extfieldsofstudy: Medicine Chemistry

s2fieldsofstudy: Biology Chemistry

text:
Structural Basis of Tau Interaction With BIN1 and Regulation by Tau Phosphorylation

Bridging integrator-1 (BIN1) gene is associated with an increased risk to develop Alzheimer’s disease, a tauopathy characterized by intra-neuronal accumulation of phosphorylated Tau protein as paired helical filaments. Direct interaction of BIN1 and Tau proteins was demonstrated to be mediated through BIN1 SH3 C-terminal domain and Tau (210–240) peptide within Tau proline-rich domain. We previously showed that BIN1 SH3 interaction with Tau is decreased by phosphorylation within Tau proline-rich domain, of at least T231. In addition, the BIN1/Tau interaction is characterized by a dynamic equilibrium between a closed and open conformations of BIN1 isoform 1, involving an intramolecular interaction with its C-terminal BIN1 SH3 domain. However, the role of the BIN1/Tau interaction, and its potential dysregulation in Alzheimer’s disease, is not yet fully understood. Here we showed that within Tau (210–240) peptide, among the two proline-rich motifs potentially recognized by SH3 domains, only motif P216TPPTR221 is bound by BIN1 SH3. A structural model of the complex between BIN1 SH3 and Tau peptide (213–229), based on nuclear magnetic resonance spectroscopy data, revealed the molecular detail of the interaction. P216 and P219 within the proline-rich motif were in direct contact with the aromatic F588 and W562 of the BIN1 SH3 domain. The contact surface is extended through electrostatic interactions between the positively charged R221 and K224 residues of Tau peptide and those negatively charged of BIN1 SH3, corresponding to E556 and E557. We next investigated the impact of multiple Tau phosphorylations within Tau (210–240) on its interaction with BIN1 isoform 1. Tau (210–240) phosphorylated at four different sites (T212, T217, T231, and S235), contrary to unphosphorylated Tau, was unable to compete with the intramolecular interaction of BIN1 SH3 domain with its CLAP domain. In accordance, the affinity of BIN1 SH3 for phosphorylated Tau (210–240) peptide was reduced, with a five-fold increase in the dissociation constant, from a Kd of 44 to 256 μM. This study highlights the complexity of the regulation of BIN1 isoform 1 with Tau. As abnormal phosphorylation of Tau is linked to the pathology development, this regulation by phosphorylation might have important functional consequences.

INTRODUCTION
Alzheimer's disease (AD) is the most common form of dementia and the most prevalent tauopathy. In genome-wide association studies, variants of the BIN1 gene coding for bridging integrator-1 (also known as amphiphysin 2) have been identified as the second highest genetic risk factor for AD after the ApoE gene, (Seshadri et al., 2010;Genin et al., 2011;Lambert et al., 2013). Several hypothesis have emerged on how BIN1 protein contributes to the increased risk of developing AD, which could involve both Aβ and Tau neurodegeneration pathways (Chapuis et al., 2013;Calafate et al., 2016;Ubelmann et al., 2017). The BIN1 gene is subject to extensive tissue-specific alternative splicing, leading to several isoforms with different cellular localizations and functions. BIN1 protein isoforms 1-7 are expressed in the brain, isoform 8 is expressed in the muscle, and isoforms 9 and 10 are expressed ubiquitously (Prokic et al., 2014). All BIN1 isoforms contain a N-terminal Bin/amphiphysin/Rvs (BAR) domain and a C-terminal SH3 domain (BIN1 SH3) (Prokic et al., 2014). The BAR domain has a role in membrane remodeling by inducing curvature (Frost et al., 2009), and the SH3 domain mediates BIN1's interactions with several partners (such as dynamin). However, only the brain-specific BIN1 isoforms contain a clathrin and AP-2-binding domain (CLAP) that mediates the interaction with clathrin and AP2 -suggesting a specific role for BIN1 in clathrin-mediated endocytosis in the brain (David et al., 1996;Takei et al., 1999). BIN1 was recently shown to negatively regulate this process and it has been proposed that lower Bin1 expression may favor Tau propagation through increased endocytosis (Calafate et al., 2016). Regulation of endosomal trafficking by BIN1 is also proposed to affect Aβ42 endocytic production by negatively regulating APP and BACE1 convergence in early endosomes (Ubelmann et al., 2017). BIN1 loss of function thus prevents APP and BACE1 segregation at early endosomes and may boost Aβ accumulation.
The BIN1 gene is the first of the sporadic AD genetic determinants to have been linked to a Tau pathology (Chapuis et al., 2013). BIN1 is upregulated in the AD brain, and its expression correlates with the neurofibrillary tangle pathology caused by intraneuronal aggregates of abnormally phosphorylated Tau proteins (Chapuis et al., 2013;Holler et al., 2014). Furthermore, the BIN1 homolog AMPH mediates Tau toxicity in a Drosophila melanogaster model overexpressing Tau (Chapuis et al., 2013;Dourlen et al., 2017). BIN1 directly interacts with Tau and the BIN1/Tau complex is observed at specific locations in primary neurons (partly co-localized with the actin cytoskeleton network) (Sottejeau et al., 2015). BIN1 was indeed shown to modulate actin dynamics by both having actin bundling activity and stabilizing Tau-induced actin bundles (Dräger et al., 2017). Deregulation of BIN1/Tau complex formation may thus impact neuronal functions.
During the disease progression, Tau presents both an increased phosphorylation level and a modification of its pattern of phosphorylation. Tau is phosphorylated by various kinases, including proline-directed kinases [like CDK5 (Baumann et al., 1993;Kimura et al., 2014), GSK3 (Ishiguro et al., 1993;Cho and Johnson, 2003), and ERK2 (Drewes et al., 1992)], that mainly target the proline rich domain (PRD) of Tau Leroy et al., 2010;Qi et al., 2016). For example, the AT8 diagnostic monoclonal antibody [pS202 + pT205 + (pS208) epitope] can be used to track the spatial progression of the pathology in the AD brain over time (Braak et al., 2006). Given that Tau has so many phosphorylation sites, characterizing the impact of these modifications on Tau functions is an analytical challenge (Lippens et al., 2012a,b). Yet, it remains of interest to decipher the functional consequences of specific Tau phosphorylation, and in the case of the present study, how it could modulate the BIN1/Tau interaction.
We have previously reported that the BIN1 SH3 binds directly to Tau PRD (Sottejeau et al., 2015). The interaction motif within the Tau PRD was narrowed down to a sequence located between amino acid residues 210-240, which contains two PXXP SH3 binding motifs (Sottejeau et al., 2015). This interaction is weakened by phosphorylation of the Tau PRD (Sottejeau et al., 2015), and in particular, phosphorylation of T231 (Sottejeau et al., 2015). Furthermore, we recently showed that the SH3 domain of the neuronal-specific BIN1 isoform 1 protein is engaged in a dynamic equilibrium between open and closed conformations (Malki et al., 2017). The closed conformation results from BIN1-SH3's interaction with the BIN1-CLAP domain. Tau (210-240) is able to shift the equilibrium toward the intermolecular interaction -indicating a subtle regulation of the Tau/BIN1 interaction.
As studying the regulation of the BIN1/Tau interaction is an important step in understanding its specific contribution to the AD process, we further pursued this investigation. Here, we first determined which PXXP motif(s) is(are) critical for the BIN1/Tau interaction. Based on the NMR data, we proposed next a structural model of the complex between BIN1 SH3 and a Tau peptide, which provides the atomic detail of the interaction. Lastly, we looked at whether Tau (210-240) phosphorylation affects the dynamic equilibrium between open and closed conformations of BIN1 isoform 1.

Proteins and Peptides
The BIN1 isoform 1 and BIN1 SH3 domain were expressed and purified as previously described (Malki et al., 2017). cDNA encoding Tau (210-240) peptide SRTPSLPTPPTREPKKVAVVRTPPKSPSSAK was cloned into the pETNKI-HisSUMO3-LIC-Kan vector following a ligation-independent cloning procedure (Luna-Vargas et al., 2011). Uniformly labeled 15 N and 13 C or 15 N protein/peptide samples were produced in M9 medium supplemented with 0.1 mM CaCl 2 and 2 mM MgSO 4 and containing 0.1% 15 NH 4 Cl and 0.2% 13 C-glucose (Sigma-Aldrich) or 0.4% glucose, with 0.5 g/l of 15 N-and 13 C-enriched or 15 N-enriched ISOGROW (Sigma-Aldrich), depending of the labeling scheme. The recombinant His 6 -SUMO Tau (210-240) was purified using a nickel affinity chromatography column and then incubated 16 h with SENP2 protease in the presence of 2 mM DTT, at 4 • C. The protein sample was buffer-exchanged using a desalting column (G25 resin, cutoff of 7 kDa; PD-10 GE Healthcare) against 50 mM sodium phosphate pH 7.3, 30 mM NaCl, 3 mM DTT, and then loaded on a nickel affinity chromatography column. The tag-cleaved peptide was then recovered in the flow-through.
The H α C α N spectra (Wang et al., 1995) of Tau (210-240) peptide at 0.5 mM were recorded at 20 • C with the standard Bruker pulse sequence in the NMR buffer containing 100% D 2 O. BIN1 isoform 1 1 H-15 N HSQC spectra were recorded at 20 • C at 900 MHz (0.08 mM sample) with 3096 points (direct) and 280 points (indirect), for spectral width of 14 ppm and 26 ppm, respectively, and 256 scans. A 3D F1 13 C/ 15 N-filtered and F3 15 Nedited NOESY-HSQC, with WATERGATE (WATER suppression by GrAdient-Tailored Excitation) scheme (Piotto et al., 1992), named noesyhsqcf3gpwgx13d, part of the Bruker pulse sequence library, was recorded at 20 • C at 950 MHz for the detection of intermolecular NOEs to amide groups. This data was used to solve the structure of the complex between BIN1 SH3 domain and Tau (210-240). Spectra were processed using TopSpin software (Bruker) and analyzed by CcpNmr Analysis (Vranken et al., 2005).  Figure S1), 13 data points were recorded, using relaxation delays between 10 and 150 ms (recycle delay was 4 s). The heteronuclear NOE experiment (Supplementary Figure S1) was recorded by including, or not, a 4 s period of 120 • 1 H saturation pulses, separated by 5 ms. Recovery delay was 4 s. Uncertainties in peak intensities were estimated from one triplicate data point.

