Phospholipid transfer protein (PLTP) deficiency impaired blood–brain barrier integrity by...

©2004 FASEB

The FASEB Journal express article 10.1096/fj.04-2400fje. Published online December 2, 2004.

Phospholipid transfer protein (PLTP) deficiency reduces brain vitamin E content and increases anxiety in mice. Catherine Desrumaux,* Pierre-Yves Risold,† Henri Schroeder,‡ Valérie Deckert,* David Masson,* Anne Athias,* Hélène Laplanche,* Naig Le Guern,* Denis Blache,* Xian-Cheng Jiang,§ Alan Tall,║ Didier Desor,‡ and Laurent Lagrost* *Laboratoire de Biochimie des Lipoprotéines, INSERM U498-IFR100, Dijon, France; †Laboratoire d’Histologie, Faculté de Médecine et de Pharmacie, Besançon, France; ‡Laboratoire de Neurosciences Comportementales, Faculté des Sciences, Vandoeuvre les Nancy, France; §State University of New York, Downstate Medical Center, Brooklyn, 11203; ║Department of Medicine, Columbia University, New York, New York 10032

Corresponding author: Dr Laurent Lagrost INSERM U498, Faculté de Médecine 7, Bd Jeanne d’Arc BP 87900 21079 DIJON Cedex. E-mail: [email protected] ABSTRACT Vitamin E supplementation constitutes a promising strategy in the prevention of neurodegenerative diseases. Here, we show that a phospholipid transfer protein (PLTP) is widely expressed in the brain where it appears to function as a transfer factor for α-tocopherol, the main isomer of vitamin E. PLTP deficiency results in significant depletion of brain α-tocopherol in both homozygous (–30.1%, P<0.0002) and heterozygous (–18.0%, P<0.05) PLTP knocked-out mice. α-tocopherol depletion in PLTP-deficient homozygotes is associated with the elevation of lipofuscin (+25% and +450% increases in cortex and substantia nigra, respectively), cholesterol oxides (+54.5%, P<0.05), and cellular peroxides (+32.3%, P<0.01) in the brain. Complete PLTP deficiency in homozygotes is accompanied by increased anxiety as shown by fewer entries (8.3% vs. 44.4% in controls, P<0.01) and less time spent (1.7% vs. 41.3% in controls, P<0.05) in the open arms of an elevated plus-maze, in the absence of locomotor deterioration. Thus, the vitamin E transfer activity of PLTP appears to be a key process in preventing oxidative damage in the brain, and PLTP-deficient mice could be a new model of the contribution of oxidative brain injury in the etiology of neurodegenerative diseases.

Key words: tocopherol • neurodegenerative diseases • α-tocopherol • PLTP-deficient mice

everal recent findings support that oxidative stress plays a key role in the pathogenesis of neurodegenerative diseases (1–9), and the use of antioxidants is currently arising as a promising strategy in the prevention and/or treatment of these disorders (10–17). Although

a number of mouse models have been created to study the relationship of high oxidative stress with aging and neurodegeneration (18–24), most of them did not directly address the contribution of selective alterations in vitamin E status to the initiation of the oxidative damage. Only in α-tocopherol transfer protein (αTTP)-deficient mice, complete vitamin E deficiency per se was proven to lead to neurological disorders (25). However, the reduction in vitamin E

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content in αTTP-deficient mice tissues was drastic and ubiquitous; the associated ataxic phenotype was early and severe; and the marked rise in the oxidative stress went far beyond the subtle, but sustained, alterations that supposedly occur along the aging process. In addition, αTTP is mainly a liver protein, which is not expressed in the brain with the exclusion of specific states of severe systemic vitamin E deficiency (26). The plasma phospholipid transfer protein (PLTP) transfers phospholipids between plasma lipoproteins and functions intracellularly in hepatocytes in the biosynthesis of apoB-containing lipoproteins (27–29). In addition, the entry and cellular incorporation of vitamin E might well constitute a key function of PLTP in vivo, although the exact molecular mechanism underlying the PLTP-mediated transfer of α-tocopherol between lipoproteins, as well as between lipoproteins and tissues remains to be fully elucidated. Recently, genetically engineered mice with PLTP deficiency were shown to accumulate vitamin E in circulating apoB-lipoprotein-containing lipoproteins at the expense of the vascular wall (30). While resulting higher antioxidative protection of plasma lipoproteins contributed to reduced atherosclerotic lesions in PLTP-deficient (PLTP−/−) mice (28), consequences of PLTP deficiency on the vitamin E content, the oxidative status, and the function of the brain─the richest source of vitamin E in the body─are unknown. Interestingly, PLTP is active in all the vertebrate species studied so far (31), and in particular, it is expressed at high levels in the brain (32–34), which has elevated vitamin E requirements.

