MitoTracker Red Research Articles

Differential timing of mitochondrial activation in rat dorsal striatum induced by procedural learning and swimming.

Stressful experiences form stronger memories due to enhanced neural plasticity mechanisms linked to glucocorticoid hormones (cortisol in humans, corticosterone in rats). Among other neural structures, the dorsal striatum plays a role in the corticosterone-induced consolidation of stressful memories, particularly in the cued water maze task. Neural plasticity is related to mitochondrial activity due to the relevance of energy production and signaling mechanisms for functional and morphological neuronal adaptations. Corticosterone has been shown to enhance brain mitochondrial activity by activating glucocorticoid receptors. In this context, striatum functions are susceptible to change in relation to mitochondrial responses. Based on this evidence, we hypothesized that training in the cued water maze would induce an increase in corticosterone levels and mitochondrial activity (mitochondrial membrane potential and calcium content) in the dorsal striatum, and that these adaptations might be related to memory consolidation of the task. We used an ELISA assay to evaluate plasma and striatal corticosterone levels; mitochondrial activity was determined with the florescent probes MitoTracker Red (mitochondrial membrane potential) and Rhod-2 (calcium content) in brain slices containing the dorsal striatum of rats trained in the cued water maze and euthanized at different times after training (0.5, 1.5, or 6.0 h). We also analyzed the effect of post-training inhibition of striatal mitochondrial activity by OXPHOS complex 1 inhibitor rotenone, on the consolidation of the cued water maze task. We found that cued water maze training induced an increase in corticosterone levels and a time-dependent elevation of mitochondrial membrane potential and mitochondrial calcium content in the dorsal striatum. Unexpectedly, rotenone administration facilitated the retention test. Altogether, our results suggest that enhanced mitochondrial activity in the dorsal striatum is relevant for cued water maze consolidation. The increase in mitochondrial activity was contextually associated with an elevation of corticosterone in plasma and the dorsal striatum. Additionally, our swimming groups also showed an increase in mitochondrial activity in the dorsal striatum, but with a different pattern, which could suggest a differential functional adaptation in this structure.

5'tiRNA-33-CysACA-1 promotes septic cardiomyopathy by targeting PGC-1α-mediated mitochondrial biogenesis

BackgroundWe revealed for the first time that the expression of 158 tRNA-derived small RNAs (tsRNAs) was altered in septic cardiomyopathy (SCM) by microarray analysis, and we selected 5'tiRNA-33-CysACA-1, which was the most significantly up-regulated, as a representative to explore the roles and mechanisms of tsRNAs in SCM. MethodsWe constructed a sepsis model by cecum ligation and puncture (CLP) in mice and detected the expression of 5'tiRNA-33-CysACA-1 using quantitative real-time PCR (qRT-PCR). The supernatant generated after LPS stimulation of macrophages was used as the conditional medium (CM) to stimulate H9C2 and established the injured cell model. CCK-8 and LDH release assays were used to detect cell viability and cell death. Mitochondrial membrane potential (MMP), ATP production, ROS production, and Mitotracker Red mitochondrial morphology were assayed to assess mitochondrial function. Expression of mRNA for molecules related to the mitochondrial quality control system was verified by qRT-PCR. The mechanism by which 5'tiRNA-33-CysACA-1 regulates peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) expression was examined by western blot, mRNA stability analysis, and rescue experiments. ResultsExpression of 5'tiRNA-33-CysACA-1 was elevated in cardiac tissue and H9C2 cells during septic myocardial injury. Stimulation of the CM resulted in cardiomyocyte injury and impaired mitochondrial function. Transfection of 5'tiRNA-33-CysACA-1 mimic in CM further downregulated PGC-1α expression, inhibited mitochondrial biogenesis thereby impairing mitochondrial function and leading to decreased cardiomyocyte activity and increased cell death. In contrast, transfection of the inhibitor ameliorated the above biological processes. In addition, mRNA stability assay and bioinformatics analysis showed that 5'tiRNA-33-CysACA-1 led to a decrease in the stability of PGC-1α mRNA, which in turn downregulated the expression of PGC-1α and promoted the development of SCM. Conclusions5'tiRNA-33-CysACA-1 expression is upregulated in SCM and inhibits mitochondrial biogenesis by targeting PGC-1α and decreasing the stability of PGC-1α mRNA, leading to mitochondrial dysfunction and promoting the development of SCM.

Comprehensive atlas of mitochondrial distribution and dynamics during oocyte maturation in mouse models

