Growth of a complex dendrite arbor is a prerequisite for neural circuit formation, and reduced arbor complexity in the cortex is a consistent finding in neurodevelopmental/neuropsychiatric disorders. For future therapeutic approaches to be conceivable, there is a critical need to elucidate the molecular mechanisms that regulate dendrite arborization. The ?-Protocadherins (?-Pcdhs), a family of 22 cell adhesion molecules encoded by the Pcdhg gene cluster, are required for normal cortical dendrite arborization; the mechanisms through which their diverse isoforms act are, however, poorly understood. The long-term goal is to identify molecular mechanisms that control neural circuit formation. The objective of this renewal application is to elucidate how both isoform diversity and common signaling mechanisms contribute to ?-Pcdh regulation of dendrite arborization. The central hypothesis is that ?-Pcdhs regulate arborization through: 1) diverse homophilic cell-cell interactions; 2) isoform-specific signaling via unique variable cytoplasmic domains (VCDs); and 3) shared signaling via a common C-terminal motif.
Three Aims are proposed, each premised on published work from the current grant period, and each utilizing novel Pcdhg alleles generated through CRISPR/Cas9 genome editing.
Aim 1 : Establish the importance of a shared ?-Pcdh C-terminal motif in dendrite arborization in vivo. PKC phosphory- lation of a serine within a lysine-rich C-terminal motif abrogates its ability to bind phospholipids, and prevents ?- Pcdhs from inhibiting FAK and promoting dendrite arborization in vitro. To address the importance of this in vivo, 2 new mouse lines will be examined for arborization, ?-Pcdh protein stability, and FAK regulation: PcdhgS/A, in which the serine is mutated to an alanine, preventing phosphorylation; and PcdhgCTD, in which a premature stop codon results in proteins lacking the motif.
Aim 2 : Identify isoform-specific mechanisms through which the ?-Pcdh-C3 VCD regulates dendrite arborization. The VCD of the ?-Pcdh-C3 isoform uniquely inhibits Wnt signaling by binding to Axin1, a known regulator of dendrite arborization. Using in utero electroporation, Axin1 knockdown, and a novel PcdhgC3KO single-isoform knockout mouse, a distinct role for C3 in dendrite arborization will be ascertained.
Aim 3 : Ascertain the importance of ?-Pcdh isoform diversity for dendrite arborization and behavior. It's unknown whether isoform diversity generated by the endogenous Pcdhg gene cluster is important arborization. To test this in vivo, multiple novel mouse lines in which varying numbers of Pcdhg exons have been disrupted, reducing potential isoform diversity, will be analyzed. Additionally, anxiety- and memory-associated tests will be carried out on Pcdhg null, single-isoform overexpression, and reduced diversity mice to link cellular phenotypes to behavioral outcomes. The proposed research is innovative, because it utilizes CRISPR/Cas9 genome editing techniques to comprehensively assess the effects of altered ?-Pcdh repertoire and signaling in vivo. It is significant, because future therapeutic approaches will rely critically on identifying the molecules and signaling mechanisms that control dendrite arbor complexity.

Public Health Relevance

The proposed research is relevant to public health because it will identify new molecular mechanisms through which a diverse family of proteins regulates dendrite arborization in the cerebral cortex, a key step in the elaboration of neural circuits that goes awry in a plethora of impactful human disorders including autism, intellectual disability, anxiety and depression. 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS055272-13
Application #
9927701
Study Section
Neurodifferentiation, Plasticity, and Regeneration Study Section (NDPR)
Program Officer
Talley, Edmund M
Project Start
2007-07-01
Project End
2021-05-31
Budget Start
2020-06-01
Budget End
2021-05-31
Support Year
13
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Iowa
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
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Mah, Kar Men; Weiner, Joshua A (2017) Regulation of Wnt signaling by protocadherins. Semin Cell Dev Biol 69:158-171
Bosch, Peter J; Fuller, Leah C; Sleeth, Carolyn M et al. (2016) Akirin2 is essential for the formation of the cerebral cortex. Neural Dev 11:21
Mah, Kar Men; Houston, Douglas W; Weiner, Joshua A (2016) The ?-Protocadherin-C3 isoform inhibits canonical Wnt signalling by binding to and stabilizing Axin1 at the membrane. Sci Rep 6:31665
Molumby, Michael J; Keeler, Austin B; Weiner, Joshua A (2016) Homophilic Protocadherin Cell-Cell Interactions Promote Dendrite Complexity. Cell Rep 15:1037-1050
Keeler, Austin B; Molumby, Michael J; Weiner, Joshua A (2015) Protocadherins branch out: Multiple roles in dendrite development. Cell Adh Migr 9:214-26
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Keeler, Austin B; Schreiner, Dietmar; Weiner, Joshua A (2015) Protein Kinase C Phosphorylation of a ?-Protocadherin C-terminal Lipid Binding Domain Regulates Focal Adhesion Kinase Inhibition and Dendrite Arborization. J Biol Chem 290:20674-86
Garrett, Andrew M; Schreiner, Dietmar; Lobas, Mark A et al. (2012) ?-protocadherins control cortical dendrite arborization by regulating the activity of a FAK/PKC/MARCKS signaling pathway. Neuron 74:269-76

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