Targeting both BDNF/TrkB pathway and delta-secretase for treating Alzheimer's disease.

Alzheimer's disease (AD) is the most common dementia, and no disease-modifying therapeutic agents are currently available. BDNF/TrkB signaling is impaired in AD and is associated with prominent delta-secretase (δ-secretase, also known as asparaginyl endopeptidase or legumain) activation, which simultaneously cleaves both APP and Tau and promotes Aß production and neurofibrillary tangles (NFT) pathologies. Here we show that the optimized δ-secretase inhibitor (#11a) or TrkB receptor agonist (CF3CN) robustly blocks δ-secretase activity separately, and their combination synergistically blunts δ-secretase, exhibiting promising therapeutic efficacy in 3xTg AD mouse model. The optimal δ-secretase inhibitor reveals demonstrable brain exposure and oral bioavailability, suppressing APP N585 and Tau N368 cleavage by δ-secretase. Strikingly, CF3CN treatment evidently escalates BDNF levels. Both #11a and CF3CN display strong in vivo PK/PD properties and ability to suppress δ-secretase activity in the brain. Orally administrated CF3CN strongly activates TrkB that triggers active Akt to phosphorylate δ-secretase T322, preventing its proteolytic activation and mitigating AD pathologies. #11a or CF3CN significantly diminishes AD pathogenesis and improves cognitive functions with the combination exhibiting the maximal effect. Thus, our data support that these derivatives are strong pharmaceutical candidates for the treatment of AD.

Apelin-13 activates the hippocampal BDNF/TrkB signaling pathway and suppresses neuroinflammation in male rats with cisplatin-induced cognitive dysfunction.

It has been established that cisplatin causes neuronal damage and cognitive impairment. However, the mechanism is not sufficiently clear. Apelin-13 is an endogenous peptide with strong neuroprotective effects through the synthesis of neurotrophic factors and suppression of inflammation. The aim of this study was to investigate the role of brain-derived neurotrophic factor/tropomyosin receptor kinase B (BDNF/TrkB) signaling pathway and the potential inhibitory effects of apelin-13 in the mechanism of cisplatin-induced hippocampal damage and cognitive impairment. Apelin-13 was administered to adult sprague dawley male rats at a dose of 20 nmol/kg every day for 4 weeks, cisplatin was administered at a dose of 5 mg/kg once a week for 4 weeks. The spatial and recognition memory tests of the rats were performed on the 5th week. BDNF and the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) levels were measured by ELISA in hippocampal homogenates. Pyramidal neuron and glial cell damage in the hippocampal CA1, CA3 and dentate gyrus (DG) were analyzed histologically. TrkB activity in the hippocampus was determined by immunohistochemical methods. Cisplatin impaired spatial and recognition memory in rats, while apelin-13 improved spatial memory but did not affect recognition memory. Cisplatin suppressed BDNF in the hippocampus while increased IL-1ß and TNF-α. In contrast, apelin-13 administration increased BDNF but significantly suppressed TNF-α and IL-1B. Cisplatin caused pyramidal neuron and glial cell damage in CA1, CA3 and DG. In the cisplatin + apelin-13 group, however, pyramidal neuron and glial cell damage was less than those without apelin-13. Cisplatin increased TrkB activity in the hippocampus, which was counteracted by apelin-13. In conclusion, apelin-13 reduced the cisplatin-induced cognitive deficiency, by suppressing inflammation and stimulating the synthesis and activation of neurotrophic factors in hippocampal tissue.

Reduced Hippocampal Dendrite Branching, Spine Density and Neurocognitive Function in Premature Rabbits, and Reversal with Estrogen or TrkB Agonist Treatment.