METHODS

Mice

PLTP knocked-out (PLTP−/−) mouse females, age-matched PLTP+/− mouse females and wild-type (PLTP+/+) C57BL/6 mouse females were used in the present study. The mice had free access to water and food, and they were fed a standard chow diet.

For histological analyses, 20 mice (10 PLTP+/+ and 10 PLTP−/−) were deeply anesthetized with pentobarbital, and then perfused through the ascending aorta with 50 ml 0.9% NaCl followed by 150 ml ice-cold 4% paraformaldehyde (PFA) fixative in 0.1 M phosphate-buffered saline (PBS) containing 10 µM butylated hydroxytoluene (BHT). The brains were removed, postfixed in the same fixative for several hours at 4°C, immersed overnight at 4°C in a 15% sucrose solution, and then quickly frozen over liquid nitrogen.

For lipid assays and measurements of lipid peroxidation, mice were sacrificed with CO2, brains were harvested, rinsed in physiologic saline solution, and immediately frozen in liquid nitrogen. Immediately before the assays, brains were homogenized in NaCl 0.9% using a T8 Ultra-Turrax.

Histological analyses

Immunohistochemistry

The brains of 8 mice (4 PLTP+/+ and 4 PLTP−/−; 6 to 12 month old) were serially cut into 10-µm coronal sections on a cryostat-microtome, mounted on gelatinated slides and stored at –40°C until treatment. After rinsing in PBS containing 0.3% Triton X100, sections were incubated for 12 h at room temperature with rabbit anti-PLTP antiserum (Abcam Ltd, Cambridge, United Kingdom) diluted 1/100 in PBS containing 0.3% Triton X100, 1% Bovine Serum Albumin, 10% lactoproteins and 0.01% sodium azide. The labeling was then revealed through an indirect


immunofluorescence procedure using a secondary antibody conjugated to Alexa Fluor (1/400, Molecular Probes – Interchim, France) for 2 h at room temperature.

Lipofuscin granules quantitation

The intraneuronal lipopigment corresponding to lipofuscin was examined in unstained 30-µm-thick sections from 12 brains (6 PLTP+/+, 6 PLTP−/−; from 18-month-old mice) with a 20× objective. Lipopigment autofluorescence was obtained by epi-illumination from a mercury arc lamp. All measurements were performed on an Olympus BX51 fluorescence microscope, through a DP 50 Olympus camera, and using the analySIS® software (Soft Imaging System-Olympus, France). Two brain areas were analyzed: the cerebral cortex at the level of the primary somatomotor field (6 to 8 consecutive sections/brain), and the pars reticulata of the substantia nigra (4 consecutive sections/brain). Using fixed parameters throughout the whole experiment, the surface of the autofluorescent particles were calculated by the software, and their mean surfaces between PLTP+/+ and PLTP−/− for each brain area were compared by the Mann-Whitney U-test.

