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Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the huntingtin (HTT) gene. When the CAG repeat exceeds 36, it results in the accumulation of the mutant HTT (mHTT) protein in neurons and glial cells. Key pathological mechanisms in HD include excitotoxicity, energy dysfunction, impaired mitochondrial function, increased oxidative stress, and neuroinflammation. The NLRP3 inflammasome is a multimeric protein complex element of NLRP3, ASC, and caspase-1, which regulates interleukin (IL)-1β and IL-18 secretion. The NLRP3 inflammasome plays an important role in inflammatory reactions and is involved in the pathogenesis of several neurodegenerative diseases. We have previously demonstrated high NLRP3 inflammasome expression levels in the striatum of R6/2 mice (a transgenic HD mouse model). Systematic administration of an NLRP3 inhibitor (MCC950) to R6/2 mice suppressed the NLRP3 inflammasome, decreased IL-1β and reactive oxygen species production, and reduced neuronal toxicity, suggesting protective effects against HD. Antrodia cinnamomea is an indigenous medicinal fungus in Taiwan, which shows diverse medicinal and pharmacological activities, but its effects in HD are not well understood. Herein, we report that systematic administration of Antcin-H isolated from A. cinnamomea to R6/2 mice suppressed the NLRP3 inflammasome, IL-1β production, and reduced neuronal toxicity. Most importantly, oral administration of Antcin-H reduced disease progression by increasing neuronal survival, reducing neuroinflammation during an extended lifespan, and improving motor dysfunction in R6/2 mice. Taken together, our data suggest that Antcin-H has therapeutic potential for treating HD., Competing Interests: Declarations. Ethics Approval and Consent to Participate: Not applicable. Consent for Publication: All the authors mentioned agreed to the publication of the manuscript. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

To explore whether the p17 protein of oncolytic avian reovirus (ARV) mediates cell migration and invadopodia formation, we applied several molecular biological approaches for studying the involved cellular factors and signal pathways. We found that ARV p17 activates the p53/phosphatase and tensin homolog (PTEN) pathway to suppress the focal adhesion kinase (FAK)/Src signaling and downstream signal molecules, thus inhibiting cell migration and the formation of invadopodia in murine melanoma cancer cell line (B16-F10). Importantly, p17-induced formation of invadopodia could be reversed in cells transfected with the mutant PTEN C124A . p17 protein was found to significantly reduce the expression levels of tyrosine kinase substrate 5 (TKs5), Rab40b, non-catalytic region of tyrosine kinase adaptor protein 1 (NCK1), and matrix metalloproteinases (MMP9), suggesting that TKs5 and Rab40b were transcriptionally downregulated by p17. Furthermore, we found that p17 suppresses the formation of the TKs5/NCK1 complex. Coexpression of TKs5 and Rab40b in B16-F10 cancer cells reversed p17-modulated suppression of the formation of invadopodia. This work provides new insights into p17-modulated suppression of invadopodia formation by activating the p53/PTEN pathway, suppressing the FAK/Src pathway, and inhibiting the formation of the TKs5/NCK1 complex.

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., Competing Interests: All the authors declare that they have no conflicts of interest., (© 2021 The Korean Society of Ginseng. Publishing services by Elsevier B.V.)

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-1β 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-1β 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., (© 2022. The Author(s).)

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., Competing Interests: None

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.

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.

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-1β 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-1β production through NLRP3-dependent pathways in lipopolysaccharide-primed macrophages. A mechanistic study revealed that S. sonnei induced IL-1β production through P 2 X 7 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-1β 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., (Copyright © 2020 Li, Chen, Chiu, Hsu, Wang, Tai, Ju, Chen, Chernikov, Tsai and Hua.)

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)-1β, 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-1β, 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., Competing Interests: None of the authors has any conflict of interest to declare.

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)-1β 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-1β 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 P 2 X 7 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.

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)-1β precursor (proIL-1β), 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-1β 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-1β, 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., (© 2018 Wiley Periodicals, Inc.)

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.

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-1β, 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.

