Larissa de Sá Lima

Chronic Unpredictable Stress Exacerbates Lipopolysaccharide-Induced Activation of Nuclear Factor-κB in the Frontal Cortex and Hippocampus via Glucocorticoid Secretion

Although the anti-inflammatory actions of glucocorticoids (GCs) are well established in the periphery, these stress hormones can increase inflammation under some circumstances in the brain. The transcription factor nuclear factor-κB (NF-κB), which is inhibited by GCs, regulates numerous genes central to inflammation. In this study, the effects of stress, GCs, and NMDA receptors on lipopolysaccharide (LPS)-induced activation of NF-κB in the brain were investigated. One day after chronic unpredictable stress (CUS), nonstressed and CUS rats were treated with saline or LPS and killed 2 h later. CUS potentiated the increase in LPS-induced activation of NF-κB in frontal cortex and hippocampus but not in the hypothalamus. This stress effect was blocked by pretreatment of rats with RU-486, an antagonist of the GC receptor. MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate], an NMDA receptor antagonist, also reduced the effect of LPS in all three brain regions. However, the combined antagonism of both GC and NMDA receptors produced no further reduction in NF-κB activation when compared with the effect of each treatment alone. Our results indicate that stress, via GC secretion, can increase LPS-induced NF-κB activation in the frontal cortex and hippocampus, agreeing with a growing literature demonstrating proinflammatory effects of GCs.

Cocaine induces cell death and activates the transcription nuclear factor kappa-B in PC12 cells.

Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-κB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-κB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-κB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-κB activation. Inhibition of NF-κB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-κB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells.

Fatty acid control of nitric oxide production by macrophages

Modulation of macrophage functions by fatty acids (FA) has been studied by several groups, but the effect of FA on nitric oxide production by macrophages has been poorly examined. In the present study the effect of palmitic, stearic, oleic, linoleic, arachidonic, docosahexaenoic and eicosapentaenoic acids on NF‐κB activity and NO production in J774 cells (a murine macrophage cell line) was investigated. All FA tested stimulated NO production at low doses (1–10 μM) and inhibited it at high doses (50–200 μM). An increase of iNOS expression and activity in J774 cells treated with a low concentration of FA (5 μM) was observed. The activity of NF‐κB was time‐dependently enhanced by the FA treatment. The inhibitory effect of FA on NO production may be due to their cytotoxicity, as observed by loss of membrane integrity and/or increase of DNA fragmentation in cells treated for 48 h with high concentrations. The results indicate that, at low concentrations FA increase NO production by J774 cells, whereas at high concentrations they cause cell death.

Age-related changes in cyclic GMP and PKG-stimulated cerebellar Na,K-ATPase activity

Energy deficiency and dysfunction of the Na,K-ATPase are common consequences of many pathological insults. Glutamate through cyclic GMP and cyclic GMP-dependent protein kinase (PKG) has been shown to stimulate alpha(2/3)-Na,K-ATPase activity in the central nervous system. Thus, a slight impairment of this pathway may amplify the disruption of ion homeostasis in the presence of a non-lethal insult. We investigate the effect of aging (4, 12 and 24 months) on the glutamate-cyclic GMP-PKG modulation of alpha1, alpha(2/3)-Na,K-ATPase activity in rat cerebellum and the stimulation of the glutamate-cyclic GMP-PKG pathway at different levels. Cyclic GMP levels and alpha(2/3)-Na,K-ATPase activity were progressively decreased from 4 and 24 month-old animals. However, PKG basal activity was reduced between 4 and 12 months, and no additional change was observed at 24 months. The ability of 8-Br-cyclic GMP to stimulate PKG activity was only reduced between 12 and 24 months. Moreover, glutamate or 8-Br-cyclic GMP promoted a smaller increase of alpha(2/3)-Na,K-ATPase activity at 24 months, when compared to 4 and 12 months. In spite of the age-related reduced basal levels of cyclic GMP, the production induced by CO or NO was not age-related. Finally, inhibition of PKG activation by KT5823 revealed a lower sensitivity of the enzyme at the older age. Taken together, these data show that basal age-related decline in sodium pump activity is a consequence of changes in different steps of the cyclic GMP-PKG pathway. On the other hand, age-related reduction in glutamate positive modulation of cerebellar alpha(2/3)-Na,K-ATPase is linked to a defective PKG signaling pathway.

