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Neurons form cell-type-specific morphologies that are shaped by cell-surface molecules and their cellular events governing dendrite growth. One growth rule is dendrite self-avoidance, whereby dendrites distribute uniformly within a neuron's territory by avoiding sibling branches. In mammalian neurons, dendrite self-avoidance is regulated by a large family of cell-recognition molecules called the clustered protocadherins (cPcdhs). Genetic and molecular studies suggest that the cPcdhs mediate homophilic recognition and repulsion between self-dendrites. However, this model has not been tested through direct investigation of self-avoidance during development. Here, we performed live imaging and four-dimensional (4D) quantifications of dendrite morphogenesis to define the dynamics and cPcdh-dependent mechanisms of self-avoidance. We focused on the mouse retinal starburst amacrine cell (SAC), which requires the gamma-Pcdhs (Pcdhgs) and self/non-self-recognition to establish a stereotypic radial morphology while permitting dendritic interactions with neighboring SACs. Through morphogenesis, SACs extend dendritic protrusions that iteratively fill the growing arbor and contact and retract from nearby self-dendrites. Compared to non-self-contacting protrusions, self-contacting events have longer lifetimes, and a subset persists as loops. In the absence of the Pcdhgs, non-self-contacting dynamics are unaffected but self-contacting retractions are significantly diminished. Self-contacting bridges accumulate, leading to the bundling of dendritic processes and disruption to the arbor shape. By tracking dendrite self-avoidance in real time, our findings establish that the γ-Pcdhs mediate self-recognition and retraction between contacting sibling dendrites. Our results also illustrate how self-avoidance shapes stochastic and space-filling dendritic outgrowth for robust pattern formation in mammalian neurons.

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In the developing human brain, only 53 stochastically expressed clustered protocadherin (cPcdh) isoforms enable neurites from individual neurons to recognize and self-avoid while simultaneously maintaining contact with neurites from other neurons. Cell assays have demonstrated that self-recognition occurs only when all cPcdh isoforms perfectly match across the cell boundary, with a single mismatch in the cPcdh expression profile interfering with recognition. It remains unclear, however, how a single mismatched isoform between neighboring cells is sufficient to block erroneous recognitions. Using systematic cell aggregation experiments, we show that abolishing cPcdh interactions on the same membrane (cis) results in a complete loss of specific combinatorial binding between cells (trans). Our computer simulations demonstrate that the organization of cPcdh in linear array oligomers, composed of cis and trans interactions, enhances self-recognition by increasing the concentration and stability of cPcdh trans complexes between the homotypic membranes. Importantly, we show that the presence of mismatched isoforms between cells drastically diminishes the concentration and stability of the trans complexes. Overall, we provide an explanation for the role of the cPcdh assembly arrangements in neuronal self/non-self-discrimination underlying neuronal self-avoidance.

