Metabolomics provide new insights into mechanisms of Wolbachia-induced paternal defects in Drosophila melanogaster

Wolbachia is a group of intracellular symbiotic bacteria that widely infect arthropods and nematodes. Wolbachia infection can regulate host reproduction with the most common phenotype in insects being cytoplasmic incompatibility (CI), which results in embryonic lethality when uninfected eggs fertilized with sperms from infected males. This suggests that CI-induced defects are mainly in paternal side. However, whether Wolbachia-induced metabolic changes play a role in the mechanism of paternal-linked defects in embryonic development is not known. In the current study, we first use untargeted metabolomics method with LC-MS to explore how Wolbachia infection influences the metabolite profiling of the insect hosts. The untargeted metabolomics revealed 414 potential differential metabolites between Wolbachia-infected and uninfected 1-day-old (1d) male flies. Most of the differential metabolites were significantly up-regulated due to Wolbachia infection. Thirty-four metabolic pathways such as carbohydrate, lipid and amino acid, and vitamin and cofactor metabolism were affected by Wolbachia infection. Then, we applied targeted metabolomics analysis with GC-MS and showed that Wolbachia infection resulted in an increased energy expenditure of the host by regulating glycometabolism and fatty acid catabolism, which was compensated by increased food uptake. Furthermore, overexpressing two acyl-CoA catabolism related genes, Dbi (coding for diazepam-binding inhibitor) or Mcad (coding for medium-chain acyl-CoA dehydrogenase), ubiquitously or specially in testes caused significantly decreased paternal-effect egg hatch rate. Oxidative stress and abnormal mitochondria induced by Wolbachia infection disrupted the formation of sperm nebenkern. These findings provide new insights into mechanisms of Wolbachia-induced paternal defects from metabolic phenotypes.

Abnormal Mitochondria-Endoplasmic Reticulum Communication Promotes Myocardial Infarction

Myocardial infarction is characterized by cardiomyocyte death, and can be exacerbated by mitochondrial damage and endoplasmic reticulum injury. In the present study, we investigated whether communication between mitochondria and the endoplasmic reticulum contributes to cardiomyocyte death after myocardial infarction. Our data demonstrated that hypoxia treatment (mimicking myocardial infarction) promoted cardiomyocyte death by inducing the c-Jun N-terminal kinase (JNK) pathway. The activation of JNK under hypoxic conditions was dependent on overproduction of mitochondrial reactive oxygen species (mtROS) in cardiomyocytes, and mitochondrial division was identified as the upstream inducer of mtROS overproduction. Silencing mitochondrial division activators, such as B cell receptor associated protein 31 (BAP31) and mitochondrial fission 1 (Fis1), repressed mitochondrial division, thereby inhibiting mtROS overproduction and preventing JNK-induced cardiomyocyte death under hypoxic conditions. These data revealed that a novel death-inducing mechanism involving the BAP31/Fis1/mtROS/JNK axis promotes hypoxia-induced cardiomyocyte damage. Considering that BAP31 is localized within the endoplasmic reticulum and Fis1 is localized in mitochondria, abnormal mitochondria-endoplasmic reticulum communication may be a useful therapeutic target after myocardial infarction.

P–129 Follicular extracellular vesicles of women with polycystic ovarian syndrome inhibit oocyte maturation

Study question Does follicular extracellular vesicles of women with polycystic ovarian syndrome (PCOS-EVs) interfere with the quality of oocytes? Summary answer PCOS-EVs induced oxidative stress in the oocytes and inhibited oocyte maturation by increasing the abnormal mitochondria distribution and abnormal spindle rates. What is known already Polycystic ovarian syndrome (PCOS) is a common endocrine disorder in women of reproductive age, with a prevalence up to 10%. Women with PCOS are characterized by a clustering of features, including hyperandrogenism, polycystic ovarian morphology, and notably, anovulation. Although international guidelines recommend assisted reproduction techniques to be an effective resort for PCOS patients to conceive. However, even after overcoming ovulatory dysfunction via ovulation induction, the pregnancy outcomes of patients with PCOS were far from satisfying with lower fertilization, cleavage, and implantation rates, implicating that theoocyte quality of these patients are affected. Whereas the mechanisms have not been elucidated yet. Study design, size, duration Follicular fluid of PCOS patients (n = 10) and healthy controls (n = 10) were collected and used for extracellular vesicles (EVs) isolation via ultracentrifugation. Germinal vesicle (GV) oocytes collected from female ICR mice were cocultured with RIF-EVs or FER-EVs, respectively, and PBS served as a blank control. GV breakdown (GVBD) rate and maturation rate were calculated at two-hour and fourteen-hour of co-culture, respectively. Besides, oocyte mitochondria distribution, meiosis spindle morphology, and oxidative status were assessed in different groups. Participants/materials, setting, methods EVs were determined by western blotting, nanoparticle tracking analysis, and transmission electron microscopy. Fluorescence labeled EVs were used to visualize internalization by oocytes. Oocytes mitochondria and mitosis spindles were stained with fluorescence, and abnormal mitochondria rate or abnormal spindle rate was calculated. Reactive oxygen species (ROS) level was detected in the differently treated oocytes. Moreover, the expression of CAT, GSS, and SOD was determined in the oocytes using quantitative reverse transcription polymerase chain reaction. Main results and the role of chance Both PCOS-EVs and CTRL-EVs are bilayered vesicles, ranging from 100 to 150 nm, and enriched in Alix, TSG101, and CD9. EVs could be internalized by oocytes within one hour. After coculture, the GVBD rate was similar among the three groups; whereas the maturation rate was significantly decreased in the PCOS-EV group compared with CTRL-EV group or PBS group. In addition, the abnormal mitochondria distribution rate or abnormal spindle rate were significantly increased in the PCOS-EV group compared with PBS or CTRL-EV group. The ROS level was increased in the PCOS-EV group compared with CTRL-EV group, and the expression of CAT, GSS, and SOD was increased in the PCOS-EV-treated oocytes. Limitations, reasons for caution Our study did not identify the contents of PCOS-EVs and CTRL-EVs, and the molecular mechanisms of dysregulations induced by PCOS-EVs need further researches to investigate. Wider implications of the findings: This work confirmed that EV-conducted cellular communication played an important role in oocyte development in women with PCOS. The dysregulation of oocytes induced by PCOS-EVs might be related to the poor oocyte quality of women with PCOS, which may provide a novel target to improve pregnancy outcomes of these patients. Trial registration number Not applicable

