Pharmacological inhibition of sphingolipid synthesis reduces ferroptosis by stimulating the HIF-1 pathway

Summary Ferroptosis is crucial to the pathology of many neurological diseases. Here, we found pre-treatment with myriocin, an inhibitor of de novo synthesis of sphingolipid, significantly decreased the erastin- or glutamate-induced ferroptosis of HT22 cells without requiring the recovery of intracellular glutathione. The transcriptome analysis of HT22 cells treated with or without myriocin identified the hypoxia-inducible factor 1 (HIF-1) pathway as a prime and novel drug target. Further study validated that HIF1α was required for the cytoprotective effects of myriocin. Myriocin treatment promoted the expression of HIF-1 pathway effectors including PDK1 and BNIP3 and altered the intracellular levels of glucose metabolites. Additionally, myriocin treatment stabilized HIF1α protein by decreasing its ubiquitination and proteasomal degradation. Similar effects of myriocin on HIF1α stabilization were also found in other mammalian cell lines indicating this is a common mechanism for the cytoprotective role of myriocin.


INTRODUCTION
Oxytosis is a form of regulated cell death (RCD) first described in nerve cells and recently recognized as ferroptosis, which is characterized by iron-dependent, lipid peroxidation, and non-apoptotic cell death (Maher et al., 2020). Ferroptosis has been shown to have significant implications in many neurologic disorders, including stroke (Alim et al., 2019), Alzheimer disease (AD) (Bao et al., 2021), Parkinson disease (PD) (Mahoney-Sanchez et al., 2021), Huntington disease (Mi et al., 2019), and multiple sclerosis (MS) (Hu et al., 2019). While enhanced ferroptosis of cancer cells may benefit patients with cancer, reduced ferroptosis is desirable for the therapies of these neurological diseases. Ferroptosis in neuronal cells can be induced by the glutamate/cystine antiporter, system x c À , inhibitors (erastin or glutamate) (Jiang et al., 2020;Zhang et al., 2020). The inhibition of system x c À results in glutathione depletion, excessive reactive oxygen species (ROS) production, and subsequent ferroptosis. It is important to note that in HT22 cells, N-methyl-d-aspartic-acid receptors (glutamate-gated cation channel) are absent and therefore this cell line is widely used to study glutamate-induced ferroptosis in neuronal cells (Maher and Davis, 1996). Although the in vivo relevance of system x c À inhibition to neurologic disorders is not clear, the in vitro model of system x c À inhibition has been extensively used for screening neuroprotective compounds targeting ferroptosis (Albrecht et al., 2010;Dixon et al., 2014).
Sphingolipids are important building blocks of cellular membranes and play key roles in the maintenance of cellular structural integrity. Additionally, sphingolipids, such as dihydrosphingosine (DHS), ceramides, and sphingosine-1-phosphate, regulate many cellular processes, including proliferation (Samarani et al., 2018), immunity (Seo et al., 2011), aging (Huang et al., 2012), auto/mitophagy (Bekhite et al., 2021), and apoptosis (Giussani et al., 2014). The de novo synthesis of sphingolipid is inhibited by myriocin, which specifically targets serine palmitoyl transferase (SPT) (Wadsworth et al., 2013). Myriocin, also called ISP-1, is a metabolite of the fungus Isaria sinclairii (Miyake et al., 1995). It resembles the transient intermediate formed by SPT in the condensation reaction of L-serine and palmitoyl-CoA (Chen et al., 1999). Our previous studies revealed that myriocin extends the lifespan of yeast cells by enhancing oxidative stress response and regulating homeostasis of intracellular amino acids (Hepowit et al., 2021;Liu et al., 2013). Recent studies have reported that limiting sphingolipid biosynthesis by myriocin had protective effects on many neurological diseases, including AD (Petit et al., 2020), PD (Lin et al., 2018), MS (Dasgupta and Ray, 2019), and Friedreich ataxia (Chen et al., 2016). However, the neuroprotective effect of myriocin against ferroptosis has not been iScience Article addressed. In the present study, we investigated the neuroprotective effect of myriocin on erastin or glutamate-induced ferroptosis in HT22 cells and discovered that the HIF-1 pathway was stimulated by myriocin to attenuate ferroptosis.

