RESUMO
Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.
Assuntos
Toxinas Bacterianas/metabolismo , Clostridium sordellii/metabolismo , Semaforinas/metabolismo , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidade , Sítios de Ligação , Sistemas CRISPR-Cas/genética , Linhagem Celular , Microscopia Crioeletrônica , Edema/patologia , Edema/prevenção & controle , Feminino , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/uso terapêutico , Semaforinas/química , Semaforinas/genéticaRESUMO
Hypothalamic melanocortin neurons play a pivotal role in weight regulation. Here, we examined the contribution of Semaphorin 3 (SEMA3) signaling to the development of these circuits. In genetic studies, we found 40 rare variants in SEMA3A-G and their receptors (PLXNA1-4; NRP1-2) in 573 severely obese individuals; variants disrupted secretion and/or signaling through multiple molecular mechanisms. Rare variants in this set of genes were significantly enriched in 982 severely obese cases compared to 4,449 controls. In a zebrafish mutagenesis screen, deletion of 7 genes in this pathway led to increased somatic growth and/or adiposity demonstrating that disruption of Semaphorin 3 signaling perturbs energy homeostasis. In mice, deletion of the Neuropilin-2 receptor in Pro-opiomelanocortin neurons disrupted their projections from the arcuate to the paraventricular nucleus, reduced energy expenditure, and caused weight gain. Cumulatively, these studies demonstrate that SEMA3-mediated signaling drives the development of hypothalamic melanocortin circuits involved in energy homeostasis.
Assuntos
Metabolismo Energético/genética , Melanocortinas/metabolismo , Semaforinas/genética , Adolescente , Adulto , Animais , Peso Corporal , Linhagem Celular , Criança , Pré-Escolar , Modelos Animais de Doenças , Ingestão de Alimentos , Feminino , Variação Genética/genética , Homeostase , Humanos , Hipotálamo/metabolismo , Leptina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Obesidade/genética , Obesidade/metabolismo , Receptores de Superfície Celular/metabolismo , Semaforinas/metabolismo , Adulto Jovem , Peixe-ZebraRESUMO
The selectivity with which proprioceptive sensory neurons innervate their central and peripheral targets implies that they exhibit distinctions in muscle-type identity. The molecular correlates of proprioceptor identity and its origins remain largely unknown, however. In screens to define muscle-type proprioceptor character, we find all-or-none differences in gene expression for proprioceptors that control antagonistic muscles at a single hindlimb joint. Analysis of three of these genes, cadherin13 (cdh13), semaphorin5a (sema5a), and cartilage-acidic protein-1 (crtac1), reveals expression in proprioceptor subsets that supply muscle groups located at restricted dorsoventral and proximodistal domains of the limb. Genetically altering the dorsoventral character of the limb mesenchyme elicits a change in the profile of proprioceptor cdh13, sema5a, and crtac1 expression. These findings indicate that proprioceptors acquire aspects of their muscle-type identity in response to mesenchymal signals expressed in restricted proximodistal and dorsoventral domains of the developing limb.
Assuntos
Extremidades/embriologia , Mesoderma/metabolismo , Propriocepção , Animais , Caderinas/genética , Proteínas de Ligação ao Cálcio/genética , Embrião de Mamíferos/metabolismo , Extremidades/fisiologia , Camundongos , Músculo Esquelético/inervação , Neurônios/metabolismo , Semaforinas/genética , Transdução de Sinais , TranscriptomaRESUMO
Visual circuit development is characterized by subdivision of neuropils into layers that house distinct sets of synaptic connections. We find that, in the Drosophila medulla, this layered organization depends on the axon guidance regulator Plexin A. In Plexin A null mutants, synaptic layers of the medulla neuropil and arborizations of individual neurons are wider and less distinct than in controls. Analysis of semaphorin function indicates that Semaphorin 1a, acting in a subset of medulla neurons, is the primary partner for Plexin A in medulla lamination. Removal of the cytoplasmic domain of endogenous Plexin A has little effect on the formation of medulla layers; however, both null and cytoplasmic domain deletion mutations of Plexin A result in an altered overall shape of the medulla neuropil. These data suggest that Plexin A acts as a receptor to mediate morphogenesis of the medulla neuropil, and as a ligand for Semaphorin 1a to subdivide it into layers. Its two independent functions illustrate how a few guidance molecules can organize complex brain structures by each playing multiple roles.
