RESUMO

Reactive oxygen species (ROS) function as signaling molecules in several physiologic and pathologic processes. In central nervous system, ROS are critical for differentiation, migration, polarization, and neurite growth. These actions are mediated by reversible oxidation of target proteins. On the other hand, PI3K/Akt signaling pathway is susceptible to be modulated by ROS and it has been implicated in neurite growth. In this study, we evaluated the participation of ROS in the neurite growth of cultured rat cerebellar granule neurons (CGN), as well as the possible regulation of the PI3K/Akt pathway by ROS during neurite outgrowth. For this purpose, CGN were treated with cellular or mitochondrial antioxidants, or an NOX inhibitor and neurite growth was evaluated. Moreover, to assess the participation Akt in this process, the p-Akt levels were measured in CGN treated with antioxidants or a NOX inhibitor. The effect of antioxidants on the neurite growth in the presence of a PI3K inhibitor was also measured. We found that cellular antioxidants and the NOX inhibitor decreased the neurite growth, but not the mitochondrial antioxidant. Interestingly, the antioxidants increased the p-Akt levels; however, the effect of antioxidants on neurite growth was no dependent on the Akt activity since the inhibitor of PI3K did not modify the antioxidant action on neurite growth. Our results show that the PI3K/Akt pathway participates in neurite growth and that ROS produced by NOX could function as signals in this process; however, this action is not mediated by a redox regulation of Akt activity.

Assuntos

RESUMO

[This corrects the article DOI: 10.3389/fnmol.2023.1210962.].

RESUMO

TXNIP is a protein sensitive to oxidant conditions whose expression is related to the progression of death in cancer, diabetes, ischemia, and neurodegenerative diseases, among others. Because of this, many studies propose TXNIP as a therapeutic target in several diseases. Exposure of cerebellar granule neurons to staurosporine or low potassium leads to apoptotic death. Both conditions generate an early production of reactive oxygen species (ROS) that induces the activation of the ASK1 pathway and the apoptotic machinery. In these models, it has been shown an increase in TXNIP protein mediated by ROS. Here, we evaluated the molecular mechanisms involved in the regulation of the Txnip expression during neuronal death, as well as the role of the protein in the progression of cell death induced by these two apoptotic conditions. In cultured cerebellar granule neurons, we observed that low potassium and staurosporine induced an early increase in ROS that correlated with an increase in Txnip mRNA. When we evaluated the promoter of the gene, we found that the JASPAR-reported FOXO1/3 transcription factor motifs are close to the transcription start site (TSS). We then verified through the Chromatin immunoprecipitation technique (ChIP) that FOXO3 interacts with the Txnip promoter after 1 h of low potassium treatment. We also detected FOXO3 nuclear translocation by low potassium and staurosporine treatments. Finally, by using shRNA in the neuroblastoma MSN cell line, we found that Txnip downregulation decreased neuronal death induced by staurosporine stimulus. Together, these results suggest that ROS promotes the expression of Txnip through the activation of the FOXO3 transcription factor mediated by Akt inhibition. We also demonstrated that TXNIP is necessary for neuronal death progression.

RESUMO

Two pore domain potassium channels (K2P) are strongly expressed in the nervous system (CNS), where they play a central role in excitability. These channels give rise to background K+ currents, also known as IKSO (standing-outward potassium current). We detected the expression in primary cultured cerebellar granule neurons (CGNs) of TWIK-1 (K2P1), TASK-1 (K2P3), TASK-3 (K2P9), and TRESK (K2P18) channels by immunocytochemistry and their association with lipid rafts using the specific lipids raft markers flotillin-2 and caveolin-1. At the functional level, methyl-ß-cyclodextrin (MßCD, 5 mM) reduced IKSO currents by ~40% in CGN cells. To dissect out this effect, we heterologously expressed the human TWIK-1, TASK-1, TASK-3, and TRESK channels in HEK-293 cells. MßCD directly blocked TASK-1 and TASK-3 channels and the covalently concatenated heterodimer TASK-1/TASK-3 currents. Conversely, MßCD did not affect TWIK-1- and TRESK-mediated K+ currents. On the other hand, the cholesterol-depleting agent filipin III did not affect TASK-1/TASK-3 channels. Together, the results suggest that neuronal background K+ channels are associated to lipid raft environments whilst the functional activity is independent of the cholesterol membrane organization.

