Deciphering tissue-induced Klebsiella pneumoniae lipid A structure.

The outcome of an infection depends on host recognition of the pathogen, hence leading to the activation of signaling pathways controlling defense responses. A long-held belief is that the modification of the lipid A moiety of the lipopolysaccharide could help Gram-negative pathogens to evade innate immunity. However, direct evidence that this happens in vivo is lacking. Here we report the lipid A expressed in the tissues of infected mice by the human pathogen Klebsiella pneumoniae. Our findings demonstrate that Klebsiella remodels its lipid A in a tissue-dependent manner. Lipid A species found in the lungs are consistent with a 2-hydroxyacyl-modified lipid A dependent on the PhoPQ-regulated oxygenase LpxO. The in vivo lipid A pattern is lost in minimally passaged bacteria isolated from the tissues. LpxO-dependent modification reduces the activation of inflammatory responses and mediates resistance to antimicrobial peptides. An lpxO mutant is attenuated in vivo thereby highlighting the importance of this lipid A modification in Klebsiella infection biology. Colistin, one of the last options to treat multidrug-resistant Klebsiella infections, triggers the in vivo lipid A pattern. Moreover, colistin-resistant isolates already express the in vivo lipid A pattern. In these isolates, LpxO-dependent lipid A modification mediates resistance to colistin. Deciphering the lipid A expressed in vivo opens the possibility of designing novel therapeutics targeting the enzymes responsible for the in vivo lipid A pattern.

Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling.

Klebsiella pneumoniae is an etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group has shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-κB canonical pathway. Our results revealed that K. pneumoniae capsule polysaccharide (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-of-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-κB. Two gene ontology categories accounted for half of the loci identified in the screening: metabolism and transport genes (32% of the mutants) and envelope-related genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression, which in turn underlined the NF-κB activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of the immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS O-polysaccharide and T2SS mutant-induced responses were dependent on TLR2-TLR4-MyD88 activation suggested that LPS O-polysaccharide and PulA perturbed Toll-like receptor (TLR)-dependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS O-polysaccharide and PulA T2SS could be new targets for the design of new antimicrobials. Increasing TLR-governed defense responses might provide also selective alternatives for the management of K. pneumoniae pneumonia.

Klebsiella pneumoniae survives within macrophages by avoiding delivery to lysosomes.

Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that Klebsiella might be able to persist intracellularly within a vacuolar compartment. This study was designed to investigate the interaction between Klebsiella and macrophages. Engulfment of K. pneumoniae was dependent on host cytoskeleton, cell plasma membrane lipid rafts and the activation of phosphoinositide 3-kinase (PI3K). Microscopy studies revealed that K. pneumoniae resides within a vacuolar compartment, the Klebsiella-containing vacuole (KCV), which traffics within vacuoles associated with the endocytic pathway. In contrast to UV-killed bacteria, the majority of live bacteria did not co-localize with markers of the lysosomal compartment. Our data suggest that K. pneumoniae triggers a programmed cell death in macrophages displaying features of apoptosis. Our efforts to identify the mechanism(s) whereby K. pneumoniae prevents the fusion of the lysosomes to the KCV uncovered the central role of the PI3K-Akt-Rab14 axis to control the phagosome maturation. Our data revealed that the capsule is dispensable for Klebsiella intracellular survival if bacteria were not opsonized. Furthermore, the environment found by Klebsiella within the KCV triggered the down-regulation of the expression of cps. Altogether, this study proves evidence that K. pneumoniae survives killing by macrophages by manipulating phagosome maturation that may contribute to Klebsiella pathogenesis.

Genome Expression Profiling-Based Identification and Administration Efficacy of Host-Directed Antimicrobial Drugs against Respiratory Infection by Nontypeable Haemophilus influenzae.

