Mitochondrial Dysfunction in Neurodegenerative Diseases Associated with Copper Imbalance

Copper is an essential transition metal ion for the function of key metabolic enzymes, but its uncontrolled redox reactivity is source of reactive oxygen species. Therefore a network of transporters strictly controls the trafficking of copper in living systems. Deficit, excess, or aberrant coordination of copper are conditions that may be detrimental, especially for neuronal cells, which are particularly sensitive to oxidative stress. Indeed, the genetic disturbances of copper homeostasis, Menkes' and Wilson's diseases, are associated with neurodegeneration. Furthermore, copper interacts with the proteins that are the hallmarks of neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, prion diseases, and familial amyotrophic lateral sclerosis. In all cases, copper-mediated oxidative stress is linked to mitochondrial dysfunction, which is a common feature of neurodegeneration. In particular we recently demonstrated that in copper deficiency, mitochondrial function is impaired due to decreased activity of cytochrome c oxidase, leading to production of reactive oxygen species, which in turn triggers mitochondria-mediated apoptotic neurodegeneration.     

Mitochondrial damage contribute to epigallocatechin-3-gallate induced death in Leishmania amazonensis

Epigallocatechin-3-gallate (EGCG), the most abundant flavonoid in green tea, has been reported to have antiproliferative effects on Trypanosoma cruzi however, the mechanism of protozoan action of EGCG has not been studied. In the present study, we demonstrate the mechanism for the antileishmanial activity of EGCG against Leishmania amazonensis promastigotes. Incubation with EGCG significantly inhibited L. amazonensis promastigote proliferation in a time- and dose-dependent manner. The IC(50) for EGCG at 120h was 0.063mM. Ultrastructural alterations of the mitochondria were observed in promastigote treated with EGCG, being the organelle injury reinforced by the decrease in rhodamine 123 fluorescence. The effects of several drugs that interfere directly with mitochondrial physiology in parasites such as Leishmania have been described. The unique mitochondrial features of Leishmania make this organelle an ideal drug target while minimizing toxicity. These data suggest mitochondrial collapse as a part of the EGCG mechanism of action and demonstrate the leishmanicidal effect of EGCG.     

GDF-15 Is Elevated in Children with Mitochondrial Diseases and Is Induced by Mitochondrial Dysfunction

BackgroundWe previously described increased levels of growth and differentiation factor 15 (GDF-15) in skeletal muscle and serum of patients with mitochondrial diseases. Here we evaluated GDF-15 as a biomarker for mitochondrial diseases affecting children and compared it to fibroblast-growth factor 21 (FGF-21). To investigate the mechanism of GDF-15 induction in these pathologies we measured its expression and secretion in response to mitochondrial dysfunction.MethodsWe analysed 59 serum samples from 48 children with mitochondrial disease, 19 samples from children with other neuromuscular diseases and 33 samples from aged-matched healthy children. GDF-15 and FGF-21 circulating levels were determined by ELISA.ResultsOur results showed that in children with mitochondrial diseases GDF-15 levels were on average increased by 11-fold (mean 4046pg/ml, 1492 SEM) relative to healthy (350, 21) and myopathic (350, 32) controls. The area under the curve for the receiver-operating-characteristic curve for GDF-15 was 0.82 indicating that it has a good discriminatory power. The overall sensitivity and specificity of GDF-15 for a cut-off value of 550pg/mL was 67.8% (54.4%-79.4%) and 92.3% (81.5%-97.9%), respectively. We found that elevated levels of GDF-15 and or FGF-21 correctly identified a larger proportion of patients than elevated levels of GDF-15 or FGF-21 alone. GDF-15, as well as FGF-21, mRNA expression and protein secretion, were significantly induced after treatment of myotubes with oligomycin and that levels of expression of both factors significantly correlated.ConclusionsOur data indicate that GDF-15 is a valuable serum quantitative biomarker for the diagnosis of mitochondrial diseases in children and that measurement of both GDF-15 and FGF-21 improves the disease detection ability of either factor separately. Finally, we demonstrate for the first time that GDF-15 is produced by skeletal muscle cells in response to mitochondrial dysfunction and that its levels correlate in vitro with FGF-21 levels.     

