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Abstract Glioblastoma is a Grade 4 primary brain tumor defined by therapy resistance, diffuse infiltration, and near-uniform lethality. The underlying mechanisms are unknown, and no treatment has been curative. Using a recently developed creatine kinase inhibitor (CKi), we explored the role of this inhibitor on GBM biology in vitro. While CKi minimally impacted GBM cell proliferation and viability, it significantly affected migration. In established GBM cell lines and patient-derived xenografts, CKi ablated both the migration and invasion of GBM cells. CKi also hindered radiation-induced migration. RNA-seq revealed a decrease in invasion-related genes, with an unexpected increase in glutathione metabolism and ferroptosis protection genes post-CKi treatment. The effects of CKi could be reversed by the addition of cell-permeable glutathione. Carbon-13 metabolite tracing indicated heightened glutathione biosynthesis post-CKi treatment. Combinatorial CKi blockade and glutathione inhibition or ferroptosis activation abrogated cell survival. Our data demonstrated that CKi perturbs promigratory and anti-ferroptotic roles in GBM, identifying the creatine kinase axis as a druggable target for GBM treatment.

It was explored the cytoprotective and antioxidant effect of MLN4924, a specific inhibitor of Nedd8-activating enzyme (NAE), against hydrogen peroxide (H2O2)-induced damage in cerebellar granule neurons (CGNs). Primary cultures of CGNs were exposed to H2O2 after preincubation with MLN4924. The compounds were removed, and CGNs were incubated in culture medium for 24 h in order to determine cell viability by 3-[4,5-dimethylthiazol-2-yl)]-2,5-diphenyl-tetrazolium bromide (MTT) and fluorescein diacetate (FDA) assays. It was demonstrated that MLN4924 remarkably attenuated H2O2-induced cell damage. Meanwhile reactive oxygen species (ROS) production was evaluated with the fluorescent probe dihydroethidium (DHE). Interestingly H2O2-induced ROS production was inhibited by pretreatment with MLN4924. MLN4924 treatment in CGNs resulted in nuclear factor E2-related factor 2 (Nrf2) protein accumulation. Intriguingly this effect was observed in the cytosolic and nuclear compartments of the CGNs. The cytoprotective effect of MLN4924 was associated with its ability to diminish ROS production induced by H2O2 and the accumulation of Nrf2 protein levels in the cytoplasm and nucleus of the CGNs.

Arsenic (As) is a metalloid naturally present in the environment, in food, water, soil, and air; however, its chronic exposure, even with low doses, represents a public health concern. For a long time, As was used as a pigment, pesticide, wood preservative, and for medical applications; its industrial use has recently decreased or has been discontinued due to its toxicity. Due to its versatile applications and distribution, there is a wide spectrum of human As exposure sources, mainly contaminated drinking water. The fact that As is present in drinking water implies chronic human exposure to this metalloid; it has become a worldwide health problem, since over 200 million people live where As levels exceed safe ranges. Many health problems have been associated with As chronic exposure including cancer, cardiovascular diseases, gastrointestinal disturbances, and brain dysfunctions. Because As can cross the blood–brain barrier (BBB), the brain represents a target organ where this metalloid can exert its long-term toxic effects. Many mechanisms of As neurotoxicity have been described: oxidative stress, inflammation, DNA damage, and mitochondrial dysfunction; all of them can converge, thus leading to impaired cellular functions, cell death, and in consequence, long-term detrimental effects. Here, we provide a current overview of As toxicity and integrated the global mechanisms involved in cognitive and behavioral impairment induced by As exposure show experimental strategies against its neurotoxicity.

