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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 phospho-Akt pro-survival signaling were abolished by SMase inhibitors.A, Diff-MN9D cells were treated with 5 ng/mL TNF in the presence or absence of SMase inhibitors Desipramine (Des 5 μM) or GW4859 (10 μM) and protein lysates were thereafter harvested for SDS-PAGE and immunoblot analysis of phospho-Akt (p-Akt) immunoreactivity. B, Quantification of western blot analysis of phospho-Akt (p-Akt) relative to total Akt. One-way ANOVA with Tukey’s post-hoc test, where ** denotes p < 0.01 compared to TNF without inhibitor.
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Figure 7: TNF-induced decreases in phospho-Akt pro-survival signaling were abolished by SMase inhibitors.A, Diff-MN9D cells were treated with 5 ng/mL TNF in the presence or absence of SMase inhibitors Desipramine (Des 5 μM) or GW4859 (10 μM) and protein lysates were thereafter harvested for SDS-PAGE and immunoblot analysis of phospho-Akt (p-Akt) immunoreactivity. B, Quantification of western blot analysis of phospho-Akt (p-Akt) relative to total Akt. One-way ANOVA with Tukey’s post-hoc test, where ** denotes p < 0.01 compared to TNF without inhibitor.

Mentions: Next, we tested the hypothesis that TNF-dependent ceramide-induced cytotoxicity in diff-MN9D cells may also result from reduced activation of pro-survival pathways, such as Akt signaling. Therefore, we investigated the effect of TNF on phosphorylation of Akt, a key step in pro-survival signaling in the majority of neurons [50,51] We found that TNF treatment reduced p-Akt levels in DA cells and SMase inhibitors robustly blocked this effect (Figure 7). Together with results from caspase inhibition experiments, these data suggest that TNF treatment leads to generation and accumulation of ceramide (and perhaps other downstream sphingolipid metabolites), leading to cytotoxicity in DA neurons via increased ER stress, compromised mitochondrial membrane potential, increased caspase-3 dependent apoptotic signaling cascades, and attenuation of phospho-Akt-dependent pro-survival signaling.


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 phospho-Akt pro-survival signaling were abolished by SMase inhibitors.A, Diff-MN9D cells were treated with 5 ng/mL TNF in the presence or absence of SMase inhibitors Desipramine (Des 5 μM) or GW4859 (10 μM) and protein lysates were thereafter harvested for SDS-PAGE and immunoblot analysis of phospho-Akt (p-Akt) immunoreactivity. B, Quantification of western blot analysis of phospho-Akt (p-Akt) relative to total Akt. One-way ANOVA with Tukey’s post-hoc test, where ** denotes p < 0.01 compared to TNF without inhibitor.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: TNF-induced decreases in phospho-Akt pro-survival signaling were abolished by SMase inhibitors.A, Diff-MN9D cells were treated with 5 ng/mL TNF in the presence or absence of SMase inhibitors Desipramine (Des 5 μM) or GW4859 (10 μM) and protein lysates were thereafter harvested for SDS-PAGE and immunoblot analysis of phospho-Akt (p-Akt) immunoreactivity. B, Quantification of western blot analysis of phospho-Akt (p-Akt) relative to total Akt. One-way ANOVA with Tukey’s post-hoc test, where ** denotes p < 0.01 compared to TNF without inhibitor.
Mentions: Next, we tested the hypothesis that TNF-dependent ceramide-induced cytotoxicity in diff-MN9D cells may also result from reduced activation of pro-survival pathways, such as Akt signaling. Therefore, we investigated the effect of TNF on phosphorylation of Akt, a key step in pro-survival signaling in the majority of neurons [50,51] We found that TNF treatment reduced p-Akt levels in DA cells and SMase inhibitors robustly blocked this effect (Figure 7). Together with results from caspase inhibition experiments, these data suggest that TNF treatment leads to generation and accumulation of ceramide (and perhaps other downstream sphingolipid metabolites), leading to cytotoxicity in DA neurons via increased ER stress, compromised mitochondrial membrane potential, increased caspase-3 dependent apoptotic signaling cascades, and attenuation of phospho-Akt-dependent pro-survival signaling.

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