Marie Randoux

Julien Jeauffre

Tatiana Thouroude

François Vasseur

Latifa Hamama

Marjorie Juchaux

Soulaiman Sakr

Fabrice Foucher
email: fabrice.foucher@angers.inra.fr

Maria Greco

Adriana Chiappetta

Leonardo Bruno

Maria Beatrice Bitonti
email: b.bitonti@unical.it

In Posidonia oceanica cadmium induces changes in DNA methylation and chromatin patterning

Keywords: 5-Methylcytosine-antibody, cadmium-stress condition, chromatin reconfiguration, CHROMOMETHYLASE, DNA-methylation, Methylation-Sensitive Amplification Polymorphism (MSAP), Posidonia oceanica (L.) Delile Floral initiation, floral repressor, gibberellins, PEBP family, polycarpic plants, rose

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In the Mediterranean coastal ecosystem, the endemic seagrass Posidonia oceanica (L.) Delile plays a relevant role by ensuring primary production, water oxygenation and provides niches for some animals, besides counteracting coastal erosion through its widespread meadows (Ott, 1980;Piazzi et al., 1999;Alcoverro et al., 2001). There is also considerable evidence that P. oceanica plants are able to absorb and accumulate metals from sediments (Sanchiz et al., 1990;Pergent-Martini, 1998;Maserti et al., 2005) thus influencing metal bioavailability in the marine ecosystem. For this reason, this seagrass is widely considered to be a metal bioindicator species (Maserti et al., 1988;Pergent et al., 1995;Lafabrie et al., 2007). Cd is one of most widespread heavy metals in both terrestrial and marine environments.

Although not essential for plant growth, in terrestrial plants, Cd is readily absorbed by roots and translocated into aerial organs while, in acquatic plants, it is directly taken up by leaves. In plants, Cd absorption induces complex changes at the genetic, biochemical and physiological levels which ultimately account for its toxicity (Valle and Ulmer, 1972;Sanitz di Toppi and Gabrielli, 1999;Benavides et al., 2005;Weber et al., 2006;[START_REF] Li | A repressor complex governs the integration of flowering signals in Arabidopsis[END_REF]. The most obvious symptom of Cd toxicity is a reduction in plant growth due to an inhibition of photosynthesis, respiration, and nitrogen metabolism, as well as a reduction in water and mineral uptake (Ouzonidou et al., 1997;Perfus-Barbeoch et al., 2000;Shukla et al., 2003;Sobkowiak and Deckert, 2003).

At the genetic level, in both animals and plants, Cd can induce chromosomal aberrations, abnormalities in ª 2011 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

Gibberellins (GAs), important plant growth regulators, are involved in different aspects of plant development such as growth promotion, seed dormancy, floral initiation, and development [START_REF] Fleet | A DELLAcate balance: the role of gibberellin in plant morphogenesis[END_REF]. GAs are cyclic diterpenoid molecules, with GA 1 , GA 3 , GA 4 , and GA 7 being the most biologically active in plants. The floral transition from vegetative growth to flowering is an important period in the life of a plant. In longday (LD) monocarpic plants, it was reported that GA is a floral stimulus and could be substituted for LDs [START_REF] Lang | Physiology of flower initiation[END_REF] whereas, in most woody plants, GA inhibits flowering [START_REF] Zeevaart | Physiology of flower formation[END_REF].

For example, GA activates flowering in Arabidopsis thaliana, whereas it represses flowering in woody fruit trees [START_REF] Wilkie | Regulation of floral initiation in horticultural trees[END_REF].

The molecular basis of floral transition and, particularly, the response to GA, was elucidated in the model plant A. thaliana, a facultative LD vernalization-mediated plant. For this transition to be successful, Arabidopsis must integrate different endogenous and environmental signals (reviewed by Srikanth and Smith, 2011). Flowering is controlled by four major genetic pathways: photoperiod, autonomous, GA, and vernalization. These different pathways converge to activate the floral integrators, FLOWERING LOCUS T (FT) and SUPPRESSOR OF CONSTANS1 (SOC1), and to repress FLOWERING LOCUS C (FLC), a floral repressor involved in the vernalization response [START_REF] Kim | Vernalization: winter and the timing of flowering in plants[END_REF]. The floral integrators then activate the meristem identity genes, LEAFY (LFY) and APETALA1 (AP1), leading to activation of the floral organ identity genes known as ABCE model genes.

In Arabidopsis, GA was required for flowering under short days (SDs) and LDs, with a weaker effect under LDs. ga1-3, a GA-deficient mutant due to a mutation in a key GA metabolism gene [START_REF] Koornneef | Induction and analysis of gibberellin sensitive mutants in Arabidopsis thaliana (L.) Heynh[END_REF], led to a delay in flowering under LDs and was unable to flower under SDs. This phenotype was reversed by exogenous application of GA 4 [START_REF] Wilson | Gibberellin is required for flowering in Arabidopsis thaliana under short days[END_REF]. Under SDs, GA 4 was accumulated in the shoot apical meristem (SAM) prior to floral transition. GA 4 may be synthesized in the leaves and then move to the apex [START_REF] Eriksson | GA4 is the active gibberellin in the regulation of LEAFY transcription and Arabidopsis floral initiation[END_REF].

GAs induce flowering through two different pathways. In leaves, the GA response was FT dependent. Under LDs, GA activated CONSTANS (CO) that in turn induced FT [START_REF] Hisamatsu | The nature of floral signals in Arabidopsis. II. Roles for FLOWERING LOCUS T (FT) and gibberellin[END_REF]. In the apex, GA acted in an FT-independent manner to induce SOC1 and LFY [START_REF] Hisamatsu | The nature of floral signals in Arabidopsis. II. Roles for FLOWERING LOCUS T (FT) and gibberellin[END_REF]. Failure of the ga1-3 mutant to flower under SDs was due to the absence of up-regulation of the floral integrator SOC1 [START_REF] Moon | The SOC1 MADS-box gene integrates vernalization and gibberellin signals for flowering in Arabidopsis[END_REF] and the meristem identity gene LFY [START_REF] Blazquez | Gibberellins promote flowering of arabidopsis by activating the LEAFY promoter[END_REF]. In ga1-3 mutants, SOC1 was accumulated after exogenous GA 4 application under SDs [START_REF] Moon | The SOC1 MADS-box gene integrates vernalization and gibberellin signals for flowering in Arabidopsis[END_REF]. GA 4 regulation of SOC1 expression could be mediated by the down-regulation of SHORT VEGETATIVE PHASE (SVP), a floral repressor involved in the vernalization and ambient temperature pathways [START_REF] Li | A repressor complex governs the integration of flowering signals in Arabidopsis[END_REF]. The GA effect on LFY expression is mediated by a transcription factor, AtMYB33, which is able to link the GAMYBbinding site in the LFY promoter [START_REF] Gocal | GAMYB-like genes, flowering, and gibberellin signaling in Arabidopsis[END_REF]. AtMYB33 expression corresponds to GA 4 accumulation in the shoot apex during floral transition [START_REF] Gocal | GAMYB-like genes, flowering, and gibberellin signaling in Arabidopsis[END_REF][START_REF] Eriksson | GA4 is the active gibberellin in the regulation of LEAFY transcription and Arabidopsis floral initiation[END_REF].

Except for Arabidopsis, few data were available about the molecular control of flowering by GAs in other species. In Lolium temulentum, an obligate LD plant, GA activated flowering [START_REF] Evans | Gibberellin structure and florigenic activity in Lolium temulentum, a long-day plant[END_REF]. The leaf GA content increased after LD exposure with transcriptional regulation of GA biosynthesis genes [START_REF] Gocal | Long-day up-regulation of a GAMYB gene during Lolium temulentum inflorescence formation[END_REF]. GA moves from leaves to the shoot apex where the increase in GA content corresponds to floral induction [START_REF] King | Longday induction of flowering in Lolium temulentum involves sequential increases in specific gibberellins at the shoot apex[END_REF]. LD treatment can be mimicked by GA application, especially GA 5 and GA 6 [START_REF] Evans | Gibberellin structure and florigenic activity in Lolium temulentum, a long-day plant[END_REF][START_REF] King | Longday induction of flowering in Lolium temulentum involves sequential increases in specific gibberellins at the shoot apex[END_REF]. Lolium perenne, a perennial near-relative of L. temulentum, requires both LDs and vernalization for flowering. LDs activate GA biosynthesis, which then stimulates the flowering of vernalized plants, whereas non-vernalized plants are unable to respond to GA for flowering [START_REF] Macmillan | Flowering of the grass Lolium perenne. Effects of vernalization and long days on gibberellin biosynthesis and signaling[END_REF]. In grapevine, the vvGAI mutant, a gene encoding a DELLA protein, a central repressor of GA signalling, was dwarf, GA-insensitive, and produced inflorescences along the length of the shoot instead of tendrils. GA represses the formation of inflorescences and promotes the initiation of tendrils [START_REF] Boss | Association of dwarfism and floral induction with a grape 'green revolution' mutation[END_REF].

Rose is an economically important ornamental crop worldwide. Flowers represent the major value of roses, and control of flowering is a major issue. Rose is a perennial woody plant with different modes of flowering. Once-flowering (OF) roses have a single annual flowering period, whereas continuous-flowering (CF) roses have the ability to flower continuously during the season. In rose, the sensitivity of plants to GA was different between OF and CF roses. Exogenous application of GA inhibited flowering in OF roses and had no effect on flowering in CF roses [START_REF] Roberts | The effect of gibberellins on flowering in roses[END_REF]. GA signalling genes such as GID1, a GA receptor, are regulated differently between OF and CF roses [START_REF] Remay | A survey of flowering genes reveals the role of gibberellins in floral control in rose[END_REF]. Furthermore, GA metabolism was involved in floral initiation. In OF and CF roses, the amount of GA in shoots before floral initiation was low and increased after floral initiation [START_REF] Roberts | The effect of gibberellins on flowering in roses[END_REF]. This GA content was correlated with GA metabolism gene regulation during floral transition. In OF and CF roses, the RoGA20ox gene, encoding an enzyme involved in GA synthesis, was transiently repressed before floral initiation. In contrast, the expression of RoGA2ox, encoding an enzyme involved in GA inactivation, increased before floral initiation and decreased later [START_REF] Remay | A survey of flowering genes reveals the role of gibberellins in floral control in rose[END_REF]. Recently, the gene encoding the continuous flowering locus was shown to correspond to a floral repressor homologue of TFL1, TERMINAL FLOWER1, and was designated as RoKSN [START_REF] Iwata | The TFL1 homologue KSN is a regulator of continuous flowering in rose and strawberry[END_REF]. CF roses had a retrotransposon inserted in the second intron of RoKSN. The presence of a retrotransposon blocked the transcription of RoKSN in CF roses, allowing continuous blooming. In OF roses, RoKSN is weakly accumulated in spring to permit blooming. Later in the season, RoKSN transcripts accumulate and shoots remain vegetative [START_REF] Iwata | The TFL1 homologue KSN is a regulator of continuous flowering in rose and strawberry[END_REF].

