RESUMO
OBJECTIVE: The dysregulation of the axon guidance pathway is common in pancreatic ductal adenocarcinoma (PDAC), yet our understanding of its biological relevance is limited. Here, we investigated the functional role of the axon guidance cue SEMA3A in supporting PDAC progression. DESIGN: We integrated bulk and single-cell transcriptomic datasets of human PDAC with in situ hybridisation analyses of patients' tissues to evaluate SEMA3A expression in molecular subtypes of PDAC. Gain and loss of function experiments in PDAC cell lines and organoids were performed to dissect how SEMA3A contributes to define a biologically aggressive phenotype. RESULTS: In PDAC tissues, SEMA3A is expressed by stromal elements and selectively enriched in basal-like/squamous epithelial cells. Accordingly, expression of SEMA3A in PDAC cells is induced by both cell-intrinsic and cell-extrinsic determinants of the basal-like phenotype. In vitro, SEMA3A promotes cell migration as well as anoikis resistance. At the molecular level, these phenotypes are associated with increased focal adhesion kinase signalling through canonical SEMA3A-NRP1 axis. SEMA3A provides mouse PDAC cells with greater metastatic competence and favours intratumoural infiltration of tumour-associated macrophages and reduced density of T cells. Mechanistically, SEMA3A functions as chemoattractant for macrophages and skews their polarisation towards an M2-like phenotype. In SEMA3Ahigh tumours, depletion of macrophages results in greater intratumour infiltration by CD8+T cells and better control of the disease from antitumour treatment. CONCLUSIONS: Here, we show that SEMA3A is a stress-sensitive locus that promotes the malignant phenotype of basal-like PDAC through both cell-intrinsic and cell-extrinsic mechanisms.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Fenótipo , Semaforina-3A , Animais , Humanos , Camundongos , Orientação de Axônios/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Neuropilina-1/metabolismo , Neuropilina-1/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Semaforina-3A/metabolismo , Semaforina-3A/genética , Transdução de SinaisRESUMO
The UNC-5 family of netrin receptor genes, predominantly expressed in brain tissues, plays a pivotal role in various neuronal processes. Mutations in genes involved in axon development contribute to a wide spectrum of human diseases, including developmental, neuropsychiatric, and neurodegenerative disorders. The NTN1/DCC signaling pathway, interacting with UNC5C, plays a crucial role in central nervous system axon guidance and has been associated with psychiatric disorders during adolescence in humans. Whole-exome sequencing analysis unveiled two compound heterozygous causative mutations within the UNC5C gene in a patient diagnosed with psychiatric disorders. In silico analysis demonstrated that neither of the observed variants affected the allosteric linkage between UNC5C and NTN1. In fact, these mutations are located within crucial cytoplasmic domains, specifically ZU5 and the region required for the netrin-mediated axon repulsion of neuronal growth cones. These domains play a critical role in forming the supramodular protein structure and directly interact with microtubules, thereby ensuring the functionality of the axon repulsion process. We emphasize that these mutations disrupt the aforementioned processes, thereby associating the UNC5C gene with psychiatric disorders for the first time and expanding the number of genes related to psychiatric disorders. Further research is required to validate the correlation of the UNC5C gene with psychiatric disorders, but we suggest including it in the genetic analysis of patients with psychiatric disorders.
