Adhesion-Mediated Mechanisms Underlying Cortical Astrocyte Development

Eroglu, Cagla

Tan, Christabel Xin

2024-03-07T18:38:58Z

2023

Cell Biology

Astrocytes, the perisynaptic glial cells of the brain, display a complex morphology that is strongly linked to their functions at the synapse. Primary processes radiating from the astrocyte cell soma branch out to secondary and tertiary processes, which further ramify into tiny perisynaptic astrocyte processes, giving a mature astrocyte its characteristic arborized structure. Astrocyte processes dynamically ensheath the pre- and post-synapse to provide instructive cues for synapse formation, maturation, and function. Perturbations in astrocyte-synapse interactions result in synaptic deficits, leading to excitation/inhibition imbalance and aberrant neural circuitry. However, the mechanisms linking astrocyte morphology and function to neuronal contact and synaptic adhesion are poorly understood. In a candidate-based reverse genetic screen utilizing rodent cortical neurons and astrocytes, I identified two genes, HepaCAM and CTNND2, as regulators of astrocyte morphogenesis in response to neuronal adhesion.HepaCAM is an astrocyte-enriched cell adhesion molecule that participates in cell-cell and cell-ECM interactions to regulate cell migration and proliferation. shRNA-mediated silencing of hepaCAM expression in astrocytes resulted in decreased astrocyte complexity in vitro and in vivo. HepaCAM stabilizes the gap junction protein connexin 43 (Cx43) at cell-cell junctions. We used stimulated emission depletion (STED) microscopy to show that hepaCAM and Cx43 colocalize at astrocyte processes in the mouse cortex and performed native affinity purifications followed by liquid chromatography-coupled high-resolution mass spectrometry (AP-MS) to demonstrate that Cx43 binds to hepaCAM. Finally, utilizing the same shRNA silencing approach, we found that hepaCAM and Cx43 were epistatic to each other in the regulation of astrocyte morphogenesis. Through mosaic analysis with double markers (MADM), we found that hepaCAM knockout astrocytes lost their ability to tile and had mislocalized Cx43. Consequently, gap junction coupling is impaired in astrocytes without hepaCAM. Additionally, we found decreased colocalization of hepaCAM puncta with synapses, a marked decrease in inhibitory synapses density, and a significant decrease in amplitude of miniature inhibitory postsynaptic currents, suggesting that loss of astrocytc hepaCAM disrupts the balance between synaptic excitation and inhibition. During development, astrocytes need to form non-overlapping territories within which they dynamically ensheathe synapses within discrete regions of neuropil. Taken together, our findings suggest that hepaCAM and Cx43 are critical proteins at the intersection of these two processes to ensure the proper molecular regulation of astrocyte self-organization and territory formation for normal circuit formation and function. Next, we identified Ctnnd2 (protein: δ-catenin) as another key regulator of astrocyte morphological complexity. δ-catenin was previously thought to be a neuron-specific protein that regulates dendrite morphology. Utilizing RNA fluorescence in situ hybridization (RNA-FISH) and immunohistochemistry, we found Ctnnd2 mRNA and δ-catenin is also highly expressed by astrocytes during the critical period of astrocyte morphological maturation and synapse formation during cortical development. shRNA-mediated silencing of Ctnnd2 expression in astrocytes resulted in decreased astrocyte complexity in vitro and in vivo. δ-catenin is hypothesized to mediate transcellular interactions through the cadherin family of cell adhesion proteins. We used structural modeling and surface biotinylation assays in both HEK293T and purified astrocyte cultures to reveal that δ-catenin interacts with N-cadherin juxtamembrane domain to promote N-cadherin surface expression. An autism-linked δ-catenin point mutation impaired N-cadherin cell surface expression and reduced astrocyte complexity. In the developing mouse cortex, only lower-layer cortical neurons express N-cadherin. Remarkably, when we silenced astrocytic N-cadherin throughout the cortex, only lower-layer astrocyte morphology was disrupted. These findings show that δ-catenin controls astrocyte-neuron cadherin interactions that regulate layer-specific astrocyte morphogenesis.

https://hdl.handle.net/10161/30256

https://creativecommons.org/licenses/by-nc-nd/4.0/

Molecular biology

Neurosciences

Cell Adhesion

Glia Biology

Neurodevelopment

Adhesion-Mediated Mechanisms Underlying Cortical Astrocyte Development

Dissertation

11

2025-02-07T18:38:58Z