MitoTracker Deep Red Research Articles

TMIC-35. MITOCHONDRIAL TRANSFER PROMOTES ASTROCYTE REACTIVITY IN GLIOBLASTOMA

Abstract Astrocytes are the most abundant glial cell in the brain and have been shown to adopt reactive phenotypes upon interaction with glioma cells. While tumor-associated astrocyte (TAA) reactivity is linked to increased inflammation, proliferation, invasion, and treatment resistance of glioblastoma (GBM), the mechanisms by which cancer cells reprogram TAA are poorly defined. We have previously shown that mitochondrial transfer from astrocytes to GBM cells via GAP43-dependent microtubes alters GBM cellular metabolism and increases tumorigenicity. However, the potential role of this communication mechanism in TAA function remains unknown. Patient-derived GBM cells (PDCs) were transduced with a mito-GFP lentiviral vector and were co-cultured with hTERT-immortalized, mito-mCherry human astrocytes. Using flow cytometry, we observed that the frequency of mitochondrial transfer to hTERT astrocytes was dependent on GBM cell type and noted transfer rates as high as ~40% in co-cultures with L1 PDCs. Utilizing high-resolution confocal microscopy, we subsequently confirmed co-localization of fluorescent protein signal with MitoTracker Deep Red and observed increases in mitochondrial biomass in transfer-positive astrocytes. To evaluate whether mitochondrial transfer occurs in the tumor microenvironment, we implanted immunocompromised mice with L1 mito-mCherry PDCs. Approximately 17.7% of mouse TAAs in the tumor core were positive for GBM mitochondria, whereas long-distance transfer was not observed in the contralateral hemisphere. Functionally, acquisition of tumor mitochondria promoted astrocyte proliferation as measured by cells in G2/M phase and significantly increased total cellular reactive oxygen species (ROS) levels. These findings highlight a process by which astrocytes may become reactive in response to increased ROS from GBM mitochondrial transfer. Future studies will investigate the cellular response to increased ROS in astrocytes and investigate potential links to inflammatory and immunosuppressive signaling. Elucidating how GBM-to-astrocyte mitochondrial transfer reprograms TAA would help identify therapeutic vulnerabilities that can be exploited by blocking mitochondrial transfer.

Effects of normal mitochondrial transplantation on proliferation, apoptosis and stemness of triple-negative breast cancer cells

Objectives: To observe the mitochondrial morphology of normal and triple-negative breast cancer cells, extract mitochondria from normal cells, and investigate the effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of triple-negative breast cancer cells. Methods: The morphology of mitochondria was observed by transmission electron microscope. Mitochondria were extracted by mitochondrial extraction kit, mitochondrial protein was identified by western blot, and mitochondrial activity was detected by mitochondrial membrane potential detection kit. MitoTracker Green or MitoTracker Deep Red fluorescent probes were used to label the mitochondria of living cells, and the degree of mitochondria entering LTT cells was observed by confocal laser microscopy at 12, 24, and 96 hours. The effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of breast cancer cells were examined by CCK8, colony formation assay, flow cytometry, and sphere formation assay after 24 hours of mitochondrial transplantation. Results: The mitochondria of normal cells were rod-shaped or elongated, while the mitochondria of triple-negative breast cancer cells were swollen and vacuolated. Western blot results showed that cytochrome c oxidase subunit I (MT-CO1) protein encoded by mitochondria was present in the isolated mitochondria. The content of heat shock protein 60 (HSP60) was higher in mitochondria than that in cytoplasm. The result of the multi-mode microplate reader showed that the content of mitochondrial J-aggregates/monomer was 1.67±0.06, which was significantly higher than 0.35±0.04 of the control group (P＜0.001). Exogenous mitochondria were observed in LTT cells at 12, 24, and 96 hours after mitochondrial transplantation. The results of the CCK8 experiment showed that OD450 of LTT cells was 0.27±0.13 after 48 hours transplantation, which was lower than 0.62±0.36 of the control group (P=0.023). The OD450 of MDA-MB-468 cells was 0.30±0.03, which was lower than 0.65±0.10 of the control group (P=0.004). After 120 hours of mitochondrial transplantation, OD450 in both groups was still significantly lower than that in the control group (P＜0.01). The number of clones formed by mitochondrial transplantation of LTT cells was 21.33±7.31, which was lower than 35.22±13.59 of the control group (P=0.016). Flow cytometry showed that the early apoptosis rate of LTT cells was (30.07±2.15)% after 24 hours of mitochondrial transplantation, which was higher than 2.07±1.58 of the control group (P＜0.001). The proportion of early apoptosis in MDA-MB-468 cells was 24.47%±5.22%, which was higher than (7.83±2.06)% in the control group (P=0.007). In addition, the number of mitochondria transplanted LTT cells into the cell sphere was 46.25±5.40, which was significantly lower than 62.58±6.43 of the control group (P＜0.001). Conclusion: Normal mitochondria can enter triple-negative breast cancer cells by co-culture, inhibit the proliferation and stemness of triple-negative breast cancer cells, and promote the apoptosis of triple-negative breast cancer cells.