The objectives of this study were to understand the molecular control of flowering by GA in rose and to decipher the link between the continuous flowering gene, RoKSN, a TFL1 homologue, and GA. In this study, it was demonstrated that GA inhibited floral initiation in rose by up-regulating the CF gene at the transcriptional level. The promoter of RoKSN was further analysed, and GA-responsive elements were found.

Materials and methods

Plant material

Rosa×wichurana (RW), an OF rose, was obtained from the rose garden, Jardin de Bagatelle (Paris, France). The mutant pair, R. hybrida 'Félicité et Perpétue' (FP), an OF rose, and 'Little White Pet' (LWP), its CF mutant, were obtained from 'Loubert Nursery' (Rosier sur Loire, France). Plants were cultivated as described [START_REF] Remay | A survey of flowering genes reveals the role of gibberellins in floral control in rose[END_REF]. Nicotiana benthamiana were maintained in a greenhouse (20 °C, 16 h light), and 6-to 8-week-old plants were used for agroinfiltration.

GA and paclobutrazol treatments on plants

In December, stems were pruned at six internodes and maintained outside for vernalization (Fig. 1A). At the beginning of the treatment (end of January), plants were placed in a tunnel. Different GA 3 concentrations (0, 30, 70, and 140 µM) were sprayed on plants three times a week during different periods and for different durations (Fig. 1B). Paclobutrazol (PCB) treatment (Bonzi ® , at 42, 70, or 112 µM) was performed every 2 weeks. After blooming, a new set of plants was used for GA 3 and PCB treatments. The inflorescences were removed and growing shoots were pruned at six nodes. Plants, maintained in a greenhouse, were sprayed with GA 3 (30 µM) three times a week for 34 d and with PCB (Bonzi ® , at 14, 56, or 112 µM) three times during the experiment [0, 15, and 29 days after the beginning of treatment (DAB)].

Phenotypic observations

Phenotypic observations were carried out on 10 plants with approximately five pruned stems, 2 months after the end of the treatment. Data were gathered from the three new emerging shoots placed on the distal part of the pruned stem (Fig. 1A). For each new emerging shoot, the percentage of flowering that represents the ratio between the number of flowering shoots and the number of shoots was determined. To calculate the average internode size, three consecutive internodes placed on the apical part of the stem were measured. Statistical analyses were done using the R software, version 2.13.1.

Histological observations

After dissection (removal of leaves), samples were fixed and sections of 2-3 µm were observed under a microscope [START_REF] Foucher | New resources for studying the rose flowering process[END_REF]. Observations were made on four different dates (42,56,63,and 70 DAB).

RNA isolation and gene expression analysis

For RNA isolation, the terminal part of the shoot was harvested from five plants per date and per treatment (Fig. 1A). Samples were dissected (removal of expanded leaves and young leaves) and frozen in liquid nitrogen. Total RNA was isolated using the NucleoSpin ® RNA plant kit (Macherey-Nagel) according to the manufacturer's instructions. Elimination of genomic DNA and reverse transcription were performed using 1 mg of total RNA; real-time PCRs were performed using 3 µl of reverse transcription product (1/25 dilution), as described by [START_REF] Remay | A survey of flowering genes reveals the role of gibberellins in floral control in rose[END_REF]. The amount of plant RNA in each sample was normalized using TCTP and UBC genes [START_REF] Klie | Identification of superior reference genes for data normalisation of expression studies via quantitative PCR in hybrid roses (Rosa hybrida)[END_REF], and the relative expression level was calculated according to [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF], from two biological replicates and three technical repetitions per replicate.

DNA constructs

The upstream genomic sequence of RoKSN (promoter region) was obtained using the Genome Walker Universal Kit (Clontech), following the manufacturer's recommendations from genomic DNA libraries of R. hybrida 'Knock Out'. Serial nested PCRs were performed using the described reverse primers (Supplementary Table S1 available at JXB online) and the forward primers of the kit. A 1200 bp fragment was amplified and sequenced. Then, using Finnzymes' Phusion ® High fidelity DNA Polymerase (Thermo Fisher Scientific), the promoter region was amplified from DNA of Rosa chinensis 'Old Blush' and cloned into the pGEM T easy vector (Promega). Next, successive promoter deletions were performed to obtain fragments of different sizes: 200, 400, 800, and 1200 bp. PCR products were cloned using the Gateway ® system (Invitrogen) with TOPO isomerase mix into the pENTR™/D-TOPO ® entry, followed by LR clonase recombination with pKGWFS7 (VIB, Gent, Belgium) [START_REF] Karimi | Modular cloning in plant cells[END_REF]. The promoter sequence was fused to a GUS::GFP (β-glucuronidase::green fluorescent protein)coding gene. Constructions were introduced by electroporation in Agrobacterium tumefaciens EHA105 with the plasmid pbbR.

Transient expression procedure

Agrobacterium tumefaciens C58C1 containing p19 [START_REF] Voinnet | An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus[END_REF] and EHA105 containing the different constructions were grown at 28 °C in LB medium supplemented with 50 mg l -1 kanamycin, 50 mg l -1 rifampicin, and 100 mg l -1 gentamicin (only for EHA105) up to the stationary phase. After centrifugation at 5000 g for 10 min, bacteria were resuspended in Murashige and Skoog (MS) medium supplemented with 5% (w/v) sucrose, acetosyringone (200 µM), pH 5.6, and with or without GA 3 (10 µM) to obtain an optical density (OD 600 ) of 0.4. Two days after agroinfiltration of 6-to 8-week-old N. benthamiana leaves, GFP fluorescence was observed using a Nikon A1 laser scanning confocal microscope.

Sequence analysis

Sequences of promoters of RoKSN (HE863824), MdTFL1, MdTFL1a (AB366639 and AB36640), FvKSN (HQ378595), PpTFL1 (AB636121), and TFL1 (At5g03837) were used to perform the alignment with MVISTA online software (http://genome.lbl.gov/vista/mvista/submit. shtml) [START_REF] Frazer | VISTA: computational tools for comparative genomics[END_REF]. The search for putative binding sites of ciselements was then carried out using Plantpan online software (http:// plantpan.mbc.nctu.edu.tw/seq_analysis.php) [START_REF] Chang | PlantPAN: plant promoter analysis navigator, for identifying combinatorial cis-regulatory elements with distance constraint in plant gene groups[END_REF].

GUS activity

To measure GUS activity quantitatively, MUG assays were conducted on each agroinfiltrated leaf (1-2 cm 2 ) as described by [START_REF] Jefferson | GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants[END_REF]. The reaction was stopped after 5, 10, 15, 20, and 30 min. Fluorescence was measured at these different times on a FLUO-star plate reader (BMG Labtechnologies Inc.) at 460 nm when excited at 355 nm. Values from the fluorescence assay were standardized by protein concentration, determined as described by [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF].

Results

Gibberellins block floral initiation in spring in once-flowering rose

To study the effect of GA treatment on plant development, exogenous application of GA 3 at different concentrations (30, 70, and 140 µM) was performed in spring on the OF roses, RW and FP, and its CF mutant LWP (Fig. 1).

On non-treated RW, 80% of the emerging shoots became floral, whereas for the GA 3 treatment (30 µM and 70 µM), almost all new emerging shoots remained vegetative (only 5.5% and 1%, respectively, were floral). At higher GA 3 concentrations, flowering was completely inhibited (Fig. 2B). GA 3 treatment also had an effect on the vegetative development of the stem. The length of internodes increased significantly: without treatment, internodes measured 3 cm, at 30 µM GA 3 they measured 4.3 cm, and they measured 4.5 cm for the higher concentrations of GA 3 (Fig. 2A). The same inhibitory effect on blooming was shown on FP (Supplementary Table S2 at JXB online). Flowering was not inhibited by GA 3 treatment in LWP, whereas internode length was reduced. The effect of the GA synthesis inhibitor PCB was tested in FP and LWP. PCB treatment significantly reduced internode length. Concerning flowering, PCB treatment has no effect on LWP, whereas at high concentrations (70 µM and 110 µM), flowering was significantly reduced in FP. Only 50% of the shoots became floral at 110 µM instead of 79% for untreated plants (Supplementary Table S2).

To evaluate the effect of GA 3 treatment on the fate of the apex, cross-sections of the apex from the vegetative to the floral stage were taken using the OF rose, RW, treated or not with 30 µM GA 3 (Fig. 3). In untreated plants after bud outgrowth (42 DAB), the vegetative meristem was narrow with leaf primordia on its flanks (Fig. 3A). The meristem then emerged and enlarged (56 DAB; Fig. 3C). This stage has been defined as the 'floral initiation' stage. The meristem then became floral and bracts were visible (63 DAB; Fig. 3E). Later, floral organs (sepals, petals, stamens, and pistils) are visible (70 DAB, Fig. 3G). Under untreated conditions, the floral initiation took place 56 DAB. In GA 3 -treated plants (Fig. 3B, 3D, 3F, 3H), the meristem was narrow and dome-shaped for all stages. Leaf primordia were visible on the flank of the meristem. This structure was typical of a vegetative meristem. These observations clearly demonstrated that GA 3 blocked floral initiation and that the meristem remained vegetative.

GA 3 is necessary during a short period after bud outgrowth to inhibit flowering.

To characterize the GA response, different durations and periods of GA 3 treatment (30 µM) were tested before blooming in spring. To modify the duration and the period of the treatment, GA 3 application was started and ended on different dates, the 'late' and 'early' treatments, respectively (Fig. 1B). The duration varied from 20 d to 70 d, and the period was from late January to the end of March.

The length of internodes significantly increased between untreated plants (3 cm) and plants treated with GA 3 (from 3.5 cm to 4.6 cm), except for the GA 3 treatment of 20 d (2.3 cm) (Fig. 2A). In general, the longer the treatment, the longer the length of the internodes.

Concerning flowering behaviour, the longer the treatment, the higher the GA inhibiting effect. For GA-treated roses, 5.5, 14.8, 25.5, and 40.5% of the shoots became floral for 70, 63, 47, and 33 d, respectively, of treatment begun on different dates (Fig. 2B). For treatment terminated on different dates ('early' treatment), the same trend was observed: 80% without treatment; 16.5, 3.6, and 3% for 20, 29, and 48 d, respectively. It is interesting to note that for the same duration of treatment, differences are observed if treatments are not carried out in the same period. For 47/48 d of treatment, the percentage of floral shoots was 3% for 'early' treatment, whereas it was 25.5% for 'late' treatment. For 33/29 d of treatment, the percentage of floral shoots was 3.6% for 'early' treatment, whereas it was 40.6% for 'late' treatment (Fig. 2B). 'Early' treatments were more effective at inhibiting flowering than 'late' treatment. For example, an 'early' treatment of 20 d had the same efficiency as a 'late' treatment of 47 d (15% blooming, Fig. 2B). In the 'early' and 'late' treatments, the non-floral shoots remain vegetative and present an indeterminate growth. GA 3 blocked the floral initiation and the meristem remained vegetative. In conclusion, a 1 month treatment in February (during floral transition) is sufficient to inhibit floral transition almost completely.

