A Peptide Uncoupling BDNF Receptor TrkB from Phospholipase Cγ1 Prevents Epilepsy Induced by Status Epilepticus.

The BDNF receptor tyrosine kinase, TrkB, underlies nervous system function in both health and disease. Excessive activation of TrkB caused by status epilepticus promotes development of temporal lobe epilepsy (TLE), revealing TrkB as a therapeutic target for prevention of TLE. To circumvent undesirable consequences of global inhibition of TrkB signaling, we implemented a novel strategy aimed at selective inhibition of the TrkB-activated signaling pathway responsible for TLE. Our studies of a mouse model reveal that phospholipase Cγ1 (PLCγ1) is the dominant signaling effector by which excessive activation of TrkB promotes epilepsy. We designed a novel peptide (pY816) that uncouples TrkB from PLCγ1. Treatment with pY816 following status epilepticus inhibited TLE and prevented anxiety-like disorder yet preserved neuroprotective effects of endogenous TrkB signaling. We provide proof-of-concept evidence for a novel strategy targeting receptor tyrosine signaling and identify a therapeutic with promise for prevention of TLE caused by status epilepticus in humans.

Study the function of the newly discovered TrkB receptor fragment (TrkB-ICD) formed by calpain cleavage

Tese de mestrado, Neurociências, Faculdade de Medicina, Universidade de Lisboa, 2016

Brain-derived neurotrophic factor and adenosine signalling on amyloid-β peptide induced toxicity : impact on hippocampal function

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2014

Molecular features of glycine-mediated neurotransmission in rat brain

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2015

Deciphering the mechanisms underlying the loss of BDNF neuroprotection in an Alzheimer’s disease model

Tese de mestrado, Neurociências, Faculdade de Medicina, Universidade de Lisboa, 2016

Enhanced nicotine-seeking behavior following pre-exposure to repeated cocaine is accompanied by changes in BDNF in the nucleus accumbens of rats

We investigated the behavioral and molecular interactions between cocaine and nicotine, through evaluating locomotor activity, nicotine intravenous self-administration and gene expression. Locomotor sensitization was induced in male Wistar rats by repeated cocaine (20 mg/kg; i.p.) or saline injections once a day over 7 days. Three days after the last injection, rats were challenged with either saline or cocaine (15 mg/kg; i.p.) and the locomotor activity was measured. The very next day animals received either saline or nicotine (0.4 mg/kg; s.c.) and the locomotor cross-sensitization was tested. Animals were then prepared with intrajugular catheters for nicotine self-administration. Nicotine self-administration patterns were evaluated using fixed or progressive ratio schedules of reinforcement and a 24-h unlimited access binge. Immediately after the binge sessions animals were decapitated, the brains were removed and the nucleus accumbens was dissected. The dynorphin (DYN), μ-opioid receptor (mu opioid), neuropeptide Y (NPY), brain-derived neurotrophic factor (BDNF), tropomyosin-related tyrosine kinase B receptor (TrkB) and corticotropin- releasing factor receptor type 1 (CRF-R1) gene expression were measured by the reverse transcription-polymerase chain reaction (RT-PCR). Pretreatment with cocaine caused sensitization of cocaine motor response and locomotor cross-sensitization with nicotine. In the self-administration experiments repeated cocaine administration caused an increase in the nicotine break point and nicotine intake during a 24 h binge session. © 2013 Elsevier Inc.

Role of BDNF secretion and signaling in the activity-dependent refinement of synaptic connections