Assuntos
Proteínas de Drosophila , Morfogênese , Proteínas do Tecido Nervoso , Neurópilo , Lobo Óptico de Animais não Mamíferos , Receptores de Superfície Celular , Semaforinas , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Semaforinas/metabolismo , Semaforinas/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Morfogênese/genética , Neurópilo/metabolismo , Lobo Óptico de Animais não Mamíferos/metabolismo , Lobo Óptico de Animais não Mamíferos/embriologia , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/embriologia , Neurônios/metabolismo , Drosophila/metabolismo , Drosophila/embriologia , Mutação/genéticaRESUMO
The precise assembly of a functional nervous system relies on axon guidance cues. Beyond engaging their cognate receptors and initiating signaling cascades that modulate cytoskeletal dynamics, guidance cues also bind components of the extracellular matrix, notably proteoglycans, yet the role and mechanisms of these interactions remain poorly understood. We found that Drosophila secreted semaphorins bind specifically to glycosaminoglycan (GAG) chains of proteoglycans, showing a preference based on the degree of sulfation. Structural analysis of Sema2b unveiled multiple GAG-binding sites positioned outside canonical plexin-binding site, with the highest affinity binding site located at the C-terminal tail, characterized by a lysine-rich helical arrangement that appears to be conserved across secreted semaphorins. In vivo studies revealed a crucial role of the Sema2b C-terminal tail in specifying the trajectory of olfactory receptor neurons. We propose that secreted semaphorins tether to the cell surface through interactions with GAG chains of proteoglycans, facilitating their presentation to cognate receptors on passing axons.
Assuntos
Orientação de Axônios , Proteínas de Drosophila , Proteoglicanas , Semaforinas , Transdução de Sinais , Animais , Semaforinas/metabolismo , Semaforinas/genética , Proteoglicanas/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Axônios/metabolismo , Drosophila melanogaster/metabolismo , Glicosaminoglicanos/metabolismo , Sítios de Ligação , Ligação Proteica , Neurônios Receptores Olfatórios/metabolismoRESUMO
After central nervous system injury, a rapid cellular and molecular response is induced. This response can be both beneficial and detrimental to neuronal survival in the first few days and increases the risk for neurodegeneration if persistent. Semaphorin4B (Sema4B), a transmembrane protein primarily expressed by cortical astrocytes, has been shown to play a role in neuronal cell death following injury. Our study shows that after cortical stab wound injury, cytokine expression is attenuated in Sema4B-/- mice, and microglia/macrophage reactivity is altered. In vitro, Sema4B enhances the reactivity of microglia following injury, suggesting astrocytic Sema4B functions as a ligand. Moreover, injury-induced microglia reactivity is attenuated in the presence of Sema4B-/- astrocytes compared to Sema4B+/- astrocytes. In vitro experiments indicate that Plexin-B2 is the Sema4B receptor on microglia. Consistent with this, in microglia/macrophage-specific Plexin-B2-/- mice, similar to Sema4B-/- mice, microglial/macrophage reactivity and neuronal cell death are attenuated after cortical injury. Finally, in Sema4B/Plexin-B2 double heterozygous mice, microglial/macrophage reactivity is also reduced after injury, supporting the idea that both Sema4B and Plexin-B2 are part of the same signaling pathway. Taken together, we propose a model in which following injury, astrocytic Sema4B enhances the response of microglia/macrophages via Plexin-B2, leading to increased reactivity.
