Mutation in the TRKB Cholesterol Recognition Site that blocks Antidepressant Binding does not Influence the Basal or BDNF-Stimulated Activation of TRKB.

Brain-derived neurotrophic factor (BDNF) acting upon its receptor Neurotrophic tyrosine kinase receptor 2 (NTRK2, TRKB) plays a central role in the development and maintenance of synaptic function and activity- or drug-induced plasticity. TRKB possesses an inverted cholesterol recognition and alignment consensus sequence (CARC), suggesting this receptor can act as a cholesterol sensor. We have recently shown that antidepressant drugs directly bind to the CARC domain of TRKB dimers, and that this binding as well as biochemical and behavioral responses to antidepressants are lost with a mutation in the TRKB CARC motif (Tyr433Phe). However, it is not clear if this mutation can also compromise the receptor function and lead to behavioral alterations. Here, we observed that Tyr433Phe mutation does not alter BDNF binding to TRKB, or BDNF-induced dimerization of TRKB. In this line, primary cultures from embryos of heterozygous Tyr433Phe mutant mice (hTRKB.Tyr433Phe) are responsive to BDNF-induced activation of TRKB, and samples from adult mice do not show any difference on TRKB activation compared to wild-type littermates (TRKB.wt). The behavioral phenotype of hTRKB.Tyr433Phe mice is indistinguishable from the wild-type mice in cued fear conditioning, contextual discrimination task, or the elevated plus maze, whereas mice heterozygous to BDNF null allele show a phenotype in context discrimination task. Taken together, our results indicate that Tyr433Phe mutation in the TRKB CARC motif does not show signs of loss-of-function of BDNF responses, while antidepressant binding to TRKB and responses to antidepressants are lost in Tyr433Phe mutants, making them an interesting mouse model for antidepressant research.

Psychedelics promote plasticity by directly binding to BDNF receptor TrkB.

Psychedelics produce fast and persistent antidepressant effects and induce neuroplasticity resembling the effects of clinically approved antidepressants. We recently reported that pharmacologically diverse antidepressants, including fluoxetine and ketamine, act by binding to TrkB, the receptor for BDNF. Here we show that lysergic acid diethylamide (LSD) and psilocin directly bind to TrkB with affinities 1,000-fold higher than those for other antidepressants, and that psychedelics and antidepressants bind to distinct but partially overlapping sites within the transmembrane domain of TrkB dimers. The effects of psychedelics on neurotrophic signaling, plasticity and antidepressant-like behavior in mice depend on TrkB binding and promotion of endogenous BDNF signaling but are independent of serotonin 2A receptor (5-HT2A) activation, whereas LSD-induced head twitching is dependent on 5-HT2A and independent of TrkB binding. Our data confirm TrkB as a common primary target for antidepressants and suggest that high-affinity TrkB positive allosteric modulators lacking 5-HT2A activity may retain the antidepressant potential of psychedelics without hallucinogenic effects.

TRKB interaction with PSD95 is associated with latency of fluoxetine and 2R,6R-hydroxynorketamine.

Brain derived neurotrophic factor (BDNF) and its receptor tropomyosin kinase receptor B (TRKB) are key regulators of activity-dependent plasticity in the brain. TRKB is the target for both slow- and rapid-acting antidepressants and BDNF-TRKB system mediates the plasticity-inducing effects of antidepressants through their downstream targets. Particularly, the protein complexes that regulate the trafficking and synapse recruitment of TRKB receptors might be crucial in this process. In the present study, we investigated the interaction of TRKB with the postsynaptic density protein 95 (PSD95). We found that antidepressants increase the TRKB:PSD95 interaction in adult mouse hippocampus. Fluoxetine, a slow-acting antidepressant, increases this interaction only after a long-term (7 days) treatment, while (2R,6R)-hydroxynorketamine (RHNK), an active metabolite of rapid-acting antidepressant ketamine, achieves this within a short treatment regimen (3 days). Moreover, the drug-induced changes of TRKB:PSD95 interaction correlate with drug latency in behaviour, observed in mice subjected to an object location memory test (OLM). While silencing of PSD95 by viral delivery of shRNA in hippocampus abolished the RHNK-induced plasticity in mice in OLM, overexpression of PSD95 shortened the fluoxetine latency. In summary, changes in the TRKB:PSD95 interaction contribute to differences observed in drug latency. This study sheds a light on a novel mechanism of action of different classes of antidepressants.