Phosphorylation of Tau (210-240)
Phosphorylation of SUMO-Tau (210-240) was obtained by incubation with recombinant CDK2/CycA3 kinase (Welburn and Endicott, 2005) (molar ratio 1/100), for 3 h at 37 • C, in the presence of 2 mM ATP, 2.5 mM MgCl 2 , 2 mM DTT, and protease inhibitors in 50 mM HEPES, pH 8.0. The incubation was repeated once after exchange into fresh buffer and upon new addition of kinase. The His-SUMO tag was removed after phosphorylation, as described here above for the unmodified peptide. Completion of phosphorylation was assessed by NMR 2D 1 H-15 N HSQC of the 15 N-Tau (210-240) peptide and MALDI-TOF (Matrix Assisted Laser Desorption Ionization -Time of Flight) mass spectrometry. Mass analysis was performed in ion-positive reflector mode on an ABI Voyager DE-Pro MALDI-TOF mass spectrometer (Applied Biosystems), using as matrix a saturated solution of α-cyano-4-hydroxycinnamic acid in CH 3 CN:H 2 O:CF 3 COOH (50:50:0.1). Phosphorylated Tau (210-240) 1 H-15 N HSQC spectrum was recorded at 600 MHz, peptide concentration 0.5 mM, resolution 2048 points in the direct dimension and 256 points in the indirect dimension, spectral width 14 and 17 ppm, respectively, and 16 scans. Based on the relative intensities of the NMR resonance signals (Supplementary Figure S3), full modification was observed for residues T212, T231, and S235 while residue T217 was halfphosphorylated. Results from MS analysis were in agreement (Supplementary Figure S4). All the purification steps were performed at 4 • C in the presence of a protease inhibitor cocktail (Sigma-Aldrich).

Dissociation Constant Determination
Titration of 70 µM 15 N-BIN1 SH3 with increasing amount of unlabeled Tau (210-240) (from 35 to 840 µM) peptides was performed, monitoring the complex formation by the gradual 1 H and 15 N chemical shift change of the resonances in 1 H, 15 N HSQC, at pH 7.3 (50 mM phosphate buffer) and 20 • C. The weighted average chemical shift differences were calculated as described by Garrett et al. (1997) -i.e., ( H 2 + ( N×0.2) 2 ) 1/2 , with H and N the chemical shift changes for 1 H and 15 N, respectively. Dissociation constants were obtained by fitting the chemical shift perturbation data to the following equation where δobs is the weighted average of the chemical shifts in the free and bound states and δmax is the maximal signal change upon saturation. K d is the dissociation constant, a and b are the total peptide and BIN1 SH3 concentrations, respectively. K d were averaged based on chemical shift perturbations of 7 distinct resonances.

Proline Assignment in Tau (210-240) Peptide
We have previously shown direct interaction of the BIN1 SH3 (BIN1 SH3) with the PRD of Tau [PRD,] (Sottejeau et al., 2015). The interaction motif within Tau PRD was narrowed-down to a sequence located between amino acid residues 210-240. In this previous work, 1 H-15 N HSQC spectra were used for chemical shift perturbation analysis. As 1 H-15 N HSQC experiment correlates the resonances of directly bound 1 H and 15 N nuclei, the majority of the amino acid residues in the peptide was represented in this analysis, except proline residues, due to the lack of the backbone amide proton. However, Tau (210-240) has eight proline residues organized in two different canonical SH3 binding sites (Feng et al., 1994;Lim et al., 1994). One of class I, PPII.1, between residues 230 to 236, and another one of class II, PPII.2, between amino acid residues 216 and 221 (Figure 1A). To investigate the role of these proline residues in Tau (210-240)/BIN1 SH3 interaction, a specific NMR strategy was used, based on H α -15 N correlation, allowing sequential assignment of molecules rich in prolines (Kanelis et al., 2000;Ahuja et al., 2016). All the proline residues in Tau (210-240) were assigned, including their minor cis conformers ( Figure 1B).

Tau (210-240) Fragment Interacts With BIN1 SH3 Domain Using PPII.2 Motif
After assignment of all the proline residues in Tau (210-240) fragment, we next looked at whether the two proline rich motifs of Tau (210-240) were involved in the interaction with BIN1 SH3 domain. Comparison of the 2D HA(CA)N spectra of Tau (210-240) in the absence and presence of an excess of BIN1 SH3 domain showed that all resonances corresponding to proline residues in the proline-rich motif PPII.2 disappeared in the presence of the BIN1 SH3 domain (Figure 2). Similarly, peaks corresponding to P223 at the C-terminal end of the PPII.2 were broadened beyond detection, whereas those of P213, in the N-terminal of PPII.2, were less affected and only showed minor chemical shift modifications. Resonances of proline residues in the proline-rich motif PPII.1 were barely affected, neither by broadening nor by chemical shift modification. This result clearly indicated that Tau (210-240) interacts with BIN1 SH3 domain using the PPII.2 proline-rich motif within its sequence.

Structural Model of the BIN1 SH3/Tau (210-240) Peptide Complex
We next used these data as a starting point to obtain a structural model of BIN1 SH3 in complex with Tau (210-240) peptide. First, NMR dynamics experiments provided insights into the complex formation. 15 N-relaxation measurements of 15 N BIN1 SH3 showed homogenous R1 and R2 values, compatible with a compact structure (Supplementary Figure S1). A correlation time τc of 6.3 ± 0.3 ns was calculated, based on the average R1 and R2 values, as expected for a monomeric BIN1 SH3 domain in the NMR samples (Supplementary Figure S1). In the presence of the peptide, average R1 values were increased while average R2 values were decreased, corresponding to an increase in the molecular weight of the system. A correlation time τc of 9.7+/−0.4 ns was calculated for BIN1 SH3/Tau (210-240) complex, based on the average R1 and R2 values, compatible with a monomeric state of a 1:1 complex. NMR data were next used to build a model of the complex, using HADDOCK program. The docking was started from an NMR structure of BIN SH3 (Tossavainen et al., 2016) and a model of Tau (213-229) peptide. Starting from these two initial individual structures, the docking was conducted under restraints derived from the NMR experimental data (Supplementary Figure S2 and Supplementary Table S3). The four best structures resulting from the docking calculation, in the cluster of conformations showing the best statistics (cluster 1 in Supplementary Table S4), were chosen as being representative of BIN1 SH3/Tau (213-229) complex (Figures 3, 4). BIN1 SH3 accommodated the P 216 xxP 219 consensus motif of Tau peptide (Figures 1, 2) into the canonical hydrophobic xP binding pocket of the SH3 domain (Figure 3). Specific hydrophobic interactions were observed between the side-chains of F588 on the BIN1 SH3 side and P216 on the Tau peptide, and between the side-chains of W562 and P219 ( Figure 3A). In contrast, P218 side-chain pointed to the opposite direction with respect to P219 and was not interacting with BIN1 SH3 domain. Moreover, residues E556 and E557 in the n-Src loop of the specificity zone of BIN1 SH3 provided an additional anchoring point. Both E556 and E557 side-chains were oriented toward the Tau (213-229) peptide, in close proximity to K224 side-chain ( Figure 3B). Mutation of K224 and K225 into alanines in Tau (210-240) peptide indeed resulted in a reduced binding affinity to the BIN1-SH3 domain, with a dissociation constant K d of 44 ± 3 µM for Tau (210-240) peptide increasing by a factor of ten to 429 ± 94 µM for the mutated peptide (Supplementary Figure S3). Finally, at the C-terminus of the docked Tau (213-229) peptide, V228/V229 H α protons were also in close contact with E557 backbone H-N, as observed by intermolecular NOEs (Supplementary Table S3). The position of Tau peptide on the surface of BIN1 SH3 showed a good match with the major chemical shift perturbations of BIN1 SH3 1 H, 15 N resonances on addition of Tau (210-240) peptide (Figures 4A,B), highlighting the β5 strand, the n-Src loop and in addition the RT loop ( Figure 4B). Reporting the electrostatic potential on the BIN1 SH3 surface additionally showed positioning of the R221 and K224 side-chain on a negatively charged region ( Figure 4C). The structure of the BIN1 SH3/Tau (213-229) peptide complex showed involvement in the binding of both the xP pocket and the specificity region of BIN1 SH3 (n-Src loop), mediated on the Tau side mainly by P216/P219 and R221/K224, respectively (Figure 4).

