Thad A. Rosenberger

Phospholipase A2 and Its Role in Brain Tissue

Abstract: Phospholipase A2 (PLA2) is the name for the class of lipolytic enzymes that hydrolyze the acyl group from the sn‐2 position of glycerophospholipids, generating free fatty acids and lysophospholipids. The products of the PLA2‐catalyzed reaction can potentially act as second messengers themselves, or be further metabolized to eicosanoids, platelet‐activating factor, and lysophosphatidic acid. All of these are recognized as bioactive lipids that can potentially alter many ongoing cellular processes. The presence of PLA2 in the central nervous system, accompanied by the relatively large quantity of potential substrate, poses an interesting dilemma as to the role PLA2 has during both physiologic and pathologic states. Several different PLA2 enzymes exist in brain, some of which have been partially characterized. They are classified into two subtypes, CA2+‐dependent and Ca2+‐independent, based on their catalytic dependence on Ca2+. Under physiologic conditions, PLA2 may be involved in phospholipid turnover, membrane remodeling, exocytosis, detoxification of phospholipid peroxides, and neurotransmitter release. However, under pathological situations, increased PLA2 activity may result in the loss of essential membrane glycerophospholipids, resulting in altered membrane permeability, ion homeostasis, increased free fatty acid release, and the accumulation of lipid peroxides. These processes, along with loss of ATP, may be responsible for the loss of membrane phospholipid and subsequent neuronal injury found in ischemia, spinal cord injury, and other neurodegenerative diseases. This review outlines the current knowledge of the PLA2 found in the central nervous system and attempts to define the role of PLA2 during both physiologic and pathologic conditions.

Chronic valproate treatment decreases the in vivo turnover of arachidonic acid in brain phospholipids: a possible common effect of mood stabilizers

Both (Li+) and valproic acid (VPA) are effective in treating bipolar disorder, but the pathway by which either works, and whether it is common to both drugs, is not agreed upon. We recently reported, using an in vivo fatty acid model, that Li+ reduces the turnover rate of the second messenger arachidonic acid (AA) by 80% in brain phospholipids of the awake rat, without changing turnover rates of docosahexaenoic or palmitic acid. Reduced AA turnover was accompanied by down‐regulation of gene expression and protein levels of an AA‐specific cytosolic phospholipase A2 (cPLA2). To see if VPA had the same effect on AA turnover, we used our in vivo fatty acid model in rats chronically administered VPA (200 mg/kg, i.p. for 30 days). Like Li+, VPA treatment significantly decreased AA turnover within brain phospholipids (by 28–33%), although it had no effect on cPLA2 protein levels. Thus, both mood stabilizers, Li+ and VPA have a common action in reducing AA turnover in brain phospholipids, albeit by different mechanisms.

Chronic lithium downregulates cyclooxygenase-2 activity and prostaglandin E(2) concentration in rat brain.

Rats treated with lithium chloride for 6 weeks have been reported to demonstrate reduced turnover of arachidonic acid (AA) in brain phospholipids, and decreases in mRNA and protein levels, and enzyme activity, of AA-selective cytosolic phospholipase A2(cPLA2). We now report that chronic lithium administration to rats significantly reduced the brain protein level and enzyme activity of cyclooxygenase-2 (COX-2), without affecting COX-2 mRNA. Lithium also reduced the brain concentration of prostaglandin E2 (PGE2), a bioactive product of AA formed via the COX reaction. COX-1 and the Ca2+-independent iPLA2 (type VI) were unaffected by lithium. These and prior results indicate that lithium targets a part of the AA cascade that involves cPLA2 and COX-2. This effect may contribute to lithiums therapeutic action in bipolar disorder.

85 kDa cytosolic phospholipase A2 is a target for chronic lithium in rat brain.

The mechanism by which chronic lithium exerts its therapeutic effect in brains of bipolar patients is not known. One possibility, suggested by our demonstration in the rat brain, is that chronic lithium inhibits turnover of arachidonic acid (AA) by reducing the activity of an AA-specific phospholipase A2 (PLA2). To test this further, mRNA levels of two AA-specific PLA2s, cytosolic PLA2 (cPLA2) type IV and intracellular PLA2 (iPLA2) type VIII, and protein level of cPLA2 were quantified in the brain of rats given lithium for 6 weeks. Chronic lithium markedly reduced brain mRNA and protein level of cPLA2, but had no effect on mRNA level of iPLA2. These results suggest that the final common path effect of chronic lithium administration is to reduce turnover of AA in brain by down-regulating cPLA2.

