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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 understood 1 . 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., (© 2024. Crown.)

NG2/CSPG4 is a complex surface-associated proteoglycan (PG) recognized to be a widely expressed membrane component of glioblastoma (WHO grade IV) cells and angiogenic pericytes. To determine the precise expression pattern of NG2/CSPG4 on glioblastoma cells and pericytes, we generated a panel of >60 mouse monoclonal antibodies (mAbs) directed against the ectodomain of human NG2/CSPG4, partially characterized the mAbs, and performed a high-resolution distributional mapping of the PG in human foetal, adult and glioblastoma-affected brains. The reactivity pattern initially observed on reference tumour cell lines indicated that the mAbs recognized 48 immunologically distinct NG2/CSPG4 isoforms, and a total of 14 mAbs was found to identify NG2/CSPG4 isoforms in foetal and neoplastic cerebral sections. These were consistently absent in the adult brain, but exhibited a complementary expression pattern in angiogenic vessels of both tumour and foetal tissues. Considering the extreme pleomorphism of tumour areas, and with the aim of subsequently analysing the distributional pattern of the NG2/CSPG4 isoforms on similar histological vessel typologies, a preliminary study was carried out with endothelial cell and pericyte markers, and with selected vascular basement membrane (VBM) components. On both tumour areas characterized by 'glomeruloid' and 'garland vessels', which showed a remarkably similar cellular and molecular organization, and on developing brain vessels, spatially separated, phenotypically diversified pericyte subsets with a polarized expression of key surface components, including NG2/CSPG4, were disclosed. Interestingly, the majority of the immunolocalized NG2/CSPG4 isoforms present in glioblastoma tissue were present in foetal brain, except for one isoform that seemed to be exclusive of tumour cells, being absent in foetal brain. The results highlight an unprecedented, complex pattern of NG2/CSPG4 isoform expression in foetal and neoplastic CNS, discriminating between phenotype-specific and neoplastic versus non-neoplastic variants of the PG, thus opening up vistas for more selective immunotherapeutic targeting of brain tumours.

Nogo is a member of the reticulon family. Our understanding of the physiological functions of the Nogo-A protein has grown over the last few years, and this molecule is now recognized as one of the most important axonal regrowth inhibitors present in central nervous system (CNS) myelin. Nogo-A plays other important roles in nervous system development, epilepsy, vascular physiology, muscle pathology, stroke, inflammation, and CNS tumors. Since the exact role of Nogo-A protein in human brain development is still poorly understood, we studied its cellular and regional distribution by immunohistochemistry in the frontal lobe of 30 human fetal brains. Nogo-A was expressed in the following cortical zones: ependyma, ventricular zone, subventricular zone, intermediate zone, subplate, cortical plate, and marginal zone. The number of positive cells decreased significantly with increasing gestational age in the subplate and marginal zone. Using different antibodies, changes in isoform expression and dimerization states could be shown between various cortical zones. The results demonstrate a significant change in the expression of Nogo-A during the development of the human brain. The effects of its time- and region-specific regulation have to be further studied in detail., (Copyright © 2012 S. Karger AG, Basel.)