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Ceramide sphingolipid signaling mediates Tumor Necrosis Factor (TNF)-dependent toxicity via caspase signaling in dopaminergic neurons.

Martinez TN, Chen X, Bandyopadhyay S, Merrill AH, Tansey MG - Mol Neurodegener (2012)

Bottom Line: Ceramide dose-dependently reduced the viability of DA neuroblastoma cells and primary DA neurons and pharmacological inhibition of sphingomyelinases (SMases) with three different inhibitors during TNF treatment afforded significant neuroprotection by attenuating increased endoplasmic reticulum (ER) stress, loss of mitochondrial membrane potential, caspase-3 activation and decreases in Akt phosphorylation.Exposure of DA neuroblastoma cells to atypical DSBs in the micromolar range reduced cell viability and inhibited neurite outgrowth and branching in primary DA neurons, suggesting that TNF-induced de novo synthesis of atypical DSBs may be a secondary mechanism involved in mediating its neurotoxicity in DA neurons.We conclude that TNF/TNFR1-dependent activation of SMases generates ceramide and sphingolipid species that promote degeneration and caspase-dependent cell death of DA neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, The University of Texas Southwestern Medical Center at Dallas, 6001 Forest Park Rd., Dallas, TX 75390, USA.

ABSTRACT

Background: Dopaminergic (DA) neurons in the ventral midbrain selectively degenerate in Parkinson's disease (PD) in part because their oxidative environment in the substantia nigra (SN) may render them vulnerable to neuroinflammatory stimuli. Chronic inhibition of soluble Tumor Necrosis Factor (TNF) with dominant-negative TNF inhibitors protects DA neurons in rat models of parkinsonism, yet the molecular mechanisms and pathway(s) that mediate TNF toxicity remain(s) to be clearly identified. Here we investigated the contribution of ceramide sphingolipid signaling in TNF-dependent toxicity.

Results: Ceramide dose-dependently reduced the viability of DA neuroblastoma cells and primary DA neurons and pharmacological inhibition of sphingomyelinases (SMases) with three different inhibitors during TNF treatment afforded significant neuroprotection by attenuating increased endoplasmic reticulum (ER) stress, loss of mitochondrial membrane potential, caspase-3 activation and decreases in Akt phosphorylation. Using lipidomics mass spectrometry we confirmed that TNF treatment not only promotes generation of ceramide, but also leads to accumulation of several atypical deoxy-sphingoid bases (DSBs). Exposure of DA neuroblastoma cells to atypical DSBs in the micromolar range reduced cell viability and inhibited neurite outgrowth and branching in primary DA neurons, suggesting that TNF-induced de novo synthesis of atypical DSBs may be a secondary mechanism involved in mediating its neurotoxicity in DA neurons.

Conclusions: We conclude that TNF/TNFR1-dependent activation of SMases generates ceramide and sphingolipid species that promote degeneration and caspase-dependent cell death of DA neurons. Ceramide and atypical DSBs may represent novel drug targets for development of neuroprotective strategies that can delay or attenuate the progressive loss of nigral DA neurons in patients with PD.

