TNFR1-mediated neuroinflammation is necessary for respiratory deficits observed in 6-hydroxydopamine mouse model of Parkinsons Disease.

Parkinson's Disease (PD) is characterized by classic motor symptoms related to movement, but PD patients can experience symptoms associated with impaired autonomic function, such as respiratory disturbances. Functional respiratory deficits are known to be associated with brainstem neurodegeneration in the mice model of PD induced by 6-hydroxydopamine (6-OHDA). Understanding the causes of neuronal death is essential for identifying specific targets to prevent degeneration. Many mechanisms can explain why neurons die in PD, and neuroinflammation is one of them. To test the influence of inflammation, mediated by microglia and astrocytes cells, in the respiratory disturbances associated with brainstem neurons death, we submitted wild-type (WT) and TNF receptor 1 (TNFR1) knockout male mice to the 6-OHDA model of PD. Also, male C57BL/6 animals were induced using the same PD model and treated with minocycline (45 mg/kg), a tetracycline antibiotic with anti-inflammatory properties. We show that degeneration of brainstem areas such as the retrotrapezoid nucleus (RTN) and the pre-Botzinger Complex (preBotC) were prevented in both protocols. Notably, respiratory disturbances were no longer observed in the animals where inflammation was suppressed. Thus, the data demonstrate that inflammation is responsible for the breathing impairment in the 6-OHDA-induced PD mouse model.

Toll-like Receptor 4 Signaling is Critical for the Adaptive Cellular Stress Response Effects Induced by Intermittent Fasting in the Mouse Brain.

Among different kinds of dietary energy restriction, intermittent fasting (IF) has been considered a dietary regimen which causes a mild stress to the organism. IF can stimulate proteins and signaling pathways related to cell stress that can culminate in the increase of the body resistance to severe stress conditions. Energy intake reduction induced by IF can induce modulation of receptors, kinases, and phosphatases, which in turn can modulate the activation of transcription factors such as NF-E2-related factor 2 (NRF2) and cAMP response element-binding (CREB) which regulate the transcription of genes related to the translation of proteins such as growth factors: brain-derived neurotrophic factor (BDNF), chaperone proteins: heat shock proteins (HSP), and so on. It has been shown that toll-like receptors (TLRs) are important molecules in innate immune response which are present not only in the periphery but also in neurons and glial cells. In central nervous system, TLRs can exert functions related to set up responses to infection, as well as influence neural progenitor cell proliferation and differentiation, being involved in cognitive parameters such as learning and memory. Little is known about the involvement of TLR4 on the beneficial effects induced by IF protocol. The present work investigated the effects of IF on memory and on the signaling mechanisms associated with NRF2 and CREB in Tlr4 knockout mice. The results suggest that TLR4 participates in the modulatory effects of IF on oxidative stress levels, on the transcription factors CREB and NRF2, and on BDNF and HSP90 expressions in hippocampus.

Activity-dependent neuronal Klotho enhances astrocytic aerobic glycolysis.

Mutations of the ß-glucuronidase protein α-Klotho have been associated with premature aging, and altered cognitive function. Although highly expressed in specific areas of the brain, Klotho functions in the central nervous system remain unknown. Here, we show that cultured hippocampal neurons respond to insulin and glutamate stimulation by elevating Klotho protein levels. Conversely, AMPA and NMDA antagonism suppress neuronal Klotho expression. We also provide evidence that soluble Klotho enhances astrocytic aerobic glycolysis by hindering pyruvate metabolism through the mitochondria, and stimulating its processing by lactate dehydrogenase. Pharmacological inhibition of FGFR1, Erk phosphorylation, and monocarboxylic acid transporters prevents Klotho-induced lactate release from astrocytes. Taken together, these data suggest Klotho is a potential new player in the metabolic coupling between neurons and astrocytes. Neuronal glutamatergic activity and insulin modulation elicit Klotho release, which in turn stimulates astrocytic lactate formation and release. Lactate can then be used by neurons and other cells types as a metabolic substrate.

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-1ß levels. The data reported here indicate a significant role of α2 NKA in regulating central LPS effects, with implications in the associated neuroinflammatory processes.

