Amyloid-beta peptide induces oligodendrocyte death by activating the neutral sphingomyelinase-ceramide pathway

Amyloid-beta peptide (Abeta) accumulation in senile plaques, a pathological hallmark of Alzheimer's disease (AD), has been implicated in neuronal degeneration. We have recently demonstrated that Abeta induced oligodendrocyte (OLG) apoptosis, suggesting a role in white matter pathology in AD. Here, we explore the molecular mechanisms involved in Abeta-induced OLG death, examining the potential role of ceramide, a known apoptogenic mediator. Both Abeta and ceramide induced OLG death. In addition, Abeta activated neutral sphingomyelinase (nSMase), but not acidic sphingomyelinase, resulting in increased ceramide generation. Blocking ceramide degradation with N-oleoyl-ethanolamine exacerbated Abeta cytotoxicity; and addition of bacterial sphingomyelinase (mimicking cellular nSMase activity) induced OLG death. Furthermore, nSMase inhibition by 3-O-methyl-sphingomyelin or by gene knockdown using antisense oligonucleotides attenuated Abeta-induced OLG death. Glutathione (GSH) precursors inhibited Abeta activation of nSMase and prevented OLG death, whereas GSH depletors increased nSMase activity and Abeta-induced death. These results suggest that Abeta induces OLG death by activating the nSMase-ceramide cascade via an oxidative mechanism.     

Protective effects of S-nitrosoglutathione against amyloid beta-peptide neurotoxicity

Amyloid beta-peptide (Abeta) is a major constituent of senile plaques in the brains of Alzheimer's disease (AD) patients. We have previously demonstrated ceramide production secondary to Abeta-induced activation of neutral sphingomyelinase (nSMase) in cerebral endothelial cells and oligodendrocytes, which may contribute to cellular injury during progression of AD. In this study, we first established the "Abeta --> nSMase --> ceramide --> free radical --> cell death" pathway in primary cultures of fetal rat cortical neurons. We also provided experimental evidence showing that S-nitrosoglutathione (GSNO), a potent endogenous antioxidant derived from the interaction between nitric oxide (NO) and glutathione, caused dose-dependent protective effects against Abeta/ceramide neurotoxicity via inhibition of caspase activation and production of reactive oxygen species (ROS). This GSNO-mediated neuroprotection appeared to involve activation of cGMP-dependent protein kinase (PKG), phosphatidylinositol 3-kinase (PI3K), and extracellular signal-regulated kinase (ERK). Activation of the cGMP/PKG pathway induced expression of thioredoxin and Bcl-2 that were beneficial to cortical neurons in antagonizing Abeta/ceramide toxicity. Consistently, exogenous application of thioredoxin exerted remarkable neuroprotective efficacy in our experimental paradigm. Results derived from the present study establish a neuroprotective role of GSNO, an endogenous NO carrier, against Abeta toxicity via multiple signaling pathways.     

Signaling and function of caspase and c-jun N-terminal kinase in cisplatin-induced apoptosis

Caspases and c-Jun N-terminal kinase (JNK) are activated in tumor cells during induction of apoptosis. We investigated the signaling cascade and function of these enzymes in cisplatin-induced apoptosis. Treatment of Jurkat T-cells with cisplatin induced cell death with DNA fragmentation and activation of caspase and JNK. Bcl-2 overexpression suppressed activation of both enzymes, whereas p35 and CrmA inhibited only the DEVDase (caspase-3-like) activity, indicating that the activation of these enzymes may be differentially regulated. Cisplatin induced apoptosis with the cytochrome c release and caspase-3 activation in both wild-type and caspase-8-deficient JB-6 cells, while the Fas antibody induced these apoptotic events only in wild-type cells. This indicates that caspase-8 activation is required for Fas-mediated apoptosis, but not cisplatin-induced cell death. On the other hand, cisplatin induced the JNK activation in both the wild-type and JB-6 cells, and the caspase-3 inhibitor Z-DEVD-fmk did not inhibit this activation. The JNK overexpression resulted in a higher JNK activity, AP-1 DNA binding activity, and metallothionein expression than the empty vector-transfected cells following cisplatin treatment. It also partially protected the cells from cisplatin-induced apoptosis by decreasing DEVDase activity. These data suggest that the cisplatin-induced apoptotic signal is initiated by the caspase-8-independent cytochrome c release, and the JNK activation protects cells from cisplatin-induced apoptosis via the metallothionein expression.     

