Expression studies of γ-Pcdh isoforms across subgroups (PcdhγA, PcdhγB, and PcdhγC) show generally overlapping patterns in large brain areas. While broader regions can express similar subsets of Pcdhα and Pcdhγ, alternative promoter selection and pre-mRNA cis splicing are used to generate specific combinations of different isoforms within individual cells (Tasic et al., 2002; Wang et al., 2002a).

Single cell RT-PCR analysis of Purkinje cells has revealed that most isoforms of these cPcdhs are monoallelically and combinatorially expressed in single neurons, whereas all five C-type isoforms are expressed biallelically and uniformly in all of these neurons (Esumi et al., 2005; Kaneko et al., 2006; Hirano et al., 2012). In contrast, C-type isoforms have been only found in a small percentage of mouse olfactory sensory neurons (OSN). Interestingly, immature OSN still express alternate and C-type isoforms, suggesting downregulation of C-type isoforms throughout their maturation (Mountoufaris et al., 2017). Studies performed on serotonergic neurons revealed an exclusive expression of Pcdh C-isoforms in these cells, with PcdhαC2 being the most prominently expressed (Chen W. V. et al., 2017; Katori et al., 2017).

Isoform expression thus seems to be cell type specific, and bound to complex regulatory mechanisms. CPcdhs expression level and specificity are also epigenetically regulated. Each variable exon contains a specific promoter that is regulated by its position within the cluster (Noguchi et al., 2009; Kaneko et al., 2014), the orientation of enhancer elements (Guo et al., 2015) and the DNA methylation status (Guo et al., 2012). Epigenetic regulation of promoter choice and alternative transcripts therefore immensely increases the diversity of cPcdhs that can be generated. For additional detailed information on the epigenetic regulation of Pcdh-α and Pcdh-γ gene expression we refer to recent excellent reviews (El Hajj et al., 2017; Mountoufaris et al., 2018; Canzio and Maniatis, 2019).

Collectively, these studies indicate that transcriptional regulation can generate a large cell-surface molecular diversity and specificity within single neurons, creating functional diversification (Esumi et al., 2005; Kaneko et al., 2006; Hirano et al., 2012; Chen W. V. et al., 2017; Katori et al., 2017; Mountoufaris et al., 2017). However, not all neuronal cell types express multiple cPcdhs, and expression can be dynamic during development. More careful mapping of expression at the single-cell level would reveal whether expression patterns are stochastic within certain cell populations or not.

Various members of all three Pcdh clusters localize on the neuronal soma, on dendrites and axons, and at growth cones and synapses in differentiating and mature neurons (Kohmura et al., 1998; Wang et al., 2002a; Kallenbach et al., 2003; Phillips et al., 2003; Junghans et al., 2008).

In the context of dendrite development, cPcdhs play important roles in dendritic self-avoidance. γ-Pcdh isoform diversity is essential for the discrimination between isoneural and heteroneural dendrites in retinal starburst amacrine cells (SAC)s, as loss of this diversity impairs dendritic self-avoidance (Lefebvre et al., 2012; Kostadinov and Sanes, 2015). In the cerebral cortex, γ-Pcdhs promote dendritic arborization complexity in layer V pyramidal neurons (Garrett et al., 2012). Recently it was shown that α- and γ-Pcdhs can functionally interact and cooperate in dendritic development in a context-dependent manner, and that together they mediate dendrite self-avoidance in Purkinje cells (Ing-Esteves et al., 2018).

cPcdhs are also implicated in spine morphogenesis. γ-Pcdhs negatively regulate mouse cortical dendritic spine morphogenesis in vivo (Molumby et al., 2017). In contrast, deletion or knockdown of the γ-Pcdh cluster has been associated with a reduction in spine density and dendritic complexity in mouse olfactory granule cells and cultured hippocampal neurons (Suo et al., 2012; Ledderose et al., 2013). CA1 pyramidal neurons and cultured hippocampal neurons of Pcdha null mutant mice display simple arbors and low dendritic spine densities. Knockdown of γ-Pcdhs and knockout of α-Pcdhs in vitro leads to similar defects as in Pcdha null mutant mice, suggesting that both γ- and α-Pcdh members contribute to dendritic arborization (Suo et al., 2012).

Although functional evidence is still lacking, the molecular diversity and isoform-specific homophilic binding properties of cPcdhs might provide a synaptic adhesive code to support synaptogenesis and proper neural connectivity (Kohmura et al., 1998; Serafini, 1999; Shapiro and Colman, 1999). α-Pcdhs are found in neocortical synapses (Kohmura et al., 1998) and in perisynaptic sites of preganglionic terminals in chicken (Blank et al., 2004). In mouse hippocampal neurons overexpression of a dominant-negative α-Pcdh ICD leads to a reduction in spine number and decrease of presynaptic synaptophysin (Suo et al., 2012). Whether α-Pcdh isoforms are involved in synaptic adhesion (Kohmura et al., 1998) as has been shown for γ-Pcdhs (Garrett and Weiner, 2009) remains to be elucidated. β-Pcdhs accumulate dendritically and post-synaptically in mammalian retinal and cerebellar neurons, suggesting a potential involvement of β-Pcdhs in synaptogenesis and synaptic refinement (Junghans et al., 2008; Puller and Haverkamp, 2011).