Myriocin protects HT22 cells against erastin-or glutamate-induced cell death
The inhibition of system x c À and the resulted oxidative damages of HT22 hippocampal neuronal cells by erastin-or glutamate-induced ferroptosis, which has been implied in the pathology of many neuronal diseases (Bueno et al., 2020;Chu et al., 2020;Neitemeier et al., 2017). To evaluate the potential neuronal cytoprotective effects of myriocin, HT22 cells were pre-treated with 0.5 mM of myriocin for different time from 6 to 48 h followed by treatment with erastin (1 mM) or glutamate (15 mM) for 24 h. The viability of HT22 cells without the pre-treatment of myriocin was significantly decreased by erastin or glutamate, while myriocin pre-treatment for 12 h or more provided significant protection ( Figures 1A and 1B). The protective effect of myriocin is also concentration-dependent from 0.1 to 0.5 mM and the concentrations higher than 0.5 mM are not more beneficial ( Figures 1C and 1D). Additionally, myriocin was able to protect HT22 cells against various concentrations of erastin or glutamate ( Figures 1E and 1F). These data demonstrate that myriocin is an effective compound against erastin-or glutamate-induced death of HT22 cells.
Myriocin is a potent suicide inhibitor of SPT, the first and rate-determining enzyme which produces 3-ketosphinganine in the pathway of the de novo biosynthesis of sphingolipids (Wadsworth et al., 2013). 3-ketosphinganine is then converted to dihydrosphingosine (DHS) by 3-ketosphinganine reductase to produce ceramides and complex sphingolipids ( Figure 1G). In order to validate the hypothesis that myriocin protects HT22 cells against ferroptosis by inhibiting the de novo biosynthesis of sphingolipids, the protective effect of myriocin on the viability of HT22 cells treated with erastin or glutamate was monitored in the presence of DHS. Indeed, the replenishment of DHS reduced the effect of myriocin on cell viability when DHS alone had no impact on cell viability ( Figures 1H and S1). The flow cytometric analysis of dying cells revealed that after 24 h of glutamate or erastin exposure, a major portion of cells was FITC Annexin V positive and PI positive (necrotic or ferroptotic) and a relatively smaller portion of cells was FITC Annexin V positive and PI negative (apoptotic) ( Figure 1I). The pre-treatment of myriocin significantly mitigated the appearance of either apoptotic or necrotic cells, suggesting that myriocin may protect cells from both ferroptosis and apoptosis. And the co-treatment of DHS with myriocin reversed the protective effect of myriocin ( Figure 1I). Therefore, the protective effect of myriocin on cell viability relies on its inhibition of DHS production.

Myriocin decreases erastin-or glutamate-induced oxidative damage independent of GSH
Erastin-and glutamate-induced ferroptosis in neuronal cells is initiated by the inhibition of the Na + -independent cystine/glutamate antiporter, system x c À , which in turn prevents the uptake of cystine and blocks the synthesis of glutathione (Murphy et al., 1989). To investigate if the cytoprotective role of myriocin against erastin-or glutamate-induced cell death is related to the homeostasis of glutathione, we monitored the effect of myriocin on the levels of oxidative and reductive glutathione in HT22 cells. Erastin or glutamate treatment greatly decreased the level of both reduced and oxidized glutathione as expected from previous studies ( Figure 2A) (Herrera et al., 2007;Wang et al., 2020). The pre-treatment of myriocin alone slightly decreased the level of glutathione in cells before erastin or glutamate treatment and it did not recover glutathione levels after erastin or glutamate treatment ( Figure 2A). Therefore, the cytoprotective role of myriocin does not rely on the restoration of GSH level.
Because most of dying cells are ferroptotic ( Figure 1I), we sought to understand how myriocin attenuates erastin-and glutamate-induced ferroptosis. We monitored lipid peroxidation, a hallmark of ferroptosis, and found that while erastin or glutamate treatment significantly enhanced lipid peroxidation 12-fold or 6-fold, respectively, pre-treatment with myriocin significantly suppressed these effects as revealed by (I) Annexin V-FITC/PI flow cytometric analysis of cells pre-treated with or without myriocin (0.5 mM) or DHS (1 mM) for 36 h before incubating with or without erastin (1 mM) or glutamate (15 mM) for 24 h. Histograms show numbers of necrotic and apoptotic cells. For the above, error bars represent the mean G SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).

