Eduard Feldbach

Luc Museur

Veera Krasnenko

Andreas Zerr

Mamoru Kitaura

Andrei Kanaev
email: andrei.kanaev@lspm.cnrs.fr

Defects induced by He+ irradiation in γ-Si3N4

Keywords: Spinel Si3N4, He + irradiation, Cathodoluminescence, Photoluminescence, Structural defects

Introduction

Optical materials will play a substantial role in various diagnostic systems of future deuterium-tritium fusion reactors. For this reason, search and characterisation of those withstanding high intensity neutron fluxes must be pursued. Formation and nature of the radiation-induced lattice defects affecting functionality of the candidate materials for optical components in fusion reactors are under investigation in order to understand their radiation tolerance and find approaches of minimising the degradation rates. It is now widely recognized that the crystalline lattice of binary oxide spinels (MgAl 2 O 4 , etc.) possesses an elevated tolerance to the ionizing radiation [START_REF] Sickafus | Radiation tolerance of complex oxides[END_REF][START_REF] Sickafus | Radiationinduced amorphization resistance and radiation tolerance in structurally related oxides[END_REF] due to an effective interstitial-vacancy recombination process and swapping of the cations between tetrahedral and octahedral sites, which take advantage of a huge number density of so-called structural vacancies in the crystal lattice: 7/8 of all available tetrahedral and 1/2 of octahedral sites. The energy cost for antisites formation in the spinel structure is much lower than that for all other elementary lattice defects [START_REF] Gilbert | A theoretical study of intrinsic point defects and defect clusters in magnesium aluminate spinel[END_REF] permitting to the cation sublattice easily accommodate the induced disorder.

The high-pressure (HP) binary nitrides with cubic spinel structure -M 3 N 4 (M=Si, Ge, Sn) [START_REF] Zerr | Recent advances in new hard high-pressure nitrides[END_REF] could be alternative radiation tolerant materials, because the cation exchange in these materials does not result in the defect creation. Among the spinel nitride family, -Si 3 N 4 [START_REF] Zerr | Synthesis of cubic silicon nitride[END_REF] is the most prominent member, not only because it exhibits outstanding mechanical properties and exceptional thermal stability but also because it can be obtained as optical windows transparent in the VUV-vis-IR ranges of light [START_REF] Mo | Interesting physical properties of the new spinel phase of Si3N4 and C3N4[END_REF]. The knowledge of the electronic band structure and structural defects is of importance in studies of its suitability for the application as radiation tolerant material.

The electronic band structure of the single-cation spinels nitrides has been predicted to be direct, with the E g value between 3.45 eV and 4.97 eV in the case of γ-Si 3 N 4 [START_REF] Ching | Theoretical prediction of the structure and properties of cubic spinel nitrides[END_REF][START_REF] Chu | Allelectron GW quasiparticle band structures of group 14 nitride compounds[END_REF][START_REF] Boyko | Class of tunable wide band gap semiconductors γ-(GexSi1-x)3N4[END_REF][START_REF] Caskey | Semiconducting properties of spinel tin nitride and other IV3N4 polymorphs[END_REF]. A slightly lower, by ~0.07 eV, indirect transitions than the direct transition of 5.16 eV have been recently calculated for this material [START_REF] Tanaka | First-Principles Calculations of anion vacancies in oxides and nitrides[END_REF]. Few calculations on the band structure distortion due to structural defects, vacancies or interstitials [START_REF] Oba | Effective doping in cubic Si3N4 and Ge3N4: A first-principles study[END_REF] and some frequent dopants [START_REF] Boyko | Electronic structure of spinel-type nitride compounds Si3N4, Ge3N4, and Sn3N4 with tunable band gaps: application to light emitting diodes[END_REF] are also available. In contrast to the theoretical predictions, experimental data on the electronic structure of the spinel nitrides are rare. The first experimental study of the solid solutions γ-(Si 1-x Ge x ) 3 N 4 , where 0≤x≤1, has been performed using soft x-ray spectroscopy [START_REF] Caskey | Semiconducting properties of spinel tin nitride and other IV3N4 polymorphs[END_REF] and later extended to γ-Sn 3 N 4 [START_REF] Museur | Photoluminescence and electronic transitions in cubic silicon nitride[END_REF]. Another detailed study of the defect and electronic band structure of γ-Si 3 N 4 by time-and energy-resolved optical spectroscopy at LHe temperatures has been published only recently [START_REF] Nishiyama | Transparent polycrystalline cubic silicon nitride[END_REF]. The band gap energy of E g =5.050.05 eV and large exciton binding energy of D e ~0.65 eV have been reported, permitting to propose lighting applications of this material under severe external impact such as mechanical and/or radiation stress. The radiation tolerance and related modifications of the defect and electronic band structure of γ-Si 3 N 4 as well as other HP spinel nitrides have not been yet considered.

In this communication we report on the first investigation of the modification of photoluminescence (PL) properties and electronic transitions in -Si 3 N 4 after irradiation with 150 keV He + ions (α-particles). The study was performed using UV-visible-near-IR time-and energyresolved spectroscopy as most relevant and sensitive experimental method for the evaluation of changes of the electronic band and defect structures of solids.

Methods

The polycrystalline γ-Si 3 N 4 sample of 3 mm diameter and 1 mm thickness was synthesized in a multi-anvil press at pressure of 15 GPa and temperature of 1700 °C by following the procedure described in Ref. [START_REF] Mo | Interesting physical properties of the new spinel phase of Si3N4 and C3N4[END_REF]. The sample was partially sintered and translucent or transparent to the visible light after polishing. The prepared sample was mounted in a stainless still frame for PL measurements (Figure 1). One side of the sample was kept untreated and another was subjected to an ionizing radiation with He + ions of 150 keV energy and total dose of 10 17 cm -². The irradiation was performed in the Accelerator Lab of Helsinki University. Both irradiated and pristine surfaces of γ-Si 3 N 4 sample were structurally characterized using a set-up consisting of an IµS high Brilliance-Incoatec Microfocus Source Mo ELM33, delivering focused Mo K 1,2 radiation, and an Imaging Plate Detector with online readout system MAR345. The two-dimensional XRD patterns were collected in the grazing incidence geometry with the angle of ~7° and converted to the standard diffractograms I(2θ) using the Fit2D software [START_REF] Hammersley | Two-dimensional detector software: From real detector to idealised image or two-theta scan[END_REF]. This set-up permitted the detection of contaminating phases (e.g. β-Si 3 N 4 ) in amounts as low as 0.5%, not accessible using standard diffractometers.

Prior to the optical measurements, the sample was removed from the stainless steel frame and fixed via silver glue on an aluminium support. Raman spectra were excited using the argon laser line λ L =514 nm and collected with the Renishaw inVia micro-Raman installation; these measurements were performed with 100 objective at the normal incidence to the sample surface. The cathodoluminescence (CL) spectra of the spinel sample mounted on a close-cycle He cryostat were recorded at 5 K temperature using a laboratory installation [START_REF] Feldbach | Radiation resistance diagnostics of wide-gap optical materials[END_REF]. The excitation was performed with an electron beam of 10 keV and 0.1 μA.

The photoluminescence (PL) experiments were carried out at the BL3B beamline [START_REF] Kitaura | Excitation process of Ce 3+ and Eu 2+ ions doped in SrGa2S4 crystals under the condition of multiplication of electronic excitations[END_REF] of the UVSOR synchrotron radiation facility with the excitation in VUV-UV-visible spectral range. The sample was mounted on the cold finger of a LHe flow type cryostat maintained during experiments at 9 K in the vacuum ~10 -7 Pa. In order to evaluate structural damage induced by the irradiation, PL and PL excitation (PLE) spectra of the pristine and irradiated samples were collected and compared. The measured PLE spectra were corrected on the detector (AXUV100 photodiode) sensitivity and optical line transmission.

The energy of the tetrahedral and octahedral sites was evaluated from the theoretical calculations of the cationic (Si) and anionic (N) defect formation energies using general gradient approximation (GGA) [START_REF] Zerr | Synthesis of cubic silicon nitride[END_REF] and 56-atoms 1x1x1 supercell, due to the low computational cost. These calculations were performed using the CASTEP module of the Materials Studio software [START_REF] Soignard | Raman spectroscopy of -Si3N4 and -Ge3N4 nitride spinel phases formed at high pressure and high temperature: evidence for defect formation in nitride spinels[END_REF]. # Although, because of a small supercell the obtained values could not expected being accurate (especially for highly charged vacancies), they reflected general trends for the defects with different charge states, which has to be compared with the experiment. The screened hybrid functional approach of Heyd, Scuseria, and Ernzerhofnd with optimized screening parameters (HSE06) [START_REF] Kröger | Relations between the concentrations of imperfections in crystalline solids[END_REF] for formation energy calculation on a primitive cell of Si 3 N 4 with neutral Si tetrahedral monovacancy (provides more accurate result for Kohn-Sham energy levels) was performed as the benchmark [START_REF] Sickafus | Cation disorder in high dose, neutronirradiated spinel[END_REF] for the GGA calculations. Complementary, GGA calculations with 448 atoms 2x2x2 supercell of some selected defects were realized in order to check general trends obtained for the smaller 56-atoms supercell. To simulate charged defects, we have removed and added electrons to the defective cells/supercells. The formation energies of a neutral vacancy/point defect of Si 3 N 4 were evaluated using the formalism described in Ref. [START_REF] Sickafus | Cation disorder in high dose, neutronirradiated spinel[END_REF]. The formation energy of a defect D in charge state q was defined as

Di i q H q D q D n E E H     , , , (11)
where E D,q is total energy of the host-defect supercell and E H,q is total energy of the neutral host (if q=0) or charged host (if q0) of supercell without defects, µ Di is the chemical potentials of the atoms removed (n i =+1) or added (n i =-1). Such approximation achieved good agreement with general trends of the charged defect formation energies by varying the supercell size [START_REF] Drouin | CASINO V2.42 -A Fast and easy-to-use modelling tool for scanning electron microscopy and microanalysis users[END_REF].