Ketamine and its metabolite 2R,6R-hydroxynorketamine promote ocular dominance plasticity and release tropomyosin-related kinase B from inhibitory control without reducing perineuronal nets enwrapping parvalbumin interneurons.

Ketamine has been described as a fast-acting antidepressant, exerting effects in depressed patients and in preclinical models with a rapid onset of action. The typical antidepressant fluoxetine is known to induce plasticity in the adult rodent visual cortex, as assessed by a shift in ocular dominance, a classical model of brain plasticity, and a similar effect has been described for ketamine and its metabolite 2R,6R-hydroxynorketamine (R,R-HNK). Here, we demonstrate that ketamine (at 3 or 20 mg/kg) and R,R-HNK facilitated the shift in ocular dominance in monocularly deprived mice, after three injections, throughout the 7-day monocular deprivation regimen. Notably, the comparison between the treatments indicates a higher effect size of R,R-HNK compared with ketamine. Treatment with ketamine or R,R-HNK failed to influence the levels of perineuronal nets (PNNs) surrounding parvalbumin-positive interneurons. However, we observed in vitro that both ketamine and R,R-HNK are able to disrupt the tropomyosin-related kinase B (TRKB) interaction with the protein tyrosine phosphatase sigma (PTPσ), which upon binding to PNNs dephosphorylates TRKB. These results support a model where diverse drugs promote the reinstatement of juvenile-like plasticity by directly binding TRKB and releasing it from PTPσ regulation, without necessarily reducing PNNs deposits.

nNOS-induced tyrosine nitration of TRKB impairs BDNF signaling and restrains neuronal plasticity.

Nitric oxide (NO) has been long recognized as an important modulator of neural plasticity, but characterization of the molecular mechanisms involved - specially the guanylyl cyclase-independent ones - has been challenging. There is evidence that NO could modify BDNF-TRKB signaling, a key mediator of neuronal plasticity. However, the mechanism underlying the interplay of NO and TRKB remains unclear. Here we show that NO induces nitration of the tyrosine 816 in the TRKB receptor in vivo and in vitro, and that post-translational modification inhibits TRKB phosphorylation and binding of phospholipase Cγ1 (PLCγ1) to this same tyrosine residue. Additionally, nitration triggers clathrin-dependent endocytosis of TRKB through the adaptor protein AP-2 and ubiquitination, thereby increasing translocation of TRKB away from the neuronal surface and directing it towards lysosomal degradation. Accordingly, inhibition of nitric oxide increases TRKB phosphorylation and TRKB-dependent neurite branching in neuronal cultures. In vivo, chronic inhibition of neuronal nitric oxide synthase (nNOS) dramatically reduced TRKB nitration and facilitated TRKB signaling in the visual cortex, and promoted a shift in ocular dominance upon monocular deprivation - an indicator of increased plasticity. Altogether, our data describe and characterize a new molecular brake on plasticity, namely nitration of TRKB receptors.

BDNF receptor TrkB as the mediator of the antidepressant drug action.

