Mitochondrial Pool Research Articles

Mitophagy-Enhanced Nanoparticle-Engineered Mitochondria Restore Homeostasis of Mitochondrial Pool for Alleviating Pulmonary Fibrosis.

Pulmonary fibrosis (PF) is an interstitial lung disease tightly associated with the disruption of mitochondrial pool homeostasis, a delicate balance influenced by functional and dysfunctional mitochondria within lung cells. Mitochondrial transfer is an emerging technology to increase functional mitochondria via exogenous mitochondrial delivery; however, the therapeutic effect on mitochondrial transfer is hampered during the PF process by the persistence of dysfunctional mitochondria, which is attributed to impaired mitophagy. Herein, we reported engineering mitochondria mediated by mitophagy-enhanced nanoparticle (Mito-MEN), which promoted synchronal regulation of functional and dysfunctional mitochondria for treating PF. Mitophagy-enhanced nanoparticles (MENs) were fabricated through the encapsulation of Parkin mRNA, and the electrostatic interaction favored MENs to anchor isolated healthy mitochondria for the construction of Mito-MEN. Mito-MEN increased the load of functional exogenous mitochondria by enhancing mitochondrial delivery efficiency and promoted mitophagy of dysfunctional endogenous mitochondria. In a bleomycin (BLM)-induced PF mouse model, Mito-MEN repaired mitochondrial function and efficiently relieved PF-related phenotypes. This study provides a powerful tool for synchronal adjustment of mitochondrial pool homeostasis and offers a translational approach for pan-mitochondrial disease therapies.

Acute contractile activity induces the activation of the mitochondrial integrated stress response and the transcription factor ATF4.

Skeletal muscle relies on mitochondria to produce energy and support its metabolic flexibility. The function of the mitochondrial pool is regulated by quality control (MQC) processes. The integrated stress response (ISR), a MQC pathway, is activated in response to various cellular stressors. The transcription factor ATF4, the main effector of the ISR, ameliorates cellular stress by upregulating protective genes, such as CHOP and ATF5. Recent literature has shown that the ISR is activated upon mitochondrial stress, however, whether this includes acute exercise-induced stress is poorly defined. To investigate this, a mouse in situ hindlimb protocol was utilized to acutely stimulate muscles at 0.25, 0.5 and 1 tetanic contraction/per second for 9 mins, followed by a 1-hour recovery period. CAMKII and JNK2 were robustly activated 6-fold immediately following the protocol. ISR activation, denoted as the ratio of phosphorylated to total-eIF2a protein levels, was also elevated following recovery. Downstream, contractile activity induced an increase in the nuclear localization of ATF4. Robust 2-fold increases in the mRNA expression of ATF4 and CHOP were also observed following the recovery period. Changes in ATF4 mRNA were independent of transcriptional activation, as assessed using an ATF4 promoter-reporter plasmid. Instead, mRNA decay assays revealed an increase in ATF4 mRNA stability post-contractile activity, as a result of enhanced stabilization by the RNA binding protein, HuR. Thus, acute contractile activity is sufficient to induce mitochondrial stress and activate the ISR, corresponding to the induction of ATF4 with potential consequences for mitochondrial phenotype adaptations in response to repeated exercise.

Guanylate Kinase 1 Deficiency: A Novel and Potentially Treatable Mitochondrial DNA Depletion/Deletions Disease.

Mitochondrial DNA (mtDNA) depletion/deletions syndrome (MDDS) comprises a group of diseases caused by primary autosomal defects of mtDNA maintenance. Our objective was to study the etiology of MDDS in 4 patients who lack pathogenic variants in known genetic causes. Whole exome sequencing of the probands was performed to identify pathogenic variants. We validated the mitochondrial defect by analyzing mtDNA, mitochondrial dNTP pools, respiratory chain activities, and GUK1 activity. To confirm pathogenicity of GUK1 deficiency, we expressed 2 GUK1 isoforms in patient cells. We identified biallelic GUK1 pathogenic variants in all 4 probands who presented with ptosis, ophthalmoparesis, and myopathic proximal limb weakness, as well as variable hepatopathy and altered T-lymphocyte profiles. Muscle biopsies from all probands showed mtDNA depletion, deletions, or both, as well as reduced activities of mitochondrial respiratory chain enzymes. GUK1 encodes guanylate kinase, originally identified as a cytosolic enzyme. Long and short isoforms of GUK1 exist. We observed that the long isoform is intramitochondrial and the short is cytosolic. In probands' fibroblasts, we noted decreased GUK1 activity causing unbalanced mitochondrial dNTP pools and mtDNA depletion in both replicating and quiescent fibroblasts indicating that GUK1 deficiency impairs de novo and salvage nucleotide pathways. Proband fibroblasts treated with deoxyguanosine and/or forodesine, a purine phosphatase inhibitor, ameliorated mtDNA depletion, indicating potential pharmacological therapies. Primary GUK1 deficiency is a new and potentially treatable cause of MDDS. The cytosolic isoform of GUK1 may contribute to the T-lymphocyte abnormality, which has not been observed in other MDDS disorders. ANN NEUROL 2024.

