RESUMEN
Wnt proteins are enzymatically lipidated by Porcupine (PORCN) in the ER and bind to Wntless (WLS) for intracellular transport and secretion. Mechanisms governing the transfer of these low-solubility Wnts from the ER to the extracellular space remain unclear. Through structural and functional analyses of Wnt7a, a crucial Wnt involved in central nervous system angiogenesis and blood-brain barrier maintenance, we have elucidated the principles of Wnt biogenesis and Wnt7-specific signaling. The Wnt7a-WLS complex binds to calreticulin (CALR), revealing that CALR functions as a chaperone to facilitate Wnt transfer from PORCN to WLS during Wnt biogenesis. Our structures, functional analyses, and molecular dynamics simulations demonstrate that a phospholipid in the core of Wnt-bound WLS regulates the association and dissociation between Wnt and WLS, suggesting a lipid-mediated Wnt secretion mechanism. Finally, the structure of Wnt7a bound to RECK, a cell-surface Wnt7 co-receptor, reveals how RECKCC4 engages the N-terminal domain of Wnt7a to activate Wnt7-specific signaling.
Asunto(s)
Receptores Acoplados a Proteínas G , Proteínas Wnt , Vía de Señalización Wnt , Barrera Hematoencefálica/metabolismo , Unión Proteica , Receptores Acoplados a Proteínas G/metabolismo , Humanos , Proteínas Wnt/química , Proteínas Wnt/metabolismoRESUMEN
The choroid plexus (ChP) is the blood-cerebrospinal fluid (CSF) barrier and the primary source of CSF. Acquired hydrocephalus, caused by brain infection or hemorrhage, lacks drug treatments due to obscure pathobiology. Our integrated, multi-omic investigation of post-infectious hydrocephalus (PIH) and post-hemorrhagic hydrocephalus (PHH) models revealed that lipopolysaccharide and blood breakdown products trigger highly similar TLR4-dependent immune responses at the ChP-CSF interface. The resulting CSF "cytokine storm", elicited from peripherally derived and border-associated ChP macrophages, causes increased CSF production from ChP epithelial cells via phospho-activation of the TNF-receptor-associated kinase SPAK, which serves as a regulatory scaffold of a multi-ion transporter protein complex. Genetic or pharmacological immunomodulation prevents PIH and PHH by antagonizing SPAK-dependent CSF hypersecretion. These results reveal the ChP as a dynamic, cellularly heterogeneous tissue with highly regulated immune-secretory capacity, expand our understanding of ChP immune-epithelial cell cross talk, and reframe PIH and PHH as related neuroimmune disorders vulnerable to small molecule pharmacotherapy.
Asunto(s)
Plexo Coroideo , Hidrocefalia , Humanos , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Plexo Coroideo/metabolismo , Hidrocefalia/líquido cefalorraquídeo , Hidrocefalia/inmunología , Inmunidad Innata , Síndrome de Liberación de Citoquinas/patologíaRESUMEN
The choroid plexus (ChP) in each brain ventricle produces cerebrospinal fluid (CSF) and forms the blood-CSF barrier. Here, we construct a single-cell and spatial atlas of each ChP in the developing, adult, and aged mouse brain. We delineate diverse cell types, subtypes, cell states, and expression programs in epithelial and mesenchymal cells across ages and ventricles. In the developing ChP, we predict a common progenitor pool for epithelial and neuronal cells, validated by lineage tracing. Epithelial and fibroblast cells show regionalized expression by ventricle, starting at embryonic stages and persisting with age, with a dramatic transcriptional shift with maturation, and a smaller shift in each aged cell type. With aging, epithelial cells upregulate host-defense programs, and resident macrophages upregulate interleukin-1ß (IL-1ß) signaling genes. Our atlas reveals cellular diversity, architecture and signaling across ventricles during development, maturation, and aging of the ChP-brain barrier.
