Female reproductive tract microbiome and early miscarriages.

Miscarriage is one of the main causes of reproductive loss, which can lead to a number of physical and psychological complications and other long-term consequences. However, the role of vaginal and uterine microbiome in such complications is poorly understood. To review the published data on the function of the female reproductive tract microbiome in the pathogenesis of early miscarriages. The articles published over the past 20 years and deposited in PubMed, Google Academy, Scopus, Elibrary, ResearchGate, and EBSCO databases were analyzed. The review presents new data on the impact of the vaginal and uterine microbiome on the local immunity, including defense against sexually transmitted infections, and its association with other factors of miscarriages. The studies on the microbiome of non-pregnant women with recurrent miscarriages in the anamnesis, patients undergoing IVF, and pregnant women with miscarriages, as well as new directions in the microbiome research are discussed. The majority of studies have demonstrated that the dominant species of the vaginal and uterine microbiome in patients with early miscarriages are non-Lactobacillus bacteria. As many of these bacteria have not previously been detected by cultural studies and their role in obstetric complications is not well defined, further research on the female reproductive tract microbiome, including the microbiome of the cervix uteri, is needed to develop new approaches for the prognosis and prevention of miscarriages.

Method for detection of mtDNA damages for evaluating of pesticides toxicity for bumblebees (Bombus terrestris L.).

Bumblebees are important for crop pollination. Currently, the number of pollinators is decreasing worldwide, which is attributed mostly to the widespread use of pesticides. The aim of this work was to develop a method for assessing the genotoxicity of pesticides for the Bombus terrestris L. bumblebee using long-range PCR of mitochondrial DNA fragments. We have developed a panel of primers and assessed the genotoxicity of the following pesticides: imidacloprid, rotenone, deltamethrin, difenocanozole, malathion, metribuzin, penconazole, esfenvalerate, and dithianon. All pesticides (except imidacloprid) inhibited mitochondrial respiration fueled by pyruvate + malate; the strongest effect was observed for rotenone and difenocanozole. Three pesticides (dithianon, rotenone, and difenocanozole) affected the rate of H2O2 production. To study the pesticide-induced DNA damage in vitro and in vivo, we used three different mtDNA. The mtDNA damage was observed for all studied pesticides. Most of the studied pesticides caused significant damage to mtDNA in vitro and in vivo when ingested. Our results indicate that all tested pesticides, including herbicides and fungicides, can have a toxic effect on pollinators. However, the extent of pesticide-induced mtDNA damage in the flight muscles was significantly less upon the contact compared to the oral administration.

Benfotiamine treatment activates the Nrf2/ARE pathway and is neuroprotective in a transgenic mouse model of tauopathy.

Impaired glucose metabolism, decreased levels of thiamine and its phosphate esters, and reduced activity of thiamine-dependent enzymes, such as pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase and transketolase occur in Alzheimer's disease (AD). Thiamine deficiency exacerbates amyloid beta (Aß) deposition, tau hyperphosphorylation and oxidative stress. Benfotiamine (BFT) rescued cognitive deficits and reduced Aß burden in amyloid precursor protein (APP)/PS1 mice. In this study, we examined whether BFT confers neuroprotection against tau phosphorylation and the generation of neurofibrillary tangles (NFTs) in the P301S mouse model of tauopathy. Chronic dietary treatment with BFT increased lifespan, improved behavior, reduced glycated tau, decreased NFTs and prevented death of motor neurons. BFT administration significantly ameliorated mitochondrial dysfunction and attenuated oxidative damage and inflammation. We found that BFT and its metabolites (but not thiamine) trigger the expression of Nrf2/antioxidant response element (ARE)-dependent genes in mouse brain as well as in wild-type but not Nrf2-deficient fibroblasts. Active metabolites were more potent in activating the Nrf2 target genes than the parent molecule BFT. Docking studies showed that BFT and its metabolites (but not thiamine) bind to Keap1 with high affinity. These findings demonstrate that BFT activates the Nrf2/ARE pathway and is a promising therapeutic agent for the treatment of diseases with tau pathology, such as AD, frontotemporal dementia and progressive supranuclear palsy.

Divalent cation chelators citrate and EDTA unmask an intrinsic uncoupling pathway in isolated mitochondria.

