Selective PPAR-Delta/PPAR-Gamma Activation Improves Cognition in a Model of Alzheimer's Disease.

Background: The continuously increasing association of Alzheimer's disease (AD) with increased mortality rates indicates an unmet medical need and the critical need for establishing novel molecular targets for therapeutic potential. Agonists for peroxisomal proliferator activating receptors (PPAR) are known to regulate energy in the body and have shown positive effects against Alzheimer's disease. There are three members of this class (delta, gamma, and alpha), with PPAR-gamma being the most studied, as these pharmaceutical agonists offer promise for AD because they reduce amyloid beta and tau pathologies, display anti-inflammatory properties, and improve cognition. However, they display poor brain bioavailability and are associated with several adverse side effects on human health, thus limiting their clinical application. Methods: We have developed a novel series of PPAR-delta and PPAR-gamma agonists in silico with AU9 as our lead compound that displays selective amino acid interactions focused upon avoiding the Tyr-473 epitope in the PPAR-gamma AF2 ligand binding domain. Results: This design helps to avoid the unwanted side effects of current PPAR-gamma agonists and improve behavioral deficits and synaptic plasticity while reducing amyloid-beta levels and inflammation in 3xTgAD animals. Conclusions: Our innovative in silico design of PPAR-delta/gamma agonists may offer new perspectives for this class of agonists for AD.

Drug Discovery and Development of Novel Therapeutics for Inhibiting TMAO in Models of Atherosclerosis and Diabetes.

Diabetes mellitus exists as a comorbidity with congestive heart failure (CHF). However, the exact molecular signaling mechanism linking CHF as the major form of mortality from diabetes remains unknown. Type 2 diabetic patients display abnormally high levels of metabolic products associated with gut dysbiosis. One such metabolite, trimethylamine N-oxide (TMAO), has been observed to be directly related with increased incidence of cardiovascular diseases (CVD) in human patients. TMAO a gut-liver metabolite, comes from the metabolic degenerative product trimethylamine (TMA) that is produced from gut microbial metabolism. Elevated levels of TMAO in diabetics and obese patients are observed to have a direct correlation with increased risk for major adverse cardiovascular events. The pro-atherogenic effect of TMAO is attributed to enhancing inflammatory pathways with cholesterol and bile acid dysregulation, promoting foam cell formation. Recent studies have revealed several potential therapeutic strategies for reducing TMAO levels and will be the central focus for the current review. However, few have focused on developing rational drug therapeutics and may be due to the gaps in knowledge for understanding the mechanism by which microbial TMA producing enzymes and hepatic flavin-containing monoxygenase (FMO) can work together in preventing elevation of TMAO levels. Therefore, it is critical to understand the advantages of developing a novel rational drug design strategy that manipulates FMO production of TMAO and TMA production by microbial enzymes. This review will focus on the inspection of FMO manipulation, as well as gut microbiota dysbiosis and its influence on metabolic disorders including cardiovascular disease and describe novel potential pharmacological therapeutic development.

The Cardioprotective Mechanism of Phenylaminoethyl Selenides (PAESe) Against Doxorubicin-Induced Cardiotoxicity Involves Frataxin.

Doxorubicin (DOX) is an anthracycline cancer chemotherapeutic that exhibits cumulative dose-limiting cardiotoxicity and limits its clinical utility. DOX treatment results in the development of morbid cardiac hypertrophy that progresses to congestive heart failure and death. Recent evidence suggests that during the development of DOX mediated cardiac hypertrophy, mitochondrial energetics are severely compromised, thus priming the cardiomyocyte for failure. To mitigate cumulative dose (5 mg/kg, QIW x 4 weeks with 2 weeks recovery) dependent DOX, mediated cardiac hypertrophy, we applied an orally active selenium based compound termed phenylaminoethyl selenides (PAESe) (QIW 10 mg/kg x 5) to our animal model and observed that PAESe attenuates DOX-mediated cardiac hypertrophy in athymic mice, as observed by MRI analysis. Mechanistically, we demonstrated that DOX impedes the stability of the iron-sulfur cluster biogenesis protein Frataxin (FXN) (0.5 fold), resulting in enhanced mitochondrial free iron accumulation (2.5 fold) and reduced aconitase activity (0.4 fold). Our findings further indicate that PAESe prevented the reduction of FXN levels and the ensuing elevation of mitochondrial free iron levels. PAESe has been shown to have anti-oxidative properties in part, by regeneration of glutathione levels. Therefore, we observed that PAESe can mitigate DOX mediated cardiac hypertrophy by enhancing glutathione activity (0.4 fold) and inhibiting ROS formation (1.8 fold). Lastly, we observed that DOX significantly reduced cellular respiration (basal (5%) and uncoupled (10%)) in H9C2 cardiomyoblasts and that PAESe protects against the DOX-mediated attenuation of cellular respiration. In conclusion, the current study determined the protective mechanism of PAESe against DOX mediated myocardial damage and that FXN is implicitly involved in DOX-mediated cardiotoxicity.

