Neurochemical Research, Vol. 20, No. 11. 1995, pp. 1329-1333

Neural Membrane Phospholipids in Alzheimer Disease*

Kimberly Wells, 1 Akhlaq A. Farooqui, 1,3 Leopold Liss, 2 and Lloyd A. Horrocks 1

(Accepted June 21, 1995)

Phospholipids form the backbone of neural membranes, providing fluidity and permeability. Two plasma membrane fractions, one from synaptosomes (SPM), the other from glial and neuronal cell bodies (PM), were prepared from different regions of autopsied Alzheimer disease (AD) brains. Corresponding fractions were prepared from age-matched control brains. All fractions from AD brains showed significantly lower levels of ethanolamine glycerophospholipids and significantly higher levels of serine glycerophospholipids than the control brain. No differences were observed in phosphatidylcholine levels among these membranes. These results suggest that altered phos- pholipid composition of plasma membranes may be involved in the abnormal signal transduction and neurodegeneration in AD.

KEY WORDS: Phospholipids; plasma membrane; signal transduction; neurodegeneration; Alzheimer disease.

INTRODUCTION

Alzheimer disease (AD) is a progressive neurode- generative disease that affects millions of individuals of all races and ethnic backgrounds. In the United States, 10% of the population over age 65 and more than 47% of the population over age 85 are afflicted. Onset before age 60 is infrequent. Numerous theories have been pro- posed to explain the degeneration of neurons in AD (1- 4) including (i) a selective vulnerability of cholinergic neurons in the basal forebrain, (ii) a viral agent, (iii) aluminum deposits, (iv) lack of trophic factors, (v) ov- erstimulation of excitatory amino acid (EAA) receptors, and (vi) altered phospholipid metabolism.

Phospholipids are essential constituents of neural membranes, providing structural integrity and functional

1 Departments of Medical Biochemistry and 2Pathology College of Medicine, The Ohio State University, 1645 Neil Avenue, Columbus, Ohio 43210.

3 Address reprint requests to: Akhlaq A. Farooqui, Ph.D., Department of Medical Biochemistry, The Ohio State University, 1645 Neit Av- enue, Rm. 479, Columbus, Ohio 43210. Telephone: 614-292-2905; fax: 614-292-5482.

* Special issue dedicated to Dr. Leon S. Wolfe.

1329

properties (5,6). Recent evidence suggests that altered membrane phospholipid metabolism may be a funda- mental component of the neurodegeneration observed in AD (3,7-9). Levels of glycerophospholipids, plasmalo- gens, choline and ethanolamine glycerophospholipids, and polyphosphoinositides are markedly decreased in patients with AD compared to age-matched normal con- trols (8,10-14). This decrease in glycerophospholipids is accompanied by marked elevations in phospholipid deg- radation metabolites such as glycerophosphocholine, phosphocholine, and phosphoethanolamine in autopsy samples of AD brain (15-19). Furthermore, marked in- creases in levels of prostaglandins and lipid peroxides have been reported in AD brain (20,21). Thus, the col- lective evidence suggests that the composition of neural membranes is altered in AD, probably resulting in func- tionally significant alterations in the biophysical prop- erties of brain cell membranes (22-24). These changes may be responsible for the neurodegeneration observed in AD (9).

Because all of the above studies were done with whole brain tissue, it was still not known whether the reported changes were associated with alterations in neu- ronal cells or their plasma membranes. The purpose of

0364-3190/95/1[00-L329507.50/0 �9 1995 Plemtm Publishing Corporation

1330 Wells, Farooqui, Liss, and Horrocks

this investigation is to compare the phospholipid com- position o f two neural membrane fractions from various regions o f AD brain to corresponding fractions from age-matched normal control brain. One plasma mem- brane fraction was from synaptosomes (SPM) and the other was from glial and neuronal cell bodies (PM).