γ-Pcdhs are found in synaptic intracellular compartments such as axonal and dendritic tubulovesicular structures within some hippocampal neurons (Phillips et al., 2003; Fernández-Monreal et al., 2009). PcdhγC5 is localized in a subset of GABAergic and glutamatergic synapses in cultured hippocampal neurons, majorly at dendrites as shown by colocalization of PcdhγC5 with specific synaptic proteins such as GABAergic presynaptic glutamate decarboxylase and vesicular glutamate transporter 1 (Li et al., 2010). In addition, it has been shown that PcdhγC5 is important for the stabilization and maintenance of some GABAergic synapses, but not for their formation (Li et al., 2012a). γ-Pcdhs were shown as well to play a role in synaptic elimination between closely spaced SACs, and in preventing autapse formation. Functional connectivity was impaired in neighboring SACs expressing a single γ-Pcdh isoform, demonstrating a necessary function of isoform diversity in establishing inter-SAC networks (Kostadinov and Sanes, 2015). In conclusion, several lines of evidence point to γ-Pcdhs as key molecular players in synapse formation, stabilization and maintenance in the mammalian nervous system. Unfortunately, current knowledge is limited to particular cPcdhs and specific neuronal cell types, hence studies focusing on different neuronal types or α and β clusters in this context might be revealing in the future. However, even for γ-Pcdhs exact roles in the regulation of synaptic function remain to be defined, and are likely to be context-dependent.

cPcdhs participate in several aspects of axonal development, and their potential to generate unique molecular codes are at the basis of both axon-target and axon-axon recognition mechanisms.

α-Pcdhs are indispensable for axon growth in cultured hippocampal neurons (Lu et al., 2018). Whether this role is unique to this cluster remains to be investigated. In the mouse spinal cord, loss of γ-Pcdhs leads to severe disorganization of Ia primary afferent projection terminals in the ventral horn, leading to a targeting defect between Ia afferents and ventral horn interneurons that suggests a critical role of γ-Pcdh-mediated recognition between the two (Prasad and Weiner, 2011; Hasegawa et al., 2016).

Other studies have revealed functions in axon targeting and branch repulsion which appear to be redundantly shared by all cPcdhs. Deletion of all three clusters, but not of single clusters, leads to the complete disruption of axonal arborization and to clumping of axonal terminals in mouse OSN. Overriding Pcdh diversity through overexpression of a fixed set of 3 cPcdhs (one α-, one β-, and one γ-Pcdh) in OSN results in the failure of axon terminals to converge and form normal glomeruli. In this case, the induced expression of an identical cPcdh membrane code seems to result in the erroneous self-avoidance between non-self axons (Mountoufaris et al., 2017).

In sharp contrast to what has been observed in the development of OSN connectivity, axonal tiling of serotonergic neurons is highly dependent on a single cPcdh isoform, PcdhαC2, that drives repulsion between neurites of distinct cells and ensures proper spatial axon distribution (Chen W. V. et al., 2017; Katori et al., 2017). To what extent other specific cPcdh isoforms have unique roles in the arrangement and targeting of projections between specific subpopulations of neuronal cells remains to be addressed.

Members of the γ-Pcdh cluster are known to prevent neuronal apoptosis of spinal cord interneurons, retinal cells, and cortical interneurons (cINs) (Wang et al., 2002b; Lefebvre et al., 2008; Prasad et al., 2008; Chen W. V. et al., 2012; Garrett et al., 2012; Hasegawa et al., 2016, 2017; Carriere et al., 2020; Leon et al., 2020). Transplantation studies and knockout mice phenotyping showed that loss of C-type γ-Pcdhs leads to increased cell death in spinal cord interneurons and cINs (Chen W. V. et al., 2012; Leon et al., 2020). Remarkably, within the C-type γ-Pcdhs, only one isoform (PcdhγC4) seems to be necessary for neuronal survival of several cell populations (Garrett et al., 2019).

While γ-Pcdhs appear to be particularly important in mouse neuronal survival, in zebrafish truncation of an α-Pcdh, Pcdh1α, has been found to lead to neuronal cell death in the developing brain and spinal cord (Emond and Jontes, 2008).

Different cPcdh clusters can also cooperate in the regulation of cell death and survival. For instance, the α-Pcdh and γ-Pcdh clusters have been demonstrated to cooperatively regulate neuronal survival in the retina (Ing-Esteves et al., 2018). In the spinal cord, interneuron apoptosis is aggravated in βγ-Pcdh and αβγ-Pcdh deficient mice compared to mice lacking only γ-Pcdhs, suggesting this process to be cPcdh dosage-dependent (Hasegawa et al., 2016). Moreover, cPcdhs seem to cooperate to prevent apoptosis in a cell type-specific manner. In chimeric mice lacking all three clusters (αβγ-Pcdh deficient mice), survival rates were found to significantly decrease in neuronal populations in the midbrain, pons and medulla, but not in the inferior olive, in sensory and motor neurons, and neuronal populations within the cerebral cortex and olfactory bulb (Wang et al., 2002b; Hasegawa et al., 2016, 2017). Therefore, context-specific combinatorial expression of cPcdhs appears to be important in the regulation of neurodevelopmental cell death versus survival.

