Pascal Croiseau
email: pascal.croiseau@jouy.inra.fr

Andres Legarra

François F Guillaume

Sébastien S Fritz

Aurélia A Baur

Carine Colombani Colombani

Christèle Robert-Granié

Didier Boichard

Vincent Ducrocq

A N D R E ´s L E G A R R A

E ´b A S T I E N F R I

C A R I N E C O L O M B A N I

D U C R O C Q

Fine tuning genomic evaluations in dairy cattle through SNP pre

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The availability of dense single-nucleotide polymorphism (SNP) arrays has considerably changed the landscape of dairy cattle selection worldwide. With such chips, it is now possible to retrieve information about quantitative trait locus (QTL) all over the genome. Genomic estimated breeding values (GEBV), which correspond to a combination of the sum of the effects of genetic markers (direct genomic value (DGV)) and estimated breeding value (EBV), can be used instead of the classical pedigree-based genetic evaluations in selection programmes. Meuwissen et al. (2001) envisioned the consequences on the estimation 40 of breeding values of a high-density marker map Comment citer ce document : Croiseau, P., Legarra , A., Guillaume, F., Fritz, S., Baur, A., colombani, C., Robert Granie, C., Boichard, D., [START_REF] Habier | Fine tuning genomic evaluations in dairy cattle through SNP pre-selection with the Elastic-Net algorithm[END_REF]. Fine tuning genomic evaluations in dairy cattle through SNP pre-selection with the Elastic-Net algorithm. Genetics Research, 93 (6), 409-417. DOI : 10.1017/S0016672311000358 others) implemented genomic selection for their national evaluations [START_REF] Hayes | Invited review: Genomic selection in dairy cattle : 635 progress and challenges[END_REF][START_REF] Van Raden | Invited review : reliability of genomic predictions for North American Holstein bulls[END_REF][START_REF] Boichard | Genomic selection in French dairy cattle[END_REF][START_REF] Harris | Genomic predictions for 630 New Zealand dairy bulls and integration with national 631 genetic evaluation[END_REF][START_REF] Liu | Dairy cattle genetic evaluation enhanced 650 with genomic information[END_REF].

Numerous methods have been proposed to perform genomic evaluations with variable resulting accuracy depending on the underlying genetic assumptions, on the trait, breed and reference population size. For instance, Habier et al. (2010 a) tested a large panel of Bayesian approaches on a data set from the Holstein breed and even though Bayes A appeared to be a nearly optimal choice in their study, they recommended determining the best method for each quantitative trait separately. Indeed, in another study on Australian Holstein Friesian dairy cattle, Bayes A provided the lowest correlation between predicted GEBV and breeding values among the set of tested methods [START_REF] Verbyla | Accuracy of genomic selection using stochastic search variable selection in Australian Holstein Friesian dairy cattle[END_REF]. On French data that [START_REF] Legarra | Improved Lasso for genomic selection[END_REF] conducted for production traits, better predictions were obtained for Bayesian LASSO than for genomic BLUP (gBLUP). For other traits like fertility, it was shown that gBLUP performed slightly better than Bayesian LASSO [START_REF] Hayes | Invited review: Genomic selection in dairy cattle : 635 progress and challenges[END_REF][START_REF] Van Raden | Invited review : reliability of genomic predictions for North American Holstein bulls[END_REF].

Hence, it is still difficult to rank the large panel of available genomic evaluation methods according to their accuracy.

In a genomic evaluation procedure where the complete set of SNP is used, the statistical challenge is to evaluate effects attached to each of the available SNPs. In a context where the number of SNPs (p) is much higher than the number of bulls (n), this may lead to a poor estimation of the SNP effects even though the sum of genotypes time effects may be adequate on this reference population. In a routine evaluation with new animals, the best way to be confident in DGV or GEBV is to attach an effect to SNP in linkage disequilibrium with a QTL which reflects the effect of the QTL and an effect regressed towards zero to the others.

An alternative is to use approaches that have been developed especially to solve the p>>n problem. This is the case of variable selection methods and, among them, we focused on the Elastic-Net (EN) algorithm [START_REF] Zou | Regularization and variable selection via the Elastic Net[END_REF] and we chose to compare it to gBLUP, which is currently the most used approach in practice. Secondly, a two-step approach was tested by adding an initial preparation step consisting of an SNP pre-selection based on results from a QTL detection analysis. The second step implements gBLUP or EN on this preselected set of SNP with the hope that individual estimates of effects of the retuned SNP would be more accurate. To compare benefits and drawbacks of these situations, a pedigree-based BLUP was used as the reference. [START_REF] Mrode | Calculating cow and daughter yield deviations and partitioning of genetic evaluations under a random regression model[END_REF].

148

To account for the varying accuracy of the DYD, they 149 were weighted by their error variance, which is pro-150 portional to the sire's effective daughters ' contri-151 bution (EDC) [START_REF] Fikse | Weighting factors of sire daughter information in international genetic evaluations[END_REF] [START_REF] Boichard | Genetic analysis of conception rate in French Holstein cattle[END_REF]. Mean results 162 over the 25 traits will also be shown. 175 [START_REF] Van Raden | Efficient methods to compute genomic predictions[END_REF] and [START_REF] Goddard | Genomic selection : prediction of accuracy and maximisation of long term response[END_REF] showed 176 that this model is equivalent to a mixed model fitting 177 the effect of the genotype score of each SNP, all SNPs 178 having a priori the same variance equal to s 2 a = 179 s 2 u =2gp i (1xp i ), where s u 2 is the polygenic variance 180 used in regular genetic evaluation and p i is the fre-181 quency of an allele of the locus i [START_REF] Gianola | Additive genetic variability and the Bayesian alphabet[END_REF]. (y i xx i b) 2 + 187 lg j jb j j} estimates lies in the form of the penalty term. 188 In both equations, b is the vector of SNP effects b j , y i 189 is the phenotype of animal i and x i is its vector of 190 genotypes. The l parameter corresponds to the in-191 tensity of the penalty. In the EN algorithm, a second 192 parameter a, taking a value in [0, 1] is used to weight 193 the RR and LASSO penalties.

b ˆEN = arg min g n i=1 (y i xx i b) 2 +l (1xa) g j b 2 j +a g j jb j j
194 With a=1, a LASSO model is defined, whereas with 195 a=0, a full RR model is chosen. Zou andHastie 196 (2003, 2005) showed that in the presence of correlated 197 explanatory variables (e.g. effects corresponding to 198 SNP in linkage disequilibrium in our case), RR re-199 tains all predictors and their corresponding coeffi-200 cients tend to be equal and no variable selection is 201 performed. On the other hand, LASSO retains only 202 one predictor and removes the others (Zou & Hastie, 203 2003[START_REF] Zou | Regularization and variable selection via the Elastic Net[END_REF]. Hence, by including RR and LASSO as 204 extreme cases, the EN algorithm provides a more 205 flexible tool.

In this study, EN procedures were used using an R package named 'glmnet' (http://cran.r-project.org/ web/packages/glmnet/index.html) implemented by [START_REF] Friedman | Regularization paths for generalized linear models via coordinate descent AQ5[END_REF]. They proposed a fast implementation of EN using cyclical coordinate descent, computed along a regularization path.

(iii) Pre-selection of the SNP For most traits, not all SNPs on the SNP chip are likely to be close to a QTL. In other words, the assumption that effects attached to each of the SNPs are non null is unrealistic. Consequently, our conjecture is that whatever the genomic evaluation method used, a pre-selection of the SNP with an attached non-null effect may help to improve the quality of genomic prediction. This was tested in the situation where preselection is based on QTL detection. QTL detection was performed using a combined linkage disequilibrium and linkage analysis (LDLA) [START_REF] Druet | Fine mapping of quantitative trait loci affecting female fertility in dairy cattle on BTA03 using a dense single-nucleotide polymorphism map[END_REF]Meuwissen & Goddard, 2001). First, the existence of a single QTL was tested in the training data set at all positions along the chromosomes defined by haplotypes of six SNPs, with a sliding window of two SNPs. From this LDLA, a value of the likelihood ratio test (LRT) was obtained for each haplotype. Positions where a potential QTL is located were defined as haplotypes each time an LRT peak higher than a threshold value of 3 or 5 was found.

These values were quite arbitrary at this stage and low enough to catch any potential QTL that can be identified through this analysis. An LRT peak was defined as the position where the highest LRT value was found within a window of 25 or 50 SNP upstream and downstream of the current haplotype.

Then, the 50 SNPs around each detected LRT peak (¡25) were included in a pre-selected set of SNPs used for genomic evaluation using either a gBLUP or EN approach. The choice of the number of SNPs to retain was based on a preliminary study where this value of 50 gave the best results (data not shown).

(iv) Quality assessment of the genomic prediction

To measure the quality of prediction equations (derived from the training set), the equations were applied to the animals of the validation data set to get DGVs. Then, the weighted correlation between DGV and observed DYD was computed using EDC as weights. The weighted Pearson product moment correlation coefficient is calculated as [START_REF] Peers | Statistical Analysis for Education and Psychology Researchers[END_REF] :

r (x, y) = gw i (x i xx ¯w)(y i xy ¯w) ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi gw i (x i xx ¯w) 2 gw i (y i xy ¯w) 2 q
where x ¯w= gw i x i gw i , y ¯w= gw i y i gw i and w i is the EDC weight of y i .

