TrkB Activation Research Articles

Upregulation vs. loss of function of NTRK2 in 44 affected individuals leads to two distinct neurodevelopmental disorders

IntroductionHeterozygous pathogenic variants in NTRK2 (HGNC: 8032) have been associated with global developmental delay. However, only scattered cases have been described in small or general studies. The aim of our work was to consolidate our understanding of NTRK2-related disorders and to delineate the clinical presentation MethodsWe report extended cohort of 44 affected individuals, of whom 19 are from the literature and 25 were previously unreported. ResultsOur analysis led to splitting the cohort into two entities. DiscussionOne group had variants in the cholesterol binding motif of the transmembrane domain, with most of these being the recurrent variant c.1301A>G p.(Tyr434Cys). These variants probably lead to upregulation of TRKB activity and to a severe phenotype of developmental delay/intellectual disability, muscular hypotonia, therapy-refractory epilepsy, visual impairment and blindness, and feeding difficulties. The second group had truncating variants or variants that presumably disturb the 3D structure of the protein leading to loss of function. These individuals had a remarkably milder phenotype of developmental delay, obesity and hyperphagia.

Neonatal hypoxia-ischemia alters the events governing the hippocampal critical period of postnatal synaptic plasticity leading to deficits in working memory in mice

The postnatal critical period of synaptic plasticity (CPsp) is characterized by profound neural network refinement, which is shaped by synaptic activity and sculpted by maturation of the GABAergic network. Even after therapeutic hypothermia (TH), neonatal hypoxia-ischemia (HI) impairs two triggers for the initiation of the CPsp in the hippocampus: i) PSA-NCAM developmental decline and ii) parvalbumin (PV) + interneuron (IN) maturation. Thus, we investigated whether neonatal HI despite TH disturbs other events governing the onset, consolidation and closure of the postnatal CPsp in the hippocampus. We induced cerebral HI in P10 C57BL6 mice with right carotid ligation and 45 m of hypoxia (FiO2 = 0.08), followed by normothermia (36 °C, NT) or TH (31 °C) for 4 h with anesthesia-exposed shams as controls. ELISA, immunoblotting and immunohistochemistry were performed at 24 h (P11), 5 days (P15), 8 days (P18) and 30 days (P40) after HI injury. We specifically assessed: i) BDNF levels and TrkB activation, controlling the CPsp, ii) Otx2 and NPTX2 immunoreactivity (IR), engaging CPsp onset and iii) NogoR1, Lynx1 IR, PNN formation and myelination (MBP) mediating CPsp closure. Pups aged to P40 also received a battery of tests assessing working memory. Here, we documented deficits in hippocampal BDNF levels and TrkB activation at P18 in response to neonatal HI even with TH. Neonatal HI impaired in the CA1 the developmental increase in PV, Otx2, and NPTX2 between P11 and P18, the colocalization of Otx2 and PV at P18 and P40, the accumulation of NPTX2 in PV+ dendrites at P18 and P40, and the expression of NogoR and Lynx1 at P40. Furthermore, neonatal HI decreased BDNF and impaired PNN development and myelination (MBP) at P40. Most of these abnormalities were insensitive to TH and correlated with memory deficits. Neonatal HI appears to disrupt many of the molecular and structural events initiating and consolidating the postnatal hippocampal CPsp, perhaps due to the early and delayed deficits in TrkB activation leading to memory deficits.

Structural basis for the transmembrane signaling and antidepressant-induced activation of the receptor tyrosine kinase TrkB.

Neurotrophin receptors of the Trk family are involved in the regulation of brain development and neuroplasticity, and therefore can serve as targets for anti-cancer and stroke-recovery drugs, antidepressants, and many others. The structures of Trk protein domains in various states upon activation need to be elucidated to allow rational drug design. However, little is known about the conformations of the transmembrane and juxtamembrane domains of Trk receptors. In the present study, we employ NMR spectroscopy to solve the structure of the TrkB dimeric transmembrane domain in the lipid environment. We verify the structure using mutagenesis and confirm that the conformation corresponds to the active state of the receptor. Subsequent study of TrkB interaction with the antidepressant drug fluoxetine, and the antipsychotic drug chlorpromazine, provides a clear self-consistent model, describing the mechanism by which fluoxetine activates the receptor by binding to its transmembrane domain.

