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Decreased phospholipase A2 activity in Alzheimer brains
Decreased Phospholipase A2 Activity in Alzheimer Brains Wagner F. Gattaz, Athanasios Maras, Nigel J. Cairns, Raymond Levy, and Hans F6rstl
Phospholipase Az (PLA2) is a key-enzyme in the metabolism of membrane phospholipids. In cholinergic neurons PLA2 controls the physico-chemical properties of neuronal membranes as well as the breakdown of phosphatidylcholine to produce choline for acetylcholine synthesis. Moreover PLA2 influences the processing and secretion of the amyloid precursor protein, which gives rise to the f3-amyloid peptide, the major component of the amyloid plaque in Alzheimer' s disease (AD). In the present study PLA2 activity was investigated in post-mortem brains from 23 patients with AD and 20 nondemented elderly controls. In AD brains PLA2 activity was significantly decreased in the parietal and to a lesser degree in the frontal cortex. Lower PLA 2 activity correlated significantly with an earlier onset of the disease, higher counts of neurofibrillary tangles and senile plaques and an earlier age at death, indicating a relationship between abnormally low PLA 2activity and a more severe form of the illness. The present results provide new evidence for a disordered phospholipid metabolism in AD brains and suggest that reduced PLAe activity may contribute to the cholinergic deficit and to the production of amyloidogenic peptides in the disease.
Key Words: Alzheimer's disease, phospholipase A2, brain phospholipids, choline, acetylcholine, amyloid precursor protein, 13-amyloid.
Introduction Alzheimer disease (AD) is the most common cause of dementia in the elderly. The clinical diagnosis of probable AD can only be confirmed at autopsy or biopsy by the neuropathological demonstration of neurofibriUary tangles (NFTs) and senile plaques (SPs) in the neocortex and hippocampus. Although the causes of AD are unknown, there is a large consensus that a preferential degeneration of choliner-
From the Central Institute of Mental Health Mannheim, Germany (WFG, AM, HF); and the MRC Alzheimer's Disease Brain Bank, Institute of Psychiatry, London, LIK (NJC, RL). Address reprint request to W.F. Gattaz, MD, Director, Neurobiology Unit, Central Institute of Mental Health, P.O. Box 12 21 20 - 68072 Mannheim - Germany. Received January 28,1994; revised April 27, 1994.
© 1995 Society of Biological Psychiatry
gic neurons is related to the memory loss and cognitive impairment in the disease (Blusztajn et a11986). A disordered phospholipid metabolism has frequently been found in the brain of AD patients. In autopsied A D brains, changes were reported in the concentrations of membrane phospholipids, their precursors, and catabolites (Bftrfiny et al 1985; Ellison et al 1987; Nitsch et al 1992). In vivo brain nuclear magnetic resonance (NMR) spectroscopy showed increased levels of phosphomonoesters, the precursors of membrane phospholipids, in some but not all cortical regions in AD patients (Brown et al 1989). Intracellular phospholipase A2 (PLA2) is a key-enzyme in the metabolism of membrane phospholipids, cleaving the sn-2-fatty acid ester to produce lysophospholipids and free fatty acids. Changes induced by PLA: on the phospholipidbilayer modify the physico-chemical properties of synaptic 0006-3223/95/$09.50 SSDI 0006-3223(94)00123-K
14
BIOLPSYCHIATRY
W.F. Gattaz et al
1995;37:13-17
membranes, such as signal transduction and membrane fluidity (Hirata and Axelrod 1980; Leprohon et al 1983). Moreover, the breakdown of membrane phospholipids by PLA2 is one of the pathways to produce choline as the precursor for acetylcholine synthesis (Blusztajn and Wurtman 1983). In a recent study Emmerling et al (1993) reported that PLA2 influences the processing and secretion of the amyloid precursor protein (APP); the latter gives rise to the 13-amyloid peptide ([3A), the major component of the amyloid plaque in AD. In the face of this finding and of the importance of PLA2 for both membrane phospholipid metabolism and cholinergic function, we explored the activity of this enzyme in brain tissue from AD patients and neurologically healthy individuals.
