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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 decreases in mitochondrial membrane potential were attenuated by SMase inhibitors.A, Treatment of diff-MN9D cells for 18 hrs with 5 or 10 μM C2-Cer or 5 ng/mL TNF comprised mitochondrial membrane potential. TMRM cytofluorescence was normalized to CCCP + Veh or CCCP + TNF, as CCCP uncouples mitochondrial membranes. Pre-treatment with the SMase inhibitors Desipramine or GW4869 attenuated the TNF-induced decreases in mitochondrial membrane potential. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post test; * denotes p < 0.05, *** denotes p < 0.001 compared to ‘Veh’; # denotes p < 0.05 compared to TNF alone. B, Treatment of diff-MN9D cells for 18 hrs with 5 or 10 μM C2-Cer or 5 ng/mL TNF induced cytotoxicity as measured by LDH release. Pre-treatment with SMase inhibitors (Desipramine or GW4869) attenuated TNF-induced cytotoxicity; the TNF inhibitor (etanercept) was used as a positive control. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post test; ### denotes p < 0.001 compared to vehicle; *** denotes p < 0.001 compared to TNF alone.
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Figure 4: TNF- induced decreases in mitochondrial membrane potential were attenuated by SMase inhibitors.A, Treatment of diff-MN9D cells for 18 hrs with 5 or 10 μM C2-Cer or 5 ng/mL TNF comprised mitochondrial membrane potential. TMRM cytofluorescence was normalized to CCCP + Veh or CCCP + TNF, as CCCP uncouples mitochondrial membranes. Pre-treatment with the SMase inhibitors Desipramine or GW4869 attenuated the TNF-induced decreases in mitochondrial membrane potential. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post test; * denotes p < 0.05, *** denotes p < 0.001 compared to ‘Veh’; # denotes p < 0.05 compared to TNF alone. B, Treatment of diff-MN9D cells for 18 hrs with 5 or 10 μM C2-Cer or 5 ng/mL TNF induced cytotoxicity as measured by LDH release. Pre-treatment with SMase inhibitors (Desipramine or GW4869) attenuated TNF-induced cytotoxicity; the TNF inhibitor (etanercept) was used as a positive control. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post test; ### denotes p < 0.001 compared to vehicle; *** denotes p < 0.001 compared to TNF alone.

Mentions: TNF has been reported to cause rapid decreases in mitochondrial membrane potential and coincident increases in reactive oxygen species [45]. Consistent with our hypothesis that ceramide is an important downstream effector of TNF cytotoxicity, ceramide itself has been shown to directly affect the mitochondrial electron transport chain [46]. To further elucidate the mechanisms of TNF and C2-Cer-induced cytotoxicity and to determine if TNF/ceramide signaling in diff-MN9D cells impinges on mitochondria, we investigated whether TNF or C2-Cer adversely impact mitochondrial membrane potential by evaluating tetramethyl rhodamine methyl ester (TMRM) cytofluorescence. TMRM is a cationic mitochondrial-selective probe that accumulates in the negatively charged mitochondrial membrane in proportion to mitochondrial membrane potential. Diff-MN9D cells treated with 5 ng/mL TNF for 36 hrs or 5 or 10 μM C2-Cer for 18 hrs exhibited compromised mitochondrial membrane potential as evidence by reduced TMRM cytofluorescence relative to vehicle treated diff-MN9D cells (Figure 4A), lending support to the interpretation that both TNF and C2-Cer adversely affect mitochondrial integrity in diff-MN9D cells. Moreover, the SMase inhibitors desipramine and GW4869 partially restored the TMRM signal in diff-MN9D cells (Figure 4A). To confirm and extend these findings we performed an additional assay to measure TNF-induced cytotoxicity. Diff-MN9D cells were treated for 18 hrs with 5 or 10 μM C2-Cer or 5 ng/mL TNF; lactate dehydrogenate (LDH) release was then measured. In agreement with results from MTS assays, pre-treatment with SMase inhibitors (Desipramine or GW4869) attenuated TNF-induced LDH release. The TNF inhibitor etanercept was used as a positive control. These data support a model in which TNF-induced cytotoxicity is mediated via ceramide-dependent signaling leading to disruption of mitochondrial membrane potential in DA 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- induced decreases in mitochondrial membrane potential were attenuated by SMase inhibitors.A, Treatment of diff-MN9D cells for 18 hrs with 5 or 10 μM C2-Cer or 5 ng/mL TNF comprised mitochondrial membrane potential. TMRM cytofluorescence was normalized to CCCP + Veh or CCCP + TNF, as CCCP uncouples mitochondrial membranes. Pre-treatment with the SMase inhibitors Desipramine or GW4869 attenuated the TNF-induced decreases in mitochondrial membrane potential. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post test; * denotes p < 0.05, *** denotes p < 0.001 compared to ‘Veh’; # denotes p < 0.05 compared to TNF alone. B, Treatment of diff-MN9D cells for 18 hrs with 5 or 10 μM C2-Cer or 5 ng/mL TNF induced cytotoxicity as measured by LDH release. Pre-treatment with SMase inhibitors (Desipramine or GW4869) attenuated TNF-induced cytotoxicity; the TNF inhibitor (etanercept) was used as a positive control. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post test; ### denotes p < 0.001 compared to vehicle; *** denotes p < 0.001 compared to TNF alone.
