RESUMO
A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains incompletely understood1. Here we performed single-cell RNA sequencing analysis of 606,380 freshly isolated endothelial cells, perivascular cells and other tissue-derived cells from 117 samples, from 68 human fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We identify extensive molecular heterogeneity of the vasculature of healthy fetal and adult human brains and across five vascular-dependent central nervous system (CNS) pathologies, including brain tumours and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated genes and pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict substantial endothelial-to-perivascular cell ligand-receptor cross-talk, including immune-related and angiogenic pathways, thereby revealing a central role for the endothelium within brain neurovascular unit signalling networks. Our single-cell brain atlas provides insights into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.
Assuntos
Neoplasias Encefálicas , Encéfalo , Malformações Vasculares do Sistema Nervoso Central , Células Endoteliais , Feto , RNA-Seq , Análise da Expressão Gênica de Célula Única , Feminino , Humanos , Masculino , Encéfalo/irrigação sanguínea , Encéfalo/patologia , Encéfalo/embriologia , Encéfalo/metabolismo , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Comunicação Celular , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais/citologia , Feto/irrigação sanguínea , Feto/citologia , Feto/embriologia , Malformações Vasculares do Sistema Nervoso Central/patologia , Antígenos HLA-D/metabolismo , Adulto , SaúdeRESUMO
The neddylation inhibitor MLN4924/Pevonedistat is in clinical trials for multiple cancers. Efficacy is generally attributed to cullin RING ligase (CRL) inhibition, but the contribution of non-CRL targets is unknown. Here, CRISPR screens map MLN4924-monotherapy sensitivity in retinoblastoma to a classic DNA damage-induced p53/E2F3/BAX-dependent death effector network, which synergizes with Nutlin3a or Navitoclax. In monotherapy-resistant cells, MLN4924 plus standard-of-care topotecan overcomes resistance, but reduces DNA damage, instead harnessing ribosomal protein nucleolar-expulsion to engage an RPL11/p21/MYCN/E2F3/p53/BAX synergy network that exhibits extensive cross-regulation. Strikingly, unneddylatable RPL11 substitutes for MLN4924 to perturb nucleolar function and enhance topotecan efficacy. Orthotopic tumors exhibit complete responses while preserving visual function. Moreover, MLN4924 plus melphalan deploy this DNA damage-independent strategy to synergistically kill multiple myeloma cells. Thus, MLN4924 synergizes with standard-of-care drugs to unlock a nucleolar death effector network across cancer types implying broad therapeutic relevance.
Assuntos
Topotecan , Proteína Supressora de Tumor p53 , Proteína X Associada a bcl-2 , Linhagem Celular Tumoral , Ciclopentanos/farmacologia , Proteínas Ribossômicas , Apoptose , Proteína NEDD8RESUMO
Glioblastomas are among the deadliest human cancers and are highly vascularized. Angiogenesis is dynamic during brain development, almost quiescent in the adult brain but reactivated in vascular-dependent CNS pathologies, including brain tumors. The oncofetal axis describes the reactivation of fetal programs in tumors, but its relevance in endothelial and perivascular cells of the human brain vasculature in glial brain tumors is unexplored. Nucleolin is a regulator of cell proliferation and angiogenesis, but its roles in the brain vasculature remain unknown. Here, we studied the expression of Nucleolin in the neurovascular unit in human fetal brains, adult brains, and human gliomas in vivo as well as its effects on sprouting angiogenesis and endothelial metabolism in vitro. Nucleolin is highly expressed in endothelial and perivascular cells during brain development, downregulated in the adult brain, and upregulated in glioma. Moreover, Nucleolin expression correlated with glioma malignancy in vivo. In culture, siRNA-mediated Nucleolin knockdown reduced human brain endothelial cell (HCMEC) and HUVEC sprouting angiogenesis, proliferation, filopodia extension, and glucose metabolism. Furthermore, inhibition of Nucleolin with the aptamer AS1411 decreased brain endothelial cell proliferation in vitro. Mechanistically, Nucleolin knockdown in HCMECs and HUVECs uncovered regulation of angiogenesis involving VEGFR2 and of endothelial glycolysis. These findings identify Nucleolin as a neurodevelopmental factor reactivated in glioma that promotes sprouting angiogenesis and endothelial metabolism, characterizing Nucleolin as an oncofetal protein. Our findings have potential implications in the therapeutic targeting of glioma.