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iScience 25, 104533, July 15, 2022 3 iScience Article flow cytometry using the fluorescent probe C11-BODIPY ( Figure 2B). The effect of myriocin on lipid peroxidation was also inhibited by DHS ( Figure 2B). Additionally, the production of ROS in HT22 cells was analyzed by using a fluorescent probe DCFH-DA. While the pre-treatment of myriocin abolished the surge of ROS production induced by erastin or glutamate treatment, the addition of DHS counteracted the effects of myriocin, and DHS alone had no impact on ROS production ( Figure 2C). Because ferroptosis is iScience Article iron-dependent, the relative labile iron pool (LIP) level in HT22 cells was monitored by the calcein fluorescence quenching method. Both erastin and glutamate treatments increased intracellular LIP in a concentration-dependent manner and the pre-treatment of myriocin significantly attenuated the increase of intracellular LIP induced by erastin or glutamate treatment ( Figures 2D and 2E). These data suggest that inhibiting the biosynthesis of sphingolipids by myriocin protected HT22 cells against erastin-or glutamateinduced ferroptosis by suppressing oxidative damage.

Myriocin activates the HIF-1 signaling pathway
To gain insight into the cytoprotective mechanism of myriocin in HT22 cells, transcriptome profiles of HT22 cells treated with or without 0.5 mM of myriocin were investigated and compared. We identified 113 upregulated and 71 downregulated differentially expressed genes (DEGs) upon myriocin treatment ( Figure S2 and Table S1). Disease and gene correlation network established by homology analysis of DEGs with Homo sapiens genome demonstrates that myriocin treatment regulated the expression of genes involved in metabolic diseases, cardiovascular diseases, cancer, and neurological diseases ( Figure 3A). Gene Ontology (GO) analysis indicates that the downregulated genes were mainly related to cell proliferation and extracellular regulation ( Figure 3B, left part). For upregulated genes, top three GO terms in biological process (BP) enrichment were ''response to hypoxia'', ''positive regulation transcription'', and ''response to cAMP''. The top three GO terms in cell component (CC) enrichment were ''membrane'', ''mitochondrion'', iScience Article and ''transcription factor complex''. And the top three GO terms in molecular function (MF) enrichment were ''transcription factor activity'', ''transcription regulatory region DNA binding'', and ''MAP kinase activity'' ( Figure 3B, right part). These results suggest that myriocin treatment activated the transcription factors related to hypoxia. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the DEGs identified 19 signaling pathways significantly altered by myriocin treatment ( Figure 3C). Among them, HIF-1 signaling pathway was the term with the lowest false discovery rate (FDR) second by the term of central carbon metabolism in cancer ( Figure 3C). Note that there was an apparent overlapping of DEGs between these two terms ( Figure 3D). These transcriptome data strongly suggest that myriocin may activate the HIF-1 signaling pathway.
To validate the DEGs regulated by HIF-1 signaling pathway, the expression levels of Eglh3, Bnip3, Glut1, Pfkl, Aldoa, Pdk1, Ldha, and Mct4 were analyzed by qPCR in HT22 cells treated from 6 to 48 h with myriocin. All of these transcripts were increased by myriocin treatment ( Figure 4A). Note that there was only a very minor increase in the mRNA level of Hif1a which is the central regulator of HIF-1 signaling pathway, consistent with the transcriptome analysis (Table S1). However, the immunofluorescence images indicate that the protein level of HIF1a was remarkably elevated by myriocin treatment (Figure 4B), suggesting that it was regulated post-transcriptionally by myriocin. Western blots also reveal that the level of HIF1a protein, together with PDK1 and BNIP3 proteins were significantly increased over the time in myriocin-treated cells ( Figures 4C and S3). Furthermore, this increase in protein levels was prevented by supplementing DHS (Figures 4B and S4).
PDK1 and BNIP3 are among the mostly studied proteins controlled by HIF-1 and play crucial roles in regulating the catabolism of glucose and mitochondrial autophagy, respectively ( Figure 4D) (Kim et al., 2006;Zhang et al., 2008). Therefore, we first asked if the catabolism of glucose was altered by myriocin. Indeed, myriocin treatment inhibited the carbon flux from glycolysis to tricarboxylic acid (TCA) cycle in HT22 cells as revealed by the elevation of intracellular levels of pyruvate and lactate and the reduction of citrate when intracellular glucose was constant ( Figure 4E). This phenomenon resembled the effects of PDK1 upregulation. Additionally, myriocin treatment decreased the content of mitochondrial ( Figure 4F) and increased LC3II, a marker of autophagy ( Figure S5). These changes are consistent to the effects of BNIP3 upregulation. These effects of myriocin were also inhibited by the addition of DHS ( Figures 4E and 4F). Together, these data indicate that myriocin induced the activation of HIF1a and its downstream signals by inhibiting the de novo synthesis of sphingolipids.