Comment citer ce document : Croiseau, P., Legarra , A., Guillaume, F., Fritz, S., Baur, A., colombani, C., Robert Granie, C., Boichard, D., [START_REF] Habier | Fine tuning genomic evaluations in dairy cattle through SNP pre-selection with the Elastic-Net algorithm[END_REF]. Fine tuning genomic evaluations in dairy cattle through SNP pre-selection with the Elastic-Net algorithm. Genetics Research, 93 (6), 409-417. DOI : 10.1017/S0016672311000358

255

The aim was to measure the accuracy of the 256 different methods to predict DYD using genomic in-257 formation (DGV). Since GEBV combine the infor-258 mation available from DGV and EBV, it is not 259 possible to know if an observed gain in accuracy is 260 due to the prediction equation or to a good combi-261 nation of DGV and EBV. This is why the correlation 262 between DGV and observed DYD was preferred in 263 this study (see e.g. [START_REF] Guillaume | Estimation by simulation of the efficiency of the French marker-assisted selection program in dairy cattle[END_REF].

(v) Parameters used for the different methods

265 For the pedigree-based BLUP, genetic parameters 266 were estimated using an AI-REML AQ2 approach (Jensen 267 et al., 1996). For the LDLA, it is necessary to incor-268 porate IBD AQ3 matrices among QTL allelic effects. 269 Software of [START_REF] Misztal | BLUPF90 and related programs[END_REF] was modified ac-270 cordingly. Heritability estimates used in pedigree-271 based BLUP and gBLUP were those used in routine 272 genetic evaluations.

273

For EN, values for the a and l penalization para-274 meters needed to be chosen and there is currently no 275 way to predict which range of values is the most ap-276 propriate for each parameter. Consequently, a large 277 range of combinations of a and l was tested by grid 278 search to find the optimal values. The search aimed at 279 finding the maximum correlation between DGV and 280 observed DYD in the validation data set. The vali-281 dation data set is consequently used to identify the 282 optimal set of parameters. This can be an advantage 283 in comparison with other methods with respect to the 284 accuracy of GEBV if this set of parameters is specific 285 to this training and validation data sets. However, by 286 looking at reference populations of increasing sizes, 287 we found that these parameters were breed-and trait-288 specific with a rather large range of combinations 289 giving similar results (data not shown). The EN ap-290 proach appears robust to moderate departures from 291 the optimal combination of parameters. To define the 292 optimal a parameter, a dichotomous search was per-293 formed on the [0, 1] interval. Initially, a values of 0, 1 294 and 0 . 5 were tested. If a=0 provided the best corre-295 lation, at the second iteration, the interval was re-296 duced to [0, 0 . 5]. If the best correlation was found with a=1, the new interval was [0 . 5, 1]. If the best correlation was found with a=0 . 5, the new interval was [0 . 25, 0 . 75]. We applied this method until the difference between two tested a was lower than 0 . 02.

The dichotomous approach requires a unimodal distribution for these correlations which is not guaranteed. Nevertheless, after testing a large panel of a values for some traits (data not shown), this unimodal distribution seems to be the rule.

For each a, 500 values of the penalty intensity l were tested in the interval [0-max(b)], where max(b) corresponds to the absolute value of the highest estimate when no penalization is applied. This research of optimal values for a and l was performed separately for the pre-selected and the full data sets. The search for the optimal a parameter is the most time-consuming step of the glmnet package and takes around 2 CPU minutes in Holstein for each tested a.

Results

Table 2 shows the optimal set of EN parameters for the six traits initially studied. Depending on the trait and breed, the optimal set of parameters differed. For instance, a complete RR approach gave the best results for Milk and Protein yield in the Montbe´liarde breed, while optimal a values of 0 . 25 for Milk yield in Holstein and of 0 . 37 for Protein yield in Normande were found, which correspond to a general EN model.

Moreover, there was a strong impact of both a and l on the number of SNPs included in the regression model. When a is near a complete LASSO procedure (a=1), there were many fewer SNPs retained compared with a complete RR procedure (a=0). Also, for a given a, high values of l led to a high intensity of penalization and consequently to a lower number of SNP (results not shown).

In the second analysis, the SNP pre-selection based on QTL detection was performed. As indicated before, this SNP pre-selection relied on two criteria : a given LRT threshold and a given window size. Table 3 reports the effect of both criteria on the number of LRT peaks identified in the case of milk yield.

Table 2. Optimal a and l parameters and corresponding number of SNPs with non-null effect for the six traits studied and for the three breeds using the EN procedure on the complete set of SNPs Comment citer ce document : Croiseau, P., Legarra , A., Guillaume, F., Fritz, S., Baur, A., colombani, C., Robert Granie, C., Boichard, D., Ducrocq, V. ( 2011). Fine tuning genomic evaluations in dairy cattle through SNP pre-selection with the Elastic-Net algorithm. Genetics Research, 93 (6), 409-417. DOI : 10.1017/S0016672311000358

339 Table 4 presents for the three breeds the results 340 obtained with the classical pedigree-based BLUP and 341 the two genomic selection methods (gBLUP and EN) 342 when either the whole set of SNP which passed the 343 quality control was used or after a pre-selection of the 344 SNP based on the LDLA approach.

345

All genomic methods improved the correlation 346 between DGV and observed DYD compared with 347 pedigree-based BLUP and the genetic architecture of 348 the trait seemed to play an important role on the gain 349 in correlation : for traits where some QTLs explain a 350 large part of the variance, such as protein content and 351 fat content (where DGAT1 gene is present), a mean gain in correlation over the three breeds of +0 . 22 and +0 . 23, respectively, was observed. In contrast, when the trait background appears to be polygenic with many QTLs explaining only a small part of the variance each, as for conception rate, the observed mean gain in correlation was more limited (+0 . 06).

Between the two genomic approaches, EN gave better results with a mean gain (compared with pedigreebased BLUP) over the six traits of 0 . 15, 0 . 13 and 0 . 20 for Montbe´liarde, Normande and Holstein, respectively, compared with 0 . 12, 0 . 08 and 0 . 18 with gBLUP.

When an SNP pre-selection was applied, the gain in correlation using gBLUP and EN was very similar to the one observed using the complete set of SNP.

Again, among the two different genomic approaches, the best results were obtained with EN. Compared with the pedigree-based BLUP, the mean gains over the six traits were 0 . 14, 0 . 15 and 0 . 20 for Montbe´liarde, Normande and Holstein, respectively, compared with 0 . 12, 0 . 11 and 0 . 18 with gBLUP.

Table 5 shows the slope of the regression of observed DYD on DGV for Holstein. A value close to 1 is expected. In dairy cattle, genomic evaluations are validated by Interbull if the slope of regression is included between 0 . 8 and 1 . 2 (Interbull, 2011). Over the three tested methods, similar ranges of values were observed for pedigree-based BLUP and EN. The slope for gBLUP deviated more from 1 than for the two other methods (on average, 0 . 22 for gBLUP compared with 0 . 11 for pedigree-based BLUP and 0 . 12 for EN). The same analysis was performed for the approach with SNP pre-selection. For EN, the SNP pre-selection had no impact on the slope. For the six presented traits, pre-selection led to a 400 reduction of the number of SNPs needed in the pre-401 diction equation. Among these traits, conception rate 402 is the one with the highest polygenic part as the 403 number of SNPs included in the EN model shows. 404 Production traits required between 1271 and 5648, 405 which is much less than the 20 904 SNPs required for 406 conception rate. The highest reduction of the number 407 of SNPs retained was for conception rate (from 20 904 408 to 9677 SNPs, which corresponds to a reduction of 409 54 %).

410

The impact of this SNP pre-selection on correla-411 tions was an absolute decrease limited to 1-2 % and 412 was relatively limited. For the 25 available traits, 413 the average number of SNPs used in the prediction equation derived from the EN algorithm applied on the whole set of SNPs was 16 334. After pre-selection, this number declined to 10 059. This important decrease in the number of SNPs used was obtained while correlations remained relatively stable (loss of 1% on average). Surprisingly, for some traits, the number of SNPs retained by EN after pre-selection was higher than when EN was applied to the whole set of SNPs. This was the case for body depth, chest width and milking speed for Holstein. Nevertheless, this phenomenon was marginal and, for most traits, preselection allowed a large decrease in SNP numbers.

The results presented in Table 6 correspond to the optimal a and l values. During the EN procedure, a large number of parameter combinations were tested and some suboptimal combinations required an even smaller number of SNPs. Table 7 presents, for the Holstein breed and for the six initial traits, the highest correlations that were observed when the total number of SNPs with non-null effect was limited to a value Comment citer ce document : Croiseau, P., Legarra , A., Guillaume, F., Fritz, S., Baur, A., colombani, C., Robert Granie, C., Boichard, D., [START_REF] Habier | Fine tuning genomic evaluations in dairy cattle through SNP pre-selection with the Elastic-Net algorithm[END_REF]. Fine tuning genomic evaluations in dairy cattle through SNP pre-selection with the Elastic-Net algorithm. Genetics Research, 93 (6), 409-417. DOI : 10.1017/S0016672311000358

434 between 2500 and 1000 SNPs. An option of the R 435 package glmnet allows the maximum number of 436 variables to be set. This option acts on the intensity of 437 the penalization to validate this constraint.