Brain-derived neurotrophic factor (BDNF) signaling through its receptor TrkB has for a long time been recognized as a critical mediator of the antidepressant drug action, but BDNF signaling has been considered to be activated indirectly through the action of typical and rapid-acting antidepressants through monoamine transporters and glutamate NMDA receptors, respectively. However, recent findings demonstrate that both typical and the fast-acting antidepressants directly bind to TrkB and thereby allosterically potentiate BDNF signaling, suggesting that TrkB is the direct target for antidepressant drugs. Increased TrkB signaling particularly in the parvalbumin-expressing interneurons orchestrates iPlasticity, a state of juvenile-like enhanced plasticity in the adult brain. iPlasticity sensitizes neuronal networks to environmental influences, enabling rewiring of networks miswired by adverse experiences. These findings have dramatically changed the position of TrkB in the antidepressant effects and they propose a new end-to-end model of the antidepressant drug action. This model emphasizes the enabling role of antidepressant treatment and the active participation of the patient in the process of recovery from mood disorders.

Nitric Oxide Synthase inhibition counteracts the stress-induced DNA methyltransferase 3b expression in the hippocampus of rats.

It has been postulated that the activation of NMDA receptors (NMDAr) and nitric oxide (NO) production in the hippocampus is involved in the behavioral consequences of stress. Stress triggers NMDAr-induced calcium influx in limbic areas, such as the hippocampus, which in turn activates neuronal NO synthase (nNOS). Inhibition of nNOS or NMDAr activity can prevent stress-induced effects in animal models, but the molecular mechanisms behind this effect are still unclear. In this study, cultured hippocampal neurons treated with NMDA or dexamethasone showed an increased of DNA methyltransferase 3b (DNMT3b) mRNA expression, which was blocked by pre-treatment with nNOS inhibitor nω -propyl-l-arginine (NPA). In rats submitted to the Learned Helplessness paradigm (LH), we observed that inescapable stress increased DNMT3b mRNA expression at 1h and 24h in the hippocampus. The NOS inhibitors 7-NI and aminoguanidine (AMG) decreased the number of escape failures in LH and counteracted the changes in hippocampal DNMT3b mRNA induced in this behavioral paradigm. Altogether, our data suggest that NO produced in response to NMDAr activation following stress upregulates DNMT3b in the hippocampus.

Facilitation of TRKB Activation by the Angiotensin II Receptor Type-2 (AT2R) Agonist C21.

Blockers of angiotensin II type 1 receptor (AT1R) exert antidepressant-like effects by indirectly facilitating the activation of the angiotensin II type 2 receptor (AT2R), which leads to increased surface expression and transactivation of tropomyosin-related kinase B receptors (TRKB). Compound 21 (C21) is a non-peptide AT2R agonist that produces neuroprotective effects. However, the behavioral effects of C21 and its involvement with the brain-derived neurotrophic factor (BDNF)-TRKB system still need further investigation. The aim of the present study was to assess the effect of C21 on the activation of TRKB and its consequences on conditioned fear. The administration of C21 (0.1-10 µM/15 min) increased the surface levels of TRKB but was not sufficient to increase the levels of phosphorylated TRKB (pTRKB) in cultured cortical neurons from rat embryos. Consistent with increased TRKB surface expression, C21 (10 µM/15 min or 3 days) facilitated the effect of BDNF (0.1 ng/mL/15 min) on pTRKB in these cells. In contextual fear conditioning, the freezing time of C21-treated (administered intranasally) wild-type mice was decreased compared to the vehicle-treated group, but no effect of C21 was observed in BDNF.het animals. We observed no effect of C21 in the elevated plus-maze test for anxiety. Taken together, our results indicate that C21 facilitated BDNF effect by increasing the levels of TRKB on the cell surface and reduced the freezing time of mice in a BDNF-dependent manner, but not through a general anxiolytic-like effect.

Urokinase plasminogen activator mediates changes in human astrocytes modeling fragile X syndrome.