Comparison of Short-Term Associations between PM2.5 Components and Mortality across Six Major Cities in South Korea.

Association between short-term exposure to fine particulate matter (PM2.5) and mortality or morbidity varies geographically, and this variation could be due to different chemical composition affected by local sources. However, there have been only a few Asian studies possibly due to limited monitoring data. Using nationwide regulatory monitoring data of PM2.5 chemical components in South Korea, we aimed to compare the associations between daily exposure to PM2.5 components and mortality across six major cities. We obtained daily 24-h concentrations of PM2.5 and 11 PM2.5 components measured from 2013 to 2015 at single sites located in residential areas. We used death certificate data to compute the daily counts of nonaccidental, cardiovascular, and respiratory deaths. Using the generalized additive model, we estimated relative risks of daily mortality for an interquartile range increase in each pollutant concentration, while controlling for a longer-term time trend and meteorology. While elemental carbon was consistently associated with nonaccidental mortality across all cities, nickel and vanadium were strongly associated with respiratory or cardiovascular mortality in Busan and Ulsan, two large port cities. Our study shows that PM2.5 components responsible for PM2.5-associated mortality differed across cities depending on the dominant pollution sources, such as traffic and oil combustion.

Virus-like nanoparticle and DNA vaccination confers protection against respiratory syncytial virus by modulating innate and adaptive immune cells.

Respiratory syncytial virus (RSV) is an important human pathogen. Expression of virus structural proteins produces self-assembled virus-like nanoparticles (VLP). We investigated immune phenotypes after RSV challenge of immunized mice with VLP containing RSV F and G glycoproteins mixed with F-DNA (FdFG VLP). In contrast to formalin-inactivated RSV (FI-RSV) causing vaccination-associated eosinophilia, FdFG VLP immunization induced low bronchoalveolar cellularity, higher ratios of CD11c(+) versus CD11b(+) phenotypic cells and CD8(+) T versus CD4(+) T cells secreting interferon (IFN)-γ, T helper type-1 immune responses, and no sign of eosinophilia upon RSV challenge. Furthermore, RSV neutralizing activity, lung viral clearance, and histology results suggest that FdFG VLP can be comparable to live RSV in conferring protection against RSV and in preventing RSV disease. This study provides evidence that a combination of recombinant RSV VLP and plasmid DNA may have a potential anti-RSV prophylactic vaccine inducing balanced innate and adaptive immune responses.

Baculovirus-expressed virus-like particle vaccine in combination with DNA encoding the fusion protein confers protection against respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a major viral agent causing significant morbidity and mortality in young infants and the elderly. There is no licensed vaccine against RSV and it is a high priority to develop a safe RSV vaccine. We determined the immunogenicity and protective efficacy of combined virus-like particle and DNA vaccines presenting RSV glycoproteins (Fd.VLP) in comparison with formalin inactivated RSV (FI-RSV). Immunization of mice with Fd.VLP induced higher ratios of IgG2a/IgG1 antibody responses compared to those with FI-RSV. Upon live RSV challenge, Fd.VLP and FI-RSV vaccines were similarly effective in clearing lung viral loads. However, FI-RSV immunized mice showed a substantial weight loss and high levels of T helper type 2 (Th2) cytokines as well as extensive lung histopathology and eosinophil infiltration. In contrast, Fd.VLP immunized mice did not exhibit Th2 type cytokines locally and systemically, which might contribute to preventing vaccine-associated RSV lung disease. These results indicate that virus-like particles in combination with DNA vaccines represent a potential approach for developing a safe and effective RSV vaccine.

Co-immunization with virus-like particle and DNA vaccines induces protection against respiratory syncytial virus infection and bronchiolitis.

This study demonstrates that immunization with non-replicating virus-like particle (FFG VLP) containing RSV F and G glycoproteins together with RSV F DNA induced T helper type 1 antibody responses to RSV F similar to live RSV infection. Upon RSV challenge 21weeks after immunization, FFG VLP vaccination induced protection against RSV infection as shown by clearance of lung viral loads, and the absence of eosinophil infiltrates, and did not cause lung pathology. In contrast, formalin-inactivated RSV (FI-RSV) vaccination showed significant pulmonary eosinophilia, severe mucus production, and extensive histopathology resulting in a hallmark of pulmonary pathology. Substantial lung pathology was also observed in mice with RSV re-infections. High levels of systemic and local inflammatory cytokine-secreting cells were induced in mice with FI-RSV but not with FFG VLP immunization after RSV challenge. Therefore, the results provide evidence that recombinant RSV FFG VLP vaccine can confer long-term protection against RSV without causing lung pathology.

