Enlightening the mechanism of ferroptosis in epileptic heart.

Epilepsy is a chronic neurological degenerative disease with a high incidence, affecting all age groups. Refractory Epilepsy (RE) occurs in approximately 30-40% of cases with a higher risk of sudden unexpected death in epilepsy (SUDEP). Recent studies have shown that spontaneous seizures developed in epilepsy can be related to an increase in oxidative stress and reactive oxygen derivatives (ROS) production. Increasing ROS concentration causes lipid peroxidation, protein oxidation, destruction of nuclear genetic material, enzyme inhibition, and cell death by a mechanism known as "ferroptosis" (Fts). Inactivation of glutathione peroxidase 4 (GPX4) induces Fts, while oxidative stress is linked with increased intracellular free iron (Fe+2) concentration. Fts is also a non-apoptotic programmed cell death mechanism, where a hypoxia-inducible factor 1 alpha (HIF-141) dependent hypoxic stress-like condition appears to occur with accumulation of iron and cytotoxic ROS in affected cells. Assuming convulsive crises as hypoxic stress, repetitive convulsive/hypoxic stress can be an effective inducer of the "epileptic heart" (EH), which is characterized by altered autonomic function and a high risk of malignant or fatal bradycardia. We previously reported that experimental recurrent seizures induce cardiomyocyte Fts associated with SUDEP. Furthermore, several genes related to Fts and hypoxia have recently been identified in acute myocardial infarction. An emerging theme from recent studies indicates that inhibition of GPX4 through modulating expression or activities of the xCT antiporter system (SLC7A11) governs cell sensitivity to oxidative stress from ferroptosis. Furthermore, during hypoxia, an increased expression of stress transcriptional factor ATF3 can promote Fts induced by erastin in a HIF-141-dependent manner. We propose that inhibition of Fts with ROS scavengers, iron chelators, antioxidants, and transaminase inhibitors could provide a therapeutic effect in epilepsy and improve the prognosis of SUDEP risk by protecting the heart from ferroptosis.

Hierro: desde la homeostasis a la muerte por ferroptosis / Iron: from normal homeostasis to death by ferroptosis

Resumen El hierro (Fe) es un elemento vital para casi todos los organismos debido a su facilidad para donar y aceptar electrones. Es cofactor de muchas proteínas y enzimas necesarias para la adecuada utilización del oxígeno y la generación de energía. Su desregulación se relaciona a procesos de estrés oxidativo y muerte celular mediada por Fe(II) denominada ferroptosis. Las células de mamíferos utilizan múltiples mecanismos para garantizar la adquisición del hierro como nutriente esencial, que se encuentra oxidado [Fe(III)], y que debe ser reducido a Fe(II) para su adecuada utilización intracelular. Cada etapa de transferencia del hierro a través de las membranas biológicas exige una reconversión de su estado de oxidado a reducido y viceversa, dependiendo del paso metabólico implicado. La distorsión de dichos procesos se asocia con varias enfermedades: desde la deficiencia de hierro debida a defectos en la adquisición o distribución del metal, que causa anemia, a la sobrecarga de hierro que resulta de una absorción excesiva de hierro o en una utilización defectuosa, que causa una sobreoferta de Fe(II) en los tejidos y que lleva a un daño oxidativo y a la muerte celular. Existen múltiples mecanismos regulatorios que en conjunto aseguran el equilibrio en la homeostasis del hierro. Esta actualización describe los avances recientes en las vías reguladoras del hierro, así como en los mecanismos subyacentes al tráfico de dicho elemento desde su absorción, principalmente biodistribución y su uso intracelular, quizás el área más importante donde se define su adecuada utilización o la muerte celular por ferroptosis.