Floral gene expression in response to GA 3 treatment

To examine whether the inhibition of flowering observed after GA 3 treatment could be associated with the modification of floral gene transcript accumulation, this accumulation in RW shoots that emerged in spring and after blooming using was analysed by qPCR (Figs 1,4). In spring, in the absence of treatment, the transcripts of the floral genes, RoFT, RoFD, RoLFY, and RoSOC1, were accumulated in shoots, which then become floral. RoFT transcripts were progressively accumulated in two steps as previously described [START_REF] Remay | A survey of flowering genes reveals the role of gibberellins in floral control in rose[END_REF]. The first accumulation occurred early during the floral transition (15-35 DAB; Fig. 4C), and the second occurred after floral initiation. RoFD was transiently accumulated early before floral initiation (15 DAB; Fig. 4B). The accumulation of RoSOC1 transcripts was progressive during floral transition and occurred before the late accumulation of the RoLFY and RoAP1 transcripts (70 DAB; Fig. 4). The accumulation of RoKSN transcripts was low in spring (Fig. 4A). Fig. 2. Effect of GA 3 treatment on average internode size in centimetres (A) and percentage of shoots that become floral (B) on R.×wichurana (RW). RW plants were treated with GA 3 (0, 30, 70, or 140 µM) for 70 d, and with GA 3 (30 µM) for different durations and periods. Data were the means of 10 plant observations. There was a statistically significant difference (Waerden's test, P < 0.05; and the χ 2 test, P < 0.0033, for the comparison of percentages) between means with different letters.

When GA 3 was exogenously applied at 30 µM, the expression patterns of floral genes were modified. GA 3 treatment strongly increased the accumulation of RoKSN transcripts. After 70 d of treatment, the RoKSN transcript level increased 40-fold in treated plants (Fig. 4A). RoFT transcript accumulation followed the same expression pattern as in untreated plants, but the accumulation was lower in treated plants than in untreated plants (Fig. 4C). The transient expression of RoFD (15 DAB) was not detected in treated plants. RoSOC1 presented a transient accumulation similar to that of untreated plants, except after 70 DAB when a 2-fold transcript decrease was observed (Fig. 4E). There was no accumulation of RoLFY and RoAP1 transcripts at the end of the experiment.

In FP, RoKSN transcripts were accumulated in response to GA 3 after a few hours. RoLFY transcripts were reduced 2-fold and GA 3 treatment had no effect on RoFT (Supplementary Table S3 at JXB online). To test whether floral gene transcription was RoKSN dependent, the expression of these floral genes was studied in the CF rose, LWP. After GA 3 treatment, the expression of RoFT, RoSOC1, and RoAP1 was not affected, whereas expression of RoLFY and RoFD was reduced 2-fold (Fig. 4G). No RoKSN transcript could be detected.

GA 3 and PCB treatment do not modify the fate of indeterminate vegetative shoots after blooming.

In OF roses, new emerging shoots were vegetative after the blooming in spring (Fig. 1A), whereas in CF roses new shoots were always terminated by an inflorescence. Since GA 3 inhibits flowering in spring, it was investigated whether a GA synthesis inhibitor, PCB, could allow new blooming later in the season in RW (Fig. 1A). Treatments with PCB were performed at different concentrations (14, 56, and 112 µM), and with GA 3 (30 µM) (Table 1). At 30 µM GA 3 , a significant increase in internode length (2.5 cm) was observed compared with untreated plants (2 cm). In contrast to the GA 3 treatment, PCB treatment significantly reduced the internode length at the three concentrations tested in RW and LWP. In RW, the length of internodes was 2 cm for untreated plants and 1.6, 1.5, and 1.4 cm for plants treated with PCB at 14, 56, and 112 µM, respectively. The same trend was observed in LWP (Table 1). Neither of the treatments (GA 3 or PCB) had an effect on flowering. Under all conditions, in RW, the shoots remained vegetative; no flowers were observed. In LWP, all the shoots were floral (Table 1).

In the absence of treatment, transcript accumulation of RoFT, RoSOC1, RoFD, RoAP1, and RoLFY remained low, and RoKSN transcripts were accumulated (Fig. 4). After exogenous application of GA 3 , RoKSN expression increased 3-fold. GA 3 treatment had no effect on RoFD and RoFT (Fig 4B). RoLFY transcript decreased 3-fold after 34 d of GA 3 treatment (Fig. 4D), while RoAP1 was accumulated later (34 DAB). No significant changes were detected after PCB (at 56 µM) treatment, except for RoFD and RoFT, which were transiently induced at 14 DAB (Fig. 4B).

RoKSN promoter contains GA-responsive cis-elements

The induction of RoKSN by GA was further investigated by studying the upstream genomic region of RoKSN. A 1200 bp sequence of RoKSN (upstream of the ATG codon) was isolated. By comparing promoters of TFL1 of other Rosaceae (Malus domestica, Prunus persica, and Fragaria vesca) and Arabidopsis thaliana, two conserved regions were detected (Fig. 5A). Block A (-350 bp to -281 bp) was specific to the Rosoideae subfamily, whereas block B (-177 bp to -61 bp) was common to the Rosaceae family. One GAMYB cis-element, known to be involved in the GA response [START_REF] Gocal | Long-day up-regulation of a GAMYB gene during Lolium temulentum inflorescence formation[END_REF], was identified in block A. No GA response elements were found in block B. The non-conserved regions of the promoter contained three PIF3, three GAMYB, 10 WRKY71, one CARE, and one GARE element (Supplementary Fig. S1 at JXB online)

To determine the functional region for GA response expression, successive promoter deletions were performed: p200, p400, p800, and p1200, which corresponded to the -200, -400, -800, and -1200 bp of the upstream sequence of RoKSN, respectively. No fluorescence or GUS activity was detected with the p200 construct that contains only a TATA box element (Fig. 5C). Weak expression was detected with the p400 promoter in the absence of GA. In the presence of GA, the promoter activity was significantly increased by 14-fold (Fig. 5C). With the p800 and p1200 constructs, the promoter activity was not significantly different between GA-treated and non-treated plants. In both conditions, GUS activity was high (Fig. 5C). The 800 bp fragment may contain a positive cis-element, which provokes a constitutive expression of the promoter under the conditions used here (heterologous system). Due to this constitutive expression, GA induction may no longer be detectable.

Discussion

Physiological studies have demonstrated that GAs are floral repressors in woody perennials such as fruit trees [START_REF] Wilkie | Regulation of floral initiation in horticultural trees[END_REF]. However, how GA mediates its inhibitory effect at the molecular level remains largely unknown. In this study, the role of GAs in flowering on OF roses was investigated at the physiological and molecular level. It was demonstrated that the inhibitory effect of GA in rose is principally mediated by a floral repressor homologue to TFL1, RoKSN.

GA treatment modified the phenology of flowering in once-flowering rose

Under the experimental conditions without GA treatment, the OF roses (RW and FP) initiated their blooming in mid-March. The histological analysis showed that the vegetative meristem was progressively converted into a floral meristem (Fig. 3). As previously described by [START_REF] Roberts | The effect of gibberellins on flowering in roses[END_REF], GA 3 treatment inhibited flowering in OF roses. High levels of GA 3 (>30 µM) completely blocked flowering (Fig. 2). The histological analysis showed that GA 3 treatment inhibited floral initiation. After GA 3 treatment, the apex remained vegetative and none of the morphological modifications observed during floral initiation (such as enlargement and doming of the meristem) was observed (Fig. 3). To specify the effect of GA 3 , different times and durations of GA 3 application were used. A 1 month treatment before floral initiation (in February) was sufficient to obtain a strong inhibition of flowering (Fig. 2A). This period was referred to as floral transition, the phase when the meristem is still vegetative but receptive to signals that stimulate or inhibit flowering. During this phase, GA 3 acts as a floral repressor. This is consistent with the role of GA, which is known to promote vegetative growth instead of reproductive development in woody plants. The exogenous application of GA in fruit trees blocked the floral process and partially reduced the intensity of flowering, for example in apple [START_REF] Tromp | Flower-bud formation in apple as affected by various gibberellins[END_REF][START_REF] Bertelsen | Effects of GA 3 and GA 4+7 on early bud development of apple[END_REF] or in avocado [START_REF] Salazar-Garcia | Use of GA3 to manipulate flowering and yield of 'Hass' avocado[END_REF]. In peach, this inhibitory effect has been linked to the date of treatment [START_REF] Painter | Peach flowering response as related to time of gibberellins application[END_REF]. In strawberry, GA treatment induced the initiation of runners and repressed the formation of crown branches [START_REF] Hytönen | Gibberellin mediates daylength-controlled differentiation of vegetative meristems in strawberry (Fragaria×ananassa Duch)[END_REF]. None of these studies explained how GA mediates its inhibitory effect on flowering.

A floral repressor, RoKSN, is transcriptionally regulated by GAs

To look for the molecular targets of GA during the rose floral process, molecular analyses were carried out in spring and after blooming in GA 3 -treated and untreated OF rose. During floral transition in untreated plants, floral activators were progressively accumulated (RoFT and RoSOC1). Later, RoLFY and RoAP1 were accumulated at the end of floral initiation when the meristem started to become floral (Figs 3,4). RoKSN transcripts remained low during floral transition and initiation. Similar patterns of transcript accumulation have already been observed in FP, an OF rose [START_REF] Remay | A survey of flowering genes reveals the role of gibberellins in floral control in rose[END_REF][START_REF] Iwata | The TFL1 homologue KSN is a regulator of continuous flowering in rose and strawberry[END_REF], and in other Rosaceae. In apple, where floral initiation takes place in June after blooming, expression patterns of floral activators (homologues of SOC1, FT, LFY, and AP1) and repressors (homologues of TFL1) are similar [START_REF] Hättasch | Isolation of flowering genes and seasonal changes in their transcript levels related to flower induction and initiation in apple (Malus domestica)[END_REF]. Data were the means of five plant observations. There was a statistically significant difference (Waerden's test; P < 0.05) between means with different letters. The experimental design is described in Fig. 1. When GA 3 was exogenously applied, the strongest effect was the large accumulation of RoKSN transcripts and the absence of the accumulation of RoLFY and RoAP1 transcripts (Fig. 4). In RW, RoKSN was accumulated 20 times more in GA 3 -treated plants than in untreated plants in spring (Fig. 4). The same pattern was observed in summer. Furthermore, RoKSN was induced by GA 3 in two independent OF roses, RW and FP, suggesting a conservation of this regulation in rose (Supplementary Table S3 at JXB online).

To analyse the induction of RoKSN by GA 3 further, promoter analysis was performed. A 200 bp fragment (located between -200 bp and -400 bp) is sufficient to induce expression of RoKSN in response to GA 3 . Indeed, the 200 bp promoter is not responsive to GA 3 , whereas the 400 bp promoter revealed a stronger activity in the presence of GA 3 (Fig. 5C). This part of the promoter contained GAMYB and PIF3 cis-elements (Supplementary Fig. S1 at JXB online). It is hypothesized that the transcription factor PIF3 (PHYTOCHROME INTERACTING FACTOR 3) integrates different endogenous and environmental factors such as light and GA to induce stem elongation [START_REF] Feng | Coordinated regulation of Arabidopsis thaliana development by light and gibberellins[END_REF]. In Arabidopsis, AtMYB33, a GAMYB transcription factor, is able to link to the GAMYB-binding site in the LFY promoter [START_REF] Gocal | GAMYB-like genes, flowering, and gibberellin signaling in Arabidopsis[END_REF]. This 200 bp fragment corresponds to block A, conserved between rose and strawberry (Fig. 5A), suggesting that GA regulation might be conserved in strawberry where GA is also known to inhibit flowering [START_REF] Thompson | Effect of gibberellic acid on the initiation of flowers and runners in the strawberry[END_REF].