Small Molecule Compound DHPA Screened by Computer-Aided Drug Design and Molecular Dynamics Simulation Inhibits Neuroblastoma Cell Proliferation by Targeting TrkB.

Neuroblastoma (NB) is a rare and malignant pediatric solid tumor. Due to its heterogeneity, it poses significant challenges for treatment, resulting in a high mortality rate. This study aimed to identify new therapeutic drugs by modeling the TrkB receptor from PDB 4AT5 and conducting virtual screening of compounds from the YaTCM database (containing 47,696 compounds derived from 6220 Traditional Chinese Medicines). The screening utilized the E-pharmacophore approach to select compounds with potential binding affinity to TrkB. The binding abilities of these compounds were tested through molecular dynamics simulations, stretch dynamics simulations, and US simulations. Among the top 11 optimized hit compounds, DHPA and 3″-demethylhexahydrocurcumin are prominent. Further simulations reveal that they form stable receptor-ligand binary complexes with TrkB. In subsequent in vitro cell experiments, 3″-demethylhexahydrocurcumin is eliminated due to its high IC50 for killing NB cells. Low concentrations of DHPA can significantly kill NB cells. Additionally, DHPA can inhibit the expression of TrkB, the activation of TrkB's downstream signaling pathways, and affect the thermal stability of TrkB protein and its response to streptase protease degradation. DHPA may be a potential TrkB inhibitor.

Nitrous oxide induces hypothermia and TrkB activation: Maintenance of body temperature abolishes antidepressant-like effects in mice

Recent studies indicate that nitrous oxide (N2O), a gaseous anesthetic and an NMDA (N-methyl-D-aspartate) receptor antagonist, produces rapid antidepressant effect in patients suffering from treatment-resistant depression. Our recent work implies that hypothermia and reduced energy expenditure are connected with antidepressant-induced activation of TrkB neurotrophin receptors — a key regulator of synaptic plasticity. In this study, we demonstrate that a brief exposure to N2O leads to a drop in body temperature following the treatment, which is linked to decreased locomotor activity; enhanced slow-wave electroencephalographic activity; reduced brain glucose utilization; and increased phosphorylation of TrkB, GSK3β (glycogen synthase kinase 3β), and p70S6K (a kinase downstream of mTor (mammalian target of rapamycin)) in the medial prefrontal cortex of adult male mice. Moreover, preventing the hypothermic response in a chronic corticosterone stress model of depression attenuated the antidepressant-like behavioral effects of N2O in the saccharin preference test. These findings indicate that N2O treatment modulates TrkB signaling and related neurotrophic signaling pathways in a temperature-dependent manner, suggesting that the phenomenon driving TrkB activation — altered thermoregulation and energy expenditure — is linked to antidepressant-like behavioral responses.

Impaired hippocampal plasticity associated with loss of recycling endosomal SLC9A6/NHE6 is ameliorated by the TrkB agonist 7,8-dihydroxyflavone

Proper maintenance of intracellular vesicular pH is essential for cargo trafficking during synaptic function and plasticity. Mutations in the SLC9A6 gene encoding the recycling endosomal pH regulator (Na+, K+)/H+ exchanger isoform 6 (NHE6) are causal for Christianson syndrome (CS), a severe form of X-linked intellectual disability. NHE6 expression is also downregulated in other neurodevelopmental and neurodegenerative disorders, such as autism spectrum disorder and Alzheimer's disease, suggesting its dysfunction could contribute more broadly to the pathophysiology of other neurological conditions. To understand how ablation of NHE6 function leads to severe learning impairments, we assessed synaptic structure, function, and cellular mechanisms of learning in a novel line of Nhe6 knockout (KO) mice expressing a plasma membrane-tethered green fluorescent protein within hippocampal neurons. We uncovered significant reductions in dendritic spines density, AMPA receptor (AMPAR) expression, and AMPAR-mediated neurotransmission in CA1 pyramidal neurons. The neurons also failed to undergo functional and structural enhancement during long-term potentiation (LTP). Significantly, the selective TrkB agonist 7,8-dihydroxyflavone restored spine density as well as functional and structural LTP in KO neurons. TrkB activation thus may act as a potential clinical intervention to ameliorate cognitive deficits in CS and other neurodegenerative disorders.