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, ß, 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-ß. 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.

Intermittent fasting attenuates lipopolysaccharide-induced neuroinflammation and memory impairment.

BACKGROUND: Systemic bacterial infections often result in enduring cognitive impairment and are a risk factor for dementia. There are currently no effective treatments for infection-induced cognitive impairment. Previous studies have shown that intermittent fasting (IF) can increase the resistance of neurons to injury and disease by stimulating adaptive cellular stress responses. However, the impact of IF on the cognitive sequelae of systemic and brain inflammation is unknown. METHODS: Rats on IF for 30 days received 1 mg/kg of lipopolysaccharide (LPS) or saline intravenously. Half of the rats were subjected to behavioral tests and the other half were euthanized two hours after LPS administration and the hippocampus was dissected and frozen for analyses. RESULTS: Here, we report that IF ameliorates cognitive deficits in a rat model of sepsis by a mechanism involving NF-κB activation, suppression of the expression of pro-inflammatory cytokines, and enhancement of neurotrophic support. Treatment of rats with LPS resulted in deficits in cognitive performance in the Barnes maze and inhibitory avoidance tests, without changing locomotor activity, that were ameliorated in rats that had been maintained on the IF diet. IF also resulted in reduced levels of mRNAs encoding the LPS receptor TLR4 and inducible nitric oxide synthase (iNOS) in the hippocampus. Moreover, IF prevented LPS-induced elevation of IL-1α, IL-1ß and TNF-α levels, and prevented the LPS-induced reduction of BDNF levels in the hippocampus. IF also significantly attenuated LPS-induced elevations of serum IL-1ß, IFN-γ, RANTES, TNF-α and IL-6 levels. CONCLUSIONS: Taken together, our results suggest that IF induces adaptive responses in the brain and periphery that can suppress inflammation and preserve cognitive function in an animal model of systemic bacterial infection.

TLR4-dependent metabolic changes are associated with cognitive impairment in an animal model of type 1 diabetes.

We investigated the role of Toll-like receptor 4 (TLR4), a major mediator of innate immune responses, on cognitive performance in a type 1 diabetes model (T1D). After administration of streptozotocin, both TLR4 knockout (TLR4 KO) and wild type (WT) diabetic mice displayed metabolic alterations similar to those observed in T1D patients, including increased levels of glucose, cholesterol, triglycerides and ketones. T1D mice exhibited cognitive impairment which was less severe in TLR4 KO mice compared to WT mice. WT mice with higher glucose and those with higher triglyceride levels exhibited significantly more anxiety and impaired memory compared to those with lower levels of glucose and triglycerides; these correlations were absent in TLR4 KO mice. Additional findings suggest roles for TLR4 signaling in modifying the expression of enzymes involved in energy metabolism in brain cells in the setting of T1D. Our data show that TLR4 contributes to the negative impact of T1D on anxiety and cognition.

Sonic hedgehog promotes autophagy in hippocampal neurons.

The Sonic hedgehog (Shh) signaling pathway is well known in patterning of the neural tube during embryonic development, but its emerging role in differentiated neurons is less understood. Here we report that Shh enhances autophagy in cultured hippocampal neurons. Microarray analysis reveals the upregulation of multiple autophagy-related genes in neurons in response to Shh application. Through analysis of the autophagy-marker LC3 by immunoblot analysis and immunocytochemistry, we confirm activation of the autophagy pathway in Shh-exposed neurons. Using electron microscopy, we find autophagosomes and associated structures with a wide range of morphologies in synaptic terminals of Shh-exposed neurons. Moreover, we show that Shh-triggered autophagy depends on class III Phosphatidylinositol 3-kinase complexes (PtdIns3K). These results identify a link between Shh and autophagy pathways and, importantly, provide a lead for further understanding the physiology of Shh signaling activity in neurons.

Evidence for miR-181 involvement in neuroinflammatory responses of astrocytes.