Abstract Iron (Fe) is a vital element for almost all organisms due to its ability to donate and accept electrons with relative ease. It serves as a cofactor for many proteins and enzymes necessary for the proper use of oxygen and energy generation, and its deregulation is related to the processes of oxidative stress and iron-mediated cell death called ferroptosis. Mammalian cells use multiple mechanisms to ensure the acquisition of iron as an essential nutrient, which is normally oxidised in the form of Fe(III) and must be reduced to Fe(II) for adequate intracellular use. Each stage of iron transfer across biological membranes requires a reconversion of its state from oxidised to reduced and vice versa, depending on the metabolic step involved. Distortion of these processes is associated with various diseases, such as iron deficiency due to defects in the acquisition or distribution of the metal that causes anemia, as well as iron overload from excessive iron absorption or defective use, which results in an oversupply of Fe(II) in tissues leading to oxidative damage and cell death. There are multiple regulatory mechanisms that together ensure the balance in iron homeostasis. This update describes the recent advances in the iron regulatory pathways, as well as in the mechanisms underlying iron trafficking from its absorption, mainly biodistribution and its intracellular use, perhaps the most important area where its adequate utilisation or cell death by ferroptosis is defined.

Resumo O ferro (Fe) é um elemento vital para quase todos os organismos devido à sua capacidade de doar e aceitar elétrons com relativa facilidade. O ferro serve como cofator para muitas proteínas e enzimas necessárias para o uso adequado do oxigênio e geração de energia, e a sua desregulação está relacionada a processos de estresse oxidativo e morte celular mediada por Fe(II) denominado ferroptose. As células de mamíferos utilizam múltiplos mecanismos para garantir a aquisição de ferro como nutriente essencial, que normalmente é oxidado na forma de Fe(III) e deve ser reduzido a Fe(II) para o uso intracelular adequado. Cada estágio de transferência de Fe através das membranas biológicas requer uma reconversão de seu estado de oxidado para reduzido e vice-versa, dependendo da etapa metabólica envolvida. A distorção desses processos está associada a várias doenças: desde a deficiência de ferro devido a defeitos na aquisição ou distribuição do metal que causa a anemia, até a sobrecarga de ferro resultante da absorção excessiva de ferro ou utilização defeituosa, que causa um excesso de oferta de Fe(II) nos tecidos levando ao dano oxidativo e morte celular. Existem múltiplos mecanismos regulatórios que juntos garantem o equilíbrio na homeostase do ferro. Esta atualização descreve os avanços recentes nas vias reguladoras do ferro, bem como nos mecanismos subjacentes ao tráfico deste elemento desde a sua absorção, principalmente biodistribuição e seu uso intracelular, talvez a área mais importante onde sua utilização adequada ou morte celular por ferroptose é definido.

Transporter hypothesis in pharmacoresistant epilepsies. Is it at the central or peripheral level?

The multidrug-resistance (MDR) phenotype is typically observed in patients with refractory epilepsy (RE) whose seizures are not controlled despite receiving several combinations of more than two antiseizure medications (ASMs) directed against different ion channels or neurotransmitter receptors. Since the use of bromide in 1860, more than 20 ASMs have been developed; however, historically ~30% of cases of RE with MDR phenotype remains unchanged. Irrespective of metabolic biotransformation, the biodistribution of ASMs and their metabolites depends on the functional expression of some ATP-binding cassette transporters (ABC-t) in different organs, such as the blood-brain barrier (BBB), bowel, liver, and kidney, among others. ABC-t, such as P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP-1), and breast cancer-resistance protein (BCRP), are mainly expressed in excretory organs and play a critical role in the pharmacokinetics (PK) of all drugs. The transporter hypothesis can explain pharmacoresistance to a broad spectrum of ASMs, even when administered simultaneously. Since ABC-t expression can be induced by hypoxia, inflammation, or seizures, a high frequency of uncontrolled seizures increases the risk of RE. These stimuli can induce ABC-t expression in excretory organs and in previously non-expressing (electrically responsive) cells, such as neurons or cardiomyocytes. In this regard, an alternative mechanism to the classical pumping function of P-gp indicates that P-gp activity can also produce a significant reduction in resting membrane potential (ΔΨ0 = -60 to -10 mV). P-gp expression in neurons and cardiomyocytes can produce membrane depolarization and participate in epileptogenesis, heart failure, and sudden unexpected death in epilepsy. On this basis, ABC-t play a peripheral role in controlling the PK of ASMs and their access to the brain and act at a central level, favoring neuronal depolarization by mechanisms independent of ion channels or neurotransmitters that current ASMs cannot control.