Assuntos
Astrócitos , Camundongos Knockout , Microglia , Proteínas do Tecido Nervoso , Semaforinas , Animais , Camundongos , Astrócitos/metabolismo , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Lesões Encefálicas/genética , Comunicação Celular , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microglia/patologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Semaforinas/metabolismo , Semaforinas/genéticaRESUMO
Semaphorin6A (Sema6A) is a repulsive guidance molecule that plays many roles in central nervous system, heart and bone development, as well as immune system responses and cell signaling in cancer. Loss of Sema6A or its receptor PlexinA2 in zebrafish leads to smaller eyes and improper retinal patterning. Here, we investigate a potential role for the Sema6A intracellular domain in zebrafish eye development and dissect which phenotypes rely on forward signaling and which rely on reverse signaling. We performed rescue experiments on zebrafish Sema6A morphants with either full-length Sema6A (Sema6A-FL) or Sema6A lacking its intracellular domain (Sema6A-ΔC). We identified that the intracellular domain is not required for eye size and retinal patterning, however it is required for retinal integrity, the number and end feet strength of Müller glia and protecting against retinal cell death. This novel function for the intracellular domain suggests a role for Sema6A reverse signaling in zebrafish eye development.
Assuntos
Domínios Proteicos , Retina , Semaforinas , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Semaforinas/metabolismo , Semaforinas/genética , Retina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Transdução de Sinais , Células Ependimogliais/metabolismo , Células Ependimogliais/citologiaRESUMO
In animals undergoing metamorphosis, the appearance of the nervous system is coincidently transformed by the morphogenesis of neurons. Such morphogenic alterations are exemplified in three types of intrinsic neurons in the Drosophila memory center. In contrast to the well-characterized remodeling of γ neurons, the morphogenesis of α/ß and α'/ß' neurons has not been adequately explored. Here, we show that mamo, a BTB-zinc finger transcription factor that acts as a terminal selector for α'/ß' neurons, controls the formation of the correct axonal pattern of α'/ß' neurons. Intriguingly, specific Mamo isoforms are preferentially expressed in α'/ß' neurons to regulate the expression of axon guidance molecule Semaphorin-1a. This action directs proper axon guidance in α'/ß' neurons, which is also crucial for wiring of α'/ß' neurons with downstream neurons. Taken together, our results provide molecular insights into how neurons establish correct axonal patterns in circuitry assembly during adult memory center construction.
Assuntos
Orientação de Axônios , Proteínas de Drosophila , Memória , Isoformas de Proteínas , Semaforinas , Animais , Axônios/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Memória/fisiologia , Metamorfose Biológica/fisiologia , Neurônios/metabolismo , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Semaforinas/metabolismo , Semaforinas/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Lymphangiogenesis is a dynamic process that involves the directed migration of lymphatic endothelial cells (LECs) to form lymphatic vessels. The molecular mechanisms that underpin lymphatic vessel patterning are not fully elucidated and, to date, no global regulator of lymphatic vessel guidance is known. In this study, we identify the transmembrane cell signalling receptor Plexin D1 (Plxnd1) as a negative regulator of both lymphatic vessel guidance and lymphangiogenesis in zebrafish. plxnd1 is expressed in developing lymphatics and is required for the guidance of both the trunk and facial lymphatic networks. Loss of plxnd1 is associated with misguided intersegmental lymphatic vessel growth and aberrant facial lymphatic branches. Lymphatic guidance in the trunk is mediated, at least in part, by the Plxnd1 ligands, Semaphorin 3AA and Semaphorin 3C. Finally, we show that Plxnd1 normally antagonises Vegfr/Erk signalling to ensure the correct number of facial LECs and that loss of plxnd1 results in facial lymphatic hyperplasia. As a global negative regulator of lymphatic vessel development, the Sema/Plxnd1 signalling pathway is a potential therapeutic target for treating diseases associated with dysregulated lymphatic growth.