By studying transcript accumulation and promoter activity, the induction by GA 3 of RoKSN, a gene of the TFL1 family, was clearly demonstrated. Further investigations are necessary to understand the signalling between GAs and RoKSN. This study represents the first demonstration of the induction of a floral repressor by GA. In Arabidopsis, TFL1 belongs to a small family [START_REF] Kobayashi | A pair of related genes with antagonistic roles in mediating flowering signals[END_REF]. It was recently demonstrated that other members of the TFL1 family were regulated by hormones. The paralogues of FT, TWIN SISTER OF FT, were activated by cytokinin (BAP) to induce flowering under SDs [START_REF] D'aloia | Cytokinin promotes flowering of Arabidopsis via transcriptional activation of the FT paralogue TSF[END_REF]. In A. thaliana, MOTHER OF FT and TFL1 regulated seed germination via abscisic acid and the GA pathway [START_REF] Xi | MOTHER OF FT AND TFL1 regulates seed germination through a negative feedback loop modulating ABA signaling in Arabidopsis[END_REF]. During floral initiation, it was hypothesized that FT was induced by GA via a positive regulation of CO [START_REF] Hisamatsu | The nature of floral signals in Arabidopsis. II. Roles for FLOWERING LOCUS T (FT) and gibberellin[END_REF][START_REF] Porri | Spatially distinct regulatory roles for gibberellins in the promotion of flowering of Arabidopsis under long photoperiods[END_REF].

RoKSN mediates floral inhibition by GA

GA blocked the floral transition in OF roses (Fig. 2), and RoKSN, a floral repressor, is induced in response to GA 3 (Fig. 4). It was uncertain whether this induction by GA might be the principal component in GA floral inhibition. During the floral process, the GA response between CF and OF roses was different: after GA 3 treatment, OF roses did not flower, while CF roses flowered [START_REF] Roberts | The effect of gibberellins on flowering in roses[END_REF]this study). The CF roses had no functional RoKSN allele, due to insertion of a retrotransposon [START_REF] Iwata | The TFL1 homologue KSN is a regulator of continuous flowering in rose and strawberry[END_REF]. As a consequence, CF roses did not accumulate RoKSN and were no longer able to respond to GA. No floral repression occurred. In OF roses, during the floral transition, RoKSN is weakly accumulated and floral initiation can happen (Fig. 4A). The low accumulation may be explained by the low GA content [START_REF] Roberts | The effect of gibberellins on flowering in roses[END_REF] and the regulation of GA metabolism genes: transient accumulation of RoGA2OX, a gene encoding an enzyme involved in GA inactivation, and low accumulation of RoGA20OX, a gene encoding an enzyme involved in the last steps of active GA synthesis [START_REF] Remay | A survey of flowering genes reveals the role of gibberellins in floral control in rose[END_REF].

After exogenous application of GA 3 , floral genes were downregulated (such as RoSOC1, RoFD, RoLFY, and RoAP1) (Fig. 4). To determine whether these regulations were RoKSN dependent, the expression of these genes in response to GA 3 was studied in a CF rose, LWP (Fig. 4G). In the absence of RoKSN, transcript accumulation of RoFT, RoSOC1, and RoAP1 was not modified after GA 3 treatment. In contrast, RoLFY and RoFD transcripts were reduced 2-fold after GA 3 treatment. These results suggest that RoFT, RoSOC1, and RoAP1 regulation by GA is RoKSN dependent, whereas the GA effect on RoFD and RoLFY is, at least partially, independent of RoKSN.

The following model is proposed to explain the floral inhibition by GA in OF roses. During the floral transition, exogenous application of GA 3 leads to a strong accumulation of RoKSN that blocks induction of floral genes such as RoAP1, RoFT, and RoSOC1 (Fig. 4). As proposed in Arabidopsis, it can be hypothesized that RoKSN is interacting with RoFD to block induction of downstream floral genes such as RoSOC1 and RoAP1 [START_REF] Hanano | Arabidopsis TERMINAL FLOWER1 is involved in the regulation of flowering time and inflorescence development through transcriptional repression[END_REF]. The RoFT repression could be due to a positive feedback loop that is blocked in the presence of RoKSN. Such a positive feedback loop has been shown for TSF, the paralogue of FT in Arabidopsis [START_REF] Yamaguchi | TWIN SISTER OF FT (TSF) acts as a floral pathway integrator redundantly with FT[END_REF]. RoKSN is not the only target of GA. RoFD and RoLFY are down-regulated in the absence of RoKSN, suggesting an independent pathway for these genes. However this RoKSN-independent pathway seems to be minor, as in roksn mutants (such as as LWP, a CF rose), no GA floral inhibition happens even at high GA 3 concentrations (Supplementary Table S2 at JXB online). The transient expression of RoFD (15 DAB, Fig. 4B) is early and may suggest a role for RoFD in processes other than floral initiation. FD is interacting with TFL1 and FT [START_REF] Wigge | Integration of spatial and temporal information during floral induction in Arabidopsis[END_REF][START_REF] Hanano | Arabidopsis TERMINAL FLOWER1 is involved in the regulation of flowering time and inflorescence development through transcriptional repression[END_REF]. However, a role for FD in other developmental process cannot be excluded as FD homologues can interact with proteins controlling processes such as growth [START_REF] Pnueli | Tomato SP-interacting proteins define a conserved signaling system that regulates shoot architecture and flowering[END_REF][START_REF] Mimida | Apple FLOWERING LOCUS T proteins interact with transcription factors implicated in cell growth and organ development[END_REF].

This is the first model that focuses on the inhibition of flowering by GA. Previous studies concerning the role of GA in flowering mainly targeted plants where GA had a stimulating effect. In these plants, for example Arabidopsis and L. perenne, GA acts via activation of floral activators [START_REF] Blazquez | Gibberellins promote flowering of arabidopsis by activating the LEAFY promoter[END_REF][START_REF] Gocal | GAMYB-like genes, flowering, and gibberellin signaling in Arabidopsis[END_REF][START_REF] Moon | The SOC1 MADS-box gene integrates vernalization and gibberellin signals for flowering in Arabidopsis[END_REF][START_REF] Macmillan | Flowering of the grass Lolium perenne. Effects of vernalization and long days on gibberellin biosynthesis and signaling[END_REF].

GA might not be the only factor that regulates RoKSN. After flowering in spring, the RoKSN level increases and blooming is blocked [START_REF] Iwata | The TFL1 homologue KSN is a regulator of continuous flowering in rose and strawberry[END_REF]this study). Treatment with PCB, a GA synthesis inhibitor, fails to induce new flowering (Table 1). Reduction of GA content is not sufficient to induce flowering. The pattern of floral gene accumulation is not modified after PCB treatment (Fig 4). It cannot be excluded that PCB treatment did not reduce GA content enough to induce blooming (as no GA dosage study has been performed). However, PCB treatment is sufficient to reduce internode length (Table 1; Supplementary Table S2 at JXB online). It is proposed that after the blooming in spring, RoKSN could be regulated by GA but also by other pathways. Vernalization is a potentially interesting pathway as OF roses maintained in a greenhouse without a cold period do not flower the next spring. Control of TFL1 homologues by another environmental pathway has already been proposed. In diploid strawberry, the expression of FvKSN, the orthologue of RoKSN, is regulated by the photoperiodic pathway. Under SDs, expression of FvKSN is repressed to permit floral initiation, whereas under LDs, expression of FvKSN increases to inhibit flowering [START_REF] Koskela | Mutation in TERMINAL FLOWER1 reverses the photoperiodic requirement for flowering in the wild strawberry, Fragaria vesca[END_REF]. Furthermore, in Arabis alpina, AaTFL1 is involved in an 'age-dependent response to vernalization' [START_REF] Wang | AaTFL1 confers an agedependent response to vernalization in perennial Arabis alpina[END_REF]. In young shoots, AaTFL1 acts as a floral repressor after vernalization and blocks LFY expression. In old shoots, AaTFL1 increases the duration of vernalization required for flowering [START_REF] Wang | AaTFL1 confers an agedependent response to vernalization in perennial Arabis alpina[END_REF].

In conclusion, a model has been proposed that reveals how GA can inhibit flowering in rose by positively regulating a floral repressor, RoKSN. Pathways other than GA such as vernalization may control blooming in rose, and further experiments are required to explore the molecular interactions between these different pathways.

Fig. 1 .

 1 Fig. 1. Management of R.×wichurana (RW) after GA 3 and PCB treatments. (A) In December, vegetative shoots were pruned at six nodes. Plants remained outside for vernalization. At the end of January, plants were transferred to a tunnel. New shoots developed from axillary buds of the previous years. These shoots were mainly terminated by an inflorescence. After floral development, new shoots grew from the base. Since RW is an OF rose, the shoots remained vegetative. In summer, these shoots were pruned at six nodes and the fate of new emerging shoots from axillary buds was studied. Under controlled conditions, these shoots remained vegetative. RW plants were treated with GA 3 (0, 30, 70, or 140 µM) during vegetative growth and floral initiation between January and March (hatched boxes). Another set of RWs was treated with GA 3 (30 µM) and PCB (14, 56, and 112 µM) during vegetative growth between August and September. Arrows and asterisks indicate the sampling carried out for RNA extraction and histological analysis, respectively. (B) Experiments with different durations and periods of GA 3 (30 µM) treatment. In the 'Late treatment', the experiment started at different times and ended at the same time, whereas in the 'Early treatment', the experiment started at the same time and ended at different times. Numbers indicate the days after the beginning of treatment (DAB). Broken lines represent the pruned shoots of the previous years, large arrows represent new indeterminate growing shoots, and black circles represent axillary buds. Open circles represent flowers. (This figure is available in colour at JXB online.)

Fig. 3 .

 3 Fig. 3. Longitudinal cross-section through apices treated with GA 3 (30 µM; B, D, F, and H) or not (A, C, E, and G). In the untreated plant, the vegetative meristem (A: 42 DAB) progressively changed into a floral meristem (C, E, and G; 56, 63, and 70 DAB, respectively), whereas in the GA 3 (30µM)treated plants, the meristem remained vegetative for the same stage of development (B, D, F, and H, 42, 56, 63, and 70 DAB, respectively). VM, vegetative meristem; LP, leaf primordia; FM, floral meristem; S, sepal; FB, floral bud; P, petal; St, stamen; Pi, pistil. Scale bar=100 µm. (This figure is available in colour at JXB online.)

Fig. 4 .

