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Generating Bona Fide Mammalian Prions with Internal Deletions Carola Munoz-Montesino,a Christina Sizun,b Mohammed Moudjou,a Laetitia Herzog,a Fabienne Reine,a Jérôme Chapuis,a Danica Ciric,a Angelique Igel-Egalon,a Hubert Laude,a Vincent Béringue,a Human Rezaei,a Michel Drona INRA U892, Virologie et Immunologie Moléculaires, Jouy-en-Josas, Francea; Institut de Chimie des Substances Naturelles, CNRS, Université Paris-Saclay, Gif-sur-Yvette, Franceb

ABSTRACT

Mammalian prions are PrP proteins with altered structures causing transmissible fatal neurodegenerative diseases. They are self-perpetuating through formation of beta-sheet-rich assemblies that seed conformational change of cellular PrP. Pathological PrP usually forms an insoluble protease-resistant core exhibiting beta-sheet structures but no more alpha-helical content, loosing the three alpha-helices contained in the correctly folded PrP. The lack of a high-resolution prion structure makes it difficult to understand the dynamics of conversion and to identify elements of the protein involved in this process. To determine whether completeness of residues within the protease-resistant domain is required for prions, we performed serial deletions in the helix H2 C terminus of ovine PrP, since this region has previously shown some tolerance to sequence changes without preventing prion replication. Deletions of either four or five residues essentially preserved the overall PrP structure and mutant PrP expressed in RK13 cells were efficiently converted into bona fide prions upon challenge by three different prion strains. Remarkably, deletions in PrP facilitated the replication of two strains that otherwise do not replicate in this cellular context. Prions with internal deletion were self-propagating and de novo infectious for naive homologous and wild-type PrP-expressing cells. Moreover, they caused transmissible spongiform encephalopathies in mice, with similar biochemical signatures and neuropathologies other than the original strains. Prion convertibility and transfer of strain-specific information are thus preserved despite shortening of an alpha-helix in PrP and removal of residues within prions. These findings provide new insights into sequence/structure/infectivity relationship for prions. IMPORTANCE

Prions are misfolded PrP proteins that convert the normal protein into a replicate of their own abnormal form. They are responsible for invariably fatal neurodegenerative disorders. Other aggregation-prone proteins appear to have a prion-like mode of expansion in brains, such as in Alzheimer’s or Parkinson’s diseases. To date, the resolution of prion structure remains elusive. Thus, to genetically define the landscape of regions critical for prion conversion, we tested the effect of short deletions. We found that, surprisingly, removal of a portion of PrP, the C terminus of alpha-helix H2, did not hamper prion formation but generated infectious agents with an internal deletion that showed characteristics essentially similar to those of original infecting strains. Thus, we demonstrate that completeness of the residues inside prions is not necessary for maintaining infectivity and the main strain-specific information, while reporting one of the few if not the only bona fide prions with an internal deletion.

P

rions are misfolded PrP proteins responsible for transmissible spongiform encephalopathies (TSE), a group of fatal neurodegenerative disorders that affect humans and animals. The correctly folded protein (PrPC), tethered at the cell surface, exhibits a structured domain with three helices and a short two-stranded beta-sheet (1, 2). The structure of the insoluble misfolded form (PrPSc) is still elusive, but low-resolution approaches indicated a high beta-sheet content (3, 4) and suggested several different models (5–12). Nevertheless, it is widely accepted that seeds of PrPSc template PrPC, leading to further aggregation and ultimately deposition in the brain (13, 14). Even though PrPC sequence and three-dimensional (3D) structure are highly conserved among mammals, prions do not transmit easily between species due to slight differences in PrP sequence (15, 16). Also, modification of only one residue can drastically affect, or may even impair, intraspecies transmission of prions (17, 18). Different strains of prions can propagate in the same host species, perpetuating specific characteristics such as the incubation time, neuropathology, the biochemical PrPSc signature, and tissue or cell tropism (19). Strain diversity is assumed to reflect a variety of more or less subtle differences in PrPSc structure and/or assemblies. Some substitutions in PrPC were reported to

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have strain-dependent effects for prion replication (20). It is thus important to investigate several prion strains to assess the impact of a particular mutation. A limited proteinase K (PK) treatment is widely used to discriminate PrPSc from PrPC. While totally digesting PrPC, this treatment cleaves about 60 to 70 N-terminal residues of PrPSc according to prion strains but preserves a protease-resistant part that remains highly infectious (14). The protease-resistant core is a polypeptide of about 135 to 150 residues, depending on PrP species and prion strains. Reverse genetic approaches using trans-

Received 25 March 2016 Accepted 14 May 2016 Accepted manuscript posted online 25 May 2016 Citation Munoz-Montesino C, Sizun C, Moudjou M, Herzog L, Reine F, Chapuis J, Ciric D, Igel-Egalon A, Laude H, Béringue V, Rezaei H, Dron M. 2016. Generating bona fide mammalian prions with internal deletions. J Virol 90:6963– 6975. doi:10.1128/JVI.00555-16. Editor: B. W. Caughey, Rocky Mountain Laboratories Address correspondence to Michel Dron, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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TABLE 1 NMR structural statistics for wtPrP, PrP⌬193-196, and PrP⌬190-197a Restraints and statistics

WT

⌬193-196

⌬190-197

No. of structures

20

20

20

No. of NOE distance constraints Total Intraresidue (i ⫽ j) Sequential |i ⫺ j| ⫽ 1 Medium range: 1 ⬍ |i ⫺ j| ⬍ 5 Long range: |i ⫺ j| ⱖ 5

1,453 437 473 283 259

1,655 447 508 332 367

1,938 536 553 383 465

10.6 90⌽, 90⌿

12.5 70⌽, 72⌿

14.5 77⌽, 79⌿

Distance violation/structure (Å) 0.1–0.2 0.2–0.5 ⬎0.5 RMS of distance violation/constraint Maximum distance violation

3.15 1.5 0.05 0.01 0.50

1.4 0.55 0 0.00 0.44

3.15 1.5 0.6 0.01 0.70

Dihedral angle violation/structure 1–10° ⬎10° RMS of dihedral angle violation/constraint (°) Maximum dihedral angle violation (°)

