Tyrosine Kinase Receptor B Research Articles

Mechanism of selenium-enriched Bacillus subtilis alleviating perfluorohexanoic acid toxicity in Carassius auratus through the microbiota–gut–brain axis

The extensive production and use of perfluorohexanoic acid (PFHxA) have established it as an emerging environmental pollutant on a global scale. Selenium has been shown to exert substantial antagonistic effects on harmful substances. Concurrently, probiotics may confer advantageous effects on the host's brain function by orchestrating modulation within the microbiota–gut–brain axis. However, despite the promising properties of selenium-enriched Bacillus subtilis, uncertainties persist regarding its impact on PFHxA and underlying mechanisms. This study evaluated the potential alleviating effects of selenium-enriched B. subtilis on brain damage in Carassius auratus exposed to PFHxA from the perspective of the microbiota–gut–brain axis. Supplementation with selenium-enriched B. subtilis significantly mitigated brain damage and memory loss in C. auratus. Nissl staining of the brain revealed that selenium-enriched B. subtilis mitigated neuronal damage induced by PFHxA. TUNEL staining of the intestine showed that selenium-enriched B. subtilis mitigated PFHxA-induced apoptosis, thus mitigating intestinal injury. Moreover, our study uncovered additional layers of complexity in the microbiota–gut–brain axis, as enhancements were noted in the gut microbiota. Concurrently, alterations in the expression of neurotransmitters and biomarkers associated with emotions, such as 5-hydroxytryptamine, Dopamine, Corticotropin Releasing Factor, Substance p, and ghrelin, highlighted the profound impact of selenium-enriched B. subtilis on the intricate interplay between gut health and emotional well-being. Expanding our focus to the molecular level, we found that supplementation with selenium-enriched B. subtilis further inhibited cellular apoptosis through the brain-derived neurotrophic factor (BDNF) / Tyrosine Kinase receptor B (TrKB) / Protein Kinase B (AKT) / Glycogen synthase kinase3β (GSK-3β) pathway. In conclusion, supplementation with selenium-enriched B. subtilis is a promising strategy to mitigate PFHxA-induced brain damage in C. auratus, offering a multifaceted approach through the microbiota–gut–brain axis. In addition, our study introduces the potential of selenium-enriched B. subtilis as a valuable source of micronutrients for fish, opening new avenues for developmental considerations in aquaculture practices.

Effect of adlay seed extract on the level of neuroprotection gene expression in human nasal orbital mesenchymal stem cells

IntroductionStem cells have gained attention for their potential as a promising approach for generating neurotrophins and advancing cell-based therapies for retinal degenerative diseases such as glaucoma. This study aimed to explore how adlay seed extract impacts the gene expression of key components within the neuroprotection pathway (NGF, TRKB, MAPK, PI3K) in human nasal orbital adipocytes mesenchymal stem cells (OAMSCs). MethodsNasal OAMSCs, with a density of 106 cells/ 10 cm2, were subjected to a 24-hour exposure to adlay seed extracts (namely the methanolic (MeOH) and residual (Res) fractions at a concentration of 1 mg/ml). The control group received an identical medium without the extract at the same time and under the same circumstances. We measured the relative expression levels of nerve growth factor (NGF), tyrosine receptor kinase B (TRKB), mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K) in the treated cells versus the control group using real-time polymerase chain reaction (PCR). ResultsOur data revealed that both the MeOH and Res extracts of adlay seed led to a significant upregulation of NGF in nasal OAMSCs. MeOH extract also led to the overexpression of TRKB (the gene coding for BDNF receptor) in OAMSCs, while the other genes understudy, were not altered. ConclusionsOur study highlighted initial documentation of the stimulating impact of adlay extract on the transcriptional level of neurotrophin NGF and the TRKB in nasal OAMSCs. We also showed that the extraction method could significantly affect the molecular-level properties of adlay. This preliminary study can pave the ground for future advancements in the treatment of retinal neurodegenerative disorders and glaucoma.

Administration of low dose intranasal ketamine exerts a neuroprotective effect on whole brain irradiation injury model in wistar rats.

