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Human Molecular Genetics Pages 1511-1516  

Pronounced impact of Th1/E47cs mutation compared with -491 AT mutation on neural APOE gene expression and risk of developing Alzheimer's disease
Introduction
Results
Discussion
Materials And Methods
   Populations
   Brain samples
   Genotype
   APOE allelic quantitation
   Statistical analysis
Acknowledgements
References

Pronounced impact of Th1/E47cs mutation compared with -491 AT mutation on neural APOE gene expression and risk of developing Alzheimer's disease

Pronounced impact of Th1/E47cs mutation compared with -491 AT mutation on neural APOE gene expression and risk of developing Alzheimer's disease

Jean-Charles Lambert, Claudine Berr1, Florence Pasquier2, André Delacourte3, Bernard Frigard4, Dominique Cottel, Jordi Pérez-Tur5, Vincent Mouroux, Michel Mohr6, Danielle Cécyre7, Douglas Galasko8, Corinne Lendon9, Judes Poirier7, John Hardy5, David Mann10, Philippe Amouyel and Marie-Christine Chartier-Harlin*

INSERM CJF95-05, Institut Pasteur de Lille, 1 rue du Professeur Calmette, BP 245, 59019 Lille Cédex, France, 1INSERM U360, Hôpital de la Salpétrière, 75651 Paris Cédex 13, France, 2CHR et U de Lille, Clinique Neurologique, Centre de la Mémoire, Hôpital Salengro, 59037 Lille Cédex, France, 3INSERM U422, Place de Verdun, 59045 Lille Cédex, France, 4Centre de Gériatrie de Wasquehal, rue Salvador Allende, BP 165, 59444 Wasquehal, France, 5Birdsall Building, Mayo Clinic Jacksonville, 4500 San Pablo Road, Jacksonville, FL 32224, USA, 6Institut de Pathologie, 1 place de l'Hôpital, BP 22, 67064 Strasbourg Cédex, France, 7McGill Centre for Studies in Aging, 6875 La Salle Boulevard, Verdun, Québec, Canada H4H 1R3, 8Department of Neurosciences, University of California, San Diego, 3350 La Jolla Village Drive, San Diego, CA 9216, USA, 9Departments of Psychiatry, Neurology and Genetics, Washington University School of Medicine, St Louis, MO 63310, USA and 10Department of Pathological Sciences, Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK

Received June 1, 1998 Revised and Accepted July 8, 1998

Possession of the apolipoprotein E (APOE) [epsis]4 allele is the most frequently associated genetic susceptibility factor for Alzheimer's disease (AD). Recently, new polymorphisms in the regulatory region of the APOE gene have been described. We analysed the effects of three of these mutations (-491 AT, -427 CT and Th1/E47cs) on disease risk in a large case-control study, and tested their impacts on APOE allelic expression in brain tissues. The Th1/E47cs T allele was associated with an increased risk of occurrence of AD, while the -491 T allele was associated with a decreased risk, independently of the APOE [epsis]2/[epsis]3/[epsis]4 polymorphism effect. However, the impact of the Th1/E47cs mutation was the strongest. The -427 CT polymorphism was not associated with the disease. In AD subjects heterozygous for the [epsis]4 allele, analysis of allelic expression showed that the relative expression levels of the [epsis]4 allele were higher than those of the corresponding controls. Consistent with epidemiological data, the relative level of expression of the [epsis]4 allele was modified accordingly to the presence or absence of the two main promoter polymorphisms, indicating, in vivo, the deleterious effect of the Th1/E47cs T allele and the protective effect of the -491 T allele in population. These data indicate that in addition to the qualitative effect of the APOE [epsis]2/[epsis]3/[epsis]4 polymorphisms on the AD occurrence, the quantitative variation of expression of these alleles due to functional APOE promoter mutations, is a key determinant of AD development.

