OPA1 Research Articles

Mitochondrial Cristae Morphology Reflecting Metabolism, Superoxide Formation, Redox Homeostasis, and Pathology.

Significance: Mitochondrial (mt) reticulum network in the cell possesses amazing ultramorphology of parallel lamellar cristae, formed by the invaginated inner mitochondrial membrane. Its non-invaginated part, the inner boundary membrane (IBM) forms a cylindrical sandwich with the outer mitochondrial membrane (OMM). Crista membranes (CMs) meet IBM at crista junctions (CJs) of mt cristae organizing system (MICOS) complexes connected to OMM sorting and assembly machinery (SAM). Cristae dimensions, shape, and CJs have characteristic patterns for different metabolic regimes, physiological and pathological situations. Recent Advances: Cristae-shaping proteins were characterized, namely rows of ATP-synthase dimers forming the crista lamella edges, MICOS subunits, optic atrophy 1 (OPA1) isoforms and mitochondrial genome maintenance 1 (MGM1) filaments, prohibitins, and others. Detailed cristae ultramorphology changes were imaged by focused-ion beam/scanning electron microscopy. Dynamics of crista lamellae and mobile CJs were demonstrated by nanoscopy in living cells. With tBID-induced apoptosis a single entirely fused cristae reticulum was observed in a mitochondrial spheroid. Critical Issues: The mobility and composition of MICOS, OPA1, and ATP-synthase dimeric rows regulated by post-translational modifications might be exclusively responsible for cristae morphology changes, but ion fluxes across CM and resulting osmotic forces might be also involved. Inevitably, cristae ultramorphology should reflect also mitochondrial redox homeostasis, but details are unknown. Disordered cristae typically reflect higher superoxide formation. Future Directions: To link redox homeostasis to cristae ultramorphology and define markers, recent progress will help in uncovering mechanisms involved in proton-coupled electron transfer via the respiratory chain and in regulation of cristae architecture, leading to structural determination of superoxide formation sites and cristae ultramorphology changes in diseases.

Upregulation of mitochondrial calcium uniporter contributes to paraquat-induced neuropathology linked to Parkinson’s disease via imbalanced OPA1 processing

Paraquat (PQ) is the most widely used herbicide in agriculture worldwide and has been considered a high-risk environmental factor for Parkinson’s disease (PD). Chronic PQ exposure selectively induces dopaminergic neuron loss, the hallmark pathologic feature of PD, resulting in Parkinson-like movement disorders. However, the underlying mechanisms remain unclear. Here, we demonstrated that repetitive PQ exposure caused dopaminergic neuron loss, dopamine deficiency and motor deficits dose-dependently in mice. Accordingly, mitochondrial calcium uniporter (MCU) was highly expressed in PQ-exposed mice and neuronal cells. Importantly, MCU knockout (KO) effectively rescued PQ-induced dopaminergic neuron loss and motor deficits in mice. Genetic and pharmacological inhibition of MCU alleviated PQ-induced mitochondrial dysfunction and neuronal death in vitro. Mechanistically, PQ exposure triggered mitochondrial fragmentation via imbalance of the optic atrophy 1 (OPA1) processing manifested by cleavage of L-OPA1 to S-OPA1, which was reversed by inhibition of MCU. Notably, the upregulation of MCU was mediated by miR-129–1–3p posttranscriptionally, and overexpression of miR-129–1–3p could rebalance OPA1 processing and attenuate mitochondrial dysfunction and neuronal death induced by PQ exposure. Consequently, our work uncovers an essential role of MCU and a novel molecular mechanism, miR-MCU-OPA1, in PQ-induced pathogenesis of PD, providing a potential target and strategy for environmental neurotoxins-induced PD treatment.