Lipid Assays

Alpha-tocopherol

α-tocopherol was assayed in brain homogenates by liquid chromatography-mass spectrometry (LC-MS). The extraction procedure was conducted as described previously (35). Briefly, 1 ml of ethanol and 300 µl of 10 M KOH were added, and saponification was conducted at 80°C for 30 min with intermittent shaking. The saponified solution was cooled down on ice water, and it was mixed with 4 ml of hexane, 2 ml of distilled water, and 10 µl of a 100 µg/ml ethanolic solution containing tocol (Spiral, France) as an internal standard. The mixture was shaken vigorously for 1 min, and the upper layer was collected after low-speed centrifugation and evaporated. The extract was finally dissolved in 100 µl of methanol containing 5 mM ammonium acetate. -Tocopherol was analyzed by LC-MS on a Nucleosil C18/5 µm, 2.0 × 250 mm column (Macherey-Nagel, Düren, Germany) using 5 mM ammonium acetate in methanol as the eluent at a flow rate of 0.4 ml/min. Positive ion electrospray ionization-mass spectrometry was performed on an MSD 1100 mass spectrometer (Agilent Technology, Waldbronn, Germany). The voltages of the aperture and capillary were set up at 80 and 3500V, respectively, and the flow rate of the drying gas was 8 L/min. Ions at m/z 502 and 460 were used to measure -tocopherol and tocol, respectively. -Tocopherol level was determined by comparison with a standard curve that was obtained with known amounts of -tocopherol (Fluka 89550).

Sterols

Cholesterol and cholesterol derivatives oxidized in position 7 were assayed in brain homogenates by gas chromatography-mass spectrometry (GC-MS). The extraction procedure was conducted as described previously (36). Briefly, homogenates were diluted in chloroform/methanol (2/1, v/v) solution containing 0.01% BHT (v/w). Epicoprostanol and 19-hydroxycholesterol (Sigma, St Louis, MO, USA) were added as internal standards for cholesterol and oxysterols, respectively. After addition of BHT (50 µg/ml) and EDTA (50 µg/ml), the mixture was centrifuged at 1800 rpm at 4°C for 5 min. The organic phase was washed twice with methanol/saline (1/1, v/v) solution and centrifuged at 1800 rpm at 4°C for 5 min. Samples were


then subjected to alkaline hydrolysis with 0.35 M KOH for 2 h at room temperature. The reaction mixture was adjusted to pH 7 with phosphoric acid and centrifuged at 1800 rpm for 5 min. After solvent evaporation, 300 µl N,O-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane (4/1, v/v) (Pierce, Rockford, IL, USA) were added, and samples were incubated at 60°C for 30 min to form trimethylsilyl ethers. After evaporation, the residue was dissolved in 100 µl hexane for GC-MS analysis. GC-MS was performed using a Hewlett Packard HP6890 GC equipped with an HP7683 Injector and a HP5973 mass selective detector. Chromatography was performed using a HP-5MS fused silica capillary column (30 m × 0.25 mm inner diameter, 0.25-µm film thickness, Agilent Technologies). GC-MS conditions were as follows: carrier gas, helium at a flow-rate of 1.1 ml/min; injector temperature, 250°C; oven temperature, 180°C increased at 10°C/min to 260°C, then at 1°C/min to 280°C and held for 5 min. The mass spectrometer was operated under electron impact mode with an electron energy of 70 eV. The ion source temperature and the quadrupole temperature were 230°C and 150°C, respectively. The ions used for analysis were epicoprostanol, 370 m/z; 7α-hydroxycholesterol, 456 m/z; cholesterol, 368 m/z; 19-hydroxycholesterol, 353 m/z; 7β-hydroxycholesterol, 456 m/z; and 7-ketocholesterol, 472 m/z. Calibration curves were obtained by using authentic standards extracted with the method used for cell samples.

Measurement of peroxide levels

Brain peroxide levels were determined by measuring the oxidation rate of dichlorofluorescein diacetate (DCFH-DA) into the fluorescent product dichlorofluorescein (DCF). Brain homogenates were diluted in Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 3.6 mM NAHCO3, 2 mM CaCl2, 10 mM D-Glucose, 5 mM HEPES) and incubated 15 min in darkness in the presence of 10 µM DCFH-DA. Fluorescence intensity (λexc 485 nm, λem 530 nm) was read in a Victor II Wallac spectrofluorimeter. DCF amounts in samples were calculated by comparison with a standard curve that was obtained with known amounts of DCF.

Neurobehavioral analysis

Locomotor activity

Locomotor activity was assessed in a circular open-field platform (diameter, 50 cm; area divided in 36 squares). After a one-minute adaptation period, mice were placed individually into the open field. Activity was videotaped for 5 min, and results were expressed as total number of squares crossed.