Inflammation is a common component of acute injuries of the central nervous system (CNS) such as ischemia, and degenerative disorders such as Alzheimer's disease. Glial cells play important roles in local CNS inflammation, and an understanding of the roles for microRNAs in glial reactivity in injury and disease settings may therefore lead to the development of novel therapeutic interventions. Here, we show that the miR-181 family is developmentally regulated and present in high amounts in astrocytes compared to neurons. Overexpression of miR-181c in cultured astrocytes results in increased cell death when exposed to lipopolysaccharide (LPS). We show that miR-181 expression is altered by exposure to LPS, a model of inflammation, in both wild-type and transgenic mice lacking both receptors for the inflammatory cytokine TNF-α. Knockdown of miR-181 enhanced LPS-induced production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß, IL-8) and HMGB1, while overexpression of miR-181 resulted in a significant increase in the expression of the anti-inflammatory cytokine IL-10. To assess the effects of miR-181 on the astrocyte transcriptome, we performed gene array and pathway analysis on astrocytes with reduced levels of miR-181b/c. To examine the pool of potential miR-181 targets, we employed a biotin pull-down of miR-181c and gene array analysis. We validated the mRNAs encoding MeCP2 and X-linked inhibitor of apoptosis as targets of miR-181. These findings suggest that miR-181 plays important roles in the molecular responses of astrocytes in inflammatory settings. Further understanding of the role of miR-181 in inflammatory events and CNS injury could lead to novel approaches for the treatment of CNS disorders with an inflammatory component.

Naphthazarin protects against glutamate-induced neuronal death via activation of the Nrf2/ARE pathway.

Nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is an important cellular stress response pathway involved in neuroprotection. We previously screened several natural phytochemicals and identified plumbagin as a novel activator of the Nrf2/ARE pathway that can protect neurons against ischemic injury. Here we extended our studies to natural and synthetic derivatives of plumbagin. We found that 5,8-dimethoxy-1,4-naphthoquinone (naphthazarin) is a potent activator of the Nrf2/ARE pathway, up-regulates the expression of Nrf2-driven genes in primary neuronal and glial cultures, and protects neurons against glutamate-induced excitotoxicity.

Nicotinamide forestalls pathology and cognitive decline in Alzheimer mice: evidence for improved neuronal bioenergetics and autophagy procession.

Impaired brain energy metabolism and oxidative stress are implicated in cognitive decline and the pathologic accumulations of amyloid ß-peptide (Aß) and hyperphosphorylated tau in Alzheimer's disease (AD). To determine whether improving brain energy metabolism will forestall disease progress in AD, the impact of the ß-nicotinamide adenine dinucleotide precursor nicotinamide on brain cell mitochondrial function and macroautophagy, bioenergetics-related signaling, and cognitive performance were studied in cultured neurons and in a mouse model of AD. Oxidative stress resulted in decreased mitochondrial mass, mitochondrial degeneration, and autophagosome accumulation in neurons. Nicotinamide preserved mitochondrial integrity and autophagy function, and reduced neuronal vulnerability to oxidative/metabolic insults and Aß toxicity. ß-Nicotinamide adenine dinucleotide biosynthesis, autophagy, and phosphatidylinositol-3-kinase signaling were required for the neuroprotective action of nicotinamide. Treatment of 3xTgAD mice with nicotinamide for 8 months resulted in improved cognitive performance, and reduced Aß and hyperphosphorylated tau pathologies in hippocampus and cerebral cortex. Nicotinamide treatment preserved mitochondrial integrity, and improved autophagy-lysosome procession by enhancing lysosome/autolysosome acidification to reduce autophagosome accumulation. Treatment of 3xTgAD mice with nicotinamide resulted in elevated levels of activated neuroplasticity-related kinases (protein kinase B [Akt] and extracellular signal-regulated kinases) and the transcription factor cyclic adenosine monophosphate (AMP) response element-binding protein in the hippocampus and cerebral cortex. Thus, nicotinamide suppresses AD pathology and cognitive decline in a mouse model of AD by a mechanism involving improved brain bioenergetics with preserved functionality of mitochondria and the autophagy system.

Role of vascular Kinin B1 and B2 receptors in endothelial nitric oxide metabolism.

