RESUMO
Western equine encephalitis virus (WEEV) is an arthropod-borne virus (arbovirus) that frequently caused major outbreaks of encephalitis in humans and horses in the early twentieth century, but the frequency of outbreaks has since decreased markedly, and strains of this alphavirus isolated in the past two decades are less virulent in mammals than strains isolated in the 1930s and 1940s1-3. The basis for this phenotypic change in WEEV strains and coincident decrease in epizootic activity (known as viral submergence3) is unclear, as is the possibility of re-emergence of highly virulent strains. Here we identify protocadherin 10 (PCDH10) as a cellular receptor for WEEV. We show that multiple highly virulent ancestral WEEV strains isolated in the 1930s and 1940s, in addition to binding human PCDH10, could also bind very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), which are recognized by another encephalitic alphavirus as receptors4. However, whereas most of the WEEV strains that we examined bind to PCDH10, a contemporary strain has lost the ability to recognize mammalian PCDH10 while retaining the ability to bind avian receptors, suggesting WEEV adaptation to a main reservoir host during enzootic circulation. PCDH10 supports WEEV E2-E1 glycoprotein-mediated infection of primary mouse cortical neurons, and administration of a soluble form of PCDH10 protects mice from lethal WEEV challenge. Our results have implications for the development of medical countermeasures and for risk assessment for re-emerging WEEV strains.
Assuntos
Vírus da Encefalite Equina do Oeste , Especificidade de Hospedeiro , Protocaderinas , Receptores Virais , Animais , Feminino , Humanos , Masculino , Camundongos , Aves/metabolismo , Aves/virologia , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/virologia , Vírus da Encefalite Equina do Oeste/classificação , Vírus da Encefalite Equina do Oeste/metabolismo , Vírus da Encefalite Equina do Oeste/patogenicidade , Encefalomielite Equina/epidemiologia , Encefalomielite Equina/virologia , Proteínas Relacionadas a Receptor de LDL/metabolismo , Neurônios/metabolismo , Neurônios/virologia , Fenótipo , Protocaderinas/metabolismo , Receptores de LDL/metabolismo , Receptores de LDL/genética , Receptores Virais/metabolismo , Proteínas do Envelope Viral/metabolismo , Zoonoses Virais/epidemiologia , Zoonoses Virais/virologiaRESUMO
Enhancers generate bidirectional noncoding enhancer RNAs (eRNAs) that may regulate gene expression. At present, the eRNA function remains enigmatic. Here, we report a 5' capped antisense eRNA PEARL (Pcdh eRNA associated with R-loop formation) that is transcribed from the protocadherin (Pcdh) α HS5-1 enhancer region. Through loss- and gain-of-function experiments with CRISPR/Cas9 DNA fragment editing, CRISPRi, and CRISPRa, as well as locked nucleic acid strategies, in conjunction with ChIRP, MeDIP, DRIP, QHR-4C, and HiChIP experiments, we found that PEARL regulates Pcdhα gene expression by forming local RNA-DNA duplexes (R-loops) in situ within the HS5-1 enhancer region to promote long-distance chromatin interactions between distal enhancers and target promoters. In particular, increased levels of eRNA PEARL via perturbing transcription elongation factor SPT6 lead to strengthened local three-dimensional chromatin organization within the Pcdh superTAD. These findings have important implications regarding molecular mechanisms by which the HS5-1 enhancer regulates stochastic Pcdhα promoter choice in single cells in the brain.
Assuntos
Elementos Facilitadores Genéticos , Protocaderinas , Cromatina , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas/genética , RNA , Transcrição GênicaRESUMO
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.
Assuntos
Regulação da Expressão Gênica , Neocórtex , Protocaderinas , Animais , Camundongos , Interneurônios/metabolismo , Neocórtex/anatomia & histologia , Neocórtex/citologia , Neocórtex/metabolismo , Neurônios/metabolismo , Protocaderinas/genética , Protocaderinas/metabolismo , Sinapses/metabolismo , Transmissão SinápticaRESUMO
The ability of neurites of the same neuron to avoid each other (self-avoidance) is a conserved feature in both invertebrates and vertebrates. The key to self-avoidance is the generation of a unique subset of cell-surface proteins in individual neurons engaging in isoform-specific homophilic interactions that drive neurite repulsion rather than adhesion. Among these cell-surface proteins are fly Dscam1 and vertebrate clustered protocadherins (cPcdhs), as well as the recently characterized shortened Dscam (sDscam) in the Chelicerata. Herein, we review recent advances in our understanding of how cPcdh, Dscam, and sDscam cell-surface recognition codes are expressed and translated into cellular functions essential for neural wiring.