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., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Artistic training is a complex learning that requires the meticulous orchestration of sophisticated polysensory, motor, cognitive, and emotional elements of mental capacity to harvest an aesthetic creation. In this study, we investigated the architecture of the resting-state functional connectivity networks from professional painters, dancers and pianists. Using a graph-based network analysis, we focused on the art-related changes of modular organization and functional hubs in the resting-state functional connectivity network. We report that the brain architecture of artists consists of a hierarchical modular organization where art-unique and artistic form-specific brain states collectively mirror the mind states of virtuosos. We show that even in the resting state, this type of extraordinary and long-lasting training can macroscopically imprint a neural network system of spontaneous activity in which the related brain regions become functionally and topologically modularized in both domain-general and domain-specific manners. The attuned modularity reflects a resilient plasticity nurtured by long-term experience.

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.

The feasibility of forming solid molecular dispersions of poorly water-soluble drugs in crosslinked poly(2-hydroethyl methacrylate) (PHEMA) hydrogel has recently been reported by our group. The purpose of the present study is to investigate the extent of enhancement of kinetic solubility of amorphous solid dispersions (ASDs) of indomethacin (IND) in crosslinked PHEMA hydrogels as compared with those based on conventional water-soluble polymer carriers. Our results show that under non-sink conditions, the initial solubility enhancement is higher for ASDs based on polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose acetate succinate (HMPCAS), but the ability to maintain this solubility enhancement at longer times is better for ASDs based on PHEMA over a period of 24h with the extent of solubility enhancement of IND ASDs in PHEMA falling between those in PVP and HPMCAS at 10.0% IND loading after 6h and outperforming those in PVP and HPMCAS at 32.9% IND loading after 8h. The observed kinetic solubility profiles reflect the fact that the amorphous IND is released from PHEMA by a different mechanism than those from water-soluble polymer carriers. In this case, the dissolution of IND ASD from water-soluble PVP and HPMCAS is almost instantaneous, resulting in an initial surge of IND concentration followed by a sharp decline due to the nucleation and crystallization events triggered by the rapid build-up of drug supersaturation. On the other hand, the dissolution of IND ASD from insoluble crosslinked PHEMA hydrogel beads is less rapid as it is regulated by a feedback-controlled diffusion mechanism, thus avoiding a sudden surge of supersaturation in the dissolution medium. The absence of an apparent decline in drug concentration during dissolution from IND-PHEMA ASD further reflects the diminished nucleation and crystallization events during IND dissolution from hydrogel-based solid molecular dispersions. Based on the XRD analyses, a threshold IND loading level of about 34% in PHEMA has been identified, above which amorphous to crystalline transition tends to occur. Also, by selecting the appropriate particle sizes, immediate to controlled release of IND from IND-PHEMA ASD can be readily achieved as the release rate increases with decreasing PHEMA bead size. Furthermore, a robust physical stability has been demonstrated in IND-PHEMA ASD with no drug precipitation for up to 8 months at IND loadings below 16.7% under direct open cup exposure to accelerated stability conditions (40°C/75% RH)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

Compromised mitochondrial function in neurons and glia has been observed in several neurodegenerative disorders, including Huntington's disease and Alzheimer's disease. Chemical/hypoxic preconditioning may afford protection against subsequently more severe oxidative damages. In this study, we tested whether induction of hypoxia inducible factor-1 (HIF-1) may exert cytoprotective effects against mitochondrial dysfunction caused by 3-nitropropionic acid (3-NP) in glial cells. Preconditioning of C6 astroglial cells with cobalt chloride, mimosine (MIM), and desferrioxamine (DFO), all of which known to activate HIF-1, significantly attenuated cytotoxicity induced by 3-NP, an irreversible inhibitor of mitochondrial complex II, and antimycin A, a mitochondrial complex III inhibitor. Application of cadmium chloride capable of neutralizing cobalt-induced HIF-1 activation, HIF-specific oligodeoxynucleotide (ODN) decoy, and antisense phosphorothioate ODN against HIF-1alpha abolished the protective effect mediated by preconditioning with cobalt chloride. Preloading of C6 cells with SN50, PD98059, or SB202190, the respective inhibitor of nuclear factor-kappaB (NF-kappaB), p44/p42 extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK), failed to affect the protection afforded by cobalt preconditioning. Taken together, these results suggest that HIF-1 induction secondary to preconditioning with cobalt chloride or iron chelators may mediate the protective effects against metabolic insult induced by the mitochondrial inhibitor 3-NP in C6 astroglial cells.

