Neuregulin-ErbB Signaling in Neuronal Development and Psychiatric Disorders

神经元发育和精神疾病中的神经调节蛋白-ErbB 信号转导

• 批准号: 8553854
• 负责人: ANDRES BUONANNO
• 金额: $ 138.42万
• 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/8553854
• 关键词: AMPA Receptors; Ablation; Accounting; Action Potentials; Acute; Address; Affect; Affinity Chromatography; Agonist; Antibodies; Antipsychotic Agents; Attention deficit hyperactivity disorder; Attenuated; Autistic Disorder; Behavior; Behavioral Symptoms; Biological; Brain; Cells; Chemicals; Chemosensitization; Clozapine; Collaborations; Complex; Cyclic AMP; Cytoplasmic Granules; DRD2 gene; Data; Development; Dopamine; Electrophysiology (science); Equilibrium; ErbB4 gene; Etiology; Failure; Fluorescence Microscopy; Frequencies; Functional disorder; Gap Junctions; Genes; Genetically Engineered Mouse; Glutamates; Glycine; Goals; Hippocampus (Brain); Histocytochemistry; Human; Immunofluorescence Immunologic; In Situ Hybridization; In Vitro; Interneurons; Ion Channel; Knock-out; Knowledge; Label; Life; Link; Liquid Chromatography; Macaca mulatta; Mediating; Mental disorders; Messenger RNA; Microdialysis; Modeling; Molecular Neurobiology; Mus; Mutant Strains Mice; Myoepithelial cell; NRG1 gene; National Institute of Mental Health; Neuregulin 1; Neuregulins; Neurobehavioral Manifestations; Neurons; Parvalbumins; Pathway interactions; Pattern; Phase; Primates; Process; Property; Proteins; Proteome; Proteomics; Pyramidal Cells; Receptor Signaling; Reporting; Resort; Reverse Transcriptase Polymerase Chain Reaction; Risk; Rodent; Role; Schizophrenia; Signal Transduction; Slice; Sodium; Sodium Channel; Stress; Synapses; Synaptic plasticity; Synaptosomes; Techniques; Time; Tissues; Transcript; Validation; behavior test; cognitive control; cognitive function; density; dopamine D4 receptor; endophenotype; extracellular; frontal lobe; granule cell; hippocampal pyramidal neuron; insight; interest; kainate; liquid chromatography mass spectrometry; mutant; neural circuit; neurochemistry; neuron development; novel; presynaptic; receptor; receptor coupling; tandem mass spectrometry; voltage; voltage clamp

1. Conservation of ErbB4 expression in GABAergic interneurons in the hippocampus and cortex, from rodents to primates: Knowledge of the cellular and subcellular localization of ErbB4 is important for understanding how Neuregulin (NRG) regulates neuronal network activity and behavior. In collaboration with Dr. McBain's and Dr. Lewis's lab, we unequivocally demonstrated, using a combination of single-cell RT-PCR and electrophysiological profiling in mice, that ErbB4 transcripts are expressed GABAergic interneurons and not in excitatory pyramidal cells. A finding confirmed with our ErbB4 mAb that showed receptor accumulates in the somato-dendritic region of cortical GABAergic interneurons in rodents, rhesus monkey and humans; presynaptic or axonal expression of ErbB4 was rarely detected. This across-species conservation validates the use of rodents to analyze cellular and neural circuit effects of abnormal ErbB4 function as a means to model endophenotypes pertinent to psychiatric disorders. 