Mehdi Amoura

Françoise Illien

Alain Joliot

Karine Guitot

John Offer

Sandrine Sagan

Fabienne Burlina
email: fabienne.burlina@sorbonne-universite.fr

Head to tail cyclisation of cell-penetrating peptides: impact on GAG-dependent internalisation and direct translocation

A series of cyclic lipidated oligo-Arg cell penetrating peptides were synthesised with varied macrocycle size and lipid chain anchoring site. The study of their cellular uptake revealed different structural requirements to promote efficient glycosaminoglycan-dependent endocytosis and direct translocation.

Cell-penetrating peptides (CPPs) are considered as promising systems for the intracellular delivery of probes and therapeutics, with several CPPs currently under clinical development. 1 They are generally short purely basic or amphipathic peptides that can enter cells by two different pathways: endocytosis and direct translocation across the plasma membrane. The contribution of endocytosis and translocation varies between CPPs and their respective extent is most probably determined by the CPP capacity to interact with key components of the plasma membrane. 2 In particular, glycosaminoglycans (GAGs) have been shown to be implicated in the endocytosis of several CPPs. 3 CPP arginine residues play an essential role in GAG binding, 4 due to their ability to form bidentate hydrogen bonds with GAG carboxylate and sulfate groups. 5 Previous studies also highlighted the critical role of Trp residues in GAG clustering, 6 most probably by forming p interactions. Phospholipids are the key partners in the translocation pathway, which involves a transient, nondeleterious, perturbation of the lipid bilayer induced by the CPP. Arg residues, which can form neutral ion-pairs with phospholipids, 5 and Trp residues, which promote interfacial and reversible interaction of the CPP with the lipid bilayer 7 play a critical role in translocation as well. Interestingly, GAGs are also involved in the mechanism of translocation of some amphipathic CPPs, 8 possibly by serving as first site of peptide accumulation on the cell-surface before their transfer to the lipid bilayer. Although for most CPPs direct translocation represents a minor route of entry compared to endocytosis, it is potentially more productive because it gives direct access to the cytosol avoiding trafficking through endosomes and possible entrapment of the CPP and its cargo in these organelles. The design of CPPs that steer translocation of their cargo to the cytosol more efficiently is therefore of particular interest.

Many different chemical modifications have been proposed in the past years to boost CPP properties. 9 Among backbone modifications, cyclisation has been found to give remarkable results and is increasingly applied to CPPs. [10][11][12][13][14][15] In particular, cyclisation of Tat was found to improve cellular uptake by inducing maximal spacing between the CPP guanidinium groups, 11 in agreement with earlier observations made with peptoid analogues of CPPs. 16 Lipidation is another interesting modification, which can improve CPP interaction with the cell membrane leading to increased translocation efficiency. [17][18][19][20] It was also found to favour GAG-dependent endocytosis of a small linear oligo-Arg CPP, most probably by increasing the local density of the CPP cationic residues through its self-assembly. 19 By changing the spatial distribution of the CPP functional groups, chemical modifications can therefore modulate CPP interaction with key membrane partners and alter their mechanism of internalisation. In this study, we have analysed the impact on GAG-dependent entry, endocytosis and translocation of head to tail cyclisation, in some cases combined with lipidation, for a series of CPPs. Cyclisations were easily performed using native chemical ligation (NCL). 21 Conveniently, the Cys residue involved in NCL, could serve after cyclisation for cargo conjugation by a disulfide bridge. Cyclisation was found to always increase the CPP internalisation efficiency, in most cases by improving their interaction with GAGs and endocytosis, but also when it was combined with lipidation, by favouring direct translocation.

A small library of CPPs was designed for this study (Chart 1). We first chose to prepare head to tail cyclic derivatives of the classical CPPs Penetratin 22 (Pen) and R6W3 23 , which had not been evaluated before under their cyclic form, and Tat, 24 one of the first CPPs cyclized. 11 It should be noted that our cyclic Tat analogue has a different structure than the previously proposed one. We also examined the impact of cyclisation of the laurylated tetra-arginine CPP C12-R4, which had previously been found to enter cells efficiently by GAG-dependent endocytosis. 19 For the cyclic derivative, the carbon chain was introduced as the (R,S)-2-amino tetradecanoic acid derivative to give CPP [C12-R4], for which all four Arg residues were kept contiguous. A series of derivatives were also designed to evaluate the impact of the lipid chain anchoring position ([R2- Cyclic peptides were synthesized by native chemical ligation (NCL) 25 introducing in the original linear sequence an N-terminal cysteine and a C-terminal thioester or a precursor corresponding to an a-methylcysteine (aMeCys) 26 residue (Scheme S1, ESI). The peptides with a preformed thioester function were prepared by Boc solid phase peptide synthesis (SPPS) using a mercaptopropionic acid leucine (mpaL) linker to the resin 27,[START_REF] Hackeng | Proc. Natl. Acad. Sci[END_REF] (Scheme S1, ESI). Cyclisations of the unprotected peptides were performed in standard conditions for NCL corresponding to phosphate buffer at final pH 7, in the presence of reducing (TCEP) and denaturing (guanidine) agents and of mercaptophenylacetic acid (MPAA) as thiol additive for thioester activation (Scheme S1, ESI). All ligations were found to proceed smoothly with no side reaction and to be complete in less than 18 h (Figure S3, ESI). Fmoc SPPS was used in our second approach to prepare the linear precursor of Pen with a Cterminal aMeCys residue for in situ generation of the thioester function by N to S acyl transfer (Scheme S1, ESI). To optimise cyclisation conditions, we examined the impact of the buffer composition and pH on the reaction rate (Table S2, ESI). In all cases, ligations went to completion. Increasing MPAA concentration from 300 to 450 mM or decreasing the pH from 7 to 5 significantly accelerated the reaction, as previously observed in two-piece ligations 26 and most likely by shifting the aMeCys amide to thioester equilibrium towards the thioester. Interestingly, we found here that among all parameters tested, the guanidine concentration had the strongest effect. Reducing it from 6 to 3 M, decreased the half-time of the reaction by a factor 2, possibly because of the decreased dielectric constant of the buffer. In the optimised conditions (3 M guanidine, 450 mM MPAA, pH 5), cyclisation was almost complete in 7 h. All CPPs, linear and cyclic, were then linked by a disulfide bridge to a non-permeant peptide cargo, which corresponds to a protein kinase C inhibitor (PKCi) (Chart 1). The cargo incorporates on its N-terminus an isotope labelling and affinity tag (Biotin(sulfone)GGGG) to allow its quantification inside cells.