Neuronal networks exhibit diverse types of plasticity, including the activity-dependent regulation of synaptic functions and refinement of synaptic connections. In addition, continuous generation of new neurons in the “adult” brain (adult neurogenesis) represents a powerful form of structural plasticity establishing new connections and possibly implementing pre-existing neuronal circuits (Kempermann et al, 2000; Ming and Song, 2005). Neurotrophins, a family of neuronal growth factors, are crucially involved in the modulation of activity-dependent neuronal plasticity. The first evidence for the physiological importance of this role evolved from the observations that the local administration of neurotrophins has dramatic effects on the activity-dependent refinement of synaptic connections in the visual cortex (McAllister et al, 1999; Berardi et al, 2000; Thoenen, 1995). Moreover, the local availability of critical amounts of neurotrophins appears to be relevant for the ability of hippocampal neurons to undergo long-term potentiation (LTP) of the synaptic transmission (Lu, 2004; Aicardi et al, 2004). To achieve a comprehensive understanding of the modulatory role of neurotrophins in integrated neuronal systems, informations on the mechanisms about local neurotrophins synthesis and secretion as well as ditribution of their cognate receptors are of crucial importance. In the first part of this doctoral thesis I have used electrophysiological approaches and real-time imaging tecniques to investigate additional features about the regulation of neurotrophins secretion, namely the capability of the neurotrophin brain-derived neurotrophic factor (BDNF) to undergo synaptic recycling. In cortical and hippocampal slices as well as in dissociated cell cultures, neuronal activity rapidly enhances the neuronal expression and secretion of BDNF which is subsequently taken up by neurons themselves but also by perineuronal astrocytes, through the selective activation of BDNF receptors. Moreover, internalized BDNF becomes part of the releasable source of the neurotrophin, which is promptly recruited for activity-dependent recycling. Thus, we described for the first time that neurons and astrocytes contain an endocytic compartment competent for BDNF recycling, suggesting a specialized form of bidirectional communication between neurons and glia. The mechanism of BDNF recycling is reminiscent of that for neurotransmitters and identifies BDNF as a new modulator implicated in neuro- and glio-transmission. In the second part of this doctoral thesis I addressed the role of BDNF signaling in adult hippocampal neurogenesis. I have generated a transgenic mouse model to specifically investigate the influence of BDNF signaling on the generation, differentiation, survival and connectivity of newborn neurons into the adult hippocampal network. I demonstrated that the survival of newborn neurons critically depends on the activation of the BDNF receptor TrkB. The TrkB-dependent decision regarding life or death in these newborn neurons takes place right at the transition point of their morphological and functional maturation Before newborn neurons start to die, they exhibit a drastic reduction in dendritic complexity and spine density compared to wild-type newborn neurons, indicating that this receptor is required for the connectivity of newborn neurons. Both the failure to become integrated and subsequent dying lead to impaired LTP. Finally, mice lacking a functional TrkB in the restricted population of newborn neurons show behavioral deficits, namely increased anxiety-like behavior. These data suggest that the integration and establishment of proper connections by newly generated neurons into the pre-existing network are relevant features for regulating the emotional state of the animal.

Tyrosine kinase receptor B (TrkB) expression in colorectal cancers highlights anoikis resistance as a survival mechanism of tumour budding cells.

AIMS Tumour buds in colorectal cancer represent an aggressive subgroup of non-proliferating and non-apoptotic tumour cells. We hypothesize that the survival of tumour buds is dependent upon anoikis resistance. The role of tyrosine kinase receptor B (TrkB), a promoter of epithelial-mesenchymal transition and anoikis resistance, in facilitating budding was investigated. METHODS AND RESULTS Tyrosine kinase receptor B immunohistochemistry was performed on a multiple-punch tissue microarray of 211 colorectal cancer resections. Membranous/cytoplasmic and nuclear expression was evaluated in tumour and buds. Tumour budding was assessed on corresponding whole tissue slides. Relationship to Ki-67 and caspase-3 was investigated. Analysis of Kirsten Ras (KRAS), proto-oncogene B-RAF (BRAF) and cytosine-phosphate-guanosine island methylator phenotype (CIMP) was performed. Membranous/cytoplasmic and nuclear TrkB were strongly, inversely correlated (P < 0.0001; r = -0.41). Membranous/cytoplasmic TrkB was overexpressed in buds compared to the main tumour body (P < 0.0001), associated with larger tumours (P = 0.0236), high-grade budding (P = 0.0011) and KRAS mutation (P = 0.0008). Nuclear TrkB was absent in buds (P <0.0001) and in high-grade budding cancers (P =0.0073). Among patients with membranous/cytoplasmic TrkB-positive buds, high tumour membranous/cytoplasmic TrkB expression was a significant, independent adverse prognostic factor [P = 0.033; 1.79, 95% confidence interval (CI) 1.05-3.05]. Inverse correlations between membranous/cytoplasmic TrkB and Ki-67 (r = -0.41; P < 0.0001) and caspase-3 (r =-0.19; P < 0.05) were observed. CONCLUSIONS Membranous/cytoplasmic TrkB may promote an epithelial-mesenchymal transition (EMT)-like phenotype with high-grade budding and maintain viability of buds themselves.

