Phospholipid composition and levels are altered in down syndrome brain

Brain Research 867 (2000) 9–18 www.elsevier.com / locate / bres

Research report

Phospholipid composition and levels are altered in down syndrome brain Eric J. Murphy, Mark B. Schapiro, Stanley I. Rapoport, H. Umesha Shetty* Section on Brain Physiology and Metabolism, National Institute on Aging, National Institutes of Health, Building 10, Room 6 C103, Bethesda, MD 20892, USA Accepted 23 February 2000

Abstract Phospholipid composition (mol %) and levels (nmol / mg protein) were determined in postmortem frontal cortical and cerebellar gray matter from older Down Syndrome (DS) patients (age range 38–68 years) and from control subjects. Neither DS nor control tissue exhibited any age-dependent alteration in phospholipid composition or levels. Total phospholipid content was significantly reduced approximately 20% in DS frontal cortex and cerebellum relative to these regions in control tissue. Individual phospholipid levels were also reduced in DS frontal cortex and cerebellum, including a specific 37% decrease in phosphatidylinositol (PtdIns) and a nearly 35% decrease in ethanolamine plasmalogen. Because of the large decrease in phospholipid content in DS brain, the cholesterol / phospholipid ratio was calculated for each group. There was no significant difference in this ratio between groups, indicative of compensatory changes to keep the cholesterol / phospholipid ratio constant. Despite the large changes in DS brain phospholipid levels, significant changes in composition were limited to a 18% decrease in PtdIns mol % and a 22% increase in the mol % of sphingomyelin. These results suggest either a decrease in membrane phospholipids due to a loss of dendrites and dendritic spines, or a general defect in brain lipid metabolism in older DS subjects. The proportionally greater alterations in PtdIns and PlsEtn levels, indicate that the metabolism of these two phospholipids was affected to a greater extent than the other phospholipids. Further, because these changes are found in both the frontal cortical and cerebellar gray matter, they likely are related to the Down syndrome condition rather than to Alzheimer neuropathology.  2000 Published by Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Developmental disorders Keywords: Phospholipids; Plasmalogens; Down syndrome; Brain; Aging; Cholesterol

1. Introduction Down Syndrome (DS) is characterized by a partial or complete trisomy of human chromosome 21, which results in mental retardation and other phenotypic abnormalities [14]. Individuals with DS have brain structural abnormalities including alterations in dendritic structure and fewer dendritic spines [14,58]. Additionally, DS subjects over the age of 40 often become demented and exhibit brain pathology similar to Alzheimer Disease (AD) [1,9,14,48,64]. Older, non-demented DS subjects have *Corresponding author. Tel.: 11-301-594-7752; fax: 11-301-4020074. E-mail address: [email protected] (H.U. Shetty)

lowered brain glucose metabolism upon audio–visual stimulation in the parietal and temporal lobes, similar to the alterations observed in AD patients [44]. Furthermore, similar to AD, there is a correlation between the apolipoprotein E4 genotype and cognitive decline in DS subjects [1]. In brains from older DS subjects, biochemical changes occur that are similar to those observed in AD. In the DS brain, choline acetyl transferase activity is decreased in the same regions which demonstrate decreased activity in the AD brain [27,67]. Serotonin levels are also decreased in regions known to be affected in AD [67]. Vasopressin, a neuropeptide which mediates limbic system function, is increased in temporal cortex in brains from both older DS and AD subjects, but unlike in AD, this peptide is also

0006-8993 / 00 / $ – see front matter  2000 Published by Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02205-8

E. J. Murphy et al. / Brain Research 867 (2000) 9 – 18

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decreased in the DS cerebellum [35]. Expression of mRNA predictors of AD are not found prior to neurofibrillary tangle formation in aged DS brain, suggesting that changes in these messages are not required for tangle formation in DS [24]. In any case, in brains from older DS subjects, cognitive, structural, and biochemical changes occur that resemble those reported in brain regions affected by AD, suggesting that DS patients over 40 years of age suffer from senile dementia of the Alzheimer type. A number of changes in brain lipids have been reported in DS. These include a significant reduction in esterified polyunsaturated fatty acids, particularly those esterified into phosphatidylinositol and phosphatidylserine [50]. In the fetal DS brain, fatty acid changes are limited to an increase in the n-3 / n-6 ratio [7]. In brains from older DS subjects, the arachidonic acid level in frontal cortex is decreased in choline glycerophospholipids and there are changes in phospholipid composition [6]. In both DS cerebellar and frontal cortex gray matter from older DS subjects, there is also an overall reduction in gangliosides [8]. However, unlike AD, there are no marked changes in glycerophosphocholine or glycerophosphoethanolamine levels in brains from older DS subjects [4,43]. Choline and myo-inositol levels, important components for phospholipid biosynthesis and signaling, are elevated in brains and cerebrospinal fluid from DS subjects [46,52]. The elevation in myo-inositol is likely linked to increased expression of the Na 1 -myo-inositol transporter, which is encoded for on chromosome 21. Thus, in DS there are a number of alterations in brain lipid metabolism. To further understand brain lipid metabolism in DS, phospholipid levels and composition were determined in brains from DS subjects, 38 years and older. Phospholipid measurements were made in frontal cortical and cerebellar gray matter from control and DS brains. Because we observed differences in DS brain phospholipid levels, we also measured cholesterol levels and calculated the cholesterol / phospholipid ratio. In brains of older DS subjects, we report a 15–37% decrease in brain phospholipid levels and altered brain phospholipid composition, in the absence of any change in the cholesterol / phospholipid ratio.