11.4 0 0.56 6.80

4.5 0 0.44 5.70

8.35 0 0.47 4.80

RMSD from ideal covalent geometry Bonds (Å) Angles (°)

0.001 0.2

0.001 0.2

0.001 0.2

RMSD to mean coordinates (residues 128 to 229) Backbone (Å) Heavy atoms (Å)

1.3 1.9

0.8 1.3

0.7 1.1

Ramachandran plotc (%) (residues 128 to 29) Most favored Additionally allowed Generously allowed Disallowed

82.6 16.4 0.9 0.2

86.3 13.2 0.1 0.0

89.0 10.8 0.2 0.1

NOE constraints per restrained residue No. of dihedral angle constraints Violation statisticsb

a

Calculated using PSVS version 1.5 (http://psvs-1_5-dev.nesg.org/). RMS, root mean square; RMSD, root mean square deviation. Average distance constraints were calculated using the sum of r⫺6. c Calculated using Procheck (69). b

genic mice demonstrated that this part of the protein (residues 88 to 231 of mouse PrP) is necessary and sufficient for prion conversion (21). Recent works and reinterpretation of previously published FTIR data indicate that there is no more alpha-helical but high beta-sheet content in the protease-resistant fragment (3). This segment includes the three helices of the correctly folded PrPC, meaning that the whole domain undergoes drastic structural changes upon conversion (9, 22). However, resolution of PrPSc structure remains elusive, and little is known about the sequence and structural requirements for conversion of PrPC into prion. To genetically delineate the critical determinants, a variety of deletions were introduced in the portion of PrP associated with infectivity, but thus far they were not compatible with prion conversion (23–25). Several deletions in PrP caused spontaneous neuropathologies in mice, but they were not associated with the production of PrPSc or transmissibility (26–32). Only one exception was reported: a PrP with two large N-terminal deletions that

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was convertible into PrPSc. However, this so-called miniprion was unable to infect wild-type mice, making it difficult to conclude which structural elements are important for prions (8, 33). Therefore, whether the entire structured domain of PrPC or all of the 140 to 150 residues forming the protease-resistant domain of PrPSc are required for prion conversion remains an important open question. Recent studies, including ours, showed that prion conversion tolerates a certain level of sequence changes at the junction of PrP helices H2 and H3, suggesting that this region might be less essential (10, 34–37). We show here that a deletion of a stretch of residues forming the C terminus of helix H2 of PrPC is plainly compatible with prion conversion and transfer of strain properties. MATERIALS AND METHODS Ethics statement. All animal experiments were carried out in strict accordance with EU directive 2010/63 and were approved by the authors’ local institution Ethics Committee, the Comité d’Ethique en Expérimen-

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FIG 1 Design and cellular expression of PrP deletion mutants. (A) Alignment of the modified sequences in helix H2 of ovine PrPC. Residues of H2 are highlighted in green, and those of helix-3 are highlighted in blue. Deletions were identified by positions of the first and last amino acids removed from wild-type PrP. (B) Cellular expression and glycosylation pattern of wild-type and mutant PrPC. Immunoblot of PrPC expressed by stably transfected RK13 cells. Di-, mono-, and unglycosylated species are indicated on the left, and molecular mass markers are indicated on the right. PrPC were detected using 4F2 MAb directed against the octarepeat region. (C) Mutant PrPC are localized at the cell surface. Immunofluorescence on living cells using the 4F2 MAb is shown. (D) ⌬ PrPs were associated with lipid rafts. The identification of DRM-associated sucrose gradient fractions was performed by immunoblotting with anti-flotilin 1 antibody, as shown in the left panel for ⌬193-196. Then, fractions from gradients with different mutants were tested for the presence of PrPC. The numbering of fractions is shown at the top. tation Animale de l’Institut National de la Recherche Agronomique et AgroParisTech (COMETHEA; permit 12/034). Generation of PrP deletion mutants. Sheep Prnp ORF encoding for PrP allotype Val136-Arg154-Gln171 was cloned into pTRE plasmid (Clontech) (38), and deletions were performed by site-directed mutagenesis (QuikChange II mutagenesis kit; Stratagene). Each mutant construct was verified by sequencing. Cell culture and isolation of Rov cells. Rov cells are epithelial RK13 cells that stably express either wild-type or mutant ovine PrP using a tetracycline-inducible system (38, 39). They were obtained by transfection and puromycin selection, grown in Opti-MEM medium (Invitrogen) supplemented with 10% fetal calf serum (FCS) and antibiotics, and split at 1:4 after trypsin dissociation once a week. To express PrPC, cells were cultivated in the continuous presence of 1 ␮g of doxycycline (Sigma)/ml. Antibodies. The anti-PrP monoclonal antibodies (MAbs) used were as follows: 4F2 directed to the octarepeat domain (residues 62 to 94 according to sheep PrP numbering) (40, 41), Sha31 (epitope; residues 148155) (40), 12B2 (residues 93 to 97) (42), 8G8 (residues 100 to 105) (40, 41), and 8F9 (residues 224 to 234) (43). Sha31 and 8G8 were biotinylated and further detected with horseradish peroxidase-conjugated streptavidin. For other MAbs, secondary antibodies were peroxidase-conjugated goat anti-mouse IgG (Abliance) used at a 1/5,000 dilution. Prion strains. The 127S strain of scrapie was isolated through serial transmission and cloning by limiting dilution of PG127 field scrapie isolate to tg338 transgenic mice expressing the V136R154Q171 allele of ovine PrP (44). T1Ov and T2Ov prions were isolated from serial transmission of human sporadic CJD (MM2, cortical form) material to tg338 mice (45).

