Induction of tyrosine kinase receptor b by retinoic acid allows brain-derived neurotrophic factor-induced amyloid precursor protein gene expression in human SH-SY5Y neuroblastoma cells

Retinoic acid (RA) is a potent regulator of morphogenesis, growth and cell differentiation. Incubation with RA causes arrest of proliferation and neurite extension in SH-SY5Y cells, a neuroblastoma cell line of human origin. In these cells, RA regulates the expression of the β-amyloid precursor protein. The retinoid increases the levels of intracellular and secreted forms of APP (amyloid precursor protein), APP–mRNA levels and the activity of the APP promoter in transient transfection studies. These responses require long periods of exposition to the ligand, thus suggesting a nondirect effect of the RA receptors on the APP gene. Also in these cells, RA induces the expression of TrkB, the tyrosine kinase receptor for brain-derived neurotrophic factor (BDNF), and 4 days of pretreatment with retinoic acid confers BDNF responsiveness to the APP promoter.     

大鼠体神经-内脏神经吻合后脑源性神经营养因子及其受体的表达变化

为了研究大鼠体神经-内脏神经吻合后脑源性神经营养因子(BDNF)及其受体酪氨酸激酶B(TrkB)在脊髓前角神经元中的表达变化,以正常大鼠为对照,运用RT-PCR技术检测大鼠体神经-内脏神经吻合术后不同时间脊髓腰4(L4)前角神经元中BDNF及TrkB mRNA的表达变化。结果显示,在正常大鼠L4前角神经元中,BDNF及TrkB的mRNA均存在一定水平的表达;体神经-内脏神经吻合术后7d、14d、1m和2m,BDNF和TrkBm RNA的表达均升高,术后7d达到高峰,14d开始下降;2m时BD-NF mRNA的表达量仍显著高于正常组(P<0.01),而TrkB mRNA的表达量到2m时虽仍高,但与正常组相比,其差异已无统计学意义(P>0.05)。上述结果表明,大鼠体神经-内脏神经反射弧建立后,脊髓L4前角神经元的内源性BDNF及其受体TrkB的表达均增高,它们可能作为保护性因子有利于受损神经元的存活和再生。     

基于BDNF/TrkB/p-CREB信号通路探讨血府逐瘀胶囊促进神经再生防治卒中后抑郁的作用及机制

目的 探讨血府逐瘀胶囊对卒中后抑郁（post-stroke depression,PSD）大鼠抑郁症状的改善作用,并基于神经再生关键通路脑源性神经营养因子（brain derived neurotrophic factor,BDNF）/酪氨酸激酶受体B(tyrosine kinase receptor,TrkB)/磷酸化环腺苷酸应答元件结合蛋白（phosphorylated cAMP response element-binding protein,p-CREB）探讨其机制。方法 采用短暂大脑中动脉梗塞（transient middle cerebral artery occlusion,tMCAO）复合慢性不可预知应激（chronic unpredictable mild stimulation,CUMS）方法复制PSD大鼠模型。大鼠随机分为假手术组、模型组及血府逐瘀低、高剂量（0.43、0.86 g/kg）组和氟西汀（1.8 mg/kg）组,每组18只。连续给予药物干预28 d,利用神经功能评分、糖水偏好实验、旷场实验、强迫游泳实验考察血府逐瘀胶囊对PSD大鼠神经功能损伤以及抑郁症...     

Effect of high-BDNF microenvironment stem cells therapy on neurogenic bladder model in rats

