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）或损伤性变体与以下疾病相关： 精神分裂症和自闭症谱系障碍的风险增加。然而，TRIO在神经元细胞中的功能作用可能与TRIO在神经元细胞中的作用有关。 生物学和电路功能还没有被很好地理解，这限制了对这些疾病的治疗的进展。 TRIO作用于细胞表面受体的下游以控制轴突和树突寻路、突触发育， 和突触传递。小鼠皮层兴奋性神经元中单个TRIO等位基因的缺失导致 在皮质神经元损伤和树突和突触发育和功能的缺陷，产生社会和运动 赤字和增加焦虑和强迫症。然而，特定的TRIO突变与 随后对皮质功能的影响尚不清楚。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 突触功能分析和皮质网络动力学的多模态体内成像来解决这个问题 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。 我们鉴定了几种新的候选TRIO信号传导伴侣（PDE 4A 5、L1 CAM和TRIO信号传导受体）。 LGI 1/ADAM 22/ADAM 23复合物），并将阐明它们如何与TRIO相互作用以调节皮质神经元 树突乔木、树突棘和突触发育。我们还产生了CRISPR小鼠， 三种与疾病相关的TRIO变体-K1431 M（自闭症），K1918 X（精神分裂症），M2145 T（双相情感障碍）- 不同的影响TRIO的生化活动，并产生不同的解剖和行为表型。 我们将使用质谱为基础的比较蛋白质组学发现新的信号伙伴差异 受到这些离散的TRIO等位基因的影响。 我们的第二个目标是确定不同的TRIO变体如何影响神经元连接和突触功能。 我们将评估我们的TRIO CRISPR变体对皮层神经元发育的影响， 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TRIO破坏和全细胞记录来测试哪些缺陷反映了细胞- 自主与网络水平的影响。 我们的第三个目标是测试TRIO的改变如何影响体内皮层网络的功能组织， 利用我们最近开发的结合单细胞和介观成像的策略， GCaMP 6标记的神经元用于测量清醒的行为小鼠中的电路组织。我们的总体目标是 了解TRIO功能的改变如何在细胞，突触和网络水平上影响神经元功能， 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.