The efficiency of the CPPs was assessed by measuring the amount of intact PKCi delivered inside cells using a protocol based on MALDI-TOF MS established previously. [START_REF] Burlina | [END_REF] Absolute quantification of the internalised PKCi peptide was performed after cell lysis in reducing conditions using as internal standard the unconjugated PKCi peptide labelled with a stable isotope (containing 4 bi-deuterated Gly residues in the tag, Table S1 in the ESI). To study the mechanism of entry of the conjugates, we compared their internalisation in CHO-K1 cells (WT cells) at 37°C (a temperature at which both endocytosis and translocation can occur) and at 4°C (a temperature at which endocytosis is inhibited and only direct translocation remains effective). The involvement of cell surface GAGs was studied by using CHO-745 cells which are deficient in GAGs (GAG neg cells). It should be noted that none of the conjugates showed toxicity at the concentration used for the internalisation experiments (Fig. S5,ESI). The linear classical CPPs, Tat, Pen and R6W3 (Chart 1) and their cyclic derivatives (denoted [Tat], [Pen] and [R6W3]) were first studied (Fig. 1, Fig. S7 and Table S4 in the ESI). Interestingly, cyclisation was found to improve cellular uptake of all CPPs in WT cells at 37°C, even though moderately. [R6W3] gave the best result by far leading to a total concentration of cargo inside cells of about 7 µM (calculated with an estimated volume of 1 pL per CHO cell), which is close to the conjugate concentration applied outside cells. Our mechanistic studies showed that internalisations of all linear and cyclic CPPs of this series mostly depend on endocytosis, amounts of PKCi delivered in WT cells being notably reduced when the temperature was decreased. However, they were found to also enter cells by translocation. For the conjugate of this series that translocates the best, R6W3-PKCi, we measured an intracellular concentration of about 1.4 μM at 4°C. It is noteworthy that conjugates that enter by translocation first reach the cytosol, from where they can eventually redistribute within the different cell compartments. In addition, conjugate internalisation was found to be principally dependent on GAGs, as cargo amounts measured in GAG neg cells at 37°C were much lower than the one obtained in WT cells. In this series of classical CPPs, cyclisation always improved endocytosis, which was found to be mostly dependent on GAGs, but no significant effect could be detected for translocation. Next, we evaluated the series of lipidated oligo-Arg CPPs (Fig. 2 S5 in the ESI). The cyclic derivative [C12-R4] enters cells more efficiently than the linear analogue C12-R4. Remarkably, in this case cyclisation improves both translocation and GAG-dependent entry. Replacement of the lipid chain by a Phe residue ([FR4]) strongly affected endocytosis and direct translocation, showing the benefit of the lipid chain for both routes of entry in the cyclic series, as also observed before in the linear one. 19 Substitution of the Phe residue by Trp ([WR4]), which has been found before to favour CPP interaction with membrane components involved in cellular uptake, 6,7 had no significant effect here (Fig. S9, ESI), suggesting that a single Trp in the CPP sequence is not sufficient. Changing the lipid chain position within the macrocycle to give compounds [R2-C12-R2] and [R3-C12-R] modifies the spatial arrangement of the 4 Arg residues and also the relative positioning of the lipid chain and disulfide linked cargo, which could both affect the uptake efficiency. For example, the cargo and lipid chain are in closer proximity in conjugate [C12-R4] compared to the 2 other cyclic analogues. Interestingly, [R2-C12-R2] shows similar uptake efficiency and mechanism as [C12-R4]. In contrast, [R3-C12-R] enter cells less efficiently in WT cells at 37°C due to a decreased ability to use GAG-dependent endocytosis but its translocation capacity is not much affected. These data suggest that the spatial arrangement of the Arg residues plays a crucial role for CPP binding to cell-surface GAGs, interaction being more favourable (in triggering internalisation by endocytosis) when the 4 Arg residues are contiguous or grouped by pairs. Increasing the number of Arg residues to 7 within the macrocycle strongly reduced the global entry inside cells at 37°C (by a factor 2 between [C12-R4] and [C12-R7]) by decreasing GAG-dependent entry. Further increasing the number of Arg residues with [C12-R9] had no effect. Interestingly, the same phenomenon, although less pronounced, was observed when the macrocycle was enlarged by the addition of a single Phe residue (Compound [C12-R2FR2]). The more compact structure resulting in higher local density in basic residues of [C12-R4] compared to [C12-R7/9] or [C12-R2FR2] might account for its improved capacity to interact with the sulfated GAGs. On the other hand, [C12-R7] and [C12-R9] show remarkable translocation efficiency, leading both to a cargo concentration of about 2 μM at 4°C. Translocation of these vectors was improved compared to [C12-R4], showing that this route of entry is favoured by increasing the number of basic residues, as previously observed with linear CPPs. 30 It should be mentioned that evaluating the efficiency of entry at 4°C gives a baseline for cytosolic concentration. Species entering by direct translocation are measured but not species that could, after endocytosis, escape from endosomes to reach the cytosol.

, Fig S8 and Table

For the most efficient derivatives identified from both nonlipidated and lipidated series (derivatives of R6W3 and C12-R4), we also analysed the cargo intracellular distribution in WT cells at 37 and 4°C (Fig. 3

-R 4 [ C 1 2 -R 9 ] [ C 1 2 -R 4 ] [ R 2 -C 1 2 -R 2 ] [ R 3 -C 1 2 -R ] [ C 1 2 -R 7 ] [ C 1 2 -R 2 F R 2 ] WT, 37°C WT, 4°C GAG neg , 37°C [ F R

and Fig S10 in the ESI). The [C12-R9]

conjugate was also chosen as example of cyclic derivative with efficient translocation properties. The 5 conjugates led at 37°C to predominant vesicular signal but diffuse cytosolic signal was also detected, in agreement with the mechanistic data. Interestingly, the R6W3 derivatives gave a more homogeneous distribution of the cargo inside cells compared to the lipidated derivatives.

In summary, cyclisation was found to improve uptake efficiency, over the whole range of chemically diverse CPPs designed here, from purely cationic to amphipathic, non lipidated or lipidated peptides. However, our study showed that improved internalisation had different origins depending on the nature of the vector. For R6W3 and C12-R4 which had been previously found to favourably interact with cell-surface GAGs, cyclisation further enhanced GAG-dependent internalisation, a result that had not been reported before to our knowledge. This might be attributed to the expected constrained structure of the cyclic vectors compared to linear analogues or to a local increased density of the critical cationic functional groups, leading to better interaction with GAGs. This is supported in the lipidated cyclic CPP series by the fact that extending the macrocycle size, introducing a Phe or even several Arg residues, disfavours GAG-dependent entry. Translocation was also improved by cyclisation in the case of C12-R4 and further enhanced by increasing the number of basic residues. This led to the identification of vectors [C12-Rn] (n=7 or 9) that translocate with their cargo into the cytosol with remarkable efficiency. Among the derivatives studied, [R6W3] and [C12-R4] performed the best for total entry at 37°C and [C12-R7/9] showed the best translocation efficiency. They appear as promising vectors for the cellular delivery of therapeutics. This study was supported by the Agence Nationale de la Recherche (ANR-16-CE16-0016-03).

General information

Reagents for peptide synthesis and protected amino acids were purchased from Iris Biotech and Merck Chemicals except for (2,2-D2, 98%)-Boc glycine, which was obtained from Euriso-top. Solvents (peptide synthesis grade) were obtained from Carlo Erba Reagents. MBHA LL resin was purchased from Bachem. (R,S)-Boc-2-amino-tetradecanoic acid (CAS 129850-62-0) was purchased from Polypeptides. Tris-HCl, Triton X-100, trypsin inhibitor and bovine serum albumin were obtained from Sigma-Aldrich. DMEM/F-12 (1:1), FBS, trypsin-EDTA (0.05 % trypsin, 0.02 % EDTA) and HBSS were purchased from Gibco. The cell counting kit (CCK8) was from Dojindo Laboratories. Streptavidin-coated magnetic beads (Dynabeads® M-280 Streptavidin) were purchased from Invitrogen. The complete mini tablets of protease inhibitors were from Roche. DAPI was from Pierce. Ultrapure water was obtained using a Milli-Q water system from Millipore. All reagents and solvents were used without further purification. HPLC purifications were carried out on a Waters system (Pump 600, Absorbance detector 2487) using ACE-5 C4 / C8 (300 Å, 5 μm, 250 × 10 mm, 5 mL/min flow rate) or Kromasil C18 (300 Å, 5 μm, 250 × 10 mm, 5 mL/min flow rate) reverse phase columns. Analytical HPLC were performed either on a Waters system (Pump 1525, Absorbance detector 2487), Agilent 1220 Infinity or Dionex Ultimate 3000. Vydac C4 / C18 (5 µm, 150 × 4.6 mm, 1 mL/min flow rate), Kromasil C18 (5 µm, 250 × 4.6 mm, 1 mL/min flow rate), ACE-5 C4 / C8 (5 µm, 250 x 4.6mm, 1 mL/min flow rate) or PROTO 200 C18 (3 µm, 100 x 4.6 mm, 1 mL/min flow rate) columns were used. Linear gradients of solvent B in A were used with A corresponding to 0.1 % TFA in H2O and B to 0.1 % TFA in CH3CN. MALDI-TOF MS analyses were performed in the linear or reflector positive ion mode on an ABI Voyager DE-Pro MALDI-TOF mass spectrometer (Applied Biosystems) or 4700 Proteomix MALDI-TOF MS/MS (Applied Biosystems), using as matrix a saturated solution of CHCA in CH3CN/H2O/TFA (50:50:0.1).

