Protocadherin clusters and cell adhesion kinase regulate dendrite complexity through Rho GTPase

Abstract

Dendritic patterning and spine morphogenesis are crucial for the assembly of neuronal circuitry to ensure normal brain development and synaptic connectivity as well as for understanding underlying mechanisms of neuropsychiatric diseases and cognitive impairments. The Rho GTPase family is essential for neuronal morphogenesis and synaptic plasticity by modulating and reorganizing the cytoskeleton. Here, we report that protocadherin (Pcdh) clusters and cell adhesion kinases (CAKs) play important roles in dendritic development and spine elaboration. The knockout of the entire Pcdhα cluster results in the dendritic simplification and spine loss in CA1 pyramidal neurons in vivo and in cultured primary hippocampal neurons in vitro. The knockdown of the whole Pcdhγ cluster or in combination with the Pcdhα knockout results in similar dendritic and spine defects in vitro. The overexpression of proline-rich tyrosine kinase 2 (Pyk2, also known as CAKβ, RAFTK, FAK2, and CADTK) recapitulates these defects and its knockdown rescues the phenotype. Moreover, the genetic deletion of the Pcdhα cluster results in phosphorylation and activation of Pyk2 and focal adhesion kinase (Fak) and the inhibition of Rho GTPases in vivo. Finally, the overexpression of Pyk2 leads to inactivation of Rac1 and, conversely, the constitutive active Rac1 rescues the dendritic and spine morphogenesis defects caused by the knockout of the Pcdhα cluster and the knockdown of the Pcdhγ cluster. Thus, the involvement of the Pcdh-CAK-Rho GTPase pathway in the dendritic development and spine morphogenesis has interesting implications for proper assembly of neuronal connections in the brain.

CTCF/cohesin-mediated DNA looping is required for protocadherin α promoter choice

“…This diversity appears to play a fundamental role in neural circuit assembly (22)(23)(24)(25). Understanding the mechanism by which this diversity is generated is, therefore, fundamental to understanding the development and function of the nervous system.…”

“…Recent studies have revealed a functional role for clustered Pcdh genes in dendritic development and self-avoidance [22][23][24][25]. Strikingly, a repertoire of heterozygous mutations in chromatid-segregating cohesin (SMC1A, SMC3, RAD21) and cohesin regulators (NIPBL, ESCO2) have been found to cause a class of developmental disorders known as Cornelia de Lange syndrome (CdLS) and Robert syndrome in humans (31).…”

Clustered protocadherins

“…Both Pcdha and Pcdhg intracellular domains also interact with the cell-adhesion kinases proline-rich tyrosin kinase 2 (Pyk2) and focal adhesion kinase (Fak), resulting in inhibition of kinase activity (Chen et al, 2009). These interactions have been implicated in the defects in cell survival and dendritic patterning observed in Pcdha-or Pcdhg-deficient neurons (Chen et al, 2009; Garrett et al, 2012;Suo et al, 2012). A large number of other potential interacting proteins, including phosphatases, kinases, adhesion molecules and synaptic proteins, have been reported (Han et al, 2010;Schalm et al, 2010), suggesting that clustered Pcdhs may form large heteromeric complexes with broad functions yet to be elucidated.…”

“…Although synaptic defects were observed in Pcdhg mutants (Wang et al, 2002b;Weiner et al, 2005; Garrett and Weiner, 2009), the issue of whether Pcdhg proteins play a direct role in synaptic interactions remains to be elucidated. Suggestive evidence for this idea was provided by the observation that Pcdhgc5 specifically interacts with the γ-aminobutyric acid A (GABA A ) receptor, which is required for stabilization and maintenance of GABAergic synapses in cultured hippocampal neurons (Li et al, 2012).In contrast to Pcdhg-deficient mice, which die as neonates, Pcdha knockouts and severe hypomorphs are viable and fertile with no obvious defects (Hasegawa et al, 2008;Katori et al, 2009;Suo et al, 2012). However, abnormal axonal projections of olfactory sensory neurons and serotonergic neurons were observed in Pcdha mutants that lack the constant region (Hasegawa et al, 2008;Katori et al, 2009).…”

“…Similar to Pcdhg mutants, Pcdha knockouts also exhibit defects in dendritic branching, as well as in spine morphogenesis. It has been proposed that Pcdha proteins regulate dendritic and spine development by inhibiting the kinase activities of associated cell-adhesion kinases Fak/Pyk2, which in turn activates small GTPases such as Ras homolog gene family member A (RhoA) and Ras-related C3 botulinum toxin substrate 1 (Rac1), leading to cytoskeleton reorganization (Suo et al, 2012).Functional studies of mice lacking the Pcdhb gene cluster have not been reported. Because there are three Pcdh gene clusters encoding 58 isoforms that are expressed broadly in overlapping brain regions, their roles in specific cellular context may not be revealed with single cluster knockouts due to functional compensation from members of the other two clusters.…”