Phloroglucinol Derivatives Exert Anti-inflammatory Effects and Attenuate Cognitive Impairment in LPS-induced Mouse Model.

Neuroinflammation is an inflammatory immune response that arises in the central nervous system. It is one of the primary causes of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. Phloroglucinol (PG) is a natural product contained in extracts of plant, algae and microbe and has been reported to have antioxidant and anti-inflammatory properties. In this study, we synthesized PG derivatives to enhance their antioxidant and anti-inflammatory activity. Among PG derivatives, 6a suppressed pro-oxidative and inflammatory molecule nitric oxide (NO) production more effectively than PG. Moreover, 6a dose-dependently reduced the expression of proinflammatory cytokines such as IL-6, IL-1ß, TNF-α, and NO producing enzyme iNOS in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Additionally, we confirmed that 6a alleviated cognitive impairment and glial activation in mouse model of LPS-induced neuroinflammation. These findings suggest that novel PG derivative, 6a, is a potential treatment for neurodegenerative diseases.

A new Caenorhabditis elegans model to study copper toxicity in Wilson disease.

Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a copper (Cu) transporting ATPase whose trafficking from the Golgi to endo-lysosomal compartments drives sequestration of excess Cu and its further excretion from hepatocytes into the bile. Loss of ATP7B function leads to toxic Cu overload in the liver and subsequently in the brain, causing fatal hepatic and neurological abnormalities. The limitations of existing WD therapies call for the development of new therapeutic approaches, which require an amenable animal model system for screening and validation of drugs and molecular targets. To achieve this objective, we generated a mutant Caenorhabditis elegans strain with a substitution of a conserved histidine (H828Q) in the ATP7B ortholog cua-1 corresponding to the most common ATP7B variant (H1069Q) that causes WD. cua-1 mutant animals exhibited very poor resistance to Cu compared to the wild-type strain. This manifested in a strong delay in larval development, a shorter lifespan, impaired motility, oxidative stress pathway activation, and mitochondrial damage. In addition, morphological analysis revealed several neuronal abnormalities in cua-1 mutant animals exposed to Cu. Further investigation suggested that mutant CUA-1 is retained and degraded in the endoplasmic reticulum, similarly to human ATP7B-H1069Q. As a consequence, the mutant protein does not allow animals to counteract Cu toxicity. Notably, pharmacological correctors of ATP7B-H1069Q reduced Cu toxicity in cua-1 mutants indicating that similar pathogenic molecular pathways might be activated by the H/Q substitution and, therefore, targeted for rescue of ATP7B/CUA-1 function. Taken together, our findings suggest that the newly generated cua-1 mutant strain represents an excellent model for Cu toxicity studies in WD.

Hypothalamic GABRA5-positive neurons control obesity via astrocytic GABA.

The lateral hypothalamic area (LHA) regulates food intake and energy balance. Although LHA neurons innervate adipose tissues, the identity of neurons that regulate fat is undefined. Here we show that GABRA5-positive neurons in LHA (GABRA5LHA) polysynaptically project to brown and white adipose tissues in the periphery. GABRA5LHA are a distinct subpopulation of GABAergic neurons and show decreased pacemaker firing in diet-induced obesity mouse models in males. Chemogenetic inhibition of GABRA5LHA suppresses fat thermogenesis and increases weight gain, whereas gene silencing of GABRA5 in LHA decreases weight gain. In the diet-induced obesity mouse model, GABRA5LHA are tonically inhibited by nearby reactive astrocytes releasing GABA, which is synthesized by monoamine oxidase B (Maob). Gene silencing of astrocytic Maob in LHA facilitates fat thermogenesis and reduces weight gain significantly without affecting food intake, which is recapitulated by administration of a Maob inhibitor, KDS2010. We propose that firing of GABRA5LHA suppresses fat accumulation and selective inhibition of astrocytic GABA is a molecular target for treating obesity.

Optimization and evaluation of pyridinyl vinyl sulfones as Nrf2 activator for the antioxidant and anti-inflammatory effects.

