Ginsenoside compound K reduces the progression of Huntington's disease via the inhibition of oxidative stress and overactivation of the ATM/AMPK pathway.

Background: Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of trinucleotide CAG repeat in the Huntingtin (Htt) gene. The major pathogenic pathways underlying HD involve the impairment of cellular energy homeostasis and DNA damage in the brain. The protein kinase ataxia-telangiectasia mutated (ATM) is an important regulator of the DNA damage response. ATM is involved in the phosphorylation of AMP-activated protein kinase (AMPK), suggesting that AMPK plays a critical role in response to DNA damage. Herein, we demonstrated that expression of polyQ-expanded mutant Htt (mHtt) enhanced the phosphorylation of ATM. Ginsenoside is the main and most effective component of Panax ginseng. However, the protective effect of a ginsenoside (compound K, CK) in HD remains unclear and warrants further investigation. Methods: This study used the R6/2 transgenic mouse model of HD and performed behavioral tests, survival rate, histological analyses, and immunoblot assays. Results: The systematic administration of CK into R6/2 mice suppressed the activation of ATM/AMPK and reduced neuronal toxicity and mHTT aggregation. Most importantly, CK increased neuronal density and lifespan and improved motor dysfunction in R6/2 mice. Conversely, CK enhanced the expression of Bcl2 protected striatal cells from the toxicity induced by the overactivation of mHtt and AMPK. Conclusions: Thus, the oral administration of CK reduced the disease progression and markedly enhanced lifespan in the transgenic mouse model (R6/2) of HD.

A selective inhibitor of the NLRP3 inflammasome as a potential therapeutic approach for neuroprotection in a transgenic mouse model of Huntington's disease.

BACKGROUND: Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of the CAG repeat in the huntingtin (HTT) gene. When the number of CAG repeats exceeds 36, the translated expanded polyglutamine-containing HTT protein (mutant HTT [mHTT]) interferes with the normal functions of many cellular proteins and subsequently jeopardizes important cellular machineries in major types of brain cells, including neurons, astrocytes, and microglia. The NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome, which comprises NLRP3, ASC, and caspase-1, is involved in the activation of IL-1ß and IL-18 and has been implicated in various biological functions. Although the existence of the NLRP3 inflammasome in the brain has been documented, the roles of the NLRP3 inflammasome in HD remain largely uncharacterized. MCC950 is a highly selective and potent small-molecule inhibitor of NLRP3 that has been used for the treatment of several diseases such as Alzheimer's disease. However, whether MCC950 is also beneficial in HD remains unknown. Therefore, we hypothesized that MCC950 exerts beneficial effects in a transgenic mouse model of HD. METHODS: To evaluate the effects of MCC950 in HD, we used the R6/2 (B6CBA-Tg[HDexon1]62Gpb/1J) transgenic mouse model of HD, which expresses exon 1 of the human HTT gene carrying 120 ± 5 CAG repeats. Male transgenic R6/2 mice were treated daily with MCC950 (10 mg/kg of body weight; oral administration) or water for 5 weeks from the age of 7 weeks. We examined neuronal density, neuroinflammation, and mHTT aggregation in the striatum of R6/2 mice vs. their wild-type littermates. We also evaluated the motor function, body weight, and lifespan of R6/2 mice. RESULTS: Systematic administration of MCC950 to R6/2 mice suppressed the NLRP3 inflammasome, decreased IL-1ß and reactive oxygen species production, and reduced neuronal toxicity, as assessed based on increased neuronal density and upregulation of the NeuN and PSD-95 proteins. Most importantly, oral administration of MCC950 increased neuronal survival, reduced neuroinflammation, extended lifespan, and improved motor dysfunction in R6/2 mice. CONCLUSIONS: Collectively, our findings indicate that MCC950 exerts beneficial effects in a transgenic mouse model of HD and has therapeutic potential for treatment of this devastating neurodegenerative disease.

Inhibition of pro-inflammatory mediator expression in macrophages using wood vinegar from griffith's ash.