Modulation of Autophagy and Mitochondrial Dynamics Gene Expression by Turmeric and Mangosteen Peel Extract

High fat diet (HFD) induces oxidative stress and mitochondrial dysfunction which culminates in fatty liver disease. Autophagy and mitochondrial dynamics are affected by HFD. Turmeric and mangosteen have potential roles as antioxidants and regulators of mitochondrial function in the liver. The study aims to examine the effect of turmeric and mangosteen peel extract on autophagy and mitochondrial dynamics in the liver after HFD induction. Five groups of animals (n=5) as used: negative control, positive control (HFD), turmeric (HFD + 270 mg/kg BW turmeric extract), mangosteen (HFD + mangosteen 270 mg/kg BW peel extract), and fenofibrate (HFD + 15 mg/kg BW fenofibrate). HFD was given for 7 weeks, continued by another 7 weeks plus treatment. Liver sections were extracted to conduct semi-quantitative PCR. Autophagy (LC3, p62), mitophagy (Pink1, Parkin, Bnip3), mitochondrial fission (Drp1, Fis1), and mitochondrial fusion (Opa1, Mfn1, Mfn2) gene expression were measured. LC3 (p=0.048), p62 (p=0.043), Pink1 (p=0.012), Bnip3 (p=0.010), Mfn1 (p=0.015), and Mfn2 (p=0.035) gene expressions were differed significantly, while Parkin (p=0.098) Drp1 (p=0.962), Fis1 (p=0.570), and Opa1 (p=0.055) gene expressions did not differ between groups. Both turmeric and mangosteen peel extract have positive effects by activating autophagy, mitophagy, and mitochondrial fusion in rat liver induced by HFD.

Buyang huanwu decoction inhibits diabetes-accelerated atherosclerosis via reduction of AMPK-Drp1-mitochondrial fission axis

Ethnopharmacological relevanceTraditional Chinese drugs, including Buyang Huanwu decoction (BYHWD), have been used in traditional practice to manage cardiovascular and cerebrovascular diseases. However, the effect and mechanisms by which this decoction alleviates diabetes-accelerated atherosclerosis are unknown and require exploration. Aim of the studyThis study aims to investigate the pharmacological effects of BYHWD on preventing diabetes-accelerated atherosclerosis, and elucidate its underlying mechanism. Materials and methodsStreptozotocin (STZ)-induced diabetic ApoE−/− mice were treated with BYHWD. Atherosclerotic aortic lesions, endothelial function, mitochondrial morphology, and mitochondrial dynamics-related proteins were evaluated in isolated aortas. High glucose-exposed human umbilical endothelial cells (HUVECs) were treated with BYHWD and its components. AMPK siRNA transfection, Drp1 molecular docking, Drp1 enzyme activity measurement, and so on were used to explore and verify the mechanism. ResultBYHWD treatment inhibited the worsening of diabetes-accelerated atherosclerosis by lessening atherosclerotic lesions in diabetic ApoE−/− mice, by impeding endothelial dysfunction under diabetic conditions, and by inhibiting mitochondrial fragmentation by lowering protein expression levels of Drp1 and mitochondrial fission-1 protein (Fis1) in diabetic aortic endothelium. In high glucose-exposed HUVECs, BYHWD treatment also downgraded reactive oxygen species, promoted nitric oxide levels, and abated mitochondrial fission by reducing protein expression levels of Drp1 and fis1, but not mitofusin-1 and optic atrophy-1. Interestingly, we found that BYHWD's protective effect against mitochondrial fission is mediated by AMPK activation-dependent reduction of Drp1 levels. The main serum chemical components of BYHWD, ferulic acid, and calycosin-7-glucoside, can reduce the expression of Drp1 by regulating AMPK, and can inhibit the activity of GTPase of Drp1. ConclusionThe above findings support the conclusion that BYHWD suppresses diabetes-accelerated atherosclerosis by reducing mitochondrial fission through modulation of the AMPK/Drp1 pathway.

Low Protein Programming Causes Increased Mitochondrial Fusion and Decreased Oxygen Consumption in the Hepatocytes of Female Rats.