Preterm-born children suffer from neurological and behavioral disorders. Herein, we hypothesized that premature birth and non-maternal care of preterm newborns might disrupt neurobehavioral function, hippocampal dendritic arborization, and dendritic spine density. Additionally, we assessed whether 17ß-estradiol (E2) replacement or the TrkB receptor agonist, 7,8-dihydroxyflavone (DHF), would reverse compromised dendritic development and cognitive function in preterm newborns. These hypotheses were tested by comparing preterm (E28.5) rabbit kits cared and gavage-fed by laboratory personnel and term-kits reared and breast-fed by their mother doe at an equivalent postconceptional age. Neurobehavioral tests showed that both premature-birth and formula-feeding with non-maternal care led to increased anxiety behavior, poor social interaction, and lack of novelty preference compared with term-kits. Dendritic branching and number of total or mushroom dendritic spines were reduced in the CA1 field of preterm-kits compared with term controls. While CDC42 and Rac1/2/3 expression levels were lower, RhoA-activity was higher in preterm-kits compared with term controls. Both E2 and DHF treatment reversed prematurity-induced reduction in spine density, reduced total RhoA-GTPase levels, and enhanced cognitive function. Hence, prematurity and non-maternal care result in cognitive deficits, and reduced dendritic arbors and spines in CA1. E2 replacement or DHF treatment might reverse changes in dendritic spines and improve neurodevelopment in premature infants.

Astrocyte-Conditioned Medium Protects Prefrontal Cortical Neurons from Glutamate-Induced Cell Death by Inhibiting TNF-α Expression.

OBJECTIVE: Both excitotoxicity and neurotrophin deficiency may contribute to the etiology of depression and neurodegeneration. Astrocytes not only regulate glutamate metabolism and clearance, they also produce neurotrophins in the brain. However, the direct interaction between neurons and astrocytes remains unknown. METHODS: This study evaluated the cellular mechanisms by which astrocyte-conditioned medium (ACM) protects prefrontal cortical neurons from glutamate-induced death by measuring cell viability and morphology as well as mRNA and protein expression of brain-derived neurotrophic factor (BDNF), BDNF receptors, glial cell line-derived neurotrophic factor (GDNF), and the proinflammatory cytokine, tumor necrosis factor (TNF)-α. Neurons and astrocytes were purified from the brains of neonatal 1-day-old Sprague-Dawley rats. ACM was harvested after exposing astrocytes to culture medium containing 100 µM glutamate for 48 h. RESULTS: Glutamate insult (100 µM for 6 h) significantly reduced neuronal cell viability and increased the mRNA expression of BDNF. Glutamate (24 h) decreased neuronal viability and the expression of BDNF, but increased mRNA expression of GFAP, p75 neurotrophin receptor (p75NTR), and TNF-α. ACM pretreatment (2 h) reversed glutamate-decreased cell viability and increased BDNF, but reduced the expression of GDNF, P75NTR, and TNF-α at the mRNA level. Western blotting generally confirmed the mRNA expression following 24 glutamate insults. Furthermore, the glutamate-induced decrease in the protein expression of BDNF and full-length TrkB receptor and increase in pro-BDNF, truncated TrkB isoform 1 receptor, p75NTR, GDNF, and TNF-α were significantly attenuated by ACM pretreatment. CONCLUSIONS: The study demonstrates that ACM exerts neuroprotective effects on cell viability, and this effect is most likely mediated through the modulation of neurotrophin and TNF-α expression.

ERK1/2-mediated disruption of BDNF-TrkB signaling causes synaptic impairment contributing to fluoride-induced developmental neurotoxicity.