Assuntos
Orientação de Axônios , Transtornos Mentais , Humanos , Orientação de Axônios/genética , Netrina-1/genética , Netrina-1/metabolismo , Receptores de Netrina/genética , Receptores de Netrina/metabolismo , Axônios/metabolismo , Transtornos Mentais/metabolismoRESUMO
Neurons migrate from their birthplaces to the destinations, and extending axons navigate to their synaptic targets by sensing various extracellular cues in spatiotemporally controlled manners. These evolutionally conserved guidance cues and their receptors regulate multiple aspects of neural development to establish the highly complex nervous system by mediating both short- and long-range cell-cell communications. Neuronal guidance genes (encoding cues, receptors, or downstream signal transducers) are critical not only for development of the nervous system but also for synaptic maintenance, remodeling, and function in the adult brain. One emerging theme is the combinatorial and complementary functions of relatively limited classes of neuronal guidance genes in multiple processes, including neuronal migration, axonal guidance, synaptogenesis, and circuit formation. Importantly, neuronal guidance genes also regulate cell migration and cell-cell communications outside the nervous system. We are just beginning to understand how cells integrate multiple guidance and adhesion signaling inputs to determine overall cellular/subcellular behavior and how aberrant guidance signaling in various cell types contributes to diverse human diseases, ranging from developmental, neuropsychiatric, and neurodegenerative disorders to cancer metastasis. We review classic studies and recent advances in understanding signaling mechanisms of the guidance genes as well as their roles in human diseases. Furthermore, we discuss the remaining challenges and therapeutic potentials of modulating neuronal guidance pathways in neural repair.
Assuntos
Orientação de Axônios , Neurônios , Humanos , Orientação de Axônios/genética , Axônios/metabolismo , Transdução de Sinais/genética , Comunicação CelularRESUMO
Beta-tubulin (TUBB) protein is one of the components of the microtubule cytoskeleton that plays a critical role in the central nervous system. Genetic variants of TUBB cause cortical dysplasia, a developmental brain defect implicated in axonal guidance and the neuron migration. In this study, we assess pathogenic variants (Q15K, Y222F, M299V, V353I, and E401K) of TUBB protein and compared with non-pathogenic variant G235S to determine their impact on protein dynamic to cause cortical dysplasia. Among the analyzed variants, Q15K, Y222F, M299V, and E401K were noticed to have deleterious effect. Then, variant structures were modeled and their affinity with their known cofactor Guanosine-5'-triphosphate (GTP) was assessed which showed diverse binding energies ranged between (-7.436 to -6.950 kcal/mol) for the variants compared to wild-type (-7.428 kcal/mol). Finally, the molecular dynamics simulation of each variant was investigated which showed difference in trajectory between the pathogenic and non-pathogenic variant. Our analysis suggests change in amino acid residue of TUBB structure has notably affects the protein flexibility and their interactions with known cofactor. Overall, our findings provide insight on the relationship between TUBB variants and their structural dynamics that may cause diverse effects leading to cortical dysplasia.
Assuntos
Malformações do Desenvolvimento Cortical , Tubulina (Proteína) , Humanos , Malformações do Desenvolvimento Cortical/genética , Simulação de Dinâmica Molecular , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Orientação de Axônios/genéticaRESUMO
In a recent study, Bonnet and colleagues leveraged in silico structure prediction and human genetic data to understand the molecular regulation of the Rac1-activating guanie nucleotide exchange factor (Rac1-GEF) domain of Trio. Their work sheds new light on the role of Trio during axon guidance and explores the mechanism by which Trio GEF function is regulated in health and dysregulated in disease.