Exposure to ionizing radiation leads to oxidative stress and neuroinflammation, resulting in neurocognitive impairments. Adverse effects are also associated with glutamate-induced excitotoxicity due to alterations in the composition of glutamate receptors. Ketamine, which is a noncompetitive NMDA glutamate receptor antagonist, has been stated to exert an impact on glutamatergic receptors. This study aims to reveal the possible alleviating or preventive effects of ketamine, which maintains glutamate homeostasis and decreases neurodegeneration, in a radiation-induced neurotoxicity model. Twenty-one female Wistar Queryrats were included in the study and 14 of these underwent whole brain irradiation (IR) with a 20 Gray single dose. Animals were allocated into three groups. Group 1: Normal control; Group 2: Placebo / IR + Saline; Group 3: IR + Ketamine. Ketamine was administered in addition to IR to rats in Group 3. The one-way ANOVA statistical test was used to compare groups. The value of p < 0.05 was considered statistically significant. When administered in addition to irradiation, ketamine treatment significantly increased scores in the three-chamber sociability test, open field test, and passive avoidance learning test. It also raised neuron counts in the hippocampal CA1 and CA3 regions as well as in Purkinje cells, and enhanced levels of brain-derived neurotrophic factor and tyrosine receptor kinase-B. Furthermore, ketamine administration resulted in decreased levels of glial fibrillary acidic protein, malondialdehyde, and tumor necrosis factor-alpha, indicating a reduction in neuroinflammation and oxidative stress. Ketamine exerted a significant protective impact on radiation-induced neurocognitive impairments and enhanced social-memory capacity by reducing neuronal loss, oxidative stress, and neuroinflammation. Our findings suggest that ketamine is beneficial in the treatment or prevention of neurodegeneration via the regulation of the BDNF/TrkB signaling pathway besides decreasing neuroinflammation and blocking NMDA receptors.

Electroacupuncture improves learning and memory impairment and enhances hippocampal synaptic plasticity through BDNF/TRKB/CREB signaling pathway in cerebral ischemia-reperfusion injury rats

To observe the effect of electroacupuncture on brain-derived neurotrophin factor (BDNF) / tyrosine kinase receptor B (TRKB) / cyclic adenosine monophosphate response element binding protein (CREB) pathway, synaptic plasticity marker protein and synaptic ultrastructure in the hippocampus of rats with learning and memory impairment induced by cerebral ischemia reperfusion (IR), so as to explore its mechanisms underlying improvement of cognitive impairment after stroke. SD rats were randomly divided into blank, sham operation, model, and EA groups, with 12 rats in each group. The model of IR was established by occlusion of the middle cerebral artery. EA (2 Hz/10 Hz, 1-3 mA) was applied to "Shenting" (GV24) and "Baihui" (GV20) for 30 min, once daily for 14 days. The neurological function was evaluated according to the Zea Longa's score criteria. Morris water maze test was used to detect the learning and memory function of the rats. Nissl staining was used to observe the pathological morphology of the hippocampus. Transmission electron microscopy was used to observe the ultrastructure of the syna-pse in the hippocampus, the synaptic gap width and postsynaptic dense substance (PSD) thickness were measured. Immunofluorescence staining was used to observe the positive expression levels of BDNF, PSD-95 and synaptophysin (SYN) in hippocampal CA1 region. The protein expression levels of BDNF, TRKB, CREB, PSD-95, and SYN in hippocampal tissue were detected by Western blot. Compared with the sham operation group, the neurological function score and escape latency (EL) were significantly increased (P<0.01), the times of crossing the original platform were decreased (P<0.01), the number of neurons in the CA1 area of the hippocampus was reduced, with incomplete morphology, widened synaptic gaps and significantly decreased PSD thickness (P<0.01), the positive expressions of BDNF, PSD-95, SYN and the protein expression levels of BDNF, TRKB, CREB, PSD-95, SYN were significantly decreased (P<0.01) in the model group. Compared with the model group, the neurological function scores and EL on the 12th and 13th day were decreased (P<0.01, P<0.05), the times of crossing the original platform were increased (P<0.01), the morphology of hippocampal CA1 neurons improved, the synaptic gaps was decreased (P<0.01), the PSD thickness was significantly increased (P<0.01), the positive expressions of BDNF, PSD-95, SYN, and the protein expression levels of BDNF, TRKB, CREB, PSD-95, SYN were increased (P<0.05, P<0.01) in the EA group. EA can alleviate cognitive impairment in IR rats, which may be related to its function in up-regulating the proteins of BDNF/TRKB/CREB pathway, promoting the expressions of synaptic plasticity marker proteins PSD-95 and SYN, thus improving the synaptic plasticity.

Chronic Cerebral Hypoperfusion-induced Dysregulations of Hyperpolarization- activated Cyclic Nucleotide-gated, KCNQ and G Protein-coupled Inwardly Rectifying Potassium Channels Correlated with Susceptibility and Unsusceptibility to Anxiety Behaviors.

