Neuroprotective action of α-Klotho against LPS-activated glia conditioned medium in primary neuronal culture.

The α-Klotho is an anti-aging protein that, when overexpressed, extends the life span in humans and mice. It has an anti-inflammatory and protective action on renal cells by inhibiting NF-κB activation and production of inflammatory cytokines in response to TNF-α. Furthermore, studies have shown the neuroprotective effect of α-Klotho against neuroinflammation on different conditions, such as aging, animal models of neurodegenerative diseases, and ischemic brain injury. This work aimed to evaluate the effects of α-Klotho protein on primary glial cell culture against the proinflammatory challenge with LPS and how this could interfere with neuronal health. Cortical mixed glial cells and purified astrocytes were pretreated with α- α-Klotho and stimulated with LPS followed by TNFα, IL-1ß, IL-6, IFN-γ levels, and NF-κB activity analysis. Conditioned medium from cortical mixed glia culture treated with LPS (glia conditioned medium (GCM) was used to induce neuronal death of primary cortical neuronal culture and evaluate if GCM-KL (medium from glia culture pretreated α-Klotho followed by LPS stimulation) or GCM + LPS in the presence of KL can reverse the effect. LPS treatment in glial cells induced an increase in proinflammatory mediators such as TNF-α, IL-1ß, IL-6, and IFN-γ, and activation of astrocyte NF-κB. GCM treated-cortical neuronal culture induced a concentration-dependent neuronal death. Pretreatment with α-Klotho decreased TNF-α and IL-6 production, reverted NF-κB activation, and decreased neuronal death induced by GCM. In addition, KL incubation together with GCM + LPS completely reverts the neuronal toxicity induced by low concentration of GCM-LPS. These data suggest an anti-inflammatory and neuroprotective effect of α-Klotho protein in the CNS. This work demonstrated the therapeutic potential of α-Klotho in pathological processes which involves a neuroinflammatory component.

Consequences of the Lack of TNFR1 in Ouabain Response in the Hippocampus of C57BL/6J Mice.

Ouabain is a cardiac glycoside that has a protective effect against neuroinflammation at low doses through Na+/K+-ATPase signaling and that can activate tumor necrosis factor (TNF) in the brain. TNF plays an essential role in neuroinflammation and regulates glutamate receptors by acting on two different receptors (tumor necrosis factor receptor 1 [TNFR1] and TNFR2) that have distinct functions and expression. The activation of constitutively and ubiquitously expressed TNFR1 leads to the expression of pro-inflammatory cytokines. Thus, this study aimed to elucidate the effects of ouabain in a TNFR1 knockout (KO) mouse model. Interestingly, the hippocampus of TNFR1 KO mice showed a basal increase in both TNFR2 membrane expression and brain-derived neurotrophic factor (BDNF) release, suggesting a compensatory mechanism. Moreover, ouabain activated TNF-α-converting enzyme/a disintegrin and metalloprotease 17 (TACE/ADAM17), decreased N-methyl-D-aspartate (NMDA) receptor subunit 2A (NR2A) expression, and induced anxiety-like behavior in both genotype animals, independent of the presence of TNFR1. However, ouabain induced an increase in interleukin (IL)-1ß in the hippocampus, a decrease in IL-6 in serum, and an increase in NMDA receptor subunit 1 (NR1) only in wild-type (WT) mice, indicating that TNFR1 or TNFR2 expression may be important for some effects of ouabain. Collectively, our results indicate a connection between ouabain signaling and TNFR1, with the effect of ouabain partially dependent on TNFR1.

The Janus face of ouabain in Na+ /K+ -ATPase and calcium signalling in neurons.

