Pharmacologically diverse antidepressant drugs disrupt the interaction of BDNF receptor TRKB and the endocytic adaptor AP-2

Antidepressant drugs activate TRKB (tropomyosin-related kinase B), however it remains unclear whether these compounds employ a common mechanism for achieving this effect. We found by using mass spectrometry that the interaction of several proteins with TRKB was disrupted in the hippocampus of fluoxetine-treated animals (single intraperitoneal injection), including members of the AP-2 complex (adaptor protein complex-2) involved in vesicular endocytosis. The interaction of TRKB with the cargo-docking mu subunit of the AP-2 complex (AP2M) was disrupted by both acute and repeated fluoxetine treatment. However, while the coupling between full length TRKB and AP2M was disrupted by fluoxetine, the interaction between AP2M and the TRKB C-terminal peptide was resistant to this drug, indicating that the binding site targeted by fluoxetine must lie outside of the TRKB:AP2M interface. In addition to fluoxetine, other pharmacologically diverse antidepressants imipramine, rolipram, phenelzine, ketamine, and the ketamine metabolite 2R,6R-hydroxynorketamine (RR-HNK) also decreased the interaction between TRKB:AP2M in vitro, as measured by ELISA. Silencing the expression of AP2M in MG87.TRKB cell line led to increased surface positioning of TRKB and to a higher response to BDNF (brain-derived neurotrophic factor), observed as the levels of active TRKB. Moreover, animals haploinsufficient for the Ap2m1 gene displayed increased levels of active TRKB in vivo, as well as an enhanced cell surface expression of the receptor in cultured hippocampal neurons. Taken together, our data suggests that disruption of the TRKB:AP2M interaction is an effect shared by several antidepressants with diverse chemical structures and canonical modes of action.


Introduction
Compromised plasticity has been suggested as one of the major causes of depression (1). Supporting this idea, morphological and functional deficits have been observed in the brains of patients with mood disorders (2,3,4). Reduced neurotrophic support, such as by BDNF signaling via TRKB, has been linked to the atrophy observed in these patients in the neuronal networks regulating mood and cognition (5,6).
Antidepressant drugs, mainly targeting monoamine neurotransmission, such as serotonin (5-HT) and noradrenaline, have been suggested to act by improving neuronal connectivity, plasticity, and information processing (4). In line with this, an intact BDNF-TRKB signaling system is crucial for the efficacy of antidepressant compounds, as mice overexpressing a dominant negative TRKB isoform (TRKB.T1) or heterozygous BDNF null (Bdnf+/−) are not responsive to antidepressant drugs (7). Furthermore, the activation of TRKB receptor signaling, and putatively the reinstatement of plasticity, is associated to the response to antidepressants (7)(8)(9).
The binding of BDNF to TRKB initiates phosphorylation of tyrosine residues in the intracellular portion of the receptor (10) which allows docking of adaptor proteins to these sites. Phosphorylated tyrosine 515, for example, serves as a docking site for SHC adaptor protein, while phospholipase C gamma (PLCg1) binds to the phosphorylated Y816 residue (11). Interestingly, the phosphorylation of TRKB receptors at Y706/7 and Y816 residues are enhanced by several antidepressants in adult mouse hippocampus while no change was detected at the Y515 residue (7,9). Despite the evidence on the necessity of TRKB receptor activation for antidepressant responses, the mechanism by which these drugs might trigger TRKB activation remains unclear.
In this scenario, proteins able to modulate TRKB transit to and from the cell surface, therefore modulating its exposure to BDNF, posed as an interesting possibility to understand the antidepressant-induced effects. In line with this idea, our mass spectrometry-based screen for TRKB interactors identified the endocytic adaptor protein complex-2 (AP-2).
Components of AP complexes 1, 2 and 3, are crucial for clathrin-dependent endocytosis, and participate in the removal of several transmembrane receptors, and their ligands (12).
AP-2 is a heterotetrameric complex which consists of two large chains (alpha-and betaadaptin), one medium chain (mu, AP2M), and one small chain (sigma) (13).TRK receptors have been identified among the targets for rapid clathrin-mediated endocytosis upon binding to their ligands (14). Earlier studies demonstrated that BDNF-induced phosphorylation of TRKB receptors recruits AP-2 complex and clathrin to the plasma membrane in cultured hippocampal neurons (15). More recently, a novel relationship has been established between TRKB receptors and AP-2, suggesting that autophagosomes containing activated TRKB are transported to the cell soma in an AP-2-dependent manner (16).
In the present study, we hypothesized that an interaction between TRKB and AP-2 might be a possible site of action for antidepressant drugs. Specifically, we identified a putative binding site on the TRKB receptor for AP2M, which provides a known interface for cargo-binding, and examined the impact of AP2M downregulation on the activation and surface exposure of TRKB. Finally, we tested the effects of several antidepressant drugs such as fluoxetine, imipramine, phenelzine, and rolipram on the interaction of TRKB receptors with AP2M.