BackgroundOocytes, the largest cells in mammals, harbor numerous mitochondria within their cytoplasm. These highly dynamic organelles are crucial for providing energy resources and serving as central regulators during oogenesis. Mitochondrial dynamics ensure proper energy distribution for various cellular processes involved in oocyte maturation. Previous studies have used alterations in mitochondrial distribution as a biomarker to assess the oocyte health. However, there are discrepancies between studies regarding mitochondrial distribution profiles in healthy oocytes. Consequently, a comprehensive mitochondrial distribution profile in oocytes during maturation has not been fully characterized. Additionally, there is a lack of objective, quantitative methods to evaluate alterations in mitochondrial distribution profiles in oocytes.MethodsThis study aims to provide an in-depth overview of mitochondrial distribution profiles in mouse oocytes at different maturation stages: germinal vesicle (GV) stage, metaphase I (MI), and mature metaphase II (MII). Freshly collected mouse GV, MI and MII oocytes were stained with MitoTracker Red. Confocal microscopy was used to obtain images of mitochondrial distribution profiles in these oocytes. Using the Imaris software, we reconstructed three-dimensional (3D) surface renderings of each oocyte and quantitatively illustrated the mitochondrial distribution profiles.ResultsAt the GV stage, mitochondria in oocytes were evenly distributed throughout the ooplasm. As oocytes progressed to MI and MII stages, mitochondria aggregated and formed clusters, the mean size of mitochondrial clusters and the proportions of clustered mitochondria increased along with the maturation of oocytes.ConclusionsOur findings reveal that mitochondria in mouse oocytes are highly dynamic, undergoing significant reorganizations during oocyte maturation. We for the first time provided comprehensive mitochondrial distribution profiles in mouse oocytes at the GV, MI and MII stages. These mitochondrial distribution profiles were further quantitatively evaluated. Our methods provide an objective and standardized approach for evaluating alterations in mitochondrial dynamics, which can be used as biomarkers to monitor oocyte conditions during maturation.

7097 The role of mitochondrial fraction locating cytoplasmic and peridroplet area in white adipocyte

Abstract Disclosure: I. Mori: None. T. Ishizuka: None. H. Morita: None. K. Kajita: None. White adipocytes (WAs) are involved in the formation of obesity and insulin resistance, but little is known about the role of WAs mitochondria. A recent study revealed that the mitochondria of brown adipocytes (BAs) consist of cytoplasmic mitochondria CM), which is responsible for β-oxidation, and peridroplet mitochondria (PDM), which supplies triglyceride to lipid droplets. However, it is unclear whether such a functional division exist in WA mitochondria. In this study, we examined whether the pellets obtained from cytoplasmic and oil layers of WAs of C57/BL/J mice by centrifugation were mitochondria. Since mitochondrial DNA, mitochondrial protein, Mitotracker Red staining, and mitochondrial images in electron microscope were detected in both fractions, they were considered to be cytoplasmic mitochondria (CMw) and peridroplet mitochondria (PDMw) in WAs, respectively. The amount of CMw and PDMw were greater in epididymal fat than in inguinal fat. CMw had higher β-oxidation activity than PDMw. PDMw was associated with perilipin 1/3 and diacylglycerol acyltransferase 2. The amount of PDMw was elevated in epididymal fat of mice fed a high-fat diet (HFD) for 2 and 4 weeks compared to normal chew-fed mice. These results suggested that CMw was involved in β-oxidation, and PDMw in droplet expansion. Although numerous researches have been shown that amount of mitochondria is suppressed in obese WAs, our results revealed that PDMw might be involved in the early stage of obesity formation. Presentation: 6/1/2024

IDH1/MDH1 deacetylation promotes NETosis by regulating OPA1 and autophagy

BackgroundAs a heterogeneous and life-threatening disease, the pathogenesis of acute liver failure (ALF) is complex. Our previous study has shown that IDH1/MDH1 deacetylation promotes ALF by regulating NETosis (a novel mode of cell death). In this article, we explore the manners of IDH1/MDH1 deacetylation regulates NETosis. MethodsIn vitro experiments, the formation of NETs was detected by immunofluorescence staining and Western blotting. LC3 fluorescence staining was used to detect autophagosome formation. To observe mitochondrial morphology, cells were stained by Mito-Tracker Red. Western blotting was used to detect the levels of autophagy protein and mitochondrial dynamin. In vivo experiments, the ALF model in mouse was established with LPS/D-gal, and the formation of NETs was detected by immunofluorescence staining and Western blotting. The autophagy levels were detected by Western blotting in liver samples. ResultsIn dHL-60 cells, Western blotting results showed that the expression of OPA1 was higher in the IDH1/MDH1 deacetylated group compared with the IDH1/MDH1 WT group. And histone deacetylase inhibitor 6 (HDAC6i, ACY1215) decreased the expression level of OPA1 in IDH1/MDH1 deacetylated group. IDH1/MDH1 deacetylation increased the expression levels of both LC3B-II and Beclin 1, while decreasing the expression level of P62. It was reversed by ACY1215. Combined with our previous experiments, IDH1/MDH1 deacetylation upregulated autophagy concomitant with the increased expression of the markers of NETs formation. In a mouse model of ALF, ACY1215 further decreased the expression levels of LC3B-II and Beclin 1, while increasing the expression level of P62 in IDH1/MDH1 deacetylated mice. ConclusionsIDH1/MDH1 deacetylation promoted NETosis by regulating autophagy and OPA1 in vitro. The regulation of neutrophil autophagy on NETosis during IDH1/MDH1 deacetylation might be masked in mice. ACY1215 might attenuate NETosis by regulating neutrophil autophagy, which alleviated ALF aggravated by IDH1/MDH1 deacetylation.