General procedures for solid phase peptide synthesis

Amounts of reagent are given in equivalents (equiv.) with respect to the peptidyl-resin. Peptide syntheses were carried out manually using Fmoc or Boc SPPS and standard protected amino acids unless otherwise mentioned. Syntheses were usually performed at a 0.1mmol scale.

Fmoc SPPS protocol: Fmoc-protected amino acids (0.6 mmol, 6 equiv.) were activated with HBTU (0.58 mmol, 5.8 equiv., 0.5 M) and DIEA (1.4 mmol, 14 equiv.) in NMP (3 min) and added to the peptide-resin (coupling for 30 min). Capping of eventual unreacted amine groups was achieved by treatment with acetic anhydride (10 %) and DIEA (0.5 mmol, 5 equiv.) in DCM for 20 min. Fmoc groups were removed by treatment with 20 % piperidine in NMP (3 x 3 min). Final peptide deprotection and cleavage from the resin were performed by treatment with TFA/H2O/iPr3SiH, 95:2.5:2.5 (3 h) (for peptides containing Met or Cys residues, the cocktail TFA/H2O/iPr3SiH/EDT, 94:2.5:1:2.5 was used). TFA was sparged under a stream of N2, peptides were precipitated in ice-cold diethyl ether, recovered by centrifugation and freeze-dried before HPLC purification.

In situ neutralization protocol for Boc SPPS [1] : Boc-protected amino acids (0.6 mmol, 6 equiv.) were activated with HBTU (0.58 mmol, 5.8 equiv., 0.5 M) and DIEA (1.4 mmol, 14 equiv.) in NMP (3 minutes) and added to the peptide-resin (coupling for 30 min). Boc groups were removed by treatment with TFA (2 x 1 min). The resin was carefully washed with NMP. Final peptide deprotection and cleavage from the resin was performed by treatment with anhydrous HF (2 h, 0°C) in the presence of the following scavengers:

-For C-terminal carboxamide peptides with no Cys residues, anisole (1.5 mL/g peptide-resin) and dimethyl sulfide (0.25 mL/g peptide-resin) were used as scavengers. -For C-terminal carboxamide peptides with a Cys residue protected with pmethoxybenzyl, p-toluenethiol (300 mg/g peptide-resin) was also added. -For C-terminal carboxamide peptides with a Cys residue protected with a NpyS, only anisole was used. -For peptide-mpaL thioesters, the only scavenger added was p-cresol (65 mg/g peptide-resin). HF was removed under vacuum and the peptide was precipitated in ice-cold diethyl ether. The precipitate was dissolved in degassed acetic acid (10 % in H2O), the resin eliminated by filtration and the peptide was freeze-dried before HPLC purification.

Linear CPPs and peptide cargo

The syntheses of the linear CPPs (Table S1), the PKCi cargo ( 1 H-PKCi) and the deuterated internal standard used for MALDI-TOF MS quantification ( 2 H-PKCi) have been described previously. [2][3]

Table S1. Name and sequences of the peptide cargo and linear CPPs

Cyclic CPPs Scheme S1. Strategies used for peptide cyclisation a. Strategy A: Synthesis of cyclic CPPs by native chemical ligation (NCL) using peptide-mpaL thioesters

Synthesis of peptide-mpaL thioesters

The procedure described by Hackeng et al. was applied. [4] Briefly, Boc-Leu was first coupled on MBHA-PS resin (0.54 mmol/g, 0.1 mmol) (see the in situ neutralization protocol for Boc SPPS p. S4). S-trityl-mercaptopropionic acid (0.2 mmol, 2 equiv.) was then coupled using HBTU (0.18 mmol, 1.8 equiv.) and DIEA (0.4 mmol, 4 equiv.) in NMP for 1h30. The peptideresin was washed with NMP. Removal of the trityl protecting group was performed by 15 min continuous flow with a mixture of TFA/H2O/TES, 95:2.5:2.5 followed by washing with NMP. The next Boc-protected amino acid was immediately coupled (HBTU/DIEA activation, 45 min coupling) and the peptides elongated using the in situ neutralization Boc SPPS protocol. Peptides were then deprotected and cleaved from the resin by HF treatment (protocol p. S4) and purified by semi-preparative RP-HPLC.

Cyclisation of the mpaL thioesters

A carefully degassed 0.2 M sodium phosphate buffer containing 2 mM EDTA, 3 or 6 M guanidine hydrochloride (see below), 3 mM MPAA and 3.5 mM TCEP was added to the peptide mpaL thioester (concentration 1 mM, final pH 6.5 or 7).The ligations were carried out at room temperature and monitored by RP-HPLC (Fig S3). For this purpose 10 µL aliquots of the reaction mixture were injected in HPLC. After completion, the reaction mixtures were diluted with 0.1 % TFA in water and purified by RP-HPLC to afford the desired cyclic products (Fig S1 andS2). NCL was performed in buffer containing 3 M Guanidine for mpaL derivatives of Tat, R6W3, C12-R7, C12-R9, FR4 and WR4, which showed good solubility in these conditions. For the other derivatives, 6 M guanidine was used. The C-terminal a-methylcysteine-containing peptide was synthesised by standard Fmoc SPPS on Wang resin functionalised by an α-methylcysteine (0.20 mmol/g, 0.1mmol) as described previously. [5] After completion of elongation, peptide-resin was deprotected and cleaved using the standard cleavage procedure (protocol p S3) and the peptide-aMeCys was purified by RP-HPLC.

Pen-aMeCys (CRQIKIWFQNRRMKWKK-aMeC) MALDI-TOF (m/z) [M+H] + calc.: 2465.3, found: 2465.7

Cyclisation of Pen-aMeCys

To optimise the conditions of cyclisation, we first evaluated the effect of different parameters (pH, guanidine and MPAA concentration) on the reaction rate. Pen-aMeCys (final concentration 1 mM) was dissolved in degassed ligation buffer containing 200 mM sodium phosphate, 2 mM EDTA, 50 mM TCEP and the guanidine and MPAA concentrations and pH indicated Table S2. The reaction was monitored by HPLC injecting aliquots of 10 µL of the ligation mixture at different reaction times (Fig S4). The extent of conversion of Pen-aMeCys into [Pen] was deduced from the relative areas of both species HPLC peaks, considering that both compounds have the same extinction coefficient at 220 nm. The

OH O N H SH O H N N H H N N H H N N H H N N H H N N H NH 2 O NH 2 O N O NH 2 O S O NH NH 2 HN O NH NH 2 HN O O NH 2 O NH 2 O O O H N N H H N N H N O O NH 2 O O H N NH 2 O O N H NH HN NH 2 O H 2 N SH FR4-mpaL [FR4]
7. Synthesis and characterisation of the CPP-PKCi disulfide conjugates used in the biological experiments

The CPP was dissolved in degassed sodium acetate buffer (50 mM, pH 4.5 to 5) and mixed with 1.1 eq. of 1 H-PKCi (final peptide concentrations: 1 to 5 mM). The reaction was monitored by HPLC. At the end of the reaction, conjugates were purified by reverse phase HPLC.

Scheme S2. Disulfide bridge formation between the CPP and the cargo

The conjugates were characterized by MALDI-TOF MS on a Voyager DE-PRO (Applied Biosystems) or on a 4700 Proteomix MALDI-TOF/TOF (Applied Biosystems) in the linear positive ion modes and reflector positive ion mode. The disulfide bond is fragile and a fragmentation is observed at this position during MALDI-TOF MS analysis, the [M+H] + peak being more difficult to detect in the reflector mode compared to the linear one.

