RESUMO
Depleting glutathione by xCT inhibition induces iron-dependent ferroptotic cell death, which is suppressed by lipophilic antioxidants. We screened food extracts with xCTKO-MEFs, identifying garlic extracts as particularly potent in inhibiting ferroptosis among the food extracts examined in this study. xCTKO-MEFs can serve as a convenient tool for identifying food extracts that are effective in inhibiting ferroptosis.
Assuntos
Sistema y+ de Transporte de Aminoácidos , Ferroptose , Fibroblastos , Camundongos Knockout , Extratos Vegetais , Ferroptose/efeitos dos fármacos , Animais , Camundongos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Sistema y+ de Transporte de Aminoácidos/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/antagonistas & inibidores , Extratos Vegetais/farmacologia , Glutationa/metabolismo , Alho/química , Ferro/metabolismo , Antioxidantes/farmacologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacosRESUMO
xCT (Slc7a11), the specific subunit of the cystine/glutamate antiporter system xc-, is present in the brain and on immune cells, where it is known to modulate behavior and inflammatory responses. In a variety of cancers -including pancreatic ductal adenocarcinoma (PDAC)-, xCT is upregulated by tumor cells to support their growth and spread. Therefore, we studied the impact of xCT deletion in pancreatic tumor cells (Panc02) and/or the host (xCT-/- mice) on tumor burden, inflammation, cachexia and mood disturbances. Deletion of xCT in the tumor strongly reduced tumor growth. Targeting xCT in the host and not the tumor resulted only in a partial reduction of tumor burden, while it did attenuate tumor-related systemic inflammation and prevented an increase in immunosuppressive regulatory T cells. The latter effect could be replicated by specific xCT deletion in immune cells. xCT deletion in the host or the tumor differentially modulated neuroinflammation. When mice were grafted with xCT-deleted tumor cells, hypothalamic inflammation was reduced and, accordingly, food intake improved. Tumor bearing xCT-/- mice showed a trend of reduced hippocampal neuroinflammation with less anxiety- and depressive-like behavior. Taken together, targeting xCT may have beneficial effects on pancreatic cancer-related comorbidities, beyond reducing tumor burden. The search for novel and specific xCT inhibitors is warranted as they may represent a holistic therapy in pancreatic cancer.
Assuntos
Doenças Neuroinflamatórias , Neoplasias Pancreáticas , Camundongos , Animais , Encéfalo , Inflamação , HipocampoRESUMO
Radiation therapy used in the treatment of cancer causes skin damage, and no method of care has been established thus far. Recently, it has become clear that sericin derived from silkworm cocoons has moisturizing and antioxidant functions. In addition, green cocoon-derived sericin, which is rich in flavonoids, may have enhanced functions. However, whether this green cocoon-derived sericin can reduce radiotherapy-induced skin damage is unclear. In the present study, we aimed at establishing care methods to reduce skin cell damage caused by X-irradiation using green cocoon-derived sericin. We investigated its effect on human keratinocytes using lactate dehydrogenase activity to indicate damage reduction. Our results showed that green cocoon-derived sericin reduced cell damage caused by X-irradiation. However, this effect was not observed when cells were treated before X-irradiation or with a sericin derived from white cocoons. In addition, green cocoon-derived sericin decreased the levels of reactive oxygen species and lipid peroxidation. Our results suggest that green cocoon sericin mitigates the damaging effect of X-irradiation on cells, hence presenting potential usefulness in reducing skin damage from radiation therapy and opening new avenues in the care of cancer patients.
