Current understanding of the interactions between neurons, and between neurons and glia, required for the formation of neural circuits is incomplete. Fundamental gaps include identifying cell adhesion molecules that can generate the diversity needed to promote specific recognition between cells of the developing mammalian CNS, and elucidating associated signaling pathways that regulate several key steps, including elaboration of dendritic arbors and synaptogenesis. The long-term goal is to identify the molecular mechanisms that control the proper formation of neural circuits during development. The objective of this renewal application is to identify the molecular mechanisms by which the ?-Pcdhs, a family of 22 cadherin superfamily adhesion molecules, regulate cortical dendrite arborization. The central hypothesis is that homophilic interactions between ?-Pcdh tetramers on cortical neurons and astrocytes promote dendrite arborization by inhibiting a PKC signaling pathway. This hypothesis is based on extensive preliminary data generated by the investigator's laboratory during the prior funding period, and will be tested by pursuing 3 Specific Aims: 1) Determine the extent to which homophilic ?-Pcdh interactions promote dendrite arborization in cortical neurons; 2) Identify roles for astrocytic ?-Pcdhs in cortical neuron dendrite arborization and 3) Identify intracellular signaling mechanisms regulating ?-Pcdhs and their role in arborization.
Under Aim 1, a model resulting from preliminary in vitro assays - combinatorially diverse ?-Pcdh cis-tetramers interact homophilically in trans - will be applied to cortical development. Neuronal ?-Pcdh tetramer composition will be manipulated using transfection and several novel Pcdh-? knock-in transgenic mouse lines to directly address the role of interaction specificity in dendrite arborization.
Aim 2 builds on preliminary data establishing astrocytic ?-Pcdhs as key regulators of circuit formation in the spinal cord. Using astrocyte-restricted Cre transgenics with conditional Pcdh-? mutants and knock-ins in vivo, the role of astrocytic ?-Pcdhs in dendrite arborization of cortical neurons will be delineated.
Aim 3 expands on preliminary data showing that a PKC signaling pathway is inhibited by the ?-Pcdhs to promote dendrite arborization. A membrane-interacting C-terminal motif shared by all ?-Pcdhs has been identified that, when phosphorylated by PKC, is hypothesized to increase ?-Pcdh ectodomain shedding and intracellular cleavage, providing a signaling feedback mechanism. This will be tested using a series of point mutant and truncation Pcdh-? constructs in biochemical assays. Also tested under this aim will be the role played by CRMP1, identified during the prior funding period as a novel ?-Pcdh intracellular binding partner, in dendrite arborization. The proposed research is significant, because it will identify molecular mechanisms that can account for diverse cell-cell interactions driving a key step in neural circuit formation, filling an important gap in current knowledge in the field. Such information will be critical to understanding, and eventually ameliorating, the many neurodevelopmental disorders that involve defective dendrite development and synaptogenesis.
The proposed research is relevant to public health because it will identify molecular mechanisms that control neural circuit formation in the developing cerebral cortex, a process that goes awry in a plethora of impactful human disorders including autism spectrum disorders and intellectual disability. Identifying such mechanisms is a critical step towards understanding these disorders and, eventually, devising therapeutic approaches to their amelioration. Thus, the proposed research is relevant to the mission of NIH in that it will expand the neuroscience knowledge base towards the understanding of human disease and the processes of human development.
