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White matter degeneration is a pathological hallmark of neurodegenerative diseases including Alzheimer's. Age remains the greatest risk factor for Alzheimer's and the prevalence of age-related late onset Alzheimer's is greatest in females. We investigated mechanisms underlying white matter degeneration in an animal model consistent with the sex at greatest Alzheimer's risk. Results of these analyses demonstrated decline in mitochondrial respiration, increased mitochondrial hydrogen peroxide production and cytosolic-phospholipase-A2 sphingomyelinase pathway activation during female brain aging. Electron microscopic and lipidomic analyses confirmed myelin degeneration. An increase in fatty acids and mitochondrial fatty acid metabolism machinery was coincident with a rise in brain ketone bodies and decline in plasma ketone bodies. This mechanistic pathway and its chronologically phased activation, links mitochondrial dysfunction early in aging with later age development of white matter degeneration. The catabolism of myelin lipids to generate ketone bodies can be viewed as a systems level adaptive response to address brain fuel and energy demand. Elucidation of the initiating factors and the mechanistic pathway leading to white matter catabolism in the aging female brain provides potential therapeutic targets to prevent and treat demyelinating diseases such as Alzheimer's and multiple sclerosis. Targeting stages of disease and associated mechanisms will be critical., Highlights • Mitochondrial dysfunction activates mechanisms for catabolism of myelin lipids to generate ketone bodies for ATP production. • Mechanisms leading to ketone body driven energy production in brain coincide with stages of reproductive aging in females. • Sequential activation of myelin catabolism pathway during aging provides multiple therapeutic targets and windows of efficacy. The mechanisms underlying white matter degeneration, a hallmark of multiple neurodegenerative diseases including Alzheimer's, remain unclear. Herein we provide a mechanistic pathway, spanning multiple transitions of aging, that links mitochondrial dysfunction early in aging with later age white matter degeneration. Catabolism of myelin lipids to generate ketone bodies can be viewed as an adaptive survival response to address brain fuel and energy demand. Women are at greatest risk of late-onset-AD, thus, our analyses in female brain address mechanisms of AD pathology and therapeutic targets to prevent, delay and treat AD in the sex most affected with potential relevance to men.

Reticulocyte-type 15-lipoxygenase is known to dioxygenate phospholipids without preceding action of phospholipases A2 (PLA2). Therefore we studied the reaction of the secretory PLA2s (sPLA2) from pancreas and snake venom, and of the human cytosolic PLA2 (cPLA2) with 1-palmitoyl-2-arachidonoyl phosphatidylcholine (PAPC) and their 15-lipoxygenated species (PAPC-OOH and PAPC-OH) either alone or as equimolar mixtures. These PLA2s cleaved PAPC-O(O)H with higher (sPLA2) or similar rates (cPLA2) as compared with native PAPC. In mixtures, however, PAPC proved to be the preferred, albeit not exclusive substrate for all three PLA2s. Thus, partial 15-lipoxygenation of phospholipids may also trigger liberation of arachidonic acid.

To determine anti-allergic effects of Koboku, a Chinese herbal medicine, we investigated its inhibitory action on the production of cysteinyl leukotrienes (LT) and LTB 4 , which are important chemical mediators in the pathogenesis of allergic diseases. A231 87-stimulated synthesis of cysteinyl LT and LTB 4 was measured by HPLC in the absence or presence of various concentrations of Koboku in rat basophilic leukemia-1 (RBL-1) cells. In a dose-dependent manner, Koboku inhibited synthesis of cysteinyl LT and LTB 4 by up to 92.3 and 100%, respectively. Immunoglobulin E-mediated release of cysteinyl LT and LTB 4 , which was measured by specific radioimmunoassay (RIA) was also inhibited by Koboku in RBL-2H3 cells. Sites of inhibition of Koboku in the metabolic pathway of LT synthesis were phospholipase A 2 (PLA 2 ) and 5-lipoxygenase (5-LO), but not LTC 4 synthase or LTA 4 hydrolase. We conclude that the anti-allergic effect of Koboku may be attributable, at least in part, to the inhibition of LT synthesis.

Prostaglandin E2 (PGE2) is an arachidonic acid (AA)-derived signaling molecule that can influence host immune responses to infection or vaccination. In this study, we investigated PGE2 production in vitro by cells infected with the poxvirus vaccine strain, modified vaccinia Ankara virus (MVA). Human THP-1 cells, murine bone marrow-derived dendritic cells, and murine C3HA fibroblasts all accumulated PGE2 to high levels in culture supernatants upon infection with MVA. We also demonstrated that MVA induced the release of AA from infected cells, and this was, most unusually, independent of host cytosolic phospholipase A2 activity. The accumulation of AA and PGE2 was dependent on viral gene expression, but independent of canonical NF-κB signaling via p65/RelA. The production of PGE2 required host cyclooxygenase-2 (COX-2) activity, and COX-2 protein accumulated during MVA infection. The results of this study provide insight into a novel aspect of MVA biology that may affect the efficacy of MVA-based vaccines.

A major part of the proinflammatory activity of tumor necrosis factor (TNF) is brought about by cytosolic phospholipase A(2) (cPLA(2)) that generates arachidonic acid, the precursor for the production of leukotrienes and prostaglandins. The activation of cPLA(2) and induction of proinflammatory lipid mediators is in striking contrast to the teleologic meaning of apoptosis, which is to avoid an inflammatory reaction. In this review we highlight the evidence for a caspase-mediated cleavage and inactivation of cPLA(2), which seems to be an important mechanism by which TNF downregulates cPLA(2) activity in cells undergoing apoptosis.