Excessive exposure to fluoride has adverse effects on neurodevelopment, but the mechanisms remain unclear. This study aimed to investigate the effects of fluoride exposure on synaptogenesis, and focused on the role of brain-derived neurotrophic factor (BDNF)-tyrosine kinase B (TrkB) signaling in these effects. Using Sprague-Dawley rats developmentally exposed to sodium fluoride (NaF) from pregnancy until 6 months of delivery as in vivo model, we showed that fluoride impaired the cognitive abilities of offspring rats, decreased the density of dendritic spines and the expression of synapse proteins synaptophysin (SYN) and postsynaptic density protein-95 (PSD-95) in hippocampus, suggesting fluoride-induced cognitive deficit associates with synaptic impairment. Consistently, NaF treatment reduced dendritic outgrowth and expression of SYN and PSD-95 in human neuroblastoma SH-SY5Y cells. Further studies demonstrated that the BDNF-TrkB axis was disrupted in vivo and in vitro, as manifested by BDNF accumulation and TrkB reduction. Importantly, fluoride treatment increased phospho-extracellular signal-regulated kinases 1 and 2 (p-ERK1/2) expression, while inhibition of p-ERK1/2 significantly attenuated the effects of NaF, indicating a regulating role of p-ERK1/2 in BDNF-TrkB signaling disruption. Collectively, these data suggest that the developmental neurotoxicity of fluoride is associated with the impairment of synaptogenesis, which is caused by ERK1/2-mediated BDNF-TrkB signaling disruption.

Arsenic trioxide induces cell cycle arrest and affects Trk receptor expression in human neuroblastoma SK-N-SH cells.

BACKGROUND: Arsenic trioxide (As2O3), a drug that has been used in China for approximately two thousand years, induces cell death in a variety of cancer cell types, including neuroblastoma (NB). The tyrosine kinase receptor (Trk) family comprises three members, namely TrkA, TrkB and TrkC. Various studies have confirmed that TrkA and TrkC expression is associated with a good prognosis in NB, while TrkB overexpression can lead to tumor cell growth and invasive metastasis. Previous studies have shown that As2O3 can inhibit the growth and proliferation of a human NB cell line and can also affect the N-Myc mRNA expression. It remains unclear whether As2O3 regulates Trks for the purposes of treating NB. METHODS: The aim of the present study was to investigate the effect of As2O3 on Trk expression in NB cell lines and its potential therapeutic efficacy. SK-N-SH cells were grown with increasing doses of As2O3 at different time points. We cultured SK-N-SH cells, which were treated with increasing doses of As2O3 at different time points. Trk expression in the NB samples was quantified by immunohistochemistry, and the cell cycle was analyzed by flow cytometry. TrkA, TrkB and TrkC mRNA expression was evaluated by real-time PCR analysis. RESULTS: Immunohistochemical and real-time PCR analyses indicated that TrkA and TrkC were over-expressed in NB, and specifically during stages 1, 2 and 4S of the disease progression. TrkB expression was increased in stage 3 and 4 NB. As2O3 significantly arrested SK-N-SH cells in the G2/M phase. In addition, TrkA, TrkB and TrkC expression levels were significantly upregulated by higher concentrations of As2O3 treatment, notably in the 48-h treatment period. Our findings suggested that to achieve the maximum effect and appropriate regulation of Trk expression in NB stages 1, 2 and 4S, As2O3 treatment should be at relatively higher concentrations for longer delivery times;however, for NB stages 3 and 4, an appropriate concentration and infusion time for As2O3 must be carefully determined. CONCLUSION: The present findings suggested that As2O3 induced Trk expression in SK-N-SH cells to varying degrees and may be a promising adjuvant to current treatments for NB due to its apoptotic effects.

Obestatin prevents H2O2-induced damage through activation of TrkB in RGC-5 cells.

BACKGROUD: In the early stage of diabetic retinopathy, the damage of retinal ganglion cells already exists, promoting the development of the disease. The aim of this study was to investigate the protective role and the mechanisms of obestatin against H2O2-induced damage in RGC-5 cells. METHODS: RGC-5 cells were incubated with various concentrations of obestatin for 24h before H2O2 added. The survival rates of RGC-5 were measured by MTT assay. The expression of apoptosis-related proteins and TrkB pathway-related proteins were detected by Western blot analysis. RESULTS: Our data showed that H2O2 evidently decreased the survival rate of RGC-5 cells. However, obestatin pretreatment reversed the decreased activity. Moreover, obestatin effectively increased the expression of Bcl-2 and decreased the expression of Bax. In addition, obestatin potentially plays a role in protecting RGC-5 by activating of TrkB. Obestatin notablely increased the phosphorylation of TrkB, AKT and ERK1/2. All these effects of obestatin can be inhibited by GLP-1R antagonist exendin (9-39). CONCLUSIONS: Obestatin prevents H2O2-induced damage in RGC-5 cells by activating TrkB pathway. Moreover, GLP-1R is closely related to the function of obestatin in RGC-5 cells.