Assuntos
Moléculas de Adesão Celular , Proteínas de Drosophila , Protocaderinas , Animais , Moléculas de Adesão Celular/metabolismo , Comunicação Celular , Proteínas de Drosophila/metabolismo , Neurônios/metabolismo , Isoformas de Proteínas/metabolismo , Invertebrados , VertebradosRESUMO
The activity-dependent transcription factor myocyte enhancer factor 2 (MEF2) induces excitatory synapse elimination in mouse neurons, which requires fragile X mental retardation protein (FMRP), an RNA-binding protein implicated in human cognitive dysfunction and autism. We report here that protocadherin 10 (Pcdh10), an autism-spectrum disorders gene, is necessary for this process. MEF2 and FMRP cooperatively regulate the expression of Pcdh10. Upon MEF2 activation, PSD-95 is ubiquitinated by the ubiquitin E3 ligase murine double minute 2 (Mdm2) and then binds to Pcdh10, which links it to the proteasome for degradation. Blockade of the Pcdh10-proteasome interaction inhibits MEF2-induced PSD-95 degradation and synapse elimination. In FMRP-lacking neurons, elevated protein levels of eukaryotic translation elongation factor 1 α (EF1α), an Mdm2-interacting protein and FMRP target mRNA, sequester Mdm2 and prevent MEF2-induced PSD-95 ubiquitination and synapse elimination. Together, our findings reveal roles for multiple autism-linked genes in activity-dependent synapse elimination.
Assuntos
Guanilato Quinases/metabolismo , Hipocampo/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Animais , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Caderinas/metabolismo , Dendritos/metabolismo , Modelos Animais de Doenças , Proteína 4 Homóloga a Disks-Large , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Hipocampo/citologia , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Protocaderinas , Sinapses/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Cortical inhibitory interneurons (cINs) are born in the ventral forebrain and migrate into the cortex where they make connections with locally produced excitatory glutamatergic neurons. Cortical function critically depends on the number of cINs, which is also key to establishing the appropriate inhibitory/excitatory balance. The final number of cINs is determined during a postnatal period of programmed cell death (PCD) when ~40% of the young cINs are eliminated. Previous work shows that the loss of clustered gamma protocadherins (Pcdhgs), but not of genes in the Pcdha or Pcdhb clusters, dramatically increased BAX-dependent cIN PCD. Here, we show that PcdhγC4 is highly expressed in cINs of the mouse cortex and that this expression increases during PCD. The sole deletion of the PcdhγC4 isoform, but not of the other 21 isoforms in the Pcdhg gene cluster, increased cIN PCD. Viral expression of the PcdhγC4, in cIN lacking the function of the entire Pcdhg cluster, rescued most of these cells from cell death. We conclude that PcdhγC4 plays a critical role in regulating the survival of cINs during their normal period of PCD. This highlights how a single isoform of the Pcdhg cluster, which has been linked to human neurodevelopmental disorders, is essential to adjust cIN cell numbers during cortical development.
Assuntos
Interneurônios , Protocaderinas , Camundongos , Animais , Humanos , Interneurônios/fisiologia , Neurônios/metabolismo , Apoptose/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Córtex Cerebral/fisiologiaRESUMO
Protocadherin19 (PCDH19)-related epilepsy syndrome is a rare disorder characterized by early-onset epilepsy, intellectual disability, and autistic behaviors. PCDH19 is located on the X chromosome and encodes a calcium-dependent single-pass transmembrane protein, which regulates cell-to-cell adhesion through homophilic binding. In human, 90% of heterozygous females, containing PCDH19 wild-type and mutant cells due to random X inactivation, are affected, whereas mutant males, containing only mutant cells, are typically not. The current view, the cellular interference, is that the altered interactions between wild-type and mutant cells during development, rather than loss of function itself, are responsible. However, studies using Pcdh19 knockout mice showed that the complete loss of function also causes autism-like behaviors both in males and females, suggesting that other functions of PCDH19 may also contribute to pathogenesis. To address whether mosaicism is required for PCDH19-related epilepsy, we generated Xenopus tropicalis tadpoles with complete or mosaic loss of function by injecting antisense morpholino oligonucleotides into the blastomeres of neural lineage at different stages of development. We found that either mosaic or complete knockdown results in seizure-like behaviors, which could be rescued by antiseizure medication, and repetitive behaviors. Our results suggest that the loss of PCDH19 function itself, in addition to cellular interference, may also contribute to PCDH19-related epilepsy.