Expression of a dominant negative TrkB receptor, T1, reveals a requirement for presynaptic signaling in BDNF-induced synaptic potentiation in cultured hippocampal neurons

We have developed a method to analyze the relative contributions of pre- and postsynaptic actions of a particular gene product in neurons in culture and potentially in slices using adenovirus-mediated gene transfer. A recombinant virus directed the expression of both a GFP reporter protein and TrkB.T1, a C-terminal truncated dominant negative TrkB neurotrophin receptor. When expressed in the presynaptic cell at synapses between embryonic hippocampal neurons in culture, the dominant negative TrkB.T1 inhibited two forms of synaptic potentiation induced by the neurotrophin brain-derived neurotrophic factor (BDNF): (i) greater evoked synaptic transmission and (ii) higher frequency of spontaneous miniature synaptic currents. These inhibition effects are not seen if the transgene is expressed only in the postsynaptic cell. We conclude that BDNF-TrkB signal transduction in the presynaptic terminal leads to both types of potentiation and is therefore the primary cause of synaptic enhancement by BDNF in these neurons.

An ancient neurotrophin code in zebrafish; the Runx/Cbfβ complex specifies the fate switch from TrkB+ to TrkC+ nociceptors and sensory receptor expression

Thesis (Ph.D.)--University of Washington, 2016-06

GABAa Receptor Mediated Signalling in the Brain: Inhibition, Shunting and Excitation

Within central nervous system, the simple division of chemical synaptic transmission to depolarizing excitation mediated by glutamate and hyperpolarizing inhibition mediated by γ-amino butyric acid (GABA), is evidently an oversimplification. The GABAa receptor (GABAaR) mediated responses can be of opposite sign within a single resting cell, due to the compartmentalized distribution of cation chloride cotransporters (CCCs). The K+/Cl- cotransporter 2 (KCC2), member of the CCC family, promotes K+ fuelled Cl- extrusion and sets the reversal potential of GABA evoked anion currents typically slightly below the resting membrane potential. The interesting ionic plasticity property of GABAergic signalling emerges from the short-term and long-term alterations in the intraneuronal concentrations of GABAaR permeable anions (Cl- and HCO3-). The short-term effects arise rapidly (in the time scale of hundreds of milliseconds) and are due to the GABAaR activation dependent shifts in anion gradients, whereas the changes in expression, distribution and kinetic regulation of CCCs are underlying the long-term effects, which may take minutes or even hours to develop. In this Thesis, the differences in the reversal potential of GABAaR mediated responses between dopaminergic and GABAergic cell types, located in the substantia nigra, were shown to be attributable to the differences in the chloride extrusion mechanisms. The stronger inhibitory effect of GABA on GABAergic neurons was due to the cell type specific expression of KCC2 whereas the KCC2 was absent from dopaminergic neurons, leading to a less prominent inhibition brought by GABAaR activation. The levels of KCC2 protein exhibited activity dependent alterations in hippocampal pyramidal neurons. Intense neuronal activity, leading to a massive release of brain derived neurotrophic factor (BDNF) in vivo, or applications of tyrosine receptor kinase B (TrkB) agonists BDNF or neurotrophin-4 in vitro, were shown to down-regulate KCC2 protein levels which led to a reduction in the efficacy of Cl- extrusion. The GABAergic transmission is interestingly involved in an increase of extracellular K+ concentration. A substantial increase in interstitial K+ tends to depolarize the cell membrane. The effects that varying ion gradients had on the generation of biphasic GABAaR mediated responses were addressed, with particular emphasis on the novel idea that the K+/Cl- extrusion via KCC2 is accelerated in response to a rapid accumulation of intracellular Cl-. The KCC2 inhibitor furosemide produced a large reduction in the GABAaR dependent extracellular K+ transients. Thus, paradoxically, both the inefficient KCC2 activity (via increased intracellular Cl-) and efficient KCC2 activity (via increased extracellular K+) may promote excitation.