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Prion infection of cell cultures. Cells were infected with the 127S prion strain using 1% (wt/vol) brain pool homogenates of terminally ill tg338 mice, as previously described (38, 39). For infections with sCJDderived T1Ov and T2Ov strains, cells were also infected with 1% brain homogenate and maintained with inoculum for 10 days. Cells were then washed, trypsinized, split and incubated for 1 week (passage 1). Cells were then split at 1:4 dilution at each following passage. To test the infectivity of cultures propagating mutant PrPSc, cells were harvested at passages 6 to 8 postexposure, pelleted, frozen and thawed, sonicated, and used as inocula to infect naive cell cultures. Cell lysis and enzymatic digestions. Cells were washed twice with cold phosphate-buffered saline and whole-cell lysates were prepared at 4°C in TL1 buffer (50 mM Tris-HCl [pH 7.4], 0.5% sodium deoxycholate, 0.5% Triton X-100). Lysates were clarified by centrifugation for 2 min at 800 ⫻ g, and protein concentrations were determined by microBCA assay (Pierce). For PrPres, lysates were incubated with 4 ␮g of PK per 1 mg of protein for 2 h at 37°C and then centrifuged for 30 min at 22,000 ⫻ g. Pellets were dissolved in Laemmli sample buffer and boiled for 5 min at 100°C. When needed, 500 U of PNGase F (New England BioLabs, Massachusetts) and 1% Nonidet P-40 were added to denatured proteins that were further incubated at 37°C. Transgenic mouse infections and tissue homogenate preparation. To characterize the levels of infectivity and the strain phenotype of PrPSc produced by mutant PrPC, infected cells were harvested by scraping at passage 8 postinfection. Cell pellets were resuspended in 150 ␮l of medium containing 5% glucose and antibiotics and then frozen and thawed three times before sonication for 1 min. A 20-␮l aliquot of these cellular

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lysates was injected intracranially to tg338 mice overexpressing the VRQ allele of ovine PrP (46). The brains of infected animals were harvested at the terminal stage of the disease and analyzed for PrPres content and distribution by immunoblots and histoblots, respectively as described previously (34, 44). Immunoblotting detection of PrPC and PrPres. Either 4 to 12% NuPage BisTris polyacrylamide gels (Invitrogen) or 12% BisTris gels (BioRad) were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For PrPC analysis, 50 ␮g of protein was deposited on the gel. For PrPres, otherwise-indicated samples corresponding to the PK-resistant PrP present in either 25 or 50 ␮g of protein of cellular lysate were loaded on the gel. The transfer of proteins, their detection, and their revelation were described previously (39, 47). Conformational stability assay. Cellular lysates were brought to the desired concentration of guanidine-HCl and incubated for 1 h at 20°C under agitation and then diluted to reach the same volume and the same concentration of guanidine-HCl (800 ␮l, 0.5 M). A total of 200 ␮l of digestion buffer (750 mM Tris-HCl [pH 7.4], 10% Sarkosyl, and 20 ␮g of PK/ml) was added, and the samples were incubated for 1 h at 37°C under agitation before methanol precipitation and analysis by immunoblotting. Immunofluorescence. Cells cultures were washed twice with OptiMEM medium (Gibco) containing 1% of heat-inactivated FCS before exposure to 4F2 MAb and secondary antibodies Alexa Fluor-conjugated anti-mouse IgG (Molecular probe) used at a 1/500 dilution as previously report (34). Images were acquired with an Axio observer Z1 microscope (Zeiss) and a CoolSnap HQ2 camera (Photometrics) driven by the Axiovision imaging system software. PMCA. Amplifications were performed according to the miniaturized bead PMCA assay (48). To test the conversion of deletion mutants in vitro, we used cell lysates to which 5% brain homogenate from PrP null mouse was added as the substrates. Then, 10-fold dilutions of 127S-infected mouse brain homogenates were used to seed the reactions. In contrast, to test the seeding potential of PrPSc propagated on mutant PrPC, brain lysate of healthy tg338 mice at 10% was used as the substrate and then seeded with diluted lysates of 127S-infected cells harvested at postinfection passage 8. Expression and purification of recombinant PrP. 15N- and 13C-labeled and unlabeled recombinant proteins were produced and purified from Escherichia coli as published previously (34, 49). Briefly, deletions were performed by site-directed mutagenesis inside the sequence encoding residues 103 to 234 of ovine PrP (VRQ allele) cloned into a pET28 expression vector downstream the His tag. Recombinant proteins were expressed and purified as previously (34). NMR experiments. Recombinant 15N-labeled wild-type PrP, PrP⌬193-196, and PrP⌬190-197, as well as 13C15N-labeled PrP⌬193-196 and PrP⌬190-197, were concentrated to 150 to 465 ␮M in 10 mM sodium acetate (pH 5.3) buffer with 10 to 100% 2H2O. Exchange into 100% deuterated buffer was done by lyophilizing the sample before solubilizing it in the same volume of 2H2O. Nuclear magnetic resonance (NMR) measurements were performed at 298 K on Bruker Avance III spectrometers at 600-, 800-, and 950-MHz 1H frequency equipped with cryogenic TCI probes. Spectra were processed with Topspin 3.1 (Bruker Biospin) or NMRPipe (50) and analyzed with CCPNMR 2.2 (51). Resonance assignments of wild-type PrP were derived from 3D 15N-TOCSY-HSQC. Assignments of PrP⌬193-196 and PrP⌬190-197 were obtained by using standard 3D triple-resonance, 3D 15N-TOCSY-HSQC, and 3D-HCCHTOCSY experiments. Distance restraints were obtained from 2D NOESY and 3D 13C- and 15N-separated NOESY spectra with 80-ms mixing time. NMR structure calculation. NMR structures were calculated with CYANA 3.0 software using ambiguous distance restraints from NOE NMR data and backbone torsion angle restraints generated with TALOS⫹ (52). The structures span residues N103-S234 of PrP and include 11 additional N-terminal residues belonging to the His tag. The disulfide bond between C182 and C217 was constrained by using upper and lower distance restraints defined in the “ssbond.cya” macro. Distance restraints for

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FIG 2 Expression and PK sensitivity of wild-type and ⌬ PrP mutants. (A) Glycosylation of wild-type and mutant PrP expressed in Rov cells. Treatment by PNGase F resolved the PrPC signal into one main band that is the unglycosylated full-length PrP protein. 4F2 MAb (B) Detection of the unglycosylated species of ⌬190-197 PrPC. A long exposure of the blot shown in “Fig. 1B” is shown. (C) ⌬ PrPC were sensitive to PK digestion. Cell lysates were treated with 4 ␮g of PK per 1 mg of protein, i.e., at the same concentration than for PrPres detection in infected cells. Sha31mAb.