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TNF-induced neurotoxicity in DA cells and neurons is attenuated by SMase inhibitors.A, TNF-induced cytotoxic cell death is dependent on SMase hydrolysis of sphingomyelin. Diff-MN9D cells were pre-treated for 30 minutes with 5 μM desipramine (Des) or 10 μM GW4869 or 1 μM ARC39 followed by 5 ng/mL TNF for 48 hrs prior to MTS viability assay. Cell viability was measured by the MTS assay described under Methods. All values represent group means +/− SEM, n =3 - 4. One-way ANOVA with Tukey’s post-hoc; # denotes difference between TNF and vehicle at p < 0.001, * and *** denote difference from TNF alone at p < 0.05 or p < 0.001, respectively. B, TNF induced dose-dependent death of primary ventral mesencephalon DA neurons with SMase inhibitors affording robust rescue; 5 μM Desipramine (Des); 10 μM GW4869. All values represent group means +/− SEM, n =3 - 4. One-way ANOVA, Tukey’s post-hoc test to compare the extent of dose-dependent cell death in response to increasing concentrations of TNF and two-way ANOVA with Tukey’s post-hoc test to compare inhibitor conditions to 10 ng/mL TNF without inhibitors. * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001.
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Figure 2: TNF-induced neurotoxicity in DA cells and neurons is attenuated by SMase inhibitors.A, TNF-induced cytotoxic cell death is dependent on SMase hydrolysis of sphingomyelin. Diff-MN9D cells were pre-treated for 30 minutes with 5 μM desipramine (Des) or 10 μM GW4869 or 1 μM ARC39 followed by 5 ng/mL TNF for 48 hrs prior to MTS viability assay. Cell viability was measured by the MTS assay described under Methods. All values represent group means +/− SEM, n =3 - 4. One-way ANOVA with Tukey’s post-hoc; # denotes difference between TNF and vehicle at p < 0.001, * and *** denote difference from TNF alone at p < 0.05 or p < 0.001, respectively. B, TNF induced dose-dependent death of primary ventral mesencephalon DA neurons with SMase inhibitors affording robust rescue; 5 μM Desipramine (Des); 10 μM GW4869. All values represent group means +/− SEM, n =3 - 4. One-way ANOVA, Tukey’s post-hoc test to compare the extent of dose-dependent cell death in response to increasing concentrations of TNF and two-way ANOVA with Tukey’s post-hoc test to compare inhibitor conditions to 10 ng/mL TNF without inhibitors. * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001.

Mentions: Ceramide can be generated either through a de novo biosynthesis pathway involving several enzymatic reactions downstream of the initial condensation of serine and palmitoyl-CoA on the cytoplasmic surface of the ER or through the sphingomyelin recycling pathway whereby acid or neutral sphingomyelinases (SMases) hydrolyze sphingomyelin (SM) to ceramide [36]. We hypothesized that activation of SMases at the plasma membrane by the activated TNFR1/TNF receptor complex is the mechanism by which TNF exposure leads to ceramide signaling and cytotoxicity in DA cells. To test this hypothesis directly, we pre-treated diff-MN9D cells with different inhibitors of SMases for 30 minutes followed by treatment with TNF for 48 hrs. We pre-treated diff-MN9D cells with three different compounds that inhibit SMases with different mechanisms of action. Pre-incubation with desipramine (Des) (an inhibitor of acid sphingomyelinase, ASMase[37]), GW4869 (an inhibitor of neutral sphingomylinase, NSMase [38]), or with 7c, also known as ARC39 (an inhibitor of lysosomal and secreted ASMase [39]) at the concentrations indicated all significantly attenuated TNF-induced cytotoxicity of diff-MN9D cells as measured by the MTS assay (Figure 2A). To confirm and extend these findings, we assayed the extent to which two of these SMase inhibitors attenuated TNF-induced death of DA neurons in primary neuron-glia cultures from rat ventral mesencephalon. Consistent with the results in MN9D cells, Des and GW4869 protected primary DA neurons from TNF-induced death (Figure 2B) to an extent comparable to that achieved in previous studies using the soluble TNF-selective inhibitor XENP345 [10]. Together these pharmacological data strongly suggest that TNF-dependent activation of SMases results in SM hydrolysis and generation of ceramide that is cytotoxic to DA neurons, compromising their viability. To confirm that the ceramide-generating pathway involved in mediating TNF-dependent cytotoxicity is due to SM hydrolysis by SMases rather than through de novo ceramide formation, we repeated these experiments using pharmacological inhibitors of the de novo ceramide biosynthesis pathway. We observed that inhibition of the enzyme serine palmitoyltransferase (the rate-limiting enzyme in de novo ceramide biosynthesis) by myriocin or inhibition of the enzyme ceramide synthase (which converts sphinganine to dihydroceramide) by Fumonisin B1 did not mitigate TNF-induced cytotoxicity in diff-MN9D cells (Additional file 2: Figure S2). Collectively, our data support a model in which SMase hydrolysis of SM to form ceramide is requisite for TNF-induced cytotoxicity in diff-MN9D cells and DA neurons.


