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!Camille Hanes thesis FINAL5.53 MB
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Dissertation
The γ-Protocadherin family of cell adhesion molecules regulates neurodevelopment via shared and isoform-specific mechanisms
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2024
DOI: 10.25820/etd.007747
Abstract
Cell-cell interactions are critical to proper neurodevelopment, as key steps including axon outgrowth, dendrite arborization, and synapse formation all require such interactions. Cell adhesion molecules (CAMs) such as the γ-Protocadherins (γ-Pcdhs)– a family of 22 CAMs encoded by the Pcdhg gene cluster that is the focus of this dissertation–mediate cell-cell interactions and are known to play key roles in neurodevelopment. Here, I delve into the different mechanisms by which γ-Pcdhs mediate processes of neurodevelopment, particularly dendrite arborization. I describe how some functions of γ-Pcdhs are shared among all 22 isoforms, whereas others are uniquely mediated by individual isoforms. Each γ-Pcdh intracellular domain contains a C-terminal “constant” domain that is identical among all 22 isoforms, and a “variable” cytoplasmic domain (VCD) that is unique to each isoform. This dissertation explores the dissociation of shared vs. unique roles among γ-Pcdhs and how these functions are carried out through these distinct protein domains.We previously showed that within a γ-Pcdh constant domain C-terminal motif is a serine residue that is a target for phosphorylation by protein kinase C (PKC). Phosphorylation of this site prevents the γ-Pcdhs from inhibiting FAK activation, resulting in increased PKC phosphorylation of MARCKS, which negatively regulates dendrite arborization. Accordingly, in vitro studies show that blocking the ability of this site to be phosphorylated results in increased dendritic arborization due to downregulation of this FAK/PKC/MARCKS signaling. Here, I explore this signaling pathway in vivo using two novel Pcdhg mutant mouse lines in which phosphorylation is prevented due to: 1) a serine-to-alanine amino acid substitution; or 2) the removal of this serine by a 15 amino acid deletion of the entire C-terminal motif. We find that both of these mutants exhibit increased dendritic arborization and hypophosphorylated MARCKS, as expected, whereas dendritic spine density was not altered. Unexpectedly, we find that the mice with the C-terminal motif deletion have decreased Pcdhg expression.
Moving on from shared roles among the γ-Pcdhs, we next elucidated a unique role for one individual γ-Pcdh isoform, γ-Pcdh C3 (γC3), in dendritic arborization of cortical pyramidal neurons. Using γC3 KO mice, we find that discrete loss of γC3 results in a ~30% decrease in dendritic arborization compared to controls, compared to a previous report from the Weiner Lab demonstrating that loss of all 22 isoforms results in a ~40% decrease. We previously demonstrated using non-neuronal cells that the γC3 isoform’s VCD binds the synaptic scaffold and Wnt signaling protein, Axin1. This interaction provided insight into the mechanism by which γC3 plays a prominent role in dendritic arborization. Using cultured γC3 KO neurons and various truncated γC3 rescue constructs, we establish an Axin1-dependent mechanism by which γC3 promotes dendritic arborization via its VCD.
Lastly, we explore potential isoform-specific roles for the γ-Pcdh C5 isoform (γC5) by characterizing a novel γC5 KO mouse we created. We find that loss of γC5 does not alter dendritic arborization of cortical pyramidal neurons or the levels of various synaptic proteins in the adult cortex. However, characterization of this novel γC5 KO mouse line is still in its early stages and more extensive studies are still required. A thorough investigation may shed light on potential mechanisms that are unique to γC5 and will further our understand of isoform-specific roles among the 22 γ-Pcdhs.
This dissertation, together with other recent work, contributes to a converging understanding of γ-Pcdh family function in which distinct roles are played by both individual isoforms and discrete protein domains. Future work aimed at identifying the full panoply of intracellular signaling partners, including both those that work with the shared constant domain and those that interact with one or more VCDs, will be important to understanding how particular γ-Pcdh functions predominate depending on the neuronal or glial cell type and the stage of neural development.
Details
- Title: Subtitle
- The γ-Protocadherin family of cell adhesion molecules regulates neurodevelopment via shared and isoform-specific mechanisms
- Creators
- Camille M. Hanes
- Contributors
- Joshua A Weiner (Advisor)Michael E Dailey (Advisor)Daniel W Summers (Committee Member)Carl A Frank (Committee Member)Gordon F Buchanan (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biology
- Date degree season
- Summer 2024
- Publisher
- University of Iowa
- DOI
- 10.25820/etd.007747
- Number of pages
- xiv, 155 pages
- Copyright
- Copyright 2024 Camille M. Hanes
- Language
- English
- Date submitted
- 06/13/2024
- Description illustrations
- illustrations (some color)
- Description bibliographic
- Includes bibliographical references (pages 137-155).
- Public Abstract (ETD)
Proper development of the central nervous system requires dendritic arborization, a process by which neurons extend their branches, or “arbors”, to form synapses with other neurons. Improper dendritic arborization is a hallmark of neurodevelopmental disorders such as autism and intellectual disabilities. Therefore, there is a critical need to understand the molecular mechanisms that contribute to proper neurodevelopment.
The γ-Protocadherins (γ-Pcdhs), a group of cell adhesion molecules that allow neurons to interact with one another, are critical to proper dendritic arborization and other processes of brain development: loss of γ-Pcdhs in the forebrain of mice results in decreased dendritic arborization and increased synapse density. There are 22 different γ-Pcdh protein isoforms, or “versions”, in mice. One domain of each γ-Pcdh protein is identical among all 22 isoforms, while other domains are unique to each isoform. Our lab has discovered that γ-Pcdhs can influence neurodevelopmental processes both through mechanisms shared by all 22 isoforms, or by mechanisms that are unique to an individual isoform.
In this dissertation, I focus on a shared role for all γ-Pcdhs in regulating dendritic arborization through a particular signaling pathway mediated by the common γ-Pcdh protein domain. Then, I describe a unique role for an individual isoform, called γC3, in regulating dendritic arborization. This function of γC3 is mediated by a protein domain that only γC3 has. Lastly, I report my preliminary efforts in seeking unique roles for a different isoform, called γC5, in neurodevelopment.
- Academic Unit
- Biology
- Record Identifier
- 9984698250602771
Metrics
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