EXPERIMENTAL PROCEDURE

The brains used for this study were from individuals aged 76 to 92. The diagnosis of AD was made on the basis of combined neuro- logical and neuropathological findings (25-27). Six AD brains were obtained at autopsy from 2 to 7.5 h after death. The following areas plus the hippocampus were dissected from each right hemisphere. The gray matter from the frontal cortex was taken from the anterior frontal region (Broca's area 11). Gray matter from the parietal region was removed from the parietal region was removed from both the primary somatosensory and somatosensory association areas (Broca's areas 3, I, 2, 5 and 7). Gray matter from the temporal region was removed from the primary auditory and auditory association areas (Broca's regions 41, 42 and 22). Tissue samples weighing 0.75 to 2 g were transported on ice to the laboratory and kept frozen at -80~ until used. The left hemispheres were fixed in buffered fonnalin for histo- logical examination.

Four control brains were kindly provided by Dr. Frank Zemlan from the University of Cincinnati Brain Bank. The post-mortem delay times ranged from 4 to 7 h.

Preparation of Plasma Membrane Fractions. Subeellular frac- tions were prepared using procedures previously described (28,29). In a previous study, we performed the biochemical characterization of PM and SPM fractions from AD and control human brain (29) and found a 2- to 3-fold enrichment of Na +, K+-ATPase and 5'-nueleotidase in those fractions. PM and SPM pellets were suspended in 0.5 ml of distilled water. Lipids were extracted by adding 9 ml of hexane:iso- propanol (3:2 v/v).

Isolation and Quantitation of Phospholipids from PM and SPM Fractions. Using an HPLC procedure described elsewhere (30), the following phospholipids were isolated: choline glycerophospholipids (ChoGpl), ethanolamine glycerophospholipids (EtuGpl), serine glycer- ophospholipids (SerGpl), inositol glycerophospholipids (InsGpl), and sphingomyelin (CerPCho). Phospholipids were quantitated by the method of Rouser (31).

Statistical Analysis. Each phospholipid class was expressed as a percentage of the total phospholipids. Unpaired Student's t-test and analysis of variance were used to compare levels of individual phos- pholipids from the different regions of AD brain to the corresponding regions from normal control subjects.

R E S U L T S

The total phospholipid content o f the PM and SPM fractions from various regions o f control human brain varied between 357.1 nmol/g wet weight and 638.79 nmol/g wet weight. In AD brain, the total phospholipid content o f the PM and SPM fractions was 118.18 and

447.56 nmol/g wet weight, respectively (data not shown).

The two largest phospholipid components of all of the PM and SPM fractions were ethanolamine glycero- phospholipids and choline glycerophospholipids, fol- lowed by sphingomyelin and serine glycerophospholi- pids (Tables I and II).

Levels o f ethanolamine glycerophospholipids were significantly reduced in the PM and SPM fractions from the hippocampal and cortical regions o f AD brain (Figs. 1 and 2). By contrast, the levels o f serine glycerophos- pholipids and sphingomyelin were significantly in- creased in these fractions. Interestingly, no differences were observed in phosphatidylcholine levels in PM and SPM fractions from parietal, temporal and frontal cor- tices. However, levels o f phosphatidylcholine were markedly decreased in the occipital cortex (Table I). In the SPM fraction from AD hippocampus, there was also a marked decrease (67%) in inositol-containing glycer- ophospholipids and phosphatidylcholine (53%).

D I S C U S S I O N

The present study is the first to report on the phos- pholipid composition o f plasma membrane fractions from different regions o f control human and AD brain. Analysis o f plasma membrane fractions is preferred over whole brain homogenate because the latter may contain necrotic areas as well as plaques and tangles. SPM frac- tions containing synaptic plasma membranes are derived entirely from neurons and are very important for func- tioning. Our data clearly show that plasma membrane fractions from the hippocampus, frontal, parietal, occip- ital, and temporal cortices o f AD brain contain signifi- cantly lower levels o f ethanolamine glycerophospholi- pids than membranes from corresponding regions of control human brain.

Further, membranes from all regions o f AD brain showed higher levels o f serine glycerophospholipids compared to membranes from corresponding regions o f control brain. No differences were observed in choline glycerophospholipid levels in these membranes. Phos- pbatidylserine stimulates protein kinase C, an enzyme that plays an important role in signal transduction, cell differentiation, and cell division (32). In spite o f the in- crease in serine glycerophospholipids in AD brain, pro- tein kinase C activity is markedly decreased in AD brain homogenates (33). This may be due to the unavailability o f diacylglycerol, which is hydrolyzed by the high level o f diacylglycerol lipase in AD tissues (29).

