Resveratrol Prevents Cell Swelling Through Inhibition of SUR1 Expression in Brain Micro Endothelial Cells Subjected to OGD/Recovery.

The SUR1-TRPM4-AQP4 complex is overexpressed in the initial phase of edema induced after cerebral ischemia, allowing the massive internalization of Na+ and water within the brain micro endothelial cells (BMEC) of the blood-brain barrier. The expression of the Abcc8 gene encoding SUR1 depends on transcriptional factors that are responsive to oxidative stress. Because reactive oxygen species (ROS) are generated during cerebral ischemia, we hypothesized that antioxidant compounds might be able to regulate the expression of SUR1. Therefore, the effect of resveratrol (RSV) on SUR1 expression was evaluated in the BMEC cell line HBEC-5i subjected to oxygen and glucose deprivation (OGD) for 2 h followed by different recovery times. Different concentrations of RSV were administered. ROS production was detected with etidine, and protein levels were evaluated by Western blotting and immunofluorescence. Intracellular Na+ levels and cellular swelling were detected by imaging; cellular metabolic activity and rupture of the cell membrane were detected by MTT and LDH release, respectively; and EMSA assays measured the activity of transcriptional factors. OGD/recovery increased ROS production induced the AKT kinase activity and the activation of SP1 and NFκB. SUR1 protein expression and intracellular Na+ concentration in the HBEC-5i cells increased after a few hours of OGD. These effects correlated with cellular swelling and necrotic cell death, responses that the administration of RSV prevented. Our results indicate that the ROS/AKT/SP1-NFκB pathway is involved in SUR1 expression during OGD/recovery in BMEC of the blood-brain barrier. Thus, RSV prevented cellular edema formation through modulation of SUR1 expression.

Glucose/Fructose Delivery to the Distal Nephron Activates the Sodium-Chloride Cotransporter via the Calcium-Sensing Receptor.

BACKGROUND: The calcium-sensing receptor (CaSR) in the distal convoluted tubule (DCT) activates the NaCl cotransporter (NCC). Glucose acts as a positive allosteric modulator of the CaSR. Under physiologic conditions, no glucose is delivered to the DCT, and fructose delivery depends on consumption. We hypothesized that glucose/fructose delivery to the DCT modulates the CaSR in a positive allosteric way, activating the WNK4-SPAK-NCC pathway and thus increasing salt retention. METHODS: We evaluated the effect of glucose/fructose arrival to the distal nephron on the CaSR-WNK4-SPAK-NCC pathway using HEK-293 cells, C57BL/6 and WNK4-knockout mice, ex vivo perfused kidneys, and healthy humans. RESULTS: HEK-293 cells exposed to glucose/fructose increased SPAK phosphorylation in a WNK4- and CaSR-dependent manner. C57BL/6 mice exposed to fructose or a single dose of dapagliflozin to induce transient glycosuria showed increased activity of the WNK4-SPAK-NCC pathway. The calcilytic NPS2143 ameliorated this effect, which was not observed in WNK4-KO mice. C57BL/6 mice treated with fructose or dapagliflozin showed markedly increased natriuresis after thiazide challenge. Ex vivo rat kidney perfused with glucose above the physiologic threshold levels for proximal reabsorption showed increased NCC and SPAK phosphorylation. NPS2143 prevented this effect. In healthy volunteers, cinacalcet administration, fructose intake, or a single dose of dapagliflozin increased SPAK and NCC phosphorylation in urinary extracellular vesicles. CONCLUSIONS: Glycosuria or fructosuria was associated with increased NCC, SPAK, and WNK4 phosphorylation in a CaSR-dependent manner.

The short form of the SUR1 and its functional implications in the damaged brain.

Sulfonylurea receptor (SUR) belongs to the adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter family; however, SUR is associated with ion channels and acts as a regulatory subunit determining the opening or closing of the pore. Abcc8 and Abcc9 genes code for the proteins SUR1 and SUR2, respectively. The SUR1 transcript encodes a protein of 1582 amino acids with a mass around 140-177 kDa expressed in the pancreas, brain, heart, and other tissues. It is well known that SUR1 assembles with Kir6.2 and TRPM4 to establish KATP channels and non-selective cation channels, respectively. Abbc8 and 9 are alternatively spliced, and the resulting transcripts encode different isoforms of SUR1 and SUR2, which have been detected by different experimental strategies. Interestingly, the use of binding assays to sulfonylureas and Western blotting has allowed the detection of shorter forms of SUR (~65 kDa). Identity of the SUR1 variants has not been clarified, and some authors have suggested that the shorter forms are unspecific. However, immunoprecipitation assays have shown that SUR2 short forms are part of a functional channel even coexisting with the typical forms of the receptor in the heart. This evidence confirms that the structure of the short forms of the SURs is fully functional and does not lose the ability to interact with the channels. Since structural changes in short forms of SUR modify its affinity to ATP, regulation of its expression might represent an advantage in pathologies where ATP concentrations decrease and a therapeutic target to induce neuroprotection. Remarkably, the expression of SUR1 variants might be induced by conditions associated to the decrease of energetic substrates in the brain (e.g. during stroke and epilepsy). In this review, we want to contribute to the knowledge of SUR1 complexity by analyzing evidence that shows the existence of short SUR1 variants and its possible implications in brain function.