the PrP mutant with the largest deletion, PrP⌬190-197, were collected from 3D 13C-NOESY-HSQC and 2D NOESY spectra in 100% 2H2O and 3D 15N-NOESY-HSQC and 2D NOESY spectra in 90% H2O/10% 2H2O. Distance restraints for wild-type PrP and recPrP⌬193-196 were collected from 3D 15N-NOESY-HSQC and 2D NOESY spectra in 90% H2O/10% 2 H2O. Structure statistics obtained from the Protein Structure Validation Server, version 1.5 (http://psvs-1_5-dev.nesg.org/), are summarized in Table 1. Visualization and graphic rendering of the protein structures were done with PyMOL (53). Accession numbers. Chemical shifts were deposited at the BioMagResBank under accession numbers 25250, 25251, and 25692 for PrP⌬190-197, PrP⌬193-196, and wild-type PrP, respectively. The coordinates of the structures of PrP⌬190-197, PrP⌬193-196, and wild-type PrP were deposited at the Protein Data Bank under accession codes 2MV8, 2MV9, and 2N53, respectively.

RESULTS

PrP deletion mutants are glycosylated and expressed at the cell surface. To test the effect of amino acids suppression in the H2 C terminus of PrP, two series of sequential deletions were performed, preserving or not the lysine residue K197 at the junction with the H2-H3 interhelix loop (Fig. 1A). Therefore, the shortest deletion was ⌬193-196 and the longest was ⌬190-197. These constructs were stably expressed in RK13 cells, a cell line previously shown to be permissive to prions upon heterologous expression of mammalian PrPs (38, 54–56). Glycosylation, correct expression at the cell surface, and association with lipid rafts are thought to be key for efficient infection of the cells (39, 57). Wild-type and mutant PrPC were mainly glycosylated (Fig. 1B), as shown by PNGase F treatment (Fig. 2A). Glycosylation patterns were roughly similar

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FIG 3 Comparison of the 3D NMR structures of wild-type PrP, PrP⌬193-196, and the nonconvertible PrP ⌬190-197. (A) The lowest energy models of NMR structure ensembles of wild-type PrP (white), PrP⌬193-196 (blue) and PrP⌬190-197 (red) are aligned and shown in cartoon representation with two views rotated by 180°. The position of helix H2 is highlighted by a green box. The disulfide bond is represented with yellow sticks. (B and C) Closeup view of PrP⌬193-196 aligned on wild-type PrP (B) and of PrP⌬190-197 aligned on PrP⌬193-196 (C). His, Phe, and Tyr side chains are drawn with sticks and annotated to illustrate the high density of aromatic residues contributing to tightly bind H3 to H1 and the reorganization of this aromatic network in the deletion mutants.

to those of wild-type PrPC among all the mutants (Fig. 1B), with the exception of PrP⌬190-197 that presented a predominant monoglycosylated band and markedly reduced unglycosylated form, detected only after overexposure of the blots (Fig. 2B). Immunofluorescence on living cells indicated that all PrP mutants were expressed at the cell surface (Fig. 1C). Mutated and wild-type PrP cosediment in the same lipid raft fractions by sucrose gradient separation (Fig. 1D) (39). Moreover, like the wild-type protein, all PrP mutants were sensitive to protease digestion (Fig. 3C), indicating no spontaneous conversion of the mutants into a PK-resistant form. The overall structure of PrP is not affected by a deletion of up to one-third of its helix H2 C terminus. To investigate the effect of deletions on PrP at the structural level, we solved the 3D structures of the wild-type and two mutant recombinant ovine PrPVRQ by NMR (Fig. 3). As expected, deletions ⌬193-196 and ⌬190-197 both significantly shortened helix H2 by four residues and up to one-third of its length, respectively (Fig. 3A). The overall fold, including the N-terminal part of H2 and the length of helix H3, were conserved in both mutants. The kink at the C terminus of H3 in wild-type PrP is probably an artifact due to scarce data, since it is straight in other wild-type PrP structures reported. The relative position of H3 and the remaining H2 did not change significantly either, apart from the slight bending of H2 toward H3 imposed by the strong C182-C217 disulfide bond constraint constituting an essential driving force for maintaining the structure. Helix H1 and

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strands B1 and B2 are stacked onto H3 like in the wild-type but, interestingly, several side chains of aromatic residues in H1 and the H1-B2 and H2-H3 loops (Y148, Y152, Y153, Y158, Y160, and F201) are rearranged in the mutants (Fig. 3B and C). In summary, our results show that the overall structure of PrP is permissive to deletions of up to eight amino acids at the C terminus of H2. Deletion of the C-terminal portion of helix H2 does not impair prion conversion. Cells expressing either wild-type PrPC or deletion mutants were exposed to 127S scrapie prions and analyzed for PrPres content at subsequent passages of cultures. Both ⌬193-196 and ⌬193-197 PrPC were consistently converted into PK-resistant proteins (15 of 15 infection assays and 5 of 5, respectively), and levels of PrPres were comparable to those observed after conversion of wild-type PrP, whatever the passage (Fig. 4A). Moreover, cells expressing ⌬PrPs were as susceptible as Rov-wt cells to prion infection as shown by exposure to serial 10-fold dilutions of 127S brain inoculum (Fig. 4C). PrPres was detected until exposure to 104-fold-diluted material. In contrast, cells expressing PrPC with larger deletions accumulated much less PrPres than the wild-type counterpart upon exposure to 127S. The ⌬192-196 PrPres levels increased with passages but remained 20-fold lower than for wild-type protein, even after the fifth passage (Fig. 4B). PrPres was still detected for the ⌬192-197 and ⌬191-196 mutants, but the amounts were 100- to 200-fold reduced. Finally, no PrPres spe-

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FIG 5 Amplification of misfolded PK-resistant ⌬PrP in cellular-PMCA. The