A Combined Computational and Experimental Approach to Studying Tropomyosin Kinase Receptor B Binders for Potential Treatment of Neurodegenerative Diseases.

Tropomyosin kinase receptor B (TrkB) has been explored as a therapeutic target for neurological and psychiatric disorders. However, the development of TrkB agonists was hindered by our poor understanding of the TrkB agonist binding location and affinity (both affect the regulation of disorder types). This motivated us to develop a combined computational and experimental approach to study TrkB binders. First, we developed a docking method to simulate the binding affinity of TrkB and binders identified by our magnetic drug screening platform from Gotu kola extracts. The Fred Docking scores from the docking computation showed strong agreement with the experimental results. Subsequently, using this screening platform, we identified a list of compounds from the NIH clinical collection library and applied the same docking studies. From the Fred Docking scores, we selected two compounds for TrkB activation tests. Interestingly, the ability of the compounds to increase dendritic arborization in hippocampal neurons matched well with the computational results. Finally, we performed a detailed binding analysis of the top candidates and compared them with the best-characterized TrkB agonist, 7,8-dyhydroxyflavon. The screening platform directly identifies TrkB binders, and the computational approach allows for the quick selection of top candidates with potential biological activities based on the docking scores.

Examination of Akt and GSK3β in BDNF-mediated reductions in BACE1 activity in neuronal cells.

Brain-derived neurotrophic factor (BDNF) content and signaling has been identified as one potential regulator of amyloid precursor protein (APP) processing. Recently published work has demonstrated that BDNF reduces BACE1 activity while also elevating the inhibition of GSK3β in the prefrontal cortex of male C57BL/6J mice. These results provide evidence that BDNF alters APP processing by reducing BACE1 activity, which may act through GSK3β inhibition. The purpose of this study was to further explore the role of GSK3β in BDNF-induced regulation on BACE1 activity. We utilized a cell culture and an invitro activity assay model to pharmacologically target BDNF and GSK3β signaling to confirm its involvement in the BDNF response. Treatment of differentiated SH-SY5Y neuronal cells with 75 ng/mL BDNF resulted in elevated pTrkB content, pAkt content, pGSK3β content, and reduced BACE1 activity. An invitro BACE1 activity assay utilizing mouse prefrontal cortex (n = 6/group) supplemented with BDNF, BDNF + ANA12 (Trkb antagonist), or BDNF + wortmannin (Akt inhibitor) demonstrated that BDNF reduced BACE1 activity; however, in the presence of TrkB or Akt inhibition, this effect was abolished. An invitro ADAM10 activity assay utilizing mouse prefrontal cortex (n = 6/group) supplemented with BDNF, BDNF + ANA12 (Trkb antagonist), or BDNF + wortmannin (Akt inhibitor) demonstrated that BDNF did not alter ADAM10 activity. However, inhibiting BDNF signaling reduced ADAM10 activity. Collectively these studies suggest that GSK3β inhibition may be necessary for BDNF-induced reductions in BACE1 activity. These findings will allow for the optimization of future therapeutic strategies by selectively targeting TrkB activation and GSK3β inhibition.

Comprehensive characterization of the neurogenic and neuroprotective action of a novel TrkB agonist using mouse and human stem cell models of Alzheimer’s disease