The anticancer potential of tetrahydrocurcumin-phytosomes against oral carcinoma progression

BackgroundHerbal medicine combined with nanotechnology offers an alternative to the increasing burden of surgery and/or chemotherapy, the main therapeutics of oral carcinoma. Phytosomes are nano-vesicular systems formed by the interaction between phospholipids and phyto-active components via hydrogen bonding, exhibiting superior efficacy over pure phytocomponents in drug delivery.MethodsTetrahydrocurcumin (THC)-phytosomes were prepared by thin film hydration method. After characterization, in vitro cytotoxicity, antiproliferative capacity, antioxidant potential and full apoptotic workup were paneled on oral squamous cell carcinoma (SCC4) in comparison with native THC-solution and cisplatin (3.58 µg/mL intravenous injection), as positive controls. In addition, we tested the three medications on normal oral keratinocytes and gingival fibroblasts to attest to their tissue-selectivity.ResultsSuccessful preparation of THC-phytosomes using 1:1 molar ratio of THC to phospholipid exhibited significantly increased aqueous solubility, good colloidal properties, and complete drug release after one hour. On SCC4 cells, THC-phytosomes, at their dose-/time-dependency at ~ 60.06 µg/mL escalated cell percentages in the S-phase with 32.5 ± 6.22% increase, as well as a startling 29.69 ± 2.3% increase in apoptotic population. Depletion of the cell colonies survival to 0.29 ± 0.1% together with restraining the migratory rate by -6.4 ± 6.8% validated THC-phytosomes’ antiproliferative capacity. Comparatively, the corresponding results of THC-solution and cisplatin revealed 12.9 ± 0.9% and 25.8 ± 1.1% for apoptosis and 0.9 ± 0.1% and 0.7 ± 0.08% for colony survival fraction, respectively. Furthermore, the nanoformulation exhibited the strongest immuno-positivity to caspase-3, which positively correlated with intense mitochondrial fluorescence by Mitotracker Red, suggesting its implication in the mitochondrial pathway of apoptosis, a finding further explained by the enormously high Bax and caspase-8 expression by RT-qPCR. Finally, the THC groups showed the lowest oxidative stress index, marking their highest free radical-scavenging potential among the test groups.ConclusionsTHC-phytosomes are depicted to be an efficient nanoformulation that enhanced the anticancer efficacy over the free drug counterpart and the conventional chemotherapeutic. Additionally, being selective to cancer cells and less cytotoxic to normal cells makes THC-phytosomes a potential candidate for tissue-targeted therapy.

Yellow Emissive Carbon Dots - A Robust Nanoprobe for Highly Sensitive Quantification of Jaundice Biomarker and Mitochondria Targeting in Cancer Cells.

The abnormally high level of bilirubin (BR) in biofluids (human serum and urine) indicates a high probability of jaundice and liver dysfunction. However, quantification of BR as the Jaundice biomarker is difficult due to the interference of various biomolecules in serum and urine. To address this issue, we developed a fluorescence-based detection strategy, for which yellow emissive carbon dots (YCDs) were produced from a one-step solvothermal process using phloroglucinol and thionin acetate as chemical precursors. The as-fabricated YCDs exhibited a strong fluorescence peak at the wavelength of 542 nm upon excitation at 390 nm. We used YCDs for detecting BR through the fluorescence turn-off mechanism, unveiling the excellent sensitivity in the linear range of 0.5-12.5 μM with a limit of detection (LOD) of 9.62 nM, which was far below the clinically relevant range. The analytical nanoprobe also offered excellent detection specificity for quantifying BR in real samples. Moreover, the biocompatible fluorescent nanoprobe was successfully employed to target mitochondria in live cancer cells. A colocalization study confirmed that YCDs possessed the ability to target mitochondria and overlapped completely with MitoTracker Red. The developed nanoprobe of YCDs turned out to be straightforward in their synthesis, noninvasive, and can be utilized for biomedical sensors to diagnose the onset of jaundice as well as for mitochondria targeting.

Ligustilide alleviates oxidative stress during renal ischemia-reperfusion injury through maintaining Sirt3-dependent mitochondrial homeostasis

BackgroundRenal ischemia-reperfusion (I/R) injury is an inevitable complication during renal transplantation and is closely related to patient prognosis. Mitochondrial damage induced oxidative stress is the core link of renal I/R injury. Ligustilide (LIG), a natural compound extracted from ligusticum chuanxiong hort and angelica sinensis, has exhibited the potential to protect mitochondrial function. However, whether LIG can ameliorate renal I/R injury requires further investigation. Delving deeper into the precise targets and mechanisms of LIG's effect on renal I/R injury is crucial. PurposeThis study aimed to elucidate the specific mechanism of LIG's protective effect on renal I/R injury. MethodsIn this study, an in vivo model of renal ischemia-reperfusion (I/R) injury was developed in mice, along with an in vitro model of hypoxia-reoxygenation (H/R) using human proximal renal tubular epithelial cells (HK-2). To assess the impact of LIG on renal injury, various methods were employed, including serum creatinine (Cr) and blood urea nitrogen (BUN) testing, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) for kidney injury molecule-1 (KIM-1). The effects of LIG on oxidative stress were examined using fluorescent probes dihydroethidium (DHE) and dichlorodihydrofluorescein diacetate (DCFH-DA), TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, and flow cytometry. Additionally, the influence of LIG on mitochondrial morphology and function was evaluated through transmission electron microscopy (TEM), Mito Tracker Red CMXRos staining, adenosine triphosphate (ATP) concentration assays, and JC-1 staining. The potential mechanism involving LIG and Sirt3 was explored by manipulating Sirt3 expression through cell transfection. ResultsThe results showed that LIG could provide protective function for mitochondria to alleviate oxidative stress induced by renal I/R. Further mechanistic studies indicated that LIG maintained mitochondrial homeostasis by targeting Sirt3. ConclusionOur findings demonstrated that LIG alleviated oxidative stress during renal I/R injury through maintaining Sirt3-dependent mitochondrial homeostasis. Overall, our data raised the possibility of LIG as a novel therapy for renal I/R injury.