FIG 4 The C terminus of helix H2 is not needed for conversion into PrPSc. Rov cells that expressed either the wild type or the PrPC mutants were infected by the 127S strain of prion and then assayed for the presence of PrPres, i.e., PKtreated samples were analyzed by Western blotting using anti-PrP Sha31 MAb. (A) Rov-⌬193-196 and Rov-⌬193-197 were permissive to 127S prion infection. The detection of PrPres from similar amount of PK-digested protein is shown. Samples were separated by electrophoresis on the same gel. The numbering of postinfection passages is indicated (P2 to P6) and ‘ni’ indicates noninfected control cells. (B) Cells that expressed PrPC with extended deletions produced less PrPres. The gels were loaded with 250 or 12.5 ␮g of PK-digested proteins per lane for the mutants or wild-type PrP-containing samples, respectively. (C) Rov-⌬193-196 and Rov-⌬193-197 are as susceptible as the Rov-wt cells to 127s prion infection. The results of Western blot analysis of PrPres accumulated in cells at passage 3 postexposure are shown. The logarithmic factor of the dilutions (10⫺2 to 10⫺5) is indicated at the top.

cific signals were detected in cells expressing PrPC with the largest deletion, ⌬190-197 (Fig. 4B). To further demonstrate that the deletion mutants were convertible, PrPC from cellular lysates was used as the substrate for PMCA. Cell lysates expressing wild type or the ⌬PrP mutants were seeded with serial dilutions of 127S infected brain homogenate and subjected to one round of PMCA reaction. The amplified products were analyzed for PrPres content by Western blot. Wildtype, ⌬193-196, and ⌬193-197 PrPC were converted into PrPres in one round of amplification after seeding with up to 106-fold dilution of 127S brain homogenates (Fig. 5A and B). PrPC with larger deletions, like ⌬192-197 or ⌬191-196, were less efficient substrates for amplification (Fig. 5C). PrP⌬190-197 was not converted even at low dilution of 127S. The results of PMCA were largely consistent with the experiments in cell cultures. Collectively, these data suggested that a portion of helix H2 C terminus is dispensable for efficient conversion of PrPC into a self-propagating protease-resistant form.

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substrate of the reaction was the PrPC provided by protein-rich cell lysates (6 mg/ml), and 10-fold dilutions of a 127S brain homogenate were used as a starter. Samples were analyzed after one round of amplification, i.e., 96 cycles of 30 s of sonication, followed by 29 min 30 s of incubation at 37°C. At the end of the reaction, aliquots from each sample were treated with PK (115-␮g/ml final concentration) at 37°C for 1 h. (A and B) PrP⌬193-196 (A) and ⌬193-197 (B) were as efficient as the wild-type PrP for the amplification. The dilution range (10⫺3 to 10⫺6) of 127S brain is indicated at the top. (C) Amplification is less efficient for the other mutants and failed for PrP⌬190-197. The PrPres signal detected at the10⫺3 dilution for PrP⌬190-197 was of the same intensity as the input127S-infected brain material (BH) and was thus due to residual seeding material.

Biochemical characteristics of PrPSc with internal deletion. The electrophoretic pattern of PrPres accumulating in the case of ⌬193-197 and ⌬193-196 constructs appears to be altered with the specific presence of a doublet of aglycosylated bands separated by 1.0 to 1.5 kDa (Fig. 4A). This raised the question of whether the biochemical characteristics of 127S PrPres were conserved upon propagation on the deletion mutants of PrPC. ⌬193-196 PrPres showed the same, or even slightly increased, resistance to PK and pronase digestion and similar sensitivity to Gdn-HCl denaturation compared to wild-type PrPres (Fig. 6A to D). The sensitivity to denaturation of ⌬193-197 PrPres was also comparable to that of wild-type PrP (Fig. 6D). However, we systematically observed a doublet of bands at the expected location for unglycosylated PrPres (Fig. 6A and 7). Treatment with PNGase F attested the coexistence of two distinct species of PrPres (Fig. 7B). The upper PrPres peptide was detected by the 12B2 and 8G8 anti-PrP MAbs as for wild-type PrPres (Fig. 7C and D). 12B2 is classically used to differentiate type 1 and type 2 prions, and the 8G8 epitope is just downstream (Fig. 7A) (42, 58). The lower band was negative for these antibodies but still positive for 8F9 that recognizes the C terminus of PrPC (Fig. 7E), thus indicating that this fragment was more N-terminally truncated than the upper one. N-terminal truncation of wild-type PrPSc is essentially due to cellular processing and preexists to PK treatment in Rov-wt cells (47). To determine whether the same observation applied to mutant PrPres, cellular lysates were centrifuged without PK treatment or, alternatively, were treated with thermolysin that is known to spare the N-terminal moiety of fulllength PrPSc. Insoluble material recovered in pellets and samples treated by thermolysin showed the same double band profiles than

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FIG 6 Biochemical analysis of wild-type and ⌬ PrPSc. (A) Sensitivity to PK digestion. Cellular lysates from 127S-infected Rov-wt () or Rov-⌬193-196 () were PK treated for 2 h at 37°C using a 2-fold dilution series in a concentration range of 2 mg to 3.9 ␮g of PK per mg of cellular protein, as indicated. (B) Immunoblot of samples from panel A; the concentration of PK used (3.9 to 500 ␮g of PK per mg of protein) is indicated above the panels. (C) Sensitivity to pronase digestion. Lysates from 127S-infected Rov-wt () or Rov-⌬193-196 () were treated for 2 h by pronase using a 2-fold dilution series in a concentration range of 2 mg to 3.9 ␮g per mg of protein. (D) Conformational stability assay. Cellular extracts from Rov-wt (rings) or Rov-⌬193-196 () and Rov-⌬193-196 (Œ) were incubated with increasing concentration of Gdn-HCl before PK digestion and blotting. The experiment was performed in triplicate, and the PrPres signals were quantified.