Macrophages are essential for host defense as they control foreign pathogens and induce acquired immune responses. Activated macrophages secrete pro-inflammatory reactive substances causing local cell and tissue inflammatory response, which helps an organism resist the invasion of foreign pathogens. Excessive or chronic inflammation can cause several diseases. Previous studies have reported that vinegar treatment decreases the levels of several inflammatory cytokines and biomarkers, including mitogen-activated protein kinases, cyclooxygenase-2, inducible nitric oxide synthase (iNOS), and nitric oxide (NO). However, the benefits of wood vinegar produced from Griffith's ash (Fraxinus formosana Hayata) in reducing inflammation have not been investigated yet. Thus, assuming that wood vinegar exerts anti-inflammatory effects in macrophages, in this study, we investigated the potential anti-inflammatory effects of the wood vinegar from Griffith's ash using a lipopolysaccharide (LPS)-induced inflammatory response model in RAW264.7 macrophages. We showed that the wood vinegar inhibited the production of iNOS, NO, and interleukin 6. In addition, we found that the wood vinegar reduced the phosphorylation levels of p38 and protein kinase C-α/δ in the LPS-stimulated RAW264.7 macrophages. Based on these results, we suggest that the produced wood vinegar can reduce inflammation in LPS-activated macrophages.

Nocturnal One-Hour Lighting Stimulates Gonadal Development and Lowers Fat Deposition in Male Mule Ducks.

In this study, the effects of a nocturnal light pulse on body weight, organ mass, gonadal function, and plasma levels of metabolites were determined in male mule ducks. In total, 32 15-week-old mule ducks were randomly allocated to either Group C (control group) or L+ (lighting group). Group C was exposed to the natural photoperiod, whereas Group L+ was provided with a 1-h lighting over 20:00-21:00 every day, in addition to the natural photoperiod. At the end of the 42-day experiment, Group L+ had significantly lower relative weights (% of live weight) of the digestive tract and abdominal fat and higher relative weights of the breast meat and testes than Group C. Moreover, Group L+ had significantly higher plasma testosterone and lower plasma glucose levels. However, no between-group differences were observed in the triacylglycerol and uric acid levels. Histological examination demonstrated that the seminiferous tubule diameter was larger in Group L+ than in Group C. Moreover, the meiosis stage in spermatogenesis had begun in Group L+ but not in Group C. In conclusion, the supplemented 1-h lighting at 20:00 stimulated gonadal development and function and reduced fat deposition.

Eucalyptus essential oils inhibit the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages through reducing MAPK and NF-κB pathways.

BACKGROUND: Eucalyptus essential oils have been used in traditional medicine for centuries. It was reported that Eucalyptus leaves possess antioxidant and antimicrobial effects. Here, we investigated the anti-inflammatory activity of the essential oils extracted from the leaves of four different Eucalyptus species in RAW264.7 macrophages. METHODS: Lipopolysaccharide (LPS)-activated RAW264.7 macrophages were used to evaluate the anti-inflammatory activity of the leaf essential oils of Eucalyptus. The cell survival was quantified by an Alamar Blue assay. Nitric oxide (NO) production was assessed by Griess reaction. TNF-α and IL-6 production were measured by enzyme-linked immunosorbent assay (ELISA). Nuclear factor-κB (NF-κB) transcriptional activity was measured by NF-κB reporter assay. Intracellular protein expression levels were determined by Western blot. The expression levels of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and NF-κB pathway were measured by western blot in LPS-activated RAW 264.7 macrophage. RESULTS: The essential oils extracted from Eucalyptus citriodora leaf exert the best NO inhibitory activity in LPS-activated RAW264.7 macrophages. The essential oils were fractionated into fractions A-H, and fraction F has been demonstrated to inhibit the expression levels of TNF-α, IL-6, NO, iNOS and COX-2 in LPS-activated RAW264.7 macrophages. Mechanistic analysis revealed that fraction F reduced the phosphorylation levels of ERK1/2, p38, PKC-α, PKC-ε and PKC-δ, and inhibited the NF-κB transcriptional activity. The chemical composition of Fraction F was determined by GC-MS. CONCLUSIONS: The discoveries made herein could help develop innovative nonsteroidal anti-inflammatory drugs with minimal side effects and strong efficacy. Clinical trials on these Eucalyptus leaf essential oils will help customize and optimize their therapeutic administration.

Critical Role for the NLRP3 Inflammasome in Mediating IL-1ß Production in Shigella sonnei-Infected Macrophages.

