Brian M. Ross

Differential alteration of phospholipase A2 activities in brain of patients with schizophrenia

We recently reported that the activity of a calcium-independent subtype of phospholipase A2 is increased in blood of patients with schizophrenia. The present investigation examined whether similar changes take place in brain of patients with this disorder, and for comparison, in patients with bipolar disorder. The activity of two classes of PLA2, calcium-stimulated and independent, were assayed in autopsied temporal, prefrontal and occipital cortices, putamen, hippocampus and thalamus of 10 patients with schizophrenia, 8 patients with bipolar disorder and 12 matched control subjects. Calcium-independent PLA2 activity was increased by 45% in the temporal cortex of patients with schizophrenia as compared with the controls but was not significantly altered in other brain areas. In contrast, calcium-stimulated PLA2 activity was decreased by 27-42% in the temporal and prefrontal cortices and putamen, with no significant alterations in other brain regions. Brain PLA2 activity was normal in patients with bipolar disorder. Calcium-stimulated PLA2 activity was normal in cortex, cerebellum and striatum of rats treated acutely or chronically with haloperidol, whereas calcium-independent PLA2 activity was decreased in striatum of chronically treated animals, indicating that altered PLA2 activity in patients with schizophrenia is unlikely to be a direct effect of medication. Studies of the cellular role played by PLA2 suggest that decreased calcium-stimulated PLA2 activity, as also occurs in striatum of chronic human cocaine users, may be due, in part, to increased dopaminergic activity in the disorder, whereas increased calcium-independent PLA2 activity may be related to abnormal fatty acid metabolism and oxidative stress in schizophrenia.

Phospholipid-metabolizing enzymes in Alzheimer's disease: increased lysophospholipid acyltransferase activity and decreased phospholipase A2 activity.

Abstract: Damage to brain membrane phospholipids may play an important role in the pathogenesis of Alzheimers disease (AD); however, the critical metabolic processes responsible for the generation and repair of membrane phospholipids affected by the disease are unknown. We measured the activity of key phospholipid catabolic and anabolic enzymes in morphologically affected and spared areas of autopsied brain of patients with AD and in matched control subjects. The activity of the major catabolic enzyme phospholipase A2 (PLA2), measured in both the presence and absence of Ca2+, was significantly decreased (−35 to −53%) in parietal and temporal cortices of patients with AD. In contrast, the activities of lysophospholipid acyltransferase, which recycles lysophospholipids into intact phospholipids, and glycerophosphocholine phosphodiesterase, which returns phospholipid catabolites to be used in phospholipid resynthesis, were increased by ∼50–70% in the same brain areas. Brain activities of enzymes involved in de novo phospholipid synthesis (ethanolamine kinase, choline kinase, choline phosphotransferase, phosphoethanolamine cytidylyltransferase, and phosphocholine cytidylyltransferase) were either normal or only slightly altered. The activities of PLA2 and acyltransferase were normal in the degenerating cerebellum of patients with spinocerebellar atrophy type 1, whereas the activity of glycerophosphocholine phosphodiesterase was reduced, suggesting that the alterations in AD brain were not nonspecific consequences of neurodegeneration. Our data suggest that compensatory phospholipid metabolic changes are present in AD brain that reduce the rate of phospholipid loss via both decreased catabolism (PLA2) and increased phospholipid resynthesis (acyltransferase and glycerophosphocholine phosphodiesterase).