BackgroundNeural stem cell (NSC) proliferation and differentiation in the mammalian brain decreases to minimal levels postnatally. Nevertheless, neurogenic niches persist in the adult cortex and hippocampus in rodents, primates and humans, with adult NSC differentiation sharing key regulatory mechanisms with development. Adult neurogenesis impairments have been linked to Alzheimer’s disease (AD) pathology. Addressing these impairments by using neurotrophic factors is a promising new avenue for therapeutic intervention based on neurogenesis. However, this possibility has been hindered by technical difficulties of using in-vivo models to conduct screens, including working with scarce NSCs in the adult brain and differences between human and mouse models or ethical limitations.MethodsHere, we use a combination of mouse and human stem cell models for comprehensive in-vitro characterization of a novel neurogenic compound, focusing on the brain-derived neurotrophic factor (BDNF) pathway. The ability of ENT-A011, a steroidal dehydroepiandrosterone derivative, to activate the tyrosine receptor kinase B (TrkB) receptor was tested through western blotting in NIH-3T3 cells and its neurogenic and neuroprotective action were assessed through proliferation, cell death and Amyloid-β (Aβ) toxicity assays in mouse primary adult hippocampal NSCs, mouse embryonic cortical NSCs and neural progenitor cells (NPCs) differentiated from three human induced pluripotent stem cell lines from healthy and AD donors. RNA-seq profiling was used to assess if the compound acts through the same gene network as BDNF in human NPCs.ResultsENT-A011 was able to increase proliferation of mouse primary adult hippocampal NSCs and embryonic cortical NSCs, in the absence of EGF/FGF, while reducing Aβ-induced cell death, acting selectively through TrkB activation. The compound was able to increase astrocytic gene markers involved in NSC maintenance, protect hippocampal neurons from Αβ toxicity and prevent synapse loss after Aβ treatment. ENT-A011 successfully induces proliferation and prevents cell death after Aβ toxicity in human NPCs, acting through a core gene network shared with BDNF as shown through RNA-seq.ConclusionsOur work characterizes a novel BDNF mimetic with preferable pharmacological properties and neurogenic and neuroprotective actions in Alzheimer’s disease via stem cell-based screening, demonstrating the promise of stem cell systems for short-listing competitive candidates for further testing.

Indole-3-Carbinol and Its Derivatives as Neuroprotective Modulators.

Brain-derived neurotrophic factor (BDNF) and its downstream tropomyosin receptor kinase B (TrkB) signaling pathway play pivotal roles in the resilience and action of antidepressant drugs, making them prominent targets in psychiatric research. Oxidative stress (OS) contributes to various neurological disorders, including neurodegenerative diseases, stroke, and mental illnesses, and exacerbates the aging process. The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant responsive element (ARE) serves as the primary cellular defense mechanism against OS-induced brain damage. Thus, Nrf2 activation may confer endogenous neuroprotection against OS-related cellular damage; notably, the TrkB/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway, stimulated by BDNF-dependent TrkB signaling, activates Nrf2 and promotes its nuclear translocation. However, insufficient neurotrophin support often leads to the downregulation of the TrkB signaling pathway in brain diseases. Thus, targeting TrkB activation and the Nrf2-ARE system is a promising therapeutic strategy for treating neurodegenerative diseases. Phytochemicals, including indole-3-carbinol (I3C) and its metabolite, diindolylmethane (DIM), exhibit neuroprotective effects through BDNF's mimetic activity; Akt phosphorylation is induced, and the antioxidant defense mechanism is activated by blocking the Nrf2-kelch-like ECH-associated protein 1 (Keap1) complex. This review emphasizes the therapeutic potential of I3C and its derivatives for concurrently activating neuronal defense mechanisms in the treatment of neurodegenerative diseases.

Retrograde adenosine/A2A receptor signaling facilitates excitatory synaptic transmission and seizures

Retrograde signaling at the synapse is a fundamental way by which neurons communicate and neuronal circuit function is fine-tuned upon activity. While long-term changes in neurotransmitter release commonly rely on retrograde signaling, the mechanisms remain poorly understood. Here, we identified adenosine/A2A receptor (A2AR) as a retrograde signaling pathway underlying presynaptic long-term potentiation (LTP) at a hippocampal excitatory circuit critically involved in memory and epilepsy. Transient burst activity of a single dentate granule cell induced LTP of mossy cell synaptic inputs, a BDNF/TrkB-dependent form of plasticity that facilitates seizures. Postsynaptic TrkB activation released adenosine from granule cells, uncovering a non-conventional BDNF/TrkB signaling mechanism. Moreover, presynaptic A2ARs were necessary and sufficient for LTP. Lastly, seizure induction released adenosine in a TrkB-dependent manner, while removing A2ARs or TrkB from the dentate gyrus had anti-convulsant effects. By mediating presynaptic LTP, adenosine/A2AR retrograde signaling may modulate dentate gyrus-dependent learning and promote epileptic activity.

Neuroligin-1 dependent phosphotyrosine signaling in excitatory synapse differentiation.

