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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- and ceramide-induced caspase-3 cleavage was attenuated by SMase inhibitors and caspase inhibitors rescued differentiated MN9D cells from TNF-induced cytotoxicity.A, Diff-MN9D cells were treated for 3 days with 5 ng/mL TNF in the presence or absence of the ASMase inhibitor Desipramine (Des, 5 μM) or the NSMase inhibitor GW4869 (10 μM) and were thereafter harvested for SDS-PAGE and immunoblot analysis of total caspase 3 or cleaved caspase 3. B, Quantification of western blot analysis of caspase 3 and cleaved caspase 3. One-Way ANOVA with Tukey’s post-hoc test to compare inhibitor conditions to TNF alone, where ** denotes p < 0.01, ***denotes p < 0.001. C, MTS assay for cell viability in diff-MN9D cells. TNF induced dose-dependent death of diff-MN9D cells and was caspase-dependent. Co-treatment with TNF plus the pan-caspase inhibitor Z-VAD (25 μM) or with TNF plus the caspase-8-specific inhibitor Z-IETD (25 μM) robustly blocked TNF-induced cell death in diff-MN9D cells. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc test to compare the effect of specified TNF concentrations on diff-MN9D viability without caspase inhibitors in the MTS assay, where # denotes p < 0.05, ## denotes p < 0.01, and ### denotes p < 0.001 compared to ‘Veh’ or between two concentrations. Two-way ANOVA with Tukey’s post-hoc to compare effects of caspase inhibitors at each TNF concentration where *** denotes p < 0.001 compared to no caspase inhibitor.
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Figure 5: TNF- and ceramide-induced caspase-3 cleavage was attenuated by SMase inhibitors and caspase inhibitors rescued differentiated MN9D cells from TNF-induced cytotoxicity.A, Diff-MN9D cells were treated for 3 days with 5 ng/mL TNF in the presence or absence of the ASMase inhibitor Desipramine (Des, 5 μM) or the NSMase inhibitor GW4869 (10 μM) and were thereafter harvested for SDS-PAGE and immunoblot analysis of total caspase 3 or cleaved caspase 3. B, Quantification of western blot analysis of caspase 3 and cleaved caspase 3. One-Way ANOVA with Tukey’s post-hoc test to compare inhibitor conditions to TNF alone, where ** denotes p < 0.01, ***denotes p < 0.001. C, MTS assay for cell viability in diff-MN9D cells. TNF induced dose-dependent death of diff-MN9D cells and was caspase-dependent. Co-treatment with TNF plus the pan-caspase inhibitor Z-VAD (25 μM) or with TNF plus the caspase-8-specific inhibitor Z-IETD (25 μM) robustly blocked TNF-induced cell death in diff-MN9D cells. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc test to compare the effect of specified TNF concentrations on diff-MN9D viability without caspase inhibitors in the MTS assay, where # denotes p < 0.05, ## denotes p < 0.01, and ### denotes p < 0.001 compared to ‘Veh’ or between two concentrations. Two-way ANOVA with Tukey’s post-hoc to compare effects of caspase inhibitors at each TNF concentration where *** denotes p < 0.001 compared to no caspase inhibitor.

Mentions: Loss of mitochondrial membrane potential and release of cytochrome C from mitochondria generally precede caspase-dependent apoptotic cell death and a wealth of data has linked TNF bioactivity to caspase activation and apoptosis in various cell types (reviewed in [47]). Similarly, ceramide has been reported to cause apoptotic cell death by altering the Bax/Bcl2 ratio which triggers cytochrome C release from the mitochondria and results in activation of the caspase-9/-3 cascade in C6 glioma cells [48]. Therefore, we investigated the extent to which addition of SMase inhibitors during TNF treatment attenuated caspase signaling. Western blot analyses showed that desipramine and GW4869 significantly attenuated caspase 3 cleavage in TNF-treated diff-MN9D cells (Figure 5A, B). To correlate this finding with TNF-induced cytotoxicity in diff-MN9D cells, we determined the extent to which pan-caspase inhibition (with Z-VAD) or caspase 8 inhibition (with Z-IETD) could ameliorate TNF dose-dependent loss of viability in diff-MN9D. We found that both caspase inhibitors robustly protected diff-MN9D cells from TNF-induced cytotoxicity at all TNF concentrations (Figure 5C), demonstrating that caspase activation is obligate for TNF-induced apoptotic cell death in terminally differentiated MN9D cells and suggesting that TNF-dependent ceramide generation promotes activation of caspase 8 and caspase 3 signaling cascades that lead to apoptotic death in DA cells and neurons. Interestingly, we also found that C2-Cer-induced cytotoxic cell death in diff-MN9D cells was not significantly blocked by Z-VAD or Z-IETD (Figure 6A), which is not entirely surprising since exogenously added C2-Cer would act downstream of TNF/TNFR1-dependent caspase 8 activation. However, we hypothesized that TNF-stimulated ceramide exerts cytotoxicity in DA cells by dysregulating intracellular Ca2+ based on reports that implicate defective Ca2+ homeostasis in apoptotic cell death of neuronal populations induced by aberrant sphingolipid metabolism [49]. To test this hypothesis directly, we pre-incubated diffMN9D cells with BAPTA-AM prior to exposure to C2-Cer and found that buffering intracellular free calcium nearly ablates C2-Cer-induced toxicity in diff-MN9D cells (Figure 6B), suggesting that elevation of [Ca2+i contributes to C2-Cer-induced neurotoxicity.


