The dual action of glioma-derived exosomes on neuronal activity: synchronization and disruption of synchrony

Seizures represent a frequent symptom in gliomas and significantly impact patient morbidity and quality of life. Although the pathogenesis of tumor-related seizures is not fully understood, accumulating evidence indicates a key role of the peritumoral microenvironment. Brain cancer cells interact with neurons by forming synapses with them and by releasing exosomes, cytokines, and other small molecules. Strong interactions among neurons often lead to the synchronization of their activity. In this paper, we used an in vitro model to investigate the role of exosomes released by glioma cell lines and by patient-derived glioma stem cells (GSCs). The addition of exosomes released by U87 glioma cells to neuronal cultures at day in vitro (DIV) 4, when neurons are not yet synchronous, induces synchronization. At DIV 7–12 neurons become highly synchronous, and the addition of the same exosomes disrupts synchrony. By combining Ca²⁺ imaging, electrical recordings from single neurons with patch-clamp electrodes, substrate-integrated microelectrode arrays, and immunohistochemistry, we show that synchronization and de-synchronization are caused by the combined effect of (i) the formation of new neuronal branches, associated with a higher expression of Arp3, (ii) the modification of synaptic efficiency, and (iii) a direct action of exosomes on the electrical properties of neurons, more evident at DIV 7–12 when the threshold for spike initiation is significantly reduced. At DIV 7–12 exosomes also selectively boost glutamatergic signaling by increasing the number of excitatory synapses. Remarkably, de-synchronization was also observed with exosomes released by glioma-associated stem cells (GASCs) from patients with low-grade glioma but not from patients with high-grade glioma, where a more variable outcome was observed. These results show that exosomes released from glioma modify the electrical properties of neuronal networks and that de-synchronization caused by exosomes from low-grade glioma can contribute to the neurological pathologies of patients with brain cancers.Subject terms: Neuroscience, Preclinical research     

Synaptic components are required for glioblastoma progression in Drosophila

Glioblastoma (GB) is the most aggressive, lethal and frequent primary brain tumor. It originates from glial cells and is characterized by rapid expansion through infiltration. GB cells interact with the microenvironment and healthy surrounding tissues, mostly neurons and vessels. GB cells project tumor microtubes (TMs) contact with neurons, and exchange signaling molecules related to Wingless/WNT, JNK, Insulin or Neuroligin-3 pathways. This cell to cell communication promotes GB expansion and neurodegeneration. Moreover, healthy neurons form glutamatergic functional synapses with GB cells which facilitate GB expansion and premature death in mouse GB xerograph models. Targeting signaling and synaptic components of GB progression may become a suitable strategy against glioblastoma. In a Drosophila GB model, we have determined the post-synaptic nature of GB cells with respect to neurons, and the contribution of post-synaptic genes expressed in GB cells to tumor progression. In addition, we document the presence of intratumoral synapses between GB cells, and the functional contribution of pre-synaptic genes to GB calcium dependent activity and expansion. Finally, we explore the relevance of synaptic genes in GB cells to the lifespan reduction caused by GB advance. Our results indicate that both presynaptic and postsynaptic proteins play a role in GB progression and lethality.     

Morphologic evidence of telocytes in human thyroid stromal tissue

Despite the evidence accumulated over the past decade that telocytes (TCs) are a distinctive, though long neglected, cell entity of the stromal microenvironment of several organs of the human body, to date their localization in the endocrine glands remains almost unexplored. This study was therefore undertaken to examine the presence and characteristics of TCs in normal human thyroid stromal tissue through an integrated morphologic approach featuring light microscopy and ultrastructural analysis. TCs were first identified by immunohistochemistry that revealed the existence of an intricate network of CD34⁺ stromal cells spread throughout the thyroid interfollicular connective tissue. Double immunofluorescence allowed to clearly differentiate CD34⁺ stromal cells lacking CD31 immunoreactivity from neighbour CD31⁺ microvascular structures, and the evidence that these stromal cells coexpressed CD34 and platelet‐derived growth factor receptor α further strengthened their identification as TCs. Transmission electron microscopy confirmed the presence of stromal cells ultrastructurally identifiable as TCs projecting their characteristic cytoplasmic processes (i.e., telopodes) into the narrow interstitium between thyroid follicles and blood microvessels, where telopodes intimately surrounded the basement membrane of thyrocytes. Collectively, these morphologic findings provide the first comprehensive demonstration that TCs are main constituents of the human thyroid stroma and lay the necessary groundwork for further in‐depth studies aimed at clarifying their putative implications in glandular homeostasis and pathophysiology.     