Locomotor coordination

The apparatus (Locotronic® Intelli-Bio, France) was composed of a starting box (10×5×20 cm, white plasticine walls, illuminated by a cold white light), a corridor (75×5×20 cm), and an arrival box (10×5×20 cm, black plasticine walls). The corridor was a flat ladder (bars: 3-mm diameter, spaced by 7 mm); three infrared sensors were placed above and under each bar space. The sensors were read 100 times per second, allowing the position of the animal to be monitored and the number of missteps and their duration to be computed.


Anxiety

Anxiety levels were assessed in a blind fashion with an elevated plus-maze consisting of two open arms (25×5 cm) and two closed arms (25×5×20 cm). The maze was 30 cm above the floor. In this system, animals were allowed to spend their time either in the safe, closed areas, or in the open areas. Mice were placed individually in the center of the maze, facing an open arm, and they were allowed free access to the arms for 5 min. Standard measurements taken were the frequency of open and closed arm entries and the time spent by the animals in open and closed sections of the maze.

RESULTS

PLTP distribution and vitamin E content in mouse brain

Immunohistochemical analysis revealed a high level of PLTP staining in the brain of PLTP+/+ mice, whereas there was no PLTP signal in the brain of PLTP−/− homozygotes (Fig. 1). The expression pattern indicates a widespread distribution of mouse PLTP among most brain structures, with a strong signal in the choroid plexus (Fig. 1). PLTP deficiency is accompanied by a significant, 30.1% decrease in the vitamin E content of the brain from PLTP−/− mice, with a lower, but still significant, 18.0% decrease in PLTP+/− heterozygotes as compared with PLTP+/+ controls (Fig. 2).

Oxidative stress markers in PLTP−/− and PLTP+/+ mouse brains

Low vitamin E content of PLTP−/− brain is associated with the elevation of oxidative stress markers. The mean index of oxidative stress of mouse brain, as measured by the oxidation rate of DCFH-DA into the fluorescent product DCF, is increased by 32.3% (P<0.01) in PLTP−/− mice, as compared with PLTP+/+ controls (Table 1). In addition, PLTP deficiency leads to the accumulation of brain cholesterol derivatives oxidized at position 7, including 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol (+54.5%, P<0.05), while the unsaturated fatty acid to total fatty acid ratio is not affected (Table 1). In further support of a worsening of chronic oxidative stress in PLTP−/− mouse brain, and as previously observed in various models of aging and age-related neurodegenerative disorders, the amount of lipofuscin, that is, an end-product of the reaction of lipid peroxides with proteins (37, 38), is significantly higher in the cortex (+25%, P<0.005) and the subtantia nigra (+450%, P<0.0001) of PLTP−/− mice as compared with PLTP+/+ controls (Fig. 3). Lipofuscin accumulation in PLTP−/− mouse brain is likely a direct result of α-tocopherol deficiency of brain tissue since lipofuscin also appears in degenerating neurons of αTTP-deficient mice or rats fed a vitamin E-deficient diet, and it returns to normal upon vitamin E dietary supplementation (25, 39).

Neurobehavioral analysis

Previous studies demonstrated that a systemic α-tocopherol deficiency in mice and humans, resulting from mutation in the αTTP gene is associated with neurological dysfunctions (25, 40, 41). In the present study, PLTP-deficient mice showed evidence of markedly increased anxiety, as demonstrated using the elevated plus-maze test. When subjected to this paradigm, PLTP−/− mice made fewer entries (8.3% vs. 44.4% for PLTP+/+ mice, P<0.01) and spent less time (1.7% vs. 41.3% for PLTP+/+ mice, P<0.05) in the open arms of the elevated plus-maze (Fig. 4).


However, unlike αTTP-deficient mice, in which the systemic and complete α−tocopherol-deficient trait led to abnormal motor performance and severe ataxia in addition to anxiety (8, 16), PLTP−/− mice showed no evidence of disorders in activity and neuromotor coordination, as observed by similar open-field activity (total number of entries, 310 (268–366) and 290 (246–363) (median and quartiles) for PLTP+/+ and PLTP−/− mice, respectively, nonsignificant, n.s.), as well as identical performance in the Locotronic apparatus (run time, 12.8 s (10.9–20.7) and 16.6 s (11.0-22.7) (median and quartiles) for PLTP+/+ and PLTP−/− mice, respectively, n.s.).