Na+ /K+ -ATPase, a transmembrane protein essential for maintaining the electrochemical gradient across the plasma membrane, acts as a receptor for cardiotonic steroids such as ouabain. Cardiotonic steroids binding to Na+ /K+ -ATPase triggers signalling pathways or inhibits Na+ /K+ -ATPas activity in a concentration-dependent manner, resulting in a modulation of Ca2+ levels, which are essential for homeostasis in neurons. However, most of the pharmacological strategies for avoiding neuronal death do not target Na+ /K+ -ATPase activity due to its complexity and the poor understanding of the mechanisms involved in Na+ /K+ -ATPase modulation. The present review aims to discuss two points regarding the interplay between Na+ /K+ -ATPase and Ca2+ signalling in the brain. One, Na+ /K+ -ATPase impairment causing illness and neuronal death due to Ca2+ signalling and two, benefits to the brain by modulating Na+ /K+ -ATPase activity. These interactions play an essential role in neuronal cell fate determination and are relevant to find new targets for the treatment of neurodegenerative diseases. LINKED ARTICLES: This article is part of a themed issue on Building Bridges in Neuropharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.8/issuetoc.

The Role of GPNMB in Inflammation.

Inflammation is a response to a lesion in the tissue or infection. This process occurs in a specific manner in the central nervous system and is called neuroinflammation, which is involved in neurodegenerative diseases. GPNMB, an endogenous glycoprotein, has been recently related to inflammation and neuroinflammation. GPNMB is highly expressed in macrophages and microglia, which are cells involved with innate immune response in the periphery and the brain, respectively. Some studies have shown increased levels of GPNMB in pro-inflammatory conditions, such as LPS treatment, and in pathological conditions, such as neurodegenerative diseases and cancer. However, the role of GPNMB in inflammation is still not clear. Even though most studies suggest that GPNMB might have an anti-inflammatory role by promoting inflammation resolution, there is evidence that GPNMB could be pro-inflammatory. In this review, we gather and discuss the published evidence regarding this interaction.

Accumulation of amyloid precursor protein C-terminal fragments triggers mitochondrial structure, function, and mitophagy defects in Alzheimer's disease models and human brains.

Several lines of recent evidence indicate that the amyloid precursor protein-derived C-terminal fragments (APP-CTFs) could correspond to an etiological trigger of Alzheimer's disease (AD) pathology. Altered mitochondrial homeostasis is considered an early event in AD development. However, the specific contribution of APP-CTFs to mitochondrial structure, function, and mitophagy defects remains to be established. Here, we demonstrate in neuroblastoma SH-SY5Y cells expressing either APP Swedish mutations, or the ß-secretase-derived APP-CTF fragment (C99) combined with ß- and γ-secretase inhibition, that APP-CTFs accumulation independently of Aß triggers excessive mitochondrial morphology alteration (i.e., size alteration and cristae disorganization) associated with enhanced mitochondrial reactive oxygen species production. APP-CTFs accumulation also elicit basal mitophagy failure illustrated by enhanced conversion of LC3, accumulation of LC3-I and/or LC3-II, non-degradation of SQSTM1/p62, inconsistent Parkin and PINK1 recruitment to mitochondria, enhanced levels of membrane and matrix mitochondrial proteins, and deficient fusion of mitochondria with lysosomes. We confirm the contribution of APP-CTFs accumulation to morphological mitochondria alteration and impaired basal mitophagy in vivo in young 3xTgAD transgenic mice treated with γ-secretase inhibitor as well as in adeno-associated-virus-C99 injected mice. Comparison of aged 2xTgAD and 3xTgAD mice indicates that, besides APP-CTFs, an additional contribution of Aß to late-stage mitophagy activation occurs. Importantly, we report on mitochondrial accumulation of APP-CTFs in human post-mortem sporadic AD brains correlating with mitophagy failure molecular signature. Since defective mitochondria homeostasis plays a pivotal role in AD pathogenesis, targeting mitochondrial dysfunctions and/or mitophagy by counteracting early APP-CTFs accumulation may represent relevant therapeutic interventions in AD.