438

Obviously, this limitation in the number of SNPs 439 led to a decrease in correlation, but this was relatively 440 limited: between 0 and 3 . 4 % depending on the trait 441 and the maximum number of SNPs defined. In com-442 plement to this table, Figure 1 presents the mean 443 change in correlation over the 25 traits for the three 444 breeds according to the number of selected SNPs.

445

The breed found to be the most sensitive to the 446 limitation of selected SNP in EN was the Holstein 447 breed, but this is also the breed in which, on average, 448 the largest number of SNPs without pre-selection are 449 retained (17 341 selected SNPs in this situation against 450 11 526 in Normande and 12 939 in Montbe´liarde). 451 When the number of selected SNPs was limited to 452 2500, the average absolute loss in correlation over the 453 25 traits ranged from 1 and 1 . 7%. This average loss in 454 correlation changed to 2 . 3 and 4 . 5% with a limit to 455 1000 selected SNPs. 456 4. Discussion 457 As for many previous studies, genomic evaluations 458 with gBLUP and EN substantially improved the 459 quality of prediction of observed DYD in the vali-460 dation data set compared with pedigree-based BLUP 461 [START_REF] Hayes | Invited review: Genomic selection in dairy cattle : 635 progress and challenges[END_REF][START_REF] Wolc | Breeding value prediction for production traits in layer chickens using pedigree or genomic relationships in a reduced animal model[END_REF]. Between these 462 two genomic evaluations, gBLUP has the advantage 463 of being conceptually simpler in the sense that there is 464 no extra parameter to define or to optimize. In theory, a method that estimates all SNP effects should ensure ' From a genetic standpoint, it is consistent with the assumption that not all SNPs are required to explain the genetic architecture of a given trait. Some of them, with non-significant effects, can still carry genetic information and particularly on genetic

  . With a minimum minor allele 120 frequency of 3 %, 38 460 SNPs were retained for the 121 Montbe´liarde breed, 38 534 SNPs for the Normande 122 breed and 39 738 SNPs for the Holstein breed. 123 Mendelian segregation was checked. The SNP pre-124 selection chosen in this study uses a QTL detection 125 method based on haplotypes which requires phased 126 data. To infer missing genotypes and phases, Dual-127 PHASE software was used (Druet & Georges, 2009). 128 The data set was divided into a training data set 129 to derive prediction equations and a validation data 130 set where predictions were compared with observed 131 phenotypes. Table 1 shows the size of training and 132 validation data sets for the three breeds. To define the 133 training and validation data sets, a cut-off date for 134 the bulls ' birth date was introduced so that 25 % of 135 the youngest genotyped bulls were included in the 136 validation dataset. Bulls without genotyped sire in 137 the training dataset were excluded. Animals from the 138 training data set were born before June 2002, while 139 animals from the validation data set were born be-140 tween June 2002 and 2004. This cross-validation 141 design corresponds to the one used in studies of the 142 EuroGenomics Consortium (Lund et al.,

182

  The EN algorithm[START_REF] Zou | Regularization and variable selection via the Elastic Net[END_REF] Croiseau 183 et al., 2009) corresponds to a combination of the ridge 184 regression (RR) and LASSO procedures. The differ-185 ence between RR b ˆRR = arg min {g n i=1 (y i xx i b) 2 + 186 lg j b 2 j } and LASSO b ˆLASSO =arg min{g n i=1

  a pre-selection of SNP in the Holstein breed. Similar results were obtained in Montbe´liarde and Normande (data not shown). The results for the six traits are given, as well as the average of the number of SNPs over the 25 traits available for the three breeds. The number of SNPs retained was dependent on the genetic architecture of the trait. Traits such as Fat

  that false-positive or uninformative effects are regressed towards zero, but in practice, these false positive or uninformative effects are not strictly equal to zero. EN, which shares some variable selection properties with other methods (like Bayes B, Cp, …) limits the number of SNPs with non-null estimated effects in the model. This property can be an advantage because it alleviates the p>>n problems, in particular for smaller breeds. Limiting the number of SNP effects to estimates becomes important for an accurate prediction equation. Since this study shows that EN provides better results than gBLUP for most traits in the three breeds studied, we tried to push further the idea of variable selection both in the case of gBLUP and EN by adding an SNP pre-selection step based on QTL detection. The resulting correlations between DGV and observed DYD and also the slopes of the regression of observed DYD on DGV were similar to the ones obtained using the complete set of SNPs. Moreover, both the EN algorithm and the pre-selection of the SNP led to a reduction of the number of SNPs included in the prediction equation with a minor effect on the quality of prediction. This procedure seems particularly relevant in the genomic selection context for two reasons :

Fig. 1 .

 1 Fig. 1. Mean change in correlation (dashed lines) over the 25 traits for Montbe´liarde (&), Normande ( ' ) and Holstein (m) when the maximum number of SNPs selected by EN is restricted to the value indicated on the x-axis. Continuous lines represent the actual number of selected SNPs.

Table 1 .

 1 Number of animals genotyped per data set for the three breeds studied

	. DYD were
	included in the analysis if EDC exceeded 20.	152
	For the three breeds, 25 traits were available : five	153
	production traits, two conception rate traits, 16 mor-	154
	phological traits, somatic cell counts and milking	155
	speed. Initially, only six traits were chosen to com-	156
	pare the different approaches and for fine tuning of	157
	different parameters. These six traits were the five	158

Table 6

 6

	presents the number of SNPs with a non-
	null effect retained by EN algorithm without or with

Table 3 .

 3 Number of LRT peaks identified for milk yield as a function of LRT threshold and window size in the Montbe´liarde, Normande and Holstein breeds

		SNP	LRT threshold
		window
		size	3	5
	Montbe´liarde	25	432	265
		50	273	180
	Normande	25	363	197
		50	219	142
	Holstein	25	481	350
		50	268	204

Table 4 .

 4 Weighted correlation between DGV and observed DYD for the three breeds obtained using pedigree-based BLUP, gBLUP and EN on the complete set of SNP (54 K) or after a pre-selection of the SNP (PS)

	Pedigree-	gBLUP	EN
	based
	BLUP	54 K PS	54 K PS
	Montbe´liarde Milk yield Fat yield Protein yield Fat content Protein content 0 . 25 0 . 28 0 . 40 0 . 27 0 . 40 Conception rate 0 . 43	0 . 44 0 . 43 0 . 45 0 . 42 0 . 50 0 . 50 0 . 50 0 . 51 0 . 46 0 . 47 0 . 46 0 . 47 0 . 51 0 . 56 0 . 59 0 . 59 0 . 44 0 . 42 0 . 44 0 . 42 0 . 43 0 . 42 0 . 47 0 . 48
	Normande Milk yield Fat yield Protein yield Fat content Protein content 0 . 33 0 . 30 0 . 27 0 . 23 0 . 58 Conception rate 0 . 24	0 . 34 0 . 38 0 . 41 0 . 42 0 . 39 0 . 38 0 . 41 0 . 41 0 . 31 0 . 33 0 . 37 0 . 40 0 . 61 0 . 63 0 . 71 0 . 75 0 . 50 0 . 55 0 . 54 0 . 53 0 . 27 0 . 30 0 . 31 0 . 31
	Holstein Milk yield Fat yield Protein yield Fat content Protein content 0 . 47 0 . 38 0 . 40 0 . 44 0 . 44 Conception rate 0 . 29	0 . 56 0 . 56 0 . 57 0 . 57 0 . 59 0 . 59 0 . 63 0 . 63 0 . 55 0 . 54 0 . 57 0 . 57 0 . 72 0 . 74 0 . 80 0 . 79 0 . 73 0 . 73 0 . 75 0 . 73 0 . 35 0 . 33 0 . 33 0 . 33

Table 5 .

 5 Slope of the regression of observed DYD on DGV for the Holstein breed obtained using pedigreebased BLUP, gBLUP and EN on the complete set of SNP (54 K) or after a pre-selection of the SNP (PS) Fine tuning genomic evaluations in dairy cattle through SNP pre-selection with the Elastic-Net algorithm. Genetics Research, 93 (6), 409-417. DOI : 10.1017/S0016672311000358394 content where DGAT1 explains a very high part of 395 the variance required fewer SNPs than conception 396 rate. The mean number of SNPs over the 25 traits 397 illustrates the impact of pre-selection on the number 398 of retained SNPs.

		Pedigree-	gBLUP		EN
		based
	Holstein	BLUP	54 K PS	54 K PS
	Milk yield Fat yield Protein yield Fat content Protein content Conception rate 0 . 80 0 . 80 0 . 96 0 . 86 0 . 98 0 . 94	0 . 68 0 . 80 0 . 65 0 . 87 0 . 90 0 . 78	0 . 68 0 . 80 0 . 61 1 . 06 0 . 76 0 . 80 0 . 89 0 . 95 0 . 83 0 . 93 0 . 69 0 . 84	0 . 80 1 . 05 0 . 78 0 . 98 0 . 92 0 . 84

Comment citer ce document : Croiseau, P., Legarra , A., Guillaume, F., Fritz, S., Baur, A., colombani, C., Robert Granie, C., Boichard, D.,

[START_REF] Habier | Fine tuning genomic evaluations in dairy cattle through SNP pre-selection with the Elastic-Net algorithm[END_REF]

.

Table 6 .

