Interestingly, in these cells, ASMase translocation has been shown to occur via two distinct mechanisms: a caspase-dependent mechanism utilized by Fas-L and a previously unrecognized caspase-independent mechanism elicited by short wave ultraviolet irradiation (UV-C). – 4. Two-way ANOVA to test for differences between TNF with inhibitor versus TNF without inhibitor for both Myrocin and Fumonisin B1; there were no significant differences between No MYR and 10 M MYR at any TNF concentration, as determined by a two-way ANOVA. There was statistically significant TNF-induced death of diff-MN9D cells, as determined by a Tukeys post-hoc test following a statistically significant one-way ANOVA, where ** denotes p? ?0.01, *** p? ?0.001 for No MYR conditions relative to vehicle, * denotes p? ?0.05 for TNF 10?+?No MYR compared to TNF 30?+?No MYR, and ### denotes p? ?0.001 for 10 M MYR conditions relative to vehicle. There were no significant differences between No FB1 and 50 M FB1 at any TNF concentration, as determined by a two-way ANOVA. There was statistically significant TNF-induced death of diff-MN9D cells, as determined by a Tukey’s post-hoc test following a statistically significant one-way ANOVA where * denotes p? ?0.05, *** denotes p? ?0.001 for No FB1 conditions relative to vehicle, ** denotes p? ?0.01 for TNF 10?+?No FB1 compared to TNF 30?+?No FB1, and ### denotes p? ?0.001 for 50uM FB1 conditions relative to vehicle, # denotes p? ?0.01 for TNF 10?+?50 M FB1 compared to TNF 30?+?50 M FB1. 1750-1326-7-45-S2.eps (614K) GUID:?B1F7A839-783D-458F-A3DE-9E5A26ED0B9A Additional file 3 Figure S3. The atypical sphingoid bases 1-deoxyMeSa and 1-deoxyMeSo did not exert cytotoxicity on 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-desoxymethylsphingosine (1-desoxyMeSo) or 1-desoxymethylsphinganine (1-desoxyMeSa) at the concentrations indicated in a complex with BSA (25 M) 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. All values represent group means +/? SEM, n?=?3C4. There were no significant effects from treatment with 1-deoxyMeSa and 1-deoxyMeSo as determined by a one-way ANOVA. 1750-1326-7-45-S3.eps (603K) GUID:?5AA911B0-ADCC-4572-BE9A-5C1CB07D2CED Abstract Background Dopaminergic (DA) neurons in the ventral midbrain selectively degenerate in Parkinsons 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 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. and model of DA neurons [26,27] because the cells GSK1838705A express high levels of tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine biosynthesis, and efficiently synthesize, store and release dopamine [28]; additionally, their sensitivity to oxidative stress and inflammatory stimuli is similar to that of primary DA neurons from ventral midbrain [25-27]. Here we report that TNF and ceramide exert dose-dependent cytotoxic effects on DA neuroblastoma cells and primary DA neurons. Functionally, inhibitors of SMase activity which block sphingomyelin hydrolysis and ceramide generation attenuated TNF-induced cytotoxicity,.Interestingly, TNF treatment also induced SM biosynthesis (Figure ?(Figure8A);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]. ANOVA to test for differences between TNF with inhibitor versus TNF without inhibitor for both Myrocin and Fumonisin B1; GSK1838705A there were no significant differences between No MYR and 10 M MYR at any TNF concentration, as determined by a two-way ANOVA. There was statistically significant TNF-induced death of diff-MN9D cells, as determined by a Tukeys post-hoc test following a statistically significant one-way ANOVA, where ** denotes p? ?0.01, *** p? ?0.001 for No MYR conditions relative to vehicle, * denotes p? ?0.05 for TNF 10?+?No MYR compared to TNF 30?+?No MYR, and ### denotes p? ?0.001 for 10 M MYR conditions relative to vehicle. There were no significant differences between No FB1 and 50 M FB1 at any TNF concentration, as determined by a two-way ANOVA. There was statistically significant TNF-induced death of diff-MN9D cells, as determined by a Tukey’s post-hoc test following a statistically significant one-way ANOVA where * denotes p? ?0.05, *** denotes p? ?0.001 for No FB1 conditions relative to vehicle, ** denotes p? ?0.01 for TNF 10?+?No FB1 compared to TNF 30?+?No FB1, and ### denotes p? ?0.001 for 50uM FB1 conditions relative to vehicle, # denotes p? ?0.01 for TNF 10?+?50 M FB1 compared to TNF 30?+?50 M FB1. 1750-1326-7-45-S2.eps (614K) GUID:?B1F7A839-783D-458F-A3DE-9E5A26ED0B9A Additional file 3 Figure S3. The atypical sphingoid bases 1-deoxyMeSa and 1-deoxyMeSo did not exert cytotoxicity on 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-desoxymethylsphingosine (1-desoxyMeSo) or 1-desoxymethylsphinganine (1-desoxyMeSa) at the concentrations indicated in a complex with BSA (25 M) 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. All values represent group means +/? SEM, n?=?3C4. There were no significant effects from treatment with 1-deoxyMeSa and 1-deoxyMeSo as determined by a one-way ANOVA. 1750-1326-7-45-S3.eps (603K) GUID:?5AA911B0-ADCC-4572-BE9A-5C1CB07D2CED Abstract Background Dopaminergic (DA) neurons in the ventral midbrain selectively degenerate in Parkinsons 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 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 Mmp14 attenuate the progressive loss of nigral DA neurons in patients with PD. and model of DA neurons [26,27] because the cells express high levels of tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine biosynthesis, and efficiently synthesize, store and release dopamine [28]; additionally, their sensitivity to oxidative stress and inflammatory stimuli is similar to that of primary DA neurons from ventral midbrain [25-27]. Here we report that TNF and ceramide exert dose-dependent cytotoxic effects on DA neuroblastoma cells and primary DA neurons. Functionally, inhibitors of SMase activity which block sphingomyelin hydrolysis and ceramide generation attenuated TNF-induced cytotoxicity, decreases in phospho-Akt, increases in caspase 3 cleavage as well as mitochondrial membrane potential changes, and ER stress GSK1838705A in DA cells..