Mechanisms Regulating Activity Dependent Synaptic Plasticity and Gene Expression

调节活动依赖性突触可塑性和基因表达的机制

• 批准号: 7968532
• 负责人: ANDRES BUONANNO
• 金额: $ 140.06万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7968532
• 关键词: Acute; Adult; Affect; Antibodies; Antipsychotic Agents; Axon; Binding Sites; Biological; Brain; Calcium; Cell surface; Cells; Characteristics; Clozapine; Cognition; Cognition Disorders; Collaborations; Contractile Proteins; Cues; DNA; DNA Sequence; Development; Differentiation and Growth; Disease; Dopamine; Dopamine Antagonists; Dopamine Receptor; Enhancers; Epigenetic Process; ErbB4 gene; Exercise; Extracellular Domain; Family; Frequencies; Gene Expression; Generations; Genes; Genetic; Genetic Polymorphism; Glutamates; Goals; Hippocampus (Brain); Homologous Gene; Immune Sera; Immunoelectron Microscopy; Institutes; Interneurons; Knockout Mice; Laboratories; Learning; Length; Link; Location; Long-Term Potentiation; Maps; Mediating; Memory; Mental disorders; Molecular; Monoclonal Antibodies; Motor Neurons; Mus; Muscle; Mutation; Myoepithelial cell; N-terminal; NRG1 gene; Neuregulin 1; Neuregulin Receptor; Neuregulins; Neuronal Plasticity; Neurons; Neurotransmitters; Pan Genus; Parvalbumins; Pathway interactions; Pattern; Persons; Phase; Physiologic pulse; Physiological; Property; Protein Isoforms; Proteins; Pyramidal Cells; Receptor Activation; Regulation; Regulatory Element; Reporting; Repression; Risk; Schizophrenia; Short-Term Memory; Signal Pathway; Signal Transduction; Single Nucleotide Polymorphism; Site; Skeletal Muscle; Slice; Small Interfering RNA; Stimulus; Synapses; Synaptic plasticity; System; Testing; Transcript; Transcription Initiation Site; Transcription Repressor/Corepressor; Troponin I; Up-Regulation; dopamine D4 receptor; endophenotype; gene repression; high risk; hippocampal pyramidal neuron; human DRD4 protein; inhibitory neuron; kainate; myelination; novel; postsynaptic; presynaptic; promoter; receptor; receptor internalization; response; transcription factor

A) NEUREGULIN/ERB-B SIGNALING REGULATES NEURONAL PLASTICITY: POSSIBLE RELEVANCE TO SCHIZOPHRENIA 1. Neuregulin-1 Regulates LTP at CA1 Hippocampal Synapses Through Activation of Dopamine D4 Receptors: Neuregulin-1 (NRG-1) and ErbB4 are genetically associated with schizophrenia, a neurodevelopmental cognitive disorder characterized by imbalances in glutamatergic and dopaminergic function. Previously we reported NRG-1 suppresses or reverses long-term potentiation (LTP) at hippocampal glutamatergic synapses. Now we demonstrate that NRG-1 stimulates dopamine release in the hippocampus, and reverses early-phase LTP via activation of D4 dopamine receptors (D4R). NRG-1 fails to depotentiate LTP in hippocampal slices treated with the antipsychotic clozapine and other more selective D4R antagonists. Moreover, LTP is not depotentiated in D4R null mice by either NRG-1 or theta-pulse stimuli. Conversely, direct D4R activation mimics NRG-1 and acts by reducing AMPAR currents and increasing receptor internalization. This novel functional link between NRG-1, dopamine and glutamate has important implications for understanding how imbalances in Neuregulin-ErbB signaling can impinge on dopaminergic and glutamatergic function, neurotransmitter pathways associated with schizophrenia. 2. Neuregulin-1 Modulates Hippocampal Gamma Oscillations: Implications for Schizophrenia: Alterations in gamma-frequency oscillations are implicated in psychiatric disorders, and their amplitude (power) have been reported to increase selectively during psychotic episodes. In collaboration with Dr. Andre Fisahn at the Karolinska Institute, we found that NRG-1 dramatically increases the power of kainate-induced gamma oscillations in acute hippocampal slices. NRG-1 effects are blocked by PD158780, a pan-specific antagonist of ErbB receptors, and are absent in slices prepared from ErbB4 null mice. Moreover, we demonstrate that 50% of GABAergic parvalbumin-positive interneurons, which heavily contribute to the generation of gamma oscillations, express ErbB4 receptors. Importantly, both the number of parvalbumin-immunoreactive interneurons and the power of kainate-induced gamma oscillations are reduced in ErbB4 knockout mice. This study provides the first plausible link between NRG-1/ErbB4 signaling and rhythmic network activity that may be altered in persons with schizophrenia. 