Beneficial Consequences of One-Month Oral Treatment with Cannabis Oil on Cardiac Hypertrophy and the Mitochondrial Pool in Spontaneously Hypertensive Rats.

Introduction: It has been demonstrated the dysregulation of the cardiac endocannabinoid system in cardiovascular diseases. Thus, the modulation of this system through the administration of phytocannabinoids present in medicinal cannabis oil (CO) emerges as a promising therapeutic approach. Furthermore, phytocannabinoids exhibit potent antioxidant properties, making them highly desirable in the treatment of cardiac pathologies, such as hypertension-induced cardiac hypertrophy (CH). Objective: To evaluate the effect of CO treatment on hypertrophy and mitochondrial status in spontaneously hypertensive rat (SHR) hearts. Methods: Three-month-old male SHR were randomly assigned to CO or olive oil (vehicle) oral treatment for 1 month. We evaluated cardiac mass and histology, mitochondrial dynamics, membrane potential, area and density, myocardial reactive oxygen species (ROS) production, superoxide dismutase (SOD), and citrate synthase (CS) activity and expression. Data are presented as mean ± SEM (n) and compared by t-test, or two-way ANOVA and Bonferroni post hoc test were used as appropriate. p < 0.05 was considered statistically significant. Results: CH was reduced by CO treatment, as indicated by the left ventricular weight/tibia length ratio, left ventricular mass index, myocyte cross-sectional area, and left ventricle collagen volume fraction. The ejection fraction was preserved in the CO-treated group despite the persistence of elevated systolic blood pressure and the reduction in CH. Mitochondrial membrane potential was improved and mitochondrial biogenesis, dynamics, area, and density were all increased by treatment. Moreover, the activity and expression of the CS were enhanced by treatment, whereas ROS production was decreased and the antioxidant activity of SOD increased by CO administration. Conclusion: Based on the mentioned results, we propose that 1-month oral treatment with CO is effective to reduce hypertrophy, improve the mitochondrial pool and increase the antioxidant capacity in SHR hearts.

Melatonin alleviates palmitic acid-induced mitochondrial dysfunction by reducing oxidative stress and enhancing autophagy in bovine endometrial epithelial cells

BackgroundNegative energy balance (NEB) typically occurs in dairy cows after delivery. Cows with a high yield are more likely to experience significant NEB. This type of metabolic imbalance could cause ketosis, which is often accompanied by a decline in reproductive performance. However, the molecular mechanisms underlying NEB have yet to be fully elucidated. During excessive NEB, the body fat is extensively broken down, resulting in the abnormal accumulation of non-esterified fatty acids (NEFAs), represented by palmitic acid (PA), within the uterus. Such an abnormal accumulation has the potential to damage bovine endometrial epithelial cells (BEECs), while the molecular mechanisms underlying its involvement in the PA-induced injury of BEECs remains poorly understood. Melatonin (MT) is recognized for its regulatory role in maintaining the homeostasis of mitochondrial reactive oxygen species (mitoROS). However, little is known as to whether MT could ameliorate the damage incurred by BEECs in response to PA and the molecular mechanism involved.ResultsAnalysis showed that 0.2 mmol/L PA stress increased the level of cellular and mitochondrial oxidative stress, as indicated by increased reactive oxygen species (ROS) level. In addition, we observed mitochondrial dysfunction, including abnormal mitochondrial structure and respiratory function, along with a reduction in mitochondrial membrane potential and mitochondrial copy number, and the induction of apoptosis. Notably, we also observed the upregulation of autophagy proteins (PINK, Parkin, LC3B and Ubiquitin), however, the P62 protein was also increased. As we expected, 100 μmol/L of MT pre-treatment attenuated PA-induced mitochondrial ROS and restored mitochondrial respiratory function. Meanwhile, MT pretreatment reversed the upregulation of P62 induced by PA and activated the AMPK-mTOR-Beclin-1 pathway, contributing to an increase of autophagy and decline apoptosis.ConclusionsOur findings indicate that PA can induce mitochondrial dysfunction and enhance autophagy in BEECs. In addition, MT is proved to not only reduce mitochondrial oxidative stress but also facilitate the clearance of damaged mitochondria by upregulating autophagy pathways, thereby safeguarding the mitochondrial pool and promoting cellular viability. Our study provides a better understanding of the molecular mechanisms underlying the effect of an excess of NEB on the fertility outcomes of high yielding dairy cows.