Methods Autopsied brain samples of parietal and frontal cortex (Brodmann areas 7 and 32, respectively) were obtained from the Medical Research Council Alzheimer's Disease Brain Bank, Institute of Psychiatry, London. Samples from 23 cases of neuropathologically confirmed AD (7 men, 16 women; mean age - SD 81.0 ___7.5 years) and 20 nondemented controls (10 men, 10 women; 75.6 --- 9.8 years) were investigated. The degree of dementia was assessed in all but 2 AD patients within 12 months before death with the Mini Mental State Exam (Folstein et al 1975). None of the AD patients had received therapy for the disease. The causes of death were similar in AD patients and controls and were usually due to a terminal circulatory or respiratory failure. In AD patients the means _ SD for the age at disease onset was 72.9 --- 6.9 years, the duration ofiUness 9.5 --- 5.8 years and the Mini Mental State score 3.1 _ 4.9.
Tissue Collection At autopsy, the brain was removed and weighed. The brainstem and cerebellum were removed prior to dividing the cerebral hemispheres in the midline. Alternate hemispheres were either fixed for histology or frozen for biochemistry. The hemisphere to be frozen was sliced in the coronal plane in approximately 1 cm thick slices. The slices were rapidly frozen by contact with a brass plate at a temperature of -70°C and stored at this temperature in airtight plastic bags to prevent desiccation. Blocks from the neocortex were subsequently dissected on a bed of dry ice. Histologic and counting methods for senile plaques and neurofibriUary tangles were described elsewhere (Ft~rstl et al 1992). Briefly, SPs and N F r s were counted in Glees and Marsland silver impregnated sections in eight adjacent, nonoverlapping columns in each neocortical site. The mean values were calculated and expressed as counts per square millimeter. Histologic counts were obtained form 16 AD patients. The mean counts/mm 2 ___ SD [range] were: SPs parietal 5.0 - 6.3 [0-22], SPs frontal 4.1 _ 7.2 [0-27],
Table 1. Demographic and Neurochemical Data in Alzheimer's Patients and Nondemented Controls (means --- SD) Alzheimer Variables Age Sex P o s t m o r t e m time (hr) P L A 2 parietal P L A 2 frontal
Controls
(n = 23)
(n = 20)
81.0 -+ 7.5 a 7 m e n : 16 w o m e n 30.7 - 14.5 27.4 ± 20.2 b 26.9 ± 16.4 '~c
75.6 ± 9.8 10 m e n : 10 w o m e n 38.1 ± 11.1 43.4 ± 23.8 37.9 - 19.8
PLA2 activity is given in pMol/mg/45 rain arachidonic acid. "p < 0.10. ~p < 0.005 (as compared to controls). On= 22.
N F r s parietal 4.7 ___4.6 [0.4-18], NFTs frontal 5.9 --- 7.6 [0.1-24]. The demographic and biochemical data and the postmortem interval are given in Table 1. There was a trend for AD patients to have a higher age at death than the control group (p < 0.10). The difference in postmortem interval, with shorter mean time from death to storage in the AD group, did not reach statistical significance (p = 0.15).
Determination of PZA 2Activity Homogenized brain tissue from parietal and frontal cortex was suspended in 0.1 Mol/L Tris-HCI-Saccharose buffer (pH 7.4). As enzyme substrate we used L-A-1-palmitoyl-2arachidonyl-phosphatidylcholine labeled in position 2 with [1-14C] arachidonic acid (New England Nuclear, Bad Homburg, Germany). The assay mixture contained 0.1 Mol/L Tris-buffer (pH 9.0), 4 mMol/L CaCI2, 200 txg of homogenate protein and 0.06 IxCi (1.035 nmol) phosphatidylcholine modified according to Flesch et al (1985). The protein concentration was measured by the Lowry method. After an incubation time of 45 rain at 37nc the reaction was stopped by adding an isopropanolhydrochloric acid mixture. The liberated arachidonic acid was then extracted by the heptane-silica method with Dole's reagent (Dole and Meinertz 1960) followed by adsorption of PLs on silica in a modified procedure according to Sundaram et al (1978). The radioactivity of [1-~4C] arachidonic acid, measured in a Beckman liquid scintillation counter, was used for calculating the PLA2 activity, which is expressed in pMol/mg protein/45 min. Data were analyzed by nonparametric tests (MannWhitney U Test, Wilcoxon and Spearman correlation coefficients = rs).