Stress-induced ceramide generation and apoptosis via the phosphorylation and activation of nSMase1 by JNK signaling

Neutral sphingomyelinase (nSMase) activation in response to environmental stress or inflammatory cytokine stimuli generates the second messenger ceramide, which mediates the stress-induced apoptosis. However, the signaling pathways and activation mechanism underlying this process have yet to be elucidated. Here we show that the phosphorylation of nSMase1 (sphingomyelin phosphodiesterase 2, SMPD2) by c-Jun N-terminal kinase (JNK) signaling stimulates ceramide generation and apoptosis and provide evidence for a signaling mechanism that integrates stress- and cytokine-activated apoptosis in vertebrate cells. An nSMase1 was identified as a JNK substrate, and the phosphorylation site responsible for its effects on stress and cytokine induction was Ser-270. In zebrafish cells, the substitution of Ser-270 for alanine blocked the phosphorylation and activation of nSMase1, whereas the substitution of Ser-270 for negatively charged glutamic acid mimicked the effect of phosphorylation. The JNK inhibitor SP600125 blocked the phosphorylation and activation of nSMase1, which in turn blocked ceramide signaling and apoptosis. A variety of stress conditions, including heat shock, UV exposure, hydrogen peroxide treatment, and anti-Fas antibody stimulation, led to the phosphorylation of nSMase1, activated nSMase1, and induced ceramide generation and apoptosis in zebrafish embryonic ZE and human Jurkat T cells. In addition, the depletion of MAPK8/9 or SMPD2 by RNAi knockdown decreased ceramide generation and stress- and cytokine-induced apoptosis in Jurkat cells. Therefore the phosphorylation of nSMase1 is a pivotal step in JNK signaling, which leads to ceramide generation and apoptosis under stress conditions and in response to cytokine stimulation. nSMase1 has a common central role in ceramide signaling during the stress and cytokine responses and apoptosis.The sphingomyelin pathway is initiated by the hydrolysis of sphingomyelin to generate the second messenger ceramide.¹ Sphingomyelin hydrolysis is a major pathway for stress-induced ceramide generation. Neutral sphingomyelinase (nSMase) is activated by a variety of environmental stress conditions, such as heat shock,^{1, 2, 3} oxidative stress (hydrogen peroxide (H₂O₂), oxidized lipoproteins),¹ ultraviolet (UV) radiation,¹ chemotherapeutic agents,⁴ and β-amyloid peptides.^{5, 6} Cytokines, including tumor necrosis factor (TNF)-α,^{7, 8, 9} interleukin (IL)-1β,¹⁰ Fas ligand,¹¹ and their associated proteins, also trigger the activation of nSMase.¹² Membrane-bound Mg²⁺-dependent nSMase is considered to be a strong candidate for mediating the effects of stress and inflammatory cytokines on ceramide.³Among the four vertebrate nSMases, nSMase1 (SMPD2) was the first to be cloned and is localized in the endoplasmic reticulum (ER) and Golgi apparatus.¹³ Several studies have focused on the potential signaling roles of nSMase1, and some reports have suggested that nSMase1 is important for ceramide generation in response to stress.^{5, 6, 14, 15} In addition, nSMase1 is responsible for heat-induced apoptosis in zebrafish embryonic cultured (ZE) cells, and a loss-of-function study showed a reduction in ceramide generation, caspase-3 activation, and apoptosis in zebrafish embryos.¹⁶ However, nSMase1-knockout mice showed no lipid storage diseases or abnormalities in sphingomyelin metabolism.¹⁷ Therefore, the molecular mechanisms by which nSMase1 is activated have yet to be elucidated.Environmental stress and inflammatory cytokines^{1, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27} stimulate stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) signaling, which involves the sequential activation of members of the mitogen-activated protein kinase (MAPK) family, including MAPK/ERK kinase kinase (MEKK)1/MAPK kinase (MKK)4, and/or SAPK/ERK kinase (SEK)1/MKK7, JNK, and c-jun. Both the JNK and sphingomyelin signaling pathways coordinately mediate the induction of apoptosis.¹ However, possible crosstalk between the JNK and sphingomyelin signaling pathways has not yet been characterized. Previously, we used SDS-PAGE to determine that nSMase1 polypeptides migrated at higher molecular masses,¹⁶ suggesting that the sphingomyelin signaling pathway might cause the production of a chemically modified phosphorylated nSMase1, which is stimulated under stressed conditions in ZE cells.¹⁶ Here, we demonstrate that JNK signaling results in the phosphorylation of Ser-270 of nSMase1, which initiates ceramide generation and apoptosis. We also provide evidence for a signaling mechanism that integrates cytokine- and stress-activated apoptosis in vertebrate cells. We studied stress-induced ceramide generation in two cell types: ZE cells and human leukemia Jurkat T-lymphoid cells. Stress-induced apoptosis has been investigated in these systems previously.^{16, 28}     

