Vincent V Planche
email: planche.vincent@gmail.com

Ismail Koubiyr

José E Romero

José V Manjon

Pierrick Coupé

Mathilde Deloire

Vincent Dousset

Bruno Brochet

Aurélie Ruet

Thomas Tourdias

Regional hippocampal vulnerability in early multiple sclerosis: Dynamic pathological spreading from dentate gyrus to CA1

Keywords: multiple sclerosis, clinically isolated syndrome, MRI, hippocampus, hippocampal subfields, dentate gyrus, CA1, memory impairment

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Introduction

Patients with multiple sclerosis (MS) are often afflicted with episodic memory impairment and, over the past decade, a growing number of studies have investigated how hippocampal abnormalities might be related to this deficit [START_REF] Sicotte | Regional hippocampal atrophy in multiple sclerosis[END_REF][START_REF] Dutta | Demyelination causes synaptic alterations in hippocampi from multiple sclerosis patients[END_REF][START_REF] Hulst | Memory impairment in multiple sclerosis: Relevance of hippocampal activation and hippocampal connectivity[END_REF]Planche et al., 2017a]. Post-mortem anatomopathological analyses of MS brains, together with studies on animal models of MS, have described early microglial activation, neuronal loss, synaptic dysfunction and demyelination within different regions of the hippocampus [START_REF] Dutta | Demyelination causes synaptic alterations in hippocampi from multiple sclerosis patients[END_REF][START_REF] Papadopoulos | Substantial archaeocortical atrophy and neuronal loss in multiple sclerosis[END_REF]Planche et al., 2017b]. However, the time course of these alterations and the inter-relations between the different types of cellular modifications during the evolution of the disease remain largely unknown.

One way to isolate pathogenic mechanisms within the hippocampal circuit is to study its regional vulnerability [START_REF] Small | Isolating pathogenic mechanisms embedded within the hippocampal circuit through regional vulnerability[END_REF]. Indeed, the hippocampus is composed of distinct subfields whose morphological, cellular, molecular, functional and connectivity profiles are very different: the dentate gyrus, the cornu ammonis (CA, with subdivisions from CA1 to CA4) and the subiculum. Initially used to study Alzheimer's disease and physiological aging [START_REF] West | Differences in the pattern of hippocampal neuronal loss in normal ageing and Alzheimer's disease[END_REF], this approach of interrogating differentially the malfunctioning hippocampal circuit has been adapted more recently to investigate MS [START_REF] Sicotte | Regional hippocampal atrophy in multiple sclerosis[END_REF][START_REF] Gold | Smaller cornu ammonis 2-3/dentate gyrus volumes and elevated cortisol in multiple sclerosis patients with depressive symptoms[END_REF][START_REF] Longoni | Deficits in memory and visuospatial learning correlate with regional hippocampal atrophy in MS[END_REF][START_REF] Rocca | In vivo evidence of hippocampal dentate gyrus expansion in multiple sclerosis[END_REF].

Regarding MRI studies, the differential vulnerability of one hippocampal subfield compared to the others during the course of MS remains controversial. Indeed, some authors have highlighted the differential vulnerability of CA1 [START_REF] Sicotte | Regional hippocampal atrophy in multiple sclerosis[END_REF][START_REF] Longoni | Deficits in memory and visuospatial learning correlate with regional hippocampal atrophy in MS[END_REF] and others the vulnerability of CA3/CA4/dentate gyrus [START_REF] Gold | Smaller cornu ammonis 2-3/dentate gyrus volumes and elevated cortisol in multiple sclerosis patients with depressive symptoms[END_REF][START_REF] Rocca | In vivo evidence of hippocampal dentate gyrus expansion in multiple sclerosis[END_REF].

The reasons for discrepancies between studies remain speculative but they might be explained by the heterogeneity of the measurement methods used (surface-based mesh modelling and volumetric analyses) and/or by the heterogeneity of the populations studied (relapsing MS with different disease durations and/or progressive MS). This latter point seems crucial if we postulate that the pattern of atrophy of hippocampal subfields changes according to disease progression. Previous studies have so far been unable to test such timing and the dynamics of differential hippocampal subfield damage because of cross-sectional design. It is also important to note that none of these works investigated clinically isolated syndrome (CIS), which is required to address the question of the differential vulnerability of hippocampal subfields to the earliest pathophysiological mechanisms in MS. Indeed, CIS is the first demyelinating event suggestive of a future relapsing-remitting MS, that will be formally defined later on by the dissemination in time and space of demyelinating lesions [START_REF] Polman | Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria[END_REF][START_REF] Miller | Clinically isolated syndromes[END_REF]. Thus, studying persons with CIS offers a unique opportunity to understand the "first steps" of the pathophysiological mechanisms leading to MS.

By analogy with other neurodegenerative diseases [START_REF] Maruszak | Why looking at the whole hippocampus is not enough-a critical role for anteroposterior axis, subfield and activation analyses to enhance predictive value of hippocampal changes for Alzheimer's disease diagnosis[END_REF][START_REF] De Flores | Structural imaging of hippocampal subfields in healthy aging and Alzheimer's disease[END_REF], we hypothesize here that hippocampal degeneration in persons with CIS and early MS is not uniform and that pathological alterations will spread from one hippocampal subfield to the others in a process leading to diffuse hippocampal atrophy. To test this hypothesis, we measured the volumes of five distinct hippocampal subfields longitudinally, using advanced 3T MRI-based automatic segmentation techniques, and analysed the dynamics of atrophy of these subfields together with their clinical and neuropsychological correlates.

Methods

Participants

Fifty-six persons with CIS (PwCIS) were prospectively enrolled between 2 to 6 months after an initial clinical event compatible with a demyelinating inflammatory syndrome: a monofocal and monophasic neurological symptom that can be related to an optic nerve, spinal cord, brainstem, cerebral hemisphere or cerebellum lesion [START_REF] Miller | Clinically isolated syndromes[END_REF]. Patients were assessed by neuropsychological testing and MRI at baseline and at 1-year follow up. At baseline, at least two clinically silent lesions with a minimum diameter of 3mm were required for inclusion. One of these lesions had to be cerebral (ovoid or periventricular), while the other could be located in the spine or brain. None of the patients were treated with diseasemodifying therapy at inclusion. Contraindications to MRI, the presence of other neurological, psychiatric or systemic diseases, steroid treatment within one month, starting or stopping antidepressants or anxiolytic treatments within 2 months of MRI and neuropsychological examination, were considered as exclusion criteria. During the follow-up period of one year, the diagnosis of multiple sclerosis was confirmed (or not) by the treating physician according to the 2010 McDonald criteria [START_REF] Polman | Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria[END_REF].

Fifty-five healthy controls (HC), free of neurological, psychiatric, or systemic diseases, and drug or alcohol abuse, were also included. They were tightly matched for age, gender and educational level to PwCIS, to calculate cognitive z-scores both at baseline and one year after (see below). Among these 55 controls, a subgroup of 38 HC (still matched with the CIS group, see Table 1) underwent MRI at baseline but they were not re-scanned at year 1.

All subjects were prospectively enrolled from 2012 to 2015 at our MS centre. Written informed consent was obtained prior to participation. This study was approved by the local institutional ethics review board and registered in Clinicaltrial.gov (NCT01865357).

Neuropsychological testing

In order to assess hippocampal functions, PwCIS and HC performed the Selective Reminding Test (SRT) for episodic verbal memory performance and the Brief Visuospatial Memory Test (BVMT-R) to test episodic visuospatial memory performance. Each PwCIS was compared with the HC group at the appropriate time point to calculate a z-score for each test at each time point. To take into account practice effects, the scores of PwCIS at baseline were compared with the mean score of the HC group obtained at baseline, while the scores of PwCIS at 1 year were compared with the mean score of the HC group obtained during their second session of neuropsychological testing at 1 year. The z-scores of each SRT and BVMT-R subtest were averaged in order to calculate one composite verbal memory score and one composite visuospatial memory score. Lower z-scores indicate lower performance. A patient was considered impaired in a given cognitive domain if his/her score was below the fifth percentile (i.e. z-score < -1.64).