Développement et radiosynthèse de ligands du récepteur tyrosine kinase neurotrophique type 2 (TrkB) marqués aux carbone-11 et fluor-18 pour l’imagerie cérébrale par tomographie d’émission de positons

Ce mémoire présente mes travaux ayant menés au développement d’une première génération de radioligands marqués au fluor-18 (t1/2 = 110 min) et au carbone-11 (t1/2 = 20.4 min) destinés à l’imagerie cérébrale in vivo du récepteur tyrosine kinase neurotrophique de type 2 (TrkB) en tomographie par émission de positons (TEP). Ces travaux reposent sur l’identification récente de ligands de TrkB non peptidiques à hautes affinités dérivés du 7,8-dihydroxyflavone. La synthèse d’une série de dérivés du 7,8-dihydroxyflavone non-radioactifs de même que des précuseurs à l’incorporation du fluro-18 et du carbone-11 a d’abord été effectuée. Partant des précurseurs adéquats synthétisés, la radiosynthèse de deux radioligands, l’un marqué au fluor-18 et l’autre au carbone-11, a été développée. Ces radiosynthèses reposent respectivement sur une 18F-radiofluorination nucléophile aromatique nouvelle et hautement efficace et sur une 11C-méthylation N-sélective. Les radiotraceurs de TrkB ainsi obtenus ont ensuite été évalués in vitro en autoradiographie et in vivo en tant que traceurs TEP dans des rats. L’évaluation des propriétés physico-chimique de même que de la stabilité in vitro des radiotraceurs sont présentées. Partant d’une série d’analogues cristallisés de ces flavones synthétiques, une étude de relation structure-activité a été menée. La combinaison de cette étude, de pair avec l’évaluation in vivo de la première génération de radiotraceurs de TrkB a aussi permis d’investiguer les pharmacophores nécessaires à l’affinité de ces ligands de même que d’identifier des fragments structurels associés au métabolisme des radiotraceurs. La radiosynthèse d’un troisième radioligand de TrkB et son évaluation TEP in vivo de même que la mise en lumière des modifications structurelles utiles au développement d’une seconde génération de radioligands de TrkB avec des propriétés optimisées pour fin d’imagerie TEP sont aussi détaillés.

Interplay between BDNF,TrkB signalling and GABAergic inhibition in the visual cortex of mice