Assuntos
Vasos Linfáticos , Semaforinas , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Células Endoteliais/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Linfangiogênese/genética , Vasos Linfáticos/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Proteínas de Transporte/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
OBJECTIVE: Genome-wide association studies have identified 1q22 as a susceptibility locus for cerebral small vessel diseases, including non-lobar intracerebral hemorrhage (ICH) and lacunar stroke. In the present study, we performed targeted high-depth sequencing of 1q22 in ICH cases and controls to further characterize this locus and prioritize potential causal mechanisms, which remain unknown. METHODS: A total of 95,000 base pairs spanning 1q22, including SEMA4A, SLC25A44, and PMF1/PMF1-BGLAP were sequenced in 1,055 spontaneous ICH cases (534 lobar and 521 non-lobar) and 1,078 controls. Firth regression and Rare Variant Influential Filtering Tool analysis were used to analyze common and rare variants, respectively. Chromatin interaction analyses were performed using Hi-C, chromatin immunoprecipitation followed by sequencing, and chromatin interaction analysis with paired-end tag databases. Multivariable Mendelian randomization assessed whether alterations in gene-specific expression relative to regionally co-expressed genes at 1q22 could be causally related to ICH risk. RESULTS: Common and rare variant analyses prioritized variants in SEMA4A 5'-UTR and PMF1 intronic regions, overlapping with active promoter and enhancer regions based on ENCODE annotation. Hi-C data analysis determined that 1q22 is spatially organized in a single chromatin loop, and that the genes therein belong to the same topologically associating domain. Chromatin immunoprecipitation followed by sequencing and chromatin interaction analysis with paired-end tag data analysis highlighted the presence of long-range interactions between the SEMA4A-promoter and PMF1-enhancer regions prioritized by association testing. Multivariable Mendelian randomization analyses demonstrated that PMF1 overexpression could be causally related to non-lobar ICH risk. INTERPRETATION: Altered promoter-enhancer interactions leading to PMF1 overexpression, potentially dysregulating polyamine catabolism, could explain demonstrated associations with non-lobar ICH risk at 1q22, offering a potential new target for prevention of ICH and cerebral small vessel disease. ANN NEUROL 2024;95:325-337.
Assuntos
Doenças de Pequenos Vasos Cerebrais , Semaforinas , Acidente Vascular Cerebral Lacunar , Humanos , Estudo de Associação Genômica Ampla , Hemorragia Cerebral/genética , Hemorragia Cerebral/complicações , Doenças de Pequenos Vasos Cerebrais/genética , Doenças de Pequenos Vasos Cerebrais/complicações , Acidente Vascular Cerebral Lacunar/complicações , Cromatina , Semaforinas/genéticaRESUMO
Synapse formation in the mammalian brain is a complex and dynamic process requiring coordinated function of dozens of molecular families such as cell adhesion molecules (CAMs) and ligand-receptor pairs (Ephs/Ephrins, Neuroligins/Neurexins, Semaphorins/Plexins). Due to the large number of molecular players and possible functional redundancies within gene families, it is challenging to determine the precise synaptogenic roles of individual molecules, which is key to understanding the consequences of mutations in these genes for brain function. Furthermore, few molecules are known to exclusively regulate either GABAergic or glutamatergic synapses, and cell and molecular mechanisms underlying GABAergic synapse formation in particular are not thoroughly understood. We previously demonstrated that Semaphorin-4D (Sema4D) regulates GABAergic synapse development in the mammalian hippocampus while having no effect on glutamatergic synapse development, and this effect occurs through binding to its high affinity receptor, Plexin-B1. In addition, we demonstrated that RNAi-mediated Plexin-B2 knock-down decreases GABAergic synapse density suggesting that both receptors function in this process. Here, we perform a structure-function study of the Plexin-B1 and Plexin-B2 receptors to identify the protein domains in each receptor which are required for its synaptogenic function. Further, we examine whether Plexin-B2 is required in the presynaptic neuron, the postsynaptic neuron, or both to regulate GABAergic synapse formation. Our data reveal that Plexin-B1 and Plexin-B2 function non-redundantly to regulate GABAergic synapse formation and suggest that the transmembrane domain may underlie functional distinctions. We also provide evidence that Plexin-B2 expression in presynaptic GABAergic interneurons, as well as postsynaptic pyramidal cells, regulates GABAergic synapse formation in hippocampus. These findings lay the groundwork for future investigations into the precise signaling pathways required for synapse formation downstream of Plexin-B receptor signaling.