MRI acquisition and analyses

Participants were scanned on a 3T MRI system at our MS centre (either Philips Achieva or GE Medical Systems Discovery MR 750w). Thirteen patients (out of 46, i.e. 28%) were not scanned on the same machine at baseline and after one year (Philips Achieva at baseline and GE Discovery after 1 year). The imaging protocol was harmonized between magnets and included the same 3D gradient-echo T1-weighted sequence (TR/TE/TI/flip angle=8.2ms/3.5ms/982ms/7°, resolution 1x1x1mm, 256mm FOV) and a 2D axial Fluid Attenuated Inversion Recovery (FLAIR) sequence (TR/TE/TI=11000ms/140ms/2800ms, resolution 1x1.1x3mm, 230mm FOV). Preliminary analyses (not shown) using the type of scanner as a covariate did not show any effect of the magnet on volumetric analyses.

Lesion load was determined by the lesion growth algorithm as implemented in the Lesion Segmentation Tool (LST) version 2.0.15 (http://www.applied-statistics.de/lst.html) in Statistical Parametric Mapping (SPM12) [START_REF] Schmidt | An automated tool for detection of FLAIRhyperintense white-matter lesions in Multiple Sclerosis[END_REF]. To do this, T1 images were co-registered with FLAIR images to calculate lesion belief maps, thresholded with the same parameters for each patient (kappa=0.3). Binary maps of lesions were reviewed and corrected manually by two blinded experts (MR engineer and neurologist), using 3D Slicer 4.4.0 (www.slicer.org).

For the volumetric analyses of brain structures and hippocampal subfields, T1-weighted images were processed using the volBrain system (http://volbrain.upv.es) [START_REF] Manjón | volBrain: An Online MRI Brain Volumetry System[END_REF]. After denoising with an adaptive non-local mean filter [START_REF] Manjón | Adaptive non-local means denoising of MR images with spatially varying noise levels[END_REF], images were affine-registered in the Montreal Neurological Institute (MNI) space using ANTS software [START_REF] Avants | A reproducible evaluation of ANTs similarity metric performance in brain image registration[END_REF], corrected for image inhomogeneities using N4 [START_REF] Tustison | N4ITK: improved N3 bias correction[END_REF] and finally intensity-normalized [START_REF] Nyúl | On standardizing the MR image intensity scale[END_REF]. Then, the hippocampal subfields were segmented based on a multi-atlas framework combining nonlinear registration and patch-based label fusion [START_REF] Romero | High Resolution Hippocampus Subfield Segmentation Using Multispectral Multiatlas Patch-Based Label Fusion[END_REF][START_REF] Romero | HIPS: A new hippocampus subfield segmentation method[END_REF].

This method uses a training library composed of 5 high resolution T1-weighted images labelled manually according to the protocol proposed by Winterburn and colleagues [START_REF] Winterburn | A novel in vivo atlas of human hippocampal subfields using highresolution 3 T magnetic resonance imaging[END_REF] which is one of the rare freely available atlases that specifically separate CA4/DG from CA2/3 on the one hand and CA1 neuritic and pyramidal layers on the other hand [START_REF] Yushkevich | Quantitative comparison of 21 protocols for labeling hippocampal subfields and parahippocampal subregions in in vivo MRI: towards a harmonized segmentation protocol[END_REF]. The 3D-T1-weighted images of the subjects considered in this study (1x1x1mm 3 ) were up-sampled to the image resolution of the training library (0.5x0.5x0.5mm 3 ) using the local adaptive super-resolution (LASR) method [START_REF] Coupé | Collaborative patchbased super-resolution for diffusion-weighted images[END_REF]. The method finally provided automatic segmentation of hippocampal subfields gathered into 5 labels: Subiculum, CA1-SP (stratum pyramidale), CA1-SRLM (stratum radiatum-lacunosum-moleculare), CA2/3 and CA4/dentate gyrus (Fig. 1), allowing us to test our a priori hypotheses regarding the selective vulnerability of the dentate gyrus or CA1, as suggested in the literature from animal, neuropsychological (using pattern separation tasks) and MRI studies [Planche et al., 2017b;Planche et al., 2017c;[START_REF] Rocca | In vivo evidence of hippocampal dentate gyrus expansion in multiple sclerosis[END_REF][START_REF] Sicotte | Regional hippocampal atrophy in multiple sclerosis[END_REF]. In our previous paper, the hippocampal subfield segmentation accuracy using T1weighted has been estimated to be 62% for the Winterburn protocol in terms of average DICE coefficient. Moreover, the whole hippocampus segmentation accuracy has been estimated to 88% in term of DICE coefficient. Finally, it has been shown that super-resolution enables to obtain an accuracy close the one obtained when using high resolution T1 [START_REF] Romero | HIPS: A new hippocampus subfield segmentation method[END_REF]. Every up-sampled image and every subfield label were quality-controlled and manually corrected by a blinded neurologist if needed (in the case of inappropriate inclusion of para-hippocampal T1-hypointense lesions (Suppl. Fig. 1), choroidal plexus and/or CSF "pockets", using 3D Slicer 4.4.0 (www.slicer.org)). To control for variations in head size, the volumes of hippocampal subfields were normalized using the intracranial cavity volume of each subject [START_REF] Manjón | Nonlocal intracranial cavity extraction[END_REF]. Normalized brain volume (NBV) was also calculated as the sum of cerebral white and grey matters, divided by the intracranial cavity volume of each subject.

Statistical Analyses

Statistical analyses were performed with Prism software 6 (Graphpad) and XLstats 19.4 (Addinsoft). The distribution of all continuous data was tested with the Shapiro-Wilk normality test. We first compared clinical, neuropsychological and imaging characteristics between HC and PwCIS at baseline by using Fisher's exact tests for categorical variables, and unpaired t-tests or Mann-Whitney tests for ordinal variables. The comparisons of baseline and 1-year characteristics of PwCIS were done with paired t-tests or Wilcoxon tests, as appropriate.

To compare the volumes of the 5 hippocampal subfields between groups, we first performed an analysis of variance, followed by a Sidak multiple comparisons test. Cohen's d was used to measure the effect size of atrophy between patients and HC whereas annualized rate of atrophy was used to compare PwCIS at year 1 and baseline.

Then, because the decreases in subfield volumes were consistent between right and left hippocampi, further statistical analyses were performed on the sum of right and left subfield volumes to avoid multiple comparisons. Relationships between neuropsychological scores, demographic, clinical and imaging variables were assessed using correlation coefficients (Pearson or Spearman according to statistical distribution). The associations were further tested in multivariate context. To this end, (i) hippocampal subfields volumes at year 1, (ii) MS diagnosis after 1-year follow-up and (iii) memory performance (dependent variables) were predicted with hierarchical regression models, including two hierarchical blocks. In the first block, relevant demographical, clinical and general MRI features known as nuisance variables were systematically forced into the model. In the second block, the volumes of hippocampal subfields were added to the variables of the first block. The predictive power of the two blocks was compared by using the Akaike information criterion (AIC). A linear regression model was used whenever possible while logistic regression was seen as the appropriate alternative for categorical/binary outcome variables. All tests were two-tailed, with a global type I error set at α=0.05.

Results

Demographic, clinical and general MRI features of participants

A total of 56 PwCIS and 55 HC were included. At baseline, all PwCIS and HC were tested with the neuropsychological battery while all PwCIS but only a subgroup 38 HCs were assessed with MRI. At year 1, because 10 patients were lost to follow-up, 46 PwCIS were retested with the same neuropsychological battery and re-scanned with the same imaging protocol. All the 55 HCs were re-tested with the same neuropsychological battery but they were not re-scanned at year 1.

The CIS and HC groups were comparable for age, sex and educational level (Table 1). In this cohort of patients, after one year, 65.2% of PwCIS were diagnosed with MS according to the 2010 McDonald criteria.