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Neuroendocrine neoplasms (NENs) encompass tumors arising from neuroendocrine cells in various organs, including the gastrointestinal tract, pancreas, adrenal gland, and paraganglia. Despite advancements, accurately predicting the aggressiveness of gastroenteropancreatic (GEP) NENs based solely on pathological data remains challenging, thereby limiting optimal clinical management. Our previous research unveiled a crucial link between hypermethylation of the protocadherin PCDHGC3 gene and neuroendocrine tumors originating from the paraganglia and adrenal medulla. This epigenetic alteration was associated with increased metastatic potential and succinate dehydrogenase complex (SDH) dysfunction. Expanding upon this discovery, the current study explored PCDHGC3 gene methylation within the context of GEP-NENs in a cohort comprising 34 cases. We uncovered promoter hypermethylation of PCDHGC3 in 29% of GEP-NENs, with a significantly higher prevalence in gastrointestinal (GI) neuroendocrine carcinomas (NECs) compared with both pancreatic (Pan) NECs and neuroendocrine tumors (NETs) of GI and Pan origin. Importantly, these findings were validated in one of the largest multi-center GEP-NEN cohorts. Mechanistic analysis revealed that PCDHGC3 hypermethylation was not associated with SDH mutations or protein loss, indicating an SDH-independent epigenetic mechanism. Clinically, PCDHGC3 hypermethylation emerged as a significant prognostic factor, correlating with reduced overall survival rates in both patient cohorts. Significantly, whereas PCDHGC3 hypermethylation exhibited a strong correlation with TP53 somatic mutations, a hallmark of NEC, its predictive value surpassed that of TP53 mutations, with an area under the curve (AUC) of 0.95 (95% CI 0.83-1.0) for discriminating GI-NECs from GI-NETs, highlighting its superior predictive performance. In conclusion, our findings position PCDHGC3 methylation status as a promising molecular biomarker for effectively stratifying patients with GI-NENs. This discovery has the potential to advance patient care by enabling more precise risk assessments and tailored treatment strategies. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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Non-clustered protocadherins (ncPcdhs) are adhesive molecules with spatio-temporally regulated overlapping expression in the developing nervous system. Although their unique role in neurogenesis has been widely studied, their combinatorial role in brain physiology and pathology is poorly understood. Using probabilistic cell typing by in situ sequencing, we demonstrate combinatorial inter- and intra-familial expression of ncPcdhs in the developing mouse cortex and hippocampus, at single-cell resolution. We discovered the combinatorial expression of Protocadherin-19 (Pcdh19), a protein involved in PCDH19-clustering epilepsy, with Pcdh1, Pcdh9 or Cadherin 13 (Cdh13) in excitatory neurons. Using aggregation assays, we demonstrate a code-specific adhesion function of PCDH19; mosaic PCDH19 absence in PCDH19+9 and PCDH19 + CDH13, but not in PCDH19+1 codes, alters cell-cell interaction. Interestingly, we found that PCDH19 as a dominant protein in two heterophilic adhesion codes could promote trans-interaction between them. In addition, we discovered increased CDH13-mediated cell adhesion in the presence of PCDH19, suggesting a potential role of PCDH19 as an adhesion mediator of CDH13. Finally, we demonstrated novel cis-interactions between PCDH19 and PCDH1, PCDH9 and CDH13. These observations suggest that there is a unique combinatorial code with a cell- and region-specific characteristic where a single molecule defines the heterophilic cell-cell adhesion properties of each code.

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Proper growth and branching of dendrites are crucial for adequate central nervous system (CNS) functioning. The neuronal dendritic geometry determines the mode and quality of information processing. Any defects in dendrite development will disrupt neuronal circuit formation, affecting brain function. Besides cell-intrinsic programmes, extrinsic factors regulate various aspects of dendritic development. Among these extrinsic factors are extracellular molecular signals which can shape the dendrite architecture during early development. This review will focus on extrinsic factors regulating dendritic growth during early neuronal development, including neurotransmitters, neurotrophins, extracellular matrix proteins, contact-mediated ligands, and secreted and diffusible cues. How these extracellular molecular signals contribute to dendritic growth has been investigated in developing nervous systems using different species, different areas within the CNS, and different neuronal types. The response of the dendritic tree to these extracellular molecular signals can result in growth-promoting or growth-limiting effects, and it depends on the receptor subtype, receptor quantity, receptor efficiency, the animal model used, the developmental time windows, and finally, the targeted signal cascade. This article reviews our current understanding of the role of various extracellular signals in the establishment of the architecture of the dendrites.

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Light touch sensation begins with activation of low-threshold mechanoreceptor (LTMR) endings in the skin and propagation of their signals to the spinal cord and brainstem. We found that the clustered protocadherin gamma (Pcdhg) gene locus, which encodes 22 cell-surface homophilic binding proteins, is required in somatosensory neurons for normal behavioral reactivity to a range of tactile stimuli. Developmentally, distinct Pcdhg isoforms mediate LTMR synapse formation through neuron-neuron interactions and peripheral axonal branching through neuron-glia interactions. The Pcdhgc3 isoform mediates homophilic interactions between sensory axons and spinal cord neurons to promote synapse formation in vivo and is sufficient to induce postsynaptic specializations in vitro. Moreover, loss of Pcdhgs and somatosensory synaptic inputs to the dorsal horn leads to fewer corticospinal synapses on dorsal horn neurons. These findings reveal essential roles for Pcdhg isoform diversity in somatosensory neuron synapse formation, peripheral axonal branching, and stepwise assembly of central mechanosensory circuitry.