INTRODUCTION

The presence of at least one apolipoprotein E (APOE) [epsis]4 allele is a major genetic susceptibility factor for late-onset familial and sporadic Alzheimer's disease (AD). This risk factor accounts for ~45-60% of cases (1). Indeed, the [epsis]4 allele is associated with an increased risk of AD in an allele dose dependant manner (2,3). Conversely, the [epsis]2 allele protects against the disease (4,5). The mechanisms by which apoE isoforms are implicated in human nervous system physiology and influence the pathogenesis of AD, are not fully understood (2,6-8). The effect of the APOE [epsis]2/[epsis]3/[epsis]4 alleles may not be due only to the intrinsic biochemical properties of the isoforms coded by these alleles. Other genetic variants influencing APOE allele expression may account for the increase or decrease in the level of risk of AD. This hypothesis is supported firstly by preliminary observations of the relative overexpression of the [epsis]4 allele compared with the [epsis]3 allele in the brain of patients with AD, compared with controls (9). Secondly, Th1/E47cs and -491 AT polymorphisms located in the APOE promoter have been described and associated with AD risk (10,11). Moreover the effect of these two polymorphisms on AD occurrence was described separately in case-control studies. This prompted us to analyse the relative contribution of these two promoter polymorphisms, and also a third, located at position -427 of the APOE promoter together with the [epsis]2/[epsis]3/[epsis]4 alleles, towards AD risk in a large case-control study (509 controls and 573 AD cases). Furthermore, we investigated the possible influences of the two main polymorphisms on APOE [epsis]2/[epsis]3/[epsis]4 allelic relative expression in brain tissues, to approach their in vivo functionality, in brain samples from 49 AD cases and 45 controls.

RESULTS

As expected, possession of the [epsis]4 allele was strongly associated with AD [odds ratio (OR) = 5.40; 95% confidence interval (CI): 4.11-7.09, P < 0.0001], while the presence of the [epsis]2 allele exhibited a protective effect (OR = 0.47; 95% CI: 0.31-0.70, P = 0.0003). The frequency of the Th1/E47cs T allele was increased in AD cases compared with controls ([chi]2 = 23.1, P < 0.0001; Table 1). The risk of developing AD for carriers of at least one T allele was 2.13 (95% CI: 1.61-2.83). Conversely, the frequency of the -491 T allele was decreased in patients, compared with controls ([chi]2 = 9.5, P = 0.002). The genotype and allele frequencies of the -491 AT polymorphism were both similar to those observed in the North American population but differed from the Spanish sample (11). The OR to develop AD in subjects bearing at least one -491 T allele was 0.67 (95% CI: 0.52-0.88, P = 0.004). No association with the disease was detected for the -427 CT polymorphism. To eliminate possible confounding effects of the APOE [epsis]4 allele on the association of the Th1/E47cs and -491 AT polymorphisms with AD, we controlled the effects of each allele using logistic regression adjusted for the presence or absence of the [epsis]4 allele. After adjustment, the risk associated with possession of at least one Th1/E47cs T allele persisted (OR = 1.56; 95% CI: 1.15-2.11, P = 0.004), while the risk associated with -491 T allele did not (OR = 0.82; 95% CI: 0.62-1.10, P = 0.19). This observation suggested that the effect of the Th1/E47cs T allele is independent of that of the [epsis]4 allele, while the -491 T allele is not. However, since the Th1/E47cs G and [epsis]2 alleles are in complete disequilibrium (Table 2), we also performed a logistic regression including both protective and deleterious effects of [epsis]2 and [epsis]4 alleles, respectively. The risk associated with the Th1/E47cs T allele still persisted (OR = 1.41; 95% CI: 1.03-1.92, P = 0.03).

If we assume that the level of expression of the APOE alleles is increased or decreased due to cis mutations in the promoter region, this hypothesis has two major implications: (i) promoter mutations may modulate the expression of the APOE alleles leading to an increase or decrease of the deleterious or protective risk associated with the [epsis]4 or [epsis]2 allele, respectively; and (ii) given that what determines the risk is the importance of the relative level of expression of both alleles of APOE, as suggested by preliminary data (12), the promoter mutations will have differential effects detectable mainly in APOE [epsis]2/[epsis]3/[epsis]4 heterozygous individuals. To verify these assumptions, we studied the OR of developing AD for APOE heterozygous and homozygous individuals, using logistic regression adjusted for age, sex and the presence of at least one -491 T and one Th1/E47 T allele. In APOE [epsis]2/[epsis]3/[epsis]4 homozygotes, no effect was found (OR = 1.13; 95% CI: 0.77-1.65, and OR = 0.99; 95% CI: 0.68-1.44, for Th1/E47 T and -491 T alleles, respectively). Conversely, in APOE [epsis]2/[epsis]3/[epsis]4 heterozygotes, individuals bearing at least one Th1/E47 T allele had an increased risk of developing AD (OR = 3.57; 95% CI: 2.22-5.75, P < 0.0001), while individuals bearing at least one -491 T allele exhibited a protective effect (OR = 0.57; 95% CI: 0.37-0.90, P = 0.014), consistent with our initial hypothesis.