Arsenic trioxide induces cell cycle arrest and affects Trk receptor expression in human neuroblastoma SK-N-SH cells

BACKGROUND: Arsenic trioxide (As2O3), a drug that has been used in China for approximately two thousand years, induces cell death in a variety of cancer cell types, including neuroblastoma (NB). The tyrosine kinase receptor (Trk) family comprises three members, namely TrkA, TrkB and TrkC. Various studies have confirmed that TrkA and TrkC expression is associated with a good prognosis in NB, while TrkB overexpression can lead to tumor cell growth and invasive metastasis. Previous studies have shown that As2O3 can inhibit the growth and proliferation of a human NB cell line and can also affect the N-Myc mRNA expression. It remains unclear whether As2O3 regulates Trks for the purposes of treating NB. METHODS: The aim of the present study was to investigate the effect of As2O3 on Trk expression in NB cell lines and its potential therapeutic efficacy. SK-N-SH cells were grown with increasing doses of As2O3 at different time points. We cultured SK-N-SH cells, which were treated with increasing doses of As2O3 at different time points. Trk expression in the NB samples was quantified by immunohistochemistry, and the cell cycle was analyzed by flow cytometry. TrkA, TrkB and TrkC mRNA expression was evaluated by real-time PCR analysis. RESULTS: Immunohistochemical and real-time PCR analyses indicated that TrkA and TrkC were over-expressed in NB, and specifically during stages 1, 2 and 4S of the disease progression. TrkB expression was increased in stage 3 and 4 NB. As2O3significantly arrested SK-N-SH cells in the G2/M phase. In addition, TrkA, TrkB and TrkC expression levels were significantly upregulated by higher concentrations of As2O3 treatment, notably in the 48-h treatment period. Our findings suggested that to achieve the maximum effect and appropriate regulation of Trk expression in NB stages 1, 2 and 4S, As2O3 treatment should be at relatively higher concentrations for longer delivery times;however, for NB stages 3 and 4, an appropriate concentration and infusion time for As2O3 must be carefully determined. CONCLUSION: The present findings suggested that As2O3 induced Trk expression in SK-N-SH cells to varying degrees and may be a promising adjuvant to current treatments for NB due to its apoptotic effects.

Antidepressant-like effects of standardized gypenosides: involvement of brain-derived neurotrophic factor signaling in hippocampus.

RATIONALE: Gypenosides have been reported to produce neuroprotective effects and increase monoamine neurotransmitter levels in the brain. OBJECTIVE: Considering that depression is involved in monoamine reduction, this study evaluated the antidepressant-like effects of gypenosides in mice exposed to chronic unpredictable mild stress (CUMS). METHODS: The sucrose preference test and forced swimming test were performed after administration of gypenosides (at 25, 50, or 100 mg/kg) for 4 weeks. Hippocampal brain-derived neurotrophic factor (BDNF) and its downstream targets were analyzed by western blot. Additionally, hippocampal neuronal proliferation was measured by immunohistochemistry. RESULTS: Four-week treatment with fluoxetine (20 mg/kg) and gypenosides (at either 50 or 100 mg/kg) increased sucrose preference and decreased the immobility time in mice exposed to CUMS. In addition, gypenosides (at either 50 or 100 mg/kg) also increased BDNF expression and neuronal proliferation in the hippocampus of CUMS animals. Further, we showed that treating CUMS mice with K252a, which is an inhibitor of the BDNF receptor TrkB, blocked the effects of gypenosides (100 mg/kg), including behavioral improvements, neuronal proliferation, and up-regulation of p-TrkB, p-ERK, and p-Akt proteins. CONCLUSIONS: This study demonstrates that gypenosides exhibit antidepressant-like effects in mice, which may be mediated by activation of the BDNF-ERK/Akt signaling pathway in the hippocampus.