Assuntos
Orientação de Axônios , Fatores de Troca do Nucleotídeo Guanina , Proteínas Serina-Treonina Quinases , Proteínas rac1 de Ligação ao GTP , Humanos , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Orientação de Axônios/genética , Modelos Genéticos , Proteínas rac1 de Ligação ao GTP/química , Domínios ProteicosRESUMO
The transcription factor hepatocyte nuclear factor 1ß (HNF-1ß) is essential for normal development of the kidney and other epithelial organs. In the developing mouse kidney, HNF-1ß is required for the differentiation and patterning of immature nephrons and branching morphogenesis of the ureteric bud (UB). Here, we used ChIP-sequencing (ChIP-seq) and RNA sequencing (RNA-seq) to identify genes that are regulated by HNF-1ß in embryonic mouse kidneys. ChIP-seq revealed that HNF-1ß binds to 8284 sites in chromatin from E14.5 mouse kidneys. Comparison with previous ATAC-seq and histone modification studies showed that HNF-1ß binding peaks colocalized with open chromatin and epigenetic marks of transcriptional activation (H3K27 acetylation, H3K4 trimethylation, H3K4 monomethylation), indicating that the binding sites were functional. To investigate the relationship between HNF-1ß binding and HNF-1ß-dependent gene regulation, RNA-seq was performed on UB cells purified from wild-type and HNF-1ß mutant embryonic kidneys. A total of 1632 genes showed reduced expression in HNF-1ß-deficient UB cells, and 485 genes contained nearby HNF-1ß binding sites indicating that they were directly activated by HNF-1ß. Conversely, HNF-1ß directly repressed the expression of 526 genes in the UB. Comparison with snATAC-seq analysis of UB-derived cells showed that both HNF-1ß-dependent activation and repression correlated with chromatin accessibility. Pathway analysis revealed that HNF-1ß binds near 68 axon guidance genes in the developing kidney. RNA-seq analysis showed that Nrp1, Sema3c, Sema3d, Sema6a, and Slit2 were activated by HNF-1ß, whereas Efna1, Epha3, Epha4, Epha7, Ntn4, Plxna2, Sema3a, Sema4b, Slit3, Srgap1, Unc5c and Unc5d were repressed by HNF-1ß. RNAscope in situ hybridization showed that Nrp1, Sema3c, Sema3d, Sema6a, and Slit2 were expressed in wild-type UB and were dysregulated in HNF-1ß mutant UB. These studies show that HNF-1ß directly regulates the expression of multiple axon guidance genes in the developing mouse kidney. Dysregulation of axon guidance genes may underlie kidney defects in HNF-1ß mutant mice.
Assuntos
Orientação de Axônios , Fator 1-beta Nuclear de Hepatócito , Animais , Camundongos , Orientação de Axônios/genética , Cromatina/genética , Cromatina/metabolismo , Efrina-A1/genética , Fator 1-beta Nuclear de Hepatócito/genética , Fator 1-beta Nuclear de Hepatócito/metabolismo , Rim/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/metabolismo , Semaforina-3A/genética , Semaforinas/genética , Fatores de Transcrição/metabolismoRESUMO
Metastatic pancreatic cancer (PDAC) has a poor clinical outcome with a 5-year survival rate below 3%. Recent transcriptome profiling of PDAC biopsies has identified 2 clinically distinct subtypes - the "basal-like" (BL) subtype with poor prognosis and therapy resistance compared with the less aggressive and drug-susceptible "classical" (CLA) subtype. However, the mechanistic events and environmental factors that promote the BL subtype identity are not very clear. Using preclinical models, patient-derived xenografts, and FACS-sorted PDAC patient biopsies, we report here that the axon guidance receptor, roundabout guidance receptor 3 (ROBO3), promotes the BL metastatic program via a potentially unique AXL/IL-6/phosphorylated STAT3 (p-STAT3) regulatory axis. RNA-Seq identified a ROBO3-mediated BL-specific gene program, while tyrosine kinase profiling revealed AXL as the key mediator of the p-STAT3 activation. CRISPR/dCas9-based ROBO3 silencing disrupted the AXL/p-STAT3 signaling axis, thereby halting metastasis and enhancing therapy sensitivity. Transcriptome analysis of resected patient tumors revealed that AXLhi neoplastic cells associated with the inflammatory stromal program. Combining AXL inhibitor and chemotherapy substantially restored a CLA phenotypic state and reduced disease aggressiveness. Thus, we conclude that a ROBO3-driven hierarchical network determines the inflammatory and prometastatic programs in a specific PDAC subtype.
Assuntos
Orientação de Axônios , Neoplasias Pancreáticas , Receptores de Superfície Celular , Orientação de Axônios/genética , Orientação de Axônios/fisiologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Prognóstico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptor Tirosina Quinase AxlRESUMO
Our lab showed that the Wnt family proteins can function as axon guidance molecules and the planar cell polarity (PCP) pathway mediates the function of Wnts in axon guidance. One of the key evidences was by identifying the axon guidance defects in knockout or conditional knockout animals. We utilized a variety of axon tracing and labeling techniques, including immunohistochemistry (IHC), DiI, BDA, and fluorescent reporters (GFP or tdTomato). These studies have primarily been conducted in spinal cord commissural axons, but have been applied to retinal ganglion cell axons, corticospinal tract axons, dopaminergic and serotonergic projections.