In PwCIS, there was no significant difference between baseline and 1-year follow-up regarding disability (Expanded Disability Status Scale, EDSS) and T2-lesion load.

Neuropsychological testing was also rather stable between baseline and 1-year follow-up, except for the episodic visuospatial memory score that had even slightly increased (p=0.02) (Table 1). Normalized brain volume (NBV) significantly decreased during this period (-1.4%, p=0.012).

Dynamics of regional hippocampal vulnerability

At baseline, hippocampal volumes were significantly lower in PwCIS compared to controls only in the CA4/dentate gyrus subfield (Fig. 2). This was consistently true for the left (-6.5%, d=0.53, p<0.05) and for the right hippocampus (-7.7%, d=0.54, p<0.01) and when both sides were pooled together (-7.2%, d=0.58, p<0.01). In PwCIS, this atrophy of CA4/dentate gyrus was not correlated with age, disability or T2 lesion-load at baseline. Follow-up data after one year compared to baseline measures showed that the normalized volumes of CA4/dentate gyrus (-6.4%, p<0.001), CA1-SP (-5,6%, p<0.01) and CA1-SRLM (-6,2%, p<0.05) significantly decreased in both sides. No significant atrophy was found in the subiculum or CA2/3 subfields (Fig. 3).

As the atrophy of CA1 subfield chronologically succeeded the atrophy of CA4/dentate gyrus, this suggests that the same pathophysiological process spreads from CA4/dentate gyrus to CA1 in individual patients. To test this hypothesis, we designed a hierarchical linear regression model to test how CA1-SP, CA1-SRLM and whole hippocampus volumes at year 1 (dependent variables) can be predicted by the volume of CA4/dentate gyrus at baseline (while first taking into account confounders such as age, T2 lesion-load and NBV in the model). We found that CA4/dentate gyrus at baseline improved the statistical prediction of CA1 volumes at year 1 from R 2 =0.19 (when considering usual factors such as age, T2 lesions and NBV) to R 2 =0.44 (AIC block2 < AIC block1 ). It also predicted the whole hippocampal volume one year afterwards independently of age, T2 lesions or NBV (Table 2). CA4/DG remained a significant and independent correlate of CA1 and whole hippocampal volumes at year 1, although other hippocampal subfields (subiculum and CA2/3) were introduced in the model (ß=0.34, p=0.018 for CA1-SP and ß=0.39, p<0.001 for the whole hippocampus, respectively).

Altogether, a smaller focal volume at baseline predicted smaller global volumes at year 1, which points to a pathological continuum starting within CA4/dentate gyrus and spreading progressively and more globally to CA1.

Relationship between the atrophy of hippocampal subfields and clinical outcomes

In order to study the link between the early vulnerability of CA4/dentate gyrus and the pathophysiological process specific to MS, we first investigated whether CA4/dentate gyrus volume at baseline would be able to predict MS diagnosis at year 1. Using univariate analyses, we found that both T2-lesion load and CA4/dentate gyrus volume were significantly associated MS diagnosis after 1-year follow-up (p=0.002 and p=0.014, respectively). A multiple logistic regression model showed that the volume of CA4/dentate gyrus at baseline was the only factor independently associated with future MS diagnosis (ß=0.57, p=0.025 and R 2 =0.28, p=0.016) while age, gender, EDSS, T2-lesion load and NBV were not predictive.

Finally, we investigated whether early hippocampal regional vulnerability had a clinical impact on episodic memory performance. The results of univariate correlations and regression models between memory scores and demographic, clinical and imaging data at baseline and year 1 are presented in Table 3. According to multivariate analyses, no model was able to explain memory performance at baseline. The volume of CA1-SP and the educational level of patients were independent predictors of the episodic verbal memory composite score at year 1 (ß=0.87, p=0.042 and ß=0.51, p=0.031, respectively).

Discussion

We demonstrated in this study that the CA4/dentate gyrus subfield of the hippocampus is the first subfield to be atrophied across the course of MS, at the stage of CIS. This pattern of regional hippocampal atrophy worsens during the first year of disease evolution and spreads within CA1 (both in the cell bodies layer CA1-SP and the neuritic layers CA1-SRLM).

CA4/dentate gyrus volume at baseline was associated with diagnosis of MS one year afterwards. Whereas isolated CA4/dentate gyrus atrophy at the stage of CIS failed to correlate with memory scores, it predicted the extension of the pathological process within CA1 one year later, which was in turn correlated with episodic verbal memory performance, independently of usual confounders.

Our main finding of a "natural history" of hippocampal subfield degeneration in MS, from dentate gyrus to CA1, is supported by anatomical and functional studies in both human and animal models of the disease. First, we previously reported that pattern separation performance -a cognitive task critically dependant on dentate gyrus function [START_REF] Bakker | Pattern separation in the human hippocampal CA3 and dentate gyrus[END_REF] -was decreased in patients with CIS and early MS, when conventional visuospatial episodic memory tests (BVMT-R) were not yet altered, suggesting an early and isolated dentate gyrus dysfunction during the course of the disease [Planche et al., 2017c]. Such functional alterations suggested by the pattern separation task are therefore consolidated by the anatomical alterations observed in the present MRI study. Second, our findings are also supported by a previous work showing that dentate gyrus structure and function are selectively disrupted by microglial activation at the early stage of experimental autoimmune encephalomyelitis (EAE, the animal model of MS) [Planche et al., 2017b]. The independent association we found here between CA4/dentate gyrus volume at the stage of CIS and the diagnosis of MS one year after CIS also suggests this link between dentate gyrus damage and the early diffuse pathophysiological process specific to MS. Third, the vulnerability of the dentate gyrus, prior to other hippocampal subfields at the early stage of MS, could be explained by its particular anatomical location. Indeed, it is adjacent to cerebrospinal fluid (CSF) spaces (third ventricle in rodents and choroidal fissure in humans) where cytokines and immune cells preferentially penetrate the hippocampus, as suggested experimentally in EAE [START_REF] Habbas | Neuroinflammatory TNFα Impairs Memory via Astrocyte Signaling[END_REF]. We postulate that the hierarchical vulnerability of hippocampal subfields in MS might be better explained by their anatomical contiguity rather than by a network-dependent disposition, as in Alzheimer's disease for instance [START_REF] Kerchner | Shared vulnerability of two synaptically-connected medial temporal lobe areas to age and cognitive decline: a seven tesla magnetic resonance imaging study[END_REF].

A gradient of infiltrating immune cells and cytokines would diffuse progressively from the CSF to the dentate gyrus, then to CA1, and probably to the whole medial temporal lobe. In this model, the progression of the disease from the dentate gyrus to CA1 might be slowed down by the presence of the vestigial intrahippocampal sulcus, potentially explaining the delay between dentate gyrus and CA1 atrophy reported in this study. Additional experiments will be needed to test these mechanistic hypotheses.

From the anatomopathological point of view, our study also highlights the striking vulnerability of the hippocampus to neurodegeneration in the context of neuroinflammation.

Indeed, the annualized rate of atrophy during the first year of the disease's evolution ranged from -5.6% to -6.4%, respectively for CA1 and CA4/dentate gyrus, whereas it was 'only' -1.4% for the normalized brain volume in our population of PwCIS (which was in the upper range of what was usually observed in the literature on CIS and relapsing MS, i.e. from -0.5% to -1.35% [START_REF] Pérez-Miralles | Clinical impact of early brain atrophy in clinically isolated syndromes[END_REF][START_REF] Stefano | Clinical relevance of brain volume measures in multiple sclerosis[END_REF]). This selective and disproportionate hippocampal volume loss, in excess to global brain atrophy, has already been observed in a seminal cross-sectional study on patients with relapsing and progressive MS [START_REF] Sicotte | Regional hippocampal atrophy in multiple sclerosis[END_REF]. The annualized rate of hippocampal atrophy we report here (-5.9%) is even in the upper range of what has been reported for patients with Alzheimer's disease or mild cognitive impairment (-3.5% to -6%/year) [START_REF] Jack | Rates of hippocampal atrophy correlate with change in clinical status in aging and AD[END_REF][START_REF] Wang | Changes in hippocampal volume and shape across time distinguish dementia of the Alzheimer type from healthy aging[END_REF][START_REF] Du | Higher atrophy rate of entorhinal cortex than hippocampus in AD[END_REF] and far beyond what has been reported for physiological aging (-0.8% to -2.3%/year) [START_REF] Jack | Steps to standardization and validation of hippocampal volumetry as a biomarker in clinical trials and diagnostic criterion for Alzheimer's disease[END_REF].