Shigella is one of the leading bacterial causes of diarrhea worldwide, affecting more than 165 million people annually. Among the serotypes of Shigella, Shigella sonnei is physiologically unique and endemic in human immunodeficiency virus-infected men who have sex with men. The NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome, a protein complex composed of NLRP3, apoptosis-associated speck-like protein, and caspase-1, recognizes, and responds to pathogen infection and diverse sterile host-derived or environmental danger signals to induce IL-1ß and IL-18 production. Although the Shigella flexneri-mediated activation of the NLRP3 inflammasome has been reported, the effect of S. sonnei on NLRP3 inflammasome activation remains unclear. We found that S. sonnei induced IL-1ß production through NLRP3-dependent pathways in lipopolysaccharide-primed macrophages. A mechanistic study revealed that S. sonnei induced IL-1ß production through P2X7 receptor-mediated potassium efflux, reactive oxygen species generation, lysosomal acidification, and mitochondrial damage. In addition, the phagocytosis of viable S. sonnei was important for IL-1ß production. Furthermore, we demonstrated that NLRP3 negatively regulated phagocytosis and the bactericidal activity of macrophages against S. sonnei. These findings provide mechanistic insight into the activation of the NLRP3 inflammasome by S. sonnei in macrophages.

Synthetic 4-Hydroxy Auxarconjugatin B, a Novel Autophagy Inducer, Attenuates Gouty Inflammation by Inhibiting the NLRP3 Inflammasome.

Gouty arthritis results from the generation of uric acid crystals within the joints. These uric acid crystals activate the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome, which is involved in chronic inflammatory diseases, including gouty arthritis. This study identified the polyenylpyrrole derivative 4-hydroxy auxarconjugatin B (4-HAB), a novel autophagy inducer, which attenuated uric acid crystals-mediated activation of the NLRP3 inflammasome in vitro and in vivo. 4-HAB dose-dependently reduced the release of interleukin (IL)-1ß, IL-18, active caspase-1 and apoptosis-associated speck-like protein (ASC) in uric acid crystals-activated macrophages. In a mechanistic study, 4-HAB was shown to inhibit uric acid crystals-induced mitochondrial damage, lysosomal rupture and ASC oligomerization. Additionally, 4-HAB inhibited the NLRP3 inflammasome through Sirt1-dependent autophagy induction. Furthermore, the anti-inflammatory properties of 4-HAB were confirmed in a mouse model of uric acid crystals-mediated peritonitis by the reduced levels of neutrophil influx, IL-1ß, active caspase-1, IL-6 and MCP-1 in lavage fluids. In conclusion, 4-HAB attenuates gouty inflammation, in part by attenuating activation of the NLRP3 inflammasome through the Sirt1/autophagy induction pathway.

Mechanistic Insight Into the Activation of the NLRP3 Inflammasome by Neisseria gonorrhoeae in Macrophages.

Gonorrhea is a type III legal communicable disease caused by Neisseria gonorrhoeae (NG), one of the most common sexually transmitted bacteria worldwide. NG infection can cause urethritis or systemic inflammation and may lead to infertility or other complications. The NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome is a protein complex composed of NLRP3, apoptosis-associated speck-like protein and caspase-1 and is an important part of the cellular machinery controlling the release of interleukin (IL)-1ß and IL-18 and the pathogenesis of numerous infectious diseases. It has been reported that NG infection activates the NLRP3 inflammasome; however, the underlying mechanism remain unclear. In this report, the signaling pathways involved in the regulation of NG-mediated NLRP3 inflammasome activation in macrophages were studied. The results indicated that viable NG, but not heat-killed or freeze/thaw-killed NG, activated the NLRP3 inflammasome in macrophages through toll-like receptor 2, but not toll-like receptor 4. NG infection provided the priming signal to the NLRP3 inflammasome that induced the expression of NLRP3 and IL-1ß precursor through the nuclear factor kappa B and mitogen-activated protein kinase pathways. In addition, NG infection provided the activation signal to the NLRP3 inflammasome that activated caspase-1 through P2X7 receptor-dependent potassium efflux, lysosomal acidification, mitochondrial dysfunction, and reactive oxygen species production pathways. Furthermore, we demonstrated that NLRP3 knockout increased phagocytosis of bacteria by macrophages and increases the bactericidal activity of macrophages against NG. These findings provide potential molecular targets for the development of anti-inflammatory drugs that could ameliorate NG-mediated inflammation.

Mechanistic insight into the attenuation of gouty inflammation by Taiwanese green propolis via inhibition of the NLRP3 inflammasome.