2. Importance of Neuregulin/ErbB4 signaling in parvalbumin-positive fast-spiking interneurons: We previously showed, using reverse-microdialysis neurochemistry and electrophysiology, that NRG1 signaling via ErbB4 potently triggers dopamine release in the hippocampus and reverses LTP at Schaeffer collateral-to-CA1 (SC-CA1) synapses through activation of dopamine D4 receptors. Importantly, we found that ErbB4 is not detectable in excitatory pyramidal neurons but is abundantly expressed in GABAergic interneurons, in particular fast-spiking parvalbumin-positive basket cells. These observations strongly suggested that effects of NRG/ErbB4 on glutamatergic synaptic plasticity are indirect. To address this question directly, we compared the effects of full vs. parvalbumin (PV) interneuron-specific Erbb4 ablation in genetically engineered mice. As expected, NRG-mediated inhibition of LTP induction and reversal of early-phase LTP were absent in full ErbB4 KO mice. Interestingly, ErbB4 ablation in PV interneurons was sufficient to reproduce the effects of the full ErbB4 knockout. We also compared these two ErbB4 mutant mouse strains in a battery of behavioral tests relevant for psychiatric disorders and found that PV-restricted mutants replicated many, albeit not all, abnormalities observed in full ErbB4 mutant mice. These findings highlight the role of PV interneurons as a critical nexus of NRG/ErbB4 signaling and support a possible contribution of this pathway, genetically linked with increased risk for schizophrenia, to the pathophysiology underlying psychiatric disorders. 3. NRG/ErbB4 and dopamine D4 receptor signaling converge in PV neurons and regulate gamma oscillations: We previously reported that NRG/ErbB4 signaling modulates the power of kainate-induced hippocampal gamma oscillations. Because NRG1 dramatically increases extracellular dopamine levels in the hippocampus, we investigated the relationship between NRG/ErbB and dopamine signaling in hippocampal gamma oscillations. In collaboration with Dr.Fisahns lab, we used selective agonists for different dopamine G-coupled receptors that increase (D1 and D5 receptors) or decrease (D2, D3 and D4 receptors) the synthesis of cAMP, and found that only an agonist specific for D4 receptors (PD168077) augmented the power of gamma oscillations. By contrast, agonists for D1/D5 and D2R/D3 receptors were without effect. The increase in gamma oscillation power induced by PD168077 was totally blocked by a highly specific D4 receptor antagonist (L-745,870), further stressing the importance of this receptor for neuronal network activity. Importantly, we found that L-745,870 and clozapine, an antipsychotic that preferentially targets D4 receptors, also blocked increases in gamma oscillation power by NRG1. Using double in situ hybridization (in collaboration with Dr. U. Winzer-Serhan) and immunofluorescence histochemistry, we showed that hippocampal D4 receptor mRNA and protein are highest in a subset of GABAergic interneurons. Importantly, D4 and ErbB4 receptors are coexpressed in PV+ fast-spiking basket cells, a type of somatic-targeting GABAergic interneurons that are critically important for regulating gamma oscillations. This novel cross-talk between D4 and ErbB4 receptor signaling to augment gamma oscillation power, and their coexpression in PV-positive interneurons, suggest a cellular mechanism that may be compromised in different psychiatric disorders affecting cognitive control. 