Exploring a New Pathway for Treating Anxiety and Mood Disorders

Definition of a Bidirectional Activity-Dependent Pathway Involving BDNF and Narp.

A Hypothesized Role for Dendritic Remodeling in the Etiology of Mood and Anxiety Disorders

The constitutive and activity-dependent components of protein synthesis are both critical for neural function. Although the mechanisms controlling extracellularly induced protein synthesis are becoming clear, less is understood about the molecular networks that regulate the basal translation rate. Here we describe the effects of chronic treatment with various neurotrophic factors and cytokines on the basal rate of protein synthesis in primary cortical neurons. Among the examined factors, brain-derived neurotrophic factor (BDNF) showed the strongest effect. The rate of protein synthesis increased in the cortical tissues of BDNF transgenic mice, whereas it decreased in BDNF knock-out mice. BDNF specifically increased the level of the active, unphosphorylated form of eukaryotic elongation factor 2 (eEF2). The levels of active eEF2 increased and decreased in BDNF transgenic and BDNF knock-out mice, respectively. BDNF decreased kinase activity and increased phosphatase activity against eEF2 in vitro. Additionally, BDNF shortened the ribosomal transit time, an index of translation elongation. In agreement with these results, overexpression of eEF2 enhanced protein synthesis. Taken together, our results demonstrate that the increased level of active eEF2 induced by chronic BDNF stimulation enhances translational elongation processes and increases the total rate of protein synthesis in neurons.

Brain derived neurotrophic factor (BDNF), signaling via its receptor Tropomyosin receptor kinase B (TrkB) plays a pivotal role in establishing and maintaining the structure and function of neurons within the central nervous system. Deficits in BDNF/TrkB signaling are reported to contribute to the pathogenesis of multiple major disorders, such as Alzheimer’s disease. While manipulating the BDNF/TrkB signaling has been shown to be a viable approach to ameliorate some of the symptoms in a variety of neurological and psychiatric disorders, effective delivery of BDNF into the brain is challenging due to its poor pharmacokinetic profile and clinical translation has fallen short. This triggered the development of BDNF mimetics which specifically activate the TrkB receptor. This work investigated whether a recently identified fully human TrkB agonist antibody (ZEB85) exerts similar biological functions to BDNF. It was shown that treatment with ZEB85 leads to TrkB phosphorylation and increases expression of the activity-dependent immediate early gene c-Fos. Further, ZEB85 significantly increases the neurite complexity of developing hippocampal neurons. Parvalbumin positive interneurons from hippocampal cultures deprived of BDNF have a severely altered dendritic phenotype which can be completely rescued by the TrkB agonist antibody. Moreover, ZEB85 leads to changes in spine number and morphology in mature neurons. Under pathological conditions, treatment with ZEB85 completely prevents amyloid-beta induced dendritic spine loss. Lastly, prolonged application of ZEB85 over several days in hippocampal organotypic slice cultures prepared from heterozygous bdnf knockout mice, known to have deficits in the induction and maintenance of long-term potentiation (LTP) improved these parameters. Taken together, ZEB85 indeed exerts some of the biological functions of BDNF as seen in TrkB activation and structural and functional changes of healthy and diseased neurons. However, when directly compared to BDNF, the effect of ZEB85 is less pronounced. Thus, this work provides an initial characterization of a new promising TrkB agonist suggesting its potential value for therapeutical applications.

Brain-derived neurotrophic factor (BDNF) and its receptor, tyrosine kinase receptor B (TrkB), play a critical role in activity-dependent synaptic plasticity and have been implicated as mediators of hippocampal-dependent learning and memory. The present study is the first to demonstrate a role for BDNF and TrkB in amygdala-dependent learning. Here, the use of Pavlovian fear conditioning as a learning model allows us to examine the concise role of BDNF in the amygdala after a single learning session and within a well understood neural circuit. Using in situ hybridization, mRNA levels of six different trophic factors [BDNF, neurotrophin (NT) 4/5, NGF, NT3, aFGF, and bFGF) were measured at varying time points during the consolidation period after fear conditioning. We found temporally specific changes only in BDNF gene expression in the basolateral amygdala after paired stimuli that supported learning but not after exposure to neutral or aversive stimuli alone. Using Western blotting, we found that the Trk receptor undergoes increased phosphorylation during this consolidation period, suggesting an activation of the receptor subsequent to BDNF release. Furthermore, disruption of neurotrophin signaling with intra-amygdala infusion of the Trk receptor antagonist K252a disrupted acquisition of fear conditioning. To address the specific role of the TrkB receptor, we created a novel lentiviral vector expressing a dominant-negative TrkB isoform (TrkB.T1), which specifically blocked TrkB activation in vitro. In vivo, TrkB.T1 lentivirus blocked fear acquisition without disrupting baseline startle or expression of fear. These data suggest that BDNF signaling through TrkB receptors in the amygdala is required for the acquisition of conditioned fear.

