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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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The atypical sphingoid base 1-deoxySa reduced neuritic branches and outgrowth in primary DA neurons. Primary neuron-glia cultures from rat ventral mesencephalon were plated in 96-well plates and exposed to treatment media alone without BSA (0) or to 1-deoxysphinganine (1-deoxySa) at the concentrations indicated in a complex with BSA (25 μM) for 48 hours prior to assessing number of branches per cell, number of processes, and number of outgrowths per cell as well as cell number using Image Xpress high-content imaging analyses. 1-DeoxySa was the only one of the sphingoid bases tested that reduced neurite outgrowth and branching. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc’ * denotes difference from treatment media alone at p < 0.05, and ** denotes p < 0.01. N.S. denotes not significant.
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Figure 10: The atypical sphingoid base 1-deoxySa reduced neuritic branches and outgrowth in primary DA neurons. Primary neuron-glia cultures from rat ventral mesencephalon were plated in 96-well plates and exposed to treatment media alone without BSA (0) or to 1-deoxysphinganine (1-deoxySa) at the concentrations indicated in a complex with BSA (25 μM) for 48 hours prior to assessing number of branches per cell, number of processes, and number of outgrowths per cell as well as cell number using Image Xpress high-content imaging analyses. 1-DeoxySa was the only one of the sphingoid bases tested that reduced neurite outgrowth and branching. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc’ * denotes difference from treatment media alone at p < 0.05, and ** denotes p < 0.01. N.S. denotes not significant.

Mentions: Based on results from lipidomics analyses (Figure 8B) which indicated that TNF exposure not only increased ceramide levels but also resulted in significant increases in the intracellular levels of several atypical deoxy-sphingoid bases (DSBs), including deoxysphinganine (deoxySa or DEOSA) and desoxymethylsphinganine (desoxyMeSa or DEOMSA), we wanted to test these atypical DSBs for direct cytotoxic effects on cells. These DSBs are devoid of the C1-hydroxyl group of sphinganine and can therefore neither be metabolized to complex sphingolipids nor degraded by the regular sphingolipid catabolism, raising the possibility that they may accumulate within DA neurons and may be cytotoxic. Therefore, we tested the extent to which 1-deoxySa, 1-desoxyMeSa, and 1-desoxyMeSo induce dose-dependent cytotoxicity in diff-MN9D cells and found that all three induced dose-dependent cytotoxicity with an IC50 around 15 μM (Figure 9). To confirm and extend the significance of these findings, we investigated the cytotoxicity of these atypical sphingoid bases on primary cultures from rat ventral mesencephalon. We found that only 1-deoxySa significantly reduced the number of neuritic branches and outgrowths per DA neuron at concentrations as low as 0.5 μM (Figure 10); a trend towards compromising DA neuron viability was also evident but it did not reach statistical significance. No significant cytotoxic effects on primary DA neurons by 1-desoxyMeSa and 1-desoxyMeSo were observed (Additional file 3: Figure S3).


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)

The atypical sphingoid base 1-deoxySa reduced neuritic branches and outgrowth in primary DA neurons. Primary neuron-glia cultures from rat ventral mesencephalon were plated in 96-well plates and exposed to treatment media alone without BSA (0) or to 1-deoxysphinganine (1-deoxySa) at the concentrations indicated in a complex with BSA (25 μM) for 48 hours prior to assessing number of branches per cell, number of processes, and number of outgrowths per cell as well as cell number using Image Xpress high-content imaging analyses. 1-DeoxySa was the only one of the sphingoid bases tested that reduced neurite outgrowth and branching. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc’ * denotes difference from treatment media alone at p < 0.05, and ** denotes p < 0.01. N.S. denotes not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 10: The atypical sphingoid base 1-deoxySa reduced neuritic branches and outgrowth in primary DA neurons. Primary neuron-glia cultures from rat ventral mesencephalon were plated in 96-well plates and exposed to treatment media alone without BSA (0) or to 1-deoxysphinganine (1-deoxySa) at the concentrations indicated in a complex with BSA (25 μM) for 48 hours prior to assessing number of branches per cell, number of processes, and number of outgrowths per cell as well as cell number using Image Xpress high-content imaging analyses. 1-DeoxySa was the only one of the sphingoid bases tested that reduced neurite outgrowth and branching. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc’ * denotes difference from treatment media alone at p < 0.05, and ** denotes p < 0.01. N.S. denotes not significant.
Mentions: Based on results from lipidomics analyses (Figure 8B) which indicated that TNF exposure not only increased ceramide levels but also resulted in significant increases in the intracellular levels of several atypical deoxy-sphingoid bases (DSBs), including deoxysphinganine (deoxySa or DEOSA) and desoxymethylsphinganine (desoxyMeSa or DEOMSA), we wanted to test these atypical DSBs for direct cytotoxic effects on cells. These DSBs are devoid of the C1-hydroxyl group of sphinganine and can therefore neither be metabolized to complex sphingolipids nor degraded by the regular sphingolipid catabolism, raising the possibility that they may accumulate within DA neurons and may be cytotoxic. Therefore, we tested the extent to which 1-deoxySa, 1-desoxyMeSa, and 1-desoxyMeSo induce dose-dependent cytotoxicity in diff-MN9D cells and found that all three induced dose-dependent cytotoxicity with an IC50 around 15 μM (Figure 9). To confirm and extend the significance of these findings, we investigated the cytotoxicity of these atypical sphingoid bases on primary cultures from rat ventral mesencephalon. We found that only 1-deoxySa significantly reduced the number of neuritic branches and outgrowths per DA neuron at concentrations as low as 0.5 μM (Figure 10); a trend towards compromising DA neuron viability was also evident but it did not reach statistical significance. No significant cytotoxic effects on primary DA neurons by 1-desoxyMeSa and 1-desoxyMeSo were observed (Additional file 3: Figure S3).

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