 6 Correlation and number of SNP used in the prediction equation using the EN algorithm on the whole set of SNP (54 K) or after a pre-selection of the SNP (PS) for the Holstein breed

		Holstein
		54 K		PS
			Number		Number	Impact on
	Traits	Correlation	of SNPs	Correlation	of SNPs	Correlation
	Milk yield Fat yield Protein yield Fat content Protein content Conception rate Mean over the 6 traits Men over 25 traits	0 . 57 0 . 63 0 . 57 0 . 79 0 . 75 0 . 33 --	1355 1271 5648 1351 2297 20 904 5471 16 334	0 . 59 0 . 62 0 . 56 0 . 78 0 . 73 0 . 34 --	1329 1211 1098 1087 1742 9677 2691 10 059	0 . 02 x0 . 01 x0 . 02 x0 . 01 x0 . 02 0 . 01 x0 . 01 x0 . 01

Table 7 .

 7 Highest correlation and corresponding number of selected SNPs when using the whole set of SNP (54 K), after a pre-selection of the SNP (PS) or when the number of selected SNPs is limited to 2500, 1500 or 1000 in the Holstein breed

			54 K	PS	2500 SNPs	1500 SNPs	1000 SNPs
	Milk yield	Correlation	0 . 569	0 . 573	0 . 573	0 . 569	0 . 551
	Fat yield	SNP Correlation	1328 0 . 631	2752 0 . 626	2422 0 . 631	1328 0 . 631	955 0 . 624
	Protein yield	SNP Correlation	1273 0 . 573	1126 0 . 568	1273 0 . 568	1273 0 . 565	991 0 . 561
	Fat content	SNP Correlation	21 716 0 . 795	2390 0 . 791	2120 0 . 795	1448 0 . 795	959 0 . 791
	Protein content	SNP Correlation	1364 0 . 748	1068 0 . 731	1364 0 . 748	1364 0 . 696	985 0 . 694
	Conception rate	SNP Correlation	2368 0 . 335	3684 0 . 328	2368 0 . 320	1419 0 . 307	996 0 . 301
		SNP	20 853	9144	2379	1141	850

Version postprint

Comment citer ce document : Croiseau, P., Legarra , A., Guillaume, F., Fritz, S., Baur, A., colombani, C., Robert Granie, C., Boichard, D., [START_REF] Habier | Fine tuning genomic evaluations in dairy cattle through SNP pre-selection with the Elastic-Net algorithm[END_REF]. Fine tuning genomic evaluations in dairy cattle through SNP pre-selection with the Elastic-Net algorithm. Genetics Research, 93 (6), 409-417. DOI : 10.1017/S0016672311000358 497 relationships [START_REF] Habier | The impact of genetic relationship information on genomeassisted breeding values[END_REF](Habier et al., , 2010 b) b). However, 534 535 In this study, we focused on one variable selection 536 method that is the EN and one pre-selection method 537 that is LDLA. Obviously, other genomic selection 538 methods (Bayesian methods for instance) and other 539 pre-selection approaches (based on 'pure ' association 540 studies instead of LDLA for instance) should be also 541 tested to complete this study. EN provided better 542 results in our study and our model assumed that all 543 genetic variation was explained by SNP. The latter 544 may be true if all causal mutations are bi-allelic and 545 if SNPs are in strong linkage disequilibrium with all 546 causal mutations. If causal mutations are multi-allelic 547 or if SNPs are in weak linkage disequilibrium with 548 this causal mutation, model based on haplotypes 549 could be more advantageous. The current French 550 genomic evaluation [START_REF] Boichard | Genomic selection in French dairy cattle[END_REF] combines 551 MAS AQ4 on QTL followed through haplotypes and geno-552 mic selection based on SNP detected with the EN 553 algorithm. EN was used as a variable selection method and prediction equations were generated for the French genomic MAS.

In conclusion, the EN algorithm appears to be a very flexible and promising tool in the genomic selection framework that can be used for genomic evaluation or as a variable selection device to provide SNP of interest to a marker-assisted evaluation method.

This work was part of the AMASGEN project within the ' UMT e´valuation ge´ne´tique ' financed by the French National Research Agency (ANR) and by ApisGene.

Labogena is gratefully acknowledged for providing the genotypes.

Declaration of Interest

None.


Agnès Petit-Paitel

Baptiste Ménard

Alice Guyon

Vincent Béringue

Jean-Louis Nahon

Nicole Zsürger

Joëlle Chabry
email: chabry@ipmc.cnrs.fr

Agne `s Petit-Paitel

Baptiste Me ´nard

Vincent Be ´ringue

Nicole Zsu ¨rger

Joe ¨lle Chabry

Prion protein

Introduction

Alcohol is among the most widely abused drugs in the world. Neuronal mechanisms responsible for the different behavioral responses to ethanol (EtOH) such as tolerance, dependence and intoxication generate intense interest to the scientific community. Alcohol has multiple effects on neurons as it modifies the physiological activity of many receptors and ion channels including c-aminobutyric acid A (GABA A ) and N-methyl-Daspartate (NMDA) [START_REF] Peoples | Lipid vs protein theories of alcohol action in the nervous system[END_REF]. NMDA receptors (NMDARs) are involved in all EtOH-associated phenotypes such as dependence, tolerance or craving indicating that they are pivotal for EtOH-induced behaviors [START_REF] Vengeliene | Neuropharmacology of alcohol addiction[END_REF]. NMDARs consist of obligatory GluN1 subunits associated with different GluN2 (A-D) subunits. GluN2B is substrate for phosphorylation by members of the Src-protein tyrosine kinases (Src-PTK), mainly fyn. Acute exposure to EtOH inhibits NMDAR activity leading to a decrease in neuronal excitability; however, within minutes after EtOH administration, activated fyn kinase specifically phosphorylates GluN2B subunit in the hippocampus, restoring the channel activity [START_REF] Yaka | Scaffolding of Fyn kinase to the NMDA receptor determines brain region sensitivity to ethanol[END_REF][START_REF] Ron | Signaling cascades regulating NMDA receptor sensitivity to ethanol[END_REF]. Consistently, mice lacking the fyn kinase gene are highly sensitive to EtOH sedative effects and do not develop tolerance [START_REF] Miyakawa | Fyn-kinase as a determinant of ethanol sensitivity: relation to NMDA-receptor function[END_REF]. Accumulating evidence shows a major role for the NMDARs in EtOH-mediated effects; therefore, all proteins or factors likely to modulate NMDAR functions exhibit an obvious interest in the pathology of alcohol-related disorders. Interestingly, growing data suggest that the prion protein (PrP) could be one of these NMDA modulating factors [START_REF] Khosravani | Prion protein attenuates excitotoxicity by inhibiting NMDA receptors[END_REF].

Widely expressed in brain, PrP is a membrane-bound, glycophosphatidylinositol (GPI)-anchored protein found primarily in lipid rafts on the cell membrane [START_REF] Vey | Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains[END_REF]. Expression of PrP is absolutely required for the neuropathogenesis of a set of fatal neurodegenerative disorders, namely prion diseases [START_REF] Prusiner | Prions[END_REF]. Because of its pivotal role in the etiology of these diseases, considerable efforts have been made to decipher the neurophysiological roles of PrP, which still remain enigmatic [START_REF] Aguzzi | The prion's elusive reason for being[END_REF]. PrP has been involved in a variety of physiological functions ranging from regulation of circadian rythm and sleep [START_REF] Tobler | Altered circadian activity rhythms and sleep in mice devoid of prion protein[END_REF][START_REF] Tobler | Sleep and sleep regulation in normal and prion protein-deficient mice[END_REF] to spatial learning [START_REF] Criado | Mice devoid of prion protein have cognitive deficits that are rescued by reconstitution of PrP in neurons[END_REF]. Neurons of PrP-null (PrP 2/2 ) mice are highly sensitive to hyperexcitability [START_REF] Collinge | Prion protein is necessary for normal synaptic function[END_REF] and excitotoxicity [START_REF] Walz | Increased sensitivity to seizures in mice lacking cellular prion protein[END_REF], both features closely linked to NMDAR activity. By binding to GluN2D subunit, PrP could prevent NMDAR sustained activity and thus excitotoxicity [START_REF] Khosravani | Prion protein attenuates excitotoxicity by inhibiting NMDA receptors[END_REF].

Since PrP can stimulate Src-PTK-dependent intracellular signaling pathway [START_REF] Mouillet-Richard | Signal transduction through prion protein[END_REF][START_REF] Santuccione | Prion protein recruits its neuronal receptor NCAM to lipid rafts to activate p59fyn and to enhance neurite outgrowth[END_REF], which in turn may regulate the NMDAR activity, we hypothesized a possible involvement of PrP in the behavioral sensitivity and acute tolerance to EtOH. We show that PrP 2/2 mice present exacerbated EtOH sensitivity compared to wild type mice. Conversely, mice over-expressing mouse, human or hamster PrP sequences are less sensitive to the EtOH-induced sedative effects than wild type mice. Electrophysiological and biochemical experiments show that PrP is a key determinant in the establishment of the acute tolerance to EtOH through a fyn-mediated GluN2B-NMDAR activation. In our attempt to decipher the molecular mechanisms underlying this process, we investigated the effects of EtOH on lipid membrane features. We established that EtOH altered lipid raft features in a different way on hippocampal homogenates prepared from EtOH-treated wt and PrP 2/2 mice, which underlies the implementation of distinct intra-cellular signaling pathways.

