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    The Role of Pals1 in Brain Development and Microcephaly

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    Sterling_temple_0225E_15148.pdf
    Embargo:
    2025-05-18
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    5.547Mb
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    Genre
    Thesis/Dissertation
    Date
    2023
    Author
    Sterling, Noelle cc
    Advisor
    Kim, Seonhee
    Committee member
    Smith, George M.
    Cho, Seo-Hee
    Thomas, Gareth
    Barbe, Mary F.
    Estaras, Conchi
    Department
    Biomedical Sciences
    Subject
    Neurosciences
    Developmental biology
    Genetics
    Cortical development
    Entosis
    Microcephaly
    p53
    Pals1
    Polarity complex proteins
    Permanent link to this record
    http://hdl.handle.net/20.500.12613/8620
    
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    DOI
    http://dx.doi.org/10.34944/dspace/8584
    Abstract
    Microcephaly is a debilitating condition in which children are born with small brains. It can be caused by a variety of factors including maternal infection, harmful substance exposure, and genetic mutation. Cerebral cortical development is often severely disrupted in human microcephaly patients. In order to form the billions of neurons which exist in the cortex, efficient and correct neural progenitor division, differentiation, and migration are key. As the center of higher brain function in mammals, reduction in cortical mass is associated with the developmental delays that are symptomatic of microcephaly. Recently, a number of microcephaly causes have been linked to P53-mediated apoptosis of neural cells. The tumor suppressor protein P53 is upregulated in response to mitotic cycle stress, and its activation can trigger cell cycle arrest or apoptotic cell death. In microcephaly, P53 can become activated by mitotic stress and trigger apoptosis to cause the loss of cortical cell numbers that leads to microcephaly. Microcephaly has often been linked to mutations in mitotic proteins that alter neural progenitor division. However, the apical polarity complex Protein Associated with Lin-7 1 (PALS1) – known as membrane palmitoylated protein (MPP)5 in people – has recently been implicated in human microcephaly. PALS1 is integral to establishing polarity in neural progenitors. Deletion of Pals1 in mouse models has also resulted in microcephaly characterized by smaller brains and a global reduction in cortical cell numbers. Interestingly, a cellular phenomenon known as entosis can be caused by polarity disruptions in epithelial cells, and P53 activation has been shown to cause entosis in MDCK cell culture. While entosis is mainly associated with cancer cells, it is a form of competitive cell cannibalism that can eliminate unfit cells from a population. The loss of PALS1 from the developing cortex is known to result in apical polarity complex disruption and microcephaly in mouse models. However, the mechanism by which the loss of PALS1 results in cortical abrogation has yet to be determined. In Chapter 1 of this dissertation, I begin by reviewing cortical development. The normal progression of cortical cells from neural progenitors to fully differentiated neurons is explained in detail. Neural progenitor mitosis in particular is addressed in detail. Furthermore, an overview of microcephaly is provided to address the similarities between known causes of microcephaly. Next, I review the polarity complex proteins and their roles in cortical development. I compare and contrast the cortical phenotypes that have been described when each of the polarity complex proteins has been genetically deleted from the mouse cortex. I go on to review studies that have shown P53-mediated apoptosis in microcephaly in order to address the phenotypic features of microcephaly that are or are not caused by P53 activation. Finally, I provide a brief history of entosis. As a newly discovered cellular process in neural progenitors, the overview of entosis highlights what is known about cell cannibalism and the contexts in which it occurs. Following this background, I describe the experimental aims, hypotheses, and methods for this project in Chapter 2. In Chapter 3, I describe our investigation of three human patients with mutations in the Pals1 gene. One of the patients, possessing a heterozygous de novo nonsense mutation in Pals1 (or MPP5), was diagnosed with microcephaly. In order to model this patient’s phenotype, we generated a heterozygous conditional knockout of Pals1 from the entire mouse nervous system with Nestin-Cre. Through behavioral analysis of these mice, I demonstrate that they are hyperactive and blind, mimicking the microcephaly patient’s symptoms. Furthermore, via analysis of the mouse cortex, I show that heterozygous deletion of Pals1 results in severe microcephaly in mice with a global reduction in cortical cell numbers at both adult and embryonic stages. Importantly, I determine that Pals1 deletion does not result in proliferation or migration defects in the mouse cortex. Instead, loss of PALS1 results in massive apoptotic cell death that affects every cell type produced in the developing cortex. In Chapter 4, I detail our investigation into the mechanism underlying cell death in the PALS1-deficient cortex. By studying dividing neural progenitors at the apical surface in both Emx1-Cre and hGFAP-Cre drive Pals1 conditional knockout models, I demonstrate an as yet undescribed neural progenitor phenotype called entosis. As has been shown in cancer cells, neural progenitor entosis is dynamic and reliant on Rho-ROCK activity to occur. Furthermore, entosis produces observable cell-in-cell structures that persist through outer cell division and cause mitotic delay. I go on to demonstrate P53 activation in Pals1 deficient mouse cortices, and show that genetic deletion of Trp53 significantly rescues microcephaly. Trp53 deletion significantly restores all cortical cell types in addition to ameliorating entosis and mitotic length. This study suggests that P53 activation is a major mechanism by which PALS1 loss results in microcephaly. Overall, these studies show that deletion of Pals1 in mice can mimic microcephaly found in a human patient with a Pals1 mutation. Furthermore, PALS1 loss promotes P53-mediated cortical cell apoptosis. These studies provide the first description of entosis in neural progenitors, and suggest that entosis could be a mechanism for unfit cell removal in the developing cortex. Furthermore, I provide evidence that ROCK inhibition can fully rescue the presence of entosis in PALS1-deficient neural progenitors, and that genetic deletion of Trp53 significantly restores microcephaly pathology after PALS1 loss. These studies open up a field of research into the causes and effects of entosis in neural progenitors, and provide further evidence that apoptotic cell death in microcephaly is largely mediated by P53 activation.
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