Cerebrovascular lesions could induce affective disorders; however, the depression- and anxiety-related symptoms caused by Chronic Cerebral Hypoperfusion (CCH) and the roles of different Hyperpolarization-activated Cyclic Nucleotide-gated (HCN), KCNQ and G protein-coupled inwardly rectifying potassium (GirK) channel subunits in these pathological processes have been poorly elucidated so far. To investigate the behavioral change and the alteration of HCN, KCNQ, and GirK subunits in amygdale rats suffering from CCH. Permanent bilateral occlusion of the common carotid arteries was used to induce CCH. Anxiety and depression levels were assessed by the elevated plus maze test, sucrose preference test and forced swimming test to classify rats as highly anxious or depressive 'susceptibility' vs. 'unsusceptibility'. The expression of brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (TrKB), HCN1/2, KCNQ2/3, and GirK1/2/3 were quantified by Western blotting. The main emotional change caused by 4 weeks of CCH is likely to be anxiety-like behavior (50%), accompanied by a down-regulation of BDNF and TrKB expression in amygdale. The increase of HCN1 and decrease of KCNQ3 expression in amygdale may be factors to blame for anxiety- like symptom caused by CCH, and the increase of KCNQ2 and Girk1 expression in amygdale may play a role in resilience to the anxiety induced by CCH. The different subunits of HCN, KCNQ and GirK channels in amygdale may contribute to distinct response to aversive stimuli or stress induced by CCH that evokes divergent influences on anxiety-like behavior in rats.

Effects of erythropoietin pretreatment on single dose pentylentetrazole-induced seizures in rats

ABSTRACT Although it is accepted that prolonged and repeated seizures can cause epileptogenesis, memory deficits and neuronal death, the precise relation between epileptic seizures and neuronal death remains unclear. Erythropoietin (EPO) exhibits neuroprotective and anti-epileptic effects. We investigated the effect of a single pentylentetrazole (PTZ) induced tonic-clonic seizure on the pyramidal neurons of the cornu ammonis 1 (CA1) and CA3 regions of hippocampus. We also investigated the effects of EPO on seizure, memory and on brain-derived neurotrophic factor (BDNF), tyrosine receptor kinase-B, sirtuin-1 (SIRT1), which are important for memory. Forty male rats were divided into four groups: control, saline treated, single 60 mg/kg dose PTZ treated, 3000 IU/kg EPO treated, and 3000 IU/kg EPO treated 24 h before PTZ administration. Seizure latency and severity were assessed following PTZ injection. A passive avoidance test was performed 24 h after seizure. BDNF, TrkB and SIRT1 levels were measured in serum, hippocampus and cortex. The hippocampus was examined histologically, and neuronal nuclear antigen (NeuN) was investigated using immunohistochemistry. EPO pretreatment decreased seizure severity and prolonged seizure latency. Single dose PTZ-induced seizures did not affect memory. Numbers of cells in the CA1 region did not change, although the number of dark stained neuron increased. Both total cell numbers and percentage of dark stained cells were elevated in the CA3 region following PTZ induced seizures. EPO pretreatment decreased the number of dark cells in both CA1 and CA3 regions and the number of cells in the CA3 region. NeuN labeling was unchanged in the CA1 and CA3 regions in the PTZ group; however, EPO pretreatment increased NeuN labeling in the CA3 region. Although EPO exhibited an anticonvulsive effect, single dose EPO pretreatment did not affect memory in either animals not exposed to PTZ or animals that had been subjected to PTZ-induced seizures. EPO pretreatment prolonged seizure latency and reduced seizure severity after PTZ-induced seizures. The anti-seizure and neuroprotective effects of EPO pretreatment may be due to the protection of CA1 and CA3 neurons, possibly owing to SIRT1 and BDNF activity.

Early intervention with gastrodin reduces striatal neurotoxicity in adult rats with experimentally‑induced diabetes mellitus.