Non-ulcerative cutaneous lesion in immunodeficient mice with Leishmania amazonensis infection

Cutaneous leishmaniasis begins as papules or nodules at the site of promastigote inoculation. The next key pathogenic event in this disease is the formation of an ulcer at this site. Leishmania infection in immunodeficient mice, however, showed non-ulcerative cutaneous lesions suggesting the involvement of the immune system in ulcer formation. Severe combined immunodeficient (SCID), recombination-activating gene 2 knockout (RAG-2-/-), and immunocompetent mice were inoculated subcutaneously with cultured L. amazonensis promastigotes. Macroscopic nodules appeared at the inoculation site within 2 weeks of infection in all the mice and gradually extended to the surrounding skin tissue. Although nodules of immunocompetent mice ulcerated within 6 weeks, immunodeficient mice did not form ulcers even after 25 weeks of inoculation. These results strongly suggest the importance of functional T and B cells in ulcer formation of cutaneous leishmaniasis and are consistent with clinical features of non-ulcerative cutaneous leishmaniasis in some AIDS patients. The present study also indicates that the L. amazonensis-infected immunodeficient mouse model might be suitable for studying the mechanisms of ulcer formation in cutaneous leishmaniasis.     

Ketamine Causes Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Neurons

Purpose

Ketamine toxicity has been demonstrated in nonhuman mammalian neurons. To study the toxic effect of ketamine on human neurons, an experimental model of cultured neurons from human induced pluripotent stem cells (iPSCs) was examined, and the mechanism of its toxicity was investigated.

Methods

Human iPSC-derived dopaminergic neurons were treated with 0, 20, 100 or 500 μM ketamine for 6 and 24 h. Ketamine toxicity was evaluated by quantification of caspase 3/7 activity, reactive oxygen species (ROS) production, mitochondrial membrane potential, ATP concentration, neurotransmitter reuptake activity and NADH/NAD⁺ ratio. Mitochondrial morphological change was analyzed by transmission electron microscopy and confocal microscopy.

Results

Twenty-four-hour exposure of iPSC-derived neurons to 500 μM ketamine resulted in a 40% increase in caspase 3/7 activity (P < 0.01), 14% increase in ROS production (P < 0.01), and 81% reduction in mitochondrial membrane potential (P < 0.01), compared with untreated cells. Lower concentration of ketamine (100 μM) decreased the ATP level (22%, P < 0.01) and increased the NADH/NAD⁺ ratio (46%, P < 0.05) without caspase activation. Transmission electron microscopy showed enhanced mitochondrial fission and autophagocytosis at the 100 μM ketamine concentration, which suggests that mitochondrial dysfunction preceded ROS generation and caspase activation.

Conclusions

We established an in vitro model for assessing the neurotoxicity of ketamine in iPSC-derived neurons. The present data indicate that the initial mitochondrial dysfunction and autophagy may be related to its inhibitory effect on the mitochondrial electron transport system, which underlies ketamine-induced neural toxicity. Higher ketamine concentration can induce ROS generation and apoptosis in human neurons.     

Leishmania (Leishmania) amazonensis: experimental cutaneous leishmaniasis associated with systemic amyloidosis in mice

We infected Swiss and C57BL/6 female mice in the left hind footpad with 10⁴Leishmania (L.) amazonensis promastigotes in stationary phase. The macroscopic examination showed a nodular non-ulcerated lesion at the site of inoculation and hepatic and spleenic enlargement. Histopathologically, the primary lesion showed an extensive liquefactive necrosis and inflammatory infiltrate, mainly consisting of macrophages filled with amastigotes, and rare lymphocytes. The inflammatory reaction in liver, spleen and kidney showed amyloid deposits. Additionally, C57BL/6 had accentuated amyloidosis in both ovarian cortical and medullar region and inflammatory infiltrates in the pancreas and adrenal gland.     

Protein kinase A activity is associated with metacyclogenesis in Leishmania amazonensis

Because of the importance of cell signalling processes in proliferation and differentiation, the adenylate cyclase pathway was studied, specifically the protein kinase A (PKA) in Leishmania amazonensis. The PKAs of soluble (SF) and enriched membrane fractions (MF) from infective/non-infective promastigotes and axenic amastigotes were assayed. In order to purify the PKA molecule, fractions were chromatographed on DEAE-cellulose columns and the phosphorylative activity was evaluated using [gamma(32)P]-ATP as the phosphate source. These experiments were performed in the presence of cyclic adenosine monophosphate (cAMP) and an inhibitor of PKA. Our data demonstrated that the PKA activity was significantly higher (about two times) in SF from promastigotes with a high concentration of metacyclic forms, when compared with the non-infective promastigotes, suggesting an association of this activity and the metacyclogenesis process. A discrete phosphorylative activity in axenic amastigotes was observed. As the adenylate cyclase/cAMP pathway would be involved in the parasite-host interiorization, the PKA activity may constitute a good intracellular target for studies of leishmanicidal drugs.     

