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Pericytes are the mural cells of the microvascular network that are in close contact with underlying endothelial cells. Endothelial-secreted PDGFB leads to recruitment of pericytes to the vessel wall, but this is disrupted in Pdgfbret/ret mice when the PDGFB retention motif is deleted. This results in severely reduced pericyte coverage on blood vessels. In this study, we investigated vascular abnormalities and hemodynamics in Pdgfbret/ret mice throughout the cerebrovascular network and in different cortical layers by in vivo two-photon microscopy. We confirmed that Pdgfbret/ret mice are severely deficient in pericytes throughout the vascular network, with enlarged brain blood vessels and a reduced number of vessel branches. Red blood cell velocity, linear density, and tube hematocrit were reduced in Pdgfbret/ret mice, which may impair oxygen delivery to the tissue. We also measured intravascular PO2 and found that concentrations were higher in cortical Layer 2/3 in Pdgfbret/ret mice, indicative of reduced blood oxygen extraction. Finally, we found that Pdgfbret/ret mice had a reduced capacity for vasodilation in response to an acetazolamide challenge during functional MRI imaging. Taken together, these results suggest that severe pericyte deficiency can lead to vascular abnormalities and altered cerebral blood flow, reminiscent of pathologies such as arteriovenous malformations.
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Células Endoteliales , Pericitos , Ratones , Animales , Proteínas Proto-Oncogénicas c-sis/metabolismo , Pericitos/metabolismo , Modelos Animales de Enfermedad , Becaplermina/metabolismo , Hemodinámica , Oxígeno/metabolismoRESUMEN
Cell transplantation is a promising approach for the reconstruction of neuronal circuits after brain damage. Transplanted neurons integrate with remarkable specificity into circuitries of the mouse cerebral cortex affected by neuronal ablation. However, it remains unclear how neurons perform in a local environment undergoing reactive gliosis, inflammation, macrophage infiltration, and scar formation, as in traumatic brain injury (TBI). To elucidate this, we transplanted cells from the embryonic mouse cerebral cortex into TBI-injured, inflamed-only, or intact cortex of adult mice. Brain-wide quantitative monosynaptic rabies virus (RABV) tracing unraveled graft inputs from correct regions across the brain in all conditions, with pronounced quantitative differences: scarce in intact and inflamed brain versus exuberant after TBI. In the latter, the initial overshoot is followed by pruning, with only a few input neurons persisting at 3 months. Proteomic profiling identifies candidate molecules for regulation of the synaptic yield, a pivotal parameter to tailor for functional restoration of neuronal circuits.
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Transplantation is a clinically relevant approach for brain repair, but much remains to be understood about influences of the disease environment on transplant connectivity. To explore the effect of amyloid pathology in Alzheimer's disease (AD) and aging, we examined graft connectivity using monosynaptic rabies virus tracing in APP/PS1 mice and in 16- to 18-month-old wild-type (WT) mice. Transplanted neurons differentiated within 4 weeks and integrated well into the host visual cortex, receiving input from the appropriate brain regions for this area. Unexpectedly, we found a prominent several-fold increase in local inputs, in both amyloid-loaded and aged environments. State-of-the-art deep proteome analysis using mass spectrometry highlights complement system activation as a common denominator of environments promoting excessive local input connectivity. These data therefore reveal the key role of the host pathology in shaping the input connectome, calling for caution in extrapolating results from one pathological condition to another.
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Microglia participate in central nervous system (CNS) development and homeostasis and are often implicated in modulating disease processes. However, less is known about the role of microglia in the biology of the neurovascular unit (NVU). In particular, data are scant on whether microglia are involved in CNS vascular pathology. In this study, we use a mouse model of primary familial brain calcification, Pdgfbret/ret , to investigate the role of microglia in calcification of the NVU. We report that microglia enclosing vessel calcifications, coined calcification-associated microglia, display a distinct activation phenotype. Pharmacological ablation of microglia with the CSF1R inhibitor PLX5622 leads to aggravated vessel calcification. Mechanistically, we show that microglia require functional TREM2 for controlling vascular calcification. Our results demonstrate that microglial activity in the setting of pathological vascular calcification is beneficial. In addition, we identify a previously unrecognized function of microglia in halting the expansion of vascular calcification.
