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Investigations into novel functions of JNK in neurons: Regulation of post-synaptic scaffold proteins by JNK3

JNK 在神经元中的新功能研究：JNK3 对突触后支架蛋白的调节

基本信息

批准号：
261102178
负责人：
Professorin Dr. Sarah A. Shoichet
金额：
--
依托单位：
Neurowissenschaftliches Forschungszentrum (CCM)
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2014
资助国家：
德国
起止时间：
2013-12-31 至 2019-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/261102178?language=en
关键词：
Investigations into novel functions JNK

项目摘要

In our previously published work, we have detected de novo truncations of the MAPK10 gene, encoding the kinase JNK3, in patients with cognitive disorders. We demonstrated that the truncated proteins lack normal catalytic activity, which supports our hypothesis that loss of normal JNK3 contributes to the disease phenotype in the patients, and provides the basis for our studies on novel functions of JNK3 in neurons, which we describe in detail in this proposal. In summary, we have shown that several disease-associated synaptic scaffold proteins, including PSD-95 and SAP102, as well as Shank- and CRMP-family proteins, are able to interact with JNK3. Moreover, we have identified a novel JNK phosphorylation site in SAP102 and generated an antibody that specifically recognises the phosphorylated form. We will use this antibody, together with phospho-specific antibodies for PSD-95, to assess the status of endogenous phosphorylated SAP102 and PSD-95 in neurons under different conditions. We will also further delineate the nature of the JNK-PSD-95/SAP102 interaction, and investigate how JNK influences the scaffolding properties of these molecules. We additionally hypothesise that phosphorylation of both of these synapse-associated proteins by JNK is critical for their subcellular localisation, potentially in response to synaptic activity. Given the location of JNK binding and phosphorylation of PSD-95 and SAP102, specific protein-protein interactions and subsequent signalling may also be affected by these events. We will investigate how JNK phosphorylation of SAP102 influences its binding to selected neuronal proteins, including e.g. Nedd4, which has been implicated in SAP102 mono-ubiquitination. Using photo-activation and photo-bleaching strategies, we will also assess the impact of JNK regulation on the mobility of this scaffold molecule into and out of dendritic spines. We will further assess the effects of JNK regulation on AMPA receptor surface expression and on AMPAR- and NMDAR-mediated currents. For all of these experiments, we will take advantage of virus-mediated gene delivery for expression of proteins in primary neurons, and we will make use of commercially available small molecule and peptide inhibitors. We will also make use of our tagged JIP1-JBD small protein inhibitor, which likewise inhibits total JNK in cell culture experiments. In the long-term, this tagged protein also can be used to generate a mouse model in which total JNK activity can be inhibited in an inducible manner, thereby establishing a system that would enable investigations into JNK regulation of synaptic scaffold molecules in vivo.

在我们以前发表的工作中，我们已经检测到在认知障碍患者中编码激酶JNK 3的MAPK 10基因的从头截短。我们证明了截短的蛋白质缺乏正常的催化活性，这支持了我们的假设，即正常JNK 3的丧失有助于患者的疾病表型，并为我们研究JNK 3在神经元中的新功能提供了基础，我们在本提案中详细描述了这一点。总之，我们已经证明了几种疾病相关的突触支架蛋白，包括PSD-95和SAP 102，以及Shank和CRMP家族蛋白，能够与JNK 3相互作用。此外，我们已经在SAP 102中鉴定了一个新的JNK磷酸化位点，并产生了特异性识别磷酸化形式的抗体。我们将使用这种抗体，与磷酸化特异性抗体PSD-95，以评估在不同条件下的神经元内源性磷酸化SAP 102和PSD-95的状态。我们还将进一步描述JNK-PSD-95/SAP 102相互作用的性质，并研究JNK如何影响这些分子的支架特性。我们还假设JNK对这两种突触相关蛋白的磷酸化对它们的亚细胞定位至关重要，可能是对突触活动的响应。考虑到JNK结合和PSD-95和SAP 102磷酸化的位置，特定的蛋白质-蛋白质相互作用和随后的信号传导也可能受到这些事件的影响。我们将研究SAP 102的JNK磷酸化如何影响其与选定的神经元蛋白的结合，包括例如Nedd 4，其与SAP 102单泛素化有关。使用光活化和光漂白策略，我们还将评估JNK调节对这种支架分子进出树突棘的流动性的影响。我们将进一步评估JNK调节对AMPA受体表面表达以及对AMPAR和NMDAR介导的电流的影响。对于所有这些实验，我们将利用病毒介导的基因递送在原代神经元中表达蛋白质，并且我们将利用商业上可获得的小分子和肽抑制剂。我们还将利用我们标记的JIP 1-JBD小蛋白抑制剂，其同样在细胞培养实验中抑制总JNK。从长远来看，这种标记的蛋白质也可以用于产生小鼠模型，其中总JNK活性可以以诱导的方式被抑制，从而建立一个系统，将能够在体内研究突触支架分子的JNK调节。