Assuntos
Orientação de Axônios , Axônios , Animais , Animais Geneticamente Modificados , Orientação de Axônios/genética , Axônios/metabolismo , Camundongos , Fenótipo , Medula Espinal/metabolismoRESUMO
During nervous system development, axons navigate complex environments to reach synaptic targets. Early extending axons must interact with guidance cues in the surrounding tissue, while later extending axons can interact directly with earlier "pioneering" axons, "following" their path. In Caenorhabditis elegans, the AVG neuron pioneers the right axon tract of the ventral nerve cord. We previously found that aex-3, a rab-3 guanine nucleotide exchange factor, is essential for AVG axon navigation in a nid-1 mutant background and that aex-3 might be involved in trafficking of UNC-5, a receptor for the guidance cue UNC-6/netrin. Here, we describe a new gene in this pathway: ccd-5, a putative cdk-5 binding partner. ccd-5 mutants exhibit increased navigation defects of AVG pioneer as well as interneuron and motor neuron follower axons in a nid-1 mutant background. We show that ccd-5 acts in a pathway with cdk-5, aex-3, and unc-5. Navigation defects of follower interneuron and motoneuron axons correlate with AVG pioneer axon defects. This suggests that ccd-5 mostly affects pioneer axon navigation and that follower axon defects are largely a secondary consequence of pioneer navigation defects. To determine the consequences for nervous system function, we assessed various behavioral and movement parameters. ccd-5 single mutants have no significant movement defects, and nid-1 ccd-5 double mutants are less responsive to mechanosensory stimuli compared with nid-1 single mutants. These surprisingly minor defects indicate either a high tolerance for axon guidance defects within the motor circuit and/or an ability to maintain synaptic connections among commonly misguided axons.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Orientação de Axônios/genética , Axônios/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Netrinas/metabolismo , Neurônios/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
OBJECTIVE: To investigate the molecular function of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta (PIK3CB) underlying Alzheimer's disease (AD). METHODS: RNA sequencing data were used to filtrate differentially expressed genes (DEGs) in AD/nondementia control and PIK3CB-low/high groups. An unbiased coexpression network was established to evaluate module-trait relationships by using weight gene correlation network analysis (WGCNA). Global regulatory network was constructed to predict the protein-protein interaction. Further cross-talking pathways of PIK3CB were identified by functional enrichment analysis. RESULTS: The mean expression of PIK3CB in AD patients was significantly lower than those in nondementia controls. We identified 2,385 DEGs from 16,790 background genes in AD/control and PIK3CB-low/high groups. Five coexpression modules were established using WGCNA, which participated in apoptosis, axon guidance, long-term potentiation (LTP), regulation of actin cytoskeleton, synaptic vesicle cycle, FoxO, mitogen-activated protein kinase (MAPK), and vascular endothelial growth factor (VEGF) signaling pathways. DEGs with strong relation to AD and low PIK3CB expression were extracted to construct a global regulatory network, in which cross-talking pathways of PIK3CB were identified, such as apoptosis, axon guidance, and FoxO signaling pathway. The occurrence of AD could be accurately predicted by low PIK3CB based on the area under the curve of 71.7%. CONCLUSIONS: These findings highlight downregulated PIK3CB as a potential causative factor of AD, possibly mediated via apoptosis, axon guidance, and FoxO signaling pathway.
Assuntos
Doença de Alzheimer/genética , Orientação de Axônios/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Proteína Forkhead Box O1/metabolismo , Doença de Alzheimer/patologia , Apoptose , Regulação para Baixo , Feminino , Humanos , Masculino , Transdução de SinaisRESUMO
Axons form the long-range connections of biological neuronal networks, which are built through the developmental process of axon guidance. Here, we describe a protocol to precisely and non-invasively control axonal growth trajectories in live zebrafish embryos using focal light activation of a photoactivatable Rac1. We outline techniques for photostimulation, time-lapse imaging, and immunohistochemistry. These approaches enable engineering of long-range axonal circuitry or repair of defective circuits in living zebrafish, despite a milieu of competing endogenous signals and repulsive barriers. For complete details on the use and execution of this protocol, please refer to Harris et al. (2020).