Ouabain attenuates oxidative stress and modulates lipid composition in hippocampus of rats in lipopolysaccharide-induced hypocampal neuroinflammation in rats.

Our study aimed to analyze the effect of ouabain (OUA) administration on lipopolysaccharide (LPS)-induced changes in hippocampus of rats. Oxidative parameters were analyzed in Wistar rats after intraperitoneal injection of OUA (1.8 µg/kg), LPS (200 µg/kg), or OUA plus LPS or saline. To reach our goal, activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), in addition to levels of reduced glutathione (GSH), protein carbonyl (PCO) and lipid peroxidation (LPO) were evaluated. We also analyzed the membrane lipid profile and some important lipids for the nervous system, such as phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid and sphingomyelin. The group that received only LPS showed increased oxidative stress, as evidenced by an increase in LPO (about twice), PCO (about three times) levels, and CAT activity (80%). Conversely, administration of LPS decreased GSH levels (55%), and GPx activity (30%), besides a reduction in the amount of PI (60%) and PC (45%). By other side, OUA alone increased the amount of PI (45%), PE (85%), and PC (70%). All harmful effects recorded were attenuated by OUA, suggesting a protective effect against LPS-induced oxidative stress. The relevance of our results extends beyond changes in oxidative parameters induced by LPS, because nanomolar doses of OUA may be useful in neurodegenerative models. Other studies on other cardenolides and substances related issues, as well as the development of new molecules derived from OUA, could also be useful in general oxidative and/or cellular stress, a condition favoring the appearance of neuronal pathologies.

Ouabain increases neuronal branching in hippocampus and improves spatial memory.

Previous research shows Ouabain (OUA) to bind Na, K-ATPase, thereby triggering a number of signaling pathways, including the transcription factors NFᴋB and CREB. These transcription factors play a key role in the regulation of BDNF and WNT-ß-catenin signaling cascades, which are involved in neuroprotection and memory regulation. This study investigated the effects of OUA (10 nM) in the modulation of the principal signaling pathways involved in morphological plasticity and memory formation in the hippocampus of adult rats. The results show intrahippocampal injection of OUA 10 nM to activate the Wnt/ß-Catenin signaling pathway and to increase CREB/BDNF and NFᴋB levels. These effects contribute to important changes in the cellular microenvironment, resulting in enhanced levels of dendritic branching in hippocampal neurons, in association with an improvement in spatial reference memory and the inhibition of long-term memory extinction.

Ouabain attenuates the oxidative stress induced by lipopolysaccharides in the cerebellum of rats.

Our study aimed to analyze the effect of ouabain administration on lipopolysaccharide (LPS)-induced changes in oxidative parameters, membrane lipid composition, and the activities of some important enzymes of the nervous system. The content of phospholipids, cholesterol, and gangliosides were analyzed in Wistar rats after intraperitoneal injection of ouabain (1.8 µg/kg), LPS (200 µg/kg), or saline. Oxidative parameters were also evaluated, including the activities of superoxide dismutase, catalase and glutathione peroxidase, the levels of glutathione and lipid peroxidation, as well as Na,K-ATPase activity and the level of glutamate transporter EAAT4. Administration of LPS resulted in increased oxidative stress, as evidenced by an increase in lipid peroxidation levels, glutathione peroxidase activity, decreased catalase activity and reduced glutathione levels. All changes recorded were attenuated by pretreatment with ouabain. Administration of ouabain plus LPS enhanced the total ganglioside content and EAAT4 levels, but failed to alter the Na,K-ATPase activity. Our data suggest a neuroprotective effect of ouabain against LPS-induced oxidative stress by promoting membrane lipid remodeling and increasing the expression of glutamate transporter EAAT4. Our results emphasize that the observed oxidative stress is not correlated with Na,K-ATPase, but with a possible ouabain-mediated effect on cellular signaling. The relevance of our results extends beyond LPS-induced changes in oxidative parameters, as nanomolar doses of ouabain might prove useful in neurodegenerative models. Further study of other cardenolides and related molecules, as well as the development of new molecules derived from ouabain, could also prove useful in the fight against the oxidative and/or general cell stress triggered by neuronal pathologies.