The function of astrocytes intertwines with the extracellular matrix, whose neuron and glial cell-derived components shape neuronal plasticity. Astrocyte abnormalities have been reported in the brain of the mouse model for fragile X syndrome (FXS), the most common cause of inherited intellectual disability, and a monogenic cause of autism spectrum disorder. We compared human FXS and control astrocytes generated from human induced pluripotent stem cells and we found increased expression of urokinase plasminogen activator (uPA), which modulates degradation of extracellular matrix. Several pathways associated with uPA and its receptor function were activated in FXS astrocytes. Levels of uPA were also increased in conditioned medium collected from FXS hiPSC-derived astrocyte cultures and correlated inversely with intracellular Ca2+ responses to activation of L-type voltage-gated calcium channels in human astrocytes. Increased uPA augmented neuronal phosphorylation of TrkB within the docking site for the phospholipase-Cγ1 (PLCγ1), indicating effects of uPA on neuronal plasticity. Gene expression changes during neuronal differentiation preceding astrogenesis likely contributed to properties of astrocytes with FXS-specific alterations that showed specificity by not affecting differentiation of adenosine triphosphate (ATP)-responsive astrocyte population. To conclude, our studies identified uPA as an important regulator of astrocyte function and demonstrated that increased uPA in human FXS astrocytes modulated astrocytic responses and neuronal plasticity.

Perineuronal Net Receptor PTPσ Regulates Retention of Memories.

Perineuronal nets (PNNs) have an important physiological role in the retention of learning by restricting cognitive flexibility. Their deposition peaks after developmental periods of intensive learning, usually in late childhood, and they help in long-term preservation of newly acquired skills and information. Modulation of PNN function by various techniques enhances plasticity and regulates the retention of memories, which may be beneficial when memory persistence entails negative symptoms such as post-traumatic stress disorder (PTSD). In this study, we investigated the role of PTPσ [receptor-type tyrosine-protein phosphatase S, a phosphatase that is activated by binding of chondroitin sulfate proteoglycans (CSPGs) from PNNs] in retention of memories using Novel Object Recognition and Fear Conditioning models. We observed that mice haploinsufficient for PTPRS gene (PTPσ+/-), although having improved short-term object recognition memory, display impaired long-term memory in both Novel Object Recognition and Fear Conditioning paradigm, as compared to WT littermates. However, PTPσ+/- mice did not show any differences in behavioral tests that do not heavily rely on cognitive flexibility, such as Elevated Plus Maze, Open Field, Marble Burying, and Forced Swimming Test. Since PTPσ has been shown to interact with and dephosphorylate TRKB, we investigated activation of this receptor and its downstream pathways in limbic areas known to be associated with memory. We found that phosphorylation of TRKB and PLCγ are increased in the hippocampus, prefrontal cortex, and amygdaloid complex of PTPσ+/- mice, but other TRKB-mediated signaling pathways are not affected. Our data suggest that PTPσ downregulation promotes TRKB phosphorylation in different brain areas, improves short-term memory performance but disrupts long-term memory retention in the tested animal models. Inhibition of PTPσ or disruption of PNN-PTPσ-TRKB complex might be a potential target for disorders where negative modulation of the acquired memories can be beneficial.

Cholesterol-recognition motifs in the transmembrane domain of the tyrosine kinase receptor family: The case of TRKB.

Cholesterol is an essential constituent of cell membranes. The discovery of cholesterol-recognition amino acid consensus (CRAC) motif in proteins indicated a putative direct, non-covalent interaction between cholesterol and proteins. In the present study, we evaluated the presence of a CRAC motif and its inverted version (CARC) in the transmembrane region (TMR) of the tyrosine kinase receptor family (RTK) in several species using in silico methods. CRAC motifs were found across all species analyzed, while CARC was found only in vertebrates. The tropomyosin-related kinase B (TRKB), a member of the RTK family, through interaction with its endogenous ligand brain-derived neurotrophic factor (BDNF) is a core participant in the neuronal plasticity process and exhibits a CARC motif in its TMR. Upon identifying the conserved CARC motif in the TRKB, we performed molecular dynamics simulations of the mouse TRKB.TMR. The simulations indicated that cholesterol interaction with the TRKB CARC motif occurs mainly at the central Y433 residue. Our binding assay suggested a bell-shaped effect of cholesterol on BDNF interaction with TRKB receptors, and our results suggest that CARC/CRAC motifs may play a role in the function of the RTK family TMR.