Mitochondrial Oxidative Stress and Dysfunction in Rat Brain Induced by Carbofuran Exposure

Repeated low-dose exposure to carbofuran exerts its neurotoxic effects by non-cholinergic mechanisms. Emerging evidence indicates that oxidative stress plays an important role in carbofuran neurotoxicity after sub-chronic exposure. The purpose of the present study is to evaluate the role of mitochondrial oxidative stress and dysfunction as a primary event responsible for neurotoxic effects observed after sub-chronic carbofuran exposure. Carbofuran was administered to rats at a dose of 1 mg/kg orally for a period of 28 days. There was a significant inhibition in the activity of acetylcholinesterase (66.6%) in brain samples after 28 days of carbofuran exposure. Mitochondrial respiratory chain functions were assessed in terms of MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) reduction and activity of succinate dehydrogenase in isolated mitochondria. It was observed that carbofuran exposure significantly inhibited MTT reduction (31%) and succinate dehydrogenase activity (57%). This was accompanied by decrease in low-molecular weight thiols (66.6%) and total thiols (37.4%) and an increase in lipid peroxidation (43.7%) in the mitochondria isolated from carbofuran-exposed rat brain. The changes in mitochondrial oxidative stress and functions were associated with impaired cognitive and motor functions in the animals exposed to carbofuran as compared to the control animals. Based on these results, it is clear that carbofuran exerts its neurotoxicity by impairing mitochondrial functions leading to oxidative stress and neurobehavioral deficits.     

Amentoflavone isolated from Selaginella sellowii Hieron induces mitochondrial dysfunction in Leishmania amazonensis promastigotes

Leishmaniasis chemotherapy is a bottleneck in disease treatment. Although available, chemotherapy is limited, toxic, painful, and does not lead to parasite clearance, with parasite resistance also being reported. Therefore, new therapeutic options are being investigated, such as plant-derived anti-parasitic compounds. Amentoflavone is the most common biflavonoid in the Selaginella genus, and its antileishmanial activity has already been described on Leishmania amazonensis intracellular amastigotes but its direct action on the parasite is controversial. In this work we demonstrate that amentoflavone is active on L. amazonensis promastigotes (IC₅₀ = 28.5 ± 2.0 μM) and amastigotes. Transmission electron microscopy of amentoflavone-treated promastigotes showed myelin-like figures, autophagosomes as well as enlarged mitochondria. Treated parasites also presented multiple lipid droplets and altered basal body organization. Similarly, intracellular amastigotes presented swollen mitochondria, membrane fragments in the lumen of the flagellar pocket as well as autophagic vacuoles. Flow cytometric analysis after TMRE staining showed that amentoflavone strongly decreased mitochondrial membrane potential. In silico analysis shows that amentoflavone physic-chemical, drug-likeness and bioavailability characteristics suggest it might be suitable for oral administration. We concluded that amentoflavone presents a direct effect on L. amazonensis parasites, causing mitochondrial dysfunction and parasite killing. Therefore, all results point for the potential of amentoflavone as a promising candidate for conducting advanced studies for the development of drugs against leishmaniasis.     

Leishmania amazonensis: effects of oral treatment with copaiba oil in mice

Leishmaniasis is a severe public-health problem, with high rates of morbidity and mortality. Efforts to find new, effective and safe oral agents for the treatment of leishmaniasis have been ongoing for several decades, in order to avoid the problems with the currently used antimonials. In the present study, we found that a copaiba oil oral treatment (Group IV) caused a significant reduction in the average lesion size (1.1 ± 0.4 mm) against Leishmania amazonensis lesions compared with untreated mice (Group I) (4.4 ± 1.3 mm). To prove the safety of the oil, the toxicity and genotoxicity were also determined. Histopathological evaluation did not reveal changes in the copaiba oil-treated animals compared to the control animals. In the mutagenicity evaluation, (micronucleus test) the dose tested (2000 mg/kg) showed no genotoxic effects. Morphological and ultrastructural analyses demonstrated notable changes in parasite cells treated with this oleoresin. The main ultrastructural effect was mitochondrial swelling. We also demonstrated that in vitro copaiba oil treatment of L. amazonensis led to an increase in plasma membrane permeability, and depolarization in the mitochondrial membrane potential in parasite cells. Although the mechanism of action of the oleoresin is still unclear, these findings indicate that copaiba oil is a possible new drug, which would provide a safer, shorter, less-expensive, and more easily administered treatment for leishmaniasis.     