Similarly to classical cadherins, ncPcdh expression is spatiotemporally regulated during brain development in several vertebrate species. In zebrafish and chicken, this mode of expression characterizes transcription of Pcdh9, Pcdh17, and Pcdh19 in the nervous system (Liu et al., 2009, 2010; Hertel et al., 2012; Lin et al., 2012). In the mouse brain, Pcdh7, Pcdh9 and Pcdh11 expression localizes to restricted regions within the neocortex, hippocampus and amygdala (Vanhalst et al., 2005). In rat, Pcdh1, Pcdh9, Pcdh10, Pcdh17, Pcdh19, and Pcdh20 are specifically expressed in limbic system structures, such as the hippocampus, the limbic cortex, the thalamus, the hypothalamus, and the amygdala. Cortical region-dependent and layer-specific expression can also be observed perinatally (Kim et al., 2007). In addition, Pcdh10 synthesis has been described in specific networks like the limbic and visual systems in mice and chicken (Hirano et al., 1999; Aoki et al., 2003; Müller et al., 2004).

Besides being present in distinct brain areas, δ-Pcdhs have been demonstrated to be combinatorially expressed in the ferret retina and in the mouse primary sensory cortex (Etzrodt et al., 2009; Krishna-K et al., 2011). Moreover, it was recently shown that one mouse OSN can express up to seven δ-Pcdhs, and that cells can adjust the number and expression levels of δ-Pcdhs on their surface to regulate their adhesivity (Bisogni et al., 2018).

Overall, the combinatorial and molecule-specific spatiotemporal expression patterns of ncPcdhs in the vertebrate central nervous system point at roles in neural circuit formation, potentially by contributing to a molecular recognition code.

Like cPcdhs, δ-Pcdhs are localized in dendrites, axons, and proximally to or within synapses (Hirano et al., 1999; Yasuda et al., 2007; Hoshina et al., 2013; Pederick et al., 2016). All members of the δ1-Pcdh family and the majority of the δ2-Pcdh family, with the exception of Pcdh18, have been associated to the regulation of dendritic initiation, growth, morphology, arbor refinement, and spine formation (Yasuda et al., 2007; Piper et al., 2008; Bruining et al., 2015; Wu et al., 2015; Schoch et al., 2017; Bassani et al., 2018; Chang et al., 2018). To date, roles in axon growth, branching, guidance, and fasciculation have been described for Pcdh7 and for almost all of the δ2-Pcdhs except Pcdh8 (Aoki et al., 2003; Uemura et al., 2007; Nakao et al., 2008; Piper et al., 2008; Leung et al., 2013, 2015; Biswas et al., 2014; Hayashi et al., 2014; Cooper et al., 2015; Asakawa and Kawakami, 2018; Guemez-Gamboa et al., 2018). While specific synaptic roles have not yet been directly demonstrated for δ1-Pcdhs, they have been suggested to participate in synaptic maintenance and plasticity based on their expression at synapses and interaction with PP1α (Yoshida et al., 1999; Vanhalst et al., 2005; Kim et al., 2007; Bruining et al., 2015). Roles in synaptogenesis, synaptic vesicle assembly and mobility, synapse elimination, and synaptic connectivity have been demonstrated for all δ2-Pcdhs (Yasuda et al., 2007; Tsai et al., 2012; Hoshina et al., 2013; Biswas et al., 2014; Bassani et al., 2018; Light and Jontes, 2019; Lv et al., 2019). While the role of cPcdhs in neuronal self-avoidance has been extensively described (see sections above), nearly no information is available for ncPcdhs in this context. Interestingly, removal of the Pcdh17 cytoplasmic domain in zebrafish has been shown to induce axon clumping in somatic motor neurons, but not in spinal interneurons, pointing to cell-type specific roles in fasciculation and guidance/targeting via ncPcdh-mediated homotypic repulsion (Asakawa and Kawakami, 2018). Table 1 summarizes currently known roles of ncPcdhs in synapse, dendrite, and axon development.

Pcdhs interactions differ significantly from those occurring between classical cadherins. Pcdhs are only partially dependent on calcium for trans binding (Schreiner and Weiner, 2010); moreover, cPcdhs lack a hydrophobic pocket and have fewer glycosylation sites (Morishita et al., 2006; Schreiner and Weiner, 2010; Thu et al., 2014; Nicoludis et al., 2015; Rubinstein et al., 2015). Analyses on γ-Pcdhs first identified the EC2 and EC3 domains (with EC1 being the most N-terminal cadherin domain) as those mediating specificity in homophilic trans interactions between Pcdhs (Schreiner and Weiner, 2010; Thu et al., 2014). Among cPcdhs, only PcdhαC1 was found to not conform to the homophilic adhesion rule, likely due to the absence of a calcium binding motif in EC3 which might affect its structure (Thu et al., 2014).