Rat brain arachidonic acid metabolism is increased by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide

Palmitate (16:0) 80.2 ± 10.9 81.9 ± 10.9 26.2 ± 0.3 27.0 ± 1.1 11.2 ± 2.6 13.4 ± 3.9 Stearate (18:0) 26.8 ± 4.3 24.6 ± 2.8 48.0 ± 8.0 59.0 ± 9.2 1.9 ± 1.5 3.2 ± 1.8 Oleate (18:1n-9) 35.3 ± 10.0 37.5 ± 8.4 36.6 ± 4.9 38.8 ± 1.4 10.6 ± 3.1 11.7 ± 3.3 Linoleate (18:2n-6) 38.5 ± 10.6 39.8 ± 7.5 1.8 ± 0.2 *3.7 ± 0.3 4.0 ± 1.2 5.5 ± 3.9 Arachidonate (20:4n-6) 8.9 ± 2.8 6.2 ± 1.8 8.2 ± 0.1 *19.7 ± 3.6 0.7 ± 0.3 0.5 ± 0.4 Docosahexaenoate (22:6n-3) 8.7 ± 3.2 8.1 ± 2.9 1.2 ± 0.8 1.7 ± 0.2 0.6 ± 0.3 0.8 ± 0.3

Valproic acid down-regulates the conversion of arachidonic acid to eicosanoids via cyclooxygenase-1 and -2 in rat brain

Sodium valproate, a mood stabilizer, when chronically administered to rats (200 mg/kg i.p. daily for 30 days) significantly reduced the brain protein levels of cyclooxygenase (COX)‐1 and COX‐2, without altering the mRNA levels of these enzymes. COX activity was decreased, as were the brain concentrations of 11‐dehydrothromboxane B2 and prostaglandin E2 (PGE2), metabolites of arachidonic acid (AA) produced via COX. In contrast, the brain protein level of 5‐lipoxygenase and the concentration of its AA metabolite leukotriene B4 were unchanged. In view of published evidence that lithium chloride administered chronically to rats, like chronic valproate, reduces AA turnover within brain phospholipids, and that lithium post‐transcriptionally down‐regulates COX‐2 but not COX‐1 protein level and enzyme activity, these observations suggest that mood stabilizers generally modulate the release and recycling of AA within brain phospholipids, and the conversion of AA via COX‐2 to PGE2 and related eicosanoids. If targeting this part of the ‘AA cascade’ accounts for their therapeutic action, non‐steroidal anti‐inflammatory drugs or selective COX‐2 inhibitors might prove effective in bipolar disorder.

Rat brain arachidonic acid metabolism is increased by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide: Neuroinflammation alters brain arachidonic acid metabolism

In a rat model of acute neuroinflammation, produced by a 6‐day intracerebral ventricular infusion of bacterial lipopolysaccharide (LPS), we measured brain activities and protein levels of three phospholipases A2 (PLA2) and of cyclo‐oxygenase‐1 and ‐2, and quantified other aspects of brain phospholipid and fatty acid metabolism. The 6‐day intracerebral ventricular infusion increased lectin‐reactive microglia in the cerebral ventricles, pia mater, and the glial membrane of the cortex and resulted in morphological changes of glial fibrillary acidic protein (GFAP)‐positive astrocytes in the cortical mantel and areas surrounding the cerebral ventricles. LPS infusion increased brain cytosolic and secretory PLA2 activities by 71% and 47%, respectively, as well as the brain concentrations of non‐esterified linoleic and arachidonic acids, and of prostaglandins E2 and D2. LPS infusion also increased rates of incorporation and turnover of arachidonic acid in phosphatidylethanolamine, plasmenylethanolamine, phosphatidylcholine, and plasmenylcholine by 1.5‐ to 2.8‐fold, without changing these rates in phosphatidylserine or phosphatidylinositol. These observations suggest that selective alterations in brain arachidonic acid metabolism involving cytosolic and secretory PLA2 contribute to early pathology in neuroinflammation.