Results AD patients showed significantly lower parietal PLA2 activity (p-PLA2) than controls (p < 0.005). Frontal PLA2 activity (f-PLA2) was also reduced in AD patients, but the difference just failed to reach statistical significance (p = 0.051; Figure 1 and Table 1). There was a significant corre-
Decreased Phospholipase A2Activity in Alzheimer Brains
BIOLPSYCHIATRY 1995;37:13-17
15
PLA~ frontal
•
parietal
9O 80 70 6O
8
5O
O O o
40
o
|,
Q
8
30
0
2O
o
o
,_!:
0
0
Figure 1. Phospholipase A: activity (in pMol/mg proteird45 rain [1-'4C] arachidonic acid) in frontal and parietal cortex in Alzheimer's disease patients and nondemented elderly controls. (Frontal lobe tissue was not available for one Alzheimer patient.)
m mm m m
in
cP
10
© 0
..
[
Alzheimer (n=22")
,I Control (n=2o)
< Alzheimer (,1=23)
lation between p-PLA2 and f-PLA2 in AD patients (rs = .55, p < 0.005) but not in controls (r~ = 0.28, NS). In the AD group both p-PLA/ and f-PLA 2 correlated positively with age at death (r~ = .37,p < 0.05 and rs = .25, NS). In controls the correlations with age were negative, but nonsignificant. Because AD patients tended to be older than controls (p < 0.10), PLA2 values were corrected for age by the regression coefficient in patients and controls for further comparisons and partial correlations. Age-correction increased the significance of the difference for p-PLA2 (p < 0.001) and the decrement of f-PLA2 in AD patients became significant (p < 0.05). Both p-PLA2 and f-PLA2 correlated positively with age at disease's onset (r, = 0.60,p < 0.005 and rs = .58,p < 0.005, respectively). F-PLA2 correlated negatively with counts of NFTs (r, ----0.56, n = 16,p < 0.01) and SPs (rs = -0.47, n = 16, p < 0.05). No significant correlations were found between p-PLA2 and histologic counts. No correlations were found between both p-PLA2 and f-PLA2 and Mini Mental State scores, duration of illness and postmortem interval. No significant differences were found between p-PLA2 and f-PLA2 within the AD group or within the control group (Table 1). Within both groups there were also no differences in the enzyme activity between men and women.
Discussion To our knowledge this is the first investigation of PLA2 in AD. Our finding of reduced PLA2 activity in the parietal
•
I
Control (n=20)
and, to a lesser degree, in the frontal cortex suggests a decreased breakdown of membrane phospholipids by PLA2 in AD patients. Moreover, our results indicate that the degree of the PLA2 reduction was related to the severity of the pathological process, as in our AD sample the low enzyme activity was correlated with an earlier onset of the disease, higher NFTs and SPs numbers and earlier age at death. Our assumption of a decreased breakdown of membrane phospholipids in AD is supported by the results of an in vivo 31p NMR spectroscopy investigation of the phosphomonoesters and phosphodiesters in AD patients (Brown et al 1989). Phosphomonoesters are the precursors and phogphodiesters are the breakdown products of membrane phospholipids in the brain. AD patients showed increased phosphomonoesters and phosphomonoesters/phosphodiesters ratios in the temporoparietal (but not in the frontal) region. Reduced PLA2 activity, as demonstrated in our study, may contribute to this phosphomoesters increment in AD. In the brain docosahexanoate (22:6, n-3) is one of the principal free fatty acids released by the deacylation of membrane phospholipids through PLA2 (Bazan et al 1986). Skinner et al (1989) found a marked reduction in the proportion of docosahexanoate in the parietal cortex (but not in the frontal cortex) from AD patients, which is compatible with reduced PLA2 as observed in the present study. Changes in the activity of other enzymes related to the membrane phospholipid turnover have already been described in AD brains, such as increased lipases and lysophospholipase activity (Farooqui et al 1990) and decreased phospholipase D activ-
16
BIOLPSYCHIATRY 1995;37:13-17