Assuntos
Bombyx , Sericinas , Animais , Humanos , Sericinas/farmacologia , Queratinócitos , Pele , Antioxidantes , SedaRESUMO
The excessive inflammatory response of macrophages plays a vital role in the pathogenesis of various diseases. The dynamic metabolic alterations in macrophages, including amino acid metabolism, are known to orchestrate their inflammatory phenotype. To explore a new metabolic pathway that regulates the inflammatory response, we examined metabolome changes in mouse peritoneal macrophages (PMs) in response to lipopolysaccharide (LPS) and found a coordinated increase of cysteine and its related metabolites, suggesting an enhanced demand for cysteine during the inflammatory response. Because Slc7a11, which encodes a cystine transporter xCT, was remarkably upregulated upon the pro-inflammatory challenge and found to serve as a major channel of cysteine supply, we examined the inflammatory behavior of Slc7a11 knockout PMs (xCT-KO PMs) to clarify an impact of the increased cysteine demand on inflammation. The xCT-KO PMs exhibited a prolonged upregulation of pro-inflammatory genes, which was recapitulated by cystine depletion in the culture media of wild-type PMs, suggesting that cysteine facilitates the resolution of inflammation. Detailed analysis of the sulfur metabolome revealed that supersulfides, such as cysteine persulfide, were increased in PMs in response to LPS, which was abolished in xCT-KO PMs. Supplementation of N-acetylcysteine tetrasulfide (NAC-S2), a supersulfide donor, attenuated the pro-inflammatory gene expression in xCT-KO PMs. Thus, activated macrophages increase cystine uptake via xCT and produce supersulfides, creating a negative feedback loop to limit excessive inflammation. Our study highlights the finely tuned regulation of macrophage inflammatory response by sulfur metabolism.
Assuntos
Cistina , Lipopolissacarídeos , Camundongos , Animais , Retroalimentação , Macrófagos/metabolismo , Acetilcisteína , Enxofre/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismoRESUMO
Glutathione (GSH) is synthesized from three amino acids and the overall process is highly dependent on the availability of l-cysteine (l-Cys). GSH serves as an essential cofactor for glutathione peroxidase 4 (Gpx4), which reduces phospholipid hydroperoxides. The inactivation of Gpx4 or an insufficient supply of l-Cys results in the accumulation of lipid hydroperoxides, eventually leading to iron-dependent cell death, ferroptosis. In this study, we investigated the anti-ferroptotic properties of d-cysteine (d-Cys) under conditions of dysfunction in cystine transporter, xCT. l-Cys supplementation completely rescued ferroptosis that had been induced by the erastin-mediated inhibition of xCT in Hepa 1-6 cells. Upon d-Cys supplementation, the erastin-treated cells remained completely viable for periods of up to 24â h but eventually died after 48â h. d-Cys supplementation suppressed the production of lipid peroxides, thereby ferroptosis. The addition of d-Cys sustained intracellular Cys and GSH levels to a certain extent. When Hepa 1-6 cells were treated with a combination of buthionine sulfoximine and erastin, the anti-ferroptotic effect of d-Cys was diminished. These collective results indicate that, although d-Cys is not the direct source of GSH, d-Cys supplementation protects cells from ferroptosis in a manner that is dependent on GSH synthesis via stimulating the uptake of l-Cys.
RESUMO
The cystine-glutamate transporter (xCT) is responsible for the transport of cystine into cells. We recently found that xCT-deficient (xCTKO) aged mice maintained a higher rate of ovulation and ovarian weight compared with wild-type (WT) mice. It has been reported that a xCT deficiency in cultured cells induces autophagy through the suppression of mTOR survival pathways. We have previously reported that starvation in neonatal mice increases the number of primordial follicles with concomitant autophagy activation. Therefore, we investigated age-related changes in follicle reserve and fertility in xCTKO mice and clarified whether the PI3K/AKT/mTOR signaling pathway contributes to this. The numbers of offspring in the xCTKO mice aged 10 and 12 months were significantly higher than those in the WT mice. The primordial follicle numbers in xCTKO neonatal mice tended to be higher than WT mice during all times evaluated. In contrast, the primary follicle number was significantly lower in the xCTKO mice at 60 h after birth. The expression of p-AKT, which promotes follicle development, was significantly lower in xCTKO mice than that in WT mice, whereas the expression ratios of LC3-II/LC3-I were significantly higher. The xCTKO mice had significantly more primordial follicles than WT mice at 2 months of age and showed a similar trend at 13-15 months of age. These results suggest that the maintenance of fertility in aged xCTKO mice can be attributed to high follicle reserve after puberty by suppression of follicle activation during the neonatal period.