Glutamate-induced excitotoxicity in the striatum has an important role in neurodegenerative diseases. It has been reported that diabetes mellitus (DM) induces excitotoxicity in striatal neurons, although the underlying mechanism remains to be fully elucidated. The present study aimed to investigate the effect of gastrodin on DM-induced excitotoxicity in the striatal neurons of diabetic rats. Adult Sprague-Dawley rats were divided into control, diabetic, and gastrodin intervention groups. Diabetes in the rats was induced with a single intraperitoneal injection of streptozotocin (65 mg/kg). In the gastrodin groups, the rats were gavaged with 60 or 120 mg/kg/day gastrodin for 6 weeks, 3 weeks following the induction of diabetes. Pathological alterations in the striatum were assessed using hematoxylin and eosin (H&E) staining. The protein expression levels of phosphorylated (p)-extracellular signal-regulated kinase (ERK)1/2, p-mitogen-activated protein kinase kinase (MEK)1/2, tyrosine receptor kinase B (TrKB) and brain-derived neurotrophic factor (BDNF) in the striatal neurons were evaluated by western blotting and double immunofluorescence. Additionally, the extracellular levels of glutamate were measured by microanalysis followed by high-pressure-liquid-chromatography. In diabetic rats, striatal neuronal degeneration was evident following H&E staining, which revealed the common occurrence of pyknotic nuclei. This was coupled with an increase in glutamate levels in the striatal tissues. The protein expression levels of p-ERK1/2, p-MEK1/2, TrKB and BDNF in the striatal tissues were significantly increased in the diabetic rats compared with those in the normal rats. In the gastrodin groups, degeneration of the striatal neurons was ameliorated. Furthermore, the expression levels of glutamate, p-ERK1/2, p-MEK1/2, TrKB and BDNF in the striatal neurons were decreased. From these findings, it was concluded that reduced neurotoxicity in striatal neurons following treatment with gastrodin may be attributed to its suppressive effects on the expression of p-ERK1/2, p-MEK1/2, BDNF and TrKB.

Dexamethasone protects against arsanilic acid‑induced rat vestibular dysfunction through the BDNF and JNK 1/2 signaling pathways.

The brain‑derived neurotrophic factor (BDNF) and c‑Jun NH 2‑terminal kinase (JNK) signaling pathways are therapeutic targets to prevent degeneration in the central nervous system. Dexamethasone (DXMS), a glucocorticoid, protects against vestibular brain injury, however, the molecular mechanisms have yet to be fully elucidated. To investigate whether the BDNF and JNK signaling pathways are involved in the protective effects of DXMS in rats with vestibular dysfunction, a rat model of severe vestibular deficits was established by middle ear injection of arsanilic acid (AA; 100 mg/ml; 0.05ml). After 3days, rat symptoms and behavior scores with vestibular disorders were detected. In brain tissues, histopathological alterations, cell apoptosis, expression levels and patterns of BDNF signaling pathway‑associated BDNF, tyrosine receptor kinase B (TrKB) and K+/Cl‑ cotransporter isoform 2 (KCC2), and the expression of apoptosis‑related cleaved‑caspase 3 and the JNK signaling pathway were detected. It was identified that DXMS relieved AA‑induced vestibular dysfunction, leading to improvement in rat behavior scores to normal levels, minimizing brain damage at the histopatholojnnkngical level, reducing cell apoptosis, enhancing the expression of BDNF, TrKB and KCC2, and downregulating cleaved‑caspase 3 and phosphorylated‑JNK1/2 in brain tissues. Together, these findings indicated the protective effect of DXMS on AA‑induced rat vestibular dysfunction, and that activating BDNF and inhibiting JNK singling pathways were the underlying mechanisms. In addition, with additional treatment of mifepristone (RU486), a specific glucocorticoid agonist, all the events elicited by DXMS mentioned above in the AA‑treated rat rats were reversed. In conclusion, DXMS was identified as a therapeutic agent targeting the BDNF and JNK singling pathways for AA‑induced rat vestibular dysfunction.

Docosahexaenoic Acid Helps to Lessen Extinction Memory in Rats.

Memory extinction is referred to as a learning process in which a conditioned response (CR) progressively reduces over time as an animal learns to uncouple a response from a stimulus. Extinction occurs when the rat is placed into a context without shock after training. Docosahexaenoic acid (DHA, C22:6, n-3) is implicated in memory formation in mammalian brains. In a two-way active shuttle-avoidance apparatus, we examined whether DHA affects the extinction memory and the expression of brain cognition-related proteins, including gastrin-releasing peptide receptor (GRPR), brain-derived neurotrophic factor receptor (BDNFR) tyrosine kinase receptor B (TrKB), and N-methyl-d-aspartate receptor (NMDAR) subunits NR2A and NR2B. Also, the protein levels of GRP, BDNF, postsynaptic density protein-95 (PSD-95), and vesicular acetylcholine transporter (VAChT), and the antioxidative potentials, in terms of lipid peroxide (LPO) and reactive oxygen species (ROS), were examined in the hippocampus. During the acquisition phase, the rats received a conditioned stimulus (CS-tone) paired with an unconditioned stimulus (UCS foot shock) for three consecutive days (Sessions S1, S2, and S3, each consisting of 30-trials) after 12 weeks of oral administration of DHA. After a three-day interval, the rats were re-subjected to two extinction sessions (S4, S5), each comprising 30 trials of CS alone. During the acquisition training in S1, the shock-related avoidance frequency (acquisition memory) was significantly higher in the DHA-administered rats compared with the control rats. The avoidance frequency, however, decreased with successive acquisition trainings in sessions S2 and S3. When the rats were subjected to the extinction sessions after a break for consolidation, the conditioned response (CR) was also significantly higher in the DHA-administered rats. Interestingly, the freezing responses (frequency and time) also significantly decreased in the DHA-administered rats, thus suggesting that a higher coping capacity was present during fear stress in the DHA-administered rats. DHA treatments increased the mRNA levels of GRPR, BDNF receptor TrKB, and NMDAR subunit NR2B. DHA also increased the protein levels of GRP, BDNF, PSD-95, and VAChT, and the antioxidative potentials in the hippocampus. These results suggest the usefulness of DHA for treating stress disorders.