Assuntos
Moléculas de Adesão Celular , Receptores de Superfície Celular , Semaforinas , Animais , Receptores de Superfície Celular/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , MamíferosRESUMO
SignificanceThe adult Drosophila mushroom body (MB) is one of the most extensively studied neural circuits. However, how its circuit organization is established during development is unclear. In this study, we provide an initial characterization of the assembly process of the extrinsic neurons (dopaminergic neurons and MB output neurons) that target the vertical MB lobes. We probe the cellular mechanisms guiding the neurite targeting of these extrinsic neurons and demonstrate that Semaphorin 1a is required in several MB output neurons for their dendritic innervations to three specific MB lobe zones. Our study reveals several intriguing molecular and cellular principles governing assembly of the MB circuit.
Assuntos
Corpos Pedunculados , Semaforinas , Animais , Neurônios Dopaminérgicos , Drosophila/fisiologia , Corpos Pedunculados/fisiologia , Neuritos , Semaforinas/genéticaRESUMO
Semaphorins and Plexins form ligand/receptor pairs that are crucial for a wide range of developmental processes from cell proliferation to axon guidance. The ability of semaphorins to act both as signaling receptors and ligands yields a multitude of responses. Here, we describe a novel role for Semaphorin-6D (Sema6D) and Plexin-A1 in the positioning and targeting of retinogeniculate axons. In Plexin-A1 or Sema6D mutant mice of either sex, the optic tract courses through, rather than along, the border of the dorsal lateral geniculate nucleus (dLGN), and some retinal axons ectopically arborize adjacent and lateral to the optic tract rather than defasciculating and entering the target region. We find that Sema6D and Plexin-A1 act together in a dose-dependent manner, as the number of the ectopic retinal projections is altered in proportion to the level of Sema6D or Plexin-A1 expression. Moreover, using retinal in utero electroporation of Sema6D or Plexin-A1 shRNA, we show that Sema6D and Plexin-A1 are both required in retinal ganglion cells for axon positioning and targeting. Strikingly, nonelectroporated retinal ganglion cell axons also mistarget in the tract region, indicating that Sema6D and Plexin-A1 can act non-cell-autonomously, potentially through axon-axon interactions. These data provide novel evidence for a dose-dependent and non-cell-autonomous role for Sema6D and Plexin-A1 in retinal axon organization in the optic tract and dLGN.SIGNIFICANCE STATEMENT Before innervating their central brain targets, retinal ganglion cell axons fasciculate in the optic tract and then branch and arborize in their target areas. Upon deletion of the guidance molecules Plexin-A1 or Semaphorin-6D, the optic tract becomes disorganized near and extends within the dorsal lateral geniculate nucleus. In addition, some retinal axons form ectopic aggregates within the defasciculated tract. Sema6D and Plexin-A1 act together as a receptor-ligand pair in a dose-dependent manner, and non-cell-autonomously, to produce this developmental aberration. Such a phenotype highlights an underappreciated role for axon guidance molecules in tract cohesion and appropriate defasciculation near, and arborization within, targets.