We demonstrate a suppression of ROS production and uncoupling of mitochondria by exogenous citrate in Mg2+ free medium. Exogenous citrate suppressed H2O2 emission and depolarized mitochondria. The depolarization was paralleled by the stimulation of respiration of mitochondria. The uncoupling action of citrate was independent of the presence of sodium, potassium, or chlorine ions, and it was not mediated by the changes in permeability of the inner mitochondrial membrane to solutes. The citrate transporter was not involved in the citrate effect. Inhibitory analysis data indicated that several well described mitochondria carriers and channels (ATPase, IMAC, ADP/ATP translocase, mPTP, mKATP) were not involved in citrate's effect. Exogenous MgCl2 strongly inhibited citrate-induced depolarization. The uncoupling effect of citrate was demonstrated in rat brain, mouse brain, mouse liver, and human melanoma cells mitochondria. We interpreted the data as an evidence to the existence of a hitherto undescribed putative inner mitochondrial membrane channel that is regulated by extramitochondrial Mg2+ or other divalent cations.

Alterations in voltage-sensing of the mitochondrial permeability transition pore in ANT1-deficient cells.

The probability of mitochondrial permeability transition (mPT) pore opening is inversely related to the magnitude of the proton electrochemical gradient. The module conferring sensitivity of the pore to this gradient has not been identified. We investigated mPT's voltage-sensing properties elicited by calcimycin or H2O2 in human fibroblasts exhibiting partial or complete lack of ANT1 and in C2C12 myotubes with knocked-down ANT1 expression. mPT onset was assessed by measuring in situ mitochondrial volume using the 'thinness ratio' and the 'cobalt-calcein' technique. De-energization hastened calcimycin-induced swelling in control and partially-expressing ANT1 fibroblasts, but not in cells lacking ANT1, despite greater losses of mitochondrial membrane potential. Matrix Ca(2+) levels measured by X-rhod-1 or mitochondrially-targeted ratiometric biosensor 4mtD3cpv, or ADP-ATP exchange rates did not differ among cell types. ANT1-null fibroblasts were also resistant to H2O2-induced mitochondrial swelling. Permeabilized C2C12 myotubes with knocked-down ANT1 exhibited higher calcium uptake capacity and voltage-thresholds of mPT opening inferred from cytochrome c release, but intact cells showed no differences in calcimycin-induced onset of mPT, irrespective of energization and ANT1 expression, albeit the number of cells undergoing mPT increased less significantly upon chemically-induced hypoxia than control cells. We conclude that ANT1 confers sensitivity of the pore to the electrochemical gradient.

Scavenging of H2O2 by mouse brain mitochondria.

Mitochondrial reactive oxygen species (ROS) metabolism is unique in that mitochondria both generate and scavenge ROS. Recent estimates of ROS scavenging capacity of brain mitochondria are surprisingly high, ca. 9-12 nmol H2O2/min/mg, which is ~100 times higher than the rate of ROS generation. This raises a question whether brain mitochondria are a source or a sink of ROS. We studied the interaction between ROS generation and scavenging in mouse brain mitochondria by measuring the rate of removal of H2O2 added at a concentration of 0.4 µM, which is close to the reported physiological H2O2 concentrations in tissues, under conditions of low and high levels of mitochondrial H2O2 generation. With NAD-linked substrates, the rate of H2O2 generation by mitochondria was ~50-70 pmol/min/mg. The H2O2 scavenging dynamics was best approximated by the first order reaction equation. H2O2 scavenging was not affected by the uncoupling of mitochondria, phosphorylation of added ADP, or the genetic ablation of glutathione peroxidase 1, but decreased in the absence of respiratory substrates, in the presence of thioredoxin reductase inhibitor auranofin, or in partially disrupted mitochondria. With succinate, the rate of H2O2 generation was ~2,200-2,900 pmol/min/mg; the scavenging of added H2O2 was masked by a significant accumulation of generated H2O2 in the assay medium. The obtained data were fitted into a simple model that reasonably well described the interaction between H2O2 scavenging and production. It showed that mitochondria are neither a sink nor a source of H2O2, but can function as both at the same time, efficiently stabilizing exogenous H2O2 concentration at a level directly proportional to the ratio of the H2O2 generation rate to the rate constant of the first order scavenging reaction.

Methylene blue upregulates Nrf2/ARE genes and prevents tau-related neurotoxicity.

