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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 and ceramide (but not dihydro-Ceramide) reduce viability of neurally differentiated MN9D dopaminergic (DA) cells.A, Dose-dependent cytotoxic cell death in diff-MN9D cells treated with TNF for 72 hrs. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc; * denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001 compared to vehicle (DMSO, 1%); for comparison between doses # denotes p < 0.05, n.s. denotes not significant. B, Dose-dependent C2-Ceramide-induced cytotoxic death in diff-MN9D cells. Cells were treated with ceramide (C2-Cer), or with equal concentrations of C2-dihydroceramide (C2-DH-Cer) as a negative control. All values represent group means +/− SEM, n = 3–4. Two-way ANOVA with Tukey’s post-hoc test for comparing C2-Cer conditions to C2-DH-Cer conditions where **denotes p < 0.01, *** denotes p < 0.001. One-way ANOVA to test for dose-dependent cell death in C2-Cer conditions where # denotes p < 0.05, ### denotes p < 0.001 relative to vehicle or different C2-Cer conditions as indicated.
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Figure 1: TNF and ceramide (but not dihydro-Ceramide) reduce viability of neurally differentiated MN9D dopaminergic (DA) cells.A, Dose-dependent cytotoxic cell death in diff-MN9D cells treated with TNF for 72 hrs. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc; * denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001 compared to vehicle (DMSO, 1%); for comparison between doses # denotes p < 0.05, n.s. denotes not significant. B, Dose-dependent C2-Ceramide-induced cytotoxic death in diff-MN9D cells. Cells were treated with ceramide (C2-Cer), or with equal concentrations of C2-dihydroceramide (C2-DH-Cer) as a negative control. All values represent group means +/− SEM, n = 3–4. Two-way ANOVA with Tukey’s post-hoc test for comparing C2-Cer conditions to C2-DH-Cer conditions where **denotes p < 0.01, *** denotes p < 0.001. One-way ANOVA to test for dose-dependent cell death in C2-Cer conditions where # denotes p < 0.05, ### denotes p < 0.001 relative to vehicle or different C2-Cer conditions as indicated.

Mentions: In light of our previous findings showing that ventral mesencephalon dopaminergic (DA) neurons are acutely sensitive to TNF in vitro and in vivo[10], we hypothesized that ceramide sphingolipids are critical effectors of TNF-induced cytotoxicity. First, we aimed to establish a correlation between TNF-dependent ceramide generation and the effect of TNF or ceramide exposure on the viability of neuronally differentiated MN9D cells or primary DA neurons. We found that TNF dose-dependently decreased the viability of diff-MN9D cells as measured by the MTS metabolic assay (Figure 1A). To test the hypothesis that elevated ceramide is directly toxic to diff-MN9D cells, we treated the cells with various concentrations of C2-Cer or C2-dihydroceramide (C2-DH-Cer) as a negative control; C2-DH-Cer is an analog of C2-Cer lacking the 4–5 trans bond in the sphingosine moiety that is incapable of activating downstream ceramide signaling [22,32]. We found that C2-Cer but not C2-DH-Cer induced dose-dependent decreases in diff-MN9D viability (Figure 1B). We previously determined that non-differentiated MN9D (non-diffMN9D) cells are not sensitive to concentrations of TNF that elicit cytotoxicity in diff-MN9D cells [26]. Similarly, C2-Cer was not cytotoxic to non-diff-MN9D cells (Additional file 1: Figure S1).


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 and ceramide (but not dihydro-Ceramide) reduce viability of neurally differentiated MN9D dopaminergic (DA) cells.A, Dose-dependent cytotoxic cell death in diff-MN9D cells treated with TNF for 72 hrs. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc; * denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001 compared to vehicle (DMSO, 1%); for comparison between doses # denotes p < 0.05, n.s. denotes not significant. B, Dose-dependent C2-Ceramide-induced cytotoxic death in diff-MN9D cells. Cells were treated with ceramide (C2-Cer), or with equal concentrations of C2-dihydroceramide (C2-DH-Cer) as a negative control. All values represent group means +/− SEM, n = 3–4. Two-way ANOVA with Tukey’s post-hoc test for comparing C2-Cer conditions to C2-DH-Cer conditions where **denotes p < 0.01, *** denotes p < 0.001. One-way ANOVA to test for dose-dependent cell death in C2-Cer conditions where # denotes p < 0.05, ### denotes p < 0.001 relative to vehicle or different C2-Cer conditions as indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3472284&req=5

Figure 1: TNF and ceramide (but not dihydro-Ceramide) reduce viability of neurally differentiated MN9D dopaminergic (DA) cells.A, Dose-dependent cytotoxic cell death in diff-MN9D cells treated with TNF for 72 hrs. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc; * denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001 compared to vehicle (DMSO, 1%); for comparison between doses # denotes p < 0.05, n.s. denotes not significant. B, Dose-dependent C2-Ceramide-induced cytotoxic death in diff-MN9D cells. Cells were treated with ceramide (C2-Cer), or with equal concentrations of C2-dihydroceramide (C2-DH-Cer) as a negative control. All values represent group means +/− SEM, n = 3–4. Two-way ANOVA with Tukey’s post-hoc test for comparing C2-Cer conditions to C2-DH-Cer conditions where **denotes p < 0.01, *** denotes p < 0.001. One-way ANOVA to test for dose-dependent cell death in C2-Cer conditions where # denotes p < 0.05, ### denotes p < 0.001 relative to vehicle or different C2-Cer conditions as indicated.
Mentions: In light of our previous findings showing that ventral mesencephalon dopaminergic (DA) neurons are acutely sensitive to TNF in vitro and in vivo[10], we hypothesized that ceramide sphingolipids are critical effectors of TNF-induced cytotoxicity. First, we aimed to establish a correlation between TNF-dependent ceramide generation and the effect of TNF or ceramide exposure on the viability of neuronally differentiated MN9D cells or primary DA neurons. We found that TNF dose-dependently decreased the viability of diff-MN9D cells as measured by the MTS metabolic assay (Figure 1A). To test the hypothesis that elevated ceramide is directly toxic to diff-MN9D cells, we treated the cells with various concentrations of C2-Cer or C2-dihydroceramide (C2-DH-Cer) as a negative control; C2-DH-Cer is an analog of C2-Cer lacking the 4–5 trans bond in the sphingosine moiety that is incapable of activating downstream ceramide signaling [22,32]. We found that C2-Cer but not C2-DH-Cer induced dose-dependent decreases in diff-MN9D viability (Figure 1B). We previously determined that non-differentiated MN9D (non-diffMN9D) cells are not sensitive to concentrations of TNF that elicit cytotoxicity in diff-MN9D cells [26]. Similarly, C2-Cer was not cytotoxic to non-diff-MN9D cells (Additional file 1: Figure S1).

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