Ana Maria Marques Orellana

Influence of N-methyl-D-aspartate receptors on ouabain activation of nuclear factor-κB in the rat hippocampus.

It has been shown that ouabain (OUA) can activate the Na,K‐ATPase complex and mediate intracellular signaling in the central nervous system (CNS). Inflammatory stimulus increases glutamatergic transmission, especially at N‐methyl‐D‐aspartate (NMDA) receptors, which are usually coupled to the activation of nitric oxide synthase (NOS). Nuclear factor‐κB (NF‐κB) activation modulates the expression of genes involved in development, plasticity, and inflammation. The present work investigated the effects of OUA on NF‐κB binding activity in rat hippocampus and the influence of this OUA‐Na,K‐ATPase signaling cascade in NMDA‐mediated NF‐κB activation. The findings presented here are the first report indicating that intrahippocampal administration of OUA, in a concentration that did not alter Na,K‐ATPase or NOS activity, induced an activation of NF‐κB, leading to increases in brain‐derived neurotrophic factor (Bdnf), inducible NOS (iNos), tumor necrosis factor‐α (Tnf‐α), and B‐cell leukemia/lymphoma 2 (Bcl2) mRNA levels. This response was not linked to any significant signs of neurodegeneration as showed via Fluoro‐Jade B and Nissl stain. Intrahippocampal administration of NMDA induced NF‐κB activation and increased NOS and α2/3‐Na,K‐ATPase activities. NMDA treatment further increased OUA‐induced NF‐κB activation, which was partially blocked by MK‐801, an antagonist of NMDA receptor. These results suggest that OUA‐induced NF‐κB activation is at least in part dependent on Na,K‐ATPase modulatory action of NMDA receptor in hippocampus. The interaction of these signaling pathways could be associated with biological mechanisms that may underlie the basal homeostatic state linked to the inflammatory signaling cascade in the brain.

Altered KLOTHO and NF-κB-TNF-α Signaling Are Correlated with Nephrectomy-Induced Cognitive Impairment in Rats

Renal insufficiency can have a negative impact on cognitive function. Neuroinflammation and changes in klotho levels associate with chronic kidney disease (CKD) and may play a role in the development of cognitive impairment (CI). The present study evaluates the correlation of cognitive deficits with neuroinflammation and soluble KLOTHO in the cerebral spinal fluid (CSF) and brain tissue of nephrectomized rats (Nx), with 5/6 renal mass ablation. Nx and sham Munich Wistar rats were tested over 4 months for locomotor activity, as well as inhibitory avoidance or novel object recognition, which started 30 days after the surgery. EMSA for Nuclear factor-κB and MILLIPLEXMAP or ELISA kit were used to evaluate cytokines, glucocorticoid and KLOTHO levels. Nx animals that showed a loss in aversive-related memory and attention were included in the CI group (Nx-CI) (n=14) and compared to animals with intact learning (Nx-M n=12 and Sham n=20 groups). CSF and tissue samples were collected 24 hours after the last behavioral test. The results show that the Nx-groups have increased NF-κB binding activity and tumor necrosis factor-alpha (TNF-α) levels in the hippocampus and frontal cortex, with these changes more pronounced in the Nx-CI group frontal cortex. In addition, the Nx-CI group showed significantly increased CSF glucocorticoid levels and TNF-α /IL-10 ratio compared to the Sham group. Klotho levels were decreased in Nx-CI frontal cortex but not in hippocampus, when compared to Nx-M and Sham groups. Overall, these results suggest that neuroinflammation mediated by frontal cortex NF-κB, TNF-α and KLOTHO signaling may contribute to Nx-induced CI in rats.

Ouabain activates NFκB through an NMDA signaling pathway in cultured cerebellar cells.

Na,K-ATPase, an ion pump, has been shown to interact with other proteins in signaling complexes in cardiac myocytes, renal and glial cells, and several other cell types. Our previous in vivo studies indicated that intrahippocampal administration of ouabain (OUA), an inhibitor of Na,K-ATPase, induces NFκB activation, leading to an increase in mRNA levels of target genes of this transcription factor in the rat hippocampus. The present work investigated whether OUA can regulate NF-κB in primary cultured rat cerebellar cells. Cells were treated with different concentrations of OUA (1, 10 or 100 μM) for different periods of time (1, 2 and 4 h). OUA induced a time- and concentration-dependent activation of NFκB (peak of activation: 10 μM, 2 h), involving both p50/p65 and p50/p50 NFκB dimers. OUA (10 μM, 2 h) induced upregulation of tumor necrosis factor α (Tnf-α), interleukin-1β (Il-1β), and brain derived neurotrophic factor (Bdnf) mRNA levels. Both NFκB activation and gene expression activation induced by OUA (10 μM) were abolished when cells were pre-treated for 20 min with MK-801 (N-Methyl-D-Aspartate (NMDA) receptor antagonist), manumycin A (farnesyltransferase inhibitor), PP-1(Src-family tyrosine kinase inhibitor) and PD98059 (mitogen-activated protein kinase (MAPK) inhibitor). OUA (10 μM) alone or in the presence of MK-801, PP-1, PD98059 did not cause cell death or DNA fragmentation. These findings suggest that OUA activates NFκB by NMDA-Src-Ras-like protein through MAPK pathways in cultured cerebellar cells. This pathway may mediate an adaptive response in the central nervous system.