Trans homophilic interfaces are formed via EC1–EC4 antiparallel domain interactions in a head-to-tail orientation, allowing binding of EC1 with EC4, and EC2 with EC3 (Nicoludis et al., 2015). Single-domain mismatches between either EC4 and EC1 or EC2 and EC3 were shown to be sufficient in, respectively, blocking trans dimerization or preventing co-aggregation (Rubinstein et al., 2015).

Several studies examining the Pcdh adhesive interface furthermore revealed that both its conformation and the interaction preferences of interface-localized residues are necessary for Pcdh binding specificity (Nicoludis et al., 2015, 2016; Cooper et al., 2016; Goodman et al., 2016a, b; Brasch et al., 2019). Trans interaction specificity is mediated by the EC1–EC4 interface, whereas the EC2–EC3 interface contributes to a greater extent to trans interaction affinity (Goodman et al., 2016b; Nicoludis et al., 2019) (Figure 3).

Cis interactions between cPcdhs are mediated via the extracellular domain and, in contrast to trans associations, are highly promiscuous between distinct members of the α-, β-, and γ-Pcdh clusters (Han et al., 2010; Schalm et al., 2010; Schreiner and Weiner, 2010; Thu et al., 2014; Rubinstein et al., 2015). These interactions also ensure proper delivery of α-Pcdh isoforms to the cell surface, as cis binding with β- and/or γ-Pcdh through the EC6 domain is crucial in this context (Thu et al., 2014; Rubinstein et al., 2015).

Additionally, cPcdhs cis multimerization allows the establishment of new homophilic specificities in trans (Schreiner and Weiner, 2010; Goodman et al., 2016b). Cis multimers can form independently of trans interactions through EC6 domains with an affinity comparable to EC1–EC4-mediated trans binding (Rubinstein et al., 2015). One recent study described the crystal structure of a PcdhγB7 cis homodimer as a model for cis cPcdh interactions. This analysis demonstrated that interfacing occurs asymmetrically between EC5 and EC6 domains of one molecule and the EC6 domain of the other, and suggested that cPcdh surface expression requires dimerization. As α-Pcdhs cannot dimerize, they can only provide the EC5-6 side of the dimer interface. This likely explains why α-Pcdhs necessitate heterodimerization with a carrier Pcdh for cell surface delivery (Goodman et al., 2017).

Evidence for inter-ncPcdhs cis binding came first from studies on Xenopus paraxial protocadherin (PAPC/Pcdh8). PAPC molecules were shown to form cis homodimers via ECD-located cysteine residues, and these interactions were proven to be essential for proper PAPC trafficking, maturation, and function (Chen et al., 2007). Pcdh19 interactions in homo- or heterodimers were also demonstrated via K562 cell assays, and cis binding disruption was suggested to underlie the developmental abnormalities characterizing epilepsy and intellectual disability linked to females (EFMR) (Pederick et al., 2018). However, recent structural evidence obtained from the investigation of δ-Pcdhs in solution was not able to substantiate any ECD-mediated cis interactions. Moreover, no conservation of cis-interface motifs characterizing cPcdh ECDs was found in δ-Pcdhs, thereby ruling out cPcdh-like cis-interactions through the ECD for δ-Pcdhs (Harrison et al., 2020). The observed cis interactions between Pcdh19 with other δ2-Pcdhs in K562 cells might therefore rely on transmembrane or ICD interactions (Figure 3).

Ectodomain shedding, whereby the ECD is cleaved by matrix metalloproteinases (MMPs) such as disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) (Figure 4), was described for cPcdhs (PcdhγC3 and Pcdhα4) and ncPcdhs (Pcdh12) (Reiss et al., 2006; Buchanan et al., 2010; Bouillot et al., 2011). Additionally, shedding of PcdhγC3 and Pcdh12 was shown to be regulated by, among others, calcium-dependent and protein kinase C-mediated pathways (Reiss et al., 2006; Bouillot et al., 2011). Endocytosis prior to shedding is required for Pcdhα4, as blocking of endocytosis prevented shedding (Buchanan et al., 2010). PcdhγC3 ECD shedding by ADAM10 can be increased through the activation of glutamate receptors via α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) stimulation, suggesting that neuronal activity can too regulate shedding. Furthermore, PcdhγC3 ECD shedding could be involved in the regulation of cell adhesion, as its inhibition increases cell aggregation (Reiss et al., 2006).

Pcdh ICD cleavage occurs at the TM level subsequently to shedding, and is mediated by the γ-secretase complex via its catalytic component presenilin (Reiss et al., 2006; Buchanan et al., 2010) (Figure 4A). This process might be regulated by cis and trans interactions between Pcdhs. For instance, the formation of cPcdh zipper arrays at the cell membrane could impede proteolysis by ADAM10 and γ-secretase (Figure 4B) (Hambsch et al., 2005; Brasch et al., 2019). The progressive reduction of Pcdh cleavage during neuronal differentiation, which comprises a gradual increase in Pcdh–Pcdh interactions, supports this hypothesis (Buchanan et al., 2010).