2. Materials and methods

2.1. Brain tissue Frontal cortical gray matter was dissected from Brod-

mann area 32 and gray matter was dissected from the cerebellum. All of the eight DS patients were clinically diagnosed with DS. Five of the eight subjects had been karyotyped and confirmed to be trisomy 21. There was no statistical difference in mean age, although the age range was wider for the control than DS group (Table 1). There was no difference in the post-mortem delay between the DS and control groups (Table 1). For data analysis, the genders were combined in the DS and control groups. DS brain tissue was obtained from the brain bank of the Laboratory of Neurosciences, National Institute on Aging; from the University of Miami Brain Bank (supported by PHS grant [P50-AG08671); University of Michigan MADRC Program (supported by NIA grant [P50AG08671); and from the University of Maryland Brain Tissue Bank for Developmental Research. Control brain tissue was obtained from the Harvard Brain Tissue Resource Center (supported by PHS grant [MH / NS 31862); University of Miami Brain and Tissue Bank for Developmental Disorders (supported by PHS grant [N01HD-3-3199); and the National Neurological Research Specimen Bank, VAMC at Los Angeles, CA (sponsored by the National Institute of Neurological Disorders and Stroke, National Institute of Mental Health, National Multiple Sclerosis Society, Hereditary Disease Foundation, and Veterans Health Services and Research Administration).

2.2. Lipid extraction Frozen brain tissue was weighed (approximately 50 mg) and homogenized using a probe sonicator (Misonex, Farmingdale, NY, USA), in a polypropylene tube containing 0.5 ml of methanol with 0.005% (w / v) butylated hydroxytoluene added. The homogenate was transferred to a tapered glass tube containing 2 ml of chloroform and the sonicator and polypropylene tube was rinsed with another 0.5 ml aliquot of methanol. This rinse was combined with the chloroform:methanol mixture for a final chloroform:methanol proportion of 2:1 (v / v). The lipid extract was mixed by vortexing for 2 min followed by the addition of 0.75 ml of KCl solution (50 mM) and mixing. Phase separation was facilitated by centrifugation at 2000 g for 10 min at 48C. The chloroform layer was transferred to another tube and residual lipids removed from the aqueous phase using 0.5 ml of chloroform and phases separated as described above. The lipid containing chloroform phases were combined and solvent evaporated using a SpeedVac

Table 1 Age and postmortem delay for control and Down Syndrome brains

Control Down Syndrome a

Values represent means6S.D.

n (male / female)

Age yr. (range)a

Post-mortem delay h (range)a

9 (3 / 6) 8 (2 / 6)

50.3623.5 (22–87) 53.0610.0 (38–68)

15.366.1 (7.5–23.5) 11.966.5 (4.5–23.0)

E. J. Murphy et al. / Brain Research 867 (2000) 9 – 18

AES 1010 (Savant Instruments, Holbrook, NY, USA). The lipids were dissolved in 0.5 ml of chloroform:methanol (2:1 (v / v)). The aqueous layer, including the protein found at the organic–aqueous interface, was evaporated using the SpeedVac and the protein residue was dissolved in a 0.25% sodium dodecyl sulfate solution. Protein levels were assayed using the BCA reagent (Pierce Chemical Co., Rockford, IL, USA) and levels quantified by converting absorbances to mass based on a bovine serum albumin standard curve.

2.2.1. Phospholipid separation Phospholipids were separated using thin layer chromatography (TLC). Sample (100 ml, 1 / 5 of the original sample) was streaked onto a TLC plate (Kieselgel 60, 0.25 mm thick, EM Science, Gibbstown, NJ, USA) and developed twice in a solvent system containing chloroform:methanol:2-propanol:potassium chloride solution (0.25% (w / v)):ethyl acetate (30:9:25:6:18, (v / v / v / v / v)). This solvent system reproducibly separates all of the major phospholipids classes [29]. Lipids were visualized with iodine vapor, and phospholipid bands corresponding to authentic commercial standards were removed by scraping and placed into acid washed test tubes for quantitation of lipid phosphorus. 2.2.2. Phosphorus assay Into tubes containing phospholipid bound to silica, 0.5 ml of water and 0.65 ml of perchloric acid (70%) were added and the tubes heated at 1858C for 1 h [45]. Following digestion, the tubes were cooled to room temperature and 0.5 ml ascorbic acid (10% (w / v)), 0.5 ml ammonium molybdate (2.5% (w / v)) and 3.3 ml water added and mixed. Color was developed by boiling the mixture for 5 min and, after cooling, the absorbance at 797 nm was measured. Absorbance was converted to mass based on a standard curve. 2.2.3. Plasmalogen analysis The ethanolamine glycerophospholipid (EtnGpl) and choline glycerophospholipids (ChoGpl) were separated using high performance liquid chromatography (HPLC) using a Phenomenex Selectosil column (5 mm, 4.53250 mm, Torrance, CA, USA) as described previously [16]. The EtnGpl and ChoGpl fractions were collected, the solvent removed under a stream of N 2 , and then exposed to HCl vapor to hydrolyze the plasmalogen vinyl ether linkage [41]. The resulting lysophosphatidylethanolamine and lysophosphatidylcholine products were separated from the acid-stable components using HPLC. The acid-stable and acid-labile components were collected and quantified by assaying for lipid phosphorus. 2.2.4. Cholesterol measurements The neutral lipid containing fraction was collected

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during the phospholipid separation described above, the solvent removed by evaporation using a stream of N 2 , and the lipids dissolved in a known volume of n-hexane:2propanol:acetic acid (98.7:1.2:0.1, (v / v / v)). Cholesterol was separated by HPLC using a Phenominex Selectosil column (5 mm, 4.53250 mm) and quantified by absorbance at 205 nm by analysis of peak area [40]. Cholesterol concentration was determined based upon a cholesterol (NuChek Prep, Elysian, MN) standard curve.

2.2.5. Statistics Statistical significance between groups was assessed using a two-tailed Student’s t-test. Phospholipid levels and age in control tissue were correlated using Spearman’s rank correlation. For all comparisons, statistical significance was defined as P,0.05. Statistical tests were done using Instat II (Graphpad, San Diego, CA, USA). All results are presented as means6S.D. unless noted otherwise.