Maternal antibodies by passive immunization with formalin inactivated respiratory syncytial virus confer protection without vaccine-enhanced disease.

Maternal immunization of mice with formalin inactivated respiratory syncytial virus (FI-RSV) resulted in the passive transfer of RSV antibodies but not cellular components to the offspring. The offspring born to FI-RSV immunized mothers showed serum RSV neutralizing activity, effectively controlled lung viral loads without vaccine-enhanced disease, did not induce pulmonary eosinophilia, and cytokine producing cells after live RSV infection. Therefore, this study provides evidence that maternal immunization provides an in vivo model in investigating the roles of antibodies independent of cellular components.

Lactobacillus plantarum DK119 as a probiotic confers protection against influenza virus by modulating innate immunity.

Lactobacillus plantarum DK119 (DK119) isolated from the fermented Korean cabbage food was used as a probiotic to determine its antiviral effects on influenza virus. DK119 intranasal or oral administration conferred 100% protection against subsequent lethal infection with influenza A viruses, prevented significant weight loss, and lowered lung viral loads in a mouse model. The antiviral protective efficacy was observed in a dose and route dependent manner of DK119 administration. Mice that were treated with DK119 showed high levels of cytokines IL-12 and IFN-γ in bronchoalveolar lavage fluids, and a low degree of inflammation upon infection with influenza virus. Depletion of alveolar macrophage cells in lungs and bronchoalveolar lavages completely abrogated the DK119-mediated protection. Modulating host innate immunity of dendritic and macrophage cells, and cytokine production pattern appeared to be possible mechanisms by which DK119 exhibited antiviral effects on influenza virus infection. These results indicate that DK119 can be developed as a beneficial antiviral probiotic microorganism.

Gpx4 ablation in adult mice results in a lethal phenotype accompanied by neuronal loss in brain.

Glutathione peroxidase 4 (Gpx4) is an antioxidant defense enzyme important in reducing hydroperoxides in membrane lipids and lipoproteins. Gpx4 is essential for survival of embryos and neonatal mice; however, whether Gpx4 is required for adult animals remains unclear. In this study, we generated a floxed Gpx4 mouse (Gpx4(f/f)), in which exons 2-4 of Gpx4 gene are flanked by loxP sites. We then cross-bred the Gpx4(f/f) mice with a tamoxifen (tam)-inducible Cre transgenic mouse (R26CreER mice) to obtain mice in which the Gpx4 gene could be ablated by tam administration (Gpx4(f/f)/Cre mice). After treatment with tam, adult Gpx4(f/f)/Cre mice (6-9 months of age) showed a significant reduction of Gpx4 levels (a 75-85% decrease) in tissues such as brain, liver, lung, and kidney. Tam-treated Gpx4(f/f)/Cre mice lost body weight and died within 2 weeks, indicating that Gpx4 is essential for survival of adult animals. Tam-treated Gpx4(f/f)/Cre mice exhibited increased mitochondrial damage, as evidenced by the elevated 4-hydroxylnonenal (4-HNE) level, decreased activities of electron transport chain complexes I and IV, and reduced ATP production in liver. Tam treatment also significantly elevated apoptosis in Gpx4(f/f)/Cre mice. Moreover, tam-treated Gpx4(f/f)/Cre mice showed neuronal loss in the hippocampus region and had increased astrogliosis. These data indicate that Gpx4 is essential for mitochondria integrity and survival of neurons in adult animals.

Cognitive impairment and increased Aß levels induced by paraquat exposure are attenuated by enhanced removal of mitochondrial H(2)O(2).

Pesticide exposure is a risk factor of Alzheimer's disease (AD). However, little is known about how pesticide exposure may promote AD pathogenesis. In this study, we investigated the effects of paraquat pesticide exposure on ß-amyloid (Aß) levels and cognition using wild-type (WT) mice and ß-amyloid precursor protein (APP) transgenic mice. Our results showed that wild-type mice and APP transgenic mice after paraquat exposure had increased oxidative damage specifically in mitochondria of cerebral cortex and exhibited mitochondrial dysfunction. Moreover, the elevated mitochondrial damage was directly correlated with impaired associative learning and memory and increased Aß levels in APP transgenic mice exposed to paraquat. Furthermore, overexpression of peroxiredoxin 3, a mitochondrial antioxidant defense enzyme important for H(2)O(2) removal, protected against paraquat-induced mitochondrial damage and concomitantly improved cognition and decreased Aß levels in APP transgenic mice. Therefore, our results demonstrate that mitochondrial damage is a key mechanism underlying cognitive impairment and elevated amyloidogenesis induced by paraquat and that enhanced removal of mitochondrial H(2)O(2) could be an effective strategy to ameliorate AD pathogenesis induced by pesticide exposure.