The liver is one of the major organs involved in the regulation of glucose and lipid homeostasis. The effectiveness of metabolic activity in hepatocytes is determined by the quality and quantity of its mitochondria. Mitochondrial function is complex, and they act via various dynamic networks, which rapidly adapt to changes in the cellular milieu. Our present study aims to investigate the effects of low protein programming on the structure and function of mitochondria in the hepatocytes of adult females. Pregnant rats were fed with a control or isocaloric low-protein diet from gestational day 4 until delivery. A normal laboratory chow was given to all dams after delivery and to pups after weaning. The rats were euthanized at 4 months of age and the livers were collected from female offspring for investigating the mitochondrial structure, mtDNA copy number, mRNA, and proteins expression of genes associated with mitochondrial function. Primary hepatocytes were isolated and used for the analysis of the mitochondrial bioenergetics profiles. The mitochondrial ultrastructure showed that the in utero low-protein diet exposure led to increased mitochondrial fusion. Accordingly, there was an increase in the mRNA and protein levels of the mitochondrial fusion gene Opa1 and mitochondrial biogenesis genes Pgc1a and Essra, but Fis1, a fission gene, was downregulated. Low protein programming also impaired the mitochondrial function of the hepatocytes with a decrease in basal respiration ATP-linked respiration and proton leak. In summary, the present study suggests that the hepatic mitochondrial dysfunction induced by an in utero low protein diet might be a potential mechanism linking glucose intolerance and insulin resistance in adult offspring.

Melatonin-mediated malic enzyme 2 orchestrates mitochondrial fusion and respiratory functions to promote odontoblastic differentiation during tooth development.

Tooth development is a complex process that is tightly controlled by circadian rhythm. Melatonin (MT) is a major hormonal regulator of the circadian rhythm, and influences dentin formation and odontoblastic differentiation during tooth development; however, the underlying mechanism remains elusive. This study investigated how MT regulates odontoblastic differentiation, with a special focus on its regulation of mitochondrial dynamics. In rat dental papilla cells (DPCs), we found that MT promotes odontoblastic differentiation concurrently with enhanced mitochondrial fusion, while disruption of mitochondrial fusion by depleting optic atrophy 1 (OPA1) impairs MT-mediated differentiation and mitochondrial respiratory functions. Through RNA sequencing, we discovered that MT significantly upregulated malic enzyme 2 (ME2), a mitochondrial NAD(P)+ -dependent enzyme, and identified ME2 as a critical MT downstream effector that orchestrates odontoblastic differentiation, mitochondrial fusion, and respiration functions. By detecting the spatiotemporal expression of ME2 in developing tooth germs, and using tooth germ reconstituted organoids, we also provided in vivo and ex vivo evidence that ME2 promotes dentin formation, indicating a possible involvement of ME2 in MT-modulated tooth development. Collectively, our findings offer novel understandings regarding the molecular mechanism by which MT affects cell differentiation and organogenesis, meanwhile, the critical role of ME2 in MT-regulated mitochondrial functions is also highlighted.

Regulation of PM2.5 on mitochondrial damage in H9c2 cells through miR-421/SIRT3 pathway and protective effect of miR-421 inhibitor and resveratrol

Fine particulate matter (PM2.5) exposure is associated with cardiovascular disease (CVD) morbidity and mortality. Mitochondria are sensitive targets of PM2.5, and mitochondrial dysfunction is closely related to the occurrence of CVD. The epigenetic mechanism of PM2.5-triggered mitochondrial injury of cardiomyocytes is unclear. This study focused on the miR-421/SIRT3 signaling pathway to investigate the regulatory mechanism in cardiac mitochondrial dynamics imbalance in rat H9c2 cells induced by PM2.5. Results illustrated that PM2.5 impaired mitochondrial function and caused dynamics homeostasis imbalance. Besides, PM2.5 up-regulated miR-421 and down-regulated SIRT3 gene expression, along with decreasing p-FOXO3a (SIRT3 downstream target gene) and p-Parkin expression and triggering abnormal expression of fusion gene OPA1 and fission gene Drp1. Further, miR-421 inhibitor (miR-421i) and resveratrol significantly elevated the SIRT3 levels in H9c2 cells after PM2.5 exposure and mediated the expression of SOD2, OPA1 and Drp1, restoring the mitochondrial morphology and function. It suggests that miR-421/SIRT3 pathway plays an epigenetic regulatory role in mitochondrial damage induced by PM2.5 and that miR-421i and resveratrol exert protective effects against PM2.5-incurred cardiotoxicity.

