PCDH17 restricts dendritic spine morphogenesis by regulating ROCK2-dependent control of the actin cytoskeleton, modulating emotional behavior.

Proper regulation of synapse formation and elimination is critical for establishing mature neuronal circuits and maintaining brain function. Synaptic abnormalities, such as defects in the density and morphology of postsynaptic dendritic spines, underlie the pathology of various neuropsychiatric disorders. Protocadherin 17 (PCDH17) is associated with major mood disorders, including bipolar disorder and depression. However, the molecular mechanisms by which PCDH17 regulates spine number, morphology, and behavior remain elusive. In this study, we found that PCDH17 functions at postsynaptic sites, restricting the number and size of dendritic spines in excitatory neurons. Selective overexpression of PCDH17 in the ventral hippocampal CA1 results in spine loss and anxiety- and depression-like behaviors in mice. Mechanistically, PCDH17 interacts with actin-relevant proteins and regulates actin filament (F-actin) organization. Specifically, PCDH17 binds to ROCK2, increasing its expression and subsequently enhancing the activity of downstream targets such as LIMK1 and the phosphorylation of cofilin serine-3 (Ser3). Inhibition of ROCK2 activity with belumosudil (KD025) ameliorates the defective F-actin organization and spine structure induced by PCDH17 overexpression, suggesting that ROCK2 mediates the effects of PCDH17 on F-actin content and spine development. Hence, these findings reveal a novel mechanism by which PCDH17 regulates synapse development and behavior, providing pathological insights into the neurobiological basis of mood disorders.

The Role of Protocadherin γ in Adult Sensory Neurons and Skin Reinnervation.

The clustered protocadherins (cPcdhs) play a critical role in the patterning of several CNS axon and dendritic arbors, through regulation of homophilic self and neighboring interactions. While not explored, primary peripheral sensory afferents that innervate the epidermis may require similar constraints to convey spatial signals with appropriate fidelity. Here, we show that members of the γ-Pcdh (Pcdhγ) family are expressed in both adult sensory neuron axons and in neighboring keratinocytes that have close interactions during skin reinnervation. Adult mice of both sexes were studied. Pcdhγ knock-down either through small interfering RNA (siRNA) transduction or AAV-Cre recombinase transfection of adult mouse primary sensory neurons from floxed Pcdhγ mice was associated with a remarkable rise in neurite outgrowth and branching. Rises in outgrowth were abrogated by Rac1 inhibition. Moreover, AAV-Cre knock-down in Pcdhγ floxed neurons generated a rise in neurite self-intersections, and a robust rise in neighbor intersections or tiling, suggesting a role in sensory axon repulsion. Interestingly, preconditioned (3-d axotomy) neurons with enhanced growth had temporary declines in Pcdhγ and lessened outgrowth from Pcdhγ siRNA. In vivo, mice with local hindpaw skin Pcdhγ knock-down by siRNA had accelerated reinnervation by new epidermal axons with greater terminal branching and reduced intra-axonal spacing. Pcdhγ knock-down also had reciprocal impacts on keratinocyte density and nuclear size. Taken together, this work provides evidence for a role of Pcdhγ in attenuating outgrowth of sensory axons and their interactions, with implications in how new reinnervating axons following injury fare amid skin keratinocytes that also express Pcdhγ.SIGNIFICANCE STATEMENT The molecular mechanisms and potential constraints that govern skin reinnervation and patterning by sensory axons are largely unexplored. Here, we show that γ-protocadherins (Pcdhγ) may help to dictate interaction not only among axons but also between axons and keratinocytes as the former re-enter the skin during reinnervation. Pcdhγ neuronal knock-down enhances outgrowth in peripheral sensory neurons, involving the growth cone protein Rac1 whereas skin Pcdhγ knock-down generates rises in terminal epidermal axon growth and branching during re-innervation. Manipulation of sensory axon regrowth within the epidermis offers an opportunity to influence regenerative outcomes following nerve injury.

WAPL functions as a rheostat of Protocadherin isoform diversity that controls neural wiring.