Animals:
Adult (7 weeks old) male C57BL/6 mice from Harlan, kept with free access to food and water, were used for TRKB immunoprecipitation and mass spectrometry analysis.
Briefly, the animals received a single intraperitoneal injection of fluoxetine (30 mg/kg) and were euthanized by CO2. The hippocampi were collected and processed as described were conducted according to the committee's guidelines. For brain dissection, animals were anesthetized with isoflurane prior to cervical dislocation. The hippocampus was dissected on ice, and processed as described below.

Sample processing and western blotting:
Samples from hippocampi of male and female AP2M.wt or AP2M.het mice were sonicated in NP lysis buffer, centrifuged (16000xg at 4 o C for 15 min), and the supernatants were submitted to SDS-PAGE. Samples from hippocampi of 7 weeks-old male mouse was used to confirm the TRKB:AP2M co-immunoprecipitation (as described above) by western blotting. Samples from MG87.TRKB cells were homogenized in NP lysis buffer and submitted to SDS-PAGE or AP2M:TRKB ELISA. For cell-free ELISA assays described below, the samples were acidified with 1M HCl (pH ~2) at room temperature for 15min, and the pH was restored to 7.4 with 1M NaOH. This procedure aims to disrupt non-covalent interactions between proteins in a given complex. The membranes were then incubated with HRP-conjugated secondary antibodies (anti-Gt, Invitrogen, #61-1620; anti-Rb, Bio-Rad, #170-5046; or mouse IgG kappa binding protein, Santa Cruz, #516102; 1:5000), and the HRP activity was detected by incubation with ECL and exposure to a CCD camera. The signal from each target was background-subtracted, normalized by GAPDH or TRKB and expressed as percentage of AP2M.wt, scrambled or vehicle-treated groups.

AP2M:TRKB interaction and surface TRKB ELISA assays:
MG87.TRKB cells were incubated with fluoxetine, imipramine, rolipram, aspirin, which was used as negative control (at 0, 1, 10 or 100 µM) or phenelzine (at 0, 0.01, 0.1, 1, The background-subtracted signal from each sample in each of the described assays was expressed as percentage of control group (vehicle-treated or WT peptide).

Structural bioinformatics data mining and docking assay:
In order to address a putative direct interaction between AP2M and TRKB, we submitted the full length, canonical sequence of rat and mouse TRKB (Uniprot: Q63604 or P15209, respectively) to Eukaryotic Linear Motif library (23). Several TRG_ENDOCYTIC_2 motifs (TRG2; a tyrosine-based sorting signal responsible for the interaction with AP2M subunit) were found for both mouse and rat TRKB. Of particular interest, the Y816 residue is also activated by BDNF and antidepressants. Thus, the sequence of the last 26aa residues of rat TRKB (TRKB.ctrl, TRKB.Y816E, and TRKB.Y816A) and full length AP2M (model generated through RaptorX server and aligned with the C-terminal structure available in RCSB databank: 2PR9) were submitted to docking simulations using CABS-docking server (24).

Statistical analysis
Data was analyzed by Student's t test, oneor two-way ANOVA (or their nonparametric equivalents) followed by Fisher's LSD post hoc test, when appropriate. Values of p<0.05 were considered significant.