TrkB is necessary for male copulatory behavior in the Syrian Hamster (Mesocricetus auratus)

The magnocellular medial preoptic nucleus (MPN mag), a subdivision of the medial preoptic area (MPOA), plays a critical role in the regulation of copulation in the male Syrian hamster; in part by mediating the effects of gonadal steroids. For example, ablation of the MPN mag eliminates mating and testosterone placed in the MPN mag restores mating in castrated males. Furthermore, testosterone treatment enhances synaptic density and dendritic spines in the MPN mag. Thus, copulatory behaviors are correlated with increases in synaptic morphology in the MPN mag. As brain derived neurotrophic factor (BDNF) and its receptor, tyrosine receptor kinase-B (TrkB), effect neuronal growth and synaptic plasticity, this study explored the role of TrkB and BDNF in mediating testosterone's effects on the MPN mag and behavior. Testosterone treatment increased BDNF expression and conversely lowered TrkB expression in the MPOA. siRNA-mediated TrkB knockdown in the MPN mag eliminated copulation two-days post injection and the behavior was restored one week later. These data indicate that testosterone influences the expression of BDNF and TrkB in the MPOA and that expression of copulation is dependent on the presence of TrkB. Taken together our findings support a role for TrkB and BDNF in mediating the effects of testosterone on copulatory behavior in the Syrian hamster.

Sexual dimorphic expression of TrkB, TrkB-T1, and BDNF in the medial preoptic area of the Syrian hamster

Neurotrophins regulate many aspects of neuronal function and activity. Specifically, the binding of Brain-derived neurotrophic factor (BDNF) to Tyrosine receptor kinase-B (TrkB) or its truncated version, TrkB-T1, can cause growth and differentiation or dominant inhibition of receptor signaling, respectively. There is evidence that these neurotropic effects on nervous tissue, in both the central and peripheral nervous system, behave differently between the sexes. This study used western blots to examine the expression of these neurotrophins in the medial preoptic area (MPOA), a sexually dimorphic region of the hamster brain that controls male sex behavior. We report that TrkB-FL and BDNF show greater expression in male MPOA tissue, when compared to female. On the contrary, TrkB-T1 is expressed in greater abundance in the female MPOA. Our results indicate a clear sexual dimorphism of neurotrophins in the MPOA of the Syrian hamster. Furthermore, the greater expression of TrkB-FL and BDNF in the male MPOA suggests that these neurotrophins could be promoting synaptic growth to facilitate male-typical copulation. In contrast, the greater TrkB-T1 expression in the female MPOA suggests a possible inhibition of synaptic growth, and may contribute to the lack of male-typical copulation. Altogether, our data suggests that neurotrophins may play a larger role sexual differentiation than previously thought.

Expression pattern and function of tyrosine receptor kinase B isoforms in rat mesenteric arterial smooth muscle cells

Tyrosine receptor kinaseB (TrkB) is a high affinity receptor for brain-derived neurotrophic factor (BDNF). TrkB isoforms involve full length TrkB (TrkB FL) and truncated TrkB type1 (TrkB T1) and type 2 (TrkB T2) in rats. The aim of present study was to explore their expression pattern and function in mesenteric arterial smooth muscle cells (MASMCs). The expression of TrkB isoform protein and mRNA was examined by Western blotting, immunofluorescence and quantitative RT-PCR analyses. Cell proliferation was measured by a bromodeoxyuridine (BrdU) incorporation assay. Cell migration was measured by a Boyden chamber assay. Cell morphology was observed with a phase-contrast microscope. Protein and mRNA expression of BDNF and TrkB isoforms was confirmed in MASMCs. Expression level of TrkB FL was less, while that of TrkB T1 was the highest in MASMCs. Although BDNF increased phosphorylation of ERK, it had no influence on migration and proliferation of MASMCs. TrkB T1 gene knockdown by a RNA interference induced morphological changes and reduced expression level of α-smooth muscle actin (α-SMA) in MASMCs. Similar morphological changes and reduced α-SMA expression were induced in MASMCs by a Rho kinase inhibitor, Y-27632. In conclusion, we for the first time demonstrate that TrkB T1 expressed highly in MASMCs contributes to maintain normal cell morphology possibly via regulation of Rho activity. This study firstly defined expression level of TrkB isoforms and partly revealed their functions in peripheral vascular cells.