Neurological Complications Associated with the Blood-Brain Barrier Damage Induced by the Inflammatory Response During SARS-CoV-2 Infection.

The main discussion above of the novel pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has focused substantially on the immediate risks and impact on the respiratory system; however, the effects induced to the central nervous system are currently unknown. Some authors have suggested that SARS-CoV-2 infection can dramatically affect brain function and exacerbate neurodegenerative diseases in patients, but the mechanisms have not been entirely described. In this review, we gather information from past and actual studies on coronaviruses that informed neurological dysfunction and brain damage. Then, we analyzed and described the possible mechanisms causative of brain injury after SARS-CoV-2 infection. We proposed that potential routes of SARS-CoV-2 neuro-invasion are determinant factors in the process. We considered that the hematogenous route of infection can directly affect the brain microvascular endothelium cells that integrate the blood-brain barrier and be fundamental in initiation of brain damage. Additionally, activation of the inflammatory response against the infection represents a critical step on injury induction of the brain tissue. Consequently, the virus' ability to infect brain cells and induce the inflammatory response can promote or increase the risk to acquire central nervous system diseases. Here, we contribute to the understanding of the neurological conditions found in patients with SARS-CoV-2 infection and its association with the blood-brain barrier integrity.

Resveratrol Prevents GLUT3 Up-Regulation Induced by Middle Cerebral Artery Occlusion.

Glucose transporter (GLUT)3 up-regulation is an adaptive response activated to prevent cellular damage when brain metabolic energy is reduced. Resveratrol is a natural polyphenol with anti-oxidant and anti-inflammatory features that protects neurons against damage induced in cerebral ischemia. Since transcription factors sensitive to oxidative stress and inflammation modulate GLUT3 expression, the purpose of this work was to assess the effect of resveratrol on GLUT3 expression levels after ischemia. Male Wistar rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) followed by different times of reperfusion. Resveratrol (1.9 mg/kg; i. p.) was administered at the onset of the restoration of the blood flow. Quantitative-PCR and Western blot showed that MCAO provoked a substantial increase in GLUT3 expression in the ipsilateral side to the lesion of the cerebral cortex. Immunofluorescence assays indicated that GLUT3 levels were upregulated in astrocytes. Additionally, an important increase in GLUT3 occurred in other cellular types (e.g., damaged neurons, microglia, or infiltrated macrophages). Immunodetection of the microtubule-associated protein 2 (MAP2) showed that MCAO induced severe damage to the neuronal population. However, the administration of resveratrol at the time of reperfusion resulted in injury reduction. Resveratrol also prevented the MCAO-induced increase of GLUT3 expression. In conclusion, resveratrol protects neurons from damage induced by ischemia and prevents GLUT3 upregulation in the damaged brain that might depend on AMPK activation.

Resveratrol reduces cerebral edema through inhibition of de novo SUR1 expression induced after focal ischemia.

Cerebral edema is a clinical problem that frequently follows ischemic infarcts. Sulfonylurea receptor 1 (SUR1) is an inducible protein that can form a heteromultimeric complex with aquaporin 4 (AQP4) that mediate the ion/water transport involved in brain tissue swelling. Transcription of the Abcc8 gene coding for SUR1 depends on the activity of transcriptional factor SP1, which is modulated by the cellular redox environment. Since oxidative stress is implicated in the induced neuronal damage in ischemia and edema formation, the present study aimed to evaluate if the antioxidant resveratrol (RSV) prevents the damage by reducing the de novo expression of SUR1 in the ischemic brain. Male Wistar rats were subjected to 2 h of middle cerebral artery occlusion followed by different times of reperfusion. RSV (1.9 mg/kg; i.v.) was administered at the onset of reperfusion. Brain damage and edema formation were recognized by neurological evaluation, time of survival, TTC (2,3,5-Triphenyltetrazolium chloride) staining, Evans blue extravasation, and water content. RSV mechanism of action was studied by SP1 binding activity measured through the Electrophoretic Mobility Shift Assay, and Abcc8 and Aqp4 gene expression evaluated by qPCR, immunofluorescence, and Western blot. We found that RSV reduced the infarct area and cerebral edema, prevented blood-brain barrier damage, improved neurological performance, and increased survival. Additionally, our findings suggest that the antioxidant activity of RSV targeted SP transcription factors and inhibited SUR1 and AQP4 expression. Thus, RSV by decreasing SUR1 expression could contribute to reducing edema formation, constituting a therapeutic alternative for edema reduction in stroke.