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We report a case of fetal diencephalic-mesencephalic junction dysplasia (DMJD) diagnosed prenatally. Prenatal ultrasound at 24 gestational weeks showed that the fetus was small, about the size at 22 weeks' gestation, with short biparietal diameter and enhanced echo at the anterior border of thalamus. Fetal MRI showed short T2 signal shadow in the left choroid plexus, and hemorrhage and midbrain dysplasia were suspected. A pathogenic homozygous mutation variant in protocadherins 12 gene (c.1558C>T) was found in this fetus by whole exome sequencing and both parents carried the same heterozygous variation revealed by Sanger sequencing. All of the above information lead to the diagnosis of fetal DMJD, and the pregnancy was terminated after genetic counseling.

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Defects in the endothelial cell barrier accompany diverse malfunctions of the central nervous system such as neurodegenerative diseases, stroke, traumatic brain injury, and systemic diseases such as sepsis, viral and bacterial infections, and cancer. Compromised endothelial sealing leads to leaking blood vessels, followed by vasogenic edema. Brain edema as the most common complication caused by stroke and traumatic brain injury is the leading cause of death. Brain microvascular endothelial cells, together with astrocytes, pericytes, microglia, and neurons form a selective barrier, the so-called blood-brain barrier, which regulates the movement of molecules inside and outside of the brain. Mechanisms that regulate blood-brain barrier permeability in health and disease are complex and not fully understood. Several newly discovered molecules that are involved in the regulation of cellular processes in brain microvascular endothelial cells have been described in the literature in recent years. One of these molecules that are highly expressed in brain microvascular endothelial cells is protocadherin gamma C3. In this review, we discuss recent evidence that protocadherin gamma C3 is a newly identified key player involved in the regulation of vascular barrier function.

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Crystal structures of many cell-cell adhesion receptors reveal the formation of linear "molecular zippers" comprising an ordered one-dimensional array of proteins that form both intercellular (trans) and intracellular (cis) interactions. The clustered protocadherins (cPcdhs) provide an exemplar of this phenomenon and use it as a basis of barcoding of vertebrate neurons. Here, we report both Metropolis and kinetic Monte Carlo simulations of cPcdh zipper formation using simplified models of cPcdhs that nevertheless capture essential features of their three-dimensional structure. The simulations reveal that the formation of long zippers is an implicit feature of cPcdh structure and is driven by their cis and trans interactions that have been quantitatively characterized in previous work. Moreover, in agreement with cryo-electron tomography studies, the zippers are found to organize into two-dimensional arrays even in the absence of attractive interactions between individual zippers. Our results suggest that the formation of ordered two-dimensional arrays of linear zippers of adhesion proteins is a common feature of cell-cell interfaces. From the perspective of simulations, they demonstrate the importance of a realistic depiction of adhesion protein structure and interactions if important biological phenomena are to be properly captured.

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The stochastic expression of fewer than 60 clustered protocadherin (cPcdh) isoforms provides diverse identities to individual vertebrate neurons and a molecular basis for self-/nonself-discrimination. cPcdhs form chains mediated by alternating cis and trans interactions between apposed membranes, which has been suggested to signal self-recognition. Such a mechanism requires that cPcdh cis dimers form promiscuously to generate diverse recognition units, and that trans interactions have precise specificity so that isoform mismatches terminate chain growth. However, the extent to which cPcdh interactions fulfill these requirements has not been definitively demonstrated. Here, we report biophysical experiments showing that cPcdh cis interactions are promiscuous, but with preferences favoring formation of heterologous cis dimers. Trans homophilic interactions are remarkably precise, with no evidence for heterophilic interactions between different isoforms. A new C-type cPcdh crystal structure and mutagenesis data help to explain these observations. Overall, the interaction characteristics we report for cPcdhs help explain their function in neuronal self-/nonself-discrimination.