 4 Fig. 4. Transcript accumulation of floral genes in spring and later in summer in GA 3 -treated (squares), PCB-treated (triangles), or untreated RW (diamonds). Transcript accumulation of floral genes was followed by qPCR in axillary shoots for (A) RoKSN, (B) RoFD, (C) RoFT, (D) RoLFY, (E) RoSOC1, and (F) RoAP1. The x-axis indicates the number of days at which apices were harvested after the beginning of the GA 3 or PCB treatment. The transcript accumulation levels are expressed in relation to the first sample, harvested in January, for each gene according to the Pfaffl ratio (Pfaffl, 2001) (base value=1);. The grey box represents the floral initiation determined according to the histological analysis (Fig. 2). Kruskall-Wallis test on two biological replicates (P < 0.05) was realized, and asterisks represent statistical differences between GA 3 -treated and untreated plants; triangles represent statistical differences between PCB-treated and untreated plants. (G) Transcript accumulation of floral genes in GA 3 -treated (grey box), and untreated (black box) LWP. The transcript accumulation levels are expressed in relation to the untreated plants. Data with different letters indicate a significant difference (Kruskall-Wallis test P < 0.05). nd, not detected

Fig. 5 .

 5 Fig. 5. Analysis of GA-responsive elements in the RoKSN promoter. (A) Alignment of the four TFL1 homologue promoters using MVISTA. Conserved regions (>70% similarity on a 100 bp window) are in red and surrounded by dotted lines (blocks A and B). Alignment was performed with TFL1 (Arabidopis), FvKSN (F. vesca), MdTFL1 (M. domestica), MdTFL1a (M. domestica), and PpTFL1 (P. persica). (B) Successive promoter deletions fused with a GFP::GUS gene. (C) GFP fluorescence and GUS activity. GFP fluorescence observed under a confocal microscope for the four different promoter deletions. Untreated and treated plants are in the left and right panel, respectively. GUS activity for the different deletions in the presence (grey) or absence (black) of GA 3 (10 µM) by transient expression in tobacco leaves after Agrobacterium infiltration. Data are expressed relative to the minimal untreated promoter (p200). Data with different letters indicate a significant difference (Kruskall-Wallis test P < 0.05). (This figure is available in colour at JXB online.)

Table 1 .

 1 Effect of GA 3 and the gibberellin inhibitor (PCB) on floral development and vegetative growth in late summer of OF rose (R.×wichurana, RW) and CF rose ('Little White Pet', LWP).

				Second treatment (August-September)
	Genotype	Treatment	Untreated	GA 3 30 µM	PCB 14 µM	PCB 56 µM	PCB 112 µM
	RW	Average internode size (cm)	2 ± 0.5 b	2.5 ± 0.7 a	1.6 ± 0.5 c	1.5 ± 0.8 c	1.4 ± 0.4 c
		Percentage of shoots that become floral	0	0	0	0	0
	LWP	Average internode size (cm)	2.3 ± 0.7 b	2.6 ± 0.5 a	2.1 ± 0.5 b	1.7 ± 0.5 c	1.5 ± 0.5 d
		Percentage of shoots that become floral	99.2 a	99.1 a	99.1 a	100 a	100 a

Downloaded from https://academic.oup.com/jxb/article/63/18/6543/502905 by guest on 27 November 2023

Acknowledgements

The research of MR was supported by a grant from Angers Loire Métropole (France). We thank N. Dousset and J. Chameau from the INEM team of IRHS (Angers, France) for taking care of the plants, M. Thellier for histological analysis, F. Dupuis for statistical analyses, G. Michel for phenotyping the plants, S. Hanteville for the tobacco, H. Iwata for his fruitful discussions on control of flowering in rose, A. Rabot for providing the gDNA library of 'Knock out ® ', A. Rolland for GUS dosage, L. Hibrand Saint-Oyant for critical reading of the manuscript, and G. Wagman for correcting the English.

Supplementary data

Supplementary data are available at JXB online.

Figure S1. Location of GA response elements in the promoter of RoKSN.

Table S1. List of the names and sequences of primers used in the study.

Table S2. Effect of GA 3 and a gibberellin inhibitor (PCB) on OF rose (Félicité et Perpétue; FP) and CF rose (Little White Pet; LWP) on floral development and vegetative growth in spring during floral transition Table S3. Induction of RoKSN, RoLFY, and RoFT by gibberellins (GA 3 ) in R. hybrida 'Félécité et Perpétue'.


Amandine Mouchard

Marie-Charlotte Boutonnet

Claire Mazzocco

Nathalie Biendon

Nathalie Macrez
email: nathalie.macrez@u-bordeaux.fr

& Neuro

Apoe-fragment/Aβ heteromers in the brain of patients with Alzheimer's disease

Identification of endogenous pathological amyloid β peptides (Aβ) forms in the brains of patients with Alzheimer's disease (AD) is still unclear. In healthy brain, Aβ can associate with Apolipoprotein e (Apoe) which is involved in its metabolism and clearance. In the brain of patients with AD, ApoE is cleaved and produces Apoe fragments. We studied the forms of Aβ and their interaction with the Apoe fragments in post-mortem brains from control and AD patients by western blots and co-immunoprecipitation. three Aβ-containing peptides and three ApoE fragments were specifically found in the brain of AD patients. Co-immunoprecipitations showed that ApoE fragments and Aβ1-42 peptides are co-partners in heteromers of 18 and 16 kDa while ApoE-fragments and Aβ peptides of 12 kDa did not interact with each other. Formation of the 18 kDa ApoE-fragment/Aβ heteromers is specifically increased in ApoE4 carriers and is a strong brain marker of AD while 16 kDa ApoE-fragment/Aβ and Aβ 12 kDa correlate to memory deficit. These data show that in patients with AD, ApoE fragmentation generates peptides that trap Aβ in the brain. Inhibiting the fragmentation or targeting Apoe fragments could be exploited to define strategies to detect or reverse AD.

 ; (iii) total post-mortem Aβ42 measured in the brains of patients by ELISA 10 . Here, we selected patients with cognitive impairment and high levels of both Aβ plaques and Tau tangles as representative cases of AD and studied Aβ and Apolipoprotein E (ApoE) expression in their brain.

 . In human ApoE4-expressing familial AD-transgenic mouse model, Aβ-ApoE interactions are lower and oligomeric Aβ levels are higher than in other human ApoE-expressing mice 25 . Targeting of nonlipidated, aggregated ApoE with antibodies inhibits amyloid accumulation in a transgenic mice model of AD 26 suggesting that targeting ApoE could be exploited to prevent or reverse the Aβ pathology of AD 27 .

ApoE fragmentation also arose as a potential AD-related pathological process. Indeed, carboxy-terminal-truncated forms of ApoE, found in AD brains and in cultured neurons, induce intracellular neurofibrillary tangle-like inclusions in neurons [START_REF] Harris | Carboxyl-terminal-truncated apolipoprotein E4 causes Alzheimer's disease-like neurodegeneration and behavioral deficits in transgenic mice[END_REF][START_REF] Huang | Apolipoprotein E fragments present in Alzheimer's disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons[END_REF][START_REF] Wang | Mass Spectrometry Quantification Revealed Accumulation of C-Terminal Fragment of Apolipoprotein E in the Alzheimer's Frontal Cortex[END_REF] . Carboxy-terminal ApoE peptides stabilize the formation of Aβ oligomers in vitro [START_REF] Wellnitz | A 13 kDa carboxy-terminal fragment of ApoE stabilizes Abeta hexamers[END_REF] and amino-terminal ApoE peptides help binding of Aβ to α7-nACh receptor [START_REF] Wang | Increased Aβ(42)-α7-like nicotinic acetylcholine receptor complex level in lymphocytes is associated with apolipoprotein E4-driven Alzheimer's disease pathogenesis[END_REF] . However, the relation between ApoE fragments and Aβ forms has not been studied so far in human brain.

Here, we studied native forms of Aβ and ApoE in post-mortem human brain tissues of AD or control patients. We used non-denaturant protein extraction and western blot analyses to characterize the peptides with a panel of antibodies targeting various sites on Aβ or ApoE. We also analyzed the Aβ-ApoE interactions in the brains of patients with AD according to their APOE genotype.

Results

Aβ forms in the brain of patients with AD. Three forms of Aβ 18, 16 and 12 kDa were specifically found in the cortex of AD versus control patients (Fig. 1A,D). The amount of each Aβ form was quantified by using different anti-Aβ antibodies and statistical analyses show that the 18 kDa Aβ peptides significantly increase in AD versus control brain (Fig. 1B; p = 0.0059 with PA3 and Fig. 1C; p = 0.0042 with 6E10), the 16 kDa Aβ peptides increase in AD versus control brain when measured with PA3 (Fig. 1B; p = 0.0007) but is not significant when measured with 6E10 (Fig. 1C; p = 0.2337). The lower molecular weight 12 kDa form is found with 6E10 antibody (Fig. 1C,D) antibody but not with PA3 (Fig. 1A,B). Statistical analysis of 12 kDa peptides show that the increase observed in AD patient is not statistically different from control patients (Fig. 1C; p = 0.1355). The majority 18 kDa Aβ form was also increased in hippocampus of AD patients (Supplementary Fig. S2).

Characterization of the 18, 16 and 12 kDa Aβ forms found in the cortex of AD patients was achieved by using 2 additional anti-Aβ antibodies which help to determine the contours of the identified Aβ peptides. N-terminus of Aβ1-42 was revealed by 6E10 positivity while G2-11 positivity revealed the C-terminus of Aβ1-42; PA3 and MOAB2 both recognized the full length human Aβ1-42. All four antibodies recognized the 18 and 16 kDa forms of Aβ (Fig. 1D,E) showing that full length Aβ1-42 is present in 16 and 18 kDa Aβ peptides. Although consisting of Aβ1-42 peptide as well, Aβ 12 kDa probably has a macromolecular structure that makes it poorly recognizable by PA3 (Fig. 1A,D,E).

Aβ1-42 monomers were not detected in the brain of patients with AD by any of the antibodies we used. In contrast, the same antibodies identified monomeric Aβ 4 kDa when purified Aβ1-42 was used as external standard or in the brain of APPxPS1 mice (Supplementary Fig. S1A). The 6E10 and PA3 antibodies also revealed Aβ 4/8 kDa in human cortex of AD patient that were either degraded by leaving the proteins overnight at room temperature or prepared in denaturant conditions with SDS 2% (Supplementary Fig. S1B,C). These control experiments show that the 4 kDa Aβ form can be observed only when human brain samples were modified by experimental conditions or in brain samples of APPxPS1 mice which overproduce Aβ.

Correlation of the Aβ forms with MMse.

We evaluated the correlation between Aβ forms and the cognitive status of ten patients. Quantitative analyses of Aβ forms (6E10/GAPDH) were plotted in function of the MMSE score for each patient (Fig. 2). The statistical analysis of Pearson correlation between the quantity of 18, 16 or 12 kDa Aβ forms and MMSE score revealed that the quantities of the 16 and 12 kDa Aβ forms are significantly correlated to the cognitive status of the patients (p = 0.0424 and p = 0.0041, respectively) while the amount of the 18 kDa Aβ form is not correlated to the MMSE. These results suggest that the main 18 kDa form could be a storage or preservative form of Aβ while the 16 and 12 kDa Aβ forms could be toxic for the brain and impair memory. The correlation graphs (Fig. 2B,C) show that Aβ 16 and 12 kDa correlate better with middle MMSE (10 < MMSE < 25) than with very low MMSE (<10).

