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. Neuregulin-1 通过激活多巴胺 D4 受体调节 CA1 海马突触的 LTP：Nuregulin-1 (NRG-1) 和 ErbB4 在遗传上与精神分裂症相关，精神分裂症是一种以谷氨酸能和多巴胺能功能失衡为特征的神经发育性认知障碍。之前我们报道过 NRG-1 抑制或逆转海马谷氨酸突触的长时程增强 (LTP)。现在我们证明 NRG-1 刺激海马体中多巴胺的释放，并通过激活 D4 多巴胺受体 (D4R) 逆转早期 LTP。在用抗精神病药物氯氮平和其他更具选择性的 D4R 拮抗剂治疗的海马切片中，NRG-1 无法减弱 LTP。此外，NRG-1 或 θ 脉冲刺激不会使 D4R 缺失小鼠中的 LTP 减弱。相反，直接激活 D4R 会模仿 NRG-1，并通过减少 AMPAR 电流和增加受体内化来发挥作用。 NRG-1、多巴胺和谷氨酸之间的这种新颖的功能联系对于理解 Neuregulin-ErbB 信号传导的不平衡如何影响多巴胺能和谷氨酸能功能以及与精神分裂症相关的神经递质途径具有重要意义。 2. Neuregulin-1 调节海马伽玛振荡：对精神分裂症的影响：伽玛频率振荡的改变与精神疾病有关，据报道，其振幅（功率）在精神病发作期间选择性增加。我们与卡罗林斯卡研究所的 Andre Fisahn 博士合作，发现 NRG-1 显着增加了急性海马切片中红藻氨酸诱导的伽马振荡的强度。 NRG-1 效应被 ErbB 受体的泛特异性拮抗剂 PD158780 阻断，并且在 ErbB4 缺失小鼠制备的切片中不存在。此外，我们证明 50% 的 GABA 能小清蛋白阳性中间神经元表达 ErbB4 受体，这些神经元对 γ 振荡的产生有很大贡献。重要的是，ErbB4 敲除小鼠中小清蛋白免疫反应性中间神经元的数量和红藻氨酸诱导的伽马振荡的强度均减少。这项研究提供了 NRG-1/ErbB4 信号传导与节律网络活动之间的第一个可能的联系，这种联系可能在精神分裂症患者中发生改变。 3.神经调节蛋白受体ErbB4的细胞和亚细胞表达：为了了解介导上述NRG-1对突触可塑性和网络活动的影响的细胞机制，我们必须鉴定表达ErbB4受体的神经元。因此，我们生成了新的单克隆抗体，并在多个应用中对其进行了严格表征，并证明其对 ErbB4 具有高度特异性。使用这些抗体，我们分析了四种功能不同的中间神经元中 ErbB4 的表达模式，这些中间神经元代表了成年小鼠海马中所有抑制性神经元的大多数。我们发现四种细胞类别中的三种细胞具有高表达水平，表明 NRG 可以调节海马体中的多种抑制途径。 ErbB4 还与发育过程中中间神经元的生成和成熟有关，与此一致，我们发现缺乏 ErbB4 的小鼠中两类中间神经元显着减少。接下来我们研究了 ErbB4 在中间神经元中的亚细胞表达，因为确切的位置对于理解 NRG-ErbB4 信号传导的生理效应再次至关重要。使用免疫电子显微镜对 CA1 中间神经元进行超微结构分析，显示体细胞树突区室中有丰富的 ErbB4 表达，它在谷氨酸能突触后位点及其附近聚集。相比之下，我们在培养的 GAD67 阳性海马中间神经元和 CA1 篮状细胞末梢中没有发现突触前表达的证据。我们的研究结果将表达 ErbB4 的中间神经元（而非锥体神经元）确定为海马 NRG 信号传导的主要靶标，并进一步表明 ErbB4 作为抑制性中间神经元上谷氨酸能突触的选择性标记物。 4. NRG-1（IV 型）的分子和细胞特征：NRG-1 编码从 I 型到 VII 型不同启动子转录的生长和分化因子家族，并且它已被可重复地鉴定为精神分裂症的“危险基因”。有趣的是，包含 HAPice 的四种单核苷酸多态性之一（称为 SNP8NRG243177 T/T）与精神分裂症相关的内表型相关，例如前额皮质功能、工作记忆、髓鞘形成和病前智商降低。由于SNP8NRG243177 T/T具有功能多态性的特征，并且图谱与编码NRG-1 IV型转录本的DNA序列接近，我们的目标是精确定位IV型转录起始位点，并研究NRG-1 IV型蛋白的特性和亚细胞分布。我们绘制了一个新的 IV 型转录起始位点，并分离了两个编码 IV 型蛋白的全长 mRNA。使用我们针对蛋白质的独特 IV 型 N 末端产生的抗血清，我们发现 IV 型 NRG-1 靶向细胞表面，并且 PKC 激活促进胞外结构域的蛋白水解切割和释放。我们还证明了 NRG1 IV 型具有与其他可释放的 NRG-1 亚型类似的生物活性。然而，不同 NRG-1 亚型的亚细胞分布不同。与在神经元的体树突和轴突区室中表达的 NRG-1 III 型不同，NRG-1 IV 型及其密切同源的 NRG-1 I 型被选择性地从轴突中排除。这些结果为理解与 SNP8NRG243177 T/T 相关的 NRG-1 IV 型表达水平的改变如何选择性地改变从体细胞树突区室释放的 NRG-1 信号传导奠定了重要的一步。 B. 肌肉类型的活动依赖性调节 1. NFAT 对快肌基因的活动依赖性抑制：钙依赖性转录因子的 NFAT 家族与响应慢模式运动神经元去极化而上调编码慢收缩蛋白的基因有关。我们与 Kristian Gundersen 博士合作，证明了 NFATc1 在慢肌中的一种新颖且意想不到的功能。利用肌钙蛋白 I 快速 (TnIf) 内含子调节元件 (FIRE)，我们确定了可选择性下调其功能的序列，以响应模仿缓慢运动神经元放电的电活动模式。通过定点突变和 EMSA 鉴定了 TnIf FIRE 中真正的 NFAT 结合位点，并显示其介导 FIRE 的活性依赖性转录抑制。成年肌肉中 siRNA 介导的 NFATc1 敲除导致慢比目鱼肌中 FIRE 的异位激活，而不影响快 EDL 肌肉中的增强子活性。这些发现证明了 NFAT 作为慢肌中快速收缩基因转录抑制因子的新功能。