The Influence of Na(+), K(+)-ATPase on Glutamate Signaling in Neurodegenerative Diseases and Senescence.

Decreased Na+, K+-ATPase (NKA) activity causes energy deficiency, which is commonly observed in neurodegenerative diseases. The NKA is constituted of three subunits: α, β, and γ, with four distinct isoforms of the catalytic α subunit (α1−4). Genetic mutations in the ATP1A2 gene and ATP1A3 gene, encoding the α2 and α3 subunit isoforms, respectively can cause distinct neurological disorders, concurrent to impaired NKA activity. Within the central nervous system (CNS), the α2 isoform is expressed mostly in glial cells and the α3 isoform is neuron-specific. Mutations in ATP1A2 gene can result in familial hemiplegic migraine (FHM2), while mutations in the ATP1A3 gene can cause Rapid-onset dystonia-Parkinsonism (RDP) and alternating hemiplegia of childhood (AHC), as well as the cerebellar ataxia, areflexia, pescavus, optic atrophy and sensorineural hearing loss (CAPOS) syndrome. Data indicates that the central glutamatergic system is affected by mutations in the α2 isoform, however further investigations are required to establish a connection to mutations in the α3 isoform, especially given the diagnostic confusion and overlap with glutamate transporter disease. The age-related decline in brain α2∕3 activity may arise from changes in the cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase (PKG) pathway. Glutamate, through nitric oxide synthase (NOS), cGMP and PKG, stimulates brain α2∕3 activity, with the glutamatergic N-methyl-D-aspartate (NMDA) receptor cascade able to drive an adaptive, neuroprotective response to inflammatory and challenging stimuli, including amyloid-β. Here we review the NKA, both as an ion pump as well as a receptor that interacts with NMDA, including the role of NKA subunits mutations. Failure of the NKA-associated adaptive response mechanisms may render neurons more susceptible to degeneration over the course of aging.

Cardiotonic Steroids as Modulators of Neuroinflammation

Cardiotonic steroids (CTS) are a class of specific ligands of the Na+, K+- ATPase (NKA). NKA is a P-type ATPase that is ubiquitously expressed and although well known to be responsible for the maintenance of the cell electrochemical gradient through active transport, NKA can also act as a signal transducer in the presence of CTS. Inflammation, in addition to importantly driving organism defense and survival mechanisms, can also modulate NKA activity and memory formation, as well as being relevant to many chronic illnesses, neurodegenerative diseases, and mood disorders. The aim of the current review is to highlight the recent advances as to the role of CTS and NKA in inflammatory process, with a particular focus in the central nervous system.

Alpha 2 Na+,K+-ATPase silencing induces loss of inflammatory response and ouabain protection in glial cells

Ouabain (OUA) is a cardiac glycoside that binds to Na+,K+-ATPase (NKA), a conserved membrane protein that controls cell transmembrane ionic concentrations and requires ATP hydrolysis. At nM concentrations, OUA activates signaling pathways that are not related to its typical inhibitory effect on the NKA pump. Activation of these signaling pathways protects against some types of injury of the kidneys and central nervous system. There are 4 isoforms of the alpha subunit of NKA, which are differentially distributed across tissues and may have different physiological roles. Glial cells are important regulators of injury and inflammation in the brain and express the α1 and α2 NKA isoforms. This study investigated the role of α2 NKA in OUA modulation of the neuroinflammatory response induced by lipopolysaccharide (LPS) in mouse primary glial cell cultures. LPS treatment increased lactate dehydrogenase release, while OUA did not decrease cell viability and blocked LPS-induced NF-κB activation. Silencing α2 NKA prevented ERK and NF-κB activation by LPS. α2 NKA also regulates TNF-α and IL-1β levels. The data reported here indicate a significant role of α2 NKA in regulating central LPS effects, with implications in the associated neuroinflammatory processes.

Ouabain increases neuronal branching in hippocampus and improves spatial memory

Abstract Previous research shows Ouabain (OUA) to bind Na, K‐ATPase, thereby triggering a number of signaling pathways, including the transcription factors NF&kgr;B and CREB. These transcription factors play a key role in the regulation of BDNF and WNT‐&bgr;‐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/&bgr;‐Catenin signaling pathway and to increase CREB/BDNF and NF&kgr;B 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. HighlightsIntrahippocampal injection of OUA 10 nM increases NF&kgr;B activation in adult rats.OUA increase BDNF expression and CREB activity in rat hippocampus.After 24 h OUA infusion, Wnt‐&bgr;‐Catenin signaling pathway is more active in CA1 neurons.10 nM OUA treatment lead increased CA1 and DG dendritic branching and improved spatial reference memory.OUA improve spatial reference memory.