Dysregulation of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome is involved in many chronic inflammatory diseases, including gouty arthritis. Activation of the NLRP3 inflammasome requires priming and activation signals: the priming signal controls the expression of NLRP3 and interleukin (IL)-1ß precursor (proIL-1ß), while the activation signal leads to the assembly of the NLRP3 inflammasome and to caspase-1 activation. Here, we reported the effects of the alcoholic extract of Taiwanese green propolis (TGP) on the NLRP3 inflammasome in vitro and in vivo. TGP inhibited proIL-1ß expression by reducing nuclear factor kappa B activation and reactive oxygen species (ROS) production in lipopolysaccharide-activated macrophages. Additionally, TGP also suppressed the activation signal by reducing mitochondrial damage, ROS production, lysosomal rupture, c-Jun N-terminal kinases 1/2 phosphorylation and apoptosis-associated speck-like protein oligomerization. Furthermore, we found that TGP inhibited the NLRP3 inflammasome partially via autophagy induction. In the in vivo mouse model of uric acid crystal-induced peritonitis, TGP attenuated the peritoneal recruitment of neutrophils, and the levels of IL-1ß, active caspase-1, IL-6 and monocyte chemoattractant protein-1 in lavage fluids. As a proof of principle, in this study, we purified a known compound, propolin G, from TGP and identified this compound as a potential inhibitor of the NLRP3 inflammasome. Our results indicated that TGP might be useful for ameliorating gouty inflammation via inhibition of the NLRP3 inflammasome.

Ergosterol peroxide from marine fungus Phoma sp. induces ROS-dependent apoptosis and autophagy in human lung adenocarcinoma cells.

As part of our ongoing search for novel therapeutic structures from microorganism, the chemical examination of marine fungus Phoma sp. resulted in the isolation of ergosterol, ergosterol peroxide (EP), and 9,11-dehydroergosterol peroxide (DEP). The bioassay results demonstrated that the three isolates reduced the viability of various cancer cells, with EP being highest in human lung cancer cell line A549 cells. EP induced caspase-dependent apoptosis through mitochondrial damage in A549 cells. Additionally, EP-induced ROS generation and apoptosis were attenuated by ROS-generating enzymes inhibitors and antioxidant N-acetylcysteine, indicated that ROS played an important role in EP-mediated apoptosis in A549 cells. Furthermore, it was observed that EP induced ROS-dependent autophagy, which attenuated apoptosis in A549 cells. On the other hand, EP reduced the LPS/ATP-induced proliferation and migration of A549 cells through attenuated NLRP3 inflammasome activity. Additionally, EP showed synergistic cytotoxic effect with antitumor drug Sorafenib in A549 cell viability inhibition. Furthermore, Micro-Western Array and Western blot analyses demonstrated that the protein levels of EGFR, HSP27, MEK5, AKT1, mTOR, Smad2, Smad3, TAB1, NF-κB, and HIF1-α decreased, while the levels of p-p38α, p-ERK1/2, p-JNK, fibronectin and p27 increased. Collectively, the results of this study demonstrated that EP might be useful to develop a therapeutic candidate for lung cancer complications.

A synthetic cationic antimicrobial peptide inhibits inflammatory response and the NLRP3 inflammasome by neutralizing LPS and ATP.

Antimicrobial peptides (AMPs) are one of the most important defense mechanisms against bacterial infections in insects, plants, non-mammalian vertebrates, and mammals. In the present study, a class of synthetic AMPs was evaluated for anti-inflammatory activity. One cationic AMP, GW-A2, demonstrated the ability to inhibit the expression levels of nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-activated macrophages. GW-A2 reduced LPS-induced increases in the phosphorylation of mitogen-activated protein kinase and protein kinase C-α/δ and the activation of NF-κB. GW-A2 also inhibited NLRP3 inflammasome activation induced by LPS and ATP. Furthermore, in the mice injected with LPS, GW-A2 reduced (1) the concentration of IL-1ß, IL-6 and TNF-α in the serum; (2) the concentration of TNF-α in the peritoneal lavage; (3) the expression levels of iNOS, COX-2 and NLRP3 in the liver and lung; (4) the infiltration of polymorphonuclear neutrophils in the liver and lung. The underlying mechanisms for the anti-inflammatory activity of GW-A2 were found to be partially due to LPS and ATP neutralization. These results provide insights into how GW-A2 inhibits inflammation and the NLRP3 inflammasome and provide a foundation for the design of rational therapeutics for inflammation-related diseases.

Activation of AMP-activated protein kinase α1 mediates mislocalization of TDP-43 in amyotrophic lateral sclerosis.

