Identifying mechanisms underlying forebrain pathology in human PAX6 haploinsufficiency

人脑是一个由数十亿个神经元组成的复杂结构，这些神经元必须以精确的方式相互连接，才能形成功能性的神经回路，使大脑正常运作。为了使这成为可能，必须产生正确数量和类型的神经元，使其成熟，然后彼此形成适当的连接。这通常发生在胎儿大脑发育期间。这些发育过程的指令由基因提供。一个众所周知的调节早期神经发育的基因是PAX6。PAX6蛋白的作用是开启或关闭正常大脑发育所需的其他基因。在PAX6单倍不足中，PAX6的两个拷贝中的一个发生突变。患者主要患有先天性无虹膜，这是一种罕见的眼部疾病，其特征是虹膜全部或部分丧失。然而，他们也患有大脑畸形，如大脑区域减少，不同大脑区域之间的连接缺陷，以及行为障碍，包括社会认知障碍，自闭症和智力残疾。到目前为止，PAX6单倍功能不全患者的脑缺陷仍未得到充分研究。先前在动物模型上的研究表明，PAX6对于确保大脑形成所需的正确数量和类型的神经元，以及神经元与适当的目标连接非常重要。我们假设人类PAX6突变在发育早期破坏了这些过程，导致PAX6单倍功能不全患者出现脑畸形和行为症状。我们建议通过研究PAX6突变对PAX6单倍功能不全患者大脑畸形的影响来确定PAX6单倍功能不全患者表现出症状的潜在原因。这包括：产生的神经元的数量和类型，大脑连接和由神经元连接产生的神经元回路。使用人类胚胎组织的实际和伦理限制使得研究人类PAX6的早期作用变得困难。因此，我们将使用先进的干细胞培养技术来生成3D胚胎脑组织，称为“脑类器官”，它概括了发育中的人类大脑的许多特征。我们已经设计了PAX6单倍体缺陷干细胞系，并将使用从中培养的脑类器官来确定PAX6突变如何影响胚胎大脑中正确数量和类型的神经元的产生。我们将通过识别类器官中单个细胞中表达的一系列基因来做到这一点，这将告诉我们是否产生了正确数量和类型的神经元。通过将类器官成对地融合在一起，我们将了解PAX6单倍不足如何影响它们之间神经连接的形成。用染料追踪神经连接将告诉我们是否与正确的目标建立了连接。此外，我们将通过对参与这些神经连接的突触蛋白进行染色来确定PAX6突变是否会导致连接缺陷，并使用自动图像分析管道对其进行分析。这将揭示PAX6突变如何影响神经连接的特征。最后，我们将通过电生理技术记录神经活动来研究PAX6突变对神经回路的影响。我们将使用专门影响兴奋性或抑制性神经元的药理学药物来剖析这些神经回路的特征，以揭示PAX6突变如何影响神经回路的状态。通过采用这种多方面的方法来确定PAX6单倍功能不全患者大脑畸形的潜在原因，我们可以帮助进一步研究这些原因，最终改善患者的症状。

The human brain is a complex structure comprised of billions of neurons which must connect to each other in a precise manner to form functional neural circuits and enable normal functioning of the brain. To make this possible, the correct numbers and types of neurons must be generated, mature, then form appropriate connections with one another. Much of this normally occurs during foetal brain development. Instructions for these developmental processes are provided by genes. One gene well known to regulate early neurodevelopment is PAX6. PAX6 protein acts by turning on or off other genes needed for normal brain development. In PAX6 haploinsufficiency, one of the two copies of PAX6 is mutated. Patients mainly suffer from congenital aniridia, a rare eye disease characterised by total or partial loss of the iris. However, they also suffer from brain malformations such as a decrease in brain areas, connectivity defects between different brain regions, and behavioural impairments, including impaired social cognition, autism and intellectual disability. As yet, the brain defects described in PAX6 haploinsufficiency patients remain mostly understudied.Previous work in animal models indicated that PAX6 is important for ensuring that the brain forms the correct numbers and types of neurons needed, and that neurons connect with their appropriate targets. We hypothesize that human PAX6 mutations disrupt these processes early in development, leading to brain malformations and behavioural symptoms found in PAX6 haploinsufficiency patients.We propose to identify the underlying causes of the symptoms displayed by PAX6 haploinsufficiency patients by investigating the effects of PAX6 mutations on the processes that we believe underlie the brain malformations found in PAX6 haploinsufficiency patients. This includes: the number and types of neurons generated, brain connectivity and neuronal circuits that result from neuronal connections. Practical and ethical constraints on use of human embryonic tissue makes studying the early actions of human PAX6 difficult. We shall therefore use advanced stem cell culture techniques to generate 3D embryonic brain tissue, termed 'cerebral organoids', which recapitulate many features of the developing human brain.We have engineered PAX6 haploinsufficient stem cell lines and will use cerebral organoids grown from these to determine how PAX6 mutations affect production of the correct numbers and types of neurons in the embryonic brain. We shall do this by identifying sets of genes expressed in individual cells in our organoids, which will tell us whether the correct number and type of neurons are being produced. By fusing organoids together in pairs, we shall how PAX6 haploinsufficiency affects the formation of neural connections between them. Tracing the neural connections using dyes will tell us whether connections are being made to the correct targets. In addition, we shall determine whether PAX6 mutations cause defective connections by staining for synaptic proteins, which are involved in these neural connections, and analyse them using an automated image analysis pipeline. This will reveal how PAX6 mutations affect the characteristics of the neural connections made. Finally, we shall study the effects of PAX6 mutations on neural circuits by recording neural activity with electrophysiological techniques. We shall dissect the characterisation of these neural circuits using pharmacological drugs that specifically affect excitatory or inhibitory neurons, to reveal how PAX6 mutations affect the state of neural circuits.By taking this multifaceted approach to identify the underlying causes of the brain malformations suffered by PAX6 haploinsufficiency patients, we can help inform further studies that target these causes, ultimately to ameliorate patient symptoms.