Therapies that are safe, effective, and not vulnerable to developing resistance are highly desirable to counteract bacterial infections. Host-directed therapeutics is an antimicrobial approach alternative to conventional antibiotics based on perturbing host pathways subverted by pathogens during their life cycle by using host-directed drugs. In this study, we identified and evaluated the efficacy of a panel of host-directed drugs against respiratory infection by nontypeable Haemophilus influenzae (NTHi). NTHi is an opportunistic pathogen that is an important cause of exacerbation of chronic obstructive pulmonary disease (COPD). We screened for host genes differentially expressed upon infection by the clinical isolate NTHi375 by analyzing cell whole-genome expression profiling and identified a repertoire of host target candidates that were pharmacologically modulated. Based on the proposed relationship between NTHi intracellular location and persistence, we hypothesized that drugs perturbing host pathways used by NTHi to enter epithelial cells could have antimicrobial potential against NTHi infection. Interfering drugs were tested for their effects on bacterial and cellular viability, on NTHi-epithelial cell interplay, and on mouse pulmonary infection. Glucocorticoids and statins lacked in vitro and/or in vivo efficacy. Conversely, the sirtuin-1 activator resveratrol showed a bactericidal effect against NTHi, and the PDE4 inhibitor rolipram showed therapeutic efficacy by lowering NTHi375 counts intracellularly and in the lungs of infected mice. PDE4 inhibition is currently prescribed in COPD, and resveratrol is an attractive geroprotector for COPD treatment. Together, these results expand our knowledge of NTHi-triggered host subversion and frame the antimicrobial potential of rolipram and resveratrol against NTHi respiratory infection.

Klebsiella pneumoniae targets an EGF receptor-dependent pathway to subvert inflammation.

The NF-κB transcriptional factor plays a key role governing the activation of immune responses. Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by lacking an early inflammatory response. Recently, we have demonstrated that Klebsiella antagonizes the activation of NF-κB via the deubiquitinase CYLD. In this work, by applying a high-throughput siRNA gain-of-function screen interrogating the human kinome, we identified 17 kinases that when targeted by siRNA restored IL-1ß-dependent NF-κB translocation in infected cells. Further characterization revealed that K. pneumoniae activates an EGF receptor (EGFR)-phosphatidylinositol 3-OH kinase (PI3K)-AKT-PAK4-ERK-GSK3ß signalling pathway to attenuate the cytokine-dependent nuclear translocation of NF-κB. Our data also revealed that CYLD is a downstream effector of K. pneumoniae-induced EGFR-PI3K-AKT-PAK4-ERK-GSK3ß signalling pathway. Our efforts to identify the bacterial factor(s)responsible for EGFR activation demonstrate that a capsule (CPS) mutant did not activate EGFR hence suggesting that CPS could mediate the activation of EGFR. Supporting this notion, purified CPS did activate EGFR as well as the EGFR-dependent PI3K-AKT-PAK4-ERK-GSK3ß signalling pathway. CPS-mediated EGFR activation was dependent on a TLR4-MyD88-c-SRC-dependent pathway. Several promising drugs have been developed to antagonize this cascade. We propose that agents targeting this signalling pathway might provide selective alternatives for the management of K. pneumoniae pneumonias.

Mitigating Age-Related Cognitive Decline and Oxidative Status in Rats Treated with Catechin and Polyphenon-60.

Aging is a normal physiological process influenced by the combination of multiple mechanisms, primarily oxidative stress and neuroinflammation, which impact general physiology and brain function. Phenolic compounds have demonstrated the ability to slow down the aging process of the brain due to their antioxidant and anti-inflammatory effects. This study assessed the protective properties of catechin and polyphenon-60 in non-pathologically aged rats regarding visuo-spatial learning and the oxidative status of the frontal cortex. Old animals were treated with catechin or green tea extract (polyphenon-60) for 36 days, daily. Healthy old and young rats were used as controls. During the first training phase, treated rats executed the test better, locating the target in less time compared with the controls. Biomarkers of oxidative stress (catalase activities, superoxide dismutase, glutathione reductase, and glutathione S-transferase) were reduced in the brain of old animals, although their activities were partially improved after both antioxidant treatments. Furthermore, the rise in the production of reactive oxygen species and malondialdehyde levels-a marker of lipid peroxidation-in the frontal cortex of aged animals was significantly ameliorated after the interventions. In conclusion, old rats exhibited enhanced cognitive function and reduced stress levels following the administration of catechin and polyphenon-60.

Modeling Klebsiella pneumoniae pathogenesis by infection of the wax moth Galleria mellonella.