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RATIONALE: Pericytes are capillary mural cells playing a role in stabilizing newly formed blood vessels during development and tissue repair. Loss of pericytes has been described in several brain disorders, and genetically induced pericyte deficiency in the brain leads to increased macromolecular leakage across the blood-brain barrier (BBB). However, the molecular details of the endothelial response to pericyte deficiency remain elusive. OBJECTIVE: To map the transcriptional changes in brain endothelial cells resulting from lack of pericyte contact at single-cell level and to correlate them with regional heterogeneities in BBB function and vascular phenotype. METHODS AND RESULTS: We reveal transcriptional, morphological, and functional consequences of pericyte absence for brain endothelial cells using a combination of methodologies, including single-cell RNA sequencing, tracer analyses, and immunofluorescent detection of protein expression in pericyte-deficient adult Pdgfbret/ret mice. We find that endothelial cells without pericyte contact retain a general BBB-specific gene expression profile, however, they acquire a venous-shifted molecular pattern and become transformed regarding the expression of numerous growth factors and regulatory proteins. Adult Pdgfbret/ret brains display ongoing angiogenic sprouting without concomitant cell proliferation providing unique insights into the endothelial tip cell transcriptome. We also reveal heterogeneous modes of pericyte-deficient BBB impairment, where hotspot leakage sites display arteriolar-shifted identity and pinpoint putative BBB regulators. By testing the causal involvement of some of these using reverse genetics, we uncover a reinforcing role for angiopoietin 2 at the BBB. CONCLUSIONS: By elucidating the complexity of endothelial response to pericyte deficiency at cellular resolution, our study provides insight into the importance of brain pericytes for endothelial arterio-venous zonation, angiogenic quiescence, and a limited set of BBB functions. The BBB-reinforcing role of ANGPT2 (angiopoietin 2) is paradoxical given its wider role as TIE2 (TEK receptor tyrosine kinase) receptor antagonist and may suggest a unique and context-dependent function of ANGPT2 in the brain.
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Barrera Hematoencefálica/metabolismo , Pericitos/citología , Animales , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/patología , Proliferación Celular , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Linfocinas/deficiencia , Linfocinas/genética , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica , Pericitos/metabolismo , Pericitos/patología , Factor de Crecimiento Derivado de Plaquetas/deficiencia , Factor de Crecimiento Derivado de Plaquetas/genética , Análisis de la Célula Individual , TranscriptomaRESUMEN
OBJECTIVE: Brain calcifications are associated with several neurodegenerative diseases. Here, we describe the occurrence of intracranial calcifications as a new phenotype in transgenic P301L mice overexpressing four repeat tau, a model of human tauopathy. MATERIALS AND METHODS: Thirty-six P301L mice (Thy1.2) and ten age-matched non-transgenic littermates of different ages were assessed. Gradient echo data were acquired in vivo and ex vivo at 7 T and 9.4 T for susceptibility-weighted imaging (SWI) and phase imaging. In addition, ex vivo micro-computed tomography (µCT) was performed. Histochemistry and immunohistochemistry were used to investigate the nature of the imaging lesions. RESULTS: SW images revealed regional hypointensities in the hippocampus, cortex, caudate nucleus, and thalamus of P301L mice, which in corresponding phase images indicated diamagnetic lesions. Concomitantly, µCT detected hyperdense lesions, though fewer lesions were observed compared to MRI. Diamagnetic susceptibility lesions in the hippocampus increased with age. The immunochemical staining of brain sections revealed osteocalcin-positive deposits. Furthermore, intra-neuronal and vessel-associated osteocalcin-containing nodules co-localized with phosphorylated-tau (AT8 and AT100) in the hippocampus, while vascular osteocalcin-containing nodules were detected in the thalamus in the absence of phosphorylated-tau deposition. DISCUSSION: SWI and phase imaging sensitively detected intracranial calcifications in the P301L mouse model of human tauopathy.