The cytoprotective effects of myriocin require HIF1a
To investigate the role of HIF1a in the cytoprotective effects of myriocin, Hif1a was knocked down in HT22 cells. Hif1a knockdown resulted in a drastic decrease of its mRNA and protein levels ( Figures 5A, 5B, and S6), and suppressed the expression of HIF-1 effectors including Pdk1, Bnip3, Glut1, Mct4, and Pfkl which were stimulated by myriocin treatment ( Figure 5A). Additionally, the expression of PDK1 and the intracellular levels of glucose, pyruvate, lactate, and citrate did not respond to myriocin treatment anymore after Hif1a knockdown ( Figures 5A, 5B, 5C and S6), indicating that the regulation of PDK1 and glycolysis by myriocin requires HIF1a. Similarly, consistent with the expression of Bnip3 (Figures 5A, 5B, and S6), the mitochondrial content of cells after Hif1a knockdown was not decreased by myriocin treatment ( Figure 5D). Additionally, Hif1a knockdown hampered the inhibitory effect of myriocin on ROS production and lipid peroxidation induced by erastin or glutamate treatment ( Figures 5E and 5F). Lastly, the protective effect of myriocin against erastin-or glutamate-induced cell death was significantly reduced by Hif1a knockdown Figure 5G). These results strongly indicate that HIF1a is crucial for the cytoprotective effects of myriocin against erastin or glutamate treatment in HT22 cells.