PK-digested samples (Fig. 7F). Collectively, these results indicate that a substantial part of 127S mutant PrPres was endogenously trimmed differently from wild-type PrPres. Removal of the C-terminal end of helix H2 facilitated Rov cells infection by ovine prions derived from sporadic CJD. Rov cells were challenged with three ovine prion strains, distinct from 127S: LA21K fast, derived from sheep scrapie (59), and two strains designated T1Ov and T2Ov and derived from human sporadic CJD adaptation to ovine PrP tg338 mice (45). Rov-⌬193-196 and Rov-wt cells challenged by LA21K fast prions sustained efficient accumulation of PrPSc with similar electrophoretic profiles as after infection with 127S strain (Fig. 8A). To maximize the chance to infect the cells with T1Ov or T2Ov prions, the time of contact between the brain inoculum and the cells was increased (see Materials and Methods). ⌬193-196 PrP was consistently converted into self-propagating PrPSc by T1Ov and T2Ov prions (Fig. 8B and C). In tg338 mouse brains, sCJD-derived T1Ov and T2Ov exhibit distinct PrPres electrophoretic pattern, the unglycosylated species migrating at 21 and 19 kDa, respectively. This difference was preserved over passages in cells expressing PrP⌬193-196 (Fig. 8B and C), as shown by antibodies directed against the central (Sha31) or N-terminal part of PrPres (12B2) (40, 60) (Fig. 8C). Cells expressing PrPs further deleted until valine 192 residue were permissive to T1Ov and T2Ov prions, while those expressing the deletion ⌬190-197 were no longer susceptible (Fig. 8E). Surprisingly Rov-wt cells did not accumulate PrPres upon exposure to any of the two sCJD-derived prions (Fig. 8B and C). This was reproducibly observed in series of three experiments in which Rov expressing wild-type or deletion mutant PrP at similar levels were challenged at the same time and under the same conditions by T1Ov or T2Ov prions in comparison with 127S prions (Fig. 8B to D). These data indicated that deletion of the C terminus of PrPC

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helix H2 facilitated the replication of MM2-sCJD derived prions in Rov cells. ⌬193-196 and ⌬193-197 PrPSc produced by the cells are bona fide prions. We first tested the capacity of PrPSc species produced in Rov-⌬193-196 and Rov-⌬193-197 to propagate in either naive homologous cells, Rov-wt or control RK13 cells. 127S-infected cells were harvested at passage six postinfection, lysed, and used for de novo infections. Both cells expressing either homologous or wild-type PrP showed productive infection, as shown by de novo PrPSc synthesis, whereas no remnant infecting material was detected in control RK13 cells (Fig. 9A). Homologous infection for ⌬193-196 mutant appeared to be more efficient than de novo infection of the wild-type counterpart, as observed in two independent experiments. ⌬193-196 PrPSc species were also potent seeds for PMCA reactions using tg338 brains lysates as PrPC source (Fig. 9B and C). Due to the very high sensitivity of the method, we tested cellular lysates from passage eight postinfection and 127S-infected RK13 cells cultivated in parallel were used as a control. 10⫺5 dilutions of lysates from Rov-⌬193-196 or Rov-wt were sufficient to seed brain PrPC (Fig. 9B), whereas RK13 control showed no seeding activity (Fig. 9C). Finally, tg338 mice were challenged with cells lysates at passage 7 postexposure. All of the mice inoculated by 127S prions passaged in Rov-wt, Rov-⌬193-196, and Rov-⌬193-197 cells succumbed to disease in 60 ⫾ 1 days, 66 ⫾ 1 days, and 77 ⫾ 2 days, respectively, whereas mice inoculated with control 127S-infected RK13 cells remained healthy for more than 250 days and were euthanized healthy (Fig. 10A). Tg338 mice inoculated by either T1Ov or T2Ov serially passaged in Rov ⌬193-196 developed a TSE with a 100% attack rate and mean incubation times of days 107 ⫾

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FIG 7 Characterization of wild-type and ⌬PrPres peptides produced upon infection by 127S prion. (A) Epitopes localization of PrP monoclonal antibodies on

the sequence of the wild-type ovine PrPres peptide. The N-terminal moiety of the misfolded PrP is removed by proteases. Cleavage of the wild-type PrPres produced by 127S-infected Rov cells was previously found to occur between residues 85 and 92 (47). Epitopes for the different MAbs used are in boldface. (B) Two PrPres species are produced after 127s infection of PrP mutants. PK-digested samples were or not treated by PNGase F, as indicated. To facilitate comparison, the gel was loaded with 3-fold-less protein per lane for the PNGase-treated samples than for the untreated, Sha31 MAb. (C and D) The shortest PrPres peptide was more N-terminally truncated. The same PrPres samples were immunoblotted with sha31 or 12B2 MAbs as specified in panel C and with sha31 or 8G8 MAbs as specified in panel D. Arrows indicate the two peptides produced by 127S-infected cells expressing ⌬PrPC. (E) The C terminus of PrP is present in mutant PrPres species. Cell lysate was left untreated or was treated by 16 ␮g/ml thermolysin (Th), and both samples were deglycosylated by PNGase F. The 8F9 MAb recognized the two ⌬193-196 PrPres peptides but also as expected the full-length PrP and the C1 fragment in the sample preserved from protease digestion. (F) N-terminal truncation of the ⌬ PrPres peptides was cell mediated. Crude cellular extracts from infected cells were analyzed (whole sample), centrifuged in the presence of protease inhibitors to analyze the insoluble materials (pellets), or treated with thermolysin or PK as described previously (47). Samples were then treated with PNGase F before immunoblotting with Sha31mAb. (G) Two PrPres peptides were identified in different populations of 127S-infected Rov-⌬193-196 cells. Four populations of Rov cells stably expressing ⌬193-196 PrPC were obtained from independent transfection (samples 2, 3, 4, and 6), and a clone was isolated from one of them (sample 5). The cells were infected by 127S and analyzed at passage 3 postinfection for PrPres content after PK and PNGase treatments (Sha31 MAb).