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Tauopatías , Proteínas tau , Animales , Modelos Animales de Enfermedad , Humanos , Imagen por Resonancia Magnética , Ratones , Ratones Transgénicos , Tauopatías/diagnóstico por imagen , Microtomografía por Rayos XRESUMEN
Primary familial brain calcification (PFBC) is an age-dependent and rare neurodegenerative disorder characterized by microvascular calcium phosphate deposits in the deep brain regions. Known genetic causes of PFBC include loss-of-function mutations in genes involved in either of three processes-platelet-derived growth factor (PDGF) signaling, phosphate homeostasis or protein glycosylation-with unclear molecular links. To provide insight into the pathogenesis of PFBC, we analyzed murine models of PFBC for the first two of these processes in Pdgfbret/ret and Slc20a2-/- mice with regard to the structure, molecular composition, development and distribution of perivascular calcified nodules. Analyses by transmission electron microscopy and immunofluorescence revealed that calcified nodules in both of these models have a multilayered ultrastructure and occur in direct contact with reactive astrocytes and microglia. However, whereas nodules in Pdgfbret/ret mice were large, solitary and smooth surfaced, the nodules in Slc20a2-/- mice were multi-lobulated and occurred in clusters. The regional distribution of nodules also differed between the two models. Proteomic analysis and immunofluorescence stainings revealed a common molecular composition of the nodules in the two models, involving proteins implicated in bone homeostasis, but also proteins not previously linked to tissue mineralization. While the brain vasculature of Pdgfbret/ret mice has been reported to display reduced pericyte coverage and abnormal permeability, we found that Slc20a2-/- mice have a normal pericyte coverage and no overtly increased permeability. Thus, lack of pericytes and increase in permeability of the blood-brain barrier are likely not the causal triggers for PFBC pathogenesis. Instead, gene expression and spatial correlations suggest that astrocytes are intimately linked to the calcification process in PFBC.
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Astrocitos/metabolismo , Encefalopatías/metabolismo , Calcinosis/metabolismo , Matriz Extracelular/metabolismo , Microglía/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Animales , Astrocitos/patología , Encefalopatías/genética , Encefalopatías/patología , Calcinosis/genética , Calcinosis/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Matriz Extracelular/patología , Femenino , Masculino , Ratones , Ratones Transgénicos , Microglía/patología , Mutación , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/metabolismoRESUMEN
Pericytes are perivascular cells along capillaries that are critical for the development of a functional vascular bed in the central nervous system and other organs. Pericyte functions in the adult brain are less well understood. Pericytes have been suggested to mediate functional hyperemia at the capillary level, regulate the blood-brain barrier and to give rise to scar tissue after spinal cord injury. Furthermore, pericyte loss has been suggested to precede cognitive decline in mouse models of Alzheimer's disease. Despite this observation, there is no convincing causality between pericyte loss and the pathogenesis of Alzheimer's disease. However, recent loss-of-function mutations in PDGFB and PDGFRB genes have implicated pericytes as the principle cell type affected in primary familiar brain calcification (PFBC), a neuropsychiatric disorder with dominant inheritance. Here we review the role of the PDGFB/PDGFRB signaling pathway in pericyte development and briefly discuss homeostatic functions of pericytes in the brain. We provide an overview of recent studies with mouse models of PFBC and discuss suggested pathogenic mechanisms for PFBC with special reference to pericytes.
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Encefalopatías , Calcinosis , Pericitos , Adulto , Animales , Encéfalo , Humanos , Ratones , Proteínas Proto-Oncogénicas c-sis , Receptor beta de Factor de Crecimiento Derivado de Plaquetas , Receptor de Retrovirus Xenotrópico y PolitrópicoRESUMEN
Brain calcifications are commonly detected in aged individuals and accompany numerous brain diseases, but their functional importance is not understood. In cases of primary familial brain calcification, an autosomally inherited neuropsychiatric disorder, the presence of bilateral brain calcifications in the absence of secondary causes of brain calcification is a diagnostic criterion. To date, mutations in five genes including solute carrier 20 member 2 (SLC20A2), xenotropic and polytropic retrovirus receptor 1 (XPR1), myogenesis regulating glycosidase (MYORG), platelet-derived growth factor B (PDGFB) and platelet-derived growth factor receptor ß (PDGFRB), are considered causal. Previously, we have reported that mutations in PDGFB in humans are associated with primary familial brain calcification, and mice hypomorphic for PDGFB (Pdgfbret/ret) present with brain vessel calcifications in the deep regions of the brain that increase with age, mimicking the pathology observed in human mutation carriers. In this study, we characterize the cellular environment surrounding calcifications in Pdgfbret/ret animals and show that cells around vessel-associated calcifications express markers for osteoblasts, osteoclasts and osteocytes, and that bone matrix proteins are present in vessel-associated calcifications. Additionally, we also demonstrate the osteogenic environment around brain calcifications in genetically confirmed primary familial brain calcification cases. We show that calcifications cause oxidative stress in astrocytes and evoke expression of neurotoxic astrocyte markers. Similar to previously reported human primary familial brain calcification cases, we describe high interindividual variation in calcification load in Pdgfbret/ret animals, as assessed by ex vivo and in vivo quantification of calcifications. We also report that serum of Pdgfbret/ret animals does not differ in calcification propensity from control animals and that vessel calcification occurs only in the brains of Pdgfbret/ret animals. Notably, ossification of vessels and astrocytic neurotoxic response is associated with specific behavioural and cognitive alterations, some of which are associated with primary familial brain calcification in a subset of patients.