ity (Kanfer et al 1986). The present finding of reduced PLA2 activity provides new evidence for a disordered membrane phospholipid metabolism in AD. Blusztajn et al (1987) stressed the uniqueness of cholinergic neurons in that they alone use choline not only as a component of membrane phospholipids such as phosphatidylcholine, but also as a precursor of their neurotransmitter acetylcholine. Because phosphatidylcholine is one of the natural substrates of PLA2, our finding of reduced PLA2 activity could produce a decreased catabolism of phosphatidylcholine to supply choline for acetylcholine synthesis, contributing thus to the cholinergic deficit in AD. Moreover, reduced PLA2 activity has been found to decrease membrane fluidity (Erin et al 1986), a mechanism that may result in functional and degenerative changes in the disorder. A characteristic neuropathological finding in AD is the deposition of fibrillar aggregates of the 13-amyloid protein, a 39-43 amino acid peptide produced by the cleavage of a large, membrane bound amyloid precursor protein (APP) (Dyrks et al 1988). Emmerling et al (1993) reported that the inhibition of PLAz decreased the carbachol-stimulated secretion of APP from cells transfected with the human m l muscarinic receptor, whereas the activation of PLA2 in-
W.F. Gattaz et al
creased APP secretion. Experimental evidence suggests that increased APP secretion by cells reduces the production of amyloidogenic peptides (Caporaso et a11992). Therefore it is conceivable that conversely the reduction of APP secretion, as caused by decreased PLA2 activity, may represent on possible route for amyloidogenesis in AD. This assumption is also supported by our finding of a correlation between the reduction of PLA2 activity and the counts of senile plaques. Taken together, the experimental evidence discussed above suggests that reduced brain PLA2 activity may be involved in neuronal degeneration, cholinergic deficit, and amyloidogenesis, three mechanisms that probably contribute to the development of AD. Our preliminary findings warrant further studies to clarify the potential role of PLA2 in the pathophysiology of the disease.
This work was supportedby the DeutscheForschungsgemeinsehaft,SFB 258. Project$4. We are indebtedto Prof. W. MOiler,CentralInstitutefor Mental Health Mannheim, to Prof. E. Ferber, Max-Planck-Institutfitr lmmunbiologie Freiburg, and to Prof. A. Burns, Institute of PsychiatryLondon for the supportgivento this work.
References B~tny M, Chang YC, Arfs C, Rustan T, Frey WH (1985): Increased glycerol-3-phosphorylcholine in post-mortem Alzheimer' s brain. Lancet 1:517. Bazan HEP, Ridenour B, Birkle DL, Bazan NG (1986): Unique metabolic features of docosahexanoate metabolism related to functional roles in brain and retina. In Horroks LA, Breysz L, Toffano G (eds): Phospholipid Research and the Nervous System. Biochemical and Molecular Pharmacology. Fidia Research Series, vol 4. Liviana Press, Padova (1986): pp 67-78.
Blusztajn JK, Wurtman RJ (1983): Choline and cholinergic neurons. Science 221:614--620. Blusztajn JK, Holbrook PG, Lakher M, et al (1986): "Autocannibalism" of membrane choline-phospholipids: Physiology and pathology. Psychopharmacol Bull 22:781-786. Blusztajn JK, Liscovitch M, Richardson UI (1987): Synthesis of acetylcholine from choline derived from phosphatidylcholine in a human neuronal cell line. Proc Natl Acad Sci USA 84:5474--5477. Brown GG, Levine SR, Gorell JM, et al (1989): In vivo 31P NMR profiles of Alzheimer's disease and multiple subcortical infarct dementia. Neurology 39:1423-1427. Coporaso GL, Gandy SE, Buxbaum JD, Ramabhadran TV, Greengard P (1992): Protein phosphorylation regulates secretion of Alzheimer I~A4amyloid precursor protein. Proc NatIAcad Sci USA 89:3055-3059. Dole VP, Meinertz H (1960): Microdetermination of long-chain fatty acids in plasma and tissues. JBiol Chem 235:2595-2599. Dyrks T, Weidemann A, Multhaup G, et al (1988): Identification, transmembrane orientation and biogenesis of the amyloid A4