Many studies have reported that chalcone-based compounds exhibit biological activities such as anticancer, antioxidant, anti-inflammatory and neuroprotective effects. Among the published chalcone derivatives, (E)-1-(3-methoxypyridin-2-yl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (VEDA-1209), which is currently undergoing preclinical study, was selected as a starting compound for the development of new nuclear factor erythroid 2-related factor 2 (Nrf2) activators. Based on our previous knowledge, we attempted to redesign and synthesize VEDA-1209 derivatives by introducing the pyridine ring and sulfone moiety to ameliorate its Nrf2 efficacy and drug-like properties. Among the synthesized compounds, (E)-3-chloro-2-(2-((3-methoxypyridin-2-yl)sulfonyl)vinyl) pyridine (10e) was found to have approximately 16-folds higher Nrf2 activating effects than VEDA-1209 (10e: EC50 = 37.9 nM vs VEDA-1209: EC50 = 625 nM) in functional cell-based assay. In addition, 10e effectively improved drug-like properties such as CYP inhibition probability and metabolic stability. Finally, 10e demonstrated excellent antioxidant and anti-inflammatory effects in BV-2 microglial cells and significantly restored spatial memory deficits in lipopolysaccharide (LPS)-induced neuroinflammatory mouse models.

Mitochondrial dysfunction reactivates α-fetoprotein expression that drives copper-dependent immunosuppression in mitochondrial disease models.

Signaling circuits crucial to systemic physiology are widespread, yet uncovering their molecular underpinnings remains a barrier to understanding the etiology of many metabolic disorders. Here, we identified a copper-linked signaling circuit activated by disruption of mitochondrial function in the murine liver or heart that resulted in atrophy of the spleen and thymus and caused a peripheral white blood cell deficiency. We demonstrated that the leukopenia was caused by α-fetoprotein, which required copper and the cell surface receptor CCR5 to promote white blood cell death. We further showed that α-fetoprotein expression was upregulated in several cell types upon inhibition of oxidative phosphorylation. Collectively, our data argue that α-fetoprotein may be secreted by bioenergetically stressed tissue to suppress the immune system, an effect that may explain the recurrent or chronic infections that are observed in a subset of mitochondrial diseases or in other disorders with secondary mitochondrial dysfunction.

Synthesis and Evaluation of Serinolamide Derivatives as Sphingosine-1-Phosphate-1 (S1P1) Receptor Agonists.

Sphingosine-1-phosphate-1 (S1P1) receptor agonists are well-known drugs for treating multiple sclerosis (MS) caused by autoreactive lymphocytes that attack the myelin sheath. Therefore, an effective therapeutic strategy is to reduce the lymphocytes in the blood by inducing S1P1 receptor internalization. We synthesized serinolamide A, a natural product of the sea, and performed S1P1 receptor internalization assay to evaluate functionally antagonistic S1P1 receptor agonist activity. In order to synthesize derivatives with better efficacy than serinolamide A and B, new derivatives were synthesized by introducing the phenyl ring moiety of fingolimod. Among them, compounds 19 and 21 had superior S1P1 agonistic effects to serinolamide. We also confirmed that compound 19 effectively inhibited lymphocyte outflow in peripheral lymphocyte count (PLC) assay.

Oral Elesclomol Treatment Alleviates Copper Deficiency in Animal Models.

Copper (Cu) is an essential trace element for key biochemical reactions. Dietary or genetic copper deficiencies are associated with anemia, cardiomyopathy, and neurodegeneration. The essential requirement for copper in humans is illustrated by Menkes disease, a fatal neurodegenerative disorder of early childhood caused by mutations in the ATP7A copper transporter. Recent groundbreaking studies have demonstrated that a copper delivery small molecule compound, elesclomol (ES), is able to substantially ameliorate pathology and lethality in a mouse model of Menkes disease when injected as an ES-Cu2+ complex. It is well appreciated that drugs administered through oral means are more convenient with better efficacy than injection methods. Here we show, using genetic models of copper-deficient C. elegans and mice, that dietary ES supplementation fully rescues copper deficiency phenotypes. Worms lacking either the homolog of the CTR1 copper importer or the ATP7 copper exporter showed normal development when fed ES. Oral gavage with ES rescued intestine-specific Ctr1 knockout mice from early postnatal lethality without additional copper supplementation. Our findings reveal that ES facilitates copper delivery from dietary sources independent of the intestinal copper transporter CTR1 and provide insight into oral administration of ES as an optimal therapeutic for Menkes disease and possibly other disorders of copper insufficiency.