Amitriptyline Activates TrkA to Aid Neuronal Growth and Attenuate Anesthesia-Induced Neurodegeneration in Rat Dorsal Root Ganglion Neurons.

Tricyclic antidepressant amitriptyline (AM) has been shown to exert neurotrophic activity on neurons. We thus explored whether AM may aid the neuronal development and protect anesthesia-induced neuro-injury in young spinal cord dorsal root ganglion (DRG) neurons.The DRG explants were prepared from 1-day-old rats. The effect of AM on aiding DRG neural development was examined by immunohistochemistry at dose-dependent manner. AM-induced changes in gene and protein expressions, and also phosphorylation states of tyrosine kinases receptor A (TrkA) and B (TrkB) in DRG, were examined by quantitative real-time polymerase chain reaction and western blot. The effect of AM on attenuating lidocaine-induced DRG neurodegeneration was examined by immunohistochemistry, and small interfering RNA (siRNA)-mediated TrkA/B down-regulation.Amitriptyline stimulated DRG neuronal development in dose-dependent manner, but exerted toxic effect at concentrations higher than 10 M. AM activated TrkA in DRG through phosphorylation, whereas it had little effect on TrkB-signaling pathway. AM reduced lidocaine-induced DRG neurodegeneration by regenerating neurites and growth cones. Moreover, the neuroprotection of AM on lidocaine-injured neurodegeneration was blocked by siRNA-mediated TrkA down-regulation, but not by TrkB down-regulation.Amitriptyline facilitated neuronal development and had protective effect on lidocaine-induced neurodegeneration, very likely through the activation of TrkA-signaling pathway in DRG.

Enriched environment reduces glioma growth through immune and non-immune mechanisms in mice.

Mice exposed to standard (SE) or enriched environment (EE) were transplanted with murine or human glioma cells and differences in tumour development were evaluated. We report that EE exposure affects: (i) tumour size, increasing mice survival; (ii) glioma establishment, proliferation and invasion; (iii) microglia/macrophage (M/Mφ) activation; (iv) natural killer (NK) cell infiltration and activation; and (v) cerebral levels of IL-15 and BDNF. Direct infusion of IL-15 or BDNF in the brain of mice transplanted with glioma significantly reduces tumour growth. We demonstrate that brain infusion of IL-15 increases the frequency of NK cell infiltrating the tumour and that NK cell depletion reduces the efficacy of EE and IL-15 on tumour size and of EE on mice survival. BDNF infusion reduces M/Mφ infiltration and CD68 immunoreactivity in tumour mass and reduces glioma migration inhibiting the small G protein RhoA through the truncated TrkB.T1 receptor. These results suggest alternative approaches for glioma treatment.

Effect of Tenuifoliside A isolated from Polygala tenuifolia on the ERK and PI3K pathways in C6 glioma cells.

Tenuifoliside A (TFSA) is a bioactive oligosaccharide ester component of Polygala tenuifolia Wild, a traditional Chinese medicine which was used to manage mental disorders effectively. The neuroprotective and anti-apoptotic effects of TFSA have been demonstrated in our previous studies. The present work was designed to study the molecular mechanism of TFSA on promoting the viability of rat glioma cells C6. We exposed C6 cells to TFSA (or combined with ERK, PI3K and TrkB inhibitors) to examine the effects of TFSA on the cell viability and the expression and phosphorylation of key proteins in the ERK and PI3K signaling pathway. TFSA increased levels of phospho-ERK and phospho-Akt, enhanced release of BDNF, which were blocked by ERK and PI3K inhibitors, respectively (U0126 and LY294002). Moreover, the TFSA caused the enhanced phosphorylation of cyclic AMP response element binding protein (CREB) at Ser133 site, the effect was revoked by U0126, LY294002 and K252a. Furthermore, when C6 cells were pretreated with K252a, a TrkB antagonist, known to significantly inhibit the activity of brain-derived neurotrophic factor (BDNF), blocked the levels of phospho-ERK, phospho-Akt and phosphor-CREB. Taking these results together, we suggested the neuroprotection of TFSA might be mediated through BDNF/TrkB-ERK/PI3K-CREB signaling pathway in C6 glioma cells.

