocytes 3 are released into the extracellular spaces in the brain. Following erythrocytes are lysed, extracellular hemoglobin is rapidly oxidized into methemoglobin and releases heme [4,5]. The free heme binds to lipids intercalating into cell membranes, leading to neuron damage [6,7,8].
Autophagy is a fundamental biological process that endows eukaryotic cells with the ability to autodigest portions of their own cytoplasm [9,10,11]. Autophagy protects cells against adverse conditions and plays important roles in aging, development, death and apoptosis [12,13,14]. Autophagy activation may contribute to ICH induced brain injury [15,16,17].
The endoplasmic reticulum (ER) is an intracellular organelle that contributes to membrane biosynthesis and the maintenance of intracellular organizational homeostasis [18,19,20]. Numerous studies demonstrate that ER stress plays an important role in many diseases, including hemorrhage stroke, other inflammatory and metabolic diseases [21,22,23]. In addition, emerging evidence suggests that ER stress can trigger autophagy [24,25,26].
However, the potential of heme to regulate ER stress and the regulation of autophagy on neuron is still unknown. Therefore, in the current study, we propose a hypothesis whether heme could induce ER stress in neurons and contribute to autophagic cell death.

Primary cell cultures
Cortical neuronal cultures were prepared from whole cerebral cortices of C57BL/6 mouse embryos (E16). Brain tissue was diced into small fragments and incubated in 0.25% trypsin and 200 μg/ml DNase I in PBS. The suspension was then filtered and 4 centrifuged. The pellet was resuspended in PBS and recentrifuged, and after a final wash in feeding medium, the cells were plated into T75 flasks coated with polyornithine (10 μg/ml). The plating density was 80 million cells in 25 ml of medium. To obtain neuron-enriched cultures, cells in the flasks were treated with at least three cycles of 25 μM cytosine arabinoside (2 d on, 3 d off) to kill dividing astrocytes. The feeding medium during this time was minimum essential medium supplemented with 10% fetal bovine serum, nonessential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, and 10% dextrose. All culture medium supplies were from Invitrogen (Burlington, Ontario, Canada). The resulting cells after three cycles of cytosine arabinoside treatment were neurons in excess of 90% purity, with astrocytes, microglia, and precursor cells forming the rest. The neuron-enriched cultures were retrypsinized and plated at 100,000 cells/well in 16-well Lab-tek slides (Nunc, Naperville, IL) in the above medium. Purity of neuronal cultures was> 95% as confirmed by random staining with neuronal and glia markers. 5 days after plating, neurons had developed a dense network of extensions.

Antibodies and reagents
The adenovirus of the GFP-LC3B fusion protein (C3007) was obtained from Beyotime Institute of Biotechnology. Some chemical reagents were purchased from Sigma,

Transmission electron microscopy
Neurons were collected and fixed in a solution containing 2.5% glutaraldehyde in 0.1 M sodium cacodylate for 2 hrs, postfixed with 1% OsO4 for 1.5 hrs, washed and stained in 3% aqueous uranyl acetate for 1h. The samples were then washed again, dehydrated with a graded alcohol series, and embedded in Epon-Araldite resin (Canemco, #034). Ultrathin sections were cut on a Reichert ultramicrotome, counterstained with 0.3% lead citrate and examined on a Philips EM420 electron microscope.

GFP-LC3 puncta formation assays
Neurons were infected with GFP-LC3B adenovirus (MOI=100:1) for 24 hs, then cultured with either vehicle or hemin (100 μM) for 24 hs, and fixed in 4% paraformaldehyde for 10 minutes at 37 °C. Confocal microscopy was performed with a Radiance 2000 laser scanning confocal microscope (Bio-Rad, San Francisco, CA), 6 followed by image analysis with LaserSharp 2000 software (Bio-Rad, San Francisco, CA). Images were acquired in a sequential scanning mode. According to methods for monitoring GFP-LC3 puncta formation assays, the average number of MAP1LC3B puncta per cell in GFP-MAP1LC3B-positive cells was determined.

Western blot analysis
Cell medium was removed and plates were washed three times with chilled PBS. The cells were quickly scraped and collected by centrifugation at 4°C, then stored at -80°C. Cell samples were sonicated with Western blot lysis buffer. Protein concentration was determined using a Bio-Rad protein assay kit (Hercules, CA, USA).

Cell viability
According to the manufacturer's instructions (Sigma), Cell viability was determined with an MTT assay. Following treatment, neurons were incubated with MTT at a final concentration of 5 mg/L for 2 hours and then dissolved in the MTT solubilization solution. The cell survival rate was measured with an absorbance at 590 nm (A590) by a microplate reader (Bio-Rad).

Cell death assay
Cell death was assessed using a PI staining assay. The cells were trypsinized, collected, and resuspended in 2 ml of PBS, then incubated with the PI staining solution at 37 °C for 30 min in the dark before being finally measured with flow cytometry.