Assuntos
Sistema y+ de Transporte de Aminoácidos , Fertilidade , Reserva Ovariana , Fosfatidilinositol 3-Quinases , Sistema y+ de Transporte de Aminoácidos/genética , Animais , Feminino , Fertilidade/genética , Camundongos , Folículo Ovariano/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Maturidade SexualRESUMO
The cystine/glutamate antiporter system xc- has been identified as the major source of extracellular glutamate in several brain regions as well as a modulator of neuroinflammation, and genetic deletion of its specific subunit xCT (xCT-/-) is protective in mouse models for age-related neurological disorders. However, the previously observed oxidative shift in the plasma cystine/cysteine ratio of adult xCT-/- mice led to the hypothesis that system xc- deletion would negatively affect life- and healthspan. Still, till now the role of system xc- in physiological aging remains unexplored. We therefore studied the effect of xCT deletion on the aging process of mice, with a particular focus on the immune system, hippocampal function, and cognitive aging. We observed that male xCT-/- mice have an extended lifespan, despite an even more increased plasma cystine/cysteine ratio in aged compared to adult mice. This oxidative shift does not negatively impact the general health status of the mice. On the contrary, the age-related priming of the innate immune system, that manifested as increased LPS-induced cytokine levels and hypothermia in xCT+/+ mice, was attenuated in xCT-/- mice. While this was associated with only a very moderate shift towards a more anti-inflammatory state of the aged hippocampus, we observed changes in the hippocampal metabolome that were associated with a preserved hippocampal function and the retention of hippocampus-dependent memory in male aged xCT-/- mice. Targeting system xc- is thus not only a promising strategy to prevent cognitive decline, but also to promote healthy aging.
Assuntos
Sistema y+ de Transporte de Aminoácidos , Cistina , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Cisteína , Cistina/metabolismo , Ácido Glutâmico , Hipocampo/metabolismo , Longevidade , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Sorafenib, a protein kinase inhibitor approved for the treatment of hepatocellular carcinoma and advanced renal cell carcinoma, has been repeatedly reported to induce ferroptosis by possibly involving inhibition of the cystine/glutamate antiporter, known as system xc-. Using a combination of well-defined genetically engineered tumor cell lines and canonical small molecule ferroptosis inhibitors, we now provide unequivocal evidence that sorafenib does not induce ferroptosis in a series of tumor cell lines unlike the cognate system xc- inhibitors sulfasalazine and erastin. We further show that only a subset of tumor cells dies by ferroptosis upon sulfasalazine and erastin treatment, implying that certain cell lines appear to be resistant to system xc- inhibition, while others undergo ferroptosis-independent cell death. From these findings, we conclude that sorafenib does not qualify as a bona fide ferroptosis inducer and that ferroptosis induced by system xc- inhibitors can only be achieved in a fraction of tumor cell lines despite robust expression of SLC7A11, the substrate-specific subunit of system xc-.