The implementation of infection models that approximate human disease is essential for understanding pathogenesis at the molecular level and for testing new therapies before they are entered into clinical stages. Insects are increasingly being used as surrogate hosts because they share, with mammals, essential aspects of the innate immune response to infections. We examined whether the larva of the wax moth Galleria mellonella could be used as a host model to conceptually approximate Klebsiella pneumoniae-triggered pneumonia. We report that the G. mellonella model is capable of distinguishing between pathogenic and nonpathogenic Klebsiella strains. Moreover, K. pneumoniae infection of G. mellonella models some of the known features of Klebsiella-induced pneumonia, i.e., cell death associated with bacterial replication, avoidance of phagocytosis by phagocytes, and the attenuation of host defense responses, chiefly the production of antimicrobial factors. Similar to the case for the mouse pneumonia model, activation of innate responses improved G. mellonella survival against subsequent Klebsiella challenge. Virulence factors necessary in the mouse pneumonia model were also implicated in the Galleria model. We found that mutants lacking capsule polysaccharide, lipid A decorations, or the outer membrane proteins OmpA and OmpK36 were attenuated in Galleria. All mutants activated G. mellonella defensive responses. The Galleria model also allowed us to monitor Klebsiella gene expression. The expression levels of cps and the loci implicated in lipid A remodeling peaked during the first hours postinfection, in a PhoPQ- and PmrAB-governed process. Taken together, these results support the utility of G. mellonella as a surrogate host for assessing infections with K. pneumoniae.

Impact of Gut Microbiota in Brain Ageing: Polyphenols as Beneficial Modulators.

Brain ageing is a complex physiological process that includes several mechanisms. It is characterized by neuronal/glial dysfunction, alterations in brain vasculature and barriers, and the decline in brain repair systems. These disorders are triggered by an increase in oxidative stress and a proinflammatory state, without adequate antioxidant and anti-inflammatory systems, as it occurs in young life stages. This state is known as inflammaging. Gut microbiota and the gut-brain axis (GBA) have been associated with brain function, in a bidirectional communication that can cause loss or gain of the brain's functionality. There are also intrinsic and extrinsic factors with the ability to modulate this connection. Among the extrinsic factors, the components of diet, principally natural components such as polyphenols, are the most reported. The beneficial effects of polyphenols in brain ageing have been described, mainly due to their antioxidants and anti-inflammatory properties, including the modulation of gut microbiota and the GBA. The aim of this review was, by following the canonical methodology for a state-of-the-art review, to compose the existing evidenced picture of the impact of the gut microbiota on ageing and their modulation by polyphenols as beneficial molecules against brain ageing.

Molecular basis of Yersinia enterocolitica temperature-dependent resistance to antimicrobial peptides.

Antimicrobial peptides (APs) belong to the arsenal of weapons of the innate immune system against infections. In the case of gram-negative bacteria, APs interact with the anionic lipid A moiety of the lipopolysaccharide (LPS). In yersiniae most virulence factors are temperature regulated. Studies from our laboratory demonstrated that Yersinia enterocolitica is more susceptible to polymyxin B, a model AP, when grown at 37°C than at 22°C (J. A. Bengoechea, R. Díaz, and I. Moriyón, Infect. Immun. 64:4891-4899, 1996), and here we have extended this observation to other APs, not structurally related to polymyxin B. Mechanistically, we demonstrate that the lipid A modifications with aminoarabinose and palmitate are downregulated at 37°C and that they contribute to AP resistance together with the LPS O-polysaccharide. Bacterial loads of lipid A mutants in Peyer's patches, liver, and spleen of orogastrically infected mice were lower than those of the wild-type strain at 3 and 7 days postinfection. PhoPQ and PmrAB two-component systems govern the expression of the loci required to modify lipid A with aminoarabinose and palmitate, and their expressions are also temperature regulated. Our findings support the notion that the temperature-dependent regulation of loci controlling lipid A modifications could be explained by H-NS-dependent negative regulation alleviated by RovA. In turn, our data also demonstrate that PhoPQ and PmrAB regulate positively the expression of rovA, the effect of PhoPQ being more important. However, rovA expression reached wild-type levels in the phoPQ pmrAB mutant background, hence indicating the existence of an unknown regulatory network controlling rovA expression in this background.

