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Cell-type Specific Mechanisms and Functional Consequences of Altered NMDA Receptor Development and Mecp2 Deficiency on Developing Cortical Circuits

Periodic Reporting for period 1 - NMDARETT (Cell-type Specific Mechanisms and Functional Consequences of Altered NMDA Receptor Development and Mecp2 Deficiency on Developing Cortical Circuits)

Reporting period: 2016-04-01 to 2018-03-31

Rett syndrome is a debilitating disorder that affects 1 out of 10,000 girls born each year. Affected children develop normally for the first 6-18 months of life prior to a devastating loss of the ability to speak, socialize and move. The genetic cause of Rett syndrome is known; however, there is no cure. To discover how the genetic changes lead to the loss of brain function, we use mice with the same genetic change. These mice also have an initial normal development after birth followed by a loss of brain function. Studying these mice will help us understand the disease and develop new treatments. One of the changes observed in the mouse brain is that the genetic change that causes Rett syndrome has opposing effects on the maturation of two different types of brain cells. In my project, I am using new single cell sequencing technologies to identify the mechanisms underlying the effect on brain development. I also grow brain cells in a dish and observed how they form networks of cells with and without the genetic change in Rett syndrome. My goal is to identify new treatments for Rett syndrome and to test their efficacy on the maturation of brain function. Increasing our knowledge of how brain dysfunction occurs in Rett syndrome and some causes of autism will also benefit patients, families and clinicians throughout Europe.
In our research, we seek to understand how brain cells form the complex networks in the brain that underlie all thought and action. How brain cells will form connections with each other over development is largely determined by our genes. Many of the genetic changes identified in different causes of autism and Rett syndrome affect the communication between brain cells. Our research is focused on understanding how the genetic changes in Rett syndrome and some causes of autism affect brain function. For this project, we employed state-of-the-art techniques for collecting single brain cells from mice that have the genetic change found in Rett syndrome and some causes of autism. We used single-cell RNA sequencing to compare which genes are turned on and off in two types of brain cells and how this is altered by the genetic change in Rett syndrome. This results will help us understand understand how the genetic change in Rett syndrome causes problems with brain function and identify new avenues for treatments. To determine how the genetic changes in Rett syndrome alter the communication in brain cells, we also grew brain cells in a dish from mice with and without the genetic change in Rett syndrome. We applied two techniques for monitoring how neurons form functional connections with each other. In the first method, we grew the cells directly on a grid of recording electrodes. This allows us to observe spontaneous activity in the network of cells at multiple time points over early development. We found that the activity in the network of brain cells increases over development and becomes more organised in brain cells. We developed new methods with other scientists from math, engineering and computer science to understand the complex dynamics of network function and how these processes are disturbed during development by the genetic changes in Rett syndrome. We also applied a technique called two-photon calcium imaging in which a fluorescent dye is applied to the brain cells that changes its brightness in relation to the level of brain cell activity. Using this technique, we also saw that network activity increases over development in the brain cells and how brain cells synchronise their responses to form clusters within the larger network of brain cells. With this research, we are identifying how the genetic changes in Rett syndrome and some causes of autism impact network development. Our ultimate goal is to combine these two parts of the research to test new drugs--targeting genes identified from the single cell RNA sequencing--on the developing brain cell networks to improve function. This may provide new treatment strategies for improving the lives of people with Rett syndrome and some causes of autism.
In this project, I developed expertise in cutting-edge single-cell RNA sequencing including using laser capture microscopy to collect two specific types of brain cells from a precise region of the brain. With these results, I will be able to identify new drug targets that may eventually lead to new treatments for people with Rett syndrome and some causes of autism. I also developed expertise in recording network function using two techniques: multi-electrode array recordings and two-photon calcium imaging. These are powerful tools for studying how brain cells for networks and for testing the effect of potential drugs to improve network function in mouse models of Rett syndrome and autism. The ultimate goal of my research is to create a platform for testing novel therapies for Rett syndrome and some cases of autism using network function at the cellular scale as an assay. This has the potential to increase our understanding of how the genetic change in Rett syndrome and some cases of autism lead to the decline in brain function. It also has the potential to identify new therapies to improve the lives of people with Rett syndrome and their families. I am continuing this research as a University Lecturer at the University of Cambridge. Through the research and training in this mentored project, I have expanded my research skills, gathered preliminary data to apply for grants to continue the research, and advanced from a postdoctoral to faculty position. I will use these skills and position to continue my research focused on understanding how the genetic changes in Rett syndrome and some causes of autism affect brain function and developing novel therapeutic strategies. I will share my findings with the scientists and clinicians through publishing the research in biomedical journals. I am also sharing my research with the general public through giving public lectures on autism at the annual Cambridge Science Festival and other events that enable members of the public to engage with researchers.
Brain cells labelled with fluorescent markers for monitoring network activity.