DISCUSSION

Neurodegenerative disorders are multifactorial in nature, and oxidative stress (1–9), genetic factors (42), and living habits contribute synergistically to cerebral aging and dementia. Overexpression or targeted disruption of specific genes in transgenic animals has offered a convenient way to determine the contribution of one given factor to the processes of aging and neurodegeneration (18–24). For instance, overexpression of either amyloid-β peptide or apolipoprotein E4, as well as a blockade of presenilin expression in the mouse, has been shown to constitute relevant Alzheimer’s disease models (21–24). However, there is still a paucity of well-suited animal models that allow researchers to address selectively the well-recognized contribution of oxidative injury to the etiology of aging and neurodegeneration. In this context, controlled alteration in the incorporation and distribution of the lipid-soluble vitamin E antioxidant into the brain tissue is likely to constitute a relevant approach. In support of the latter view, it was recently reported that early vitamin E supplementation in young mice reduces amyloid-β peptide levels and amyloid deposition in a transgenic model of Alzheimer’s disease (43). In the present study, we show that complete PLTP deficiency in PLTP knocked-out homozygotes is associated with a significant decrease in brain vitamin E content, producing a chronic and moderate oxidative stress. These observations come in addition to previous studies which demonstrated that complete PLTP deficiency leads to a profound decrease in HDL phospholipids and apolipoprotein A1, and to the emergence of structurally abnormal HDL particles (44). Not only complete PLTP deficiency, but also partial PLTP deficiency in PLTP knocked-out heterozygotes, is characterized by a significant reduction in brain vitamin E content, constituting the first evidence for a clear phenotype of partial PLTP deficiency in the mouse. Because earliest observations reported no change in lipoprotein metabolism of PLTP+/− mice as compared with PLTP+/+ controls (44), the contribution of the derangement of HDL itself to the alteration in vitamin E distribution in this situation is unlikely. Overall, it appears that the entry and cellular incorporation of vitamin E into the brain might well constitute a dominant, key function of PLTP in vivo. Whereas remodeling of HDL from cerebrospinal fluid, cholesterol efflux from neurons, redistribution of phospholipid species, and removal of cholesterol oxides from brain might still be directly affected by PLTP, neither brain cholesterol nor brain phospholipids, [i.e., two other amphipathic compounds transferred by PLTP] are modified by PLTP deficiency in the mouse (personal data). Interestingly, the PLTP-deficient trait led to a significant decrease in tissue α-tocopherol content, but not in the vitamin E concentration of PLTP−/− mouse plasma (30). The latter point is in contrast with the severe α-tocopherol deficiency, which is known to occur at a systemic level in αTTP-deficient homozygous mice, with no detectable α-tocopherol levels in the plasma and tissues including cortex, cerebellum, and spinal cord (25). Thus, the present data suggest that in contrast to αTTP, which is mainly involved at a systemic level in the early lipoprotein incorporation and secretion of α−tocopherol by the liver, PLTP acts at a distal level as a local transporter of vitamin E, offering a putative,


previously unreported route of transfer of α-tocopherol across the blood–brain barrier. Whether the level of PLTP expression may help in predicting the impact of vitamin E supplementation in the prevention and treatment of neurodegenerative disorders will deserve particular attention.