The synaptic adhesion molecule neuroligin-1 (NLGN1) is involved in the differentiation of excitatory synapses, but the precise underlying molecular mechanisms are still debated. Here, we explored the role of NLGN1 tyrosine phosphorylation in this process, focusing on a subset of receptor tyrosine kinases (RTKs), namely FGFR1 and Trks, that were previously described to phosphorylate NLGN1 at a unique intracellular residue (Y782). We used pharmacological inhibitors and genetic manipulation of those RTKs in dissociated hippocampal neurons, followed by biochemical measurement of NLGN1 phosphorylation and immunocytochemical staining of excitatory synaptic scaffolds. This study shows that: (i) the accumulation of PSD-95 at de novo NLGN1 clusters induced by neurexin crosslinking is reduced by FGFR and Trk inhibitors; (ii) the increase in PSD-95 puncta caused by NLGN1 over-expression is impaired by FGFR and Trk inhibitors; (iii) TrkB activation by BDNF increases NLGN1 phosphorylation; and (iv) TrkB knock-down impairs the increase of PSD-95 puncta caused by NLGN1 over-expression, an effect which is not seen with the NLGN1 Y782A mutant. Together, our data identify TrkB as one of the major RTKs responsible for NLGN1 tyrosine phosphorylation, and reveal that TrkB activity is necessary for the synaptogenic effects of NLGN1.

BDNF/TrkB Induces pCREBS133 Phosphorylation and Activates Mitochondrial Biogenesis in Rat Phrenic Motor Neurons

Neural control of the diaphragm muscle (DIAm) is essential to sustain breathing. During breathing, fatigue resistant DIAm motor units are recruited comprising smaller phrenic motor neurons (PhMNs) and type I and IIa muscle fibers. Larger PhMNs comprise more fatigable DIAm motor units that are infrequently active during expulsive behaviors. The difference in activation history is reflected in the higher mitochondrial volume density in smaller PhMNs. In PhMNs, brain-derived neurotrophic factor (BDNF) signals through its high-affnity receptor, tropomyosin receptor kinase B (TrkB.FL). Previously, we found that intrathecal BDNF treatment induces CREB phosphorylation at Serine 133 (pCREBs133). We hypothesized that pCREBs133 binds to the PGC1a promoter, transcriptionally targeting PGC1a expression, mediating downstream increased expression of NRF1, NRF2, and TFAM and mitochondrial biogenesis. To test this hypothesis, we used a chemosensitive TrkBF616 rat model with a knock-in allele sensitive to 1NMPP1 in that TrkB kinase is inhibited following 1NMPP1, a small molecule derivative of the protein kinase inhibitor protein phosphatase 1 that plays a significant role inhibiting the TrkB phosphorylation and activation. Systemic treatment of 1NMPP1 in TrkBF616 rats inhibits TrkB kinase activity. A bioinformatic analysis was performed to identify a putative binding site for pCREBs133 in the PGC1a promoter sequence in rats. Using a chromatin immunoprecipitation (ChIP) assay we found that pCREBS133 binds to the PGC1a promoter in rat cervical ventral horn. We showed that intrathecal BDNF treatment increased pCREBs133 phosphorylation, which was blocked by 1NMPP1 treatment in TrkBF616 rats. PGC1a, NRF1/2, and TFAM protein was compared in cervical ventral horn lysates from BDNF- and BDNF+1NMPP1-treated TrkBF616 rats by qRT-PCR and Western Blot. Relative mtDNA copy number (normalized to nuclear DNA) was quantified by qPCR to assess the mitochondrial biogenesis. All results were analyzed by paired t-test. Following 1NMPP1-induced inhibition of TrkB kinase, PGC1a, NRF1/2, and TFAM mRNA and protein expression was reduced. Consistent with these findings, mtDNA copy number was decreased following TrkB kinase inhibition by 1NMPP1. We conclude that inhibition of TrkB kinase by 1NMPP1 reduces PGC1a-mediated mitochondrial biogenesis in rat cervical ventral horn. Supported by NIH grants AG44615 and HL146114. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Brain-derived neurotrophic factor scales presynaptic calcium transients to modulate excitatory neurotransmission