After release by cleavage, the ICD can translocate to the nucleus to regulate gene expression, be intracellularly trafficked within the endosomal system, or be degraded by the proteasome (Phillips et al., 2003; Haas et al., 2005; Fernández-Monreal et al., 2009; Hanson et al., 2010; Bouillot et al., 2011; O’Leary et al., 2011; Shonubi et al., 2015) (Figure 4A). While nuclear localization was substantiated for γ- and α-Pcdhs (Haas et al., 2005; Bonn et al., 2007; Emond and Jontes, 2008), the target genes or nuclear interactions of Pcdh ICDs are so far poorly characterized.

Regarding γ-Pcdhs, Ca²⁺/calmodulin-dependent protein kinase phosphatase (CaMPK) was shown to bind to PcdhγC5 and inhibit the nuclear translocation of the PcdhγC5 ICD. Once in the nucleus, the γ-Pcdh ICD was suggested to mediate autoregulatory γ-Pcdh expression processes by binding to the γ-Pcdh locus (Hambsch et al., 2005).

Among ncPcdhs, the ICD of human Pcdh FAT1 was found to translocate to the nucleus owing to a juxta-membrane nuclear translocation signal (NLS) (Magg et al., 2005). Moreover, the PCDH19 ICD, which contains several predicted NLSs, was shown to nuclearly localize and interact with the nuclear paraspeckle protein NONO in human cell lines (Pham et al., 2017).

Studies have shown that Pcdhs can be found in presynaptic and postsynaptic endosomal vesicles. Pcdhs can also regulate intracellular trafficking of synapse-associated proteins. For instance, Pcdh8 was demonstrated to associate with N-cad and induce its endocytosis at synapses, while Pcdh10 was shown to mediate associations between PSD-95 and the proteasome to initiate PSD-95 degradation (Yasuda et al., 2007; Tsai et al., 2012; Chal et al., 2017).

In the case of cPcdhs, evidence highlights the importance of the ICD for endosomal trafficking. For instance, the ICD of α-Pcdhs was found to interact with the endosomal sorting complex required for transport (ESCRT) in undifferentiated neuronal cells (Buchanan et al., 2010). Moreover, the ICD of γ-Pcdhs was demonstrated to be required for intracellular trafficking and cell surface delivery of these Pcdhs, and a conserved 26–residue ICD segment, known as the variable cytoplasmic domain (VCD) motif, was proven to be crucial for endolysosomal targeting (Fernández-Monreal et al., 2009, 2010; O’Leary et al., 2011; Shonubi et al., 2015). Furthermore, it was shown that colocalization of γ-PcdhA and γ-PcdhB isoforms with the endolysosomal markers autophagy protein LC3 and lysosome associated membrane protein 2 (LAMP-2) are ICD-dependent (Buchanan et al., 2010; Hanson et al., 2010). Initially, Pcdhs that do not engage in trans binding at the synaptic membrane were postulated to be endocytosed and stored in intracellular organelles to be eventually recycled (Phillips et al., 2003; Jontes and Phillips, 2006; Fernández-Monreal et al., 2010). Later, cPcdh trafficking was speculated to be involved in self-avoidance. According to the latter hypothesis, cPcdh-mediated matching between cell surfaces might induce endolysosomal trafficking of adhesive molecules, leading to the transition from transmembrane adhesion to detachment (Phillips et al., 2017; LaMassa et al., 2019).

cPcdhs can regulate the activity of cytoskeletal regulators such as Pyk2, focal adhesion kinase (FAK), and Rho-GTPases (e.g., Rac1) in processes such as dendritic arborization (α-, γ-Pcdhs) and cortical neuron migration (α-Pcdhs) by binding through their cytoplasmic tails (Figure 5). This binding inhibits the kinase activity, thus resulting in the activation of Rho GTPases capable of modulating neuronal cytoskeletal reorganization via both WRC- and non-WRC-mediated mechanisms (Chen et al., 2009; Garrett et al., 2012; Suo et al., 2012; Fan et al., 2018).

In γ-Pcdh-deficient mice defective dendritic arborization is the result of elevated phosphorylation of myristoylated alanine rich protein kinase C substrate (MARCKS). In the absence of γ-Pcdhs, FAK is activated through autophosphorylation. FAK in turn phosphorylates and activates protein kinase C (PKC) and phospholipase C (PLC). Active PKC phosphorylates MARCKS, leading to its dissociation from the membrane and actin (Hartwig et al., 1992), and consequently to a decrease in arbor complexity (Garrett et al., 2012). Additionally, PKC can contribute to the negative regulation of dendritic arborization by phosphorylating the ICD of γ-Pcdhs and allowing FAK release (Keeler et al., 2015b).