Assuntos
Neoplasias Encefálicas , Glioma , Adulto , Humanos , Glioma/metabolismo , Fosfoproteínas/metabolismo , Encéfalo/metabolismo , Neoplasias Encefálicas/patologia , NucleolinaRESUMO
The CNS critically relies on the formation and proper function of its vasculature during development, adult homeostasis and disease. Angiogenesis - the formation of new blood vessels - is highly active during brain development, enters almost complete quiescence in the healthy adult brain and is reactivated in vascular-dependent brain pathologies such as brain vascular malformations and brain tumours. Despite major advances in the understanding of the cellular and molecular mechanisms driving angiogenesis in peripheral tissues, developmental signalling pathways orchestrating angiogenic processes in the healthy and the diseased CNS remain incompletely understood. Molecular signalling pathways of the 'neurovascular link' defining common mechanisms of nerve and vessel wiring have emerged as crucial regulators of peripheral vascular growth, but their relevance for angiogenesis in brain development and disease remains largely unexplored. Here we review the current knowledge of general and CNS-specific mechanisms of angiogenesis during brain development and in brain vascular malformations and brain tumours, including how key molecular signalling pathways are reactivated in vascular-dependent diseases. We also discuss how these topics can be studied in the single-cell multi-omics era.
Assuntos
Neoplasias Encefálicas , Malformações Vasculares do Sistema Nervoso Central , Humanos , Neovascularização Fisiológica/fisiologia , Encéfalo , Transdução de SinaisRESUMO
Cancer heterogeneity impacts therapeutic response, driving efforts to discover over-arching rules that supersede variability. Here, we define pan-cancer binary classes based on distinct expression of YAP and YAP-responsive adhesion regulators. Combining informatics with in vivo and in vitro gain- and loss-of-function studies across multiple murine and human tumor types, we show that opposite pro- or anti-cancer YAP activity functionally defines binary YAPon or YAPoff cancer classes that express or silence YAP, respectively. YAPoff solid cancers are neural/neuroendocrine and frequently RB1-/-, such as retinoblastoma, small cell lung cancer, and neuroendocrine prostate cancer. YAP silencing is intrinsic to the cell of origin, or acquired with lineage switching and drug resistance. The binary cancer groups exhibit distinct YAP-dependent adhesive behavior and pharmaceutical vulnerabilities, underscoring clinical relevance. Mechanistically, distinct YAP/TEAD enhancers in YAPoff or YAPon cancers deploy anti-cancer integrin or pro-cancer proliferative programs, respectively. YAP is thus pivotal across cancer, but in opposite ways, with therapeutic implications.
