Genetic and Optic Dissection of AMPK Dynamics in Neurotransmission

神经传递中 AMPK 动力学的遗传和光学解剖

• 批准号: 9165641
• 负责人: Dong Kong
• 金额: $ 24.75万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9165641
• 关键词: 5' -AMP-activated protein kinase; AMP-activated protein kinase kinase; Accounting; Adult; Affect; Anorexia; Brain; Calcium; Cells; Cellular biology; Cultured Cells; Diabetes Mellitus; Dissection; Electrophysiology (science); Energy Metabolism; Fasting; Feeding behaviors; Fluorescence; Fluorescence Resonance Energy Transfer; Functional disorder; Genetic; Genetic Screening; Glutamates; Hunger; Hypothalamic structure; Image; Knockout Mice; Knowledge; Laser Scanning Microscopy; Lasers; Left; Leptin; Life; Measures; Mediating; Metabolic Diseases; Metabolism; Microscopy; Monitor; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; Neurologic; Neurons; Obesity; Optics; Pathway interactions; Peptides; Pharmacogenetics; Physiological; Physiology; Play; Process; Protein-Serine-Threonine Kinases; Regulation; Reporter; Reporting; Resolution; Role; Scanning; Signal Transduction; Slice; Specificity; Synapses; Synaptic Transmission; Synaptic plasticity; Technology; Testing; Time; Transgenic Mice; Viral Vector; base; brain metabolism; chemical genetics; energy balance; feeding; ghrelin; innovation; mTOR Signaling Pathway; nervous system disorder; neurobiological mechanism; neurotransmission; novel; p21 activated kinase; patch clamp; postsynaptic; presynaptic; sensor; spatiotemporal; synaptogenesis; transmission process; two-photon

AMP-activated protein kinase (AMPK), an evolutionarily conserved serine/threonine kinase stimulated by both decreased cellular energy status and increased calcium, is an important player acting at the interface between metabolism and brain function. In addition to metabolic diseases like obesity and diabetes, abnormal AMPK activities have been implicated in a variety of neurological disorders with dysfunctional neurotransmission. The neurobiological mechanisms of AMPK responsible for these effects, however, are largely unknown. Recent studies have suggested that agouti-related peptide (AgRP)-expressing neurons in the hypothalamus, a master controller of feeding and energy balance, receive intense glutamatergic input and their excitatory synaptic plasticity plays an essential role in regulating AgRP neuron firing and related feeding. Importantly, our prior findings demonstrate that fasting significantly induces dendritic spinogenesis, glutamatergic synaptogenesis, and firing in AgRP neurons, and this fasting-induced plasticity requires postsynaptic NMDA receptors on AgRP neurons and contributes essentially to their fasting-induced activation. The neurobiological mechanism that underlies fasting-induced plasticity in AgRP neurons, however, is left unknown. In this context, AMPK in the hypothalamus is activated by fasting and manipulation of AMPK activity in this region affects feeding. In addition, when stimulated pharmacologically in brain slices, AMPK increases glutamatergic input to AgRP neurons. These findings suggest that AMPK likely trigger this fasting-induced plasticity. However, given the wide expression of AMPK in the brain and its multi-faceted roles in cellular biology, whether AMPK in AgRP neurons mediates fasting-induced feeding is still in debate. How fasting modulates AMPK dynamics is also unclear. By employing a battery of neuron-specific approaches, including neuron-specific transgenic and knockout mouse lines, cre-dependent AAV viral vectors, 2-photon laser scanning microscopy (2PLSM) combined with whole cell patch-clamp electrophysiology, and particularly 2PLSM-based fluorescence lifetime imaging (FLIM), this proposal aims to provide a unique, multi-faceted study to understand AMPK signaling and its physiology in the neurotransmission of AgRP neurons. Based on our compelling preliminary findings, we hypothesize that a postsynaptic pathway engaged by AMPK in AgRP neurons drives fasting induced excitatory synaptic plasticity and the plasticity brought about by this pathway accounts for the effects of AMPK on energy balance (Aim 1). We further hypothesize that AMPK functions as a critical integrator of diverse inputs (such as fasting, ghrelin, and leptin) of AgRP neurons and mediates both synaptic and cellular changes (Aim 2). Our novel findings on synaptic plasticity and AMPK will provide innovative knowledge in the feeding circuits. Given the wide distribution of AMPK and its substrates, the uncovered pathway engaged by AMPK in AgRP neurons will likely operate both within and beyond the hypothalamus, and have important implications for many processes where synaptic plasticity plays a key regulatory role.

AMP激活的蛋白激酶（AMPK），这是一种进化保守的丝氨酸/苏氨酸激酶，均被两者刺激 细胞能量状态降低和钙的增加，是一个重要的参与者，在 代谢和大脑功能。除了肥胖和糖尿病等代谢疾病外，异常AMPK 活动与各种神经疾病有关，具有功能失调的神经传递。这 但是，负责这些作用的AMPK的神经生物学机制在很大程度上尚不清楚。最近的 研究表明，与Agouti相关的肽（AGRP）表达神经元， 喂养和能量平衡的控制器，接收强烈的谷氨酸能输入及其兴奋性突触 可塑性在调节AGRP神经元点火和相关喂养方面起着至关重要的作用。重要的是，我们的先验 发现表明，禁食显着诱导树突状旋转生成，谷氨酸能突触发生， 并在AGRP神经元中发射，这种禁食诱导的可塑性需要在AGRP上突触后NMDA受体 神经元并基本上有助于其禁食引起的激活。神经生物学机制 但是，尚不清楚AGRP神经元中禁食诱导的可塑性的基础。在这种情况下，AMPK在 通过禁食和操纵AMPK活性在该区域会影响下丘脑会影响进食。在 此外，当在大脑切片中刺激药理学时，AMPK将谷氨酸能输入增加到AGRP 神经元。这些发现表明AMPK可能会触发这种禁食引起的可塑性。但是，给定 AMPK在大脑中的广泛表达及其在细胞生物学中的多面角色，是否在AGRP中AMPK 神经元介导禁食引起的喂养仍在争论中。禁食如何调制AMPK动力学 不清楚。通过采用一系列神经元特异性方法，包括神经元特异性转基因和 敲除小鼠系，依赖CRE的AAV病毒载体，2光子激光扫描显微镜（2PLSM） 结合全细胞贴片钳电生理学，特别是基于2PLSM的荧光寿命 成像（FLIM），该提案旨在提供一项独特的多面研究，以了解AMPK信号和 它在AGRP神经元神经传递中的生理学。根据我们引人入胜的初步发现，我们 假设AMPK参与AGRP神经元中的突触后途径驱动禁食引起兴奋性 突触可塑性和该途径带来的可塑性是AMPK对能量的影响 平衡（目标1）。我们进一步假设AMPK是各种投入的关键集成商（例如 AGRP神经元的禁食，生长素和瘦素）并介导突触和细胞变化（AIM 2）。我们的 关于突触可塑性和AMPK的新发现将在喂养电路中提供创新的知识。给出 AMPK及其底物的广泛分布，AMPK参与AGRP神经元中的未发现途径 可能会在下丘脑内外运作，并对许多人具有重要意义 突触可塑性起关键调节作用的过程。