The ICD of α-Pcdhs was found to not only regulate dendritic morphology, but also to control cortical radial migration by inhibiting the autophosphorylation of Pyk2, and by recruiting the WRC via its WAVE interacting receptor sequence (WIRS) (Chen et al., 2009; Garrett et al., 2012; Suo et al., 2012; Fan et al., 2018). This WIRS motif is present and highly conserved in several other Pcdhs, including Pcdh10, Pcdh17, Pcdh18b, and Pcdh19 (Chen et al., 2014). Aberrant dendritic development and spine morphogenesis have also been connected to loss of α-Pcdhs. When α-Pcdhs are present, Pyk2 autophosphorylation is prevented, thus Rac1 is phosphorylated and activates the WRC; ultimately, this influences the formation of lamellipodial and filopodial protrusions (Chen et al., 2009; Garrett et al., 2012; Suo et al., 2012; Fan et al., 2018).

Nap1 is a core component of the WAVE complex and an important actin regulator (Chen et al., 2010). Several δ2-Pcdh ICDs (10, 17, 18b and 19) bind Nap1 through a conserved binding site, enabling δ2-Pcdh to regulate actin dynamics (Nakao et al., 2008; Tai et al., 2010; Biswas et al., 2014; Hayashi et al., 2014).

The interaction of δ2-Pcdhs with Nap1 plays a role in axon development. In deletion models, defects in axon initiation, outgrowth, pathfinding and branching have been reported in zebrafish motor neurons, mouse striatal axons, and Xenopus retinal ganglion cells (RGCs) (Uemura et al., 2007; Nakao et al., 2008; Piper et al., 2008; Biswas et al., 2014). The influence of these interactions on growth cone dynamics are exemplified by Pcdh17-expressing amygdala neuronal projections. Pcdh17 ICD can associate with Nap1, WAVE1 and Abi1, and recruit the WRC at inter-axonal contact sites. Additionally, Pcdh17 can recruit Lamellipodin (LPD)/MIG10 and Ena/VASP proteins via Nap1 to these sites, facilitating growth cone migration along other axons and thereby supporting collective axon extension. The GTPase Rac seems to be necessary for the recruitment of Nap1 by Pcdh17 ICD, as Rac inhibition blocks Nap1 and VASP agglomeration at contact sites (Hayashi et al., 2014). Rac is proposed to bind and regulate LPD interaction with the WRC and recruit Ena/VASP proteins to facilitate actin filament elongation (Pula and Krause, 2008; Law et al., 2013; Krause and Gautreau, 2014).

NcPcdh interactions with the WRC can also regulate cell migration processes. For instance, the Pcdh10 ICD can recruit Nap1 and WAVE1 to cell-cell contacts, and was shown to stimulate cell migration in human astrocytoma cells via F-actin and N-cad reorganization at contact sites (Nakao et al., 2008).

Thus, while cPcdhs facilitate Rho GTPases-mediated cytoskeletal reorganization principally by inhibiting cell adhesion kinases, ncPcdhs directly bind WRC components, which then promotes actin polymerization through actin regulators (Figure 5).

PCDH10 is a tumor suppressor gene that reduces cell proliferation in hepatocellular carcinoma (HCC), and can induce cancer cell apoptosis via several routes. First, PCDH10 can negatively regulate the PI3K/Akt signaling pathway, resulting in tumor suppressor gene 53 (p53) degradation (Zhao et al., 2014; Ye et al., 2017). Second, PCDH10 can induce apoptosis by inhibiting the NF-κB pathway, thus reducing anti-apoptotic proteins such as B-cell lymphoma (Bcl)-2 and survivin (Li et al., 2014). PCDH10 expression blocks NF-κB phosphorylation and nuclear translocation via IκB kinase (IKK) inhibition, hence preventing NF-κB constitutive activation. Third, PCDH10 can directly activate caspases to trigger apoptosis (Li et al., 2014; Yang et al., 2016).

Furthermore, PCDH9 was shown to act as a tumor suppressor by eliciting apoptosis and G0/G1 cell cycle arrest in glioma cells. In these cells, PCDH9 expression, respectively, upregulated and downregulated the synthesis of BAX and BCL-2 (Wang C. et al., 2014). Similarly, in gastric cancer cells PCDHGA9 overexpression induced apoptosis, cell cycle arrest, and autophagy. In this case, PCDHGA9 blocked TGF-β-induced epithelial-mesenchymal-transition (EMT) by inhibiting SMAD2/3 phosphorylation and nuclear translocation (Weng et al., 2018). In colorectal cancer restoring PCDH17 expression was found to enhance apoptotic pathway activation, and to induce autophagy by upregulating autophagic proteins such as Atg-5 and LC3BII (Hu et al., 2013).

In conclusion, current evidence suggests that several Pcdhs mediate pro-apoptotic functions through different signaling pathways.

In the developing brain both loss and overexpression of Pcdhs can elicit neuronal apoptosis. Therefore, the maintenance of proper Pcdh levels is crucial to preserve the balance between neuronal death and survival.

Excessive Pcdh7 causes primary cortical neuron apoptosis via downregulation of the apoptotic inhibitor survivin (BIRC5), and effect that is mediated by the cytoplasmic CM2 domain of Pcdh7 (Xiao et al., 2018).