The endoplasmic reticulum pool of Bcl-xL prevents cell death through IP3R-dependent calcium release

Apoptosis plays a role in cell homeostasis in both normal development and disease. Bcl-xL, a member of the Bcl-2 family of proteins, regulates the intrinsic mitochondrial pathway of apoptosis. It is overexpressed in several cancers. Bcl-xL has a dual subcellular localisation and is found at the mitochondria as well as the endoplasmic reticulum (ER). However, the biological significance of its ER localisation is unclear. In order to decipher the functional contributions of the mitochondrial and reticular pools of Bcl-xL, we generated genetically modified mice expressing exclusively Bcl-xL at the ER, referred to as ER-xL, or the mitochondria, referred to as Mt-xL. By performing cell death assays, we demonstrated that ER-xL MEFs show increased vulnerability to apoptotic stimuli but are more resistant to ER stress. Furthermore, ER-xL MEFs displayed reduced 1,4,5-inositol trisphosphate receptor (IP3R)-mediated ER calcium release downstream of Phospholipase C activation. Collectively, our data indicate that upon ER stress, Bcl-xL negatively regulates IP3R-mediated calcium flux from the ER, which prevents ER calcium depletion and maintains the UPR and subsequent cell death in check. This work reveals a moonlighting function of Bcl-xL at the level of the ER, in addition to its well-known role in regulating apoptosis through the mitochondria.

Targeting valine catabolism to inhibit metabolic reprogramming in prostate cancer

Metabolic reprogramming and energetic rewiring are hallmarks of cancer that fuel disease progression and facilitate therapy evasion. The remodelling of oxidative phosphorylation and enhanced lipogenesis have previously been characterised as key metabolic features of prostate cancer (PCa). Recently, succinate-dependent mitochondrial reprogramming was identified in high-grade prostate tumours, as well as upregulation of the enzymes associated with branched-chain amino acid (BCAA) catabolism. In this study, we hypothesised that the degradation of the BCAAs, particularly valine, may play a critical role in anapleurotic refuelling of the mitochondrial succinate pool, as well as the maintenance of intracellular lipid metabolism. Through the suppression of BCAA availability, we report significantly reduced lipid content, strongly indicating that BCAAs are important lipogenic fuels in PCa. This work also uncovered a novel compensatory mechanism, whereby fatty acid uptake is increased in response to extracellular valine deprivation. Inhibition of valine degradation via suppression of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) resulted in a selective reduction of malignant prostate cell proliferation, decreased intracellular succinate and impaired cellular respiration. In combination with a comprehensive multi-omic investigation that incorporates next-generation sequencing, metabolomics, and high-content quantitative single-cell imaging, our work highlights a novel therapeutic target for selective inhibition of metabolic reprogramming in PCa.