Table 1. APOE, Th1/E47cs and -491 AT allele and genotype distributions
  Allele Genotype
APOE [epsis]2 [epsis]3 [epsis]4 [epsis]2[epsis]2a [epsis]2[epsis]3 [epsis]2[epsis]4 [epsis]3[epsis]3 [epsis]3[epsis]4 [epsis]4[epsis]4
Controls 75 (0.07) 832 (0.82) 111 (0.11) 4 (0.01) 57 (0.11) 10 (0.02) 344 (0.68) 88 (0.17) 6 (0.01)
AD 40 (0.03) 699 (0.61) 407 (0.36) - 16 (0.03) 24 (0.04) 223 (0.39) 237 (0.41) 73 (0.13)
Th1/[Egr]47cs G T GGa GT TT  
Controls 562 (0.55) 456 (0.45) 162 (0.32) 238 (0.47) 109 (0.21)
AD 515 (0.45) 631 (0.55) 103 (0.18) 308 (0.54) 162 (0.28)
-427 TCb T C TT TC CC
Controls 874 (0.91) 88 (0.09) 396 (0.82) 82 (0.17) 3 (0.01)
AD 1000 (0.92) 86 (0.08) 458 (0.84) 84 (0.16) 1 (0.00)
-491 AT A T AAc AT TT
Controls 833 (0.82) 185 (0.18) 343 (0.67) 147 (0.29) 19 (0.04)
AD 993 (0.87) 153 (0.13) 432 (0.75) 129 (0.23) 12 (0.02)
Allele and genotype numbers (frequencies) are presented.The genotypic distributions in control population were in Hardy-Weinberg equilibrium.
aP < 10-4.
bNot significant.
cP = 0.005.

Table 2. Combination of the -491 AT, Th1/E47cs and APOE polymorphism alleles in the controls and AD cases
APOE and Th1/E47cs polymorphism combination
  n [epsis]2/[epsis]3a [epsis]3/[epsis]3 [epsis]3/[epsis]4 [epsis]4/[epsis]4 [epsis]2/[epsis]4
AD cases
GG 103 5 60 30 3 5
GT 306 11 113 136 27 19
TT 139 - 49 70 43 -
n 571 16 222 236 73 24
Control cases
GG 162 41 100 16 - 5
GT 238 20 170 40 2 5
TT 109 - 73 32 4 -
n 509 61 343 88 6 10
APOE and -491 AT polymorphism combination
  n [epsis]2/[epsis]3a [epsis]3/[epsis]3 [epsis]3/[epsis]4 [epsis]4/[epsis]4 [epsis]2/[epsis]4
AD cases
AA 431 6 157 190 67 11
AT 128 8 58 44 6 12
TT 12 2 7 2 - 1
n 261 16 222 102 28 24
Control cases
AA 342 24 242 65 5 6
AT 147 34 89 19 1 4
TT 20 3 12 4 - -
n 509 61 343 88 6 10
Th1/E47cs and -491 AT polymorphism combination
  n [Agr][Agr] [Agr][Sigma] [Sigma][Sigma]  
AD cases
GG 103 79 22 2
GT 307 226 75 6
TT 162 127 31 4
n 572 432 128 12
Control cases
GG 162 111 48 3
GT 238 162 67 9
TT 109 69 32 8
n 509 342 147 20
aThree individuals were [epsis]2/[epsis]2 in control.
The -491 A and [epsis]4 alleles present a degree of linkage disequilibrium of 26 and 75% in control and AD populations, respectively. The Th1/E47cs G and [epsis]2 alleles are in complete linkage disequilibrium in both populations, while the Th1/E47cs T and [epsis]4 alleles show a linkage disequilibrium of 44% in both populations. No linkage disequilibrium was found between -491 AT and Th1/E47cs polymorphisms.