Assuntos
Orientação de Axônios , Optogenética/métodos , Imagem com Lapso de Tempo/métodos , Animais , Orientação de Axônios/genética , Orientação de Axônios/efeitos da radiação , Embrião não Mamífero , Feminino , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Masculino , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
Variants in the high confident autism spectrum disorder (ASD) gene ANK2 target both ubiquitously expressed 220 kDa ankyrin-B and neurospecific 440 kDa ankyrin-B (AnkB440) isoforms. Previous work showed that knock-in mice expressing an ASD-linked Ank2 variant yielding a truncated AnkB440 product exhibit ectopic brain connectivity and behavioral abnormalities. Expression of this variant or loss of AnkB440 caused axonal hyperbranching in vitro, which implicated AnkB440 microtubule bundling activity in suppressing collateral branch formation. Leveraging multiple mouse models, cellular assays, and live microscopy, we show that AnkB440 also modulates axon collateral branching stochastically by reducing the number of F-actin-rich branch initiation points. Additionally, we show that AnkB440 enables growth cone (GC) collapse in response to chemorepellent factor semaphorin 3 A (Sema 3 A) by stabilizing its receptor complex L1 cell adhesion molecule/neuropilin-1. ASD-linked ANK2 variants failed to rescue Sema 3A-induced GC collapse. We propose that impaired response to repellent cues due to AnkB440 deficits leads to axonal targeting and branch pruning defects and may contribute to the pathogenicity of ANK2 variants.
Assuntos
Anquirinas/genética , Orientação de Axônios/genética , Axônios/fisiologia , Semaforina-3A/genética , Transdução de Sinais/genética , Animais , Anquirinas/metabolismo , Camundongos , Semaforina-3A/metabolismoRESUMO
Response Gene to Complement 32 (RGC-32) is an important mediator of the TGF-ß signaling pathway, and an increasing amount of evidence implicates this protein in regulating astrocyte biology. We showed recently that spinal cord astrocytes in mice lacking RGC-32 display an immature phenotype reminiscent of progenitors and radial glia, with an overall elongated morphology, increased proliferative capacity, and increased expression of progenitor markers when compared to their wild-type (WT) counterparts that make them incapable of undergoing reactive changes during the acute phase of experimental autoimmune encephalomyelitis (EAE). Here, in order to decipher the molecular networks underlying RGC-32's ability to regulate astrocytic maturation and reactivity, we performed next-generation sequencing of RNA from WT and RGC-32 knockout (KO) neonatal mouse brain astrocytes, either unstimulated or stimulated with the pleiotropic cytokine TGF-ß. Pathway enrichment analysis showed that RGC-32 is critical for the TGF-ß-induced up-regulation of transcripts encoding proteins involved in brain development and tissue remodeling, such as axonal guidance molecules, transcription factors, extracellular matrix (ECM)-related proteins, and proteoglycans. Our next-generation sequencing of RNA analysis also demonstrated that a lack of RGC-32 results in a significant induction of WD repeat and FYVE domain-containing protein 1 (Wdfy1) and stanniocalcin-1 (Stc1). Immunohistochemical analysis of spinal cords isolated from normal adult mice and mice with EAE at the peak of disease showed that RGC-32 is necessary for the in vivo expression of ephrin receptor type A7 in reactive astrocytes, and that the lack of RGC-32 results in a higher number of homeodomain-only protein homeobox (HOPX)+ and CD133+ radial glia cells. Collectively, these findings suggest that RGC-32 plays a major role in modulating the transcriptomic changes in astrocytes that ultimately lead to molecular programs involved in astrocytic differentiation and reactive changes during neuroinflammation.