Contrarily, in the absence of γ-Pcdhs Pyk2 autophosphorylates and accumulates in the cells, triggering their death (Chen et al., 2009). In addition, the γ-Pcdh ICD interacts with the intracellular adaptor protein programmed cell death 10 (PDCD10), and PDCD10 depletion attenuates chicken spinal neuron apoptosis caused by knockdown of γ-Pcdhs, implicating PDCD10 as an inducer of apoptosis downstream of these cPcdhs. In this context, γ-Pcdhs might protect neurons from apoptosis by sequestering PCDC10. Moreover, PDCD10 and Pyk2 cooperate to mediate the γ-Pcdhs-induced neuronal apoptosis (Lin et al., 2010).

Recently, γ-Pcdhs-deficient cINs were shown to have reduced phosphorylated serine-threonine kinase (AKT) levels. Numerous anti-apoptotic/pro-survival actions have been attributed to the PI3K-AKT pathway (Brunet et al., 2001), and cytoplasmic phospho-AKT is known to act as an anti-apoptotic factor. As loss of γ-Pcdhs increased apoptosis in cINs, γ-Pcdhs appear to have a role in cIN survival mediated by AKT (Carriere et al., 2020).

Overall, regulating Pcdh surface expression appears to be important for neuronal survival. Neuronal apoptosis is induced by ncPcdh overexpression or cPcdh loss. In oncogenesis, most Pcdhs seem to have a tumor suppressor function, as for several cancer types their downregulation [with the exception of PCDHB9 (Mukai et al., 2017; Sekino et al., 2019), and PCDH9 (Robbins et al., 2018)] correlates with tumor survival. Taken together, the precise dosage control of Pcdhs might therefore be crucial for cell survival in different contexts.

Over the last decade, evidence suggestive of a functional relationship between the Wnt signaling pathway and Pcdhs has been collected from a variety of studies. Intriguingly, the effect of Pcdh expression on Wnt signaling seems to be Pcdh- and context-dependent. The relationship between the Wnt pathway and Pcdhs has been mostly examined in cancer, where loss of Pcdhs often increases Wnt signaling, which in its turn stimulates cellular proliferation. A comprehensive overview of the different ways Pcdhs can regulate canonical Wnt signaling is shown in Figure 7.

Pcdhs appear to affect canonical Wnt signaling primarily by changing the ratio of nuclear versus cytoplasmic β-catenin. The subcellular distribution of β-catenin can be regulated at different levels. Reported mechanisms include: (1) the retention of β-catenin at the nucleus, the cytoplasm, or the plasma membrane; (2) the degradation of β-catenin in the cytoplasm by the destruction complex; (3) the independent or guided nuclear import/export of β-catenin, for example via TCF4 and BCL9 (import) or APC and Axin1 (export) (Behrens et al., 1996; Huber et al., 1996; Henderson, 2000; Neufeld et al., 2000; Rosin-Arbesfeld et al., 2000; Tolwinski and Wieschaus, 2001; Kramps et al., 2002; Cong and Varmus, 2004; Townsley et al., 2004; Krieghoff et al., 2006).

Wnt signaling regulation by β-catenin sequestration might not be limited to classical cadherins. Although it was generally accepted that all Pcdhs lack a β-catenin binding site, small serine-rich domains homologous to the β-catenin binding site of classical cadherins have been identified in the C-terminus of Pcdh7 and Pcdh11Y (Chen et al., 2002; Yang et al., 2005; Ren et al., 2018). Furthermore, mass spectrometric analysis provided direct evidence for a physical interaction between γ-Pcdhs and α- and β-catenin (Han et al., 2010).

Even without a β-catenin binding site, Pcdhs can affect the ratio of nuclear versus cytoplasmic β-catenin. PCDH18 knockdown in a human colon mucosal epithelial cell line promotes the nuclear accumulation of β-catenin and LEF/TCF transcriptional activity, while the overexpression of PCDH10, PCDH20, or PCDHGA9 in RPMI-8226, CNE1, or SGC-7901 cells, respectively, promotes the translocation of β-catenin from the nucleus to the cytoplasm and its accumulation at the membrane, thereby decreasing LEF/TCF activity (Chen et al., 2015; Xu et al., 2015; Zhou D. et al., 2017; Weng et al., 2018). The observed negative relationship between the expression of several Pcdhs and the nuclear accumulation of β-catenin contradicts findings describing increased Wnt signaling and TCF/LEF activity due to nuclear accumulation of β-catenin with PCDH11Y overexpression in human prostate cancer cells (LNCaP) (Yang et al., 2005). This discrepancy might be related to the proto-oncogenic role of Pcdh11Y.