Antidepressant drugs act by directly binding to TRKB neurotrophin receptors.

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.

Role of 5-HT1A and 5-HT2C receptors of the dorsal periaqueductal gray in the anxiety- and panic-modulating effects of antidepressants in rats.

Antidepressant drugs are first-line treatment for panic disorder. Facilitation of 5-HT1A receptor-mediated neurotransmission in the dorsal periaqueductal gray (dPAG), a key panic-associated area, has been implicated in the panicolytic effect of the selective serotonin reuptake inhibitor fluoxetine. However, it is still unknown whether this mechanism accounts for the antipanic effect of other classes of antidepressants drugs (ADs) and whether the 5-HT interaction with 5-HT2C receptors in this midbrain area (which increases anxiety) is implicated in the anxiogenic effect caused by short-term treatment with ADs. The results showed that previous injection of the 5-HT1A receptor antagonist WAY-100635 in the dPAG blocked the panicolytic-like effect caused by chronic systemic administration of the tricyclic AD imipramine in male Wistar rats tested in the elevated T-maze. Neither chronic treatment with imipramine nor fluoxetine changed the expression of 5-HT1A receptors in the dPAG. Treatment with these ADs also failed to significantly change ERK1/2 (extracellular-signal regulated kinase) phosphorylation level in this midbrain area. Blockade of 5-HT2C receptors in the dPAG with the 5-HT2C receptor antagonist SB-242084 did not change the anxiogenic effect caused by a single acute injection of fluoxetine or imipramine in the Vogel conflict test. These results reinforce the view that the facilitation of 5-HT1A receptor-mediated neurotransmission in the dPAG is a common mechanism involved in the panicolytic effect caused by chronic administration of ADs. On the other hand, the anxiogenic effect observed after short-term treatment with these drugs does not depend on 5-HT2C receptors located in the dPAG.

Antidepressant and Antipsychotic Drugs Reduce Viral Infection by SARS-CoV-2 and Fluoxetine Shows Antiviral Activity Against the Novel Variants in vitro.

Repurposing of currently available drugs is a valuable strategy to tackle the consequences of COVID-19. Recently, several studies have investigated the effect of psychoactive drugs on SARS-CoV-2 in cell culture models as well as in clinical practice. Our aim was to expand these studies and test some of these compounds against newly emerged variants. Several antidepressants and antipsychotic drugs with different primary mechanisms of action were tested in ACE2/TMPRSS2-expressing human embryonic kidney cells against the infection by SARS-CoV-2 spike protein-dependent pseudoviruses. Some of these compounds were also tested in human lung epithelial cell line, Calu-1, against the first wave (B.1) lineage of SARS-CoV-2 and the variants of concern, B.1.1.7, B.1.351, and B.1.617.2. Several clinically used antidepressants, including fluoxetine, citalopram, reboxetine, imipramine, as well as antipsychotic compounds chlorpromazine, flupenthixol, and pimozide inhibited the infection by pseudotyped viruses with minimal effects on cell viability. The antiviral action of several of these drugs was verified in Calu-1 cells against the B.1 lineage of SARS-CoV-2. By contrast, the anticonvulsant carbamazepine, and novel antidepressants ketamine, known as anesthetic at high doses, and its derivatives as well as MAO and phosphodiesterase inhibitors phenelzine and rolipram, respectively, showed no activity in the pseudovirus model. Furthermore, fluoxetine remained effective against pseudoviruses with common receptor binding domain mutations, N501Y, K417N, and E484K, as well as B.1.1.7 (alpha), B.1.351 (beta), and B.1.617.2 (delta) variants of SARS-CoV-2. Our study confirms previous data and extends information on the repurposing of these drugs to counteract SARS-CoV-2 infection including different variants of concern, however, extensive clinical studies must be performed to confirm our in vitro findings.