Materials and Methods

Animals

In the present study, we used transgenic mouse lines knockout for the PrP gene (PrP 2/2 [START_REF] Bueler | Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein[END_REF]), over-expressing (5-8 fold) mouse PrP (Prnp-a allele, tga20 line [START_REF] Fisher | Prion protein (PrP) with amino-terminal deletions restoring susceptibility of PrP knockout mice to crapie[END_REF]), human PrP (M129 allele, tg650 line [START_REF] Be ´ringue | Prominent and persistent extraneural infection in human PrP transgenic mice infected with variant CJD[END_REF]) or hamster PrP (tg7 line [START_REF] Cronier | Prion strain-and species-dependent effects of antiprion molecules in primary neuronal cultures[END_REF]) on a PrP 2/2 background. Because PrP 2/2 mice are on a C57BL6/Jx129/Sv mixed genetic background, we used the hybrid strain of C57Bl6/J x 129/Sv as wt controls. To control for the possible influence of the genetic background, homozygous knockout and over-expressing mouse PrP gene mice onto pure C57BL6/J background were also used [START_REF] Gourdain | Adoptive transfer of T lymphocytes sensitized against the prion protein attenuates prion invasion in scrapie-infected mice[END_REF][START_REF] Loeuillet | Prion replication in the hematopoietic compartment is not required for neuroinvasion in scrapie mouse model[END_REF]. To do so, heterozygote mating pairs, inbred for at least ten generations onto C57BL6/J background, were mated to produce homozygous (noted as PrP 2/2 B6 and tga20 B6 ) and wt were approved by the Nice Sophia Antipolis University regional animal safety committee (CIEPAL-Azur).

Loss of righting reflex (LORR)

For all behavioral and biochemical experiments, 8-to10-week old male mice were housed in individual cages for a 30 min habituation period then injected i.p. with EtOH (2.4 or 3.2 g/kg, 30% or 40% solutions in NaCl 0.9% respectively). After the mice lost the righting reflex, they were put on their backs in their home cage. The duration of LORR was defined as the time from the loss of the righting reflex to the time at which it was regained. Recovery was defined as the time at which mice could right themselves twice in a 30 second period after being placed on their backs [START_REF] Harris | Mutant mice lacking the gamma isoform of protein kinase C show decreased behavioral actions of ethanol and altered function of gamma-aminobutyrate type A receptors[END_REF]. The behavioral room was illuminated with a soft light, and external noise was attenuated. Animals that failed to react to hypnotic effects of EtOH and those presenting a LORR greater than 180 min were excluded from the statistical analysis. Sensitivity to hypnotic effect of 4,5,6,7-tetrahydroisoxazolo [5,4c]pyridin-3-ol (THIP) and diazepam was assessed using same procedure. Blood samples of ,50 ml were obtained from tail vein 15, 90 and 180 min after administration of 3.2 g/kg EtOH from independent experiments. Blood EtOH concentration was quantified using the DXC600, Beckmann Coulter.

Preparation of hippocampus homogenates and western blotting

Subjects were decapitated 5, 20 and 60 min after 3.2 g/Kg EtOH or 5 min after saline (NaCl 0.9%, Ctrl) injection. Hippocampi were dissected and immediately frozen on dry ice. Tissues were homogenized in cold lysis buffer containing 10 mM Tris-HCl pH 7.4, 1% triton X-100, 0.1% CHAPS, 140 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, and protease and phosphatase inhibitor cocktails, then centrifuged at 4000 rpm for 20 min at 4uC. Supernatants (100 mg of proteins) were mixed with the 2X denaturing loading buffer (20 mM Tris-HCl pH 6.8, 10% SDS, 10% glycerol, 10% ß-mercaptoethanol) boiled for 5 min, loaded onto a 10% polyacrylamide gel and then blotted onto a nitrocellulose membrane. Rabbit polyclonal antibodies were used to detect flotilin-1, GluN2B-containing NMDAR and phosphorylated-Tyr1472 GluN2B (p-Y1472) (AbCam, cat. ab41927, ab65875 and ab59205 respectively). Goat monoclonal antibodies to detect fyn were from Santa Cruz (cat. sc-434). Rabbit polyclonal antibodies from Cell Signaling against phospho-Y416Src-PTK cross-react with all Src-PTK members when phosphorylated at the equivalent site (cat. 2101S). Anti-PrP mouse monoclonal antibody, SAF83, was provided by Dr. J. Grassi (CEA, France).

Blots were developed using an enhanced chemoluminescence system with a LAS3000 detector (Fuji). Densitometry analyses were performed with a ''National Institutes of Health'' IMAGE software.

Isolation of lipid-raft microdomains by sucrose-gradient centrifugation

Hippocampi were lysed for 30 min at 4uC in 0.5 ml of 25 mM Tris-HCl pH 7.5 containing 150 mM NaCl, 1% Triton X-100. Lysates were resuspended in an equal volume of 85% sucrose and placed beneath a discontinuous gradient of sucrose consisting of 4 ml of 35% sucrose and 4 ml of 5% sucrose. Samples were centrifuged at 200 000 g for 16 h at 4uC in a SW 41 rotor, then fractions of 1 ml (9 fractions in total) were collected from the top of the gradient tube (fraction 1) to the bottom (fraction 9). Thirty ml of each fraction were analyzed by SDS-PAGE and western blot.

Immuno-stainings and confocal laser microscopy on hippocampal neurons in primary culture

Hippocampal neurons from embryonic day 16-17 mice were prepared as described previously [START_REF] Marella | Neurons and astrocytes respond to prion infection by inducing microglia recruitment[END_REF]. Briefly, dissociated neurons were plated onto glass coverslips pre-coated with polylysine and used after 15-18 days of culture. Neurons were washed three times in PBS and then incubated at 37uC in Earle's buffer (HEPES 25 mM Tris buffered to pH 7.4, NaCl 140 mM, KCl 5 mM, CaCl 2 1.8 mM, MgCl 2 0.8 mM, glucose 5 mM, BSA 0.01%) in the absence or in the presence of 100 mM EtOH for times indicated in the results. After two washes on ice with ice-cold PBS, neurons were fixed with 4% paraformaldehyde in PBS, 5% sucrose for 30 min at 4uC then washed three times with PBS. After 20 min in PBS, 5% BSA, cells were permeabilized in PBS, 5% BSA containing 0.2% triton X-100. Coverslips were incubated with 1:200 primary antibodies for 3 h at room temperature in PBS, 1% BSA, followed by incubation with the appropriate fluorescent-conjugated secondary antibody (1:1000). Coverslips were then mounted on glass slices with Mowiol containing 1 mg/ ml Hoescht, a fluorescent specific DNA dye. Each fluorochrome was independently captured with an FV10i scanning confocal microscope (Olympus, France). Images were acquired as single transcellular optical sections and analyzed using Image J software ("National Institutes of Health'').

Hippocampal slice preparation and electrophysiological recordings

Brains were rapidly collected into ice-cold artificial cerebrospinal fluid (aCSF), containing (in mM): 125 NaCl, 2.5 KCl, 1 MgCl 2 , 2 CaCl 2 , 1.25 NaH 2 PO 4 , 26 NaHCO 3 , pH 7.4, 25 glucose. Sagital slices of hippocampus (300 mM) were cut using a HM650V vibratome (Microm, Germany) and placed in a holding chamber at 34uC for 1 h and bubbled with 95% O 2 , 5% CO 2. Individual slices were placed in a submerged recording chamber and continuously superfused with ''low Mg 2+'' aCSF (Mg 2+ concentration lowered to 0.1 mM to unblock the NMDA receptors) at a flow rate of ,2 ml/min (23uC61). In response to stimulation of the Schaffer collateral-commissural pathway by a steel bipolar microelectrode, field EPSPs (fEPSPs) from the stratum radiatum of the hippocamal CA1 region were recorded extra cellularly using a glass micropipette filled with aCSF (5-10 MV). The stimulus intensity was set to evoke responses which were about half of the magnitude at which a population spike started to appear. Control responses were recorded in the presence of 10 mM 6-cyano-7-nitroquinoxalin 2,3 dione (CNQX, which blocks the non-NMDA component of the fEPSP), for 10 min (one single pulse every 10 s, averaged by groups of 6). Field potentials were recorded using a Axopatch 200B and peaks and slopes amplitudes of fEPSPs were analyzed using pCLAMP software (Axon Instruments). To determine the changes in fEPSP peak and slope amplitudes following 100 mM EtOH, fEPSPs were normalized to control (average peak or slope of 5 min pre-EtOH). Traces were filtered at 1000 Hz with Clampfit software and smoothed by adjacent averaging using Microcal Origin software. CNQX, EtOH and DL-2-aminophosphonovalerate (APV, 10 mM) dissolved in aCSF were applied in the perfusion medium.

Statistics

Statistical analysis was performed using Statistica software TM . For behavior experiments, Mann-Whitney test was used for comparison between groups, each dot representing an animal. For western blot analysis, comparison of mice was performed using the one-way analysis of variance followed by a Dunnett's post hoc analysis comparing all data to these obtained with the wt mice in the absence of EtOH taken as control.