Assuntos
Antineoplásicos/farmacologia , Ferroptose/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Sorafenibe/farmacologia , Sistema y+ de Transporte de Aminoácidos/antagonistas & inibidores , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Células HEK293 , Humanos , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Piperazinas/farmacologia , Sulfassalazina/farmacologiaRESUMO
The knockout (KO) of the cystine transporter xCT causes ferroptosis, a type of iron-dependent necrotic cell death, in mouse embryonic fibroblasts, but this does not occur in macrophages. In this study, we explored the gene that supports cell survival under a xCT deficiency using a proteomics approach. Analysis of macrophage-derived peptides that were tagged with iTRAQ by liquid chromatography-mass spectrometry revealed a robust elevation in the levels of carnosine dipeptidase II (CNDP2) in xCT KO macrophages. The elevation in the CNDP2 protein levels was confirmed by immunoblot analyses and this elevation was accompanied by an increase in hydrolytic activity towards cysteinylglycine, the intermediate degradation product of glutathione after the removal of the γ-glutamyl group, in xCT KO macrophages. Supplementation of the cystine-free media of Hepa1-6 cells with glutathione or cysteinylglycine extended their survival, whereas the inclusion of bestatin, an inhibitor of CNDP2, counteracted the effects of these compounds. We established CNDP2 KO mice by means of the CRISPR/Cas9 system and found a decrease in dipeptidase activity in the liver, kidney, and brain. An acetaminophen overdose (350 mg/kg) showed not only aggravated hepatic damage but also renal injury in the CNDP2 KO mice, which was not evident in the wild-type mice that were receiving the same dose. The aggravated renal damage in the CNDP2 KO mice was consistent with the presence of abundant levels of CNDP2 in the kidney, the organ prone to developing ferroptosis. These collective data imply that cytosolic CNDP2, in conjugation with the removal of the γ-glutamyl group, recruits Cys from extracellular GSH and supports redox homeostasis of cells, particularly in epithelial cells of proximal tubules that are continuously exposed to oxidative insult from metabolic wastes that are produced in the body.
Assuntos
Carnosina , Dipeptidases , Animais , Cisteína , Dipeptidases/genética , Fibroblastos , Glutationa , CamundongosRESUMO
Despite ample evidence for the therapeutic potential of inhibition of the cystine/glutamate antiporter system xc - in neurological disorders and in cancer, none of the proposed inhibitors is selective. In this context, a lot of research has been performed using the EMA- and FDA-approved drug sulfasalazine (SAS). Even though this molecule is already on the market for decades as an anti-inflammatory drug, serious side effects due to its use have been reported. Whereas for the treatment of the main indications, SAS needs to be cleaved in the intestine into the anti-inflammatory compound mesalazine, it needs to reach the systemic circulation in its intact form to allow inhibition of system xc -. The higher plasma levels of intact SAS (or its metabolites) might induce adverse effects, independent of its action on system xc -. Some of these effects have however been attributed to system xc - inhibition, calling into question the safety of targeting system xc -. In this study we chronically treated system xc - - deficient mice and their wildtype littermates with two different doses of SAS (160 mg/kg twice daily or 320 mg/kg once daily, i.p.) and studied some of the adverse effects that were previously reported. SAS had a negative impact on the survival rate, the body weight, the thermoregulation and/or stress reaction of mice of both genotypes, and thus independent of its inhibitory action on system xc -. While SAS decreased the total distance travelled in the open-field test the first time the mice encountered the test, it did not influence this parameter on the long-term and it did not induce other behavioral changes such as anxiety- or depressive-like behavior. Finally, no major histological abnormalities were observed in the spinal cord. To conclude, we were unable to identify any undesirable system xc --dependent effect of chronic administration of SAS.
RESUMO
Ferroptosis is a type of iron-dependent, non-apoptotic cell death, which is typically induced by cysteine starvation or by the inhibition of glutathione peroxidase 4 (GPX4) activity with the accompanying elevation in lipid peroxidation product levels. Despite the central role of mitochondria in oxidative metabolism and hence, as main sources of superoxide, the issue of whether mitochondrial superoxide participates in the execution of ferroptosis remains unclear. To gain additional insights into this issue, we employed suppressors of the site IQ electron leak (S1QEL) and suppressors of the site IIIQo electron leak (S3QEL), small molecules that suppress mitochondrial superoxide production from complex I and III, respectively. The findings indicate that S3QEL, but not S1QEL, significantly protected mouse hepatoma Hepa 1-6 cells from lipid peroxidation and the subsequent ferroptosis induced by cysteine (Cys) starvation (cystine deprivation from culture media or xCT inhibition by erastin). The intracellular levels of Cys and GSH remained low irrespective of life or death. Moreover, S3QEL also suppressed ferroptosis in xCT-knockout mouse-derived embryonic fibroblasts, which usually die under conventional cultivating conditions due to the absence of intracellular Cys and GSH. Although it has been reported that erastin induces the hyperpolarization of the mitochondrial membrane potential, no correlation was observed between hyperpolarization and cell death in xCT-knockout cells. Collectively, these results indicate that superoxide production from complex III plays a pivotal role in the ferroptosis that is induced by Cys starvation, suggesting that protecting mitochondria is a promising therapeutic strategy for the treatment of multiple diseases featuring ferroptosis.