3. Results

3.1. Total phospholipid content The effect of DS on the total phospholipid level was determined in both frontal cortical and cerebellar gray matter from both DS and control subjects. In the frontal cortical gray matter, the total phospholipid levels (nmol / mg protein) were 495644 and 677667 for DS and control subjects, respectively. There was a significant 27% (P5 0.0008) reduction in DS total phospholipid levels relative to control in this brain region. In cerebellar gray matter, the total phospholipid levels were 452691 and 563626 for DS and control subjects, respectively. There was a significant 20% (P50.0068) reduction in DS total phospholipid levels relative to control. Because of the large changes in total phospholipid normalized to protein, the protein to wet weight ratios were calculated for each sample. These ratios were found not to be significant between groups irrespective of brain region. Furthermore, the total protein content did not vary between groups. Collectively, these results are indicative that neither the hydration state nor the protein content varied between the control and DS samples. Thus, these results demonstrate that the change in total phospholipid normalized to protein reflects a decrease in total phospholipid as opposed to alterations in protein content. This large change in total phospholipid content also suggests that the levels of one or more of the individual phospholipid classes may also be decreased.

3.2. Individual phospholipid levels Changes in total phospholipid content do not necessarily reflect similar changes in all phospholipid classes. Therefore, the phospholipid classes were resolved by TLC and

E. J. Murphy et al. / Brain Research 867 (2000) 9 – 18

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Table 2 Phospholipid levels in frontal cortex and cerebellum from control and Down Syndrome brains Phospholipid levels (nmol / mg protein)a Frontal cortex

EtnGpl PtdIns PtdSer ChoGpl CerPCho ] Other a b

Cerebellum

Control

Down Syndrome

227.7614.4 26.863.6 82.7612.1 256.7642.5 62.667.7 20.861.3 n59

158.4612.6* 17.062.9* 58.864.0* 189.0628.4* 55.164.6* 16.465.7* n58

b

Control

Down Syndrome b

213.3615.7 20.462.1 66.466.0 210.367.2 36.364.5 16.466.3 n58

149.4635.9* 13.463.2* 56.768.2* 172.8638.2* 48.766.8* 12.164.4 n55

Phospholipid levels are normalized to total protein (nmol / mg protein) and are expressed as means6S.D. The * signifies statistical significance from control samples as determined using a two-tailed Student’s t-test.

individual phospholipid class levels determined. Surprisingly, in DS frontal cortex there was a marked decrease in the levels of each glycerophospholipid class examined (Table 2). Phosphatidylserine (PtdSer) and ethanolamine glycerophospholipid (EtnGpl) levels were decreased nearly 30% relative to control, with the choline glycerophospholipid (ChoGpl) level decreased 26% compared to control. The phosphatidylinositol (PtdIns) level was decreased 37%, thereby accounting for the observed specific decrease in PtdIns proportions. Similarly, the sphingomyelin (CerPCho) level was decreased 12%. Hence, in ] DS frontal cortex, the level of PtdIns was decreased to a greater extent than that of the other phospholipids, although there was a net decrease in all phospholipid levels. To assess whether these alterations in phospholipid levels were the result of an AD-like process, which would be expected to spare the cerebellum, cerebellar phospholipid levels were analyzed. Similar to frontal cortex, the PtdIns level in DS cerebellum was decreased by a larger extent than were the levels of the other phospholipids.

However, the magnitude of the decrease in the PtdSer (15% decrease) and ChoGpl (18% decrease) levels was less than in the frontal cortex. Unlike frontal cortex, CerPCho levels were increased nearly 34% in the cere] bellum, accounting for the 61% increase found in the proportion of this phospholipid compared to control. Thus, similar to frontal cortex, there was a general decrease in cerebellar phospholipid levels, although the extent of the decrease was not as large as in frontal cortex. Because plasmalogens, a phospholipid subclass containing a vinyl ether linkage at the sn-1 position, have a role in lipid-mediated signal transduction [30,31,36,37], the effect of DS on these phospholipids was also determined. There was no significant change in the choline plasmalogen (PlsCho) level in either DS frontal cortex or cerebellum (Table 3a). However, there was a substantial decrease in the PlsEtn level in both frontal cortex and cerebellum. PlsEtn was decreased by about 35% in both regions relative to control. This decrease in PlsEtn levels were substantially greater than that observed for the other

Table 3 Plasmalogen levels and composition in frontal cortex and cerebellum from control and Down Syndrome brains Plasmalogen levels (nmol / mg protein)a Frontal cortex

PlsEtn PlsCho

Cerebellum

Control

Down Syndrome

Control

Down Syndrome

123.2614.0 23.765.0 n57

80.165.4* 24.169.0 n58

105.369.1 17.366.9 n56

67.1618.8* 19.262.5 n55

Plasmalogen composition (mol %)b Frontal cortex

PlsEtn PlsCho a

Cerebellum

Control

Down Syndrome

Control

Down Syndrome

53.363.7 8.961.0 n57

48.366.6 12.063.9 n58

49.162.2 8.263.0 n56

44.663.0* 11.663.1 n55

Values represent nmoles / mg protein and are expressed as means6S.D. The * signifies statistical significance from control as determined using a two-tailed Student’s t-test, P,0.05. b Values represent the mol % of each respective glycerophospholipid class and represents means6S.D. The * signifies statistical significance from control as determined using a two-tailed Student’s t-test, P,0.05.

E. J. Murphy et al. / Brain Research 867 (2000) 9 – 18

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Table 4 Phospholipid composition in frontal cortex and cerebellum from control and Down Syndrome brains Phospholipid composition (mol %)a Frontal cortex

EtnGpl PtdIns PtdSer ChoGpl CerPCho ] Other a b

Cerebellum

Control

Down Syndrome

33.862.1 4.060.5 12.261.2 37.862.9 9.260.5 3.560.3 n59

32.362.5 3.360.5* 11.861.0 38.362.5 11.260.9* 3.161.1 n58

b

Control

Down Syndrome b

37.761.7 3.660.4 12.161.3 36.662.2 6.861.4 3.560.5 n59

32.962.4* 3.060.2* 12.761.2 38.061.7 11.061.8* 2.560.8 n55

Values represent mol % of total phospholipid and are expressed as means6S.D. The * signifies statistical significance from control samples as determined using a two-tailed Student’s t-test, P,0.05.

phospholipids, except for PtdIns, suggesting that there was a specific decrease in the PlsEtn level in DS brain.