Objectives: Brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) protein have been previously associated with mood disorders. Here, we aimed to investigate the roles of BDNF and TrkB in suicidality and obesity in patients with major depressive disorder (MDD). Methods: The serum protein levels of BDNF and TrkB were determined in 60 MDD in-and outpatients. We classified men and women with a body mass index (BMI) of ≧26.4 kg/m^2 as obese. We examined the differences in serum BDNF and TrkB protein levels between obese/non-obese and suicidal/non-suicidal MDD patients. Results: We found no significant difference in serum BDNF protein levels between obese and non-obese MDD patients or between MDD patients with or without suicide history. The TrkB protein levels were found to be significantly increased in obese MDD patients compared with those in non-obese MDD patients (F=4.137, p=0.047), but no significant difference was found in the TrkB protein levels between MDD patients with and without suicide history. Conclusion: We found in this study that obesity in our study MDD patients was associated with increased serum TrkB levels. Increased TrkB levels may play a role in the pathophysiology of obese MDD patients.

Plasma brain-derived neurotrophic factor in women after bariatric surgery: a pilot study

Cell Survival through Trk Neurotrophin Receptors Is Differentially Regulated by Ubiquitination

Throughout our entire life, memory is central to our ability to carry out everyday tasks. Therefore, diseases that affect cognition have severe consequences, that influence patients' daily lives. However, not only do deficits in forming or recalling memories impact peoples' lives but also the inability to forget traumatic events or fear towards specific stimuli, such as with post-traumatic stress disorder (PTSD), anxiety disorders and phobias. Hence, it is essential to understand the cellular and molecular mechanisms in order to help people with any of these disorders. In the first study of this thesis, I investigated if nuclear calcium, which is known to control cAMP response element binding protein (CREB)/CREB binding protein (CBP)-mediated transcription is required for the formation of memory. Indeed, I found that inhibition of hippocampal nuclear calcium impairs memory formation in two hippocampus-dependent tasks. I further investigated the possible mechanisms that contribute to these deficits and found that dysregulation of nuclear calcium leads to alterations in dendritic morphology. I was able to rescue these morphological alterations via overexpression of the vascular endothelial growth factor D (VEGFD). VEGFD is regulated by nuclear calcium under basal conditions and is known to be important for the maintenance of dendritic morphology. Additionally, I could show that overexpression of VEGFD rescued memory deficits caused by nuclear calcium inhibition, probably due to the restoration of the dendritic architecture. In the second study, we aimed to investigate whether DNA methyltransferases (DNMTs) are required for memory formation. We found that DNMT3a2 is regulated by nuclear calcium and is induced upon neuronal activity. Additionally, we were able to show that the level of DNMT3a2 determines memory performance in mice. Downregulation of DNMT3a2 caused memory deficits and, in addition, a decrease in DNMT3a2 expression was associated with age-dependent memory decline. Further, restoring the level of DNMT3a2 in aged mice rescued memory impairments. In addition, we showed that DNMT3a2 expression correlates with global methylation levels, and we were able to identify two of the target genes of DNMT3a2, namely, activity-regulated cytoskeleton-associated protein (ARC) and brain-derived neurotrophic factor (BDNF). In the third part of this thesis, I studied if nuclear calcium is also required for fear memory extinction, which is routinely used as a model of PTSD. It is widely accepted that during fear extinction a new memory is built up that inhibits the previous, acquired memory. Though it is known that memory formation and fear extinction share common mechanisms, studies investigating the role of transcription in fear extinction are partly controversial. Here, I have shown that nuclear calcium is involved in fear extinction, pointing to a requirement of gene transcription. As I have shown in the first study of this thesis, nuclear calcium, which maintains the dendritic architecture primarily via alterations in the dendritic tree, probably also contributes to fear memory extinction. In summary, I have shown in this thesis that nuclear calcium mediates two forms of cognition, memory formation and fear extinction. I provide evidence that nuclear calcium-regulated VEGFD maintains the dendritic structure, which is important for the permissiveness of the neuron to process information required for long-term adaptations. Additionally, nuclear calcium regulates DNMT3a2, and we show that the level of DNMT3a2 has an impact on memory formation. Overexpression of DNMT3a2 restored the level of DNMT3a2 in aged mice and rescued age-dependent memory deficits.