1 days and days 91 ⫾ 1, respectively (Fig. 10A), i.e., values compatible to that found for the original strains (45). For strain typing purposes, the brains of terminally sick animals were analyzed by Western blotting and histoblots. Properties of 127S strain did not change after replication in Rov-⌬193-196 or Rov-⌬193-197 compared to passage in Rov-wt cells or to direct inoculation of the prion to tg338 mice (44). The PrPres electrophoretic profile was strictly conserved (Fig. 10B), and key features of 127S PrPres regionalization in the brain were observed, such as PrPres deposition in the corpus callosum, habenular nuclei, lateral hypothalamic area, and raphe nuclei of the brain stem (Fig. 10C). The biochemical strain features of T1Ov and T2Ov prions (45) were also conserved in tg338 mice upon intermediate passage onto Rov-⌬193-196 cells. Mice challenged with T2Ov accumulated characteristic T2Ov PrPres with an unglycosylated form migrating at 19 kDa, whereas mice challenged with T1Ov exhibited a 21-kDa signature (Fig. 10B). After challenge with cell-passaged T1Ov, PrPres deposited preferentially in lateral hypothalamic areas, corpus callosum, habenula, and raphe nuclei, as the original T1Ov strain (Fig. 10C). After challenge with T2Ov replicated on mutant PrP, PrPres deposition was prominent in the dorsal thalamic nuclei and in the external capsule/cingulum areas, as for the original

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T2Ov (Fig. 10C). Altogether, these data indicate that, despite propagation of the prions on a PrPC with a C-terminally truncated helix H2, neuropathological and biochemical strain properties were retained. DISCUSSION

We describe here using a reverse-genetic approach and a cellular model of infection that a stretch of residues in the middle of the protease-resistant domain is not required for the formation of prions and preservation of their neuropathological and biochemical strain-specific characteristics. Indeed, PrP lacking four to five highly conserved C-terminal residues of the second helix were efficiently converted into prions upon infection by three distinct strains. This also demonstrated that shortening of an alpha-helix does not impair the molecular process of conformational change of the protein and might even facilitate replication of certain strains, otherwise difficult to pass in the cellular model. So far, deletions performed inside the protease-resistant domain prevented prion conversion, even though several of them induced nontransmissible fatal neuropathies or produced inhibitory proteins of the conversion (23–25, 27–32, 61). The only ex-

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FIG 8 ⌬PrPs were converted into PrPSc by other prion strains, including T1Ov and T2Ov, two sCJD-derived prions. All of the samples were PK treated, except those for panel D. (A) Rov-⌬193-196 were susceptible to the LA21K (fast) prion strain. Cells that expressed ⌬193-196 PrPC were infected (passages 1 to 7) or not infected (ni) by LA21K and tested for protease-resistant PrP (Sha31 MAb). (B) PrP⌬193-196, but not the wild-type PrP, conferred to Rov cells susceptibility to the infection by T1Ov and T2Ov. In the same experiment Rov-⌬193-196 and Rov-wt were infected in parallel by each of the two sCJD-derived strains adapted to tg338 mice. Cell lysates were obtained at passages 1 to 7 postexposure, and the PK digestion results are shown. (C) Infection of cells by three different strains of prion in parallel. Tests for PrPres detection by immunoblotting with either the Sha31 or 12B2 MAbs are shown. (D) Detection of PrPC in infected cells. The same cellular lysates used to obtain the PK-digested samples shown in the left panel of C were analyzed without PK treatment to ascertain the expression of PrPC in Rov-wt and Rov-⌬193-196-infected cells (4F2 MAb). (E) Infection assay for the set of ⌬ PrPs indicated. Rov cells were infected either by T1Ov and T2Ov sCJD-derived prions, and PK-treated samples were analyzed by immunoblotting with Sha31 MAb.

ception is mouse PrP106, a mutant with two deletions (⌬23-88 and ⌬141-176) that adopted spontaneously a moderate proteaseresistance in N2A or ScN2A cells and was converted into a miniprion in PrP106 transgenic mice upon infection by RML prion

FIG 9 PrPSc with internal deletions are infectious for cells and are potent seeds for PMCA. (A) Infection of Rov cells by cellular homogenates from 127Sinfected cultures. Naive Rov-wt, Rov-(⌬193-196), and RK13 cells were challenged using frozen and sonicated pellets of 127S-infected cells harvested at passage 7. An immunoblot shows PrPres detection in de novo-infected cell cultures (passage 3 postinfection). (B) PMCA amplification of wild-type brain PrPC seeded by 127S-infected cells. For the PMCA reactions, lysates from127Sinfected cells at passage 8 were serially diluted as indicated to seed 10% tg338 brain lysate. The dot blot shows analysis of protease-resistant PrP generated after one round of PMCA. (C) Western blot analysis of samples from a PMCA amplification equivalent to that shown in “Fig. 9B.” 10⫺3 and 10⫺4 dilutions of cell lysates from infected cells were used as seeding material. The amplified material shows the expected PrPres profile, whereas no signal was detected using cell lysate from 127S-infected RK13 cells at passage 8 to seed the PMCA. Sha31 MAb was used for each panel of the figure.

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strain (33, 62, 63). The miniprion was infectious for homologous transgenic mice but not for mice expressing the wild-type PrP and was thought to lack a large part of the original prion structure (8). In contrast, we report here that ⌬PrPSc produced upon infection were self-replicating through multiple passages in cell culture, had strong cell-free seeding activity, were infectious for naive cells expressing homologous or wild-type PrPC and caused typical transmissible spongiform encephalopathies in transgenic mice expressing wild-type ovine PrP. These data conclusively demonstrate that PrPSc harboring deletions ⌬193-196 or ⌬193-197 exhibit typical prion hallmark. This study is thus one of the few, if not the only one reporting generation of bona fide prions with an internal deletion. Extension of deletions to residue 192 reduced PrPC conversion efficiency and ⌬190-197 deletion was not compatible with generation of prions in cell culture or with amplification by PMCA. This suggests that the upstream part of helix H2 might be critical for the conversion, even though structural analysis provides alternative or complementary explanations. The tridimensional structure of recombinant PrP⌬193-196 revealed preservation of H2 alpha-helical structure despite reduced size. This is consistent with the conversion of PrP⌬193-196 in cell culture. While the overall structure was maintained after removal of up to one-third of the helix by ⌬190-197 deletion, this induced some rearrangement of side chain interaction between aromatic residues and a slight bending of H3 toward H2 due to increased tense in the H2-H3 hairpin. Accentuation of the bending and differences in rearrangement of side chain inter-