Neural type-specific expression of clustered Protocadherin (Pcdh) proteins is essential for the establishment of connectivity patterns during brain development. In mammals, deterministic expression of the same Pcdh isoform promotes minimal overlap of tiled projections of serotonergic neuron axons throughout the brain, while stochastic expression of Pcdh genes allows for convergence of tightly packed, overlapping olfactory sensory neuron axons into targeted structures. How can the same gene locus generate opposite transcriptional programs that orchestrate distinct spatial arrangements of axonal patterns? Here, we reveal that cell type-specific Pcdh expression and axonal behavior depend on the activity of cohesin and its unloader, WAPL (wings apart-like protein homolog). While cohesin erases genomic-distance biases in Pcdh choice, WAPL functions as a rheostat of cohesin processivity that determines Pcdh isoform diversity.

E3 ubiquitin ligase RNF180 prevents excessive PCDH10 methylation to suppress the proliferation and metastasis of gastric cancer cells by promoting ubiquitination of DNMT1.

BACKGROUND: Downregulation of certain tumor-suppressor genes (TSGs) by aberrant methylation of CpG islands in the promoter region contributes a great deal to the oncogenesis and progression of several cancers, including gastric cancer (GC). Protocadherin 10 (PCDH10) is a newly identified TSG in various cancers and is downregulated in GC; however, the specific mechanisms of PCDH10 in GC remain elusive. Here, we elucidated a novel epigenetic regulatory signaling pathway involving the E3 ubiquitin ligase RNF180 and DNA methyltransferase 1 (DNMT1), responsible for modulating PCDH10 expression by affecting its promoter methylation. RESULTS: We revealed that PCDH10 was downregulated in GC cells and tissues, and low PCDH10 expression was correlated with lymph node metastasis and poor prognosis in patients with GC. Additionally, PCDH10 overexpression suppressed GC cell proliferation and metastasis. Mechanistically, DNMT1-mediated promoter hypermethylation resulted in decreased expression of PCDH10 in GC tissues and cells. Further analysis revealed that RNF180 can bind directly to DNMT1 and was involved in DNMT1 degradation via ubiquitination. Additionally, a positive correlation was found between RNF180 and PCDH10 expression and an inverse association between DNMT1 and PCDH10 expression showed considerable prognostic significance. CONCLUSION: Our data showed that RNF180 overexpression upregulated PCDH10 expression via ubiquitin-dependent degradation of DNMT1, thus suppressing GC cell proliferation, indicating that the RNF180/DNMT1/PCDH10 axis could be a potential therapeutic target for GC treatment.

Patterned cPCDH expression regulates the fine organization of the neocortex.

The neocortex consists of a vast number of diverse neurons that form distinct layers and intricate circuits at the single-cell resolution to support complex brain functions1. Diverse cell-surface molecules are thought to be key for defining neuronal identity, and they mediate interneuronal interactions for structural and functional organization2-6. However, the precise mechanisms that control the fine neuronal organization of the neocortex remain largely unclear. Here, by integrating in-depth single-cell RNA-sequencing analysis, progenitor lineage labelling and mosaic functional analysis, we report that the diverse yet patterned expression of clustered protocadherins (cPCDHs)-the largest subgroup of the cadherin superfamily of cell-adhesion molecules7-regulates the precise spatial arrangement and synaptic connectivity of excitatory neurons in the mouse neocortex. The expression of cPcdh genes in individual neocortical excitatory neurons is diverse yet exhibits distinct composition patterns linked to their developmental origin and spatial positioning. A reduction in functional cPCDH expression causes a lateral clustering of clonally related excitatory neurons originating from the same neural progenitor and a significant increase in synaptic connectivity. By contrast, overexpression of a single cPCDH isoform leads to a lateral dispersion of clonally related excitatory neurons and a considerable decrease in synaptic connectivity. These results suggest that patterned cPCDH expression biases fine spatial and functional organization of individual neocortical excitatory neurons in the mammalian brain.

SNHG22 promotes migration and invasion of trophoblasts via miR-128-3p/PCDH11X axis and activates PI3K/Akt signaling pathway.