Oxidative Stress in Antibiotic Toxic Optic Neuropathy Mimicking Acute LHON in a Patient with Exacerbation of Cystic Fibrosis

The striking similarity of disc edema without leakage on fluorescein angiography, which is pathognomonic of Leber hereditary optic neuropathy (LHON), was present in a patient with cystic fibrosis with antibiotic toxic optic neuropathy. This similarity suggested the common effect of oxidative stress on retinal ganglion cells in inherited mitochondrial and antibiotic optic neuropathies. We present the case of a patient with advanced cystic fibrosis on chronic antibiotic treatment who experienced a rapid painless bilateral visual decline over a course of a few weeks. At examination, his corrected visual acuity was reduced to 0.3 in both eyes, with dyschromatopsia and central scotoma. The appearance of the fundus resembled the typical clinical features of acute LHON with hyperemic optic discs and tortuous vessels with no dye leakage from the optic discs on fluorescein angiography. Ganglion cell layer loss was seen on optic coherence tomography, with all findings pointing to LHON. Genetic testing did not reveal any LHON-specific mutations. After extended genetic testing, a heterozygous variant c.209C>T in the OPA3 gene on chromosome 19, g.46032648G>A, classified as a variant of unknown significance, was also found. After discontinuing antibiotics and general improvements in his health, surprisingly, his visual function completely improved. Later, he also received a bilateral lung transplant that further improved his general condition, and his vision remained normal. Excluding LHON, the transient optic neuropathy in our patient could be mainly due to antibiotic toxicity of linezolid and ciprofloxacin, which have been linked to mitochondrial dysfunction and advanced cystic fibrosis with hypoxic status. We suggest the possibility that patients with cystic fibrosis may be more prone to developing mitochondrial optic neuropathy, especially with additional risk factors such as chronic antibiotic therapy, which affect mitochondrial function, and can perhaps serve as a model for LHON.

Humic substances from composted fennel residues control the inflammation induced by Helicobacter pylori infection in AGS cells.

Helicobacter pylori (H. pylori) is a common human pathogen causing inflammation. Recent studies have suggested a sophisticated interplay between mitochondria, innate immunity and inflammatory response, thus proposing mitochondrial disfunction as the hallmark of severe inflammatory disorders. In this study, humic substances isolated from composted fennel residues (HS-FEN) were tested as potential therapeutical strategy to restore the mitochondrial physiology and control the inflammation associated with H. pylori infection. The molecular features of HS-FEN were characterized by infrared spectrometry, thermochemolysis-GC/MS, NMR spectroscopy, and high-performance size-exclusion chromatography (HPSEC), which revealed the presence of aromatic polyphenolic components arranged in a rather stable conformation. In vitro results showed antioxidant and anti-inflammatory properties of HS-FEN, that was found to increase the expression level of OPA-1 and SOD-2 genes and in AGS cells stimulated with H. pylori culture filtrate (Hpcf) and concomitantly decrease the expression level of Drp-1 gene and IL-12, IL-17 and G-CSF proteins. The hydrophobic features of HS, their conformational arrangement and large content of bioactive molecules may explain the beneficial effects of HS-FEN, that may potentially become an interesting source of anti-inflammatory agents capable to counteract or prevent the H. pylori-related inflammatory disorders.

Evaluation of a Less Invasive Cochlear Implant Surgery in OPA1 Mutations Provoking Deafblindness.