Besides these anatomical considerations, we also questioned the link between the early regional vulnerability of the hippocampus and memory impairment in MS. On the one hand, the CA4/dentate gyrus atrophy observed at baseline was not correlated with episodic memory performance in our cohort. Although we have to take into account that the memory abilities of the PwCIS included in this study were not severely affected (regarding the median z-scores and the percentage of impaired patients), we postulate that CA4/dentate gyrus atrophy at the stage of CIS is "not enough" to explain the memory decline observed in "global" episodic memory tests such as the SRT or the BVMT-R. Perhaps more specific tests such as the behavioural pattern separation task [START_REF] Stark | A task to assess behavioral pattern separation (BPS) in humans: Data from healthy aging and mild cognitive impairment[END_REF] would have allowed us to pinpoint such a subtle memory decline related to CA4/dentate gyrus damage [Planche et al., 2017c] and future studies should address this point. On the other hand, we found that CA1 atrophy explained part of the "global" episodic verbal memory decline one year after inclusion, when diagnosis of MS was finally observed in 65.2% of the patients. This suggests that CA1 atrophy is the best anatomical correlate of memory performance in MS, as previously described in cross-sectional studies including patients with relapsing and progressive MS [START_REF] Sicotte | Regional hippocampal atrophy in multiple sclerosis[END_REF][START_REF] Longoni | Deficits in memory and visuospatial learning correlate with regional hippocampal atrophy in MS[END_REF].

A recent cross-sectional study found an "expansion" of the dentate gyrus during MS [Rocca et al., 2015]. The divergences between our findings and this previous work may be attributed to various points. Indeed, contrary to our work, this study investigated patients at a later stage of the disease, with relapsing and progressive MS, and it used a surface-based mesh modelling technique to study the shape of the hippocampus [START_REF] Thompson | Mapping hippocampal and ventricular change in Alzheimer disease[END_REF]. Using this technique, the authors found an outward displacement of the supero-medial hippocampal surface and concluded on a larger radial distance of the dentate gyrus in patients with MS.

However, it should be borne in mind that when using such a method, measuring external surface modifications does not enable the direct characterization of the inner alterations of a particular subfield. It is not clear how the atrophy of the deepest regions of the hippocampus (such as the dentate gyrus, which is curled inside the Cornu Ammonis) would impact the outer surface of the structure. For instance, the expansion of CSF pockets due to atrophy within the hippocampal fissure/sulcus might result in tissue "expansion" on the supero-medial side of the hippocampus despite there being no real dentate gyrus "hypertrophy". We also hypothesized that a shift of rotation of the hippocampus due to atrophy would induce an outward displacement of part of the surface, introducing contradictory results. Thus, radial mapping provides valuable information on hippocampal surface change (i.e. CA1 and the subiculum) but should be interpreted with caution for inner structures such as the dentate gyrus [START_REF] Joie | Differential effect of age on hippocampal subfields assessed using a new high-resolution 3T MR sequence[END_REF]. The limits of these surface-based modelling strategies to assess hippocampal subfield anatomy have been discussed extensively in the field of Alzheimer's disease and aging [START_REF] De Flores | Structural imaging of hippocampal subfields in healthy aging and Alzheimer's disease[END_REF][START_REF] Morra | Automated 3D mapping of hippocampal atrophy and its clinical correlates in 400 subjects with Alzheimer's disease, mild cognitive impairment, and elderly controls[END_REF], even by the authors who pioneered the method [START_REF] Thompson | Mapping hippocampal and ventricular change in Alzheimer disease[END_REF][START_REF] Frisoni | Mapping local hippocampal changes in Alzheimer's disease and normal ageing with MRI at 3 Tesla[END_REF]. Therefore, proper volumetric analyses with manual or automatic segmentation are now considered more relevant than radial mapping to investigate hippocampal regional vulnerability, although they have their own limitations and require protocol harmonization to clearly define subfield boundaries [Wisse et al., 2017]. For instance, we acknowledge that the Winterburn protocol we used here mainly delineates CA2/3 in its dorsal portion to increase segmentation reliability, although it can lead to volume underestimations. Therefore, our results might have differed by using other definitions of the hippocampal subfields boundaries. From the technical point of view, we also acknowledge that we only used T1-weighted images to label hippocampal subfields, whereas both T1 and T2-weighted images are often preferred to delineate internal boundaries, especially for very thin layers such as CA1-SRLM. However pooling CA1-SRLM with the bigger CA1-SP wouldn't have changed our main conclusions. Furthermore, we have recently validated that the single use of T1-weighted images upsampled with local adaptive super-resolution leads to reliable hippocampal subfield labeling, compared to the combination of T1 and T2-weighted images [START_REF] Romero | HIPS: A new hippocampus subfield segmentation method[END_REF].

The main limitation of our study is the lack of MRI follow-up for our healthy control group to clearly disentangle the contribution of time-dependant versus disease-dependant processes in our longitudinal measures of atrophy in the CIS group. However, our results on the dynamics of progressive hippocampal subfield atrophy are likely related to the disease process because they are specific (i.e. CA4/dentate gyrus and CA1 but not CA2/3 and the subiculum) and because the annualized rate of hippocampal atrophy measured (-5.9%/year) is clearly above what can be observed on average in the healthy population (-0.4%/year in people less than 55 years old) [START_REF] Fraser | A systematic review and meta-analysis of longitudinal hippocampal atrophy in healthy human ageing[END_REF]. Another limitation is the short follow-up period (1-year), which was nonetheless long enough to capture the sequential progression of hippocampal subfield atrophy. We also acknowledge that we did not assess the potential microstructural damage that underlies or precedes hippocampal regional atrophy, with sequences such as diffusion-tensor imaging or magnetization transfer MRI, but these techniques are difficult to implement at the spatial resolution required to study hippocampal subfields. The final limitation was that, although no area of T2-hypersignal or T1-black hole was clearly detected within the hippocampus on our conventional sequences, we did not assess potential subtle demyelinating hippocampal lesions with specific double inversion recovery sequences.

Conclusion

We demonstrated that CA4/dentate gyrus is the first subfield of the hippocampus to be atrophied during the course of MS, from the stage of CIS. This regional pattern of hippocampal atrophy rapidly spread to CA1. This dynamic vulnerability is associated with future diagnosis of MS and contributes to hippocampal-dependant memory performance.

Figure 2: Comparison of the normalized volumes of hippocampal subfields between healthy controls and persons with clinically isolated syndrome (CIS) at baseline (y0).

Histograms represent the cumulative volumes of all the left hippocampal subfields (in red), of all the right hippocampal subfields (in blue) and of all right&left hippocampal subfields (in orange). The colour gradient represents individual hippocampal subfields. ns = nonsignificant, *p<0.05, **p<0.01 (corrected for multiple comparisons).

Figure 3: Comparison of the normalized volumes of hippocampal subfields between persons with clinically isolated syndrome (CIS) at baseline (y0) and 1-year follow-up (y1).

The histograms represent the cumulative volumes of all the left hippocampal subfields (in red), of all the right hippocampal subfields (in blue) and of all right&left hippocampal subfields (in orange). The colour gradient represents individual hippocampal subfields. ns = non-significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (corrected for multiple comparisons).