Klebsiella pneumoniae outer membrane protein A is required to prevent the activation of airway epithelial cells.

Outer membrane protein A (OmpA) is a class of proteins highly conserved among the Enterobacteriaceae family and throughout evolution. Klebsiella pneumoniae is a capsulated gram-negative pathogen. It is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by a lack of an early inflammatory response. Data from our laboratory indicate that K. pneumoniae CPS helps to suppress the host inflammatory response. However, it is unknown whether K. pneumoniae employs additional factors to modulate host inflammatory responses. Here, we report that K. pneumoniae OmpA is important for immune evasion in vitro and in vivo. Infection of A549 and normal human bronchial cells with 52OmpA2, an ompA mutant, increased the levels of IL-8. 52145-Δwca(K2)ompA, which does not express CPS and ompA, induced the highest levels of IL-8. Both mutants could be complemented. In vivo, 52OmpA2 induced higher levels of tnfα, kc, and il6 than the wild type. ompA mutants activated NF-κB, and the phosphorylation of p38, p44/42, and JNK MAPKs and IL-8 induction was via NF-κB-dependent and p38- and p44/42-dependent pathways. 52OmpA2 engaged TLR2 and -4 to activate NF-κB, whereas 52145-Δwca(K2)ompA activated not only TLR2 and TLR4 but also NOD1. Finally, we demonstrate that the ompA mutant is attenuated in the pneumonia mouse model. The results of this study indicate that K. pneumoniae OmpA contributes to attenuate airway cell responses. This may facilitate pathogen survival in the hostile environment of the lung.

Host cell kinases, α5 and ß1 integrins, and Rac1 signalling on the microtubule cytoskeleton are important for non-typable Haemophilus influenzae invasion of respiratory epithelial cells.

Non-typable Haemophilus influenzae (NTHi) is a common commensal of the human nasopharynx, but causes opportunistic infection when the respiratory tract is compromised by infection or disease. The ability of NTHi to invade epithelial cells has been described, but the underlying molecular mechanisms are poorly characterized. We previously determined that NTHi promotes phosphorylation of the serine-threonine kinase Akt in A549 human lung epithelial cells, and that Akt phosphorylation and NTHi cell invasion are prevented by inhibition of phosphoinositide 3-kinase (PI3K). Because PI3K-Akt signalling is associated with several host cell networks, the purpose of the current study was to identify eukaryotic molecules important for NTHi epithelial invasion. We found that inhibition of Akt activity reduced NTHi internalization; differently, bacterial entry was increased by phospholipase Cγ1 inhibition but was not affected by protein kinase inhibition. We also found that α5 and ß1 integrins, and the tyrosine kinases focal adhesion kinase and Src, are important for NTHi A549 cell invasion. NTHi internalization was shown to be favoured by activation of Rac1 guanosine triphosphatase (GTPase), together with the guanine nucleotide exchange factor Vav2 and the effector Pak1. Also, Pak1 might be associated with inactivation of the microtubule destabilizing agent Op18/stathmin, to facilitate microtubule polymerization and NTHi entry. Conversely, inhibition of RhoA GTPase and its effector ROCK increased the number of internalized bacteria. Src and Rac1 were found to be important for NTHi-triggered Akt phosphorylation. An increase in host cyclic AMP reduced bacterial entry, which was linked to protein kinase A. These findings suggest that NTHi finely manipulates host signalling molecules to invade respiratory epithelial cells.

Klebsiella pneumoniae subverts the activation of inflammatory responses in a NOD1-dependent manner.

Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Subversion of inflammation is essential for pathogen survival during infection. Evidence indicates that K. pneumoniae infections are characterized by lacking an early inflammatory response although the molecular bases are currently unknown. Here we unveil a novel strategy employed by a pathogen to counteract the activation of inflammatory responses. K. pneumoniae attenuates pro-inflammatory mediators-induced IL-8 secretion. Klebsiella antagonizes the activation of NF-κB via the deubiquitinase CYLD and blocks the phosphorylation of mitogen-activated protein kinases (MAPKs) via the MAPK phosphatase MKP-1. Our studies demonstrate that K. pneumoniae has evolved the capacity to manipulate host systems dedicated to control the immune balance. To exert this anti-inflammatory effect, Klebsiella engages NOD1. In NOD1 knock-down cells, Klebsiella neither induces the expression of CYLD and MKP-1 nor blocks the activation of NF-κB and MAPKs. Klebsiella inhibits Rac1 activation; and inhibition of Rac1 activity triggers a NOD1-mediated CYLD and MKP-1 expression which in turn attenuates IL-1ß-induced IL-8 secretion. A capsule (CPS) mutant does not attenuate the inflammatory response. However, purified CPS neither reduces IL-1ß-induced IL-8 secretion nor induces the expression of CYLD and MKP-1 thereby indicating that CPS is necessary but not sufficient to attenuate inflammation.

Effects of pollutants and microplastics ingestion on oxidative stress and monoaminergic activity of seabream brains.

Nowadays, microplastics (MPs) and adsorbed pollutants are considered a global thread to marine ecosystems. This study describes the effects of pollutants and MPs ingestion on fish brains through the assessment of oxidative stress biomarkers and monoaminergic neurotransmitters using gilthead seabream (Sparus aurata) as fish model. Juveniles were experimentally exposed to three different dietary treatments for 90 days: Control treatment (C) consisted of standard feed; Virgin treatment (V) contained feed enriched with 10% of MPs; and Exposed treatment (E) consisted of feed with 10% of MPs that were exposed to seawater in an anthropogenically impacted area for 2 months in order to enrich the plastic with the pollutants within the water column. Sampling was made at the start of the experiment (T0), at the end of the dietary treatments (T90) and after a posterior detoxification period of 30 days (T120). Results evidenced that a MPs and pollutants enriched diet increases the activity of some of the oxidative stress biomarkers (e.g. CAT and GST), and it was shown for the first time alterations on dopaminergic and serotonergic system activity on seabream brains, indicating potential neurofunctional effects associated to MPs and pollutants ingestion. In addition, results showed a tendency to recover enzymatic and brain monoaminergic neurotransmitter levels after a 30-day detoxification period. In conclusion, MPs and pollutants exposure for 90 days induced oxidative stress and changes on monoaminergic activity in the brain of S. aurata.

Analysis of the networks controlling the antimicrobial-peptide-dependent induction of Klebsiella pneumoniae virulence factors.

Antimicrobial peptides (APs) impose a threat to the survival of pathogens, and it is reasonable to postulate that bacteria have developed strategies to counteract them. Polymyxins are becoming the last resort to treat infections caused by multidrug-resistant Gram-negative bacteria and, similar to APs, they interact with the anionic lipopolysaccharide. Given that polymyxins and APs share the initial target, it is possible that bacterial defense mechanisms against polymyxins will be also effective against host APs. We sought to determine whether exposure to polymyxin will increase Klebsiella pneumoniae resistance to host APs. Indeed, exposure of K. pneumoniae to polymyxin induces cross-resistance not only to polymyxin itself but also to APs present in the airways. Polymyxin treatment upregulates the expression of the capsule polysaccharide operon and the loci required to modify the lipid A with aminoarabinose and palmitate with a concomitant increase in capsule and lipid A species containing such modifications. Moreover, these surface changes contribute to APs resistance and also to polymyxin-induced cross-resistance to APs. Bacterial loads of lipid A mutants in trachea and lungs of intranasally infected mice were lower than those of wild-type strain. PhoPQ, PmrAB, and the Rcs system govern polymyxin-induced transcriptional changes, and there is a cross talk between PhoPQ and the Rcs system. Our findings support the notion that Klebsiella activates a defense program against APs that is controlled by three signaling systems. Therapeutic strategies directed to prevent the activation of this program could be a new approach worth exploring to facilitate the clearance of the pathogen from the airways.

Neurochemical and Cognitive Beneficial Effects of Moderate Physical Activity and Catechin in Aged Rats.