Assuntos
Caderinas , Epilepsia , Protocaderinas , Animais , Feminino , Masculino , Comportamento Animal , Caderinas/genética , Caderinas/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Mosaicismo , XenopusRESUMO
Clustered protocadherin (Pcdh) functions as a cell recognition molecule through the homophilic interaction in the central nervous system. However, its interactions have not yet been visualized in neurons. We previously reported PcdhγB2-Förster resonance energy transfer (FRET) probes to be applicable only to cell lines. Herein, we designed γB2-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single γB2 molecule and succeeded in visualizing γB2 homophilic interaction in cultured hippocampal neurons. The γB2-FRET probe localized in the soma and neurites, and FRET signals, which were observed at contact sites between neurites, eliminated by ethylene glycol tetraacetic acid (EGTA) addition. Live imaging revealed that the FRET-negative γB2 signals rapidly moved along neurites and soma, whereas the FRET-positive signals remained in place. We observed that the γB2 proteins at synapses rarely interact homophilically. The γB2-FRET probe might allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism.
Assuntos
Neurônios , Protocaderinas , Neuritos , Corpo Celular , Comunicação CelularRESUMO
Lung cancer is the leading cause of cancer deaths. Its high mortality is associated with high metastatic potential. Here, we show that the RAC1-selective guanine nucleotide exchange factor T cell invasion and metastasis-inducing protein 1 (TIAM1) promotes cell migration and invasion in the most common subtype of lung cancer, non-small-cell lung cancer (NSCLC), through an unexpected nuclear function. We show that TIAM1 interacts with TRIM28, a master regulator of gene expression, in the nucleus of NSCLC cells. We reveal that a TIAM1-TRIM28 complex promotes epithelial-to-mesenchymal transition, a phenotypic switch implicated in cell migration and invasion. This occurs through H3K9me3-induced silencing of protocadherins and by decreasing E-cadherin expression, thereby antagonizing cell-cell adhesion. Consistently, TIAM1 or TRIM28 depletion suppresses the migration of NSCLC cells, while migration is restored by the simultaneous depletion of protocadherins. Importantly, high nuclear TIAM1 in clinical specimens is associated with advanced-stage lung adenocarcinoma, decreased patient survival, and inversely correlates with E-cadherin expression.
Assuntos
Adenocarcinoma de Pulmão , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/genética , Protocaderinas , Carcinoma Pulmonar de Células não Pequenas/genética , Caderinas/genética , Epigênese Genética , Proteína 28 com Motivo Tripartido , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/genéticaRESUMO
Cell division requires dramatic reorganization of the cell cortex, which is primarily driven by the actomyosin network. We previously reported that protocadherin 7 (PCDH7) gets enriched at the cell surface during mitosis, which is required to build up the full mitotic rounding pressure. Here, we report that PCDH7 interacts with and is palmitoylated by the palmitoyltransferase, ZDHHC5. PCDH7 and ZDHHC5 colocalize at the mitotic cell surface and translocate to the cleavage furrow during cytokinesis. The localization of PCDH7 depends on the palmitoylation activity of ZDHHC5. Silencing PCDH7 increases the percentage of multinucleated cells and the duration of mitosis. Loss of PCDH7 expression correlates with reduced levels of active RhoA and phospho-myosin at the cleavage furrow. This work uncovers a palmitoylation-dependent translocation mechanism for PCDH7, which contributes to the reorganization of the cortical cytoskeleton during cell division.