Interaction of BIN1 Isoform 1 With Tau (210-240) Is Weakened by Phosphorylation
We previously showed that BIN1 SH3 domain was engaged in an intramolecular interaction with BIN1 isoform 1 own CLAP domain (Malki et al., 2017). Tau (210-240) peptide was FIGURE 2 | Critical proline residues involved in Tau binding to BIN1. The 2D HA(CA)N spectrum of Tau (210-240) peptide free or in the presence of BIN1 SH3 domain molar ratio 1:4 is shown in blue and green, respectively. The proline residues affected by the presence of BIN1 SH3 domain are indicated by red circles. In addition, the position of each affected proline residue within Tau (210-240) sequence is numbered in the sequence below the spectrum. able to compete with this interaction, shifting the dynamic equilibrium from a closed to an open conformation. As NMR is sensitive to mobility, this equilibrium translated into detection of BIN1 SH3 domain signals only upon its release from the intramolecular interaction (Malki et al., 2017). We further addressed whether such equilibrium between open and closed states of BIN1 isoform 1 might be affected by phosphorylation of Tau (210-240). A 1 H-15 N HSQC spectrum of BIN1 isoform1 was thus acquired in the absence or presence of an excess of Tau (210-240) phosphorylated at residues T212, T217, T231, and S235 (Supplementary Figures S4), all proline-directed sites within this peptide. Addition of phosphorylated Tau (210-240) peptide to BIN1 isoform1 sample did not result in the same changes in the spectrum (Figure 5, compare A with B), although some weak signals from BIN1 SH3 were still visible. This indicated that Tau (210-240) phosphorylated at multiple sites was not able to compete with the intra-molecular interaction of BIN1 SH3 with the CLAP domain as efficiently as its unphosphorylated counterpart. Indeed, comparison of the affinity of the isolated BIN1 SH3 domain for the Tau peptide (210-240), unphosphorylated or not, showed an increase of the dissociation constant K d , from 44 ± 3 µM to 256 ± 12 µM for the phosphorylated peptide (Figure 6 and Supplementary Figure S5). Consequently, in the presence of the phosphorylated Tau peptide (210-240), BIN1 SH3 domain remained immobilized by the intramolecular interaction with BIN1 isoform1 CLAP domain, mainly adopting the closed conformation.

DISCUSSION
BIN1 gene was found as a genetic determinant of AD, allowing to address new patho-physiological hypothesis (Lambert et al., 2009;Seshadri et al., 2010). A first step in deciphering the role of BIN1 in AD dysfunctions was taken by the discovery of its direct interaction with Tau protein (Chapuis et al., 2013), a well-known actor of the disease. The specific function of the interaction of BIN1 with Tau, however, remained to date to be uncovered, and in this context emphasized the need to grasp its molecular detail. Here we first sought the atomic detail of the interaction of BIN1 SH3 with Tau. The BIN1 interaction motif within Tau PRD is a sequence located between amino acid residues 210-240, containing two potential SH3-binding motifs (Sottejeau et al., 2015;Malki et al., 2017). The binding site for the BIN1 SH3 within this sequence was found here to be specific for a canonical SH3 class II binding motif PXXPX+ (Feng et al., 1994;Lim et al., 1994) corresponding to P 216 TPPTR 221 . The identification was based on HA(CA)N spectra allowing for the detection of the resonances specific for the proline residues, which are absent in a classical 2D 1 H, 15 N HSQC spectrum. Assignment of the Pro residues matched previous assignment of Pro residue resonances in peptides and fragments of Tau encompassing  Tau (210-240) (Smet et al., 2004(Smet et al., , 2005Ahuja et al., 2016). Our analysis also confirmed that the proline residues mainly adopt the trans conformation (Smet et al., 2004(Smet et al., , 2005Ahuja et al., 2016). The BIN1 SH3/Tau (213-229) peptide complex structure further showed that the consensus motif is bound to the hydrophobic xP binding pocket of BIN1 SH3, in which P216 and P219 are in direct contact with the aromatic F588 and W562 residues on the domain side. The contact surface is increased through electrostatic interactions between the positively charged residues on the Tau peptide side, namely R221 and K224, and those negatively charged on the protein side within the n-Src loop, corresponding to E556 and E557. These negatively charged residues, located in the so-called specificity region of BIN SH3 (Pineda-Lucena et al., 2005), explain the importance of basic residues in the binding affinity. Further interactions involved residues V228 and V229 of Tau peptide, which appeared to be oriented toward the protein.
We have previously showed that BIN1 interaction with Tau is decreased by phosphorylation of Tau, of at least T231 (Sottejeau et al., 2015). In addition, a competition for BIN1 SH3 domain binding to Tau was shown by BIN1 Isoform 1 own CLAP domain, in an intramolecular mechanism of regulation (Malki et al., 2017). Addition of an excess of Tau to BIN1 is able to displace the intramolecular interaction, in profit of an intermolecular interaction with Tau proline-rich motif (Malki et al., 2017). These results demonstrated the complex regulation of the BIN1/Tau interaction and the need to understand all the key elements of this regulation mechanism. Here, we looked at both aspects of the regulation of this interaction: how phosphorylation of Tau (210-240) peptide on sites surrounding the proline-rich SH3binding consensus motif (pT212, pT231, and pS235) or within this motif (pT217) would impact the competition for BIN1 SH3 domain binding. This phosphorylation corresponded to specific epitopes of Tau antibodies used to detect the pathological Tau protein, namely the AT180 epitope (pT231/pS235) (Goedert et al., 1994) and the AT100 epitope (pT212/pS214) (Zheng-Fischhofer et al., 1998). pT231/pS235 residues affect the interaction by themselves, because full-length Tau phosphorylated on T231-S235, but not on T217, has a decreased affinity for BIN1 SH3 (Sottejeau et al., 2015). However, according to the structural model of BIN1 SH3/Tau (213-229) peptide (Figures 3, 4), T231-S235 residues did not make direct contact with BIN1 SH3 surface. Additional effects might thus be involved, and may be associated with the conformational modification of Tau induced by phosphorylation of T231 and S235 (Luna-Munoz et al., 2005;Sibille et al., 2012;Schwalbe et al., 2015). Alternatively, phosphorylation of T231 and S235 might act by a global electrostatic effect, partially neutralizing the K224-K225 basic motif that contacts the acidic BIN1 SH3 specificity region ( Figure 4C). Substitution of K224 and K225 basic residues with neutral alanines indeed reduced the binding affinity by a factor of 10, confirming that K224 and/or K225 contribute significantly to the binding (Supplementary Figure S3). In addition, the structural model of BIN1 SH3/Tau (213-229) showed that T217 is accommodated by the proline-binding pocket of BIN1 SH3 (Figure 4). T217 phosphorylation in Tau (210-240) peptide may thus additionally weaken the interaction because of an unfavorable steric effect of the phosphate group.
Our results showed that conversely to Tau (210-240) peptide, phosphorylated Tau (210-240) peptide was not able to displace the intramolecular interaction of BIN1 SH3 with BIN1 CLAP domain, as its affinity for BIN1 SH3 was decreased compared to the unphosphorylated peptide. This confirmed our previous results that pT231 (together with other residues) weakens the BIN1/Tau interaction both in vitro and in primary neuronal cultures (Sottejeau et al., 2015). Here, a structure of the complex, based on NMR measurements, highlighted that T212, T231, and S235 did not make direct contact with BIN1 SH3 domain. T217, on the other hand, located in the bound proline-rich motif, but had however, its side-chain oriented in the opposite direction with respect to the domain surface (Figure 4). However, these combined phosphorylations decreased the affinity of BIN1 SH3 for the Tau peptide, either from their global electrostatic contribution, or by their impact on the peptide structure (Luna-Munoz et al., 2005;Sibille et al., 2012;Schwalbe et al., 2015).
Addition of Tau excess to BIN1 isoform 1 disfavors the intramolecular interaction whereas it favors an intermolecular interaction with a Tau proline-rich motif (Malki et al., 2017). Conversely, phosphorylation of Tau decreased the BIN1-SH3 domain's affinity for Tau and pushed the dynamic equilibrium toward the intramolecular interaction, and might thus indirectly influence the availability of BIN1-CLAP domain. The prolinedirected sites within Tau (210-240) might thus be the target of a transduction pathway; the activation of Pro-directed kinases would result in a decrease of BIN1's affinity for Tau and (for BIN1 isoform 1) modify the availability of the CLAP domain.

AUTHOR CONTRIBUTIONS
AL and IM performed the biochemical experiments. AL, IM, and F-XC performed the NMR experiments. AL solved the structure of the complex. EB participated in NMR data treatment and HM in protein sample preparation. AL, IM, and IL wrote the manuscript. J-CL and IL conceived the study.

FUNDING
The NMR facilities were funded by the Nord Region Council, CNRS, Institut Pasteur de Lille, the European Union with the European Regional Development Fund (ERDF), the French Ministry of Research and the University of Lille and by the CTRL CPER co-funded by the European Union (ERDF), by the Hauts de France Regional Council (contrat no. 17003781), Métropole Européenne de Lille (Contract N • 2016_ESR_05) and French State (Contract No. 2017-R3-CTRL-Phase 1). Financial support from the TGIR-RMN-THC Fr3050 CNRS and from FRABio (Univ. Lille, CNRS, FR-3688) for conducting the research is gratefully acknowledged. This study was supported by a grant from the LabEx (Laboratory of Excellence) DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to Alzheimer's disease), by the French government funding agency Agence Nationale de la Recherche ANR-15-CE16-0002 BinAlz and by the Alzheimer's association (BFG-14-318355).


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Psychology

text:
Maternal Regulation of Pups’ Cortical Activity: Role of Serotonergic Signaling

Visual Abstract

IN OUT
Maternal location in relation to the nest LFP low frequency oscillation power in pups' PFC Pup in isolation + SSRI + 5-HT2R antagonist +SSRI + 5-HT2R antagonist A developing brain shows intense reorganization and heightened neuronal plasticity allowing for environmental modulation of its development. During early life, maternal care is a key factor of this environment and defects in this care can derail adaptive brain development and may result in susceptibility to neuropsychiatric disorders. Nevertheless, the mechanisms by which those maternal interactions immediately impact the offspring's brain activity to initiate the pathway to pathology are not well understood. We do know that multiple neurotransmitter

Significance Statement
Maternal care is a major environmental factor influencing brain development, and deficits in this care can result in maladaptive behaviors in the offspring. While maternal interaction is crucial, little is known about the underlying mechanisms by which it modulates infant/pup brain activity. In this study, using wireless electrophysiological recordings, we show that the mother's contact regulates the activity of key prefrontal regions of 10-to 12-d-old rat pups. Regarding mechanisms, we found that the serotonergic system contributes to this modulation, notably through 5-HT2 receptors (5-HT2Rs). These results suggest maternal care affects serotonergic neural activity during early life and provide key insights into how maternal care affects the adaptive/maladaptive development of brain circuits implicated in adult pathology.