Results and discussion

# The plane-wave basis energy cutoff was chosen to be 350 eV. The Monkhorst-Pack scheme k-points grid sampling was set at 6×6×6 for the Brillouin zone. The convergence parameters were set as follows: total energy tolerance 1.0•10 -5 eV/atom, maximum force tolerance 0.03 eV/Å, maximum stress 0.05 GPa and maximum displacement 1.0•10 -3 Å.

The XRD patterns of the pristine and He + -irradiated sides of -Si 3 N 4 crystal were identical (Fig. 2), showing no effect of the energetic ions on the crystalline structure and cubic cell parameters. In overall, the measured patterns confirmed an almost pure single phase sample with spinel structure, although a negligible admixture (up to 2 vol.%) of -phase could be noticed. The relative amounts of β-Si 3 N 4 and γ-Si 3 N 4 phases did not change after irradiation. The examination by Raman spectroscopy (Fig. 3) confirmed the previous conclusion about the sample purity. The Raman spectra of pristine -Si 3 N 4 (Fig. 3a) were dominated by two sharp intense bands at 524 and 847 cm -1 , in agreement with the earlier reports [START_REF] Gritsyna | Structure and electronic states of defects in spinel of different compositions MgOnAl2O3:Me[END_REF]. The higher frequency peak has been assigned to the T 2g asymmetric stretching mode of SiN 4 units and the lower frequency one to the E g deformation vibration of SiN 4 groups (also SiN 6 stretching/bending); the somewhat weaker sharp band at 982 cm -1 has been assigned to the symmetric A 1g stretching vibration [START_REF] Feldbach | Optical characteristics of virgin and proton-irradiated ceramics of magnesium aluminate spinel[END_REF]. In contrast, Raman spectra of the irradiated sample (Fig. 3b) were strongly modified, indicating structural damage induced by H + ions. Indeed, sharp E g , T 2g

-Si 3 N 4 (331) -Si 3 N 4 (301) -Si 3 N 4 (130) -Si 3 N 4 (201) -Si 3 N 4 (210) -Si 3 N 4 (101) -Si 3 N 4 (200) -Si 3 N 4 (110) -Si 3 N 4 (531) -Si 3 N 4 (220) -Si 3 N 4 (311) + -Si 3 N 4 (400) -Si 3 N 4 (422) -Si 3 N 4 (511) -Si 3 N 4 (440) -Si 3 N 4 (
and A 1g modes weakened and broad bands appeared, which may be interpreted as a partial amorphisation of the material. However, Raman intensities cannot serve as a quantitative measure of the material phase composition. In fact, the absence of any significant amount of the amorphous material on the irradiated area is supported by XRD pattern in Fig. 2. The identical backgrounds of the XRD patterns before and after irradiation indicated that the amount of an amorphous material, if present at all, is minimal. The latter conclusion is further strengthened by the fact that the XRD measurements were performed in the grazing geometry which strongly enhances contribution of surface layers to the Bragg reflections. The additional broad features around 300-450, 550-650 and 900-1050 cm -1 have been earlier attributed to N 3-vacancies in the spinel structure, or to Si-N stretching/bending vibrations of SiN 3 units, associated with the formation of N 3-vacancies [START_REF] Feldbach | Optical characteristics of virgin and proton-irradiated ceramics of magnesium aluminate spinel[END_REF], which can be defined as V N ''' defects in Kröger-Vink notation [START_REF] Lushchik | Creation and thermal annealing of structural defects in neutron-irradiated MgAl2O4 single crystals[END_REF]. The appearance of these negatively charged vacancies strongly indicates the creation of positively charged cation defects after the irradiation. A wealth of information about cation defects can be obtained from the spectral analysis of the materials. The low-temperature CL spectra of the pristine and irradiated -Si 3 N 4 samples are shown in Fig. 4. They contain two principal emission bands at 312 nm and 470 nm, which have been previously assigned to, respectively, self-trapped exciton and mixed contribution of exciton bound to neutral cation X * Si Si

x and metastable paramagnetic nitrogen anion radical N N '* [START_REF] Nishiyama | Transparent polycrystalline cubic silicon nitride[END_REF]. A strong decrease of the CL intensity was observed after irradiation with He + ions, which is common to the spinel materials: the UV band was found totally suppressed while the visible band was attenuated by a factor of ~25. According to the previous analysis [START_REF] Nishiyama | Transparent polycrystalline cubic silicon nitride[END_REF], longwave part of the visible band ( PL 500 nm) is formed by the bound exciton X * Si Si

x transitions and shortwave part ( PL 450 nm) by the anion radical N N '* . The spectrum of the pristine sample (a) in Fig. 4 corresponds to a stronger contribution of the anion radicals, which can be formed after an electron capture by neutral sites. The electron beam in CL measurements supplies electrons, hence favoring creation of such centers. The irradiation with positively charged ions can induce the suppression of such structural defect. Indeed, the spectrum in Fig. 4b indicates a significant attenuation of the shortwave part of this band compared to the longwave one, which, most probably, evidences this effect. The remaining luminescence belongs mostly to X * Si Si x centers. Resuming, we can conclude about the rapid quenching of excitons, which energy can be only partially transferred to the bound exciton luminescence.

The results of a SRIM (Stopping and Range of Ions in Matter) simulation indicated quite a high level of damage up to 5 dpa in the irradiated layer of 0.6 µm thickness (Fig. 5), which might signify the material amorphisation. Instead, a significant part of these displacements is expected to recombine, which results in a much smaller real concentration of various structural defects [START_REF] Spiridigliozzi | Photoluminescence of MgAl2O4 spinel ceramics doped with Ta2O5 and irradiated with He + ions[END_REF]. As CASINO calculations showed [START_REF] Perdew | Generalized Gradient Approximation Made Simple[END_REF], the penetration depth of 10 keV electron beam matches the damaged layer, making CL a valuable source of information about its electronic structure. On the other hand, XRD patterns and Raman spectra also contain information from deep non-damaged layers (d≥1 µm) of the crystal. In particular, x-ray and Raman laser radiation in their respective experimental geometries could penetrate to the depth of about 3-10 µm and, consequently, result in ~80% of the signal from the pristine sample behind the damage layer. Even though these analyses cannot be considered as exclusively relevant to the damaged material, they quantitatively showed almost no long-range structural modification and a considerable short-range perturbation around the radiation induced defects. More detailed information about the material electronic structure provide excited state-selective PL and PLE measurements at cryogenic temperatures. Since characteristic absorption coefficients at the interband and excitonic transitions are k>10 4 cm -1 , the penetration depth of the excitation photons in the UV spectral range d<1/k~1 µm corresponds to the penetration depth of He + ions and therefore supplies state-selective information from the damaged material. The PL spectra of pristine and irradiated -Si 3 N 4 sample sides are shown in Fig. 6. The emission of bound exciton X * Si Si

x with the maximum at 495 nm (2.51 eV) dominates the spectra of the pristine sample (Fig. 6a). In contrast to CL, this spectrum seems to be free of N N '* emission, in agreement with the e-beam induced centers formation. After the irradiation (Fig. 6b), its spectral maximum was red shifted to 525 nm (2.36 eV) by E=-150 meV and a new near-IR band appeared with the maximum at 725 nm (1.71 eV). The near-IR band may be connected to that previously reported in pristine -Si 3 N 4 sample at 860 nm (1.44 eV) tentatively assigned to Si Si '* center excited by the exciton dissociation on Si Si

x defect [START_REF] Nishiyama | Transparent polycrystalline cubic silicon nitride[END_REF]. Obviously, any modification of the Si site would provoke simultaneous modifications of the trapped exciton and near-IR luminescence spectra. Conclusively, both the trapped exciton PL band shift and the new near-IR band appearance could indicate a modification of the cation sub-lattice. We notice that He bubbles, which might be produced in the spinel structure after the He + ions irradiation would induce the spectral shift of the PL bands to higher energies, because of the dielectric relaxation of an emitting dipole in the vicinity of the excluded volume. PLE spectra of the pristine and irradiated -Si 3 N 4 sample sides are shown in Fig. 7. The pristine sample (Fig. 7a) is characterized by the interband transitions above E g =5.05 eV and direct exciton transitions at about 4.35 eV (285 nm). In contrast, the interband transitions vanish after the irradiation (Fig. 7b) and a new band appeared at ~3.9 eV (320 nm). The corresponding absorption band due to these new PLE transitions is most probable reason for the strong screening and disappearance of the self-localized exciton PL at 4.0 eV (310 nm). We also notice that the red wing of the excitonic band at 4.5 eV in our non-irradiate sample extends to the lower energies and is stronger compared to that in a previously studied pristine γ-Si 3 N 4 [START_REF] Nishiyama | Transparent polycrystalline cubic silicon nitride[END_REF], as respectively shown by solid and dotted lines in Fig. 7a. Moreover, the 3.9 eV photons excite the same PL band of the trapped exciton as via direct exciton transitions at 4.35 eV, as shown in pristine (Fig. 8a) and irradiated (Fig. 8b) samples, though the energetic position of the last one is red shifted by E=74 meV. This refers to different environments of the emitting center. We observed from PLE spectra that, after the irradiation, the interband transitions (h≥5.05 eV) were stronger attenuated than the excitonic transitions (4.35 eV), which is most probably connected to charged defects, which provoke an effective trapping and non-radiative recombination of the conduction band (CB) electrons and valence band (VB) holes. The strong inhibition of interband transitions is common for spinel materials [START_REF]Accelrys Software Inc, Material Studio Release Notes[END_REF][START_REF] Krukau | Influence of the exchange screening parameter on the performance of screened hybrid functionals[END_REF][31][START_REF] Hine | Supercell size scaling of density functional theory formation energies of charged defects[END_REF]. In contrast, for non-charged particles, such as excitons, the radiative decay could be much more probable. We adopted the following scheme of photoinduced processes in -Si 3 N 4 explaining the observed PL bands:

      Si X Si X tr * * ( PL =495 nm) (1) * * ' Si h Si X       ( PL =860 nm) (2)        Vs Si He Si Si Si Si , ( 3
)        Si He Si Si V Si ' ( 4
) ) ( ½ 2 ' ' ' g N V N N He N       (5)       Vs Vs Si X Si X * * ( PL =510 nm) (6)         Vs Si X Si h e * ' ( PL =530 nm) (7) ) ( * IR near Si X Vs      (8) * ) 1 ( ' '      V Vs Si Si e
( PL =725 nm) (9) where subscript Vs stands for structural vacancies inherent to the spinel structure. The processes ( 1)-( 2) occur in the pristine sample and those ( 3)-( 9) substitute ( 1)-( 2) in the irradiated layer. The collisions of He + ion with Si Si ionise (3) and displace cations to available structural vacancies Si Vs (4) by simultaneously producing compensating cation vacancies V Si '. The processes ( 6), ( 7) and ( 9) replace ( 1) and ( 2) in the PL activation, involving respectively Si Vs and Si Vs (1-) ' * defect states. According to our observations, in contrast to the pristine material, the exciton cannot excite the near-IR emission in the irradiated layer [START_REF] Chu | Allelectron GW quasiparticle band structures of group 14 nitride compounds[END_REF]. Moreover, red shifts of the trapped exciton PL are different after the band gap (Fig. 6b) and direct exciton (Fig. 8b) excitations. The explanation of these results will be given below based on the previous assignment of PL and PLE bands [START_REF] Nishiyama | Transparent polycrystalline cubic silicon nitride[END_REF] and correlations between their spectral shifts after the irradiation.

In fact, the formation of several cation defects Si Vs can be assumed after the irradiation, which can be shared between the tetrahedral (1/8 occupied) and octahedral (1/2 occupied) voids as depicted in Fig. 9 below. The energetic cost of permutations a and d (respectively b and c) may be different but they result in the similar cationic defect states in the tetrahedral (Si Vt and octahedral (Si Vo ) voids. These states are expected to be partially positively charged (0<<1) because of the local dielectric polarisation, which alters their energetic positions. In contrast, the permutations e and f within the same voids do not result in any appreciable energetic shift because of a short-range charge relaxation. The remaining vacancies V Si ' (partially negatively charged) weaken N N bonds favouring the formation of compensating V N ''' defects. Because of a higher energy cost, the permutations a-d (Fig. 9) are expected to take place under the ionized irradiation, while those e-f may appear in the material during the synthesis and, therefore, depend on the synthesis conditions.

Our experimental observation of different spectral shifts of the trapped exciton PL after the band gap [START_REF] Ching | Theoretical prediction of the structure and properties of cubic spinel nitrides[END_REF] and direct exciton (6) excitations suggests a significant energy gap between the cation defect states Si Vt and Si Vo . Consequently, one of these states can receive a CB electron forming the near-IR emitting state [START_REF] Boyko | Class of tunable wide band gap semiconductors γ-(GexSi1-x)3N4[END_REF]; alternatively, this state can attract exciton leading to the visible PL [START_REF] Ching | Theoretical prediction of the structure and properties of cubic spinel nitrides[END_REF]. Another cation state can effectively trap an exciton leading to the visible PL [START_REF] Mo | Interesting physical properties of the new spinel phase of Si3N4 and C3N4[END_REF], however cannot excite the near-IR emitting state [START_REF] Chu | Allelectron GW quasiparticle band structures of group 14 nitride compounds[END_REF]. If this supposition is true, the energetic positions of the visible and near-IR emission bands would be expected to correlate. Indeed, two PL spectra in Fig. 6b obtained with the band gap excitation manifest opposite shifts of the visible and near-IR bands:  near-IR = - VIS (where  VIS E 2 -E 1 60 meV). We notice that (in contrast to the relative bands shifts in the irradiated spinel) the absolute energy shifts of the visible and near-IR bands between pristine and irradiated samples are much different because of different natures of the cation sites. In contrast to the neutral cation defect state Si  , the charged defect Si Vs  suggests an additional energy decrease of the trapped exciton due to the charge-induced dipole interaction during its formation. On the other hand, the weak PL intensity after the band gap excitation (h≥E g ) could be explained by the dominant quenching channel relating the He + beam induced formation of charged cation defects (3):

      Vs Si Vs Si Si Si Si Si e , , ' (10)
The results of the theoretical calculations of the formation energies of the cation and anion vacancies in γ-Si 3 N 4 are shown in Fig. 10. The formation energy of cation vacancies is lower than that of anion vacancy, which is a known feature of the spinel lattice caused by the presence of a huge amount of structural stoichiometric cation vacancies. The energy needed for swapping the cation position between octahedral and tetrahedral voids was estimated to be 0.95 eV for the neutral vacancies, which confirmed the tendency in MgAl 2 O 4 spinel, where the formation energy for anti-sites defects is much smaller than any other point defect of the lattice [START_REF] Sickafus | Radiation tolerance of complex oxides[END_REF][START_REF] Smith | Dynamical simulations of radiation damage in magnesium aluminate spinel MgAl2O4[END_REF]. This low energy affords effective permutations of Si between the octahedral and tetrahedral positions during the irradiation by He + ions and, consequently, affects optical spectra of the spinel. The formation energy of Si defects (for the same charge) in the octahedral void are lower than that in the tetrahedral void, which can set the defect states ordering: E(Si Vt ) > E(Si Vo ). Although cation permutations within the same tetrahedral or octahedral voids, as depicted in Fig. 9 (e,f) might also result in some energy variation, they do not produce charged states; the same defect states are produced after permutations within a couple of voids to / to. Moreover, as our calculations showed, the new cation positions do not present local minima of energy and readily relax to the stable initial positions. Consequently, the permutations a-d (Fig. 9) between different voids could be expected to induce appreciable energy changes. In the opposite limit, we simulated cation permutations over a long distance after the irradiation. # For displacements, native 16c and 8b and perturbed 48f, 16d and 8a Wyckoff positions were chosen. For a convenience, the 56-atoms supercell was transformed (using VESTA program [START_REF] Momma | VESTA 3 for three-dimensional visualization of crystal, volumetric and morphology data[END_REF]) in a way to set the relevant Wyckoff positions in the cell center and the transformed cells were fully relaxed. Then, the 57 th Si atom was placed in this Wyckoff position, the cell was positively charged +4 in order to compensate for extra electrons, and the atomic position were additionally relaxed in a fixed cell. The big 448-atoms supercells, having enough place to consider the displacement of atoms over relatively long distances ~1 nm, made from transformed and optimized 56-atoms supercells were also modelled, and the results on similar defects in the big and small supercells were compared. These calculations showed that the most energetically favourable position for permuted atom is 48f and least favourable is tetrahedral 8a with the energy difference of 7.25 eV. The energy difference between structures with permuted atom into 48f and 8a Wyckoff positions in 2x2x2 supercells were 7.62 and 9.23 eV, depending on which atom 8b or 16c was removed. The permutation from "native" 16c cell position to 8a was energetically more favourable than that from 8b to 8a with the energy difference of 1.61 eV. The energy difference between structures with permutated atom into 48f and 16d Wyckoff positions was 1.32 eV and 1 eV for 57-atoms and 448-atoms supercell, respectively. The calculations showed that negative charging of Si Vs defect moves its energetic position downwards, which suggests a higher energy of Si Vs (1-) ' * defect level compared # Due to a low computational cost, the plane-wave basis energy cutoff was chosen to be 250 eV. The Monkhorst-Pack scheme k-points grid sampling was set at 2×2×2 and 1x1x1 for the Brillouin zone respectively for small and big supercells. The convergence parameters were conserved.

with Si Vs ' * . This could explain its weak interaction with exciton (Eq. ( 8)) and blue shift of the near-IR emission band (Eq. ( 9)). Based on the obtained results, an explanation of the defect related PL in the irradiated spinel crystal is the following. (i) The lower energy octahedral Si Vo site can receive CB electron e' and VB hole h  , which annihilate forming trapped exciton X * -Si Vo . The free exciton X * preferentially interacts with the energetically closelying (quasi-resonant) defect state, which is tetrahedral Si Vt , and annihilate forming trapped exciton X * -Si Vt . (ii) The visible PL shifts to lower energies (compared to that in pristine spinel) because the charged cation site Si Vt/Vo induces an additional energy lowering due to a charge-dipole interaction with the exciton. The energetic position of the exciton bound to Si Vo is lower than that bound to Si Vt . (iii) The near-IR PL band is formed after the e' localisation on Si Vo and, therefore, its energetic position correlates with that of the trapped exciton at the same Si Vo site. The Si Vt (1-) ' * state does not show up in PL since its energetic position is in the CB continuum of the spinel. The low energy of cation permutations may result in a non-negligible, from spectroscopic point of view, number density of structural defects in the pristine sample. Indeed, the number density of such structural defects as large as 10 20 cm -3 could be expected in the synthesised spinels [START_REF] Ball | Predicting lattice parameter as a function of cation disorder in MgAl2O4 spinel[END_REF], which may strongly affect PL spectra of even nonirradiated samples. Because of different intensities of the self-trapped exciton PL (4.35 eV) in different pristine spinel samples, as well as strong PL/PLE spectra modifications before and after irradiation, it can be assumed that natures of structural defects by the synthesis and irradiation different, as respectively depicted by permutations a-d and e-f in Fig. 9. This confirms Fig. 7a, which compares PLE spectra of the bound exciton in two different pristine γ-Si 3 N 4 samples: that from the present work (solid line) with non-appreciable PL of the self-trapped exciton absorbing at ~4 eV, that is in the range of the self-trapped exciton transitions, while that from Ref. [START_REF] Nishiyama | Transparent polycrystalline cubic silicon nitride[END_REF] (dotted line) with a strong PL of the selftrapped exciton did not show any absorption in this spectral range. In particular, the cation permutations e-f in Fig. 9 within a tetrahedral and/or octahedral site may be a reason of the local lattice deformation, which induced absorption in the range of the self-trapped exciton radiative transitions, making this PL weaker or even cancelled. Future molecular dynamics calculations will shed light on the addressed issue.