The mechanisms by which translocation and accumulation occurs in Pcdhs lacking a β-catenin binding site are currently unknown. Perhaps they could indirectly sequester β-catenin through a common binding partner, and regulate the release and translocation of β-catenin via phosphorylation events or ICD cleavage. One cPcdh (PCDHGC3) was found to modulate Wnt signaling by directly binding the scaffold protein Axin1 at the cell membrane (Mah et al., 2016). Thus, sequestration of Wnt signaling molecules at the plasma membrane might be common within the Pcdh family. PCDHGC3 was found to compete with Disheveled to bind the DIX domain of Axin1, resulting in its stabilization, reduced phosphorylation of LRP6 and a decrease of Wnt signaling in luciferase TOP FLASH assays (Mah et al., 2016) (Figure 7A). Intriguingly, none of the other PCDHGs can bind Axin1. In contrast, individual overexpression of other PCDHG isoforms (PCDHGA1, PCDHGA3, PCDHGA7-10, PCDHGB1-7 and PCDHGC5) was observed to significantly increase β-catenin/TCF transcriptional activity (Mah et al., 2016). Similar to in vitro models, in vivo overexpression of PCDHGA1- or PCDHGC3-mCherry in Emx1-positive cells in the murine cerebral cortex significantly increased and decreased reporter activity, respectively (Mah et al., 2016). These findings further support the hypothesized Pcdh-subtype dependent nature of the effects of Pcdh expression on Wnt signaling.

The intracellular availability of β-catenin can also be directly regulated by its degradation in the cytoplasm. Multiple studies link the overexpression or silencing of ncPcdhs (PCDH8, PCDH10, PCDH18, PCDH20) in primary tumors and tumor cell lines to reduced or increased levels of phosphorylated GSK3β, respectively (Lv et al., 2015; Xu et al., 2015; Zhou D. et al., 2017; Zong et al., 2017). These changes in phosphorylated GSK3β levels were accompanied by altered β-catenin levels and expression of Wnt target genes (Figure 7B) (Lv et al., 2015; Xu et al., 2015; Zhou D. et al., 2017; Zong et al., 2017). Pcdhs could also modulate the activity of kinases and phosphatases that play a role in Wnt signaling. For instance, signaling downstream of Fak and Pyk2 has been linked to several Wnt pathway modules (Chen et al., 2002; Gao et al., 2015, 2019; Sun et al., 2016; Zheng et al., 2019).

Finally, some Pcdhs might regulate the expression of nuclear β-catenin importers/exporters. One study described a strongly reduced expression of BCL9 when PCDH10 was overexpressed in RPMI-8226 and KM3 cells (Xu et al., 2015). BCL9 is a necessary transcriptional co-activator of Wnt target genes, and a complex of BCL9 and Pygopus has been shown to recruit β-catenin to the nuclear compartment (Figure 7C) (Kramps et al., 2002; Townsley et al., 2004).

One of the best characterized Pcdh/Wnt interactions is the activation of the Wnt/Planar Cell Polarity (PCP) pathway by Pcdh8 to regulate convergent extension and tissue separation/morphogenesis during Xenopus laevis gastrulation (Figure 8). In the vertebrate Wnt/PCP pathway, binding of Wnt ligands to Frizzled (Fz) receptors recruits Disheveled (Dvl) to the membrane, resulting in the formation of complexes with either Disheveled-associated activator of morphogenesis 1 (Daam1) or small GTPase Rac1 that activate downstream signaling via RhoA or c-jun N-terminal kinase (JNK), respectively (Strutt et al., 1997; Axelrod et al., 1998; Boutros et al., 1998; Wallingford et al., 2000; Habas et al., 2001, 2003; Mah and Weiner, 2017). The extracellular domain of Pcdh8 is able to directly bind Frizzled7 to coordinate cellular polarity (Medina et al., 2004; Unterseher et al., 2004; Kraft et al., 2012). Both components are necessary for the initiation of Wnt/PCP signaling, as loss of Pcdh8 function was found to specifically block JNK activation via Rac1 (Unterseher et al., 2004). Furthermore, four intracellular Pcdh8 interaction partners related to the Wnt/PCP-associated molecular network have been recently discovered. The intracellular domain of Pcdh8 can sequester Sprouty to inhibit its antagonistic effect on Wnt/PCP signaling (Wang et al., 2008). Moreover, a direct physical interaction between Xenopus (x)ANR5 and Pcdh8 can activate the downstream effector molecules JNK and Rho, strengthening the output of the PCP pathway (Chung et al., 2007), while the interaction between Pcdh8 and Nemo-like Kinase1 (NLK1) is necessary for the stabilization of Pcdh8 (Kumar et al., 2017). Lastly, an interaction with casein kinase 2β (CK2β) blocks CK2β-mediated stabilization of β-catenin, thereby reducing canonical Wnt/β-catenin signaling (Kietzmann et al., 2012). Expression of Pcdh8 itself is regulated by Wnt/PCP signaling, providing a feedback loop into this pathway (Schambony and Wedlich, 2007).

In summary, although the majority of Pcdhs do not possess a β-catenin binding site, the interplay between cadherin-mediated adhesion and Wnt signaling seems to be conserved across the cadherin superfamily. Identifying a general interaction modality between Pcdh- and Wnt-mediated signaling pathways might prove difficult, as available evidence highlights Pcdh- and context-dependent effects on Wnt signaling. Clearly, additional research is required to better understand the complex relationship between individual Pcdhs and Wnt-related pathways. Moreover, since most studies so far have been performed in cancer cells, whether and how their findings might translate contextually to neural development is currently poorly understood. The comprehensive investigation of binding partners and molecular action mechanisms of individual Pcdhs will therefore be a crucial step in the complete elucidation of Pcdh functions across multiple contexts.