Tauroursodeoxycholic acid prevents amyloid-beta peptide-induced neuronal death via a phosphatidylinositol 3-kinase-dependent signaling pathway

Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, modulates cell death by interrupting classic pathways of apoptosis. Amyloid-beta (Abeta) peptide has been implicated in the pathogenesis of Alzheimer's disease, where a significant loss of neuronal cells is thought to occur by apoptosis. In this study, we explored the cell death pathway and signaling mechanisms involved in Abeta-induced toxicity and further investigated the anti-apoptotic effect(s) of TUDCA. Our data show significant induction of apoptosis in isolated cortical neurons incubated with Abeta peptide. Apoptosis was associated with translocation of pro-apoptotic Bax to the mitochondria, followed by cytochrome c release, caspase activation, and DNA and nuclear fragmentation. In addition, there was almost immediate but weak activation of the serine/threonine protein kinase Akt. Inhibition of the phosphatidylinositide 3 prime-OH kinase (PI3K) pathway with wortmannin did not markedly affect Abeta-induced cell death, suggesting that this signaling pathway is not crucial for Abeta-mediated toxicity. Notably, co-incubation with TUDCA significantly modulated each of the Abeta-induced apoptotic events. Moreover, wortmannin decreased TUDCA protection against Abeta-induced apoptosis, reduced Akt phosphorylation, and increased Bax translocation to mitochondria. Together, these findings indicate that Abeta-induced apoptosis of cortical neurons proceeds through a Bax mitochondrial pathway. Further, the PI3K signaling cascade plays a role in regulating the anti-apoptotic effects of TUDCA.     

Amyloid beta induces neuronal cell death through ROS-mediated ASK1 activation

Amyloid beta (Abeta) is a main component of senile plaques in Alzheimer's disease and induces neuronal cell death. Reactive oxygen species (ROS), nitric oxide and endoplasmic reticulum (ER) stress have been implicated in Abeta-induced neurotoxicity. We have reported that apoptosis signal-regulating kinase 1 (ASK1) is required for ROS- and ER stress-induced JNK activation and apoptosis. Here we show the involvement of ASK1 in Abeta-induced neuronal cell death. Abeta activated ASK1 mainly through production of ROS but not through ER stress in cultured neuronal cells. Importantly, ASK1-/- neurons were defective in Abeta-induced JNK activation and cell death. These results indicate that ROS-mediated ASK1 activation is a key mechanism for Abeta-induced neurotoxicity, which plays a central role in Alzheimer's disease.     

Activation of the c-Jun N-terminal kinase signaling cascade mediates the effect of amyloid-beta on long term potentiation and cell death in hippocampus: a role for interleukin-1beta?