Assuntos
Células Ganglionares da Retina , Semaforinas , Animais , Camundongos , Axônios/fisiologia , Ligantes , Células Ganglionares da Retina/metabolismo , Semaforinas/genética , Semaforinas/metabolismoRESUMO
Idiopathic pulmonary fibrosis (IPF) is marked by the activation of fibroblasts, leading to excessive production and deposition of extracellular matrix (ECM) within the lung parenchyma. Despite the pivotal role of ECM overexpression in IPF, potential negative regulators of ECM production in fibroblasts have yet to be identified. Semaphorin class 3B (SEMA3B), a secreted protein highly expressed in lung tissues, has established roles in axonal guidance and tumor suppression. However, the role of SEMA3B in ECM production by fibroblasts in the pathogenesis of IPF remains unexplored. Here, we show the downregulation of SEMA3B and its cognate binding receptor, neuropilin 1 (NRP1), in IPF lungs compared with healthy controls. Notably, the reduced expression of SEMA3B and NRP1 is associated with a decline in lung function in IPF. The downregulation of SEMA3B and NRP1 transcripts was validated in the lung tissues of patients with IPF, and two alternative mouse models of pulmonary fibrosis. In addition, we show that transforming growth factor-ß (TGFß) functions as a negative regulator of SEMA3B and NRP1 expression in lung fibroblasts. Furthermore, we demonstrate the antifibrotic effects of SEMA3B against TGFß-induced ECM production in IPF lung fibroblasts. Overall, our findings uncovered a novel role of SEMA3B in the pathogenesis of pulmonary fibrosis and provided novel insights into modulating the SEMA3B-NRP1 axis to attenuate pulmonary fibrosis.NEW & NOTEWORTHY The excessive production and secretion of collagens and other extracellular matrix proteins by fibroblasts lead to the scarring of the lung in severe fibrotic lung diseases. This study unveils an antifibrotic role for semaphorin class 3B (SEMA3B) in the pathogenesis of idiopathic pulmonary fibrosis. SEMA3B functions as an inhibitor of transforming growth factor-ß-driven fibroblast activation and reduced levels of SEMA3B and its receptor, neuropilin 1, are associated with decreased lung function in idiopathic pulmonary fibrosis.
Assuntos
Proteínas da Matriz Extracelular , Fibroblastos , Fibrose Pulmonar Idiopática , Pulmão , Neuropilina-1 , Semaforinas , Fator de Crescimento Transformador beta , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Células Cultivadas , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/genética , Pulmão/metabolismo , Pulmão/patologia , Glicoproteínas de Membrana , Camundongos Endogâmicos C57BL , Neuropilina-1/metabolismo , Neuropilina-1/genética , Semaforinas/metabolismo , Semaforinas/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Intellectual disability (ID) is a neurodevelopmental disorder frequently caused by monogenic defects. In this study, we collected 14 SEMA6B heterozygous variants in 16 unrelated patients referred for ID to different centers. Whereas, until now, SEMA6B variants have mainly been reported in patients with progressive myoclonic epilepsy, our study indicates that the clinical spectrum is wider and also includes non-syndromic ID without epilepsy or myoclonus. To assess the pathogenicity of these variants, selected mutated forms of Sema6b were overexpressed in Human Embryonic Kidney 293T (HEK293T) cells and in primary neuronal cultures. shRNAs targeting Sema6b were also used in neuronal cultures to measure the impact of the decreased Sema6b expression on morphogenesis and synaptogenesis. The overexpression of some variants leads to a subcellular mislocalization of SEMA6B protein in HEK293T cells and to a reduced spine density owing to loss of mature spines in neuronal cultures. Sema6b knockdown also impairs spine density and spine maturation. In addition, we conducted in vivo rescue experiments in chicken embryos with the selected mutated forms of Sema6b expressed in commissural neurons after knockdown of endogenous SEMA6B. We observed that expression of these variants in commissural neurons fails to rescue the normal axon pathway. In conclusion, identification of SEMA6B variants in patients presenting with an overlapping phenotype with ID and functional studies highlight the important role of SEMA6B in neuronal development, notably in spine formation and maturation and in axon guidance. This study adds SEMA6B to the list of ID-related genes.