Methylene blue (MB, methylthioninium chloride) is a phenothiazine that crosses the blood brain barrier and acts as a redox cycler. Among its beneficial properties are its abilities to act as an antioxidant, to reduce tau protein aggregation and to improve energy metabolism. These actions are of particular interest for the treatment of neurodegenerative diseases with tau protein aggregates known as tauopathies. The present study examined the effects of MB in the P301S mouse model of tauopathy. Both 4 mg/kg MB (low dose) and 40 mg/kg MB (high dose) were administered in the diet ad libitum from 1 to 10 months of age. We assessed behavior, tau pathology, oxidative damage, inflammation and numbers of mitochondria. MB improved the behavioral abnormalities and reduced tau pathology, inflammation and oxidative damage in the P301S mice. These beneficial effects were associated with increased expression of genes regulated by NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE), which play an important role in antioxidant defenses, preventing protein aggregation, and reducing inflammation. The activation of Nrf2/ARE genes is neuroprotective in other transgenic mouse models of neurodegenerative diseases and it appears to be an important mediator of the neuroprotective effects of MB in P301S mice. Moreover, we used Nrf2 knock out fibroblasts to show that the upregulation of Nrf2/ARE genes by MB is Nrf2 dependent and not due to secondary effects of the compound. These findings provide further evidence that MB has important neuroprotective effects that may be beneficial in the treatment of human neurodegenerative diseases with tau pathology.

PGC-1α overexpression exacerbates ß-amyloid and tau deposition in a transgenic mouse model of Alzheimer's disease.

The peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) interacts with various transcription factors involved in energy metabolism and in the regulation of mitochondrial biogenesis. PGC-1α mRNA levels are reduced in a number of neurodegenerative diseases and contribute to disease pathogenesis, since increased levels ameliorate behavioral defects and neuropathology of Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis. PGC-1α and its downstream targets are reduced both in postmortem brain tissue of patients with Alzheimer's disease (AD) and in transgenic mouse models of AD. Therefore, we investigated whether increased expression of PGC-1α would exert beneficial effects in the Tg19959 transgenic mouse model of AD; Tg19959 mice express the human amyloid precursor gene (APP) with 2 familial AD mutations and develop increased ß-amyloid levels, plaque deposition, and memory deficits by 2-3 mo of age. Rather than an improvement, the cross of the Tg19959 mice with mice overexpressing human PGC-1α exacerbated amyloid and tau accumulation. This was accompanied by an impairment of proteasome activity. PGC-1α overexpression induced mitochondrial abnormalities, neuronal cell death, and an exacerbation of behavioral hyperactivity in the Tg19959 mice. These findings show that PGC-1α overexpression exacerbates the neuropathological and behavioral deficits that occur in transgenic mice with mutations in APP that are associated with human AD.

Bezafibrate administration improves behavioral deficits and tau pathology in P301S mice.

Peroxisome proliferator-activated receptors (PPARs) are ligand-mediated transcription factors, which control both lipid and energy metabolism and inflammation pathways. PPARγ agonists are effective in the treatment of metabolic diseases and, more recently, neurodegenerative diseases, in which they show promising neuroprotective effects. We studied the effects of the pan-PPAR agonist bezafibrate on tau pathology, inflammation, lipid metabolism and behavior in transgenic mice with the P301S human tau mutation, which causes familial frontotemporal lobar degeneration. Bezafibrate treatment significantly decreased tau hyperphosphorylation using AT8 staining and the number of MC1-positive neurons. Bezafibrate treatment also diminished microglial activation and expression of both inducible nitric oxide synthase and cyclooxygenase 2. Additionally, the drug differentially affected the brain and brown fat lipidome of control and P301S mice, preventing lipid vacuoles in brown fat. These effects were associated with behavioral improvement, as evidenced by reduced hyperactivity and disinhibition in the P301S mice. Bezafibrate therefore exerts neuroprotective effects in a mouse model of tauopathy, as shown by decreased tau pathology and behavioral improvement. Since bezafibrate was given to the mice before tau pathology had developed, our data suggest that bezafibrate exerts a preventive effect on both tau pathology and its behavioral consequences. Bezafibrate is therefore a promising agent for the treatment of neurodegenerative diseases associated with tau pathology.

Mitochondrial permeability transition pore component cyclophilin D distinguishes nigrostriatal dopaminergic death paradigms in the MPTP mouse model of Parkinson's disease.