BackgroundThe purpose of this study is to explore the effects of high-BDNF microenvironment produced by engineered immortalized mesenchymal stem cells (imMSCs) on the neurogenic bladder (NB) and investigate underlying mechanism.MethodsMale Sprague-Dawley rat (12-week-old, weighing about 370–400 g) were purchased from a Korean company (Orient Bio Co. Seongnam, Korea) and divided into the following groups (n=32): sham control group (n=8), NB group (n=8), NB + ImMSCs group (n=8), NB + ImMSCs (BDNF) group (n=8). The major pelvic ganglion (MPG) was observed under anesthesia. Three NB groups of rats were then subjected to bilateral MPG injury. The sham control group of rats was treated with sham surgery. Cystometry were performed before the rats were sacrificed, and then MPG and bladder were collected for histochemical and Western blot analysis.ResultsMSCs treatment improves lower urinary tract function, and the NB + ImMSCs (BDNF) group is better than the NB + ImMSCs group (P<0.01). MSCs treatment accelerates recovery of injured nerve tissue, and the NB + ImMSCs (BDNF) group is better than the NB + ImMSCs group (P<0.01). In high BDNF environment, apoptosis was reduced more significantly and muscle tissue recovered more rapidly (P<0.01). High-BDNF microenvironment activates more BDNF/TrkB/CREB signaling pathways (P<0.01).ConclusionsIn a rat NB model caused by nerve injury, imMSCs have certain effects on nerve tissue repair. At the same time, it was proved that increasing the expression of BDNF which had specific effect on nerve injury repair could more effectively repair injured MPG in local microenvironment. The mechanism may be related to the activation of the BDNF/TrkB/CREB signaling pathway and the reduction of apoptosis by highly expressed BDNF.     

Brain-derived neurotrophic factor mediates neuroprotection against Aβ-induced toxicity through a mechanism independent on adenosine 2A receptor activation

Abstract

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival through TrkB-FL activation. The activation of adenosine A_2A receptors (A_2AR) is essential for most of BDNF-mediated synaptic actions, such as synaptic plasticity, transmission and neurotransmitter release. We now aimed at evaluating the A_2AR influence upon BDNF-mediated neuroprotection against Aβ_25–35 toxicity in cultured neurons. Results showed that BDNF increases cell survival and reduces the caspase-3 and calpain activation induced by amyloid-β (Aβ) peptide, in a mechanism probably dependent on PLCγ pathway. This BDNF-mediated neuroprotection is not affected by A_2AR activation or inhibition. Moreover neither activation nor inhibition of A_2AR, per se, significantly influenced Aβ-induced neuronal death on calpain-mediated cleavage of TrkB induced by Aβ. In conclusion, these results suggest that, in opposition to the fast synaptic actions of BDNF, the neuroprotective actions of this neurotrophin against a strong Aβ insult do not require the activation of A_2AR.     

Presenilin mediates neuroprotective functions of ephrinB and brain-derived neurotrophic factor and regulates ligand-induced internalization and metabolism of EphB2 and TrkB receptors

Activation of EphB receptors by ephrinB (efnB) ligands on neuronal cell surface regulates important functions, including neurite outgrowth, axonal guidance, and synaptic plasticity. Here, we show that efnB rescues primary cortical neuronal cultures from necrotic cell death induced by glutamate excitotoxicity and that this function depends on EphB receptors. Importantly, the neuroprotective function of the efnB/EphB system depends on presenilin 1 (PS1), a protein that plays crucial roles in Alzheimer's disease (AD) neurodegeneration. Furthermore, absence of one PS1 allele results in significantly decreased neuroprotection, indicating that both PS1 alleles are necessary for full expression of the neuroprotective activity of the efnB/EphB system. We also show that the ability of brain-derived neurotrophic factor (BDNF) to protect neuronal cultures from glutamate-induced cell death depends on PS1. Neuroprotective functions of both efnB and BDNF, however, were independent of γ-secretase activity. Absence of PS1 decreases cell surface expression of neuronal TrkB and EphB2 without affecting total cellular levels of the receptors. Furthermore, PS1-knockout neurons show defective ligand-dependent internalization and decreased ligand-induced degradation of TrkB and Eph receptors. Our data show that PS1 mediates the neuroprotective activities of efnB and BDNF against excitotoxicity and regulates surface expression and ligand-induced metabolism of their cognate receptors. Together, our observations indicate that PS1 promotes neuronal survival by regulating neuroprotective functions of ligand-receptor systems.     