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BACKGROUND: Adverse cardiac remodeling and tissue damage following heart disease is strongly associated with chronic low grade inflammation. The mechanisms underlying persisting inflammatory signals are not fully understood, but may involve defective and/or non-responsive transcriptional and post-transcriptional regulatory mechanisms. In the current study, we aimed to identify novel mediators and pathways involved in processes associated with inflammation in the development and maintenance of cardiac disease. METHODS AND RESULTS: We performed RNA sequencing analysis of cardiac tissue from patients undergoing coronary artery bypass grafting (CABG) or aortic valve replacement (AVR) and compared with control tissue from multi-organ donors. Our results confirmed previous findings of a marked upregulated inflammatory state, but more importantly, we found pronounced reduction of non-protein coding genes, particularly long non-coding RNAs (lncRNA), including several lncRNAs known to be associated with inflammation and/or cardiovascular disease. In addition, Gene Set Enrichment Analysis revealed markedly downregulated microRNA pathways, resulting in aberrant expression of other genes, particularly γ-protocadherins. CONCLUSIONS: Our data suggest that aberrant expression of non-coding gene regulators comprise crucial keys in the progression of heart disease, and may be pivotal for chronic low grade inflammation associated with cardiac dysfunction. By unmasking atypical γ-protocadherin expression as a prospective genetic biomarker of myocardial dysfunction, our study provides new insight into the complex molecular framework of heart disease. Creating new approaches to modify non-coding gene regulators, such as those identified in the current study, may define novel strategies to shift γ-protocadherin expression, thereby normalizing part of the molecular architecture associated with heart disease.

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The structure of eukaryotic genes is generally a combination of exons interrupted by intragenic non-coding DNA regions (introns) removed by RNA splicing to generate the mature mRNA. A fraction of genes, however, comprise a single coding exon with introns in their untranslated regions or are intronless genes (IGs), lacking introns entirely. The latter code for essential proteins involved in development, growth, and cell proliferation and their expression has been proposed to be highly specialized for neuro-specific functions and linked to cancer, neuropathies, and developmental disorders. The abundant presence of introns in eukaryotic genomes is pivotal for the precise control of gene expression. Notwithstanding, IGs exempting splicing events entail a higher transcriptional fidelity, making them even more valuable for regulatory roles. This work aimed to infer the functional role and evolutionary history of IGs centered on the mouse genome. IGs consist of a subgroup of genes with one exon including coding genes, non-coding genes, and pseudogenes, which conform approximately 6% of a total of 21,527 genes. To understand their prevalence, biological relevance, and evolution, we identified and studied 1,116 IG functional proteins validating their differential expression in transcriptomic data of embryonic mouse telencephalon. Our results showed that overall expression levels of IGs are lower than those of MEGs. However, strongly up-regulated IGs include transcription factors (TFs) such as the class 3 of POU (HMG Box), Neurog1, Olig1, and BHLHe22, BHLHe23, among other essential genes including the ß-cluster of protocadherins. Most striking was the finding that IG-encoded BHLH TFs fit the criteria to be classified as microproteins. Finally, predicted protein orthologs in other six genomes confirmed high conservation of IGs associated with regulating neural processes and with chromatin organization and epigenetic regulation in Vertebrata. Moreover, this study highlights that IGs are essential modulators of regulatory processes, such as the Wnt signaling pathway and biological processes as pivotal as sensory organ developing at a transcriptional and post-translational level. Overall, our results suggest that IG proteins have specialized, prevalent, and unique biological roles and that functional divergence between IGs and MEGs is likely to be the result of specific evolutionary constraints.