Brain-derived neurotrophic factor (BDNF) plays a critical role in synaptic physiology, as well as mechanisms underlying various neuropsychiatric diseases and their treatment. Despite its clear physiological role and disease relevance, BDNF's function at the presynaptic terminal, a fundamental unit of neurotransmission, remains poorly understood. In this study, we evaluated single synapse dynamics using optical imaging techniques in hippocampal cell cultures. We find that exogenous BDNF selectively increases evoked excitatory neurotransmission without affecting spontaneous neurotransmission. However, acutely blocking endogenous BDNF has no effect on evoked or spontaneous release, demonstrating that different approaches to studying BDNF may yield different results. When we suppressed BDNF-Tropomyosin receptor kinase B (TrkB) activity chronically over a period of days to weeks using a mouse line enabling conditional knockout of TrkB, we found that evoked glutamate release was significantly decreased while spontaneous release remained unchanged. Moreover, chronic blockade of BDNF-TrkB activity selectively downscales evoked calcium transients without affecting spontaneous calcium events. Via pharmacological blockade by voltage-gated calcium channel (VGCC) selective blockers, we found that the changes in evoked calcium transients are mediated by the P/Q subtype of VGCCs. These results suggest that BDNF-TrkB activity increases presynaptic VGCC activity to selectively increase evoked glutamate release.

Abstract 2437 Neuroplasticity in Alzheimer's Treatment: The Impact of Psychedelic-Induced TrkB Activation

Abstract 2437 Neuroplasticity in Alzheimer's Treatment: The Impact of Psychedelic-Induced TrkB Activation

Vagus nerve stimulation inhibits cortical spreading depression via glutamate-dependent TrkB activation mechanism in the nucleus tractus solitarius.

Vagus nerve stimulation (VNS) was recently found to inhibit cortical spreading depression (CSD), the underlying mechanism of migraine aura, through activation of the nucleus tractus solitarius (NTS), locus coeruleus (LC) and dorsal raphe nucleus (DRN). The molecular mechanisms underlying the effect of VNS on CSD in these nuclei remain to be explored. We hypothesized that VNS may activate glutamate receptor-mediated tropomyosin kinase B (TrkB) signaling in the NTS, thereby facilitating the noradrenergic and serotonergic neurotransmission to inhibit CSD. To investigate the role of TrkB and glutamate receptors in non-invasive VNS efficacy on CSD, a validated KCl-evoked CSD rat model coupled with intra-NTS microinjection of selective antagonists, immunoblot and immunohistochemistry was employed. VNS increased TrkB phosphorylation in the NTS. Inhibition of intra-NTS TrkB abrogated the suppressive effect of VNS on CSD and CSD-induced cortical neuroinflammation. TrkB was found colocalized with glutamate receptors in NTS neurons. Inhibition of glutamate receptors in the NTS abrogated VNS-induced TrkB activation. Moreover, the blockade of TrkB in the NTS attenuated VNS-induced activation of the LC and DRN. VNS induces the activation of glutamate receptor-mediated TrkB signaling in the NTS, which might modulate serotonergic and norepinephrinergic innervation to the cerebral cortex to inhibit CSD and cortical inflammation.

TrkB-mediated sustained neuroprotection is sex-specific and text{ER}alpha-dependent in adult mice following neonatal hypoxia ischemia

BackgroundNeonatal hypoxia ischemia (HI) related brain injury is one of the major causes of life-long neurological morbidities that result in learning and memory impairments. Evidence suggests that male neonates are more susceptible to the detrimental effects of HI, yet the mechanisms mediating these sex-specific responses to neural injury in neonates remain poorly understood. We previously tested the effects of treatment with a small molecule agonist of the tyrosine kinase B receptor (TrkB), 7,8-dihydroxyflavone (DHF) following neonatal HI and determined that females, but not males exhibit increased phosphorylation of TrkB and reduced apoptosis in their hippocampi. Moreover, these female-specific effects of the TrkB agonist were found to be dependent upon the expression of text{ER}alpha. These findings demonstrated that TrkB activation in the presence of text{ER}alpha comprises one pathway by which neuroprotection may be conferred in a female-specific manner. The goal of this study was to determine the role of text{ER}alpha-dependent TrkB-mediated neuroprotection in memory and anxiety in young adult mice exposed to HI during the neonatal period.MethodsIn this study, we used a unilateral hypoxic ischemic (HI) mouse model. text{ER}alpha+/+ or text{ER}alpha−/− mice were subjected to HI on postnatal day (P) 9 and mice were treated with either vehicle control or the TrkB agonist, DHF, for 7 days following HI. When mice reached young adulthood, we used the novel object recognition, novel object location and open field tests to assess long-term memory and anxiety-like behavior. The brains were then assessed for tissue damage using immunohistochemistry.ResultsNeonatal DHF treatment prevented HI-induced decrements in recognition and location memory in adulthood in females, but not in males. This protective effect was absent in female mice lacking text{ER}alpha. The female-specific improved recognition and location memory outcomes in adulthood conferred by DHF therapy after neonatal HI tended to be or were text{ER}alpha-dependent, respectively. Interestingly, DHF triggered anxiety-like behavior in both sexes only in the mice that lacked text{ER}alpha. When we assessed the severity of injury, we found that DHF therapy did not decrease the percent tissue loss in proportion to functional recovery. We additionally observed that the presence of text{ER}alpha significantly reduced overall HI-associated mortality in both sexes.ConclusionsThese observations provide evidence for a therapeutic role for DHF in which TrkB-mediated sustained recovery of recognition and location memories in females are text{ER}alpha-associated and dependent, respectively. However, the beneficial effects of DHF therapy did not include reduction of gross tissue loss but may be derived from the enhanced functioning of residual tissues in a cell-specific manner.

