Mitochondrial diseases: Drosophila melanogaster as a model to evaluate potential therapeutics

While often presented as a single entity, mitochondrial diseases comprise a wide range of clinical, biochemical and genetic heterogeneous disorders. Among them, defects in the process of oxidative phosphorylation are the most prevalent. Despite intense research efforts, patients are still without effective treatment. An important part of the development of new therapeutics relies on predictive models of the pathology in order to assess their therapeutic potential. Since mitochondrial diseases are a heterogeneous group of progressive multisystemic disorders that can affect any organ at any time, the development of various in vivo models for the different diseases-associated genes defects will accelerate the search for effective therapeutics. Here, we review existing Drosophila melanogaster models for mitochondrial diseases, with a focus on alterations in oxidative phosphorylation, and discuss the potential of this powerful model organism in the process of drug target discovery.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.     

Residues required for phosphorylation of translation initiation factor eIF2α under diverse stress conditions are divergent between yeast and human

PERK, PKR, HRI and GCN2 are the four mammalian kinases that phosphorylate the α subunit of the eukaryotic translation initiation factor 2 (eIF2α) on Ser51. This phosphorylation event is conserved among many species and attenuates protein synthesis in response to diverse stress conditions. In contrast, Saccharmyces cerevisiae expresses only the GCN2 kinase. It was demonstrated previously in S. cerevisiae that single point mutations in eIF2α’s N-terminus severely impaired phosphorylation at Ser51. To assess whether similar recognition patterns are present in mammalian eIF2α, we expressed human eIF2α’s with these mutations in mouse embryonic fibroblasts and assessed their phosphorylation under diverse stress conditions. Some of the mutations prevented the stress-induced phosphorylation of eIF2α by all mammalian kinases, thus defining amino acid residues in eIF2α (Gly 30, Leu 50, and Asp 83) that are required for substrate recognition. We also identified residues that were less critical or not required for recognition by the mammalian kinases (Ala 31, Met 44, Lys 79, and Tyr 81), even though they were essential for recognition of the yeast eIF2α by GCN2. We propose that mammalian eIF2α kinases evolved to maximize their interactions with the evolutionarily conserved Ser51 residue of eIF2α in response to diverse stress conditions, thus adding to the complex signaling pathways that mammalian cells have over simpler organisms.     

Organelle communication: Signaling crossroads between homeostasis and disease

Cellular organelles do not function as isolated or static units, but rather form dynamic contacts between one another that can be modulated according to cellular needs. The physical interfaces between organelles are important for Ca²⁺ and lipid homeostasis, and serve as platforms for the control of many essential functions including metabolism, signaling, organelle integrity and execution of the apoptotic program. Emerging evidence also highlights the importance of organelle communication in disorders such as Alzheimer's disease, pulmonary arterial hypertension, cancer, skeletal and cardiac muscle dysfunction. Here, we provide an overview of the current literature on organelle communication and the link to human pathologies.     

Tumour progression and cancer-induced pain: A role for protease-activated receptor-2?

The role of proteases in modifying the microenvironment of tumour cells has long been recognised. With the discovery of the protease-activated receptor family of G protein-coupled receptors a mechanism for cells to sense and respond directly to proteases in their microenvironment was revealed. Many early studies described the roles of protease-activated receptors in the cellular events that occur during blood coagulation and inflammation. More recently, studies have begun to focus on the roles of protease-activated receptors in the establishment, progression and metastasis of a variety of tumours. This review will focus on the expression of protease-activated receptor-2 and its activators by normal and neoplastic tissues, and describe current evidence that activation of protease-activated receptor-2 is an important event at multiple stages of tumour progression and in pain associated with cancer.     

Identification of critical regions within the TIR domain of IL-1 receptor type I

Interleukin-1 receptor type I (IL-1RI) belongs to a superfamily of proteins characterized by an intracellular Toll/IL-1 receptor (TIR) domain. This domain harbors three conserved regions called boxes 1-3 that play crucial roles in mediating IL-1 responses. Boxes 1 and 2 are considered to be involved in binding of adapter molecules. Amino acids possibly crucial for IL-1RI signaling were predicted via homology models of the IL-1RI TIR domain based on the crystal structure of IL-1RAPL. The role of ten of these residues was investigated by site-directed mutagenesis and a functional luciferase assay reflecting NF-κB activity in transiently transfected Jurkat cells. In particular, the mutants E437K/D438K, E472A/E473A and S465A/S470A/S471A/E472A/E473A showed decreased and the mutant E437A/D438A increased IL-1 responsiveness compared to the mouse IL-1RI wild type. In conclusion, the αC′ helix (Q469-E473 in mouse IL-1RI) is probably involved in heterotypic interactions of IL-1RI with IL-1RAcP or MyD88.     

