Myelin contains neutral sphingomyelinase activity that is stimulated by tumor necrosis factor‐α

Abstract

Purified myelin from mouse brain was found to contain two forms of neutral sphingomyelinase, one Mg2+ dependent and the other Mg2+ independent. The former had a pH optimum of 7.5 and Km of 0.35 mM, whereas the corresponding values for the latter were pH 8.0 and Km 3.03 mM. Specific activity of the Mg(2+)-dependent enzyme showed a rostral-caudal gradient, ranging from 75 nmol/mg protein/hr in myelin from cerebral hemispheres to 21 nmol/mg protein/hr in myelin from spinal cord. Relative specific activity was approximately 20% that of brain stem or cerebral hemisphere homogenate. Treatment of myelin with taurocholate or high salt concentration did not significantly reduce activity of the Mg(2+)-dependent enzyme. The activity of that enzyme did not change with time or in the presence or absence of protease inhibitors; by contrast, that of Mg(2+)-independent enzyme decreased sharply in the absence of protease inhibitors but rose in their presence. To test for the effect of tumor necrosis factor-alpha (TNF alpha) on myelin sphingomyelinase, mouse brain myelin was labeled in vivo by intracerebral injection of [3H]acetate into 18-20-day-old mice. After 40 hr, brain stems were removed, minced, and treated with TNF alpha in Krebs-Ringer solution, after which myelin was immediately isolated. Separation and counting of individual lipids revealed TNF alpha treatment to cause increased labeling of myelin ceramide and cholesterol ester with concomitant decrease in myelin sphingomyelin. Western blotting of myelin proteins using antibodies to the two TNF alpha receptors as probes revealed the presence of the p75 receptor. Implications of these findings in relation to possible mechanisms of autoimmune demyelination are discussed.

Intraneuronal N‐acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin‐associated aspartoacylase

“…1981). A cocktail of protease inhibitors was present throughout isolation (Chakraborty et al . 1997).…”

“…To determine distribution, other subcellular fractions were subjected to aspartoacylase assay by the same procedure used for myelin. These were prepared as previously described (Chakraborty et al . 1997), utilizing cerebral hemispheres and brain stems.…”

“…The above neutral/zwitterionic lipids solubilized with ether/ethanol were resolved into individual components by two previously described thin layer chromatography (TLC) systems (Chakraborty et al . 1997) employing Merck silica gel 60‐coated plates (Fisher Scientific, Springfield, NJ, USA).…”

Mechanisms of damage to myelin and oligodendrocytes and their relevance to disease

“…Ceramide, a sphingolipid generated by neutral or acidic sphingomyelinase action on sphingomyelin, is a second messenger implicated in apoptotic cell death. In the absence of functional trkA (as in mature oligodendrocytes or cells where the ratio of p75–trk is greater than 1:1), NGF causes a sustained intracellular ceramide increase, elevation of c‐jun via c‐Jun amino‐terminal kinase (JNK) activity, and apoptotic cell death ([ 30]; see below). In addition to its induction by the low affinity NGFR, ceramide is also inducible in cells via TNFR1 and IL1βR; however, these receptor‐mediated mechanisms of increasing intracellular ceramide do not automatically lead to oligodendrocyte cell death.…”

Identification of Multiple Forms of Membrane‐Associated Neutral Sphingomyelinase in Bovine Brain

“…Among these isoforms, because transient and rapid activation of N‐SMase is observed in response to several extracellular stimuli, increasing attention has been focused on this N‐SMase in various cells. Many recent studies show that this ceramide‐dependent signaling pathway is attributable to several pathophysiologic processes in brain, such as Parkinson's disease (Hunot et al, 1997) and Alzheimer's disease (Fiebich et al, 1995), ischemic brain injury (Kubota et al, 1989), and autoimmune demyelination (Chakraborty et al., 1997). Neurotrophin (p75NT) and tumor necrosis factor‐α‐induced signaling pathways are known to utilize this ceramide as a signaling molecule for downstream effectors in many neuronal cells, including oligodendrocytes (Casaccia‐Bonnefil et al, 1996; Larocca et al, 1997), PC12 cells (Dobrowsky et al., 1995), and T9 glioma cells (MacPhee and Barker, 1997).…”