Melatonin synthesis impairment as a new deleterious outcome of diabetes‐derived hyperglycemia

Melatonin is a neurohormone that works as a nighttime signal for circadian integrity and health maintenance. It is crucial for energy metabolism regulation, and the diabetes effects on its synthesis are unresolved. Using diverse techniques that included pineal microdialysis and ultrahigh‐performance liquid chromatography, the present data show a clear acute and sustained melatonin synthesis reduction in diabetic rats as a result of pineal metabolism impairment that is unrelated to cell death. Hyperglycemia is the main cause of several diabetic complications, and its consequences in terms of melatonin production were assessed. Here, we show that local high glucose (HG) concentration is acutely detrimental to pineal melatonin synthesis in rats both in vivo and in vitro. The clinically depressive action of high blood glucose concentration in melatonin levels was also observed in type 1 diabetes patients who presented a negative correlation between hyperglycemia and 6‐sulfatoxymelatonin excretion. Additionally, high‐mean‐glycemia type 1 diabetes patients presented lower 6‐sulfatoxymelatonin levels when compared to control subjects. Although further studies are needed to fully clarify the mechanisms, the present results provide evidence that high circulating glucose levels interfere with pineal melatonin production. Given the essential role played by melatonin as a powerful antioxidant and in the control of energy homeostasis, sleep and biological rhythms and knowing that optimal glycemic control is usually an issue for patients with diabetes, melatonin supplementation may be considered as an additional tool to the current treatment.

Amyloid β‐peptide activates nuclear factor‐κB through an N‐methyl‐D‐aspartate signaling pathway in cultured cerebellar cells

Amyloid β‐peptide (Aβ) likely causes functional alterations in neurons well prior to their death. Nuclear factor‐κB (NF‐κB), a transcription factor that is known to play important roles in cell survival and apoptosis, has been shown to be modulated by Aβ in neurons and glia, but the mechanism is unknown. Because Aβ has also been shown to enhance activation of N‐methyl‐D‐aspartate (NMDA) receptors, we investigated the role of NMDA receptor‐mediated intracellular signaling pathways in Aβ‐induced NF‐κB activation in primary cultured rat cerebellar cells. Cells were treated with different concentrations of Aβ1–40 (1 or 2 μM) for different periods (6, 12, or 24 hr). MK‐801 (NMDA antagonist), manumycin A and FTase inhibitor 1 (farnesyltransferase inhibitors), PP1 (Src‐family tyrosine kinase inhibitor), PD98059 [mitogen‐activated protein kinase (MAPK) inhibitor], and LY294002 [phosphatidylinositol 3‐kinase (PI3‐k) inhibitor] were added 20 min before Aβ treatment of the cells. Aβ induced a time‐ and concentration‐dependent activation of NF‐κB (1 μM, 12 hr); both p50/p65 and p50/p50 NF‐κB dimers were involved. This activation was abolished by MK‐801 and attenuated by manumycin A, FTase inhibitor 1, PP1, PD98059, and LY294002. Aβ at 1 μM increased the expression of inhibitory protein IκB, brain‐derived neurotrophic factor, inducible nitric oxide synthase, tumor necrosis factor‐α, and interleukin‐1β as shown by RT‐PCR assays. Collectively, these findings suggest that Aβ activates NF‐κB by an NMDA‐Src‐Ras‐like protein through MAPK and PI3‐k pathways in cultured cerebellar cells. This pathway may mediate an adaptive, neuroprotective response to Aβ.

