D-Cysteine Activates Chaperone-Mediated Autophagy in Cerebellar Purkinje Cells via the Generation of Hydrogen Sulfide and Nrf2 Activation.

Chaperone-mediated autophagy (CMA) is a pathway in the autophagy-lysosome protein degradation system. CMA impairment has been implicated to play a role in spinocerebellar ataxia (SCA) pathogenesis. D-cysteine is metabolized by D-amino acid oxidase (DAO), leading to hydrogen sulfide generation in the cerebellum. Although D-cysteine alleviates the disease phenotypes in SCA-model mice, it remains unknown how hydrogen sulfide derived from D-cysteine exerts this effect. In the present study, we investigated the effects of D-cysteine and hydrogen sulfide on CMA activity using a CMA activity marker that we have established. D-cysteine activated CMA in Purkinje cells (PCs) of primary cerebellar cultures where DAO was expressed, while it failed to activate CMA in DAO-deficient AD293 cells. In contrast, Na2S, a hydrogen sulfide donor, activated CMA in both PCs and AD293 cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) is known to be activated by hydrogen sulfide and regulate CMA activity. An Nrf2 inhibitor, ML385, prevented CMA activation triggered by D-cysteine and Na2S. Additionally, long-term treatment with D-cysteine increased the amounts of Nrf2 and LAMP2A, a CMA-related protein, in the mouse cerebellum. These findings suggest that hydrogen sulfide derived from D-cysteine enhances CMA activity via Nrf2 activation.

Glucocorticoids negatively regulates chaperone mediated autophagy and microautophagy.

Glucocorticoids are released from the adrenal cortex and are important for regulating various physiological functions. However, a persistent increase in glucocorticoids due to chronic stress causes various dysfunctions in the central nervous system which can lead to mental disorders such as depression. Macroautophagy, one of the pathways of the autophagy-lysosome protein degradation system, is dysregulated in psychiatric disorders, implicating a disturbance of protein degradation in the pathogenesis of psychiatric disorders. In the present study, we investigated whether glucocorticoids affect the activity of chaperone-mediated autophagy (CMA) and microautophagy (mA), the other two pathways of the autophagy-lysosome system. Treatment of human-derived AD293 cells and primary cultured rat cortical neurons with dexamethasone, a potent glucocorticoid receptor agonist, and endogenous glucocorticoids decreased both CMA and mA activities. However, this decrease was significantly suppressed by treatment with RU-486, a glucocorticoid receptor antagonist. In addition, dexamethasone significantly decreased lysosomal Hsc70. These findings suggest that glucocorticoids negatively regulate CMA and mA in a glucocorticoid receptor-dependent manner, and provide evidence for CMA and mA as novel therapeutic targets for depression.

Role of 6-O-α-maltosyl-ß-cyclodextrin in lysosomal cholesterol deprivation in Npc1-deficient Chinese hamster ovary cells.

We aimed to investigate whether 6-O-α-maltosyl-ß-cyclodextrin (Mal-ßCD) is incorporated into cells and lysosomes during the release of unesterified cholesterol in Npc1-deficient Chinese hamster ovary (CHO) cells (Npc1 KO cells) and CHO-JP17 cells (JP17 cells). Internalization of Mal-ßCD in cells and lysosomes and extracellular release of lysosomal unesterified cholesterol were demonstrated by LC/MS/MS and LC/MS, respectively. Internalization of Mal-ßCD was greater in Npc1 KO cells than in JP17 cells. The majority of internalized Mal-ßCD in both cell types was metabolized by lysosomal α-glucosidase to 6-O-α-D-glucosyl-ß-cyclodextrin (Glc-ßCD). However, Mal-ßCD did not directly enter the lysosomes prepared from cell homogenates. Mal-ßCD-treated Npc1 KO cells and JP17 cells both released Mal-ßCD and Glc-ßCD, together with unesterified cholesterol, out of cells. The release of unesterified cholesterol by Mal-ßCD in Npc1 KO cells was much greater than that in JP17 cells. This study is the first to report the influx of Mal-ßCD into the lysosomes of Npc1 KO cells and JP17 cells, followed by generation of Glc-ßCD, and the efflux of Mal-ßCD/Glc-ßCD and unesterified cholesterol to the extracellular fluid, based on the quantitative LC/MS analysis.

Collaborative Action of Surface Chemistry and Topography in the Regulation of Mesenchymal and Epithelial Markers and the Shape of Cancer Cells.

Malignant transformation is associated with enhancement of cell plasticity, which allows cancer cells to survive under different conditions by adapting to their microenvironment during growth and metastatic spread. Much effort has been devoted to understanding the molecular mechanisms of these processes. Although the importance of the extracellular matrix and of surface properties in these mechanisms is evident, the direct impact of distinct physical and chemical surfaces characteristics on cell fate remains unclear. Here, we have addressed this question using HT1080 fibrosarcoma cells as a model. To examine the relationship between surface topography, chemistry, and cell behavior, hydrophobic poly(butyl methacrylate-co-ethylene dimethacrylate) (BMA-EDMA) and hydrophilic poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (HEMA-EDMA) surfaces with three different topographies (microporous, nanoporous, and nonporous) were generated. These surfaces were then modified by photoinitiated grafting of three different methacrylate monomers to create surface chemistry gradients of either negatively (AMPS) or positively (META) charged or zwitterionic (MDSA) functionalities. Our results show that AMPS promotes cell spreading, but that META abolishes cell growth. META and MDSA grafted on microporous BMA-EDMA produced superhydrophilic surfaces with high globularity and elasticity, which modified the cell phenotype by inhibiting cell spreading, followed by loss of mesenchymal characteristics and a reduction in protein levels of the mesenchymal markers N-cadherin, beta-catenin, p120 catenin, and also of the adaptor proteins vinculin and paxillin that are associated with adhesion and cancer cell invasion. The effect was strengthened along the gradient, suggesting that the density of the functional groups plays a role in this process. On the nanoporous surface, only MDSA grafting resulted in a significant increase in cell number, a reduction in N-cadherin expression, increased beta-catenin and p120 catenin levels, as well as the appearance of the epithelial marker E-cadherin. This indicates that the cancer cells have a high plasticity that is triggered by the collaborative effect of physical and chemical surface properties.