Conclusion

Defects states created by He + ions irradiation in a polycrystalline sample of -Si 3 N 4 synthesised via highpressure method were investigated. The defects analysis was based on previous assignment of the PL bands in the pristine material [START_REF] Nishiyama | Transparent polycrystalline cubic silicon nitride[END_REF], correlation of their spectral positions and theoretical calculations of the formation energies of cation and anion vacancies in the octahedral and tetrahedral voids.

We observed strong changes of cathodoluminescence, photoluminescence, photoluminescence excitation and Raman spectra after the irradiation, which can be interpreted as a partial disorder due to the formation of V N ''' anion vacancies and permutation of cations between filled Si Si and empty V Vs structural sites in the octahedral and tetrahedral voids.

The visible PL of the trapped exciton after the irradiation was significantly inhibited and red shifted and a new near-IR band appeared with the band gap excitation h≥E g =5.05 eV, which is connected to the formation of the octahedral void defect Si Vo . After the direct exciton excitation, the visible PL was explained by the formation of exciton bound to the tetrahedral void defect Si Vt , which position is energetically higher than that of Si Vo . The near-IR and visible PL bands are formed after the interaction of respectively e' and exciton with the common Si Vo site and their energetic positions correlate. In contrast, the tetrahedral site Si Vt does not show up in PL of the near-IR spectral region, because its energetic position is out of the spinel band gap. The local deformation of the spinel lattice may induce absorption in the spectral range of the selftrapped exciton transitions, which attenuates or even cancels the respective PL.

Fig. 1 .

 1 Fig. 1. View of -Si3N4 polycrystalline sample in a stainless steel ring mounting.

Fig. 2 .

 2 Fig. 2. X-ray diffraction patterns of -Si3N4 before (blue) and after (magenta) irradiation with He + ions. Intensities of Al-support peaks are different because of different illuminated areas after the sample rotation.

Fig. 3 .

 3 Fig. 3. Raman spectra of -Si3N4 before (a) and after (b) irradiation with He + ions.

Fig. 4 .

 4 Fig. 4. CL spectra of -Si3N4 at T=5 K before (a) and after (b) irradiation with He + ions.

Fig. 5 .

 5 Fig. 5. Damage level induced by the He + ions (maximum value ~4-5 dpa) and normalised number of CL electrons (10 keV) in -Si3N4 sample.

Fig. 6 .

 6 Fig.6. PL spectra of -Si3N4 at T=9 K before (a) and after (b) irradiation with He + ions, with excitation at 5.39 eV (230 nm) (1) and 6.89 eV (180 nm) (2); E1=150 meV and E2=210 meV. The energy shifts of the visible and near-IR bands are opposite: near-IR -VIS (where VIS  E2 -E1  60 meV).

Fig. 7 .

 7 Fig.7. PLE spectra at PL=500 nm of -Si3N4 at T=9 K before (a) and after (b) irradiation with He + ions; dotted line shows PLE spectrum of the trapped exciton from Ref[START_REF] Nishiyama | Transparent polycrystalline cubic silicon nitride[END_REF].

Fig. 8 .

 8 Fig. 8. PL spectra of -Si3N4 at T=9 K before (a) and after (b) irradiation with He + ions; excitation wavelengths are indicated; the spectral shift by E=74 eV is indicated.

Fig. 9 .

 9 Fig. 9. Possible permutations of a cation between tetrahedral (∆) and octahedral (□) voids resulting in formation of defects VSi (○) and SiVs (•).

Fig. 10 .

 10 Fig. 10. Calculated trends in formation energy of charged cation (Si) and anoin (N) vacancies in the spinel structure of -Si3N4.

Acknowledgments

This work has been carried out within the framework of the EUROfusion Consortium and has received funding from the Euratom research and training programme 2014-2018 and 2019-2020 under grant agreement No 633053. The views and opinions expressed herein do not necessarily reflect those of the European Commission. The authors are indebted to Norimasa Nishiyama for providing the sample.


D Kassis

Jl Gueddouri

Paul

Vanessa Baloula

Marta Fructuoso

Nadim Kassis

Dalale Gueddouri

Jean-Louis Paul

Nathalie Janel
email: janel@univ-paris-diderot.fr

Homocysteine-lowering gene therapy rescues signaling pathways in brain of mice with intermediate hyperhomocysteinemia

Keywords: Hyperhomocysteinemia DYRK1A AAV Brain RTK pathway NFkB AD, Alzheimer's disease, AAV, adeno-associated viral, BDNF, brain-derived neurotrophic factor, CBS, Cystathionine beta-synthase, hcy, homocysteine, hhcy, hyperhomocysteinemia, HPLC, high-performance liquid chromatography, NMDA, N-methyl-D-aspartate, NMDAR, NMDA receptor, PI3K, Phosphatidylinositol 3-kinase, PP2A, Protein phosphatase 2A

published or not. The documents may come

Homocysteine-lowering gene therapy rescues signaling pathways in brain of mice with intermediate hyperhomocysteinemia

V Baloula, M Fructuoso, N

Introduction

Cystathionine beta-synthase (CBS) deficiency is an inborn error of sulfur amino acid metabolism. CBS operates at the intersection of the transmethylation, transsulfuration, and remethylation pathways. CBS catalyzes the conversion of homocysteine (hcy), an intermediate amino acid derived from the metabolism of dietary methionine, to cystathionine in the first step of the transsulfuration pathway. Cystathionine can be converted to cysteine, thus CBS is critical for the regulation of methionine and cysteine metabolism [START_REF] Mudd | Disorders in transsulfuration[END_REF].

Elevated plasma hcy, or hyperhomocysteinemia (hhcy), is categorized by range as moderate (15-30 μM), intermediate (30-100 μM), and severe (above 100 μM). The most common form of severe hhcy is that by CBS deficiency. Patients with CBS deficiency suffer from numerous clinical complications including not only vascular disease, ectopia lentis, osteoporosis, fatty liver but also cognitive dysfunctions such as intellectual disabilities, seizures and psychiatric disorders including personality disorder, anxiety, depression, obsessive-compulsive behavior, and psychotic episodes [START_REF] Mudd | Disorders in transsulfuration[END_REF][START_REF] Yap | The intellectual abilities of earlytreated individuals with pyridoxine-nonresponsive homocystinuria due to cystathionine beta-synthase deficiency[END_REF]. Hhcy is also a risk factor for neurodegenerative diseases such as Alzheimer's disease (AD), even if hhcy is moderate [START_REF] Hooshmand | Plasma Homocysteine, Alzheimer and cerebrovascular pathology: a population-based autopsy study[END_REF]. Moreover, elevated hcy level has been associated with neuropsychiatric disorders such as schizophrenia, depression, bipolar disorder, and autism [START_REF] Kinoshita | Plasma total homocysteine is associated with DNA methylation in patients with schizophrenia[END_REF][START_REF] Nabi | Association of serum homocysteine with major depressive disorder: results from a large population-based study[END_REF][START_REF] Vuksan-Cusa | Association between C-reactive protein and homocysteine with the subcomponents of metabolic syndrome in stable patients with bipolar disorder and schizophrenia[END_REF][START_REF] Józefczuk | Homocysteine as a Diagnostic and Etiopathogenic Factor in Children with Autism Spectrum Disorder[END_REF]. We showed that hhcys is accompanied by alterations in signaling pathways initiated by receptor tyrosine kinases (RTK), the ERK and Phosphatidylinositol 3-kinase (PI3K)/Akt pathways, and brain-derived neurotrophic factor/TrkB signaling pathway, in brain of CBS deficient mice, a murine model of hhcy [START_REF] Watanabe | Mice deficient in cystathionine beta-synthase: animal models for mild and severe homocyst(e)inemia[END_REF][START_REF] Robert | The neuronal JNK MAP kinase pathway is altered in a murine model of hyperhomocysteinemia[END_REF][START_REF] Robert | Regulation of extracellular signal-regulated kinase by homocysteine in hippocampus[END_REF][START_REF] Abekhoukh | Dyrk1A, a serine/threonine kinase, is involved in ERK and Akt activation in brain of hyperhomocysteinemic mice[END_REF][START_REF] Souchet | Molecular rescue of DYRK1A overexpression in cystathionine beta synthase-deficient mouse brain by enriched environment combined with voluntary exercise[END_REF].

We also demonstrated the relationship between increased DYRK1A level and these signaling pathways in brain of CBS deficient mice [START_REF] Abekhoukh | Dyrk1A, a serine/threonine kinase, is involved in ERK and Akt activation in brain of hyperhomocysteinemic mice[END_REF][START_REF] Souchet | Molecular rescue of DYRK1A overexpression in cystathionine beta synthase-deficient mouse brain by enriched environment combined with voluntary exercise[END_REF][START_REF] Planque | Mice deficient in cystathionine beta synthase display increased Dyrk1A and SAHH activities in brain[END_REF]. DYRK1A, for dual specificity tyrosine-phosphorylation-regulated kinase 1A, is a serine/threonine kinase that, when overexpressed in brain, contributes to the neurodegeneration, neuronal death, and loss of function observed in multiple neurodegenerative diseases [START_REF] Wegiel | The role of DYRK1A in neurodegenerative diseases[END_REF]. These results suggest that those could be some of the molecular mechanisms linking hhcy to these neuronal abnormalities. In this line, we observed that enriched environment combined with voluntary exercise enriched environment combined with voluntary exercise restored the altered levels of DYRK1A and brain-derived neurotrophic factor (BDNF), a neurotrophin contributing to hippocampusdependent forms of learning and memory, in hippocampus of CBS deficient mice [START_REF] Souchet | Molecular rescue of DYRK1A overexpression in cystathionine beta synthase-deficient mouse brain by enriched environment combined with voluntary exercise[END_REF]. However, no effect was found on plasma hcy level.