Effects of olive leaf polyphenols on male mouse brain NGF, BDNF and their receptors TrkA, TrkB and p75.

In this study, we evaluated, in the mouse, the effects of 20 mg/kg i.p. daily administration for 15 consecutive days of a blend of polyphenols, containing mostly oleuropein, extracted from the olive leaves (Olea europaea) on brain nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and on the expression of their receptors, TrkA, TrkB and p75. Polyphenols decreased the levels of reduced glutathione (GSH) and increased the levels of NGF and BDNF in the serum. In the brain, we found decreased levels of NGF and BDNF in the hippocampus and striatum but elevated levels of NGF in the olfactory lobes and hypothalamus and again BDNF potentiation in the olfactory lobes. No changes in TrkA, TrkB and p75 expression were observed. In conclusion, olive polyphenols may not only elicit an activation of the rodent olfactory system by increasing the levels of NGF and BDNF but also be stressing for the animal by reducing both the levels of hippocampal NGF/BDNF and serum GSH and increasing serum levels of NGF and BDNF.

Prenatal buprenorphine exposure decreases neurogenesis in rats.

Perinatal opioid exposure has a negative effect on neurogenesis and produces neurological consequences. However, its mechanisms of action are incompletely understood. Buprenorphine, a mixed opioid agonist/antagonist, is an alternative medication for managing pregnant opioid addicts. This study provides evidence of decreased neurogenesis and depression-like consequences following prenatal exposure to buprenorphine and sheds light on mechanisms of action in a rat model involving administration of intraperitoneal injection to pregnant rats starting from gestation day 7 and lasting for 14 days and a cultured neurosphere model. Results of forced swimming test and tail suspension test showed that pups at postnatal day 21 had worse parameters of depression-like neurobehaviors, independent of gender. Neurobehavioral changes were accompanied by reduction of neuronal composition, biochemical parameters of neural stem/progenitor cells, brain-derived neurotrophic factor (BDNF) expression, tropomyosin-related kinase receptor type B phosphorylation, protein kinase A (PKA) activity, and cAMP response element-binding protein phosphorylation. Results of parallel cell studies further demonstrated a negative impact of buprenorphine on cultured neurospheres, including proliferation, differentiation, BDNF expression and signaling, and PKA activity. Taken together, our results suggest that prenatal exposure to buprenorphine might result in depression-like phenotypes associated with impaired BDNF action and decreased neurogenesis in the developing brain of weanlings.

MicroRNA-204 increases sensitivity of neuroblastoma cells to cisplatin and is associated with a favourable clinical outcome.

BACKGROUND: Neuroblastoma remains a major cause of cancer-linked mortality in children. miR-204 has been used in microRNA expression signatures predictive of neuroblastoma patient survival. The aim of this study was to explore the independent association of miR-204 with survival in a neuroblastoma cohort, and to investigate the phenotypic effects mediated by miR-204 expression in neuroblastoma. METHODS: Neuroblastoma cell lines were transiently transfected with miR-204 mimics and assessed for cell viability using MTS assays. Apoptosis levels in cell lines were evaluated by FACS analysis of Annexin V-/propidium iodide-stained cells transfected with miR-204 mimics and treated with chemotherapy drug or vehicle control. Potential targets of miR-204 were validated using luciferase reporter assays. RESULTS: miR-204 expression in primary neuroblastoma tumours was predictive of patient event-free and overall survival, independent of established known risk factors. Ectopic miR-204 expression significantly increased sensitivity to cisplatin and etoposide in vitro. miR-204 direct targeting of the 3' UTR of BCL2 and NTRK2 (TrkB) was confirmed. CONCLUSION: miR-204 is a novel predictor of outcome in neuroblastoma, functioning, at least in part, through increasing sensitivity to cisplatin by direct targeting and downregulation of anti-apoptotic BCL2. miR-204 also targets full-length NTRK2, a potent oncogene involved with chemotherapy drug resistance in neuroblastoma.