3.4. Cholesterol levels and cholesterol /phospholipid ratio

3.3. Phospholipid composition

The large decrease in phospholipid levels in DS suggest a generalized alteration in lipid metabolism, which might also be reflected in cholesterol levels. Furthermore, because cholesterol levels and the cholesterol / phospholipid ratio are indicators of membrane dynamics [47,65], alterations in this ratio might indicate that compensatory changes occur in cholesterol levels in DS to maintain a normal cholesterol / phospholipid ratio. The mean cholesterol level was not significantly altered in either brain region in DS relative to control; possibly reflecting the large variance in the samples (Table 5). Additionally, there was also no difference in the mean cholesterol / phospholipid ratio between groups (Table 5). A large change in the level of DS brain phospholipid and the lack of a significant change in the cholesterol / phospholipid ratio suggests that there were alterations in cholesterol levels in DS brain that kept this ratio at a value similar to that observed in control brains.

Although in DS frontal cortical and cerebellar gray matter the total phospholipid content and individual phospholipid levels were altered, these data do not provide any information regarding the relative distribution of the individual phospholipid classes. Using the data from Table 2, the phospholipid composition (mol %) of frontal cortex and cerebellar gray matter was determined for both DS and control tissue. Compositional changes in either region were limited. In frontal cortex, changes in phospholipid composition were limited to a 17% decrease in the mol % of PtdIns and a 21% increase in the CerPCho mol % in DS ] compared to control (Table 4). In cerebellum, a similar decrease (17%) was seen in the PtdIns mol %; but this difference was accompanied by a 13% decrease in EtnGpl mol % in DS compared to control cerebellum (Table 4). Part of this decrease in the EtnGpl mol % was accounted for by a 9% decrease in the mole % of PlsEtn in DS compared to control (Table 3b). In contrast to the cerebellum, there was no alteration in the mol % of the EtnGpl subclasses in frontal cortex (Table 3b). Similar to frontal cortex, cerebellar CerPCho mol % were increased 61% in ] DS relative to control (Table 3b). Hence, in both frontal cortex gray matter and cerebellum, PtdIns mol % were decreased and CerPCho mol % were increased in DS brain ] as compared to control brain.

3.5. Age-correlations with phospholipid levels Because of the wide age range in the control samples (Table 1), Spearman’s rank correlation analysis was used to test for significant correlations between age and phospholipid levels. There was no significant correlation in control frontal cortex phospholipid levels with age (Figs. 1 and 2). No change was observed in control cerebellar gray

Table 5 Cholesterol levels and cholesterol:phospholipid ratio in frontal cortex and cerebellum from control and Down Syndrome brains Control

Cholesterol Cholesterol / phospholipid b a b

Frontal cortex a

Cerebellum a

Control

Down Syndrome

Control

Down Syndrome

413678 0.7160.10 n58

358676 0.6860.08 n58

3886149 0.6160.13 n56

288698 0.6260.18 n55

Values represent means6S.D. Cholesterol levels are expressed as nmol / mg protein. The cholesterol / phospholipid ratio represents total nmoles of cholesterol divided by the total nmoles of phospholipid.

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E. J. Murphy et al. / Brain Research 867 (2000) 9 – 18

Fig. 1. Correlation of frontal cortex gray matter phospholipid levels with age. Phospholipid masses are expressed as nmol / mg protein. Correlation analysis was done using Spearman’s Rank Correlation analysis and correlation was considered significant if P,0.05. The abbreviations are as follows: EtnGpl – ethanolamine glycerophospholipids; ChoGpl – choline glycerophospholipids; and PtdSer – phosphatidylserine.

matter phospholipid levels with age (data not shown). These data are indicative that in aging, brain phospholipid levels were maintained at the same level over a wide age range. In DS frontal cortex and cerebellar gray matter, no significant correlation was observed in either cerebellum or frontal cortex (data not shown). As no significant correlation was detected between age and phospholipid levels in either DS or control tissue, data from these two groups can be reliably compared.

4. Discussion Only limited changes in brain phospholipids have been reported in adult DS brain [6,32,54] or in fetal DS brain [7]. However, reported increases in both choline and myoinositol levels in brain and cerebrospinal fluid from young adult DS subjects, suggest that lipid metabolism is altered in DS [46,52], even though increases in catabolic lipid intermediates such as glycerophosphocholine and glycerophosphoethanolamine have not been found [4,8]. To further examine brain phospholipid metabolism in DS, we determined phospholipid composition (mol %) and levels (nmol / mg protein) in brains from older DS subjects and controls. Phospholipid levels were decreased 12–37%

Fig. 2. Correlation of frontal cortex gray matter phospholipid levels with age. Phospholipid masses are expressed as nmol / mg protein. Correlation analysis was done using Spearman’s Rank Correlation and considered significant if P,0.05. The abbreviations are as follows: PtdIns – phosphatidylinositol; CerPCho – sphingomyelin. ]

in frontal cortex gray matter and 15–35% in cerebellum from DS brain. Despite these large changes, the mean cholesterol / phospholipid ratio remained unchanged, suggesting that compensatory changes occur in the DS brain to maintain a normal ratio. We report a large decrease in phospholipid levels in both cerebellum and frontal cortex gray matter from adult DS subjects. In both regions, the PtdIns level was reduced nearly 37% and the PlsEtn level was reduced by 35% (Tables 2 and 3 a). The mol % of these phospholipids also was reduced (Tables 3 b and 4), indicating these two phospholipids were decreased to a greater extent than the other phospholipids. The EtnGpl level has been reported to be reduced (13%) in frontal cortex from adult DS subjects, although reductions in the corresponding PlsEtn levels were not mentioned [6]. No phospholipid change has been reported for myelin in DS, including any change in plasmalogen levels [32]. However, the samples from these studies were obtained from young adult DS subjects (18– 30 years old) and may not have had the same reductions in phospholipids that we observed in brains from older subjects. In platelets isolated from DS subjects (12–30 years old), a statistically insignificant 15–30% decrease in the phospholipid levels is reported [54]. This decrease is similar in magnitude to the statistically significant change that we report in this paper. The mechanisms underlying these decreases in phos-