To rectify Alzheimer's disease etiology, excessive mitochondrial division might be stopped or mitophagy might be promoted

In both in vitro and in vivo Alzheimer's disease (AD) models, mitochondrial dysfunction is a crucial feature that limits neuronal activity and results in A and phosphorylated Tau toxicity. To rectify AD etiology, excessive mitochondrial division might be stopped or mitophagy might be promoted. However, there are still unexplained mysteries surrounding the formation of senile plaques and NFTs, and the pathophysiology of Alzheimer's disease lacks fundamental unifying principles. Some scientists believe A toxicity and Tau toxicity are upstream processes in mitochondrial dysfunction, while others feel it is a downstream chain of events involving abnormal mitochondria. There are several mitophagy mechanisms for the clearance of dead mitochondria in PINK1 signaling; some are regulated by Parkin, while others are not. Drp1, Mfn1/2, PINK1, or Parkin, according to some researchers, have no role in mitophagy cleaning dysfunctional mitochondria; so, additional study is needed to solve the puzzle of mitophagy signaling pathways for clearing dead mitochondria and conserving high-quality mitochondria. Therapeutic techniques targeting mitophagy activity might be useful in reversing AD etiology.

Exploiting the Role of Hypoxia-Inducible Factor 1 and Pseudohypoxia in the Myelodysplastic Syndrome Pathophysiology

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem (HSCs) and/or progenitor cells disorders. The established dependence of MDS progenitors on the hypoxic bone marrow (BM) microenvironment turned scientific interests to the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 facilitates quiescence maintenance and regulates differentiation by manipulating HSCs metabolism, being thus an appealing research target. Therefore, we examine the aberrant HIF-1 stabilization in BMs from MDS patients and controls (CTRLs). Using a nitroimidazole–indocyanine conjugate, we show that HIF-1 aberrant expression and transcription activity is oxygen independent, establishing the phenomenon of pseudohypoxia in MDS BM. Next, we examine mitochondrial quality and quantity along with levels of autophagy in the differentiating myeloid lineage isolated from fresh BM MDS and CTRL aspirates given that both phenomena are HIF-1 dependent. We show that the mitophagy of abnormal mitochondria and autophagic death are prominently featured in the MDS myeloid lineage, their severity increasing with intra-BM blast counts. Finally, we use in vitro cultured CD34+ HSCs isolated from fresh human BM aspirates to manipulate HIF-1 expression and examine its potential as a therapeutic target. We find that despite being cultured under 21% FiO2, HIF-1 remained aberrantly stable in all MDS cultures. Inhibition of the HIF-1α subunit had a variable beneficial effect in all <5%-intra-BM blasts-MDS, while it had no effect in CTRLs or in ≥5%-intra-BM blasts-MDS that uniformly died within 3 days of culture. We conclude that HIF-1 and pseudohypoxia are prominently featured in MDS pathobiology, and their manipulation has some potential in the therapeutics of benign MDS.