Discovery of Novel Sphingosine-1-Phosphate-1 Receptor Agonists for the Treatment of Multiple Sclerosis.

The sphingosine-1-phosphate-1 (S1P1) receptor agonists have great potential for the treatment of multiple sclerosis (MS) because they can inhibit lymphocyte egress through receptor internalization. We designed and synthesized triazole and isoxazoline derivatives to discover a novel S1P1 agonist for MS treatment. Of the two scaffolds, the isoxazoline derivative was determined to have excellent in vitro efficacy and drug-like properties. Among them, compound 21l was found to have superior drug-like properties as well as excellent in vitro efficacies (EC50 = 7.03 nM in ß-arrestin recruitment and EC50 = 11.8 nM in internalization). We also confirmed that 21l effectively inhibited lymphocyte egress in the peripheral lymphocyte count test and significantly improved the clinical score in the experimental autoimmune encephalitis MS mouse model.

Elesclomol alleviates Menkes pathology and mortality by escorting Cu to cuproenzymes in mice.

Loss-of-function mutations in the copper (Cu) transporter ATP7A cause Menkes disease. Menkes is an infantile, fatal, hereditary copper-deficiency disorder that is characterized by progressive neurological injury culminating in death, typically by 3 years of age. Severe copper deficiency leads to multiple pathologies, including impaired energy generation caused by cytochrome c oxidase dysfunction in the mitochondria. Here we report that the small molecule elesclomol escorted copper to the mitochondria and increased cytochrome c oxidase levels in the brain. Through this mechanism, elesclomol prevented detrimental neurodegenerative changes and improved the survival of the mottled-brindled mouse-a murine model of severe Menkes disease. Thus, elesclomol holds promise for the treatment of Menkes and associated disorders of hereditary copper deficiency.

An extracellular histidine-containing motif in the zinc transporter ZIP4 plays a role in zinc sensing and zinc-induced endocytosis in mammalian cells.

Zinc is an essential trace element that serves as a cofactor for enzymes in critical biochemical processes and also plays a structural role in numerous proteins. Zinc transporter ZIP4 (ZIP4) is a zinc importer required for dietary zinc uptake in the intestine and other cell types. Studies in cultured cells have reported that zinc stimulates the endocytosis of plasma membrane-localized ZIP4 protein, resulting in reduced cellular zinc uptake. Thus, zinc-regulated trafficking of ZIP4 is a key means for regulating cellular zinc homeostasis, but the underlying mechanisms are not well understood. In this study, we used mutational analysis, immunoblotting, HEK293 cells, and immunofluorescence microscopy to identify a histidine-containing motif (398HTH) in the first extracellular loop that is required for high sensitivity to low zinc concentrations in a zinc-induced endocytic response of mouse ZIP4 (mZIP4). Moreover, using synthetic peptides with selective substitutions and truncated mZIP4 variants, we provide evidence that histidine residues in this motif coordinate a zinc ion in mZIP4 homodimers at the plasma membrane. These findings suggest that 398HTH is an important zinc-sensing motif for eliciting high-affinity zinc-stimulated endocytosis of mZIP4 and provide insight into cellular mechanisms for regulating cellular zinc homeostasis in mammalian cells.

Elesclomol restores mitochondrial function in genetic models of copper deficiency.

Copper is an essential cofactor of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Inherited loss-of-function mutations in several genes encoding proteins required for copper delivery to CcO result in diminished CcO activity and severe pathologic conditions in affected infants. Copper supplementation restores CcO function in patient cells with mutations in two of these genes, COA6 and SCO2, suggesting a potential therapeutic approach. However, direct copper supplementation has not been therapeutically effective in human patients, underscoring the need to identify highly efficient copper transporting pharmacological agents. By using a candidate-based approach, we identified an investigational anticancer drug, elesclomol (ES), that rescues respiratory defects of COA6-deficient yeast cells by increasing mitochondrial copper content and restoring CcO activity. ES also rescues respiratory defects in other yeast mutants of copper metabolism, suggesting a broader applicability. Low nanomolar concentrations of ES reinstate copper-containing subunits of CcO in a zebrafish model of copper deficiency and in a series of copper-deficient mammalian cells, including those derived from a patient with SCO2 mutations. These findings reveal that ES can restore intracellular copper homeostasis by mimicking the function of missing transporters and chaperones of copper, and may have potential in treating human disorders of copper metabolism.