Der visuelle Kortex ist eine der attraktivsten Modellsysteme zur Untersuchung der molekularen Mechanismen der synaptischen Plastizität im Gehirn. Es hat sich gezeigt, dass der Wachstumsfaktor brain-derived-neurotrophic-factor (BDNF) und die GABAerge Hemmung während der Entwicklung eine essentielle Funktion in der Regulierung der synaptischen Plastizität im visuellen Kortex besitzen. BDNF bindet u.a. an TrkB Rezeptoren, die das Signal intrazellular an unterschiedliche Effektormoleküle weiter vermitteln. Außer BDNF sind auch andere TrkB-Rezeptor Agonisten in der Literatur beschrieben. Einer davon ist das kürzlich identifizierte Flavonoid 7,8-Dihydroxyflavone (7,8-DHF), welchem eine neurotrophe Wirkung zugeschrieben wird. Im ersten Abschnitt der vorliegenden Doktorarbeit wurde der Effekt dieses Agonisten auf die synaptische Übertragung und intrinsischen Zelleigenschaften im visuellen Kortex der Maus untersucht. Dies wurde mit Hilfe der whole-cell patch clamp Methode durchgeführt, wobei die synaptischen Eingänge der Pyramidalzellen der kortikalen Schicht 2/3 von besonderem Interesse waren.rnEine 30 minütige Inkubationszeit der kortikalen Schnitte mit 7,8 DHF (20µM) erzielte eine signifikante Reduktion der GABAergen Hemmung, während die glutamaterge synaptische Übertragung unverändert blieb. Des weiteren konnte in Gegenwart von 7,8 DHF eine Veränderung der intrinsischen neuronalen Zellmembraneigenschaften beobachtet werden. Dies wurde deutlich in der Erhöhung des Eingangwiderstandes und der Frequenz der induzierten Aktionspotentiale. Die chronische Applikation von 7,8 DHF in vivo bestätigte die selektive Wirkung von 7,8 DHF auf das GABAerge System. rnDie Rolle des BDNF-TrkB-Signalweges in der GABAergen Hemmung nach kortikalen Verletzungen ist bisher wenig verstanden. Eine häufig beschriebene elektrophysiologische Veränderung nach kortikaler Verletzung ist eine Reduktion in der GABAergen Hemmung. Im zweiten Abschnitt dieser Doktorarbeit wurde hierzu die Funktion des BDNF-TrkB-Signalweges auf die GABAerge Hemmung nach kortikaler Verletzung untersucht. Es wurde ein "ex-vivo/in-vitro“ Laser-Läsions Modell verwendet, wobei mittels eines Lasers im visuellen Kortex von WT und heterozygoten BDNF (+/−) Mäusen eine definierte, reproduzierbare Läsion induziert wurde. Nachfolgende elektrophysiologische Messungen ergaben, dass die Auswirkung einer Verletzung des visuellen Kortex auf die GABAerge Funktion signifikant von der basalen BDNF Konzentration im Kortex abhängt. Des weiteren konnte beobachtet werden, dass nach kortikaler Verletzung in WT Mäusen sowohl die Frequenz der basalen inhibitorischen, postsynaptischen Potentiale (mIPSCs) reduziert war, als auch ein erhöhtes Paired-Pulse Verhältnis vorlag. Diese Ergebnisse deuten auf Veränderungen der präsynaptischen Funktion inhibitorischer Synapsen auf Pyramidalneurone hin. Im Gegensatz dazu konnte in BDNF (+/−) mice Mäusen eine erhöhte und gleichzeitig verlängerte mIPSC-Amplitude beobachtet werden, induziert durch Reizung afferenter Nervenfasern. Hieraus lässt sich schließen, dass kortikale Verletzungen in BDNF (+/−) mice Mäusen Auswirkungen auf die Eigenschaften von postsynaptischen GABAA-Rezeptoren haben. Die nachfolgende Gabe eines TrkB-Rezeptor Antagonisten bestätigte diese Ergebnisse für das GABAerge System post-Läsion. Dies zeigt auch, dass die Änderungen der synaptischen Hemmung nicht auf eine Reduktion der BDNF-Konzentration zurückzuführen sind. Zusammengefasst zeigen die Ergebnisse der vorliegenden Arbeit, dass der BDNF-TrkB Signalweg eine wichtige Rolle in der Reorganisation der GABAergen Hemmung nach kortikalen Verletzungen spielt. So könnte ein TrkB-Rezeptor Agonist, wie das kürzlich entdeckte 7,8-DHF, über eine Modulation der BDNF-TrB Signalkaskade pharmakologisch die funktionelle Reorganisation des Kortex nach einer fokalen Gehirnverletzung fördern. rnrn

Suppression of trkB expression by antisense oligonucleotides alters a neuronal phenotype in the rod pathway of the developing rat retina.