Results

PrP-null and over-expressing PrP gene mice present altered sedation in response to an acute administration of EtOH

To investigate the involvement of PrP in the sedative effect induced by EtOH, we measured the duration of the loss of righting reflex (LORR) in PrP 2/2 , wt and over-expressing the mouse PrP gene (tga20). For both concentrations of EtOH administrated (i.e 2.4 and 3.2 g/kg), the duration of LORR was significantly longer for PrP 2/2 mice than for wild type (wt) control (Fig. 1A). Conversely, the LORR duration was shorter for tga20 mice compared to wt mice (Fig. 1A). Difference between the three mouse strains does not result from differential EtOH metabolism, since no significant difference in the blood EtOH concentration was found over time after EtOH administration (Fig. 1B). To examine the effect of different PrP genes, we also measured the LORR duration in mice over-expressing human and hamster PrP, namely tg650 and tg7 respectively (Fig. 1C). Mice over-expressing similar levels (i.e. 5-8 fold over-expression) of mouse, human and hamster PrP presented LORR durations significantly shorter than that of wt mice (Fig. 1A,C). Nevertheless, to control for a possible effect of the genetic background, we performed LORR assay on PrP 2/2 and tga20 back-crossed on a pure C57BL6/J genetic background (Fig. 1D). PrP 2/2 B6 mice slept much longer and tga20 B6 mice shorter than wt B6 mice suggesting that the EtOHinduced phenotype is due to the Prnp gene rather than to other genetic factors (Fig. 1D).

In order to examine whether the observed effects of alcohol could involve the NMDA receptors, mice were pretreated with MK-801, a selective NMDA open channel blocker [START_REF] Silveri | The effects of NMDA and GABAA pharmacological manipulations on ethanol sensitivity in immature and mature animals[END_REF][START_REF] Wilson | NMDA agonists and antagonists alter the hypnotic response to ethanol in LS and SS mice[END_REF] or ifenprodil, a selective blocker of GluN2B. Strikingly, pre-treatment with MK-801 or ifenprodil abolished the difference of LORR between PrP 2/2 and wt mice suggesting the involvement of NMDAR activity in the PrP-dependent sensibility to EtOH (Fig. 1E). Unlike ifenprodil, pre-treatment with MK-801 markedly potentiated the sedative effect of 3.2 g/kg EtOH both in PrP 2/2 and wt mice (Fig. 1A,E). In agreement with previous studies, we found that i.p. injection of MK-801 (0.2 mg/kg) or ifenprodil (3 mg/kg) never produced sedative effects per se ( [START_REF] Yaka | Fyn kinase and NR2B-containing NMDA receptors regulate acute ethanol sensitivity but not ethanol intake or conditioned reward[END_REF][START_REF] Palachick | Role of major NMDA or AMPA receptor subunits in MK-801 potentiation of ethanol intoxication[END_REF] and data not shown).

The GABAergic neurotransmitter system has also been involved in many EtOH's behavioral effects, therefore it was of interest to assess whether sensitivity to GABAergic drugs was altered by the Prnp gene deletion. To do so, duration of LORR was measured following administration of diazepam (a non subunit-selective GABA A receptor modulator) and THIP (a selective GABA A receptor agonist) in PrP 2/2 and wt mice (Fig. 1F). The mouse strains did not differ in their sensitivity to GABA receptor modulators suggesting that Prnp gene deletion did not alter significantly the GABA receptor functions. Taken together, our data suggest that the level of PrP expression may control EtOHinduced sedative effects probably through the regulation of the GluN2B-containing NMDAR activity.

Altered electrophysiological activity of NMDAR in hippocampal slices from PrP 2/2 mice following EtOH exposure To ascertain whether exacerbated EtOH sensitivity of PrP 2/2 mice was due to altered GluN2B activity, we carried out electrophysiological recordings on EtOH-treated hippocampal slices. Exposure of hippocampal slices to EtOH is known to transiently inhibit GluN2B-mediated excitatory post-synaptic potentials (EPSPs) [START_REF] Grover | Acute tolerance to ethanol inhibition of NMDA-mediated EPSPs in the CA1 region of the rat hippocampus[END_REF]. An acute tolerance caused by the Src-PTK-mediated phosphorylation of GluN2B is rapidly set up, resulting in the potentiation of NMDAR currents [START_REF] Miyakawa | Fyn-kinase as a determinant of ethanol sensitivity: relation to NMDA-receptor function[END_REF]. We hypothesized that the difference of LORR phenotype between PrP 2/2 and wt mice may be due to altered acute tolerance kinetics. To test this hypothesis, we compared the effects of EtOH on NMDA-mediated fEPSPs recorded with extracellular microelectrodes in hippocampal slices from PrP 2/2 and wt mice (Fig. 2A-D). Bath application of EtOH (100 mM) rapidly depressed NMDAR-mediated fEPSPs in slices from both mice (Fig. 2A-D). The fEPSPs amplitude partially recovered in wt mice after 8 min EtOH indicating the establishment of the acute tolerance, whereas recovery was delayed and lowered in hippocampal slices of PrP 2/2 mice. After EtOH wash out, fEPSP were gradually increased to the baseline for both mice, although the kinetic of recovery was slower for PrP 2/2 than for wt mice. In average, there was a significant difference between fEPSPs (both peak and slope) from PrP 2/2 and wt hippocampal slices in EtOH washout (Fig. 2C,D). As previously described [START_REF] Miyakawa | Fyn-kinase as a determinant of ethanol sensitivity: relation to NMDA-receptor function[END_REF], ifenprodil eliminated the acute tolerance to EtOH inhibition in hippocampal slices from wt mice (Fig. 2E,F). In summary, the acute tolerance to EtOH inhibition of NMDAR-mediated EPSPs developed more slowly in hippocampus of PrP 2/2 than in wt mice.

Altered GluN2B and Src-PTK phosphorylation patterns in hippocampus of PrP 2/2 mice following EtOH exposure Since PrP modifies the NMDAR function in hippocampal slices probably via a fyn kinase-dependent activation, cell colocalization between these proteins is expected in hippocampal neurons. Employing double immunofluorescence labeling in hippocampal slices, we showed that PrP was strongly co-localized with both GluN2B and fyn in CA1 of mouse hippocampus (Fig. 3A).

The sedative effects of EtOH and the NMDAR activity are closely linked to the phosphorylation of the GluN2B subunit mediated by members of the Src-PTK family [START_REF] Yaka | Fyn kinase and NR2B-containing NMDA receptors regulate acute ethanol sensitivity but not ethanol intake or conditioned reward[END_REF]. We postulated that the phosphorylation level (i.e. activation) of GluN2B and Src-PTK might be differentially modulated in the hippocampus of PrP 2/2 and tga20 mice as compared to wt after EtOH administration. Mice were i.p. injected with saline solution (Ctrl) or 3.2 g/kg of EtOH and euthanized 5, 20 or 60 min later. The phosphorylation levels of GluN2B and Src-PTK rapidly increased in the hippocampus of wt and tga20 mice as soon as 5 min post-EtOH injection, and reached a maximal value in 20 min (Fig. 3B,C). In marked contrast, no significant increase was observed in PrP 2/2 mice (Fig. 3B,C). The anti-phospho-Y416Src-PTK antibody used here does not distinguish between fyn and src; however, the signal is virtually absent in fyn-null mice suggesting that fyn is the predominant kinase in lysates of mouse hippocampus [START_REF] Bjarnadottir | Neuregulin1 (NRG1) signaling through Fyn modulates NMDA receptor phosphorylation: differential synaptic function in NRG1+/2 knock-outs compared with wild-type mice[END_REF]. Interestingly, in basal conditions, both GluN2B and Src-PTK phosphorylation levels were significantly reduced in the hippocampus of PrP 2/2 and increased in that of tga20 as compared to wt mice (Fig. 3B,C, Ctrl) suggesting that PrP is a major regulator of fyn-mediated GluN2B phosphorylation. By quantitative PCR and Western blot experiments, we checked that fyn and GluN2B mRNAs and proteins were indeed expressed at similar levels in hippocampus of the three strains of mice (data not shown).

In summary, we showed that the PrP expression is required to induce fyn-dependent GluN2B phosphorylation in response to acute in vivo administration of EtOH.

Sub-cellular co-localization of PrP, fyn and GluN2B in cultured hippocampal neurons: kinetics of EtOH-induced phosphorylation of Src-PTK and GluN2B

Immunohistochemistry and confocal microscopy observations were then performed on cultured hippocampal neurons to refine the sub-cellular location of proteins of interest (Fig. 4). PrP colocalized almost perfectly on neuronal extension with both fyn and GluN2B subunit and their phosphorylated isoforms (Fig. 4A). Immunolabelings of PrP and phosphorylated isoform of GluN2B overlapped with PSD95, a specific post-synaptic marker (Fig. 4B). Interestingly, immunolabeling of PrP, overlapped partially with Thy1, a specific marker of lipid rafts (Fig. 4B). Fyn and GluN2B staining were perfectly co-localized with Thy1 (Fig. 4B). Our results indicate that all the proteins of interest are co-localized in lipid rafts of the plasma membrane at a post-synaptic level.