Apoe forms in the brains of patients with AD. Since ApoE can increase Aβ oligomer formation [START_REF] Hashimoto | Especially Apolipoprotein E4, Increases the Oligomerization of Amyloid β Peptide[END_REF][START_REF] Jones | Isoform Specific Differences in Tertiary Structure and Interaction with Amyloid-β in Human Alzheimer Brain[END_REF] and truncated forms of ApoE occur in AD brains [START_REF] Wang | Mass Spectrometry Quantification Revealed Accumulation of C-Terminal Fragment of Apolipoprotein E in the Alzheimer's Frontal Cortex[END_REF] , we investigated the multiple forms of ApoE present in the brain of control and AD patients. The full length ApoE protein (35 kDa) and a shorter ApoE protein of 30 kDa were found in all samples but three additional forms (18, 16 and 12 kDa) were specifically found in the cortex of AD patients as shown in Fig. 3A. Statistical analyses show that only the ApoE 18 kDa was significantly increased in AD patients (Fig. 3B, p = 0.0066) while the increases in 16 and 12 kDa ApoE were not significant (p = 0.3673 and p = 0.1179, respectively). As for Aβ, the forms of ApoE fragments were altered when the proteins were degraded after overnight at room temperature which results in the loss of the 18 and 16 kDa forms (Supplementary Fig. S1D). The ApoE 12 kDa form seems less sensitive to degradation.

Four different antibodies (ApoE 126-191, ApoE FL, ApoE Cter and ApoE 262-293) were used to characterize the ApoE fragments observed in cortex of AD patients (Fig. 4A). All fours antibodies recognized the 35 kDa form corresponding to the full length ApoE while none of the short ApoE fragments were recognized by the ApoE 126-191 antibody showing that all three fragments come from the COOH-half part of ApoE. The 16 and 18 kDa ApoE fragments lack the very C-terminus end of ApoE as shown by the lack of immunoreactivity with anti-ApoE Cter (Fig. 4A-C). The 12 kDa form is a C-terminal end form that is revealed by anti-ApoE FL, anti-ApoE 262-293 and anti-ApoE Cter antibodies (Fig. 4A-C).

We analyzed the fragments of ApoE found in ApoE2/3 carriers (including APOE2/2, 2/3 and 3/3 genotypes) versus ApoE4 carriers (including APOE3/4 and APOE4/4 genotypes). The bar graph illustrated Fig. 4D show no difference between ApoE2/3 and ApoE4 carriers suggesting that the production of fragments of ApoE is not dependent on APOE genotype.

Mixed Apoe-fragment/Aβ heteromers in AD brain. Intrigued by the similarities of ApoE and Aβ profiles, we studied the interaction of ApoE fragments with Aβ in AD brains by co-immunoprecipitation (Fig. 5A). Anti-ApoE (FL and 262-293) were better capture antibodies than anti-Aβ antibodies to resolve Aβ forms bound to ApoE fragments. The full ApoE (35 kDa) and the 18, 16 and 12 kDa ApoE fragments were revealed by anti-ApoE Cter and anti-ApoE FL in the ApoE immunoprecipitates (Fig. 5A,B).The 18 and 16 kDa proteins precipitated with ApoE were also positive for Aβ western blotting (PA3 and 6E10) thereby revealing that they are composed of ApoE and Aβ. The 12 kDa form precipitated with ApoE was not recognized by any of the anti-Aβ antibodies (Fig. 6A,B). To sum up: in AD patients ApoE is fractioned and the ApoE fragments associate with Aβ to produce heteromers whose molecular weights are 18 and 16 kDa. In addition, small ApoE fragments of 12 kDa do not bind Aβ (Fig. 5C).

More Apoe-fragment/Aβ heteromers in AD patient expressing APOE4 allele. Since APOE4

allele is a high risk factor for developing AD, we analyzed the impact of APOE genotype on the quantity of Aβ in the cortex of patients (Fig. 6). ApoE4 carriers had significantly higher ApoE-fragment/Aβ 18 kDa levels measured with two different Aβ antibodies (PA3 in Fig. 6A, p < 0.0001 and 6E10 in Fig. 6B, p < 0.0001) while ApoE-fragment/Aβ 16 kDa was not different between APOE genotypes (Fig. 6A,B). These results show that the forms of ApoE-fragment/Aβ heteromers are not equally sensitive to the various APOE genotypes with the APOE4 genotype producing more ApoE-fragment/Aβ 18 kDa. This result was unexpected when considering the fact that APOE4 genotype did not increase ApoE 18 kDa fragments (Fig. 4D).

Since gender matters in AD (women with APOE4 are more likely to develop AD than men with the same allele) [START_REF] Altmann | Alzheimer's Disease Neuroimaging Initiative, I. Sex modifies the APOErelated risk of developing Alzheimer disease[END_REF] and since our population of control patients was deficient in women and in APOE4 allele carriers (Supplementary Table 1), we did similar analyze of ApoE fragment/Aβ heteromers restricted to the patients with AD (55.6% women and 55.6% APOE4). These analyzes confirm that APOE4 genotype produces more ApoE-fragment/Aβ 18 kDa than APOE2/3 (p = 0,0021 for analysis with PA3 and p = 0,0403 for analysis with 6E10; data not shown).

Discussion

Physiological and pathological states of Aβ and ApoE in brain remain unclear, but here we show that 2 of the 3 forms of Aβ peptides specifically identified in the brain of AD patients are hybrid heteromers formed of Aβ1-42 peptides and ApoE middle fragments.

18, 16 and 12 kDa Aβ forms in the cortex of AD patients. Studies of post-mortem brain samples have revealed the increase of Aβ dimers and trimers [START_REF] Lesné | Brain amyloid-β oligomers in ageing and Alzheimer's disease[END_REF][START_REF] Shankar | Amyloid-[beta] protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory[END_REF] , dodecamers [START_REF] Lesné | Brain amyloid-β oligomers in ageing and Alzheimer's disease[END_REF] or tetra/pentamers [START_REF] Bao | Different beta-amyloid oligomer assemblies in Alzheimer brains correlate with age of disease onset and impaired cholinergic activity[END_REF][START_REF] Pham | Progressive accumulation of amyloid-β oligomers in Alzheimer's disease and in amyloid precursor protein transgenic mice is accompanied by selective alterations in synaptic scaffold proteins[END_REF] in AD patients compared to control patients. Our results show specific increase of 16 and 18 kDa (which match tetra/pentamer's size) and 12 kDa (which match trimer's size) but no dodecamers in the brain of patients with AD. We did not detect Aβ monomer peptides (4 kDa) in their brain while we observed Aβ 4 kDa when purified Aβ42 peptides were used as external standard or when analyzing Aβ forms in the brain of APPxPS1 mice. Our results are consistent with other studies of human brain Aβ species [START_REF] Bao | Different beta-amyloid oligomer assemblies in Alzheimer brains correlate with age of disease onset and impaired cholinergic activity[END_REF][START_REF] Pham | Progressive accumulation of amyloid-β oligomers in Alzheimer's disease and in amyloid precursor protein transgenic mice is accompanied by selective alterations in synaptic scaffold proteins[END_REF][START_REF] Savioz | A Study of Abeta Oligomers in the Temporal Cortex and Cerebellum of Patients with Neuropathologically Confirmed Alzheimer's Disease Compared to Aged Controls[END_REF] and with study suggesting that Aβ appears as a tetramer that undergoes breakdown before plaque formation [START_REF] Klementieva | Pre-plaque conformational changes in Alzheimer's disease-linked Aβ and APP[END_REF] . The 18 kDa Aβ form was also found in the hippocampus which confirms the importance of Aβ 18 kDa as a biomarker for AD. On the contrary, the absence of 16 kDa Aβ in the hippocampus is intriguing and requires larger hippocampal samples in control and AD patients to determine if the cortex and hippocampus have different Aβ signatures.

Aβ1-42 peptides in AD patients. Antibodies targeting both the N-terminus and the C-terminus of Aβ1-42 identified the 18, 16 and 12 kDa Aβ forms indicating that all three Aβ forms in the brain of AD patients contain Aβ1-42. Previous trials aimed at identification of pathological Aβ forms have identified an assortment of Aβ species in oligomers [START_REF] Selkoe | The amyloid hypothesis of Alzheimer's disease at 25 years[END_REF][START_REF] Alzforum | Series -2016 Zilkha Symposium on Alzheimer Disease & Related Disorders[END_REF] . Full Aβ1-42 peptides appear to be the earliest form to accumulate in the brain, and are thought to be modified over time into a complex array of truncated, isomerized, and/or phosphorylated peptides [START_REF] Mori | Mass spectrometry of purified amyloid beta protein in Alzheimer's disease[END_REF][START_REF] Nussbaum | Prion-like behaviour and tau-dependent cytotoxicity of pyroglutamylated amyloid-β[END_REF][START_REF] Wildburger | Diversity of Amyloid-beta Proteoforms in the Alzheimer's Disease Brain[END_REF] . The diverse experimental procedures for protein preparations used in the different studies may modify the peptides and explain part of the diversity in Aβ species observed. Our results show that Aβ1-42 peptides are found in the three forms of Aβ observed in the brain of AD patient, which is consistent with previous work [START_REF] Portelius | Mass spectrometric characterization of brain amyloid beta isoform signatures in familial and sporadic Alzheimer's disease[END_REF] , but we cannot exclude that other Aβ species may be part of the extracted peptides. Future experiments of immunoprecipitation combined with mass spectrometry should unmask more composites which could include several of the N-and C-terminally-truncated Aβ peptides identified in the brain of AD patients [START_REF] Portelius | Mass spectrometric characterization of brain amyloid beta isoform signatures in familial and sporadic Alzheimer's disease[END_REF][START_REF] Dunys | Are N-and C-terminally truncated Aβ species key pathological triggers in Alzheimer's disease?[END_REF] . Different functions for the different Aβ forms. Correlative analyses between the quantities of each one of the Aβ forms with the MMSE of the patients showed that ApoE-fragment/Aβ 16 kDa heteromer and the Aβ oligomer 12 kDa correlate to the memory deficit while the ApoE-fragment/Aβ 18 kDa heteromer do not directly impact the memory. The difference between ApoE-fragment/Aβ heteromers 18 and 16 kDa probably retains important information relative to alteration of neuronal function. These forms may differ by the number and/or nature of Aβ they can bind [START_REF] Carter | Alzheimer's Disease: Cellular and Molecular Aspects of Amyloid β[END_REF] . Moreover, we could not exclude that ApoE-fragment/Aβ 18 kDa could play other pathogenic roles such as being intermediate Aβ storage forms or initiating tau hyperphosphorylation and neurodegeneration [START_REF] Harris | Carboxyl-terminal-truncated apolipoprotein E4 causes Alzheimer's disease-like neurodegeneration and behavioral deficits in transgenic mice[END_REF] .

We noticed that ApoE-fragment/Aβ 16 and Aβ 12 kDa correlate better with mid MMSE scores than with very low ones (MMSE < 10). This led us to speculate that in end-stage AD patients with lowest MMSE, neurodegeneration might play a major role in cognitive deficit. Whereas for early stages less demented patients (10 < MMSE < 24), the cognitive impairment might be due to Aβ-mediated alteration of neuronal function and might thus correlate with the quantity of Aβ forms.