Amyloid-beta (Abeta) is a major constituent of the neuritic plaque found in the brain of Alzheimer's disease patients, and a great deal of evidence suggests that the neuronal loss that is associated with the disease is a consequence of the actions of Abeta. In the past few years, it has become apparent that activation of c-Jun N-terminal kinase (JNK) mediates some of the effects of Abeta on cultured cells; in particular, the evidence suggests that Abeta-triggered JNK activation leads to cell death. In this study, we investigated the effect of intracerebroventricular injection of Abeta(1-40) on signaling events in the hippocampus and on long term potentiation in Schaffer collateral CA1 pyramidal cell synapses in vivo. We report that Abeta(1-40) induced activation of JNK in CA1 and that this was coupled with expression of the proapoptotic protein, Bax, cytosolic cytochrome c, poly-(ADP-ribose) polymerase cleavage, and Fas ligand expression in the hippocampus. These data indicate that Abeta(1-40) inhibited expression of long term potentiation, and this effect was abrogated by administration of the JNK inhibitor peptide, D-JNKI1. In parallel with these findings, we observed that Abeta-induced changes in caspase-3 activation and TdT-mediated dUTP nick-end labeling staining in neuronal cultured cells were inhibited by D-JNKI1. We present evidence suggesting that interleukin (IL)-1beta plays a significant role in mediating the effects of Abeta(1-40) because Abeta(1-40) increased hippocampal IL-1beta and because several effects of Abeta(1-40) were inhibited by the caspase-1 inhibitor Ac-YVAD-CMK. On the basis of our findings, we propose that Abeta-induced changes in hippocampal plasticity are likely to be dependent upon IL-1beta-triggered activation of JNK.     

Up-regulation of Bak and Bim via JNK downstream pathway in the response to nitric oxide in human glioblastoma cells

Nitric oxide (NO) is a chemical messenger implicated in neuronal damage associated with ischemia neurodegenerative disease and excitotoxicity. In the present study, we examined the biological effects of NO and its mechanisms in human malignant glioblastoma cells. Addition of a NO donor, S-nitroso-N-acetyl-penicillamine (SNAP), induced apoptosis in U87MG human glioblastoma cells, accompanied by opening mitochondrial permeability transition pores, release of cytochrome c and AIF, and subsequently by caspase activation. NO-induced apoptosis occurred concurrently with significantly increased levels of the Bak and Bim. Treatment with SNAP resulted in sustained activation of JNK and its downstream pathway, c-Jun/AP-1. The expression of dominant-negative (DN)-JNK1 and DN-c-Jun suppressed the activation of AP-1, the induction of Bak and Bim, and the SNAP-induced apoptosis. In addition, de novo protein synthesis was required for the initiation of apoptosis in that the protein synthesis inhibitor, cycloheximide (CHX), inhibited NO-induced apoptotic cell death as well as up-regulation of Bak and Bim. These results suggest that NO activates an apoptotic cascade, involving sustained JNK activation, AP-1 DNA binding activity, and subsequent Bak and Bim induction, followed by cytochrome c and AIF releases and caspases cascade activation, resulting in human malignant brain tumor cell death.     

Signaling events in amyloid beta-peptide-induced neuronal death and insulin-like growth factor I protection

Amyloid beta-peptide (Abeta) is implicated as the toxic agent in Alzheimer's disease and is the major component of brain amyloid plaques. In vitro, Abeta causes cell death, but the molecular mechanisms are unclear. We analyzed the early signaling mechanisms involved in Abeta toxicity using the SH-SY5Y neuroblastoma cell line. Abeta caused cell death and induced a 2- to 3-fold activation of JNK. JNK activation and cell death were inhibited by overexpression of a dominant-negative SEK1 (SEK1-AL) construct. Butyrolactone I, a cdk5 inhibitor, had an additional protective effect against Abeta toxicity in these SEK1-AL-expressing cells suggesting that cdk5 and JNK activation independently contributed to this toxicity. Abeta also weakly activated ERK and Akt but had no effect on p38 kinase. Inhibitors of ERK and phosphoinositide 3-kinase (PI3K) pathways did not affect Abeta-induced cell death, suggesting that these pathways were not important in Abeta toxicity. Insulin-like growth factor I protected against Abeta toxicity by strongly activating ERK and Akt and blocking JNK activation in a PI3K-dependent manner. Pertussis toxin also blocked Abeta-induced cell death and JNK activation suggesting that G(i/o) proteins were upstream activators of JNK. The results suggest that activation of the JNK pathway and cdk5 may be initial signaling cascades in Abeta-induced cell death.     