Compartmentalized mitochondrial ferroptosis converges with optineurin-mediated mitophagy to impact airway epithelial cell phenotypes and asthma outcomes

A stable mitochondrial pool is crucial for healthy cell function and survival. Altered redox biology can adversely affect mitochondria through induction of a variety of cell death and survival pathways, yet the understanding of mitochondria and their dysfunction in primary human cells and in specific disease states, including asthma, is modest. Ferroptosis is traditionally considered an iron dependent, hydroperoxy-phospholipid executed process, which induces cytosolic and mitochondrial damage to drive programmed cell death. However, in this report we identify a lipoxygenase orchestrated, compartmentally-targeted ferroptosis-associated peroxidation process which occurs in a subpopulation of dysfunctional mitochondria, without promoting cell death. Rather, this mitochondrial peroxidation process tightly couples with PTEN-induced kinase (PINK)−1(PINK1)-Parkin-Optineurin mediated mitophagy in an effort to preserve the pool of functional mitochondria and prevent cell death. These combined peroxidation processes lead to altered epithelial cell phenotypes and loss of ciliated cells which associate with worsened asthma severity. Ferroptosis-targeted interventions of this process could preserve healthy mitochondria, reverse cell phenotypic changes and improve disease outcomes.

Mitochondrial respiratory function is preserved under cysteine starvation via glutathione catabolism in NSCLC

Cysteine metabolism occurs across cellular compartments to support diverse biological functions and prevent the induction of ferroptosis. Though the disruption of cytosolic cysteine metabolism is implicated in this form of cell death, it is unknown whether the substantial cysteine metabolism resident within the mitochondria is similarly pertinent to ferroptosis. Here, we show that despite the rapid depletion of intracellular cysteine upon loss of extracellular cystine, cysteine-dependent synthesis of Fe-S clusters persists in the mitochondria of lung cancer cells. This promotes a retention of respiratory function and a maintenance of the mitochondrial redox state. Under these limiting conditions, we find that glutathione catabolism by CHAC1 supports the mitochondrial cysteine pool to sustain the function of the Fe-S proteins critical to oxidative metabolism. We find that disrupting Fe-S cluster synthesis under cysteine restriction protects against the induction of ferroptosis, suggesting that the preservation of mitochondrial function is antagonistic to survival under starved conditions. Overall, our findings implicate mitochondrial cysteine metabolism in the induction of ferroptosis and reveal a mechanism of mitochondrial resilience in response to nutrient stress.

Deoxyguanosine kinase deficiency: natural history and liver transplant outcome.

Autosomal recessive pathogenetic variants in the DGUOK gene cause deficiency of deoxyguanosine kinase activity and mitochondrial deoxynucleotides pool imbalance, consequently, leading to quantitative and/or qualitative impairment of mitochondrial DNA synthesis. Typically, patients present early-onset liver failure with or without neurological involvement and a clinical course rapidly progressing to death. This is an international multicentre study aiming to provide a retrospective natural history of deoxyguanosine kinase deficient patients. A systematic literature review from January 2001 to June 2023 was conducted. Physicians of research centres or clinicians all around the world caring for previously reported patients were contacted to provide followup information or additional clinical, biochemical, histological/histochemical, and molecular genetics data for unreported cases with a confirmed molecular diagnosis of deoxyguanosine kinase deficiency. A cohort of 202 genetically confirmed patients, 36 unreported, and 166 from a systematic literature review, were analyzed. Patients had a neonatal onset (≤ 1 month) in 55.7% of cases, infantile (>1 month and ≤ 1 year) in 32.3%, pediatric (>1 year and ≤18 years) in 2.5% and adult (>18 years) in 9.5%. Kaplan-Meier analysis showed statistically different survival rates (P < 0.0001) among the four age groups with the highest mortality for neonatal onset. Based on the clinical phenotype, we defined four different clinical subtypes: hepatocerebral (58.8%), isolated hepatopathy (21.9%), hepatomyoencephalopathy (9.6%), and isolated myopathy (9.6%). Muscle involvement was predominant in adult-onset cases whereas liver dysfunction causes morbidity and mortality in early-onset patients with a median survival of less than 1 year. No genotype-phenotype correlation was identified. Liver transplant significantly modified the survival rate in 26 treated patients when compared with untreated. Only six patients had additional mild neurological signs after liver transplant. In conclusion, deoxyguanosine kinase deficiency is a disease spectrum with a prevalent liver and brain tissue specificity in neonatal and infantile-onset patients and muscle tissue specificity in adult-onset cases. Our study provides clinical, molecular genetics and biochemical data for early diagnosis, clinical trial planning and immediate intervention with liver transplant and/or nucleoside supplementation.