Apoe fragments in the brains of AD patients. Beside Aβ accumulation, ApoE fragmentation arose

as a potential AD-related pathological process. We found three proteolytic fragments of ApoE in the brain of patient with AD: 18 and 16 kDa fragments coming from the COOH-half of ApoE lacking both the NH2-half and the C-terminus end and the 12 kDa fragment lacking the NH2-half but including the C-terminus end of ApoE. Previous studies have shown 12-20 kDa ApoE fragments in the brain of AD patients [START_REF] Huang | Apolipoprotein E fragments present in Alzheimer's disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons[END_REF][START_REF] Cho | Quantitation of apoE domains in Alzheimer disease brain suggests a role for apoE in Abeta aggregation[END_REF] and a 19kDa C-terminal ApoE fragment that promote Aβ42 accumulation in human neuroblastoma and primary mouse neuronal cells [START_REF] Dafnis | The ability of apolipoprotein E fragments to promote intraneuronal accumulation of amyloid beta peptide 42 is both isoform and size-specific[END_REF] .

We did not detect N-terminal ApoE fragment in the brain of patient with AD suggesting that they are cleared, removed from the brain or degraded.

Our results show that the fragmentation of ApoE is independent of APOE genotype which disagrees with previous data showing that ApoE4 is more susceptible to proteolytic cleavage than ApoE3. These results were based on in vitro essays with recombinant ApoE3 or ApoE4 and in vivo in transgenic mice expressing ApoE3 or ApoE4 [START_REF] Harris | Carboxyl-terminal-truncated apolipoprotein E4 causes Alzheimer's disease-like neurodegeneration and behavioral deficits in transgenic mice[END_REF][START_REF] Huang | Apolipoprotein E fragments present in Alzheimer's disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons[END_REF] . In the studied population, which includes 75% heterozygous APOE genotypes, similar cleavage has been observed in APOE4 and APOE2/3 carriers. This is in agreement with statement that in addition to ApoE4, ApoE3 can also be cleaved by chymotrypsin-like serine protease which is elevated in AD [START_REF] Alzforum | Does ApoE Fragmentation Drive Pathology?[END_REF] .

Apoe-fragment/Aβ heteromers in human brain of patient with AD. Among the three forms of Aβ found in the brain of AD patients, the 18 and 16 kDa are ApoE-fragment/Aβ heteromers while the 12 kDa forms seem to be made of Aβ (probably trimers) which do not interact with the 12 kDa C-terminal ApoE fragments. The binding of ApoE fragments to Aβ could explain the higher molecular weight of ApoE 16/18 kDa compared to ApoE 12 kDa. Truncation of the C-terminus of ApoE (in ApoE-fragment/Aβ heteromers 16 and 18 kDa) seems to be an essential feature for the ability of the ApoE fragment to bind Aβ as it is for induction of AD-like neurodegeneration and behavioral deficits in transgenic mice [START_REF] Harris | Carboxyl-terminal-truncated apolipoprotein E4 causes Alzheimer's disease-like neurodegeneration and behavioral deficits in transgenic mice[END_REF] . After cleavage, ApoE fragments lacking the ApoE receptor-binding domain and the C-terminus, can still bind to Aβ and trap Aβ within the brain.

Molecular interactions between Aβ peptides and ApoE full length proteins have been described [START_REF] Kim | The Role of Apolipoprotein E in Alzheimer's Disease[END_REF][START_REF] Kanekiyo | ApoE and Aβ in Alzheimer's Disease: Accidental Encounters or Partners?[END_REF] . In brief, residues 12-28 of Aβ appear to contain the binding site for ApoE and residues 244-272 of ApoE are required for interaction with Aβ [START_REF] Strittmatter | Apolipoprotein E: high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease[END_REF][START_REF] Strittmatter | Binding of human apolipoprotein E to synthetic amyloid beta peptide: isoform-specific effects and implications for late-onset Alzheimer disease[END_REF] . The site of Aβ-ApoE interaction is still present in fragments of ApoE produced in AD patients, but the N-terminal part of ApoE is lacking which invalidates the Aβ-clearance and favor Aβ accumulation. Subtle differences in ApoE fragments or Aβ sequences composing the 18 and 16 kDa heteromers remain to be uncovered to explain their differences in molecular weight, in affinity for ApoE4 versus other ApoE and in ability to affect memory. The interaction between ApoE and Aβ seems to depend on ApoE isoform, its lipidation status, and the cellular compartment generating it [START_REF] Tai | Levels of Soluble Apolipoprotein E/Amyloid-β (Aβ) Complex Are Reduced and Oligomeric Aβ Increased with APOE4 and Alzheimer Disease in a Transgenic Mouse Model and Human Samples[END_REF] , suggesting that the role of the ApoE-fragment/Aβ heteromers could be determined by their intrinsic feature. The methods chosen for evaluating ApoE/Aβ complexes may greatly influence the results, for example ApoE purification, which delipidates ApoE, modifies the ApoE to Aβ binding [START_REF] Ladu | Preferential interactions between ApoE-containing lipoproteins and Aβ revealed by a detection method that combines size exclusion chromatography with non-reducing gel-shift[END_REF] . It is likely that any protein preparation that either purify or denature proteins will end up with low or no detection of ApoE-fragment/Aβ heteromers.

Role of APOE4 in ApoE-fragment/Aβ interaction.

Our data show that the APOE4 genotype has a great propensity to produce 18 kDa ApoE-fragment/Aβ forms and suggest that the fragments derived from ApoE4 carriers could better associate with Aβ to form ApoE/Aβ 18 kDa heteromers. A simple explanation could have been that ApoE4 carrier's produce more ApoE fragments [START_REF] Harris | Carboxyl-terminal-truncated apolipoprotein E4 causes Alzheimer's disease-like neurodegeneration and behavioral deficits in transgenic mice[END_REF][START_REF] Huang | Apolipoprotein E fragments present in Alzheimer's disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons[END_REF] , but this hypothesis is ruled out by our results showing no significant difference of ApoE fragment production between ApoE4 and ApoE2/3 carriers. Another bias could be the low representation of women and ApoE4 carriers in the control group, however, analyzes performed on a population restricted to AD patients and composed of half women and half carrier ApoE4, also argue that ApoE4 increases the ApoE fragment/Aβ 18 kDA heteromers.

Low lipidation of ApoE4 has been suggested to result in reduced ApoE4/Aβ levels and increased accumulation of Aβ [START_REF] Tai | Soluble apoE/Abeta complex: mechanism and therapeutic target for APOE4-induced AD risk[END_REF] . Our results suggest that, if lipidation of ApoE4 fragments is altered, it is rather favoring its binding to Aβ resulting in increased formation of ApoE-fragment/Aβ heteromers and increased AD risk. Understanding the interplay between ApoE4 and Aβ seems important because neither ApoE4 nor Aβ on alone does drive risk of memory loss in human [START_REF] Mormino | Amyloid and APOE ε4 interact to influence short-term decline in preclinical Alzheimer disease[END_REF] .

Conclusions

Our results show that ApoE fragments lacking N-and C-termini are partners of Aβ in inducing AD pathology. Because they lack the Aβ transporter-binding domain, ApoE-fragment/Aβ heteromer formation slow down Aβ clearance and promote Aβ accumulation within the brain of patients with AD. APOE4 is more efficient than other APOE genotype for ApoE-fragment/Aβ heteromer formation. Although N-terminal fragments of ApoE is not correlated with AD in the plasma or CSF [START_REF] Gause | Evaluation of Apolipoprotein E Fragmentation as a Biomarker for Alzheimer's Disease[END_REF] , other ApoE fragments and ApoE-fragment/Aβ heteromers might be targeted in brain and peripheral fluids of AD patients to define strategies to detect or reverse AD. A promising strategy to decrease AD progression could be to control ApoE fragmentation which should in turn revive the clearance of Aβ and decrease deleterious effects of Aβ and ApoE fragments in the brain.

Methods

Brain collection. Post-mortem human cortical brain tissues were obtained from Neuro-CEB brain bank of Hôpital de la Pitié-Salpétrière (Paris, France) after informed consent of all participants during their life and/or their legal guardians, human hippocampal brain tissues were obtained from the Bio-bank of the anatomo-pathology department of CHU (Bordeaux, France) after ethics approval and consent. All the experiments were done according to the legislation in force for the use of biological samples in scientific research and respecting the anonymity of the donors.

Since definitions of AD are based on the co-occurrence of Aβ and tau pathologies, post-mortem brain tissues have been classified after neurofibrillar tau and Aβ plaque scoring by members of the Neuro-CEB network as follow: Braak stage 4/6 and Thal stage 2/5 for AD patients and Braak stage 0/2 and Thal stage 0/1 for control patients. Four samples displaying other pathologies (Parkinson disease, Fronto-temporal dementia) and ambiguous staging (high Braak and low Thal or High Thal and low Braak) were discarded from the study. Characteristics of the population of patients whom brain were analyzed in this study were listed in Supplementary table 1. Ten of these cases had been clinically diagnosed according to MMSE score (Mini-Mental State Examination). The MMSE and psychiatric history of others patients are not known. All brain samples were collected at Neuro-CEB or CHU Bordeaux and immediately stored at -80 °C until use.

We also used brain samples from 5 APPxPS1 male mice aged 8-9 months old. These mice overexpress the human PS1dE9 mutant plus the human APP displaying the double Swedish mutation and show memory deficits and Aβ plaques starting around 4-5 months old [START_REF] Lagadec | Early temporal short-term memory deficits in double transgenic APP/PS1 mice[END_REF] . The brain were collected after cervical dislocation of the mouse and stored at -80 °C immediately after collection and until use.

Biochemical analyses of brain tissues.

We designed protein extraction and Western blot conditions to preserve the native structure of the Aβ (no SDS, no formic acid, keep 4 °C temperature throughout the duration of protein preparation). In these conditions, the β-sheet folded Aβ plaques stained by Thioflavine S could not migrate into the gel and were found in deposit pits only. Similar post-mortem intervals between death and brain sampling were respected in AD and control patients (see Supplementary Table 1). Therefore post-mortem interval was not identified as a confounding factor and we did not exclude any sample on this basis. ApoE fragment/Aβ heteromers were observed in all AD patients but they were never observed in control patients even those with the longer post-mortem intervals. These observations exclude the possibility that ApoE fragment/Aβ heteromers could be generated during post-mortem protein degradation. For some control experiments (see Supplementary Fig. S1) the protein extraction were performed in the presence of 2% SDS, 10% formic acid or the native proteins were leave overnight at room temperature for gentle degradation.

Frozen brain tissues were homogenized in two volumes of ice cold lysis buffer (HEPES 20 mM pH 7.2; NaCl 100 mM; Triton 0.5%, Protease Inhibitor Cocktail 1%) and shaken with silicon beads (diameter 1,4 mm) in Minilys (Bertin; 5000 rpm, 4 cycles of 15 s separated by ice cooling). After testing centrifugations of the samples, we decided to keep the whole homogenates to preserve all forms of Aβ and store the preparations at -80 °C. The protein concentration was determined by DC protein assay kit (Bio-Rad) and by direct 280 nm UV measurement (Biodrop, Fisher Scientific), it was adjusted to 10 µg/µL and stored at -20 °C before electrophoresis.