Table 3: Univariate correlations and hierarchical regression models between memory composite scores at baseline or after 1-year follow-up (dependent variables) and demographical, clinical and MRI features.

Age, T2LL, NBV and educational level were entered into an initial model (block 1) as nuisance variables. Covariables related to hippocampal subfields were added in a second model (block 2) according to univariate correlations (p-value <0.2) to predict memory scores. CA1-SP = CA1stratum pyramidale, CA1-SRLM = CA1-stratum radiatum-lacunosum-moleculare, T2LL = T2 Lesion-Load. NBV = Normalized Brain Volume. ns = non-significant, *p<0.05. § : Block 2 was equivalent to Block 1 because no correlation (p<0.2) was found between the volume of any hippocampal subfields and the memory score.

Figure 1 :

 1 Figure 1: Segmentation of the five hippocampal subfields.

  correlations and hierarchical linear regression models between volumes of CA1 subfields or whole hippocampal volume at year 1 (dependent variables) and volume of CA4/dentate gyrus at baseline. Age, T2LL and NBV were entered into an initial model (block 1) as nuisance variables. CA1-SP = CA1-stratum pyramidale, CA1-SRLM = CA1-stratum radiatum-lacunosummoleculare, CA4/DG = CA4/dentate gyrus, NBV = Normalized Brain Volume, T2LL = T2 Lesion-Load. ns = non-significant, *p<0.05, **p<0.01, ***p<0.001. # AIC block2 < AIC block1 .

Imaging features Median T2 lesion volume, mL [range]

		Controls with MRI (n=38)	CIS baseline (n=56)	CIS 1-year (n=46)	p-value
	Demographic and clinical features
	Mean age, years [SD] 36.6 [10.7]	36.5 [11.2]	-	0.94
	Sex ratio (F/M)	26/12	46/10	-	0.14
	Education level, (High/Low § )	27/11	39/17	-	0.88
	Median EDSS score [range]	-	1.0 [1.0 to 6.0]	1.0 [1.0 to 6.0]	0.63
	Conversion to MS	-	-	30/46 (65.2%)	-
	Neuropsychological features
	Median verbal memory z-score [range]		0.10 [-4.9 to 1.0]	-0.13 [-3.7 to 1.2]	0.91
	{% impaired}		{7.4%}	{10.7%}
	Median visuospatial memory z-score [range]
			-0.20 [-5.2 to 0.82]	0.10 [-3.5 to 1.0]	0.02
	{% impaired}		{17.9%}	{6.5%}
		-	0.73 [0.02-63.12]	1.08 [0.06-67.74]	0.67
	Mean normalized brain volume, % [SD]	86.4 [3.2]	85.1 [3.9]	83.9 [4.1]	0.21 # /0.012 †

Table 1 : Clinical, neuropsychological and general MRI features of the studied populations. CIS

 1 = Clinically Isolated Syndrome; EDSS = Expanded Disability Status Scale; MS: Multiple Sclerosis; SD = Standard Deviation. § Education level was considered as high or low according to French baccalaureate (equivalent to A-level). # Controls vs CIS baseline. † CIS baseline vs CIS 1-year.

Acknowledgments

The authors thank Julie Charré-Morin and Aurore Saubusse (Bordeaux University Hospital) for neuropsychological testing and the ARSEP Foundation for its support.

Study funding

This study was funded by the ARSEP Foundation, Bordeaux University Hospital and TEVA laboratories. The work was further supported by public grants from the French Agence Nationale de la Recherche within the context of the Investments for the Future program referenced ANR-10-LABX-57, named TRAIL (project IBIO-NI, GM-COG and HR-DTI), ANR-10-LABX-43, named BRAIN (Project MEMO-MS), ANR-10-IDEX-03-02, named IdEx Bordeaux -CPU, ANR-10-COHO-002, named French Observatoire for multiple sclerosis (OFSEP) and the CNRS multidisciplinary project "Défi ImagIn" HL-MRI. This research was also funded by Spanish UPV2016-0099 and TIN2013-43457-R grants from UPV and the Ministerio de Economia y competitividad. The sponsors did not participate in any aspect of the design or performance of the study, including data collection, management, analysis, and the interpretation or preparation, review, and approval of the manuscript.

Disclosures

VP received a research grant from the ARSEP Foundation to conduct this study; he received travel expenses and/or consulting fees from Biogen, Teva-Lundbeck and Merk-Serono unrelated to the submitted work. BB serves on scientific advisory boards on behalf of his institution and has received honoraria or research support from Biogen-Idec, Merck-Serono, Novartis, Genzyme, Teva, Roche, Medday and Bayer unrelated to the submitted work. AR received research grants and/or consulting fees from Novartis, Biogen, Merck-Serono, Bayer Healthcare, Roche, Teva, and Genzyme unrelated to the submitted work.


Jens Januschke

Emmanuelle Nicolas

Julien Compagnon

Etienne Formstecher

Bruno Goud

Antoine Guichet
email: guichet@ijm.jussieu.fr

Drab6 and the secretory pathway affect oocyte polarity in Drosophila

Keywords: Rab6, secretory pathway, RNA transport, Gurken, microtubules, Bicaudal D, oskar mRNA

INTRODUCTION

By regulating the transport of proteins and lipids toward the plasma membrane, the secretory pathway plays an important function in cell polarity. Rab GTPases are important regulatory factors of vesicular traffic. Members of the Rab6 family regulate protein transport between the Golgi, endoplasmic reticulum, plasma membrane and endosome [START_REF] Del Nery | Rab6A and Rab6A' GTPases play non-overlapping roles in membrane trafficking[END_REF][START_REF] Mallard | Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform[END_REF][START_REF] Martinez | GTP-bound forms of rab6 induce the redistribution of Golgi proteins into the endoplasmic reticulum[END_REF][START_REF] Martinez | The small GTP-binding protein rab6 functions in intra-Golgi transport[END_REF][START_REF] Opdam | The small GTPase Rab6B, a novel Rab6 subfamily member, is cell-type specifically expressed and localised to the Golgi apparatus[END_REF]. The role of Rab6 in establishing cell polarity was, however, unclear. We chose the Drosophila oocyte as a model to study cell polarity in vivo. The oocyte lies at the posterior of the egg chamber, which consists of a cluster of 16 interconnected germ cells surrounded by a monolayer of follicular epithelium. During egg chamber formation, the germ line forms a sixteen cell cluster and in which one cell is singled out to become the oocyte, while its 15 sister cells develop into nurse cells (for review, see [START_REF] Huynh | The origin of asymmetry: early polarisation of the Drosophila germline cyst and oocyte[END_REF]).During early oogenesis, microtubules (MTs) are nucleated from the microtubule-organizing center at the posterior of the oocyte. Towards stage 7, an unidentified signal from the posterior follicle cells triggers the organization of perpendicular MT subsets controlling the dorsoventral axis (DV) and anteroposterior axis (AP) in the oocyte [START_REF] Januschke | The centrosome-nucleus complex and microtubule organization in the Drosophila oocyte[END_REF][START_REF] Macdougall | Drosophila gurken (TGFalpha) mRNA localizes as particles that move within the oocyte in two dyneindependent steps[END_REF]. Bicoid, oskar and gurken mRNAs, which determine the embryonic axes, are then localized, respectively, to the anterior, posterior and antero-dorsal poles of the oocyte [START_REF] Riechmann | Axis formation during Drosophila oogenesis[END_REF].