Phospholipid biosynthetic enzymes in human brain

Growing evidence suggests an involvement of brain membrane phospholipid metabolism in a variety of neurodegenerative and psychiatric conditions. This has prompted the use of drugs (e.g., CDPcholine) aimed at elevating the rate of neural membrane synthesis. However, no information is available regarding the human brain enzymes of phospholipid synthesis which these drugs affect. Thus, the objective of our study was to characterize the enzymes involved, in particular, whether differences existed in the relative affinity of substrates for the enzymes of phosphatidylethanolamine (PE) compared to those of phosphatidylcholine (PC) synthesis. The concentration of choline in rapidly frozen human brain biopsies ranged from 32–186 nmol/g tissue, a concentration similar to that determined previously for ethanolamine. Since human brain ethanolamine kinase possessed a much lower affinity for ethanolamine (Km=460 μM) than choline kinase did for choline (Km=17 μM), the activity of ethanolamine kinase in vivo may be more dependent on substrate availability than that of choline kinase. In addition, whereas ethanolamine kinase was inhibited by choline, and to a lesser extent by phosphocholine, choline kinase activity was unaffected by the presence of ethanolamine, or phosphoethanolamine, and only weakly inhibited by phosphocholine. Phosphoethanolamine cytidylyl-transferase (PECT) and phosphocholine cytidylyltransferase (PCCT) also displayed dissimilar characteristics, with PECT and PCCT being located predominantly in the cytosolic and particulate fractions, respectively. Both PECT and PCCT exhibited a low affinity for CTP (Km approximately 1.2 mM), suggesting that the activities of these enzymes, and by implication, the rate of phospholipid synthesis, are highly dependent upon the cellular concentration of CTP. In conclusion, our data indicate different regulatory properties of PE and PC synthesis in human brain, and suggest that the rate of PE synthesis may be more dependent upon substrate (ethanolamine) availability than that of PC synthesis.

Characterization of a Novel Phospholipase A2 Activity in Human Brain

Abstract: Phospholipases A2 (PLA2) are a family of enzymes that catalyze the removal of fatty acid residues from phosphoglycerides. The enzyme is postulated to be involved in several human brain disorders, although little is known regarding the status of PLA2 activity in human CNS. We therefore have characterized some aspects of the PLA2 activity present in the temporal cortex of human brain. More PLA2 activity was found in the membrane (particulate) fraction than in the cytosolic fraction. The enzyme could be solubilized from particulate material using 1 M potassium chloride, and was capable of hydrolyzing choline phosphoglyceride (CPG) and ethanolamine phosphoglyceride (EPG), with a preference (approximately eightfold) for EPG over CPG. When the solubilized particulate enzyme was subjected to gel filtration chromatography, PLA2 activity eluted in a high molecular mass fraction (∼180 kDa). PLA2 activity was weakly stimulated by dithiothreitol, strongly stimulated by millimolar concentrations of calcium ions, and inhibited by brief heat treatment at 57°C, bromophenacyl bromide, the arachidonic acid derivative AACOCF3, γ‐linolenoyl amide, and N‐methyl γ‐linolenoyl amide. Thus, whereas the human brain enzyme(s) characterized in our study displays some of the characteristics of previously characterized PLA2s, it differs in several key features.