CHCA-1 is a copper-regulated CTR1 homolog required for normal development, copper accumulation, and copper-sensing behavior in Caenorhabditis elegans.

Copper plays key roles in catalytic and regulatory biochemical reactions essential for normal growth, development, and health. Dietary copper deficiencies or mutations in copper homeostasis genes can lead to abnormal musculoskeletal development, cognitive disorders, and poor growth. In yeast and mammals, copper is acquired through the activities of the CTR1 family of high-affinity copper transporters. However, the mechanisms of systemic responses to dietary or tissue-specific copper deficiency remain unclear. Here, taking advantage of the animal model Caenorhabditis elegans for studying whole-body copper homeostasis, we investigated the role of a C. elegans CTR1 homolog, CHCA-1, in copper acquisition and in worm growth, development, and behavior. Using sequence homology searches, we identified 10 potential orthologs to mammalian CTR1 Among these genes, we found that chca-1, which is transcriptionally up-regulated in the intestine and hypodermis of C. elegans during copper deficiency, is required for normal growth, reproduction, and maintenance of systemic copper balance under copper deprivation. The intestinal copper transporter CUA-1 normally traffics to endosomes to sequester excess copper, and we found here that loss of chca-1 caused CUA-1 to mislocalize to the basolateral membrane under copper overload conditions. Moreover, animals lacking chca-1 exhibited significantly reduced copper avoidance behavior in response to toxic copper conditions compared with WT worms. These results establish that CHCA-1-mediated copper acquisition in C. elegans is crucial for normal growth, development, and copper-sensing behavior.

The Function of ATPase Copper Transporter ATP7B in Intestine.

BACKGROUND & AIMS: Wilson disease is a disorder of copper (Cu) misbalance caused by mutations in ATP7B. ATP7B is highly expressed in the liver-the major site of Cu accumulation in patients with Wilson disease. The intestine also expresses ATP7B, but little is known about the contribution of intestinal ATP7B to normal intestinal copper homeostasis or to Wilson disease manifestations. We characterized the role of ATP7B in mouse intestinal organoids and tissues. METHODS: We collected intestinal tissues from ATP7B-knockout (Atp7b-/-) and control mice, and established 3-dimensional enteroids. Immunohistochemistry and x-ray fluorescence were used to characterize the distribution of ATP7B and Cu in tissues. Electron microscopy, histologic analyses, and immunoblotting were used to determine the effects of ATP7B loss. Enteroids derived from control and ATP7B-knockout mice were incubated with excess Cu or with Cu-chelating reagents; effects on cell fat content and ATP7B levels and localization were determined by fluorescent confocal microscopy. RESULTS: ATP7B maintains a Cu gradient along the duodenal crypt-villus axis and buffers Cu levels in the cytosol of enterocytes. These functions are mediated by rapid Cu-dependent enlargement of ATP7B-containing vesicles and increased levels of ATP7B. Intestines of Atp7b-/- mice had reduced Cu storage pools in intestine, Cu depletion, accumulation of triglyceride-filled vesicles in enterocytes, mislocalization of apolipoprotein B, and loss of chylomicrons. In primary 3-dimensional enteroids, administration of excess Cu or Cu chelators impaired assembly of chylomicrons. CONCLUSIONS: ATP7B regulates vesicular storage of Cu in mouse intestine. ATP7B buffers Cu levels in enterocytes to maintain a range necessary for formation of chylomicrons. Misbalance of Cu and lipid in the intestine could account for gastrointestinal manifestations of Wilson disease.

The mitochondrial metallochaperone SCO1 maintains CTR1 at the plasma membrane to preserve copper homeostasis in the murine heart.