Cochlear implantation (CI) for deafblindness may have more impact than for non-syndromic hearing loss. Deafblind patients have a double handicap in a society that is more and more empowered by fast communication. CI is a remedy for deafness, but requires revision surgery every 20 to 25 years, and thus placement should be minimally invasive. Furthermore, failed reimplantation surgery will have more impact on a deafblind person. In this context, we assessed the safety of minimally invasive robotically assisted cochlear implant surgery (RACIS) for the first time in a deafblind patient. Standard pure tone audiometry and speech audiometry were performed in a patient with deafblindness as part of this robotic-assisted CI study before and after surgery. This patient, with an optic atrophy 1 (OPA1) (OMIM#165500) mutation consented to RACIS for the second (contralateral) CI. The applicability and safety of RACIS were evaluated as well as her subjective opinion on her disability. RACIS was uneventful with successful surgical and auditory outcomes in this case of deafblindness due to the OPA1 mutation. RACIS appears to be a safe and beneficial intervention to increase communication skills in the cases of deafblindness due to an OPA1 mutation. The use of RACIS use should be widespread in deafblindness as it minimizes surgical trauma and possible failures.

Mitochondrial structure and function adaptation in residual triple negative breast cancer cells surviving chemotherapy treatment.

Neoadjuvant chemotherapy (NACT) used for triple negative breast cancer (TNBC) eradicates tumors in ~45% of patients. Unfortunately, TNBC patients with substantial residual cancer burden have poor metastasis free and overall survival rates. We previously demonstrated mitochondrial oxidative phosphorylation (OXPHOS) was elevated and was a unique therapeutic dependency of residual TNBC cells surviving NACT. We sought to investigate the mechanism underlying this enhanced reliance on mitochondrial metabolism. Mitochondria are morphologically plastic organelles that cycle between fission and fusion to maintain mitochondrial integrity and metabolic homeostasis. The functional impact of mitochondrial structure on metabolic output is highly context dependent. Several chemotherapy agents are conventionally used for neoadjuvant treatment of TNBC patients. Upon comparing mitochondrial effects of conventional chemotherapies, we found that DNA-damaging agents increased mitochondrial elongation, mitochondrial content, flux of glucose through the TCA cycle, and OXPHOS, whereas taxanes instead decreased mitochondrial elongation and OXPHOS. The mitochondrial effects of DNA-damaging chemotherapies were dependent on the mitochondrial inner membrane fusion protein optic atrophy 1 (OPA1). Further, we observed heightened OXPHOS, OPA1 protein levels, and mitochondrial elongation in an orthotopic patient-derived xenograft (PDX) model of residual TNBC. Pharmacologic or genetic disruption of mitochondrial fusion and fission resulted in decreased or increased OXPHOS, respectively, revealing longer mitochondria favor oxphos in TNBC cells. Using TNBC cell lines and an in vivo PDX model of residual TNBC, we found that sequential treatment with DNA-damaging chemotherapy, thus inducing mitochondrial fusion and OXPHOS, followed by MYLS22, a specific inhibitor of OPA1, was able to suppress mitochondrial fusion and OXPHOS and significantly inhibit regrowth of residual tumor cells. Our data suggest that TNBC mitochondria can optimize OXPHOS through OPA1-mediated mitochondrial fusion. These findings may provide an opportunity to overcome mitochondrial adaptations of chemoresistant TNBC.

CryoEM of OPA1 helical structures gives perspective to mitochondrial dysfunction.

The leading cause of childhood blindness, Dominant Optic Atrophy (DOA), is primarily caused by mutation of the gene encoding the Optic Atrophy 1 (OPA1) protein. These OPA1 mutations putatively dysregulate the inner-membrane (IM) fusion processes of the mitochondria, leading to mitochondrial network disruption and DOA pathology. OPA1 exists in two forms in vivo: the long-form which is tethered to the inner mitochondrial membrane through a single transmembrane domain, and the short form that results from proteolytic cleavage in the inner membrane space as a response to mitochondrial membrane depolarization. Using cryo-electron microscopy, we solved helical structures of the short form of human OPA1 isoform-1 (sOPA1) with and without nucleotide. These helical assemblies formed around a lipid core, with densely packed protein rungs marked by minimal inter-rung connectivity. Notably, we also observed nucleotide-dependent dimerization of the GTPase domains, a hallmark of dynamin superfamily proteins. The sOPA1 assemblies contained several unique secondary structures to strengthen membrane association, pointing to the importance of sOPA1 membrane interaction to its function. The novel structural features and interfaces also shed light on the effects of pathogenic point mutations on protein folding, inter-protein assembly, and membrane interactions. Further, the biological relevance of the sOPA1 helical assemblies was supported by in-cell assays, where disruption of the structurally identified interfaces and membrane binding regions caused fragmentation of the mitochondrial network.