Objetivo: Determinar el efecto de los metabolitos producidos por la actividad de la kinurenina monooxigenasa en células de glioma sobre la proliferación de los linfocitos T efectores. Antecedentes: El glioblastoma (GBM) es el tumor astrocítico más frecuente y agresivo del sistema nervioso central debido a su heterogeneidad celular, metabolismo alterado, alta proliferación y evasión del sistema inmune. Recientemente se demostró que las células tumorales de glioma expresan kinurenina monooxigenasa (KMO), y no así los astrocitos. KMO es una enzima de la vía de las kinureninas que induce la formación de 3-hidroxikinurenina (3-HK), 3-hidroxiantranilato (3-HANA) y ácido quinolínico (QUIN); metabolitos que han sido descritos con propiedades redox, neuroactivas e inmunomoduladoras. Por lo que el propósito de este trabajo fue determinar el efecto de estos metabolitos en la proliferación de linfocitos T efectores activados. Metodología: Se realizó un implante intracraneal de células C6 en el cerebro de ratas para caracterizar la actividad de KMO y kinurenina aminotransferasa (KAT) en tumores de 10 y 20 días. Adicionalmente, linfocitos T activados con fitohematoglutinina (PHA) fueron incubados con concentraciones crecientes (0-250 µM) de 3-HK, 3-HANA y QUIN para determinar su efecto en la proliferación, ciclo celular e inducción de la apoptosis. Resultados: Al día 10 posterior al implante, los tumores mostraron una menor actividad de KAT, mientras que la actividad de KMO aumentó comparando con tejido cerebral sano; este efecto ya no se observó a los 20 días post-implante, lo que sugiere que la activación de KMO en etapas tempranas podría favorecer al establecimiento del tumor a través de la modulación de la respuesta inmune. En este sentido, los metabolitos derivados de la actividad de KMO: 3-HK, 3-HANA y QUIN, disminuyeron la proliferación de linfocitos T activados, y eso se relacionó con arresto del ciclo celular e inducción de muerte celular. Conclusión: La producción de metabolitos derivados de la expresión y actividad aberrante de KMO en células tumorales de glioma promueve un microambiente inmunosupresor mediante el arresto del ciclo celular e inducción de muerte celular en los linfocitos activados. [ABSTRACT FROM AUTHOR]

Objetivo: evaluar el mimetismo molecular entre antígenos de T, gondii y proteínas del neurodesarrollo y su impacto en la conducta. Antecedentes: el neurodesarrollo es un proceso complejo que se encuentra bajo una estricta regulación genética, ambiental e inmunológica; insultos o infecciones durante esta etapa pueden alterar de manera permanente el desarrollo normal del cerebro, dando lugar a la posterior aparición de desórdenes neurológicos y psiquiátricos. Estudios epidemiológicos reportan la presencia de altos niveles de IgG anti-T. gondii en neonatos que luego desarrollaron esquizofrenia. La ausencia física del parásito y los altos niveles de IgG anti-T.gondii en estos pacientes sugieren la transferencia transplacentaria de estos anticuerpos así como su posible unión a estructuras cerebrales capaces resultando en alteraciones durante el neurodesarrollo. Métodos: inmunización subcutánea en ratas hembras con el lisado de T. gondii (HITG, 100 g de proteína) o PBS 3 semanas previas a la gestación: Evaluación conductual en la progenie (60 días postnatales): actividad locomotora espontánea y prueba de interacción social. Identificación de la presencia de IgG en cortes histológicos de cerebros de fetos de madres inmunizadas HITG así como de la reactividad entre el suero de HITG y las estructuras fetales de la progenie de madres control (sin inmunizar) por marcaje inmunofluorescente. Análisis bioinformático comparativo entre las secuencias peptídicas altamente antigénicas de T. gondii y aquellas secuencias de proteínas del neurodesarrollo. Núm. de registro del protocolo: 90/17 Resultados: 1) La progenie de HITG mostró alteraciones conductuales tanto en la conducta exploratoria como en la capacidad de interaccionar con otros individuos. 2) Fueron identificados anticuerpos IgG en el cerebro de fetos de HITG; además, se observó que los anticuerpos presentes en el suero de HITG reconocen y se unen a estructuras cerebrales de fetos control de madres no inmunizadas 3) Se identificaron múltiples secuencias peptídicas compartidas entre los antígenos de T. gondii y proteínas del neurodesarrollo temprano (Musashi1, Otx2, Cd29, Ncam1, Brg1, Dcx, Nest y Pax6). Conclusiones: el análisis bioinformático y la reactividad inmunogénica observada en cortes cerebrales de fetos de rata confirmaron el mimetismo molecular existente entre proteínas antigénicas de T. gondii y proteínas del neurodesarrollo. Por lo tanto, los anticuerpos producidos en madres expuestas pre-gestacionalmente a T. gondii pueden reconocer estructuras cerebrales fetales como blancos antigénicos y dar lugar posteriormente a alteraciones conductuales en la progenie. [ABSTRACT FROM AUTHOR]

