RESUMEN

Patients affected by glioma frequently experience epileptic discharges; however, the causes of brain tumor-related epilepsy (BTRE) are still not completely understood. We investigated the mechanisms underlying BTRE by analyzing the effects of exosomes released by U87 glioma cells and by patient-derived glioma cells. Rat hippocampal neurons incubated for 24 hours with these exosomes exhibited increased spontaneous firing, while their resting membrane potential shifted positively by 10-15 mV. Voltage clamp recordings demonstrated that the activation of the Na+ current shifted toward more hyperpolarized voltages by 10-15 mV. To understand the factors inducing hyperexcitability, we focused on exosomal cytokines. Western blot and ELISAs showed that TNF-α was present inside glioma-derived exosomes. Remarkably, incubation with TNF-α fully mimicked the phenotype induced by exosomes, with neurons firing continuously, while their resting membrane potential shifted positively. Real-time PCR revealed that both exosomes and TNF-α induced overexpression of the voltage-gated Na+ channel Nav1.6, a low-threshold Na+ channel responsible for hyperexcitability. When neurons were preincubated with infliximab, a specific TNF-α inhibitor, the hyperexcitability induced by exosomes and TNF-α was drastically reduced. We propose that infliximab, an FDA-approved drug to treat rheumatoid arthritis, could ameliorate the conditions of glioma patients with BTRE.

Asunto(s)

Neoplasias Encefálicas , Exosomas , Glioma , Canal de Sodio Activado por Voltaje NAV1.6 , Neuronas , Factor de Necrosis Tumoral alfa , Exosomas/metabolismo , Exosomas/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/genética , Humanos , Animales , Ratas , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/genética , Glioma/metabolismo , Glioma/patología , Glioma/genética , Neuronas/metabolismo , Neuronas/patología , Línea Celular Tumoral , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/genética , Epilepsia/metabolismo , Epilepsia/genética , Epilepsia/patología , Hipocampo/metabolismo , Hipocampo/patología , Regulación Neoplásica de la Expresión Génica

RESUMEN

Glioblastoma (GBM) is amongst the deadliest types of cancers, with no resolutive cure currently available. GBM cell proliferation in the patient's brain is a complex phenomenon controlled by multiple mechanisms. The aim of this study was to determine whether the ionic fluxes controlling cell duplication could represent a target for GBM therapy. In this work, we combined multi-channel Ca2+ and Cl- imaging, optical tweezers, electrophysiology, and immunohistochemistry to describe the role of ion fluxes in mediating the cell volume changes that accompany mitosis of U87 GBM cells. We identified three main steps: (i) in round GBM cells undergoing mitosis, during the transition from anaphase to telophase and cytokinesis, large Ca2+ flares occur, reaching values of 0.5 to 1 µmol/L; (ii) these Ca2+ flares activate Ca2+-dependent Cl- channels, allowing the entry of Cl- ions; and (iii) to maintain osmotic balance, GBM cells swell to complete mitosis. This sequence of steps was validated by electrophysiological experiments showing that Cl- channels are activated either directly or indirectly by Ca2+, and by additional live-cell imaging experiments. Cl- channel blockers with different molecular structures, such as niflumic acid and carbenoxolone, blocked GBM replication by arresting GBM cells in a round configuration. These results describe the central role of Ca2+ flares and Cl- fluxes during mitosis and show that inhibition of Ca2+-activated Cl- channels blocks GBM replication, opening the way to new approaches for the clinical treatment of GBM. Implications: Our work identifies ionic fluxes occurring during cell division as targets for devising novel therapies for glioblastoma treatment.

Asunto(s)

Calcio , Glioblastoma , Mitosis , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Calcio/metabolismo , Línea Celular Tumoral , Canales de Cloruro/metabolismo , Proliferación Celular , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología

RESUMEN

The COVID-19 pandemic brought attention to our limited understanding of human olfactory physiology. While the cellular composition of the human olfactory epithelium is similar to that of other vertebrates, its functional properties are largely unknown. We prepared acute slices of human olfactory epithelium from nasal biopsies and used the whole-cell patch-clamp technique to record electrical properties of cells. We measured voltage-gated currents in human olfactory sensory neurons and supporting cells, and action potentials in neurons. Additionally, neuronal inward current and action potentials responses to a phosphodiesterase inhibitor suggested a transduction cascade involving cAMP as a second messenger. Furthermore, responses to odorant mixtures demonstrated that the transduction cascade was intact in this preparation. This study provides the first electrophysiological characterization of olfactory sensory neurons in acute slices of the human olfactory epithelium, paving the way for future research to expand our knowledge of human olfactory physiology.

RESUMEN

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.

Asunto(s)

Neoplasias Encefálicas , Exosomas , Glioma , Neoplasias Encefálicas/patología , Exosomas/metabolismo , Glioma/patología , Humanos , Neuronas/patología , Calidad de Vida , Convulsiones/metabolismo , Microambiente Tumoral

RESUMEN

Although lithium has long been one of the most widely used pharmacological agents in psychiatry, its mechanisms of action at the cellular and molecular levels remain poorly understood. One of the targets of Li+ is the phosphoinositide pathway, but whereas the impact of Li+ on inositol lipid metabolism is well documented, information on physiological effects at the cellular level is lacking. We examined in two mammalian cell lines the effect of acute Li+ exposure on the mobilization of internal Ca2+ and phospholipase C (PLC)-dependent membrane conductances. We first corroborated by Western blots and immunofluorescence in HEK293 cells the presence of key signaling elements of a muscarinic PLC pathway (M1AchR, Gq, PLC-ß1, and IP3Rs). Stimulation with carbachol evoked a dose-dependent mobilization of Ca, as determined with fluorescent indicators. This was due to release from internal stores and proved susceptible to the PLC antagonist U73122. Li+ exposure reproducibly potentiated the Ca response in a concentration-dependent manner extending to the low millimolar range. To broaden those observations to a neuronal context and probe potential Li modulation of electrical signaling, we next examined the cell line SHsy5y. We replicated the potentiating effects of Li on the mobilization of internal Ca, and, after characterizing the basic properties of the electrical response to cholinergic stimulation, we also demonstrated an equally robust upregulation of muscarinic membrane currents. Finally, by directly stimulating the signaling pathway at different links downstream of the receptor, the site of action of the observed Li effects could be narrowed down to the G protein and its interaction with PLC-ß. These observations document a modulation of Gq/PLC/IP3-mediated signaling by acute exposure to lithium, reflected in distinct physiological changes in cellular responses.