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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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Proposed model for cellular mechanisms and signaling pathways activated by TNF and ceramide/sphingolipid signaling to induce neurotoxicity in DA neurons. We propose a model by which TNF/TNFR1-dependent activation of SMases triggers production of ceramide and other downstream lipid metabolites that promote activation of caspase-8/3 signaling, decreased Akt activation and mitochondrial membrane potential, and increased endoplasmic reticulum (ER) stress in DA cells.
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Figure 11: Proposed model for cellular mechanisms and signaling pathways activated by TNF and ceramide/sphingolipid signaling to induce neurotoxicity in DA neurons. We propose a model by which TNF/TNFR1-dependent activation of SMases triggers production of ceramide and other downstream lipid metabolites that promote activation of caspase-8/3 signaling, decreased Akt activation and mitochondrial membrane potential, and increased endoplasmic reticulum (ER) stress in DA cells.

Mentions: The purpose of these studies was to test the hypothesis that ceramide-dependent signaling mediates TNF-induced cytotoxicity and degeneration of DA neurons. Our results indicate that exposure of neurally differentiated DA neuroblastoma cells to soluble TNF induced activation of membrane-bound sphingomyelinases (SMases) and sphingomyelin (SM) turnover resulting in generation of ceramide as measured by lipidomics mass spectrometry. Direct addition of C2-ceramide to DA neuroblastoma cells or primary DA neurons in vitro resulted in dose-dependent cytotoxicity, and pharmacological inhibition of SMases with three different inhibitors of SMase function to block ceramide generation during TNF exposure (but not inhibitors of de novo ceramide synthesis) afforded significant protection from TNF-induced cytotoxicity. Although desipramine can exert SMase-independent effects on cells [52], two other inhibitors with greater specificity for SMase (GW4869 and ARC39) afforded similar protection against TNF-induced cytotoxicity. Based on these findings, we propose a model by which binding of soluble TNF to TNFR1 on the cell surface of DA neurons activates SMases to generate ceramide and trigger downstream signaling cascades that compromise survival of DA neurons by eliciting ER stress, reducing mitochondria membrane potential, leading to activation of caspase-3-dependent pro-apoptotic signaling and inhibition of Akt-dependent pro-survival signaling cascades which combine to compromise survival of DA neurons (Figure 11). Interestingly, TNF treatment also induced SM biosynthesis (Figure 8A); the significance of this novel finding is unknown, but TNF and lipopolysaccharide (LPS) have both been reported to induce sphingolipid biosynthesis in liver [53] and macrophages [54]. It is also worth noting that increases in atypical deoxy-sphingoid bases (DSBs) were detectable in DA cells after prolonged exposure to TNF (Figure 8B), the potential significance of which is discussed below.


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)

Proposed model for cellular mechanisms and signaling pathways activated by TNF and ceramide/sphingolipid signaling to induce neurotoxicity in DA neurons. We propose a model by which TNF/TNFR1-dependent activation of SMases triggers production of ceramide and other downstream lipid metabolites that promote activation of caspase-8/3 signaling, decreased Akt activation and mitochondrial membrane potential, and increased endoplasmic reticulum (ER) stress in DA cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 11: Proposed model for cellular mechanisms and signaling pathways activated by TNF and ceramide/sphingolipid signaling to induce neurotoxicity in DA neurons. We propose a model by which TNF/TNFR1-dependent activation of SMases triggers production of ceramide and other downstream lipid metabolites that promote activation of caspase-8/3 signaling, decreased Akt activation and mitochondrial membrane potential, and increased endoplasmic reticulum (ER) stress in DA cells.
Mentions: The purpose of these studies was to test the hypothesis that ceramide-dependent signaling mediates TNF-induced cytotoxicity and degeneration of DA neurons. Our results indicate that exposure of neurally differentiated DA neuroblastoma cells to soluble TNF induced activation of membrane-bound sphingomyelinases (SMases) and sphingomyelin (SM) turnover resulting in generation of ceramide as measured by lipidomics mass spectrometry. Direct addition of C2-ceramide to DA neuroblastoma cells or primary DA neurons in vitro resulted in dose-dependent cytotoxicity, and pharmacological inhibition of SMases with three different inhibitors of SMase function to block ceramide generation during TNF exposure (but not inhibitors of de novo ceramide synthesis) afforded significant protection from TNF-induced cytotoxicity. Although desipramine can exert SMase-independent effects on cells [52], two other inhibitors with greater specificity for SMase (GW4869 and ARC39) afforded similar protection against TNF-induced cytotoxicity. Based on these findings, we propose a model by which binding of soluble TNF to TNFR1 on the cell surface of DA neurons activates SMases to generate ceramide and trigger downstream signaling cascades that compromise survival of DA neurons by eliciting ER stress, reducing mitochondria membrane potential, leading to activation of caspase-3-dependent pro-apoptotic signaling and inhibition of Akt-dependent pro-survival signaling cascades which combine to compromise survival of DA neurons (Figure 11). Interestingly, TNF treatment also induced SM biosynthesis (Figure 8A); the significance of this novel finding is unknown, but TNF and lipopolysaccharide (LPS) have both been reported to induce sphingolipid biosynthesis in liver [53] and macrophages [54]. It is also worth noting that increases in atypical deoxy-sphingoid bases (DSBs) were detectable in DA cells after prolonged exposure to TNF (Figure 8B), the potential significance of which is discussed below.

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