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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 R. Tall, Didier Desor^* and Laurent Lagrost^#,1

^# Laboratoire de Biochimie des Lipoprotéines, INSERM, 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, New York, USA;
Department of Medicine, Columbia University, New York, New York, USA

¹ Correspondence: INSERM U498-Faculté de Médecine, 7, Bd Jeanne d’Arc BP 87900, 21079 DIJON Cedex. E-mail: Laurent.Lagrost{at}u-bourgogne.fr

SPECIFIC AIMS

Chronic oxidative stress and aging are major risk factors of neurodegenerative disorders, and the use of antioxidants constitutes a promising strategy in the prevention of brain damage. Although plasma phospholipid transfer protein (PLTP) promotes the exchange of vitamin E (an essential lipid-soluble antioxidant) between lipoproteins and cells, it is unknown whether PLTP functions as a transfer factor for vitamin E in the brain. The effect of PLTP deficiency on the vitamin E content of the brain was addressed in the present study and consequences in terms of brain oxidative status and behavior were investigated.

PRINCIPAL FINDINGS

1. PLTP is highly expressed in the brain of wild-type (PLTP^+/+) mice, where it functions as a vitamin E transfer factor
Immunohistochemical analysis of 10 µm coronal brain sections incubated with a rabbit anti-PLTP antiserum revealed a high and widespread level of PLTP staining in PLTP^+/+ mouse brain, with a strong signal in the choroid plexus (not shown). There was no PLTP signal in the brain of PLTP^–/– homozygotes. Vitamin E was assayed in brain homogenates by liquid chromatography/mass spectrometry (LC/MS). Complete PLTP deficiency is accompanied by a 30.1% decrease in vitamin E content of the brain from PLTP^–/– homozygotes compared with PLTP^+/+ controls (Fig. 1 ). A 30% decrease in mean plasma PLTP activity in PLTP^+/– heterozygotes is associated with a significant 18.0% decrease in brain vitamin E content compared with PLTP^+/+ controls (not shown).

View larger version (10K): [in this window] [in a new window]   Figure 1. Decreased brain vitamin E content in PLTP–/– mice. Vitamin E levels (mean ±SD) in brains from PLTP+/+ (n=16), PLTP+/– (n=10) and PLTP–/– (n=6) mice were measured by LC/MS. *P < 0.05; **P < 0.0002 vs. PLTP+/+ animals (unpaired Student’s t test).

2. PLTP deficiency is accompanied by the accumulation of oxidative stress markers in the brain
Decreased vitamin E content of PLTP^–/– brain is associated with significant increases in oxidative stress markers in mouse brain. When the amount of cholesterol derivatives oxidized in position 7 was quantified in brain homogenates by GC/MS, a significant 54.5% increase was observed in PLTP^–/– homozygotes compared with PLTP^+/+ controls [cholesterol derivatives/cholesterol ratios, 1.36±0.57 and 0.88±0.36 for PLTP^–/– homozygotes and PLTP^+/+ controls, respectively, P<0.05 (Student’s t test)]. The oxidative stress index in brain homogenates was assessed as the oxidation rate of dichlorofluorescein-diacetate into the fluorescent product dichlorofluorescein. The oxidative stress index was significantly higher in brain homogenates from PLTP^–/– homozygotes than PLTP^+/+ controls [2.17±0.55 and 1.64±0.46 (arbitrary units) for PLTP^–/– homozygotes and PLTP^+/+ controls, respectively; P<0.01 (Student’s t test)].

To further confirm the elevation of chronic oxidative stress in PLTP^–/– mouse brain, the intraneuronal lipopigment corresponding to lipofuscin (an end product of the reaction of lipid peroxides with proteins) was examined by fluorescence microscopy in unstained 30 µm thick sections from PLTP^+/+ (n=6) and PLTP^–/– (n=6) brains. As observed in various models of age-related neurodegenerative disorders, the amount of lipofuscin is significantly higher in the cortex and substantia nigra of PLTP^–/– than of PLTP^+/+ mice [cortex: 480.1±138.7 and 383.3±62.1 pixels², P<0.005; substantia nigra: 1196.4±264.6 and 122.0±6.3 pixels², P<0.0001 (Mann-Whitney U test) in PLTP^–/– and PLTP^+/+ brains, respectively].

3. PLTP deficiency leads to a behavior of anxiety
To evaluate the effect of PLTP deficiency on mouse behavior, a neurobehavioral analysis was performed in a blind fashion on nine PLTP^+/+ and nine PLTP^–/– mice. PLTP^–/– mice showed no evidence of locomotor activity and neuromotor coordination disorders as assessed by open-field activity [total number of entries, 310 (268-366) and 290 (246-363) (median and quartiles) for PLTP^+/+ and PLTP^–/– mice, respectively, n.s.] and performance in the Locotronic® apparatus [run time, 12.8 s (10.9–20.7) and 16.6 s (11.0–22.7) for PLTP^+/+ and PLTP^–/– mice, respectively, n.s.]. Anxiety levels were assessed with an elevated plus maze consisting of two open arms and two closed arms. Animals were allowed to spend 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 allowed free access to the arms for 5 min. Standard measures comprised the frequency of open and closed arm entries and the time spent by the animals in open and closed sections of the maze. Strikingly, PLTP^–/– mice showed evidence of markedly increased anxiety, making fewer entries (8.3% vs. 44.4% for PLTP^+/+ mice, P<0.01) and spending less time (1.7% vs. 41.3% for PLTP^+/+ mice, P <0.05) in the open arms of the maze (Fig. 2 ).

View larger version (22K): [in this window] [in a new window]   Figure 2. Increased anxiety in PLTP–/– mice. Anxiety levels of PLTP+/+ (n=9) and PLTP–/– (n=9) mice were assessed in an elevated plus maze. Recorded parameters (median and quartiles) are the % of entries in closed and open arms (left panel) and % of time spent in closed and open arms (right panel). *P < 0.05; **P < 0.01 vs. PLTP+/+ animals (Mann-Whitney U test).

CONCLUSIONS AND SIGNIFICANCE

Recent findings support a key role of high oxidative stress/low antioxidant protection in the pathogenesis of brain neurodegenerative disorders. The present work demonstrates for the first time that PLTP is ubiquituously expressed in mouse brain, where it functions as a vitamin E transfer factor. Although moderate, the vitamin E-deficient state of PLTP knockout mouse brain produces a subtle but sustained increase in brain oxidative stress and lipofuscin content. PLTP knockout mice constitute a new model of chronic vitamin E deficiency in the brain; the associated phenotype resembles in several ways the aging process. In contrast to -tocopherol transfer protein knockout mice, in which the drastic and ubiquitous vitamin E deficiency led to an early and severe ataxic phenotype, we show that the behavioral phenotype of PLTP knockout mice is characterized only by increased anxiety, with no abnormalities in locomotor performance.

A significant increase in brain PLTP mRNAs has recently been 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 pathological condition. The present study strongly supports vitamin E transfer into tissues as a key function of PLTP in vivo. This activity might constitute a key process in preventing oxidative damage in the brain; PLTP knockout mice may be a new model to analyze the contribution of oxidative brain injury in the etiology of neurodegenerative diseases.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2400fje;

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