RESUMO

Some studies have shown that silicon dioxide nanoparticles (SiO2-NPs) can reach different regions of the brain and cause toxicity; however, the consequences of SiO2-NPs exposure on the diverse brain cell lineages is limited. We aimed to investigate the neurotoxic effects of SiO2-NP (0-100 µg/mL) on rat astrocyte-rich cultures or neuron-rich cultures using scanning electron microscopy, Attenuated Total Reflection-Fourier Transform Infrared spectroscopy (ATR-FTIR), FTIR microspectroscopy mapping (IQ mapping), and cell viability tests. SiO2-NPs were amorphous particles and aggregated in saline and culture media. Both astrocytes and neurons treated with SiO2-NPs showed alterations in cell morphology and changes in the IR spectral regions corresponding to nucleic acids, proteins, and lipids. The analysis by the second derivative revealed a significant decrease in the signal of the amide I (α-helix, parallel ß-strand, and random coil) at the concentration of 10 µg/mL in astrocytes but not in neurons. IQ mapping confirmed changes in nucleic acids, proteins, and lipids in astrocytes; cell death was higher in astrocytes than in neurons (10-100 µg/mL). We conclude that astrocytes were more vulnerable than neurons to SiO2-NPs toxicity. Therefore, the evaluation of human exposure to SiO2-NPs and possible neurotoxic effects must be followed up.

RESUMO

In this study we evaluated the effect of the reduction in the endoplasmic reticulum calcium concentration ([Ca2+]ER), changes in the cytoplasmic calcium concentration ([Ca2+]i), alteration of the mitochondrial membrane potential, and the ER stress in the activation of caspase-3 in neonatal cerebellar granule cells (CGN). The cells were loaded with Fura-2 to detect changes in the [Ca2+]i and with Mag-fluo-4 to measure variations in the [Ca2+]ER or with TMRE to follow modifications in the mitochondrial membrane potential in response to five different inducers of CGN cell death. These inducers were staurosporine, thapsigargin, tunicamycin, nifedipine and plasma membrane repolarization by switching culture medium from 25 mM KCl (K25) to 5 mM KCl (K5). Additionally, different markers of ER stress were determined and all these parameters were correlated with the activation of caspase-3. The different inducers of cell death in CGN resulted in three different levels of activation of caspase-3. The highest caspase-3 activity occurred in response to K5. At the same time, staurosporine, nifedipine, and tunicamycin elicited an intermediate activation of caspase-3. Importantly, thapsigargin did not activate caspase-3 at any time. Both K5 and nifedipine rapidly decreased the [Ca2+]i, but only K5 immediately reduced the [Ca2+]ER and the mitochondrial membrane potential. Staurosporine and tunicamycin increased the [Ca2+]i and they decreased both the [Ca2+]ER and mitochondrial membrane potential, but at a much lower rate than K5. Thapsigargin strongly increased the [Ca2+]i, but it took 10 min to observe any decrease in the mitochondrial membrane potential. Three cell death inducers -K5, staurosporine, and thapsigargin- elicited ER stress, but they took 30 min to have any effect. Thapsigargin, as expected, displayed the highest efficacy activating PERK. Moreover, a specific PERK inhibitor did not have any impact on cell death triggered by these cell death inducers. Our data suggest that voltage-gated Ca2+ channels, that are not dihydropyridine-sensitive, load the ER with Ca2+ and this Ca2+ flux plays a critical role in keeping the mitochondrial membrane potential polarized. A rapid decrease in the [Ca2+]ER resulted in rapid mitochondrial membrane depolarization and strong activation of caspase-3 without the intervention of the ER stress in CGN.