4. Neuregulin directly decreases voltage-gated sodium currents in hippocampal ErbB4-expresssing interneurons: One of our primary goals has been to investigate if and how NRG1 may directly affect the intrinsic properties of ErbB4-positive (ErbB4+) interneurons. To this end, we resorted to using dissociated hippocampal cultures, which are devoid of all hippocampal-projecting afferent connections, and to blocking all synaptic activity pharmacologically. We initially analyzed the effects of NRG1 on the excitability of ErbB4+ and ErbB4- neurons, identified in live-labeling by an ectodomain-targeting antibody, using whole-cell current-clamp recordings. We found that NRG1 decreased firing of ErbB4+ but not ErbB4- neurons by shifting the action potential threshold. Using voltage-clamp recordings, we determined that these effects are primarily attributable to decreased voltage-gated sodium channel activity, as current density was attenuated by 60% after 20 minutes of NRG1 treatment. This was the first study to identify direct actions of NRG1 on voltage-gated sodium channel function in ErbB4-expressing interneurons, thereby offering novel insights into how NRG1/ErbB4 signaling can directly impact GABAergic interneuron activity and potentially affect excitatory/inhibitory balance. 5. NRG1 signaling directly influences AMPA receptor (AMPAR) activity in glutamatergic ErbB4-expressing cerebellar granule cells (CGCs): Previous studies demonstrated how NRG1 indirectly affects plasticity at glutamatergic synapses in principal glutamatergic neurons in the hippocampus and frontal cortex. We analyzed the effects of NRG1 on developing cultured CGCs in collaboration with Dr. C. Fenster. These cultures consist predominantly of granule neurons that express ErbB4. We found that NRG1 does not affect whole-cell AMPAR or NMDAR mediated currents, nor the frequency or amplitude of spontaneous NMDAR- or AMPAR-mediated miniature excitatory post-synaptic currents, in baseline conditions of CGCs grown for 10-12 days in vitro. However, we found that high-glycine induces a form of chemical potentiation (chemLTP) in CGCs characterized by an increase in AMPAR-mEPSC frequency that is decreased by NRG1 treatment. Our data suggest that the NRG1 effect is mediated via GluA4 subunits in CGCs. This study for the first time shows that high-glycine can induce plasticity at glutamatergic synapses in CGCs, and that acute NRG1/ErbB signaling can regulate glutamatergic plasticity in CGCs in a fashion similar to SC-CA1 synapses. 6. Proteomics approach to understanding NRG/ErbB4 proximal signaling targets: To discover potentially novel and direct biological targets of NRG/ErbB4, in collaboration with Dr. S. Markeys group (NIMH), we are employing an unbiased liquid chromatography-tandem mass spectrometry (LC/MS/MS) proteomics approach to determine the composition of the ErbB4 receptor proteome. We recently reported validation of using our receptor mAb for affinity purification ErbB4 complexes and their analysis by LC/MS/MS. We are using a similar approach to isolate endogenous ErbB4 complexes from metabolically active synaptosomes following NRG-1 stimulation. Using this approach, we discovered that NRG/ErbB4 signaling alters the ion channel composition of cellular microdomains associated with ErbB4. Our goal is to determine the functional consequences of these alterations.