Analysis of the Expressions of TrkB and GLT-1 in Brain Tissues on a Rat Pentylenetetrazol Kindling Model of Chronic Epilepsy.

Tyrosine kinase receptor-B (TrkB), a high-affinity receptor for brain-derived neurotrophic factor (BDNF), functions via specific binding with the BDNF. Although studies have shown that BDNF promotes the expression of glutamate transporter-1 (GLT-1) in the glial cells during neurodegeneration, evidence is lacking regarding whether BDNF/TrkB pathway is associated with the abnormal function of GLT-1.The aim of this study is to analyze the expressions of TrkB and GLT-1 in the brain of pentylenetetrazol (PTZ) kindling model of chronic epilepsy in rats, and to provide evidence for the correlation between the BDNF/TrkB pathway and GLT-1 and clues for the prevention and treatment targets of epilepsy. In total, 40 Sprague-Dawley rats were randomly classified as model (n= 30) and control (n= 10) groups respectively. Western blotting was used for analyzing the expression of TrkB and GLT-1, and double immunofluorescence staining was used for detecting the cells positive for both glial fibrillary acidic protein (GFAP) and TrkB in the hippocampus and temporal cortex 7 days after establishing the kindling model. Compared with the control group, protein level expression of TrkB and GLT-1, and mean optical density (OD) value of the cells positive for both GFAP and TrkB in the hippocampus and temporal cortex were significantly increased after the onset of epilepsy (P < 0.05). The BDNF/TrkB pathway may participate in the epileptogenesis through modulating the biological effect of GLT-1 in the glial cells of PTZ kindling rats, though the specific mechanisms require further investigations.

Agonistic TAM-163 antibody targeting tyrosine kinase receptor-B

TAM-163, an agonist monoclonal antibody targeting tyrosine receptor kinase-B (TrkB), is currently being investigated as a potential body weight modulatory agent in humans. To support the selection of the dose range for the first-in-human (FIH) trial of TAM-163, we conducted a mechanistic analysis of the pharmacokinetic (PK) and pharmacodynamic (PD) data (e.g., body weight gain) obtained in lean cynomolgus and obese rhesus monkeys following single doses ranging from 0.3 to 60 mg/kg. A target-mediated drug disposition (TMDD) model was used to describe the observed nonlinear PK and Emax approach was used to describe the observed dose-dependent PD effect. The TMDD model development was supported by the experimental determination of the binding affinity constant (9.4 nM) and internalization rate of the drug-target complex (2.08 h−1). These mechanistic analyses enabled linking of exposure, target (TrkB) coverage, and pharmacological activity (e.g., PD) in monkeys, and indicated that ≥ 38% target coverage (time-average) was required to achieve significant body weight gain in monkeys. Based on the scaling of the TMDD model from monkeys to humans and assuming similar relationship between the target coverage and pharmacological activity between monkey and humans, subcutaneous (SC) doses of 1 and 15 mg/kg in humans were projected to be the minimally and the fully pharmacologically active doses, respectively. Based on the minimal anticipated biological effect level (MABEL) approach for starting dose selection, the dose of 0.05 mg/kg (3 mg for a 60 kg human) SC was recommended as the starting dose for FIH trials, because at this dose level < 10% target coverage was projected at Cmax (and all other time points). This study illustrates a rational mechanistic approach for the selection of FIH dose range for a therapeutic protein with a complex model of action.

Combination treatment with progesterone and vitamin D hormone is more effective than monotherapy in ischemic stroke: The role of BDNF/TrkB/Erk1/2 signaling in neuroprotection

We investigated whether combinatorial post-injury treatment with progesterone (P4) and vitamin D hormone (VDH) would reduce ischemic injury more effectively than P4 alone in an oxygen glucose deprivation (OGD) model in primary cortical neurons and in a transient middle cerebral artery occlusion (tMCAO) model in rats. In the OGD model, P4 and VDH each showed neuroprotection individually, but combination of the “best” doses did not show substantial efficacy; instead, the lower dose of VDH in combination with P4 was the most effective. In the tMCAO model, P4 and VDH were given alone or in combination at different times post-occlusion for 7 days. In vivo data confirmed the in vitro findings and showed better infarct reduction at day 7 and functional outcomes (at 3, 5 and 7 days post-occlusion) after combinatorial treatment than when either agent was given alone. VDH, but not P4, upregulated heme oxygenase-1, suggesting a pathway for the neuroprotective effects of VDH differing from that of P4. The combination of P4 and VDH activated brain-derived neurotrophic factor and its specific receptor, tyrosine kinase receptor-B. Under specific conditions VDH potentiates P4's neuroprotective efficacy and should be considered as a potential partner of P4 in a low-cost, safe and effective combinatorial treatment for stroke.