Promising anti-inflammatory effects of chalcones via inhibition of cyclooxygenase,prostaglandin E2, inducible NO synthase and nuclear factor κb activities

Chalcones (1, 3-Diphenyl-2-propen-1-one) consist of a three carbon α, β-unsaturated carbonyl system and act as precursors for the biosynthesis of flavonoids in plants. However, laboratory synthesis of various chalcones has also been reported. Both natural and synthetic chalcones are known to exhibit a variety of pharmacological activities such as anti-inflammatory, antitumor, antibacterial, antifungal, antimalarial and antituberculosis. These promising activities, ease of synthesis and simple chemical structure have awarded chalcones considerable attraction. This review focuses on the anti-inflammatory effects of chalcones, caused by their inhibitory action primarily against the activities and expressions of four key inflammatory mediators viz., cyclooxygenase, prostaglandin E₂, inducible NO synthase, and nuclear factor κB. Various methodologies for the synthesis of chalcones have been discussed. The potency of recently synthesized chalcones is given in terms of their IC₅₀ values. Structure-Activity Relationships (SARs) of a variety of chalcone derivatives have been discussed. Computational methods were applied to calculate the ideal orientation of a typical chalcone scaffold against three enzymes, namely, cyclooxygenase-1, cyclooxygenase-2 and inducible NO synthase for the formation of stable complexes. The global market of anti-inflammatory drugs and its expected growth (from 2018 to 2026) have been discussed. SAR analysis, docking studies, and future prospects all together provide useful clues for the synthesis of novel chalcones of improved anti-inflammatory activities.     

Norepinephrine stimulation of alpha1D-adrenoceptor promotes proliferation of pulmonary artery smooth muscle cells via ERK-1/2 signaling

It has been shown that the sympathetic nervous system is activated in pulmonary arterial hypertension (PAH). Norepinephrine (NE) levels are increased by chemoreflex-dependent sympathetic overactivation and involved in pulmonary vascular remodeling. However, the underlying mechanisms of the remodeling induced by NE are poorly understood. In this study, we found that, in vivo, the expression of tyrosine hydroxylase and the concentration of plasma NE were increased in PAH rats compared with normal rats. Increases in ventricular hypertrophy and medial width of the pulmonary arteries were reversed by prazosin, α₁-adrenoceptor (α₁-AR) antagonists, in PAH rats. Elevated expression of α_1D-AR was detected in PAH rats. In addition, prazosin reduced the increasing expression of PCNA, CyclinA and CyclinE induced by hypoxia. In vitro, MTT assay, flow cytometry, Western blotting and immunofluorescence were performed to investigate the effects of NE on proliferation of pulmonary artery smooth muscle cells (PASMCs). We revealed that NE promoted PASMCs viability, increased the expression of PCNA, CyclinA and CyclinE, made more cells from G₀/G₁ phase to G₂/M + S phase and enhanced the microtubule formation. Above NE-induced changes could be suppressed by BMY 7378, an inhibitor of α_1D-AR. Furthermore, ERK-1/2 pathway was activated by NE. U0126, a specific inhibitor for ERK-1/2, attenuated the NE-induced proliferation of PASMCs under normoxia and hypoxia. Taken together, our results suggest that NE which stimulates α_1D-AR promotes proliferation of PASMCs and the effect is, at least in part, mediated via the ERK-1/2 pathway.     