Treatment strategies for CBS deficiency generally focus on ways to lower hcy in patients, as this seems to be the best predictor of clinical severity. As the central organ of metabolism, many metabolic diseases originate in the liver. However, clinical manifestations can be extrahepatic. In the case of hhcy, the liver plays a central role in the metabolism of methionine and contributes the high levels of plasma hcy. Recently, we were able to lower plasma hcy levels in mice with moderate and intermediate hhcy using an adenoviral construct designed to restrict the expression of DYRK1A, involved in methionine metabolism (and therefore homocysteine production) [START_REF] Noll | DYRK1A, a novel determinant of the methionine-homocysteine cycle in different mouse models overexpressing this Down-syndrome-associated kinase[END_REF], to hepatocytes [START_REF] Tlili | Hepatocytespecific Dyrk1a gene transfer rescues plasma apolipoprotein A-I levels and aortic Akt/GSK3 pathways in hyperhomocysteinemic mice[END_REF][START_REF] Latour | Corrective effects of hepatotoxicity by hepatic Dyrk1a gene delivery in mice with intermediate hyperhomocysteinemia[END_REF]. Interestingly, we found that targeted hepatic expression of DYRK1A abolished the increased DYRK1A level in brain of intermediate hhcy mice, which may be the result of hcy lowering [START_REF] Latour | Corrective effects of hepatotoxicity by hepatic Dyrk1a gene delivery in mice with intermediate hyperhomocysteinemia[END_REF].

However, since this adenoviral construct is not suitable for human research, we decided to proceed with an adeno-associated viral (AAV) vector to gain insight about brain molecular readout of DYRK1A overexpression as a hcy lowering therapy. For this, we here used a hepatocyte-specific recombinant AAV serotype 8-mediated Dyrk1A gene transfer (AAV2/8-DYRK1A), AAV serotype 8 having high affinity for hepatocytes without inducing any significant innate immune response. The central nervous system effects of the Dyrk1A gene transfer in hhcy mice were evaluated through the level and activity of proteins related to neuropathological and inflammatory processes.

Materials and methods

Construction, generation, and production of AVV2/8-DYRK1A

Plasmid pEGFP-DYRK1A containing the 2.6 kb rat Dyrk1a cDNA sequence [START_REF] Becker | Structural and functional characteristics of Dyrk, a novel subfamily of protein kinases with dual specificity, Progress[END_REF] was used by the Therapeutic Research Institute (Institut de Recherche en Santé de l'Université de Nantes (IRS-UN), Centre de Production de Vecteurs viraux (CPV), Institut National de la santé et de la recherche médicale (INSERM), Unité Mixte de Recherche (UMR) 1089, Nantes, France), to generate and purify AAV2/8-DYRK1A. The expression cassette of this vector consists of the human α1-antitrypsin promoter and one copy of a human apolipoprotein hepatic control region, upstream of the bovine growth hormone polyadenylation signal. Final viral preparations were stored in Dulbecco's Phosphate-Buffered Saline (DPBS) (with Ca2 + and Mg2 +), aliquoted in small volumes (50 µL), and stored at -80 °C. Particle titer (number of viral genomes) was determined by quantitative PCR poly A. Production and purification processes by this technological platform are consistent with production requirements of clinical grade vectors [START_REF] Nathwani | Adenovirus-associated virus vector-mediated gene transfer in hemophilia B[END_REF].

Experimental animals

Mice heterozygous for targeted disruption of the Cbs gene (Cbs +/-) were generously donated by Dr. N. Maeda (Department of Pathology, University of North Carolina, Chapel Hill, NC, USA) [START_REF] Watanabe | Mice deficient in cystathionine beta-synthase: animal models for mild and severe homocyst(e)inemia[END_REF]. Cbs +/-mice, on a C57BL/6 background, were obtained by mating male Cbs +/-mice with female wild-type C57BL/6 (Cbs +/+ ) mice. DNA isolated from tail biopsies of 4-week-old mice was subjected to genotyping of the targeted Cbs allele using polymerase chain reaction (PCR) [START_REF] Watanabe | Mice deficient in cystathionine beta-synthase: animal models for mild and severe homocyst(e)inemia[END_REF]. Mice were maintained in a controlled environment with unlimited access to food and water on a 12 h light/dark cycle. Mice were fed a standard laboratory diet (CRM, Special Diets Services, Dietex, France Usine). This diet has a protein content of 19%, a methionine content of 2.700 mgKg -1 , a folic acid content of 4.41 mgKg -1 , and a vitamin B 12 content of 0.082 mgKg -1 . Number of mice and suffering were minimized as possible. Before the experiments and to induce intermediate hhcy the same number of male and female Cbs +/-mice of 3 months of age were maintained for three months on the standard diet supplemented with 0.5% L-methionine (Sigma-Aldrich, France) in drinking water.

We were not powering for rigourously testing sex differences, but preliminary analysis showed no differences for plasma hcy level. Therefore, here we combined data from male and female. Cbs +/-mice and mice with intermediate hhcy (Cbs +/-mice having the methionine enriched diet) were divided into two groups for the last month, with one group (hhcy/AAV) receiving injection in the retro-orbital sinus with the AAV2/8-DYRK1A to have 2 × 10 12 viral particles/kg body weight and the second group receiving an equivalent dose of saline buffer. Control mice (CTL), healthy control Cbs +/+ mice also received injection in the retro-orbital sinus with the AAV2/8-DYRK1A or an equivalent dose of saline buffer and were used as references to monitor hyperhomocysteinemic development.

All procedures were carried out in accordance with the ethical standards of French and European regulations (European Communities Council Directive, 86/609/EEC). Official authorization from the French Ministry of Agriculture was granted to perform research and experiments on animals (authorization number 75-369) and the experimental protocol was approved by the institutional animal care and use committee of the Paris Diderot University (CEEA40).

Preparation of serum samples, tissue collection, and plasma assays

Mice were anesthetized by Ketamine/Xylazine intraperitoneal injection, and blood samples were obtained by retro-orbital sinus sampling with heparinized capillaries, collected into tubes containing a 1/ 10 vol of 3.8% sodium citrate, and immediately placed on ice. Plasma was isolated by centrifugation at 2500 × g for 15 min at 4 °C. Livers and brains were harvested, snap-frozen, and stored at -80 °C until use. Plasma total hcy, defined as the total concentration of hcy after quantitative reductive cleavage of all disulfide bonds, was assayed using the fluorimetric high-performance liquid chromatography (HPLC) method as previously described [START_REF] Ducros | Society for Clinical Biology, Methods for homocysteine analysis and biological relevance of the results[END_REF]. The DYRK1A levels were assessed by a solid phase immobilized epitope immunoassay, as described [START_REF] Janel | Combined assessment of DYRK1A, BDNF and homocysteine levels as diagnostic marker for Alzheimer's disease[END_REF].

Protein extraction and analysis

Total protein samples were prepared by homogenizing liver and brain in 500 µL phosphate-buffered saline with a cocktail of proteases inhibitors. Cytoplasmic and nuclear proteins were obtained by using the NE-PER Nuclear Protein Extraction Kit (Thermo Scientific). Protein concentrations were detected with the Bio-Rad Protein Assay reagent (Bio-Rad). To assess the relative amount of proteins, we used a slot blot method after testing the specificity of antibodies by western blotting. Protein preparations were blotted on Hybond-C Extra membrane (GE Healthcare Europe GmbH) using Bio-Dot SF Microfiltration Apparatus (Bio-Rad). After transfer, membranes were saturated by incubation in 10% w/v nonfat milk powder or 5% w/v BSA in Tris-saline buffer (1.5 mM Tris base, pH 8; 5 mM NaCl; 0.1% Tween-20), and incubated overnight at 4 °C with an antibody directed against Akt1/ 2/3 (1/1000; Santa Cruz Biotechnology, Tebu, France), Phospho-Akt1/2/3 (Ser 473; 1/1000; Santa Cruz Biotechnology, Tebu, France), p44/42 MAPK (ERK1/2)(137F5) (1/2000; Cell Signaling, Ozyme, France), Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1/2000; Cell Signaling, Ozyme, France), IkBα (1/1000, Cell Signaling Technology), P-IkBα (Ser32) (1/1000, Cell Signaling Technology), Calpain 2 Domain I (1/ 5000, abcam, France), Calpastatin (1/1000, Cell Signaling Technology), DYRK1A (1/500, Abnova corporation, Tebu, France), NFkB p65 (1/ 2000, abcam, France). Binding of the primary antibody was detected by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody using the Western Blotting Luminol Reagent (Santa Cruz Biotechnology, Tebu, France). Ponceau-S coloration (Sigma-Aldrich, France) was used as an internal control. Digitized images of the immunoblots obtained using LAS-3000 imaging system (Fuji Photo Film Co., Ltd.) were used for densitometric measurements with an image analyzer (UnScan It software, Silk Scientific Inc.).