Asunto(s)
Plexo Coroideo/embriología , Plexo Coroideo/metabolismo , Factores de Edad , Envejecimiento/fisiología , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Encéfalo/fisiología , Encefalopatías/genética , Encefalopatías/fisiopatología , Diferenciación Celular/genética , Linaje de la Célula/genética , Plexo Coroideo/fisiología , Células Epiteliales/metabolismo , Femenino , Masculino , Ratones/embriología , Ratones Endogámicos C57BL , Transducción de Señal , Análisis de la Célula IndividualRESUMEN
Blood protein extravasation through a disrupted blood-brain barrier and innate immune activation are hallmarks of neurological diseases and emerging therapeutic targets. However, how blood proteins polarize innate immune cells remains largely unknown. Here, we established an unbiased blood-innate immunity multiomic and genetic loss-of-function pipeline to define the transcriptome and global phosphoproteome of blood-induced innate immune polarization and its role in microglia neurotoxicity. Blood induced widespread microglial transcriptional changes, including changes involving oxidative stress and neurodegenerative genes. Comparative functional multiomics showed that blood proteins induce distinct receptor-mediated transcriptional programs in microglia and macrophages, such as redox, type I interferon and lymphocyte recruitment. Deletion of the blood coagulation factor fibrinogen largely reversed blood-induced microglia neurodegenerative signatures. Genetic elimination of the fibrinogen-binding motif to CD11b in Alzheimer's disease mice reduced microglial lipid metabolism and neurodegenerative signatures that were shared with autoimmune-driven neuroinflammation in multiple sclerosis mice. Our data provide an interactive resource for investigation of the immunology of blood proteins that could support therapeutic targeting of microglia activation by immune and vascular signals.
Asunto(s)
Enfermedad de Alzheimer , Microglía , Ratones , Animales , Microglía/metabolismo , Multiómica , Barrera Hematoencefálica/metabolismo , Enfermedad de Alzheimer/genética , FibrinógenoRESUMEN
CD19-directed immunotherapies are clinically effective for treating B cell malignancies but also cause a high incidence of neurotoxicity. A subset of patients treated with chimeric antigen receptor (CAR) T cells or bispecific T cell engager (BiTE) antibodies display severe neurotoxicity, including fatal cerebral edema associated with T cell infiltration into the brain. Here, we report that mural cells, which surround the endothelium and are critical for blood-brain-barrier integrity, express CD19. We identify CD19 expression in brain mural cells using single-cell RNA sequencing data and confirm perivascular staining at the protein level. CD19 expression in the brain begins early in development alongside the emergence of mural cell lineages and persists throughout adulthood across brain regions. Mouse mural cells demonstrate lower levels of Cd19 expression, suggesting limitations in preclinical animal models of neurotoxicity. These data suggest an on-target mechanism for neurotoxicity in CD19-directed therapies and highlight the utility of human single-cell atlases for designing immunotherapies.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Células Epiteliales/metabolismo , Inmunoterapia Adoptiva/efectos adversos , Animales , Anticuerpos Biespecíficos/inmunología , Antígenos CD19/inmunología , Linfocitos B/inmunología , Barrera Hematoencefálica/inmunología , Encéfalo/inmunología , Encéfalo/metabolismo , Línea Celular Tumoral , Citotoxicidad Inmunológica , Humanos , Inmunoterapia/efectos adversos , Inmunoterapia/métodos , Inmunoterapia Adoptiva/métodos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Músculo Liso Vascular/metabolismo , Neoplasias , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Quiméricos de Antígenos/inmunología , Análisis de la Célula Individual/métodos , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Brain disorders are at the leading edge of global disease burden worldwide. Effective therapies are lagging behind because most drugs cannot reach their targets in the brain because of the blood-brain barrier (BBB). The new development of a BBB transport vehicle may bring us a step closer to solve this problem.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Encefalopatías/patología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Barrera Hematoencefálica/efectos de los fármacos , Encefalopatías/metabolismo , Portadores de Fármacos/química , Humanos , Inmunoconjugados/química , Inmunoconjugados/inmunología , Inmunoconjugados/farmacología , Receptores de Transferrina/inmunología , Receptores de Transferrina/metabolismo , TranscitosisRESUMEN
Bacterial lipopolysaccharide (LPS) is implicated in disrupting the blood-brain barrier (BBB). In a recent issue of Nature, Wei et al. now show that LPS activates the inflammatory caspases (4, 5, and 11) and gasdermin D (GSDMD) in brain endothelial cells, which triggers their pyroptotic cell death and disrupts the BBB.