Dual oxidase 2 generated reactive oxygen species selectively mediate the induction of mucins by epidermal growth factor in enterocytes

Dual oxidase 2 enzyme is a member of the reactive oxygen species-generating cell membrane NADPH oxidases involved in mucosal innate immunity. It is not known if the biological activity of dual oxidase 2 is mediated by direct bacterial killing by reactive oxygen species produced by the enzyme or by the same reactive oxygen species acting as second messengers that stimulate novel gene expression. To uncover the role of reactive oxygen species and dual oxidases as signaling molecules, we have dissected the pathway triggered by epidermal growth factor to induce mucins, the principal protective components of gastrointestinal mucus. We show that dual oxidase 2 is essential for selective epidermal growth factor induction of the transmembrane MUC3 and the secreted gel-forming MUC5AC mucins. Reactive oxygen species generated by dual oxidase 2 stabilize tyrosine phosphorylation of epidermal growth factor receptor and induce MUC3 and MUC5AC through persistent activation of extracellular signal-regulated kinases 1/2–protein kinase C. Knocking down dual oxidase 2 by selective RNA targeting (siRNA) reduced epidermal growth factor receptor phosphorylation, and MUC3 and MUC5AC gene expression. Extracellular reactive oxygen species produced by dual oxidase 2, upon stimulation by epidermal growth factor, stabilize epidermal growth factor receptor phosphorylation and activate extracellular signal-regulated kinases 1/2–protein kinase C which induce MUC5AC and MUC3. Extracellular reactive oxygen species produced by dual oxidase 2 that are known to directly kill bacteria, also contribute to the maintenance of the epidermal growth factor-amplification loop, which induces mucins. These data suggest a new function of dual oxidase 2 protein in the luminal protection of the gastrointestinal tract through the induction of mucin expression by growth factors.     

Cyanobacterial bioactive metabolites—A review of their chemistry and biology

Cyanobacterial blooms occur when algal densities exceed baseline population concentrations. Cyanobacteria can produce a large number of secondary metabolites. Odorous metabolites affect the smell and flavor of aquatic animals, whereas bioactive metabolites cause a range of lethal and sub-lethal effects in plants, invertebrates, and vertebrates, including humans. Herein, the bioactivity, chemistry, origin, and biosynthesis of these cyanobacterial secondary metabolites were reviewed. With recent revision of cyanobacterial taxonomy by Anagnostidis and Komárek as part of the Süβwasserflora von Mitteleuropa volumes 19(1–3), names of many cyanobacteria that produce bioactive compounds have changed, thereby confusing readers. The original and new nomenclature are included in this review to clarify the origins of cyanobacterial bioactive compounds.Due to structural similarity, the 157 known bioactive classes produced by cyanobacteria have been condensed to 55 classes. This review will provide a basis for more formal procedures to adopt a logical naming system. This review is needed for efficient management of water resources to understand, identify, and manage cyanobacterial harmful algal bloom impacts.     

Characterization of three serotonin receptors from the small white butterfly,Pieris rapae

Serotonin (5-hydroxytryptamine, 5-HT) plays a key role in modulating diverse physiological processes and behaviors in both protostomes and deuterostomes. These functions are mediated through the binding of serotonin to its receptors, which are recognized as potential insecticide targets. We investigated the sequence, pharmacology and tissue distribution of three 5-HT receptors (Piera5-HT_1A, Piera5-HT_1B, Piera5-HT₇) from the small white butterfly Pieris rapae, an important pest of cultivated cabbages and other mustard family crops. Activation of Piera5-HT_1A or Piera5-HT_1B by 5-HT inhibited the production of cAMP in a dose-dependent manner. Stimulation of Piera5-HT₇ with 5-HT increased cAMP level significantly. Surprisingly, with the exception of 5-methoxytryptamine, agonists including α-methylserotonin, 8-Hydroxy-DPAT and 5-carboxamidotryptamine activated these receptors poorly. The results are consistent with previous findings in Manduca sexta. All three receptors were blocked by methiothepin, but ketanserin and yohimbine were not effective. The selective mammalian 5-HT receptor antagonists SB 216641 and SB 269970 displayed potent inhibition effects on Piera5-HT_1B and Piera5-HT₇ respectively. The results we achieved here indicate that the pharmacological properties of Lepidoptera 5-HT receptors are quite different from those in other insects and vertebrates and may contribute to development of new selective pesticides. This study offers important information on three 5-HT receptors from P. rapae that will facilitate further analysis of the functions of 5-HT receptors in insects.     

DNA repair mechanisms in dividing and non-dividing cells

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.     