E. J. Murphy et al. / Brain Research 867 (2000) 9 – 18

pholipid levels are unknown. Others have reported changes in n-3 and n-6 fatty acid levels in fetal and, to a lesser extent, in adult DS brain [6,7]. In synaptosomes prepared from DS brain, polyunsaturated fatty acids are decreased, while only monounsaturated fatty acids are decreased in myelin [50]. The reported changes in n-3 and n-6 fatty acids reflect a decrease in arachidonic acid (20:4 n-6) with an increase in docosahexaenoic acid (22:6 n-3). This discrimination between these two fatty acids might reflect an increase in activation of a phospholipase A 2 selective for arachidonic acid, or an alteration in CoA-dependent and CoA-independent acyl transferase or transacylases [66]. Regardless, the fatty acid results support the idea that lipid metabolism is deranged in DS. A plausible explanation for our observations and published results is a central defect in phospholipid synthesis de novo affecting Kennedy pathway enzymes in DS brain [63]. A general decrease in all phospholipid classes suggests a defect in the initial steps leading to phosphatidic acid formation. In addition, the minimal change in CerPCho levels suggests that CerPCho synthesis is mini] ] mally compromised. Because CerPCho synthesis goes ] through a pathway independent of the Kennedy pathway [60,63], these data support an alteration specifically in the Kennedy pathway affecting the glycerophospholipids. A very low level of lysophospholipids was observed in the DS brain extracts during our HPLC analysis tends to discount the possibility of accelerated phospholipid breakdown by one or more acylhydrolases. This is further supported by reports that phospholipid catabolic products such as glycerophosphocholine and glycerophosphoethanolamine are not increased in DS [4,43]. Additionally, evidence indicates that both PlsEtn and PtdIns are selectively decreased relative to the other phospholipids. Because plasmalogen formation proceeds through both peroxisomal [26,53] and microsomal [25,61,62] steps, one or both of these enzyme steps also may be altered in DS. On the other hand, PtdIns proceeds through a CDP-diacylglycerol intermediate [3,34], which is a critical branch point for glycerophospholipid biosynthesis [28]. The specific decrease in PtdIns suggests that synthesis of PtdIns is compromised in DS. In any case, the significant changes in phospholipid levels could be accounted for by a decrease in general phospholipid synthesis. An increase in lipid peroxidation is another possible explanation for the observed decrease in phospholipid levels. Both superoxide dismutase activity and lipid peroxidation are increased in fetal DS cerebral cortex [5,11]. Although lipid peroxidation products, measured using thiobarbituric acid, are elevated in fetal DS brain, there is no change in polyunsaturated fatty acid content or increased conjugated diene formation [7]. These reported results do not support a massive increase in lipid peroxidation. Furthermore, in brains from adult DS subjects, there is no evidence that superoxide dismutase activity or lipid peroxidation products are altered relative to control [27].

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Vitamin E, an endogenous anti-oxidant found in membranes, is not decreased in DS brain [38]. Although plasmalogens can serve as free radical scavenging molecules [19,39,68], the 35% decrease in ethanolamine plasmalogen suggests a mechanism other than lipid peroxidation. This is supported by the lack of a decrease in total cholesterol levels, because cholesterol is subjected to lipid peroxidation during central nervous system injury [2,15]. A large amount of oxidative stress on the membrane, capable of causing large decreases in phospholipid content, would be expected to decrease vitamin E and cholesterol levels [2,15]. The lack of an effect on vitamin E, and lack of evidence for increased lipid peroxidation in adult DS brain, indicate that there is not enough oxidative stress in adult DS brain to cause the decrease in phospholipid levels that we report. The decrease in phospholipid levels may be the result of an AD-like process occurring in the brain from DS subjects older than 38 years. Phospholipid levels are decreased in AD, although the scope and magnitude of this change is much less pronounced than what we are reporting for DS [18,21–23,57]. One hallmark of lipid metabolic changes in AD is the increase in glycerophosphocholine and glycerophosphoethanolamine levels, two catabolic intermediates from ChoGpl and EtnGpl respectively, which are not changed in DS brain [43]. Furthermore, the cerebellum is usually unaffected in AD [10], but in DS there were large decreases in cerebellar phospholipid levels. Lastly, if the AD-like processes documented in DS brain [1,9,14,48,64] are an age-dependent process, one could speculate that the changes in DS brain phospholipid levels would reflect an age-dependent decrease. However, no correlation between age and phospholipid levels was observed in the DS subjects. Because of the magnitude of the changes as well as our results documenting changes in the cerebellum, it is unlikely that these changes are consistent with an AD-like process. Analysis of brain tissue from younger DS subjects is required to confirm these suggestions. The biological implications of the changes in phospholipid levels are numerous. First, there are fundamental changes in PtdIns and PlsEtn levels relative to the other phospholipids. Changes in plasmalogen proportions are associated with alterations in the critical temperature for membrane formation [21]. Because membrane critical temperature is not affected by changes in membrane protein and is an indicator of membrane stability [20], the decreased plasmalogen levels in DS brain suggest unstable cell membranes. This instability may account for the abnormal electric membrane properties seen in fetal DS dorsal root ganglion neurons [13,42]. While brain membrane acyl chain order has not been reported for DS, both leukocyte and erythrocyte membranes from DS patients display altered acyl chain order, indicative of membrane instability [33,49]. However, in DS brain, the cholesterol to phospholipid ratio is unchanged, suggesting that com-

