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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 treatment induces accumulation of ceramide and several atypical sphingoid bases (DBS) in MN9D dopaminergic (DA) neuroblastoma cells derived from mouse ventral mesencephalon. MN9D cells were treated with PBS or 5 ng/mL TNF for 24 or 48 hours as indicated prior to cell harvest for lipid extraction as described in Methods. A, Lipidomic analyses indicate time-dependent accumulation of ceramide (Cer), sphingomyelin (SM), and hexosylceramide (HexCer) in TNF-treated cells relative to PBS-treated cells. B, Lipidomic analyses also revealed time-dependent accumulation of sphingosine (So), sphinganine (Sa), sphingosine-phosphate (SoP), sphinganine-phosphate (SaP), 1-deoxysphinganine (1-deoxySa), and 1-desoxymethylsphinganine (1-desoxyMeSa) after treatment with TNF. All values represent group means +/− SEM, n = 3. One-way ANOVA with Tukey’s post-hoc test; *** denotes p < 0.001 and * denotes p < 0.05 compared to PBS at 48 hrs. N.S. denotes not significant.
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Figure 8: TNF treatment induces accumulation of ceramide and several atypical sphingoid bases (DBS) in MN9D dopaminergic (DA) neuroblastoma cells derived from mouse ventral mesencephalon. MN9D cells were treated with PBS or 5 ng/mL TNF for 24 or 48 hours as indicated prior to cell harvest for lipid extraction as described in Methods. A, Lipidomic analyses indicate time-dependent accumulation of ceramide (Cer), sphingomyelin (SM), and hexosylceramide (HexCer) in TNF-treated cells relative to PBS-treated cells. B, Lipidomic analyses also revealed time-dependent accumulation of sphingosine (So), sphinganine (Sa), sphingosine-phosphate (SoP), sphinganine-phosphate (SaP), 1-deoxysphinganine (1-deoxySa), and 1-desoxymethylsphinganine (1-desoxyMeSa) after treatment with TNF. All values represent group means +/− SEM, n = 3. One-way ANOVA with Tukey’s post-hoc test; *** denotes p < 0.001 and * denotes p < 0.05 compared to PBS at 48 hrs. N.S. denotes not significant.

Mentions: Given that SMase inhibition affords significant protection from TNF-dependent toxicity in DA neuroblastoma cells and primary DA neurons, it was of interest to confirm that TNF treatment results in detectable formation of ceramide in vivo. We used a lipidomics approach to enable quantitative analysis of complex sphingolipids and sphingoid bases in lipid extracts of MN9D cells exposed to PBS or soluble TNF for up to 48 hours. We chose to use DA neuroblastoma cells for our analysis because a homogeneous population of cells is needed for a meaningful result and primary DA neurons only make up a small percentage of total neurons in ventral midbrain cultures. Our analyses indicated that TNF exposure significantly increased the intracellular levels of total ceramide (Cer), sphingomyelin (SM), and hexosylceramide (HexCer) (Figure 8A) as well as several sphingoid bases including sphingosine (So), sphinganine (Sa), sphingosine-1-P (SoP), sphinganine-1-P (SaP), and the atypical sphingoid bases deoxy-sphinganine (deoxySa or DEOSA) and desoxymethylsphinganine (desoxyMeSa or DEOMSA) (Figure 8B). TNF-induced increases in the levels of other complex sphingolipids including deoxydihydro-Ceramide (deoxyDH-Cer) and deoxyceramide (deoxyCer) were not consistently or reproducibly detected (data not shown). These data raise the possibility that in addition to ceramide, any of these additional sphingolipids could be critical second messengers involved in mediating TNF cytotoxicity in DA neuroblastoma cells.


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 treatment induces accumulation of ceramide and several atypical sphingoid bases (DBS) in MN9D dopaminergic (DA) neuroblastoma cells derived from mouse ventral mesencephalon. MN9D cells were treated with PBS or 5 ng/mL TNF for 24 or 48 hours as indicated prior to cell harvest for lipid extraction as described in Methods. A, Lipidomic analyses indicate time-dependent accumulation of ceramide (Cer), sphingomyelin (SM), and hexosylceramide (HexCer) in TNF-treated cells relative to PBS-treated cells. B, Lipidomic analyses also revealed time-dependent accumulation of sphingosine (So), sphinganine (Sa), sphingosine-phosphate (SoP), sphinganine-phosphate (SaP), 1-deoxysphinganine (1-deoxySa), and 1-desoxymethylsphinganine (1-desoxyMeSa) after treatment with TNF. All values represent group means +/− SEM, n = 3. One-way ANOVA with Tukey’s post-hoc test; *** denotes p < 0.001 and * denotes p < 0.05 compared to PBS at 48 hrs. N.S. denotes not significant.
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Figure 8: TNF treatment induces accumulation of ceramide and several atypical sphingoid bases (DBS) in MN9D dopaminergic (DA) neuroblastoma cells derived from mouse ventral mesencephalon. MN9D cells were treated with PBS or 5 ng/mL TNF for 24 or 48 hours as indicated prior to cell harvest for lipid extraction as described in Methods. A, Lipidomic analyses indicate time-dependent accumulation of ceramide (Cer), sphingomyelin (SM), and hexosylceramide (HexCer) in TNF-treated cells relative to PBS-treated cells. B, Lipidomic analyses also revealed time-dependent accumulation of sphingosine (So), sphinganine (Sa), sphingosine-phosphate (SoP), sphinganine-phosphate (SaP), 1-deoxysphinganine (1-deoxySa), and 1-desoxymethylsphinganine (1-desoxyMeSa) after treatment with TNF. All values represent group means +/− SEM, n = 3. One-way ANOVA with Tukey’s post-hoc test; *** denotes p < 0.001 and * denotes p < 0.05 compared to PBS at 48 hrs. N.S. denotes not significant.
Mentions: Given that SMase inhibition affords significant protection from TNF-dependent toxicity in DA neuroblastoma cells and primary DA neurons, it was of interest to confirm that TNF treatment results in detectable formation of ceramide in vivo. We used a lipidomics approach to enable quantitative analysis of complex sphingolipids and sphingoid bases in lipid extracts of MN9D cells exposed to PBS or soluble TNF for up to 48 hours. We chose to use DA neuroblastoma cells for our analysis because a homogeneous population of cells is needed for a meaningful result and primary DA neurons only make up a small percentage of total neurons in ventral midbrain cultures. Our analyses indicated that TNF exposure significantly increased the intracellular levels of total ceramide (Cer), sphingomyelin (SM), and hexosylceramide (HexCer) (Figure 8A) as well as several sphingoid bases including sphingosine (So), sphinganine (Sa), sphingosine-1-P (SoP), sphinganine-1-P (SaP), and the atypical sphingoid bases deoxy-sphinganine (deoxySa or DEOSA) and desoxymethylsphinganine (desoxyMeSa or DEOMSA) (Figure 8B). TNF-induced increases in the levels of other complex sphingolipids including deoxydihydro-Ceramide (deoxyDH-Cer) and deoxyceramide (deoxyCer) were not consistently or reproducibly detected (data not shown). These data raise the possibility that in addition to ceramide, any of these additional sphingolipids could be critical second messengers involved in mediating TNF cytotoxicity in DA neuroblastoma cells.

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