Amyloid-beta peptide enhances tumor necrosis factor-alpha-induced iNOS through neutral sphingomyelinase/ceramide pathway in oligodendrocytes

Although accumulating evidence demonstrates that white matter degeneration contributes to pathology in Alzheimer's disease (AD), the underlying mechanisms are unknown. In order to study the roles of the amyloid-beta peptide in inducing oxidative stress damage in white matter of AD, we investigated the effects of amyloid-beta peptide 25-35 (Abeta) on proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha)-induced inducible nitric oxide synthase (iNOS) in cultured oligodendrocytes (OLGs). Although Abeta 25-35 by itself had little effect on iNOS mRNA, protein, and nitrite production, it enhanced TNF-alpha-induced iNOS expression and nitrite generation in OLGs. Abeta, TNF-alpha, or the combination of both, increased neutral sphingomyelinase (nSMase) activity, but not acidic sphingomyelinase (aSMase) activity, leading to ceramide accumulation. Cell permeable C2-ceramide enhanced TNF-alpha-induced iNOS expression and nitrite generation. Moreover, the specific nSMase inhibitor, 3-O-methyl-sphingomyelin (3-OMS), inhibited iNOS expression and nitrite production induced by TNF-alpha or by the combination of TNF-alpha and Abeta. Overexpression of a truncated mutant of nSMase with a dominant negative function inhibited iNOS mRNA production. 3-OMS also inhibited nuclear factor kappaB (NF-kappaB) binding activity induced by TNF-alpha or by the combination of TNF-alpha and Abeta. These results suggest that neutral sphingomyelinase/ceramide pathway is required but may not be sufficient for iNOS expression induced by TNF-alpha and the combination of TNF-alpha and Abeta.     

Role of the JNK/c-Jun/AP-1 signaling pathway in galectin-1-induced T-cell death

Galectin-1 (gal-1), an endogenous β-galactoside-binding protein, triggers T-cell death through several mechanisms including the death receptor and the mitochondrial apoptotic pathway. In this study we first show that gal-1 initiates the activation of c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 4 (MKK4), and MKK7 as upstream JNK activators in Jurkat T cells. Inhibition of JNK activation with sphingomyelinase inhibitors (20 μM desipramine, 20 μM imipramine), with the protein kinase C-δ (PKCδ) inhibitor rottlerin (10 μM), and with the specific PKCθ pseudosubstrate inhibitor (30 μM) indicates that ceramide and phosphorylation by PKCδ and PKCθ mediate gal-1-induced JNK activation. Downstream of JNK, we observed increased phosphorylation of c-Jun, enhanced activating protein-1 (AP-1) luciferase reporter, and AP-1/DNA-binding in response to gal-1. The pivotal role of the JNK/c-Jun/AP-1 pathway for gal-1-induced apoptosis was documented by reduction of DNA fragmentation after inhibition JNK by SP600125 (20 μM) or inhibition of AP-1 activation by curcumin (2 μM). Gal-1 failed to induce AP-1 activation and DNA fragmentation in CD3-deficient Jurkat 31-13 cells. In Jurkat E6.1 cells gal-1 induced a proapoptotic signal pattern as indicated by decreased antiapoptotic Bcl-2 expression, induction of proapoptotic Bad, and increased Bcl-2 phosphorylation. The results provide evidence that the JNK/c-Jun/AP-1 pathway plays a key role for T-cell death regulation in response to gal-1 stimulation.     