We then investigated whether acute EtOH exposure on cultured hippocampal neurons induced GluN2B phosphorylation in a Src-PTK-dependent manner. In wt hippocampal neurons, phosphorylation levels of both GluN2B and Src-PTK were increased within 2 min of EtOH exposure and returned to prestimulation levels within 20 min (Fig. 4C, left). When EtOH was co-incubated with PP2, a specific inhibitor of Src-PTK, no change in GluN2B phosphorylation was observed indicating that Src-PTK mediated the EtOH-induced GluN2B phosphorylation (Fig. 4C, right). EtOH failed to increase Src-PTK and GluN2B phosphorylation levels in PrP 2/2 hippocampal neurons confirming that PrP is a major contributor of the EtOH effects in cultured hippocampal neurons (Fig. 4C, middle).

EtOH induced lipid rafts rearrangements on hippocampus of wt but not PrP 2/2 mice Rearrangement and alterations of lipid membrane have been well documented as early events in EtOH-induced effects [START_REF] Szabo | TLR4, ethanol, and lipid rafts: a new mechanism of ethanol action with implications for other receptormediated effects[END_REF][START_REF] Nourissat | Ethanol induces oxidative stress in primary rat hepatocytes through the early involvement of lipid raft clustering[END_REF]. We postulated that EtOH might modify the distribution of proteins of interest differently in hippocampal membranes from PrP 2/2 and wt mice. To test this hypothesis, lipid raft fractions were prepared by discontinuous sucrose gradient ultracentrifugation from hippocampus of PrP 2/2 and wt mice in basal conditions and after i.p. injection of EtOH, then were analyzed by western blotting. In control conditions, PrP was mainly found in the lowdensity membrane fraction (Fig. 5A andC, ctrl, fraction 4), together with the phosphorylated forms of GluN2B and Src-PTK and the lipid raft marker, flotilin-1. Similar distribution of these proteins was observed in flotation gradients prepared from PrP 2/2 mouse hippocampus in control conditions (Fig. 5B andC, ctrl). When flotation gradients were performed on EtOH-treated wt mouse hippocampus, the flotilin-1 labeling shifted toward a lower buoyancy fraction, which might suggest a protein clustering into lipid rafts (Fig. 5A and C 5 min, fraction 5). Five min post-EtOH administration, the immunolabeling of PrP, phosphorylated isoforms of GluN2B and Src-PTK also shifted toward the fraction 5. Twenty min post-EtOH administration, all proteins co-distributed in the fraction 4 as in the control conditions indicating that the EtOH effect on the lipid membrane was transient (Fig. 5A andC, 20 min). Interestingly, no change in buoyancy of proteins of interest was observed after EtOH administration to PrP 2/2 mice (Fig. 5B), suggesting that PrP could contribute to the formation of such clustering.

Discussion

Expression of PrPc is absolutely required for the neuropathogenesis of a set of dramatic and fatal neurodegenerative disorders, namely TSEs. Because prion-induced neurodegeneration might be a consequence of its loss of function(s), one can assume that neurophysiological function(s) of PrPc must be of prime importance. Despite considerable efforts made in many directions to understand PrPc functions, a clear comprehensive view failed to be achieved. Our results demonstrate that PrP is involved in the neurological effects of acute EtOH exposure by regulating the GluN2B-NMDAR function via a fyn-dependent activation pathway, a previously unknown physiological role for this protein. Moreover, our data strongly suggest that PrP plays a pivotal role in the organization of lipid rafts making possible the transduction of intracellular signalings upon acute EtOH administration (Fig. 6).

Based on behavioral experiments, we showed that the EtOHinduced sedative effect is inversely correlated with the level of expression of PrP. Indeed, PrP 2/2 mice presented a greater sensitivity while tga20 showed a relative insensitivity to the sedative effect of EtOH as compared to wt mice. The phenotype was manifest in mouse lines on two different genetic backgrounds providing strong evidence that the phenotype is caused by the level of PrP expression per se. Like tga20, transgenic mice overexpressing human or hamster PrP gene present a relative insensitivity to the EtOH-induced sedative effect as compared to wt mice suggesting that the phenotype is poorly dependent on the PrP sequence.

As expected, in the hippocampus of wt mice, an acute tolerance was rapidly set up through Src-PTK-mediated phosphorylation of the tyrosine residue 1472 of the GluN2B subunit. In PrP 2/2 mice, EtOH exposure only lead to the inhibition of the channel activity but did not result in Src-PTK and GluN2B-NMDAR phosphorylation. Moreover, acute desensitization or rebond potentiation failed to occur in hippocampal slices from PrP 2/2 mice after the EtOH wash out. The lack of ethanol-induced intracellular pathway activation may explain the enhanced sensitivity to sedative effects of EtOH in PrP 2/2 mice. Taken together, we demonstrated that PrP is as key determinant in the EtOH sensitivity through the regulation of GluN2B-NMDAR activity. Deletion and over-expression of PrP profoundly altered behavioral effects of acute EtOH exposure underlining the functional importance of PrP-dependent intracellular signaling cascade. To our knowledge, we provide the first evidence of in vivo PrPmediated intracellular signaling pathway activation.

Proteins, such as PrP, attached to the outer leaflet of the cell membrane via GPI anchors are often receptors that mediate cell activation [START_REF] Lisanti | Caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis[END_REF]. Because such proteins lack trans-membrane and cytoplasmic domains and therefore cannot directly transduce an intracellular signal, there has been considerable interest in determining how these proteins function. a-PrP antibodiesmediated cross-linking induced fyn activation in a caveolindependent pathway [START_REF] Mouillet-Richard | Signal transduction through prion protein[END_REF]. It is possible that EtOH induces the oligomerization of PrP in specific membrane microdomains leading to PrP-dependent transduction. Enriched in potent effectors on both sides of the plasma membrane, lipid rafts are thought as functional platforms favoring signal transduction [START_REF] Tansey | GFRalpha-mediated localization of RET to lipid rafts is required for effective downstream signaling, differentiation, and neuronal survival[END_REF]. An accurate regulation of recruitment into and exclusion outside lipid rafts of signaling proteins is expected for efficient intracellular signaling. Owing to its interaction with lipids, EtOH could alter protein recruitment and thus, could provoke subsequently an aberrant downstream response. As an example, EtOH inhibits lipid raft-mediated TCR-signaling in T lymphocytes, resulting in suppression of immune responses [START_REF] Ghare | Ethanol Inhibits Lipid Raft-Mediated TCR Signaling and IL-2 Expression: Potential Mechanism of Alcohol-Induced Immune Suppression[END_REF]? Hence, we demonstrated that EtOH provokes transient lipid rafts rearrangement and thus may trigger specific transduction in neuronal cell. Thus, EtOH may trigger the transient clustering of PrP leading to its oligomerization or alternatively induce interplay with lipid raft components. Remarkably, no buoyancy changes of lipid rafts were observed after acute administration of EtOH to PrP 2/2 mice suggesting that PrP favors cohesion and interplays of proteins of interest. PrP could be an important factor for the establishment and/or the maintenance of a correct positioning of the different partners, allowing the spatio-temporal sequence of events occurring after an acute EtOH exposure. It is conceivable that the over-expression of PrP (tga20 mice) would trigger its constitutive oligomerization at the neuronal surface resulting in sustained fyn activation. As a consequence, GluN2B would be constitutively hyper-phosphorylated in hippocampus of tga20 mice explaining at least in part the relative insensitivity to EtOHsedative effects (Fig. 6). Supporting this hypothesis, mice overexpressing fyn present a low sensitivity to EtOH-induced sedation, as tga20 mice [START_REF] Boehm | Over-expression of the fyn-kinase gene reduces hypnotic sensitivity to ethanol in mice[END_REF].

How PrP and fyn are linked across the membrane remains an intriguing mystery. The neural cell adhesion molecules (N-CAM) could ensure this role. Indeed, the trans-membrane N-CAM isoforms interact both with PrP [START_REF] Schmitt-Ulms | Binding of neural cell adhesion molecules (N-CAMs) to the cellular prion protein[END_REF] and fyn [START_REF] Beggs | NCAM140 interacts with the focal adhesion kinase p125(fak) and the SRC-related tyrosine kinase p59(fyn)[END_REF]. Interestingly, PrP recruits to and stabilizes N-CAM in lipid rafts leading to the activation of fyn [START_REF] Santuccione | Prion protein recruits its neuronal receptor NCAM to lipid rafts to activate p59fyn and to enhance neurite outgrowth[END_REF]. Specific binding sites of EtOH have been recently characterized on the extra-cellular domain of a member of N-CAM family, namely L1 [START_REF] Arevalo | An alcohol binding site on the neural cell adhesion molecule L1[END_REF]. Further investigations will be required to determine whether N-CAM or other well-known PrP partners such as caveolin or laminin receptor participate to PrPdependent transduction under EtOH stimulus.