Maf/ham1-like pyrophosphatases of non-canonical nucleotides are host-specific partners of viral RNA-dependent RNA polymerases

Cassava brown streak disease (CBSD), dubbed the “Ebola of plants”, is a serious threat to food security in Africa caused by two viruses of the family Potyviridae: cassava brown streak virus (CBSV) and Ugandan (U)CBSV. Intriguingly, U/CBSV, along with another member of this family and one secoviridae, are the only known RNA viruses encoding a protein of the Maf/ham1-like family, a group of widespread pyrophosphatase of non-canonical nucleotides (ITPase) expressed by all living organisms. Despite the socio-economic impact of CDSD, the relevance and role of this atypical viral factor has not been yet established. Here, using an infectious cDNA clone and reverse genetics, we demonstrate that UCBSV requires the ITPase activity for infectivity in cassava, but not in the model plant Nicotiana benthamiana. HPLC-MS/MS experiments showed that, quite likely, this host-specific constraint is due to an unexpected high concentration of non-canonical nucleotides in cassava. Finally, protein analyses and experimental evolution of mutant viruses indicated that keeping a fraction of the yielded UCBSV ITPase covalently bound to the viral RNA-dependent RNA polymerase (RdRP) optimizes viral fitness, and this seems to be a feature shared by the other members of the Potyviridae family expressing Maf/ham1-like proteins. All in all, our work (i) reveals that the over-accumulation of non-canonical nucleotides in the host might have a key role in antiviral defense, and (ii) provides the first example of an RdRP-ITPase partnership, reinforcing the idea that RNA viruses are incredibly versatile at adaptation to different host setups.     

Intermittent Anoxia Reduces Oxygen Free Radicals Formation During Reoxygenation in Rat Hepatocytes

The sensitivity of liver cells to anoxia is a major problem afflicting liver preservation and transplantation. Intermittent ischemia has been proposed to reduce reperfusion injury. The aim of the study was to assess oxygen free radical formation and cell injury during continuous or intermittent anoxia/reoxygenation in rat hepatocytes. Anion superoxide was measured by lucigenin-enhanced chemiluminescence and cell damage by LDH release and trypan blue uptake. During anoxia, superoxide generation dropped to background level in both groups; trypan blue uptake and LDH release, which increased progressively, were significantly greater in hepatocytes exposed to continuous compared to intermittent anoxia. During reoxygenation, a massive generation of superoxide anion formation, followed by a sharp increase in LDH release, was observed in both groups. However, both oxyradical generation and cell injury were significantly greater in cells exposed to continuous compared to intermittent anoxia. The data, showing that intermittent oxygen deprivation reduce liver cell injury and oxygen free radical formation determined by anoxia/reoxygenation, suggest a novel possible approach to the reduction of reperfusion injury.     

Effects of selective modulation of the central melanocortin-3-receptor on food intake and hypothalamic POMC expression

Hypothalamic POMC neurons regulate energy balance via interactions with brain melanocortin receptors (MC-Rs). POMC neurons express the MC3-R which can function as an inhibitory autoreceptor in vitro. We now demonstrate that central activation of MC3-R with ICV infusion of the specific MC3-R agonist, [D-Trp(8)]-gamma-MSH, transiently suppresses hypothalamic Pomc expression and stimulates food intake in rats. Conversely, we also show that ICV infusion of a low dose of a selective MC3-R antagonist causes a transient decrease in feeding and weight gain. These data support a functional inhibitory role for the MC3-R on POMC neurons that leads to changes in food intake.