Introduction
The postnatal period is a critical stage during which the brain undergoes major changes: new wiring, organization, and intense plasticity. This synaptic plasticity serves a dual function of organizing the infant's immediate behavior, while programming the brain for future adaptive behaviors. Thus, any environmental factor that can affect the developing brain during this period can have long-lasting repercussions (Chaudhury et al., 2016). At this early stage of life, one of the major environmental inputs is maternal care. Maternal presence, the quality of maternal care, and the removal of that care during prolonged separation during early-life has been shown, in humans and rodents alike, to regulate fear (Routh et al., 1978;Barr et al., 2009), to mediate life-long changes in mood (Vetulani, 2013), to modulate response to stress (Hellstrom et al., 2012;Rincón-Cortés et al., 2015), and influence drug consumption (Delavari et al., 2016). Maternal interactions during early-life are thus critical for individuals' life-long mental health (Nelson et al., 2011;Cameron et al., 2017;Sale, 2018). Recently, pup cortical activity has been found to be influenced by maternal interactions. Using local-field potential (LFP) recordings in behaving rat pups (P12-P19), maternal presence was found to be related to an increase in low-frequency neural activity, and norepinephrine was found to mediate the pup's neural responses to maternal milk ejection (Sarro et al., 2014). While norepinephrine can mediate the pup's neural responses to milk ejection, the exact mechanisms by which maternal presence/absence regulates the developing brain's activity are not yet understood. Considering that maternal neglect is the most prevalent form of child abuse, with important societal consequences (U.S. Department of Health and Human Services 2010), it is critical to dissect and understand these mechanisms.
Serotonin (5-HT) is a key neuromodulator known to regulate expression of maternal behaviors (Angoa-Pérez et al., 2014). In developing rodents, serotonin levels peak during the perinatal phase (Hohmann et al., 1988). Importantly, modifying serotonergic signaling during early-life leads to changes in cortical pyramidal neuron morphology as well as emotional deficits in adulthood (Ansorge et al., 2004(Ansorge et al., , 2008Rebello et al., 2014). Those deficits appear to be mediated through 5-HT2 receptors (5-HT2Rs;Sarkar et al., 2014). One of the highest density of serotonergic terminals in the neocortex is found in the prefrontal cortex (PFC), where multiple serotonergic receptors, particularly 5-HT1A and 5-HT2R, are abundant (Celada et al., 2013). Importantly, in the developing PFC, there is a dramatic shift in 5-HT receptor function and expression (Beique et al., 2004). Beique et al. (2004) show that in slices of P6 -P19 rats, 5-HT elicits depolarization of pyramidal neurons that shifts to hyperpolarization starting in the third postnatal week. A change in receptor expression occurs with a decrease in 5-HT7R-mediated depolarization and the appearance of 5-HT1AR-mediated hyperpolarization. At the same time, shifts in the effectiveness, rather than shifts in receptor expression, are associated with a decline in 5-HT2AR-mediated depolarization (Beique et al., 2004). The PFC is a critical structure subserving various cognitive functions (Bechara et al., 1998;Miller and Cohen, 2001;Schoenbaum et al., 2002), including behavioral flexibility and working memory (Tottenham et al., 2011;Parnaudeau et al., 2013;Funahashi, 2017). The PFC has a prolonged developmental period, extending high levels of plasticity into adolescence, which allows it to adapt to environmental changes but also makes it more susceptible to environmental insults that can lead to maladaptive development and consequent behavioral deficits (Kolb et al., 2012). Serotonin is thus a good candidate to par-ticipate in the regulation of developing brain activity, especially PFC activity, associated with maternal care.
Using wireless LFP recording, we recorded pups' PFC [specifically in the anterior cingulate cortex (ACC) region] activity in their home cage with littermates and dam. We found that the dam's presence and absence from the nest modulated the PFC activity of rat pups. First, we found that LFP power was increased in low-frequency bands during periods when the dam was in contact with the pup. Second, we found that blocking 5-HT2R blocked the increase in low-frequency oscillations associated with the maternal contact. Third, we observed that increasing serotonergic signaling, using a selective-serotonin-reuptake-inhibitor (SSRI), was sufficient to increase LFP power in low-frequency bands compared to controls and that this increase was mediated through 5-HT2R. Our results reveal the contribution of the serotonergic system in the regulation of pups' cortical activity in response to maternal interactions.

Animals
Long Evans rats (dam and pups) were used for these experiments (Taconic Farms). Twenty-four male rat pups (P9 -P12) from 12 litters were used for the LFP experiments, 12 male pups (P11-P16) from six litters were used for the nipple-attachment experiment, and 24 pups (P7-P12) from six litters were used for the ultrasonic vocalizations (USVs) experiment. The pups were bred in our colony and were maintained on a 12/12 h light/dark cycle with access to food and water ad libitum. For the LFP experiments, litters were culled to four to six pups and habituated to the cage and experimental room for 3 d before surgery to ensure a calm dam during recordings. Surgeries were performed on P9 -P11 rat pups and recordings were performed the day after surgery (P10 -P12). This range of ages was chosen because maternal regulation of pups' brain activity is maximal before P16 (Sarro et al., 2014), and P9 was the earliest time at which this surgery could be reliably performed by us.
Experiments were conducted blind to pup treatment condition. All animal procedures were performed in ac-cordance with the local animal care committee's regulations and NIH guidelines.

Surgery for LFP recording
Under isoflurane anesthesia (5% for induction and 1.5-3% for maintenance), a stainless-steel electrode (A-M Systems) was implanted in the ACC region of the PFC (AP: ϩ2.0 mm, ML: -0.5 mm, DV: -2 mm; Fig. 1A,B) and cemented to the skull. The electrode was previously connected to a telemetry device (ETA-F10, DSI). The telemetry device was placed subcutaneously under the dorsal skin. The incision was closed using surgical suture (Stoelting) and the area of the incision was cleaned using sterile water to remove as much extraneous odors as possible. After surgery, the pups were kept warm until fully recovered from anesthesia (ϳ2 min). The pups were then gently covered with some used bedding and placed back in the nest. Surgeries were performed in two male pups per litter.

Drugs
To test the contribution of the serotonergic system on pups' neural activity associated with maternal presence, pharmacological modifications were performed using drugs injected intraperitoneally.
To block serotonergic signaling, we targeted 5-HT2R using the 5-HT 2A/2C R antagonist ketanserin (2.5 mg/kg; Tocris, catalog #0908; Sarkar et al., 2014). Ketanserin is a strong 5-HT2AR and 5-HT2CR antagonist with weak associated ␣᎑adrenergic blocking and anti-histamine activity (Awouters, 1985). To enhance serotonergic signaling, we used the SSRI fluoxetine (10 mg/kg; Sigma, catalog #1279804; Ansorge et al., 2004). These drugs and vehicle (0.9% saline) were injected at a volume of 0.02 ml per 10 g. Fluoxetine does not readily dissolve in saline solution so fluoxetine was first diluted in sterile water and then NaCl was added. Average pup weight was 25.46 Ϯ 4.7 g. To ensure that the experimenter was blind to the treatment of the animals, the solutions were prepared by a different experimenter and color coded. The color code was only revealed after histologic and data analyses.

Experimental design (LFP recordings)
Twenty-four hours following surgery, pups' neural activity was recorded during the different stages of the behavioral experiments. LFP signals were acquired, digitized and stored using Spike 2 software (CED). Twenty-four male pups were implanted and recorded for these experiments (two were excluded due to frequent signal artifacts). Two pups were recorded per day, one in the morning and one in the afternoon. The drug treatment administered in the morning or the afternoon was counterbalanced. The wireless LFP recording approach chosen allows recording during undisturbed maternal interactions in the home cage. Neural activity of P10 -P12 pups was recorded in two housing setups: in the home cage with the dam and littermates present, and in isolation in a 500-ml warm plastic beaker. The home cage (47 ϫ 25 cm) was covered with a layer of pine shaving bedding and included a plastic hutch under which the nest was generally located. During home cage periods, the presence (in nest) and absence (out of nest) of the dam from the nest were observed directly and time stamped onto the LFP recording file. "In nest" behavior was recorded as the time when the dam was touching the nest. "Out of nest" was recorded when the dam was not in physical contact with the nest. During this period, the motorically immature pups mainly remained huddled in the nest, while the dams, engaged in eating/drinking, roaming the cage and resting either on top of the hutch or elsewhere in the cage. Maternal in nest and out of nest transitions were not analyzed to ensure uninterrupted and consistent recordings related to maternal presence or absence.
Three experiments were performed on each pup in a serial sequence:

Experiment 1
LFP recordings were obtained during an hour in the home cage during which the presence or absence of the dam in the nest was time stamped. After this period, the pups were placed in isolation in a beaker containing home cage bedding for 15 min ( Fig. 2A). This period of isolation was used as baseline for data analysis. Within the ϳ1-h homecage recording, LFP signals were only analyzed in pups in which we obtained uninterrupted periods of presence or absence of the dam in the nest of at least 5 min.