Chondroitinase and Antidepressants Promote Plasticity by Releasing TRKB from Dephosphorylating Control of PTPσ in Parvalbumin Neurons.

Perineuronal nets (PNNs) are an extracellular matrix structure rich in chondroitin sulfate proteoglycans (CSPGs), which preferentially encase parvalbumin-containing (PV+) interneurons. PNNs restrict cortical network plasticity but the molecular mechanisms involved are unclear. We found that reactivation of ocular dominance plasticity in the adult visual cortex induced by chondroitinase ABC (chABC)-mediated PNN removal requires intact signaling by the neurotrophin receptor TRKB in PV+ neurons. Additionally, we demonstrate that chABC increases TRKB phosphorylation (pTRKB), while PNN component aggrecan attenuates brain-derived neurotrophic factor (BDNF)-induced pTRKB in cortical neurons in culture. We further found that protein tyrosine phosphatase σ (PTPσ, PTPRS), receptor for CSPGs, interacts with TRKB and restricts TRKB phosphorylation. PTPσ deletion increases phosphorylation of TRKB in vitro and in vivo in male and female mice, and juvenile-like plasticity is retained in the visual cortex of adult PTPσ-deficient mice (PTPσ+/-). The antidepressant drug fluoxetine, which is known to promote TRKB phosphorylation and reopen critical period-like plasticity in the adult brain, disrupts the interaction between TRKB and PTPσ by binding to the transmembrane domain of TRKB. We propose that both chABC and fluoxetine reopen critical period-like plasticity in the adult visual cortex by promoting TRKB signaling in PV+ neurons through inhibition of TRKB dephosphorylation by the PTPσ-CSPG complex.SIGNIFICANCE STATEMENT Critical period-like plasticity can be reactivated in the adult visual cortex through disruption of perineuronal nets (PNNs) by chondroitinase treatment, or by chronic antidepressant treatment. We now show that the effects of both chondroitinase and fluoxetine are mediated by the neurotrophin receptor TRKB in parvalbumin-containing (PV+) interneurons. We found that chondroitinase-induced visual cortical plasticity is dependent on TRKB in PV+ neurons. Protein tyrosine phosphatase σ (PTPσ, PTPRS), a receptor for PNNs, interacts with TRKB and inhibits its phosphorylation, and chondroitinase treatment or deletion of PTPσ increases TRKB phosphorylation. Antidepressant fluoxetine disrupts the interaction between TRKB and PTPσ, thereby increasing TRKB phosphorylation. Thus, juvenile-like plasticity induced by both chondroitinase and antidepressant treatment is mediated by TRKB activation in PV+ interneurons.

Anti-Inflammatory Treatment with FTY720 Starting after Onset of Symptoms Reverses Synaptic Deficits in an AD Mouse Model.