In the present study, the complete PLTP-deficient trait resulting in only partial α-tocopherol depletion in the brain was characterized by a significant increase in anxiety as shown by fewer entries and less time spent in the open arms of an elevated plus-maze (Fig. 4). In contrast however, no evidence of musculoskeletal alterations was noted, as observed by similar open-field activity, as well as normal muscular and locomotor coordination. The latter point comes in contrast with observations in αTTP-deficient mice in which the systemic and complete α-tocopherol-deficient trait led to abnormal motor performance and severe ataxia in addition to anxiety. Moreover, and unlike αTTP-deficient mice, PLTP−/− mice show a peculiar sensitivity to moderate α−tocopherol deficiency. Indeed, the partial restoration of brain vitamin E content in αTTP-deficient mice, not exceeding 10% of normal, was sufficient to reverse detectable neurological symptoms (25), whereas abnormal behavior of PLTP−/− mice is observable with only a 30% decrease in brain α−tocopherol content (Fig. 2). These observations suggest that a subtle redistribution of α-tocopherol pools through the PLTP-mediated transfer reaction may be of major pathophysiological relevance within the central nervous system, and PLTP−/− mice may be a new model to analyze the contribution of partial brain vitamin E depletion to the etiology of neurodegenerative diseases. A significant increase in brain PLTP mRNAs has been recently reported in patients with established Alzheimer’s disease, but not in patients in the earliest phase of the disease, suggesting that induction of PLTP could represent an adaptive response to oxidative stress in this condition (45).

Recently, decreased atherosclerosis susceptibility has been described in PLTP−/− mice (28), and independent studies reported increased atherosclerosis lesions in transgenic mice overexpressing PLTP (46–48). These observations in genetically engineered mice provide a rationale to the inverse, independent relationship between plasma PLTP levels and the risk of coronary artery disease in humans (49). Results of the present study suggest that forthcoming evaluations of PLTP inhibition as a new cardioprotective strategy should ideally integrate possible consequences on vitamin E in the central nervous system.

ACKNOWLEDGMENTS

This study was supported by INSERM, the Conseil Régional de Bourgogne, the Université de Bourgogne and the Fondation de France. The authors thank Andrée Morel and Daniel Moncotel for excellent technical assistance.

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Received July 31, 2004; accepted October 21, 2004.


Table 1 Oxidative stress markers in PLTP-/- and PLTP+/+ mouse brains

PLTP-/- PLTP+/+

Cholesterol derivatives oxidized in position 7/Cholesterol 1.36 ± 0.57* 0.88 ± 0.36 Peroxide levels (DCFH oxidation, arbitrary units) 2.17 ± 0.55** 1.64 ± 0.46 Unsaturated fatty acids/total fatty acids 0.552 ± 0.003 0.556 ± 0.004 _*P < 0.05; **P < 0.01 vs. PLTP+/+, Student’s t-test.


Fig. 1

Figure 1. PLTP distribution in mouse brain. Immunohistochemical analysis of PLTP expression in mouse brain. Immunostaining was performed using a polyclonal PLTP specific antiserum (1/100) on coronal sections at the level of the hippocampus. The right panel illustrates the cytoarchitecture of the corresponding brain region on a Nissl-stained section. Magnification, ×630. CA1-3: field 1 or 3 of the Ammon’s horn; cc: corpus callosum; ch: choroid plexus; fi: fimbria.


Fig. 2

Figure 2. Vitamin E content in mouse brain. Vitamin E levels in brains from 16 PLTP+/+ mice, 10 PLTP+/− heterozygotes, and 6 PLTP−/− homozygotes were measured by LC/MS. *P < 0.05, **P < 0.0002, statistically significant differences from PLTP+/+ animals (unpaired Student’s t test).


Fig. 3

Figure 3. Increased lipofuscin levels in PLTP−/− mouse brain. Lipofuscin granules were examined in unstained 30-µm thick sections of 6 PLTP+/+ and 6 PLTP−/− homozygote brains by fluorescence microscopy and quantified using the analySIS® software. *P < 0.005, **P < 0.0001, statistically significant differences from PLTP+/+ animals (Mann-Whitney U-test).


Fig. 4

Figure 4. Increased anxiety in PLTP−/− mice. Anxiety levels of PLTP+/+ and PLTP−/− homozygous mice were assessed in an elevated plus-maze. Recorded parameters are (A) the percentage of entries in closed and open arms and (B) The percentage of time spent in closed and open arms. Results are expressed as median and quartiles of 9 PLTP+/+ and 9 PLTP−/− homozygous mice. *P < 0.05, **P < 0.01, statistically significant differences from PLTP+/+ animals (Mann-Whitney U-test).


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