AP2M is an interaction partner of TRKB receptors
Samples from adult mouse hippocampus were processed for co-immunoprecipitation with TRKB-specific antibody and analyzed by mass spectrometry to investigate the TRKB interactome. The list of identified interactors comprises several components of the synaptic and endocytic machinery (Fig. 1a)  binding site YLDI, which is located within the C-terminal portion of TRK, is phosphorylated following antidepressant administration (9).
In order to assess the supposed interaction mechanism between AP2M and TRKB, we generated a model of AP2M in the RaptorX server (27,28) and aligned it with AP2M Cterminal chain crystal structure from RCSB databank (2PR9) (Fig. 1e). Since the alignment indicated a low root-mean squared deviation (RMSD) from the crystal structure (2Å), whereby proposing a reliable structure for docking, we proceeded to the simulations. To this aim, the AP2M model and the last 26aa residues from the C-terminal portion of TRKB; including the mutations: Y816A and Y816E (phosphomimetic), were uploaded to CABS-dock server (24). We obtained the Gibbs' free energy for the AP2M:TRKB C-terminus peptide interaction, and the lowest 10 values from each group was used in statistical analysis. The interaction energy of AP2M:TRKB.Y816E complex was lower, suggesting a more stable interaction, while it was significantly higher for AP2M:TRKB.Y816A, compared to AP2M:TRKB.ctrl [Kruskal-Wallis= 25.83; p<0.0001] (Fig. 1c). Based on these in silico results, we proceeded to analyze the AP2M:TRKB C-terminus interaction in vitro by ELISA.
For this purpose, the N-biotinylated synthetic peptides (ctrl, pY or Y816A) of the last 26 aa of rat TRKB were used. The interaction between AP2M and the phosphorylated TRKB peptide (TRKB.pY) was stronger compared to control peptide. In line with our earlier evidence, the AP2M:TRKB.Y816A interaction was weaker in comparison with the control peptide [Kruskal-Wallis= 20.48; p<0.0001] (Fig. 1g).

Role of AP2M in TRKB function
Previous studies indicate that depleting AP2M from mammalian cells prevents the assembly of functional AP-2 complexes (29) and corresponds to an AP-2 loss of function (17). Thus, we investigated the impact of AP2M downregulation on TRKB receptor signaling.
However, we also observed reduced levels of total TRKB in the hippocampus of these animals as shown by western blotting [t(10)=2.794; p=0.019] (Fig. 2g). This result was unexpected, especially as brain lysates of neuron-specific AP2M null mice were reported to have increased TRKB expression levels (16). However, the complete loss of an essential endocytic protein might cause very different compensatory changes than its partial reduction. total TRKB in the hippocampus, associated with (h) increased levels of phosphorylated TRKB at Y706 and Y816 residues (n=5-6/group). *p<0.05 for comparison with ctrl/wild-type group; #p<0.05 for comparison with ctrl/siAP2M group.
The administration of fluoxetine or imipramine also increased the levels of surface exposed TRKB as measured in surface ELISA [F(1,54)=62.54; p<0.0001] (Fig. 3c, d).
Interestingly, the disruptive effect of fluoxetine on the TRKB:AP2M interaction is not dependent on TRKB activation, since pretreatment with the TRK inhibitor k252a was not able to prevent such effect [interaction effect: F(1,20)=0.3066; p=0.586] (Fig. 3e). Contrary to what was observed for antidepressants, BDNF administration (25 ng/ml) resulted in an increased interaction of TRKB receptors with AP2M, which was dependent on TRKBactivation, since pretreatment with k252a prevented this effect [interaction effect: F(1,20)=4.528; p=0.046] (Fig. 3f).