Discrete TrkB-expressing neurons of the dorsomedial hypothalamus regulate feeding and thermogenesis

Mutations in the TrkB neurotrophin receptor lead to profound obesity in humans, and expression of TrkB in the dorsomedial hypothalamus (DMH) is critical for maintaining energy homeostasis. However, the functional implications of TrkB-fexpressing neurons in the DMH (DMH^TrkB) on energy expenditure are unclear. Additionally, the neurocircuitry underlying the effect of DMH^TrkB neurons on energy homeostasis has not been explored. In this study, we show that activation of DMH^TrkB neurons leads to a robust increase in adaptive thermogenesis and energy expenditure without altering heart rate or blood pressure, while silencing DMH^TrkB neurons impairs thermogenesis. Furthermore, we reveal neuroanatomically and functionally distinct populations of DMH^TrkB neurons that regulate food intake or thermogenesis. Activation of DMH^TrkB neurons projecting to the raphe pallidus (RPa) stimulates thermogenesis and increased energy expenditure, whereas DMH^TrkB neurons that send collaterals to the paraventricular hypothalamus (PVH) and preoptic area (POA) inhibit feeding. Together, our findings provide evidence that DMH^TrkB neuronal activity plays an important role in regulating energy expenditure and delineate distinct neurocircuits that underly the separate effects of DMH^TrkB neuronal activity on food intake and thermogenesis.

Impairments in energy homeostasis resulting from the compound effects of overeating and sedentary lifestyles have led to a profound increase in the rate of obesity around the world (1). Therapeutic strategies aimed at combating obesity by increasing energy expenditure or decreasing appetite have commonly failed due to counterregulatory mechanisms (2) and adverse side effects on cardiovascular physiology (3–5). To achieve safe and sustained weight loss, it will be essential to understand the mechanisms that govern and coordinate discrete physiological processes that contribute to energy homeostasis.Adaptive thermogenesis is the process by which energy is converted into heat and occurs primarily in brown adipose tissue (BAT) in response to environmental cues (6). BAT has a particularly high capacity for dissipating energy from fat and thus represents an important component of energy homeostasis. The dorsomedial hypothalamus (DMH) in the brain is centrally positioned in an established thermoregulatory neurocircuit, receiving inputs from the preoptic area (POA) (7–9) and sending excitatory projections to preautonomic neurons in the raphe pallidus (RPa) (10–13) that promote sympathetic activity in BAT, leading to increased thermogenesis. Direct chemical stimulation of the DMH (14) or activation of select populations of thermogenic DMH neurons (9, 11, 12, 15) leads to increased body temperature and energy expenditure but also significantly increases heart rate and blood pressure (12, 13, 15, 16). An inability to target increased sympathetic tone specifically in BAT without affecting other target tissues has greatly hampered strategies to treat obesity by targeting thermogenesis (4, 5).In addition to its influence on energy expenditure, the DMH also represents an important brain region in the regulation of feeding (17–19). Lesioning studies support an orexigenic role for the DMH (17), which can promote food intake through inhibitory projections to either the paraventricular hypothalamus (PVH) (18) or the arcuate nucleus (ARC) (20). Despite these early findings, evidence has also emerged that demonstrates the importance of anorexigenic populations of DMH neurons (19, 21, 22). We previously established that the activity of DMH neurons expressing the neurotrophin receptor TrkB (DMH^TrkB) is important for regulating feeding, showing that activation of DMH^TrkB neurons suppresses feeding and that deletion of the TrkB-encoding Ntrk2 gene in the DMH results in hyperphagia and obesity (21). Furthermore, humans with mutations in the TrkB-encoding NTRK2 gene exhibit severe obesity and impaired thermoregulation (23). However, it is unclear whether activation of DMH^TrkB neurons has a direct influence on adaptive thermogenesis. Additionally, the neurocircuitry through which DMH^TrkB neurons govern feeding or energy expenditure is unknown.Here, we demonstrate that DMH^TrkB neuronal activity potently promotes energy expenditure by elevating thermogenesis and physical activity with a notable lack of influence on heart rate and blood pressure. We further reveal that DMH^TrkB neurons send diverging projections to the RPa or the POA and PVH to differentially regulate energy expenditure and food intake, respectively.     