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Related In: Results  -  Collection

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Figure 4: TNF- induced decreases in mitochondrial membrane potential were attenuated by SMase inhibitors.A, Treatment of diff-MN9D cells for 18 hrs with 5 or 10 μM C2-Cer or 5 ng/mL TNF comprised mitochondrial membrane potential. TMRM cytofluorescence was normalized to CCCP + Veh or CCCP + TNF, as CCCP uncouples mitochondrial membranes. Pre-treatment with the SMase inhibitors Desipramine or GW4869 attenuated the TNF-induced decreases in mitochondrial membrane potential. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post test; * denotes p < 0.05, *** denotes p < 0.001 compared to ‘Veh’; # denotes p < 0.05 compared to TNF alone. B, Treatment of diff-MN9D cells for 18 hrs with 5 or 10 μM C2-Cer or 5 ng/mL TNF induced cytotoxicity as measured by LDH release. Pre-treatment with SMase inhibitors (Desipramine or GW4869) attenuated TNF-induced cytotoxicity; the TNF inhibitor (etanercept) was used as a positive control. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post test; ### denotes p < 0.001 compared to vehicle; *** denotes p < 0.001 compared to TNF alone.
Mentions: TNF has been reported to cause rapid decreases in mitochondrial membrane potential and coincident increases in reactive oxygen species [45]. Consistent with our hypothesis that ceramide is an important downstream effector of TNF cytotoxicity, ceramide itself has been shown to directly affect the mitochondrial electron transport chain [46]. To further elucidate the mechanisms of TNF and C2-Cer-induced cytotoxicity and to determine if TNF/ceramide signaling in diff-MN9D cells impinges on mitochondria, we investigated whether TNF or C2-Cer adversely impact mitochondrial membrane potential by evaluating tetramethyl rhodamine methyl ester (TMRM) cytofluorescence. TMRM is a cationic mitochondrial-selective probe that accumulates in the negatively charged mitochondrial membrane in proportion to mitochondrial membrane potential. Diff-MN9D cells treated with 5 ng/mL TNF for 36 hrs or 5 or 10 μM C2-Cer for 18 hrs exhibited compromised mitochondrial membrane potential as evidence by reduced TMRM cytofluorescence relative to vehicle treated diff-MN9D cells (Figure 4A), lending support to the interpretation that both TNF and C2-Cer adversely affect mitochondrial integrity in diff-MN9D cells. Moreover, the SMase inhibitors desipramine and GW4869 partially restored the TMRM signal in diff-MN9D cells (Figure 4A). To confirm and extend these findings we performed an additional assay to measure TNF-induced cytotoxicity. Diff-MN9D cells were treated for 18 hrs with 5 or 10 μM C2-Cer or 5 ng/mL TNF; lactate dehydrogenate (LDH) release was then measured. In agreement with results from MTS assays, pre-treatment with SMase inhibitors (Desipramine or GW4869) attenuated TNF-induced LDH release. The TNF inhibitor etanercept was used as a positive control. These data support a model in which TNF-induced cytotoxicity is mediated via ceramide-dependent signaling leading to disruption of mitochondrial membrane potential in DA 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