背景:蛋白激酶A(PKA)增强神经肌肉接头(NMJ)的神经传递,受神经诱导的肌肉收缩的逆行调节,通过参与突触小泡胞吐作用的分子(SNAP-25和Synapsin-1)的磷酸化来促进乙酰胆碱(ACh)的释放。然而,BDNF/TrkB通路和毒蕈碱信号对PKA亚基及其靶标的逆行调节的分子机制至今尚未得到证实。在NMJ,逆行控制主要与BDNF/TrkB信号相关,因为肌肉收缩增强BDNF水平并控制参与神经传递的特定激酶。NMJ的神经传递也受到毒蕈碱受体M1和M2(mAChRs)的高度调节,与PKA和TrkB信号有关。这里,我们研究了TrkB的假设,与MAChRs合作,调节PKA亚基的活性依赖性动力学以磷酸化SNAP-25和Synapsin-1。
方法:要探索这一点,我们以1Hz(30分钟)刺激大鼠膈神经,有或没有随后的收缩(被µ-conotoxinGIIIB废除)。用抗TrkB抗体克隆47/TrkB进行药物治疗,以抑制TrkB和外源性h-BDNF;用盐酸哌仑西平和盐酸甲辛四胺对M1和M2mAChRs进行毒蕈碱抑制,分别。Westernblotting检测diaphragm蛋白水平和磷酸化变化。使用免疫组织化学证实靶蛋白的位置。
结果:虽然TrkB不直接影响PKA催化亚基Cα和Cβ的水平,它调节PKA调节亚基RIα和RIβ,促进关键胞吐靶标如SNAP-25和Synapsin-1的磷酸化。此外,毒蕈碱受体通路在这一调控过程中维持着微妙的平衡.这些发现解释了受BDNF/TrkB信号影响的PKA亚基的动态相互作用,M1和M2mAChRs途径,受突触前和突触后活动不同的调节,证明了BDNF/TrkB和毒蕈碱受体途径在逆行调节中的特定作用。
结论:这种复杂的分子相互作用与PKA-突触调节中的两个基本途径相关:一个是逆行(神经营养),另一个是自分泌(毒蕈碱)。这加深了对神经传递的神经肌肉生理学的基本理解,该神经传递赋予突触可塑性,并具有在以神经肌肉沟通受损为特征的疾病中确定治疗策略的潜力。
BACKGROUND: Protein kinase A (PKA) enhances neurotransmission at the neuromuscular junction (NMJ), which is retrogradely regulated by nerve-induced muscle contraction to promote Acetylcholine (ACh) release through the phosphorylation of molecules involved in synaptic vesicle exocytosis (SNAP-25 and Synapsin-1). However, the molecular mechanism of the retrograde regulation of PKA subunits and its targets by BDNF/TrkB pathway and muscarinic signalling has not been demonstrated until now. At the NMJ, retrograde control is mainly associated with BDNF/TrkB signalling as muscle contraction enhances BDNF levels and controls specific kinases involved in the neurotransmission. Neurotransmission at the NMJ is also highly modulated by muscarinic receptors M1 and M2 (mAChRs), which are related to PKA and TrkB signallings. Here, we investigated the hypothesis that TrkB, in cooperation with mAChRs, regulates the activity-dependent dynamics of PKA subunits to phosphorylate SNAP-25 and Synapsin-1.
METHODS: To explore this, we stimulated the rat phrenic nerve at 1Hz (30 minutes), with or without subsequent contraction (abolished by µ-conotoxin GIIIB). Pharmacological treatments were conducted with the anti-TrkB antibody clone 47/TrkB for TrkB inhibition and exogenous h-BDNF; muscarinic inhibition with Pirenzepine-dihydrochloride and Methoctramine-tetrahydrochloride for M1 and M2 mAChRs, respectively. Diaphragm protein levels and phosphorylation\' changes were detected by Western blotting. Location of the target proteins was demonstrated using immunohistochemistry.
RESULTS: While TrkB does not directly impact the levels of PKA catalytic subunits Cα and Cβ, it regulates PKA regulatory subunits RIα and RIIβ, facilitating the phosphorylation of critical exocytotic targets such as SNAP-25 and Synapsin-1. Furthermore, the muscarinic receptors pathway maintains a delicate balance in this regulatory process. These findings explain the dynamic interplay of PKA subunits influenced by BDNF/TrkB signalling, M1 and M2 mAChRs pathways, that are differently regulated by pre- and postsynaptic activity, demonstrating the specific roles of the BDNF/TrkB and muscarinic receptors pathway in retrograde regulation.
CONCLUSIONS: This complex molecular interplay has the relevance of interrelating two fundamental pathways in PKA-synaptic modulation: one retrograde (neurotrophic) and the other autocrine (muscarinic). This deepens the fundamental understanding of neuromuscular physiology of neurotransmission that gives plasticity to synapses and holds the potential for identifying therapeutic strategies in conditions characterized by impaired neuromuscular communication.