Kinetoplastid membrane protein-11 exacerbates infection with Leishmania amazonensis in murine macrophages

In Leishmania amazonensis, kinetoplastid membrane protein-11 (KMP-11) expression increases during meta-cyclogenesis and is higher in amastigotes than in promastigotes, suggesting a role for this protein in the infection of the mammalian host. We show that the addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide (NO) production. The doses of KMP-11, the IL-10 levels and the intracellular amastigote loads were strongly, positively and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10 neutralising antibodies, but not by isotype controls. The neutralising antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. In this study, the exacerbating effect of KMP-11 on macrophage infection with Leishmania is for the first time demonstrated, implicating it as a virulence factor in L. amazonensis. The stimulation of IL-10 production and arginase activity and the inhibition of NO synthesis are likely involved in this effect.     

Vaccination with the Leishmania infantum ribosomal proteins induces protection in BALB/c mice against Leishmania chagasi and Leishmania amazonensis challenge

Leishmania chagasi and Leishmania amazonensis are the etiologic agents of different clinical forms of human leishmaniasis in South America. In an attempt to select candidate antigens for a vaccine protecting against different Leishmania species, the efficacy of vaccination using Leishmania ribosomal proteins and saponin as adjuvant was examined in BALB/c mice against challenge infection with both parasite species. Mice vaccinated with parasite ribosomal proteins purified from Leishmania infantum plus saponin showed a specific production of IFN-γ, IL-12 and GM-CSF after in vitro stimulation with L. infantum ribosomal proteins. Vaccinated mice showed a reduction in the liver and spleen parasite burdens after L. chagasi infection. After L. amazonensis challenge, vaccinated mice showed a decrease of the dermal pathology and a reduction in the parasite loads in the footpad and spleen. In both models, protection was correlated to an IL-12-dependent production of IFN-γ by CD4⁺ and CD8⁺ T cells that activate macrophages for the synthesis of NO. In the protected mice a decrease in the parasite-mediated IL-4 and IL-10 responses was also observed. In mice challenged with L. amazonensis, lower levels of anti-parasite-specific antibodies were detected. Thus, Leishmania ribosomal proteins plus saponin fits the requirements to compose a pan-Leishmania vaccine.     

Low and high-dose intradermal infection with Leishmania major and Leishmania amazonensis in C57BL/6 mice

A model of skin infection with Leishmania amazonensis with low doses of parasites is compared to infection with high doses of L. amazonensis and low and high doses of Leishmania major. C57BL/6 mice were infected with 103 or 10(6) parasites in the ear and the outcome of infection was assessed. The appearance of lesions in mice infected with 103 parasites was delayed compared to mice infected with 10(6) Leishmania and parasites were detectable at the infection site before lesions became apparent. Mice infected with L. amazonensis displayed persistent lesions, whereas infection with L. major spontaneously healed in all groups, although lymphocytes persisted at the site of infection after healing. Macrophages persisted only in L. amazonensis-infected mice. High-dose L. amazonensis-infected mice produced lower levels of IFN-γ and TNF than mice infected with L. major. No correlation between the persistence of parasites and IL-10 levels and the production of nitric oxide or urea by macrophages was found. We conclude that infection with low doses of L. amazonensis in the dermis changes the course of infection by delaying the appearance of lesions. However, low-dose infection does not change the outcomes of susceptibility and cytokine production described for subcutaneous infection with high numbers of parasites.     

Late cutaneous metastases in C3H SCID mice infected with Leishmania amazonensis

The biological behavior of Leishmania amazonensis in the mammalian host is highly variable, resulting in local to diffuse cutaneous lesions that sometimes metastasize. Inflammation and, more specifically, CD4+ T cells have been shown to enhance metastases in mice infected with L. amazonensis, suggesting that the process may be lymphocyte mediated. However, we document, in this study, the development of multiple cutaneous metastases in C3H SCID mice infected with L. amazonensis. This shows that functional T and B cells are not required for metastases to occur.     