Apoptosis
The ratio of apoptotic cells was evaluated by staining 5×10 5

Acridine orange staining
In acridine orange-stained cells, the cytoplasm and nucleus appear bright green and dim red, respectively, and acidic compartments appear bright red. The intensity of the red fluorescence is proportional to the degree of acidity. After receiving the 8 specified treatments, cells were incubated with acridine orange solution (1 mg/ml) for 15 min in drug-free medium at 37 °C and washed with PBS. Then, cells were trypsinised and analysed by flow cytometry using a FACScan cytometer and CellQuest software. Statistical analyses were performed as described above.

Statistical analysis
The results are expressed as the mean ± standard error (SEM). Two group data were analyzed by Student's t-test, and multiple group data were analyzed using one-way analysis of variance (ANOVA). Statistically significant differences are indicated by asterisks (*P<0.05).

Heme induced cell death in neurons
To investigate whether heme could induce cell death in neurons, we performed a cell death assay using a PI staining assay. As shown in Fig. 1A, the cell death ratio of heme group was much higher than control groups. However, the cell survival 9 ratio of heme group was much lower than control groups (P<0.05) (Fig. 1B). These data indicate that heme induced cell death in neurons.

Heme induced cell death on autophagy
To investigate whether heme induced cell death in time-dependent manner, we performed a cell death assay at 1h, 6hs or 12hs. As shown in Fig. 2A, there were time-dependent increases in cell death ratio after heme treatment. Similar timedependent decreases in cell viability ratio were observed in MTT assays (Fig. 2B).
These data indicate that heme induces cell death in time-dependent manner. In addition, to further analyze whether autophagy contributed to heme induced cell death, we performed a cell death assay and cell viability after the cells were pretreated with 3-MA. The results demonstrated that autophagy inhibitor 3-MA decreased the cell death ratio, while increased cell viability compared with control groups (P<0.05). In addition, the apoptosis inhibitor Z-VAD had the similar effects (P<0.05) (Fig. 2C-D).

Heme treatment induced cell autophagy
To determine whether heme treatment induced autophagy, we utilized acridine orange staining and MDC staining assays to analyze the number of autophagosomes. The data demonstrated that heme revealed an increase in the number of autophagosomes ( Fig. 3A and 3B). MAP1LC3B (using Actin as a loading control), which is considered an accurate indicator of autophagy. We also observed a gradual increase in the ratio of MAP1LC3B-II to Actin in cells treated with heme compared to control cells after 6 hs (Fig. 3C). Furthermore, Baf A1 challenge resulted in the further accumulation of MAP1LC3B-II in neurons after 6 hrs (Fig. 3D), suggesting that heme promotes cellular autophagic flux. To further investigate that heme induces autophagy in neurons, we used a GFP-MAPLC3B puncta formation assay to monitor autophagy. As shown in Fig. 3E, heme-treated neurons demonstrated a significant increase in the percentage of cells with autophagosomes (GFP-MAPLC3B puncta) compared with control cells. TEM of neurons treated with heme revealed an increase in the number of autophagosomes (Fig. 3F). The results suggest that heme induces a complete autophagic response in neurons.

Heme treatment induced cell autophagy via ER stress
To detect whether heme induced ER stress in neurons, we analyzed the ultrastructure of heme-treated neurons using electron microscopy. The data demonstrated that there was more dilated ERs in the heme treatment group than in the control group (P < 0.05) (Fig. 4A). To further analyze the effect of ER stress on antophagy, neurons were pretreated with ER stress activator Thap, and administrated with heme. We observed a gradual increase in the ratio of MAP1LC3B-II to Actin in cells pretreated with Thap compared to control cells after 6 hs (Fig.   4B). In addition, acridine orange staining assay demonstrated that Thap revealed an increase in the number of autophagosomes (Fig. 4C). Furthermore, PI staining assay suggested that ER stress activator Thap increased the cell death ratio following heme treatment (Fig.4D). These data revealed that heme treatment induced cell autophagy via ER stress.

DDIT3/ATF4 pathway
To detect whether heme induced ER stress in time-dependent manner, we analyzed the ER stress marker p-EIF2S1 of heme-treated neurons by western blot assay. The data demonstrated that heme treatment increased p-EIF2S1 levels from 0h to 12 hrs (Fig. 5A). In addition, we analyzed the number of autophagosomes and cell death ratio of heme-treated neurons. The data demonstrated that heme treatment increased the number of autophagosomes and promoted cell death ratio of neurons from 0h to 12 hrs (Fig. 5C and 5E). To further address the possibility that the inhibition of ER stress is responsible for the cell autophagy induced by heme, we assessed the effects of DDIT3 and ATF4 silencing and autophagy and cell death by RNA interference (Fig. 5B and 5D and 5F). The siRNA-mediated knockdown of DDIT3 and ATF4, which are required for ER stress, decreased heme-induced autophagy and cell death, suggesting that ER stress promotes autophagy and the cell death induced by heme through DDIT3/ATF4 pathway.