Therapeutic approaches providing effective medication for Alzheimer's disease (AD) patients after disease onset are urgently needed. Previous studies in AD mouse models suggested that physical exercise or changed lifestyle can delay AD-related synaptic and memory dysfunctions when treatment started in juvenile animals long before onset of disease symptoms, while a pharmacological treatment that can reverse synaptic and memory deficits in AD mice was thus far not identified. Repurposing food and drug administration (FDA)-approved drugs for treatment of AD is a promising way to reduce the time to bring such medication into clinical practice. The sphingosine-1 phosphate analog fingolimod (FTY720) was approved recently for treatment of multiple sclerosis patients. Here, we addressed whether fingolimod rescues AD-related synaptic deficits and memory dysfunction in an amyloid precursor protein/presenilin-1 (APP/PS1) AD mouse model when medication starts after onset of symptoms (at five months). Male mice received intraperitoneal injections of fingolimod for one to two months starting at five to six months. This treatment rescued spine density as well as long-term potentiation in hippocampal cornu ammonis-1 (CA1) pyramidal neurons, that were both impaired in untreated APP/PS1 animals at six to seven months of age. Immunohistochemical analysis with markers of microgliosis (ionized calcium-binding adapter molecule 1; Iba1) and astrogliosis (glial fibrillary acid protein; GFAP) revealed that our fingolimod treatment regime strongly down regulated neuroinflammation in the hippocampus and neocortex of this AD model. These effects were accompanied by a moderate reduction of Aß accumulation in hippocampus and neocortex. Our results suggest that fingolimod, when applied after onset of disease symptoms in an APP/PS1 mouse model, rescues synaptic pathology that is believed to underlie memory deficits in AD mice, and that this beneficial effect is mediated via anti-neuroinflammatory actions of the drug on microglia and astrocytes.

Inactivation of the GATA Cofactor ZFPM1 Results in Abnormal Development of Dorsal Raphe Serotonergic Neuron Subtypes and Increased Anxiety-Like Behavior.

Serotonergic neurons in the dorsal raphe (DR) nucleus are associated with several psychiatric disorders including depression and anxiety disorders, which often have a neurodevelopmental component. During embryonic development, GATA transcription factors GATA2 and GATA3 operate as serotonergic neuron fate selectors and regulate the differentiation of serotonergic neuron subtypes of DR. Here, we analyzed the requirement of GATA cofactor ZFPM1 in the development of serotonergic neurons using Zfpm1 conditional mouse mutants. Our results demonstrated that, unlike the GATA factors, ZFPM1 is not essential for the early differentiation of serotonergic precursors in the embryonic rhombomere 1. In contrast, in perinatal and adult male and female Zfpm1 mutants, a lateral subpopulation of DR neurons (ventrolateral part of the DR) was lost, whereas the number of serotonergic neurons in a medial subpopulation (dorsal region of the medial DR) had increased. Additionally, adult male and female Zfpm1 mutants had reduced serotonin concentration in rostral brain areas and displayed increased anxiety-like behavior. Interestingly, female Zfpm1 mutant mice showed elevated contextual fear memory that was abolished with chronic fluoxetine treatment. Altogether, these results demonstrate the importance of ZFPM1 for the development of DR serotonergic neuron subtypes involved in mood regulation. It also suggests that the neuronal fate selector function of GATAs is modulated by their cofactors to refine the differentiation of neuronal subtypes.SIGNIFICANCE STATEMENT Predisposition to anxiety disorders has both a neurodevelopmental and a genetic basis. One of the brainstem nuclei involved in the regulation of anxiety is the dorsal raphe, which contains different subtypes of serotonergic neurons. We show that inactivation of a transcriptional cofactor ZFPM1 in mice results in a developmental failure of laterally located dorsal raphe serotonergic neurons and changes in serotonergic innervation of rostral brain regions. This leads to elevated anxiety-like behavior and contextual fear memory, alleviated by chronic fluoxetine treatment. Our work contributes to understanding the neurodevelopmental mechanisms that may be disturbed in the anxiety disorder.

Pharmacologically diverse antidepressants facilitate TRKB receptor activation by disrupting its interaction with the endocytic adaptor complex AP-2.