A Novel mouse model of enhanced proteostasis: Full-length human heat shock factor 1 transgenic mice.

The heat shock response (HSR) is controlled by the master transcriptional regulator heat shock factor 1 (HSF1). HSF1 maintains proteostasis and resistance to stress through production of heat shock proteins (HSPs). No transgenic model exists that overexpresses HSF1 in tissues of the central nervous system (CNS). We generated a transgenic mouse overexpressing full-length non-mutant HSF1 and observed a 2-4-fold increase in HSF1 mRNA and protein expression in all tissues studied of HSF1 transgenic (HSF1(+/0)) mice compared to wild type (WT) littermates, including several regions of the CNS. Basal expression of HSP70 and 90 showed only mild tissue-specific changes; however, in response to forced exercise, the skeletal muscle HSR was more elevated in HSF1(+/0) mice compared to WT littermates and in fibroblasts following heat shock, as indicated by levels of inducible HSP70 mRNA and protein. HSF1(+/0) cells elicited a significantly more robust HSR in response to expression of the 82 repeat polyglutamine-YFP fusion construct (Q82YFP) and maintained proteasome-dependent processing of Q82YFP compared to WT fibroblasts. Overexpression of HSF1 was associated with fewer, but larger Q82YFP aggregates resembling aggresomes in HSF1(+/0) cells, and increased viability. Therefore, our data demonstrate that tissues and cells from mice overexpressing full-length non-mutant HSF1 exhibit enhanced proteostasis.

Short form glutathione peroxidase 4 is the essential isoform required for survival and somatic mitochondrial functions.

Glutathione peroxidase 4 (Gpx4) is an essential antioxidant enzyme having multiple functions. A long form Gpx4 protein and a short form Gpx4 protein, which are distinguishable by the presence or lack of a mitochondrial signal peptide at the N terminus, are generated from the Gpx4 gene. In this study, we generated transgenic mice using mutated GPX4 genes encoding either the long form Gpx4 (lGPX4 gene) or the short form Gpx4 (sGPX4 gene). Our results showed that transgenic mice with the sGPX4 gene had increased Gpx4 protein in all tissues and were protected against diquat-induced apoptosis in liver. Moreover, the sGPX4 gene was able to rescue the lethal phenotype of the mouse Gpx4-null mutation. In contrast, transgenic mice with the lGPX4 gene had increased Gpx4 protein only in the testes, and the lGPX4 gene failed to rescue the lethal phenotype of the mouse Gpx4-null mutation. In Gpx4-null mice rescued by the sGPX4 gene, the Gpx4 protein was present in mitochondria isolated from somatic tissues, and the submitochondrial distribution pattern of the Gpx4 protein in these mice was identical to that in wild-type mice. Interestingly, the male Gpx4-null mice rescued by the sGPX4 gene were infertile and exhibited sperm malformation. Together, our results demonstrated for the first time that the short form Gpx4 protein is present in somatic tissue mitochondria and is essential for survival and protection against apoptosis in mice, whereas the long form Gpx4 protein is important for male fertility.

HMCO5, herbal extract, inhibits NF-kappaB expression in lipopolysaccharide treated macrophages and reduces atherosclerotic lesions in cholesterol fed mice.

HMCO5 is a herbal extract which comprises of eight different herbs. We studied whether this extract has anti-atherosclerotic effects. In lipopolysaccharide (LPS) stimulated RAW264.7 cells, HMCO5 inhibited NF-kappaB activation as well as iNOS promoter activity, inhibited the secretion of TNF-alpha and IL-1beta, and directly inhibited the intracellular accumulation of reactive oxygen species. ApoE knock-out mice fed a high-fat high-cholesterol diet with HMCO5 for 10 weeks showed a significant reduction in atherosclerotic lesions. A notable finding was the preservation of the smooth muscle cell layer in the media of aorta in the HMCO5 co-treated mice. HMCO5 treated mice did not show significant decrease in serum level of cholesterol. These results suggest that HMCO5 has anti-atherosclerotic effects which in part may be attributable to the inhibition of production of NF-kappaB dependent pro-inflammatory cytokines.