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E. J. Murphy et al. / Brain Research 867 (2000) 9 – 18

pensatory changes occurred in cholesterol levels to maintain a normal ratio. Furthermore, alterations in PtdIns may cause a decrease in polyphosphoinositide levels, thereby perturbing polyphosphoinositide mediated signal transduction [51]. Ultimately, these changes may result in both altered neuroplasticity and membrane function in DS brain. Lastly, brain phospholipid levels are reported to change [12,55,56] or remain unchanged [17] during normal aging. Several groups agree that changes in phospholipid content occur only after 90 years of age [17,55], although Svennerholm and colleagues also report a progressive decrease in phospholipid levels with increasing age [56]. Others have reported no age-change using whole brain analysis, but indicate that synaptic plasma membrane phospholipid levels do decrease with increasing age [12]. We report no correlation between age with decreased phospholipid levels in brain tissue (Figs. 1 and 2). Hence, we conclude that phospholipid levels were not altered between 22 and 87 years of age. In summary, we report a large decrease in phospholipid levels in DS frontal cortical and cerebellar gray matter. These changes are also consistent with an age-dependent decline in dendritic branching, length and spine frequency [58,59], thus a change in the neuronal membrane surface to volume ratio. Furthermore, a reduction in the amount of dendritic plasma membrane relative to the cell body membrane may account for the compositional changes observed in DS. However, at this time, it is difficult to ascertain whether this change in the cell surface to volume ratio would lead to only a selective decrease in PlsEtn and PtdIns. The general decrease in glycerophospholipid levels also suggest a decrease in the synthesis of phospholipid levels through the Kennedy pathway. PtdIns and PlsEtn appear to be selectively decreased beyond that observed for the other phospholipids, suggesting a secondary defect in the synthetic pathways for PlsEtn and PtdIns, above and beyond the proposed defect in the Kennedy pathway. The lack of increased lysophospholipids in these DS brain regions suggest no increase in acylhydrolase activity. Thus, although the mechanisms causing the decrease in brain phospholipid content in DS brain are unknown, the involvement of the cerebellum clearly indicate that these changes are not the result of Alzheimer disease in these older DS subjects, but instead are the direct result of DS pathology.

Acknowledgements We thank Ruth Seeman for help in the dissection of the brain tissue, Sirkka Lahtivirta for performing several brain autopsies, and Cindy Murphy for typed preparation of the manuscript. This work was supported by a senior fellowship to EJM by the National Research Council.

References [1] G.E. Alexander, A.M. Saunders, J. Szczepanik, T.L. Strassburger, P. Pietrini, A. Dani, M.L. Furey, M.J. Mentis, A.D. Roses, S.I. Rapoport, M.B. Schapiro, Relation of age and apolipoprotein E to cognitive function in Down syndrome adults, NeuroReport 8 (1997) 1835–1840. [2] D.K. Anderson, R.D. Saunders, P. Demediuk, L.L. Dugan, J.M. Braughler, E.D. Hall, E.D. Means, L.A. Horrocks, Lipid hydrolysis and peroxidation in injured spinal cord: partial protection with methylprednisolone or vitamin E and selenium, Central Nervous System Trauma 2 (1985) 257–267. [3] B. Antonsson, Phosphatidylinositol synthase from mammalian tissues, Biochim. Biophys. Acta 1348 (1997) 179–186. [4] J.K. Blusztajn, I.L. Gonzalez-Coviella, M. Logue, J.H. Growdon, R.J. Wurtman, Levels of phospholipid catabolic intermediates, glycerophosphocholine and glycerophosphoethanolamine, are elevated in brains of Alzheimer’s disease but not of Down’s syndrome patients, Brain Res. 536 (1990) 240–244. [5] B.W. Brooksbank, R. Balazs, Superoxide dismutase, glutathione peroxidate and lipoperoxidation in Down’s syndrome fetal brain, Brain Res. 318 (1984) 37–44. [6] B.W.L. Brooksbank, M. Martinez, Lipid abnormalities in the brain in adult Down’s syndrome and Alzheimer’s disease, Mol. Chem. Neuropath. 11 (1989) 157–185. [7] B.W.L. Brooksbank, M. Martinez, R. Balazs, Altered composition of polyunsaturated fatty acyl groups in phosphoglycerides of Down’s syndrome fetal brain, J. Neurochem. 44 (1985) 869–874. [8] B.W.L. Brooksbank, J. McGovern, Gangliosides in the brain in adult Down’s syndrome and Alzheimer’s disease, Mol. Chem. Neuropath. 11 (1989) 143–156. [9] K.L. Brugge, S.L. Nichols, D.P. Salmon, L.R. Hill, D.C. Delis, L. Aaron, D.A. Trauner, Cognitive impairment in adults with Down’s syndrome: similarities to early cognitive changes in Alzheimer’s disease, Neurology 44 (1994) 232–238. [10] A. Brun, L. Gustafson, Distribution of cerebral degeneration in Alzheimer’s disease, Arch. Psychiatr. Nervenkrank. 223 (1976) 15–33. [11] J. Busciglio, B.A. Yankner, Apoptosis and increased generation of reactive oxygen species in Down’s syndrome neurons in vitro, Nature 378 (1995) 776–779. [12] G. Calderini, A.C. Bonetti, A. Battistella, F.T. Crews, G. Toffano, Biochemical changes of rat brain membranes with aging, Neurochem. Res. 8 (1983) 483–492. [13] P. Caviedes, B. Ault, S.I. Rapoport, The role of altered sodium currents in action potential abnormalities of cultured dorsal root ganglion neurons from trisomy 21 (Down syndrome) human fetuses, Brain Res. 510 (1990) 229–236. [14] J.T. Coyle, M.L. Oster-Granite, J.D. Gearhart, The neurobiologic consequences of down syndrome, Brain Res. Bull. 16 (1986) 773– 787. [15] P. Demediuk, R.D. Saunders, D.K. Anderson, E.D. Means, L.A. Horrocks, Membrane lipid changes in laminectomized and traumatized cat spinal cord, Proc. Natl. Acad. Sci. USA 82 (1985) 7071– 7075. [16] L.L. Dugan, P. Demediuk, C.E. Pendley II, L.A. Horrocks, Separation of phospholipids by high pressure liquid chromatography: all major classes including ethanolamine and choline plasmalogens, and most minor classes, including lysophosphatidylethanolamine, J. Chromatogr. 378 (1986) 317–327. [17] C. Edlund, M. Soderberg, K. Kristensson, G. Dallner, Ubiquinone, dolichol, and cholesterol metabolism in aging and Alzheimer’s disease, Biochem. Cell Biol. 70 (1992) 422–428. [18] A.A. Farooqui, S.I. Rapoport, L.A. Horrocks, Membrane phospholipid alterations in Alzheimer’s disease: deficiency of ethanolamine plasmalogens, Neurochem. Res. 22 (1997) 523–527.