Experiment 2
After experiment 1, pups were removed from the litter, weighed, injected with ketanserin or vehicle, and immediately placed back in the nest. This procedure took Ͻ1 min. Pup treatment order was alternated between litters, so half the time the first pup recorded received saline and the other half, ketanserin.
Fifteen minutes after the injection, LFP recording was started and the presence/absence of the dam in the nest was registered and time stamped onto the recording file ( Fig. 3A). Within the ϳ1 h of recording, LFP signals were only analyzed in pups in which we obtained uninterrupted period of presence or absence of the dam in the nest of at least 5 min.

Experiment 3
Following the ketanserin or saline injection and subsequent ϳ1 h home cage period of experiment 2, pups were subdivided to receive a second drug administration of fluoxetine or vehicle, in a pseudo-random order, to produce four treatment groups: saline-saline, saline-fluoxetine, ketanserinsaline, and ketanserin-fluoxetine. Immediately after the second injection, pups were placed in isolation and after a 15-min wait period, LFPs were recorded for 30 min (Fig. 4A).

Experimental design (nipple-attachment)
To test whether ketanserin affects the behavior of the pups in the nest we assessed nursing behavior. Two male pups (P11-P16) per litter, from six litters, were randomly selected and one was injected with saline and one with ketanserin. The pups were returned to the home cage with only the mother present. In this setup, all the other littermates and the plastic hutch were removed two hours before the testing to facilitate observation of nursing. Thirty minutes after injection, the duration each pup was nipple-attached was recorded by an observer.

Experimental design (USVs)
Four male pups (P7-P12) per litter were randomly selected and injected with saline or ketanserin and returned to their home cage for 1 h. They were then injected with saline or fluoxetine and placed back in their home cage to produce four treatment groups: saline-saline, saline-fluoxetine, ketanserin-saline, and ketanserin-fluoxetine. Six litters were tested in this experiment. Thirty minutes later, the pups were removed from the litter and placed in isolation in a beaker, positioned under a ULTRAMIC200K (Dodotronic) microphone, inside a sound proof box. USVs were recorded for 30 min and analyzed using the SeaWave (CIPRA and AEST) software. The number of vocalizations in frequencies between 30 and 65 Hz (Simola, 2015) was quantified.

LFP analysis
LFP signals were analyzed offline using Spike 2 during the different behavioral epochs. Fast Fourier transform (FFT) power analyses were performed over periods of several minutes (defined by the markers for each behavioral period) using a Hanning window with 0.9766-Hz frequency bins. To correct for interanimal variability, LFP power was expressed as a ratio relative to the first period when the pup was isolated in the beaker (experiments 1 and 3, maternal location/isolation001ء and treatment/iso-lation,001ء respectively) or relative to the out of nest period (experiment 2, in nest/out of nest.)001ء We then calculated the average power in the different frequency bands: ␦ (0 -3.9 Hz), (3.9 -7.8 Hz), and ␤ (15.6 -31.3 Hz).

Statistical analysis
Statistical analysis was performed using GraphPad Prism software with ␣ of 0.05. To test differences between groups across frequencies, we used two-way repeated measures (RM) ANOVA. To compare the normalized LFP power between in nest and out nest conditions within animals in experiment 1, we performed paired t tests. To test for differences between treatment groups composed of independent animals in experiment 2, we used unpaired t tests. To test for differences between treatment groups in experiment 3, we used one-way ANOVA followed by LSD post hoc test. Data are reported as mean Ϯ SEM.

Anatomic location of electrode placement
At the completion of the experiments, pups were overdosed with urethane (1.25 g/kg), brains were removed, fixed, and cryoprotected in a 4% PFA ϩ 30% sucrose solution. Coronal brain sections (40 -50 m thick) were cut on a microtome (Leica). Sections were mounted on slides, stained with cresyl violet, and electrode placements were examined under a light microscope. Electrode placement predominantly targeted the ACC portion of the PFC (Schweimer and Hauber, 2005; Fig. 1A,B). The tracks of some electrodes passed through the motor cortex, with some electrode tips being located close to the motor cortex.
Six pups in which the electrode tracks showed damage to the corpus callosum were removed from analyses.

Maternal presence/absence from the nest affects PFC activity in pups
Male rat pups (P10 Ϯ 1 d) were implanted with an electrode targeted to the ACC region of the PFC and connected to a telemetry device.
The day after surgery, cortical LFPs were recorded in the pups' home cage for 1 h. After this recording, baseline activity was obtained by recording the pups in isolation for  LFPs recorded within the same session during In and Out of the nest period for a pup injected with saline (top) and another pup injected with ketanserin (bottom). C, Ketanserin blocked the effect of maternal presence (ratio of "In nest"/"Out of nest")001ء on pups' PFC activity in the ␦ frequency band compared to saline injection. D, There was no significant difference between saline and ketanserin treated animals in the band. N ϭ 6 ketanserin; N ϭ 7 saline. E, There were no differences in the percentage of time spent in the nest with the mother in the saline and ketanserin recordings. F, In a separate group of animals, the time spent nipple-attached was quantified. There were no differences between saline and ketanserin injected animals (N ϭ 6 per group). ء p Ͻ 0.05.

Figure 4.
Fluoxetine administration increases LFP power in the low-frequency bands during isolation compared to controls. A, Experimental design. An implanted pup was placed in isolation for 15 min while baseline LFP was recorded. The pup was injected with ketanserin or saline for the first injection and returned to their home cage. One hour later, the pup was injected with fluoxetine or saline. The pup was then placed in isolation and 15 min later, the LFPs were recorded for 30 min. B, Example of sonograms and LFPs recorded within the same session during the first (left) and second (right, after the treatments) isolated periods in pups injected (from top to bottom) with Sal/Sal, Sal/FLX, Ket/Sal, and Ket/FLX. C-D, Fluoxetine administration increased LFP power in ␦ and bands during isolation. This effect was blocked by the 5-HT2R antagonist ketanserin N ϭ 4 in each group. E, USVs in isolated pups (N ϭ 6 per group). Sal ϭ saline, Ket ϭ ketanserin, FLX ϭ fluoxetine. ء p Ͻ 0.05. 15 min ( Fig. 2A). LFP low-frequency band oscillations (␦, , and ␤ bands) were normalized to baseline and analyzed as they appear to be critical in long-range functional connectivity in early life (Del Rio-Bermudez et al., 2017).
We observed an overall tendency for increased LFP power across frequencies when the dam was in the nest compared to out of the nest (0-30 Hz; dam location: F (1,24) ϭ 3.261, p ϭ 0.08, two-way RM ANOVA; Fig. 2B,C). When data were analyzed by specific oscillatory frequency bands (i.e., ␦: 0 -3.9 Hz; : 3.9 -7.8 Hz; and ␤: 15.6 -30 Hz), we observed a significant increase in power in ␦ (t (12) ϭ 2.580, p ϭ 0.02, paired t test; Fig. 2D) during maternal presence compared to the out of the nest condition, as well as a trend in (t (12) ϭ 1.934, p ϭ 0.08, paired t test; Fig. 2E) and no significant difference in ␤ (t (12) ϭ 1.639, p ϭ 0.13, paired t test; Fig. 2F; Table 1). The current results as well as a previous report (Sarro et al., 2014) highlight the relevance of lower frequency oscillations at an early age in response to maternal presence.

The effect of maternal presence on pups' PFC activity is mediated through 5-HT2R
We next tested whether the serotonergic system contributes to the modulation of PFC activity in the pup in response to maternal presence. We used ketanserin to inhibit 5-HT2R. In each recording session, one pup was selected randomly and injected (intraperitoneally) with either vehicle (0.9% saline) or ketanserin (2.5 mg/kg). Fifteen minutes after injection, LFP was recorded for 1 h while simultaneously registering whether the dam was in or out of the nest (Fig. 3A). Data were expressed as a ratio of LFP power in/out of the nest.001ء As shown in Figure  4C,D and described below, there was no effect of ketanserin on LFP activity during baseline (isolation).
When we analyzed the data within the oscillatory frequency bands of interest, we observed a significant reduction of the effect of maternal presence in ketanserin injected animals in the ␦ (t (11) ϭ 2.353, p ϭ 0.04, unpaired t test; Fig. 3B,C) but not in band (t (11) ϭ 1.264, p ϭ 0.23, unpaired t test; Fig. 3B-D). We verified that this difference was not related to changes in the dams' behavior. To assess that, we measured the percentage of time the dam spent in the nest, as well as the number of times the dam entered the nest and we found no difference between the two groups (t (11) ϭ 0.09022, p ϭ 0.9297, unpaired t test; Fig. 3E). Furthermore, in a separate set of animals, we tested whether the nursing behavior was affected in ketanserin injected pups. We found no differences in the time nipple-attached between saline and ketanserin injected animals (t (10) ϭ 0.4373, p ϭ 0.6712, unpaired t test; Fig.  3F).
We thus demonstrated that blocking 5-HT2R inhibits the effect of maternal presence on the lowest LFP oscillatory frequency.