Assuntos
Cisteína/deficiência , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Ferroptose , Potencial da Membrana Mitocondrial , Membranas Mitocondriais/metabolismo , Superóxidos/metabolismo , Animais , Células HeLa , Humanos , CamundongosRESUMO
The astrocytic cystine/glutamate antiporter system x c - (with xCT as the specific subunit) imports cystine in exchange for glutamate and has been shown to interact with multiple pathways in the brain that are dysregulated in age-related neurological disorders, including glutamate homeostasis, redox balance, and neuroinflammation. In the current study, we investigated the effect of genetic xCT deletion on lactacystin (LAC)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced degeneration of the nigrostriatal pathway, as models for Parkinson's disease (PD). Dopaminergic neurons of adult xCT knock-out mice (xCT-/-) demonstrated an equal susceptibility to intranigral injection of the proteasome inhibitor LAC, as their wild-type (xCT+/+) littermates. Contrary to adult mice, aged xCT-/- mice showed a significant decrease in LAC-induced degeneration of nigral dopaminergic neurons, depletion of striatal dopamine (DA) and neuroinflammatory reaction, compared to age-matched xCT+/+ littermates. Given this age-related protection, we further investigated the sensitivity of aged xCT-/- mice to chronic and progressive MPTP treatment. However, in accordance with our previous observations in adult mice (Bentea et al., 2015a), xCT deletion did not confer protection against MPTP-induced nigrostriatal degeneration in aged mice. We observed an increased loss of nigral dopaminergic neurons, but equal striatal DA denervation, in MPTP-treated aged xCT-/- mice when compared to age-matched xCT+/+ littermates. To conclude, we reveal age-related protection against proteasome inhibition-induced nigrostriatal degeneration in xCT-/- mice, while xCT deletion failed to protect nigral dopaminergic neurons of aged mice against MPTP-induced toxicity. Our findings thereby provide new insights into the role of system x c - in mechanisms of dopaminergic cell loss and its interaction with aging.
RESUMO
The astrocytic cystine/glutamate antiporter system xc- represents an important source of extracellular glutamate in the central nervous system, with potential impact on excitatory neurotransmission. Yet, its function and importance in brain physiology remain incompletely understood. Employing slice electrophysiology and mice with a genetic deletion of the specific subunit of system xc-, xCT (xCT-/- mice), we uncovered decreased neurotransmission at corticostriatal synapses. This effect was partly mitigated by replenishing extracellular glutamate levels, indicating a defect linked with decreased extracellular glutamate availability. We observed no changes in the morphology of striatal medium spiny neurons, the density of dendritic spines, or the density or ultrastructure of corticostriatal synapses, indicating that the observed functional defects are not due to morphological or structural abnormalities. By combining electron microscopy with glutamate immunogold labeling, we identified decreased intracellular glutamate density in presynaptic terminals, presynaptic mitochondria, and in dendritic spines of xCT-/- mice. A proteomic and kinomic screen of the striatum of xCT-/- mice revealed decreased expression of presynaptic proteins and abnormal kinase network signaling, that may contribute to the observed changes in postsynaptic responses. Finally, these corticostriatal deregulations resulted in a behavioral phenotype suggestive of autism spectrum disorder in the xCT-/- mice; in tests sensitive to corticostriatal functioning we recorded increased repetitive digging behavior and decreased sociability. To conclude, our findings show that system xc- plays a previously unrecognized role in regulating corticostriatal neurotransmission and influences social preference and repetitive behavior.