Discussion
In the last years, several pieces of evidence have indicated that sub-chronic (30) and chronic fluoxetine treatment (31,32) can induce changes in the proteomics profile of rodent brains. Fluoxetine was found to alter the expression of proteins involved in neuroprotection, serotonin biosynthesis, and axonal transport in hippocampus and frontal cortex (31), as well as in endocytosis and transport in visual cortex (32).
The present study identified the endocytic complex AP-2 as a target whose interaction with TRKB is reduced following acute fluoxetine treatment. Since AP-2 appeared as a promising candidate for the modulation of TRKB surface levels and activity putatively taking part as downstream of antidepressant treatment, we selected it for further investigation. In line with a robust interaction between AP-2 and TRKB we found all subunits of the AP-2 in the same complex with TRKB, while in case of the related proteins, ie AP-3 and AP-1, only one subunit was identified for each Ap3d1 and Ap1b1. Furthermore, we identified tyrosine-based motifs in the TRKB receptor as putative binding sites for the AP2M subunit of the AP-2 adaptor complex. Especially the region following the Y816 residue of TRKB conformed to the classical consensus for an AP2M-interacting motif. In line with its classical role as endocytic adaptor protein, AP-2 appears to regulate the surface localization of its interactor TRKB, and therefore its signaling. Decreased levels of AP2M resulted in increased surface localization of TRKB, facilitating the activation by its ligand BDNF.
Consistent with this, we found increased levels of activated TRKB in the hippocampus of AP2M heterozygous mice. Finally, we observed that antidepressant drugs such as fluoxetine, imipramine, rolipram, and phenelzine are able to disrupt the binding between AP2M and TRKB resulting in increased exposure of TRKB at the cell surface.
It has been established that activation, endocytosis, and trafficking of TRKB receptors regulate the propagation and fate of the downstream signaling response (33). The post-endocytic sorting machinery plays a major role during this fate-determination process (34). In this scenario, some of the vesicles carrying the receptors are destined for retrograde transport towards the cell body (35,36), while some are recycled back to the cell surface (37). For instance, Pincher, a membrane trafficking protein, has been shown to regulate the formation of TRK-carrying endosomes from the plasma membrane ruffles in soma, axon, and dendrite (38). Pincher-mediated retrograde transport of signaling TRK receptors are rescued from lysosomal degradation (39). Moreover, BDNF-induced survival signaling in hippocampal neurons has been shown to be regulated by Endophilin-A, that is involved in the endosomal sorting of active TRKB receptors (40). Evidence suggests that the maintenance of long-term potentiation in hippocampal slices depends on the recycling and re-secretion of BDNF, thus, emphasizing the necessity of TRKB receptor recycling back to the cell surface (41). Another study indicates that Slitrk5, by coupling to TRKB receptors, promotes its recycling to the cell surface in striatal neurons (42).
AP-2, as a member of the endocytic machinery (43), contributes to the formation of clathrin-coated vesicles around cargo proteins for endocytosis (44). In line with this idea, our results indicate a BDNF-induced enhancement of TRKB and AP2M interaction, suggesting that the receptor is endocytosed upon activation. Although acute treatment (e.g. 30 min) with antidepressant drugs can induce a rapid phosphorylation of TRKB receptor in the mouse brain (7), these drugs disrupted the interaction of TRKB:AP2M. Thus, we suggest that the differential regulation of TRKB:AP2M by BDNF and antidepressant drugs may underlie the differential surface expression of the receptor. Zheng and colleagues (22) have shown that BDNF quickly reduces the TRKB receptor surface level, while our data indicates that fluoxetine and imipramine enhance cell surface expression of TRKB. Interestingly, AMPA receptors, also committed to AP-2 complex-dependent endocytosis in the brain (45,46), are internalized upon activation by glutamate. However, while the activation of NMDA receptors results in rapid reinsertion of AMPAR subunits to the cell surface, AMPARs activated in the absence of NMDAR activation are destined for degradation in lysosomes (47). Thus, the source of the activation signal recruits alternative machineries to differentially regulate the fate of the target receptor.
