Imaging macroscopic cortical dynamics to understand sensorimotor dysfunction and recovery in a mouse model of Rett Syndrome

Rett综合征是一种严重的神经系统疾病，由编码甲基CpG结合蛋白2（MeCP 2）的X连锁基因突变引起，甲基CpG结合蛋白2是一种调节大脑发育和功能的DNA结合蛋白。Rett综合征的患病率估计为1/10 000活产女婴，受影响的女孩通常在出生后第二年经历停滞期之前表现出正常的早期产后发育。雷特综合征的标志性症状包括交流和运动技能的丧失，包括有目的的手部运动、呼吸异常和早发性癫痫发作。虽然大多数Rett综合征患者都能活到成年，但他们的生活质量受到严重影响，需要24小时护理。啮齿动物模型的产生和可用性加速了对Rett综合征病理生理学的理解，这些模型概括了患者表型的发育时间轴和谱。概念验证临床前研究表明，重新引入有缺陷的Mecp 2基因逆转了与Rett综合征相关的一些衰弱表型，给家庭带来了希望，即基因治疗可以提供“金子弹”。然而，有迹象表明，重新表达缺失的MeCP 2蛋白可以恢复一些身体和大脑功能，但不是全部，这表明逆转可能只是表面的。在我们对Rett综合征的理解中，缺少的是MeCP 2的丢失如何影响全脑交流和行为，以及它的重新引入是否可以驱动高水平的脑功能和表型拯救。解决这个问题的一个主要进展是最近开发的光学方法，允许同时可视化的神经活动跨多个大脑区域的啮齿动物使用遗传编码的钙传感器。我们的目标是使用这种方法来确定MeCP 2的丢失如何影响休息时的区域间神经动力学，同时小鼠学习一种新的基于触摸屏的视觉到达任务。通过对野生型和MeCP 2突变小鼠的活动进行成像，我们将揭示导致高水平脑功能和行为缺陷的全脑通信故障的主要机制。由于雌性MeCP 2突变小鼠显示MeCP 2在整个大脑中的镶嵌表达（即~40-80%的细胞表达MeCP 2），我们将使用第二种成像方法，该方法使用光片来生成全脑MeCP 2表达图。这将使我们能够将蛋白质表达的变化与破坏的全脑神经动力学和行为缺陷联系起来。我们将通过询问使用病毒介导的重新激活重新表达MeCP 2是否可以驱动全脑神经动力学的重组以及高水平脑功能和行为的恢复来跟进。为此，我们将优化整个大脑神经元中非侵入性基因再激活的方法，并将采用光学成像方法来绘制MeCP 2重新表达，神经活动重组和感觉运动学习恢复的程度。最终，我们的研究将提供新的见解，了解MeCP 2的丢失如何导致学习过程中的区域间沟通障碍，以及它的重新引入在多大程度上挽救了高水平的大脑功能和行为。这项工作有可能通过我们与国际Rett综合征基因治疗联盟（International Rett Syndrome Gene Therapy Consortium）的持续联系直接影响转化医学，该联盟致力于开发Rett综合征的改进和有效治疗方法。

Rett syndrome is a severe neurological disorder resulting from mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2), a DNA binding protein that regulates brain development and function. The prevalence of Rett syndrome is estimated at 1 in 10,000 live female births where affected girls typically exhibit normal early postnatal development before experiencing a stagnation phase during the second year of life. The hallmark symptoms of Rett syndrome include loss of communication and motor skills, including purposive hand movements, breathing abnormalities and early onset seizures. Although most individuals with Rett syndrome live into adulthood their quality of life is severely impacted and they require 24-hour care. Progress in understanding the pathophysiology of Rett syndrome has been accelerated by the generation and availability of rodent models that recapitulate the developmental timeline and spectrum of patient phenotypes. Proof-of-concept pre-clinical studies demonstrate that re-introduction of the defective Mecp2 gene reverses some of the debilitating phenotypes associated with Rett syndrome giving hope to families that gene therapy could provide the 'golden bullet'. However, there are indications that re-expressing the missing MeCP2 protein recovers some body and brain functions, but not all, suggesting reversal may only be superficial. What is missing in our understanding of Rett syndrome is how loss of MeCP2 affects brainwide communication and behaviour and whether its reintroduction can drive high level brain function and phenotypic rescue. A major advance towards addressing this issue has been the recent development of optical methods which allow the simultaneous visualisation of neural activity across multiple brain regions in rodents using genetically encoded calcium sensors. We aim to use this method to establish how loss of MeCP2 affects inter-areal neural dynamics both at rest and while mice learn a novel touchscreen-based visuomotor reaching task. By imaging activity in wild type and MeCP2-mutant mice we will uncover the principal mechanism of brainwide communication breakdown that lead to deficits in high-level brain function and behaviour. Since female MeCP2-mutant mice show a mosaic expression of MeCP2 across the brain (i.e. ~40-80% of cells express MeCP2), we will use a second imaging approach that uses sheets of light to generate whole brain MeCP2 expression maps. This will allow us correlate changes in protein expression with disrupted brainwide neural dynamics and behavioural deficits. We will follow this up by asking whether reexpression of MeCP2 using viral-mediated reactivation can drive reorganisation of brainwide neural dynamics and recovery of high-level brain function and behaviour. To do this we will optimise methods for non-invasive gene reactivation in neurons across the brain and will employ optical imaging methods to map the extent of MeCP2 re-expression, reorganisation of neural activity and recovery of sensorimotor learning. Ultimately our research will provide new insights into how loss of MeCP2 leads to inter-areal communication breakdown during learning and the extent to which its reintroduction rescues high-level brain function and behaviour. This work has the potential to directly influence translational medicine through our ongoing links with the International Rett Syndrome Gene Therapy Consortium who strive to develop improved and effective treatments for Rett syndrome.