Bavachin and Corylifol A Improve Muscle Atrophy by Enhancing Mitochondria Quality Control in Type 2 Diabetic Mice.

Type 2 diabetes reduces muscle mass and function. Chronic inflammation and mitochondrial dysfunction play critical roles in muscle atrophy pathogenesis. Here, we investigated the effects of bavachin and corylifol A from Psoralea corylifolia L. seeds on muscle atrophy in dexamethasone-treated mice and in db/db mice. Bavachin and corylifol A enhanced muscle strength and muscle mass in dexamethasone-treated mice. In diabetic mice, they enhanced muscle strength and cross-sectional areas. Bavachin and corylifol A suppressed inflammatory cytokine (interleukin-6 and tumor necrosis factor-α) expression levels by downregulating nuclear factor-κB phosphorylation. They decreased the muscle atrophic factor (myostatin, atrogin-1, and muscle RING finger-1) expression levels. They activated the AKT synthetic signaling pathway and induced a switch from fast-type glycolytic fibers (type 2B) to slow-type oxidative fibers (types I and 2A). They increased mitochondrial biogenesis and dynamic factor (optic atrophy-1, mitofusin-1/2, fission, mitochondrial 1, and dynamin 1-like) expression levels via the AMP-activated protein kinase-peroxisome proliferator-activated receptor gamma coactivator 1-alpha signaling pathway. They also improved mitochondrial quality by upregulating the mitophagy factor (p62, parkin, PTEN-induced kinase-1, and BCL2-interacting protein-3) expression levels. Therefore, bavachin and corylifol A exert potential therapeutic effects on muscle atrophy by suppressing inflammation and improving mitochondrial function.

OPA1 Dominant Optic Atrophy: Diagnostic Approach in the Pediatric Population

A clinical and genetic study was conducted with pediatric patients and their relatives with optic atrophy 1 (OPA1) mutations to establish whether there is a genotype-phenotype correlation among the variants detected within and between families. Eleven children with a confirmed OPA1 mutation were identified during the study period. The main initial complaint was reduced visual acuity (VA), present in eight patients of the cohort. Eight of eleven patients had a positive family history of optic atrophy. The mean visual acuity at the start of the study was 0.40 and 0.44 LogMAR in the right and left eye, respectively. At the end of the study, the mean visual acuity was unchanged. Optical coherence tomography during the first visit showed a mean retinal nerve fiber layer thickness of 81.6 microns and 80.5 microns in the right and left eye, respectively; a mean ganglion cell layer of 52.5 and 52.4 microns, respectively, and a mean central macular thickness of 229.5 and 233.5 microns, respectively. The most common visual field defect was a centrocecal scotoma, and nine out of eleven patients showed bilateral temporal disc pallor at baseline. Sequencing of OPA1 showed seven different mutations in the eleven patients, one of which, NM_130837.3: c.1406_1407del (p.Thr469LysfsTer16), has not been previously reported. Early diagnosis of dominant optic atrophy is crucial, both for avoiding unnecessary consultations and/or treatments and for appropriate genetic counseling.

High-fat diet causes mitochondrial damage and downregulation of mitofusin-2 and optic atrophy-1 in multiple organs.