precursor of Alzheimer' s disease. EMBO J 7:949-957. Ellison DW, Beal MF, Martin JB (1987): Phosphoethanolamine and ethanolamine are decreased in Alzheimer's disease and Huntington's disease. Brain Res 417:389-392. Emmerling MR, Moore CJ, Doyle PD, Carroll RT, Davis RE (1993): Phospholipase A2 activation influences the processing and secretion of the amyloid precursor protein. Biochem Biophys Res Commun 197:292-297. Erin AN, Gorbunov NV, Brusovanik VI, Tyurin VA, Prilipko LL (1986): Stabilization of synaptic membranes by et-tocopherol against the damaging action of phospholipases: Possible mechanism of biological action of vitamin E. Brain Res 398:85-90. Farooqui AA, Liss L, Horroks LA (1990): Elevated activities of lipases and lysophospholipase in Alzheimer's disease. Dement/a 1:208-214. Flesch I, Schmidt B, Ferber E (1985): Acyl chain specificity and kinetic properties of phospholipase A 1 and A2 of bone marrow derived macrophages. Z Naturforsch 40:356-363. Folstein M, Folstein S, McHugh P (1975): Mini Mental State. J Psychiat Res 12:189--198. FOrstl H, Bums A, Levy R, Cairns N, Luthert P, Lantos P (1992): Neurologic signs in Alzheimer's disease: Results of a prospective clinical and neuropathologic study. Arch Neurology 49:1038--1042. Hiram F, Axelrod G (1980): Phospholipid methylation and biological signal transmission. Science 209:1082-1090. Kanfer JN, Hattori H, Orihel D (1986): Reduced phospholipase D activity in brain tissue samples from Alzheimer's disease patients. Ann Neuro120:265-267.
Decreased Phospholipase A2 Activity in Alzheimer Brains
Leprohon CE, Blusztajn JH, Wurtman RJ (1983): Dopamine stimulation of phosphafidycholine (lecithine) biosynthesis in brain neurons. Proc Natl Acad Sci USA 80:2063-2066. Nitsch RM, Blusztajn JK, Pittas AG, Slack BE, Growdon JH, Wurtman RJ (1992): Evidence for a membrane defect in Alzheimer disease brain. Proc Natl Acad Sci USA 89:1671-1675.
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17
Skinner ER, Watt C, Besson JAO, Best PV (1989): Lipid composition of different regions of the brain in patients with Alzheimer' s disease. Biochem Soc Trans 17:213-214. Sundaram GS, Shakir KMM, Barnes G. Margolis S (1978): Release of phospholipase A and triglyceride lipase from rat liver. J Biol Chem 253:7703-7710.
Phospholipase Az (PLA2) is a key-enzyme in the metabolism of membrane phospholipids. In cholinergic neurons PLA2 controls the physico-chemical properties of neuronal membranes as well as the breakdown of phosphatidylcholine to produce choline for acetylcholine synthesis. Moreover PLA2 influences the processing and secretion of the amyloid precursor protein, which gives rise to the f3-amyloid peptide, the major component of the amyloid plaque in Alzheimer' s disease (AD). In the present study PLA2 activity was investigated in post-mortem brains from 23 patients with AD and 20 nondemented elderly controls. In AD brains PLA2 activity was significantly decreased in the parietal and to a lesser degree in the frontal cortex. Lower PLA 2 activity correlated significantly with an earlier onset of the disease, higher counts of neurofibrillary tangles and senile plaques and an earlier age at death, indicating a relationship between abnormally low PLA 2activity and a more severe form of the illness. The present results provide new evidence for a disordered phospholipid metabolism in AD brains and suggest that reduced PLAe activity may contribute to the cholinergic deficit and to the production of amyloidogenic peptides in the disease.
Key Words: Alzheimer's disease, phospholipase A2, brain phospholipids, choline, acetylcholine, amyloid precursor protein, 13-amyloid.
Introduction Alzheimer disease (AD) is the most common cause of dementia in the elderly. The clinical diagnosis of probable AD can only be confirmed at autopsy or biopsy by the neuropathological demonstration of neurofibriUary tangles (NFTs) and senile plaques (SPs) in the neocortex and hippocampus. Although the causes of AD are unknown, there is a large consensus that a preferential degeneration of choliner-
From the Central Institute of Mental Health Mannheim, Germany (WFG, AM, HF); and the MRC Alzheimer's Disease Brain Bank, Institute of Psychiatry, London, LIK (NJC, RL). Address reprint request to W.F. Gattaz, MD, Director, Neurobiology Unit, Central Institute of Mental Health, P.O. Box 12 21 20 - 68072 Mannheim - Germany. Received January 28,1994; revised April 27, 1994.