Assuntos
Neoplasias Pulmonares/genética , Carcinoma de Pequenas Células do Pulmão/genética , Fatores de Transcrição de Domínio TEA/genética , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/genética , Proteínas de Sinalização YAP/genética , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Humanos , Integrinas/metabolismo , Masculino , Camundongos Transgênicos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Retina/genética , Neoplasias da Retina/patologia , Retinoblastoma/genética , Retinoblastoma/patologia , Proteínas de Ligação a Retinoblastoma/genética , Fatores de Transcrição de Domínio TEA/metabolismo , Ubiquitina-Proteína Ligases/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Leber congenital amaurosis (LCA), a form of autosomal recessive severe early-onset retinal degeneration, is an important cause of childhood blindness. This may be associated with systemic features or not. Here we identified COG5 compound-heterozygous variants in patients affected with a complex LCA phenotype associated with microcephaly and skeletal dysplasia. COG5 is a component of the COG complex, which facilitates retrograde Golgi trafficking; if disrupted this can result in protein misfolding. To date, variants in COG5 have been associated with a distinct congenital disorder of glycosylation (type IIi) and with a variant of Friedreich's ataxia. We show that COG5 variants can also result in fragmentation of the Golgi apparatus and upregulation of the UPR modulator, PKR-like endoplasmic reticulum kinase (PERK). In addition, upregulation of PERK induces DNA damage in cultured cells and in murine retina. This study identifies a novel role for COG5 in maintaining ER protein homeostasis and that disruption of that role results in activation of PERK and early-onset retinal degeneration, microcephaly and skeletal dysplasia. These results also highlight the importance of the UPR pathway in early-onset retinal dystrophy and as potential therapeutic targets for patients.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Amaurose Congênita de Leber/genética , eIF-2 Quinase/metabolismo , Doenças do Desenvolvimento Ósseo/genética , Dano ao DNA , Estresse do Retículo Endoplasmático , Feminino , Humanos , Amaurose Congênita de Leber/metabolismo , Masculino , Microcefalia/genética , Retina/metabolismo , Degeneração Retiniana/genética , Resposta a Proteínas não Dobradas , Sequenciamento Completo do GenomaRESUMO
Inherited retinal degenerations (IRDs) are characterized by the progressive loss of photoreceptors and represent one of the most prevalent causes of blindness among working-age populations. Cyclic nucleotide dysregulation is a common pathological feature linked to numerous forms of IRD, yet the precise mechanisms through which this contributes to photoreceptor death remain elusive. Here we demonstrate that cAMP induced upregulation of the dependence receptor neogenin in the retina. Neogenin levels were also elevated in both human and murine degenerating photoreceptors. We found that overexpressing neogenin in mouse photoreceptors was sufficient to induce cell death, whereas silencing neogenin in degenerating murine photoreceptors promoted survival, thus identifying a pro-death signal in IRDs. A possible treatment strategy is modeled whereby peptide neutralization of neogenin in Rd1, Rd10, and Rho P23H-knockin mice promotes rod and cone survival and rescues visual function as measured by light-evoked retinal ganglion cell recordings, scotopic/photopic electroretinogram recordings, and visual acuity tests. These results expose neogenin as a critical link between cAMP and photoreceptor death, and identify a druggable target for the treatment of retinal degeneration.
Assuntos
Proteínas de Membrana/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Degeneração Retiniana/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Linhagem Celular Tumoral , AMP Cíclico/genética , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Células Fotorreceptoras de Vertebrados/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Células Ganglionares da Retina/patologiaRESUMO
In the immune system, degranulation/exocytosis from lymphocytes is crucial for life through facilitating eradication of infected and malignant cells. Dysfunction of the NK cell exocytosis process has been implicated with devastating immune diseases, such as familial hemophagocytic lymphohistiocytosis, yet the underlying molecular mechanisms of such processes have remained elusive. In particular, although the lytic granule exocytosis from NK cells is strictly Ca2+-dependent, the molecular identity of the Ca2+ sensor has yet to be identified. In this article, we show multiple lines of evidence in which point mutations in aspartic acid residues in both C2 domains of human Munc13-4, whose mutation underlies familial hemophagocytic lymphohistiocytosis type 3, diminished exocytosis with dramatically altered Ca2+ sensitivity in both mouse primary NK cells as well as rat mast cell lines. Furthermore, these mutations within the C2 domains severely impaired NK cell cytotoxicity against malignant cells. Total internal reflection fluorescence microscopy analysis revealed that the mutations strikingly altered Ca2+ dependence of fusion pore opening of each single granule and frequency of fusion events. Our results demonstrate that both C2 domains of Munc13-4 play critical roles in Ca2+-dependent exocytosis and cytotoxicity by regulating single-granule membrane fusion dynamics in immune cells.