Ceramide sphingolipid signaling mediates Tumor Necrosis Factor (TNF)-dependent toxicity via caspase signaling in dopaminergic neurons.

Martinez TN, Chen X, Bandyopadhyay S, Merrill AH, Tansey MG - Mol Neurodegener (2012)

TNF-induced neurotoxicity in DA cells and neurons is attenuated by SMase inhibitors.A, TNF-induced cytotoxic cell death is dependent on SMase hydrolysis of sphingomyelin. Diff-MN9D cells were pre-treated for 30 minutes with 5 μM desipramine (Des) or 10 μM GW4869 or 1 μM ARC39 followed by 5 ng/mL TNF for 48 hrs prior to MTS viability assay. Cell viability was measured by the MTS assay described under Methods. All values represent group means +/− SEM, n =3 - 4. One-way ANOVA with Tukey’s post-hoc; # denotes difference between TNF and vehicle at p < 0.001, * and *** denote difference from TNF alone at p < 0.05 or p < 0.001, respectively. B, TNF induced dose-dependent death of primary ventral mesencephalon DA neurons with SMase inhibitors affording robust rescue; 5 μM Desipramine (Des); 10 μM GW4869. All values represent group means +/− SEM, n =3 - 4. One-way ANOVA, Tukey’s post-hoc test to compare the extent of dose-dependent cell death in response to increasing concentrations of TNF and two-way ANOVA with Tukey’s post-hoc test to compare inhibitor conditions to 10 ng/mL TNF without inhibitors. * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001.
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Related In: Results  -  Collection

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Figure 2: TNF-induced neurotoxicity in DA cells and neurons is attenuated by SMase inhibitors.A, TNF-induced cytotoxic cell death is dependent on SMase hydrolysis of sphingomyelin. Diff-MN9D cells were pre-treated for 30 minutes with 5 μM desipramine (Des) or 10 μM GW4869 or 1 μM ARC39 followed by 5 ng/mL TNF for 48 hrs prior to MTS viability assay. Cell viability was measured by the MTS assay described under Methods. All values represent group means +/− SEM, n =3 - 4. One-way ANOVA with Tukey’s post-hoc; # denotes difference between TNF and vehicle at p < 0.001, * and *** denote difference from TNF alone at p < 0.05 or p < 0.001, respectively. B, TNF induced dose-dependent death of primary ventral mesencephalon DA neurons with SMase inhibitors affording robust rescue; 5 μM Desipramine (Des); 10 μM GW4869. All values represent group means +/− SEM, n =3 - 4. One-way ANOVA, Tukey’s post-hoc test to compare the extent of dose-dependent cell death in response to increasing concentrations of TNF and two-way ANOVA with Tukey’s post-hoc test to compare inhibitor conditions to 10 ng/mL TNF without inhibitors. * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001.
Mentions: Ceramide can be generated either through a de novo biosynthesis pathway involving several enzymatic reactions downstream of the initial condensation of serine and palmitoyl-CoA on the cytoplasmic surface of the ER or through the sphingomyelin recycling pathway whereby acid or neutral sphingomyelinases (SMases) hydrolyze sphingomyelin (SM) to ceramide [36]. We hypothesized that activation of SMases at the plasma membrane by the activated TNFR1/TNF receptor complex is the mechanism by which TNF exposure leads to ceramide signaling and cytotoxicity in DA cells. To test this hypothesis directly, we pre-treated diff-MN9D cells with different inhibitors of SMases for 30 minutes followed by treatment with TNF for 48 hrs. We pre-treated diff-MN9D cells with three different compounds that inhibit SMases with different mechanisms of action. Pre-incubation with desipramine (Des) (an inhibitor of acid sphingomyelinase, ASMase[37]), GW4869 (an inhibitor of neutral sphingomylinase, NSMase [38]), or with 7c, also known as ARC39 (an inhibitor of lysosomal and secreted ASMase [39]) at the concentrations indicated all significantly attenuated TNF-induced cytotoxicity of diff-MN9D cells as measured by the MTS assay (Figure 2A). To confirm and extend these findings, we assayed the extent to which two of these SMase inhibitors attenuated TNF-induced death of DA neurons in primary neuron-glia cultures from rat ventral mesencephalon. Consistent with the results in MN9D cells, Des and GW4869 protected primary DA neurons from TNF-induced death (Figure 2B) to an extent comparable to that achieved in previous studies using the soluble TNF-selective inhibitor XENP345 [10]. Together these pharmacological data strongly suggest that TNF-dependent activation of SMases results in SM hydrolysis and generation of ceramide that is cytotoxic to DA neurons, compromising their viability. To confirm that the ceramide-generating pathway involved in mediating TNF-dependent cytotoxicity is due to SM hydrolysis by SMases rather than through de novo ceramide formation, we repeated these experiments using pharmacological inhibitors of the de novo ceramide biosynthesis pathway. We observed that inhibition of the enzyme serine palmitoyltransferase (the rate-limiting enzyme in de novo ceramide biosynthesis) by myriocin or inhibition of the enzyme ceramide synthase (which converts sphinganine to dihydroceramide) by Fumonisin B1 did not mitigate TNF-induced cytotoxicity in diff-MN9D cells (Additional file 2: Figure S2). Collectively, our data support a model in which SMase hydrolysis of SM to form ceramide is requisite for TNF-induced cytotoxicity in diff-MN9D cells and DA neurons.

