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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 ER stress responses in MN9D dopaminergic (DA) cells were attenuated by SMase inhibitors.A, Diff-MN9D cells were pre-treated with 5 μM Desipramine (Des) or 10 μM GW4869 followed by treatment with 5 ng/mL TNF for 24 hours. Cell lysates were harvested for SDS-PAGE and were analyzed by immunoblot using antibodies against the ER stress proteins ATF6, IRE1, and PERK or GAPDH for normalization. Dihydro-ceramide (DH-C2-Cer at 10 μM) and tunicamycin (Tunicam. at 0.1 μg/mL) were used as negative and positive controls, respectively. B, Quantification of western blots in A. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc test; * denotes p < 0.05, *** denotes p < 0.001 compared to TNF alone.
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Figure 3: TNF-induced ER stress responses in MN9D dopaminergic (DA) cells were attenuated by SMase inhibitors.A, Diff-MN9D cells were pre-treated with 5 μM Desipramine (Des) or 10 μM GW4869 followed by treatment with 5 ng/mL TNF for 24 hours. Cell lysates were harvested for SDS-PAGE and were analyzed by immunoblot using antibodies against the ER stress proteins ATF6, IRE1, and PERK or GAPDH for normalization. Dihydro-ceramide (DH-C2-Cer at 10 μM) and tunicamycin (Tunicam. at 0.1 μg/mL) were used as negative and positive controls, respectively. B, Quantification of western blots in A. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc test; * denotes p < 0.05, *** denotes p < 0.001 compared to TNF alone.

Mentions: TNF and ceramide have been shown to impinge on ER stress mechanisms in non-neuronal cells types [40,41] and ER stress has been implicated as a potentially important pathway in PD pathogenesis [42], being coupled to the cell death program in DA cells in response to the toxin paraquat [43]. Therefore, we investigated the extent to which activation of ER stress pathways by TNF are dependent on ceramide generation by SMase activity in diff-MN9D cells. We used immunoblots to ascertain if TNF treatment of diff-MN9D cells increased protein expression of key ER stress transducers, including activating transcription factor 6 (ATF6), ER- resident PKR-like eIF2α kinase (PERK), and inositol requiring enzyme-1 (IRE). We found that the increased expression of ER stress proteins by TNF and C2-Cer was comparable to increased protein levels caused by the positive control tunicamycin, (Figure 3) which is known to potently induce ER stress by inhibiting protein N-glycosylation [44]. These results support a model in which TNF employs ceramide signaling to elicit ER stress 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 ER stress responses in MN9D dopaminergic (DA) cells were attenuated by SMase inhibitors.A, Diff-MN9D cells were pre-treated with 5 μM Desipramine (Des) or 10 μM GW4869 followed by treatment with 5 ng/mL TNF for 24 hours. Cell lysates were harvested for SDS-PAGE and were analyzed by immunoblot using antibodies against the ER stress proteins ATF6, IRE1, and PERK or GAPDH for normalization. Dihydro-ceramide (DH-C2-Cer at 10 μM) and tunicamycin (Tunicam. at 0.1 μg/mL) were used as negative and positive controls, respectively. B, Quantification of western blots in A. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc test; * denotes p < 0.05, *** denotes p < 0.001 compared to TNF alone.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: TNF-induced ER stress responses in MN9D dopaminergic (DA) cells were attenuated by SMase inhibitors.A, Diff-MN9D cells were pre-treated with 5 μM Desipramine (Des) or 10 μM GW4869 followed by treatment with 5 ng/mL TNF for 24 hours. Cell lysates were harvested for SDS-PAGE and were analyzed by immunoblot using antibodies against the ER stress proteins ATF6, IRE1, and PERK or GAPDH for normalization. Dihydro-ceramide (DH-C2-Cer at 10 μM) and tunicamycin (Tunicam. at 0.1 μg/mL) were used as negative and positive controls, respectively. B, Quantification of western blots in A. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc test; * denotes p < 0.05, *** denotes p < 0.001 compared to TNF alone.
Mentions: TNF and ceramide have been shown to impinge on ER stress mechanisms in non-neuronal cells types [40,41] and ER stress has been implicated as a potentially important pathway in PD pathogenesis [42], being coupled to the cell death program in DA cells in response to the toxin paraquat [43]. Therefore, we investigated the extent to which activation of ER stress pathways by TNF are dependent on ceramide generation by SMase activity in diff-MN9D cells. We used immunoblots to ascertain if TNF treatment of diff-MN9D cells increased protein expression of key ER stress transducers, including activating transcription factor 6 (ATF6), ER- resident PKR-like eIF2α kinase (PERK), and inositol requiring enzyme-1 (IRE). We found that the increased expression of ER stress proteins by TNF and C2-Cer was comparable to increased protein levels caused by the positive control tunicamycin, (Figure 3) which is known to potently induce ER stress by inhibiting protein N-glycosylation [44]. These results support a model in which TNF employs ceramide signaling to elicit ER stress 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