E. J. Murphy et al. / Brain Research 867 (2000) 9 – 18 [19] T.A. Foglia, E. Nungesser, W.N. Marmer, Oxidation of 1-o-(alk-1enyl)-2,3-di-o-acylglycerols: models for plasmalogen oxidation, Lipids 23 (1988) 430–434. [20] N.L. Gershfeld, L. Ginsberg, Membrane bilayer instability as a pathogenetic mechanism for neurological disease, Reviews in the Neurosciences 6 (1995) 1–13. [21] L. Ginsberg, J.R. Atack, S.I. Rapoport, N.L. Gershfeld, Regional specificity of membrane instability in Alzheimer’s disease brain, Brain Res. 615 (1993) 355–357. [22] L. Ginsberg, S. Rafique, J.H. Xuereb, S.I. Rapoport, N.L. Gershfeld, Disease and anatomic specificity of ethanolamine plasmalogen deficiency in Alzheimer’s disease brain, Brain Res. 698 (1995) 223–226. [23] L. Ginsberg, J.H. Xuereb, N.L. Gershfeld, Membrane instability, plasmalogen content, and Alzheimer’s disease, J. Neurochem. 70 (1998) 2533–2538. [24] K.L. Goodison, I.M. Parhad, C.L. White, A.A. Sima, A.W. Clark, Neuronal and glial gene expression in neocortex of Down’s syndrome and Alzheimer’s disease, J. Neuropathol. Exp. Neurol. 52 (1993) 192–198. [25] A.K. Hajra, J.E. Bishop, Glycerolipid biosynthesis in peroxisomes via the acyl dihydroxyacetone phosphate pathway, Ann. NY Acad. Sci. 386 (1992) 170–182. [26] A.K. Hajra, C.L. Burke, C.L. Jones, Subcellular localization of acyl coenzyme A: dihydroxyacetone phosphate acyltransferase in rat liver peroxisomes (microbodies), J. Biol. Chem. 254 (1979) 10896– 10900. [27] M. Hayn, K. Kremser, N. Singewald, N. Cairns, M. Nemethova, B. Lubec, G. Lubec, Evidence against the involvement of reactive oxygen species in the pathogenesis of neuronal death in Down’s syndrome and Alzheimer’s disease, Life Sci. 59 (1996) 537–544. [28] A.M. Heacock, B.W. Agranoff, CDP-diacylglycerol synthase from mammalian tissues, Biochim. Biophys. Acta 1348 (1997) 166–172. [29] E. Hedegaard, B. Jensen, Nano-scale densitometric quantitation of phospholipids, J. Chromatogr. 225 (1981) 450–454. [30] L.A. Horrocks, H.W. Harder, R. Mozzi, G. Goracci, E. Francescangeli, S. Porcellati, G.G. Nenci, Receptor mediated degradation of choline plasmalogen and glycerophospholipid methylation: a new hypothesis, in: L. Freysz, H. Dreyfus, R. Massarelli, S. Gatt (Eds.), Enzymes of Lipid Metabolism, Vol. 2, Plenum Press, New York, 1986, pp. 707–711. [31] L.A. Horrocks, Y.K. Yeo, H.W. Harder, R. Mozzi, G. Goracci, Choline plasmalogens, glycerophospholipid methylation, and receptor-mediated activation of adenylate cyclase, in: P. Greengard, G.A. Robinson (Eds.), Advances in Cyclic Nucleotide Protein Phosphorylation Research, Vol. 20, Raven Press, New York, 1986, pp. 263–292. [32] R.C. Johnson, C.M. McKean, S.N. Shah, Fatty acid composition of lipids in cerebral myelin and synaptosomes in phenylketonuria and Down syndrome, Arch. Neurol. 34 (1977) 288–294. [33] A. Kantar, P.L. Giorgi, G. Curatola, R. Fiorini, Alterations in erythrocyte membrane fluidity in children with trisomy 21: a fluorescence study, Biol. Cell 75 (1992) 135–138. [34] O. Kuge, M. Nishijima, Phosphatidylserine synthase I and II of mammalian cells, Biochim. Biophys. Acta 1348 (1997) 151–156. [35] O. Labudova, S. Fang-Kircher, N. Cairns, H. Moenkemann, K. Yeghiazaryan, G. Lubec, Brain vasopressin levels in Down syndrome and Alzheimer’s disease, Brain Res. 806 (1998) 55–59. [36] J. McHowat, S. Liu, Interleukin-1(beta) stimulates phospholipase A2 activity in adult rat ventricular myocytes, Am. J. Physiol. 272 (Cell Phys. 41) (1997) C450–C456. [37] J. McHowat, S. Liu, M.H. Creer, Selective hydrolysis of plasmalogen phospholipids by Ca21-independent PLA2 in hypoxic ventricular myocytes, Am. J. Physiol. 274 (Cell Phys. 43) (1998) C1727–C1737. [38] T. Metcalfe, D.M. Bowen, D.P.R. Muller, Vitamin E concentrations

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48] [49]

[50]

[51] [52]

[53]

[54] [55]

[56]

[57]