Brain homogenates (50 µg of total protein) or purified Aβ42 peptides (ref 1428, Tocris, 0.1 µg) were electrophoretically resolved in a precast 4-20% Tris-Glycine Criterion gels (TGX, BioRad) for 45 min at 180 V. Proteins were transferred from gel onto PVDF membranes (Transblot, BioRad). Before 6E10 immunostaining, membranes were left during 15 min in PBS at 85 °C for epitope unsmasking and wash in PBS-Tween. For all immunostaining, membranes were incubated for 1 h at room temperature in 5% non-fat dry milk to block unspecific binding.

Immunodetection of the proteins was performed by incubating the membranes for 24 h at 4 °C with primary antibodies against Aβ: 6E10 (AB_662799, Covance), PA3 (AB_2258328, Thermo Scientific), G2-11 (AB_10562244, Millipore), MOAB2 (AB_2313888, Millipore) or antibodies against ApoE: ApoE FL (AB_564230, Calbiochem,), ApoE 126-191 and ApoE C-ter (sc-393302, SantaCruz), ApoE 262-293 (AB_2057990, Abgent). Secondary antibodies, dilutions and conditions used for IR imaging are summarized in Supplementary Table 2.

Western blots were imaged with the Odyssey scanner (LiCor) and analyzed with Image Studio Software (LiCor). Anti-GAPDH antibody (AB_10615768, Millipore) was used as loading control and allowed the quantification of Aβ and ApoE as x/GAPDH ratios.

Co-immunoprecipitation.

For co-immunoprecipitation of ApoE and Aβ, we followed the Pierce classic Magnetic IP Kit instructions. Briefly, equal amounts of total homogenates (1500 μg) were incubated with 10 µg of IP antibody (anti-ApoE FL antibody, anti-ApoE 262-293, PA3 or 6E10 antibodies) for 1-2 h at room temperature (RT) or overnight at 4 °C. Antigen/antibody complex was bound to Protein A/G magnetic beads for 1 h at RT. Non-bound sample components were washed away, and antibody and antigen were eluted with a buffer that disrupts the binding interactions. The antigens were further characterized by classical ApoE or Aβ Western blot as described above.

Apolipoprotein-E genotyping. We have used restriction enzyme isoform genotyping for rapid typing of APOE (E2, E3, E4) alleles in the human brain samples used in this study. APOE restriction isotyping used oligonucleotides to amplify APOE gene sequences encoding for amino acid positions 112 and 158. The amplification products were digested with Cfo1 and subjected to electrophoresis on polyacrylamide gels. Each of the isoforms was distinguished by a unique combination of Cfo1 fragment sizes as previously reported [START_REF] Hixson | Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI[END_REF] .

Statistical analyses. GraphPad Prism 6.0 was used for all statistical analysis. The peptides observed in the brain of control patients (Braak 0/2 and Thal 0/1 which express few if any tangles and plaques) were compared to those of AD patients (Braak 4/6 and Thal 2/5 which display significant amount of tangles and plaques). Data were analyzed with the Mann-Whitney test for comparison of the same peptide between control and AD and with two-way ANOVA for comparison of different forms of peptides between APOE2/3 versus APOE4 genotypes. Correlation analysis was conducted using Pearson's correlation. All Data were presented as the mean ± SEM (Standard Error of the Mean) or in plots where each data point represent one subject, p values < 0.05 were considered to be significant.

Figure 1 .

 1 Figure 1. Quantification and characterization of the Aβ forms found in the cortex of AD patients. (A) Representative western blot of cortex proteins extracted from control and AD patients (each pit representing a different patient) and revealed with an anti-Aβ full length (PA3-16761, green). GAPDH (red) was revealed as an internal control for each deposit. (B) Bar graph representing the statistical analysis of Aβ/GAPDH ratio measured with PA3 antibody for 18 and 16 kDa forms in control versus AD brain samples. (C) Bar graphs representing the statistical analysis of the Aβ/GAPDH ratio measured with 6E10 antibody for the 18, 16 and 12 kDa forms in control versus AD brain samples. The number of samples analyzed is indicated in parentheses, significant statistical differences are marked by *(p < 0.05) and **(p < 0.005) as calculated by Mann-Whitney test. (D) Western blots of cortex from a control (Braak 1, Thal 0) and an AD patient (Braak 6, Thal 4) revealed with 3 different anti-Aβ antibodies (G2-11, MOAB2, 6E10). All fours antibodies recognized the 18 and 16 kDa forms of Aβ as compiled in (E) while the PA3 antibody poorly recognize the 12 kDa Aβ form. The lanes illustrated in Fig. 1D have been cropped from Supplementary Fig. S3A.

Figure 2 .

 2 Figure 2. Correlation between Aβ forms and the cognitive status of the patients evaluated by MMSE. Quantitative analyses of Aβ forms (6E10 relative to the quantity of GAPDH for each sample) were plotted in function of the MMSE score for each patient. The statistical analysis of correlation between the quantity of 18, 16 and 12 kDa Aβ forms and MMSE as well as the linear regressions of the data are plotted in (A-C) respectively. Pearson analyses reveal that the quantities of the 16 kDa and 12 kDa Aβ forms are significantly correlated to the cognitive status of the patients while the amount of the 18 kDa Aβ form is not correlated to the MMSE. Significant correlation are marked by *(p < 0.05) and **(p < 0.005).

Figure 3 .

 3 Figure 3. ApoE protein and fragments detected in the cortex of AD patients. (A) Representative western blot of cortex proteins extracted from control and AD patients and revealed with an anti-ApoE full length antibody (178479 Calbiochem). The full length ApoE protein (35 kDa) was found in all samples and 3 additional forms (18, 16 and 12 kDa) were found only in the cortex of AD patients. (B) Bar graphs representing the statistical analysis of the quantification of various ApoE forms for each sample. The number of samples analyzed is indicated in parentheses, significant statistical differences between control and AD are marked by *(p < 0.05) as calculated by Mann-Whitney Test.

Figure 4 .

 4 Figure 4. Characterization of ApoE fragments detected in the cortex of AD patients. (A) Illustrations of western blots of cortex from 2 AD patients revealed with 4 different anti-ApoE antibodies (ApoE 126-191, ApoE FL, ApoE Cter and ApoE 262-293). All fours antibodies recognized the 35 kDa form corresponding to the full length ApoE but only the anti-ApoE FL and the anti-ApoE 262-293 identified the 18 and 16 kDa forms, the smaller 12 kDa form was identified by three ApoE antibodies as compiled in (B,C). Schematic illustration of ApoE in control patient and of ApoE fragments formed only in AD patients. (D) Bar graph illustrating the effect of ApoE2/3 carriers versus ApoE4 carriers on the production of ApoE fragments. The number of samples analyzed is indicated in parentheses.

Figure 5 .

 5 Figure 5. Co-immunoprecipitation of ApoE and Aβ revealed heteromers of 18 and 16 kDa composed of ApoE fragments and Aβ in the cortex of AD patients. (A) Proteins extracted from human cortex of AD patients (Braak 6, Thal 4) were immunoprecipitated (IP) with an anti-ApoE FL or an anti-ApoE 262-293 antibody and western blotted (WB) with five anti-ApoE or anti-Aβ antibodies. The full length ApoE protein (35 kDa) was found in all samples revealed with anti-ApoE antibodies. In addition the 18, 16 and 12 kDa forms of ApoE were identified in the immunoprecipitates western blotted with appropriate anti-ApoE antibodies. The 18 and 16 kDa of these immunoprecipitates were also identified by PA3 and 6E10 antibodies as summarized in (B). The lanes illustrated in (A) have been cropped from western blots illustrated in Supplementary Fig. S3B. (C) Schematic illustration of ApoE regions involved in interaction with Aβ. In AD patients, ApoE fragments form heteromers with Aβ whose molecular weight are 18 and 16 kDa. In addition ApoE fragments of 12 kDa do not bind Aβ.

Figure 6 .

 6 Figure 6. Effect of APOE genotype on the quantity of Aβ forms in the cortex of AD patients. Aβ 18 and 16 kDa measured with PA3 antibody (A) or with 6E10 antibody (B) in the cortex of patients carrying APOE2/3 or APOE4. The number of samples analyzed is indicated in parentheses. Two way ANOVA and Bonferroni posthoc analyses reveal a significant effect of APOE4-carriers on the production of the 18 kDa form of Aβ/ApoE heteromer with both Aβ antibodies (***p < 0.001).

  ethics approval. Human brain tissues were obtained from Neuro-CEB brain bank of Hôpital de la Pitié-Salpétrière, Paris, France and from the Bio-bank of the anatomo-pathology department of CHU Bordeaux, France after ethics approval and consent of the Committee for the Protection of Persons (CPP No. CEBH 2009/03; MESR: DC-2008-337).

Scientific RepoRts | (2019) 9:3989 | https://doi.org/10.1038/s41598-019-40438-4

Scientific RepoRts |

responsible for acquisition of Aβ data and pathological classification. N.M., A.M. and N.B. were responsible for acquisition of APOE and co-immunoprecipitation data. A.M., M.C.B. and N.M. were responsible for statistical analysis, creating figures and data interpretation. Members of Neuro-CEB Neuropathology Network were responsible for collecting brains and clinical data. All authors read and approved the final manuscript.

Acknowledgements

The authors want to thank (1) the patients who donated their brain to science and their family, this was the primary and essential step allowing this study; (2) the technicians and engineers working at NeuroCEB network for the enormous task of collecting/preserving brain tissues and collecting/compiling/archiving clinical and pathological data concerning donors; special thanks to Sabrina Turbant for collection and transmission of data relative to the present study. (3) Physicians who collected many precious data. (4) All members of the Neuro-CEB Neuropathology Network which includes: Dr. Franck Letournel (CHU Angers), Pr Anne Vital (CHU Bordeaux), Pr Françoise Chapon (CHU Caen), Pr Catherine Godfraind (CHU Clermont-Ferrand), Pr Claude-Alain Maurage (CHU Lille), Dr. Vincent Deramecourt (CHU Lille), Dr. Mathilde Duchesne (Limoges), Dr. David Meyronnet (CHU Lyon), Dr. Nathalie Streichenberger (CHU Lyon), Dr. André Maues de Paula (CHU Marseille), Pr Valérie Rigau (CHU Montpellier), Dr. Fanny Vandenbos-Burel (Nice), Pr Charles Duyckaerts (CHU PS Paris), Pr Danielle Seilhean (CHU PS, Paris), Dr. Véronique Sazdovitch (CHU PS Paris), Dr. Serge Milin (CHU Poitiers), Dr. Dan Christian Chiforeanu (CHU Rennes), Pr Annie Laquerrière (CHU Rouen), Dr. Béatrice Lannes (CHU Strasbourg). This work was supported by the Centre National de la Recherche Scientifique (CNRS), the University of Bordeaux, the Agence Nationale pour la Recherche (ANR-10-CESA-002-02 to NM, ANR-10-MALZ-001-03 to NM) and doctoral fellowships from Ministère de l'Enseignement Supérieur to MCB.

Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Author Contributions

Additional Information

Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-40438-4.

Competing Interests:

The authors declare no competing interests.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Consortia Neuro-CEB Neuropathology Network