High-fat consumption promotes the development of obesity, which is associated with various chronic illnesses. Mitochondria are the energy factories of eukaryotic cells, maintaining self-stability through a fine-tuned quality-control network. In the present study, we evaluated high-fat diet (HFD)-induced changes in mitochondrial ultrastructure and dynamics protein expression in multiple organs. C57BL/6J male mice were fed HFD or normal diet (ND) for 24 weeks. Compared with ND-fed mice, HFD-fed mice exhibited increased body weight, cardiomyocyte enlargement, pulmonary fibrosis, hepatic steatosis, renal and splenic structural abnormalities. The cellular apoptosis of the heart, liver, and kidney increased. Cellular lipid droplet deposition and mitochondrial deformations were observed. The proteins related to mitochondrial biogenesis (TFAM), fission (DRP1), autophagy (LC3 and LC3-II: LC3-I ratio), and mitophagy (PINK1) presented different changes in different organs. The mitochondrial fusion regulators mitofusin-2 (MFN2) and optic atrophy-1 (OPA1) were consistently downregulated in multiple organs, even the spleen. TOMM20 and ATP5A protein were enhanced in the heart, skeletal muscle, and spleen, and attenuated in the kidney. These results indicated that high-fat feeding caused pathological changes in multiple organs, accompanied by mitochondrial ultrastructural damage, and MFN2 and OPA1 downregulation. The mitochondrial fusion proteins may become promising targets and/or markers for treating metabolic disease.

Abstract 1888: The protein optic atrophy 1 (OPA1) functions as a novel assembly factor for the respiratory complex V, and its deletion is tolerated in the liver

Abstract 1888: The protein optic atrophy 1 (OPA1) functions as a novel assembly factor for the respiratory complex V, and its deletion is tolerated in the liver

The effects of nuclear DNA mutations on mitochondrial function

The multiple functions of mitochondria, including adenosine triphosphate synthesis, are controlled by the coordination of both the mitochondrial DNA (mtDNA) and the nuclear DNA (nDNA) genomes. Mitochondrial disorders manifest because of impairment of energy metabolism. This article focuses on mutations in two nuclear genes and their effect on mitochondrial function. Mutations in the polymerase gamma, or POLG, gene are associated with multisystemic disease processes, including Alpers Syndrome, a severe childhood-onset syndrome. Mutations in the OPA1 gene are associated with autosomal dominant optic atrophy and other neurologic, musculoskeletal, and ophthalmologic symptoms. When assessing for disorders affecting energy metabolism, sequencing of both the mtDNA genome and the nDNA whole exome sequencing is necessary.

Kaempferol counteracts toxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in D. melanogaster: An implication of its mitoprotective activity

The current study aimed to investigate whether kaempferol (KMP), the major bioactive component of green leafy vegetables, could counteract the toxicity elicited by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in Drosophila melanogaster or not. First, we performed a dose-response curve, where adult wild-type flies were fed on diet-containing different concentrations of KMP throughout their lifespan. Afterward, flies were fed on a diet containing MPTP (500 μM) and KMP (20 and 40 μM) for 7 days. The MPTP- fed flies presented a higher mortality rate, lower emergence rate, locomotor deficits, and disruption in circadian rhythm when compared to the control. MPTP exposure induced severe oxidative stress, which was marked by reduction in thiol content, overproduction of reactive species, lipid and protein oxidation, and disruption of enzymes of antioxidant and neurotransmission pathways. MPTP also compromised the mitochondrial dynamics and respiration of flies, affecting the electron transport chain, oxidative phosphorylation, and fusion/fission processes. Besides extending per se the lifespan of flies, KMP counteracted the toxic effects of MPTP on the circadian cycle, survival, climbing, and hatching rates. KMP was also effective in restoring the activities of acetylcholinesterase (AChE) and monoamine oxidase (MAO) enzymes, as well as in normalizing the levels of all oxidant/antioxidant markers disrupted in MPTP-fed flies. Indeed, KMP reestablished the mitochondrial functionality in MPTP- fed flies, restoring the electron transport system linked to mitochondrial complex I and II, and rescuing the mRNA transcription of genes associated with mitochondrial fusion and fission, namely OPA-1 (Optic atrophy 1) and DRP-1 (Dynamin related protein 1). Our results showed the efficacy of KMP in hindering the toxicity induced by MPTP in D. melanogaster and suggest that the mitoprotective action of flavonoid may be boosting its anti-parkinsonism activity in the model. Besides, the study showed that wild-type strains of D. melanogaster proved to be reproducible in vivo model to mimic parkinsonian phenotypes through exposure to the neurotoxin MPTP.