Conjugate C12-R4-PKCi has been previously described. [3] 8. Cell culture and cell viability assays CHO cells were cultured in sterile conditions in DMEM/F-12 medium supplemented with 10 % heat-inactivated FBS in a humidified atmosphere of 5 % CO2, at 37 °C. Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8). Cells were seeded in 96-well plates to obtain 5 000 cells/well the day of the cell viability experiment. Cells were incubated for 75 min at 37 °C with 100 µL of the conjugate solution (7.5 µM in DMEM) and for the control experiment with 100 µL of DMEM. The supernatant was removed and 100 µL of CCK8 (10 % in DMEM) was added. After 2 h incubation at 37 °C, the absorbance was measured at 450 nm using a microplate reader (FLUOstar OPTIMA, BMG LABTECH) with a reference wavelength at 620 nm. The experiments were performed in triplicates and repeated twice independently. For each conjugate, the percentage of viable cells compared to the control conditions (no conjugate) was calculated. Mean values +/-SEM are given in the histogram.

Elimination of the membrane-bound peptide:

After incubation, cells were washed 3 times with 1 mL HBSS, treated for 3 min at rt with 200 µL TCEP (2 mM in 50 mM Tris-HCl buffer pH 7.5) to reduce all cell-surface disulfide conjugates and washed again once with 1 mL HBSS.

-When internalisation experiments had been performed at 37 °C: membrane-bound peptide was eliminated by cell treatment for 5 min at 37 °C with 500 µL trypsin-EDTA. Trypsin inhibitor (100 µL, 5 mg/mL) and BSA (100 µL, 1 mg/mL) were then added and the cell suspension was transferred to a micro-tube.

-When internalisation experiments had been performed at 4°C: membrane-bound peptide was eliminated by cell treatment for 10 min with pronase (0.5 mg/mL in 100 mM Tris-HCl pH 7.5). A cocktail of protease inhibitors (100 µL of a 7x solution of Complete mini, Roche) and BSA (100 µL, 1 mg/mL) were then added and the cell suspension was transferred to a microtube.

In both cases, wells were washed with 500 µL of 50 mM Tris-HCl buffer (pH 7.5). The combined suspensions were centrifuged for 2 min at 640 g. The pellet was washed with 1 mL 50 mM Tris-HCl pH 7.5, 0.1 % BSA (buffer A) and centrifuged again in the same conditions.

Cell lysis and extraction of the biotinylated peptide:

The cell pellet was mixed with a known amount of 2 H-PKCi internal standard (typically between 0.1 to 10 pmol) and 150 µL of a solution containing 0. The efficiency of endocytosis was estimated by subtracting the amount of intact PKCi cargo (pmol) delivered in 10 6 WT cells at 4°C from the values obtained in WT cells at 37°C. 2 The efficiency of translocation is directly given by the amount of intact PKCi cargo measured in WT cells at 4°C. 3 The efficiency of GAG-dependent entry was estimated by subtracting the amount of intact PKCi cargo (pmol) delivered in 10 6 GAG neg cells at 37°C from the values obtained in WT cells at 37°C. Table S5. Effect of cyclisation on the efficiency of endocytosis, translocation and GAGdependent entry

Route of internalisation

C12-R4 [C12-R4] [R2-C12-R2] [R3-C12-R] [C12-R2FR2] [C12-R7] [C12-R9]

Endocytosis The efficiency of endocytosis was estimated by subtracting the amount of intact PKCi cargo (pmol) delivered in 10 6 WT cells at 4°C from the values obtained in WT cells at 37°C. 2 The efficiency of translocation is directly given by the amount of intact PKCi cargo measured in WT cells at 4°C. 3 The efficiency of GAG-dependent entry was estimated by subtracting the amount of intact PKCi cargo (pmol) delivered in 10 6 GAG neg cells at 37°C from the values obtained in WT cells at 37°C.

  C12-R2] and [R3-C12-R]) and macrocycle size. The macrocycle was either enlarged by incorporating 3 or 5 additional Arg residues ([C12-R7] and [C12-R9]) or by introducing a single Phe residue ([C12-R2FR2]). Finally, to evaluate the importance of the lipid chain (in the [C12-R4] CPP), it was replaced by an aromatic amino acid (Phe or Trp) to give [FR4] and [WR4].

Chart 1

 1 Synthesized CPP-Cargo conjugates. Cyclic CPPs are marked under square brackets, biotin(O2): biotin sulfone, Ac: acetyl.

Figure 1

 1 Figure 1 Amount of intact PKCi cargo delivered inside cells by the linear classical CPPs and their cyclic derivatives. The cyclic derivatives are marked under square brackets. Free PKCi (no CPP) and CPP-PKCi conjugates (7.5 µM) were incubated with 10 6 WT or GAG neg cells for 75 min at 4°C or 37°C.

Figure 2

 2 Figure 2 Amount of intact PKCi cargo delivered inside cells by the oligo-Arg derivatives. The cyclic derivatives are marked under square brackets. Free PKCi (no CPP) and CPP-PKCi conjugates (7.5 µM) were incubated with 10 6 WT or GAG neg cells for 75 min at 4°C or 37°C.

Figure 3

 3 Figure 3 Intracellular distribution of the PKCi cargo at 37°C. Free PKCi (no CPP) and CPP-PKCi conjugates (7.5 µM) were incubated with 10 5 WT cells for 75 min at 37°C. The internalised biotinylated PKCi was revealed with streptavidin-Alexa 488 (green) after saturation of the membrane-bound species with unlabelled avidin. Nuclei were stained with DAPI (blue). Scale bar represents 15 µm.

  )GGGGC(NpyS)RFARKGALRQKNV-NH2 Biotin(sulfone)GGGGCRFARKGALRQKNV-NH2 * Ac-CGRKKRRQRRR-NH2 Ac-CRRWWRRWRR-NH2 Ac-CRQIKIWFQNRRMKWKK-NH2 Ac = acetyl. NpyS, 3-nitro-2-pyridinesulphenyl. * Gly residues shown in red in the sequence are bideuterated.

  (CGRKKRRQRRR-mpaL) MALDI-TOF m/z [M+H] + calcd.: 1701.1, found: 1701.4 R6W3-mpaL (CRRWWRRWRR-mpaL) MALDI-TOF (m/z) [M+H] + calcd.: 1817.9, found: 1818.2 C12-R4-mpaL (CC12RRRR-mpaL) MALDI-TOF (m/z) [M+H] + calcd.: 1171.7, found: 1771.6 K(C12)-R4-mpaL (CK(C12:0)RRRR-mpaL) MALDI-TOF (m/z) [M+H] + calcd.: 1257.8, found: 1258.9

  -R-mpaL (CRRRC12R-mpaL) MALDI-TOF (m/z) [M+H] + calcd.: 1171.7, found: 1173.4 R2-C12-R2-mpaL (CRRC12RR-mpaL) MALDI-TOF (m/z) [M+H] + calcd.: 1171.6, found: 1174.7 C12-R2FR2-mpaL (CC12RRFRR-mpaL) MALDI-TOF (m/z) [M+H] + calcd.: 1318.8, found: 1320.5 C12-R7-mpaL (CC12RRRRRRR-mpaL) MALDI-TOF (m/z) [M+H] + calcd.: 1640.0, found: 1638.2

  -mpaL (CC12RRRRRRRRR-mpaL) MALDI-TOF (m/z) [M+H] + calcd.: 1952.2, found: 1954.6 FR4-mpaL (CFRRRR-mpaL) MALDI-TOF (m/z) [M+H] + calcd.: 1093.6, found: 1093.7 WR4-mpaL (CWRRRR-mpaL) MALDI-TOF (m/z) [M+H] + calcd.: 1132.6, found: 1132.8

Figure S3 .