Mitochondrial transfer mediates endothelial cell engraftment through mitophagy.

Ischaemic diseases such as critical limb ischaemia and myocardial infarction affect millions of people worldwide1. Transplanting endothelial cells (ECs) is a promising therapy in vascular medicine, but engrafting ECs typically necessitates co-transplanting perivascular supporting cells such as mesenchymal stromal cells (MSCs), which makes clinical implementation complicated2,3. The mechanisms that enable MSCs to facilitate EC engraftment remain elusive. Here we show that, under cellular stress, MSCs transfer mitochondria to ECs through tunnelling nanotubes, and that blocking this transfer impairs EC engraftment. We devised a strategy to artificially transplant mitochondria, transiently enhancing EC bioenergetics and enabling them to form functional vessels in ischaemic tissues without the support of MSCs. Notably, exogenous mitochondria did not integrate into the endogenous EC mitochondrial pool, but triggered mitophagy after internalization. Transplanted mitochondria co-localized with autophagosomes, and ablation of the PINK1-Parkin pathway negated the enhanced engraftment ability of ECs. Our findings reveal a mechanism that underlies the effects of mitochondrial transfer between mesenchymal and endothelial cells, and offer potential for a new approach for vascular cell therapy.

Human mitochondrial glutathione transferases: Kinetic parameters and accommodation of a mitochondria-targeting group in substrates

Glutathione-S-transferases are key to the cellular detoxification of xenobiotics and products of oxidative damage. GSTs catalyse the reaction of glutathione (GSH) with electrophiles to form stable thioether adducts. GSTK1-1 is the main GST isoform in the mitochondrial matrix, but the GSTA1-1 and GSTA4-4 isoforms are also thought to be in the mitochondria with their distribution altering in transformed cells, thus potentially providing a cancer specific target. A mitochondria-targeted version of the GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), MitoCDNB, has been used to manipulate the mitochondrial GSH pool. To finesse this approach to target particular GST isoforms in the context of cancer, here we have determined the kcat/Km for the human isoforms of GSTK1-1, GSTA1-1 and GSTA4-4 with respect to GSH and CDNB. We show how the rate of the GST-catalysed reaction between GSH and CDNB analogues can be modified by both the electron withdrawing substituents, and by the position of the mitochondria-targeting triphenylphosphonium on the chlorobenzene ring to tune the activity of mitochondria-targeted substrates. These findings can now be exploited to selectively disrupt the mitochondrial GSH pools of cancer cells expressing particular GST isoforms.

Sulfide oxidation promotes hypoxic angiogenesis and neovascularization.

Angiogenic programming in the vascular endothelium is a tightly regulated process for maintaining tissue homeostasis and is activated in tissue injury and the tumor microenvironment. The metabolic basis of how gas signaling molecules regulate angiogenesis is elusive. Here, we report that hypoxic upregulation of ·NO in endothelial cells reprograms the transsulfuration pathway to increase biogenesis of hydrogen sulfide (H2S), a proangiogenic metabolite. However, decreased H2S oxidation due to sulfide quinone oxidoreductase (SQOR) deficiency synergizes with hypoxia, inducing a reductive shift and limiting endothelial proliferation that is attenuated by dissipation of the mitochondrial NADH pool. Tumor xenografts in whole-body (WBCreSqorfl/fl) and endothelial-specific (VE-cadherinCre-ERT2Sqorfl/fl) Sqor-knockout mice exhibit lower mass and angiogenesis than control mice. WBCreSqorfl/fl mice also exhibit decreased muscle angiogenesis following femoral artery ligation compared to control mice. Collectively, our data reveal the molecular intersections between H2S, O2 and ·NO metabolism and identify SQOR inhibition as a metabolic vulnerability for endothelial cell proliferation and neovascularization.