OBJECTIVES: Long non-coding RNAs (LncRNAs) act as an indispensable role in the Preeclampsia (PE)-related trophoblast function, while its relationship with Small Nucleolar RNA Host Gene 22 (SNHG22) remains unknown. Hence, this study aimed to investigate the roles of lncRNA SNHG22 in the Preeclampsia (PE)-related trophoblasts function and the underlying mechanism. METHOD: Normal placentas and placentas from PE patients were collected to detect the expression of lncRNA SNHG22. Then, trophoblasts HTR-8/Svneo and JEG-3 were purchased, cultured, and treated to investigate the roles of lncRNA SNHG22 on cell migration and invasion as well as its underlying regulatory mechanism. RESULTS: The SNHG22 was downregulated in PE patients, and it was found that SNHG22 overexpression could drive migration and invasion of trophoblasts, while SNHG22 depletion exerted a suppressive effect. Mechanistically, SNHG22 was validated to regulate microRNA-128-3p (miR-128-3p), and Protocadherin 11 X-Linked (PCDH11X) was identified as the target gene of miR-128-3p. Furthermore, it was found that SNHG22 acted as a promoter in the migration and invasion of trophoblast cells in a miR-128-3p/PCDH11X dependent manner, and SNHG22 silencing weakened the activation of PCDH11X-mediated PI3K/Akt signaling pathways through inhibiting miR-128-3p, thereby preventing migration and invasion of trophoblasts. CONCLUSION: SNHG22 acted as a driver in the migration and invasion of trophoblasts and may be considered a candidate for the amelioration of PE.

PCDHB14 promotes ferroptosis and is a novel tumor suppressor in hepatocellular carcinoma.

Liver cancer, a result of multifactorial interplay between heredity and the environment, is one of the leading causes of cancer-related death worldwide. Hepatocellular carcinoma (HCC) is the most common histologic type of primary liver cancer. Here, we reported that deficiency in PCDHB14, a member of the cadherin superfamily, participates in the progression of HCC. We found that PCDHB14 is inactivated by aberrant methylation of its promoter in HCC patients and that PCDHB14 functions as a tumor suppressor to promote cell cycle arrest, inhibit cell proliferation, and induce ferroptosis. Furthermore, PCDHB14 ablation dramatically enhanced diethylenenitrite-induced HCC development. Mechanistically, PCDHB14 is induced by p53, and increased PCDHB14 downregulates the expression of SLC7A11, which is critical for ferroptosis. This effect is mediated by accelerated p65 protein degradation resulting from PCDHB14 promoting E3 ubiquitin ligase RNF182-mediated ubiquitination of p65 to block p65 binding to the promoter of SLC7A11. This study reports the new discovery that PCDHB14 serves as a potential prognostic marker for HCC.

The epilepsy-associated protein PCDH19 undergoes NMDA receptor-dependent proteolytic cleavage and regulates the expression of immediate-early genes.

Protocadherin-19 (PCDH19) is a synaptic cell-adhesion molecule encoded by X-linked PCDH19, a gene linked with epilepsy. Here, we report a synapse-to-nucleus signaling pathway through which PCDH19 bridges neuronal activity with gene expression. In particular, we describe the NMDA receptor (NMDAR)-dependent proteolytic cleavage of PCDH19, which leads to the generation of a PCDH19 C-terminal fragment (CTF) able to enter the nucleus. We demonstrate that PCDH19 CTF associates with chromatin and with the chromatin remodeler lysine-specific demethylase 1 (LSD1) and regulates expression of immediate-early genes (IEGs). Our results are consistent with a model whereby PCDH19 favors maintenance of neuronal homeostasis via negative feedback regulation of IEG expression and provide a key to interpreting PCDH19-related hyperexcitability.

Comparative distribution of Arcadlin/Protocadherin-8 mRNA in the intact and ischemic brains of adult mice.