Chronic nutritional deprivation of n-3 α-linolenic acid does not affect n-6 arachidonic acid recycling within brain phospholipids of awake rats

Using an in vivo fatty acid model and operational equations, we reported that esterified and unesterified concentrations of docosahexaenoic acid (DHA, 22 : 6 n‐3) were markedly reduced in brains of third‐generation (F3) rats nutritionally deprived of α‐linolenic acid (18 : 3 n‐3), and that DHA turnover within phospholipids was reduced as well. The concentration of docosapentaenoic acid (DPA, 22 : 5 n‐6), an arachidonic acid (AA, 20 : 4 n‐6) elongation/desaturation product, was barely detectable in control rats but was elevated in the deprived rats. In the present study, we used the same in vivo model, involving the intravenous infusion of radiolabeled AA to demonstrate that concentrations of unesterified and esterified AA, and turnover of AA within phospholipids, were not altered in brains of awake F3‐generation n‐3‐deficient rats, compared with control concentrations. Brain DPA‐CoA could be measured in the deprived but not control rats, and AA‐CoA was elevated in the deprived animals. These results indicated that AA and DHA are recycled within brain phospholipids independently of each other, suggesting that recycling is regulated independently by AA‐ and DHA‐selective enzymes, respectively. Competition among n‐3 and n‐6 fatty acids within brain probably does not occur at the level of recycling, but at levels of elongation and desaturation (hence greater production of DPA during n‐3 deprivation), or conversion to bioactive eicosanoids and other metabolites.

α‐Synuclein gene ablation increases docosahexaenoic acid incorporation and turnover in brain phospholipids

Previously, we demonstrated that ablation of α‐synuclein (Snca) reduces arachidonate (20:4n‐6) turnover in brain phospholipids through modulation of an endoplasmic reticulum‐localized acyl‐CoA synthetase (Acsl). The effect of Snca ablation on docosahexaenoic acid (22:6n‐3) metabolism is unknown. In the present study, we examined the effect of Snca gene ablation on brain 22:6n‐3 metabolism. We determined 22:6n‐3 uptake and incorporation into brain phospholipids by infusing awake, wild‐type and Snca−/− mice with [1‐14C]22:6n‐3 using steady‐state kinetic modeling. In addition, because Snca modulates 20:4n‐6‐CoA formation, we assessed microsomal Acsl activity using 22:6n‐3 as a substrate. Although Snca gene ablation does not affect brain 22:6n‐3 uptake, brain 22:6n‐3‐CoA mass was elevated 1.5‐fold in the absence of Snca. This is consistent with the 1.6‐ to 2.2‐fold increase in the incorporation rate and turnover in ethanolamine glycerophospholipid, phosphatidylserine, and phosphatidylinositol pools. Increased 22:6n‐3‐CoA mass was not the result of altered Acsl activity, which was unaffected by the absence of Snca. While Snca bound 22:6n‐3, Kd = 1.0 ± 0.5 μmol/L, it did not bind 22:6n‐3‐CoA. These effects of Snca gene deletion on 22:6n‐3 brain metabolism are opposite to what we reported previously for brain 20:4n‐6 metabolism and are likely compensatory for the decreased 20:4n‐6 metabolism in brains of Snca−/− mice.

Energy consumption by phospholipid metabolism in mammalian brain.

Until recently, brain phospholipid metabolism was thought to consume only 2% of the ATP consumed by the mammalian brain as a whole. In this paper, however, we calculate that 1.4% of total brain ATP consumption is consumed for the de novo synthesis of ether phospholipids and that another 5% is allocated to the phosphatidylinositide cycle. When added to previous estimates that fatty acid recycling within brain phospholipids and maintenance of membrane lipid asymmetries of acidic phospholipids consume, respectively, 5% and 8% of net brain ATP consumption, it appears that phospholipid metabolism can consume up to 20% of net brain ATP consumption. This new estimate is consistent with recent evidence that phospholipids actively participate in brain signaling and membrane remodeling, among other processes.