Asunto(s)
Barrera Hematoencefálica , Células Endoteliales , Lipopolisacáridos , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/inmunología , Animales , Humanos , Células Endoteliales/metabolismo , Células Endoteliales/inmunología , Lipopolisacáridos/inmunología , Caspasas/metabolismo , Piroptosis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Unión a Fosfato/metabolismo , RatonesRESUMEN
Low-grade gliomas almost invariably progress into secondary glioblastoma (sGBM) with limited therapeutic option and poorly understood mechanism. By studying the mutational landscape of 188 sGBMs, we find significant enrichment of TP53 mutations, somatic hypermutation, MET-exon-14-skipping (METex14), PTPRZ1-MET (ZM) fusions, and MET amplification. Strikingly, METex14 frequently co-occurs with ZM fusion and is present in â¼14% of cases with significantly worse prognosis. Subsequent studies show that METex14 promotes glioma progression by prolonging MET activity. Furthermore, we describe a MET kinase inhibitor, PLB-1001, that demonstrates remarkable potency in selectively inhibiting MET-altered tumor cells in preclinical models. Importantly, this compound also shows blood-brain barrier permeability and is subsequently applied in a phase I clinical trial that enrolls MET-altered chemo-resistant glioma patients. Encouragingly, PLB-1001 achieves partial response in at least two advanced sGBM patients with rarely significant side effects, underscoring the clinical potential for precisely treating gliomas using this therapy.
Asunto(s)
Neoplasias Encefálicas , Exones , Glioblastoma , Mutación , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas c-met , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Sistemas de Liberación de Medicamentos , Femenino , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Ratas Sprague-Dawley , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Endogenous circadian rhythms are thought to modulate responses to external factors, but mechanisms that confer time-of-day differences in organismal responses to environmental insults/therapeutic treatments are poorly understood. Using a xenobiotic, we find that permeability of the Drosophila "blood"-brain barrier (BBB) is higher at night. The permeability rhythm is driven by circadian regulation of efflux and depends on a molecular clock in the perineurial glia of the BBB, although efflux transporters are restricted to subperineurial glia (SPG). We show that transmission of circadian signals across the layers requires cyclically expressed gap junctions. Specifically, during nighttime, gap junctions reduce intracellular magnesium ([Mg2+]i), a positive regulator of efflux, in SPG. Consistent with lower nighttime efflux, nighttime administration of the anti-epileptic phenytoin is more effective at treating a Drosophila seizure model. These findings identify a novel mechanism of circadian regulation and have therapeutic implications for drugs targeted to the central nervous system.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Relojes Circadianos , Drosophila/metabolismo , Rodaminas/metabolismo , Xenobióticos/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/metabolismo , Relojes Circadianos/efectos de los fármacos , Conexinas/metabolismo , Proteínas de Drosophila/metabolismo , Femenino , Uniones Comunicantes/metabolismo , Magnesio/metabolismo , Neuroglía/metabolismo , Fenitoína/farmacología , Fenitoína/uso terapéutico , Convulsiones/tratamiento farmacológico , Convulsiones/patología , Convulsiones/veterinariaRESUMEN
The vertebrate vasculature displays high organotypic specialization, with the structure and function of blood vessels catering to the specific needs of each tissue. A unique feature of the central nervous system (CNS) vasculature is the blood-brain barrier (BBB). The BBB regulates substance influx and efflux to maintain a homeostatic environment for proper brain function. Here, we review the development and cell biology of the BBB, focusing on the cellular and molecular regulation of barrier formation and the maintenance of the BBB through adulthood. We summarize unique features of CNS endothelial cells and highlight recent progress in and general principles of barrier regulation. Finally, we illustrate why a mechanistic understanding of the development and maintenance of the BBB could provide novel therapeutic opportunities for CNS drug delivery.