Myocardial Iron Overload in an Experimental Model of Sudden Unexpected Death in Epilepsy.

Uncontrolled repetitive generalized tonic-clonic seizures (GTCS) are the main risk factor for sudden unexpected death in epilepsy (SUDEP). GTCS can be observed in models such as Pentylenetetrazole kindling (PTZ-K) or pilocarpine-induced Status Epilepticus (SE-P), which share similar alterations in cardiac function, with a high risk of SUDEP. Terminal cardiac arrhythmia in SUDEP can develop as a result of a high rate of hypoxic stress-induced by convulsions with excessive sympathetic overstimulation that triggers a neurocardiogenic injury, recently defined as "Epileptic Heart" and characterized by heart rhythm disturbances, such as bradycardia and lengthening of the QT interval. Recently, an iron overload-dependent form of non-apoptotic cell death called ferroptosis was described at the brain level in both the PTZ-K and SE-P experimental models. However, seizure-related cardiac ferroptosis has not yet been reported. Iron overload cardiomyopathy (IOC) results from the accumulation of iron in the myocardium, with high production of reactive oxygen species (ROS), lipid peroxidation, and accumulation of hemosiderin as the final biomarker related to cardiomyocyte ferroptosis. Iron overload cardiomyopathy is the leading cause of death in patients with iron overload secondary to chronic blood transfusion therapy; it is also described in hereditary hemochromatosis. GTCS, through repeated hypoxic stress, can increase ROS production in the heart and cause cardiomyocyte ferroptosis. We hypothesized that iron accumulation in the "Epileptic Heart" could be associated with a terminal cardiac arrhythmia described in the IOC and the development of state-potentially in the development of SUDEP. Using the aforementioned PTZ-K and SE-P experimental models, after SUDEP-related repetitive GTCS, we observed an increase in the cardiac expression of hypoxic inducible factor 1α, indicating hypoxic-ischemic damage, and both necrotic cells and hemorrhagic areas were related to the possible hemosiderin production in the PTZ-K model. Furthermore, we demonstrated for the first time an accumulation of hemosiderin in the heart in the SE-P model. These results suggest that uncontrolled recurrent seizures, as described in refractory epilepsy, can give rise to high hypoxic stress in the heart, thus inducing hemosiderin accumulation as in IOC, and can act as an underlying hidden mechanism contributing to the development of a terminal cardiac arrhythmia in SUDEP. Because iron accumulation in tissues can be detected by non-invasive imaging methods, cardiac iron overload in refractory epilepsy patients could be treated with chelation therapy to reduce the risk of SUDEP.

The role of P-glycoprotein (P-gp) and inwardly rectifying potassium (Kir) channels in sudden unexpected death in epilepsy (SUDEP).

Sudden unexpected death in epilepsy (SUDEP) is the major cause of death that affects patients with epilepsy. The risk of SUDEP increases according to the frequency and severity of uncontrolled seizures; therefore, SUDEP risk is higher in patients with refractory epilepsy (RE), in whom most antiepileptic drugs (AEDs) are ineffective for both seizure control and SUDEP prevention. Consequently, RE and SUDEP share a multidrug resistance (MDR) phenotype, which is mainly associated with brain overexpression of ABC-transporters such as P-glycoprotein (P-gp). The activity of P-gp can also contribute to membrane depolarization and affect the normal function of neurons and cardiomyocytes. Other molecular regulators of membrane potential are the inwardly rectifying potassium channels (Kir), whose genetic variants have been related to both epilepsy and heart dysfunctions. Although it has been suggested that dysfunctions of the cardiac, respiratory, and brainstem arousal systems are the causes of SUDEP, the molecular basis for explaining its dysfunctions remain unknown. In rats, repetitive seizures or status epilepticus induced high expression of P-gp and loss Kir expression in the brain and heart, and promoted membrane depolarization, malignant bradycardia, and the high rate of mortality. Here we reviewed clinical and experimental evidences suggesting that abnormal expression of depolarizing/repolarizing factors as P-gp and Kir could favor persistent depolarization of membranes without any rapid functional recovery capacity. This condition induced by convulsive stress could be the molecular mechanism leading to acquired severe bradycardia, as an ineffective heart response generating the appropriate scenario for SUDEP development. This article is part of the Special Issue "NEWroscience 2018".