Effects of intermittent fasting on age-related changes on Na,K-ATPase activity and oxidative status induced by lipopolysaccharide in rat hippocampus.

Chronic neuroinflammation is a common characteristic of neurodegenerative diseases, and lipopolysaccharide (LPS) signaling is linked to glutamate-nitric oxide-Na,K-ATPase isoforms pathway in central nervous system (CNS) and also causes neuroinflammation. Intermittent fasting (IF) induces adaptive responses in the brain that can suppress inflammation, but the age-related effect of IF on LPS modulatory influence on nitric oxide-Na,K-ATPase isoforms is unknown. This work compared the effects of LPS on the activity of α1,α2,3 Na,K-ATPase, nitric oxide synthase gene expression and/or activity, cyclic guanosine monophosphate, 3-nitrotyrosine-containing proteins, and levels of thiobarbituric acid-reactive substances in CNS of young and older rats submitted to the IF protocol for 30 days. LPS induced an age-related effect in neuronal nitric oxide synthase activity, cyclic guanosine monophosphate, and levels of thiobarbituric acid-reactive substances in rat hippocampus that was linked to changes in α2,3-Na,K-ATPase activity, 3-nitrotyrosine proteins, and inducible nitric oxide synthase gene expression. IF induced adaptative cellular stress-response signaling pathways reverting LPS effects in rat hippocampus of young and older rats. The results suggest that IF in both ages would reduce the risk for deficits on brain function and neurodegenerative disorders linked to inflammatory response in the CNS.

Signaling function of Na,K-ATPase induced by ouabain against LPS as an inflammation model in hippocampus.

BACKGROUND: Ouabain (OUA) is a newly recognized hormone that is synthesized in the adrenal cortex and hypothalamus. Low doses of OUA can activate a signaling pathway by interaction with Na,K-ATPase, which is protective against a number of insults. OUA has central and peripheral anti-inflammatory effects. Lipopolysaccharide (LPS), via toll-like receptor 4 activation, is a widely used model to induce systemic inflammation. This study used a low OUA dose to evaluate its effects on inflammation induced by LPS injection in rats. METHODS: Adult male Wistar rats received acute intraperitoneal (ip) OUA (1.8 µg/kg) or saline 20 minutes before LPS (200 µg/kg, ip) or saline injection. Some of the animals had their femoral artery catheterized in order to assess arterial blood pressure values before and after OUA administration. Na,K-ATPase activity, cytokine mRNA levels, apoptosis-related proteins, NF-κB activation brain-derived neurotrophic factor BDNF, corticosterone and TNF-α levels were measured. RESULTS: OUA pretreatment decreased mRNA levels of the pro-inflammatory cytokines, inducible nitric oxide synthase (iNOS) and IL-1ß, which are activated by LPS in the hippocampus, but with no effect on serum measures of these factors. None of these OUA effects were linked to Na,K-ATPase activity. The involvement of the inflammatory transcription factor NF-κB in the OUA effect was indicated by its prevention of LPS-induced nuclear translocation of the NF-κB subunit, RELA (p65), as well as the decreased cytosol levels of the NF-κB inhibitor, IKB, in the hippocampus. OUA pretreatment reversed the LPS-induced glial fibrillary acidic protein (GFAP) activation and associated inflammation in the dentate gyrus. OUA also prevented LPS-induced increases in the hippocampal Bax/Bcl2 ratio suggesting an anti-apoptotic action in the brain. CONCLUSION: Our results suggest that a low dose of OUA has an important anti-inflammatory effect in the rat hippocampus. This effect was associated with decreased GFAP induction by LPS in the dentate gyrus, a brain area linked to adult neurogenesis.