AIMS: Mitochondrial damage due to Ca(2+) overload-induced opening of permeability transition pores (PTP) is believed to play a role in selective degeneration of nigrostriatal dopaminergic neurons in Parkinson's disease (PD). Genetic ablation of mitochondrial matrix protein cyclophilin D (CYPD) has been shown to increase Ca(2+) threshold of PTP in vitro and to prevent cell death in several in vivo disease models. We investigated the role of CYPD in a mouse model of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced PD. RESULTS: We demonstrate that in vitro, brain mitochondria isolated from CYPD knockout mice were less sensitive to MPP+ (1-methyl-4-phenyl-pyridinium ion)-induced membrane depolarization, and free radical generation compared to wild-type mice. CYPD knockout mitochondria isolated from ventral midbrain of mice treated with MPTP in vivo exhibited less damage as judged from respiratory chain Complex I activity, State 3 respiration rate, and respiratory control index than wild-type mice, whereas assessment of apoptotic markers showed no differences between the two genotypes. However, CYPD knockout mice were significantly resistant only to an acute regimen of MPTP neurotoxicity in contrast to the subacute and chronic MPTP paradigms. INNOVATION: Inactivation of CYPD is beneficial in preserving mitochondrial functions only in an acute insult model of MPTP-induced dopaminergic neurotoxicity. CONCLUSION: Our results suggest that CYPD deficiency distinguishes the modes of dopaminergic neurodegeneration in various regimens of MPTP-neurotoxicity.

Behavioral deficit, oxidative stress, and mitochondrial dysfunction precede tau pathology in P301S transgenic mice.

Abnormal tau accumulation can lead to the development of neurodegenerative diseases. P301S mice overexpress the human tau mutated gene, resulting in tau hyperphosphorylation and tangle formation. Mice also develop synaptic deficits and microglial activation prior to any neurodegeneration and tangles. Oxidative stress can also affect tauopathy. We studied the role of oxidative stress in relationship to behavioral abnormalities and disease progression in P301S mice at 2, 7, and 10 mo of age. At 7 mo of age, P301S mice had behavioral abnormalities, such as hyperactivity and disinhibition. At the same age, we observed increased carbonyls in P301S mitochondria (∼215 and 55% increase, males/females), and deregulation in the activity and content of mitochondrial enzymes involved in reactive oxygen species formation and energy metabolism, such as citrate synthase (∼19 and ∼5% decrease, males/females), MnSOD (∼16% decrease, males only), cytochrome C (∼19% decrease, females only), and cytochrome C oxidase (∼20% increase, females only). These changes in mitochondria proteome appeared before tau hyperphosphorylation and tangle formation, which were observed at 10 mo and were associated with GSK3ß activation. At that age, mitochondria proteome deregulation became more apparent in male P301S mitochondria. The data strongly suggest that oxidative stress and mitochondrial abnormalities appear prior to tau pathology.

Protein phosphatase 2C binds selectively to and dephosphorylates metabotropic glutamate receptor 3.

Cell surface receptor membrane localization is strongly dependent on protein-protein interactions often involving regulation by phosphorylation/dephosphorylation of the intracellular domains of membrane proteins. The present study was carried out to identify metabotropic glutamate receptor (mGluR) 3 regulatory binding proteins. Using the yeast two-hybrid technique, we found that the 50-aa C-terminal cytoplasmic tail of mGluR3 interacts specifically with protein phosphatase 2Calpha (PP2Calpha). This interaction was confirmed by GST pull-down and coimmunoprecipitation assays. mGluR3 interacts with PP2Calpha, beta, gamma, and delta isoforms; however, among the mGluR family only mGluR3 interacted with PP2C. The minimal interacting domain of mGluR3 comprised residues 836-855. Alignment between mGluR3 and mGluR2, a closely related group II receptor, indicated that this domain is not conserved between the two receptors. The mGluR3 cytoplasmic C-terminal tail contains one phosphorylation site for protein kinase A (Ser-845), but the phosphatase that dephosphorylates this site has not been previously identified. We find that PP2C, but not PP1, PP2A, or PP2B, dephosphorylates the mGluR3 cytoplasmic tail in vitro. The dephosphorylated form of the mGluR3 cytoplasmic tail, but not the equivalent region of mGluR2, inhibited PP2C assayed by using [32P]casein as a substrate. However, phosphorylation of the mGluR3 cytoplasmic tail at Ser-845 inhibits the interaction with PP2C. These results indicate distinct functions for mGluR2 and mGluR3 and suggest a dynamic regulation of mGluR3 by PP2C.