Amyloid beta-peptide (Abeta) is a major constituent of senile plaques in the brains of Alzheimer's disease (AD) patients. We have previously demonstrated ceramide production secondary to Abeta-induced activation of neutral sphingomyelinase (nSMase) in cerebral endothelial cells and oligodendrocytes, which may contribute to cellular injury during progression of AD. In this study, we first established the "Abeta --> nSMase --> ceramide --> free radical --> cell death" pathway in primary cultures of fetal rat cortical neurons. We also provided experimental evidence showing that S-nitrosoglutathione (GSNO), a potent endogenous antioxidant derived from the interaction between nitric oxide (NO) and glutathione, caused dose-dependent protective effects against Abeta/ceramide neurotoxicity via inhibition of caspase activation and production of reactive oxygen species (ROS). This GSNO-mediated neuroprotection appeared to involve activation of cGMP-dependent protein kinase (PKG), phosphatidylinositol 3-kinase (PI3K), and extracellular signal-regulated kinase (ERK). Activation of the cGMP/PKG pathway induced expression of thioredoxin and Bcl-2 that were beneficial to cortical neurons in antagonizing Abeta/ceramide toxicity. Consistently, exogenous application of thioredoxin exerted remarkable neuroprotective efficacy in our experimental paradigm. Results derived from the present study establish a neuroprotective role of GSNO, an endogenous NO carrier, against Abeta toxicity via multiple signaling pathways.

Mitochondrial dysfunction and oxidative stress are often linked to various neurodegenerative disorders including ischemic stroke and Huntington's disease (HD). S-Nitrosoglutathione (GSNO) is an endogenous nitric oxide carrier recently identified as a potent antioxidant capable of neutralizing oxidative stress. In the present study, we explore the neuroprotective effects of GSNO against metabolic insults induced by 3-nitropropionic acid (3-NP), a mitochondrial complex II inhibitor commonly used as a pharmacological model for HD, in primary culture of fetal rat cortical and striatal neurons. Application of GSNO (1-5 microM) substantially reduced neuronal loss caused by 3-NP (1-5 mM) exposure based on MTT reduction, lactate dehydrogenase (LDH) release, and Hoechst staining assays. The protective effect of GSNO appeared to be more potent than N-acetyl-l-cysteine (NAC), a glutathione precursor, at the same concentrations. These results suggest that manipulation of GSNO metabolism may exert protective effects against mitochondrial dysfunction often observed in neurodegenerative disorders., (Copyright 2004 Elsevier Ireland Ltd.)

Inducible nitric oxide synthase (iNOS or NOS2) is expressed in malignant glioma. Previously we noted that C6 glioma cells overexpressing NOS2 displayed chemoresistance against 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and other chloroethylnitrosourea derivatives with carbamoylating action. Herein we report experimental evidence supporting the contention that this NOS2 effect is mediated, at least in part, by S-nitrosoglutathione (GSNO), a potent antioxidant derived from interaction of NO and glutathione. Out of three NO donors tested, only GSNO was effective in protecting glioma cells against BCNU cytotoxicity. Furthermore, the protective effect of GSNO, similar to that of NOS2, was confined to carbamoylating, but not alkylating action. Experimental manipulations that were expected to increase or decrease cellular GSNO stores, as confirmed by immunocytochemical staining using a GSNO-specific antibody and HPLC analysis of GSNO contents in culture medium, led respectively to enhanced or reduced chemoresistance against carbamoylating cytotoxicity. Finally, neocuproine, a selective cuprous ion chelator known to neutralize GSNO actions, abolished NOS2-mediated chemoresistance against carbamoylating agents. Our results reveal a novel action of NOS2/GSNO that may potentially contribute to the development of chemoresistance against BCNU, which remains a mainstay in chemotherapy for glioblastoma multiforme.