Owing to its localization at the synaptic contacts, PrP could participate to the synaptic physiology [START_REF] Sale `s N | Cellular prion protein localization in rodent and primate brain[END_REF][START_REF] Herms | Evidence of presynaptic location and function of the prion protein[END_REF]. Our study is consistent with such assumption, inasmuch as the deletion of Prnp gene results in altered synaptic NMDAR function in response to EtOH exposure. Altogether, our results reinforce the concept of a prominent role of PrP in synaptic function. In addition, the identification of a new partner, namely the PrP, involved in neuronal responses to alcohol should allow the identification of new targets for future drug development for the treatment of alcohol disorders. . Such interplay could be pivotal to activate the Src-PTK (i.e. phosphorylated on tyrosine Y416) that in turn restores NMDAR activity by phosphorylation on tyrosine residue Y1472. In PrP 2/2 mice, the acute tolerance to EtOH is established slowly because the lack of PrP hinders fyn interplay. Thus, no EtOH-induced activation of the fyn kinase is observed in hippocampus of PrP 2/2 . As a consequence, GluN2B phosphorylation does not occur resulting in the lack of repotentiation of NMDAR currents that may explain, at least partly, the hypersensitivity of PrP -/2 mice to EtOH. In tga20 mice, the over-expression of PrP and thus its high concentration into lipid rafts would lead to a constitutive activation of fyn and subsequently to the hyper-phosphorylation of GluN2B-NMDAR. doi:10.1371/journal.pone.0034691.g006

Figure 1 .

 1 Figure 1. Altered sedative effect of EtOH in PrP 2/2 , PrP-over-expressing mice: relation to NMDAR function. EtOH sensitivity was evaluated by measuring the duration of LORR after i.p. injection of 2.4 (A, left) or 3.2 g/kg EtOH (A, right and B, C, D, E). In panel E, the EtOH injection was preceded by an i.p. injection of 0.2 mg/kg of the NMDAR antagonist, MK-801 or 3 mg/kg of ifenprodil. In panel F, sedation induced by GABA modulators was evaluated by measuring the duration of LORR after i.p. injection of 50 mg/kg diazepam (left) or 30 g/kg THIP (right). For LORR experiments (panels A, C, D, E and F), each symbol represents a mouse; the number of mice tested is shown below each condition; the dark line is the median value. The wt mice i.e. C57BL6/J x 129/Sv for panels A, B, C, E and F or C57BL6/J for panel D (noted Wt B6 ) were taken as controls. Significance (p) was calculated using the nonparametric Mann-Whitney test; *p,0.05; **p,0.01; ***p,0.001; n.s; not significant. Blood EtOH concentration (B) was performed on four mice of each genotype for each time point and represented as the mean 6 SD (student t test). doi:10.1371/journal.pone.0034691.g001

Figure 2 .

 2 Figure 2. Effect of acute exposure to EtOH on GluN2B-containing NMDAR function in hippocampal slices of PrP 2/2 and wt mice. Panel A represents typical traces of: NMDAR fEPSP recorded in the presence of CNQX of wt (left) and PrP 2/2 (right) hippocampal slices (average of 6 single sweeps) obtained before (baseline, black), during EtOH application for 12 min (red), 10 min after EtOH wash out (grey) or in the presence of the NMDAR antagonist, APV (blue). Time course for normalized maximal peaks (B) for fEPSPs in the hippocampus of PrP 2/2 (open) and wt (grey) mice measured before and during the bath application of 100 mM EtOH. Data are presented as mean 6SEM, percentage of baseline. Curves were significantly different from times 3-25 min using exact and Monte Carlo resampling for the Wilcoxon-Mann-Whitney test (p,0.05). Histograms show the comparison of fEPSPs peaks (C) and slopes (D) during EtOH application (0-12 min) and after EtOH wash out (17227 min in B). Mean 6SEM, N = 66, 33 in C for wt and PrP 2/2 respectively. Comparison of fEPSPs peaks (E) and slopes (F) in wt mouse during 15 min of EtOH application and after wash out with and without 5 mM ifenprodil. Mean 6 s.e.m, n = 33, 66 with and without ifenprodil respectively. **p,0.01, ***p,0.001 using a one-way ANOVA followed by a Dunnett's post hoc analysis. doi:10.1371/journal.pone.0034691.g002

Figure 3 .

 3 Figure 3. Acute exposure to EtOH transiently increased phosphorylation levels of GluN2B and Src-PTK in hippocampus of PrPexpressing mice but not in that of PrP 2/2 mice. A. Fixed and permeabilized hippocampus slices were incubated with 1:200 primary antibodies in PBS 1%BSA containing 0.05% triton X-100 for 3 h, followed by incubation with the appropriate fluorescent-conjugated secondary antibody (1:1000). Co-labeling with antibodies against PrP (green, left) and fyn or GluN2B (red, middle) are presented in A. The merge images (right) correspond to the superposition of the two fluorescence signals (yellow). B, C. PrP 2/2 , tga20 and wt mice were injected i.p. with saline and euthanized 5 min later (Ctrl in B and C) or with EtOH (3.2 g/kg) and euthanized 5, 20 or 60 min later (B, C). Solubilized proteins from hippocampus were submitted to SDS-PAGE, blots were then probed with antibodies directed against phospho(p)-Y416Src-PTK, p-Y1472GluN2B, and actin (B). Histograms (C) depict quantification of the levels of phosphorylation of Src-PTK (left) and GluN2B (right) normalized to actin. Results are representative of 2 animals/ condition in 6 independent experiments and presented as mean 6 SD. Relative ratio of hippocampus from saline injected wt mouse taken as 1 (N = 6; Dunnett's post hoc; *p,0.05; **p,0.01; ***p,0.001). doi:10.1371/journal.pone.0034691.g003

Figure 4 .

 4 Figure 4. Co-localization of PrP, fyn, GluN2B and their phosphorylated forms in hippocampal neurons in primary culture: Effects of an acute exposure to EtOH on GluN2B phosphorylation level. (A) Hippocampal neurons were co-labeled with antibodies against PrP (green, left) and fyn, p-Y416Src-PTK, GluN2B and p-Y1472GluN2B (red, middle). The merge images (right) correspond to the superposition of the two fluorescence signals (yellow). Nuclei were labeled with the Hoescht reagent and appeared in blue. (B) High magnification of neuronal extensions labeled with antibodies against PrP (green), p-Y1472 GluN2B (red) and PSD95 or Thy1 (blue). The merge images (right) correspond to the overlap of the three fluorescence signals (white). Scale bar = 10 mm. (C) Hippocampal neurons from wt and PrP 2/2 mouse embryos were incubated with saline solution (Ctrl) or 100 mM EtOH (in NaCl 0.9%) for the indicated times. Alternatively, PP2 was pre-incubated 15 min before the addition of EtOH. Neurons were washed twice with PBS, lysed and prepared for SDS-PAGE and western blotting analysis. Blots were probed with the antibodies directed against p-Y1472GluN2B, p-Y416Src-PTK and actin to ensure correct protein loading in the different conditions. One representative of four independent experiments is shown. doi:10.1371/journal.pone.0034691.g004

Figure 5 .

 5 Figure 5. EtOH induced changes in the buoyancy of lipid-raft associated proteins. Lipid-rafts were prepared by sucrose-gradient centrifugation from hippocampus of saline (Ctrl) and EtOH-treated wt (A) and PrP 2/2 (B) mice (5, 20 min post-EtOH i.p injection). An aliquot of each fraction was analysed by SDS-PAGE and blots were probed with the indicated antibodies. One representative of four independent experiments is shown in panels A and B. Amounts of immunoreactivity of PrP, p-Y1472GluN2B, p-Y416Src-PTK and flotilin-1 were determined in fractions 4 and 5 and expressed as the percent of total immunoreactivity measured in all fractions for each protein of interest. Results are representative of 2 mice/ condition in 4 independent experiments and presented as mean 6 SD (N = 4; Dunnett's post hoc; EtOH injections versus saline injections: *p,0.05; **p,0.01). doi:10.1371/journal.pone.0034691.g005

Figure 6 .

 6 Figure 6. Proposed model of PrP-dependent EtOH effects. In wt mice, acute administration of EtOH inhibits GluN2B-NMDAR currents. An acute tolerance (i.e. reversion) is rapidly set up leading to the repotentiation of NMDAR currents mediated by Src-PTK-dependent phosphorylation of the GluN2B subunit. EtOH provokes the clustering of proteins into lipid rafts allowing interplay of PrP/PrP or PrP/putative trans-membrane partner (TM). Such interplay could be pivotal to activate the Src-PTK (i.e. phosphorylated on tyrosine Y416) that in turn restores NMDAR activity by phosphorylation on tyrosine residue Y1472. In PrP 2/2 mice, the acute tolerance to EtOH is established slowly because the lack of PrP hinders fyn interplay. Thus, no EtOH-induced activation of the fyn kinase is observed in hippocampus of PrP 2/2 . As a consequence, GluN2B phosphorylation does not occur resulting in the lack of repotentiation of NMDAR currents that may explain, at least partly, the hypersensitivity of PrP -/2 mice to EtOH. In tga20 mice, the over-expression of PrP and thus its high concentration into lipid rafts would lead to a constitutive activation of fyn and subsequently to the hyper-phosphorylation of GluN2B-NMDAR. doi:10.1371/journal.pone.0034691.g006

PLoS ONE | www.plosone.org

April 2012 | Volume 7 | Issue 4 | e34691

Acknowledgments

The authors thank Drs. C. Weissmann, J-Y Cesbron, C. Aucouturier for the use of PrP 2/2 and tga20 mice and Ms. Raynaud from the "Ho ˆpital d'Antibes" (France) for the blood alcohol concentration assays. We are grateful to Drs. P. Chavis, C. Lemaire, S. Martin and P. Haghighi for critical reading of the manuscript and fruitful discussions.

This work was supported by grants from the Agence Nationale de Recherche (ANR-06-NEURO-007-01) and by the IREB (France). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions