A novel role of acid sphingomyelinase in mitochondrial dysfunction due to glutamate-induced programmed necrosis

The glutamate/cystine antiporter system xc - plays an important role in regulating extracellular glutamate levels and in maintaining intracellular cysteine/glutathione (GSH)-dependent antioxidant defense mechanisms in the brain. Blocking the system xc - -dependent pathway could trigger a novel form of programmed necrosis, ferroptosis, which has demonstrated an exciting potential for cancer therapy. Yet, the mechanisms of regulated necrosis in the neural cells and its relevance to neurological disease remain unexplored. Primary oligodendrocytes (OLs), unique myelin-forming cells in the CNS, were treated with glutamate to provoke system xc --mediated cell death. Pharmacological analysis revealed ferroptosis as a major contributing factor to glutamate-initiated OL death. Although RIP1 kinase inhibitor necrostatin-1 protected OLs from glutamate toxicity, there was no activation of the RIP1 and/or RIP3 kinase-mediated necroptosis signaling pathway, suggesting an offtarget effect of necrostatin-1. A quantitative lipidomics analysis showed a significant elevation of bioactive sphingolipids (ceramide and sphingosine) that was prevented by Reclast, a potent inhibitor of acid sphingomyelinase (ASM) activity. OL survival was enhanced by both down-regulating ASM expression and by blocking ASM activity, which suggests ASM plays a crucial role of in OL response to glutamate. Glutamate-induced ASM activation seems to involve post-transcriptional mechanisms and was associated with a decreased GSH level. Further investigation of the mechanisms of OL response to glutamate revealed enhanced ROS production, augmented lipid peroxidation and opening of the mitochondrial permeability transition pore (MPTP) that were attenuated by hindering ASM. Of note, knocking down SIRT3, a deacetylase governing the mitochondrial antioxidant system, attenuated OL survival, highlighting the protective impact of SIRT3 on glutamate-induced necrotic cell death. The data shed more light on the mechanisms of programmed necrotic cell death and accentuate the novel role of ASM in disturbing mitochondrial functions during OL response to glutamate toxicity, which could be important for pathobiology in stroke and traumatic brain injury

Isc1 regulates sphingolipid metabolism in yeast mitochondria

The Saccharomyces cerevisiae inositol sphingolipid phospholipase C (Isc1p), a homolog of mammalian neutral sphingomyelinases, hydrolyzes complex sphingolipids to produce ceramide in vitro. Epitope-tagged Isc1p associates with the mitochondria in the post-diauxic phase of yeast growth. In this report, the mitochondrial localization of Isc1p and its role in regulating sphingolipid metabolism were investigated. First, endogenous Isc1p activity was enriched in highly purified mitochondria, and western blots using highly purified mitochondrial membrane fractions demonstrated that epitope-tagged Isc1p localized to the outer mitochondrial membrane as an integral membrane protein. Next, LC/MS was employed to determine the sphingolipid composition of highly purified mitochondria which were found to be significantly enriched in α-hydroxylated phytoceramides (21.7 fold) relative to the whole cell. Mitochondria, on the other hand, were significantly depleted in sphingoid bases. Compared to the parental strain, mitochondria from isc1Δ in the post-diauxic phase showed drastic reduction in the levels of α-hydroxylated phytoceramide (93.1% loss compared to WT mitochondria with only 2.58 fold enrichment in mitochondria compared to whole cell). Functionally, isc1Δ showed a higher rate of respiratory-deficient cells after incubation at high temperature and was more sensitive to hydrogen peroxide and ethidium bromide, indicating that isc1Δ exhibits defects related to mitochondrial function. These results suggest that Isc1p generates ceramide in mitochondria, and the generated ceramide contributes to the normal function of mitochondria. This study provides a first insight into the specific composition of ceramides in mitochondria

A woolly mammoth (Mammuthus primigenius) carcass from Maly Lyakhovsky Island (New Siberian Islands, Russian Federation)

A partial carcass of an adult woolly mammoth (Mammuthus primigenius) found in 2012 on Maly Lyakhovsky Island presents a new opportunity to retrieve associated anatomical, morphological, and life history data on this important component of Pleistocene biotas. In addition, we address hematological, histological, and microbiological issues that relate directly to quality of preservation. Recovered by staff from North-Eastern Federal University in Yakutsk, this individual is a relatively old female preserving soft tissue of the anteroventral portion of the head, most of both fore-quarters, and the ventral aspect of much of the rest of the body. Both tusks were recovered and subjected to computed tomographic analysis in which annual dentin increments were revealed as cycles of variation in X-ray attenuation. Measurements of annual increment areas (in longitudinal section) display a pulsed pattern of tusk growth showing cycles of growth rate variation over periods of 3-5 years. These intervals are interpreted as calving cycles reflecting regular shifts in calcium and phosphate demand for tusk growth vs. fetal ossification and lactation. Brown liquid associated with the frozen carcass turned out to include remains of hemolyzed blood, and blood samples examined microscopically included white blood cells with preserved nuclei. Muscle tissue from the trunk was unusually well preserved, even at the histological level. Intestinal contents and tissue samples were investigated microbiologically, and several strains of lacticacid bacteria (e.g., Enterococcus faecium, Enterococcus hirae) that are widely distributed as commensal organisms in the intestines of herbivores were isolated. (C) 2017 Elsevier Ltd and INQUA. All rights reserved

Long-chain Ceramide Is a Potent Inhibitor of the Mitochondrial Permeability Transition Pore*

The sphingolipid ceramide has been implicated in mediating cell death that is accompanied by mitochondrial functional alterations. Moreover, ceramide has been shown to accumulate in mitochondria upon induction of apoptotic processes. In this study, we sought to evaluate the effects of natural, highly hydrophobic long-chain ceramides on mitochondrial function in vitro. Ceramide in a dodecane/ethanol delivery system inhibited the opening of the mitochondrial permeability transition pore (PTP) induced by either oxidative stress, SH group cross-linking, or high Ca2+ load, suggesting that the inhibitory point is at a level at which major PTP regulatory pathways converge. Moreover, ceramide had no effect on well known mitochondrial components that modulate PTP activity, such as cyclophilin D, voltage-dependent anion channel, adenine nucleotide transporter, and ATP synthase. The inhibitory effect of ceramide on PTP was not stereospecific, nor was there a preference for ceramide over dihydroceramide. However, the effect of ceramide on PTP was significantly influenced by the fatty acid moiety chain length. These studies are the first to show that long-chain ceramide can influence PTP at physiologically relevant concentrations, suggesting that it is the only known potent natural inhibitor of PTP. These results suggest a novel mechanism of ceramide regulation of mitochondrial function