Regulation of Axonal Transport by the JNK (c-Jun N-terminal Kinase) Pathway

描述（由申请人提供）：神经元中的轴突转运对于将功能蛋白传递到突触，清除受损或错误折叠的蛋白，以及从远端轴突到细胞体的长距离信号传递至关重要。然而，轴突运输在货物识别和运输行为（速度，方向，暂停）方面的调节知之甚少。这项研究提出细胞器的运输可以通过细胞内信号的变化来调节，例如JNK （c-Jun n -末端激酶）的激活，这与应激反应和神经元死亡有关。我从神经元培养中获得的初步数据表明，JNK的药理抑制抑制了两个方向的囊泡运输，而JNK的激活增加了逆行运输的速度。介导这些转运反应的一个极好的候选者是JIP1 （JNK相互作用蛋白），它具有与JNK、顺行和逆行运动蛋白以及囊泡（通过跨膜蛋白）结合的能力。此外，JNK激活也导致JIP1磷酸化的初步观察支持了JNK诱导的JIP1磷酸化通过改变JIP1与运动蛋白之间的相互作用导致轴突转运改变的假设。为了验证这一假设，拟议的实验将使用活细胞显微镜来表征JNK信号的药理操作对特定荧光标记囊泡群体运输的影响。此外，siRNA敲低实验将用于确定这些jnk诱导的变化是否由JIP1介导。此外，共免疫沉淀将更清楚地阐明JIP1与逆行运动复合物dynein/dynactin之间的相互作用。最后，突变体非磷酸化JIP1构建体将被用来确定JIP1磷酸化是否为jnk诱导的转运变化的机制。这些实验将进一步证明轴突运输可以在货物水平上通过响应信号通路激活的翻译后修饰来调节。更好地了解损伤信号和转运之间的联系将为神经退行性疾病中转运失调的作用提供新的见解。

DESCRIPTION (provided by applicant): Axonal transport in neurons is essential for the delivery of functional proteins to the synapse, for clearance of damaged or misfolded proteins, and for long-distance signaling from the distal axon to the cell body. However, regulation of axonal transport in terms cargo recognition and transport behavior (speed, direction, pausing) is poorly understood. This study proposes the idea that transport of organelles can be modulated by changes in intracellular signaling, such as activation of JNK (c-Jun N-terminal kinase), which has been implicated in stress response and neuronal death. My preliminary data from neuronal cultures shows that pharmacological inhibition of JNK inhibits vesicular transport in both directions and that activation of JNK increases the speed of retrograde transport. An excellent candidate for mediating these transport responses is JIP1 (JNK-interacting protein), which has the ability to associate with JNK, both anterograde and retrograde motor proteins, and vesicles (via transmembrane proteins). In addition, the preliminary observation that JNK activation also leads to phosphorylation of JIP1 supports the hypothesis that JNK-induced phosphorylation of JIP1 leads to changes in axonal transport by altering the interaction between JIP1 and motor proteins. To verify this hypothesis, the proposed experiments will use live-cell microscopy to characterize the effects of pharmacological manipulation of JNK signaling on the transport of specific fluorescently labeled vesicle populations. Also, siRNA knockdown experiments will be used to determine whether these JNK-induced changes are mediated by JIP1. Further, coimmunoprecipitations will more clearly elucidate the interaction between JIP1 and the retrograde motor complex, dynein/dynactin. Finally, mutant nonphosphorylatable JIP1 constructs will be used to determine whether JIP1 phosphorylation is the mechanism responsible for these JNK-induced changes in transport. These experiments will further the idea that axonal transport can be regulated at the cargo level via post-translational modification in response to signaling pathway activation. A better understanding of the connection between injury signaling and transport will provide new insight into the role of transport dysregulation in neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: Neurons are specialized cells that contain long processes called axons along which electrical information travels. In order to maintain normal neuronal function, proteins and organelles must be transported at great distances along these axons to and from the cell body. This project investigates the role of an injury signaling pathway in regulating axonal transport properties, such as which cargos are transported and how fast they move, and will provide insight into understanding the role of axonal transport in neuronal injury and neurodegeneration.