The location of both polymorphisms within the regulatory region of the APOE gene, and their potential effect in vitro reported in hepatoma cells, suggested that these polymorphisms may play a role in the control of APOE expression levels in brain. In order to test this hypothesis, we obtained 94 AD or control brain tissues heterozygous for the APOE and measured the percentage of the expression level of the three APOE alleles. In controls, the [epsis]4 allele was strongly underexpressed compared with the [epsis]3 or the [epsis]2 allele in the [epsis]3[epsis]4 and [epsis]2[epsis]4 brain samples, respectively, while the [epsis]2 and [epsis]3 expressions were almost similar in the [epsis]2[epsis]3 samples (Fig. 1). As suggested by preliminary data (9), there was a clear and consistent increase in the relative [epsis]4 allelic expression between [epsis]3[epsis]4 patients and [epsis]3[epsis]4 controls (34.9 ± 2.7 versus 22.9 ± 2.4%, respectively, P < 10-4). A significant decrease in the relative [epsis]2 expression was demonstrated in [epsis]2[epsis]3 AD brains compared with [epsis]2[epsis]3 controls (32.8 ± 4.5 versus 47.8 ± 3.9%, respectively, P = 0.002). An increased expression of the [epsis]4 allele was also detected between the [epsis]2[epsis]4 AD and controls (46.0 ± 5.4 versus 28.0 ± 2.8%, respectively, P = 0.01). Measured values in [epsis]2[epsis]4 AD cases and controls corresponded to the expected values estimated from the other genotypes reinforcing our observations (see Materials and Methods; arrows in Fig. 1).


Figure 1. Differential expression of the APOE mRNA for the three heterozygous genotypes in AD and control cases. The average level of expression is indicated by a bold line in each category. For each brain sample, RT-PCR and semi-quantitation was repeated at least twice. Bold arrows indicate the expected values of the [epsis]4 allele percentage in the [epsis]2[epsis]4 samples, calculated from the [epsis]2 and [epsis]4 percentage in the [epsis]2[epsis]3 and [epsis]3[epsis]4 AD and control cases, respectively.

The previous epidemiological data suggest that the -491 AT polymorphism and the Th1/E47cs polymorphism may be good candidates to mediate these allelic distortions. In particular, estimation of the OR associated with the combination of the [epsis]4 and the Th1/E47cs T allele on the same chromosome indicate an estimated increased risk of 1.7 for the heterozygous [epsis]3/[epsis]4 individuals presenting this haplotype, as described previously (10). Thus, if the [epsis]4 allele is associated with the Th1/E47cs T allele on the same chromosome, while the [epsis]3 allele is associated with the Th1/E47cs G allele on the other, the relative [epsis]4/[epsis]3 mRNA ratio should be significantly increased and associated with AD. Conversely, if both [epsis]4 and [epsis]3 alleles are under the control of the same Th1/E47cs allele, either G or T, the level of expression should be similar. Thus, we expected a stronger distortion in Th1/E47cs heterozygotes than in homozygotes. This argument is clearly illustrated by our experimental data (Table 3). The same approach is valid for the -491 AT mutation.

The influence of both polymorphisms on relative mRNA expression level could be observed only in cases (Fig. 2 and Table 3). Affected patients carrying the Th1/E47cs GT or the -491 AT genotypes had the highest and lowest relative levels of expression of [epsis]4 expression, respectively (Fig. 1 and Table 3). The difference in the levels of expression in cases was more pronounced for the Th1/E47cs GT bearers than for the -491 AT bearers (36.6 ± 2.2 versus 33.5 ± 1.3%, respectively, P = 0.004; Fig. 2).


Figure 2. Differential expression of the [epsis]4 allele in the [epsis]3[epsis]4 AD and control cases, according to the Th1/E47cs genotype. AD cases are denoted by triangles and controls by circles. Closed symbols and open symbols correspond to the -491 AT and AA genotypes, respectively.

DISCUSSION

The deleterious effect of the Th1/E47cs T allele and the protective effect of the -491 T allele described recently (10,11) were confirmed by this large case-control study. These effects were obvious in the APOE heterozygous population, but not in the homozygous population. These data are not surprising for two reasons: (i) we would expect that APOE [epsis]4 homozygotes with high expressing promoter alleles on both chromosomes, had a higher risk of developing AD than [epsis]4 homozygotes with low expressing promoter alleles. However, the small size of the [epsis]4/[epsis]4 control population did not allow such an impact to be brought forward; (ii) as a consequence, the measured OR in [epsis]2/[epsis]3/[epsis]4 homozygous individuals mainly depended on the [epsis]3/[epsis]3 population. No effect was found in the latter population whatever the studied promoter polymorphism. Furthermore, the apoE3 isoform is considered as the isoform having the lesser effect on AD pathology.