Neural Membrane Phospholipids in Alzheimer Disease 1331

Table I. Phospholipid Content of SPM and PM Fractions from AD and Control Cerebral Cortex (nmol/g wet weight _+ SEM)

SPM Fractions PM Fractions

Alzheimer Control Alzheimer Control

Parietal PtdIns CerPCho PtdCho PtdSer PtdEtn

n = 4 n = 3 n = 5 n = 3 9.03 _+ 2.6,1. 7.52 4- 3,50 8.62 +_ 3.74 11.45 4- 2.37

31.66 4- 8.77 48,01 _ 5.73 27.98 4- 4.66 54.26 _+_ 6.62 146.43 +_ 50.9 151.49 4- 18.37 111.91 + 22.04 198.8I 4- 22.92 20.05 4- 4.41 10.12 4- 4.90 21.30 _+ 6.08 18.29 4- 2.68

t23.70 4- 41.93 162.43 4- 11.89 90.95 _+ 18.16 203.86 4- 23.87

Temporal n = 6 n = 4 n = 6 n = 4 PtdIns 11.12 _+ 5.04 7.93 _+ 1.26 12.10 4- 4.42 15.59 4- 5.16 CerPCho 55.24 4- 17.86 54.17 4- 10.39 54.85 4- 10.68 71.90 4- 6.97 PtdCho 163.92 4- 52.96 180.77 4- 32.56 177.07 4- 32.79 235.52 + 17.83 PtdSer 38.05 4- 10.79 24.22 4- 6.41 45.16 _+ 10.16 26.91 4- 5.00 PtdEtn 140.46 4- 47.45 184.37 4- 31.53 153.23 4- 29.54 232.19 _+ 15.19

Frontal n = 5 n = 4 n = 5 n = 3 PtdIns 7.40 _-2- 3.74 8.03 4- 3.66 6.37 _+ 1.75 14.74 +_ 2.94 CerPCho 36.07 _+ 12.52 47.21 4- 15.18 34.60 4- 8.11 81.36 4- 13.02 PtdCho 124.39 _+ 48.30 149.71 _+ 48.29 117.28 _+ 19.28 239.76 _+ 43.58 PtdSer 30.84 _+ 11.00 21.72 4- 9.53 31.21 +_ 15.44 32.65 4- 7,46 PtdEtn 104.48 4- 39.51 158.91 _+ 52.16 104.16 _+ 18.54 246.54 _+ 46.78

Occipital n = 4 n = 3 n = 4 n = 2 PtdIns 2.93 4- 1.65 4.80 _+ 1.96 2.93 _+ 1.65 6.92 +_ 0.66 CerPCho 13.76 +_ 2.73 48.87 _ 8.14 13.76 +_ 2.73 85.18 +_ 7.02 PtdCho 47.07 4- 12.66 137.32 4- 20.51 47.07 +_ 12.66 252.87 4- 20.08 PtdSer 10.87 +_ 1.74 10.65 _+ 4.03 10.87 + 1.74 22.55 4- 3.36 PtdEtn 43.10 _+ 6.72 155.07 _+ 21.20 43.10 _+ 6.72 269.91 + 30.10 i

Our findings of altered phospholipid composition in AD brain are consistent with earlier studies (1,11,13) that reported, on the basis of whole brain region analy- sis, decreased levels of ethanolamine and choline gly- cerophospholipids. Levels of inositol-containing glycer- ophospholipids are also decreased in AD (12). The loss of ethanolamine and inositol-containing phospholipids may be related to the loss of synapses in AD (1,34).