TAR DNA-binding protein-43 (TDP-43) is a nuclear RNA-binding protein involved in many cellular pathways. TDP-43-positive inclusions are a hallmark of amyotrophic lateral sclerosis (ALS). The major clinical presentation of ALS is muscle weakness due to the degeneration of motor neurons. Mislocalization of TDP-43 from the nucleus to the cytoplasm is an early event of ALS. In this study, we demonstrate that cytoplasmic mislocalization of TDP-43 was accompanied by increased activation of AMP-activated protein kinase (AMPK) in motor neurons of ALS patients. The activation of AMPK in a motor neuron cell line (NSC34) or mouse spinal cords induced the mislocalization of TDP-43, recapitulating this characteristic of ALS. Down-regulation of AMPK-α1 or exogenous expression of a dominant-negative AMPK-α1 mutant reduced TDP-43 mislocalization. Suppression of AMPK activity using cAMP-simulating agents rescued the mislocalization of TDP-43 in NSC34 cells and delayed disease progression in TDP-43 transgenic mice. Our findings demonstrate that activation of AMPK-α1 plays a critical role in TDP-43 mislocalization and the development of ALS; thus, AMPK-α1 may be a potential drug target for this devastating disease.

AMPK-α1 functions downstream of oxidative stress to mediate neuronal atrophy in Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurological disorder that is induced by a CAG trinucleotide expansion in exon 1 of the Huntingtin (HTT) gene. We previously reported that the abnormal activation of an important energy sensor, AMP-activated protein kinase α1 (AMPK-α1), occurs in the brains of mice and patients with HD, which suggests that this abnormal activation may contribute to neuronal degeneration in HD. In the present study, we demonstrated that the elevated oxidative stress that was evoked by a polyQ-expanded mutant HTT (mHTT) caused the abnormal activation of AMPK-α1 and, subsequently, resulted in neurotoxicity in a striatal progenitor cell line (STHdh(Q109)) and in the striatum of a transgenic mouse model of HD (R6/2). The systematic administration of an antioxidant (N-acetyl-cysteine, NAC) to R6/2 mice suppressed the activation of AMPK-α1, reduced neuronal toxicity, which was assessed by the activation of caspases, increased neuronal density, ameliorated ventricle enlargement, and improved motor dysfunction. This beneficial effect of NAC in vivo appears to be direct because NAC also reduced the activation of AMPK-α1 and the death of STHdh(Q109) cells upon elevated oxidative stress. Moreover, the activation of AMPK enhanced the level of oxidative stress in STHdh(Q109) cells, in primary neurons of R6/2 mice, and in the striatum of two different HD mouse models (R6/2 and Hdh(150Q/+)), whereas the inhibition of AMPK reduced the level of oxidative stress. Collectively, our findings suggest that positive feedback regulation between the elevated oxidative stress and the activation of AMPK-α1 contributes to the progression of HD.

Energy dysfunction in Huntington's disease: insights from PGC-1α, AMPK, and CKB.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in the Huntingtin (Htt) gene. When the number of CAG repeats exceeds 36, the translated polyglutamine-expanded Htt protein interferes with the normal functions of many types of cellular machinery and causes cytotoxicity. Clinical symptoms include progressive involuntary movement disorders, psychiatric signs, cognitive decline, dementia, and a shortened lifespan. The most severe brain atrophy is observed in the striatum and cortex. Besides the well-characterized neuronal defects, recent studies showed that the functions of mitochondria and several key players in energy homeostasis are abnormally regulated during HD progression. Energy dysregulation thus is now recognized as an important pathogenic pathway of HD. This review focuses on the importance of three key molecular determinants (peroxisome proliferator-activated receptor-γ coactivator-1α, AMP-activated protein kinase, and creatine kinase B) of cellular energy homeostasis and their possible involvement in HD pathogenesis.

Nuclear translocation of AMPK-alpha1 potentiates striatal neurodegeneration in Huntington's disease.

Adenosine monophosphate-activated protein kinase (AMPK) is a major energy sensor that maintains cellular energy homeostasis. Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of CAG repeats in the huntingtin (Htt) gene. In this paper, we report that activation of the α1 isoform of AMPK (AMPK-α1) occurred in striatal neurons of humans and mice with HD. Overactivation of AMPK in the striatum caused brain atrophy, facilitated neuronal loss, and increased formation of Htt aggregates in a transgenic mouse model (R6/2) of HD. Such nuclear accumulation of AMPK-α1 was activity dependent. Prevention of nuclear translocation or inactivation of AMPK-α1 ameliorated cell death and down-regulation of Bcl2 caused by mutant Htt (mHtt). Conversely, enhanced expression of Bcl2 protected striatal cells from the toxicity evoked by mHtt and AMPK overactivation. These data demonstrate that aberrant activation of AMPK-α1 in the nuclei of striatal cells represents a new toxic pathway induced by mHtt.