© 1995 Society of Biological Psychiatry
gic neurons is related to the memory loss and cognitive impairment in the disease (Blusztajn et a11986). A disordered phospholipid metabolism has frequently been found in the brain of AD patients. In autopsied A D brains, changes were reported in the concentrations of membrane phospholipids, their precursors, and catabolites (Bftrfiny et al 1985; Ellison et al 1987; Nitsch et al 1992). In vivo brain nuclear magnetic resonance (NMR) spectroscopy showed increased levels of phosphomonoesters, the precursors of membrane phospholipids, in some but not all cortical regions in AD patients (Brown et al 1989). Intracellular phospholipase A2 (PLA2) is a key-enzyme in the metabolism of membrane phospholipids, cleaving the sn-2-fatty acid ester to produce lysophospholipids and free fatty acids. Changes induced by PLA: on the phospholipidbilayer modify the physico-chemical properties of synaptic 0006-3223/95/$09.50 SSDI 0006-3223(94)00123-K
14
BIOLPSYCHIATRY
W.F. Gattaz et al
1995;37:13-17
membranes, such as signal transduction and membrane fluidity (Hirata and Axelrod 1980; Leprohon et al 1983). Moreover, the breakdown of membrane phospholipids by PLA2 is one of the pathways to produce choline as the precursor for acetylcholine synthesis (Blusztajn and Wurtman 1983). In a recent study Emmerling et al (1993) reported that PLA2 influences the processing and secretion of the amyloid precursor protein (APP); the latter gives rise to the 13-amyloid peptide ([3A), the major component of the amyloid plaque in AD. In the face of this finding and of the importance of PLA2 for both membrane phospholipid metabolism and cholinergic function, we explored the activity of this enzyme in brain tissue from AD patients and neurologically healthy individuals.
Methods Autopsied brain samples of parietal and frontal cortex (Brodmann areas 7 and 32, respectively) were obtained from the Medical Research Council Alzheimer's Disease Brain Bank, Institute of Psychiatry, London. Samples from 23 cases of neuropathologically confirmed AD (7 men, 16 women; mean age - SD 81.0 ___7.5 years) and 20 nondemented controls (10 men, 10 women; 75.6 --- 9.8 years) were investigated. The degree of dementia was assessed in all but 2 AD patients within 12 months before death with the Mini Mental State Exam (Folstein et al 1975). None of the AD patients had received therapy for the disease. The causes of death were similar in AD patients and controls and were usually due to a terminal circulatory or respiratory failure. In AD patients the means _ SD for the age at disease onset was 72.9 --- 6.9 years, the duration ofiUness 9.5 --- 5.8 years and the Mini Mental State score 3.1 _ 4.9.
Tissue Collection At autopsy, the brain was removed and weighed. The brainstem and cerebellum were removed prior to dividing the cerebral hemispheres in the midline. Alternate hemispheres were either fixed for histology or frozen for biochemistry. The hemisphere to be frozen was sliced in the coronal plane in approximately 1 cm thick slices. The slices were rapidly frozen by contact with a brass plate at a temperature of -70°C and stored at this temperature in airtight plastic bags to prevent desiccation. Blocks from the neocortex were subsequently dissected on a bed of dry ice. Histologic and counting methods for senile plaques and neurofibriUary tangles were described elsewhere (Ft~rstl et al 1992). Briefly, SPs and N F r s were counted in Glees and Marsland silver impregnated sections in eight adjacent, nonoverlapping columns in each neocortical site. The mean values were calculated and expressed as counts per square millimeter. Histologic counts were obtained form 16 AD patients. The mean counts/mm 2 ___ SD [range] were: SPs parietal 5.0 - 6.3 [0-22], SPs frontal 4.1 _ 7.2 [0-27],
Table 1. Demographic and Neurochemical Data in Alzheimer's Patients and Nondemented Controls (means --- SD) Alzheimer Variables Age Sex P o s t m o r t e m time (hr) P L A 2 parietal P L A 2 frontal
Controls
(n = 23)
(n = 20)
81.0 -+ 7.5 a 7 m e n : 16 w o m e n 30.7 - 14.5 27.4 ± 20.2 b 26.9 ± 16.4 '~c
75.6 ± 9.8 10 m e n : 10 w o m e n 38.1 ± 11.1 43.4 ± 23.8 37.9 - 19.8
PLA2 activity is given in pMol/mg/45 rain arachidonic acid. "p < 0.10. ~p < 0.005 (as compared to controls). On= 22.