The decrease in ethanolamine glycerophospholipids can be explained by three possible mechanisms. First, the catabolism of ethanolamine glycerophospholipids may be significantly increased in AD due to the stimu- lation of phospholipases and lipases. This possibility is

Table II. Phospholipid Content of SPM and PM Fractions from AD and Control Hippocampus (mnol/g wet weight + SD)

SPM Fractions PM Fractions

Alzheimer Control Alzheimer Control ( n = 4) (n = 3) ( n = 4 ) ( n = 3)

Ptdlns 2 _-4_- 0.8 6 _+ 1.5 10 _+ 1.8 17 _+ 5.3 CerPCho 32 4-_ 10.1 48 _+ 10.2 60 + 15.6 61 _+ 7.3 PtdCho 76 _ 22.7 162 _+ 35.2 154 +_ 28.2 217 _+ 23.9 PtdSer 17 _+ 4.2 11 4- 2.6 46 _+ 10.9 31 _+ 4.2 PtdEtn 65 _+ 16.3 t49 + 28.2 I34 _+ 25.4 211 -4- 20.8

supported by earlier studies from our laboratory that have indicated a marked stimulation of diacylglycerol and monoacylglycerol lipases in AD brain (29,35). Sec- ond, the synthesis of ethanolamine glycerophospholi- pids may be decreased. The de novo synthesis of phosphatidylethanolamine from CDP-ethanolamine and diacylglycerol is catalyzed by 1,2-diradyl-sn-glycerol: CDPethanolamine phosphoethanolamine transferase.

5o

30

20

5

Hippocarnpus

E ~ ] Pk4 Contro

PtdCho PtdEtn PtdSer

[ ~ SPM CDn~ro

SPM ALZ

_ii 0 PtdCho P~dEtn PtdSer

Fig. 1. Phospholipid compositions of PM and SPM fractions from control and AD hippocampus. Phospholipids are expressed as a per- centage of the total phospholipids. Control n = 3, and AD n = 4. Data are means _+ SEM. Student's t-test was used to compare each phospholipid class in the control hippocampus to the co]Tesponding class in the AD hippocampus. PM - PtdEtu (p = 0.0027); PtdSer (p = 0.0465) and SPM - PtdEtu (p = 0.084); PtdSer (p = 0.0001).

1332 Wells, Farooqui, Liss, and Horroeks

5O

4 5 [ :g

40

3 5 -

~. 30

-6 25

fi- 2o ~ -

�9

o. 5 -

o

Ternporol

PM Contro PM AkZ

PtdCho PtdEtn PtdSer PtdCho PtdEtn PtdSer

Fig. 2. Phospholipid compositions of PM and SPM fractions from control and AD temporal cortex. Phospholipids are expressed as a percentage of the total phospholipids. Control n = 4, and AD n = 6. Data are means + SEM. Student's t-test was used to compare each phospholipid class in the control temporal cortex to the corresponding class in the AD temporal cortex. PM - PtdEtn (p = 0.0041); PtdSer (13 = 0.0011) and SPM - PtdEtu (p = 0.0001); PtdSer (p = 0.0160).

Unavailability of diacylglycerol due to the high dia- cylglycerol lipase activity found in AD may result in decreased synthesis of ethanolamine glycerophospho- lipids. Finally, ethanolamine glycerophospholipids may be converted to serine glycerophospholipids by base exchange (36). The rate of base exchange depends on the intracellular Ca 2+ concentration (37,38), which may be high due to excitotoxicity. This possibility would also explain the noticeable increase in serine glycero- phospholipids in AD.

Phosphatidylserine has been used for the treatment of AD (39-41). It acts by incorporating into the plasma membrane and stimulating the production of acetylcho- line and dopamine in the cerebral cortex (42,43). It also restores NMDA receptor density and function in mem- branes (44). This receptor may be involved in the neu- rodegeneration of AD (6). Alterations in levels of mem- brane phospholipids are accompanied by marked elevations in phosphoethanolamine, glycerophosphoe- thanolamine, and phosphocholine in AD brain (16, 17,45). Our data support the hypothesis that alterations in membrane phospholipid composition may be respon- sible for changes in membrane fluidity, integrity, and permeability. These processes, along with compromised energy metabolism (46), may be involved in the abnor- mal signal transduction and neurodegeneration in AD.

ACKNOWLEDGMENTS

We thank Dr. Frank Zemlan of the University of Cincinnati for providing control human brain from the University of Cincinnati Brain Bank. We also thallk Mr. Thad Rosenberger for statistical analysis of phospholipid composition. This work was supported in part by NIH grants NS-10165 and NS-29441.

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