Myriocin treatment stabilizes HIF1a by decreasing its ubiquitination
HIF1a undergoes rapid degradation in normoxia, and its accumulation is controlled by the rates of both synthesis and degradation. Our results demonstrate that myriocin significantly stimulates the protein level of HIF1a with only very minor effect in the level of Hif1a mRNA ( Figures 4A-4C), indicating that myriocin regulates HIF1a level mainly via post-transcriptional events. It has been suggested that the MAPK and PI3K pathways may promote translation of HIF1a ( Figure 6A) (Befani et al., 2012;Masoud and Li, 2015;Mi et al., 2017). We found that inhibiting the MAPK pathway did not interfere with HIF1a expression in HT22 cells ( Figure S7A). On the other hand, when HT22 cells were treated with LY294002, AZD5363, or rapamycin, inhibitors of PI3K, AKT, and mTOR, respectively, HIF1a was significantly decreased even when ll OPEN ACCESS 6 iScience 25, 104533, July 15, 2022 iScience Article cells were co-treated with myriocin ( Figures 6B and S7B). However, myriocin treatment did not increase, but instead decreased the phosphorylation of AKT and P70S6K ( Figures 6C and S7C), indicating that myriocin inhibits the PI3K/AKT/mTOR axis. Therefore, although the level of HIF1a is indeed regulated by PI3K/AKT/ mTOR axis, myriocin did not increase HIF1a by activating this pathway.
Because HIF1a is degraded by proteasome, a proteasome inhibitor MG-132 was added into cell culture with or without myriocin treatment. The co-treatment of both MG-132 and myriocin did not further increase the HIF1a level which was elevated by MG-132 or myriocin alone ( Figures 6D and S7D), indicating that there are some overlapping effects between myriocin and MG132. A cycloheximide chase assay also  iScience Article revealed that HIF1a protein is much more stable in cells pre-treated with myriocin ( Figure S7E). These data indicate that myriocin may stabilize HIF1a by preventing its proteasomal degradation.
To investigate if the ubiquitination of HIF1a is regulated by myriocin, HIF1a protein was immunoprecipitated and its ubiquitination level was analyzed. While myriocin treatment increased the level of the un-ubiquitinated form of HIF1a, ubiquitinated HIF1a was decreased to about 50% ( Figures 6E and S7F). And the addition of DHS counteracted this effect ( Figure S7G). Together, these results suggest that myriocin inhibits the proteasomal degradation of HIF1a by partially preventing its ubiquitination.
Because HIF-1 pathway plays important roles in many physiological and pathological processes in all metazoan organisms, we asked if myriocin also upregulates HIF1a in cells other than HT22. Therefore, the levels of HIF1a in four other cell lines including PC-12 (rat neural cell), HT1080 (human fibrosarcoma cell), GES-1 (human gastric epithelial cell), and SK-Hep-1 (human hepatoma cell) were monitored with or without myriocin treatment. Myriocin treatment increased the protein level of HIF1a in all of these cell types ( Figures 7A and S8A) with no effect on the mRNA levels ( Figure 7B). Additionally, the ubiquitination of HIF1a in PC-12, HT1080, and GES-1 cells was inhibited by myriocin ( Figures 7C and S8B). The baseline ubiquitination of HIF1a in SK-Hep-1 was too low to observe a further decrease ( Figures 7C and S8B). Pre-treatment with myriocin decreased the erastin-induced cell death of PC-12 and HT1080 cells, but it did not promote the survival of GES-1 and SK-Hep-1 cells which had higher basal levels of HIF1a than PC-12, HT1080, and HT22 cells ( Figures 7D and S8C). Therefore, the effect of myriocin on HIF1a is not limited in HT22 although its effects on cell fate are cell-type-dependent.