Asunto(s)
Transporte Biológico/fisiología , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/crecimiento & desarrollo , Sistema Nervioso Central/citología , Células Endoteliales/citología , Animales , Astrocitos/citología , Membrana Basal/citología , Membrana Basal/metabolismo , Transporte Biológico/genética , Barrera Hematoencefálica/metabolismo , Encéfalo/citología , Encéfalo/fisiología , Sistema Nervioso Central/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Homeostasis , Humanos , Leucocitos , Acoplamiento Neurovascular/fisiología , Pericitos/citología , Uniones Estrechas , Transcitosis/fisiología , Vía de Señalización Wnt/genética , Vía de Señalización Wnt/fisiologíaRESUMEN
Injury is a key driver of inflammation, a critical yet necessary response involving several mediators that is aimed at restoring tissue homeostasis. Inflammation in the central nervous system can be triggered by a variety of stimuli, some intrinsic to the brain and others arising from peripheral signals. Fine-tuned regulation of this response is crucial in a system that is vulnerable due to, for example, aging and ongoing neurodegeneration. In this context, seemingly harmless interventions like a common surgery to repair a broken limb can overwhelm the immune system and become the driver of further complications such as delirium and other perioperative neurocognitive disorders. Here, we discuss potential mechanisms by which the immune system affects the central nervous system after surgical trauma. Together, these neuroimmune interactions are becoming hallmarks of and potential therapeutic targets for multiple neurologic conditions, including those affecting the perioperative space.
Asunto(s)
Inflamación/etiología , Inflamación/metabolismo , Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/metabolismo , Complicaciones Posoperatorias , Alarminas/genética , Alarminas/metabolismo , Animales , Coagulación Sanguínea , Factores de Coagulación Sanguínea/genética , Factores de Coagulación Sanguínea/metabolismo , Barrera Hematoencefálica/metabolismo , Terapia Combinada , Activación de Complemento/inmunología , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Humanos , Sistema Inmunológico/inmunología , Sistema Inmunológico/metabolismo , Inflamación/diagnóstico , Inflamación/terapia , Enfermedades del Sistema Nervioso/diagnóstico , Enfermedades del Sistema Nervioso/terapia , Neuroglía/inmunología , Neuroglía/metabolismo , Neuroinmunomodulación , Resultado del TratamientoRESUMEN
Neurodegeneration is a devastating complication of Langerhans cell histiocytosis (LCH), but it is not clear how it develops. In this issue of Immunity, Wilk et al. demonstrate that circulating BRAFV600E+ myeloid cells damage the blood-brain barrier and infiltrate the brain. Dual inhibition of the MAPK and senescence pathways can block parenchymal injury, providing a potential therapeutic avenue for histiocytic neurodegeneration.
Asunto(s)
Histiocitosis de Células de Langerhans , Monocitos , Humanos , Monocitos/metabolismo , Histiocitosis de Células de Langerhans/metabolismo , Encéfalo/metabolismo , Barrera Hematoencefálica/metabolismo , Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismoRESUMEN
Astrocyte endfeet enwrap the entire vascular tree within the central nervous system, where they perform important functions in regulating the blood-brain barrier (BBB), cerebral blood flow, nutrient uptake, and waste clearance. Accordingly, astrocyte endfeet contain specialized organelles and proteins, including local protein translation machinery and highly organized scaffold proteins, which anchor channels, transporters, receptors, and enzymes critical for astrocyte-vascular interactions. Many neurological diseases are characterized by the loss of polarization of specific endfoot proteins, vascular dysregulation, BBB disruption, altered waste clearance, or, in extreme cases, loss of endfoot coverage. A role for astrocyte endfeet has been demonstrated or postulated in many of these conditions. This review provides an overview of the development, composition, function, and pathological changes of astrocyte endfeet and highlights the gaps in our knowledge that future research should address.