1。从啮齿动物到灵长类动物的海马和皮质中GABA能中神经元中ERBB4表达的保护：ERBB4细胞和亚细胞定位的知识对于理解神经蛋白（NRG）如何调节神经元网络的活性和行为很重要。通过与McBain博士和Lewis博士的实验室合作，我们明确地证明了小鼠单细胞RT-PCR和电​​生理分析的组合，ERBB4转录本是GABA能中间神经元而不是兴奋性倍增性细胞。我们的ERBB4 mAb证实了这一发现，该发现表明受体在啮齿动物，恒河猴和人类的皮质Gabaergic中间神经元的Somato树枝状区域积累。 ERBB4的突触前或轴突表达很少被检测到。这种跨物种的保护验证了啮齿动物的使用来分析异常ERBB4的细胞和神经回路效应，作为模拟与精神疾病有关的末日型的一种手段。 2。神经调节蛋白/ERBB4信号传导在白蛋白阳性阳性快速刺激性神经元中的重要性：我们先前使用反向微透析神经化学和电生理学的反向微透析性神经化学和电生理学表明，NRG1信号通过ERBB4发出ERBB4在Hippocampus and-caeses ltpp中的多巴胺释放（collebbb4）通过激活多巴胺D4受体的突触。重要的是，我们发现在兴奋性锥体神经元中无法检测到ERBB4，但在GABA能中间神经元，尤其是快速刺激性的白细胞蛋白阳性篮细胞中表达了大量表达。这些观察结果强烈表明，NRG/ERBB4对谷氨酸能突触可塑性的影响是间接的。为了直接解决这个问题，我们比较了基因工程小鼠中完整与白细胞蛋白（PV）间含膜特异性ERBB4消融的影响。正如预期的那样，在全ERBB4 KO小鼠中不存在NRG介导的LTP诱导抑制和早期LTP的逆转。有趣的是，PV中间神经元中的ERBB4消融足以再现全ERBB4敲除的影响。我们还比较了这两个与精神疾病相关的行为测试中的ERBB4突变小鼠菌株，发现PV限制的突变体在全ERBB4突变体中复制了许多，尽管不是全部，尽管并非全部。这些发现突出了PV中间神经元作为NRG/ERBB4信号的关键联系的作用，并支持该途径的可能贡献，其基因与精神分裂症的风险增加有关，与基本的精神疾病的病理生理学联系在一起。 3。NRG/ERBB4和多巴胺D4受体信号在PV神经元中收敛并调节γ振荡：我们先前报道了NRG/ERBB4信号传导调节海马诱导的海马γ振荡的功率。由于NRG1在海马中大大增加了细胞外多巴胺水平，因此我们研究了海马γ振荡中NRG/ERBB与多巴胺信号传导之间的关系。在与Fisahns Lab博士合作的情况下，我们使用了选择性的激动剂来用于增加（D1和D5受体）或减少（D2，D3和D4受体）营地的合成的受体，并发现仅D4受体的激动剂（PD168077）（PD168077）的增强功率。相比之下，D1/D5和D2R/D3受体的激动剂无效。 PD168077诱导的γ振荡能力的增加完全被高度特异性的D4受体拮抗剂（L-745,870）所阻断，进一步强调了该受体对神经元网络活性的重要性。重要的是，我们发现L-745,870和氯氮平是一种优先针对D4受体的抗精神病药，也阻止了NRG1的γ振荡能力的增加。使用双重原位杂交（与U. Winzer-Serhan博士合作）和免疫荧光组织化学，我们表明海马D4受体mRNA和蛋白质在GABA能中神经元的子集中最高。重要的是，D4和ERBB4受体在PV+快速加速篮细胞中共表达，这是一种靶向的GABA能中间神经元，对于调节γ振荡至关重要。 D4和ERBB4受体信号之间的这种新颖的串扰，以增强γ振荡能力，以及它们在PV阳性中间神经元中的共表达，这表明一种细胞机制，可能在影响认知控制的不同精神疾病中受到损害。 4。神经结合蛋白直接降低海马ERBB4-表达中间神经元中的电压门控钠电流：我们的主要目标之一是研究NRG1是否以及如何直接影响ERBB4阳性（ERBB4+）interneurons的内在特性。为此，我们诉诸于使用分离的海马培养物，这些海马培养物没有所有海马预注射传入连接，并在药理学上阻止了所有突触活动。我们最初使用整个细胞电流钳记录分析了NRG1对Ectodomain靶向抗体在实时标记中鉴定的ERBB4+和ERBB4-神经元的兴奋性的影响。我们发现NRG1通过移动动作电位阈值而降低了ERBB4+的发射，但不能减少ERBB4-神经元。使用电压钳记录，我们确定这些作用主要归因于降低电压门控钠通道活性，因为在NRG1处理20分钟后，电流密度减弱了60％。这是第一个确定NRG1在表达ERBB4表达中间神经元电压门控钠通道功能的直接作用的研究，从而为NRG1/ERBB4信号如何直接影响GABA能中神经元的活性并潜在影响兴奋性/抑制性平衡。 5。NRG1信号传导直接影响谷氨酸能ERBB4表达小脑颗粒细胞（CGC）的AMPA受体（AMPAR）活性：先前的研究表明，NRG1在主要谷氨酸性神经元中的谷氨酸性神经元中的谷氨酸突出中的可塑性如何间接影响Hippipocampus和Hippipocampus和Hippocampus的谷氨酸性突触。我们分析了NRG1与C. Fenster博士合作开发培养的CGC的影响。这些培养物主要由表达ERBB4的颗粒神经元组成。我们发现，NRG1不会影响全细胞AMPAR或NMDAR介导的电流，也不会影响自发的NMDAR-或AMPAR介导的微型兴奋性兴奋性兴奋性后突触电流，在体外生长10-12天的CGCS的基线条件下。 然而，我们发现高甘氨酸在CGC中诱导了一种化学增强（ChemLTP）的形式，其特征是AMPAR-MEPSC频率增加，而NRG1处理降低。我们的数据表明，NRG1效应是通过CGC中的GLUA4亚基介导的。 这项研究首次表明，高甘氨酸可以在CGC中诱导谷氨酸能突触的可塑性，并且急性NRG1/ERBB信号传导可以以类似于SC-CA1突触的方式调节CGC中的谷氨酸能塑性。 6。理解NRG/ERBB4近端信号传导目标的蛋白质组学方法：与S. Markeys Group（NIMH）合作，发现NRG/ERBB4的潜在新颖和直接生物学靶标，我们正在采用无偏置的液态色谱量表质量光谱法（LC/MS/MS/MS）的方法来确定ORB ORB 4的构图。我们最近报道了使用我们的受体mAb进行亲和力纯化ERBB4复合物及其分析的验证。我们正在使用类似的方法来分离NRG-1刺激后代谢活性突触体的内源性ERBB4复合物。使用这种方法，我们发现NRG/ERBB4信号传导改变了与ERBB4相关的细胞微区的离子通道组成。我们的目标是确定这些改变的功能后果。