Acquisition of stem associated-features on metastatic osteosarcoma cells and their functional effects on mesenchymal stem cells.

BACKGROUND: Osteosarcoma (OS) is the most frequent malignant bone tumor, affecting predominantly children and young adults. Metastases are a major clinical challenge in OS. In this context, 20% of OS patients are diagnosed with metastatic OS, but near 80% of all OS patients could present non-detectable micrometastases at the moment of diagnosis. METHODS: Osteogenic differentiation; doxorubicin exclusion assay; fluorescence microscopy; RT-qPCR; proteomic analysis. RESULTS: Our results suggest that metastatic OS cells possess a diminished osteoblastic differentiation potential with a gain of metastatic traits like the capacity to modify intracellular localization of chemodrugs and higher levels of expression of stemness-related genes. On the opposite hand, non-metastatic OS cells possess bone-associated traits like higher osteoblastic differentiation and also an osteoblastic-inducer secretome. OS cells also differ in the nature of their interaction with mesenchymal stem cells (MSCs), with opposites impacts on MSCs phenotype and behavior. CONCLUSIONS: All this suggests that a major trait acquired by metastatic cells is a switch into a stem-like state that could favor its survival in the pulmonary niche, opening new possibilities for personalized chemotherapeutic schemes. GENERAL SIGNIFICANCE: Our work provides new insights regarding differences among metastatic and non-metastatic OS cells, with particular emphasis on differentiation potential, multidrug resistance and interaction with MSCs.

Bayesian approach to model CD137 signaling in human M. tuberculosis in vitro responses.

Immune responses are qualitatively and quantitatively influenced by a complex network of receptor-ligand interactions. Among them, the CD137:CD137L pathway is known to modulate innate and adaptive human responses against Mycobacterium tuberculosis. However, the underlying mechanisms of this regulation remain unclear. In this work, we developed a Bayesian Computational Model (BCM) of in vitro CD137 signaling, devised to fit previously gathered experimental data. The BCM is fed with the data and the prior distribution of the model parameters and it returns their posterior distribution and the model evidence, which allows comparing alternative signaling mechanisms. The BCM uses a coupled system of non-linear differential equations to describe the dynamics of Antigen Presenting Cells, Natural Killer and T Cells together with the interpheron (IFN)-γ and tumor necrosis factor (TNF)-α levels in the media culture. Fast and complete mixing of the media is assumed. The prior distribution of the parameters that describe the dynamics of the immunological response was obtained from the literature and theoretical considerations Our BCM applies successively the Levenberg-Marquardt algorithm to find the maximum a posteriori likelihood (MAP); the Metropolis Markov Chain Monte Carlo method to approximate the posterior distribution of the parameters and Thermodynamic Integration to calculate the evidence of alternative hypothesis. Bayes factors provided decisive evidence favoring direct CD137 signaling on T cells. Moreover, the posterior distribution of the parameters that describe the CD137 signaling showed that the regulation of IFN-γ levels is based more on T cells survival than on direct induction. Furthermore, the mechanisms that account for the effect of CD137 signaling on TNF-α production were based on a decrease of TNF-α production by APC and, perhaps, on the increase in APC apoptosis. BCM proved to be a useful tool to gain insight on the mechanisms of CD137 signaling during human response against Mycobacterium tuberculosis.