Asunto(s)
Astrocitos , Barrera Hematoencefálica , Astrocitos/fisiología , Barrera Hematoencefálica/metabolismo , Sistema Nervioso Central , Biosíntesis de Proteínas , Encéfalo/patologíaRESUMEN
High-fat diet (HFD) feeding induces rapid reprogramming of systemic metabolism. Here, we demonstrate that HFD feeding of mice downregulates glucose transporter (GLUT)-1 expression in blood-brain barrier (BBB) vascular endothelial cells (BECs) and reduces brain glucose uptake. Upon prolonged HFD feeding, GLUT1 expression is restored, which is paralleled by increased expression of vascular endothelial growth factor (VEGF) in macrophages at the BBB. In turn, inducible reduction of GLUT1 expression specifically in BECs reduces brain glucose uptake and increases VEGF serum concentrations in lean mice. Conversely, myeloid-cell-specific deletion of VEGF in VEGF(Δmyel) mice impairs BBB-GLUT1 expression, brain glucose uptake, and memory formation in obese, but not in lean mice. Moreover, obese VEGF(Δmyel) mice exhibit exaggerated progression of cognitive decline and neuroinflammation on an Alzheimer's disease background. These experiments reveal that transient, HFD-elicited reduction of brain glucose uptake initiates a compensatory increase of VEGF production and assign obesity-associated macrophage activation a homeostatic role to restore cerebral glucose metabolism, preserve cognitive function, and limit neurodegeneration in obesity.
Asunto(s)
Encéfalo/metabolismo , Dieta Alta en Grasa , Glucosa/metabolismo , Obesidad/fisiopatología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Cognición , Células Endoteliales/metabolismo , Ácidos Grasos/metabolismo , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Ratones , Células Mieloides/metabolismoRESUMEN
During development, the central nervous system (CNS) vasculature grows to precisely meet the metabolic demands of neurons and glia. In addition, the vast majority of the CNS vasculature acquires a unique set of molecular and cellular properties-collectively referred to as the blood-brain barrier-that minimize passive diffusion of molecules between the blood and the CNS parenchyma. Both of these processes are controlled by signals emanating from neurons and glia. In this review, we describe the nature and mechanisms-of-action of these signals, with an emphasis on vascular endothelial growth factor (VEGF) and beta-catenin (canonical Wnt) signaling, the two best-understood systems that regulate CNS vascular development. We highlight foundational discoveries, interactions between different signaling systems, the integration of genetic and cell biological studies, advances that are of clinical relevance, and questions for future research.
Asunto(s)
Factor A de Crecimiento Endotelial Vascular , Vía de Señalización Wnt , Barrera Hematoencefálica/metabolismo , Sistema Nervioso Central , Factor A de Crecimiento Endotelial Vascular/metabolismo , Vía de Señalización Wnt/fisiologíaRESUMEN
COVID-19 can cause severe neurological symptoms, but the underlying pathophysiological mechanisms are unclear. Here, we interrogated the brain stems and olfactory bulbs in postmortem patients who had COVID-19 using imaging mass cytometry to understand the local immune response at a spatially resolved, high-dimensional, single-cell level and compared their immune map to non-COVID respiratory failure, multiple sclerosis, and control patients. We observed substantial immune activation in the central nervous system with pronounced neuropathology (astrocytosis, axonal damage, and blood-brain-barrier leakage) and detected viral antigen in ACE2-receptor-positive cells enriched in the vascular compartment. Microglial nodules and the perivascular compartment represented COVID-19-specific, microanatomic-immune niches with context-specific cellular interactions enriched for activated CD8+ T cells. Altered brain T-cell-microglial interactions were linked to clinical measures of systemic inflammation and disturbed hemostasis. This study identifies profound neuroinflammation with activation of innate and adaptive immune cells as correlates of COVID-19 neuropathology, with implications for potential therapeutic strategies.