The structural plasticity of dendritic spines serves as the adaptive capabilities of the central nervous system to various stimuli. Among these stimuli, cerebral ischemia induces dynamic alterations in neuronal network activity. Arcadlin/Paraxial protocadherin/Protocadherin-8 (Acad), a regulator of dendritic spine density, is strongly induced by activating stimuli to the neurons. However, the detailed distribution of Acad in normal and ischemic adult brains remains unclear. We comprehensively described Acad expression patterns in normal and ischemic adult brains by in situ hybridization histochemistry. We found that intact adult brains expressed Acad in the piriform cortex, dentate gyrus, hippocampal CA3, entorhinal cortex, amygdala, and hypothalamus. Acad expression was dramatically upregulated in the piriform cortex, olfactory area, dentate gyrus, entorhinal cortex, prefrontal cortex, insular cortex, amygdala, and septohippocampal nucleus 4 h after cerebral ischemia. Cerebral ischemia induced widespread neuronal activation, which was required for Acad upregulation. Our data suggested the involvement of Acad in the adaptive plasticity and remodeling of the neuronal network in the limbic and paralimbic systems.

Cadherins and the pathogenesis of epilepsy.

Epilepsy is a nervous system disease caused by abnormal discharge of brain neurons, which is characterized by recurrent seizures. The factors that induce epilepsy include genetic and environmental factors. Genetic factors are important pathogenic factors of epilepsy, such as epilepsy caused by protocadherin-19 (PCDH-19) mutation, which is an X-linked genetic disease. It is more common in female heterozygotes, which are caused by mutations in the PCDH-19 gene. Epilepsy caused by environmental factors is mainly caused by brain injury, which is commonly caused by brain tumors, brain surgery, or trauma to the brain. In addition, the pathogenesis of epilepsy is closely related to abnormalities in some signaling pathways. The Wnt/ß-catenin signaling pathway is considered a new target for the treatment of epilepsy. This review summarizes these factors inducing epilepsy and the research hypotheses regarding the pathogenesis of epilepsy. The focus of this review centers on cadherins and the pathogenesis of epilepsy. We analyzed the pathogenesis of epilepsy induced by N-cadherin and PCDH-19 in the cadherin family members. Finally, we expect that in the future, new breakthroughs will be made in the study of the pathogenesis and mechanism of epilepsy at the cellular and molecular levels.

Protocadherin-7 Regulates Osteoclast Differentiation through Intracellular SET-Binding Domain-Mediated RhoA and Rac1 Activation.

Protocadherin-7 (Pcdh7) is a member of the non-clustered protocadherin δ1 subgroup of the cadherin superfamily. Although the cell-intrinsic role of Pcdh7 in osteoclast differentiation has been demonstrated, the molecular mechanisms of Pcdh7 regulating osteoclast differentiation remain to be determined. Here, we demonstrate that Pcdh7 contributes to osteoclast differentiation by regulating small GTPases, RhoA and Rac1, through its SET oncoprotein binding domain. Pcdh7 is associated with SET along with RhoA and Rac1 during osteoclast differentiation. Pcdh7-deficient (Pcdh7-/-) cells showed abolished RANKL-induced RhoA and Rac1 activation, and impaired osteoclast differentiation. Impaired osteoclast differentiation in Pcdh7-/- cells was restored by retroviral transduction of full-length Pcdh7 but not by a Pcdh7 mutant that lacks SET binding domain. The direct crosslink of the Pcdh7 intracellular region induced the activation of RhoA and Rac1, which was not observed when Pcdh7 lacks the SET binding domain. Additionally, retroviral transduction of the constitutively active form of RhoA and Rac1 completely restored the impaired osteoclast differentiation in Pcdh7-/- cells. Collectively, these results demonstrate that Pcdh7 controls osteoclast differentiation by regulating RhoA and Rac1 activation through the SET binding domain.

Development of FRET-based indicators for visualizing homophilic trans interaction of a clustered protocadherin.