Assuntos
Transtorno do Espectro Autista , Ácido Glutâmico , Animais , Antiporters , Transtorno do Espectro Autista/genética , Cistina , Camundongos , Proteômica , Interação SocialRESUMO
The cystine/glutamate antiporter, system xc- , is essential for the efficient uptake of cystine into cells. Interest in the mechanisms of system xc- function soared with the recognition that system xc- presents the most upstream node of ferroptosis, a recently described form of regulated necrosis relevant for degenerative diseases and cancer. Since targeting system xc- hold the great potential to efficiently combat tumor growth and metastasis of certain tumors, we disrupted the substrate-specific subunit of system xc- , xCT (SLC7A11) in the highly metastatic mouse B16F10 melanoma cell line and assessed the impact on tumor growth and metastasis. Subcutaneous injection of tumor cells into the syngeneic B16F10 mouse melanoma model uncovered a marked decrease in the tumor-forming ability and growth of KO cells compared to control cell lines. Strikingly, the metastatic potential of KO cells was markedly reduced as shown in several in vivo models of experimental and spontaneous metastasis. Accordingly, survival rates of KO tumor-bearing mice were significantly prolonged in contrast to those transplanted with control cells. Analyzing the in vitro ability of KO and control B16F10 cells in terms of endothelial cell adhesion and spheroid formation revealed that xCT expression indeed plays an important role during metastasis. Hence, system xc- emerges to be essential for tumor metastasis in mice, thus qualifying as a highly attractive anticancer drug target, particularly in light of its dispensable role for normal life in mice.
Assuntos
Sistema y+ de Transporte de Aminoácidos/genética , Técnicas de Inativação de Genes/métodos , Melanoma/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Taxa de SobrevidaRESUMO
Ferroptosis is characterized by an iron-dependent cell death with increased lipid peroxidation and is typically induced by either a decrease in glutathione (GSH) levels due to an insufficient supply of cysteine (Cys) or the inhibition of phospholipid hydroperoxide glutathione peroxidase (Gpx4). While lipid peroxides are the direct trigger for ferroptosis, the issue of how radical species involve in the cytocidal process remains unclear. To gain insights into this issue, we employed edaravone, a free radical scavenger that is clinically approved for the treatment of acute ischemic strokes and amyotrophic lateral sclerosis (ALS), against ferroptotic cell death caused by various situations, notably under cystine deprivation. We initially investigated the effects of edaravone on ferroptosis in mouse hepatoma Hepa 1-6â¯cells cultivated in cystine-free medium and found that edaravone largely suppressed ferroptosis. Ferroptosis that was induced in the cells by the use of inhibitors for xCT or Gpx4 was also suppressed by edaravone. Moreover, edaravone also suppressed ferroptosis in xCT-knockout mouse-derived embryonic fibroblasts, which usually die in normal cultivating conditions due to the depletion of intracellular Cys and GSH. Although the edaravone treatment had no effects on the intracellular levels of Cys and GSH, both of which remained low in Hepa 1-6â¯cells under conditions of cystine deprivation, the causative factors for ferroptosis, including ferrous iron and lipid peroxide levels, were significantly suppressed. Collectively, these results indicate that radical species produced at the initial stage of the cytocidal process under Cys-deprived conditions trigger ferroptosis and scavenging these radicals by edaravone represents a promising treatment.
