Thesis Format
Integrated Article
Degree
Doctor of Philosophy
Program
Neuroscience
Supervisor
Bérubé, Nathalie G.
Abstract
Mutations in the ATRX gene cause forms of intellectual disability. As with other neurodevelopmental disorder genes, ATRX has largely been studied with a focus on neurons. Astrocytes, the most abundant glial cells, are now recognized as major players in synaptic transmission and cognition. However, the role of ATRX in astrocytes has not yet been investigated. We initially optimized a fast and reliable method to obtain fluorescent-tagged glial nuclei suitable for RNA sequencing and chromatin related techniques. In the second portion of the study, mice with inducible Atrx deletion in astrocytes (Atrxf/y;GlastCreERor “ATRX aiKO”) were generated to understand the contribution of astrocytic ATRX in cognition. Analysis of the hippocampal transcriptome of control and mutant mice revealed potential disruption in astrocytic cytoskeletal dynamics regulation and suggested a mild inflammatory response. Moreover, genes involved in neuronal function were also altered, suggesting that loss of ATRX in astrocytes influences neighbouring neurons at the transcriptional level. Evaluation of the astrocytic transcriptome confirmed changes in mRNA levels of genes involved in cytoskeletal regulation, immune activation, and metabolic function. Genome-wide chromatin accessibility profiles of ATRX-null astrocytes revealed a generally more open chromatin state. Chromatin accessibility was increased in genic regulatory regions like promoters and in binding sites for CTCF (a regulator of chromatin topology) and transcription factors implicated in interferon signaling. At the electrophysiological level, we showed that loss of ATRX in astrocytes leads to hyperexcitability and decreased capacitance of hippocampal CA1 neurons but no changes in dendrite number, length, and branching and only minor alterations in spine shape. Finally, behaviour testing of mutant mice demonstrated that loss of ATRX in astrocytes does not affect learning in the animals and disrupts specific types of long-term spatial and recognition memory. Together this study identifies important neuronal and cognitive dysfunction caused upon disruption of ATRX-mediated chromatin topology in astrocytes. This work also emphasizes the contributions of glial cells and particularly astrocytes to disorders like ATR-X syndrome.
Summary for Lay Audience
Chromatin is a DNA and protein complex that regulates how genetic information is packaged in cells. Chromatin is usually in two configurations: 1) open and available, when access to the information is required and 2) compact and inaccessible to prevent damage or miscommunication. ATRX is a chromatin remodeler that can regulate the balance between these configurations. Mutations in the ATRX gene cause intellectual disability, and the role of ATRX in brain development has been previously studied with a focus on neurons. However, the brain is made of different cell types including astrocytes. These cells do several jobs to support neuronal function, like providing nutrients, regulating neuronal communication, and maintaining brain structure. In this study, I hypothesized that ATRX is necessary for the normal function of astrocytes in the context of learning and memory. For my experiments, I used a mouse in which ATRX is absent only in astrocytes (an astrocyte-specific ATRX knockout mouse). To study astrocytic chromatin, I first optimized an approach that uses genetic tools to mark the nuclei of astrocytes allowing purification and analysis of the astrocyte nuclei. Using this approach, I determined that in the absence of ATRX the chromatin in astrocytes is overall more open, and that expression of genes linked to cytoskeleton regulation, immune response and metabolism are deregulated. We used brain slices to show that astrocytes lacking ATRX affect the neighbouring neurons, making them hyperresponsive to incoming signals. Finally, a battery of behaviour tests identified long-term memory deficits in the astrocyte-specific ATRX knockout mice. Collectively, this work shows that ATRX deficiency in astrocytes may contribute to some aspects of the ATR-X syndrome and could be potential therapeutic targets.
Recommended Citation
Pena-Ortiz, Miguel A., "Exploring the role of the ATRX chromatin remodeling protein in mouse astrocytes" (2023). Electronic Thesis and Dissertation Repository. 9666.
https://ir.lib.uwo.ca/etd/9666
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.
Included in
Behavioral Neurobiology Commons, Cell Biology Commons, Molecular and Cellular Neuroscience Commons, Molecular Biology Commons