Kynurenine 3-monooxygenase (KMO), a key player in the kynurenine pathway (KP) of tryptophan degradation, regulates the synthesis of the neuroactive metabolites 3-hydroxykynurenine (3-HK) and kynurenic acid (KYNA). KMO activity has been implicated in several major brain diseases including Huntington’s disease (HD) and schizophrenia. In the brain, KMO is widely believed to be predominantly localized in microglial cells, but verification in vivo has not been provided so far. Here, we examined KP metabolism in the brain after depleting microglial cells pharmacologically with the colony stimulating factor 1 receptor inhibitor PLX5622. Young adult mice were fed PLX5622 for 21 days and were euthanized either on the next day or after receiving normal chow for an additional 21 days. Expression of microglial marker genes was dramatically reduced on day 22 but had fully recovered by day 43. In both groups, PLX5622 treatment failed to affect Kmo expression, KMO activity or tissue levels of 3-HK and KYNA in the brain. In a parallel experiment, PLX5622 treatment also did not reduce KMO activity, 3-HK and KYNA in the brain of R6/2 mice (a model of HD with activated microglia). Finally, using freshly isolated mouse cells ex vivo, we found KMO only in microglia and neurons but not in astrocytes. Taken together, these data unexpectedly revealed that neurons contain a large proportion of functional KMO in the adult mouse brain under both physiological and pathological conditions.

L-kynurenine (L-KYN) is an endogenous metabolite, that has been used as a neuroprotective strategy in experimental models. The protective effects of L-KYN have been attributed mainly to kynurenic acid (KYNA). However, considering that L-KYN is prone to oxidation, this redox property may play a substantial role in its protective effects. The aim of this work was to characterize the potential impact of the redox properties of L-KYN, in both synthetic and biological systems. First, we determined whether L-KYN scavenges reactive oxygen species (ROS) and prevents DNA and protein oxidative degradation in synthetic systems. The effect of L-KYN and KYNA (0.1–100 µM) on redox markers (ROS production, lipoperoxidation and cellular function) was compared in rat brain homogenates when exposed to FeSO4 (10 µM). Then, the effect of L-KYN administration (75 mg/kg/day for 5 days) on the GSH content and the enzymatic activity of glutathione reductase (GR) and glutathione peroxidase (GPx) was determined in rat brain tissue. Finally, brain homogenates from rats pretreated with L-KYN were exposed to pro-oxidants and oxidative markers were evaluated. The results show that L-KYN is an efficient scavenger of ●OH and ONOO−, but not O2●– or H2O2 and that it prevents DNA and protein oxidative degradation in synthetic systems. L-KYN diminishes the oxidative effect induced by FeSO4 on brain homogenates at lower concentrations (1 µM) when compared to KYNA (100 µM). Furthermore, the sub-chronic administration of L-KYN increased the GSH content and the activity of both GR and GPx, and also prevented the oxidative damage induced by the ex vivo exposure to pro-oxidants. Altogether, these findings strongly suggest that L-KYN can be considered as a potential endogenous antioxidant.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. The enzyme indoleamine-2,3-dioxygenase (IDO), which participates in the rate-limiting step of tryptophan catabolism through the kynurenine pathway (KP), is associated with poor prognosis in patients with GBM. The metabolites produced after tryptophan oxidation have immunomodulatory properties that can support the immunosuppressor environment. In this study, mRNA expression, protein expression, and activity of the enzyme kynurenine monooxygenase (KMO) were analyzed in GBM cell lines (A172, LN-18, U87, U373) and patient-derived astrocytoma samples. KMO mRNA expression was assessed by real-time RT-qPCR, KMO protein expression was evaluated by flow cytometry and immunofluorescence, and KMO activity was determined by quantifying 3-hydroxykynurenine by HPLC. Heterogenous patterns of both KMO expression and activity were observed among the GBM cell lines, with the A172 cell line showing the highest KMO expression and activity. Higher KMO mRNA expression was observed in glioma samples than in patients diagnosed with only a neurological disease; high KMO mRNA expression was also observed when using samples from patients with GBM in the TCGA program. The KMO protein expression was localized in GFAP+ cells in tumor tissue. These results suggest that KMO is a relevant target to be explored in glioma since it might play a role in supporting tumor metabolism and immune suppression.