Assuntos
Astrócitos/metabolismo , Gliose/genética , Doenças Neuroinflamatórias/genética , Proteínas Nucleares/fisiologia , Transcriptoma , Animais , Orientação de Axônios/genética , Encéfalo/patologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Regulação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Gliose/etiologia , Gliose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/metabolismo , Neurogênese , Doenças Neuroinflamatórias/metabolismo , Proteínas Nucleares/deficiência , Organismos Livres de Patógenos Específicos , Medula Espinal/patologiaRESUMO
ABSTRACT: Hirschsprung disease (HD) is a common form of digestive tract malformation in children. However, the pathogenesis of HD is not very clear. This study aimed to investigate the expression of slit guidance ligand 2 (Slit2) and roundabout 1 (Robo1) in patients with HD.From January 2018 to January 2019, 30 colon specimens from children with HD undergoing surgical resection at the Department of Surgery in Qilu Children's Hospital of Shandong University were obtained. These specimens were divided into the normal segment group, the transitional segment group and the spastic segment group. Immunohistochemical staining, Western blotting, and real-time polymerase chain reaction were used to measure the expression of Slit2 and Robo1 in the intestinal walls of normal, transitional, and spastic segments.Immunohistochemical staining and Western blot analyses showed high levels of the Slit2 and Robo1 proteins in normal ganglion cells in children with HD, lower levels in transitional ganglion cells, and no expression in spastic segments, with significant differences between groups (Pâ<â.05). Similarly, the real-time polymerase chain reaction results were consistent with the Western blot analysis results.The expression of Slit2 and Robo1 decreases significantly in the spastic segment of the intestinal tract in patients with HD.
Assuntos
Orientação de Axônios/genética , Doença de Hirschsprung/genética , Peptídeos e Proteínas de Sinalização Intercelular/análise , Proteínas do Tecido Nervoso/análise , Receptores Imunológicos/análise , Orientação de Axônios/imunologia , Expressão Gênica/genética , Expressão Gênica/imunologia , Doença de Hirschsprung/patologia , Humanos , Proteínas RoundaboutRESUMO
Patients with Tuberous Sclerosis Complex (TSC) show aberrant wiring of neuronal connections formed during development which may contribute to symptoms of TSC, such as intellectual disabilities, autism, and epilepsy. Yet models examining the molecular basis for axonal guidance defects in developing human neurons have not been developed. Here, we generate human induced pluripotent stem cell (hiPSC) lines from a patient with TSC and genetically engineer counterparts and isogenic controls. By differentiating hiPSCs, we show that control neurons respond to canonical guidance cues as predicted. Conversely, neurons with heterozygous loss of TSC2 exhibit reduced responses to several repulsive cues and defective axon guidance. While TSC2 is a known key negative regulator of MTOR-dependent protein synthesis, we find that TSC2 signaled through MTOR-independent RHOA in growth cones. Our results suggest that neural network connectivity defects in patients with TSC may result from defects in RHOA-mediated regulation of cytoskeletal dynamics during neuronal development.