Studies in cultured mammalian cells have revealed a molecular mechanism whereby BicD modulates MT based Golgi trafficking by recruiting cytoplasmic Dynein to transport vesicles [START_REF] Hoogenraad | Mammalian Golgiassociated Bicaudal-D2 functions in the dynein-dynactin pathway by interacting with these complexes[END_REF][START_REF] Matanis | Bicaudal-D regulates COPIindependent Golgi-ER transport by recruiting the dynein-dynactin motor complex[END_REF]. The recruitment of Dynein to vesicles is mediated by the interaction of BicD with the small Golgi localized GTPase Rab6 [START_REF] Matanis | Bicaudal-D regulates COPIindependent Golgi-ER transport by recruiting the dynein-dynactin motor complex[END_REF][START_REF] Short | Golgins and GTPases, giving identity and structure to the Golgi apparatus[END_REF]. In Drosophila, and more specifically in the oocyte, the Golgi apparatus is not organized into stacked cisternae arranged into "Golgi ribbons". Instead, it is organized into mini stacks of tER-Golgi units evenly distributed throughout the cell [START_REF] Herpers | mRNA localization and ER-based protein sorting mechanisms dictate the use of transitional endoplasmic reticulum-golgi units involved in gurken transport in Drosophila oocytes[END_REF][START_REF] Kondylis | Biogenesis of Golgi stacks in imaginal discs of Drosophila melanogaster[END_REF]. Whereas three Rab6 isoforms have been characterized in mammals, [START_REF] Del Nery | Rab6A and Rab6A' GTPases play non-overlapping roles in membrane trafficking[END_REF][START_REF] Mallard | Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform[END_REF][START_REF] Martinez | GTP-bound forms of rab6 induce the redistribution of Golgi proteins into the endoplasmic reticulum[END_REF][START_REF] Martinez | The small GTP-binding protein rab6 functions in intra-Golgi transport[END_REF][START_REF] Opdam | The small GTPase Rab6B, a novel Rab6 subfamily member, is cell-type specifically expressed and localised to the Golgi apparatus[END_REF], only one has been identified in Drosophila [START_REF] Shetty | Rab6 regulation of rhodopsin transport in Drosophila[END_REF]. So far, Drab6 has been shown to be involved in Rhodopsin transport in photoreceptor cells and bristle morphogenesis [START_REF] Purcell | The developmental role of warthog, the notch modifier encoding Drab6[END_REF][START_REF] Shetty | Rab6 regulation of rhodopsin transport in Drosophila[END_REF]. In this study, we present the characterization of the function of Drab6 during oogenesis and propose a possible connection between Rab protein-mediated secretion, the organization of the cytoskeleton and mRNA transport.

Materials and Methods

Fly stocks

w 1118 used as wild-type; rab6 D23D [START_REF] Purcell | The developmental role of warthog, the notch modifier encoding Drab6[END_REF] was recombined to FRT-40A (Bloomington). rab6 D23D and khc 7.288 germ line and follicle cells clones were generated as described in [START_REF] Januschke | Polar transport in the Drosophila oocyte requires Dynein and Kinesin I cooperation[END_REF]. GFP-trap, GalT and PDI (A. Debec). Khc-LacZ (I. Clark). BicD-GFP and BicD mom (B. Suter). Over-expression of Dynamitin and Colchicine treatment was performed as described in [START_REF] Januschke | Polar transport in the Drosophila oocyte requires Dynein and Kinesin I cooperation[END_REF].

Transgenes, biochemistry and immunohistochemistry.

Drab6 was cloned into maternally expressed tubGFP [START_REF] Januschke | Polar transport in the Drosophila oocyte requires Dynein and Kinesin I cooperation[END_REF] and Polyubiquitin mRFP vectors. Details of protocols for biochemistry experiments can be obtained upon request. Yeast two-hybrid screen was carried out as described [START_REF] Formstecher | Protein interaction mapping: a Drosophila case study[END_REF].

Electron microscopy and immunodetections were performed as described in [START_REF] Januschke | The centrosome-nucleus complex and microtubule organization in the Drosophila oocyte[END_REF]. Antibodies: Stau [START_REF] St Johnston | Staufen, a gene required to localize maternal RNAs in the Drosophila egg[END_REF]; Osk [START_REF] Hachet | Drosophila Y14 shuttles to the posterior of the oocyte and is required for oskar mRNA transport[END_REF]; β-Galactosidase (Roche); Grk (DSHB); Lav (W. Sullivan); BicD, Syntaxin-5 (DSHB); KDEL (Stressgen); Dynactin (EL. Holzbaur); GFP (Roche). LE Lectin (Vector). WGA, Phalloidin, Lysotracker (Molecular Probe).

RESULTS AND DISCUSSION

In vertebrate cells, Rab6 is associated with the Golgi and the trans-Golgi network (TGN) membranes [START_REF] Del Nery | Rab6A and Rab6A' GTPases play non-overlapping roles in membrane trafficking[END_REF][START_REF] Mallard | Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform[END_REF][START_REF] Martinez | GTP-bound forms of rab6 induce the redistribution of Golgi proteins into the endoplasmic reticulum[END_REF][START_REF] Martinez | The small GTP-binding protein rab6 functions in intra-Golgi transport[END_REF][START_REF] Opdam | The small GTPase Rab6B, a novel Rab6 subfamily member, is cell-type specifically expressed and localised to the Golgi apparatus[END_REF]. To investigate the sub-cellular localization of Drab6 in the Drosophila germline, we monitored the expression pattern of transgenic lines expressing Drab6 fused to GFP (Fig. 1A) and RFP (Fig. 1B). We observed that during oogenesis, the global distribution of Drab6 evolved. Drab6 first accumulated transiently in a central position during stages 7/8, then was uniformly distributed at the beginning of stage 9 to end up juxtaposed to the entire oocyte cortex (Fig. 1A). It is noteworthy that promoters of different strengths gave similar expression patterns. In addition, the genomic null allele rab6 D23D [START_REF] Purcell | The developmental role of warthog, the notch modifier encoding Drab6[END_REF] was fully rescued by the different lines expressing Drab6.

Drab6 did not co-localize extensively with ER membranes (labelled with PDI-GFP, [START_REF] Bobinnec | Dynamics of the endoplasmic reticulum during early development of Drosophila melanogaster[END_REF] (Fig. 1B). Instead, it seemed to be differentially associated with two types of Golgi unit (Fig. 1C). Lava Lamp (Lav), a cis-Golgi marker [START_REF] Papoulas | The golgin Lava lamp mediates dyneinbased Golgi movements during Drosophila cellularization[END_REF] colocalized with Drab6, mainly at the cortex of the oocyte and in nurse cells. A GFP trap protein coding for an UDP-galactose beta-N-acetylglucosamine beta-1,3-galactosyltransferase (GalT) [START_REF] Morin | A protein trap strategy to detect GFPtagged proteins expressed from their endogenous loci in Drosophila[END_REF])), predominantly enriched in Golgi membranes (Fig. 1D), exhibited a distribution similar to that of GFP-Drab6: it accumulated in the center of the oocyte at stage 8, where it co-localized with Drab6, and was later confined to the cortex (Fig. 1C). Importantly, the distribution of Lav and GalT was similar in both mat αtubGFP-Drab6, ubiRFP-Drab6 and control oocytes (Fig. S3). Given that Lav and GalT markers are not present in the Golgi cisternae that are evenly distributed throughout the oocyte, as documented by electron microscopy (EM) analysis [START_REF] Herpers | mRNA localization and ER-based protein sorting mechanisms dictate the use of transitional endoplasmic reticulum-golgi units involved in gurken transport in Drosophila oocytes[END_REF], they may be the hallmark of distinct functional Golgi units, with Drab6 being able to interact with both of them. Unlike Lav, whose distribution is only mildly affected (Fig. 1C), GalT and acetyl-glucosamin modified proteins (detected by the wheat germ agglutinin lectin (WGA)) expressed by Golgi structures were abnormally distributed in Drab6 mutants (Fig. 1C). Moreover, ultrastructural analysis with EM in Drab6 mutant oocytes revealed that the ER was abnormally swollen (Fig. 2 A' vs B'), and that the Golgi mini-stacks were markedly curved with partially inflated cisternae (Fig. 2 A'' vs B'').