Asunto(s)
Encéfalo/inmunología , Linfocitos T CD8-positivos/inmunología , COVID-19/inmunología , Microglía/inmunología , Barrera Hematoencefálica/inmunología , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Encéfalo/metabolismo , Encéfalo/patología , Linfocitos T CD8-positivos/metabolismo , COVID-19/patología , Comunicación Celular , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Humanos , Proteínas de Punto de Control Inmunitario/metabolismo , Inflamación , Activación de Linfocitos , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Bulbo Olfatorio/inmunología , Bulbo Olfatorio/metabolismo , Bulbo Olfatorio/patología , Insuficiencia Respiratoria/inmunología , Insuficiencia Respiratoria/patología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain remains unknown1-3. The major facilitator superfamily transporter FLVCR1 (also known as MFSD7B or SLC49A1) was recently determined to be a choline transporter but is not highly expressed at the blood-brain barrier, whereas the related protein FLVCR2 (also known as MFSD7C or SLC49A2) is expressed in endothelial cells at the blood-brain barrier4-7. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus and embryonic lethality, but the physiological role of FLVCR2 is unknown4,5. Here we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in both inward-facing and outward-facing states using cryo-electron microscopy. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of therapeutic agents into the brain.
Asunto(s)
Encéfalo , Colina , Proteínas de Transporte de Membrana , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Transporte Biológico , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Colina/metabolismo , Microscopía por Crioelectrón , Técnicas In Vitro , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/ultraestructura , Modelos MolecularesRESUMEN
Vertebrate organs require locally adapted blood vessels1,2. The gain of such organotypic vessel specializations is often deemed to be molecularly unrelated to the process of organ vascularization. Here, opposing this model, we reveal a molecular mechanism for brain-specific angiogenesis that operates under the control of Wnt7a/b ligands-well-known blood-brain barrier maturation signals3-5. The control mechanism relies on Wnt7a/b-dependent expression of Mmp25, which we find is enriched in brain endothelial cells. CRISPR-Cas9 mutagenesis in zebrafish reveals that this poorly characterized glycosylphosphatidylinositol-anchored matrix metalloproteinase is selectively required in endothelial tip cells to enable their initial migration across the pial basement membrane lining the brain surface. Mechanistically, Mmp25 confers brain invasive competence by cleaving meningeal fibroblast-derived collagen IV α5/6 chains within a short non-collagenous region of the central helical part of the heterotrimer. After genetic interference with the pial basement membrane composition, the Wnt-ß-catenin-dependent organotypic control of brain angiogenesis is lost, resulting in properly patterned, yet blood-brain-barrier-defective cerebrovasculatures. We reveal an organ-specific angiogenesis mechanism, shed light on tip cell mechanistic angiodiversity and thereby illustrate how organs, by imposing local constraints on angiogenic tip cells, can select vessels matching their distinctive physiological requirements.