Assuntos
Orientação de Axônios/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Rede Nervosa/metabolismo , Neurogênese/genética , Neurônios/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Esclerose Tuberosa/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Adolescente , Orientação de Axônios/efeitos dos fármacos , Biópsia , Sistemas CRISPR-Cas , Linhagem Celular , Efrinas/farmacologia , Transferência Ressonante de Energia de Fluorescência , Haploinsuficiência , Heterozigoto , Humanos , Masculino , Miosinas/metabolismo , Rede Nervosa/patologia , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Organoides/citologia , Organoides/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismo , Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/genéticaRESUMO
The microtubule (MT) cytoskeleton plays a critical role in axon growth and guidance. Here, we identify the MT-severing enzyme fidgetin-like 2 (FL2) as a negative regulator of axon regeneration and a therapeutic target for promoting nerve regeneration after injury. Genetic knockout of FL2 in cultured adult dorsal root ganglion neurons resulted in longer axons and attenuated growth cone retraction in response to inhibitory molecules. Given the axonal growth-promoting effects of FL2 depletion in vitro, we tested whether FL2 could be targeted to promote regeneration in a rodent model of cavernous nerve (CN) injury. The CNs are parasympathetic nerves that regulate blood flow to the penis, which are commonly damaged during radical prostatectomy (RP), resulting in erectile dysfunction (ED). Application of FL2-siRNA after CN injury significantly enhanced functional nerve recovery. Remarkably, following bilateral nerve transection, visible and functional nerve regeneration was observed in 7 out of 8 animals treated with FL2-siRNA, while no control-treated animals exhibited regeneration. These studies identify FL2 as a promising therapeutic target for enhancing regeneration after peripheral nerve injury and for mitigating neurogenic ED after RP - a condition for which, at present, only poor treatment options exist.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/fisiologia , Orientação de Axônios/genética , Axônios/metabolismo , Gânglios Espinais/citologia , Proteínas Associadas aos Microtúbulos/fisiologia , Regeneração Nervosa/genética , Neurônios/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Células Cultivadas , Masculino , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos , Pênis/inervação , Prostatectomia , Interferência de RNA , RNA Interferente PequenoRESUMO
BACKGROUND: Fetal alcohol syndrome (FAS) due to gestational alcohol exposure represents one of the most common causes of nonheritable lifelong disability worldwide. In vitro and in vivo models have successfully recapitulated multiple facets of the disorder, including morphological and behavioral deficits, but far less is understood regarding the molecular and genetic mechanisms underlying FAS. METHODS: In this study, we utilized an in vitro human pluripotent stem cell-based (hPSC) model of corticogenesis to probe the effects of early, chronic intermittent alcohol exposure on the transcriptome of first trimester-equivalent cortical neurons. RESULTS: We used RNA sequencing of developing hPSC-derived neurons treated for 50 days with 50 mM ethanol and identified a relatively small number of biological pathways significantly altered by alcohol exposure. These included cell-type specification, axon guidance, synaptic function, and regional patterning, with a notable upregulation of WNT signaling-associated transcripts observed in alcohol-exposed cultures relative to alcohol-naïve controls. Importantly, this effect paralleled a shift in gene expression of transcripts associated with regional patterning, such that caudal forebrain-related transcripts were upregulated at the expense of more anterior ones. Results from H9 embryonic stem cells were largely replicated in an induced pluripotent stem cell line (IMR90-4), indicating that these patterning alterations are not cell line-specific. CONCLUSIONS: We found that a major effect of chronic intermittent alcohol on the developing cerebral cortex is an overall imbalance in regionalization, with enrichment of gene expression related to the production of posterodorsal progenitors and a diminution of anteroventral progenitors. This finding parallels behavioral and morphological phenotypes observed in animal models of high-dose prenatal alcohol exposure, as well as patients with FAS.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Córtex Cerebral/efeitos dos fármacos , Etanol/farmacologia , Transtornos do Espectro Alcoólico Fetal/genética , Expressão Gênica/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Orientação de Axônios/efeitos dos fármacos , Orientação de Axônios/genética , Diferenciação Celular/genética , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Humanos , Técnicas In Vitro , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Células-Tronco Pluripotentes , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/embriologia , Prosencéfalo/metabolismo , RNA-Seq , Via de Sinalização Wnt/genéticaRESUMO
In response to inflammatory cytokines and chemokines, monocytes differentiate into macrophages. Comprehensive analysis of gene expression regulation of neuronal guidance cue (NGC) ligands and receptors in the monocyte-to-macrophage differentiation process is not available yet. We performed transcriptome profiling in both human primary PBMCs/PBMC-derived macrophages and THP-1 cells/THP-1-macrophages using microarray or RNA sequencing methods. Pathway analysis showed that the axonal guidance pathway is significantly regulated upon monocyte differentiation. We confirmed NGC ligands and receptors which were consistently regulated, including SEMA4D, SEMA7A, NRP1, NRP2, PLXNA1 and PLXNA3. The involvement of RNA-binding protein quaking (QKI) in the regulation of NGC expression was investigated using monocytes and macrophages from a QKI haplo-insufficient patient and her healthy sibling. This revealed a positive correlation of SEMA7A expression with QKI expression. In silico analysis of 3'UTRs of NGCs proposed the competitive binding of QKI to proximal microRNA targeting sites as the mechanism of QKI-dependent regulation of SEMA7A. RNA immunoprecipitation confirmed an interaction of QKI with the 3'UTR of SEMA7A. Loss of SEMA7A resulted in monocyte differentiation towards a more anti-inflammatory macrophage. Taken together, the axonal guidance pathway is regulated during monocyte-to-macrophage differentiation, and the regulation is in line with the necessary functional adaption for the specialised role of macrophages.