A healthy aging process is a requirement for good life quality. A relationship between physical activity, the consumption of antioxidants and brain health has been stablished via the activation of pathways that reduce the harmful effects of oxidative stress, by inducing enzymes such as SIRT1, which is a protector of brain function. We analyzed the cognitive and neurochemical effects of applying physical exercise in elderly rats, alone or in combination with the antioxidant catechin. Several tests of spatial and episodic memory and motor coordination were evaluated. In addition, brain monoaminergic neurotransmitters and SIRT1 protein levels were assessed in the brains of the same rats. The results show that physical activity by itself improved age-related memory and learning deficits, correlating with the restoration of brain monoaminergic neurotransmitters and SIRT1 protein levels in the hippocampus. The administration of the antioxidant catechin along with the exercise program enhanced further the monoaminergic pathways, but not the other parameters studied. These results agree with previous reports revealing a neuroprotective effect of physical activity, probably based on its ability to improve the redox status of the brain, demonstrating that exercise at an advanced age, combined with the consumption of antioxidants, could produce favorable effects in terms of brain health.

Klebsiella pneumoniae capsule polysaccharide impedes the expression of beta-defensins by airway epithelial cells.

Human beta-defensins (hBDs) contribute to the protection of the respiratory tract against pathogens. It is reasonable to postulate that pathogens have developed countermeasures to resist them. Klebsiella pneumoniae capsule polysaccharide (CPS), but not the lipopolysaccharide O antigen, mediated resistance against hBD1 and hBD2. hBD3 was the most potent hBD against Klebsiella. We investigated the possibility that as a strategy for survival in the lung, K. pneumoniae may not activate the expression of hBDs. Infection of A549 and normal human bronchial cells with 52145-Deltawca(K2), a CPS mutant, increased the expression of hBD2 and hBD3. Neither the wild type nor the lipopolysaccharide O antigen mutant increased the expression of hBDs. In vivo, 52145-Deltawca(K2) induced higher levels of mBD4 and mBD14, possible mouse orthologues of hBD2 and hBD3, respectively, than the wild type. 52145-Deltawca(K2)-dependent upregulation of hBD2 occurred via NF-kappaB and mitogen-activated protein kinases (MAPKs) p44/42, Jun N-terminal protein kinase (JNK)-dependent pathways. The increase in hBD3 expression was dependent on the MAPK JNK. 52145-Deltawca(K2) engaged Toll-like receptors 2 and 4 (TLR2 and TLR4) to activate hBD2, whereas hBD3 expression was dependent on NOD1. K. pneumoniae induced the expression of CYLD and MKP-1, which act as negative regulators for 52145-Deltawca(K2)-induced expression of hBDs. Bacterial engagement of pattern recognition receptors induced CYLD and MKP-1, which may initiate the attenuation of proinflammatory pathways. The results of this study indicate that K. pneumoniae CPS not only protects the pathogen from the bactericidal action of defensins but also impedes their expression. These features of K. pneumoniae CPS may facilitate pathogen survival in the hostile environment of the lung.

Chronic melatonin treatment and its precursor L-tryptophan improve the monoaminergic neurotransmission and related behavior in the aged rat brain.

Melatonin has an important role in the aging process as a potential drug to relieve oxidative damage, a likely cause of age-associated brain dysfunction. As age advances, the nocturnal production of melatonin decreases potentially causing physiological alterations. The present experiments were performed to study in vivo the effects of exogenously administered melatonin chronically on monoaminergic central neurotransmitters serotonin (5-HT), dopamine (DA) and norepinephrine (NE) and behavioral tests in old rats. The accumulation of 5-hydroxy-tryptophan (5-HTP) and L-3,4-dihydroxyphenylalanine (DOPA) after decarboxylase inhibition was used as a measure of the rate of tryptophan and tyrosine hydroxylation in rat brain. Also neurotransmitters 5-HT, DA and NE and some metabolites were quantified by HPLC. In control rats, an age-related decline was observed in neurochemical parameters. However, chronic administration of melatonin (1 mg/kg/day, diluted in drinking water, 4 wk) significantly reversed the age-induced deficits in all the monoaminergic neurotransmitters studied. Also, neurochemical parameters were analyzed after administration of melatonin biosynthesis precursor L-tryptophan (240 mg/kg/day, i.p., at night for 4 wk) revealing similar improvement effects to those induced by melatonin. Behavioral data corresponded well with the neurochemical findings since spatial memory test in radial-maze and motor coordination in rota-rod were significantly improved after chronic melatonin treatment. In conclusion, these in vivo findings suggest that melatonin and L-tryptophan treatments exert a long-term effect on the 5-HT, DA and NE neurotransmission by enhancing monoamine synthesis in aged rats, which might improve the age-dependent deficits in cognition and motor coordination.