Characterization of Lysophospholipid Metabolizing Enzymes in Human Brain

Abstract: Lysophospholipids are generated during the turnover and breakdown of membrane phospholipids. We have identified and partially characterized three enzymes involved in the metabolism of lysophospholipids in human brain, namely, lysophospholipase, lysophospholipid:acyl‐CoA acyltransferase (acyltransferase), and lysophospholipid:lysophospholipid transacylase (transacylase). Each enzyme displayed comparable levels of activity in biopsied and autopsied human brain, although in all cases the activity was somewhat lower in human than that in rat brain. All three enzymes were localized predominantly in the particulate fraction, with lysophospholipase possessing the greatest activity followed by acyltransferase and transacylase. Lysophosphatidylcholine possessed a Km in the micromolar range for lysophospholipase and transacylase, and in the millimolar range for acyltransferase, whereas arachidonyl‐CoA displayed a Km in the micromolar range for acyltransferase. The three enzymes differed in their pH optima, with lysophospholipase being most active at pH 8.0, transacylase at pH 7.5, and acyltransferase at pH 6.0. Both bromophenacyl bromide and N‐ethylmaleimide inhibited lysophospholipase activity and, to a lesser extent, that of acyltransferase and transacylase. None of the enzyme activities were affected by the presence of dithiothreitol or EDTA, although particulate lysophospholipase was activated approximately two‐fold by the addition of 5 mM MgCl2 or CaCl2 but not KCl. Transacylating activity was stimulated by CoA, the EC50 of activation being 6.8 µM. Acyltransferase displayed an approximately threefold preference for arachidonyl‐CoA over palmitoyl‐CoA, whereas the acylation rate of different lysophospholipids was in the order lysophosphatidylinositol > 1‐palmitoyl lysophosphatidylcholine > 1‐oleoyl lysophosphatidylcholine ≫ lysophosphatidylserine > lysophosphatidylethanolamine. This, and the preference of human brain phospholipase A2 for phosphatidylinositol, suggests that this phospholipid may possess a higher turnover rate than the other phospholipid classes examined. Human brain homogenates also possessed the ability to transfer fatty acid from lysophosphatidylcholine to lysophosphatidylethanolamine. In addition, we also present evidence that diacylglycerophospholipids can act as acyl donors for the transacylation of lysophospholipids. We have therefore demonstrated the presence of, and partially characterized, three enzymes that are involved in the metabolism of lysophospholipids in human brain. Our results suggest that lysophospholipase may be the major route by which lysophospholipids are removed from the cell membrane in human brain. However, all three enzymes likely play an important role in the remodeling of membrane composition and thereby contribute to the overall functioning of membrane‐associated processes.

An animal model of nicotinic-acid-induced vasodilation: effect of haloperidol, caffeine and nicotine upon nicotinic acid response

BACKGROUND The normal vasodilatory response to ingestion of nicotinic acid (NA) is impaired in some patients with schizophrenia. It is unclear whether the impairment is a feature of the disorder itself or to a confounding factor such as neuroleptics, caffeine or nicotine use. METHODS To address this question in a controlled manner, we have developed an animal (rat) model of NA-induced vasodilation, in which response is monitored by measuring change in skin temperature. RESULTS We observed that (i) acute administration of acetylsalicylic acid (100mg/kg), caffeine (2.5mg/kg) and haloperidol (0.1 or 0.5mg/kg) and (ii) chronic administration of haloperidol (0.2mg/kg/day) significantly inhibited NA (30 mg/kg) response, whereas neither acute (0.25mg/kg) or chronic (0.5mg/kg/day for 14 days) administration of nicotine, or chronic administration of caffeine (5mg/kg/day for 14 days) had any significant effect upon NA response. CONCLUSIONS Our data suggest that at least one drug commonly used to treat schizophrenia (haloperidol) can interfere with the vasodilatory response to NA. Studies using non-medicated patients with schizophrenia are required to determine whether reduced vasodilatory response to NA in schizophrenia is a feature of the disorder or a consequence of treatment.

Low activity of key phospholipid catabolic and anabolic enzymes in human substantia nigra : Possible implications for Parkinson's disease

To determine whether increased oxidative stress in substantia nigra of patients with idiopathic Parkinsons disease might be related to decreased ability of nigral cells to detoxify oxidized membrane phospholipids, we compared levels of the major phospholipid metabolizing enzymes in autopsied substantia nigra with those in non-nigral (n = 11) brain areas of the normal human brain. Whereas most enzymes possessed a relatively homogeneous distribution, the activity of the major phospholipid catabolizing enzyme phospholipase A2, assayed in the presence of calcium ions, varied amongst different regions, with substantia nigra possessing the lowest activity. Similarly, calcium-independent phospholipase A2 activity, although possessing a relatively homogeneous regional distribution, was also low in the substantia nigra. This, coupled with low activity of phosphoethanolamine- and phosphocholine-cytidylyltransferases, major regulatory enzymes of phospholipid synthesis, in this brain region, suggest that the rate of phospholipid turnover is low in the substantia nigra. Low activity of key phospholipid catabolic and anabolic enzymes in human substantia nigra might result in reduced ability to repair oxidative membrane damage, as may occur in Parkinsons disease.