Dynamic identification of H2 epitopes from Leishmania (Leishmania) amazonensis cysteine proteinase B with potential immune activity during murine infection

Peptides from the COOH‐terminal extension of cysteine proteinase B from Leishmania (Leishmania) amazonensis (cyspep) can modulate immune responses in vertebrate hosts. With this hypothesis as base, we used the online analysis tool SYFPEITHI to predict seven epitopes from this region with potential to bind H2 proteins. We performed proliferation tests and quantified reactive T lymphocytes applying a cytometry analysis, using samples from draining lymph node of lesions from L. (L.) amazonensis‐infected mice. To define reactivity of T cells, we used complexes of DimerX (H2 D^b:Ig and H2 L^d:Ig) and the putative epitopes. Additionally, we applied surface plasmon resonance to verify real time interactions between the putative epitopes and DimerX proteins. Five peptides induced blastogenesis in BALB/c cells, while only two presented the same property in C57BL/6 mouse cells. In addition, our data indicate the existence of CD8⁺ T lymphocyte populations able to recognize each tested peptide in both murine strains. We observed an overlapping of results between the peptides that induced lymphocyte proliferation and those capable of binding to the DimerX in the surface plasmon resonance assays thus indicating that using these recombinant proteins in biosensing analyses is a promising tool to study real time molecular interactions in the context of major histocompatibility complex epitopes. The data gathered in this study reinforce the hypothesis that cyspep‐derived peptides are important factors in the murine host infection by L. (L.) amazonensis. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification of three proteins that associate in vitro with the Leishmania (Leishmania) amazonensis G-rich telomeric strand.

The chromosomal ends of Leishmania (Leishmania) amazonensis contain conserved 5'-TTAGGG-3' telomeric repeats. Protein complexes that associate in vitro with these DNA sequences, Leishmania amazonensis G-strand telomeric protein (LaGT1-3), were identified and characterized by electrophoretic mobility shift assays and UV cross-linking using protein fractions purified from S100 and nuclear extracts. The three complexes did not form (a) with double-stranded DNA and the C-rich telomeric strand, (b) in competition assays using specific telomeric DNA oligonucleotides, or (c) after pretreatment with proteinase K. LaGT1 was the most specific and did not bind a Tetrahymena telomeric sequence. All three LaGTs associated with an RNA sequence cognate to the telomeric G-rich strand and a complex similar to LaGT1 is formed with a double-stranded DNA bearing a 3' G-overhang tail. The protein components of LaGT2 and LaGT3 were purified by affinity chromatography and identified, after renaturation, as approximately 35 and approximately 52 kDa bands, respectively. The 相似文献   

Leishmania amazonensis: Anionic currents expressed in oocytes upon microinjection of mRNA from the parasite

Transport mechanisms involved in pH homeostasis are relevant for the survival of Leishmania parasites. The presence of chloride conductive pathways in Leishmania has been anticipated since anion channel inhibitors limit the proton extrusion mediated by the H+ATPase, which is the major regulator of intracellular pH in amastigotes. In this study, we used Xenopus laevis oocytes as a heterologous expression system in which to study the expression of ion channels upon microinjection of polyA mRNA from Leishmania amazonensis. After injection of polyA mRNA into the oocytes, we measured three different types of currents. We discuss the possible origin of each, and propose that Type 3 currents could be the result of the heterologous expression of proteins from Leishmania since they show different pharmacological and biophysical properties as compared to endogenous oocyte currents.     

Redox Reactions Induced by Nitrosative Stress Mediate Protein Misfolding and Mitochondrial Dysfunction in Neurodegenerative Diseases

Overstimulation of N-methyl-d-aspartate (NMDA)-type glutamate receptors accounts, at least in part, for excitotoxic neuronal damage, potentially contributing to a wide range of acute and chronic neurologic diseases. Neurodegenerative disorders including Alzheimer’s disease (AD) and Parkinson’s disease (PD), manifest deposits of misfolded or aggregated proteins, and result from synaptic injury and neuronal death. Recent studies have suggested that nitrosative stress due to generation of excessive nitric oxide (NO) can mediate excitotoxicity in part by triggering protein misfolding and aggregation, and mitochondrial fragmentation in the absence of genetic predisposition. S-Nitrosylation, or covalent reaction of NO with specific protein thiol groups, represents a convergent signal pathway contributing to NO-induced protein misfolding and aggregation, compromised dynamics of mitochondrial fission-fusion process, thus leading to neurotoxicity. Here, we review the effect of S-nitrosylation on protein function under excitotoxic conditions, and present evidence suggesting that NO contributes to protein misfolding and aggregation via S-nitrosylating protein-disulfide isomerase or the E3 ubiquitin ligase parkin, and mitochondrial fragmentation through β-amyloid-related S-nitrosylation of dynamin-related protein-1. Moreover, we also discuss that inhibition of excessive NMDA receptor activity by memantine, an uncompetitive/fast off-rate (UFO) drug can ameliorate excessive production of NO, protein misfolding and aggregation, mitochondrial fragmentation, and neurodegeneration.