Competitive binding can also be a crucial factor for determining the fate of a receptor as it was shown by Lavezzari and colleagues (48). These authors found that the interaction of postsynaptic density (PSD) proteins: PSD-95, PSD-93 and SAP-97 with NMDA receptor prevents endocytosis via blocking the binding of AP-2 to the designated motif on the Cterminus of the NR2B subunit. According to these authors, due to binding to PSD proteins, the surface expression of NR2B subunits was significantly upregulated. Furthermore, the competitive binding of N-ethylmaleimide-sensitive fusion protein (NSF) and AP-2 to GluR2 subunit of AMPA receptors regulates the cell surface level of this subunit, thus NSF acts as a steric blocker for the AP-2 complex (45). Based on this evidence, it is plausible to suggest that the antidepressant drug-induced prevention of AP-2 complex recruitment to TRKB receptors (or the disruption of formed complexes) allows binding of other proteins such as PLCg1 upon activation by BDNF, resulting in enhanced TRKB signaling. This argument is supported with our finding that the AP2M binding site on TRKB receptors, Y816, is the same one which is also targeted by PLCg binding and favored by antidepressants (9).
Although the AP-2 complex is best known for its major role in endocytosis (49), it remains doubtful whether the main purpose of the identified AP-2:TRKB interaction in the present study is the facilitated endocytosis of TRKB. A recent study performing endocytosis assays with WT and AP2M null neurons overexpressing EGFP-TRKB and treated with BDNF could not detect a significant defect in the uptake of TRKB upon loss of AP-2. Instead, the authors showed that the retrograde axonal transport of autophagosomes containing TRKB receptor is dependent on AP-2 linking the autophagy adaptor LC3 to p150 Glued , an activator of the dynein motor required for autophagosome transport (16). Interesting to highlight, LC3 (UniProt: Q91VR7) was co-precipitated with TRKB, and fluoxetine treatment decreased (in fact abolished) such interaction as shown by our mass spectrometry data.
This led us to conclude that the action of the AP-2 complex is not necessarily restricted to the neuronal surface, but can take place in different neuronal compartments. Therefore, there is not only the possibility that the antidepressant drugs disrupt the interaction of TRKB receptors with AP2M at the cell surface, but they might also uncouple the interaction while both TRKB and AP2M are present on these autophagosomes which are transported towards the cell soma. Disruption of the AP2M:TRKB interaction either at the surface or on internal vesicles could prevent the interaction of the complex with the members of the autophagylysosome pathway that can lead to degradation of TRKB receptors (16). The delayed delivery of the signaling endosomes to soma may locally enhance the specific signaling pathways that can recruit more plasticity-related proteins to the synapses. However, the two above mentioned scenarios are complementary rather than mutually exclusive. Recently, a study led by Eran Perlson described that BDNF-induced activation of TRKB leads to the internalization and dimerization of this receptor (50). These authors argue that TRKB is expressed in the cell surface as a monomer and only after BDNF-binding and internalization, in a process dependent on dynamin, the dimerization and consequent signaling is triggered.
In the present study we combined in vivo, in vitro and in silico methods to gain insight into putative interaction domains between TRKB and AP-2. The in silico techniques allowed us to obtain a priori knowledge about possible interactions, especially involving the Y816 residue and the AP2M subunit. In fact, in the present study, the in silico results on the AP2M interaction with the TRKB C-terminus matched the in vitro observations very well. As predicted by the CABS-docking server, AP2M displayed an increased interaction with phosphorylated TRKB (simulated using the phosphomimetic mutation Y816E). Interestingly, the Y816A mutation led to a decrease below the levels of control peptide (ctrl, nonphosphorylated) both observed in in silico (interpreted as a less stable complex) and in vitro assays. This suggests a putative interaction between AP2M and non-activated TRKB. The role of such interaction, if any, or its occurrence in vivo, remains to be investigated.
At this point, we speculated that the interaction of AP2M and TRKB could be a putative target for antidepressant drugs. Supporting this idea, cell-free assays indicated that the disruption of the TRKB:AP2M by fluoxetine happens in a dose-dependent manner.
However, the same effect was not replicated in the AP2M:TRKB.peptide interaction assay.
Therefore, although our data supports a putative binding site for antidepressant drugs in the TRKB complex, it is unlikely to be located between the receptor C-terminal portion and AP2M subunit. Moreover, such elusive site would present a low affinity for fluoxetine, given the effective doses are found above 0.3 µM.
Altogether, in the present study we suggest a putative novel mechanism where the drug-induced disruption of the TRKB:AP2M regulates TRKB receptor signaling. However, further investigation will be necessary to address the precise mechanism and the long-term consequences of such a disruption.