Assuntos
Células Matadoras Naturais/imunologia , Linfo-Histiocitose Hemofagocítica/imunologia , Mastócitos/imunologia , Proteínas de Membrana/metabolismo , Vesículas Secretórias/metabolismo , Animais , Ácido Aspártico/genética , Sinalização do Cálcio , Degranulação Celular , Células Cultivadas , Citotoxicidade Imunológica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação/genética , Domínios Proteicos/genética , RatosRESUMO
Developing strategies that promote axonal regeneration within the injured CNS is a major therapeutic challenge, as axonal outgrowth is potently inhibited by myelin and the glial scar. Although regeneration can be achieved using the genetic deletion of PTEN, a negative regulator of the mTOR pathway, this requires inactivation prior to nerve injury, thus precluding therapeutic application. Here, we show that, remarkably, fibroblast-derived exosomes (FD exosomes) enable neurite growth on CNS inhibitory proteins. Moreover, we demonstrate that, upon treatment with FD exosomes, Wnt10b is recruited toward lipid rafts and activates mTOR via GSK3ß and TSC2. Application of FD exosomes shortly after optic nerve injury promoted robust axonal regeneration, which was strongly reduced in Wnt10b-deleted animals. This work uncovers an intercellular signaling pathway whereby FD exosomes mobilize an autocrine Wnt10b-mTOR pathway, thereby awakening the intrinsic capacity of neurons for regeneration, an important step toward healing the injured CNS.
Assuntos
Comunicação Autócrina , Axônios/metabolismo , Exossomos/metabolismo , Regeneração Nervosa , Traumatismos do Nervo Óptico/metabolismo , Proteínas Wnt/metabolismo , Animais , Axônios/fisiologia , Células COS , Células Cultivadas , Chlorocebus aethiops , Fibroblastos/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HEK293 , Humanos , Microdomínios da Membrana/metabolismo , Camundongos , Nervo Óptico/metabolismo , Nervo Óptico/fisiologia , Células PC12 , Ratos , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/metabolismo , Proteínas Wnt/genéticaRESUMO
Although originally discovered as neuronal growth cone-collapsing factors, repulsive guidance molecules (RGMs) are now known as key players in many fundamental processes, such as cell migration, differentiation, iron homeostasis, and apoptosis, during the development and homeostasis of many tissues and organs, including the nervous, skeletal, and immune systems. Furthermore, three RGMs (RGMa, RGMb/DRAGON, and RGMc/hemojuvelin) have been linked to the pathogenesis of various disorders ranging from multiple sclerosis (MS) to cancer and juvenile hemochromatosis (JHH). While the molecular details of these (patho)biological effects and signaling modes have long remained unknown, recent studies unveil several exciting and novel aspects of RGM processing, ligand-receptor interactions, and downstream signaling. In this review, we highlight recent advances in the mechanisms-of-action and function of RGM proteins.
Assuntos
Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Animais , Humanos , Ligantes , Modelos Biológicos , Família Multigênica , Proteínas do Tecido Nervoso/genética , Receptores de Superfície Celular/metabolismoRESUMO
The tumor microenvironment is a critical modulator of carcinogenesis; however, in many tumor types, the influence of the stroma during preneoplastic stages is unknown. Here we explored the relationship between pre-tumor cells and their surrounding stroma in malignant progression of the cerebellar tumor medulloblastoma (MB). We show that activation of the vascular regulatory signalling axis mediated by Norrin (an atypical Wnt)/Frizzled4 (Fzd4) inhibits MB initiation in the Ptch+/- mouse model. Loss of Norrin/Fzd4-mediated signalling in endothelial cells, either genetically or by short-term blockade, increases the frequency of pre-tumor lesions and creates a tumor-permissive microenvironment at the earliest, preneoplastic stages of MB. This pro-tumor stroma, characterized by angiogenic remodelling, is associated with an accelerated transition from preneoplasia to malignancy. These data expose a stromal component that regulates the earliest stages of tumorigenesis in the cerebellum, and a novel role for the Norrin/Fzd4 axis as an endogenous anti-tumor signal in the preneoplastic niche.