The effect of exercise and oxidant–antioxidant intervention on the levels of neurotrophins and free radicals in spinal cord of rats

This study was designed to investigate the effects of oxidant and antioxidant treatment, as well as regular exercise, on neurotrophin levels in the spinal cord of rats. Reactive oxygen species (ROS) play a role in neurodegenerative diseases, but ROS at moderate levels could stimulate biochemical processes through redox-sensitive transcription. Exercised or sedentary animals were injected subcutaneously with hydrogen peroxide (H(2)O(2)), N-tert butyl-alpha-phenyl nitrone (PBN) or saline for the last 2 weeks of a 10-week experimental period to challenge redox balance. Free radical (FR) concentration was evaluated in the spinal cord by electron spin resonance, protein carbonyls, brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) levels and the mRNA expression of BDNF receptor and tyrosine kinase receptor B (TrKB). Research Institute of Sport Science, Semmelweis University, Budapest, Hungary. Exercise or PBN decreased the concentration of FR, whereas the carbonyl content did not change. BDNF was significantly decreased in exercised sham and sedentary PBN-treated groups, and its content correlated with the level of FR. GDNF was significantly increased in sedentary H(2)O(2)-treated groups. No differences were observed in TrkB mRNA expression among groups. Results suggest that regular exercise alone and PBN in sedentary animals can successfully decrease FR levels in the spinal cord. Redox alteration seems to affect the levels of GDNF and BDNF, which might have clinical consequences, as neurotrophins play an important role in cellular resistance and regeneration.

587 POSTER Understanding the role of Raf signaling in B-Raf V600E mutant versus wildtype tumors

To investigate whether genes that had been found to be differentially expressed in deep-infiltrating endometriosis and matched eutopic endometrium in our previous complementary DNA microarray study also are differentially expressed in ovarian endometriosis and matched eutopic endometrium.Prospective study.University hospital in France.Patients with ovarian endometriosis.During surgery, paired samples of tissue representing ovarian endometriosis and eutopic endometrium were obtained from 12 patients.Expression levels of messenger RNA for heat shock protein 90 alpha (HSP90A), chicken ovalbumin upstream promoter transcription factor 2 (COUP-TF2), prostaglandin E2 receptor subtype EP3 (PGE2EP3), tyrosine kinase receptor B (TrKB), and 17β-hydroxysteroid dehydrogenase type 2 (17βHSD2; epithelial cells) and of platelet-derived growth factor receptor alpha (PDGFRA), protein kinase C beta 1 (PKCβ1), Janus kinase 1 (JAK1), mitogen-activated protein kinase kinase (MKK7), Sprouty2, μ-opioid receptor (MOR), and 5HTT (stromal cells) from ovarian endometriosis and matched eutopic endometrium were determined by using laser capture microdissection and real-time reverse-transcription polymerase chain reaction (RT-PCR) techniques.Expression of PDGFRA, PKCβ1, JAK1, HSP90A, COUP-TF2, MOR, and 17βHSD2 was significantly higher in ovarian endometriosis than in eutopic endometrium, whereas that of Sprouty2 and PGE2EP3 was significantly lower. There was no significant difference in mitochondrial RNA expression of MKK 7, TrKB, and 5HTT.Ovarian endometriosis might share several common molecules with deep-infiltrating endometriosis that act to sustain endometriotic lesions, whereas molecules involved in local endocrine control might be different between these two types of endometriosis.