Assuntos
Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Substâncias Protetoras/farmacologia , Animais , Linhagem Celular Tumoral , Cisteína/metabolismo , Edaravone/farmacologia , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Ferro/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismoRESUMO
OBJECTIVE: The cystine/glutamate antiporter system xc- could represent a new target for antiepileptogenic treatments due to its crucial roles in glutamate homeostasis and neuroinflammation. To demonstrate this, we compared epilepsy development and seizure susceptibility in xCT knockout mice (xCT-/- ) and in littermate controls (xCT+/+ ) in different chronic models of epilepsy. METHODS: Mice were surgically implanted with electrodes in the basolateral amygdala and chronically stimulated to develop self-sustained status epilepticus (SSSE); continuous video-electroencephalography monitoring was performed for 28 days after SE and hippocampal histopathology was assessed. Corneal kindling was induced by twice daily electrical stimulation at 6 Hz and maintenance of the fully kindled state was evaluated. Next, messenger RNA (mRNA) and protein levels of xCT and of the proteins involved in the phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase 3ß (GSK-3ß)/eukaryotic initiation factor 2α (eIF2α)/activating transcription factor 4 (ATF4) signaling pathway were measured at different time points during epileptogenesis in NMRI mice treated with pilocarpine. Finally, the anticonvulsant effect of sulfasalazine (SAS), a nonselective system xc- inhibitor, was assessed against 6 Hz-evoked seizures in pilocarpine-treated mice. RESULTS: In the SSSE model, xCT-/- mice displayed a significant delayed epileptogenesis, a reduced number of spontaneous recurrent seizures, and less pronounced astrocytic and microglial activation. Moreover, xCT-/- mice showed reduced seizure severity during 6 Hz kindling development and a lower incidence of generalized seizures during the maintenance of the fully kindled state. In pilocarpine-treated mice, protein levels of the PI3K/Akt/GSK-3ß/eIF2α/ATF4 pathway were increased during the chronic phase of the model, consistent with previous findings in the hippocampus of patients with epilepsy. Finally, repeated administration of SAS protected pilocarpine-treated mice against acute 6 Hz seizure induction, in contrast to sham controls, in which system xc- is not activated. SIGNIFICANCE: Inhibition of system xc- could be an attractive target for the development of new therapies with a potential for disease modification in epilepsy.
Assuntos
Sistema y+ de Transporte de Aminoácidos/efeitos dos fármacos , Anticonvulsivantes/farmacologia , Epilepsia/tratamento farmacológico , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Anticonvulsivantes/uso terapêutico , Modelos Animais de Doenças , Eletroencefalografia , Epilepsia/etiologia , Epilepsia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pilocarpina/farmacologia , Estado Epiléptico/tratamento farmacológico , Estado Epiléptico/etiologia , Estado Epiléptico/metabolismoRESUMO
Post-septic neurological and psychiatric illness (PSNPI) including dementia and depression may be observed after sepsis. However, the etiology of PSNPI and therapeutic treatment of PSNPI are unclear. We show that glutamate produced from microglia through the activity of system xc- plays a role in PSNPI. We established a mouse model of PSNPI by lipopolysaccharide (LPS) treatment that shows a disturbance of short/working memory and depression-like hypoactivity. Glutamate receptor antagonists (MK801 and DNQX) reduced these phenotypes, and isolated microglia from LPS-treated mice released abundant glutamate. We identified system xc- as a source of the extracellular glutamate. xCT, a component of system xc-, was induced and expressed in microglia after LPS treatment. In xCT knockout mice, PSNPI were decreased compared to those in wildtype mice. Moreover, TNF-α and IL-1ß expression in wildtype mice was increased after LPS treatment, but inhibited in xCT knockout mice. Thus, system xc- in microglia may be a therapeutic target for PSNPI. The administration of sulfasalazine, an inhibitor of xCT, in symptomatic and post-symptomatic mice improved PSNPI. Our results suggest that glutamate released from microglia through system xc- plays a critical role in the manifestations of PSNPI and that system xc- may be a therapeutic target for PSNPI.