COX-2 optimizes cardiac mitochondrial biogenesis and exerts a cardioprotective effect during sepsis

BackgroundSeptic cardiomyopathy is a component of multiple organ dysfunction in sepsis. Mitochondrial dysfunction plays an important role in septic cardiomyopathy. Studies have shown that cyclooxygenase-2 (COX-2) had a protective effect on the heart, and prostaglandin E2 (PGE2), the downstream product of COX-2, was increasingly recognized to have a protective effect on mitochondrial function. ObjectiveThis study aims to demonstrate that COX-2/PGE2 can protect against septic cardiomyopathy by regulating mitochondrial function. MethodsCecal ligation and puncture (CLP) was used to establish a mouse model of sepsis and RAW264.7 macrophages and H9C2 cells were used to simulate sepsis in vitro. The NS-398 and celecoxib were used to inhibit the activity of COX-2. ZLN005 and SR18292 were used to activate or inhibit the PGC-1α activity. The mitochondrial biogenesis was examined through the Mitotracker Red probe, mtDNA copy number, and ATP content detection. ResultsThe experimental data suggested that COX-2 inhibition attenuated PGC-1α expression thus decreasing mitochondrial biogenesis, whereas increased PGE2 could promote mitochondrial biogenesis by activating PGC-1α. The results also showed that the effect of COX-2/PGE2 on PGC-1α was mediated by the activation of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB). Finally, the effect of COX-2/PGE2 on the heart was also verified in the septic mice. ConclusionCollectively, these results suggested that COX-2/PGE2 pathway played a cardioprotective role in septic cardiomyopathy through improving mitochondrial biogenesis, which has changed the previous understanding that COX-2/PGE2 only acted as an inflammatory factor.

FDX1 downregulation activates mitophagy and the PI3K/AKT signaling pathway to promote hepatocellular carcinoma progression by inducing ROS production

BackgroundMitochondrial dysfunction and metabolic reprogramming can lead to the development and progression of hepatocellular carcinoma (HCC). Ferredoxin 1 (FDX1) is a small mitochondrial protein and recent studies have shown that FDX1 plays an important role in tumor cuproptosis, but its role in HCC is still elusive. In this study, we aim to investigate the expression and novel functions of FDX1 in HCC. MethodsFDX1 expression was first analyzed in publicly available datasets and verified by immunohistochemistry, qRT-PCR and Western blot. In vitro and in vivo experiments were applied to explore the functions of FDX1. Non-targeted metabolomics and RNA-sequencing were used to determine molecular mechanism. mRFP-GFP-LC3 lentivirus transfection, Mito-Tracker Red and Lyso-Tracker Green staining, transmission electron microscopy, flow cytometry, JC-1 staining, etc. were used to analyze mitophagy or ROS levels. Hydrodynamic tail vein injection (HTVi) and patient-derived organoid (PDO) models were used to analyze effect of FDX1 overexpression. ResultsFDX1 expression is significantly downregulated in HCC tissues. FDX1 downregulation promotes HCC cell proliferation, invasion in vitro and growth, metastasis in vivo. In addition, FDX1 affects metabolism of HCC cells and is associated with autophagy. We then confirmed that FDX1 deficiency increases ROS levels, activates mitophagy and the PI3K/AKT signaling pathway in HCC cells. Interestingly, scavenging ROS attenuates the tumor-promoting role and mitophagy of FDX1 downregulation. The results of HTVi and PDO models both find that FDX1 elevation significantly inhibits HCC progression. Moreover, low FDX1 expression is associated with shorter survival and is an independent risk factor for prognosis in HCC patients. ConclusionsOur research had investigated novel functions of FDX1 in HCC. Downregulation of FDX1 contributes to metabolic reprogramming and leads to ROS-mediated activation of mitophagy and the PI3K/AKT signaling pathway. FDX1 is a potential prognostic biomarker and increasing FDX1 expression may be a potential therapeutic approach to inhibit HCC progression.