Clustered protocadherins (Pcdhs), which are cell adhesion molecules, play a fundamental role in self-recognition and non-self-discrimination by conferring diversity on the cell surface. Although systematic cell-based aggregation assays provide information regarding the binding properties of Pcdhs, direct visualization of Pcdh trans interactions across cells remains challenging. Here, we present Förster resonance energy transfer (FRET)-based indicators for directly visualizing Pcdh trans interactions. We developed the indicators by individually inserting FRET donor and acceptor fluorescent proteins (FPs) into the ectodomain of Pcdh molecules. They enabled successful visualization of specific trans interactions of Pcdh and revealed that the Pcdh trans interaction is highly sensitive to changes in extracellular Ca2+ levels. We expect that FRET-based indicators for visualizing Pcdh trans interactions will provide a new approach for investigating the roles of Pcdh in self-recognition and non-self-discrimination processes.

Long non­coding RNA HHIP­AS1 inhibits lung cancer epithelial­mesenchymal transition and stemness by regulating PCDHGA9.

The aim of the present study was to investigate the effect of hedgehog­interacting protein antisense RNA 1 (HHIP­AS1) on epithelial­mesenchymal transition (EMT) and cellular stemness of human lung cancer cells by regulating the microRNA (miR)­153­3p/PCDHGA9 axis. Reverse transcription­quantitative PCR was used to compare the expression of HHIP­AS1 in lung cancer and adjacent normal lung tissues. In addition, the correlation of HHIP­AS1 with E­cadherin, Vimentin, N­cadherin and Twist1 was analyzed. HHIP­AS1 overexpression vector was transfected into lung cancer A549 and NCI­H1299 cell lines. Cell Counting Kit­8 and Transwell and clonogenic assays were used to detect the proliferation, invasion and clonogenesis of the lung cancer cells, respectively. The associations among HHIP­AS1, miR­153­3p and PCDHGA9 were predicted by bioinformatics analysis and verified by a dual­luciferase reporter system. The results showed that the expression of HHIP­AS1 in lung cancer tissues was significantly lower than that in normal tissues (P<0.001). HHIP­AS1 was positively correlated with E­cadherin and negatively correlated with Vimentin, N­cadherin and Twist1. HHIP­AS1 overexpression inhibited the proliferation, invasion and clonal formation of the A549 and NCI­H1299 cells. The luciferase reporter system verified that HHIP­AS1 could adsorb miR­153­3p and that PCDHGA9 was the target gene of miR­153­3p. A549 cells were transfected with HHIP­AS1 overexpression vector and miR­153­3p mimic, and the miR­153­3p mimic had a mitigating effect on HHIP­AS1 inhibition (P<0.001). In conclusion, HHIP­AS1 inhibits the EMT and stemness of lung cancer cells by regulating the miR­153­3p/PCDHGA9 axis. Thus, HHIP­AS1 may be a new potential target for lung cancer treatment.

A complete Protocadherin-19 ectodomain model for evaluating epilepsy-causing mutations and potential protein interaction sites.

Cadherin superfamily members play a critical role in differential adhesion during neurodevelopment, and their disruption has been linked to several neurodevelopmental disorders. Mutations in protocadherin-19 (PCDH19), a member of the δ-protocadherin subfamily of cadherins, cause a unique form of epilepsy called PCDH19 clustering epilepsy. While PCDH19 and other non-clustered δ-protocadherins form multimers with other members of the cadherin superfamily to alter adhesiveness, the specific protein surfaces responsible for these interactions are unknown. Only portions of the PCDH19 extracellular domain structure had been solved previously. Here, we present a structure of the missing segment from zebrafish Protocadherin-19 (Pcdh19) and create a complete ectodomain model. This model shows the structural environment for 97% of disease-causing missense mutations and reveals two potential surfaces for intermolecular interactions that could modify Pcdh19's adhesive strength and specificity.

Decreased expression of the clock gene Bmal1 is involved in the pathogenesis of temporal lobe epilepsy.