Developmental changes in the BDNF‐induced modulation of inhibitory synaptic transmission in the Kölliker–Fuse nucleus of rat

The Kölliker-Fuse nucleus (KF), part of the pontine respiratory group, is involved in the control of respiratory phase duration, and receives both excitatory and inhibitory afferent input from various other brain regions. There is evidence for developmental changes in the modulation of excitatory inputs to the KF by the neurotrophin brain-derived neurotrophic factor (BDNF). In the present study we investigated if BDNF exerts developmental effects on inhibitory synaptic transmission in the KF. Recordings of inhibitory postsynaptic currents (IPSCs) in KF neurons in a pontine slice preparation revealed general developmental changes. Recording of spontaneous and evoked IPSCs (sIPSCs, eIPSCS) revealed that neonatally the gamma-aminobutyric acid (GABA)ergic fraction of IPSCs was predominant, while in later developmental stages glycinergic neurotransmission significantly increased. Bath-application of BDNF significantly reduced sIPSC frequency in all developmental stages, while BDNF-mediated modulation on eIPSCs showed developmental differences. The eIPSCs mean amplitude was uniformly and significantly reduced following BDNF application only in neurons from rats younger than postnatal day 10. At later postnatal stages the response pattern became heterogeneous, and both augmentations and reductions of eIPSC amplitudes occurred. All BDNF effects on eIPSCs and sIPSCs were reversed with the tyrosine kinase receptor-B inhibitor K252a. We conclude that developmental changes in inhibitory neurotransmission, including the BDNF-mediated modulation of eIPSCs, relate to the postnatal maturation of the KF. The changes in BDNF-mediated modulation of IPSCs in the KF may have strong implications for developmental changes in synaptic plasticity and the adaptation of the breathing pattern to afferent inputs.

Both GnRH agonist and continuous oral progestin treatments reduce the expression of the tyrosine kinase receptor B and mu-opioid receptor in deep infiltrating endometriosis

Deep infiltrating endometriosis (DIE) is commonly associated with severe pain. The pain can be managed successfully with GnRH agonists or continuous progestins. The precise molecular mechanism by which DIE causes pain or why hormonal treatment is effective, however, remains unclear. We recently identified three potential candidate genes that might be involved in DIE pain pathways: tyrosine kinase receptor B (TrKB), mu-opioid receptor (MOR) and serotonin transporter (5HTT). We hypothesized that if these three genes were involved in DIE-associated pain, their expression levels would probably be modulated by GnRH agonist or progestin. In this study, we compared mRNA expression levels of TrKB, MOR and 5HTT in DIE among patients pre-operatively treated with GnRH agonist, progestin or without pre-operative medical treatments. The expression levels of TrKB, MOR and 5HTT mRNA in DIE were determined using laser capture microdissection and real-time RT-PCR techniques. The expression levels of TrKB in epithelial cells and MOR in stromal cells from DIE were significantly decreased in patients with pre-operative GnRH agonist or progestin. There was no significant difference in 5HTT expression levels among untreated, GnRH agonist- and progestin-treated patients. The expression levels of TrKB and MOR genes in DIE appeared to be modulated by GnRH agonist or progestin. However, the functional roles of TrKB and MOR in DIE remain to be clarified.

Differential expression of genes in eutopic and ectopic endometrium from patients with ovarian endometriosis

To investigate whether genes that had been found to be differentially expressed in deep-infiltrating endometriosis and matched eutopic endometrium in our previous complementary DNA microarray study also are differentially expressed in ovarian endometriosis and matched eutopic endometrium. Prospective study. University hospital in France. Patients with ovarian endometriosis. During surgery, paired samples of tissue representing ovarian endometriosis and eutopic endometrium were obtained from 12 patients. Expression levels of messenger RNA for heat shock protein 90 alpha (HSP90A), chicken ovalbumin upstream promoter transcription factor 2 (COUP-TF2), prostaglandin E(2) receptor subtype EP3 (PGE(2)EP3), tyrosine kinase receptor B (TrKB), and 17beta-hydroxysteroid dehydrogenase type 2 (17betaHSD2; epithelial cells) and of platelet-derived growth factor receptor alpha (PDGFRA), protein kinase C beta 1 (PKCbeta1), Janus kinase 1 (JAK1), mitogen-activated protein kinase kinase (MKK7), Sprouty2, mu-opioid receptor (MOR), and 5HTT (stromal cells) from ovarian endometriosis and matched eutopic endometrium were determined by using laser capture microdissection and real-time reverse-transcription polymerase chain reaction (RT-PCR) techniques. Expression of PDGFRA, PKCbeta1, JAK1, HSP90A, COUP-TF2, MOR, and 17betaHSD2 was significantly higher in ovarian endometriosis than in eutopic endometrium, whereas that of Sprouty2 and PGE(2)EP3 was significantly lower. There was no significant difference in mitochondrial RNA expression of MKK 7, TrKB, and 5HTT. Ovarian endometriosis might share several common molecules with deep-infiltrating endometriosis that act to sustain endometriotic lesions, whereas molecules involved in local endocrine control might be different between these two types of endometriosis.