Assuntos
Citocinese , Protocaderinas , Lipoilação , Ciclo Celular , Mitose , Caderinas/genéticaRESUMO
Hematopoietic stem and progenitor cells emerge from the aorta and migrate to the caudal hematopoietic tissue (CHT) of zebrafish larvae, the hematopoietic equivalent of the mammalian fetal liver, for their proliferation and differentiation. We previously reported that somite-derived stromal cells were a key component of the CHT niche. Here, we found that the cell adhesion protein Protocadherin 18a (Pcdh18a) is expressed in the stromal cell progenitors (SCPs) emigrating from somites toward the future CHT. Deletion of most of the Pcdh18a intracellular domain caused a decrease in the number of SCPs, the directionality of their migration, and the cell-contact mediated repulsion that normally occurs between migrating SCPs. These defects were followed by abnormal morphogenesis of the venous plexus that forms the CHT framework, and the inability of the CHT to function as a niche for hematopoietic stem and progenitor cells. Finally, we found that the extracellular domain of Pcdh18a mediates trans heterophilic adhesion of stromal cells to endothelial cells in vivo and thereby the reticular versus perivascular fate of SCPs. Thus, Pcdh18a expression in SCPs is essential for the proper development of the hematopoietic niche.
Assuntos
Células-Tronco Hematopoéticas , Peixe-Zebra , Animais , Células Endoteliais/metabolismo , Mamíferos , Protocaderinas , Nicho de Células-Tronco , Células EstromaisRESUMO
Coxsackievirus B3 (CVB3) is known to cause acute myocarditis and pancreatitis in humans. We investigated the microRNAs (miRNAs) that can potentially govern the viral life cycle by binding to the untranslated regions (UTRs) of CVB3 RNA. MicroRNA-22-3p was short-listed, as its potential binding site overlapped with the region crucial for recruiting internal ribosome entry site trans-acting factors (ITAFs) and ribosomes. We demonstrate that miR-22-3p binds CVB3 5' UTR, hinders recruitment of key ITAFs on viral mRNA, disrupts the spatial structure required for ribosome recruitment, and ultimately blocks translation. Likewise, cells lacking miR-22-3p exhibited heightened CVB3 infection compared to wild type, confirming its role in controlling infection. Interestingly, miR-22-3p level was found to be increased at 4 hours post-infection, potentially due to the accumulation of viral 2A protease in the early phase of infection. 2Apro enhances the miR-22-3p level to dislodge the ITAFs from the SD-like sequence, rendering the viral RNA accessible for binding of replication factors to switch to replication. Furthermore, one of the cellular targets of miR-22-3p, protocadherin-1 (PCDH1), was significantly downregulated during CVB3 infection. Partial silencing of PCDH1 reduced viral replication, demonstrating its proviral role. Interestingly, upon CVB3 infection in mice, miR-22-3p level was found to be downregulated only in the small intestine, the primary target organ, indicating its possible role in influencing tissue tropism. It appears miR-22-3p plays a dual role during infection by binding viral RNA to aid its life cycle as a viral strategy and by targeting a proviral protein to restrict viral replication as a host response.IMPORTANCECVB3 infection is associated with the development of end-stage heart diseases. Lack of effective anti-viral treatments and vaccines for CVB3 necessitates comprehensive understanding of the molecular players during CVB3 infection. miRNAs have emerged as promising targets for anti-viral strategies. Here, we demonstrate that miR-22-3p binds to 5' UTR and inhibits viral RNA translation at the later stage of infection to promote viral RNA replication. Conversely, as host response, it targets PCDH1, a proviral factor, to discourage viral propagation. miR-22-3p also influences CVB3 tissue tropism. Deciphering the multifaced role of miR-22-3p during CVB3 infection unravels the necessary molecular insights, which can be exploited for novel intervening strategies to curb infection and restrict viral pathogenesis.