These morphological effects led us to investigate the role of Drab6 in the secretory pathway. We monitored the polarized secretion of the TGFα-like growth factor Grk [START_REF] Neuman-Silberberg | The Drosophila dorsoventral patterning gene gurken produces a dorsally localized RNA and encodes a TGF-alpha-like protein[END_REF]. Grk secretion is restricted to the antero-dorsal corner through a rapid transit from the ER towards the Golgi apparatus [START_REF] Herpers | mRNA localization and ER-based protein sorting mechanisms dictate the use of transitional endoplasmic reticulum-golgi units involved in gurken transport in Drosophila oocytes[END_REF]. In GFP-Drab6 rescued egg chambers, Grk and Drab6 co-localized (Fig. 2C).

In Drab6 mutant oocytes, grk mRNA localization is the same as in wild type (Fig. S1). The protein, however, was slightly more abundant than in controls and an important fraction extended ventrally (Fig. 2E). Polarized secretion of Grk led to the formation of two dorsal appendages on the egg shell (Fig. 2D). In the absence of Drab6, mislocalized Grk induced ventralization, (Fig. 2E, inset, 22 % absent dorsal appendages, 28 % fused, n=199), instead of a dorsalization (multiple dorsal appendages on the egg shell) as observed when Grk is ectopically secreted [START_REF] Neuman-Silberberg | Dorsoventral axis formation of Drosophila depends on the correct dosage of the gene gurken[END_REF]. Hence, this argues for a specific failure of Grk delivery to the plasma membrane. This phenotype is specific to Drab6 since it could be fully rescued by the GFP-Drab6 transgene (Fig. 2C).

Next, we analysed the intracellular localization of Grk in absence of Drab6. Grk accumulated frequently in large ring-likeparticles in the Drab6 mutant but not in control oocytes (Fig. 2D,E). Those Grk "rings", similar to those of yolk granules [START_REF] Bokel | Drosophila Cornichon acts as cargo receptor for ER export of the TGF-like growth factor Gurken[END_REF][START_REF] Queenan | The transmembrane region of Gurken is not required for biological activity, but is necessary for transport to the oocyte membrane in Drosophila[END_REF] did not contain Lav (Fig. 2G), suggesting that Grk is not blocked in the Golgi. Grk actually accumulated in Drab6 mutants on vesicles stained by Lysotracker (Fig. 2H vs. I) which labels either lysosomes or late endosomes containing yolk granules [START_REF] Dermaut | Aberrant lysosomal carbohydrate storage accompanies endocytic defects and neurodegeneration in Drosophila benchwarmer[END_REF]. Hence, two independent approaches suggest that Grk is not blocked in the Golgi but is rather mis-localized to post-Golgi compartments, probably ensodomes.

Interestingly, the secretory impairment was also confirmed by Lycopersicon Esculentum Tomato Lectin (LE), detecting modified proteins in the Golgi. In absence of Drab6, LE revealed abnormal vesicular structures in the oocyte and nurse cells that had failed to reach the cortex (Fig. S2). EM analysis also demonstrated rupture of the plasma membrane between neighbouring nurse cells (Fig. S2). Finally, we observed that GFP-Drab6 rescued egg chambers exhibited an accumulative enrichment at the plasma membrane during oogenesis, which was particularly evident in nurse cells (Fig. S2). This is consistent with the involvement of Drab6 in secretion towards the plasmalemma.

We have established the existence of three important and novel aspects of Drab6 function during oogenesis: i) Consistent with its localization in vertebrate cells, Drab6 is predominantly localized to the Golgi complex in Drosophila, but overlaps with Golgi markers that have distinct localizations, suggesting that Drab6 may associate with distinct functional Golgi units. Drab6 might also play a role in membrane exchange between Golgi and ER and in Golgi organization according to our EM analysis, which is again consistent with known functions of mammalian Rab6 [START_REF] Del Nery | Rab6A and Rab6A' GTPases play non-overlapping roles in membrane trafficking[END_REF][START_REF] Martinez | GTP-bound forms of rab6 induce the redistribution of Golgi proteins into the endoplasmic reticulum[END_REF][START_REF] Young | Regulation of microtubule-dependent recycling at the trans-Golgi network by Rab6A and Rab6A[END_REF].

ii) By controlling the migration of Golgi units towards the cell cortex, Drab6 controls the delivery of membrane to the plasmalemma as shown in Drab6 mutants in which glycosylated proteins labelled by WGA and LE Lectins accumulate in large vesicular structures. This pattern is similar to the mis-localization profile of Grk in the absence of Drab6.

iii) In the oocyte, Drab6 is required for the antero-dorsal secretion of Grk, which leads to the differentiation of the follicle cells required for the morphogenesis of the dorsal appendages of the egg shell. In the absence of Drab6, we observed that Grk is mis-localized to late endosomal or lysosomal compartments, demonstrating that Drab6 also affects post-Golgi traffic. In vertebrate cells, one of the Rab6 isoforms (Rab6A') is also involved in endosome to Golgi transport [START_REF] Del Nery | Rab6A and Rab6A' GTPases play non-overlapping roles in membrane trafficking[END_REF][START_REF] Utskarpen | Transport of ricin from endosomes to the Golgi apparatus is regulated by Rab6A and Rab6A[END_REF]. Additionally, a role for Ypt6p (the only copy of Rab6 in the yeast S. cerevisiae) has also been documented for fusion of endosome-derived vesicles with the late Golgi [START_REF] Siniossoglou | An effector of Ypt6p binds the SNARE Tlg1p and mediates selective fusion of vesicles with late Golgi membranes[END_REF]. It remains to be established whether Drab6 functions directly in the secretory pathway or if the effects observed in Drab6 mutants on post-Golgi trafficking are a consequence of defects in endosome to Golgi trafficking.

In order to identify potential Drab6 binding proteins, we performed a yeast two-hybrid screen [START_REF] Formstecher | Protein interaction mapping: a Drosophila case study[END_REF], using as bait Drab6Q71L, a GTP-ase deficient mutant. Sixty-two distinct truncated clones of BicD, lacking parts of the amino terminus, interacted with Drab6Q71L (data not shown). The intersection of all identified fragments defined a minimal interacting domain, mapping BicD amino acids 699-772 in the coiled-coil motif H4 (Fig. 3A), shown for murine BicD to interact with the mammalian Rab6 [START_REF] Matanis | Bicaudal-D regulates COPIindependent Golgi-ER transport by recruiting the dynein-dynactin motor complex[END_REF]. In order to validate this interaction, we performed glutathione S-transferase (GST) pull-down assays, using lysates from wild type ovaries. GST-Drab6 specifically retained BicD, since GST alone andGST-Rab1 did not bind BicD. Furthermore, preloading GST-Drab6 with the nonhydrolyzable GTP analog, GTP-γ-S yielded an improved interaction with BicD (Fig. 3B). We conclude, therefore, that in vitro, BicD interacts through its carboxy terminus preferentially with the active form of Drab6 (GTP-bound), like mammalian Rab6 [START_REF] Matanis | Bicaudal-D regulates COPIindependent Golgi-ER transport by recruiting the dynein-dynactin motor complex[END_REF][START_REF] Short | The Rab6 GTPase regulates recruitment of the dynactin complex to Golgi membranes[END_REF]. Time lapse recording showed that in the oocyte and nurse cells, RFP-Drab6 and BicD-GFP [START_REF] Pare | Subcellular localization of Bic-D::GFP is linked to an asymmetric oocyte nucleus[END_REF] co-localize to multiple large aggregates with low dynamics (supp. MOV2 and Fig. 3B). Further GFP-Drab6 accumulation in the center depended on the presence of BicD during stage 8 as observed in a BicD mom background (see Swan and Suter, 1996, Fig. 3C). Interestingly, in such BicD mom oocytes, Grk was found in ring-likestructures remote from the nucleus as in Drab6 mutant oocytes (Fig. 3C).