SIRT1-activating butein inhibits arecoline-induced mitochondrial dysfunction through PGC1α and MTP18 in oral cancer

BackgroundMitochondrial dysfunction associated with mitochondrial DNA mutations, enzyme defects, generation of ROS, and altered oxidative homeostasis is known to induce oral carcinogenesis during exposure to arecoline. Butein, a natural small molecule from Butea monosperma, possesses anti-inflammatory, anti-diabetic, and anti-cancer effects. However, the role of butein in the mitochondrial quality control mechanism has not been illuminated clearly. PurposeThis study aimed to explore the role of butein in preserving mitochondrial quality control during arecoline-induced mitochondrial dysfunction in oral cancer to curtail the early onset of carcinogenesis. MethodsCell viability was evaluated by MTT assay. The relative protein expressions were determined by western blotting. Immunofluorescence and confocal imaging were used to analyze the relative fluorescence and co-localization of proteins. Respective siRNAs were used to examine the knockdown-based studies. ResultsButein, in the presence of arecoline, significantly caused a decrease in mitochondrial hyperpolarization and ROS levels in oral cancer cells. Mechanistically, we found an increase in COXIV, TOM20, and PGC1α expression during butein treatment, and inhibition of PGC1α blunted mitochondrial biogenesis and decreased the mitochondrial pool. Moreover, the fission protein MTP18, and its molecular partners DRP1 and MFF were dose-dependently increased during butein treatment to maintain mitochondria mass. In addition, we also found increased expression of various mitophagy proteins, including PINK1, Parkin, and LC3 during butein treatment, suggesting the clearance of damaged mitochondria to maintain a healthy mitochondrial pool. Interestingly, butein increased the activity of SIRT1 to enhance the functional mitochondrial pool, and inhibition of SIRT1 found to reduce the mitochondrial levels, as evident from the decrease in the expression of PGC1α and MTP18 in oral cancer cells. ConclusionOur study proved that SIRT1 maintains a functional mitochondrial pool through PGC1α and MTP18 for biogenesis and fission of mitochondria during arecoline exposure and could decrease the risk of mitochondria dysfunctionality associated with the onset of oral carcinogenesis.

CLU (clusterin) and PPARGC1A/PGC1α coordinately control mitophagy and mitochondrial biogenesis for oral cancer cell survival

ABSTRACT Mitophagy involves the selective elimination of defective mitochondria during chemotherapeutic stress to maintain mitochondrial homeostasis and sustain cancer growth. Here, we showed that CLU (clusterin) is localized to mitochondria to induce mitophagy controlling mitochondrial damage in oral cancer cells. Moreover, overexpression and knockdown of CLU establish its mitophagy-specific role, where CLU acts as an adaptor protein that coordinately interacts with BAX and LC3 recruiting autophagic machinery around damaged mitochondria in response to cisplatin treatment. Interestingly, CLU triggers class III phosphatidylinositol 3-kinase (PtdIns3K) activity around damaged mitochondria, and inhibition of mitophagic flux causes the accumulation of excessive mitophagosomes resulting in reactive oxygen species (ROS)-dependent apoptosis during cisplatin treatment in oral cancer cells. In parallel, we determined that PPARGC1A/PGC1α (PPARG coactivator 1 alpha) activates mitochondrial biogenesis during CLU-induced mitophagy to maintain the mitochondrial pool. Intriguingly, PPARGC1A inhibition through small interfering RNA (siPPARGC1A) and pharmacological inhibitor (SR-18292) treatment counteracts CLU-dependent cytoprotection leading to mitophagy-associated cell death. Furthermore, co-treatment of SR-18292 with cisplatin synergistically suppresses tumor growth in oral cancer xenograft models. In conclusion, CLU and PPARGC1A are essential for sustained cancer cell growth by activating mitophagy and mitochondrial biogenesis, respectively, and their inhibition could provide better therapeutic benefits against oral cancer.

MT-TN mutations lead to progressive mitochondrial encephalopathy and promotes mitophagy

Mitochondrial encephalopathy is a neurological disorder caused by impaired mitochondrial function and energy production. One of the genetic causes of this condition is the mutation of MT-TN, a gene that encodes the mitochondrial transfer RNA (tRNA) for asparagine. MT-TN mutations affect the stability and structure of the tRNA, resulting in reduced protein synthesis and complex enzymatic deficiency of the mitochondrial respiratory chain. Our patient cohort manifests with epileptic encephalopathy, ataxia, hypotonia, and bilateral basal ganglia calcification, which differs from previously reported cases. MT-TN mutation deficiency leads to decreased basal and maximal oxygen consumption rates, disrupted spare respiratory capacity, declined mitochondrial membrane potential, and impaired ATP production. Moreover, MT-TN mutations promote mitophagy, a process of selective degradation of damaged mitochondria by autophagy. Excessive mitophagy further leads to mitochondrial biogensis as a compensatory mechanism. In this study, we provided evidence of pathogenicity for two MT-TN mutations, m.5688 T > C and m.G5691A, explored the molecular mechanisms, and summarized the clinical manifestations of MT-TN mutations. Our study expanded the genotype and phenotypic spectrum and provided new insight into mt-tRNA (Asn)-associated mitochondrial encephalopathy.