 S3 Figure S3. HPLC monitoring of the cyclisation of FR4-mpaL to give [FR4]The reaction was performed in 200 mM sodium phosphate buffer containing 2 mM EDTA, 3 M Guanidine, 3.5 mM TCEP, 3 mM MPAA, final pH 6.5 using 1 mM FR4-mpaL. HPLC gradient: 0-30 % B in 30 min; column: Vydac C18; detection wavelength: 214 nm. *MPAA.

Figure S5 . 9 .

 S59 Figure S5. Viability of cells incubated with the different CPP-PKCi conjugates (Ctr = control, no conjugate added to the cells)

Figure S8 .

 S8 Figure S8. Amount of intact PKCi cargo delivered inside cells by the lipidated CPP and its cyclic derivatives. The cyclic derivatives are marked under square brackets. Conjugates CPP-PKCi (7.5 µM) and unconjugated PKCi (No CPP) were incubated with 10 6 WT (CHO-K1) or GAG neg (CHO 745) cells for 75 min at 4°C or 37°C. Mean values +/-SEM are given in the histogram. Mean values are also shown in the table below (pmol for 10 6 cells). ND: not determined. Significance was tested using a Welch's corrected t test ( ns p > 0.05, * 0.05< p > 0.01, ** 0.01< p > 0.001, *** p<0.001).

Amount of intact PKCi cargo delivered inside cells by the linear classical CPPs and their cyclic derivatives.

  3 % Triton X-100, 1 M NaCl, 2 mM DTT. The mixture was immediately heated for 15 min at 100 °C. The lysate was centrifuged for 5 min at 7080 g. The supernatant was mixed with 850 µL of buffer A containing 2 mM DTT. Streptavidin-coated magnetic beads (10 µL of Dynabeads® M-280 Streptavidin, Invitrogen) were added to the sample and the mixture was incubated for 1 h at rt to capture the biotinylated peptides (intact 1 H and 2 H-PKCi and potential digests). After bead immobilisation with a magnet, the supernatant was removed and beads were washed twice with 200 µL buffer A, they were incubated 10 min at 50 ºC with 20 µL Laemmli buffer (d10)*, then washed twice with 200 µL buffer A containing 1 M NaCl, twice with 200 µL H2O, incubated 2 min with 50 µL biotin (10 µM in H2O), washed with 50 µL H2O and finally 50 µL CH3CN/H2O (1:1). The supernatant was removed and 3 µL of matrix (saturated solution of CHCA in CH3CN/H2O/TFA, 50:50:0.1) was added. The beads were incubated 10 min at rt to elute the biotinylated peptides and 1 µL of the supernatant was deposited on the MALDI-TOF plate. MALDI-TOF MS analyses were performed in the ion positive reflector mode on an ABI Voyager DE-Pro MALDI-TOF mass spectrometer (Applied Biosystems). On the MALDI-TOF mass spectrum corresponding to the average of several hundreds laser shots, the area of the [M+H] + signals (including all isotopes) of the 1 H-PKCi and 2 H-PKCi signals were measured and the amount of intact internalised cargo was calculated from the area ratio. All internalisation experiments were performed in triplicates and repeated at least twice independently. The cyclic derivatives are marked under square brackets. Conjugates CPP-PKCi (7.5 µM) or unconjugated PKCi (No CPP) were incubated with 10 6 WT (CHO-K1) or GAG neg (CHO 745) cells for 75 min at 4°C or 37°C. Mean values +/-SEM are given in the histogram. Mean values are also shown in the table just below the histogram (pmol for 10 6 cells). Significance was tested using a Welch's corrected t test ( ns p > 0.05, * 0.05< p > 0.01, ** 0.01< p > 0.001, *** p<0.001)

		No CPP	Tat	[Tat]	Pen	[Pen]	R6W3	[R6W3]
	WT 37°C	0.19		0.81	1.15	1.99	3.44	5.24	7.06
	WT 4°C	0.14		0.27	0.43	0.77	0.87	1.44	1.01
	GAG neg 37°C	0.15		0.20	0.22	0.28	0.30	0.44	0.87
	Figure S7.
						,
		100		Internalised:,	Internal, standard:,	2140.5
					6.8,pmol,	10,pmol,,	2132.5
		% Intensity	50
			920 0	1189	1458	1727 Mass (m/z)	1996	2265

*Diluted (d10) Laemmli buffer composition: 1 % SDS, 6 % glycerol, 1 % ß-mercaptoethanol, 0.025 % bromophenol blue, 25 mM Tris-HCl, pH 6.8.

MALDI-TOF MS analysis:

Figure S6. Mass spectrum obtained for the cellular uptake of [R6W3]-PKCi conjugate at 37 °C in CHO-K1 cells. Insert: zoom on the intact internalised cargo ( 1 H-PKCi) and the internal standard ( 2 H-PKCi) peaks, which are used for quantification.

Table S4 .

 S4 Effect of cyclisation on the efficiency of endocytosis, translocation and GAGdependent entry

	Route of internalisation	Tat	[Tat]	Pen	[Pen]	R6W3	[R6W3]
	Endocytosis 1	0.54	0.72	1.22	2.57	3.8	6.05
	Translocation 2	0.27	0.43	0.77	0.87	1.44	1.01
	GAG-dependent entry 3	0.61	0.93	1.71	3.14	4.8	6.19
	1

Conflicts of interest

"There are no conflicts to declare".

Head to tail cyclisation of cell-penetrating peptides: impact on GAGdependent internalisation and direct translocation M. Amoura, F. Illien, A. Joliot, K. Guitot, J. Offer, S. Sagan and F. Burlina reaction was repeated in larger scale at pH 6 using 3 M Guanidine and 450 mM MPAA and the compound purified by reverse phase HPLC.

Electronic Supplementary Information (ESI)

Table S2: Effect of pH, guanidine and MPAA concentrations on the half-time of ligation

Guanidine (M) MPAA (mM) pH t ½ (h)


Keywords: urban biodiversity, pets, passerines, predator, garden birds, Domestic Cat

The impact of Domestic Cats Felis silvestris catus, Cat from hereon, on biodiversity and particularly on bird populations, has been documented worldwide [START_REF] Parmalee | Food habits of the feral house cat in eastcentral Texas[END_REF][START_REF] Churcher | Predation by domestic cats in an English village[END_REF][START_REF] Barratt | Predation by house cats, Felis catus (L.), in Canberra, Australia. I: prey composition and preference[END_REF][START_REF] Loss | Population impacts of free-ranging domestic cats on mainland vertebrates[END_REF]. However, the majority of currently available studies address closed ecosystems, such as small islands [START_REF] Medina | A global review of the impacts of invasive cats on island endangered vertebrates[END_REF]. The impact of Cat populations can be very strong in these cases, even leading to local extinctions of species [START_REF] Veitch | Methods of eradicating feral cats from offshore island in New Zealand[END_REF][START_REF] Dowding | The impact of predation by introduced mammals on endemic shorebirds in New Zealand: a conservation perspective[END_REF][START_REF] Medway | The land bird fauna of Stephens Island, New Zealand in the early 1890s, and the cause of its demise[END_REF]). The impact of Cat populations appears to be less clear in continental ecosystems, especially in North America and Europe. The presence of Cats may significantly impact the number of birds through both predation events [START_REF] Woods | Predation of wildlife by domestic cats Felis catus in Great Britain[END_REF][START_REF] Winter | Impacts of feral and free-ranging cats on bird species of conservation concern: a five-state review of New York, New Jersey, Florida, California, and Hawaii[END_REF][START_REF] Dauphiné | Impacts of free-ranging domestic cats (Felis catus) on birds in the United States: a review of recent research with conservation and management recommendations[END_REF]) and lower fertility rates linked to the disturbances induced by the presence of these predators [START_REF] Beckerman | Urban bird declines and the fear of cats[END_REF][START_REF] Bonnington | Fearing the feline: domestic cats reduce avian fecundity through traitmediated indirect effects that increase nest predation by other species[END_REF]). In the United Kingdom, [START_REF] Baker | Cats about town: is predation by free-ranging pet cats (Felis catus) likely to affect urban bird populations?[END_REF] suggested that Cats have likely been a major cause of mortality for some species of birds.