Enzyme activity assays

CBS activity assay was performed on 300 µg of protein extracts as described previously [START_REF] Miller | Folate-deficiency-induced homocysteinaemia in rats: disruption of S-adenosylmethionine's co-ordinate regulation of homocysteine metabolism[END_REF]. Proteins were incubated for 1 h at 37 °C with 1 mM DL-propargylglycine, 0.2 mM pyridoxal 5'-phosphate, 10 mM L- serine, 10 mM DL-Hcy, and 0.8 mM S-(5'-adenosyl)-L-methionine, using 5,5'-dithiobis-(2-nitrobenzoic acid) based-assay. The reaction was performed at 37 °C by measuring the absorbance at 412 nm over 10 min, using a spectrophotometer (Lambda XLS, PerkinElmer). Calpain activity was measured using the fluorogenic peptide N-Succinyl-Leu-Tyr-7-Amido-4-Methylcoumarin as described by Kohli et al. [START_REF] Kohli | Calpain is a mediator of preservationreperfusion injury in rat liver transplantation[END_REF]. Briefly, 60 µg of brain extract in a final volume of 40 µL was added to 160 µL of 50 µM N-Succinyl-Leu-Tyr-7-Amido-4-Methylcoumarin dissolved in dimethyl sulfoxide and Tris buffer (100 mM Tris-HCl, 145 mM NaCl at pH 7.3). Proteolysis of the substrate was monitored for 21 min at room temperature with a FlexStation3 multi-mode microplate reader (excitation: 380 nm, emission: 460 nm; Molecular Devices) either in presence of 10 mM Ca 2+ or 10 mM EGTA to determine calcium-independent activity, thus excluding cathepsin activity. Protein phosphatase 2 A (PP2A) activity assay was performed using the PP2A immunoprecipitation phosphatase assay kit (Millipore) using 5 mg of protein extract as described by the manufacturer.

BDNF protein analysis

BDNF protein levels were measured in the brain using the BDNF EMax Immunoassay (ELISA E-Max, Promega, Madison, WI, USA). Protein preparations were incubated on a 96-well polystyrene ELISA plate previously coated with anti-BDNF monoclonal antibody. A standard curve was generated from serial dilutions of a human recombinant BDNF solution at 1 μg/mL. The captured neurotrophin was bound by a second specific anti-human BDNF polyclonal antibody, which was detected using a species-specific antibody conjugated to horseradish peroxidase (HRP). After removal of unbound conjugates, bound enzyme activity was assessed by chromogenic substrate for measurement at 450 nm by a microplate reader (Flex Station3, Molecular Device). All assays were performed in duplicate.

RNA extraction, cDNA synthesis, and real-time PCR using SYBR-Green chemistry

Total RNA was isolated from the brain and liver using RNeasy Lipid kit (Qiagen). The concentration of RNA samples was ascertained by measuring optical density at 260 nm. The quality of RNA was verified by optical density absorption ratio OD 260 nm/OD 280 nm. To remove residual DNA contamination, the RNA samples were treated with RNAse-free DNAse (Qiagen) and purified with an RNeasy mini column (Qiagen). For each sample, 4 µg of total RNA from each sample was reverse transcribed with 200 U of M-MLV Reverse Transcriptase (Invitrogen, Life Technologies) using random hexamer primers. Real time quantitative PCR amplification reactions were carried out in a LightCycler 480 detection system (Roche) using the LightCycler FastStart DNA Master plus SYBR Green I kit (Roche). Primer sequences are 5' TCAGTCTTCAGGCACCACCT 3' (sense primer) and 5' TGTTACTCG TTCCCGAGGAT 3' (antisense primer) for DYRK1A. For each reaction, 40 ng of reverse transcribed RNA was used as template. All reactions were carried out in duplicate with a no template control. The PCR conditions were: 95 °C for 5 min, followed by 45 cycles of 95 °C for 10 s, 60 °C for 10 s and 72 °C for 10 s. The mRNA transcript level was normalized against the mean of two genes: H1a and rpL19. Primer sequences are 5' AGAAGAACAACAGCCGCATC3' (sense primer) and 5' TGCACCAGTGTGCCTTTATT 3' (antisense primer) for H1a, and 5' GGGC AGGCATATGGGCATA 3' (sense primer) and 5' GGCGGTCAATCTTCTTG GATT 3' (antisense primer) for rpL19. To compare the target gene level, relative quantification was performed as outlined in Pfaffl et al. [START_REF] Pfaffl | Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in realtime PCR[END_REF].

Data analysis

For multiple pairwise comparisons between genotypes and treatments, statistical analysis was done with two-way ANOVA followed by Fisher's post hoc test using Statview software. The results are expressed as mean ± SEM (standard error of the mean). Data were considered significant when p < 0.05. Correlations were determined by using Spearman's rank correlation, as data were not normally distributed according to Shapiro-Wilk test.

Results

Effect of AAV2/8-DYRK1A gene transfer on plasma hcy level in mice with hhcy

Similar to previous reports [START_REF] Tlili | Hepatocytespecific Dyrk1a gene transfer rescues plasma apolipoprotein A-I levels and aortic Akt/GSK3 pathways in hyperhomocysteinemic mice[END_REF][START_REF] Latour | Corrective effects of hepatotoxicity by hepatic Dyrk1a gene delivery in mice with intermediate hyperhomocysteinemia[END_REF][START_REF] Hamelet | Effect of hyperhomocysteinemia on the protein kinase DYRK1A in liver of mice[END_REF], mean DYRK1A protein level was decreased in the liver of Cbs +/-mice (Fig. 1A) and mice with intermediate hhcy (Fig. 1B) when compared with control mice. However, after injection of AAV2/8-DYRK1A, we observed an increase in DYRK1A protein level when compared with mice without treatment. The mRNA expression of DYRK1A was also examined by Q-PCR and, as previously described [START_REF] Hamelet | Effect of hyperhomocysteinemia on the protein kinase DYRK1A in liver of mice[END_REF], even if the difference is not significant, the mRNA expression was increased in liver of mice with intermediate hhcy (hhcy), when compared with control (CTL) mice (Table 1). Moreover, the increased expression of protein DYRK1A was confirmed by Q-PCR analysis in liver of mice with intermediate hhcy after injection of AAV2/8-DYRK1A (hhcy/AAV) (Table 1). We therefore determined the effect of AAV2/8-DYRK1A on plasma hcy levels, and found that control mice, Cbs +/-mice (Fig. 2A) and mice with hhcy (Fig. 2B) had significantly lower plasma hcy level compared to mice without treatment. We interestingly observed an increased level of plasma DYRK1A in mice with intermediate hhcy after AAV2/8-DYRK1A injection (Fig. 2C).

Effect of AAV2/8-DYRK1A gene transfer on DYRK1A protein level and CBS activity in brain of mice with intermediate hhcy

Similar to our previous reports [START_REF] Souchet | Molecular rescue of DYRK1A overexpression in cystathionine beta synthase-deficient mouse brain by enriched environment combined with voluntary exercise[END_REF][START_REF] Planque | Mice deficient in cystathionine beta synthase display increased Dyrk1A and SAHH activities in brain[END_REF], mean DYRK1A protein level was increased in brain of mice with intermediate hhcy when compared with control (CTL) mice (Fig. 3A). However, the mRNA expression of DYRK1A was not changed in brain of mice with intermediate hhcy (hhcy) when compared with control (CTL) mice (Table 1), as previously described [START_REF] Planque | Mice deficient in cystathionine beta synthase display increased Dyrk1A and SAHH activities in brain[END_REF]. Furthermore, after injection of AAV2/8-DYRK1A, we observed a decrease in DYRK1A protein level when compared with mice with hhcy and without treatment (hhcy) (Fig. 3A), but without effect on mRNA expression of DYRK1A (Table 1). Taken together, the results on brain DYRK1A protein and mRNA levels also emphasize the liver specific expression after AAV2/8-DYRK1A injection. Moreover, treatment can also increase the CBS activity in brain of mice with hhcy (Fig. 3B).

Effect of AAV2/8-DYRK1A gene transfer on pathways initiated by RTK in brain of mice with intermediate hhcy

PI3K can become activated by at least three independent pathways, all of which start with binding of ligand to RTK. Activation of Ras can coordinate activation of PI3K and Raf/Mek/Erk. Therefore, Akt and ERK activation was analyzed using phospho-specific antibodies. Phospho-Akt (P-Akt) and phospho-ERK (P-ERK) levels were increased in brain of mice with intermediate hhcy compared to control mice, as previously described [START_REF] Abekhoukh | Dyrk1A, a serine/threonine kinase, is involved in ERK and Akt activation in brain of hyperhomocysteinemic mice[END_REF] (Fig. 4). However, after injection of AAV2/8-DYRK1A, we observed reduced Phospho-Akt and phospho-ERK levels when compared with mice with intermediate hhcy and without treatment (Fig. 4).

Effect of AAV2/8-DYRK1A gene transfer on BDNF level in brain of mice with intermediate hhcy

BDNF levels were decreased in brain of mice with intermediate hhcy compared to control mice, as previously described [START_REF] Souchet | Molecular rescue of DYRK1A overexpression in cystathionine beta synthase-deficient mouse brain by enriched environment combined with voluntary exercise[END_REF] (Fig. 5). Following AAV2/8-DYRK1A injection, BDNF protein levels significantly increased when compared with mice with intermediate hhcy and without treatment (Fig. 5).

Effect of AAV2/8-DYRK1A gene transfer on NFkB pathway in brain of mice with intermediate hhcy

To determine whether the NF-kB pathway is activated in brain of mice with intermediate hhcy, we studied the level of IkBα and found a decrease compared to control (CTL) mice (Fig. 6A). The first possible way for IκBα degradation implicates the balance of IKKβ /PP2A. We quantified the ratio of phospho-IκBα/IκBα (Fig. 6B) and PP2A activity (Fig. 6C) and found an increase and a decrease respectively compared to control (CTL) mice. Following AAV2/8-DYRK1A injection, IkBα protein levels significantly increased when compared with mice with intermediate hhcy and without treatment, with a decrease of phospho-IκBα/IκBα ratio but no difference for PP2A activity (Fig. 6).

