The role of TRIO signaling in neuronal development, synaptic function, and circuit connectivity

摘要 TRIO基因中的杂合性功能丧失(LOF)或破坏性变异与 精神分裂症和自闭症谱系障碍的风险增加。然而，TRIO在神经元中的功能作用 生物学和电路功能还不是很清楚，这限制了这些疾病的治疗进展。 TRIO作用于细胞表面受体的下游，控制轴突和树突的寻路，突触的发育， 和突触传递。小鼠皮质兴奋性神经元中单个三对等位基因的缺失导致神经元数量减少 在皮质神经纤维和树突和突触的发育和功能缺陷，产生社会和运动 缺陷、焦虑和强迫症的增加。然而，特定的Trio突变和 皮质功能的后续后果尚不清楚。在这里，我们将整合广泛的高度 互补的跨学科方法，包括遗传学、生物化学和蛋白质组学、光遗传学 突触功能的分析，以及解决这一问题的皮质网络动力学的多模式活体成像 问题。 我们的第一个目标是确定TRIO调节皮质神经元发育的生化机制。 我们确定了几个新的候选Trio信令合作伙伴(PDE4A5、L1CAM和 LGI1/ADAM22/ADAM23复合体)，并将阐明它们如何与TRIO相互作用来调节皮质神经元 树枝、树突棘和突触发育。我们还培育出了杂合子的CRISPR小鼠 三个与障碍相关的三重变种-K1431M(自闭症)，K1918X(精神分裂症)，M2145T(双相情感障碍)- 这对TRIO的生化活动产生了不同的影响，并产生了不同的解剖和行为表型。 我们将使用基于质谱学的比较蛋白质组学来发现不同的新的信号伙伴 受这些离散的三对等位基因的影响。 我们的第二个目标将确定不同的Trio变体如何影响神经元连接和突触功能。 我们将通过测量来评估我们的三个CRISPR变体对皮质神经元发育的影响 它们如何影响轴突、树突和突触发育、突触传递和可塑性。我们还将 使用病毒Cre介导的稀疏三联体干扰和全细胞录音来测试哪些缺陷反映了细胞- 自主与网络级别的影响。 我们的第三个目标是测试TRIO的变化如何影响体内皮质网络的功能组织， 利用我们最近开发的将单细胞和介观成像相结合的策略 GCaMP6标记的神经元用来测量清醒的、行为正常的小鼠的电路组织。我们的总体目标是 了解TRIO功能改变如何在细胞、突触和网络水平影响神经元功能， 为理解遗传失调如何驱动行为变化提供了一个广泛的框架。

Abstract Heterozygous loss-of-function (LOF) or damaging variants in the TRIO gene are associated with increased risk for schizophrenia and autism spectrum disorders. However, the functional role of TRIO in neuronal biology and circuit function are not well understood, which limits the advance of therapies for these disorders. TRIO acts downstream of cell surface receptors to control axon and dendrite pathfinding, synapse development, and synaptic transmission. Deletion of a single TRIO allele in mouse cortical excitatory neurons drives reductions in cortical neuropil and defects in dendrite and synapse development and function, yielding social and motor deficits and increased anxiety and compulsivity. However, the links between specific TRIO mutations and subsequent consequences for cortical function are unknown. Here, we will integrate a broad array of highly complementary, interdisciplinary approaches including genetics, biochemistry and proteomics, optogenetic analysis of synaptic function, and multimodal in vivo imaging of cortical network dynamics to address this question. Our first aim will identify the biochemical mechanisms by which TRIO regulates cortical neuron development. We identified several new candidate TRIO signaling partners (PDE4A5, L1CAM, and the LGI1/ADAM22/ADAM23 complex) and will elucidate how they interact with TRIO to regulate cortical neuron dendritic arbor, dendritic spine, and synapse development. We also generated CRISPR mice heterozygous for three disorder-related TRIO variants - K1431M (autism), K1918X (schizophrenia), M2145T (bipolar disorder) - that differentially impact TRIO’s biochemical activities and yield different anatomical and behavioral phenotypes. We will use mass spectrometry-based comparative proteomics to discover new signaling partners differentially impacted by these discrete TRIO alleles. Our second aim will determine how different TRIO variants impact neuronal connectivity and synaptic function. We will assess the consequences of our TRIO CRISPR variants for cortical neuron development by measuring how they impact axon, dendrite, and synapse development, synaptic transmission and plasticity. We will also use viral Cre-mediated sparse TRIO disruption and whole cell recordings to test which deficits reflect cell- autonomous versus network level effects. Our third aim will test how alterations in TRIO impact the functional organization of cortical networks in vivo, taking advantage of our recently developed strategies for combining single cell and mesoscopic imaging of GCaMP6-labeled neurons to measure circuit organization in awake, behaving mice. Our overall goal is to understand how altered TRIO function impacts neuronal function at the cellular, synaptic, and network levels, providing a broad framework for understanding how genetic dysregulation drives changes in behavior.