Bottom Line: Ceramide dose-dependently reduced the viability of DA neuroblastoma cells and primary DA neurons and pharmacological inhibition of sphingomyelinases (SMases) with three different inhibitors during TNF treatment afforded significant neuroprotection by attenuating increased endoplasmic reticulum (ER) stress, loss of mitochondrial membrane potential, caspase-3 activation and decreases in Akt phosphorylation.Exposure of DA neuroblastoma cells to atypical DSBs in the micromolar range reduced cell viability and inhibited neurite outgrowth and branching in primary DA neurons, suggesting that TNF-induced de novo synthesis of atypical DSBs may be a secondary mechanism involved in mediating its neurotoxicity in DA neurons.We conclude that TNF/TNFR1-dependent activation of SMases generates ceramide and sphingolipid species that promote degeneration and caspase-dependent cell death of DA neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, The University of Texas Southwestern Medical Center at Dallas, 6001 Forest Park Rd., Dallas, TX 75390, USA.

ABSTRACT

Background: Dopaminergic (DA) neurons in the ventral midbrain selectively degenerate in Parkinson's disease (PD) in part because their oxidative environment in the substantia nigra (SN) may render them vulnerable to neuroinflammatory stimuli. Chronic inhibition of soluble Tumor Necrosis Factor (TNF) with dominant-negative TNF inhibitors protects DA neurons in rat models of parkinsonism, yet the molecular mechanisms and pathway(s) that mediate TNF toxicity remain(s) to be clearly identified. Here we investigated the contribution of ceramide sphingolipid signaling in TNF-dependent toxicity.

Results: Ceramide dose-dependently reduced the viability of DA neuroblastoma cells and primary DA neurons and pharmacological inhibition of sphingomyelinases (SMases) with three different inhibitors during TNF treatment afforded significant neuroprotection by attenuating increased endoplasmic reticulum (ER) stress, loss of mitochondrial membrane potential, caspase-3 activation and decreases in Akt phosphorylation. Using lipidomics mass spectrometry we confirmed that TNF treatment not only promotes generation of ceramide, but also leads to accumulation of several atypical deoxy-sphingoid bases (DSBs). Exposure of DA neuroblastoma cells to atypical DSBs in the micromolar range reduced cell viability and inhibited neurite outgrowth and branching in primary DA neurons, suggesting that TNF-induced de novo synthesis of atypical DSBs may be a secondary mechanism involved in mediating its neurotoxicity in DA neurons.

Conclusions: We conclude that TNF/TNFR1-dependent activation of SMases generates ceramide and sphingolipid species that promote degeneration and caspase-dependent cell death of DA neurons. Ceramide and atypical DSBs may represent novel drug targets for development of neuroprotective strategies that can delay or attenuate the progressive loss of nigral DA neurons in patients with PD.

Show MeSH
Related in: MedlinePlus