IkB inhibits NFkB by sequestering the NFkB p65 subunit in the cytoplasm. Into the nucleus, p65 provides the activation of the transcription factor complex. We therefore analyzed the subcellular p65 localization in brain of mice and found a decrease in the cytoplasmic cortex fraction (Fig. 7A) but an increase in the cytoplasmic hippocampus fraction (Fig. 7B) of mice with intermediate hhcy compared to control (CTL) mice. The nuclear p65 fraction was increased in cortex and hippocampus of mice with intermediate hhcy (Figs. 7C and7D). However, after injection of AAV2/8-DYRK1A, we observed reduced nuclear p65 levels both in cortex and hippocampus when compared with mice with intermediate hhcy and without treatment (Figs. 7C and Fig. 6. Effect of AVV2/8-DYRK1A gene transfer on brain IkBα degradation. A) IkBα protein level, B) Ser32-phosphorylated IkBα and C) PP2A activity in brain of Cbs +/-mice with intermediate hyperhomocysteinemia (hhcy) with (hhcy/AAV) or without (hhcy) AAV2/8-DYRK1A treatment, and control (CTL) mice. Proteins were subjected to slot blot analysis using antibodies specific to P-IkBα. After stripping, the membranes were reprobed with anti IkBα antibody. Relative protein expression was determined by normalization of the density of images from P-IkBα with that of IkBα of the same blot. Values of IkBα were obtained by normalization of images from IkBα to total proteins colored with Ponceau-S. PP2A activity was determined by immunoprecipitation phosphatase assay as described in material and methods Data were normalized to the mean of CTL mice. The results are represented as means ± SEM. n = number of mice.

7D).

The second possible way for IκBα degradation implicates the balance of calpain/calpastatin. We found an increased calpain activity (Fig. 8A), with a decreased calpastatin level (Fig. 8B), in brain of mice with intermediate hhcy. Following AAV2/8-DYRK1A injection, calpain activity significantly decreased (Fig. 8A) when compared with mice with intermediate hhcy and without treatment, with an increase of calpastatin level (Fig. 8B). We also analyzed the protein level of calpain 1 and calpain 2, the two major calpain isoforms in the brain. Calpain are calcium-dependent proteases, with a large subunit containing domains I-IV. No difference was found for domain I, the propeptide domain, of calpain 1, nor for domain IV, a Ca2 + -binding domain, of calpain 1 and 2 (data not shown). However, an increase of calpain 2 domain I protein level was found in brain of mice with intermediate hhcy after AAV2/8-DYRK1A injection compared to control and mice with intermediate hhcy (Fig. 8C).

Discussion

High levels of plasma hcy have been linked to the development of neurological disorders associated with neuronal loss such as AD, vascular dementia or mild cognitive impairment [START_REF] Seshadri | Plasma homocysteine as a risk factor for dementia and Alzheimer's disease[END_REF][START_REF] Haan | Homocysteine, B vitamins, and the incidence of dementia and cognitive impairment: results from the Sacramento Area Latino Study on Aging[END_REF]. We found a positive correlation between plasma hcy level and brain DYRK1A protein level (r = 0.278, p < 0.05), as well as a negative correlation between brain CBS activity and plasma hcy (r = -0.409, p < 0.03). We have previously produced moderate and intermediate hyperhomocysteinaemia in mice in order to monitor the levels of hepatic CBS activity and found that not only methionine enriched diet increased plasma hcy levels but also decreased hepatic CBS activity in Cbs +/-mice [START_REF] Hamelet | Homocysteine threshold value based on cystathionine beta synthase and paraoxonase 1 activities in mice, EUr[END_REF]. All together, these results underline the effect of intermediate hhcy on brain protein DYRK1A level and CBS activity. We showed that increasing hepatic expression of DYRK1A in mice with intermediate hhcy results in plasma hcy lowering. The decrease brain DYRK1A level and the increase brain CBS activity would result from plasma hcy lowering [START_REF] Latour | Corrective effects of hepatotoxicity by hepatic Dyrk1a gene delivery in mice with intermediate hyperhomocysteinemia[END_REF].

The study on transgenic mice overexpressing DYRK1A [START_REF] Abekhoukh | Dyrk1A, a serine/threonine kinase, is involved in ERK and Akt activation in brain of hyperhomocysteinemic mice[END_REF] established a relationship between DYRK1A protein and RTK-mediated signaling pathways such as MAPK/ERK and PI3K/Akt pathways as well as the BDNF-dependent TrkB pathway in brain of mice [START_REF] Guedj | Green tea polyphenols rescue of brain defects induced by overexpression of DYRK1A[END_REF]. We confirmed the activation of ERK and Akt, as well as the decreased BDNF content in brain of mice with hhcy [START_REF] Abekhoukh | Dyrk1A, a serine/threonine kinase, is involved in ERK and Akt activation in brain of hyperhomocysteinemic mice[END_REF][START_REF] Souchet | Molecular rescue of DYRK1A overexpression in cystathionine beta synthase-deficient mouse brain by enriched environment combined with voluntary exercise[END_REF][START_REF] Matté | Acute homocysteine administration impairs memory consolidation on inhibitory avoidance task and decreases hippocampal brain-derived neurotrophic factor immunocontent: prevention by folic acid treatment[END_REF]. Positive correlations were found between brain DYRK1A protein level and ERK and Akt activation (r = 0.601, p < 0.0001). We previously found decreased activation of ERK and Akt pathways associated with decreased DYRK1A protein level in liver of mice with hhcy [START_REF] Latour | Corrective effects of hepatotoxicity by hepatic Dyrk1a gene delivery in mice with intermediate hyperhomocysteinemia[END_REF][START_REF] Hamelet | Effect of hyperhomocysteinemia on the protein kinase DYRK1A in liver of mice[END_REF][START_REF] Hamelet | Inhibition of extracellular signalregulated kinase in liver of hyperhomocysteinemic mice[END_REF]. The observed negative correlation between plasma hcy level and brain BDNF level (r = -0.444, p < 0.004) suggest a negative effect of hhcy on BDNF/TrkB signaling pathway, as previously described [START_REF] Souchet | Molecular rescue of DYRK1A overexpression in cystathionine beta synthase-deficient mouse brain by enriched environment combined with voluntary exercise[END_REF]. Taken together, our It has been shown in primary cortical neurons that glutamate-induced triggering of the ionotropic N-methyl-D-aspartate (NMDA) receptor (NMDAR) was required for the enhanced and persistent Pi3k/ Akt-dependent NF-kappa B activation by the 75 kDa tumor necrosis factor receptor (TNFR2), indicating a positive cooperation of TNF and neurotransmitter-induced signal pathways [START_REF] Marchetti | Tumor necrosis factor (TNF)-mediated neuroprotection against glutamate-induced excitotoxicity is enhanced by N-methyl-D-aspartate receptor activation. Essential role of a TNF receptor 2-mediated phosphatidylinositol 3-kinase-dependent NF-kappa B pathway[END_REF]. We previously demonstrated that both mGluRs, NMDAR and non-NMDAR, cooperatively mediate hcy-induced ERK activation, indicating complex cellular mechanisms that render the hippocampus sensitive to hcy [START_REF] Robert | Regulation of extracellular signal-regulated kinase by homocysteine in hippocampus[END_REF]. Expression of serine / threonine phosphatase PP2A, a phosphatase, that supress IkB phosphorylation by IKK, is inhibited by the PI3K/Akt pathway in primary cultures of neurons [START_REF] Didonato | A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB[END_REF][START_REF] Meske | Coupling of mammalian target of rapamycin with phosphoinositide 3-kinase signaling pathway regulates protein phosphatase 2A-and glycogen synthase kinase-3 -dependent phosphorylation of Tau[END_REF]. PP2A may also be involved in the inactivation of MEK and ERK [START_REF] Junttila | Phosphatase-mediated crosstalk between MAPK signaling pathways in the regulation of cell survival[END_REF]. Previous study demonstrated that impaired hcy metabolism and reduced PP2A methylation levels could trigger the accumulation of phosphorylation of tau and APP in the brain, a process that may favor neurofibrillary tangle formation and amyloidogenesis [START_REF] Sontag | Protein phosphatase 2A methyltransferase links homocysteine metabolism with tau and amyloid precursor protein regulation[END_REF]. We therefore analyzed the PP2A activity in brain of mice with intermediate hhcy and found that, commensurate with the decreased PP2A activation, they present decreased level of IkBα with an increased level of its phosphorylation. In addition, a negative and a positive correlation were found between plasma hcy level and IkBα protein level (r = -0.415, p < 0.03) and phosphorylation of IkBα (r = 0.404, p < 0.04) respectively. In absence of stimuli, NFkB is sequestered in the cytoplasm by the protein IkBα. Following a stimulus, IkBα is phosphorylated, resulting in the dissociation of the complex NFkB/IkBα and the translocation of NFkB in the nucleus [START_REF] Oeckinghaus | The NF-kappaB family of transcription factors and its regulation, Cold Spring Harb[END_REF]. We therefore analyzed the p65 level and found an increase in nuclear fraction in cortex and hippocampus of mice with intermediate hhcy. This increase was found to be associated with a decrease cytoplasmic p65 level in cortex. An increase cytoplasmic p65 level was found in hippocampus of mice with intermediate hhcy, as previously described in rats after chronic hcy administration [START_REF] Da Cunha | chronic hyperhomocysteinemia increases inflammatory markers in hippocampus and serum of rats[END_REF], with a positive correlation between cytoplasmic and nuclear p65 levels in mice hippocampus (r = 0.653, p < 0.02). The increased nuclear p65 level emphasizes the activation of NFkB pathway in brain of mice with intermediate hhcy.