The Notch γ-secretase inhibitor ameliorates kidney fibrosis via inhibition of TGF-β/Smad2/3 signaling pathway activation

Kidney fibrosis is a common feature of chronic kidney disease (CKD). A recent study suggests that abnormal Notch signaling activation contributes to the development of renal fibrosis. However, the molecular mechanism that regulates this process remains unexplored. Unilateral ureteral obstruction (UUO) or sham-operated C57BL6 mice (aged 10 weeks) were randomly assigned to receive dibenzazepine (DBZ, 250 μg/100 g/d) or vehicle for 7 days. Histologic examinations were performed on the kidneys using Masson's trichrome staining and immunohistochemistry. Real-time PCR and western blot analysis were used for detection of mRNA expression and protein phosphorylation. The expression of Notch 1, 3, and 4, Notch intracellular domain (NICD), and its target genes Hes1 and HeyL were upregulated in UUO mice, while the increase in NICD protein was significantly attenuated by DBZ. After 7 days, the severity of renal fibrosis and expression of fibrotic markers, including collagen 1α1/3α1, fibronectin, and α-smooth muscle actin, were markedly increased in UUO compared with sham mice. In contrast, administration of DBZ markedly attenuated these effects. Furthermore, DBZ significantly inhibited UUO-induced expression of transforming growth factor (TGF)-β, phosphorylated Smad 2, and Smad 3. Mechanistically, Notch signaling activation in tubular epithelial cells enhanced fibroblast proliferation and activation in a coculture experiment. Our study provides evidence that Notch signaling is implicated in renal fibrogenesis. The Notch inhibitor DBZ can ameliorate this process via inhibition of the TGF-β/Smad2/3 signaling pathway, and might be a novel drug for preventing chronic kidney disease.     

Inhibition of vascular smooth muscle cell calcification by vasorin through interference with TGFβ1 signaling

Elevated transforming growth factor β1 (TGFβ1) levels are frequently observed in chronic kidney disease (CKD) patients. TGFβ1 contributes to development of medial vascular calcification during hyperphosphatemia, a pathological process promoted by osteo−/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). Vasorin is a transmembrane glycoprotein highly expressed in VSMCs, which is able to bind TGFβ to inhibit TGFβ signaling. Thus, the present study explored the effects of vasorin on osteo−/chondrogenic transdifferentiation and calcification of VSMCs. Primary human aortic smooth muscle cells (HAoSMCs) were treated with recombinant human TGFβ1 or β-glycerophosphate without or with recombinant human vasorin or vasorin gene silencing by siRNA. As a result, TGFβ1 down-regulated vasorin mRNA expression in HAoSMCs. Vasorin supplementation inhibited TGFβ1-induced pathway activation, SMAD2 phosphorylation and downstream target genes expression in HAoSMCs. Furthermore, treatment with exogenous vasorin blunted, while vasorin knockdown augmented TGFβ1-induced osteo−/chondrogenic transdifferentiation of HAoSMCs. In addition, phosphate down-regulated vasorin mRNA expression in HAoSMCs. Phosphate-induced TGFβ1 expression was not affected by addition of exogenous vasorin. Nonetheless, the phosphate-induced TGFβ1 signaling, osteo−/chondrogenic transdifferentiation and calcification of HAoSMCs were all blunted by vasorin. Conversely, silencing of vasorin aggravated osteoinduction in HAoSMCs during high phosphate conditions. Aortic vasorin expression was reduced in the hyperphosphatemic klotho-hypomorphic mouse model of CKD-related vascular calcification. In conclusion, vasorin, which suppresses TGFβ1 signaling and protects against osteo−/chondrogenic transdifferentiation and calcification of VSMCs, is reduced by pro-calcifying conditions. Thus, vasorin is a novel key regulator of VSMC calcification and may represent a potential therapeutic target for vascular calcification during CKD.     