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FIG 10 PrPSc with internal deletions are infectious for tg338 mice. (A) Inoculation of cell extracts from Rov-⌬193-196 or Rov-⌬193-197 propagating proteaseresistant mutant PrP caused TSE in mice. The survival rate curves of tg338 mice inoculated with homogenates of 127S-infected cells harvested at passage 7 postinfection are shown: Rov-wt (), Rov-⌬193-196 (), Rov-⌬193-197 (Œ), and RK13 cells (}). Two other groups of mice also received extracts of Rov⌬193-196 cells infected by either T2Ov (䊐) or T1Ov (〫) sCJD-derived prions and harvested after passage 7 postexposure of the cultures. (B) Immunoblot analysis of PrPres in the brains of terminally ill mice. All of the mice analyzed exhibited PrPres in the brain. The molecular weight of the unglycosylated band was 19K for mice receiving cells originally infected by the T2Ov prion and 21K for T1Ov. (C) PrPres deposition in brains of mice inoculated with the three prion strains propagated on the ⌬PrP mutant. Representative histoblots of anteroposterior coronal sections of brains of tg338 mice inoculated with prions, at the level of septum (I), hippocampus (II), midbrain (III), and brainstem (IV), are shown. For the upper panels, mice were inoculated with 127S propagated for seven passages in RK13 cells that expressed the wild-type PrP (line1) or deletion mutant PrP, either ⌬193-197 (line 2) or ⌬193-196 (line 3), as indicated. The lower panels show brain sections of mice inoculated by either T2Ov or T1Ov prions propagated in Rov-⌬190-196 cells for seven passages.

action compared to ⌬193-196 might contribute to the loss of convertibility of PrP ⌬190-197. While the 193-197 segment can be omitted, deletion of this region generated changes in some aspects of replication. Deleting these residues facilitated propagation of MM2-sCJD derived T1ov and T2ov prions in RK13 cells compared to wild-type PrPC. The

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reasons for this remain elusive, but one possible explanation would be that deletions favor compatibility of mutant PrP with specific structural requirements for the CJD-derived prions. However, these strains replicate efficiently in Tg338 mice, and we cannot rule out potential differential interactions of mutant versus wild-type PrPC with RK13 cell-specific factors. In line with this

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hypothesis, a specific subclone constitutively expressing the wildtype PrP and spotted for its marked susceptibility to 127S strain was finally found permissive to MM2-sCJD derived prions (45). For the 127S strain, the levels of PrPres accumulated in the cells and the degree of cell susceptibility to the infection were similar for wild-type and mutant PrPC, but the pattern of PrPres was modified for mutants with the emergence of species more N-terminally truncated than expected for this strain (47). This might reflect differential cellular processing of mutant PrPSc rather than the formation of novel structures given that 127S strain phenotype was restored on back-passage to tg338 mice. Altogether, our data lead to the main conclusion that deletions ⌬193-196 and ⌬193197 did not inhibit and could even facilitate prion replication depending on the prion strain considered. The main strain properties of 127S, T1ov, and T2ov were restored in tg338 mice despite intermediate passage onto cells expressing ⌬PrPC. Such preservation of prion strain characteristics through passage on deletion mutant PrPs is in line with the prion ability to propagate onto a new PrP sequence while retaining its strain-specific properties in some cases of interspecies transmission (64). The specificity of prion strains is thought to rely on more or less subtle 3D and/or 4D structural differences (65, 66), suggesting that deletions 193-196 and 193-197 have few, if any impact on prion structure. What do these findings bring to our knowledge in the context of the proposed PrPSc structural models? For decades, the most popular model speculated that helices H2 and H3 were preserved in PrPSc (8); however, recent works strongly suggest a complete absence of alpha-helical content in PrPSc (3, 22), and several authors have suggested that that a conformational change of the H2-H3 region, to which 193 to 197 residues belong, is essential to generate prions (4, 6, 49). The fact that the removal of the 193-197 region does not inhibit prion formation and appears to have little impact on their structure, as discussed above, led us to suggest that 193 to 197 amino acids are not likely to be inside a beta strand element essential for prion structure. We propose instead that the 193-197 area, which is alpha-helical in PrPC, is converted into an element that might be less critical for PrPSc structure, i.e., an unstructured loop segment. This is also supported by different observations: the region tolerates insertion of unrelated heptapeptide or octapeptide (34, 36) or replacement of one of the four threonines by a cysteine (10) and also individual replacement of each amino acids by any other one (37); this region is also significantly more accessible to deuterium exchange than surrounding areas (4); and, finally, in the particular example of the HETs prion of Podospora anserina whose structure is resolved, a series of short deletions was performed, and the only ones compatible with prion conversion were found to localize in the loop connecting the two stacked rings of beta strands forming the amyloid fiber (67). Therefore, our results are incompatible with PrPSc models including residues 193 to 197 into a beta strand (11, 68). In contrast, they are congruent with recent models that place them in a loop region joining two beta strands formed by upstream H2 residues and downstream H3 residues (9, 12). According to this hypothesis, the removal, or at least a size reduction, of this loop does not appear to significantly impact the structural organization of PrPSc. To conclude, we generated bona fide prions lacking up to five residues, providing the first evidence that a short PrP section located inside the protease-resistant domain is unnecessary for prion conversion and transmission of strain-specific characteris-

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tics. Moreover, we showed that prion replication is compatible with shortening one of the main structural elements of the PrPC protein. Both observations are valuable contribution to our knowledge on these aggregation-prone pathogenic agents. ACKNOWLEDGMENTS We thank the staff of Animalerie Rongeurs (INRA, Jouy-en-Josas, France) for excellent animal care and for kindly providing anti-PrP antibodies. We also acknowledge the Ile-de-France region (DIM MALINF) and the French Fondation pour la Recherche Médicale (Equipe FRM DEQ20150331689) for their support. Christina Sizun thanks the TGIR-RMN-THC Fr3050 CNRS for access to the 950 MHz equipment at Gif-sur-Yvette.

FUNDING INFORMATION This work, including the efforts of Vincent Béringue and Human Rezaei, was funded by Fondation pour la Recherche Medicale (FRM) (FRM DEQ20150331689). This work, including the efforts of Vincent Béringue and Michel Dron, was funded by Conseil Regional, Ile-de-France (Ile-deFrance Regional Council) (DIM MALINF). Carola Munoz-Montesino acknowledges CONICYT-Becas Chile and DEFRA (United Kingdom) for postdoctoral fellowship support. Christina Sizun acknowledges the TGIR-RMN-THC Fr3050 CNRS for financial support.

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