Clock genes not only regulate the circadian rhythm of physiological activities but also participate in the pathogenesis of many diseases. Previous studies have documented the abnormal expression of clock genes in epilepsy. However, the molecular mechanism of brain and muscle Arnt-like protein 1 (Bmal1), one of the core clock genes, in the epileptogenesis and seizures of temporal lobe epilepsy (TLE) remain unclear. We first investigated the levels of Bmal1 and other clock proteins in the hippocampus of subjects with epilepsy to define the function of Bmal1. The levels of Bmal1 were decreased during the latent and chronic phases in the experimental group compared with those in the control group. Knockout of Bmal1 in hippocampal dentate gyrus (DG) neurons of Bmal1flox/flox mice by Synapsin 1 (Syn1) promoter AAV (adeno-associated virus) lowered the threshold of seizures induced by pilocarpine administration. High-throughput sequencing analysis showed that PCDH19 (protocadherin 19), a gene associated with epilepsy, was regulated by Bmal1. PCDH19 expression was also decreased in the hippocampus of epileptic mice. Furthermore, the higher levels of Bmal1 and PCDH19 were detected in patients with no hippocampal sclerosis (no HS) than in patients with HS International League Against Epilepsy (ILAE) type I and III. Altogether, these data suggest that decreased expression of clock gene Bmal1 may participate in epileptogenesis and seizures via PCDH19 in TLE.

Morphological characterization of Etv2 vascular explants using fractal analysis and atomic force microscopy.

The rapid engraftment of vascular networks is critical for functional incorporation of tissue explants. However, existing methods for inducing angiogenesis utilize approaches that yield vasculature with poor temporal stability or inadequate mechanical integrity, which reduce their robustness in vivo. The transcription factor Ets variant 2 (Etv2) specifies embryonic hematopoietic and vascular endothelial cell (EC) development, and is transiently reactivated during postnatal vascular regeneration and tumor angiogenesis. This study investigates the role for Etv2 upregulation in forming stable vascular beds both in vitro and in vivo. Control and Etv2+ prototypical fetal-derived human umbilical vein ECs (HUVECs) and adult ECs were angiogenically grown into vascular beds. These vessel beds were characterized using fractal dimension and lacunarity, to quantify their branching complexity and space-filling homogeneity, respectively. Atomic force microscopy (AFM) was used to explore whether greater complexity and homogeneity lead to more mechanically stable vessels. Additionally, markers of EC integrity were used to probe for mechanistic clues. Etv2+ HUVECs exhibit greater branching, vessel density, and structural homogeneity, and decreased stiffness in vitro and in vivo, indicating a greater propensity for stable vessel formation. When co-cultured with colon tumor organoid tissue, Etv2+ HUVECs had decreased fractal dimension and lacunarity compared to Etv2+ HUVECs cultured alone, indicating that vessel density and homogeneity of vessel spacing increased due to the presence of Etv2. This study sets forth the novel concept that fractal dimension, lacunarity, and AFM are as informative as conventional angiogenic measurements, including vessel branching and density, to assess vascular perfusion and stability.

FOXM1-induced miR-552 expression contributes to pancreatic cancer progression by targeting multiple tumor suppressor genes.

Dysregulation of microRNAs (miRNAs) plays important roles during carcinogenesis. Forkhead box M1 (FOXM1), a well-known oncogenic transcription factor, has been implicated in the progression of multiple cancer types. To find out FOXM1-induced abnormal miRNAs in pancreatic cancer, we analyzed TCGA database and figured out miR-552 as the most relevant miRNA with FOXM1. Molecular experimental results demonstrated that FOXM1 transcriptionally activated miR-552 expression by directly binding to the promoter region of miR-552. In a pancreatic cancer tissue microarray, miR-552 expression was positively correlated with FOXM1 and high expression of miR-552 could predict poor patient outcome. Functionally, overexpression of miR-552 promoted pancreatic cancer cell migration and inhibition of miR-552 attenuated this phenotype. The inhibitory effect on cell migration caused by FOXM1 knockdown could be restored by exogenous expression of miR-552. By informatics analysis, we identified three tumor suppressor genes: DACH1, PCDH10 and SMAD4, all of which were negatively associated with FOXM1 and validated as functionally relevant targets of miR-552. Taken together, our findings provide a new FOXM1-miR-552-DACH1/PCDH10/SMAD4 axis to regulate pancreatic cancer cell progression and new opportunities for therapeutic intervention against this disease.