Brain‐derived neurotrophic factor but not vesicular zinc promotes TrkB activation within mossy fibers of mouse hippocampus in vivo

The neurotrophin receptor, TrkB receptor tyrosine kinase, is critical to central nervous system (CNS) function in health and disease. Elucidating the ligands mediating TrkB activation in vivo will provide insights into its diverse roles in the CNS. The canonical ligand for TrkB is brain‐derived neurotrophic factor (BDNF). A diversity of stimuli also can activate TrkB in the absence of BDNF, a mechanism termed transactivation. Zinc, a divalent cation packaged in synaptic vesicles along with glutamate in axons of mammalian cortical neurons, can transactivate TrkB in neurons and heterologous cells in vitro. Yet the contributions of BDNF and zinc to TrkB activation in vivo are unknown. To address these questions, we conducted immunohistochemical (IHC) studies of the hippocampal mossy fiber axons and boutons using an antibody selective for pY816 of TrkB, a surrogate measure of TrkB activation. We found that conditional deletion of BDNF resulted in a reduction of pY816 in axons and synaptic boutons of hippocampal mossy fibers, thereby implicating BDNF in activation of TrkB in vivo. Unexpectedly, pY816 immunoreactivity was increased in axons but not synaptic boutons of mossy fibers in ZnT3 knockout mice that lack vesicular zinc. Marked increases of BDNF content were evident within the hippocampus of ZnT3 knockout mice and genetic elimination of BDNF reduced pY816 immunoreactivity in these mice, implicating BDNF in enhanced TrkB activation mediated by vesicular zinc depletion. These findings support the conclusion that BDNF but not vesicular zinc activates TrkB in hippocampal mossy fiber axons under physiological conditions. J. Comp. Neurol. 522:3885–3899, 2014. © 2014 Wiley Periodicals, Inc.     

TrkB影响结肠癌细胞SW620侵袭能力及与ERK信号通路活化关系的研究

目的:探讨干扰结肠癌细胞TrkB蛋白表达及抑制ERK活化对细胞增殖、凋亡和侵袭以及细胞内ERK磷酸化水平的影响。方法:应用特异性TrkB-siRNA瞬时转染及ERK特异性抑制剂处理SW620结肠癌细胞,观察SW620细胞增殖、凋亡和侵袭情况以及细胞内ERK磷酸化水平的变化。结果:特异性siRNA转染高表达TrkB的SW620细胞,TrkB蛋白表达减少,ERK的磷酸化水平降低,且转染组细胞数显著低于对照组(P=0.001),转染组的细胞凋亡率显著高于对照组(P=0.000 1),24 h后侵袭至下层小室的细胞数转染组显著低于对照组(P=0.001);ERK抑制剂明显降低SW620细胞ERK磷酸化水平(P=0.001),而对ERK蛋白表达没有影响,且应用ERK抑制剂作用SW620细胞,对照组和处理组中细胞数没有显著差异(P=0.544),对照组和处理组中SW620细胞的凋亡率差异无统计学意义(P=0.103),但对照组24 h后侵袭至下层小室的细胞数显著高于处理组(P=0.0001)。结论:干扰TrkB蛋白表达能够降低高转移结肠腺癌SW620细胞内ERK磷酸化水平,促进细胞凋亡并抑制细胞增殖和侵袭。同时应用ERK特异性抑制剂也显著抑制细胞侵袭。因此ERK信号转导通路可能与TrkB介导的结肠腺癌细胞抗凋亡和侵袭能力的增加相关,沉默TrkB表达可能成为阻断结肠癌转移的新靶点。     

A dopamine D1 receptor agonist improved learning and memory in morphine-treated rats

ABSTRACT

Objective: The objective of this article is to study the role of the dopamine (DA) D1 receptor in the midbrain periaqueductal grey (PAG) on learning and memory in morphine-addicted rats.

Methods: DA D1 receptor agonist SKF81297 and D1 receptor antagonist SCH SCH23390 were administrated into the PAG, respectively, and the learning and memory behavioral changes of morphine addicted rats were detected by water maze. Western blot and immunohistochemistry were used to detect glutamate decarboxylase 67 (GAD67) and tyrosine receptor kinase B (TrkB) in PAG.

Results: D1 receptor agonist shortened the latency to platform and increased the number of platform crossings, indicating improved learning and memory ability of morphine addict rat. D1 receptor agonist increased GAD67 expression and decreased TrkB in PAG.

Conclusion: (1) The PAG is involved in the learning and memory changes of the addicted rats; (2) the activation of DA D1 receptor will increase the GAD67, reduce the damage to peripheral neurons, and improve the learning and memory of the addicted rats; and (3) D1 receptor agonists further reduced TrkB expression in morphine-addicted rats, whereas TrkB levels deviated from changes in rat behavior.