In addition to the link between PI3K/Akt pathway and PP2A activity on IkBα stability, Ras (a small GTP-binding protein) activated by RTK, enhances NFkB transcriptional activity through a Raf-independent mechanism [START_REF] Margolis | Activation of Ras by receptor tyrosine kinases[END_REF][START_REF] Norris | Oncogenic Ras enhances NF-kappaB transcriptional activity through Raf-dependent and Raf-independent mitogen-activated protein kinase signaling pathways[END_REF]. We previously demonstrated that hcy-mediated IkBα enhanced proteolysis occurred via calcium-dependent calpains, which was supported by an increased level of calpain activity and a reduced expression of calpastatin in liver of mice with hhcy [START_REF] Hamelet | Calpain activation is required for homocysteine-mediated hepatic degradation of inhibitor I kappa B alpha[END_REF]. Mice with intermediate hhcy also showed increased brain calpain activity, with a decreased calpastatin level, suggesting the involvement of the calpain-calpastatin system in reducing the level of IkBα in response to intermediate hhcy. Taken together, our results emphasize the deleterious effect of hhcy on NFkB pathway, due not only by RTK activation but also in parallel by NMDAR activation and calpain inducing effect.

Low plasma BDNF level has been associated with high plasma hcy level in drug naïve, first episode schizophrenia patients compared to healthy control group, which may play an important role in the neurodevelopmental process and clinical manifestation of schizophrenia [START_REF] Song | Serum levels of BDNF, folate and homocysteine: in relation to hippocampal volume and psychopathology in drug naïve, first episode schizophrenia[END_REF]. Interestingly, there was a notable increase in plasma hcy level and significant decrease in serum BDNF level in amnestic mild cognitive impairment patients that converts to AD patients, especially in those with the APOE ε4 allele [START_REF] Zheng | Conversion from MCI to AD in patients with the APOE ε4 genotype: prediction by plasma HCY and serum BDNF[END_REF]. Cognitive impairment is also closely related to inflammatory responses, which is highly regulated by many factors, including NFkB pathway [START_REF] Silva | Memory deficit associated with increased brain proinflammatory cytokine levels and neurodegeneration in acute ischemic stroke[END_REF][START_REF] Kohman | Neurogenesis, inflammation and behavior[END_REF]. Exacerbated activation of calpain has been implicated as a major component in the signaling cascade that leads to β-amyloid (Aβ) production and tau hyperphosphorylation in AD, leading to a hypothesis that selective calpain inhibitors are potential therapeutic strategies for AD [START_REF] Liang | Calpain activation promotes BACE1 expression, amyloid precursor protein processing, and amyloid plaque formation in a transgenic mouse model of Alzheimer disease[END_REF][START_REF] Rao | Specific calpain inhibition by calpastatin prevents tauopathy and neurodegeneration and restores normal lifespan in tau P301L mice[END_REF]. ERK kinases can phosphorylate tau and induce tau-dependent and tau-independent pathogenic effects, including synaptic plasticity defects and cognitive impairment [START_REF] Medeiros | Calpain inhibitor A-705253 mitigates Alzheimer's disease-like pathology and cognitive decline in aged 3xTgAD mice[END_REF]. Therefore, we analyzed the effect of this selective hcy lowering therapy upon molecular mechanisms implicated not only in neuropathological but also in inflammatory processes. After treatment, we found an increased BDNF level compared to mice with intermediate hhcy and without treatment. Previous study also demonstrated a positive effect of hcy lowering on BDNF level in hippocampus and shortand long-term memories in rats [START_REF] Matté | Acute homocysteine administration impairs memory consolidation on inhibitory avoidance task and decreases hippocampal brain-derived neurotrophic factor immunocontent: prevention by folic acid treatment[END_REF]. ERK, Akt and IkBα phosphorylation were found to be decreased. Commensurate with the decreased level of IkBα phosphorylation, we found increased IkBα level and decreased level of nuclear p65 after AAV2/8-DYRK1A gene transfer. However, no effect was found for PP2A activity in brain of mice with intermediate hhcy after treatment. The increased IκBα protein level is associated with a reduction of calpain activity, which is associated with increased calpastatin level in brain of mice with intermediate hhcy after AAV2/8-DYRK1A gene transfer. The increase of calpain 2 domain I suggests a positive feedback to maintain its homeostasis.

Conclusion

Taken together, our results demonstrate that targeting hepatic DYRK1A expression can correct plasma DYRK1A and hcy level in mice with intermediate hhcy. The consequences of plasma hcy lowering therapy are restoration of brain DYRK1A levels, BDNF levels, ERK, Akt and IkBα phosphorylation levels as well as calpain-calpastatin system and CBS activity, therefore, thus protecting from neuropathological and inflammatory processes. The inhibition of calpain activity and RTKmediated signaling pathways after AAV2/8-DYRK1A gene transfer suggest a potential therapeutic effect of hcy lowering in the treatment of tauopathies and neurodegenerative states.

Fig. 1 .

 1 Fig. 1. Effect of AVV2/8-DYRK1A gene transfer on hepatic DYRK1A level. A) Liver DYRK1A protein level in control (CTL) mice, Cbs +/-mice, and B) Cbs +/-mice with intermediate hhcy (hhcy) with or without AAV2/8-DYRK1A treatment (AAV). Liver DYRK1A was determined by slot blotting, and values were obtained by normalization of images from DYRK1A to total proteins colored with Ponceau-S. Data were normalized to the mean of control (CTL) mice. The results are represented as means ± SEM. n = number of mice.

Table 1 4 )

 14 Relative liver and brain mRNA expression of DYRK1A based upon Q-PCR data obtained from control (CTL), Cbs +/-mice with intermediate hyperhomocysteinemia (hhcy) with (hhcy/AAV) or without (hhcy) AAV2/8-DYRK1A treatment. Data were normalized to the mean of control (CTL) mice. Data are presented as mean ± SEM. n = number of mice. * p < 0.01 (compared to CTL mice); $ p < 0.02 (compared to hhcy mice).

Fig. 2 .Fig. 3 .

 23 Fig. 2. Effect of AVV2/8-DYRK1A gene transfer on plasma hcy and DYRK1A level. A) Plasma hcy level in A) control (CTL) mice, Cbs +/-mice and B) Cbs +/-mice with intermediate hhcy (hhcy) with or without AAV2/8-DYRK1A treatment (AAV). C) Plasma DYRK1A level in mice with intermediate hhcy with or without AAV2/ 8-DYRK1A treatment (AAV), determined by ELISA. The results are represented as means ± SEM. n = number of mice.

Fig. 4 .

 4 Fig. 4. Effect of AVV2/8-DYRK1A gene transfer on brain RTK activation. A) Akt and B) ERK activation in brain of Cbs +/-mice with intermediate hyperhomocysteinemia (hhcy) with (hhcy/AAV) or without (hhcy) AAV2/8-DYRK1A treatment, and control (CTL) mice. Proteins were subjected to slot blot analysis using antibodies specific to P-akt or P-ERK. After stripping, the membranes were reprobed with anti Akt or anti ERK antibody for the control. Relative protein expression was determined by normalization of the density of images from P-Akt or P-ERK with that of Akt or ERK of the same blot. Data were normalized to the mean of CTL mice. The results are represented as means ± SEM. n = number of mice.

Fig. 5 .

 5 Fig. 5. Effect of AVV2/8-DYRK1A gene transfer on brain BDNF level. BDNF levels were measured by ELISA in brain of Cbs +/-mice with intermediate hyperhomocysteinemia (hhcy) with (hhcy/AAV) or without (hhcy) AAV2/8-DYRK1A treatment, and control (CTL) mice. The results are represented as means ± SEM. n = number of mice.

Fig. 7 .

 7 Fig. 7. Effect of AVV2/8-DYRK1A gene transfer on brain p65 localization. Cytoplasmic p65 levels in A) cortex and B) hippocampus, and nuclear p65 levels in C) cortex and D) hippocampus in brain of Cbs +/-mice with intermediate hyperhomocysteinemia (hhcy) with (hhcy/AAV) or without (hhcy) AAV2/8-DYRK1A treatment, and control (CTL) mice. Values were obtained by normalization of images from nuclear and cytoplasmic p65 to total proteins colored with Ponceau-S. Data were normalized to the mean of CTL mice. The results are represented as means ± SEM. n = number of mice.

Fig. 8 .

 8 Fig. 8. Effect of AVV2/8-DYRK1A gene transfer on brain calpain activity. A) Calpain activity, B) calpastatin and C) calpain 2 domain I protein levels in brain of Cbs +/-mice with intermediate hyperhomocysteinemia (hhcy) with (hhcy/AAV) or without (hhcy) AAV2/8-DYRK1A treatment, and control (CTL) mice. Calpain activity was determined by measuring the proteolysis of the substrate for 21 min. Calpastatin and calpain 2 domain I protein values were obtained by normalization of images from calpastatin and calpain 2 domain I to total proteins marked with Ponceau-S. Data were normalized to the mean of CTL mice. The results are represented as means ± SEM. n = number of mice.

Acknowledgments

This work was supported by the Lagafe association and Fondation Jerome Lejeune (grant number 2014a-1273). We acknowledge the platform accommodation and animal testing of the animal house at the Institute Jacques-Monod (University Paris Diderot), and the FlexStation3 Facility of the Functional and Adaptative Biology (BFA) laboratory. We thank Tiphaine Bringel for technical assistance.

Author contributions

V.B. and M.F. performed the majority of experiments; D.G. performed additional experiments; J.L.P. performed HPLC analysis. N.K. performed Q-PCR analysis. V.B. and N.J. performed data analysis and interpretation; N.J. conceived and designed this work, and wrote the paper.

Conflict of interest

None