Metformin Alleviates Epirubicin-Induced Endothelial Impairment by Restoring Mitochondrial Homeostasis

Vascular endothelial injury is important in anthracycline-induced cardiotoxicity. Anthracyclines seriously damage the mitochondrial function and mitochondrial homeostasis. In this study, we investigated the damage of epirubicin to vascular endothelial cells and the protective role of metformin from the perspective of mitochondrial homeostasis. We found that epirubicin treatment resulted in DNA double-strand breaks (DSB), elevated reactive oxygen species (ROS) production, and excessive Angiotensin II release in HUVEC cells. Pretreatment with metformin significantly mitigated the injuries caused by epirubicin. In addition, inhibited expression of Mitochondrial transcription factor A (TFAM) and increased mitochondria fragmentation were observed in epirubicin-treated cells, which were partially resumed by metformin pretreatment. In epirubicin-treated cells, knockdown of TFAM counteracted the attenuated DSB formation due to metformin pretreatment, and inhibition of mitochondrial fragmentation with Mdivi-1 decreased DSB formation but increased TFAM expression. Furthermore, epirubicin treatment promoted mitochondrial fragmentation by stimulating the expression of Dynamin-1-like protein (DRP1) and inhibiting the expression of Optic atrophy-1(OPA1) and Mitofusin 1(MFN1), which could be partially prevented by metformin. Finally, we found metformin could increase TFAM expression and decrease DRP1 expression in epirubicin-treated HUVEC cells by upregulating the expression of calcineurin/Transcription factor EB (TFEB). Taken together, this study provided evidence that metformin treatment was an effective way to mitigate epirubicin-induced endothelial impairment by maintaining mitochondrial homeostasis.

Cardiac sympathetic innervation is an unrecognized disease target in autosomal dominant optic atrophy

The Optic Atrophy-1 protein (Opa1) controls mitochondrial (mt) fusion and cristae morphology, ATP production, maintenance of mtDNA, and apoptosis. Mutations in Opa1 gene cause Autosomal Dominant Optic Atrophy (ADOA), a neurodegenerative disease characterized by “retinal ganglion cell” death and visual loss. Recently, ADOA has been described as a “multi-systemic disease” also featuring myopathy, peripheral neuropathy, Parkinsonism and cardiac rhythm alterations. Sympathetic neurons (SNs) are the main extrinsic regulators of heart activity in basal and stress conditions, and their homeostasis strikingly depend on the correct mitochondrial dynamics. We here determined the effect of ADOA genetics on cardiac sympathetic innervation. The majority of Opa1 gene mutations result in truncated forms of the protein, leading to reduction in its cellular content. To study the impact of Opa1 haploinsufficiency on cardiac innervation, we generated transgenic mice with SN-specific Opa1 deletion (TH-Opa1+/− mice), which, in heterozygosity, causes 50% reduction of protein expression. Heart function of TH-Opa1+/− mice was assessed by ECHO and ECG telemetry. Cardiac innervation was evaluated by confocal IF, and whole-mount IF in tissue clarified heart blocks. In vitro assays achieved insights on the effects of Opa1 haploinsufficiency on SNs. SNs were also evaluated in skin biopsies from ADOA patients, and age- and sex-matched controls. We here found that SN density is significantly reduced in the skin of ADOA patients, indicating thatSNs are additional, previously neglected, targets of ADOA. To isolate the effects of Opa1 haploinsufficiency in SNs, we analyzed hearts of adult and aged TH-Opa1+/− mice, which showed intact morphology and contractile function. Looking at cardiac innervation, confocal IF and 3-D reconstruction of the SN network revealed a significant reduction in innervation density, which appeared evident already in adult mice, and worsened during ageing. Consistently, TH-Opa1+/− mice showed decreased Heart Rate Variability and increased incidence of arrhythmic beats. In vitro assays demonstrated the causal role of Opa1 haploinsufficiency in affecting SNs, by compromising TrkA/NGF signaling, which is key to sustain neuronal viability. Our data demonstrates that Opa1 haploinsufficiency also affects peripheral autonomic innervation, thus explaining extra-retinal symptoms experienced by ADOA patients.