Antioxidant Properties and Protective Effects of Some Species of the Annonaceae, Lamiaceae, and Geraniaceae Families against Neuronal Damage Induced by Excitotoxicity and Cerebral Ischemia.

This study aimed to compare the antioxidant activities of extracts obtained from three plant families and evaluate their therapeutic effect on strokes. Ethanol extracts were obtained from either the leaf or the aerial parts of plants of the families Annonaceae (Annona cherimola, A. diversifolia, A. muricata, A. purpurea, and A. reticulata), Lamiaceae (Salvia amaríssima and S. polystachya), and Geraniaceae (Geranium niveum and G. mexicanum). Extracts were analyzed in terms of hydroxyl radical (OH•), peroxyl radical (ROO•), and superoxide anion (O2•-). The efficiency of the extracts to prevent neuronal death induced by excitotoxicity was tested with the tetrazolium assay, the O2•- scavenging capacity was evaluated with the dihydroethidium dye, and the protective effect of the extracts with the highest antioxidant activity was tested on a stroke experimental model. The extracts' IC50 values (µg/mL) of scavenging varied from 98.9 to 155.04, 4.5 to 102.4, and 20.2 to 118.97 for OH•, ROO•, and O2•-, respectively. In the excitotoxicity model, Annonaceae extracts were highly cytotoxic while Lamiaceae and Geraniaceae reduced intracellular O2•- production and protect neurons against oxidative stress. Salvia polystachya reduced cerebral damage, as well as improved survival and behavior after ischemia. Our results encouraged the use of plant extracts as natural antioxidants to minimize neuronal injury following stroke.

Resveratrol Activates Neuronal Autophagy Through AMPK in the Ischemic Brain.

During cerebral ischemia, oxygen and glucose levels decrease, producing many consequences such as the generation of reactive oxygen species, tissue injury, and the general metabolism collapse. Resveratrol triggers signaling dependent on the protein kinase activated by adenosine monophosphate (AMPK), the sensor of cellular energy metabolism that regulates autophagy, eliminates damaged mitochondria, and increases energy sources. In the present study, we investigated the participation of AMPK activation in the protective effect of resveratrol on cerebral ischemia and excitotoxicity. We found that resveratrol increased the levels of phosphorylated AMPK in the cerebral cortex of rats subjected to middle cerebral artery occlusion (MCAO) and in primary cultured neurons exposed to glutamate-induced excitotoxicity. Resveratrol (1.8 mg/Kg; i. v.; administered at the beginning of reperfusion) decreased the infarct area and increased survival of rats subjected to MCAO. In neuronal cultures, resveratrol treatment (40 µM, after excitotoxicity) reduced the production of superoxide anion, prevented the overload of intracellular Ca+2 associated to mitochondrial failure, reduced the release of the lactate dehydrogenase enzyme, and reduced death. It also promoted mitophagy (increased Beclin 1 level, favored the recruitment of LC3-II, reduced LAMP1, and reduced mitochondrial matrix protein HSP60 levels). In both models, inhibition of AMPK activation with Compound C obstructed the effect of resveratrol, showing that its protective effect depends, partially, on the activation of the AMPK/autophagy pathway.

Glial Excitatory Amino Acid Transporters and Glucose Incorporation.

Excitatory amino acid transporters (EAATs) expressed in astrocytes remove the glutamate released by neurons in and around the synaptic cleft. In this manner, astrocytes preserve the signaling functions mediated by glutamate on synapses and prevent excitotoxicity. Additionally, EAAT activation stimulates glucose utilization in astrocytes, linking neuronal activity with astrocyte metabolism. In this chapter, we briefly review the characteristics of the EAATs and the glucose transporters (GLUTs) expressed in the brain. Thereafter, we focus on the effect of EAATs activation and its association with glucose utilization in astrocytes, specifically addressing the role played by Na+ and Ca2+ ions. Next, we analyze evidence that proposes mechanisms by which the activity of GLUTs could be modulated after EAAT activation (e.g., kinases altering GLUTs traffic to cell membrane). Finally, we analyzed the current knowledge on EAAT function during energy deficiency as a possible inducer of GLUT expression to prevent neuronal damage.