Chronic Polyphenon-60 or Catechin Treatments Increase Brain Monoamines Syntheses and Hippocampal SIRT1 Levels Improving Cognition in Aged Rats.

Polyphenolic compounds from green tea have great interest due to its large consumption and therapeutic potential on the age-associated brain decline. The current work compares a similar dose regimen of a whole-green-tea extract and catechin in old rats over the course of 36 days. Results showed a significant improvement in visuo-spatial working memory and episodic memory of old rats after polyphenolic compounds administration assessed by behavioral tests. No effects were observed on the age-associated motor coordination decline. Statistically, results were correlated with significant improvements, mainly in hippocampal and striatal noradrenergic and serotonergic systems, but also with the striatal dopaminergic system. Both polyphenolic treatments also reverted the age-associated reduction of the neuroinflammation by modulating protein sirtuin 1 (SIRT1) expression in hippocampus, but no effects were observed in the usual reduction of the histone-binding protein RBAP46/48 protein linked to aging. These results are in line with previous ones obtained with other polyphenolic compounds, suggesting a general protective effect of all these compounds on the age-associated brain decline, pointing to a reduction of the oxidative stress and neuroinflammatory status reduction as the leading mechanisms. Results also reinforce the relevance of SIRT1-mediated mechanism on the neuroprotective effect and rule out the participation of RBAP46/48 protein.

Cognitive and Neurochemical Changes Following Polyphenol-Enriched Diet in Rats.

Dietary recommendations are frequently developed based on nutrient deficiency or prevention of disease, but less attention has been paid to the dietary guidelines to promote brain health. Active and healthy aging is a prerequisite for improving quality of life as people age, and evidence is establishing a relationship between diet and brain health. This work studied the effect of a diet based on foods rich in antioxidants, especially polyphenols, in rats, three days a week for 20 months starting at 14 months. Behavioral analysis testing working memory, spatial and episodic memory, as well as brain monoaminergic neurotransmitters involved in these processes but also in general brain health were analyzed. In addition, hippocampal SIRT1 protein which has an important role in regulating normal brain function was evaluated. The results show that long-term intake of polyphenol-enriched diet improves memory and learning, correlating with restoration of brain monoaminergic neurotransmitters and hippocampal SIRT1 levels in aged rats. These results agree with reports revealing a neuroprotective effect of different polyphenolic compounds on age-related brain decline, based on its antioxidant and anti-inflammatory properties; and demonstrate that consumption of antioxidant-rich foods, a few days a week, gives good long-term results in terms of brain health.

Grape Polyphenols Ameliorate Muscle Decline Reducing Oxidative Stress and Oxidative Damage in Aged Rats.

A large number of studies have demonstrated the implication of oxidative stress (OxS) in the pathogenesis of ageing-related muscle decline and atrophy. The key mechanism is related to the OxS-induced production of free radicals, with the consequent increase in oxidative damage, resulting in affected muscle quality and strength. The present study aimed to evaluate the efficacy of a grape polyphenol-based nutraceutical formulation (Taurisolo®) in reducing the OxS in muscle of aged rats. A group of 16 aged (20 months) rats were orally administered with Taurisolo® (n = 8; 100 mg/kg Taurisolo®) or placebo (n = 8; 50 mg/kg maltodextrin); an additional group of eight young (three months) rats were also treated with placebo. All the treatments were orally administered for 30 days. The activities of antioxidant enzymes, the levels of malondialdehyde (MDA) and nitrotyrosine (N-Tyr) and the expression of OxS- and inflammation-related genes were evaluated on the gastrocnemius muscle. In muscle samples of the treated-group, increased activity of antioxidant enzymes, reduced MDA and N-Tyr levels and increased expression of antioxidant and anti-inflammatory genes were observed in respect to the placebo. Data herein presented suggest that the chronic treatment with Taurisolo® significantly reduces oxidative damage and improves muscle performance in aged rats.