Analyses of α-amylase and α-glucosidase in the malaria vector mosquito,Anopheles gambiae,as receptors of Cry11Ba toxin of Bacillus thuringiensis subsp. jegathesan

Bacillus thuringiensis subsp. jegathesan produces Cry11Ba crystal protein with high toxicity to mosquito larvae. The Cry11Ba toxicity is dependent on its receptors on mosquito larval midgut epithelial cells. Previously, a cadherin-like protein (AgCad2), aminopeptidase (AgAPN2) and alkaline phosphatase (AgALP1) were reported to be involved in regulation of Cry11Ba toxicity on Anopheles gambiae larvae. Here, the cDNAs encoding α-amylase (AgAmy1) and α-glucosidase (Agm3) were cloned from A. gambiae larva midgut. Both are glycophosphatidylinositol (GPI) anchored proteins on brush border membranes (BBMV). Immunohistochemistry revealed their localization on different regions of the larval midgut. AgAmy1 and Agm3 bound Cry11Ba with high affinity, 37.6 nM and 21.1 nM respectively. Cry11Ba toxicity against A. gambiae larvae was neutralized by both AgAmy1 and Agm3. The results provide evidence that both AgAmy1 and Agm3 function as receptors of Cry11Ba in A. gambiae.     

Glutenase and collagenase activities of wheat cysteine protease Triticain-α: Feasibility for enzymatic therapy assays

Insufficient and/or improper protein degradation is associated with the development of various human pathologies. Enzymatic therapy with proteolytic enzymes aimed to improve insufficient proteolytic activity was suggested as a treatment of protease deficiency-induced disorders. Since in many cases human degradome is incapable of degrading the entire target protein(s), other organisms can be used as a source of proteases exhibiting activities distinct from human enzymes, and plants are perspective candidates for this source. In this study recombinant wheat cysteine protease Triticain-α was shown to refold in vitro into an autocatalytically activated proteolytic enzyme possessing glutenase and collagenase activities at acidic (or close to neutral) pH levels at the temperature of human body. Mass-spectrometry analysis of the products of Triticain-α-catalyzed gluten hydrolysis revealed multiple cleavage sites within the sequences of gliadin toxic peptides, in particular, in the major toxic 33-mer α-gliadin-derived peptide initiating inflammatory responses to gluten in celiac disease (CD) patients. Triticain-α was found to be relatively stable in the conditions simulating stomach environment. We conclude that Triticain-α can be exploited as a basic compound for development of (i) pharmaceuticals for oral administration aimed at release of the active enzyme into the gastric lumen for CD treatment, and (ii) topically active pharmaceuticals for wound debridement applications.     

Translational studies on regulation of brain docosahexaenoic acid (DHA) metabolism in vivo

One goal in the field of brain polyunsaturated fatty acid (PUFA) metabolism is to translate the many studies that have been conducted in vitro and in animal models to the clinical setting. Doing so should elucidate the role of PUFAs in the human brain, and effects of diet, drugs, disease and genetics on this role. This review discusses new in vivo radiotracer kinetic and neuroimaging techniques that allow us to do this, with a focus on docosahexaenoic acid (DHA). We illustrate how brain PUFA metabolism is influenced by graded reductions in dietary n-3 PUFA content in unanesthetized rats. We also show how kinetic tracer techniques in rodents have helped to identify mechanisms of action of mood stabilizers used in bipolar disorder, how DHA participates in neurotransmission, and how brain DHA metabolism is regulated by calcium-independent iPLA₂β. In humans, regional rates of brain DHA metabolism can be quantitatively imaged with positron emission tomography following intravenous injection of [1-¹¹C]DHA.     

An atypical residue in the pore of Varroa destructor GABA-activated RDL receptors affects picrotoxin block and thymol modulation

GABA-activated RDL receptors are the insect equivalent of mammalian GABA_A receptors, and play a vital role in neurotransmission and insecticide action. Here we clone the pore lining M2 region of the Varroa mite RDL receptor and show that it has 4 atypical residues when compared to M2 regions of most other insects, including bees, which are the major host of Varroa mites. We create mutant Drosophila RDL receptors containing these substitutions and characterise their effects on function. Using two electrode voltage clamp electrophysiology we show that one substitution (T6′M) ablates picrotoxin inhibition and increases the potency of GABA. This mutation also alters the effect of thymol, which enhances both insect and mammalian GABA responses, and is widely used as a miticide. Thymol decreases the GABA EC₅₀ of WT receptors, enhancing responses, but in T6′M-containing receptors it is inhibitory. The other 3 atypical residues have no major effects on either the GABA EC₅₀, the picrotoxin potency or the effect of thymol. In conclusion we show that the RDL 6′ residue is important for channel block, activation and modulation, and understanding its function also has the potential to prove useful in the design of Varroa-specific insecticidal agents.