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Neurodevelopment in humans is a long, elaborate, and highly coordinated process involving three trimesters of prenatal development followed by decades of postnatal development and maturation. Throughout this period, the brain is highly sensitive and responsive to the external environment, which may provide a range of inputs leading to positive or negative outcomes. Fetal alcohol spectrum disorders (FASD) result from prenatal alcohol exposure (PAE). Although the molecular mechanisms of FASD are not fully characterized, they involve alterations to the regulation of gene expression via epigenetic marks. As in the prenatal stages, the postnatal period of neurodevelopment is also sensitive to environmental inputs. Often this sensitivity is reflected in children facing adverse conditions, such as maternal separation. This exposure to early life stress (ELS) is implicated in the manifestation of various behavioral abnormalities. Most FASD research has focused exclusively on the effect of prenatal ethanol exposure in isolation. Here, we review the research into the effect of prenatal ethanol exposure and ELS, with a focus on the continuum of epigenomic and transcriptomic alterations. Interestingly, a select few experiments have assessed the cumulative effect of prenatal alcohol and postnatal maternal separation stress. Regulatory regions of different sets of genes are affected by both treatments independently, and a unique set of genes are affected by the combination of treatments. Notably, epigenetic and gene expression changes converge at the clustered protocadherin locus and oxidative stress pathway. Functional studies using epigenetic editing may elucidate individual contributions of regulatory regions for hub genes and further profiling efforts may lead to the development of non-invasive methods to identify children at risk. Taken together, the results favor the potential to improve neurodevelopmental outcomes by epigenetic management of children born with FASD using favorable postnatal conditions with or without therapeutic interventions.

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BACKGROUND: Although the genomes of monozygotic twins are practically identical, their methylomes may evolve divergently throughout their lifetime as a consequence of factors such as the environment or aging. Particularly for young and healthy monozygotic twins, DNA methylation divergence, if any, may be restricted to stochastic processes occurring post-twinning during embryonic development and early life. However, to what extent such stochastic mechanisms can systematically provide a stable source of inter-individual epigenetic variation remains uncertain until now. RESULTS: We enriched for inter-individual stochastic variation by using an equivalence testing-based statistical approach on whole blood methylation microarray data from healthy adolescent monozygotic twins. As a result, we identified 333 CpGs displaying similarly large methylation variation between monozygotic co-twins and unrelated individuals. Although their methylation variation surpasses measurement error and is stable in a short timescale, susceptibility to aging is apparent in the long term. Additionally, 46% of these CpGs were replicated in adipose tissue. The identified sites are significantly enriched at the clustered protocadherin loci, known for stochastic methylation in developing neurons. We also confirmed an enrichment in monozygotic twin DNA methylation discordance at these loci in whole genome bisulfite sequencing data from blood and adipose tissue. CONCLUSIONS: We have isolated a component of stochastic methylation variation, distinct from genetic influence, measurement error, and epigenetic drift. Biomarkers enriched in this component may serve in the future as the basis for universal epigenetic fingerprinting, relevant for instance in the discrimination of monozygotic twin individuals in forensic applications, currently impossible with standard DNA profiling.

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There are more than a thousand trillion specific synaptic connections in the human brain and over a million new specific connections are formed every second during the early years of life. The assembly of these staggeringly complex neuronal circuits requires specific cell-surface molecular tags to endow each neuron with a unique identity code to discriminate self from non-self. The clustered protocadherin (Pcdh) genes, which encode a tremendous diversity of cell-surface assemblies, are candidates for neuronal identity tags. We describe the adaptive evolution, genomic structure, and regulation of expression of the clustered Pcdhs. We specifically focus on the emerging 3-D architectural and biophysical mechanisms that generate an enormous number of diverse cell-surface Pcdhs as neural codes in the brain.

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There are more than a thousand trillion specific synaptic connections in the human brain and over a million new specific connections are formed every second during the early years of life. The assembly of these staggeringly complex neuronal circuits requires specific cell-surface molecular tags to endow each neuron with a unique identity code to discriminate self from non-self. The clustered protocadherin (Pcdh) genes, which encode a tremendous diversity of cell-surface assemblies, are candidates for neuronal identity tags. We describe the adaptive evolution, genomic structure, and regulation of expression of the clustered Pcdhs. We specifically focus on the emerging 3-D architectural and biophysical mechanisms that generate an enormous number of diverse cell-surface Pcdhs as neural codes in the brain.