DISCUSSION
It has been demonstrated that inhibiting de novo synthesis of sphingolipids by myriocin induces stress responses and increases lifespan in yeast cells. This cytoprotective effect of myriocin seems to be conserved in eukaryotes because myriocin treatment also increases lifespan in Caenorhabditis elegans (Cutler et al., 2014). In rodent models, myriocin treatment mitigates age-related diseases including Alzheimer disease, motor neuron degeneration, and Parkinson disease (Geekiyanage et al., 2013;Lin et al., 2019;Petit et al., 2020). The cytoprotective effects of myriocin on yeast cells are established by inducing a state resembling amino acid restriction and the subsequent inhibition of mTOR pathway (Hepowit et al., 2021). However, it is unclear how inhibiting sphingolipid synthesis by myriocin promotes cell survival in metazoan organisms. To facilitate the therapeutic usage of myriocin or myriocin-like compounds, determining the mechanistic basis of the cytoprotective effect induced by myriocin in mammalian cells is critical.
Here, we report that myriocin inhibits ferroptosis induced by glutathione depletion in neuronal HT22 cells. This is so far the first study demonstrating that inhibiting the synthesis of sphingolipids protects cells against ferroptosis. Because ferroptosis induced by oxidative stress in neuronal cells is relevant to aging and age-related neurodegeneration, our work provides a new approach for developing therapeutic or preventive means against those disorders.
Erastin or glutamate treatment in HT22 cells results in glutathione depletion which in turn induces ferroptosis. Glutathione is required for the removal of lipid peroxides by glutathione peroxidase 4 (GPx4), a key enzyme to protect cells from ferroptotic death under oxidative stress. The fact that myriocin treatment iScience Article barely prevents the drop of intracellular levels of glutathione, suggests that the protective effect of myriocin do not rely on the scavenging of lipid peroxide by GPx4. Recent studies have revealed that lipid peroxides can be removed by FSP1 through ubiquinone (CoQ10) or by nitric oxide synthase (iNOS)/ NO independent of GPx4 (Doll et al., 2019;Kapralov et al., 2020), which shares a similar mechanical character as the protective effects of myriocin. However, myriocin pre-treatment significantly suppressed ROS production under glutathione depletion. Additionally, the level of LIP was also decreased, most likely due to the less ROS which leads to the releasement of iron from its storage proteins (Di Tano et al., 2020;Lu and Imlay, 2019;Torti and Torti, 2013). Unlike GPx4 or FSP1, the protective role of myriocin against ferroptosis relies on the prevention of lipid peroxide accumulation caused by the oxidative reaction of polyunsaturated fatty acid with ROS and iron (Boada-Romero et al., 2020;Tang et al., 2019). Therefore, it is likely that the removal of lipid peroxides contributes very little, if any, to the protective effects of myriocin against ferroptosis. To address how myriocin prevents the buildup of oxidative stress induced by glutathione depletion, we performed transcriptome analysis and surprisingly identified the HIF-1 pathway. Although it has been well established that this pathway is involved in cell survival and the inhibition of ferroptosis Poloznikov et al., 2020;Yang et al., 2019), it has never been reported that inhibiting biosynthesis of sphingolipids activates HIF-1 pathway. Additionally, although some studies have demonstrated that HIF-1 pathway or hypoxia plays substantial roles in sphingolipid metabolism (Novgorodov and Gudz, 2011;Testai et al., 2014), if and how sphingolipid metabolism affects HIF-1 pathway is not well established. By validating the activation effect of myriocin on the HIF-1 pathway, this work identifies a novel and interesting connection between sphingolipid metabolism and the HIF-1 pathway. There are many downstream effectors in the HIF-1 pathway which impact many aspects of cell physiology. In this study, we examined PDK1 and BNIP3 because they are key regulators of glycolysis and mitochondrial homeostasis, respectively. Because glycolysis and mitochondrial homeostasis are crucial for energy metabolism and ROS production, their alteration by myriocin is likely to be cytoprotective. (B) Cells treated with myriocin (0.5 mM) for 36 h were subjected to qPCR to measure the relative Hif1a mRNA expression. Error bars represent the mean G SD (n = 3). (C) Ubiquitin/HIF1a co-immunoprecipitation in cells extracts. Cells treated with or without myriocin (0.5 mM) were lysed and immunoprecipitated with anti-HIF1a antibody or IgG as negative control. The immunocomplexes were then immunoblotted using anti-ubiquitin or anti-HIF1a antibody (Output). The levels of HIF1a in cell lysates before immunoprecipitation were also monitored (Input). (D) Cell viability analysis of cells pre-treated with myriocin (0.5 mM) for 36 h before incubating with erastin for 24 h. Error bars represent the mean G SD (n = 3, **p < 0.01, ***p < 0.001).

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iScience 25, 104533, July 15, 2022 11 iScience Article Finally, the regulatory role of myriocin on the level of HIF1a is pinpointed to the proteolysis process because the regulation at transcriptional or translational level has been ruled out. The proteolysis of HIF-1a is initiated by the binding of the von Hippel-Lindau tumor-suppressor protein (VHL), which in turn favors the ubiquitination of HIF1a and targets it to proteasomal degradation (Paltoglou and Roberts, 2007). Although further studies are required to reveal the details about how myriocin alters this process, our results indicate that the ubiquitination of HIF1a is decreased by myriocin treatment which results in the upregulation of HIF1a.
In conclusion, our work demonstrates that inhibiting the de novo synthesis of sphingolipid by myriocin protects neuronal cells against ferroptosis via the HIF-1 pathway. Mechanistically, myriocin increases HIF1a protein by inhibiting its ubiquitination and subsequent proteasomal degradation. Elevated HIF1a in turn stimulates the HIF-1 pathway which promotes cell survival against ferroptosis by rewiring carbon metabolism and promoting redox balance. Future works will aim to understand the cytoprotective roles of myriocin on in vivo models of neurodegeneration to evaluate the potential of myriocin or similar compounds for the prevention or treatment of multiple age-associated neurological disorders.

Limitations of the study
Our data revealed the link between sphingolipid, HIF-1 pathway, and ferroptosis, but the exact underlying mechanism remains to be explored, especially the relationship between sphingolipid synthesis and HIF1a ubiquitination. And in vivo experiments are needed to evaluate the systemic effect of myriocin.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: