Preclinical evidence for mitochondrial DNA as a potential blood biomarker for chemotherapy-induced peripheral neuropathy

Chemotherapy-induced peripheral neuropathy (CIPN) is a serious dose-limiting side effect of several first-line chemotherapeutic agents including paclitaxel, oxaliplatin and bortezomib, for which no predictive marker is currently available. We have previously shown that mitochondrial dysfunction is associated with the development and maintenance of CIPN. The aim of this study was to evaluate the potential use of mitochondrial DNA (mtDNA) levels and complex I enzyme activity as blood biomarkers for CIPN. Real-time qPCR was used to measure mtDNA levels in whole blood collected from chemotherapy- and vehicle-treated rats at three key time-points of pain-like behaviour: prior to pain development, at the peak of mechanical hypersensitivity and at resolution of pain-like behaviour. Systemic oxaliplatin significantly increased mtDNA levels in whole blood prior to pain development. Furthermore, paclitaxel- and bortezomib-treated animals displayed significantly higher levels of mtDNA at the peak of mechanical hypersensitivity. Mitochondrial complex I activity in whole blood was assessed with an ELISA-based Complex I Enzyme Activity Dipstick Assay. Complex I activity was not altered by any of the three chemotherapeutic agents, either prior to or during pain-like behaviour. These data demonstrate that blood levels of mtDNA are altered after systemic administration of chemotherapy. Oxaliplatin, in particular, is associated with higher mtDNA levels before animals show any pain-like behaviour, thus suggesting a potential role for circulating mtDNA levels as non-invasive predictive biomarker for CIPN.

Bicalutamide Exhibits Potential to Damage Kidney via Destroying Complex I and Affecting Mitochondrial Dynamics

Bicalutamide (Bic) is an androgen deprivation therapy (ADT) for treating prostate cancer, while ADT is potentially associated with acute kidney injury. Previously, we recognized Bic induced renal mitochondria dysfunction in vitro and in vivo via the ROS -HIF1α pathway. Whether OXPHOS complex, as well as mitochondrial dynamics, can be influenced by Bic via modulation of peroxisome proliferator-activated receptor coactivator 1α (PGC1α), NADPH oxidase 4 (Nox4), mitofusins 1/2 (MFN 1/2), optic atrophy 1 (OPA1), and sirtuins (SIRTs) has not been documented. Renal mesangial cell line was treated with Bic (30~60 μM) for the indicated time. SIRTs, complex I, mitochondrial dynamics- and oxidative stress-related proteins were analyzed. Bic dose-dependently reduced mitochondrial potential, but dose- and time-dependently suppressed translocase of the outer mitochondrial membrane member 20 (Tomm 20), complex I activity. Nox4 and glutathione lead to decreased NAD+/NADH ratio, with upregulated superoxide dismutase 2. SIRT1 was initially stimulated and then suppressed, while SIRT3 was time- and dose-dependently downregulated. PGC1α, MFN2, and OPA1 were all upregulated, with MFN1 and pro-fission dynamin-related protein I downregulated. Bic exhibits potential to damage mitochondria via destroying complex I, complex I activity, and mitochondrial dynamics. Long-term treatment with Bic should be carefully followed up.

Blood-based mitochondrial respiratory chain function in major depression

Mitochondrial dysfunction has been implicated in major depressive disorder (MDD). A measure of mitochondrial respiratory chain (RC) enzymatic activity—the Mitochondrial Health Index (MHI)—has previously been found to correlate with stress and emotional states in caregivers. We here report mitochondrial RC activities, mitochondrial DNA copy number (mtDNAcn), and the composite MHI in unmedicated and somatically healthy subjects with MDD (n = 47) and healthy controls (HC) (n = 11). We also explore, in a subset of the MDD sample (n = 33), whether these markers are associated with response to 8 weeks of SSRI treatment. Mitochondrial RC complexes I, II, IV, citrate synthase (CS), mtDNAcn, and the MHI were assayed in peripheral blood mononuclear cells. Treatment response was defined as >50% decrease on the 25-item Hamilton Depression Rating Scale (HRDS-25). There were no significant differences in MHI or any of the mitochondrial markers between MDD subjects and HCs. Compared to SSRI nonresponders, SSRI responders had significantly higher baseline mitochondrial content markers CS (p = 0.02) and mtDNAcn (p = 0.02), and higher complex I activity (p = 0.01). Complex II activity increased significantly over treatment, irrespective of clinical response (p = 0.03). Complex I activity decreased in responders (n = 9), but increased in nonresponders (n = 18) (group x time interaction, p = 0.02). Absolute treatment-associated change in HDRS-25 scores correlated significantly with change in complex I activity between baseline and week 8 (r = 0.47, p = 0.01). Although mitochondrial markers did not distinguish MDD from controls, they did distinguish SSRI responders from nonresponders. If larger studies validate these mitochondrial differences, they may become useful biomarkers and identify new drug targets.

CFM6 is an Essential CRM Protein Required for the Splicing of nad5 Transcript in Arabidopsis Mitochondria

Plant CRM domain-containing proteins are capable of binding RNA to facilitate the splicing of group I or II introns in chloroplasts, but their functions in mitochondria are less clear. In the present study, Arabidopsis thaliana CFM6, a protein with a single CRM domain, was expressed in most plant tissues, particularly in flower tissues, and restricted to mitochondria. Mutation of CFM6 causes severe growth defects, including stunted growth, curled leaves, delayed embryogenesis, and pollen development. CFM6 functions specifically in the splicing of group II intron 4 of nad5, which encodes a subunit of mitochondrial complex I, as evidenced by the loss of nad5 intron 4 splicing and high accumulation of its pretranscripts in cfm6 mutants. The phenotypic and splicing defects of cfm6 were rescued in transgenic plants overexpressing 35S::CFM6-YFP. Splicing failure in cfm6 also led to the loss of complex I activity and to its improper assembly. Moreover, dysfunction of complex I induced the expression of proteins or genes involved in alternative respiratory pathways in cfm6. Collectively, CFM6, a previously uncharacterized CRM domain-containing protein, is specifically involved in the cis-splicing of nad5 intron 4 and plays a pivotal role in mitochondrial complex I biogenesis and normal plant growth.

Mitochondrial Telomerase Reverse Transcriptase Protects from Myocardial Ischemia/reperfusion Injury by Improving Complex I Composition and Function

Background: The catalytic subunit of telomerase, Telomerase Reverse Transcriptase (TERT) has protective functions in the cardiovascular system. TERT is not only present in the nucleus, but also in mitochondria. However, it is unclear whether nuclear or mitochondrial TERT is responsible for the observed protection and appropriate tools are missing to dissect this. Methods: We generated new mouse models containing TERT exclusively in the mitochondria (mitoTERT mice) or the nucleus (nucTERT mice) to finally distinguish between the functions of nuclear and mitochondrial TERT. Outcome after ischemia/reperfusion, mitochondrial respiration in the heart as well as cellular functions of cardiomyocytes, fibroblasts, and endothelial cells were determined. Results: All mice were phenotypically normal. While respiration was reduced in cardiac mitochondria from TERT-deficient and nucTERT mice, it was increased in mitoTERT animals. The latter also had smaller infarcts than wildtype mice, whereas nucTERT animals had larger infarcts. The decrease in ejection fraction after one, two and four weeks of reperfusion was attenuated in mitoTERT mice. Scar size was also reduced and vascularization increased. Mitochondrial TERT protected a cardiomyocyte cell line from apoptosis. Myofibroblast differentiation, which depends on complex I activity, was abrogated in TERT-deficient and nucTERT cardiac fibroblasts and completely restored in mitoTERT cells. In endothelial cells, mitochondrial TERT enhanced migratory capacity and activation of endothelial NO synthase. Mechanistically, mitochondrial TERT improved the ratio between complex I matrix arm and membrane subunits explaining the enhanced complex I activity. In human right atrial appendages, TERT was localized in mitochondria and there increased by remote ischemic preconditioning. The Telomerase activator, TA-65 evoked a similar effect in endothelial cells, thereby increasing their migratory capacity, and enhanced myofibroblast differentiation. Conclusions: Mitochondrial, but not nuclear TERT, is critical for mitochondrial respiration and during ischemia/reperfusion injury. Mitochondrial TERT improves complex I subunit composition. TERT is present in human heart mitochondria, and remote ischemic preconditioning increases its level in those organelles. TA-65 has comparable effects ex vivo and improves migratory capacity of endothelial cells and myofibroblast differentiation. We conclude that mitochondrial TERT is responsible for cardioprotection and its increase could serve as a therapeutic strategy.

The ER protein Creld regulates ER-mitochondria contact dynamics and respiratory complex 1 activity

Dynamic contacts are formed between endoplasmic reticulum (ER) and mitochondria that enable the exchange of calcium and phospholipids. Disturbed contacts between ER and mitochondria impair mitochondrial dynamics and are a molecular hallmark of Parkinson’s disease. Cystein-rich with EGF-like domain (Creld) are ER-proteins associated with atrioventricular septal defects, but human CRELD1 is also a poorly characterized risk gene for Parkinson’s disease. Here we show that Creld is required for ER-mitochondria communication. Loss of Creld leads to mitochondrial hyperfusion and reduced ROS signaling in Drosophila melanogaster, Xenopus tropicalis and human cells. We found that reduced respiratory complex I activity lowers hydrogen peroxide levels, which disturbs neuronal activity and leads to impaired locomotion in Creld mutants. Our study presents a new paradigm of neuron dysfunction as a result of impaired ER-mitochondria communication and a new model for Parkinson’s disease.

Spatiotemporal AMPKα2 deletion in mice induces cardiac dysfunction, fibrosis and cardiolipin remodeling associated with mitochondrial dysfunction in males only

Background The AMP-activated protein kinase (AMPK) is a major regulator of cellular energetics which plays key role in acute metabolic response and in long-term adaptation to stress. Recent works have also suggested non-metabolic effects. Methods To decipher AMPK roles in the heart, we generated a cardio-specific inducible model of gene deletion of the main cardiac catalytic subunit of AMPK (Ampkα2) in mice. This allowed us to avoid the eventual impact of AMPK-KO in peripheral organs. Results Cardio-specific Ampkα2 deficiency led to a progressive left ventricular systolic dysfunction and the development of cardiac fibrosis in males. We observed a reduction in complex I-driven respiration without change in mitochondrial mass or in vitro complex I activity, associated with a rearrangement of the cardiolipins and reduced integration of complex I into the electron transport chain supercomplexes. Strikingly, none of these defects were present in females. Interestingly, suppression of estradiol signaling by ovariectomy partially mimicked the male sensitivity to AMPK loss, notably the cardiac fibrosis and the rearrangement of cardiolipins, but not the cardiac function that remained protected. Conclusion Our results confirm the close link between AMPK and cardiac mitochondrial function, but also highlight links with cardiac fibrosis. Importantly, we show that AMPK is differently involved in these processes in males and females, which may have clinical implications for the use of AMPK activators in the treatment of heart failure.

Elevated METTL9 is associated with peritoneal dissemination in human scirrhous gastric cancers

Methylation, the most common chemical modification of cellular components such as DNA, RNA, and proteins, impacts biological processes including transcription, RNA processing, and protein dynamics. Although abnormal expression of methyltransferase can lead to various diseases including cancers, little is known about the relationship between methyltransferase and cancers. Here we aimed to understand the role of methyltransferase in cancer metastasis. We found that elevated methyltransferase-like 9 (METTL9) is closely associated with the acquisition of metastatic activity in human scirrhous gastric cancers. The stable knockdown of METTL9 via an shRNA vector technique in our original metastatic cells from scirrhous gastric cancer patients significantly inhibited migration and invasion. In metastatic cells, METTL9 protein is predominantly localized in mitochondria, and the METTL9 knockdown significantly reduced mitochondrial Complex I activity. METTL9 can be a promising molecular target to inhibit peritoneal dissemination of scirrhous gastric cancers. This report is the first to describe the relationship between METTL9 and cancer metastasis.

PERM1 interacts with the MICOS-MIB complex to connect the mitochondria and sarcolemma via ankyrin B

Skeletal muscle subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria subpopulations have distinct metabolic activity and sensitivity, though the mechanisms that localize SSM to peripheral areas of muscle fibers are poorly understood. A protein interaction study and complexome profiling identifies PERM1 interacts with the MICOS-MIB complex. Ablation of Perm1 in mice reduces muscle force, decreases mitochondrial membrane potential and complex I activity, and reduces the numbers of SSM in skeletal muscle. We demonstrate PERM1 interacts with the intracellular adaptor protein ankyrin B (ANKB) that connects the cytoskeleton to the plasma membrane. Moreover, we identify a C-terminal transmembrane helix that anchors PERM1 into the outer mitochondrial membrane. We conclude PERM1 functions in the MICOS-MIB complex and acts as an adapter to connect the mitochondria with the sarcolemma via ANKB.