Biology subjects

Casarotto, P. C.

Publications and source records attributed to Casarotto, P. C..

11 recordsLinked to original sources

bioRxiv2026

Fingolimod acutely facilitates the activation of TRKB

Fingolimod (FNG) is a sphingosine-1-phosphate receptor agonist currently prescribed for the treatment of remitting-relapsing multiple sclerosis. However, an increasing body of evidence indicates that FNG has a variety of other effects on the central nervous system, making it a good candidate to target other brain disorders that display loss of neuronal cells and synaptic plasticity. FNG treatments induce production of brain-derived neurotrophic factor (BDNF), which promotes neuronal plasticity, arborization and survival via signaling through its cognate receptor TRKB. In this study we characterize the relationship between FNG and TRKB in vitro and in vivo following acute treatments. We found that FNG induces TRKB activation in primary neuronal cultures in a BDNF-dependent way, indicating a rapid effect of FNG. This effect is different from the one elicited by antidepressants and is likely mediated by modulation of plasma membrane properties, as the enhancement of fluoxetine binding and dimerization of the cholesterol-insensitive TRKB mutant Y433F mimic the effects of cholesterol. Moreover, acute FNG treatment normalizes the generalization of conditioned fear response seen in heterozygous BDNF null female mice without affecting the wild-type littermates. Taken together, our data indicate that FNG allosterically promotes TRKB signaling and thereby induces the increase in BDNF production, which mediates the therapeutic effects of the drug on neuronal plasticity.

neuroscience↗

bioRxiv2022

Ketamine and its metabolite 2R,6R-hydroxynorketamine promote ocular dominance plasticity and release TRKB from inhibitory control without changing 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 3 injections, throughout the 8-days regimen. Notably, the comparison between the treatments indicates a higher effect size of R,R-HNK compared to 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 (PTP{sigma}), 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 PTP{sigma} regulation, without necessarily affecting PNNs deposits.

neuroscience↗

bioRxiv2021

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 (0.1-10M/15min) increased the surface levels of TRKB but was not sufficient to increase the levels of phosphorylated TRKB (pTKB) in cultured cortical neurons from rat embryos. Consistent with increased TRKB surface expression, C21 (10M/15min or 3 days) facilitated the effect of BDNF (0.1ng/ml/15min) 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.

pharmacology and toxicology↗

bioRxiv2021

Perineuronal net receptor PTPσ regulates retention of memories

Perineuronal nets (PNNs) have an important physiological role in 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 new acquired skills and information. Modulation of PNN function by various techniques enhances plasticity and regulates 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 PTP{sigma} (receptor-type tyrosine-protein phosphatase S, a phosphatase that is activated by binding of chondroitin sulfate proteoglycans from PNNs) in retention of memories using novel object recognition and fear conditioning rodent models. We observed that mice haploinsufficient for PTPRS gene (PTP{sigma}+/-), 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, PTP{sigma}+/- mice didnt 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 PTP{sigma} 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 PLC{gamma} are increased in the hippocampus, prefrontal cortex and amygdala of PTP{sigma}+/- mice, but other TRKB-mediated signaling pathways are not affected. Our data suggest that disruption of PNN-PTP{sigma} complex facilitates short-term memory by promoting TRKB phosphorylation in different brain areas, but that PTP{sigma} activity is required for the retention of long-term memories. Inhibition of PTP{sigma} or disruption of PNN-PTP{sigma}-TRKB complex might be a potential target for disorders where negative modulation of the acquired memories can be beneficial. Conflict of interestThe authors declare no conflict of interest. FundingThis work was supported by Doctoral Program in Integrative Life Science, Jalmari ja Rauha Ahokkaan Saatio grant, Centre for International Mobility (CIMO) Grant TM-16-10112, and by grants from European Research Council (#322742), EU Joint Programme - Neurodegenerative Disease Research (JPND) CircProt (#301225 and #643417), Sigrid Juselius Foundation, Jane and Aatos Erkko Foundation, and the Academy of Finland (#294710 and #307416). None of the funders had a role in the data acquisition, analysis or manuscript preparation. Author contributionsPC, CB and EC designed the study; AL, PC and CB performed the experiments and analyzed the data; AL wrote the manuscript draft; AL, PC, CB and EC revised the manuscript. Contribution to the Field StatementPlasticity of neuronal networks increases brains ability to adapt and it is compromised in various conditions including psychiatric diseases, brain injuries, neurodevelopmental and neurodegenerative disorders. Perineuronal nets, through their receptor PTP{sigma} restrict neuronal plasticity in adult brain, which is considered important for the retention of long-term memories. We have previously shown that this restricted plasticity is mediated by inhibition by PTP{sigma} of the TRKB neurotrophin receptor. We have now investigated the biochemical and behavioral phenotype of mice with reduced PTP{sigma} expression. We observed that PTP{sigma}+/- mice have increased phosphorylation of TRKB and its downstream partner PLC{gamma}1 in the prefrontal cortex, hippocampus and amygdala, demonstrating chronic overactivation of plasticity-related pathways. Consistently, PTP{sigma}+/- mice demonstrated facilitated learning but impaired long-term memory retention. Unexpectedly, long-term memory of PTP{sigma}+/- mice was impaired, suggesting that perneuronal net-stimulated PTP{sigma} activity is important in memory retention. This effect of neuronal "hyperplasticity" induced by PTP{sigma} knockout may be both beneficial and harmful in the treatment of human patients, which should be taken into account when developing therapeutic strategies.

neuroscience↗

bioRxiv2020

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 sulphate 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 (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 BDNF-induced pTRKB in cortical neurons in culture. We further found that protein tyrosine phosphatase sigma (PTP{sigma}, PTPRS), receptor for CSPGs, interacts with TRKB and restricts TRKB phosphorylation. PTP{sigma} 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 PTP{sigma} deficient mice (PTP{sigma}+/-). 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 PTP{sigma} 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 PTP{sigma}-CSPG complex. Significance statementCritical 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 type S (PTP{sigma}, PTPRS), a receptor for PNNs, interacts with TRKB and inhibits its phosphorylation, and chondroitinase treatment or deletion of PTP{sigma} increases TRKB phosphorylation. Antidepressant fluoxetine disrupts the interaction between TRKB and PTP{sigma}, thereby increasing TRKB phosphorylation. Thus, juvenile-like plasticity induced by both chondroitinase and antidepressant treatment is mediated by TRKB activation in PV+ interneurons.

neuroscience↗

bioRxiv2020

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

It has been postulated that 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 increased of DNA methyltransferase 3b (DNMT3b) mRNA expression, which was blocked by pre-treatment with nNOS inhibitor n{omega}-propyl-L-arginine (NPA). In rats submitted to the Learned Helplessness paradigm (LH), we observed that inescapable stress increased of 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.

pharmacology and toxicology↗

bioRxiv2019

Antidepressants act by binding to the cholesterol-interaction site at TRKB neurotrophin receptor

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 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 the allosteric facilitation of BDNF signaling is the common mechanism for antidepressant action, which proposes a framework for how molecular effects of antidepressants are translated into clinical mood recovery.

neuroscience↗

bioRxiv2019

Cholesterol recognition motifs in the transmembrane domain of the tyrosine kinase receptor family: the case for TRKB

Cholesterol is an essential constituent of cell membranes. Recently, the discovery of cholesterol recognition amino acid consensus (CRAC) on 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, is a core participant in the neuronal plasticity process and exhibits a CARC motif in its TMR. Upon recognition of the conserved CARC motif in the TRKB, we performed molecular dynamics simulations with the mouse TRKB TMR sequence, which indicated that cholesterol interaction with the TRKB CARC motif occurs mainly at the central Y433 residue. Binding assay suggested a bell-shaped effect of cholesterol on BDNF interaction with TRKB receptors. Therefore, CARC/CRAC motifs may have a role in the function of the RTK family TMR.

neuroscience↗

bioRxiv2019

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.\n\nTaken 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.

pharmacology and toxicology↗

bioRxiv2016

Antidepressant-like effects of P2 purinergic antagonist PPADS is dependent on serotonergic and noradrenergic integrity

Depression is a common mental disorder affecting around 350 million of individuals globally. The available antidepressant drug monotherapy is far from ideal since it has an efficiency of approximately 60% and takes around 3-4 week to achieve clinical improvement. Attention has been paid to the purinergic signaling regarding neuropathological mechanisms, since it might be involved in psychiatric disorders, such as depression. In fact, blockade of purinergic P2X receptors induces antidepressant-like effects in preclinical models. However, the mechanisms involved in this effect are not yet completely understood. The present work investigated the interplay between a P2X receptor antagonist (PPADS) and clinically used antidepressant drugs on the forced swimming test, an animal model predictive of antidepressant effect. We observed significant synergistic effect of PPADS combined with sub-effective doses of fluoxetine or reboxetine in the FST. Moreover, depletion of serotonergic or noradrenergic systems, with PCPA and DSP-4 treatment, respectively, blocked the antidepressant-like effect of PPADS. No increase in locomotion, a possible source of confusion on FST data, was detected in any of the treated groups. Our results indicate the antidepressant-like effect of PPADS depends on the integrity of serotonergic and noradrenergic transmission.

pharmacology and toxicology↗

bioRxiv2016

Inducible nitric oxide synthase (NOS2) knockout mice as a model of trichotillomania

Trichotillomania (TTM) is an impulse control disorder characterized by repetitive hair pulling/trimming. Barbering behavior (BB) has been observed in laboratory animals and proposed as TTM model. The neurobiological basis of TTM is not clear, but it seems to involve striatal hyperactivity parallel to hypoactivation of prefrontal cortex. In this study we observed that knockout mice to the inducible isoform of nitric oxide synthase (NOS2) exhibit exacerbated BB following the 4th week of age, as well as increased repetitive movements compared to wild-type mice (WT). These behaviors are associated to decreased levels of NMDA receptor subunit (NR1) in prefrontal cortex, while an increase was observed in striatum of NOS2KO compared to WT. Striatal neurons from NOS2KO also exhibited increased number of branches compared to WT. The repeated treatment with clomipramine, a clinically approved drug to treat TTM in humans, or memantine, an antagonist of NMDA receptors, as well as partial rescue of NOS2 expression in haploinsufficient animals, attenuated the expression of BB. The silencing of NOS2 expression reduced the MAP2 (microtubule-associated protein 2) levels in activity-induced differentiated PC12 cells. Our data led us to propose that NOS2 regulates the neuronal maturation of the inhibitory afferent pathways to striatum during neurodevelopment, and such inadequate inhibition of striatal motor programs might be associated to the observed phenotype.

animal behavior and cognition↗