Assuntos
Regiões 3' não Traduzidas/genética , Macrófagos/fisiologia , MicroRNAs/genética , Monócitos/fisiologia , Proteínas de Ligação a RNA/genética , RNA/genética , Semaforinas/metabolismo , Orientação de Axônios/genética , Diferenciação Celular , Feminino , Perfilação da Expressão Gênica , Haploinsuficiência , Humanos , Cultura Primária de Células , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Semaforinas/genética , Irmãos , Células THP-1RESUMO
Intra-retinal axon guidance involves a coordinated expression of transcription factors, axon guidance genes, and secretory molecules within the retina. Pax6, the master regulator gene, has a spatio-temporal expression typically restricted till neurogenesis and fate-specification. However, our observation of persistent expression of Pax6 in mature RGCs led us to hypothesize that Pax6 could play a major role in axon guidance after fate specification. Here, we found significant alteration in intra-retinal axon guidance and fasciculation upon knocking out of Pax6 in E15.5 retina. Through unbiased transcriptome profiling between Pax6fl/fl and Pax6-/- retinas, we revealed the mechanistic insight of its role in axon guidance. Our results showed a significant increase in the expression of extracellular matrix molecules and decreased expression of retinal fate specification and neuron projection guidance molecules. Additionally, we found that EphB1 and Sema5B are directly regulated by Pax6 owing to the guidance defects and improper fasciculation of axons. We conclude that Pax6 expression post fate specification of RGCs is necessary for regulating the expression of axon guidance genes and most importantly for maintaining a conducive ECM through which the nascent axons get guided and fasciculate to reach the optic disc.
Assuntos
Fasciculação Axônica/fisiologia , Orientação de Axônios/fisiologia , Fator de Transcrição PAX6/fisiologia , Células Ganglionares da Retina/fisiologia , Animais , Fasciculação Axônica/genética , Orientação de Axônios/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Matriz Extracelular/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurogênese/genética , Neurogênese/fisiologia , Fator de Transcrição PAX6/deficiência , Fator de Transcrição PAX6/genética , Gravidez , RNA-Seq , Receptor EphB1/genética , Receptor EphB1/fisiologia , Retina/embriologia , Retina/crescimento & desenvolvimento , Retina/fisiologia , Células Ganglionares da Retina/citologia , Semaforinas/genética , Semaforinas/fisiologiaRESUMO
Human brain is anatomically and functionally asymmetric. How brain asymmetry is initiated and established during fetal development is poorly understood. Accumulating evidence has shown that microRNAs (miRNAs) play crucial roles in brain development and function. In this study, we investigate miRNA expression profiles in left and right hemispheres of human fetal brains at 12 weeks post conception (PC), and identify 42 miRNAs showing differential expression between two hemispheres using Affymetrix microarray analyses. Target genes for left- and right-biased miRNAs are largely involved in developmental and functional regulations in the cortex such as axon guidance, GABAergic synapse and dopaminergic synapse pathways. Moreover, we find that predicted targets associated with canonical and non-canonical WNT signaling pathway show variations and differential expression between two hemispheres in response to left- and right-biased miRNAs. Our results highlight a potential role of miRNAs in regulating asymmetric development of human fetal brains.