Asunto(s)
Encéfalo , Neovascularización Fisiológica , Animales , Membrana Basal/metabolismo , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/citología , Encéfalo/citología , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Movimiento Celular , Colágeno Tipo IV/metabolismo , Sistemas CRISPR-Cas/genética , Células Endoteliales/metabolismo , Células Endoteliales/citología , Meninges/citología , Meninges/irrigación sanguínea , Meninges/metabolismo , Especificidad de Órganos , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
The blood-brain barrier (BBB) protects the central nervous system from infections or harmful substances1; its impairment can lead to or exacerbate various diseases of the central nervous system2-4. However, the mechanisms of BBB disruption during infection and inflammatory conditions5,6 remain poorly defined. Here we find that activation of the pore-forming protein GSDMD by the cytosolic lipopolysaccharide (LPS) sensor caspase-11 (refs. 7-9), but not by TLR4-induced cytokines, mediates BBB breakdown in response to circulating LPS or during LPS-induced sepsis. Mice deficient in the LBP-CD14 LPS transfer and internalization pathway10-12 resist BBB disruption. Single-cell RNA-sequencing analysis reveals that brain endothelial cells (bECs), which express high levels of GSDMD, have a prominent response to circulating LPS. LPS acting on bECs primes Casp11 and Cd14 expression and induces GSDMD-mediated plasma membrane permeabilization and pyroptosis in vitro and in mice. Electron microscopy shows that this features ultrastructural changes in the disrupted BBB, including pyroptotic endothelia, abnormal appearance of tight junctions and vasculature detachment from the basement membrane. Comprehensive mouse genetic analyses, combined with a bEC-targeting adeno-associated virus system, establish that GSDMD activation in bECs underlies BBB disruption by LPS. Delivery of active GSDMD into bECs bypasses LPS stimulation and opens the BBB. In CASP4-humanized mice, Gram-negative Klebsiella pneumoniae infection disrupts the BBB; this is blocked by expression of a GSDMD-neutralizing nanobody in bECs. Our findings outline a mechanism for inflammatory BBB breakdown, and suggest potential therapies for diseases of the central nervous system associated with BBB impairment.
Asunto(s)
Barrera Hematoencefálica , Encéfalo , Células Endoteliales , Gasderminas , Inflamación , Animales , Femenino , Humanos , Masculino , Ratones , Membrana Basal/metabolismo , Membrana Basal/ultraestructura , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/ultraestructura , Barrera Hematoencefálica/virología , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/ultraestructura , Caspasas Iniciadoras/metabolismo , Dependovirus , Células Endoteliales/metabolismo , Células Endoteliales/ultraestructura , Gasderminas/antagonistas & inhibidores , Gasderminas/metabolismo , Inflamación/patología , Inflamación/metabolismo , Klebsiella pneumoniae/fisiología , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/sangre , Lipopolisacáridos/farmacología , Ratones Endogámicos C57BL , Piroptosis , Sepsis/metabolismo , Sepsis/patología , Sepsis/microbiología , Análisis de la Célula Individual , Uniones Estrechas/metabolismo , Uniones Estrechas/ultraestructuraRESUMEN
Pathogen infection causes a stereotyped state of sickness that involves neuronally orchestrated behavioural and physiological changes1,2. On infection, immune cells release a 'storm' of cytokines and other mediators, many of which are detected by neurons3,4; yet, the responding neural circuits and neuro-immune interaction mechanisms that evoke sickness behaviour during naturalistic infections remain unclear. Over-the-counter medications such as aspirin and ibuprofen are widely used to alleviate sickness and act by blocking prostaglandin E2 (PGE2) synthesis5. A leading model is that PGE2 crosses the blood-brain barrier and directly engages hypothalamic neurons2. Here, using genetic tools that broadly cover a peripheral sensory neuron atlas, we instead identified a small population of PGE2-detecting glossopharyngeal sensory neurons (petrosal GABRA1 neurons) that are essential for influenza-induced sickness behaviour in mice. Ablating petrosal GABRA1 neurons or targeted knockout of PGE2 receptor 3 (EP3) in these neurons eliminates influenza-induced decreases in food intake, water intake and mobility during early-stage infection and improves survival. Genetically guided anatomical mapping revealed that petrosal GABRA1 neurons project to mucosal regions of the nasopharynx with increased expression of cyclooxygenase-2 after infection, and also display a specific axonal targeting pattern in the brainstem. Together, these findings reveal a primary airway-to-brain sensory pathway that detects locally produced prostaglandins and mediates systemic sickness responses to respiratory virus infection.