Since BicD and Rab6 have been shown to be involved in MT-based transport, we checked whether Drab6 positive structures require MTs to move. Time lapse microscopy revealed that: big aggregates were less dynamic than the highly motile small particles. Colchicine MT depolymerization severely reduced the movement of Drab6 particles movements which formed big clusters (Fig. 4B), indicating that Drab6 is actively transported along MT. The MT motors Kinesin I and Dynein have been shown to be involved in polarizing the Drosophila oocyte [START_REF] Brendza | A function for kinesin I in the posterior transport of oskar mRNA and Staufen protein[END_REF][START_REF] Brendza | Posterior localization of dynein and dorsal-ventral axis formation depend on kinesin in Drosophila oocytes[END_REF][START_REF] Duncan | The cytoplasmic dynein and kinesin motors have interdependent roles in patterning the Drosophila oocyte[END_REF][START_REF] Januschke | Polar transport in the Drosophila oocyte requires Dynein and Kinesin I cooperation[END_REF]. Inactivating the Dynein complex by the over-expression of Dynamitin, [START_REF] Januschke | Polar transport in the Drosophila oocyte requires Dynein and Kinesin I cooperation[END_REF] prevented accumulation of Drab6 at the oocyte cortex (Fig. 4C) but did not significantly reduce Drab6 movements (Fig. 4C'). In contrast, in khc 7.288 germ line clones, Drab6 did not localize in the centre of the oocyte during stage 7/8 and formed abnormal aggregates around the mispositioned nucleus. For reasons we currently do not fully understand, the speed of Drab6 particles was significantly reduced when compared to controls or over-expressing Dynamitin oocytes (Fig. 4D).

We observed that Drab6 and BicD interact in a yeast two hybrid screen and in GST pull down assays and co-localize in vivo. Moreover, there were indications that Drab6 requires BicD for correct sub-cellular localization, which suggests that Drab6 interacts with BicD in Drosophila as it does in mammals. Strikingly, we found that lack of both proteins compromises Grk secretion in a very similar way. Overexpression of Dynamitin, to impair Dynein function, induces ectopic accumulation of Grk and ventralization of the egg shell as well [START_REF] Januschke | Polar transport in the Drosophila oocyte requires Dynein and Kinesin I cooperation[END_REF]. Therefore, in Drosophila, BicD/Dynein and Drab6 are likely involved together in Grk secretion to the antero dorsal corner of the oocyte.

It is important to mention that co-localization of the two proteins was limited. Moreover lack of BicD or Drab6 yields two different phenotypes. BicD mutation affects oocyte determination and the position of the oocyte nucleus [START_REF] Swan | Drosophila Lissencephaly-1 functions with Bic-D and dynein in oocyte determination and nuclear positioning[END_REF], but has no impact on MT organization in mid-oogenesis [START_REF] Swan | Role of Bicaudal-D in patterning the Drosophila egg chamber in mid-oogenesis[END_REF], which is not the case in the Drab6 mutant (Fig. S1). A genetic interaction between BicD's co-factor Egalitarian and Kinesin I has already been demonstrated [START_REF] Navarro | Egalitarian binds dynein light chain to establish oocyte polarity and maintain oocyte fate[END_REF], hence suggesting that Drab6 may interact with Dynein and Kinesin I via BicD.

Interestingly, we noticed that, in absence of Drab6, oskar mRNA was not correctly localized in the oocyte (Fig. S1) (42% dot, 31% diffuse, 10% undetectable, remainder wildtype, n=75). gurken and bicoid mRNAs were, however, unaffected (Fig. S1), and osk mRNA localization to the oocyte center is frequent when the MT network is not correctly polarized [START_REF] St Johnston | Moving messages: the intracellular localization of mRNAs[END_REF]. In Drab6 mutant oocytes, the defective posterior localization of the MT plus end marker Khc-βGal [START_REF] Clark | Reciprocal localization of Nod and kinesin fusion proteins indicates microtubule polarity in the Drosophila oocyte, epithelium, neuron and muscle[END_REF] indicates a defect in MT organization (Fig. S1). Similar observations have been recently reported [START_REF] Coutelis | Rab6 mediates membrane organization and determinant localization during Drosophila oogenesis[END_REF].

Given that Drab6 is required for late Grk signaling at the anterodorsal corner of the oocyte, it might also be involved in early germ line to soma signaling mediated by Grk, which controls MT organization [START_REF] Gonzalez-Reyes | Polarization of both major body axes in Drosophila by gurken-torpedo signalling[END_REF][START_REF] Roth | Cornichon and the EGF receptor signaling process are necessary for both anterior-posterior and dorsalventral pattern formation in Drosophila[END_REF]. Nevertheless, we think that this is unlikely. In absence of this signaling, posterior follicle cells differentiate into anterior follicle cells, and, as a consequence, the posterior structure of the eggshell, the aeropyle, is substituted with an anterior structure, the micropyle [START_REF] Gonzalez-Reyes | Polarization of both major body axes in Drosophila by gurken-torpedo signalling[END_REF][START_REF] Roth | Cornichon and the EGF receptor signaling process are necessary for both anterior-posterior and dorsalventral pattern formation in Drosophila[END_REF]. We always observed an aeropyle at the posterior of eggs derived from Drab6 mutant oocytes (Fig. S2). Additionally, removing Drab6 from the posterior follicle cells did not affect oocyte polarity. Drab6 is, hence, possibly involved in MT organization at the posterior pole.

Interestingly, Rab6 family interactors such as Rab6IP2/ELKS [START_REF] Monier | Characterization of novel Rab6-interacting proteins involved in endosome-to-TGN transport[END_REF] are capable of interacting with CLASPs at the cortex of HeLa cells [START_REF] Lansbergen | CLASPs attach microtubule plus ends to the cell cortex through a complex with LL5beta[END_REF] suggesting a link between Rab6 protein and MT organization at the cortex. Preloading GST-Drab6 with the non-hydrolyzable GTP analog, GTP-γ-S, yielded an improved interaction. In addition to BicD (~ 89kDa), a polypeptide of lower molecular weight (probably a degradation product, ~ 60kDa) was specifically retained on GST-Drab6 beads and could be revealed with the 1B11 but not the 4C2 antibody (II). (III-V) Frames taken from time lapse recording on BicD-GFP (III) and RFP-Drab6 (IV) co-expressing egg chamber(supp. MOV1), which co-localized to several aggregates in nurse cells and the oocyte

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Grk is mis-localized to post Golgi compartments in Drab6 mutants.

Figure 3

 3 Figure 3 Drab6 interacts with BicD in Drosophila.

Acknowledgements

J.J. is very grateful to C. González in whose lab this work was finished. We thank S. Lepanse for EM analysis, E. Poisot for her work on BicD. C. Braendle and P. Dominguez for comments on the manuscript, A Kropfinger for correcting the English language, the Bloomington stock centre and the Developmental Studies Hybridoma Bank for providing reagents. J. J. was supported by the "Association pour la Recherche sur le Cancer (ARC)". This work was supported by : ARC (grant 4446 and 3297), a GenHomme Network Grant (02490-6088) to Hybrigenics and Institut Curie, ACI "Biologie cellulaire ","Jeune chercheur, grant 035117", ANR « Blanche » (grant Cymempol, Blan06-3-139786).

(V). (C) Drab6 accumulates in the centre (arrow) in GFP-Drab6 rescued egg chambers (I). In BicD mom egg chambers of equal age (II), GFP-Drab6 accumulation in the centre was abolished (arrow). In BicD mom oocytes, Grk protein accumulated in ring-like structures remote from the nucleus (III, arrow in lower inset, i.e. magnification of boxed area). Asterisk, position of oocyte nucleus, scale bar 20µm, probes with anti α-Tubulin for loading control. In Drab6 D23D /Cyo; ubi-RFP-Drab6 / Tm2 and Drab6 D23D / Cyo; mat αtub-GFP-Drab6 / Tm2 ovaries, the amount of Drab6 is approximately twice that in wild type (W 1118 ). Signal quantification was done with MultiGauge software (Fujifilm). We used α-Tubulin as a loading reference. Drab6 antibody (Eurogentec) was obtained from rabbit after immunization with two peptides (sequences can be obtained upon request); Rab6 antibody does not function in whole mount.