GFAP and vimentin during human brain development: A focus on Alexander Disease

Abstract

This dissertation focuses on GFAP and Alexander disease. GFAP (glial fibrillary acidic protein) is a cytoskeletal protein of the intermediate filament type that supports cell structure and stability. Mutations in the GFAP gene cause Alexander disease, a severe neurological disorder affecting primarily young children, but it can occur at any age. In younger patients, symptoms include epilepsy, muscle weakness, and loss of developmental milestones like sitting and walking. Older patients experience milder symptoms, mainly affecting posture, movement, sleep, and swallowing. The severity of the disease is related to age of onset, with younger patients experiencing more rapid brain degeneration. This degeneration results from dysfunctional astrocytes, cells that rely on GFAP to support neuronal function. In Alexander disease, these astrocytes malfunction, leading to severe brain dysfunction. While much research focuses on the defective astrocytes in Alexander disease, less is known about how GFAP mutations affect early brain development. GFAP is also present in neural stem cells called radial glia, which can proliferate or differentiate into neurons. The decision to remain a stem cell or differentiate is crucial for proper brain development. In this dissertation, I investigated how mutant GFAP affects early brain development using human models. Specifically, I used induced pluripotent stem cells (iPSCs) derived from patients with Alexander disease. iPSCs are reprogrammed patient cells that resemble embryonic stem cells and can differentiate into various cell types, making them ideal for studying disease mechanisms. I generated brain organoids—3D tissue structures that mimic early brain development—from these patient-derived iPSCs. We discovered that GFAP mutations disrupt the development of these organoids, causing them to mistakenly differentiate into heart cells rather than brain cells. This phenomenon was observed in organoids up to nine days old. Additionally, I found that this developmental issue could be modulated by using fewer iPSCs or by applying specific compounds to direct the cells toward becoming brain cells. In Chapter 3, I adapted the organoid generation method to create older organoids, as astrocytes, which are critical for GFAP function, develop later in brain organoids (around 100 days). Analysis of these older organoids revealed that they also struggled to develop the correct cell types, due to a lack of key brain developmental transcription factors. In collaboration with a lab in Sweden, we added these transcription factors to the iPSCs and found that they still had difficulty differentiating into brain cells. These results confirm that mutations in GFAP impair the development of iPSCs into brain organoids. In Chapter 4, I used CRISPR/Cas9 gene editing to disrupt the GFAP and VIM genes in healthy stem cells, preventing them from producing the GFAP and vimentin proteins. Vimentin, like GFAP, is an intermediate filament found in radial glia cells. We found a synergistic relationship between GFAP and vimentin—while the absence of one did not significantly affect development, the loss of both proteins impaired the development of brain organoids, particularly in the cortex. This effect was influenced by the organoid generation method. Organoids lacking GFAP and vimentin had difficulty developing cortex tissue, especially when I specifically aimed to generate cortex, but had different outcomes when allowed to develop freely into various brain regions. In conclusion, this dissertation highlights the critical role of intermediate filaments like GFAP and vimentin in brain development. The findings also emphasize the importance of the model and methods used in research, as they can greatly impact the results. The research contributes to understanding the molecular mechanisms of Alexander disease and the broader role of cytoskeletal proteins in brain development.