Elevated activity of phospholipid biosynthetic enzymes in substantia nigra of patients with Parkinson's disease

We reported that the activities of phospholipase A2, phosphocholine cytidylyltransferase and phosphoethanolamine cytidylyltransferase, key phospholipid metabolic enzymes, are low in substantia nigra of normal human brain and that this might reduce the ability of nigral neurons to repair damage to cell membranes. To determine whether adaptive changes in nigral phospholipid metabolism can occur in idiopathic Parkinsons disease we compared activities of 11 catabolic and anabolic enzymes in autopsied brain of 10 patients with Parkinsons disease to those in control subjects. Nigral activity of the catabolic enzyme phospholipase A2 was normal in the Parkinsons disease group, whereas that of the biosynthetic enzymes phosphoethanolamine cytidylyltransferase, phosphocholine cytidylyltransferase, and phosphatidylserine synthase were elevated 193, 48 and 38%, respectively, possibly representing a compensatory response to repair membrane phospholipids. Enzyme activities were normal in all other brain areas with the exception of increased (+26%) activity of calcium-stimulated phospholipase A2 in putamen, a change which could be consequent to either decreased dopaminergic striatal input or to a dopamine nerve terminal degenerative process. Our data indicate that the normally low rate of membrane phospholipid synthesis in the substantia nigra, the primary area of neurodegeneration in Parkinsons disease, is increased during the course of the disorder. We suggest that pharmacotherapies which augment this compensatory response might have utility as a treatment for Parkinsons disease.

The human nucleus accumbens is highly susceptible to G protein down-regulation by methamphetamine and heroin.

Abstract: Although the nucleus accumbens is assumed to be a critical brain “pleasure center,” its function in humans is unknown. As animal data suggest that a unique feature of this small brain area is its high sensitivity to down‐regulation of an inhibitory G protein by drugs of abuse, we compared G protein levels in postmortem nucleus accumbens with those in seven other brain regions of chronic users of cocaine, methamphetamine, and heroin, and of matched controls. Biochemical changes were restricted to the nucleus accumbens in which concentrations of Gαi1 and/or Gαi2 were reduced by 32‐49% in the methamphetamine and heroin users. This selective responsiveness to these abused drugs implies a special role for the human nucleus accumbens in mechanisms of drug reinforcement and suggests that some features of the drug‐dependent state (e.g., tolerance) might be related to inhibition of Gαi‐linked receptor activity.

Alterations in the activity of adenylate cyclase and high affinity GTPase in Alzheimer's disease

The aim of this study was to assess the effect of Alzheimers disease has on the functional integrity of several signal transduction proteins. The relative levels of the G-protein alpha subunits Gs alpha-L, Gs alpha-S, Gi alpha-2 and G(o) alpha were measured by western blotting and found to be unchanged in membranes prepared from Alzheimer-diseased frontal cortex or hippocampus compared to control brains. However the activity of the G-protein associated enzyme, high affinity GTPase, was found to be reduced in the frontal cortex (reduced by 25%) and by a similar magnitude in the hippocampus (reduced by 27%) of Alzheimer subjects. The same membrane preparations were also assayed for the activity of adenylate cyclase. Basal enzyme activity was not significantly altered in Alzheimer diseased hippocampus, but was markedly reduced (by 45%) in the frontal cortex. The ability of fluoride and aluminium ions to stimulate adenylate cyclase was not significantly changed in either brain region. This suggests that G-proteins, especially Gs, are still able to interact with this enzyme. These results indicate that although the presence of Alzheimers disease does not significantly alter G-protein levels, changes have taken place in the overall activity of these proteins. However this alteration does not affect their ability to stimulate adenylate cyclase activity.