ReviewStructural origins of clustered protocadherin-mediated neuronal barcoding
Introduction
The establishment of functional neural circuits in the human brain involves highly specific connections among billions of neurons through trillions of synapses [1]. The formation of such complex neural circuits depends on a limited repertoire of guidance cues and cell surface receptors. Clustered protocadherins (Pcdhs) are a family of highly diverse cell-surface receptors that are thought to provide individual neurons with single-cell-specific molecular “barcodes”, which provide unique cell surface identities required for neurite self-avoidance [2], [3], [4]. Although recent studies have demonstrated that Pcdhs have additional roles in regulating neuronal survival, synaptogenesis, dendritic arborization, and neuronal tiling [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17] —this review focuses primarily on the role of Pcdhs in neuronal self-avoidance that in turn requires that neurons be able to distinguish “self” from “non-self”.
Mammalian genomes contain 50–60 Pcdh genes that are arranged in three contiguous gene clusters designated α, β, and γ [18], [19]. Other vertebrates, such as the fugu and elephant shark, also have Pcdh genes but with varying numbers of isoforms and distinct cluster organizations [20], [21]. Each Pcdh isoform has a distinct extracellular region, single pass transmembrane helix, and short cytoplasmic region encoded by a single “variable” exon. Additionally, the Pcdh α- and γ- gene clusters each contain three constant exons that encode a cluster-specific constant cytoplasmic region. Phylogenetic analysis of the 58 clustered Pcdh mouse isoforms revealed that they fall into five distinct subfamilies (Fig. 1): alternate α-Pcdhs (1–12), alternate β-Pcdhs (1–22), alternate γA-Pcdhs (1–12), alternate γB-Pcdhs (1–2 & 4–8), and C-type Pcdhs (αC1, αC2, γC3, γC4, and γC5) (Fig. 1). Alternate (non-C-type) Pcdh isoforms are chosen for expression in each neuron by a stochastic promoter choice mechanism [19], [22], [23], [24], [25], [26]. Individual neurons appear to express a small subset of the ∼50 alternate isoforms [19], [22], [23], [24], [25], [26]. The C-type Pcdhs are expressed ‘deterministically’ rather than stochastically [22], [24].
In neurite self-avoidance, an essential feature of neural circuit assembly, branching neurites (axons and dendrites) from the same neuron avoid one another, while neurites from different neurons do not. This assures that neurites from the same neuron can arborize extensively without crossing or clumping, while neurites from different neurons can interdigitate and occupy the same field. This phenomenon requires a mechanism that allows individual neurons to distinguish self from non-self interactions [27], [28]. It appears that, for both vertebrates and insects, neuronal self-avoidance relies on generating unique individual cell surface identities through the stochastic expression of diverse repertoires of cell surface protein isoforms [27], [28], [29], [30]. In the fly neuronal identity is defined by the expression of single-cell-specific subsets of Dscam1 protein isoforms, generated by stochastic alternative RNA splicing [31], [32], [33], [34]. In vertebrates, neuronal identity is provided by stochastic expression of single-cell-specific subsets of Pcdh isoforms [4], [22], [23], [24].
Counter-intuitively, in both insects and vertebrates the process of self-avoidance begins with adhesive homophilic interactions required for recognition [27], [35], [36], [37], [38]. In the fly, there are 19,008 possible Dscam isoforms with distinct extracellular domains, of which ∼10–50 are expressed in each neuron [31], [33], [34], [35], [39]. The majority of these isoforms bind in trans in a strictly homophilic manner [35], [36]. In mammals, the 50–60 Pcdh isoforms have been shown to bind with homophilic specificity, as will be discussed below. Current thinking posits that identical Dscam/Pcdh isoforms located on the surface of neurites emanating from the same cell bind to each other homophilically in trans (different neurites) and this interaction triggers a signaling process that requires the intracellular domains [40], which leads to repulsion. In contrast, when two neurons expressing a sufficiently diverse set of Dscam/Pcdh isoforms come into contact, their different isoform composition will not lend itself to homophilic binding and hence an avoidance mechanism will not be triggered [27].
The large number of distinct Dscam1 isoforms generated in individual neurons by alternative RNA splicing decreases the probability that any two interacting fly neurons have an identical or even a similar isoform repertoire [29]. Assuming, for example, that 15 distinct isoforms are produced per cell, the probabilities that two cells will express three or more isoforms in common (thereby presumably leading to inappropriate initial adhesion and then repulsion) is ∼10−7 (Table 1). These numbers are small enough to ensure that inappropriate repulsion will be a rare event [29]. How do Pcdhs, with far fewer isoforms, provide sufficient diversity for a single-cell identity within mammalian nervous systems, which are far more complex than that of the fly? Recent structural and biophysical studies combined with cell-based aggregation assays have provided a surprising mechanism that appears to solve this problem. Here, we review recent studies that have transformed our understanding of Pcdh structure and function, and have led to the proposal of a structure-based mechanism for neuronal barcoding, which provides greater neuronal diversity than that of Drosophila Dscam1.
Section snippets
Homophilic cell–cell recognition specificity
In common with many other cadherin superfamily members [41], Pcdhs function in cell–cell recognition through calcium-dependent binding between their extracellular regions [37]. The Pcdh extracellular region contains six extracellular cadherin (EC) domains, each of which is composed of approximately 100 residues that form a two-layered anti-parallel β-sheet structure. Binding three Ca2+ ions to cadherin-conserved calcium-binding motifs stabilizes Pcdh EC interdomain junctions.