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During brain development, neurons need to form the correct connections with one another in order to give rise to a functional neuronal circuitry. Mistakes during this process, leading to the formation of improper neuronal connectivity, can result in a number of brain abnormalities and impairments collectively referred to as neurodevelopmental disorders. Cell adhesion molecules (CAMs), present on the cell surface, take part in the neurodevelopmental process regulating migration and recognition of specific cells to form functional neuronal assemblies. Among CAMs, the members of the protocadherin (PCDH) group stand out because they are involved in cell adhesion, neurite initiation and outgrowth, axon pathfinding and fasciculation, and synapse formation and stabilization. Given the critical role of these macromolecules in the major neurodevelopmental processes, it is not surprising that clinical and basic research in the past two decades has identified several PCDH genes as responsible for a large fraction of neurodevelopmental disorders. In the present article, we review these findings with a focus on the non-clustered PCDH sub-group, discussing the proteins implicated in the main neurodevelopmental disorders.

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The clustered protocadherins (cPcdhs) are a subfamily of type I single-pass transmembrane cell adhesion molecules predominantly expressed in the brain. Their stochastic and combinatorial expression patterns encode highly diverse neural identity codes which are central for neuronal self-avoidance and non-self discrimination in brain circuit formation. In this review, we first briefly outline mechanisms for generating a tremendous diversity of cPcdh cell-surface assemblies. We then summarize the biological functions of cPcdhs in a wide variety of neurodevelopmental processes, such as neuronal migration and survival, dendritic arborization and self-avoidance, axonal tiling and even spacing, and synaptogenesis. We focus on genetic, epigenetic, and 3D genomic dysregulations of cPcdhs that are associated with various neuropsychiatric and neurodevelopmental diseases. A deeper understanding of regulatory mechanisms and physiological functions of cPcdhs should provide significant insights into the pathogenesis of mental disorders and facilitate development of novel diagnostic and therapeutic strategies.

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Inhibitory interneurons integrate into developing circuits in specific ratios and distributions. In the neocortex, inhibitory network formation occurs concurrently with the apoptotic elimination of a third of GABAergic interneurons. The cell surface molecules that select interneurons to survive or die are unknown. Here, we report that members of the clustered Protocadherins (cPCDHs) control GABAergic interneuron survival during developmentally-regulated cell death. Conditional deletion of the gene cluster encoding the γ-Protocadherins (Pcdhgs) from developing GABAergic neurons in mice of either sex causes a severe loss of inhibitory populations in multiple brain regions and results in neurologic deficits such as seizures. By focusing on the neocortex and the cerebellar cortex, we demonstrate that reductions of inhibitory interneurons result from elevated apoptosis during the critical postnatal period of programmed cell death (PCD). By contrast, cortical interneuron (cIN) populations are not affected by removal of Pcdhgs from pyramidal neurons or glial cells. Interneuron loss correlates with reduced AKT signaling in Pcdhg mutant interneurons, and is rescued by genetic blockade of the pro-apoptotic factor BAX. Together, these findings identify the PCDHGs as pro-survival transmembrane proteins that select inhibitory interneurons for survival and modulate the extent of PCD. We propose that the PCDHGs contribute to the formation of balanced inhibitory networks by controlling the size of GABAergic interneuron populations in the developing brain.SIGNIFICANCE STATEMENT A pivotal step for establishing appropriate excitatory-inhibitory ratios is adjustment of neuronal populations by cell death. In the mouse neocortex, a third of GABAergic interneurons are eliminated by BAX-dependent apoptosis during the first postnatal week. Interneuron cell death is modulated by neural activity and pro-survival pathways but the cell-surface molecules that select interneurons for survival or death are unknown. We demonstrate that members of the cadherin superfamily, the clustered γ-Protocadherins (PCDHGs), regulate the survival of inhibitory interneurons and the balance of cell death. Deletion of the Pcdhgs in mice causes inhibitory interneuron loss in the cortex and cerebellum, and leads to motor deficits and seizures. Our findings provide a molecular basis for controlling inhibitory interneuron population size during circuit formation.

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