Today, a tendency exists to enhance biodiversity in towns, favouring the quality of green spaces and species dispersion from peri-urban areas to city parks and gardens. This trend implies changing management strategies resulting in an increase in vertebrate diversity Roman Pavisse 1 , Didier Vangeluwe 2 & Philippe Clergeau 1,* Domestic Cat Felis silvestris catus populations have been increasing since the 1970s in Western Europe. This increase is particularly notable in human habitats, such as gardens and parks, where Cats have locally become the most abundant carnivore. In urban contexts, understanding Cat behaviour could be important for biodiversity management. In this study, we analysed Cat predation on garden birds, using existing data from ringing programmes in France and Belgium. These types of data permit us to (1) investigate the bird species killed by Cats, (2) analyse changes in the proportion of birds killed by Cats over the years 2000-2015, and (3) compare the causes of bird mortality as reported by observers. The most affected species were passerines, both granivorous and insectivorous (Europian Robin Erithacus rubecula, Dunnock Prunella modularis, Greenfinch Carduelis chloris), who can frequently be found feeding on the ground or visiting feeders in the winter. Perched birds (Chiffchaff Phylloscopus collybita, Blackcap Sylvia atricapilla) were less represented. Ground-dwelling habits were significantly correlated to predation rates. Between 2000 and 2015, Cat-related mortality in garden birds increased by at least 50%, depending on the ringing programme studied, which coincided with an increase in Cat populations. Cat-related mortality is one of the leading causes of death reported by observers (12.8-26.3% of total dead birds) and occurs at the same order of magnitude as deaths due to collisions with windows. These results underline the need to consider Cats and their potential impacts on urban biodiversity management at garden and urban levels.

and number [START_REF] Baker | Urban mammals: what does the future hold? An analysis of the factors affecting patterns of use of residential gardens in Great Britain[END_REF][START_REF] Pellissier | Birds are also sensitive to landscape composition and configuration within the city centre[END_REF]. As Cats are the main predator in towns and peri-urban landscapes (e.g. [START_REF] Coleman | Cats and wildlife: a conservation dilemma[END_REF], Lepczyk et al. 2004[START_REF] Thomas | Spatiotemporal variation in predation by urban domestic cats (Felis catus) and the acceptability of possible management actions in the UK[END_REF], it is important to assess to what extent Cats impact biodiversity in these habitats. The available studies (e.g. [START_REF] Baker | Impact of predation by domestic cats Felis catus in an urban area[END_REF][START_REF] Woods | Predation of wildlife by domestic cats Felis catus in Great Britain[END_REF][START_REF] Tschanz | Hunters and non-hunters: skewed predation rate by domestic cats in a rural village[END_REF] show that small mammals, mainly rodents, are the most abundant prey in the diet of Domestic Cats in urban and rural landscapes, but that birds can represent 12 to 24% of observed prey species.

In Europe, house Cat populations (in contrast to feral Cats) have significantly increased over the past few decades to an estimated 99 million in 2014 (FEDIAF 2014). In France, the number of house Cats has increased since 1970since , and doubled between 1990since and 2015since (FACCO 2017)). In Belgium, the house Cat population increased by 200,000 between 2008 and 2014 (SPF Economie 2016). The number of feral or stray Cats remains completely unknown.

To assess the impact of Cats on urban bird populations, we focus here on garden bird data from French and Belgian ringing programmes where the causes of death are noted. We wanted to (1) investigate the bird species killed by Domestic Cats, (2) analyse changes in the proportion of birds killed by Cats over time (2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015), and (3) compare the causes of bird mortality as reported by observers.

Methods

To study Domestic Cat predation on garden birds and examine how it has changed over time, we analysed the French and Belgian ring-recovery programme data collected from 2000 to 2015. In these two countries, the rings from birds found dead are returned with a response indicating the probable cause of death including predation by Cats (see below). In two thirds of these cases, information on ringed birds was precisely recorded allowing a quantitative analysis of causes of mortality including the prevalence of Cat predation. Those reporting these dead birds were not only ornithologists but also people who directly observed a cat eat or kill the bird and/or found a whole or partial bird body near their house. The remaining third of returns were without a cause of death.

Only data including birds recovered in France and Belgium were analysed. We assume that the probability of reporting a ringed bird killed by a Cat is the same regardless of the bird species being considered and whether it is ringed. There was no indication that reporting effort varied over the study period (Oliver Dehorter, CRBPO, pers. comm.).

To study Cat predation, we separately analysed three datasets where the causes of death were mentioned (Table 1): (1) The SPOL Mangeoire programme in France (feeder-centred monitoring of local wintering bird populations) is a capture-based monitoring programme that has been operational since 2007. This programme aims to monitor wintering birds visiting bird feeders. This monitoring is focused on specific species and habitats, i.e. garden birds, in environments where observers are numerous and where recovery rates are potentially higher. Data were collected at more than 150 sites across the country between 2007 and 2015. Hereafter, this programme is referred to as Prog1.

(2) The STOC capture (temporal monitoring of common birds through capture) protocol is for monitoring common birds in France in generally diverse environments and at the initiative of the ringers (approximately 120 locations monitored between 2000 and 2015), and a great number of protocols are dedicated to specific processes or habitats that display relatively homogeneous sensitivity to predation by Cats. This dataset is not specifically dedicated to garden birds. Hereafter, this programme is referred to as Prog2.

(3) All of the Belgian data collection programmes are included in this category. In Belgium, there are no programmes specifically dedicated to species or habitats. Data from Belgium essentially relate to the activity of workgroups that have autonomy in ringing practices. This dataset is also not specifically dedicated to garden birds. Hereafter, this programme is referred to as Prog3 (data also from 2000 to 2015).

We focused on 43 species known to be common in gardens (selected from Hagemeijer & Blair 1997; Table 1). Due to the very large difference in ringing effort between species, we avoided the few species that had less than 10,000 ringed individuals, i.e. Spotted Flycatcher Muscicapa striata, European Serin Serinus serinus, European Crested Tit Lophophanes cristatus, Great Spotted Woodpecker Dendrocopos major and Cirl Bunting Emberiza cirlus.

First, to analyse Cat predation on each of the remaining 38 species, we determined the rate of predation over the entire study period. In agreement with the very variable rate of ring returns depending on the species (Oliver Dehorter, CRBPO, pers. comm.), we have chosen to calculate the predation rate on the total of ringed individuals of a species and not on the total of returns of ringed birds. As a result, the values will be very low. For each programme, we tested the hypothesis that the annual change of the Cat-related death rate for all birds followed a linear model using the R v. 3.2.5 software (R Development Core Team 2016). Second, to determine if ground-feeding was a trait of most predated birds, we characterized each species into Group 1: bird species rarely feeding on the ground, Group 2: bird species sometimes feeding on the ground, or Group 3: bird species frequently feeding on the ground (from [START_REF] Voous | Atlas of European birds[END_REF][START_REF] Yeatman-Berthelot | Atlas des oiseaux en hiver[END_REF][START_REF] Mullarney | The complete guide to the birds of Europe[END_REF][START_REF] Dubois | Inventaire des oiseaux de France[END_REF]. We used a Wilcoxon non-parametric test to compare pairs in each Group; it is assumed that there is no heterogeneity of variance in the samples (the absence of outliers and the very low dependence between observations has been verified).