1. We tested whether pretreatment of reagents known to induce hypoxia-inducible factor-1 (HIF-1) may confer chemoresistance against cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to rat C6 glioma cells. We also studied which cytotoxic mechanism(s) of chloroethylnitrosoureas could be neutralized by cobalt preconditioning. 2. Preconditioning of rat C6 glioma cells with cobalt chloride (300 microm, 2 h) induced HIF-1 binding activity based on electrophoretic mobility shift assay (EMSA). Results from Western blotting confirmed a heightened HIF-1alpha level upon cobalt chloride exposure (300-400 microm, 2 h). Cobalt chloride (300 microm) pretreatment for 2 h substantially neutralized BCNU toxicity, leading to increases in glioma cell survival based on MTT assay. In addition, pre-exposure of C6 cells with desferrioxamine (DFO; 400 microm, 3 h), an iron chelator known to activate HIF-1, also induced HIF-1 binding and rendered the glioma cells resistant to cytotoxicity of BCNU. 3. Pre-incubation with cobalt chloride abolished the cytotoxicity of several carbamoylating agents including 2-chloroethyl isocyanate and cyclohexyl isocyanate, the respective carbamoylating metabolites of BCNU and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. The protective effect of cobalt exposure, however, was not observed when cells were challenged with alkylating agents including temozolomide. 4. Cadmium chloride (50 microm) effectively reversed cobalt-induced HIF-1 activation. Correspondingly, cadmium chloride suppressed carbamoylating chemoresistance mediated by cobalt chloride pretreatment. Furthermore, both double-stranded oligodeoxynucleotide (ODN) decoy with HIF-1 cognate sequence and antisense phosphorothioate ODNs against HIF-1alpha partially abolished the carbamoylating chemoresistance associated with cobalt preconditioning. 5. Our results suggest that cobalt- or DFO-preconditioning may enhance glioma carbamoylating chemoresistance that is dependent, at least in part, on induction of HIF-1.

The goal of this study was to examine the phagocytosis of alginate based microspheres with different surface properties. Favorable interaction with macrophages is critical for uptake subsequent processing of the microspheres used for oral vaccine delivery. We examined the effects of size of alginate microspheres and hydrophobicity on cellular uptake. We also examined the toxicity of formulation components to phagocytic cells. Alginate microspheres were made by the emulsion-cross-linking technique. Five different formulations of microspheres were evaluated for size, hydrophobicity, cellular uptake and toxicity to macrophages. The formulations examined were: alginate alone (A), alginate with methylcellulose (AA) AA with Pluronic L61 (AA61), alginate with hydroxypropyl methylcellulose (AK3), and AK3 with Pluronic (L61 (AK3 61). Microspheres with without poly-L-lysine (PLL) coating were tested. The mean volume sizes of A, AA, AA61, AK3, AK3 61 microspheres (MS) were 11, 10.5, 3.8, 8.7 and 3.9 mocrom, respectively. After coating them with PLL the mean volume sizes were 10.4, 10, 3.7, 8.8 and 3.5 microm, respectively. Hydrophobicity of the microspheres was evaluated by measuring contact angle on a glass slide coated with the microspheres. The contact angles measured using a goniometer on A, AA, AA61, AK3, AK3 61 MS were 20, 34.8, 71, 29 and 80 degrees, respectively whereas those MS coated with PLL were 49.7, 55.8, 91, 48.25 and 84.4 degrees, respectively. Cellular uptake studies using flow cytometery revealed that AA61 MS coated with PLL were phagocytosed most often by mouse macrophages. There was no statistically significant difference in cellular uptake among those MS without PLL coating. Toxicity to macrophages was shown to depend on the ratio of microspheres to cells. These studies suggest that formulation can dramatically affect the physical characteristics of alginate MS in ways that can affect how they will interact with cells in the body when administered as a vaccine delivery system.