Annexin-I (ANX-I) is a 37-kDa protein with a calcium-dependent phospholipid-binding property. Previously we have observed the inhibition of cytosolic phospholipase A2 (cPLA2) by ANX-I in the studies using purified recombinant ANX-I, and proposed a specific interaction model for the mechanism of cPLA2 inhibition by ANX-I [Kim et al. (1994) FEBS Lett. 343, 251–255]. Here we have studied the role of ANX-I in the cPLA2 signaling pathway by transient transfection assay. The stimulation of Rat2 fibroblast cells with phorbol 12-myristate 13-acetate (PMA) induced the c-fos serum response element (SRE). The SRE stimulation by PMA was dramatically reduced by (1) pretreatment with a cPLA2-specific inhibitor, arachidonyltrifluoromethyl ketone, or (2) co-transfection with antisense cPLA2 oligonucleotide, indicating that the SRE activation was through cPLA2 activation. Co-transfection with an ANX-I expression vector also reduced the SRE stimulation by PMA, suggesting the inhibition of cPLA2 by ANX-I. The active domain of ANX-I was mapped using various deletion mutants. ANX-I(1–113) and ANX-I(34–346) were fully active, whereas ANX-I(114–346) abolished the activity. Therefore the activity was in the amino acid 34 to 113 region, which corresponds to the conserved domain I of ANX-I.

A series of fatty alkyl trifluoromethyl ketones and methyl fluorophosphonates have been prepared and tested as inhibitors and inactivators of human groups IV and VI phospholipases A(2) (cPLA(2) and iPLA(2)). Compounds were analyzed with phospholipid vesicle-, detergent-phospholipid mixed-micelle-, and natural membrane-based assays, and, with few exceptions, the relative inhibitor potencies measured with the three assays were similar. Ph(CH(2))(4)COCF(3) and Ph(CH(2))(4)PO(OMe)F emerged as a potent inhibitor and inactivator, respectively, of iPLA(2), and both are poorly effective against cPLA(2). Of all 13 fatty alkyl trifluoromethyl ketones tested, the trifluoromethyl ketone analog of arachidonic acid is the most potent cPLA(2) inhibitor, and structurally similar compounds including the trifluoromethyl ketone analog of docosahexenoic acid are much poorer cPLA(2) inhibitors. Inactivation of cPLA(2) by fatty alkyl fluoromethylphosphonates is greatly promoted by binding of enzyme to the interface. The use of both vesicles and mixed micelles to assay phospholipase A(2) inhibitors and inactivators present at low mol fraction in the interface provides reliable rank order potencies of a series of compounds that correlate with their behavior in a natural membrane assay.

Background Phospholipase A2 (PLA2)-derived proinflammatory lipid mediators such as prostaglandin E2 (PGE2), leukotrienes B4 (LTB4), lysophosphatidylcholine (LPC), and free fatty acids (FFA) are implicated in spinal cord injury (SCI) pathologies. Reducing the levels of these injurious bioactive lipid mediators is reported to ameliorate SCI. However, the specific role of the group IVA isoform of PLA2 cytosolic PLA2 (cPLA2) in lumbar spinal canal stenosis (LSS) due to cauda equina compression (CEC) injury is not clear. In this study, we investigated the role of cPLA2 in a rat model of CEC using a non-toxic cPLA2-preferential inhibitor, arachidonyl trifluoromethyl ketone (ATK). Methods LSS was induced in adult female rats by CEC procedure using silicone blocks within the epidural spaces of L4 to L6 vertebrae. cPLA2 inhibitor ATK (7.5 mg/kg) was administered by oral gavage at 2 h following the CEC. cPLA2-derived injurious lipid mediators and the expression/activity of cPLA2, 5-lipoxygenase (5-LOX), and cyclooxygenase-2 (COX-2) were assessed. ATK-treated (CEC + ATK) were compared with vehicle-treated (CEC + VEH) animals in terms of myelin levels, pain threshold, and motor function. Results ATK treatment of CEC animals reduced the phosphorylation of cPLA2 (pcPLA2) determined by Western blot, improved locomotor function evaluated by rotarod task, and reduced pain threshold evaluated by mechanical hyperalgesia method. Levels of FFA and LPC, along with PGE2 and LTB4, were reduced in CEC + ATK compared with CEC + VEH group. However, ATK treatment reduced neither the activity/expression of 5-LOX nor the expression of COX-2 in CEC + VEH animals. Increased cPLA2 activity in the spinal cord from CEC + VEH animals correlated well with decreased spinal cord as well as cauda equina fiber myelin levels, which were restored after ATK treatment. Conclusion The data indicate that cPLA2 activity plays a significant role in tissue injury and pain after LSS. Reducing the levels of proinflammatory and tissue damaging eicosanoids and the deleterious lipid mediator LPC shows therapeutic potential. ATK inhibits cPLA2 activity, thereby decreasing the levels of injurious lipid mediators, reducing pain, improving functional deficits, and conferring protection against LSS injury. Thus, it shows potential for preclinical evaluation in LSS.