Interactions of three neurotrophin dimers, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and a NT-3.BDNF heterodimer with extracellular, soluble TrkB and TrkC receptors were studied using native gels, light scattering, and sedimentation equilibrium. These three neurotrophins showed binding of two TrkB receptors per neurotrophin dimer, with a tendency to dissociate into one TrkB per dimer for NT-3 and the heterodimer, as determined by native gels, light scattering, and sedimentation equilibrium. For TrkC, native gels suggested binding of NT-3, heterodimer, and BDNF but not of nerve growth factor. Sedimentation equilibrium revealed that all three neurotrophin molecules bind to TrkC at two receptors per dimer but that BDNF binds much more weakly and that the heterodimer has an intermediate binding strength. Light scattering/size exclusion chromatography showed complexes with two TrkC receptors per NT-3 dimer and one TrkC per heterodimer but did not detect binding of BDNF to TrkC. This latter result is not inconsistent with the sedimentation data, because the weak binding of BDNF to TrkC may be easily dissociated by nonspecific interactions of BDNF with the size exclusion column. The relative binding constants for these neurotrophins and the soluble receptor extracellular domains, as determined by sedimentation equilibrium, are correlated with their biological activity. However, the magnitude of these binding constants is insufficient by approximately 3 orders of magnitude to promote receptor dimerization at physiologically active concentrations.

Background Recent studies showed that diabetic retinal neuropathy is an earlier and more dangerous complication and neurotrophin has a protective effect on retina. Objective The present study was to observe the changes of brain derived neurotrophic factor (BDNF),its receptor TrkB,signal pathway protein phosphatized extracellular signal-regulated protein kinase1/2 (p-ERK1/2) and c-fos in the retina after injection of BDNF into the vitreous in STZ induced Wistar diabetic rats. Methods Wistar rats aged 9 weeks-old were randomly divided into BDNF injection group,diabetes mellitus (DM) control group and normal control group and 20 rats for each group.STZ was intraperitoneally injected in the rats of BDNF injection group and DM control group to create the experimental DM.BDNF was intravitreously injected in the rats of BDNF group 2 weeks after administration of STZ in three-day interval for 5 times,and BSS containing O.1％ bovine serum albumin (BSA) was used at the same way in the DM control group and normal control group.The retina was isolated for hybridization in situ for BDNF,and TrkB,p-ERK1/2 and c-fos.Levels in retina were detected using sandwich method ELISA. Results The number of BDNF positive cells and the gray scale were lower obviously in the rat retina of DM control group than those of BDNF injection group and normal control group,showing significant differences among the 3 groups ( F =102.36,92.55 ;P＜0.05 ).ELISA assay showed that TrkB,p-ERK1/2 and c-fos values in retina were statistically significantly different among the 3 groups ( F =92.54,95.46,94.84,P＜0.05 ).The TrkB level in retina was statistically reduced,but the p-ERK1/2 and c-fos levels in retina were increased statistically in DM control group compared with BDNF injection group and normal control group( P＜0.05 ).No statistical difference was found in TrkB,p-ERK1/2 and c-fos values between the BDNF injection group and normal control group(P＞0.05). Conclusions The injection of BDNF into the vitreous cavity can protect retina from downregulating BDNF and TrkB levels and up-regulating the p-ERK1/2 and c-fos protein levels in the early stage of DM. Key words: Diabetes mellitus/diabetic retinopathy; Brain derived neurotrophic factor; Signal cascade pathway