Assuntos
Regiões 5' não Traduzidas , Infecções por Coxsackievirus , Enterovirus Humano B , Interações entre Hospedeiro e Microrganismos , MicroRNAs , Biossíntese de Proteínas , RNA Viral , Animais , Humanos , Camundongos , Regiões 5' não Traduzidas/genética , Antivirais/metabolismo , Infecções por Coxsackievirus/genética , Infecções por Coxsackievirus/virologia , Enterovirus Humano B/genética , Enterovirus Humano B/patogenicidade , Enterovirus Humano B/fisiologia , Células HeLa , Intestino Delgado/metabolismo , Intestino Delgado/virologia , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Tropismo Viral/genética , Replicação Viral/genética , Cisteína Endopeptidases/metabolismo , Protocaderinas/deficiência , Protocaderinas/genética , Miocardite , Interações entre Hospedeiro e Microrganismos/genéticaRESUMO
BACKGROUND AND AIMS: Sustained inflammation and hepatocyte injury in chronic liver disease activate HSCs to transdifferentiate into fibrogenic, contractile myofibroblasts. We investigated the role of protocadherin 7 (PCDH7), a cadherin family member not previously characterized in the liver, whose expression is restricted to HSCs. APPROACH AND RESULTS: We created a PCDH7 fl/fl mouse line, which was crossed to lecithin retinol acyltransferase-Cre mice to generate HSC-specific PCDH7 knockout animals. HSC contraction in vivo was tested in response to the HSC-selective vasoconstrictor endothelin-1 using intravital multiphoton microscopy. To establish a PCDH7 null HSC line, cells were isolated from PCDH7 fl/fl mice and infected with adenovirus-expressing Cre. Hepatic expression of PCDH7 was strictly restricted to HSCs. Knockout of PCDH7 in vivo abrogated HSC-mediated sinusoidal contraction in response to endothelin-1. In cultured HSCs, loss of PCDH7 markedly attenuated contractility within collagen gels and led to altered gene expression in pathways governing adhesion and vasoregulation. Loss of contractility in PCDH7 knockout cells was impaired Rho-GTPase signaling, as demonstrated by altered gene expression, reduced assembly of F-actin fibers, and loss of focal adhesions. CONCLUSIONS: The stellate cell-specific cadherin, PCDH7, is a novel regulator of HSC contractility whose loss leads to cytoskeletal remodeling and sinusoidal relaxation.
Assuntos
Caderinas , Células Estreladas do Fígado , Camundongos Knockout , Animais , Camundongos , Caderinas/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/fisiologia , Protocaderinas , Endotelina-1/metabolismo , Células CultivadasRESUMO
Neurite self-recognition and avoidance are fundamental properties of all nervous systems1. These processes facilitate dendritic arborization2,3, prevent formation of autapses4 and allow free interaction among non-self neurons1,2,4,5. Avoidance among self neurites is mediated by stochastic cell-surface expression of combinations of about 60 isoforms of α-, ß- and γ-clustered protocadherin that provide mammalian neurons with single-cell identities1,2,4-13. Avoidance is observed between neurons that express identical protocadherin repertoires2,5, and single-isoform differences are sufficient to prevent self-recognition10. Protocadherins form isoform-promiscuous cis dimers and isoform-specific homophilic trans dimers10,14-20. Although these interactions have previously been characterized in isolation15,17-20, structures of full-length protocadherin ectodomains have not been determined, and how these two interfaces engage in self-recognition between neuronal surfaces remains unknown. Here we determine the molecular arrangement of full-length clustered protocadherin ectodomains in single-isoform self-recognition complexes, using X-ray crystallography and cryo-electron tomography. We determine the crystal structure of the clustered protocadherin γB4 ectodomain, which reveals a zipper-like lattice that is formed by alternating cis and trans interactions. Using cryo-electron tomography, we show that clustered protocadherin γB6 ectodomains tethered to liposomes spontaneously assemble into linear arrays at membrane contact sites, in a configuration that is consistent with the assembly observed in the crystal structure. These linear assemblies pack against each other as parallel arrays to form larger two-dimensional structures between membranes. Our results suggest that the formation of ordered linear assemblies by clustered protocadherins represents the initial self-recognition step in neuronal avoidance, and thus provide support for the isoform-mismatch chain-termination model of protocadherin-mediated self-recognition, which depends on these linear chains11.
Assuntos
Caderinas/metabolismo , Caderinas/ultraestrutura , Microscopia Crioeletrônica , Neurônios/química , Neurônios/metabolismo , Animais , Caderinas/química , Caderinas/genética , Cristalografia por Raios X , Lipossomos/química , Lipossomos/metabolismo , Camundongos , Modelos Moleculares , Neurônios/ultraestrutura , Domínios Proteicos , Multimerização Proteica , ProtocaderinasRESUMO
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.