N F r s parietal 4.7 ___4.6 [0.4-18], NFTs frontal 5.9 --- 7.6 [0.1-24]. The demographic and biochemical data and the postmortem interval are given in Table 1. There was a trend for AD patients to have a higher age at death than the control group (p < 0.10). The difference in postmortem interval, with shorter mean time from death to storage in the AD group, did not reach statistical significance (p = 0.15).
Determination of PZA 2Activity Homogenized brain tissue from parietal and frontal cortex was suspended in 0.1 Mol/L Tris-HCI-Saccharose buffer (pH 7.4). As enzyme substrate we used L-A-1-palmitoyl-2arachidonyl-phosphatidylcholine labeled in position 2 with [1-14C] arachidonic acid (New England Nuclear, Bad Homburg, Germany). The assay mixture contained 0.1 Mol/L Tris-buffer (pH 9.0), 4 mMol/L CaCI2, 200 txg of homogenate protein and 0.06 IxCi (1.035 nmol) phosphatidylcholine modified according to Flesch et al (1985). The protein concentration was measured by the Lowry method. After an incubation time of 45 rain at 37nc the reaction was stopped by adding an isopropanolhydrochloric acid mixture. The liberated arachidonic acid was then extracted by the heptane-silica method with Dole's reagent (Dole and Meinertz 1960) followed by adsorption of PLs on silica in a modified procedure according to Sundaram et al (1978). The radioactivity of [1-~4C] arachidonic acid, measured in a Beckman liquid scintillation counter, was used for calculating the PLA2 activity, which is expressed in pMol/mg protein/45 min. Data were analyzed by nonparametric tests (MannWhitney U Test, Wilcoxon and Spearman correlation coefficients = rs).
Results AD patients showed significantly lower parietal PLA2 activity (p-PLA2) than controls (p < 0.005). Frontal PLA2 activity (f-PLA2) was also reduced in AD patients, but the difference just failed to reach statistical significance (p = 0.051; Figure 1 and Table 1). There was a significant corre-
Decreased Phospholipase A2Activity in Alzheimer Brains
BIOLPSYCHIATRY 1995;37:13-17
15
PLA~ frontal
•
parietal
9O 80 70 6O
8
5O
O O o
40
o
|,
Q
8
30
0
2O
o
o
,_!:
0
0
Figure 1. Phospholipase A: activity (in pMol/mg proteird45 rain [1-'4C] arachidonic acid) in frontal and parietal cortex in Alzheimer's disease patients and nondemented elderly controls. (Frontal lobe tissue was not available for one Alzheimer patient.)
m mm m m
in
cP
10
© 0
..
[
Alzheimer (n=22")
,I Control (n=2o)
< Alzheimer (,1=23)
lation between p-PLA2 and f-PLA2 in AD patients (rs = .55, p < 0.005) but not in controls (r~ = 0.28, NS). In the AD group both p-PLA/ and f-PLA 2 correlated positively with age at death (r~ = .37,p < 0.05 and rs = .25, NS). In controls the correlations with age were negative, but nonsignificant. Because AD patients tended to be older than controls (p < 0.10), PLA2 values were corrected for age by the regression coefficient in patients and controls for further comparisons and partial correlations. Age-correction increased the significance of the difference for p-PLA2 (p < 0.001) and the decrement of f-PLA2 in AD patients became significant (p < 0.05). Both p-PLA2 and f-PLA2 correlated positively with age at disease's onset (r, = 0.60,p < 0.005 and rs = .58,p < 0.005, respectively). F-PLA2 correlated negatively with counts of NFTs (r, ----0.56, n = 16,p < 0.01) and SPs (rs = -0.47, n = 16, p < 0.05). No significant correlations were found between p-PLA2 and histologic counts. No correlations were found between both p-PLA2 and f-PLA2 and Mini Mental State scores, duration of illness and postmortem interval. No significant differences were found between p-PLA2 and f-PLA2 within the AD group or within the control group (Table 1). Within both groups there were also no differences in the enzyme activity between men and women.