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Astrocitos/metabolismo , Osificación Heterotópica/patología , Proteínas Proto-Oncogénicas c-sis/metabolismo , Anciano , Animales , Encéfalo/patología , Encefalopatías/genética , Calcinosis/patología , Femenino , Humanos , Masculino , Ratones , Mutación , Osteogénesis/fisiología , Estrés Oxidativo , Linaje , Proteínas Proto-Oncogénicas c-sis/genética , Proteínas Proto-Oncogénicas c-sis/fisiología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/genética , Receptor de Retrovirus Xenotrópico y PolitrópicoRESUMEN
Vascular diseases are major causes of death, yet our understanding of the cellular constituents of blood vessels, including how differences in their gene expression profiles create diversity in vascular structure and function, is limited. In this paper, we describe a single-cell RNA sequencing (scRNA-seq) dataset that defines vascular and vessel-associated cell types and subtypes in mouse brain and lung. The dataset contains 3,436 single cell transcriptomes from mouse brain, which formed 15 distinct clusters corresponding to cell (sub)types, and another 1,504 single cell transcriptomes from mouse lung, which formed 17 cell clusters. In order to allow user-friendly access to our data, we constructed a searchable database (http://betsholtzlab.org/VascularSingleCells/database.html). Our dataset constitutes a comprehensive molecular atlas of vascular and vessel-associated cell types in the mouse brain and lung, and as such provides a strong foundation for future studies of vascular development and diseases.
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Vasos Sanguíneos , Encéfalo/irrigación sanguínea , Pulmón/irrigación sanguínea , Transcriptoma , Animales , Vasos Sanguíneos/citología , Vasos Sanguíneos/fisiología , Bases de Datos Factuales , Células Endoteliales/fisiología , Ratones , Miocitos del Músculo Liso/fisiología , Pericitos/fisiología , Análisis de Secuencia de ARN , Análisis de la Célula IndividualRESUMEN
The exocyst is a molecular tether that retains secretory vesicles at the plasma membrane prior to SNARE-mediated docking and fusion. However, individual exocyst complex components (EXOCs) may also function independently of exocyst assembly. Alternative splice variants of EXOC mRNA and paralogs of EXOC genes have been described and several have been attributed functions that may be independent of the exocyst complex. Here we describe a novel splice variant of murine Exoc3l2, which we term Exoc3l2a. We discuss possible functional implications of the resulting domain excision from this isoform of EXOC3L2 based on structural similarities with its paralog M-Sec (EXOC3L3), which is implicated in tunneling nanotube formation. The identification of this Exoc3l2 splice variant expands the potential for subunit diversity within the exocyst and for alternative functionality of this component independently of the exocyst.
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Empalme Alternativo , Factores de Necrosis Tumoral/química , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Animales , Ratones , Modelos Moleculares , Conformación Proteica , ARN/metabolismo , Análisis de Secuencia de ARN , Factores de Necrosis Tumoral/genéticaRESUMEN
In Fig. 1b of this Article, 'Csf1r' was misspelt 'Csfr1'. In addition, in Extended Data Fig. 11b, owing to an error during figure formatting, the genes listed in the first column shifted down three rows below the first gene on the list, causing a mismatch between the gene names and their characteristics. These errors have been corrected online, and the original Extended Data Fig. 11b is provided as Supplementary Information to the accompanying Amendment.
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Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited. Here, using vascular single-cell transcriptomics, we provide molecular definitions for the principal types of blood vascular and vessel-associated cells in the adult mouse brain. We uncover the transcriptional basis of the gradual phenotypic change (zonation) along the arteriovenous axis and reveal unexpected cell type differences: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells. We also provide insight into pericyte organotypicity and define a population of perivascular fibroblast-like cells that are present on all vessel types except capillaries. Our work illustrates the power of single-cell transcriptomics to decode the higher organizational principles of a tissue and may provide the initial chapter in a molecular encyclopaedia of the mammalian vasculature.