Assuntos
Epilepsia , Deficiência Intelectual , Semaforinas , Animais , Orientação de Axônios , Embrião de Galinha , Espinhas Dendríticas , Epilepsia/genética , Células HEK293 , Humanos , Deficiência Intelectual/genética , Semaforinas/genéticaRESUMO
Semaphorin ligands interact with plexin receptors to contribute to functions in the development of myriad tissues including neurite guidance and synaptic organisation within the nervous system. Cell-attached semaphorins interact in trans with plexins on opposing cells, but also in cis on the same cell. The interplay between trans and cis interactions is crucial for the regulated development of complex neural circuitry, but the underlying molecular mechanisms are uncharacterised. We have discovered a distinct mode of interaction through which the Drosophila semaphorin Sema1b and mouse Sema6A mediate binding in cis to their cognate plexin receptors. Our high-resolution structural, biophysical and in vitro analyses demonstrate that monomeric semaphorins can mediate a distinctive plexin binding mode. These findings suggest the interplay between monomeric vs dimeric states has a hereto unappreciated role in semaphorin biology, providing a mechanism by which Sema6s may balance cis and trans functionalities.
Assuntos
Moléculas de Adesão Celular/química , Proteínas de Drosophila/química , Proteínas do Tecido Nervoso/química , Semaforinas/química , Animais , Células COS , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Chlorocebus aethiops , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Estrutura Quaternária de Proteína , Semaforinas/genética , Semaforinas/metabolismo , Relação Estrutura-AtividadeRESUMO
Long noncoding RNAs (lncRNAs) are crucial regulators of tumor-initiating cells (TICs) and hold particular importance in triple negative breast cancer (TNBC). Yet, the precise mechanisms by which TIC-associated lncRNAs influence TNBC remain unclear. Our research utilized The Cancer Genome Atlas Breast Cancer (BC) data set to identify prognostic lncRNAs. We then conducted extensive assays to explore their impact on the tumor-initiating phenotype of TNBC cells and the underlying mechanisms. Notably, we found that low expression of lncRNA SEMA3B-AS1 correlated with unfavorable survival in BC patients. SEMA3B-AS1 was also downregulated in TNBC and linked to advanced tumor stage. Functional experiments confirmed its role as a TIC-suppressing lncRNA, curtailing mammosphere formation, ALDH + TIC cell proportion, and impairing clonogenicity, migration, and invasion. Mechanistic insights unveiled SEMA3B-AS1's nuclear localization and interaction with MLL4 (mixed-lineage leukemia 4), triggering H3K4 methylation-associated transcript activation and thus elevating the expression of SEMA3B, a recognized tumor suppressor gene. Our findings emphasize SEMA3B-AS1's significance as a TNBC-suppressing lncRNA that modulates TIC behavior. This study advances our comprehension of lncRNA's role in TNBC progression, advocating for their potential as therapeutic targets in this aggressive BC subtype.