In an important
Crystal structures of Pcdh trans dimers
The most thoroughly characterized cadherins are the classical cadherins, which mediate calcium dependent cell–cell adhesion through trans (cell-cell) homodimerization of their membrane-distal EC1 domains. In contrast to classical cadherins the first Pcdh structures obtained, which included the membrane-distal EC1 domains [47] or EC1–EC3 domain fragments [48], [49], were found to be monomeric in solution. Consistently, constructs of corresponding size did not mediate cell–cell binding in cell
Structural basis of Pcdh homophilic specificity
In order to identify the Pcdh trans-homophilic specificity-determining domains Pcdh chimeras with shuffled EC domains between different isoforms were used [38], [49]. Studies using Pcdh chimeras with multiple domains shuffled simultaneously demonstrated that chimeras with non-matching EC1 and EC4 domains do not bind to each other even when their EC2 and EC3 domains are identical [49]. Similarly, chimeras with non-matching EC2 and EC3 domains do not bind to each other even when their EC1 and EC4
Interference and tolerance
It is critical that two interacting neurons do not erroneously recognize each other as “self” and avoid each other. However, since both Dscams and Pcdhs are stochastically expressed, there is a finite probability that any pair of neurons will express one or more common isoform, which will then bind to each other and potentially signal both cells to move apart. How can this inappropriate repulsion be avoided? Table 1 reports probabilities that two cells will randomly express one or more of the
cis-Dimeric recognition units
In addition to their homophilic trans interactions, Pcdhs also interact in cis [37], [38], [49], [53]. Solution biophysical measurements of purified recombinant Pcdh ectodomains and cell aggregation studies showed that Pcdhs form cis dimers mediated by EC5 and EC6. Specifically, ectodomain fragments containing the EC5–EC6 domains of β and γΒ isoforms dimerize in solution independent of the EC1–EC4 trans dimer interactions, but do not aggregate cells indicating that this interaction occurs in cis
Two models of the Pcdh recognition complex and associated functional implications
Two molecular models have been proposed to account for the role of Pcdhs in neuronal recognition. The first was based on the assumption (which we now know to be incorrect) that Pcdhs form cis-tetrameric recognition units that interact in trans to form discrete octamers between apposed cells. In this model, interference is caused by the dilution of matched isoform pairs on different cells through their incorporation into a large number of cis tetramers with isoforms that are not matched [60].
Conclusion
Comparison of the molecular logic of Dscams and Pcdhs reveals a number of remarkable insights as to how vertebrates and many invertebrates have evolved to solve the problem of neuronal barcoding. Drosophila use alternative splicing to generate diversity that is coded on three independent domains, each of which exhibits homophilic binding specificity. Since each domain presents a separate interface, the 19,008 distinct isoforms simply correspond to the product of the number of alternative exons
Acknowledgments
This work was supported by a National Science Foundation grant to B.H. (MCB-1412472), an NIH grant to L.S. (R01GM062270), and a joint NIH grant to T.M. and L.S. (R01GM107571). The computing in this project was supported by two NIH instrumentation grants (S10OD012351 and S10OD021764) received by the Department of Systems Biology at Columbia University.
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Collective mechanical responses of cadherin-based adhesive junctions as predicted by simulations
2022, Biophysical JournalCitation Excerpt :The predicted mechanical strength of the PCDHγB4 trans dimers, isolated and in junctions, is intriguing given that the clustered PCDHs mainly play a role in neuronal recognition (82,83), while the adherens junction and the desmosome maintain epithelial cell-cell adhesion must withstand significant mechanical stress. The γ-PCDHs, of which PCHDγB4 is a member, might play a role in adhesion at the synapse (98,152), but it is difficult to say if the clustered PCDHs are under or respond to any mechanical strain in vivo. Perhaps this brittle response is appropriate for more subtle types of mechanical stimuli that clustered PCDHs may experience in neurons during development, normal function, and in brain injury (66–73, 76).
Novel ultra-rare exonic variants identified in a founder population implicate cadherins in schizophrenia
2021, NeuronCitation Excerpt :The PCDHA3 gene is a member of the clustered protocadherin gene family. This family comprises three gene clusters, α, β, and γ, which are stochastically expressed in individual neurons, generating a cell surface “barcode” required for neuronal self-recognition (Canzio and Maniatis, 2019; Mountoufaris et al., 2018; Rubinstein et al., 2017; Wu and Jia, 2021). Individual PCDH isoforms from all three gene clusters form nearly random cis-dimers (αβ, αγ, β/γ,β/β,γ/γ − but not α/α), which assemble on the cell surface, and engage in highly specific homophilic interactions with cis-dimers on apposing plasma membranes of neurites, which leads to self-recognition and neurite repulsion (Goodman et al., 2016, 2017; Rubinstein et al., 2015; Thu et al., 2014).
The role of clustered protocadherins in neurodevelopment and neuropsychiatric diseases
2020, Current Opinion in Genetics and DevelopmentCitation Excerpt :In contrast, the regulated (as opposed to stochastic) expression of Pcdhαc2 and the stochastic expression of the Pcdhβ and Pcdhγ gene clusters are regulated by an independent, unidentified mechanism, as these genes do not generate an antisense lncRNA and the variable exons do not contain CTCF binding sites. While the protocadherin gene cluster encodes only 52 unique isoforms in humans, detailed structural and biochemical studies have demonstrated that complex, heterophilic cis-PCDH dimerization and homophilic trans-PCDH interactions confer the high degree of cellular specificity required proper self-avoidance [11,32–35,36•]. The cPCDH proteins consist of six extracellular cadherin domains (EC1-6), which are required for specific cPCDH interactions.
The generation of a protocadherin cell-surface recognition code for neural circuit assembly
2019, Current Opinion in Neurobiology
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Co-first authors.