The SMA Modifier Plastin 3 Targets Cell Membrane-Associated Proteins in Motoneurons.

Loss of the Survival Motor Neuron (SMN) gene inevitably leads to spinal muscular atrophy (SMA), one of the most common fatal neuromuscular diseases in children with FDA and EMA approved therapies. However, the cellular mechanisms leading to neuromuscular junction (NMJ) dysfunction due to impaired Ca2+ homeostasis in the presynaptic compartment remain largely unexplained. In the last decade, the so-called SMA modifiers have gained attention. The F-actin bundler Plastin 3 (PLS3) is one of them and counteracts neurotransmission defects, including altered vesicle endocytosis, in Smn-deficient NMJs. Properly bundled F-actin is the basis for the translocation and arrangement of transmembrane proteins at the cell surface. Our recently published data by Hennlein et al., J Cell Biol. (2023) clearly showed that Smn deficiency impairs the F-actin dependent translocation of the high-affinity BDNF receptor TrkB to the cell surface resulting in reduced BDNF-mediated TrkB activation in motor axon terminals. Strikingly, the overexpression of PLS3 restores TrkB availability, and significantly improves the clustering of the active zone-associated voltage-gated calcium channel Cav2.2 in growth cones of Smn-deficient motoneurons. These observations raise the question of how PLS3 mediates the proper cell surface localization and cluster-like formation of Cav2.2 in motor axon terminals.

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.

Genetic knockout of NTRK2 by CRISPR/Cas9 decreases neurogenesis and favors glial progenitors during differentiation of neural progenitor stem cells.

The tropomyosin receptor kinase B (TrkB) is encoded by the NTRK2 gene. It belongs to the family of transmembrane tyrosine kinases, which have key roles in the development and maintenance of the nervous system. Brain-derived neurotrophic factor (BDNF) and the neurotrophins NT3 and NT4/5 have high affinity for TrkB. Dysregulation of TrkB is associated to a large spectrum of diseases including neurodegeneration, psychiatric diseases and some cancers. The function of TrkB and its role in neural development have mainly been decrypted using transgenic mouse models, pharmacological modulators and human neuronal cell lines overexpressing NTRK2. In this study, we identified high expression and robust activity of TrkB in ReNcell VM, an immortalized human neural progenitor stem cell line and generated NTRK2-deficient (NTRK2-/-) ReNcell VM using the CRISPR/Cas9 gene editing technology. Global transcriptomic analysis revealed major changes in expression of specific genes responsible for neurogenesis, neuronal development and glial differentiation. In particular, key neurogenic transcription factors were massively down-regulated in NTRK2-/- cells, while early glial progenitor markers were enriched in NTRK2-/- cells compared to NTRK2+/+. This indicates a previously undescribed inhibitory role of TrkB on glial differentiation in addition to its well-described pro-neurogenesis role. Altogether, we have generated for the first time a human neural cell line with a loss-of-function mutation of NTRK2, which represents a reproducible and readily available cell culture system to study the role of TrkB during human neural differentiation, analyze the role of TrkB isoforms as well as validate TrkB antibodies and pharmacological agents targeting the TrkB pathway.