Enhancing serotonergic transmission in isolated pups is sufficient to enhance LFP power in lowfrequency bands compared to controls
In this last experiment, we tested whether an artificial increase of 5-HT is sufficient to increase low-frequency oscillation power in isolated pups compared to controls. To do so, pups were injected with the SSRI-fluoxetine (10 mg/kg). To test the specificity of fluoxetine action on serotonergic transmission, ketanserin was used to inhibit 5-HT2R (2.5 mg/kg). In the first part of this experiment, each individual pup was placed and recorded for 15 min in isolation in a 500-ml plastic beaker, then they were selected randomly to receive vehicle or ketanserin, injected, and placed back in the home cage. One hour later, each pup was selected pseudo-randomly to receive fluoxetine or vehicle. Following a wait time of 15 min, pups' PFC activity was recorded for 30 min while they were isolated (Fig. 4A). This design resulted in four treatment  Figure 2D, ␦ Paired t test t (12) ϭ 2.580, p ϭ 0.02 Figure 2E, Paired t test t (12) ϭ 1.934, p ϭ 0.08 Figure 2F, ␤ Paired t test t (12) ϭ 1.639, p ϭ 0.13 Figure 3C, ␦ Unpaired t test t (11) ϭ 2.353, p ϭ 0.04 Figure 3D, Unpaired t test t (11) ϭ 1.264, p ϭ 0.23 Figure 3E, % time in nest Unpaired t test t (11) ϭ 0.09022, p ϭ 0.9297 Figure 3F groups: saline-saline; saline-fluoxetine; ketanserin-saline; and a ketanserin-fluoxetine (Fig. 4A). When each frequency band of interest (␦ and bands) was analyzed, we observed an increase in the salinefluoxetine group in the ␦ (F (3,12) ϭ 4.704, p ϭ 0.0215, one-way ANOVA; LSD post hoc test; Fig. 4B,C; Table 1) and bands compared to the other treatment groups (F (3,12) ϭ 3.668, p ϭ 0.0439, one-way ANOVA; LSD post hoc test; Fig. 4B-D; Table 1). These data suggest that increasing serotonergic signaling, when pups are isolated from the dam, is sufficient to increase LFP low-frequency power compared to controls. Furthermore, this effect was completely abolished by 5-HT2R blockage further supporting that the effects of serotonergic neurotransmission on cortical LFPs in our model is mediated through 5-HT2R. Importantly, 5-HT2R antagonism alone did not change LFPs during baseline conditions (i.e., saline/saline vs ketanserin/saline did not differ significantly; Table 1). We thus demonstrated in isolated pups, that PFC activity in the low-frequency band can be artificially increased by increasing 5-HT compared to controls, an increase also observed when the dam was in the nest. When separated from their mother and littermates, rat pups emit USVs in frequencies between 30 and 65 kHz, a range generally referred to as distress calls (Simola, 2015). These call have been found to be reduced by several antidepressant drugs, including fluoxetine (Simola, 2015). Consistent with the literature, although not statistically significant, we found a tendency for a reduction in the number of USVs in the fluoxetine treated group. Furthermore, ketanserin does not seem to block the reduction in USVs in fluoxetine treated pups and by itself tends to reduce the number USVs (F (3,20) ϭ 2.039, p ϭ 0.1407, one-way ANOVA).

Discussion
Neuronal activity during development shapes neuronal anatomy, connectivity and function. Extensive work has been done, especially in the visual system, establishing how environmental experience and neural oscillations during critical developmental periods affects neuronal development and function (Katz and Shatz, 1996). During early life, maternal behavior is a major source of environmental input for the pup. Models of maternal abuse/ neglect have implicated poor maternal care as critical in the development of later-life cognitive and emotional deficits in the offspring, which has been supported by the human developmental literature and causally linked within the animal literature since the 1950s using a myriad of paradigms, including maternal separation, high/low licking of pups, and reduction of maternal resources for nest building (Hofer, 1994;Liu et al., 1997;Krugers et al., 2017;Walker et al., 2017). However, minimal research has focused on how maternal presence impacts the immediate response of the infant brain. By using a wireless LFP recording system that permitted undisturbed, direct, and simultaneous assessment of the effect of maternal presence on the brain activity of young pups, our results pinpoint maternal contact with pups in the nest as a major regulator of neuronal activity.
This study establishes a contribution of 5-HT in the regulation of pups' cortical oscillations. Specifically, we demonstrated that maternal contact is correlated to an increase of low-frequency oscillations in the ACC region of the PFC, and this increase is mediated by 5-HT2R. Furthermore, we found that enhancing serotonergic transmission using a SSRI is sufficient to enhance LFP power in low-frequency bands in a pattern similar to the one obtained when the dam is in contact with the pup. Importantly, this regulation was also abolished by blocking 5-HT2R. Together, these results establish that the serotonergic system is part of the network involved in the modulation of pups' brain activity in response to maternal presence.

Influence of maternal presence/absence in the nest on offsprings' brain activity: importance of lowfrequency oscillations and the PFC
Maternal presence/absence can critically affect developing animals. Among other things, it modulates the release of stress hormones, changes USVs and provides olfacto-tactile inputs to the pups (Kuhn and Schanberg, 1998;Ise and Ohta, 2009;Hanganu-Opatz, 2010).
Relative to neuronal activity, we found that maternal presence in the nest increases LFP power in low-frequency bands in the PFC (Fig. 2). This increase of cortical LFP power can be related to various factors during the in nest period. Although, we did not monitor the specific activity of the dam in the nest, we can hypothesize, based on previous work, that this increase is related to (1) nippleattachment, shown to induce an increased in lowfrequency band in the somatosensory cortex (Sarro et al., 2014); (2) tactile stimulation arising from the mother/littermates, shown to drive somatosensory cortex activity (Yang et al., 2009;Akhmetshina et al., 2016a); (3) maternal olfactory stimulation (Moriceau andSullivan, 2005, 2006) and most probably a combination of these and other factors.
Our results support the importance of LFP low-frequency band oscillations in young pups. Slow oscillatory activity is thought to be important in the coordination between long distance brain regions and is also proposed to be critical for development (Khazipov and Luhmann, 2006;Uhlhaas et al., 2010). Notably, in premature humans, using electroencephalography, rhythmic activity has been observed and ␦ oscillatory band has been described as the main oscillatory frequency band on top of which faster rhythms, namely ␦ brushes, can be superimposed (for review, see Khazipov and Luhmann, 2006). Interestingly, ␦ brushes have been proposed as biomarkers to detect neurocognitive deficits in human infants (Whitehead et al., 2017). In rodents, few studies have recorded neuronal activity in pups in vivo but in P1-P8 rat pups, Khazipov et al. (2004) demonstrated the emergence of spindles in somatosensory cortex related to sensorimotor integration thought to be analogous to ␦ brushes in humans. In addition, Adelsberger et al. (2005) also revealed, in vivo, an intrinsic neocortical calcium slow rhythmic activity in resting mouse pups. The effect of maternal presence we observed on LFP slow oscillation power might thus have critical effects on the developing brain.
The effect on the PFC is of high interest as it is a structure involved in a wide range of functions such as executive control or working memory (Miller and Cohen, 2001). As highlighted by Kolb et al. (2012), the PFC has an extended developmental period and early life experience, such as maternal care, can critically shape this development leading to long-term consequences in adulthood. Indeed, it has been shown that maternal separation leads to modification of the dendritic arborization in PFC pyramidal neurons (Monroy et al., 2010;González-Mariscal and Melo, 2017). In addition, Chugani et al. (2001) showed that institutionalized orphans display reduced glucose metabolism in prefrontal areas associated with neurocognitive deficits. Critically, it has also been shown that early maternal separation modifies the prefrontal-hippocampal network in rats recorded at P21-P22, especially in males (Reincke and Hanganu-Opatz, 2017). Notably, the authors show a decrease of LFP -␤ oscillatory band power in the prelimbic part of the PFC as well as a decrease of prelimbic neurons spikes phase-locking to oscillation recorded in the hippocampus. This study highlights the fact that a wide network of structures can be impacted by maternal interactions.

Role of serotonin in the modulation of pups' neural activity by maternal care
Young altricial animals rely on maternal regulation of their physiologic function and homeostasis through sensory stimuli received during maternal care, including somatosensory and chemosensory stimuli (Hofer, 1994). The influence of sensory stimulation on the infant's immediate behavior and later-life neurobehavioral function has been documented by decades of research. This work has highlighted an important role for glucocorticoid levels (Levine, 1967;. Subsequent studies have shown that handling/licking/grooming regulate glucocorticoid receptor transcription via the serotonergic system (Mitchell et al., 1990(Mitchell et al., , 1992Laplante et al., 2002;Hellstrom et al., 2012). Interestingly, in rats, maternal separation decreases the 5-HIAA/5-HT ratio in PFC (Xue et al., 2013;González-Mariscal and Melo, 2017). Relative to the different 5HT receptor subtypes, it has notably been shown that animals subjected to maternal separation exhibit deregulation of 5-HT2R function (Benekareddy et al., 2010). Along with these lines of evidence, showing a contribution of serotonergic signaling to the effect of maternal presence on pups' brain activity, we found, in vivo in behaving pups, that blocking serotonergic signaling via 5-HT2R abolished the increase in power in low-frequency bands observed when the dam was in the nest (Fig. 3). This effect was not due to a general lowering of LFP power due to the 5-HT2R antagonist ketanserin as we did not observe a change in cortical activity between saline and ketanserin injected animals (Fig. 4C,D). The effect of ketanserin we observed might be indirect and linked to a change of the dam's behavior in regard to the injected pup (i.e., grooming, nursing) known to differentially modulate pup's LFP activity (Sarro et al., 2014). We did not record the specific dam's behaviors in the nest, however we did not find differences in the frequency and duration of time the dam spent in the nest, nor the time the pups spent nipple-attached (performed in a different set of animals), between ketanserin and saline injected groups.
In the future, it would be interesting to measure realtime release of 5-HT during maternal interaction using microdialysis or cyclic voltammetry. Unfortunately, these techniques, in the current state of the art, cannot be performed wirelessly, thus impairing analyses during active maternal-pup interaction.
Finally, we found that enhancing 5-HT signaling using a SSRI induces an increase of low LFP frequency band oscillatory power in isolated pups compared to controls. This effect may be correlated for example with changes in the pup's behavior (e.g., USVs), or/and activity in other brain areas; for example, the SSRI citalopram was been shown to reduce the spontaneous, as well as the sensory evoked, activities in the barrel cortex of P2-P5 rat pups (Akhmetshina et al., 2016b). However, from our data, it does not seem that the modulation of cortical activity can only be ascribed to changes in USVs. Indeed and consistent with previous studies, we found a tendency for a reduction in the number of USVs in pups injected with fluoxetine (Simola, 2015). However, we found that ketanserin does not seem to block the reduction in USVs in fluoxetine treated pups and furthermore, as previously described, ketanserin by itself seems to reduce USVs (Schreiber et al., 1998). These data thus suggest a dissociation between USVs and LFP power in our treatment conditions.
The tight regulation of 5-HT levels during development is of extreme importance, with both low and high levels of serotonergic signaling seeming to be detrimental. Indeed, it appears that there is a critical period and an optimal range of 5-HT level and that any deviation from this range can alter normal brain development Suri et al., 2015;Gingrich et al., 2017). For example, administration of the SSRI fluoxetine to rodent pups has been shown to lead to behavioral deficits in adulthood (Ansorge et al., 2004;Sarkar et al., 2014;Rebello et al., 2014). Interestingly, these behavioral deficits are avoided by concomitant administration of fluoxetine and ketanserin (Sarkar et al., 2014). Furthermore, treatment with the 5-HT2R antagonist, ketanserin, prevented the emergence of anxiety behaviors and changes in 5-HT2R expression associated with maternal separation (Benekareddy et al., 2011).