SCO1 is a ubiquitously expressed, mitochondrial protein with essential roles in cytochrome c oxidase (COX) assembly and the regulation of copper homeostasis. SCO1 patients present with severe forms of early onset disease, and ultimately succumb from liver, heart or brain failure. However, the inherent susceptibility of these tissues to SCO1 mutations and the clinical heterogeneity observed across SCO1 pedigrees remain poorly understood phenomena. To further address this issue, we generated Sco1hrt/hrt and Sco1stm/stm mice in which Sco1 was specifically deleted in heart and striated muscle, respectively. Lethality was observed in both models due to a combined COX and copper deficiency that resulted in a dilated cardiomyopathy. Left ventricular dilation and loss of heart function was preceded by a temporal decrease in COX activity and copper levels in the longer-lived Sco1stm/stm mice. Interestingly, the reduction in copper content of Sco1stm/stm cardiomyocytes was due to the mislocalisation of CTR1, the high affinity transporter that imports copper into the cell. CTR1 was similarly mislocalized to the cytosol in the heart of knockin mice carrying a homozygous G115S substitution in Sco1, which in humans causes a hypertrophic cardiomyopathy. Our current findings in the heart are in marked contrast to our prior observations in the liver, where Sco1 deletion results in a near complete absence of CTR1 protein. These data collectively argue that mutations perturbing SCO1 function have tissue-specific consequences for the machinery that ultimately governs copper homeostasis, and further establish the importance of aberrant mitochondrial signaling to the etiology of copper handling disorders.

Organ-specific regulation of ATP7A abundance is coordinated with systemic copper homeostasis.

Copper (Cu) is an essential cofactor for various enzymatic activities including mitochondrial electron transport, iron mobilization, and peptide hormone maturation. Consequently, Cu dysregulation is associated with fatal neonatal disease, liver and cardiac dysfunction, and anemia. While the Cu transporter ATP7A plays a major role in both intestinal Cu mobilization to the periphery and prevention of Cu over-accumulation, it is unclear how regulation of ATP7A contributes to Cu homeostasis in response to systemic Cu fluctuation. Here we show, using Cu-deficient mouse models, that steady-state levels of ATP7A are lower in peripheral tissues (including the heart, spleen, and liver) under Cu deficiency and that subcutaneous administration of Cu to these animals restore normal ATP7A levels in these tissues. Strikingly, ATP7A in the intestine is regulated in the opposite manner - low systemic Cu increases ATP7A while subcutaneous Cu administration decreases ATP7A suggesting that intestine-specific non-autonomous regulation of ATP7A abundance may serve as a key homeostatic control for Cu export into the circulation. Our results support a systemic model for how a single transporter can be inversely regulated in a tissue-specific manner to maintain organismal Cu homeostasis.

The Intestinal Copper Exporter CUA-1 Is Required for Systemic Copper Homeostasis in Caenorhabditis elegans.

Copper plays key catalytic and regulatory roles in biochemical processes essential for normal growth, development, and health. Defects in copper metabolism cause Menkes and Wilson's disease, myeloneuropathy, and cardiovascular disease and are associated with other pathophysiological states. Consequently, it is critical to understand the mechanisms by which organisms control the acquisition, distribution, and utilization of copper. The intestinal enterocyte is a key regulatory point for copper absorption into the body; however, the mechanisms by which intestinal cells transport copper to maintain organismal copper homeostasis are poorly understood. Here, we identify a mechanism by which organismal copper homeostasis is maintained by intestinal copper exporter trafficking that is coordinated with extraintestinal copper levels in Caenorhabditis elegans Specifically, we show that CUA-1, the C. elegans homolog of ATP7A/B, localizes to lysosome-like organelles (gut granules) in the intestine under copper overload conditions for copper detoxification, whereas copper deficiency results in a redistribution of CUA-1 to basolateral membranes for copper efflux to peripheral tissues. Worms defective in gut granule biogenesis exhibit defects in copper sequestration and increased susceptibility to toxic copper levels. Interestingly, however, a splice isoform CUA-1.2 that lacks a portion of the N-terminal domain is targeted constitutively to the basolateral membrane irrespective of dietary copper concentration. Our studies establish that CUA-1 is a key intestinal copper exporter and that its trafficking is regulated to maintain systemic copper homeostasis. C. elegans could therefore be exploited as a whole-animal model system to study regulation of intra- and intercellular copper trafficking pathways.