Assuntos
Carcinogênese , Proteínas do Olho/metabolismo , Receptores Frizzled/metabolismo , Meduloblastoma/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , CamundongosRESUMO
Syntaxin-1 is the central SNARE protein for neuronal exocytosis. It interacts with Munc18-1 through its cytoplasmic domains, including the N-terminal peptide (N-peptide). Here we examine the role of the N-peptide binding in two conformational states ("closed" vs. "open") of syntaxin-1 using PC12 cells and Caenorhabditis elegans. We show that expression of "closed" syntaxin-1A carrying N-terminal single point mutations (D3R, L8A) that perturb interaction with the hydrophobic pocket of Munc18-1 rescues impaired secretion in syntaxin-1-depleted PC12 cells and the lethality and lethargy of unc-64 (C. elegans orthologue of syntaxin-1)-null mutants. Conversely, expression of the "open" syntaxin-1A harboring the same mutations fails to rescue the impairments. Biochemically, the L8A mutation alone slightly weakens the binding between "closed" syntaxin-1A and Munc18-1, whereas the same mutation in the "open" syntaxin-1A disrupts it. Our results reveal a striking interplay between the syntaxin-1 N-peptide and the conformational state of the protein. We propose that the N-peptide plays a critical role in intracellular trafficking of syntaxin-1, which is dependent on the conformational state of this protein. Surprisingly, however, the N-peptide binding mode seems dispensable for SNARE-mediated exocytosis per se, as long as the protein is trafficked to the plasma membrane.
Assuntos
Proteínas de Caenorhabditis elegans/química , Exocitose , Proteínas Munc18/metabolismo , Neurônios/fisiologia , Sintaxina 1/química , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Técnicas de Silenciamento de Genes , Dados de Sequência Molecular , Neurônios/metabolismo , Células PC12 , Peptídeos/química , Peptídeos/metabolismo , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Sintaxina 1/genética , Sintaxina 1/metabolismoRESUMO
The nervous system is enormously complex, yet the number of cues that control axonal growth is surprisingly meager. Posttranslational modifications amplify diversity, but the degree to which they are employed is unclear. Here, we show that Furin and SKI-1 combine with autocatalytic cleavage and a disulfide bridge to generate four membrane-bound and three soluble forms of the repulsive guidance molecule (RGMa). We provide in vivo evidence that these proprotein convertases are involved in axonal growth and that RGMa cleavage is essential for Neogenin-mediated outgrowth inhibition. Surprisingly, despite no sequence homology, N- and C-RGMa fragments bound the same Fibronectin-like domains in Neogenin and blocked outgrowth. This represents an example in which unrelated fragments from one molecule inhibit outgrowth through a single receptor domain. RGMa is a tethered membrane-bound molecule, and proteolytic processing amplifies RGMa diversity by creating soluble versions with long-range effects as well.
Assuntos
Axônios/fisiologia , Proteínas de Ligação a DNA/metabolismo , Furina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Células COS , Chlorocebus aethiops , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Furina/genética , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Inativação Gênica , Imunoprecipitação , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genéticaRESUMO
Although Munc18-1 was originally identified as a syntaxin1-interacting protein, the physiological significance of this interaction remains unclear. In fact, recent studies of Munc18-1 mutants have suggested that Munc18-1 plays a critical role for docking of secretory vesicles, independent of syntaxin1 regulation. Here we investigated the role of Munc18-1 in syntaxin1 localization by generating stable neuroendocrine cell lines in which Munc18-1 was strongly down-regulated. In these cells, the secretion capability, as well as the docking of dense-core vesicles, was significantly reduced. More importantly, not only was the expression level of syntaxin1 reduced, but the localization of syntaxin1 at the plasma membrane was also severely perturbed. The mislocalized syntaxin1 resided primarily in the perinuclear region of the cells, in which it was highly colocalized with Secretogranin II, a marker protein for dense-core vesicles. In contrast, the expression level and the plasma membrane localization of SNAP-25 were not affected. Furthermore, the syntaxin1 localization and the secretion capability were restored upon transfection-mediated reintroduction of Munc18-1. Our results indicate that endogenous Munc18-1 plays a critical role for the plasma membrane localization of syntaxin1 in neuroendocrine cells and therefore necessitates the interpretation of Munc18-1 mutant phenotypes to be in terms of mislocalized syntaxin1.