17

in human brain of patients with Alzheimer’s disease, fetuses with Down’s syndrome, centenarians, and controls, Neurochem. Res. 14 (1989) 1209–1212. O.H. Morand, R.A. Zoeller, C.R.H. Raetz, Disappearance of plasmalogens from membranes of animal cells subjected to photosensitized oxidation, J. Biol. Chem. 263 (1988) 11597–11606. E.J. Murphy, T.A. Rosenberger, L.A. Horrocks, Separation of neutral lipids by high performance liquid chromatography: quantification by ultraviolet, light scattering and fluorescent detectors, J. Chromatogr. B 685 (1996) 9–14. E.J. Murphy, R. Stephens, M. Jurkowitz-Alexander, L.A. Horrocks, Acidic hydrolysis of plasmalogens followed by high-performance liquid chromatography, Lipids 28 (1993) 565–568. K. Nieminen, B.A. Suarez-Isla, S.I. Rapoport, Electrical properties of cultured dorsal root ganglion neurons from normal and trisomy 21 human fetal tissue, Brain Res. 474 (1988) 246–254. R.M. Nitsch, J.K. Blusztajn, A.G. Pittas, B.E. Slack, J.H. Growdon, R.J. Wurtman, Evidence for a membrane defect in Alzheimer disease brain, Proc. Natl. Acad. Sci. USA 89 (1992) 1671–1675. P. Pietrini, A. Dani, M.L. Furey, G.E. Alexander, U. Freo, C.L. Grady, M.J. Mentis, D. Mangot, E.W. Simon, B. Horwitz, J.V. Haxby, M.B. Schapiro, Low glucose metabolism during brain stimulation in older Down’s syndrome subjects at risk for Alzheimer’s disease prior to dementia, Am. J. Psychiatry 154 (1997) 1063– 1069. G. Rouser, A. Siakotos, S. Fleischer, Quantitative analysis of phospholipids by thin layer chromatography and phosphorus analysis of spots, Lipids 1 (1969) 85–86. M.B. Schapiro, J.R. Atack, I. Hanin, C. May, J.V. Haxby, S.I. Rapoport, Lumbar cerebrospinal fluid choline in healthy aging and in Down’s syndrome, Arch. Neurol. 47 (1990) 977–980. F. Schroeder, A.A. Frolov, E.J. Murphy, B.P. Atshaves, L. Pu, W.G. Wood, W.B. Foxworth, A.B. Kier, Recent advances in membrane cholesterol domain dynamics and intracellular cholesterol trafficking, Proc. Soc. Exp. Biol. Med. 213 (1996) 150–177. M.S. Schweber, Alzheimer’s disease and Down’s syndrome, Prog. Clin. Biol. Res. 317 (1989) 247–267. R.B. Scott, J.M. Collins, P.A. Hunt, Alzheimer’s disease and Down’s syndrome: leukocyte membrane fluidity alterations, Mech. Aging Develop. 75 (1994) 1–10. S.N. Shah, Fatty acid composition of lipids of human brain myelin and synaptosomes: changes in phenylketonuria and Down’s syndrome, J. Biochem. 10 (1979) 477–482. S.B. Shears, Review: The versatility of inositol phosphates as cellular signals, Biochim. Biophys. Acta 1436 (1998) 49–67. H.U. Shetty, M.B. Schapiro, H.W. Holloway, S.I. Rapoport, Polyol profiles in Down syndrome: myo-inositol, specifically, is elevated in the cerebrospinal fluid, J. Clin. Invest. 95 (1995) 542–546. H. Singh, K. Beckman, A. Poulos, Exclusive localization in peroxisomes of dihydroxyacetone phosphate acyltransferase and alkyldihydroxyacetone phosphate synthase in rat liver, J. Lipid Res. 34 (1993) 467–477. K. Strynadka, E.E. McCoy, Phospholipid composition of Down’s syndrome and normal platelets, Clin. Biochem. 11 (1978) 35–37. L. Svennerholm, K. Bostrom, C.G. Helander, B. Jungbjer, Membrane lipids in the aging human brain, J. Neurochem. 56 (1991) 2051–2059. L. Svennerholm, K. Bostrom, B. Jungbjer, L. Olsson, Membrane lipids of adult human brain: lipid composition of frontal and temporal lobe in subjects of age 20 to 100 years, J. Neurochem. 63 (1994) 1802–1811. L. Svennerholm, C.G. Gottfries, Membrane lipids, selectively diminished in Alzheimer brains, suggest synapse loss as a primary event in early-onset form (Type I) and demyelination in late-onset form (Type II), J. Neurochem. 62 (1994) 1039–1047.

18

E. J. Murphy et al. / Brain Research 867 (2000) 9 – 18

[58] S. Takashima, A. Ieshima, H. Nakamura, L.E. Becker, Dendrites, dementia and the Down syndrome, Brain Dev. 11 (1989) 131–133. [59] S. Takashima, K. Iida, T. Mito, M. Arima, Dendritic and histochemical development and ageing in patients with Down’s syndrome, J. Intellect. Disabil. Res. 38 (1994) 265–273. [60] G.A. Thompson, Jr., Phospholipid metabolism in animal tissues, in: G.B. Ansell, J.N. Hawthorne, R.M.C. Dawson, (Eds.), Form and Function of Phospholipids (B.B.A. Library, Vol. 3), Elsevier Scientific Publishing Co., Amsterdam, 1973, pp. 67–96. [61] H. van den Bosch, G. Schrakamp, D. Hardeman, A.W.M. Zomer, R.J.A. Wanders, R.B.H. Schutgens, Ether lipid synthesis and its deficiency in peroxisomal disorders, Biochimie 75 (1993) 183–189. [62] H. van den Bosch, R.B.H. Schutgens, R.J.A. Wanders, J.M. Tager, Biochemistry of peroxisomes, Ann, Rev. Biochem. 61 (1992) 157– 197. [63] H. van den Bosch, D.E. Vance, Editorial, Biochim. Biophys. Acta 1348 (1997) 1–2.

[64] K.E. Wisniewski, H.M. Wisniewski, G.Y. Wen, Occurrence of neuropathological changes and dementia of Alzheimer’s disease in Down’s syndrome, Ann. Neurol. 17 (1985) 278–282. [65] W.G. Wood, F. Schroeder, N.A. Avdulov, S.V. Chochina, U. Igbauboa, Recent advances in brain cholesterol dynamics: transport, domains, and Alzheimer’s disease, Lipids 34 (1999) 225–234. [66] A. Yamashita, T. Sugiura, K. Waku, Acyltransferases and transacylases involved in fatty acid remodeling of phospholipids and metabolism of bioactive lipids in mammalian cells, J. Biochem. 122 (1997) 1–16. [67] C.M. Yates, J. Simpson, A. Gordon, Regional brain 5-hydroxytryptamine levels are reduced in senile Down’s syndrome as in Alzheimer’s disease, Neurosci. Lett. 65 (1986) 189–192. [68] R.A. Zoeller, O.H. Morand, C.R.H. Raetz, A possible role for plasmalogens in protecting animal cells against photosensitized killing, J. Biol. Chem. 263 (1988) 11590–11596.