Third, to assess the rate of Cat predation in comparison with other causes of mortality, we selected the following 10 categories that grouped different causes of death, selected from EURING and CRBPO databases:

(1) shooting, (2) deliberate trapping (lime traps, glue traps and boxes to obtain song birds or bird meat), (3) involuntary trapping (crop-protection nets, drowning in artificial basins), (4) collisions with traffic (road, rail), ( 5) collisions with buildings (window panes, high-rise buildings), ( 6) predation by Cats, (7) predation of unknown origin/by other species, (8) natural causes (diseases, weather), ( 9) other causes and (10) causes unknown or undetermined. Here, we assessed the importance of Cat predation in comparison with the total of causes of mortality (in percentages).

Results

BIRD SPECIES KILLED BY CATS

Across the three programmes, the number of bird species killed by Cat predation was 83. How ever, focusing on our 38 garden bird species (Table 1 In Prog3, the most preyed upon species were European Greenfinch, European Robin and Blackbird.

The differences among the other species were minimal, and the distribution of predated species was more homogeneous. Based on the ranking process, several common species confirmed this trend, such as the Dunnock and Eurasian Collared Dove.

Overall (proportion of all returns on all the ringed birds of the three programmes), the most predated species were European Greenfinch, Eurasian Collared Dove and House Sparrow, which are three granivorous species. Comparing between bird species with different levels of ground-feeding showed that in the group of species that usually feeds or moves on the ground, Group 3, mortality rate by Cats was significantly higher (42 out of 10,000 ringed birds) than Group 2 (19 from 10,000) and Group 1 (12 from 10,000; Wilcoxon tests, both P < 0.0001).

TEMPORAL CHANGES IN CAT PREDATION

For each of the three programmes, an increasing trend in the proportion of birds killed by Cats was observed. For Prog1, we noted an average increase of 102% between 2007 and 2015. This increase was strong and significant (linear model, r 2 = 0.674, df = 38, P = 0.015). For Prog2, the proportion of birds killed by Cats during 2000-2015 increased by 82% (r 2 = 0.597, df = 439, P = 0.022). Data from Prog3 also showed an increasing trend in Cat predation, although at 52% it was less strong (r 2 = 0.422, df = 314, P = 0.044). The analysis of the three datasets describes mortality caused by Cats as increasing over the last ten years.

COMPARISON OF CAUSES OF DEATH

The Prog1 data showed a preponderance of predation by Cats (26.3% of the total, n = 146), followed by mortality due to collisions with window panes and buildings and then mortality from trapping, predominantly intentional trapping. Prog2 was distinguished by its very high incidence of mortality from shooting (39.6%, n = 3439), followed by predation by other species and by Domestic Cats. Mortality from shooting was very variable over time and was irregularly reported by observers. Prog3 showed a strong prevalence of mortality due to collisions with windows and buildings (25.7%, n = 1933) followed by shooting and by mortality caused by Cats. The cause of predation by Cats always appeared as one of the three first causes of mortality in each programme (Table 2).

Discussion

BIRD SPECIES KILLED BY CATS Our results demonstrate higher predation rates for ground dwelling birds, whether they are granivorous (House Sparrow, European Greenfinch and Eurasian Collared Dove) or insectivorous (European Robin, Blackbird and Dunnock). In addition, these species regularly visited bird feeders where they are more vulnerable [START_REF] Dunn | Predation of birds at feeders in winter[END_REF]. [START_REF] Mead | Ringed birds killed by cats[END_REF] showed an especially high recovery (31%) of ringed Robins and Dunnocks in England. The population modelling study of [START_REF] Thomas | Spatiotemporal variation in predation by urban domestic cats (Felis catus) and the acceptability of possible management actions in the UK[END_REF] in the UK suggested that predation rates could significantly reduce the size of local bird populations for common urban species.

It appears that the habit of dwelling or visiting gardens is not the principal risk factor in terms of predation by Cats. The Eurasian Blue Tit Cyanistes caeruleus and Great Tit Parus major are abundant in urban areas rich in Cats, and they frequently visit bird feeders where they are predated on to a lower extent. Similarly, birds that are abundant in gardens, but which use mainly bushes and trees, such as Chiffchaff Phylloscopus collybita and Blackcap Sylvia atricapilla, were rarely predated on by Cats. [START_REF] Woods | Predation of wildlife by domestic cats Felis catus in Great Britain[END_REF], despite using a different research method (prey brought by Cats into houses), also documented a strong representation of ground-foraging species such as House Sparrow and Blackbird, but they also cited tit species. Data given by different authors in different continents also underlined the important proportion of groundfeeding passerines in the diet of Cats (e.g. [START_REF] Baker | Impact of predation by domestic cats Felis catus in an urban area[END_REF][START_REF] Blancher | Estimated number of birds killed by house cats (Felis catus) in Canada[END_REF]).

TEMPORAL CHANGE IN CAT PREDATION Cat predation on birds increased by 52 to 102% over the past 15 years. This increase appears connected with the parallel increase in the number of Cats in the two countries. Over the past 15 years (2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014), the number of house Cats grew from 9.67 to 12.7 million in France (+30.1%; FACCO 2017). Whereas between 2008 and 2014, they increased by 10% in Belgium (SPF Economie 2016). We do not have information about the number and species of birds killed by feral Cats, which could have greatly increased over the same period in western European cities. However, feral Cats seem to remain a problem for cities since sterilization and capture programmes continue to be carried out by most municipalities: the feeding of feral Cats by city dwellers is still a common practise and kitten litters are often abandoned.

COMPARISON OF THE CAUSES OF DEATH In each program, Cat predation appears as one of the three main causes of death in garden birds. Cat predation was a particularly strong factor in the programme dedicated to garden birds. In the USA, [START_REF] Loss | The impact of free-ranging domestic cats on wildlife of the United States[END_REF] also reported the importance of predation by Cats; they extrapolated results and suggested that billions of birds were caught by Cats and hundreds of millions of birds were killed by collisions with buildings and vehicles. Collisions, mainly with windows, are unanimously recognized by ornithologists as a significant hazard for birds either living near inhabited areas or migrating [START_REF] Machtans | A first estimate for Canada of the number of birds killed by colliding with building windows[END_REF]. [START_REF] Erickson | A summary and comparison of bird mortality from anthropogenic causes with an emphasis on collisions[END_REF] estimated that buildings, power lines and Cats were the causes of approximately 82% of the anthropogenic mortality of birds in the USA. To date, there has been a lack of data to identify house Cats as a factor in the decline of some bird populations throughout large areas. Moreover, Cat predation seems difficult to generalize across biotopes, regions or periods [START_REF] Thomas | Spatiotemporal variation in predation by urban domestic cats (Felis catus) and the acceptability of possible management actions in the UK[END_REF][START_REF] Krauze-Gryz | Annual variation in prey composition of domestic cats in rural and urban environment[END_REF]. The decline of bird populations clearly involves numerous factors requiring new studies.

In fragile ecosystems such as urban areas, several solutions, more or less severe, have been proposed to reduce predation by Cats. For example, one solution is keeping house Cats indoors as much as possible [START_REF] Mcdonald | Reconciling actual and perceived rates of predation by domestic cats[END_REF]. Devices to reduce Cat predation on birds and other vertebrates are already available, and some have already been proven to be effective: colourful collars, making Cats more obvious to prey [START_REF] Calver | Reducing the rate of predation on wildlife by pet cats: the efficacy and practicability of collar-mounted pounce protectors[END_REF], collars emitting specific sounds [START_REF] Clark | A pilot field trial into the effectiveness of the CatAlert™ collar at reducing predation by domestic cats[END_REF] or belled collars [START_REF] Ruxton | Bells reduce predation of wildlife by domestic cats (Felis catus)[END_REF]. The issue of their acceptability by Cat owners and the possibility of developing them at a large scale remains unknown. Limiting Cats is another issue. An official programme targeting feral Cats is currently being developed in Belgium that includes systematic catching and sterilizing campaigns. In numerous French cities, Cats are also being controlled by mandatory individual identification and now systematic sterilization. This subject is not only a technical issue but also a social issue.