Autophagy facilitates mitochondrial rebuilding after acute heat stress via a DRP-1–dependent process

Acute heat stress (aHS) can induce strong developmental defects in Caenorhabditis elegans larva but not lethality or sterility. This stress results in transitory fragmentation of mitochondria, formation of aggregates in the matrix, and decrease of mitochondrial respiration. Moreover, active autophagic flux associated with mitophagy events enables the rebuilding of the mitochondrial network and developmental recovery, showing that the autophagic response is protective. This adaptation to aHS does not require Pink1/Parkin or the mitophagy receptors DCT-1/NIX and FUNDC1. We also find that mitochondria are a major site for autophagosome biogenesis in the epidermis in both standard and heat stress conditions. In addition, we report that the depletion of the dynamin-related protein 1 (DRP-1) affects autophagic processes and the adaptation to aHS. In drp-1 animals, the abnormal mitochondria tend to modify their shape upon aHS but are unable to achieve fragmentation. Autophagy is induced, but autophagosomes are abnormally elongated and clustered on mitochondria. Our data support a role for DRP-1 in coordinating mitochondrial fission and autophagosome biogenesis in stress conditions.

α-synuclein impairs autophagosome maturation through abnormal actin stabilization

Vesicular trafficking defects, particularly those in the autophagolysosomal system, have been strongly implicated in the pathogenesis of Parkinson’s disease and related α-synucleinopathies. However, mechanisms mediating dysfunction of membrane trafficking remain incompletely understood. Using a Drosophila model of α-synuclein neurotoxicity with widespread and robust pathology, we find that human α-synuclein expression impairs autophagic flux in aging adult neurons. Genetic destabilization of the actin cytoskeleton rescues F-actin accumulation, promotes autophagosome clearance, normalizes the autophagolysosomal system, and rescues neurotoxicity in α-synuclein transgenic animals through an Arp2/3 dependent mechanism. Similarly, mitophagosomes accumulate in human α-synuclein-expressing neurons, and reversal of excessive actin stabilization promotes both clearance of these abnormal mitochondria-containing organelles and rescue of mitochondrial dysfunction. These results suggest that Arp2/3 dependent actin cytoskeleton stabilization mediates autophagic and mitophagic dysfunction and implicate failure of autophagosome maturation as a pathological mechanism in Parkinson’s disease and related α-synucleinopathies.

Lysosomal SLC46A3 modulates hepatic cytosolic copper homeostasis

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic toxicity associated with prominent lipid accumulation in humans. Here, the authors report that the lysosomal copper transporter SLC46A3 is induced by TCDD and underlies the hepatic lipid accumulation in mice, potentially via effects on mitochondrial function. SLC46A3 was localized to the lysosome where it modulated intracellular copper levels. Forced expression of hepatic SLC46A3 resulted in decreased mitochondrial membrane potential and abnormal mitochondria morphology consistent with lower copper levels. SLC46A3 expression increased hepatic lipid accumulation similar to the known effects of TCDD exposure in mice and humans. The TCDD-induced hepatic triglyceride accumulation was significantly decreased in Slc46a3−/− mice and was more pronounced when these mice were fed a high-fat diet, as compared to wild-type mice. These data are consistent with a model where lysosomal SLC46A3 induction by TCDD leads to cytosolic copper deficiency resulting in mitochondrial dysfunction leading to lower lipid catabolism, thus linking copper status to mitochondrial function, lipid metabolism and TCDD-induced liver toxicity.

Focal Segmental Glomerulosclerosis with a Mutation in the Mitochondrially Encoded NADH Dehydrogenase 5 Gene: A Case Report

Background: NADH dehydrogenase 5 (ND5) is one of 44 subunits forming Complex I in the mitochondrial respiratory chain. Therefore, a mitochondrially encoded ND5 (MT-ND5) gene mutation causes mitochondrial oxidative phosphorylation (OXPHOS) disorder, resulting in the development of mitochondrial diseases, such as mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. Although focal segmental glomerulosclerosis (FSGS) is induced by several mitochondrial diseases, no reports have yet confirmed that an MT-ND5 mutation causes FSGS. Case presentation: A Japanese woman at 29 years old was found to have proteinuria and renal dysfunction in an annual health check-up for the first time. Because her proteinuria and renal dysfunction were persistent, she was admitted for a kidney biopsy to investigate the reason at 33 years of age. Although there were no abnormal neurological findings, she was diagnosed with sensorineural hearing loss by an otolaryngologist. Renal histology showed perihilar variant-type FSGS with podocytes filled with abnormal mitochondria. In addition, the renal pathological findings showed granular swollen epithelial cells (GSECs) in tubular cells, age-inappropriately disarranged and irregularly sized vascular smooth muscle cells (AiDIVs), and red-colored podocytes (ReCPos) by acidic dye, which are assumed to be characteristic changes of mitochondrial nephropathy. Genetic analysis using peripheral mononuclear blood cells and urine sediment cells detected the m.13513 G>A variant, which has already been confirmed as a pathogenic variant of the MT-ND5 gene. The heteroplasmy rates of the blood cells and urine sediments were 10.3% and 62.2%, respectively. Therefore, this patient was diagnosed with FSGS due to an MT-ND5 mutation. Conclusion: This is the first case report to show that a gene mutation encoding subunits of Complex I causes FSGS with podocytes filled with abnormal mitochondria. The routine evaluation of GSECs, AiDIVs, and ReCPos is expected to be useful for detecting mitochondrial nephropathies, which could be latent in etiology-unknown FSGS or glomerulosclerosis cases.