Assuntos
Neurônios , Conformação Proteica , Protocaderinas , Animais , Tomografia com Microscopia Eletrônica , Método de Monte Carlo , Neurônios/metabolismo , Ligação Proteica , Protocaderinas/química , VertebradosRESUMO
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.
Assuntos
Regeneração Nervosa , Protocaderinas , Células Receptoras Sensoriais , Animais , Feminino , Masculino , Camundongos , Axônios/fisiologia , Regeneração Nervosa/fisiologia , Protocaderinas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Células Receptoras Sensoriais/metabolismoRESUMO
The establishment of a functional cerebral cortex depends on the proper execution of multiple developmental steps, culminating in dendritic and axonal outgrowth and the formation and maturation of synaptic connections. Dysregulation of these processes can result in improper neuronal connectivity, including that associated with various neurodevelopmental disorders. The γ-Protocadherins (γ-Pcdhs), a family of 22 distinct cell adhesion molecules that share a C-terminal cytoplasmic domain, are involved in multiple aspects of neurodevelopment including neuronal survival, dendrite arborization, and synapse development. The extent to which individual γ-Pcdh family members play unique versus common roles remains unclear. We demonstrated previously that the γ-Pcdh-C3 isoform (γC3), via its unique "variable" cytoplasmic domain (VCD), interacts in cultured cells with Axin1, a Wnt-pathway scaffold protein that regulates the differentiation and morphology of neurons. Here, we confirm that γC3 and Axin1 interact in the cortex in vivo and show that both male and female mice specifically lacking γC3 exhibit disrupted Axin1 localization to synaptic fractions, without obvious changes in dendritic spine density or morphology. However, both male and female γC3 knock-out mice exhibit severely decreased dendritic complexity of cortical pyramidal neurons that is not observed in mouse lines lacking several other γ-Pcdh isoforms. Combining knock-out with rescue constructs in cultured cortical neurons pooled from both male and female mice, we show that γC3 promotes dendritic arborization through an Axin1-dependent mechanism mediated through its VCD. Together, these data identify a novel mechanism through which γC3 uniquely regulates the formation of cortical circuitry.SIGNIFICANCE STATEMENT The complexity of a neuron's dendritic arbor is critical for its function. We showed previously that the γ-Protocadherin (γ-Pcdh) family of 22 cell adhesion molecules promotes arborization during development; it remained unclear whether individual family members played unique roles. Here, we show that one γ-Pcdh isoform, γC3, interacts in the brain with Axin1, a scaffolding protein known to influence dendrite development. A CRISPR/Cas9-generated mutant mouse line lacking γC3 (but not lines lacking other γ-Pcdhs) exhibits severely reduced dendritic complexity of cerebral cortex neurons. Using cultured γC3 knock-out neurons and a variety of rescue constructs, we confirm that the γC3 cytoplasmic domain promotes arborization through an Axin1-dependent mechanism. Thus, γ-Pcdh isoforms are not interchangeable, but rather can play unique neurodevelopmental roles.
Assuntos
Dendritos , Protocaderinas , Animais , Feminino , Masculino , Camundongos , Proteína Axina/metabolismo , Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Dendritos/fisiologia , Camundongos Knockout , Plasticidade Neuronal , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
The sensory epithelium of the inner ear, found in all extant lineages of vertebrates, has been subjected to over 500 million years of evolution, resulting in the complex inner ear of modern vertebrates. Inner-ear adaptations are as diverse as the species in which they are found, and such unique anatomical variations have been well studied. However, the evolutionary details of the molecular machinery that is required for hearing are less well known. Two molecules that are essential for hearing in vertebrates are cadherin-23 and protocadherin-15, proteins whose interaction with one another acts as the focal point of force transmission when converting sound waves into electrical signals that the brain can interpret. This "tip-link" interaction exists in every lineage of vertebrates, but little is known about the structure or mechanical properties of these proteins in most non-mammalian lineages. Here, we use various techniques to characterize the evolution of this protein interaction. Results show how evolutionary sequence changes in this complex affect its biophysical properties both in simulations and experiments, with variations in interaction strength and dynamics among extant vertebrate lineages. Evolutionary simulations also characterize how the biophysical properties of the complex in turn constrain its evolution and provide a possible explanation for the increase in deafness-causing mutants observed in cadherin-23 relative to protocadherin-15. Together, these results suggest a general picture of tip-link evolution in which selection acted to modify the tip-link interface, although subsequent neutral evolution combined with varying degrees of purifying selection drove additional diversification in modern tetrapods.