Assuntos
Sistema y+ de Transporte de Aminoácidos/genética , Ácido Glutâmico/metabolismo , Transtornos Mentais/etiologia , Microglia/metabolismo , Doenças do Sistema Nervoso/etiologia , Sistema y+ de Transporte de Aminoácidos/antagonistas & inibidores , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Modelos Animais de Doenças , Maleato de Dizocilpina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Interleucina-1beta/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , Transtornos Mentais/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças do Sistema Nervoso/tratamento farmacológico , Quinoxalinas/farmacologia , Sepse/psicologia , Sulfassalazina/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The contribution of glial transporters to glutamate movement across the membrane has been identified as a potential target for anti-seizure therapies. Two such glutamate transporters, GLT-1 and system xc-, are expressed on glial cells, and modulation of their expression and function have been identified as a means by which seizures, neuronal injury, and gliosis can be reduced in models of brain injury. While GLT-1 is responsible for the majority of glutamate uptake in the brain, system xc- releases glutamate in the extracellular cleft in exchange for cystine and represents as such the major source of hippocampal extracellular glutamate. Using the Theiler's Murine Encephalomyelitis Virus (TMEV) model of viral-induced epilepsy, we have taken two well-studied approaches, one pharmacological, one genetic, to investigate the potential role(s) of GLT-1 and system xc- in TMEV-induced pathology. Our findings suggest that the methods we utilized to modulate these glial transporters, while effective in other models, are not sufficient to reduce the number or severity of behavioral seizures in TMEV-infected mice. However, genetic knockout of xCT, the specific subunit of system xc-, may have cellular effects, as we observed a slight decrease in neuronal injury caused by TMEV and an increase in astrogliosis in the CA1 region of the hippocampus. Furthermore, xCT knockout caused an increase in GLT-1 expression selectively in the cortex. These findings have significant implications for both the characterization of the TMEV model as well as for future efforts to discover novel and effective anti-seizure drugs.
Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Convulsões/metabolismo , Animais , Encéfalo/patologia , Infecções por Cardiovirus/complicações , Infecções por Cardiovirus/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Convulsões/patologia , Convulsões/virologia , TheilovirusRESUMO
Objectives: The cystine/glutamate antiporter (system xc-) is believed to contribute to nonvesicular glutamate release from glial cells in various brain areas. Although recent investigations implicate system xc- in mood disorders, unambiguous evidence has not yet been established. Therefore, we evaluated the possible role of system xc- in the depressive state. Methods: We conducted a protein expression analysis of the specific subunit of system xc- (xCT) in brain regions of the corticosterone mouse model, Flinders Sensitive Line rat model and post-mortem tissue of depressed patients. We next subjected system xc- deficient mice to the corticosterone model and analysed their behaviour in several tests. Lastly, we subjected additional cohorts of xCT-deficient and wild-type mice to N-acetylcysteine treatment to unveil whether the previously reported antidepressant-like effects are dependent upon system xc-. Results: We did not detect any changes in xCT expression levels in the animal models or patients compared to proper controls. Furthermore, loss of system xc- had no effect on depression- and anxiety-like behaviour. Finally, the antidepressant-like effects of N-acetylcysteine are not mediated via system xc-. Conclusions: xCT protein expression is not altered in the depressed brain and system xc- deficiency does not affect depression-associated behaviour in the corticosterone mouse model.
Assuntos
Sistema y+ de Transporte de Aminoácidos/deficiência , Encéfalo/metabolismo , Depressão/genética , Depressão/fisiopatologia , Idoso de 80 Anos ou mais , Sistema y+ de Transporte de Aminoácidos/genética , Animais , Anti-Inflamatórios , Encéfalo/patologia , Corticosterona , Modelos Animais de Doenças , Comportamento Exploratório , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora , RatosRESUMO
Macrophages manifest distinct phenotype according to the organs in which they reside. In addition, they flexibly switch their character in adaptation to the changing environment. However, the molecular basis that explains the conversion of the macrophage phenotype has so far been unexplored. We find that CD169+ macrophages change their phenotype by regulating the level of a transcription factor Maf both in vitro and in vivo in C57BL/6J mice. When CD169+ macrophages were exposed to bacterial components, they expressed an array of acute inflammatory response genes in Maf-dependent manner and simultaneously start to downregulate Maf. This Maf suppression is dependent on accelerated degradation through proteasome pathway and microRNA-mediated silencing. The downregulation of Maf unlocks the NF-E2-related factor 2-dominant, cytoprotective/antioxidative program in the same macrophages. The present study provides new insights into the previously unanswered question of how macrophages initiate proinflammatory responses while retaining their capacity to repair injured tissues during inflammation.