Table 3. Relative [epsis]4 allele expression according to the Th1/E47cs and -491 AT polymorphisms in AD and control brain samples
  n AD (%) n Controlsa (%)
Th1/E47cs
GG 4 33.5 ± 1.7 4 22.5 ± 2.5
GT 19 36.3 ± 2.1b 11 22.6 ± 1.9
TT 10 32.4 ± 1.4 10 22.8 ± 2.7
491 AT
AA 26 35.3 ± 2.7 15 22.8 ± 2.2
AT 7 33.5 ± 1.3c 10 22.5 ± 2.3
aNot significant.
bP < 10-4.
cP = 0.11.

We did not confirm the deleterious effect of the -491 AA genotype described in the [epsis]3[epsis]3 Spanish population (11). This discrepancy may be explained by the significant difference in the -491 AT genotype distribution observed between the Spanish and the French or American control populations. Since the AD genotypic frequency was similar in the three studied populations, the deleterious effect of the AA genotype in the [epsis]3[epsis]3 group may only be due to the distribution in the Spanish control population.

When the impacts of these two promoter polymorphisms and the APOE [epsis]4 allele were studied simultaneously in a multiple logistic regression model, we demonstrated that only the Th1/E47cs T allele modified the risk of developing AD independently of the [epsis]4 allele, suggesting that the -491 polymorphisms may play a weaker role compared with Th1/E47cs in the development of AD. The location of both polymorphisms in the regulatory region of the APOE gene, their effects on the transcriptional activity in hepatoma cells (12) and their impacts in AD population, prompted us to test the hypothesis that they may modulate the APOE expression levels in brain and, therefore, be partly responsible for the marked allelic distortion of the APOE mRNA we observed in AD brain compared with controls in all the heterozygous genotypes. Indeed, the [epsis]4 allele was overexpressed in [epsis]3[epsis]4 and [epsis]2[epsis]4 AD cases and the [epsis]2 allele underexpressed in [epsis]2[epsis]3 AD cases compared with their respective controls. Consistent with the effects deduced from the case-control study, the [epsis]4 allele expression in AD brain was also correlated with the polymorphisms of the regulatory region of the APOE (Fig. 2): the Th1/E47cs T allele may increase the relative expression of the [epsis]4 allele in AD, while the -491 T allele may decrease it.

Because numerous studies reported contradictory findings in brain and cerebrospinal fluid (CSF) concerning apoE protein level, it is difficult to speculate on the link between APOE mRNA and apoE protein levels (13-17). However, as suggested by Yamada et al., the APOE expression may be decreased by an [epsis]4 gene dosage in AD: the [epsis]3 homozygous AD cases expressed more APOE mRNA than the [epsis]4[epsis]4 AD cases, the [epsis]3[epsis]4 presenting an intermediate mRNA level (18). A similar trend was found for proteins in AD brains (14) or in plasma (J. Poirier, personal communication), indicating that both the protein and mRNA levels may be directly correlated during the disease. Underlining the importance of apoE level, the APOE expression has been shown to correlate with A[beta] peptide deposition in transgenic mice (19,20).

This observation may be very important, since previous reports showed that the APOE gene expression was increased between 2- and 3-fold in AD brains compared with controls regardless of the APOE genotype (18,21). In [epsis]3[epsis]4 AD cases, both the [epsis]3 and [epsis]4 alleles would be overexpressed, but the [epsis]4 allele, due to the promoter mutations, being still more overexpressed than the [epsis]3 allele. Exacerbation of APOE [epsis]4 expression level may foster the intrinsic deleterious effect of the apoE4 isoform in AD for instance in promoting A[beta] aggregation (2,6,22).

This expression level modulation may be intended not only for neurodegenerative disease, but also for neural development. Indeed, we observed a strong underexpression of the [epsis]4 allele compared with the [epsis]3 allele in controls. Although the role of apoE during maturation and ageing is not completely understood yet, the fact that apoE deficient homozygous mice exhibit reversible dendritic alterations and significant learning deficits in the Morris water maze, suggests neurotrophic capabilities for apoE (23-25). However, the abilities of each isoform may be different since the apoE4 isoform does not have the same effects as the apoE3 isoform on neurite outgrowth (26). For instance, apoE3, but not apoE4, may stabilize the neuronal cytoskeleton (7).