Prevention of TNF-alpha-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2

It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-alpha/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by alpha-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-alpha/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-alpha/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-alpha/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-alpha/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-alpha/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-alpha/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-alpha/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.     

Induction of apoptosis by shikonin through a ROS/JNK-mediated process in Bcr/Abl-positive chronic myelogenous leukemia （CML） cells

This study examined the signaling events induced by shikonin that lead to the induction of apoptosis in Bcr/ Abl-positive chronic myelogenous leukemia （CML） cells （e.g., K562, LAMA84）. Treatment of K562 cells with shikonin （e.g., 0.5 pM） resulted in profound induction of apoptosis accompanied by rapid generation of reactive oxygen species （ROS）, striking activation of c-Jun-N-terminal kinase （JNK） and p38, marked release of the mitochondrial proteins cytochrome c and Smac/DIABLO, activation of caspase-9 and -3, and cleavage of PARP. Scavenging of ROS completely blocked all of the above-mentioned events （i.e., JNK and p38 phosphorylation, cytochrome c and Smac/DIABLO release, caspase and PARP cleavage, as well as the induction of apoptosis） following shikonin treatment. Inhibition of JNK and knock-down of JNK1 significantly attenuated cytochrome c release, caspase cleavage and apoptosis, but did not affect shikonin-mediated ROS production. Additionally, inhibition of caspase activation completely blocked shikonin-induced apoptosis, but did not appreciably modify shikonin-mediated cytochrome c release or ROS generation. Altogether, these findings demonstrate that shikonin-induced oxidative injury operates at a proximal point in apoptotic signaling cascades, and subsequently activates the stress-related JNK pathway, triggers mitochondrial dysfunction, cytochrome c release, and caspase activation, and leads to apoptosis. Our data also suggest that shikonin may be a promising agent for the treatment of CML, as a generator of ROS.     

MGLuR5 activation reduces beta-amyloid-induced cell death in primary neuronal cultures and attenuates translocation of cytochrome c and apoptosis-inducing factor

Activation of metabotropic glutamate receptor 5 (mGluR5) has been shown to reduce caspase-dependent apoptosis in primary neuronal cultures induced by staurosporine and etoposide. beta-Amyloid (Abeta)-induced neurotoxicity in culture appears to be in part caspase mediated. In the present studies the effects of treatment with an mGluR5 agonist or antagonist on Abeta-induced neuronal apoptosis were examined in rat cortical neuronal cultures. Pretreatment with the selective mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) markedly reduced the number of apoptotic cells after exposure to Abeta (25-35), as well as associated LDH release. Blockade of mGluR5 by the selective antagonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP) attenuated these effects of CHPG. A similar neuroprotective effect of mGluR5 activation by CHPG was observed in cultures treated with full-length Abeta peptide (1-42). CHPG attenuated Abeta (25-35)-induced cytochrome c release and decreased levels of active caspase-3 protein. CHPG also reduced translocation of apoptosis-inducing factor (AIF) induced by Abeta (25-35). Thus, mGluR5 activation limits the release of mitochondrial proteins associated with induction of both caspase-dependent and -independent apoptosis.     

Neutral sphingomyelinase 2 activity and protein stability are modulated by phosphorylation of five conserved serines

We previously presented that the neutral sphingomyelinase 2 (nSMase2) is the only SMase activated in human airway epithelial (HAE) cells following exposure to oxidative stress (ox-stress), yielding ceramide accumulation and thereby inducing apoptosis. Furthermore, we reported that nSMase2 is a phospho-protein in which the level of phosphorylation controls nSMase2 activation induced by ox-stress. Here we identify five specific serines that are phosphorylated in nSMase2 and demonstrate that their phosphorylation controls the nSMase2 activity upon ox-stress exposure in an interdependent manner. Furthermore, we show that the nSMase2 protein stability and thus its level of expression is also post-translationally regulated by these five serine phosphorylation sites. This study provides initial structure/function insights regarding nSMase2 phosphorylation sites and offers some new links for future studies aiming to fully elucidate nSMase2 regulatory machinery.