Summary It is unclear how binding of antidepressant drugs to their targets gives rise to the clinical antidepressant effect. We discovered that the transmembrane domain of tyrosine kinase receptor 2 (TRKB), the brain-derived neurotrophic factor (BDNF) receptor that promotes neuronal plasticity and antidepressant responses, has a cholesterol-sensing function that mediates synaptic effects of cholesterol. We then found that both typical and fast-acting antidepressants directly bind to TRKB, thereby facilitating synaptic localization of TRKB and its activation by BDNF. Extensive computational approaches including atomistic molecular dynamics simulations revealed a binding site at the transmembrane region of TRKB dimers. Mutation of the TRKB antidepressant-binding motif impaired cellular, behavioral, and plasticity-promoting responses to antidepressants in vitro and in vivo . We suggest that binding to TRKB and allosteric facilitation of BDNF signaling is the common mechanism for antidepressant action, which may explain why typical antidepressants act slowly and how molecular effects of antidepressants are translated into clinical mood recovery.

It is unclear how binding of antidepressant drugs to their targets gives rise to the clinical antidepressant effect. We found that both typical and fast-acting antidepressants bind to a cholesterol interaction motif in the BDNF receptor TRKB, a known mediator of neuronal plasticity and antidepressant responses. Cholesterol stabilized a cross-shaped configuration of TRKB transmembrane domain dimers and prolonged TRKB cell surface expression and activation by BDNF. Mutation of the TRKB cholesterol interaction site or cholesterol depletion by pravastatin impaired BDNF-mediated plasticity and cellular and behavioral responses to antidepressants in vitro and in vivo . We suggest that binding to and facilitation of TRKB activity is the common mechanism for antidepressant action, and propose a framework for how molecular effects of antidepressants are translated into clinical mood recovery.

The molecular pathomechanisms of major depressive disorder (MDD) are still not completely understood. Here, we follow the hypothesis, that mitochondria dysfunction which is inevitably associated with bioenergetic disbalance is a risk factor that contributes to the susceptibility of an individual to develop MDD. Thus, we investigated molecular mechanisms related to mitochondrial function in induced neuronal progenitor cells (NPCs) which were reprogrammed from fibroblasts of eight MDD patients and eight non-depressed controls. We found significantly lower maximal respiration rates, altered cytosolic basal calcium levels, and smaller soma size in NPCs derived from MDD patients. These findings are partially consistent with our earlier observations in MDD patient-derived fibroblasts. Furthermore, we differentiated MDD and control NPCs into iPS-neurons and analyzed their passive biophysical and active electrophysiological properties to investigate whether neuronal function can be related to altered mitochondrial activity and bioenergetics. Interestingly, MDD patient-derived iPS-neurons showed significantly lower membrane capacitance, a less hyperpolarized membrane potential, increased Na+ current density and increased spontaneous electrical activity. Our findings indicate that functional differences evident in fibroblasts derived from MDD patients are partially present after reprogramming to induced-NPCs, could relate to altered function of iPS-neurons and thus might be associated with the aetiology of major depressive disorder.

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.

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.

BACKGROUND: The molecular pathomechanisms of major depressive disorder (MDD) are still not completely understood. Here, we follow the hypothesis, that mitochondrial dysfunction which is inevitably associated with bioenergetic misbalance is a risk factor that contributes to the susceptibility of an individual to develop MDD. MDD can be regarded as disease of the mind as well as of the body. Thus, we focused on mitochondrial and cellular function in reprogrammed neural cells from MDD patients and healthy controls. METHODS: We investigated molecular mechanisms related to mitochondrial and cellular functions in induced neuronal progenitor cells (NPCs) as well as in neurons, which were reprogrammed from fibroblasts of depressed patients and non-depressed controls, respectively. RESULTS: We found significantly lower basal respiration rates and a less hyperpolarized mitochondrial membrane potential in NPCs derived from MDD patients. These findings are in line with our earlier observations in patient-derived fibroblasts (1). Furthermore, we differentiated MDD and control NPCs into iPS-neurons and analyzed their passive biophysical and active electrophysiological properties. Interestingly, MDD patient-derived iPS-neurons showed significantly lower membrane capacitance, a more depolarized membrane potential, and increased spontaneous electrical activity. This is the first report showing functional differences in MDD patient-derived and control NPCs and iPS-neurons. CONCLUSION: Our findings indicate that functional differences evident in fibroblasts from depressed patients are also present after reprogramming and differentiation to induced-NPCs and iPS-neurons and might be associated with the aetiology of major depressive disorder.