Kinin B(1) and B(2) receptors play an essential role in inflammatory process and cardiovascular homeostasis. The present study investigated the vascular reactivity and nitric oxide (NO) generation in the isolated mesenteric arteriolar bed from B(1) (B(1)(-/-)) and B(2) receptor (B(2)(-/-)) knockout mice. Endothelial-dependent relaxation was significantly decreased in arterioles from both B(1)(-/-) and B(2)(-/-) in comparison to wild type (WT) mice, with no differences for endothelial-independent relaxating or vasoconstrictor agents. Plasmatic and vascular NO production were markedly reduced in both B(1)(-/-) and B(2)(-/-). In contrast, in the presence of l-arginine, Ca(2+) and co-factors for the enzyme, NO synthase activity was higher in homogenates of mesenteric vessels of B(1)(-/-) and B(2)(-/-). The present study demonstrated that targeted deletion of B(1) or B(2) receptor gene in mice induces important alterations in the vascular reactivity of resistance vessels and NO metabolism. The severe impairment in the endothelial-mediated vasodilation accompanied by decreased NO bioavailability, despite the augmented NOS activity, strongly indicates an exacerbation of NO inactivation in B(1)(-/-) and B(2)(-/-) vessels. The present data provide valuable information in order to clarify the relevance of kinin receptors in regulating vascular physiology and may point to new approaches regarding its correlation with endothelial dysfunction, oxidative stress and NO availability.

Altered reactivity of gastric fundus smooth muscle in the mouse with targeted disruption of the kinin B1 receptor gene.

Relaxing action of sodium nitroprusside (SNP) was significantly reduced in the stomach fundus of mice lacking the kinin B(1) receptor (B(1)(-/-)). Increased basal cGMP accumulation was correlated with attenuated SNP induced dose-dependent relaxation in B(1)(-/-) when compared with wild type (WT) control mice. These responses to SNP were completely blocked by the guanylate cyclase inhibitor ODQ (10 microM). It was also found that Ca(2+)-dependent, constitutive nitric oxide synthase (cNOS) activity was unchanged but the Ca(2+)-independent inducible NOS (iNOS) activity was greater in B(1)(-/-) mice than in WT animals. Zaprinast (100 microM), a specific phosphodiesterase inhibitor, increased the nitrergic relaxations and the accumulation of the basal as well as the SNP-stimulated cGMP in WT but not in B(1)(-/-) stomach fundus. From these findings it is concluded that the inhibited phosphodiesterase activity and high level of cGMP reduced the resting muscle tone, impairing the relaxant responses of the stomach in B(1)(-/-) mice. In addition, it can be suggested that functional B(2) receptor might be involved in the NO compensatory mechanism associated with the deficiency of kinin B(1) receptor in the gastric tissue of the transgenic mice.

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-kappaB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-kappaB 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-kappaB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-kappaB activation. Inhibition of NF-kappaB 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-kappaB 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.

Apolipoprotein E genotype is related to nitric oxide production in platelets.

The presence of the epsilon4 allele of apolipoprotein E (APOE) is considered a risk factor for sporadic Alzheimer's disease (AD). Our recent data demonstrated that the systemic modulation of oxidative stress in platelets and erythrocytes is disrupted in aging and AD. In this study, the relationship between APOE genotype and oxidative stress markers, both in AD patients and controls, was evaluated. The AD group showed an increase in the content of thiobarbituric acid-reactive substances (TBARS) and in the activities of nitric oxide synthase (NOS) and Na, K-ATPase, when compared to controls. Both groups had a similar cGMP content and superoxide dismutase activity. APOE epsilon4 allele carriers showed higher NOS activity than non-carriers. These results suggest a possible influence of APOE genotype on nitric oxide (NO) production that might enhance the effects of age-related specific factor(s) associated with neurodegenerative disorders.

Biomarkers of oxidative stress and antioxidant status in children born small for gestational age: evidence of lipid peroxidation.

