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

在我们之前发表的工作中，我们已经在认知障碍患者中检测到编码JNK3激酶的MAPK10基因的从头截断。我们证明了截断的蛋白缺乏正常的催化活性，这支持了我们的假设，即正常JNK3的缺失导致了患者的疾病表型，并为我们研究JNK3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SAP102，以及Shank和crmp家族蛋白，能够与JNK3相互作用。此外，我们在SAP102中发现了一个新的JNK磷酸化位点，并产生了一种特异性识别磷酸化形式的抗体。我们将使用该抗体与PSD-95的磷酸化特异性抗体一起，评估不同条件下神经元内源性磷酸化的SAP102和PSD-95的状态。我们还将进一步描述JNK- psd -95/SAP102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，这可能是对突触活动的反应。考虑到JNK结合的位置以及PSD-95和SAP102的磷酸化，特异性蛋白-蛋白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SAP102的JNK磷酸化如何影响其与选定的神经元蛋白的结合，包括Nedd4，它与SAP102的单泛素化有关。利用光激活和光漂白策略，我们还将评估JNK调控对支架分子进出树突棘的移动性的影响。我们将进一步评估JNK调控对AMPA受体表面表达以及AMPAR和nmda介导电流的影响。对于所有这些实验，我们将利用病毒介导的基因传递来表达初级神经元中的蛋白质，我们将利用市售的小分子和肽抑制剂。我们还将使用我们标记的JIP1-JBD小蛋白抑制剂，同样在细胞培养实验中抑制总JNK。从长远来看，该标记蛋白也可用于生成以诱导方式抑制JNK总活性的小鼠模型，从而建立一个能够在体内研究JNK对突触支架分子调节的系统。

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.