Ctr2 regulates biogenesis of a cleaved form of mammalian Ctr1 metal transporter lacking the copper- and cisplatin-binding ecto-domain.

Copper is an essential catalytic cofactor for enzymatic activities that drive a range of metabolic biochemistry including mitochondrial electron transport, iron mobilization, and peptide hormone maturation. Copper dysregulation is associated with fatal infantile disease, liver, and cardiac dysfunction, neuropathy, and anemia. Here we report that mammals regulate systemic copper acquisition and intracellular mobilization via cleavage of the copper-binding ecto-domain of the copper transporter 1 (Ctr1). Although full-length Ctr1 is critical to drive efficient copper import across the plasma membrane, cleavage of the ecto-domain is required for Ctr1 to mobilize endosomal copper stores. The biogenesis of the truncated form of Ctr1 requires the structurally related, previously enigmatic copper transporter 2 (Ctr2). Ctr2(-/-) mice are defective in accumulation of truncated Ctr1 and exhibit increased tissue copper levels, and X-ray fluorescence microscopy demonstrates that copper accumulates as intracellular foci. These studies identify a key regulatory mechanism for mammalian copper transport through Ctr2-dependent accumulation of a Ctr1 variant lacking the copper- and cisplatin-binding ecto-domain.

A novel role for copper in Ras/mitogen-activated protein kinase signaling.

Copper (Cu) is essential for development and proliferation, yet the cellular requirements for Cu in these processes are not well defined. We report that Cu plays an unanticipated role in the mitogen-activated protein (MAP) kinase pathway. Ablation of the Ctr1 high-affinity Cu transporter in flies and mouse cells, mutation of Ctr1, and Cu chelators all reduce the ability of the MAP kinase kinase Mek1 to phosphorylate the MAP kinase Erk. Moreover, mice bearing a cardiac-tissue-specific knockout of Ctr1 are deficient in Erk phosphorylation in cardiac tissue. in vitro investigations reveal that recombinant Mek1 binds two Cu atoms with high affinity and that Cu enhances Mek1 phosphorylation of Erk in a dose-dependent fashion. Coimmunoprecipitation experiments suggest that Cu is important for promoting the Mek1-Erk physical interaction that precedes the phosphorylation of Erk by Mek1. These results demonstrate a role for Ctr1 and Cu in activating a pathway well known to play a key role in normal physiology and in cancer.

Ctr1 is an apical copper transporter in mammalian intestinal epithelial cells in vivo that is controlled at the level of protein stability.

Copper is an essential trace element that functions in a diverse array of biochemical processes that include mitochondrial respiration, neurotransmitter biogenesis, connective tissue maturation, and reactive oxygen chemistry. The Ctr1 protein is a high-affinity Cu(+) importer that is structurally and functionally conserved in yeast, plants, fruit flies, and humans and that, in all of these organisms, is localized to the plasma membrane and intracellular vesicles. Although intestinal epithelial cell-specific deletion of Ctr1 in mice demonstrated a critical role for Ctr1 in dietary copper absorption, some controversy exists over the localization of Ctr1 in intestinal epithelial cells in vivo. In this work, we assess the localization of Ctr1 in intestinal epithelial cells through two independent mechanisms. Using immunohistochemistry, we demonstrate that Ctr1 localizes to the apical membrane in intestinal epithelial cells of the mouse, rat, and pig. Moreover, biotinylation of intestinal luminal proteins from mice fed a control or a copper-deficient diet showed elevated levels of both total and apical membrane Ctr1 protein in response to transient dietary copper limitation. Experiments in cultured HEK293T cells demonstrated that alterations in the levels of the glycosylated form of Ctr1 in response to copper availability were a time-dependent, copper-specific posttranslational response. Taken together, these results demonstrate apical localization of Ctr1 in intestinal epithelia across three mammalian species and suggest that increased Ctr1 apical localization in response to dietary copper limitation may represent an adaptive response to homeostatically modulate Ctr1 availability at the site of intestinal copper absorption.