In conclusion, new research on cat populations and on the different aspects of its impact on biodiversity (especially in highly anthropized areas such as cities) needs to be developed. For example, we still know little about the health status of the birds killed by Cats and whether predation by Cats is additive or compensatory to natural mortality in birds.

Samenvatting

Sinds de jaren zeventig is het aantal Huiskatten Felis silvestris catus in West-Europa toegenomen. Deze toename is vooral merkbaar in tuinen en parken in bewoond gebied, waar de huiskat lokaal de meest talrijke carnivoor is geworden. In het stedelijk gebied kan inzicht in het gedrag van katten belangrijk zijn voor het beheer van de biodiversiteit. In dit onderzoek hebben wij de predatie van katten op tuinvogels geanalyseerd met behulp van bestaande gegevens uit ringprogramma's in België en Frankrijk. Deze gegevens maken het mogelijk om (1) de door katten gedode vogelsoorten vast te stellen, (2) veranderingen in het aantal door katten gedode vogels te analyseren in de jaren 2000-2015 en (3) de doodsoorzaken, zoals gerapporteerd door waarnemers, te analyseren. De meest gepredeerde soorten waren zangvogels, zowel zaadetende als insectenetende (Roodborst Erithacus rubecula, Heggenmus Prunella modularis, Groenling Carduelis chloris), die vaak in de winter op de grond foerageren of voedertafels bezoeken. Vogels die niet op de grond foerageren (Tjiftjaf Phylloscopus collybita, Zwartkop Sylvia atricapilla) waren minder vertegenwoordigd. Grondgebonden gedrag was significant gecorreleerd met de mate van predatie. Tussen 2000 en 2015 nam de sterfte door kattenpredatie bij tuinvogels met minstens 50% toe, afhankelijk van het onderzochte ringprogramma. Dit viel samen met een groei van de kattenpopulaties. De sterfte door kattenpredatie is een van de belangrijkste doodsoorzaken blijkens de waarnemers (12,8-26,3% van alle dode vogels), wat in dezelfde orde van grootte is als sterfte als gevolg van botsingen tegen ramen. Het resultaat van ons onderzoek benadrukt de noodzaak om de gevolgen van het mogelijke effect van katten voor het beheer van de stedelijke biodiversiteit in overweging te nemen, zowel op het niveau van tuinen als dat van steden in het algemeen.

Table 1 .

 1 The 38 passerine and dove species obtained from the three programmes (Prog1 for wintering birds in French gardens, Prog2 for STOC in France and Prog3 for Belgian data) and category of ground-feeding (1: rarely, 2: sometimes, and 3: frequently on the ground). For each programme the number of ringed birds is listed. Species are ordered according to decreasing predations rate, which is the percentage of ringed birds predated by Cats over all years combined.

	Species	Category of		Number ringed		Predation rate
		ground-feeding	Prog1	Prog2	Prog3	Total	(% of ringend birds, ×10 -5 )
	European Greenfinch Chloris chloris	3	43,300	32,501	185,700	261,501	56.21
	Eurasian Collared Dove Streptopelia decaocto	2	239	16,978	18,119	35,336	50.94
	House Sparrow Passer domesticus	3	8969	72,138	54,300	135,407	48.74
	Common Blackbird Turdus merula	3	4479	79,311	142,140	225,930	48.24
	European Robin Erithacus rubecula	3	3204	115,229	397,000	515,433	39.97
	European Goldfinch Carduelis carduelis	3	15,941	20,747	31,552	68,240	39.57
	Dunnock Prunella modularis	3	2042	38,790	272,500	313,332	36.38
	Eurasian Siskin Spinus spinus	2	17,219	11,221	134,200	162,640	35.66
	Black Redstart Phoenicurus ochruros	2	178	5414	6800	12,392	32.28
	Eurasian Blue Tit Cyanistes caeruleus	1	48,179	140,223	646,500	834,902	32.10
	Eurasian Wren Troglodytes troglodytes	2	595	17,645	42,280	60,520	31.39
	Great Tit Parus major	1	47,709	110,792	801,000	959,501	27.72
	Hawfinch Coccothraustes coccothraustes	2	999	1591	8990	11,580	25.91
	Common Starling Sturnus vulgaris	3	849	14,019	165,166	180,034	22.22
	Eurasian Bullfinch Pyrrhula pyrrhula	2	391	6021	7426	13,838	21.68
	Song Thrush Turdus philomelos	2	339	42,842	133,051	176,232	20.99
	Coal Tit Periparus ater	1	1289	11,757	73,600	86,646	17.31
	Long-Tailed Tit Aegithalos caudatus	2	1530	20,114	43,357	65,001	16.92
	Common Redstart Phoenicurus phoenicurus	2	25	8184	23,400	31,609	15.82
	Eurasian Nuthatch Sitta europaea	1	970	2224	22,600	25,794	15.51
	Common Chaffinch Fringilla coelebs	2	9986	20,556	147,350	177,892	13.49
	Eurasian Tree Sparrow Passer montanus	2	1181	6585	105,300	113,066	13.28
	Eurasian Blackcap Sylvia atricapilla	1	1269	352,517 1,646,000 1,999,786	12.60
	Yellowhammer Emberiza citrinella	2	157	4385	12,836	17,378	11.51
	Common Swift Apus apus	1	1	3155	7290	10,446	9.57
	Common Wood Pigeon Columba palumbus	2	16	43,699	11,200	54,915	9.11
	Barn Swallow Hirundo rustica	1	29	213,935	276,000	489,964	8.57
	Short-Toed Treecreeper Certhia brachydactyla	1	286	3871	9031	13,188	7.58
	White Wagtail Motacilla alba	2	21	2917	63,700	66,638	7.50
	Goldcrest Regulus regulus	2	589	12,064	82,100	94,753	7.39
	Brambling Fringilla montifringilla	2	4795	4438	85,662	94,895	7.38
	Marsh Tit Poecile palustris	2	1989	4439	7900	14,328	6.99
	European Pied Flycatcher Ficedula hypoleuca	2	2	14,405	46,300	60,707	6.59
	Common Chiffchaff Phylloscopus collybita	1	541	124,081	328,200	452,822	6.62
	Common Linnet Linaria cannabina	1	25	6416	122,052	128,493	5.45
	Common House Martin Delichon urbicum	1	14	13,359	27,500	40,873	4.89
	Redwing Turdus iliacus	2	108	9066	182,650	191,824	3.65
	Eurasian Magpie Pica pica	1	23	1551	61,110	62,684	0
	Total		219,478 1,609,180 6,431,862 8,260,520	22.26

Shooting Deliberate Involuntary Collisions Collisions Predation Predation by Natural Other trapping trapping with traffic with buildings by Cats other species causes causes

	Prog1	1.26	21.08	1.98	8.65	21.62	26.31	12.79	6.13	0.18
	Prog2	39.64	7.85	1.90	5.92	8.94	12.76	14.85	8.07	0.06
	Prog3	18.59	17.60	1.43	8.96	25.67	16.31	5.62	3.88	1.93

Table 2 .

 2 Causes of death of ringed birds according to the three ringing programmes (in percentage of dead birds found): Prog1 for wintering birds in French gardens, Prog2 for Programme STOC in France, and Prog3 for Belgian data.

We are grateful to Frédéric Jiguet, Pierre-Yves Henry and Olivier Dehorter from Centre de Recherche sur la Biologie des Populations d'Oiseaux (CRBPO) for their advice. Thank you also to the Belgian Ringing Scheme, the Royal Belgian Institute of Natural Sciences (Scientific Political SPP) and all the voluntary ringers who collected the data. We are grateful to the Region Ilede-France for supporting this project (AAP PICRI 110971).