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- and ceramide-induced caspase-3 cleavage was attenuated by SMase inhibitors and caspase inhibitors rescued differentiated MN9D cells from TNF-induced cytotoxicity.A, Diff-MN9D cells were treated for 3 days with 5 ng/mL TNF in the presence or absence of the ASMase inhibitor Desipramine (Des, 5 μM) or the NSMase inhibitor GW4869 (10 μM) and were thereafter harvested for SDS-PAGE and immunoblot analysis of total caspase 3 or cleaved caspase 3. B, Quantification of western blot analysis of caspase 3 and cleaved caspase 3. One-Way ANOVA with Tukey’s post-hoc test to compare inhibitor conditions to TNF alone, where ** denotes p < 0.01, ***denotes p < 0.001. C, MTS assay for cell viability in diff-MN9D cells. TNF induced dose-dependent death of diff-MN9D cells and was caspase-dependent. Co-treatment with TNF plus the pan-caspase inhibitor Z-VAD (25 μM) or with TNF plus the caspase-8-specific inhibitor Z-IETD (25 μM) robustly blocked TNF-induced cell death in diff-MN9D cells. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc test to compare the effect of specified TNF concentrations on diff-MN9D viability without caspase inhibitors in the MTS assay, where # denotes p < 0.05, ## denotes p < 0.01, and ### denotes p < 0.001 compared to ‘Veh’ or between two concentrations. Two-way ANOVA with Tukey’s post-hoc to compare effects of caspase inhibitors at each TNF concentration where *** denotes p < 0.001 compared to no caspase inhibitor.
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Figure 5: TNF- and ceramide-induced caspase-3 cleavage was attenuated by SMase inhibitors and caspase inhibitors rescued differentiated MN9D cells from TNF-induced cytotoxicity.A, Diff-MN9D cells were treated for 3 days with 5 ng/mL TNF in the presence or absence of the ASMase inhibitor Desipramine (Des, 5 μM) or the NSMase inhibitor GW4869 (10 μM) and were thereafter harvested for SDS-PAGE and immunoblot analysis of total caspase 3 or cleaved caspase 3. B, Quantification of western blot analysis of caspase 3 and cleaved caspase 3. One-Way ANOVA with Tukey’s post-hoc test to compare inhibitor conditions to TNF alone, where ** denotes p < 0.01, ***denotes p < 0.001. C, MTS assay for cell viability in diff-MN9D cells. TNF induced dose-dependent death of diff-MN9D cells and was caspase-dependent. Co-treatment with TNF plus the pan-caspase inhibitor Z-VAD (25 μM) or with TNF plus the caspase-8-specific inhibitor Z-IETD (25 μM) robustly blocked TNF-induced cell death in diff-MN9D cells. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc test to compare the effect of specified TNF concentrations on diff-MN9D viability without caspase inhibitors in the MTS assay, where # denotes p < 0.05, ## denotes p < 0.01, and ### denotes p < 0.001 compared to ‘Veh’ or between two concentrations. Two-way ANOVA with Tukey’s post-hoc to compare effects of caspase inhibitors at each TNF concentration where *** denotes p < 0.001 compared to no caspase inhibitor.
Mentions: Loss of mitochondrial membrane potential and release of cytochrome C from mitochondria generally precede caspase-dependent apoptotic cell death and a wealth of data has linked TNF bioactivity to caspase activation and apoptosis in various cell types (reviewed in [47]). Similarly, ceramide has been reported to cause apoptotic cell death by altering the Bax/Bcl2 ratio which triggers cytochrome C release from the mitochondria and results in activation of the caspase-9/-3 cascade in C6 glioma cells [48]. Therefore, we investigated the extent to which addition of SMase inhibitors during TNF treatment attenuated caspase signaling. Western blot analyses showed that desipramine and GW4869 significantly attenuated caspase 3 cleavage in TNF-treated diff-MN9D cells (Figure 5A, B). To correlate this finding with TNF-induced cytotoxicity in diff-MN9D cells, we determined the extent to which pan-caspase inhibition (with Z-VAD) or caspase 8 inhibition (with Z-IETD) could ameliorate TNF dose-dependent loss of viability in diff-MN9D. We found that both caspase inhibitors robustly protected diff-MN9D cells from TNF-induced cytotoxicity at all TNF concentrations (Figure 5C), demonstrating that caspase activation is obligate for TNF-induced apoptotic cell death in terminally differentiated MN9D cells and suggesting that TNF-dependent ceramide generation promotes activation of caspase 8 and caspase 3 signaling cascades that lead to apoptotic death in DA cells and neurons. Interestingly, we also found that C2-Cer-induced cytotoxic cell death in diff-MN9D cells was not significantly blocked by Z-VAD or Z-IETD (Figure 6A), which is not entirely surprising since exogenously added C2-Cer would act downstream of TNF/TNFR1-dependent caspase 8 activation. However, we hypothesized that TNF-stimulated ceramide exerts cytotoxicity in DA cells by dysregulating intracellular Ca2+ based on reports that implicate defective Ca2+ homeostasis in apoptotic cell death of neuronal populations induced by aberrant sphingolipid metabolism [49]. To test this hypothesis directly, we pre-incubated diffMN9D cells with BAPTA-AM prior to exposure to C2-Cer and found that buffering intracellular free calcium nearly ablates C2-Cer-induced toxicity in diff-MN9D cells (Figure 6B), suggesting that elevation of [Ca2+i contributes to C2-Cer-induced neurotoxicity.

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