3. Cellular and Subcellular Expression of the Neuregulin Receptor ErbB4: In order to understand the cellular mechanisms that mediate the above-mentioned effects of NRG-1 on synaptic plasticity and network activity we have to identify the neurons that express the ErbB4 receptor. We therefore generated new monoclonal antibodies that we stringently characterized in several applications and that proved to be highly specific for ErbB4. Using these antibodies we analyzed the expression pattern of ErbB4 in four functionally distinct classes of interneurons that represent the majority of all inhibitory neurons in the adult hippocampus of mice. We found high expression levels in three of the four cell classes, indicating that NRG can modulate several inhibitory pathways in the hippocampus. ErbB4 has also been implicated in the generation and maturation of interneurons during development, and consistent with this we found significant reductions of two classes of interneurons in mice that lack ErbB4. We next investigated the subcellular expression of ErbB4 in interneurons, because the exact location is again crucial for understanding the physiological effects of NRG-ErbB4 signaling. Ultrastructural analysis in CA1 interneurons using immunoelectron microscopy revealed abundant ErbB4 expression in the somatodendritic compartment where it accumulates at, and adjacent to, glutamatergic postsynaptic sites. By contrast, we found no evidence for presynaptic expression in cultured GAD67-positive hippocampal interneurons and in CA1 basket cell terminals. Our findings identify ErbB4-expressing interneurons, but not pyramidal neurons, as a primary target of NRG signaling in the hippocampus, and furthermore implicate ErbB4 as a selective marker for glutamatergic synapses on inhibitory interneurons. 4. Molecular and Cellular Characterization of NRG-1 (type IV): NRG-1 encodes a family of growth and differentiation factors transcribed from distinct promoters designated type I through type VII, and it has been reproducibly identified as an "at risk gene" for schizophrenia. Interestingly one of the four single nucleotide polymorphisms comprising HAPice designated as SNP8NRG243177 T/T, is associated with endophenotypes related to schizophrenia, such as reduced prefrontal cortical function, working memory, myelination and premorbid IQ. Since SNP8NRG243177 T/T has characteristics of a functional polymorphism and maps close to DNA sequences encoding NRG-1 type IV transcripts, our goal was to precisely map the type IV transcription initiation site and to investigate the properties and subcellular distribution of NRG-1 type IV protein. We mapped a novel type IV transcription initiation site and isolated two full-length mRNAs encoding type IV proteins. Using an antiserum we raised against the unique type IV N-terminal end of the protein, we found that NRG-1 type IV is targeted to the cell surface and proteolytic cleavage and release of the extracellular domain is promoted by PKC activation. Also we demonstrated that NRG1 type IV is possessed biological activity similar to other releasable NRG-1 isoforms. However, the subcellular distributions of distinct NRG-1 isoforms differ. Unlike NRG-1 type III which are expressed in the somato-dendritic and axonal compartments of neurons, NRG-1 type IV and its close homolog NRG-1 type I are excluded selecctively from axons. These results constitute an important step for understanding how alterations in NRG-1 type IV expression levels associated with SNP8NRG243177 T/T could selectively modify signaling from NRG-1 released from somato-dendritic compartments. B. ACTIVITY-DEPENDENT REGULATION OF MUSCLE TYPES 1. Activity-Dependent Repression of Fast Muscle Genes by NFAT: The NFAT family of calcium-dependent transcription factors has been implicated in the upregulation of genes encoding slow contractile proteins in response to slow-patterned motoneuron depolarization. In collaboration with Dr. Kristian Gundersen, we demonstrated a novel, and unexpected, function of NFATc1 in slow-twitch muscles. Utilizing the Troponin I fast (TnIf) intronic regulatory element (FIRE), we identified sequences that downregulate its function selectively in response to patterns of electrical activity that mimic slow motoneuron firing. A bona fide NFAT binding site in the TnIf FIRE was identified by site directed mutations and EMSAs, and shown to mediated the activity-dependent transcriptional repression of FIRE. siRNA-mediated knockdown of NFATc1 in adult muscles resulted in ectopic activation of the FIRE in the slow soleus, without affecting enhancer activity in the fast EDL muscle. These findings demonstrate a novel function of NFAT as a repressor of transcription of fast contractile genes in slow muscles.