A global assessment of the modulation of APOE expression is difficult. Indeed, to date, five polymorphisms have been described in the APOE promoter region (10,12). Two of the three polymorphisms studied, seem to play a role in AD. The two remaining have allelic frequencies <1% (12), and therefore cannot explain the effects observed in the case-control and mRNA studies. However, it remains possible that other unknown polymorphisms modulate the APOE mRNA level. These new mutations may be far away from the promoter polymorphisms, for instance in the brain tissue specific element (27,28). The interaction between all the putative polymorphisms modifying APOE expression will be very difficult to model. Various combinations of different mutations may confer a large APOE expression heterogeneity at two levels: (i) the absolute level of APOE expression and (ii) the percentage of expression of one allele compared with the other in heterozygous individuals, adding to the complexity of the association between APOE and AD. Therefore, the variation of the APOE expression induced by the APOE promoter polymorphisms may partly explain the heterogeneity of the impact of the [epsis]4 allele in different ethnic groups (1,29).

MATERIALS AND METHODS

Populations

The AD and control samples were Caucasians originating from France. Diagnoses of probable AD were established according to the DSM-III-R and NINDCS-ADRDA criteria (n = 573; age = 73.8 ± 8.1 years; age of onset = 70.4 ± 7.9 years; 35.9% of men) (30,31). The Caucasian controls were defined as subjects without DMS-III-R dementia criteria and with integrity of their cognitive functions (n = 509; age = 74.3 ± 9.9 years; 35.9% of men). Each individual or their relatives signed an informed consent.

Brain samples

These consisted of 45 controls (age = 80.3 ± 8.6 years; 44.9% of men) and 49 late-onset AD cases (age = 76.5 ± 9.3 years; age of onset = 68.4 ± 10.0 years; 44.4% of men), selected according to their APOE genotype. Eighty-eight samples were extracted from frontal cortex and six from occipital cortex, no difference of relative expressions being observed between those regions. The mean age was 78.4 ± 8.7 years for the AD cases genotyped [epsis]3[epsis]4 and 75.9 ± 7.5 years for the controls. Diagnoses were confirmed by neuropathological examination.

Genotype

The -491 AT, -427 CT, Th1E47cs and APOE genotypes were produced by PCR followed by restriction enzyme digestion of the amplified DNA as described (10-12,32).

APOE allelic quantitation

Total RNA extraction from brain tissues, heterozygous for APOE genotype, was performed as described (33) or using RNeasy Mini kit (Qiagen, Germany) and then digested by DNase (Eurogentec, Belgium). No DNA contamination was observed after DNase digestion as detected by PCR of the digested product. APOE mRNAs were amplified by RT-PCR and allele quantitation was performed as described previously (9) by measuring a ratio of expression of two alleles in heterozygotes in order to avoid problems due to post-mortem delay, agonal state and cell population density.

Statistical analysis

The SAS software release 6.10 was used (SAS Institute, Cary, NC). Univariate analysis was performed using Pearson's [chi]2 test. In the multivariate analysis, we coded the genotypes of each subject as dummy variables according to the tested hypotheses (at least one Th1/E47cs T allele: TT+TG/GG, at least one -491 T allele: TT+AT/AA and at least one [epsis]4 allele). The effects of these variables on the disease were assessed with a multiple logistic regression model adjusted for age and gender. Comparison of the allelic expression was performed using Wilcoxon test.

We calculated the expected range of values of [epsis]4 percentage in [epsis]2[epsis]4 AD and control individuals using the extreme values obtained for the [epsis]2 and the [epsis]4 percentages in the [epsis]3[epsis]4 and [epsis]2[epsis]3 AD and control samples, respectively (the [epsis]3 allele being used as reference).

ACKNOWLEDGEMENTS

J.-C.L. is a recipient of the Ministère de l'Enseignement Supérieur et de la Recherche (MESR). This work was supported by the Institut National pour la Santé et la Recherche Médicale (INSERM), the Institut Pasteur de Lille, the Conseil Régional du Nord-Pas de Calais `axe régional de recherche sur les maladies neurodégénératives et le vieillissement cérébral' (M.-C.C.-H., P.A. and F.P.) and the Fondation pour la Recherche Médicale.

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*To whom correspondence should be addressed. Tel: +33 3 20 87 72 28; Fax: + 33 3 20 87 78 94; Email: marie-christine.chartier{at}pasteur-lille.fr

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