Several antidepressant drugs activate tropomyosin-related kinase B (TRKB) receptor, but it remains unclear whether these compounds employ a common mechanism for TRKB activation. Here, using MS, we found that a single intraperitoneal injection of fluoxetine disrupts the interaction of several proteins with TRKB in the hippocampus of mice. These proteins included members of adaptor protein complex-2 (AP-2) involved in vesicular endocytosis. The interaction of TRKB with the cargo-docking µ subunit of the AP-2 complex (AP2M) was confirmed to be disrupted by both acute and repeated fluoxetine treatments. Of note, fluoxetine disrupted the coupling between full-length TRKB and AP2M, but not the interaction between AP2M and the TRKB C-terminal region, indicating that the fluoxetine-binding site in TRKB lies outside the TRKB:AP2M interface. ELISA experiments revealed that in addition to fluoxetine, other chemically diverse antidepressants, such as imipramine, rolipram, phenelzine, ketamine, and its metabolite 2R,6R-hydroxynorketamine, also decreased the interaction between TRKB and AP2M in vitro Silencing the expression of AP2M in a TRKB-expressing mouse fibroblast cell line (MG87.TRKB) increased cell-surface expression of TRKB and facilitated its activation by brain-derived neurotrophic factor (BDNF), observed as levels of phosphorylated TRKB. Moreover, animals haploinsufficient for the Ap2m1 gene displayed increased levels of active TRKB, along with enhanced cell-surface expression of the receptor in cultured hippocampal neurons. Taken together, our results suggest that disruption of the TRKB:AP2M interaction is a common mechanism underlying TRKB activation by several chemically diverse antidepressants.

Reduced P2X receptor levels are associated with antidepressant effect in the learned helplessness model.

Purinergic receptors, especially P2RX, are associated to the severity of symptoms in patients suffering from depressive and bipolar disorders, and genetic deletion or pharmacological blockade of P2RX7 induces antidepressant-like effect in preclinical models. However, there is scarce evidence about the alterations in P2RX7 or P2RX4 levels and in behavioral consequences induced by previous exposure to stress, a major risk factor for depression in humans. In the present study, we evaluated the effect of imipramine (IMI) on P2RX7 and P2RX4 levels in dorsal and ventral hippocampus as well as in the frontal cortex of rats submitted to the pretest session of learned helplessness (LH) paradigm. Repeated, but not acute administration of IMI (15 mg/kg ip) reduced the levels of both P2RX7 and P2RX4 in the ventral, but not in dorsal hippocampus or frontal cortex. In addition, we tested the effect of P2RX7/P2RX4 antagonist brilliant blue G (BBG: 25 or 50 mg/kg ip) on the LH paradigm. We observed that repeated (7 days) but not acute (1 day) treatment with BBG (50 mg) reduced the number of failures to escape the shocks in the test session, a parameter mimicked by the same regimen of IMI treatment. Taken together, our data indicates that pharmacological blockade or decrease in the expression of P2RX7 is associated to the antidepressant-like behavior observed in the LH paradigm after repeated drug administration.

Activation of the TRKB receptor mediates the panicolytic-like effect of the NOS inhibitor aminoguanidine.

Nitric oxide (NO) triggers escape reactions in the dorsal periaqueductal gray matter (dPAG), a core structure mediating panic-associated response, and decreases the release of BDNF in vitro. BDNF mediates the panicolytic effect induced by antidepressant drugs and produces these effects per se when injected into the dPAG. Based on these findings, we hypothesize that nitric oxide synthase (NOS) inhibitors would have panicolytic properties associated with increased BDNF signaling in the dPAG. We observed that the repeated (7 days), but not acute (1 day), systemic administration of the NOS inhibitor aminoguanidine (AMG; 15 mg/kg/day) increased the latency to escape from the open arm of the elevated T-maze (ETM) and inhibited the number of jumps in hypoxia-induced escape reaction in rats, suggesting a panicolytic-like effect. Repeated, but not acute, AMG administration (15 mg/kg) also decreased nitrite levels and increased TRKB phosphorylation at residues Y706/7 in the dPAG. Notwithstanding the lack of AMG effect on total BDNF levels in this structure, the microinjection of the TRK antagonist K252a into the dPAG blocked the anti-escape effect of this drug in the ETM. Taken together our data suggest that the inhibition of NO production by AMG increases the levels of pTRKB, which is required for the panicolytic-like effect observed.