a）神经调节蛋白/erb-b信号传导调节神经元可塑性：可能与精神分裂症相关 1。神经蛋白-1通过激活多巴胺D4受体在CA1海马突触调节LTP：Neuregulin-1（NRG-1）和ERBB4在遗传上与精神分裂症相关，该精神分裂症是一种神经发育性认知障碍，以胶原氨基糖浆和多巴胺敏感的功能为单位。以前，我们报道了NRG-1在海马谷氨酸能突触下抑制或逆转长期增强（LTP）。现在，我们证明NRG-1刺激海马中的多巴胺释放，并通过激活D4多巴胺受体（D4R）逆转早期LTP。 NRG-1未能在用抗精神病药氯氮平和其他更具选择性的D4R拮抗剂处理的海马切片中分配LTP。此外，NRG-1或theta-Pulse刺激在D4R NULL小鼠中均未将LTP屈服。相反，直接D4R激活模仿NRG-1，并通过减少AMPAR电流和增加受体内在化来起作用。 NRG-1，多巴胺和谷氨酸之间的这种新型功能联系对于理解神经蛋白 - erbb信号的失衡如何影响多巴胺能和谷氨酸能功能，神经递质途径与schizizophrenia相关。 2. Neuregulin-1调节海马γ振荡：对精神分裂症的影响：γ-频率振荡的改变与精神疾病有关，据报道，在精神病发作期间，它们的振幅（功率）已有选择性增加。与Karolinska研究所的Andre Fisahn博士合作，我们发现NRG-1大大提高了急性海马切片中海藻酸盐诱导的γ振荡的功能。 NRG-1的效应被PD158780（ERBB受体的PAN特异性拮抗剂）阻塞，并且在ERBB4无效小鼠中制备的切片中不存在。此外，我们证明了50％的Gabaergic白蛋白阳性中间神经元，这有助于产生γ振荡，这表达ERBB4受体。重要的是，在ERBB4基因敲除小鼠中，白蛋白蛋白 - 免疫反应性中间神经元的数量和海藻酸盐诱导的伽马振荡的功率都减少了。这项研究提供了在精神分裂症患者中可能改变的NRG-1/ERBB4信号传导与节奏网络活动之间的第一个合理联系。 3。神经调节蛋白受体ERBB4的细胞和亚细胞表达：为了了解介导NRG-1对突触可塑性和网络活性上述作用的细胞机制，我们必须识别表达ERBB4受体的神经元。因此，我们生成了新的单克隆抗体，这些抗体在几种应用中进行了严格的特征，事实证明对ERBB4非常具体。使用这些抗体，我们分析了四个在功能上不同类别的中间神经元中ERBB4的表达模式，这些神经元代表小鼠成人海马中所有抑制性神经元中的大多数。我们在四个细胞类别中的三个中发现了高表达水平，表明NRG可以调节海马中的几种抑制途径。 ERBB4在发育过程中也与中间神经元的产生和成熟有关，并且与此相一致，我们发现缺乏ERBB4的小鼠中两类中间神经元的显着降低。接下来，我们研究了中间神经元中ERBB4的亚细胞表达，因为确切的位置再次对于理解NRG-ERBB4信号的生理效应至关重要。使用免疫电子显微镜在CA1中间神经元中进行的超微结构分析显示，在体内室中的ERBB4表达丰富，在该室中积累了谷氨酸能突触后地点，并在其中积聚并毗邻。相比之下，我们没有发现在培养的GAD67阳性海马中间神经元和CA1篮细胞末端中突触前表达的证据。我们的发现将表达ERBB4的中间神经元而不是金字塔神经元确定为海马中NRG信号传导的主要靶标，此外，ERBB4将ERBB4作为谷氨酸能突触的选择性标记在抑制性含量中。 4。NRG-1的分子和细胞表征（IV型）：NRG-1编码从指定I型通过VII型的不同启动子转录的生长和分化因子的家族，并且已被重现为Schizophrenia的“ AT风险基因”。有趣的是，指定为SNP8NRG243177 T/T的HAPICE的四种单核苷酸多态性之一与与精神分裂症相关的内酚型有关，例如降低前额叶皮质功能，工作记忆，骨髓，骨髓和前质体IQ。由于SNP8NRG243177 T/T具有功能性多态性的特征和接近编码NRG-1 IV型转录本的DNA序列的地图，因此我们的目标是精确映射IV型转录起始位点并研究NRG-1型IV型蛋白质的特性和亚细胞分布。我们绘制了一个新型的IV型转录起始位点，并分离了两个编码IV型蛋白的全长mRNA。使用抗血清，我们针对蛋白质的独特IV型N端端提出，我们发现NRG-1 IV型针对细胞表面，蛋白水解裂解和细胞外结构域的释放是通过PKC激活促进的。我们还证明，NRG1 IV具有类似于其他可释放的NRG-1同工型的生物学活性。但是，不同NRG-1同工型的亚细胞分布有所不同。与NRG-1型III不同，在神经元的somato树突和轴突室中表达，NRG-1 IV型及其近距离同源性NRG-1类型I类型从轴突中选择性排除在外。这些结果构成了了解如何与SNP8NRG243177 T/T相关的NRG-1类型表达水平的变化可以选择性地从Somato-Dendritic室释放的NRG-1中选择性修改信号传导。 B.肌肉类型的活动依赖性调节 1。NFAT对快速肌肉基因的抑制：钙依赖性转录因子的NFAT家族与编码缓慢的收缩蛋白的基因上调有关，响应缓慢的运动神经元去极化。在与克里斯蒂安·冈德森（Kristian Gundersen）博士合作的情况下，我们展示了NFATC1在慢速肌肉中的小说且出乎意料的功能。利用肌钙蛋白I快速（TNIF）内含子调节元件（FIR），我们确定了序列，这些序列可选择性地下调其功能，以响应模拟慢速运动神经元激发的电活动模式。 TNIF火灾中的真正的NFAT结合位点通过位置的突变和EMSA鉴定出来，并证明可以介导了依赖活性的火对火的转录抑制。 siRNA介导的成年肌肉中NFATC1的敲低导致慢镜头中火的异位激活，而不会影响快速EDL肌肉的增强剂活性。这些发现证明了NFAT是缓慢肌肉中快速收缩基因转录的抑制剂的新功能。