Objective To study the expression of brain-derived neurotrophic factor (BDNF) and Tyrosine Kinase B Receptor (TrkB) in pancreatic ductal adenocarcinoma (PDAC) and it’s relationship in PDAC. Methods Collected 62 cases of PDAC paraffin samples which surgical resection was performed in Anhui provincial hospital from August 2003 to December 2015. In the study, First, according to the postoperative pathology of nerve invasion into two groups in 62 cases of pancreatic cancer specimens: Perineural invasion (PNI)-positive group (32/62) and PNI-negative group (30/62), comparative analysis of the clinical pathological characteristics, especially the relationship with perineural invasion, at the same time with 10 cases of normal pancreas tissues as control, then using immunohistochemical SP method, the Real-time polymerase chain reaction (PCR)and Western blotting separately from gene transcription level and protein level of BDNF and TrkB validated positive expression in pancreatic cancer. Results PDAC nerve invasion in the group of BDNF positive expression rate was 56.2% (18/32 cases), nerve not infringe tissues was 30.0% (9/30 cases); Nerve invasion group TrkB positive expression rate was 62.5% (20/32 cases), nerve not infringe tissues was 36.7% (11/30 cases), but not seen in 10 cases of normal pancreatic tissues of BDNF and TrkB both positive expression, and nerve infiltrating group is significantly higher than without nerve infiltration (P 0.05), adjacent to cancer cells of the nervous tissue of BDNF and TrkB positive degree higher than cancer cells from nervous tissue; Statistical analysis found that BDNF protein positive expression and the tumor diameter, tumor location, closely related to neural invasion (P<0.05); TrkB protein positive expression and the tumor location, nerve infiltration, clinical stage, lymph node metastasis (P<0.05); BDNF mRNA expression levels in pancreatic cancer, nerve invasion in the group to express quantity are not nerve invasion and normal pancreas group 2.06 times and 4.07 times, the difference was statistically significant (P<0.05); TrkB mRNA expression levels in pancreatic cancer, nerve invasion in the group to express quantity are not nerve invasion and normal pancreas group 5.41 times and 1.64 times that of comparison between groups was statistically significant (P<0.05). Conclusion This study shows that PDAC with evidence of PNI shows increased expression of BDNF and TrkB and suggests that BDNF and TrkB are involved with the mechanism leading to PNI. Key words: Pancreatic ductal adenocarcinoma; Brain-derived neurotrophic factor; Tyrosine kinase B receptor; Perineural invasion

Neurotrophins are a family of growth factors playing key roles in the survival, development, and function of neurons. The neurotrophins brain-derived neurotrophic factor (BDNF) and NT4 both bind to and activate TrkB receptors, however, they mediate distinct neuronal functions. The molecular mechanism of how TrkB activation by BDNF and NT4 leads to diverse outcomes is unknown. Here, we report that BDNF and NT4 lead to differential endocytic sorting of TrkB receptors resulting in diverse biological functions in cultured cortical neurons. Fluorescent microscopy and surface biotinylation experiments showed that both neurotrophins stimulate internalization of TrkB with similar kinetics. Exposure to BDNF for 2-3h reduced the surface pool of TrkB receptors to half, whereas a longer treatment (4-5h) with NT4 was necessary to achieve a similar level of down-regulation. Although BDNF and NT4 induced TrkB phosphorylation with similar intensities, BDNF induced more rapid ubiquitination and degradation of TrkB than NT4. Interestingly, TrkB receptor ubiquitination by these ligands have substantially different pH sensitivities, resulting in varying degrees of receptor ubiquitination at lower pH levels. Consequently, NT4 was capable of maintaining longer sustained downstream signaling activation that correlated with reduced TrkB ubiquitination at endosomal pH. Thus, by leading to altered endocytic trafficking itineraries for TrkB receptors, BDNF and NT4 elicit differential TrkB signaling in terms of duration, intensity, and specificity, which may contribute to their functional differences invivo. The neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4), both bind to and activate TrkB receptors, however, they mediate distinct neuronal functions. Here, we propose that BDNF and NT4 lead to differential endocytic sorting of TrkB receptors resulting in diverse biological functions. BDNF induces more rapid ubiquitination and degradation of TrkB than NT4. Consequently, NT4 is capable of maintaining more sustained signaling downstream of TrkB receptors.

Metabotropic Glutamate Receptors of Subtype 5 (mGluR5) and Sleep Homeostasis: Effects of Gene Knock-out and of Selective Negative Allosteric Modulation on EEG, Behavioral and Molecular Variables in Mice

Airway-related vagal preganglionic neurons express brain-derived neurotrophic factor and TrkB receptors: Implications for neuronal plasticity