Assuntos
Orelha Interna , Protocaderinas , Animais , Orelha Interna/metabolismo , Audição , Caderinas/genética , Caderinas/química , Caderinas/metabolismoRESUMO
Protocadherin 8 (PCDH8), a calcium-dependent transmembrane protein in the protocadherin family, regulates cell adhesion and signal transduction. While some studies have provided indirect evidence that PCDH8 has cancer-promoting properties, this association is controversial. In particular, its involvement in thyroid cancer (THCA) remains unclear. We aimed to elucidate the role of PCDH8 in THCA using bioinformatic analysis. Subsequently, the results were experimentally validated. The analysis conducted using the R programming language and online web tools explored PCDH8 expression levels, prognostic, and clinical implications, and its relationship with the tumor immune microenvironment in THCA. Furthermore, we examined the association between PCDH8 and co-expressed genes, highlighting their involvement in several biological processes relevant to THCA. The potential of PCDH8 as a therapeutic target for this pathology was also explored. Immunohistochemical (IHC) staining was performed on samples from 98 patients with THCA, and experimental validation was carried out. PCDH8 was significantly elevated in cancer tissues and associated with poor prognosis, several clinical factors, and immune cell and checkpoint abundance. Cox regression and survival analyses, together with Receiver Operating Curves (ROC) indicated that PCDH8 was an independent prognostic factor for THCA. Furthermore, PCDH8 impacts cell viability and proliferation, promoting tumorigenesis. Also, it influences tumor cell sensitivity to various drugs. Thus, PCDH8 might be a potential therapeutic target for THCA. IHC, cell culture, MTT, and colony formation experiments further confirmed our findings. This analysis provided insights into the potential carcinogenic role of PCDH8 in THCA, as it impacts cell viability and proliferation. Thus, PCDH8 might play an important role in its prognosis, immune infiltration, and diagnosis.
Assuntos
Protocaderinas , Neoplasias da Glândula Tireoide , Humanos , Prognóstico , Neoplasias da Glândula Tireoide/genética , Proliferação de Células , Carcinogênese , Biomarcadores , Microambiente TumoralRESUMO
OBJECTIVE: We aimed to identify common genes and recurrent causative variants in a large group of Asian patients with different epilepsy syndromes and subgroups. METHODS: Patients with unexplained pediatric-onset epilepsy were identified from the in-house Severance Neurodevelopmental Disorders and Epilepsy Database. All patients underwent either exome sequencing or multigene panels from January 2017 to December 2019, at Severance Children's Hospital in Korea. Clinical data were extracted from the medical records. RESULTS: Of the 957 patients studied, 947 (99.0%) were Korean and 570 were male (59.6%). The median age at testing was 4.91 years (interquartile range, 1.53-9.39). The overall diagnostic yield was 32.4% (310/957). Clinical exome sequencing yielded a diagnostic rate of 36.9% (134/363), whereas the epilepsy panel yielded a diagnostic rate of 29.9% (170/569). Diagnostic yield differed across epilepsy syndromes. It was high in Dravet syndrome (87.2%, 41/47) and early infantile developmental epileptic encephalopathy (60.7%, 17/28), but low in West syndrome (21.8%, 34/156) and myoclonic-atonic epilepsy (4.8%, 1/21). The most frequently implicated genes were SCN1A (n = 49), STXBP1 (n = 15), SCN2A (n = 14), KCNQ2 (n = 13), CDKL5 (n = 11), CHD2 (n = 9), SLC2A1 (n = 9), PCDH19 (n = 8), MECP2 (n = 6), SCN8A (n = 6), and PRRT2 (n = 5). The recurrent genetic abnormalities included 15q11.2 deletion/duplication (n = 9), Xq28 duplication (n = 5), PRRT2 deletion (n = 4), MECP2 duplication (n = 3), SCN1A, c.2556+3A>T (n = 3), and 2q24.3 deletion (n = 3). SIGNIFICANCE: Here we present the results of a large-scale study conducted in East Asia, where we identified several common genes and recurrent variants that varied depending on specific epilepsy syndromes. The overall genetic landscape of the Asian population aligns with findings from other populations of varying ethnicities.