Probes for INS

Abstract

The factors responsible for the low detection rate of cell-free tumor DNA (ctDNA) in the plasma of glioblastoma (GB) patients are currently unknown. In this study, we measured circulating nucleic acids in patient-derived orthotopically implanted xenograft (PDOX) models of GB (n=64) and show that tumor size and cell proliferation, but not the integrity of the blood-brain barrier or cell death, affect the release of ctDNA in treatment naïve GB PDOX. Analysis of fragment length profiles by shallow genome-wide sequencing (<0.2x coverage) of host (rat) and tumor (human) circulating DNA identified a peak at 145 bp in the human DNA fragments, indicating a difference in the origin or processing of the ctDNA. The concentration of ctDNA correlated with cell death only after treatment with Temozolomide and radiotherapy. Digital PCR detection of plasma tumor mitochondrial DNA (tmtDNA), an alternative to detection of nuclear ctDNA, improved plasma DNA detection rate (82% versus 24%) and allowed detection in cerebrospinal fluid (CSF) and urine. Mitochondrial mutations are prevalent across all cancers and can be detected with high sensitivity, at low cost and without prior knowledge of tumor mutations via capture-panel sequencing. Coupled with the observation that mitochondrial copy number increases in glioma, these data suggest analyzing tmtDNA as a more sensitive method to detect and monitor tumor burden in cancer, specifically in GB where current methods have largely failed.

Pericytes are mural cells of blood microvessels which play a crucial role at the neurovascular interface of the central nervous system. They are involved in the regulation of blood-brain barrier integrity, angiogenesis, clearance of toxic metabolites, capillary hemodynamic responses, and neuroinflammation, and they demonstrate stem cell activity. Morphological and molecular studies to characterize brain pericytes recently pointed out some heterogeneity in pericyte population. Nevertheless, a clear definition of pericyte subtypes is still lacking. Here, we demonstrate that a fraction of brain pericytes express Connexin 30 (Cx30), a gap junction protein, which, in the brain parenchyma, was thought to be exclusively found in astrocytes. Cx30 could thus be a candidate protein in the composition of the gap junction channels already described between endothelial cells and pericytes. It could also form hemichannels or acts in a channel-independent manner to regulate pericyte morphology, as already observed in astrocytes. Altogether, our results suggest that Cx30 defines a novel brain pericyte subtype.

Activity-dependent transcriptional responses shape cortical function. However, a comprehensive understanding of the diversity of these responses across the full range of cortical cell types, and how these changes contribute to neuronal plasticity and disease, is lacking. To investigate the breadth of transcriptional changes that occur across cell types in the mouse visual cortex after exposure to light, we applied high-throughput single-cell RNA sequencing. We identified significant and divergent transcriptional responses to stimulation in each of the 30 cell types characterized, thus revealing 611 stimulus-responsive genes. Excitatory pyramidal neurons exhibited inter- and intralaminar heterogeneity in the induction of stimulus-responsive genes. Non-neuronal cells showed clear transcriptional responses that may regulate experience-dependent changes in neurovascular coupling and myelination. Together, these results reveal the dynamic landscape of the stimulus-dependent transcriptional changes occurring across cell types in the visual cortex; these changes are probably critical for cortical function and may be sites of deregulation in developmental brain disorders.

Hyperactivity and disturbances of attention are common behavioral disorders whose underlying cellular and neural circuit causes are not understood. We report the discovery that striatal astrocytes drive such phenotypes through a hitherto unknown synaptic mechanism. We found that striatal medium spiny neurons (MSNs) triggered astrocyte signaling via γ-aminobutyric acid B (GABAB) receptors. Selective chemogenetic activation of this pathway in striatal astrocytes in vivo resulted in acute behavioral hyperactivity and disrupted attention. Such responses also resulted in upregulation of the synaptogenic cue thrombospondin-1 (TSP1) in astrocytes, increased excitatory synapses, enhanced corticostriatal synaptic transmission, and increased MSN action potential firing in vivo. All of these changes were reversed by blocking TSP1 effects. Our data identify a form of bidirectional neuron-astrocyte communication and demonstrate that acute reactivation of a single latent astrocyte synaptogenic cue alters striatal circuits controlling behavior, revealing astrocytes and the TSP1 pathway as therapeutic targets in hyperactivity, attention deficit, and related psychiatric disorders.

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.