High-level biosynthesis of enantiopure germacrene D in yeast.

Germacrene D, a sesquiterpenoid compound found mainly in plant essential oils at a low level as (+) and/or (-) enantiomeric forms, is an ingredient for the fragrance industry, but a process for the sustainable supply of enantiopure germacrene D is not yet established. Here, we demonstrate metabolic engineering in yeast (Saccharomyces cerevisiae) achieving biosynthesis of enantiopure germacrene D at a high titer. To boost farnesyl pyrophosphate (FPP) flux for high-level germacrene D biosynthesis, a background yeast chassis (CENses5C) was developed by genomic integration of the expression cassettes for eight ergosterol pathway enzymes that sequentially converted acetyl-CoA to FPP and by replacing squalene synthase promoter with a copper-repressible promoter, which restricted FPP flux to the competing pathway. Galactose-induced expression of codon-optimized plant germacrene D synthases led to 13-30 fold higher titers of (+) or (-)-germacrene D in CENses5C than the parent strain CEN.PK2.1C. Furthermore, genomic integration of germacrene D synthases in GAL80, LPP1 and rDNA loci generated CENses8(+D) and CENses8(-D) strains, which produced 41.36µg/ml and 728.87µg/ml of (+) and (-)-germacrene D, respectively, without galactose supplementation. Moreover, coupling of mitochondrial citrate pool to the cytosolic acetyl-CoA, by expressing a codon-optimized ATP-citrate lyase of oleaginous yeast, resulted in 137.71µg/ml and 815.81µg/ml of (+) or (-)-germacrene D in CENses8(+D)* and CENses8(-D)* strains, which were 67-120 fold higher titers than in CEN.PK2.1C. In fed-batch fermentation, CENses8(+D)* and CENses8(-D)* produced 290.28µg/ml and 2519.46µg/ml (+) and (-)-germacrene D, respectively, the highest titers in shake-flask fermentation achieved so far. KEY POINTS: • Engineered S. cerevisiae produced enantiopure (+) and (-)-germacrene D at high titers • Engineered strain produced up to 120-fold higher germacrene D than the parental strain • Highest titers of enantiopure (+) and (-)-germacrene D achieved so far in shake-flask.

A mitochondrial-targeted activity-based sensing probe for ratiometric imaging of formaldehyde reveals key regulators of the mitochondrial one-carbon pool.

Formaldehyde (FA) is both a highly reactive environmental genotoxin and an endogenously produced metabolite that functions as a signaling molecule and one-carbon (1C) store to regulate 1C metabolism and epigenetics in the cell. Owing to its signal-stress duality, cells have evolved multiple clearance mechanisms to maintain FA homeostasis, acting to avoid the established genotoxicity of FA while also redirecting FA-derived carbon units into the biosynthesis of essential nucleobases and amino acids. The highly compartmentalized nature of FA exposure, production, and regulation motivates the development of chemical tools that enable monitoring of transient FA fluxes with subcellular resolution. Here we report a mitochondrial-targeted, activity-based sensing probe for ratiometric FA detection, MitoRFAP-2, and apply this reagent to monitor endogenous mitochondrial sources and sinks of this 1C unit. We establish the utility of subcellular localization by showing that MitoRFAP-2 is sensitive enough to detect changes in mitochondrial FA pools with genetic and pharmacological modulation of enzymes involved in 1C and amino acid metabolism, including the pervasive, less active genetic mutant aldehyde dehydrogenase 2*2 (ALDH2*2), where previous, non-targeted versions of FA sensors are not. Finally, we used MitoRFAP-2 to comparatively profile basal levels of FA across a panel of breast cancer cell lines, finding that FA-dependent fluorescence correlates with expression levels of enzymes involved in 1C metabolism. By showcasing the ability of MitoRFAP-2 to identify new information on mitochondrial FA homeostasis, this work provides a starting point for the design of a broader range of chemical probes for detecting physiologically important aldehydes with subcellular resolution and a useful reagent for further studies of 1C biology.