Conclusion
In this report, we reveal that maternal presence modulates the pups' cortical activity via the serotonergic system stressing the importance of maternal care in the activity of this tightly regulated system. 5-HT is one of the neuromodulators involved in the regulation of the offspring's brain activity by maternal care, other neuromodulators and brain regions are probably jointly or in parallel involved such as the noradrenaline in the somatosensory cortex (Sarro et al., 2014) or the acetylcholine in the prefrontal-hippocampal network (Janiesch et al., 2011). Further studies will help us dissect the whole extent of this modulation.


s2fieldsofstudy: Biology Medicine

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Role of Interleukin- (IL-) 17 in the Pathogenesis and Targeted Therapies in Spondyloarthropathies

Spondyloarthropathy (SpA) is a unique type of joint inflammation characterized by coexisting erosive bone damage and pathological new bone formation. Previous genetic association studies have demonstrated that several cytokine pathways play a critical role in the pathogenesis of ankylosing spondylitis (AS), psoriatic arthritis (PsA), and other types of SpA. In addition to several well-known proinflammatory cytokines, recent studies suggest that IL-17 plays a pivotal role in the pathogenesis of SpA. Further evidence from human and animal studies have defined that IL-17 and IL-17-producing cells contribute to tissue inflammation, autoimmunity, and host defense, leading to the following pathologic events associated with SpA. Recently, several clinical trials targeting IL-17 pathways demonstrated the positive response of IL-17 blockade in treating AS, indicating a great potential of IL-17-targeting therapy in SpA. In this review article, we have discussed the contributing role of IL-17 and different IL-17-producing cells in the pathogenesis of SpA and provided an outline of therapeutic application of the IL-17 blockade in the treatment of SpA. Other targeted cytokines associated with IL-17 axis in SpA will also be included.

Introduction
Spondyloarthropathies (SpA) represent a group of chronic inflammatory conditions, involving mainly the axial skeleton (spine and sacroiliac joints), and share a close association with HLA-B27. Ankylosing spondylitis (AS) is the prototypical form of human SpA and characterized in acute and chronic spinal inflammation initiating from sacroiliac joints eventually leading to joint ankylosis [1]. Extensive genomewide association studies have demonstrated a great number of novel genetic associations beyond HLA-B27 pointing toward inflammatory cytokine pathways in the pathogenesis of AS and other types of human SpA [2,3], suggesting the role of inflammatory cytokines in the pathogenesis and potential therapeutic application in SpA. Tumor necrosis factor (TNF), interleukin-(IL-) 1, IL-6, and IL-23/IL-17 are major inflammatory cytokine pathways of great interest from serial studies of single nucleotide polymorphisms, cytokine receptors, and associated intracellular signaling molecules. Among these inflammatory cytokines, IL-17 plays a dominant role in the inflammatory and proliferative cascades of human SpA [4,5]. In this review, we have discussed the contributing role of the IL-17 pathways in the pathophysiology of SpA from current evidence of genetic associations, studies in animal models, and expression of IL-17 in human SpA. Therapeutic effects of the IL-17 blockade in human SpA have also been discussed.

Signal Transduction of IL-17
It is now well known that IL-17 can activate the nuclear factor-(NF-) κB pathway [16], and the IL-17A-mediated NF-κB activation is dependent on tumor necrosis factor receptor-associated factor 6 (TRAF6) [17]. Act1, a critical intermediate adaptor between IL-17RA and TRAF6, is recruited after IL-17A stimulation and binds to IL-17RA through SEFIR-dependent interactions [18]. In addition, the TRAF6-binding motif of Act1 further enables it to bind TRAF6 and TGF-β-activated kinase 1 to deliver downstream signals, resulting in activation of the canonical NF-κB pathway. Moreover, Act1 can also activate the mitogenactivated protein kinase pathway to stabilize several mRNAs encoding proinflammatory cytokines and chemokines [19]. IL-17-mediated signaling not only promotes proinflammatory cascades but also triggers several regulatory pathways. IL-17A stimulation can recruit TRAF4 to the IL-17 receptor complex, and TRAF4 can be a negative modulator of IL-17mediated signaling by competing with TRAF6 for Act1 binding [20]. TRAF3 has also been shown to be another negative regulator with a similar action on the IL-17 signaling cascade [21]. Other phosphorylation events, such as phosphorylation of C/EBPβ by ERK and glycogen synthase kinase 3β (GSK3β), can also lead to inhibition of IL-17-dependent proinflammatory gene induction [22,23].

Sources of IL-17
IL-17A was first cloned from CD4+ T cells, and subsequent evidence suggested the IL-17A-producing CD4+ T cells were distinct from interferon-γ-(IFNγ-) producing effector T cells [24,25]. Moreover, these unique subpopulations of CD4+ T cells that produce IL-17 were driven by IL-23 and thus named as Th17 cells [26,27]. Indeed, these IL-17-producing CD4+ T cells (Th17 cells) were distinct from Th1 and Th2 cells and could develop independently of STAT4 and STAT6, confirming this new T cell subset in the production of IL-17 [28,29]. Besides Th17 cells, another T cell subset, γδ T cells, can respond to IL-23, thus amplifying Th17 responses [30] and producing IL-17 in certain circumstances [31]. In addition to T cells, other IL-17-producing cells include natural killer (NK) cells, mast cells, neutrophils, and innate lymphoid cells; these IL-17producing cells can produce IL-17 in a specific inflammatory condition possibly through genetic programming [32][33][34][35]. Although several animal and human studies suggest these cells produce IL-17 in response to inflammation, the respective contributions of these different cell types to the disease pathology remain to be explored. This raises a critical question for the targeted role of IL-17 in inflammation according to cell-type and disease-type specificity.

Function of IL-17
The major biological activity of IL-17 is involved in promoting inflammation as earlier studies demonstrated that IL-17 triggers the IL-6 production of synoviocytes from patients with rheumatoid arthritis and this effect is even more enhanced when in synergy with other proinflammatory cytokines (IL-1, TNF) [24,36,37]. In addition, IL-17 in chronic inflammation contributes to inhibition of matrix production in chondrocytes and osteoblasts through activating MMPs, resulting in joint destruction and defective tissue repair [38]. IL-17 also increases the expression of receptor activator of NF-κB ligand (RANKL) on osteoblasts and in turn increases RANK signaling in osteoclasts [39][40][41]. These studies link IL-17 activity to bone destruction, suggesting the potential role of IL-17 in osteoimmunology. In intestinal inflammation, IL-17 also stimulates MMPs, IL6, and IL-8 production from colonic subepithelial myofibroblasts in vitro [42]. In the central nervous system, IL-17 can disrupt the blood-brain barrier tight junction and further facilitates local migration of CD4 T cells, leading to neuroinflammation [43]. All these results support the role of IL-17 in the pathogenesis of tissue-specific and systemic autoimmune diseases.