trkB is the high-affinity receptor for brain-derived neurotrophic factor (BDNF), a trophic molecule with demonstrated effects on the survival and differentiation of a wide variety of neuronal populations. In the mammalian retina, trkB is localized to both ganglion cells and numerous cells in the inner nuclear layer. Much information on the role of BDNF in neuronal development has been derived from the study of trkB- and BDNF-deficient mutant mice. This includes an attenuation of the numbers of cortical neurons immunopositive for the calcium-binding proteins, parvalbumin, and calbindin. Unfortunately, these mutant animals typically fail to survive for > 24-48 hr after birth. Since most retinal neuronal differentiation occurs postnatally, we have devised an alternative scheme to suppress the expression of trkB in the retina to examine the role of BDNF on the postnatal development of neurons of the inner retina. Neonatal rats were treated with intraocular injection of an antisense oligonucleotide (1-2 microliters of 10-100 microM solution) targeted to the trkB mRNA. Immunohistochemistry with a polyclonal antibody to trkB showed that the expression of trkB in retinal neurons was suppressed 48-72 hr following a single injection. Northern blot analysis demonstrated that antisense treatment had no effect on the level of trkB mRNA, even after multiple injections. This suggests an effect of trkB antisense treatment on protein translation, but not on RNA transcription. No alterations were observed in the thickness of retinal cellular or plexiform layers, suggesting that BDNF is not the sole survival factor for these neurons. There were, however, alterations in the patterns of immunostaining for parvalbumin, a marker for the narrow-field, bistratified AII amacrine cell-a central element of the rod (scotopic) pathway. This was evidenced by a decrease in both the number of immunostained somata (> 50%) and in the intensity of immunolabeling. However, the immunostaining pattern of calbindin was not affected. These studies suggest that the ligands for trkB have specific effects on the neurochemical phenotypic expression of inner retinal neurons and in the development of a well-defined retinal circuit.

BDNF-TrkB Signaling in Single-Spine Structural Plasticity

Multiple lines of evidence reveal that activation of the tropomyosin related kinase B (TrkB) receptor is a critical molecular mechanism underlying status epilepticus (SE) induced epilepsy development. However, the cellular consequences of such signaling remain unknown. To this point, localization of SE-induced TrkB activation to CA1 apical dendritic spines provides an anatomic clue pointing to Schaffer collateral-CA1 synaptic plasticity as one potential cellular consequence of TrkB activation. Here, we combine two-photon glutamate uncaging with two photon fluorescence lifetime imaging microscopy (2pFLIM) of fluorescence resonance energy transfer (FRET)-based sensors to specifically investigate the roles of TrkB and its canonical ligand brain derived neurotrophic factor (BDNF) in dendritic spine structural plasticity (sLTP) of CA1 pyramidal neurons in cultured hippocampal slices of rodents. To begin, we demonstrate a critical role for post-synaptic TrkB and post-synaptic BDNF in sLTP. Building on these findings, we develop a novel FRET-based sensor for TrkB activation that can report both BDNF and non-BDNF activation in a specific and reversible manner. Using this sensor, we monitor the spatiotemporal dynamics of TrkB activity during single-spine sLTP. In response to glutamate uncaging, we report a rapid (onset less than 1 minute) and sustained (lasting at least 20 minutes) activation of TrkB in the stimulated spine that depends on N-methyl-D-aspartate receptor (NMDAR)-Ca2+/Calmodulin dependent kinase II (CaMKII) signaling as well as post-synaptically synthesized BDNF. Consistent with these findings, we also demonstrate rapid, glutamate uncaging-evoked, time-locked release of BDNF from single dendritic spines using BDNF fused to superecliptic pHluorin (SEP). Finally, to elucidate the molecular mechanisms by which TrkB activation leads to sLTP, we examined the dependence of Rho GTPase activity - known mediators of sLTP - on BDNF-TrkB signaling. Through the use of previously described FRET-based sensors, we find that the activities of ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) require BDNF-TrkB signaling. Taken together, these findings reveal a spine-autonomous, autocrine signaling mechanism involving NMDAR-CaMKII dependent BDNF release from stimulated dendritic spines leading to TrkB activation and subsequent activation of the downstream molecules Rac1 and Cdc42 in these same spines that proves critical for sLTP. In conclusion, these results highlight structural plasticity as one cellular consequence of CA1 dendritic spine TrkB activation that may potentially contribute to larger, circuit-level changes underlying SE-induced epilepsy.