A high cholesterol diet given to apolipoprotein E-knockout mice has a differential effect on the various neurotrophin systems in the hippocampus.

Apolipoprotein E (apoE) is one of the major transporters of cholesterol in the body and is essential for maintaining various neural functions in the brain. Given that hypercholesterolemia is a risk factor in Alzheimer's disease (AD), it has been suggested that altered cholesterol metabolism may be involved in the development of the pathogenesis, including neural degeneration, commonly observed in AD patients. Neurotrophic factors and their receptors, which are known to regulate various neural functions, are also known to be altered in various neurodegenerative diseases. We therefore hypothesized that cholesterol metabolism may itself influence the neurotrophin system within the brain. We decided to investigate this possibility by modulating the amount of dietary cholesterol given to apoE-knockout (apoE-KO) and wild-type (WT) mice, and examining the mRNA expression of various neurotrophin ligands and receptors in their hippocampal formations. Groups of eight-week-old apoE-KO and WT mice were fed a diet containing either "high" (HCD) or "normal" (ND) levels of cholesterol for a period of 12 weeks. We found that high dietary cholesterol intake elevated BDNF mRNA expression in both apoE-KO and WT mice and TrkB mRNA expression in apoE-KO animals. On the other hand, NGF and TrkA mRNA levels remained unchanged irrespective of both diet and mouse type. These findings indicate that altered cholesterol metabolism induced by HCD ingestion combined with apoE deficiency can elicit a differential response in the various neurotrophin ligand/receptor systems in the mouse hippocampus. Whether such changes can lead to neural degeneration, and the mechanisms that may be involved in this, awaits further research.

Lestaurtinib enhances the antitumor efficacy of chemotherapy in murine xenograft models of neuroblastoma.

PURPOSE: Neuroblastoma, a common pediatric tumor of the sympathetic nervous system, is characterized by clinical heterogeneity. The Trk family neurotrophin receptors play an important role in this behavior. Expression of TrkA is associated with favorable clinical features and outcome, whereas TrkB expression is associated with an unfavorable prognosis. We wanted to determine if the Trk-selective inhibitor lestaurtinib had therapeutic efficacy in a preclinical neuroblastoma model. EXPERIMENTAL DESIGN: We performed intervention trials of lestaurtinib alone or in combination with other agents in TrkB-overexpressing neuroblastoma xenograft models. RESULTS: Lestaurtinib alone significantly inhibited tumor growth compared to vehicle-treated animals [P = 0.0004 for tumor size and P = 0.011 for event-free survival (EFS)]. Lestaurtinib also enhanced the antitumor efficacy of the combinations of topotecan plus cyclophosphamide (P < 0.0001 for size and P < 0.0001 for EFS) or irinotecan plus temozolomide (P = 0.011 for size and P = 0.012 for EFS). There was no additive benefit of combining either 13-cis-retinoic acid or fenretinide with lestaurtinib compared to lestaurtinib alone. There was dramatic growth inhibition combining lestaurtinib with bevacizumab (P < 0.0001), but this combination had substantial systemic toxicity. CONCLUSIONS: We show that lestaurtinib can inhibit the growth of neuroblastoma both in vitro and in vivo and can substantially enhance the efficacy of conventional chemotherapy, presumably by inhibition of the Trk/brain-derived neurotrophic factor autocrine survival pathway. It may also enhance the efficacy of selected biological agents, but further testing is required to rule out unanticipated toxicities. Our data support the incorporation of Trk inhibitors, such as lestaurtinib, in clinical trials of neuroblastoma or other tumors relying on Trk signaling pathways for survival.

Acceleration of methylmercury-induced cell death of rat cerebellar neurons by brain-derived neurotrophic factor in vitro.