Assuntos
MicroRNAs , RNA Longo não Codificante , Semaforinas , Neoplasias de Mama Triplo Negativas , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , MicroRNAs/genética , Histona-Lisina N-Metiltransferase/genética , Regulação Neoplásica da Expressão Gênica , Proliferação de Células/genética , Linhagem Celular Tumoral , Glicoproteínas de Membrana/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Semaforinas/uso terapêuticoRESUMO
BACKGROUND AND AIMS: Chronic liver disease is a growing epidemic, leading to fibrosis and cirrhosis. TGF-ß is the pivotal profibrogenic cytokine that activates HSC, yet other molecules can modulate TGF-ß signaling during liver fibrosis. Expression of the axon guidance molecules semaphorins (SEMAs), which signal through plexins and neuropilins (NRPs), have been associated with liver fibrosis in HBV-induced chronic hepatitis. This study aims at determining their function in the regulation of HSCs. APPROACH AND RESULTS: We analyzed publicly available patient databases and liver biopsies. We used transgenic mice, in which genes are deleted only in activated HSCs to perform ex vivo analysis and animal models. SEMA3C is the most enriched member of the semaphorin family in liver samples from patients with cirrhosis. Higher expression of SEMA3C in patients with NASH, alcoholic hepatitis, or HBV-induced hepatitis discriminates those with a more profibrotic transcriptomic profile. SEMA3C expression is also elevated in different mouse models of liver fibrosis and in isolated HSCs on activation. In keeping with this, deletion of SEMA3C in activated HSCs reduces myofibroblast marker expression. Conversely, SEMA3C overexpression exacerbates TGF-ß-mediated myofibroblast activation, as shown by increased SMAD2 phosphorylation and target gene expression. Among SEMA3C receptors, only NRP2 expression is maintained on activation of isolated HSCs. Interestingly, lack of NRP2 in those cells reduces myofibroblast marker expression. Finally, deletion of either SEMA3C or NRP2, specifically in activated HSCs, reduces liver fibrosis in mice. CONCLUSION: SEMA3C is a novel marker for activated HSCs that plays a fundamental role in the acquisition of the myofibroblastic phenotype and liver fibrosis.
Assuntos
Células Estreladas do Fígado , Semaforinas , Animais , Humanos , Camundongos , Células Estreladas do Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/patologia , Fosforilação , Semaforinas/genética , Semaforinas/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
The accessibility of cell surface proteins makes them tractable for targeting by cancer immunotherapy, but identifying suitable targets remains challenging. Here we describe plasma membrane profiling of primary human myeloma cells to identify an unprecedented number of cell surface proteins of a primary cancer. We used a novel approach to prioritize immunotherapy targets and identified a cell surface protein not previously implicated in myeloma, semaphorin-4A (SEMA4A). Using knock-down by short-hairpin RNA and CRISPR/nuclease-dead Cas9 (dCas9), we show that expression of SEMA4A is essential for normal myeloma cell growth in vitro, indicating that myeloma cells cannot downregulate the protein to avoid detection. We further show that SEMA4A would not be identified as a myeloma therapeutic target by standard CRISPR/Cas9 knockout screens because of exon skipping. Finally, we potently and selectively targeted SEMA4A with a novel antibody-drug conjugate in vitro and in vivo.
Assuntos
Mieloma Múltiplo , Semaforinas , Membrana Celular/metabolismo , Humanos , Fatores Imunológicos , Imunoterapia , Proteínas de Membrana , Mieloma Múltiplo/genética , Mieloma Múltiplo/terapia , Proteômica , Semaforinas/genética , Semaforinas/metabolismoRESUMO
One of the major functions of the semaphorin signaling system is the regulation of cell shape. In the nematode Caenorhabditis elegans, membrane-bound semaphorins SMP-1/2 (SMPs) regulate the morphology of epidermal cells via their receptor plexin, PLX-1. In the larval male tail of the SMP-PLX-1 signaling mutants, the border between two epidermal cells, R1.p and R2.p, is displaced anteriorly, resulting in the anterior displacement of the anterior-most ray, ray 1, in the adult male. To elucidate how the intercellular signaling mediated by SMPs regulates the position of the intercellular border, we performed mosaic gene expression analyses by using infrared laser-evoked gene operator (IR-LEGO). We show that PLX-1 expressed in R1.p and SMP-1 expressed in R2.p are required for the proper positioning of ray 1. The result suggests that SMP signaling promotes extension, rather than retraction, of R1.p. This is in contrast to a previous finding that SMPs mediate inhibition of cell extension of vulval precursor cells, another group of epidermal cells of C. elegans, indicating the context dependence of cell shape control via the semaphorin signaling system.