Autophagy is upstream of apoptosis in heme-induced cell death
Furthermore, we determined the relationship between autophagy and apoptosis in heme-induced cell death. We utilized acridine orange staining and WB assays to analyze autophagy level. The data demonstrated that 3-MA decreased heme induced neuron autophagy (Fig. 6A and 6B). In addition, 3-MA decreased heme induced capase-3 levels of neurons (Fig. 6C). To further address the possibility that inhibition of autophagy is responsible for the cell apoptosis induced by heme, we assessed the effects of BECN1 and ATG5 silencing and autophagy and cell death by RNA interference (Fig. 6D-F). The siRNA-mediated knockdown of BECN1 and ATG5, which are required for autophagy, decreased heme-induced PARP1 levels and cell death, suggesting that autophagy promotes cell apoptosis and cell death induced by heme through BECN1/ ATG5 pathway. Moreover, we assessed the effects of apoptosis inhibitor Z-VAD on cell autophagy. The results demonstrated that inhibition of cell apoptosis could not decreased cell autophagy levels (Fig. 6G-I).
These data revealed that autophagy is upstream of apoptosis in heme-induced cell death.

Discussion
In this study, we firstly represented that heme induces autophagic cell death via ER stress in neurons. The data is supported by the following evidence: (1) heme treatment induced cell autophagy; (2) heme treatment induced cell autophagy via ER stress; (3) heme treatment induced ER stress mediated cell autophagy through DDIT3/ATF4 pathway; and (4) autophagy is upstream of apoptosis in heme-induced cell death.
Intracerebral hemorrhage (ICH) is a common and serious acute cerebrovascular disease, and most of the survivors suffer from apparent disability [27,28,29]. Free heme is released during erythrocyte lysis, and then degraded by heme oxygenase to form iron. Accumulation of heme and iron in the perihematoma region is characterized by neurotoxicity, and causes acute inflammation resulting in neurology dysfunction [30,31]. To investigate the neurotoxicity effect of heme on neurons, we cocultured neurons and heme together, and detected the neuron death and viability. The data indicated that heme increased cell death and decreased cell viability in a time-dependent manner. However, the molecular mechanism of heme mediated neurotoxicity on neurons has not been well reported.
Autophagy is the process of bulk degradation and recycling of long-lived proteins, macromolecular aggregates, and damaged intracellular organelles [32,33,34].
Cellular homeostasis requires continuous removal of worn-out components and 13 replacement with newly synthesized proteins. Recent studies have enlarged the knowledge of the molecular mechanism of autophagy and the effect of autophagy in different pathological conditions. Autophagy has been identified in a number of pathological conditions, including cancer, myopathies, and neurodegenerative disorders [35,36,37]. Autophagy has also been associated with both cell survival and cell death, but the role of autophagy in cell death has been controversial [38,39,40]. Therefore, to further identify whether autophagy could be induced after heme treatment, we investigated the autophagy formation and the specific role in neuron survival or death. We utilized acridine orange staining and MDC staining assays to analyze the number of autophagosomes, and found that heme induced a complete autophagic response in neurons. In addition, we found that autophagy inhibitor 3-MA decreased the cell death ratio, while increased cell viability.
The endoplasmic reticulum (ER) is an intracellular organelle where the protein molecule folding, transportation, or modification takes place and also a place for calcium storage, lipid synthesis, and carbohydrate metabolism [41,42,43]. Much evidences observed that the homeostasis of ER alters under certain pathological conditions leading to the accumulation of misfolded or unfolded proteins and ER stress. Recent evidence reveals that ER stress can stimulate autophagy [44].
However, the role of heme in ER stress-induced autophagy and the related signal events remain to be fully illustrated. We assessed the effects of DDIT3 and ATF4 silencing and autophagy and cell death by RNA interference. We found that knockdown of DDIT3 and ATF4 decreased heme-induced autophagy and cell death, suggesting that ER stress promotes autophagy and the cell death induced by heme through DDIT3/ATF4 pathway.
The interactions between apoptotic and autophagic proteins via the proteolytic 14 systems are known mechanisms through which autophagy and apoptosis regulate each other [45,46]. Finally, we determined the relationship between autophagy and apoptosis in heme-induced cell death. We found that autophagy promoted cell apoptosis and cell death induced by heme through BECN1/ ATG5 pathway. However, inhibition of cell apoptosis could not decreased cell autophagy levels. These data revealed that autophagy is upstream of apoptosis in heme-induced cell death.

Conclusion
Our findings suggested that heme initiated neuron autophagy via ER stress, in turn inducing cell death via BECN1/ATG5 pathway. Targeting ER stress mediated autophagy might be a promising therapeutic strategy for ICH.  Heme treatment induced ER stress mediated cell autophagy through DDIT3/ATF4 pathway (A 25 Figure 6 Autophagy is upstream of apoptosis in heme-induced cell death (A) Neurons (1×105) were st