Children born small for gestational age are known to be at increased risk for adult diseases such as hypertension, diabetes, and coronary heart disease. Oxidative stress is a common feature of these pathogenic conditions and can be the key link between size at birth and increased morbidity later in life. The purpose of this study was to analyze the parameters of lipoperoxidation and changes in antioxidant defense system as well as assess their relationship to birth weight. Concentrations of thiobarbituric-acid-reactive-substances and F2-isoprostanes, total antioxidant status, and the activity of both superoxide dismutase and glutathione peroxidase were measured in 65 children (33 boys, 32 girls; ages 8-13 y). Thiobarbituric-acid-reactive-substances and F2-isoprostane levels were significantly elevated in children born small for gestational age. Nevertheless, superoxide dismutase activity was significantly elevated in these children and the levels of both glutathione peroxidase activity and total antioxidant status were unchanged. Moreover, we found that systolic blood pressure was positively associated with thiobarbituric-acid-reactive-substances levels in race- and gender-adjusted models but not in a multivariable regression model. In conclusion, the current study revealed that there is evidence of oxidative stress in children born small for gestational age as supported by increased lipid peroxidation.

Correction of endothelial dysfunction in diabetic female rats by tetrahydrobiopterin and chronic insulin.

Diabetes-induced vascular dysfunction has mainly been studied in males. However, the mechanisms involved may not correspond to those in females. Here we analyzed the effects of tetrahydrobiopterin (BH(4)) and chronic insulin on the physiology of mesenteric arterioles of alloxan-diabetic female rats. The parameters studied were the mesenteric arteriolar reactivity (intravital microscopy), nitric oxide synthase (NOS) activity (conversion of L-arginine to L-citrulline), eNOS gene expression (RT-PCR), NO production (diaminofluorescein), reactive oxygen species (ROS) generation (intravital fluorescence microscopy) and Cu/Zn superoxide dismutase (SOD) activity (spectrophotometry) and gene expression (RT-PCR). The reduced endothelium-dependent vasodilation of diabetic females was corrected by both BH(4) and insulin. NOS activity was decreased by diabetes, but insulin did not correct it. However, NOS expression was not modified by either diabetes or insulin. Arterioles of diabetic rats exhibited lower NO production, which was fully corrected by BH(4) and only partially by insulin. ROS generation was increased in diabetic rats, and both BH(4) and insulin normalized it. Diabetes did not change SOD activity and gene expression. However, insulin increased SOD activity but not its expression. Our data suggest that, similarly to males, endothelial dysfunction in female diabetic rats involves an altered ROS/NO imbalance. In contrast to males, however, insulin does not regulate NOS in the microcirculation of diabetic females.

Tetrahydrobiopterin improves endothelial dysfunction and vascular oxidative stress in microvessels of intrauterine undernourished rats.

In the present study, we investigated the effects of the exogenous application of tetrahydrobiopterin on the endothelium-dependent vasorelaxation and superoxide anion generation in the mesenteric microvessels of intrauterine undernourished rats. In addition, we investigated the presence of peroxynitrite in these rats by evaluation of nitrotyrosine-containing proteins, a stable end-product of peroxynitrite oxidation. For this, female pregnant Wistar rats were fed either normal or 50% of the normal intake diets during the whole gestational period. Male offspring (16 weeks of age) were studied to assess microvascular reactivity, superoxide production using a hydroethidine staining assay, nitric oxide synthase (NOS) activity and nitric oxide (NO) production. Western blot analysis was used to quantify nitrotyrosine-containing proteins and relative multiplex RT-PCR analysis for endothelial NOS (eNOS) mRNA expression. Superfusion with tetrahydrobiopterin significantly decreased superoxide generation and improved vascular function. Intrauterine malnutrition induced a decrement of NOS activity and NO production without affecting the gene expression of eNOS. However, incubation with tetrahydrobiopterin significantly improved NO production after stimulation with acetylcholine or bradykinin in intrauterine undernourished rats. The fact that the nitrotyrosine-containing proteins were increased could, at first sight, suggest that the peroxynitrite is the mediator responsible for the excessive oxidation and depletion of tetrahydrobiopterin. Our study shows that exogenous application of tetrahydrobiopterin leads to a significant improvement of endothelium-dependent vasodilatation, enhanced NO production and decreased superoxide generation in microvessels of intrauterine undernourished rats. Since we found a decrease in NOS activity without an alteration in the gene expression of eNOS, we suggest that impaired NOS-dependent responses of mesenteric arterioles are related to the impairment of tetrahydrobiopterin pathways.