Discussion To our knowledge this is the first investigation of PLA2 in AD. Our finding of reduced PLA2 activity in the parietal
•
I
Control (n=20)
and, to a lesser degree, in the frontal cortex suggests a decreased breakdown of membrane phospholipids by PLA2 in AD patients. Moreover, our results indicate that the degree of the PLA2 reduction was related to the severity of the pathological process, as in our AD sample the low enzyme activity was correlated with an earlier onset of the disease, higher NFTs and SPs numbers and earlier age at death. Our assumption of a decreased breakdown of membrane phospholipids in AD is supported by the results of an in vivo 31p NMR spectroscopy investigation of the phosphomonoesters and phosphodiesters in AD patients (Brown et al 1989). Phosphomonoesters are the precursors and phogphodiesters are the breakdown products of membrane phospholipids in the brain. AD patients showed increased phosphomonoesters and phosphomonoesters/phosphodiesters ratios in the temporoparietal (but not in the frontal) region. Reduced PLA2 activity, as demonstrated in our study, may contribute to this phosphomoesters increment in AD. In the brain docosahexanoate (22:6, n-3) is one of the principal free fatty acids released by the deacylation of membrane phospholipids through PLA2 (Bazan et al 1986). Skinner et al (1989) found a marked reduction in the proportion of docosahexanoate in the parietal cortex (but not in the frontal cortex) from AD patients, which is compatible with reduced PLA2 as observed in the present study. Changes in the activity of other enzymes related to the membrane phospholipid turnover have already been described in AD brains, such as increased lipases and lysophospholipase activity (Farooqui et al 1990) and decreased phospholipase D activ-
16
BIOLPSYCHIATRY 1995;37:13-17
ity (Kanfer et al 1986). The present finding of reduced PLA2 activity provides new evidence for a disordered membrane phospholipid metabolism in AD. Blusztajn et al (1987) stressed the uniqueness of cholinergic neurons in that they alone use choline not only as a component of membrane phospholipids such as phosphatidylcholine, but also as a precursor of their neurotransmitter acetylcholine. Because phosphatidylcholine is one of the natural substrates of PLA2, our finding of reduced PLA2 activity could produce a decreased catabolism of phosphatidylcholine to supply choline for acetylcholine synthesis, contributing thus to the cholinergic deficit in AD. Moreover, reduced PLA2 activity has been found to decrease membrane fluidity (Erin et al 1986), a mechanism that may result in functional and degenerative changes in the disorder. A characteristic neuropathological finding in AD is the deposition of fibrillar aggregates of the 13-amyloid protein, a 39-43 amino acid peptide produced by the cleavage of a large, membrane bound amyloid precursor protein (APP) (Dyrks et al 1988). Emmerling et al (1993) reported that the inhibition of PLAz decreased the carbachol-stimulated secretion of APP from cells transfected with the human m l muscarinic receptor, whereas the activation of PLA2 in-
W.F. Gattaz et al
creased APP secretion. Experimental evidence suggests that increased APP secretion by cells reduces the production of amyloidogenic peptides (Caporaso et a11992). Therefore it is conceivable that conversely the reduction of APP secretion, as caused by decreased PLA2 activity, may represent on possible route for amyloidogenesis in AD. This assumption is also supported by our finding of a correlation between the reduction of PLA2 activity and the counts of senile plaques. Taken together, the experimental evidence discussed above suggests that reduced brain PLA2 activity may be involved in neuronal degeneration, cholinergic deficit, and amyloidogenesis, three mechanisms that probably contribute to the development of AD. Our preliminary findings warrant further studies to clarify the potential role of PLA2 in the pathophysiology of the disease.
This work was supportedby the DeutscheForschungsgemeinsehaft,SFB 258. Project$4. We are indebtedto Prof. W. MOiler,CentralInstitutefor Mental Health Mannheim, to Prof. E. Ferber, Max-Planck-Institutfitr lmmunbiologie Freiburg, and to Prof. A. Burns, Institute of PsychiatryLondon for the supportgivento this work.
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