IL-17 in AS
Accumulated evidences have demonstrated IL-17 and Th17 effector responses are involved in inflammatory spondyloarthritic condition, including AS, psoriatic arthritis (PsA), reactive arthritis, and undifferentiated spondyloarthritis, suggesting Th17-associated pathways including IL-23R are genetically associated with AS susceptibility [44][45][46]. A key question to IL-17 in the pathogenesis of SpA is how IL-17 drives inflammation leading to erosive bone damage and pathological new bone formation.
AS is the prototypical form of human SpA and is characterized by joint inflammation, leading to the formation of ectopic new bone and eventually progressive ankylosis of sacroiliac joints. AS has shown a strong genetic association with IL-23R polymorphisms with functional relevance in T cell immune response [44,47,48], suggesting genetic variations in the IL-23/IL-17 axis may influence the effector function of Th17 cells in patients with AS. In addition, TNFSF15, TRADD, and CARD9 genes were also reported to be important in the regulation of Th17 cell development and proliferation [49,50]. Uddin et al. used multiple integrated genomic approaches and identified AS risk alleles that overlap with global immune-related pathways and Th17-related genes [51]. These genetic data results indicate that IL-17 and Th17 effector responses are influenced by both genetic and upstream signal modulation to amplify and perpetuate inflammation.
The importance of IL-17 in the pathogenesis of AS was further defined in several animal and human observational studies. Mice with overexpression of IL-23 represented axial and peripheral enthesitis, and entheseal new bone formation, which is similar to human AS; blockade of the downstream effector cytokine IL-17 significantly reduced disease [52]. IL-17-deficient mice exhibit less joint inflammation in an inflammatory arthritis model [53], and IL-17 receptordeficient mice also showed decreased proinflammatory cytokine production in the synovium which prevents cartilage destruction [54]. In human AS, IL-17 and IL-23 were elevated in the serum of patients compared to controls and the production IL-17 was even more enhanced when stimulated with IL-23 [55,56]. Chen et al. also reported similar results of elevated levels of IL-17 and IL-23 in AS patients and further observed that the levels correlate to disease activity measured by Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) scores [57]. These observation studies suggest IL-17 may contribute to the pathogenesis of AS.
Th17 cells are the main cell type in the production of IL-17, and Th17 cells were observed to increase in the blood from patients with AS [58,59]. Th17 cells specifically expressing KIR3DL2 could respond to the HLA-B27 homodimer and increased in number in AS patients [60]. In addition to Th17 cells, other IL-17-producing T cell subsets have also been identified to be specifically upregulated in AS. For example, increased circulating IL-17-producing IL-23R+ γδ T cells were observed in patients with active AS [61]. Furthermore, mast cells were observed to express more IL-17 in SpA and constituted the major IL-17-expressing cell population in the SpA synovium [62]. Increased expression of IL-17 was found predominantly in MPO+ cells and in CD15+ neutrophils in the subchondral bone marrow of inflamed spine from AS patients [4]. All these results suggest that both the innate and adaptive mechanisms drive AS pathogenesis and IL-17-mediated inflammation of AS joints may be tissue-and cell-type specific.
Several antibodies blocking the IL-17/IL-17 receptors have been developed and examined in clinical trials in AS patients over the past few years. At least three monoclonal antibodies have been developed to neutralize IL-17 or block IL-17 receptor signaling: secukinumab (anti-IL-17A), ixekizumab (anti-IL-17A), and brodalumab (anti-IL-17RA). Secukinumab is a fully human monoclonal antibody that selectively binds and neutralizes IL-17. In an earlier phase II clinical trial, secukinumab showed good efficacy in controlling symptoms of AS [63]. Subsequent two large placebo-controlled phase III trials provide further proof of its efficacy in the inhibition of disease activity, similar to what has been seen in previous TNF-blocker trials [64]. Moreover, the drug was superior to placebo both in patients that were naive and in those that had not responded to previous anti-TNF treatment, suggesting the IL-17 pathway inhibits inflammation distinct from that in TNF [65]. Similarly to secukinumab, ixekizumab is another humanized monoclonal antibody that binds and neutralizes IL-17A, and a phase III trial in AS is ongoing (identifier: NCT02696798). Brodalumab is a fully human anti-IL17 RA antibody that inhibits the biological activity of IL-17A, IL-17F, and other IL-17 family members, and a phase III trial in AS is ongoing (identifier: NCT02985983).
Patients with spondyloarthritis exhibit excessive bone formation followed by joint inflammation that can occur at entheses (enthesophytes), near joints on the periosteal surface (osteophytes), or in the spine (syndesmophytes). Whether IL-17 inhibitor treatment might have an effect on the radiographic progression of bone formation and destruction in AS remains to be determined. A recent randomised phase III study on the evaluation of radiographic outcomes of secukinumab in AS shows a low mean progression of spinal radiographic changes over 2 years [66]. However, this secukinumab study did not include a control group. In addition, the effects of blocking IL-17A on periosteal bone formation have not yet been evaluated. Therefore, there is still no firm conclusion about the effects of IL-17 inhibition therapy on radiographic disease progression.

IL-17 in PsA
PsA belongs to SpA and is characterized by the involvement of the skin (psoriatic plaque), nails, and peripheral and axial joints as well as occasional enthesitis [67,68]. PsA develops in approximately 25% of psoriasis patients [69], suggesting that the link of psoriasis and PsA may be through a similar immune mechanism and that they share collective pathological events and clinical features. The genetic variations on the IL-23/IL-17 signaling pathway affect the effector function of Th1, and Th17 cells may contribute to the pathogenesis of AS and other SpA [70,71], and a single nucleotide polymorphism (SNP) in IL-23A and IL-23R has also been shown in association with PsA [72,73], suggesting that the IL-23/ IL-17 pathway may share common genetic background in developing SpA.
Accumulating studies have evidenced the critical role of IL-17 in the pathogenesis of PsA. There were an increased expression of IL-17 observed from psoriatic skin compared to the nonlesioned psoriatic skin, and the upregulation of IL-17 was positively associated with psoriatic disease severity [74,75]. Fibroblast-like synoviocytes (FLS) from PsA synovium produce more proinflammatory cytokines and MMPs upon IL-17 stimulation [76]. As mentioned earlier, IL-17 can promote osteoclastogenesis resulting in bone erosion through activating the expression of RANKL [77,78], which can be partially explained by the destructive bone lesions in PsA. All these results suggest IL-17 triggers both skin and joint pathology and inflammation in PsA. The source of IL-17 in psoriatic skin and inflamed joint has been investigated from several observational study results. Th17 cells were observed to have an increased number in both blood and skin lesions from PsA patients, which was also positively correlated with disease activity [79,80]. In PsA synovium, there were increased IL-17-producing CD4+ effector memory T cell aggregation and expression of functionally active IL-17RA [76]. Other IL-17-producing cells, including mast cells and neutrophils, also represent additional sources of IL-17-mediating psoriasis progression [81]. IL-17-producing mast cells are increased in the synovial fluid of patients with psoriatic arthritis [82] and NKp44+ ILC3 cells are also increased in the peripheral blood and skin in psoriasis [83] and produce IL-17 [84], indicating the IL-17-rich milieu in the inflamed skin and joints of PsA resulting from multiple innate and adaptive IL-17-producing cells. This concept is further confirmed in the murine model of aldara-induced psoriasis, where both skin-invading population of γδ T cells and RORγt CD3-innate lymphocytes which could produce IL-17 contribute to the initiation of psoriasiform plaque formation [85]. Taken together, both IL-17 and IL-17-producing cells contribute to multiple cytokine network and play a pivotal role in the pathogenesis of PsA.
Therapeutic blockade of IL-17 works very well for psoriasis and is relatively good for PsA. In psoriasis, phase III clinical trials for secukinumab have demonstrated that IL-17 inhibition therapy can achieve a 75% improvement of the Psoriasis Area and Severity Index (PASI75) in more than 75% of patients with plaque psoriasis response, and a significant portion of the patients also achieved PASI90 and PASI100 responses [86,87]. Similar to secukinumab, around 80% to 90% of patients treated with ixekizumab [88,89] and brodalumab [90,91] achieved a PASI75 response, indicating that the IL-17 pathway is critical for keratinocytes and infiltrating immune cells in the skin of psoriasis. As for PsA, IL-17 inhibition therapy seems to have a modest effect on the inhibition of joint inflammation and bone destruction. In a phase II clinical trial for secukinumab in patients with moderate-to-severe PsA, a trend towards improvement was demonstrated in the secukinumab group although it did not meet statistical significance [92]; a subsequent phase III clinical trial is ongoing to verify its efficacy in PsA (identifier: NCT02771210). Although a phase III clinical trial for brodalumab in PsA was halted initially because of increased suicidal ideation and suicide events in previous psoriasis clinical trials [90], a phase III trial for brodalumab in axial SpA is ongoing (identifier: NCT02985983).

Other Targeted Cytokines Associated with IL-17 Axis in SpA
Previous studies have shown that IL-23 can trigger and drive Th17 responses by expansion of Th17 cells and thus an increase in IL-17 levels. Ustekinumab, a monoclonal antibody against the p40 subunit of IL-12 and IL-23, has shown to be beneficial in the treatment of AS, with improvement of clinical disease score, patient-reported outcome parameters, and MRI score [93], suggesting IL-23/ IL-17-targeted therapies are very promising for SpA. IL-17 is the main cytokine of Th17 cells; nevertheless, Th17 cells can also produce some other cytokines including IL-22 [94,95]. IL-22 belongs to the IL-10 family and is released by several types of CD4+ and CD8+ T cells as well as natural killer T cells, γδ T cells, and type 3 ILCs [96]. The IL-17 and IL-22 expression is associated with many autoimmune processes, such as psoriasis and inflammatory bowel disease [97,98]. In an SpA animal model, although the development of enthesitis and entheseal new bone formation are largely influenced by IL-17/IL-23 axis, enthesitis is specifically dependent on IL-22 [99], and the IL-22producing entheseal resident cells have shown to activate osteoblast-mediated bone remodeling [52], suggesting IL-22 may coordinate with the IL-17/IL-23 pathway in inflammatory bone remodeling. Furthermore, synovial fluid of PsA patients have shown higher concentration of IL-22, and exogenous administration of IL-22 can induce marked proliferation of PsA-derived fibroblast-like synoviocytes [100]. A recent study has further demonstrated that IL-22 increases the proliferation and migration of the human mesenchymal stem cell (MSC) in inflammatory environments, and the MSC osteogenesis is enhanced following IL-22 exposure [101]. All these results suggest the potential role of IL-22 in addition to the IL-17/IL-23 axis in bone remodeling and provide a novel pathway for exploring pathological and inflammatory osteogenesis in SpA.

Conclusions
In summary, accumulated evidence supports the critical role of IL-17 and IL-17-producing cells in the joint inflammation, cartilage damage, and bone remodeling associated with SpA. Animal models as well as human genetic and observation studies provided strong mechanistic rationales for targeting IL-17-associated pathways in the treatment of SpA. Several clinical trials showed that blockade of IL-17 successfully restores joint inflammation and remodeling in SpA, providing effective and well-tolerated alternatives to conventional treatment in SpA.

Conflicts of Interest
The authors have no conflicts of interest to declare.