Influence of sesamin on CYP2C-mediated diclofenac metabolism: in vitro and in vivo analysis.

Our previous studies revealed that sesamin caused a mechanism-based inhibition (MBI) of CYP2C9 in human liver microsomes. Additionally, we observed a similar MBI of CYP2C by sesamin in the rat liver microsomes. Sesamin-induced difference spectra of rat or human liver microsomes in the presence of NADPH showed a peak at 459 nm, suggesting the formation of a metabolic-intermediate (MI) complex of cytochrome P450 and the methylenedioxyphenyl group of sesamin. However, the peak disappeared in both liver microsomes within 30 min after the termination of the metabolism. These results suggest that the MI complex of cytochrome P450 and sesamin is unstable, and the effects of sesamin on human CYP2C9- or rat CYP2C-mediated drug metabolism may be small. To confirm this, in vivo studies using rats were performed. The pharmacokinetics of diclofenac, which is mainly metabolized by CYP2C11 in male rats, were investigated after a 3-days administration of sesamin (0, 10, and 100 mg/kg bw). No significant differences were observed among the three groups in the pharmacokinetic parameters, C max, T max, and AUC. Furthermore, administration of sesamin to rats for 7 days had no significant effects on diclofenac hydroxylation activity in rat liver microsomes. These results demonstrate that no significant interaction occurs between diclofenac and sesamin in rats. Moreover, the results of these in vitro and in vivo studies suggest that no significant interaction may occur between sesamin and diclofenac when sesamin is administered to humans as a supplement, since the standard sesamin dose in humans is much lower than that administered to rats in this study.

Neuromodulatory effect of creatine on extracellular action potentials in rat hippocampus: role of NMDA receptors.

The creatine (Cr) and phosphocreatine (PCr) system is essential for the buffering and transport of high-energy phosphates. Although achievements made over the last years have highlighted the important role of creatine in several neurological diseases, the adaptive processes elicited by this guanidino compound in hippocampus are poorly understood. In the present study, we showed that creatine (0.5-25mM) gradually increases the amplitude of first population spike (PS) and elicits secondary PS in stratum radiatum of the CA1 region, in hippocampal slices. Creatine also decreased the intensity of the stimulus to induce PS, when compared with hippocampal slices perfused with artificial cerebrospinal fluid (ACSF). The competitive NMDA receptor antagonist, 2-amino-5-phosphonopentanoic acid (AP5; 100microM) attenuated creatine-induced increase of amplitude of PS and appearance of secondary PS, providing pharmacological evidence of the involvement of NMDA receptors in the electrophysiological effects of creatine. Accordingly, creatine (0.01-1mM) increased [3H]MK-801 binding to hippocampal membranes by 55%, further indicating that this compound modulates NMDA receptor function. These results implicate the NMDA receptor in amplitude and population spike increase elicited by creatine in hippocampus. Furthermore, these data suggest that this guanidino compound may also play a putative role as a neuromodulator in the brain, and that at least some of its effects may be mediated by an increase in glutamatergic function.

Avaliação eletrofisiológica in vitro de drogas antiepilépticas em fatias hipocampais humanas provenientes de pacientes portadores de epilepsia do lobo temporal refratária ao tratamento medicamentoso / In vitro electrophysiological assessment of antiepileptic drugs in human hippocampal slices originating from patients with refractory temporal lobe epilepsy

The absence of a satisfactory response to antiepileptic drug (AED) therapy, is an unresolved problem in a significant number of epileptic patients. Mechanisms of intractability are not well understood but may include a combination of poor penetration of AED across a functionally altered blood-brain barrier owing to increased expression of multiple drug resistance transporters. Therefore, the aim of this work was to assess the in vitro efficacy of antiepileptic drugs through human hippocampal slices originating from patients with refractory temporal lobe epilepsy submitted to corticoamygdalohippocampectomy. Slices was prepared from a 1 cm3 block of the hippocampus body 30 min after resection. Briefly, hippocampal slices of 400 µM thickness was cut coronally. Extracellular field potentials was recorded from the st. Granulosum of the dentate gyrus. The antiepileptic drugs added in the bath were Carbamazepine, Topiramate and Phenytoin. The phenytoin was effective reducing the hyperexcitability (polispikes) in 60% of the experiments (n = 5). On the other hand, the carbamazepine promoted a decrease in evoked epileptiform activity in 37,5% of the cases (n = 8). The application of topiramate in the bath reduced in 30% the number of polispikes (n = 10). Our results showed that the phenytoin application resulted in a significant reduction in neuronal excitability, however, the carbamazepine and topiramate were not able to control of the hiperexcitability, suggesting that local neuronal alterations, as well changes in blood brain barrier, could be responsible for such behaviors.