RESUMO

The guanine-based purines (GBPs) have essential extracellular functions such as modulation of glutamatergic transmission and trophic effects on neurons and astrocytes. We previously showed that GBPs, such as guanosine-5'-monophosphate (GMP) or guanosine (GUO), promote the reorganization of extracellular matrix proteins in astrocytes, and increase the number of neurons in a neuron-astrocyte co-culture protocol. To delineate the molecular basis underlying these effects, we isolated cerebellar neurons in culture and treated them with a conditioned medium derived from astrocytes previously exposed to GUO or GMP (GBPs-ACM) or, directly, with GUO or GMP. Agreeing with the previous studies, there was an increase in the number of ß-tubulin III-positive neurons in both conditions, compared with controls. Interestingly, the increase in the number of neurons in the neuronal cultures treated directly with GUO or GMP was more prominent, suggesting a direct interaction of GBPs on cerebellar neurons. To investigate this issue, we assessed the role of adenosine and glutamate receptors and related intracellular signaling pathways after GUO or GMP treatment. We found an involvement of A2A adenosine receptors, ionotropic glutamate N-methyl-D-aspartate (NMDA), and non-NMDA receptors in the increased number of cerebellar neurons. The signaling pathways extracellular-regulated kinase (ERK), calcium-calmodulin-dependent kinase-II (CaMKII), protein kinase C (PKC), phosphatidilinositol-3'-kinase (PI3-K), and protein kinase A (PKA) are also potentially involved with GMP and GUO effect. Such results suggest that GMP and GUO, and molecules released in GBPs-ACM promote the survival or maturation of primary cerebellar neurons or both via interaction with adenosine and glutamate receptors.

Assuntos

RESUMO

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated pore loop channels. In mammals, this family consists of four different subunits (HCN1-4) and their ion channels activity have been proposed to play an essential role in regulating the membrane potential of excitable cells. Here, we describe the expression and relative abundances of HCN channels in cerebellum and primary cultures of cerebellar granule neurons (CGN). Quantitative determination of mRNA expression levels demonstrated the existence of an accumulation pattern of transcripts in cerebellum that encode HCN2 > HCN3 = HCN4 > HCN1 subunits. Immunolocalization analyses of HCN channels in cerebella revealed positive staining in Purkinje and granule cell layers. The presence of the HCN subunits in the cerebellar granule cell layer was then confirmed in primary cultures of CGN by quantitative real-time PCR (qPCR), as well as western blot and immunofluorescence analysis, demonstrating the presence of all four channel proteins.

Assuntos

RESUMO

Nitric oxide (NO) regulates numerous physiological process and is the main source of reactive nitrogen species (RNS). NO promotes cell survival, but it also induces apoptotic death having been involved in the pathogenesis of several neurodegenerative diseases. NO and superoxide anion react to form peroxynitrite, which accounts for most of the deleterious effects of NO. The mechanisms by which these molecules regulate the apoptotic process are not well understood. In this study, we evaluated the role of NO and peroxynitrite in the apoptotic death of cultured cerebellar granule neurons (CGN), which are known to experience apoptosis by staurosporine (St) or potassium deprivation (K5). We found that CGN treated with the peroxynitrite catalyst, FeTTPs were completely rescued from St-induced death, but not from K5-induced death. On the other hand, the inhibition of the inducible nitric oxide synthase partially protected cell viability in CGN treated with K5, but not with St, while the inhibitor L-NAME further reduced the cell viability in St, but it did not affect K5. Finally, an inhibitor of the soluble guanylate cyclase (sGC) diminished the cell viability in K5, but not in St. Altogether, these results shows that NO promotes cell survival in K5 through sGC-cGMP and promotes cell death by other mechanisms, while in St NO promotes cell survival independently of cGMP and peroxynitrite results critical for St-induced death. Our results suggest that RNS are differentially handled by CGN during cell death depending on the death-inducing conditions.

Assuntos

RESUMO

Two pore domain potassium (K2P) channels are mostly present in the central nervous system (CNS) where they play important roles in modulating neuronal excitability. K2P channels give rise to background K(+) currents (IKSO) a key component in setting and maintaining the resting membrane potential in excitable cells. Here, we studied the expression and relative abundances of K2P channels in cerebellar granule neurons (CGNs), combining molecular biology, electrophysiology and immunologic techniques. The CGN IKSO was very sensitive to external pH, as previously reported. Quantitative determination of mRNA expression level demonstrated the existence of an accumulation pattern of transcripts in CGN that encode K2P9>K2P1>K2P3>K2P18>K2P2=K2P10>K2P4>K2P5 subunits. The presence of the major K2P subunits expressed was then confirmed by Western blot and immunofluorescence analysis, demonstrating robust expression of K2P1 (TWIK-1), K2P3 (TASK-1), K2P9 (TASK-3) and K2P18 (TRESK) channel protein. Based, on these results, it is concluded that K2P1, -3, -9 and -18 subunits represent the majority component of IKSO current in CGN.

Assuntos