How does the autism-related protein Shank contribute to the regulation of neuronal spine plasticity?

神经元棘突是一种稳定的结构，也可以在突触激活时迅速重排。可塑性和稳定性之间的微妙平衡支持学习和记忆，而即使是微小的平衡失调也会导致神经疾病，如自闭症谱系障碍(ASD)。脊椎的可塑性是由突触后密度(PSD)及其邻近区域中复杂的分子相互作用网络控制的，但人们对这些分子的相互作用知之甚少。SHANK3是支持突触受体的PSD核心的结构部分。它的缺失和突变与不同形式的ASD有关，在培养的神经元中，Shank3对脊椎和PSD的结构和动力学有影响。我们在Shank3中发现了一个新的SPN结构域，解决了结构域的问题，并证明了它与RAS家族GTP酶的相互作用。Shank3与RAS和Rap信号通路的这种意想不到的直接联系导致了一个令人兴奋的假说，即Shank3参与了信号传递过程，我们在非神经细胞中进行了跟踪，以证明整合素黏附受体调节的新机制。这项博士研究的重点是神经元中的Shank3信号。我们假设Shank3与RAS家族GTP酶的连接是一种新的脊椎调节的分子机制。我们将结合神经元的结构分析和显微镜实验来验证这一假说。1.Shank3与Ras GTP酶是如何相互作用的？我们将利用核磁共振和X射线结晶学来解析Shank3 SPN与GTP酶的复合体的结构。我们将使用等温量热法(ITC)来定义其他Shank3结构域是否影响相互作用。我们将设计调节细胞内Shank3相互作用的突变体，并设计通过荧光来监测细胞内相互作用的探针。Shank3与Ras GTP酶的相互作用如何影响脊柱的可塑性？我们将Shank3突变体和荧光探针导入不同发育阶段的皮质神经元培养物中，并像以前一样通过荧光显微镜监测蛋白质的定位和相互作用，以及对脊柱的结构影响。然后，我们将使用透射电子显微镜和SBFSEM来进一步量化脊柱的依赖性，以及PSD、形态和数量对Shank3与GTP酶相互作用的影响。战略研究优先该项目符合BBSRC的研究优先事项2--健康的生物科学，研究控制中枢神经系统早期发育和在整个生命周期中维持健康状态的基本神经机制。该项目的重点是支持学习和记忆的突触可塑性和稳态，通常随着年龄的增长而恶化。该项目通过加深我们对神经元脊柱重塑的分子基础的理解，将为开发治疗策略和药物以纠正与年龄或疾病相关的神经退行性变提供洞察力。该项目完全符合要求或优先事项4-通过提供从分子到神经细胞的广泛研究方法的培训，探索新的工作方式，并为学生提供操作利物浦和纽卡斯尔研究设施的最新仪器的实用技能。这名学生将与核磁共振中心(利物浦)的结构生物学家团队和纽卡斯尔(NewCastle)的ION神经生物学家团队一起工作，并与国际合作研究团队互动，并参观他们在德国和法国的实验室。该项目的培训将解决目前缺乏的多学科专家，这些专家拥有现代生物学研究所需的分子和细胞知识，并将基础知识转化为新的疗法。

Neuronal spines are stable structures that can also rapidly rearrange in response to synaptic activation. The fine balance between plasticity and stability supports learning and memory, while even a small upset of the balance leads to neurological diseases such as autism spectrum disorder (ASD). Spine plasticity is controlled by a complex network of molecular interactions in the postsynaptic density (PSD) and its proximity that are poorly understood. Shank3 forms a structural part of the PSD core supporting synaptic receptors. Its deletion and mutations are associated with different forms of ASD, and Shank3 effect on spine and PSD structure and dynamics was demonstrated in cultured neurons. We have discovered a new SPN domain in Shank3 and solved the domain structure and demonstrated its interaction with Ras-family GTPases. This unexpected direct connection of Shank3 to Ras and Rap signalling pathways lead to an exciting hypothesis of Shank3 involvement in signalling processes that we followed up in non-neuronal cells to demonstrate a novel mechanism of integrin adhesion receptor regulation. This PhD proposal focuses on Shank3 signalling in neurons.We hypothesise that Shank3 connection to Ras-family GTPases serves as a novel molecular mechanism of spine regulation. We will use a combination of structural analysis and microscopy experiments in neurons to test this hypothesis. 1. How does Shank3 interact with Ras GTPases?We will use NMR and X-ray crystallography to solve the structures of Shank3 SPN complexes with GTPases. We will use isothermal calorimetry (ITC) to define whether other Shank3 domains affect the interactions. We will design mutants that modulate Shank3 interactions in cells, and probes to monitor the interactions in cells by fluorescence.2. How does Shank3 interaction with Ras GTPases affect spine plasticity?We will transfect Shank3 mutants and fluorescent probes into cortical neuronal cultures at different stages of development and monitor the protein localisation and interactions, and the structural effects on spines by fluorescent microscopy as we have done previously. We will then use transmission electron microscopy and SBFSEM to further quantify the dependence of spine, as well as PSD, morphology and number on Shank3 interactions with GTPases.Strategic Research PrioritiesThe project fits into BBSRC research priority 2 - bioscience for health, investigating fundamental neuronal mechanisms that control development of central nervous system early in life and maintenance of the healthy state throughout the lifespan. The project focuses on the synaptic plasticity and homoeostasis that support learning and memory, often deteriorating with age. The project, by furthering our understanding of the molecular basis of neuronal spine remodelling, will provide insight to the development of therapeutic strategies and agents to correct age- or disease-related neurodegeneration. The project fully meets the requirements or the priority 4 - exploring new ways of working by delivering training in the wide range research methods spanning from molecules to neuronal cells and giving student practical skills in operating latest instruments of the research facilities of Liverpool and Newcastle. The student will work alongside teams of structural biologists in the NMR Centre (Liverpool) and neurobiologists in IoN (Newcastle), as well as interacting with the international collaborative research teams and visiting their laboratories in Germany and France. The training in the project will address the current shortage of multidisciplinary experts that have both molecular and cellular knowledge required to modern biology research and translation of fundamental knowledge into new therapies.