Mitochondria-derived Vesicle Packaging as a Novel Therapeutic Mechanism in Pulmonary Hypertension.

Increasing evidence suggests that mitochondrial dysfunction in pulmonary endothelial cells (ECs) plays a causative role in the initiation and progression of pulmonary hypertension (PH); how mitochondria become dysfunctional in PH remains elusive. Mitochondria-derived vesicles (MDVs) are small subcellular vesicles that excise from mitochondria. Whether MDV deregulation causes mitochondrial dysfunction in PH is unknown. The aim of this study was to determine MDV regulation in ECs and to elucidate how MDV deregulation in ECs leads to PH. MDV formation and mitochondrial morphology/dynamics were examined in ECs of EC-specific liver kinase B1 (LKB1) knockout mice (LKB1ec-/-), in monocrotaline-induced PH rats, and in lungs of patients with PH. Pulmonary ECs of patients with PH and hypoxia-treated pulmonary ECs exhibited increased mitochondrial fragmentation and disorganized mitochondrial ultrastructure characterized by electron lucent-swelling matrix compartments and concentric layering of the cristae network, together with defective MDV shedding. MDVs actively regulated mitochondrial membrane dynamics and mitochondrial ultrastructure via removing mitofission-related cargoes. The shedding of MDVs from parental mitochondria required LKB1-mediated mitochondrial recruitment of Rab9 GTPase. LKB1ec-/- mice spontaneously developed PH with decreased mitochondrial pools of Rab9 GTPase, defective MDV shedding, and disequilibrium of the mitochondrial fusion-fission cycle in pulmonary ECs. Aerosol intratracheal delivery of adeno-associated virus LKB1 reversed PH, together with improved MDV shedding and mitochondrial function in rats invivo. We conclude that LKB1 regulates MDV shedding and mitochondrial dynamics in pulmonary ECs by enhancing mitochondrial recruitment of Rab9 GTPase. Defects of LKB1-mediated MDV shedding from parental mitochondria instigate EC dysfunction and PH.

The role of TFE3 in mediating skeletal muscle mitochondrial adaptations to exercise training.

Transcription factor E3 (TFE3) is a transcription factor that activates the expression of lysosomal genes involved in the clearance of dysfunctional mitochondria, termed mitophagy. With exercise, TFE3 is presumed to optimize the mitochondrial pool through the removal of organelles via lysosomes. However, the molecular mechanisms of the involved pathways remain unknown. Wild-type (WT) and TFE3 knockout (KO) mice were subjected to 6 wk of voluntary wheel running as an endurance training regimen. This was followed by a 45-min bout of in situ stimulation of the sciatic nerve innervating hindlimb muscles to evaluate muscle fatigue and contractile properties. A subset of animals was treated with colchicine to measure autophagy and mitophagy flux. Fatigability during stimulation was reduced with training in WT animals, as seen by a 13% increase in the percentage of maximum force at 5 min of stimulation, and a 30% increase at 30 minutes. Permeabilized fiber oxygen consumption was also improved with training. Concurrent with improved muscle and mitochondrial function, cytochrome c oxidase (COX) activity and COX I protein expression were increased in trained WT animals compared to untrained animals, signifying an increase in mitochondrial content. These training adaptations were abolished with the loss of TFE3. Surprisingly, the absence of TFE3 did not affect lysosomal content nor did it blunt the induction of mitophagy flux with contractile activity compared to WT mice. Our results suggest that the loss of TFE3 compromises beneficial training adaptations that lead to improved muscle endurance and mitochondrial function.NEW & NOTEWORTHY Our understanding of the role of transcription factor E3 (TFE3) in skeletal muscle is very limited. This research shows that TFE3 plays a direct role in skeletal muscle mitochondrial enhancement with exercise training, thereby introducing a paradigm shift in our perception of the function of TFE3 in mitochondrial maintenance, beyond mitophagy. This research serves to introduce TFE3 as a protein that holds promise as a future therapeutic target for metabolic diseases and skeletal muscle dysfunction.