Brain-derived neurotrophic factor (BDNF) is a member of the nerve growth factor (NGF) family and has been shown to promote neuronal survival and contribute to neural development. Although methylmercury, a neurotoxin, induces the cell death of neurons in vitro, there is little information regarding the effects of neurotrophins on the methylmercury-induced cell death of neurons. In the present study, we investigated the effect of BDNF on methylmercury-induced cell death in a primary culture of rat cerebellar granular cells. BDNF increased the viability of the cultured cells when treated alone, but unexpectedly accelerated the cell death induced by administration of methylmercury. Among other growth factors tested, only neurotrophin-4 (NT-4) demonstrated a similar acceleration of methylmercury-induced cell death. The cell death-accelerating effect of BDNF was inhibited by a BDNF-neutralizing antibody or a MAPK inhibitor. To determine whether the effect of BDNF occurs via TrkB, a receptor of BDNF and NT-4, we investigated the effects of BDNF and methylmercury in a TrkB transformant of rat neuroblastoma B35 cells. The methylmercury-induced cell death of the TrkB transformant was accelerated by BDNF, while that of the mock transformant was not. These results indicate that BDNF accelerates methylmercury-induced cell death via TrkB, at least in vitro, and suggest that BDNF and TrkB may also contribute to the sensitivity of neurons to methylmercury toxicity.

Progesterone reverses 17beta-estradiol-mediated neuroprotection and BDNF induction in cultured hippocampal slices.

Due to the many similarities in mechanisms of action, targets and effects, progesterone (P4), estrogen and neurotrophins have been implicated in synaptic plasticity as well as in neuroprotection and neurodegeneration. In this study, we examined the interactions between 17beta-estradiol (E2) and P4 and brain-derived neurotrophic factor (BDNF) on both plasticity and excitotoxicity in rat cultured hippocampal slices. First, we evaluated the neuroprotective effects of E2 and P4 against N-methyl-D-aspartate (NMDA) toxicity in cultured rat hippocampal slices. As previously reported, pretreatment with 10 nm E2 (24 h) was neuroprotective against NMDA toxicity. However, P4 (10 nm) added 20 h after E2 treatment for 4 h reversed its protective effect. In addition, the same E2 treatment resulted in an increase in BDNF protein levels as well as in activation of its receptor, TrkB, while addition of P4 attenuated E2-mediated increase in BDNF and TrkB levels. Furthermore, E2-mediated neuroprotection was eliminated by a BDNF scavenger, TrkB-Fc. Our results indicate that E2 neuroprotective effects are mediated through the BDNF pathway and that, under certain conditions, P4 antagonizes the protective effect of estrogen.

Neurotrophin-dependent dendritic filopodial motility: a convergence on PI3K signaling.

Synapse formation requires contact between dendrites and axons. Although this process is often viewed as axon mediated, dendritic filopodia may be actively involved in mediating synaptogenic contact. Although the signaling cues underlying dendritic filopodial motility are mostly unknown, brain-derived neurotrophic factor (BDNF) increases the density of dendritic filopodia and conditional deletion of tyrosine receptor kinase B (TrkB) reduces synapse number in vivo. Here, we report that TrkB associates with dendritic growth cones and filopodia, mediates filopodial motility, and does so via the phosphoinositide 3 kinase (PI3K) pathway. We used genetic and pharmacological manipulations of mouse hippocampal neurons to assess signaling downstream of TrkB. Conditional knock-out of two downstream negative regulators of TrkB signaling, Pten (phosphatase with tensin homolog) and Nf1 (neurofibromatosis type 1), enhanced filopodial motility. This effect was PI3K-dependent and correlated with synaptic density. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) was preferentially localized in filopodia and this distribution was enhanced by BDNF application. Thus, intracellular control of filopodial dynamics converged on PI3K activation and PIP3 accumulation, a cellular paradigm conserved for chemotaxis in other cell types. Our results suggest that filopodial movement is not random, but responsive to synaptic guidance molecules.