Thalidomide suppresses inflammation in adenine-induced CKD with uraemia in mice

BACKGROUND Persistent systemic inflammation has been widely recognized in patients with chronic kidney disease (CKD), and is associated with increased risk of morbidity and mortality. Intervention therapies aiming for the blockade of inflammatory cytokines are considered attractive approaches for CKD patients with signs of chronic inflammation. In this context, thalidomide, due to its potent anti-inflammatory and immunomodulatory properties, may represent an alternative strategy of treatment. In the present study, we developed an experimental model of CKD with uraemia in mice, induced by a diet rich in adenine, which causes progressive renal dysfunction, resembling the human uraemic features. Inflammatory parameters were analysed in this model of CKD and the potential beneficial effects of thalidomide as an anti-inflammatory drug was also investigated. METHODS C57/BL-6 mice were fed with an adenine-containing diet during a period of 6 weeks. Thirty mice were divided into three groups: Control group (animals receiving normal diet), ADE group (mice receiving adenine-containing diet) and ADE + TLD group (CKD mice receiving thalidomide, 30 mg/kg/day, by gavage). Besides biochemical and histopathological changes, local and systemic inflammatory parameters were also analysed, including expression of cytokines interleukin (IL)-1β, tumour necrosis factor-α, IL-6, IL-4 and IL-10 in kidney samples by real-time RT-PCR and quantification of serum levels of cytokines. Finally, the electrophoretic mobility shift assay (EMSA) for NF-κB was also examined. RESULTS Adenine-fed mice developed advanced CKD characterized by a marked increase in serum urea, creatinine, phosphorus and intact parathyroid hormone (iPTH) levels. In addition, histological changes of tubulointerstitial injury, characterized by deposition of crystals in the kidney, accompanied by tubular dilatation, degeneration of proximal tubular epithelium with loss of the brush border, inflammatory cellular infiltration, foreign-body granuloma formation and interstitial fibrosis were also evident. By immunohistochemistry, Mac-2- and α-SMA-positive cells were identified in the tubulointerstitial compartment. Treatment with thalidomide significantly reduced serum urea, creatinine, phosphorus and iPTH levels and protected against tubulointerstitial injury. Local and systemic inflammation in the mice model of adenine-induced CKD was confirmed by the findings of significantly high expression of cytokine mRNA levels and NF-κB activation in the kidney tissue as well as marked increased serum levels of inflammatory cytokines. Thalidomide treatment significantly reduced gene expression of these cytokines and the activation of the NF-κB in the renal tissue and the circulating levels of cytokines. CONCLUSIONS Dietary adenine caused advanced CKD with uraemia in mice providing a useful experimental model to study molecular and morphological changes associated with this disease. The negative impact of inflammation in this CKD model was overcome by the marked anti-inflammatory effects of thalidomide, promoting renal protection.

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.

Microglial Cells Are Involved in the Susceptibility of NADPH Oxidase Knockout Mice to 6-Hydroxy-Dopamine-Induced Neurodegeneration

We explored the impact of Nox-2 in modulating inflammatory-mediated microglial responses in the 6-hydroxydopamine (6-OHDA)-induced Parkinson’s disease (PD) model. Nox1 and Nox2 gene expression were found to increase in striatum, whereas a marked increase of Nox2 expression was observed in substantia nigra (SN) of wild-type (wt) mice after PD induction. Gp91phox-/- 6-OHDA-lesioned mice exhibited a significant reduction in the apomorphine-induced rotational behavior, when compared to wt mice. Immunolabeling assays indicated that striatal 6-OHDA injections reduced the number of dopaminergic (DA) neurons in the SN of wt mice. In gp91phox-/- 6-OHDA-lesioned mice the DA degeneration was negligible, suggesting an involvement of Nox in 6-OHDA-mediated SN degeneration. Gp91phox-/- 6-OHDA-lesioned mice treated with minocycline, a tetracycline derivative that exerts multiple anti-inflammatory effects, including microglial inhibition, exhibited increased apomorphine-induced rotational behavior and degeneration of DA neurons after 6-OHDA injections. The same treatment also increased TNF-α release and potentiated NF-κB activation in the SN of gp91phox-/--lesioned mice. Our results demonstrate for the first time that inhibition of microglial cells increases the susceptibility of gp91phox-/- 6-OHDA lesioned mice to develop PD. Blockade of microglia leads to NF-κB activation and TNF-α release into the SN of gp91phox-/- 6-OHDA lesioned mice, a likely mechanism whereby gp91phox-/- 6-OHDA lesioned mice may be more susceptible to develop PD after microglial cell inhibition. Nox2 adds an essential level of regulation to signaling pathways underlying the inflammatory response after PD induction.

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.