MitoTracker Red for isolation of zone-specific hepatocytes and characterization of hepatic sublobular metabolism

BackgroundThe liver lobule is divided into three zones or regions: periportal (PP or Zone 1) that is highly oxidative and active in ureagenesis, pericentral (PC or Zone 3) that is more glycolytic, and midzonal (MZ or Zone 2) with intermediate characteristics. AimOur goal was to isolate and metabolically characterize hepatocytes from specific sublobular zones. MethodsMice were administered rhodamine123 (Rh123) or MitoTracker Red (MTR) prior to intravital imaging, liver fixation, or hepatocyte isolation. After in vivo MTR, hepatocytes were isolated and sorted based on MTR fluorescence intensity. Alternatively, E-cadherin (Ecad) and cytochrome P450 2E1 (CYP2E1) immunolabeling was performed in fixed liver slices. Ecad and CYP2E1 gene expression in sorted hepatocytes was assessed by qPCR. Oxygen consumption rates (OCR) of sorted hepatocytes were also assessed. ResultsMultiphoton microscopy showed Rh123 and MTR fluorescence distributed zonally, decreasing from PP to PC in a flow-dependent fashion. In liver cross-sections, Ecad was expressed periportally and CYP2E1 pericentrally in association with high and low MTR labeling, respectively. Based on MTR fluorescence, hepatocytes were sorted into PP, MZ, and PC populations with PP and PC hepatocytes enriched in Ecad and CYP2E1, respectively. OCR of PP hepatocytes was ∼4 times that of PC hepatocytes. ConclusionsMTR treatment in vivo delineates sublobular hepatic zones and can be used to sort hepatocytes zonally. PP hepatocytes have substantially greater OCR compared to PC and MZ. The results also indicate a sharp midzonal demarcation between hepatocytes with PP characteristics (Ecad) and those with PC features (CYP2E1). This new method to sort hepatocytes in a zone-specific fashion holds the potential to shed light on sublobular hepatocyte metabolism and regulatory pathways in health and disease.

P-354 Advanced Paternal Age (APA) amplifies cryopreservation-induced stress in human spermatozoa

Abstract Study question Does paternal age intensify the negative effects of cryopreservation on sperm quality? Summary answer Paternal age exacerbates the adverse impact of cryopreservation on sperm quality. What is known already Despite mounting evidence indicating age-related declines in sperm quality, the impact of APA on male fertility remains unclear. APA raises questions about DNA fragmentation and mitochondrial activity, a robust indicator of sperm functionality. While sperm cryopreservation is extensively employed in fertility preservation and assisted reproductive technology (ART), its potentially detrimental effects on cells compartments, including mitochondria, persist. The mechanisms of APA on sperm susceptibility to cryopreservation-induced cellular damage still need to be addressed. Study design, size, duration This prospective multicentric study, conducted between January and October 2023, comprised 200 men. Only normozoospermic samples were included in the research (n = 81). Then, based on their age, the participants were categorised into two groups: ages ≤35 (non-APA, n = 40) and ages ≥42 (APA, n = 41). The cohorts were stratified into distinct subgroups, encompassing both fresh and frozen/thawed specimens of APA and non-APA sperm, and subjected to evaluations encompassing mitochondrial functionality, acrosomal reaction, DNA fragmentation and Tyrosine phosphorylation. Participants/materials, setting, methods Semen samples were collected after 2-7 days abstinence and analysed according to WHO 2021 guidelines. Acrosomal status was determined using FITC-conjugate PSA. DNA fragmentation was examined with the Halosperm kit. Mitochondrial functionality was gauged using MitoTracker Red CMXRos through the percentage of stained cells and fluorescence intensity and PCR for mitochondrial DNA copy number (mtDNAcn). Tyrosine phosphorylation was assessed through immunofluorescence and Western Blotting. Main results and the role of chance Non-APA group exhibited significantly superior fresh semen characteristics. DNA fragmentation was notably elevated in fresh semen from APA group, reaching 21.7%, compared to 15.4% non-APA individuals. After cryopreservation, the DNA fragmentation index further increased in men over 42 years to 26.7%. Mitochondrial functionality analysis indicated a significant decrease in staining intensity in cryopreserved sperm, notably in the APA group, with mean fluorescence intensity decreasing from 3.35±1.5 to 1.8±0.5. In contrast, the non-APA group experienced a drop from 3.33±1.6 to 2.53±1.6. Additionally, in the APA group, a significant increase in mtDNAcn was observed in both fresh and frozen/thawed sperm compared to the younger counterparts, indicative of poor mitochondrial quality. Cryopreservation negatively impacted acrosome integrity in both age groups, with a significant decrease observed in unreacted acrosomes from 71.5% to 57.7% in the non-APA group and from 75% to 63% in the APA group. Furthermore, exploration of capacitation-like changes, like tyrosine phosphorylation, demonstrated a significant decrease with age and a negative impact of cryopreservation on tyrosine phosphorylation in sperm from young males. Limitations, reasons for caution Main limitations are the small number of examined samples and the inclusion of only normozoospermic patients. Therefore, future investigations are needed to provide a more comprehensive understanding of the impact of paternal age on cryopreservation-induced stress in human spermatozoa. Wider implications of the findings Our research sheds light on the influence of paternal age on sperm cryopreservation, revealing higher vulnerability of mitochondria and elevated DNA fragmentation in APA. These findings should be considered when advising older men on choosing cryopreservation techniques in assisted reproductive treatments. Trial registration number Not applicable

Cell Death in Leishmania donovani promastigotes in response to Mammalian Aurora Kinase B Inhibitor– Hesperadin

Deciphering how hesperadin, a repurposed mammalian aurora kinase B inhibitor, affects the cellular pathways in Leishmania donovani might be beneficial. This investigation sought to assess the physiological effects of hesperadin on promastigotes of L. donovani, by altering the duration of treatment following exposure to hesperadin. Groups pre-treated with inhibitors such as EGTA, NAC, and z-VAD-fmk before hesperadin exposure were also included. Morphological changes by microscopy, ATP and ROS changes by luminometry; DNA degradation using agarose gel electrophoresis and metacaspase levels through RT-PCR were assessed. Flow cytometry was used to study mitochondrial depolarization using JC-1 and MitoTracker Red; mitochondrial-superoxide accumulation using MitoSOX; plasma membrane modifications using Annexin-V and propidium iodide, and lastly, caspase activation using ApoStat. Significant alterations in promastigote morphology were noted. Caspase activity and mitochondrial-superoxide rose early after exposure whereas mitochondrial membrane potential demonstrated uncharacteristic variations, with significant functional disturbances such as leakage of superoxide radicals after prolonged treatments. ATP depletion and ROS accumulation demonstrated inverse patterns, genomic DNA showed fragmentation and plasma membrane showed Annexin-V binding, soon followed by propidium iodide uptake. Multilobed macronuclei and micronuclei accumulated in hesperadin exposed cells before they disintegrated into necrotic debris. The pathologic alterations were unlike the intrinsic or extrinsic pathways of classical apoptosis and suggest a caspase-mediated cell death most akin to mitotic-catastrophe. Most likely, a G2/M transition block caused accumulation of death signals, disorganized spindles and mechanical stresses, causing changes in morphology, organellar functions and ultimately promastigote death. Thus, death was a consequence of mitotic-arrest followed by ablation of kinetoplast functions, often implicated in L. donovani killing.

Activation of the Nrf-2 pathway improves the mitochondrial phenotype in skeletal muscle cells

Aging muscle displays a reduction in mitochondrial content, along with elevated reactive oxygen species (ROS) emission and reduced levels of antioxidant enzymes. These characteristics enhance tissue susceptibility to oxidative damage, facilitating sub-optimal cellular metabolism and subsequent muscle atrophy. While these perturbations to metabolic homeostasis are a natural course of the aging process, there has been ongoing research investigating how to implement interventions to reduce this decline in muscle health. Regular exercise is one possible treatment modality, however this may be unsuitable for some populations who are unable to participate due to their health status. The objective of this study was to examine the activation of the Nrf-2 pathway using the nutraceutical Sulforaphane (SFN) and evaluate its role as a potential exercise mimetic. We hypothesized that SFN would act through similar pathways activated with chronic exercise to improve mitochondrial content, respiration, and ROS emission. To address our objective, C2C12 myotubes were treated with SFN for various timepoints basally, and in the presence of the Complex-I inhibitor rotenone to induce oxidative stress. Western blotting revealed an 8-fold increase in Nrf-2 nuclear localization following 24hrs of SFN treatment, which subsequently led to the upregulation of antioxidant enzymes NQO1, Heme Oxygenase-1, catalase, glutathione reductase and Glucose-6-Phosphate Dehydrogenase by 2-to 3-fold. These protein changes, were accompanied by dramatic reductions in both total cellular and mitochondrial ROS emission with SFN, detected with CellROX Green and MitoSOX Red respectively. Interestingly, this led to an increase in mitochondrial membrane potential, detected via MitoTracker Red (MTR), despite rotenone-induced oxidative stress. Western blotting revealed TFAM upregulation at 24hrs, suggesting an increase in mitochondrial DNA (mtDNA) replication and transcription. This was corroborated by an increase in COX subunit I protein, a mitochondrially-encoded subunit of cytochrome c oxidase, as well as MitoTracker Green and MTR staining by 48hrs. Mitochondrial respiration was evaluated using Seahorse analysis, demonstrating a marked increase in ATP production as well as basal and maximal respiration. Together these data suggest that activation of the Nrf-2 pathway via SFN may lead to adaptations similar to chronic exercise. Additionally, SFN may have applications in disorders characterized by mitochondrial loss and high ROS emission. To fully elucidate the precise mechanisms underlying this agent, SFN will be combined with chronic contractile activity to determine if these two interventions operate through independent pathways. This work was supported by funds from the Natural Science and Engineering Research Council (NSERC). Sabrina Champsi is the recipient of the NSERC Alexander Graham Bell Canada Graduate Scholarship-Master’s (CGS-M). David A. Hood is the holder of a Canadian Research Chair in Cell Physiology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

C2 Spinal Cord Hemisection Reduces Mitochondrial Volume Density in Phrenic Motor Neurons

The diaphragm muscle (DIAm) is the major inspiratory pump for breathing. Neural control of the DIAm during breathing involves the recruitment of fatigue resistant motor units comprising smaller phrenic motor neurons (PhMNs), which innervate type I and IIa fibers. More fatigable DIAm motor units comprise larger PhMNs that innervate type IIx/IIb fibers, which generate greater forces but are more fatigable. The fatigable motor units are only infrequently recruited for expulsive behaviors of the DIAm. Thus, there are marked differences in the activation history of DIAm motor units. Consistent with their higher activity levels, previous studies from our lab have shown that smaller PhMNs and type I and IIa DIAm fibers have greater mitochondrial volume density (MVD). PhMNs located in the cervical spinal cord (C3-C5) receive direct excitatory input from medullary premotor neurons, which is primarily ipsilateral. Thus, C2 spinal cord hemisection (C2SH) disrupts the neural pathway essential for breathing and significantly reduces DIAm activity on the same side. Therefore, we hypothesized that C2SH significantly reduces MVD of smaller PhMNs. In Sprague Dawley rats, PhMNs on both sides were retrogradely labeled by intrapleural injection of cholera toxin B (CTB) 3 days prior to C2SH (right side). In addition, DIAm EMG activity was recorded on both sides by inserting bipolar electrodes. Mitochondria were labeled by intraspinal injection of MitoTracker Red and imaged in identified PhMNs using 3D confocal microscopy. A series of optical slices (0.5 mm) of PhMNs were deconvolved to improve contrast, then thresholded for MitoTracker labeling followed by 3D reconstruction, providing detailed images of mitochondrial morphometry and distribution within PhMNs. In sham rats, the MVD of smaller (lower somal surface area) PhMNs was higher compared to larger PhMNs. C2SH reduced DIAm EMG activity on the ipsilateral side across a 14-day period, while activity on the contralateral side increased. After 14 days, C2SH reduced MVD by ~25% in smaller PhMNs on the ipsilateral side. We conclude that the reduced activity of smaller PhMNs induced by C2SH induced mitochondrial morphological changes, most likely due to reduced mitochondrial biogenesis. These results indicate that reduced DIAm activity associated with mechanical ventilation may impose mitochondrial remodeling that reduces mitochondrial oxidative capacity in PhMNs. These adaptations may disrupt mitochondrial function and impair weaning from mechanical ventilation. Research support by NIH grants HL166204, HL146114 and AG44615. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Spinning Disk Confocal Microscopy for Optimized and Quantified Live Imaging of 3D Mitochondrial Network.

Mitochondria are the energy factories of a cell, and depending on the metabolic requirements, the mitochondrial morphology, quantity, and membrane potential in a cell change. These changes are frequently assessed using commercially available probes. In this study, we tested the suitability of three commercially available probes-namely 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), MitoTracker Red CMX Rox (CMXRos), and tetramethylrhodamine methyl ester (TMRM)-for assessing the mitochondrial quantity, morphology, and membrane potential in living human mesoangioblasts in 3D with confocal laser scanning microscope (CLSM) and scanning disk confocal microscope (SDCM). Using CLSM, JC-1, and CMXRos-but not TMRM-uncovered considerable background and variation. Using SDCM, the background signal only remained apparent for the JC-1 monomer. Repetitive imaging of CMXRos and JC-1-but not TMRM-demonstrated a 1.5-2-fold variation in signal intensity between cells using CLSM. The use of SDCM drastically reduced this variation. The slope of the relative signal intensity upon repetitive imaging using CLSM was lowest for TMRM (-0.03) and highest for CMXRos (0.16). Upon repetitive imaging using SDCM, the slope varied from 0 (CMXRos) to a maximum of -0.27 (JC-1 C1). Conclusively, our data show that TMRM staining outperformed JC-1 and CMXRos dyes in a (repetitive) 3D analysis of the entire mitochondrial quantity, morphology, and membrane potential in living cells.

In vivo 3-photon fluorescence microscopy of white matter in mouse brain excited at the 1700 nm window

White matter, a densely packed collection of myelinated axons, plays an essential part in neural networks. With high spatial resolution and deep penetration, multi-photon microscopy (MPM) is promising for white matter imaging in animal models in vivo. The third harmonic generation (THG) signal can be generated from white matter, but the bottom part of the white matter layer generates weak THG due to its high scattering. Here, we demonstrate an in vivo labeling and imaging technology, capable of visualizing the white matter layer in the mouse brain, combining fluorescence labeling with MitoTracker Red and three-photon fluorescence (3PF) microscopy excited at the 1700 nm window. 3PF signals are several times higher than THG signals, resulting in deeper imaging of the white matter layer with the former. Our results indicate that 3PF microscopy is a promising technology for white matter imaging in the deep brain in vivo.

Nucleoside supplements as treatments for mitochondrial DNA depletion syndrome.

Introduction: In mitochondrial DNA (mtDNA) depletion syndrome (MDS), patients cannot maintain sufficient mtDNA for their energy needs. MDS presentations range from infantile encephalopathy with hepatopathy (Alpers syndrome) to adult chronic progressive external ophthalmoplegia. Most are caused by nucleotide imbalance or by defects in the mtDNA replisome. There is currently no curative treatment available. Nucleoside therapy is a promising experimental treatment for TK2 deficiency, where patients are supplemented with exogenous deoxypyrimidines. We aimed to explore the benefits of nucleoside supplementation in POLG and TWNK deficient fibroblasts. Methods: We used high-content fluorescence microscopy with software-based image analysis to assay mtDNA content and membrane potential quantitatively, using vital dyes PicoGreen and MitoTracker Red CMXRos respectively. We tested the effect of 15 combinations (A, T, G, C, AT, AC, AG, CT, CG, GT, ATC, ATG, AGC, TGC, ATGC) of deoxynucleoside supplements on mtDNA content of fibroblasts derived from four patients with MDS (POLG1, POLG2, DGUOK, TWNK) in both a replicating (10% dialysed FCS) and quiescent (0.1% dialysed FCS) state. We used qPCR to measure mtDNA content of supplemented and non-supplemented fibroblasts following mtDNA depletion using 20µM ddC and after 14- and 21-day recovery in a quiescent state. Results: Nucleoside treatments at 200µM that significantly increased mtDNA content also significantly reduced the number of cells remaining in culture after 7days of treatment, as well as mitochondrial membrane potential. These toxic effects were abolished by reducing the concentration of nucleosides to 50µM. In POLG1 and TWNK cells the combination of ATGC treatment increased mtDNA content the most after 7days in non-replicating cells. ATGC nucleoside combination significantly increased the rate of mtDNA recovery in quiescent POLG1 cells following mtDNA depletion by ddC. Conclusion: High-content imaging enabled us to link mtDNA copy number with key read-outs linked to patient wellbeing. Elevated G increased mtDNA copy number but severely impaired fibroblast growth, potentially by inhibiting purine synthesis and/or causing replication stress. Combinations of nucleosides ATGC, T, or TC, benefited growth of cells harbouring POLG mutations. These combinations, one of which reflects a commercially available preparation, could be explored further for treatment of POLG patients.

Isoquercitrin alleviates pirarubicin-induced cardiotoxicity in vivo and in vitro by inhibiting apoptosis through Phlpp1/AKT/Bcl-2 signaling pathway.

Introduction: Due to the cardiotoxicity of pirarubicin (THP), it is necessary to investigate new compounds for the treatment of THP-induced cardiotoxicity. Isoquercitrin (IQC) is a natural flavonoid with anti-oxidant and anti-apoptosis properties. Thus, the present study aimed to investigate the influence of IQC on preventing the THP-induced cardiotoxicity in vivo and in vitro. Methods: The optimal concentration and time required for IQC to prevent THP-induced cardiomyocyte damage were determined by an MTT assay. The protective effect was further verified in H9c2 and HCM cells using dichlorodihydrofluorescein diacetate fluorescent probes, MitoTracker Red probe, enzyme-linked immunosorbent assay, JC-1 probe, and real time-quantitative polymerase chain reaction (RT-qPCR). Rats were administered THP to establish cardiotoxicity. An electrocardiogram (ECG) was performed, and cardiac hemodynamics, myocardial enzymes, oxidative stress indicators, and hematoxylin-eosin staining were studied. Voltage-dependent anion channel 1 (VDAC1), adenine nucleotide translocase 1 (ANT1), and cyclophilin D (CYPD) were detected by qRT-PCR, and the Phlpp1/AKT/Bcl-2 axis proteins were detected by western blot, confirming that IQC markedly increased cell viability and superoxide dismutase (SOD) levels, diminished the levels of ROS and MDA, and elevated mitochondrial function and apoptosis in vivo and in vitro. Results: Results showed that IQC reduced THP-induced myocardial histopathological injury, electrocardiogram (ECG) abnormalities, and cardiac dysfunction in vivo. IQC also decreased serum levels of MDA, BNP, CK-MB, c-TnT, and LDH, while increasing levels of SOD and GSH. We also found that IQC significantly reduced VDAC1, ANT1, and CYPD mRNA expression. In addition, IQC controlled apoptosis by modulating Phlpp1/AKT/Bcl-2 signaling pathways. IQC markedly increased H9c2 and HCM cell viability and SOD levels, diminished the levels of ROS and MDA, and elevated mitochondrial function in H9c2 and HCM cells to defend against THP-induced cardiomyocyte apoptosis in vitro. The AKT inhibitor IMQ demonstrated that IQC lacked antioxidant and anti-apoptotic properties. Moreover, our data showed that IQC regulates Phlpp1 expression, thereby influencing the expression levels of p-AKT, cytochrome c, caspase-3, caspase-9, Bcl-2, and Bax. Discussion: In conclusion, our results indicate that IQC protects the changes in mitochondrial membrane permeability in cardiomyocytes by regulating the Phlpp1/AKT/Bcl-2 signaling pathway, inhibits the release of cytc from the mitochondrial inner membrane to the cytoplasm, forms apoptotic bodies, induces cell apoptosis, and reduces THP induced cardiotoxicity.

Exploring the limitations of mitochondrial dye as a genuine horizontal mitochondrial transfer surrogate

Rosamine-based mitochondrial dyes, such as Mitotracker Red, have commonly been employed to visualize mitochondrial localization within cells due to their preferential accumulation in organelles with membrane potential. Consequently, Mitotracker Red has often served as a surrogate indicator for tracking mitochondrial movement between neighboring cells. However, it is important to note that the presence of membrane potential in the cell membrane and other organelles may lead to the non-specific partial enrichment of Mitotracker Red in locations other than mitochondria. This study comprehensively investigates the reliability of mitochondrial dye as a marker for studying horizontal mitochondrial transfer (HMT). By meticulous replicating of previous experiments and comparing the efficiency of mitochondrial dye transfer with that of mito-targeted GFP, our findings confirm that HMT occurs at significantly lower efficiency than previously indicated by Mitotracker dye. Subsequent experiments involving mitochondria-deficient cells robustly demonstrates the non-specificity of mitochondrial dye as indicator for mitochondria. We advocate for a thorough reevaluation of existing literature in this field and propose exploration of alternative techniques to enhance the investigation of HMT. By addressing these pivotal aspects, we can advance our understanding of cellular dynamics and pave the way for future explorations in this captivating field.