Chimeric Cell Therapy Transfers Healthy Donor Mitochondria in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a severe X-linked disorder characterized by dystrophin gene mutations and mitochondrial dysfunction, leading to progressive muscle weakness and premature death of DMD patients. We developed human Dystrophin Expressing Chimeric (DEC) cells, created by the fusion of myoblasts from normal donors and DMD patients, as a foundation for DT-DEC01 therapy for DMD. Our preclinical studies on mdx mouse models of DMD revealed enhanced dystrophin expression and functional improvements in cardiac, respiratory, and skeletal muscles after systemic intraosseous DEC administration. The current study explored the feasibility of mitochondrial transfer and fusion within the created DEC cells, which is crucial for developing new therapeutic strategies for DMD. Following mitochondrial staining with MitoTracker Deep Red and MitoTracker Green dyes, mitochondrial fusion and transfer was assessed by Flow cytometry (FACS) and confocal microscopy. The PEG-mediated fusion of myoblasts from normal healthy donors (MBN/MBN) and normal and DMD-affected donors (MBN/MBDMD), confirmed the feasibility of myoblast and mitochondrial fusion and transfer. The colocalization of the mitochondrial dyes MitoTracker Deep Red and MitoTracker Green confirmed the mitochondrial chimeric state and the creation of chimeric mitochondria, as well as the transfer of healthy donor mitochondria within the created DEC cells. These findings are unique and significant, introducing the potential of DT-DEC01 therapy to restore mitochondrial function in DMD patients and in other diseases where mitochondrial dysfunction plays a critical role.Graphical

Identification of cardioprotective substances in Panax ginseng/P. notoginseng based on mitochondrial morphological characteristics and UPLC-Triple-TOF-MS

Panax ginseng is reputed to be capable of replenishing healthy Qi and bolstering physical strength, and P. notoginseng can resolve blood stasis and alleviate pain. P. ginseng and P. notoginseng are frequently employed to treat ischemic heart diseases caused by blockages in the heart vessels. Mitochondrial dysfunction often coexists with abnormal mitochondrial morphology, and mitochondrial plasticity and dynamics play key roles in cardiovascular diseases. In this study, primary neonatal rat cardiomyocytes were exposed to 4 hours of hypoxia(H) followed by 2 hours of reoxygenation(R). MitoTracker Deep Red and Hoechst 33342 were used to label mitochondria and nuclei, respectively. Fluorescence images were then acquired using ImageXpress Micro Confocal. Automated image processing and parameter extraction/calculation were carried out using ImagePro Plus. Subsequently, representative parameters were selected as indicators to assess alterations in mitochondrial morphology and function. The active compounds of P. ginseng and P. notoginseng were screened out and identified based on the UPLC-Triple-TOF-MS results and mitochondrial morphometric parameters. The findings demonstrated that RS-2, RS-4, SQ-1, and SQ-4 significantly increased the values of three key morphometric parameters, including mitochondrial length, branching, and area, which might contribute to rescuing morphological features of myocardial cells damaged by H/R injury. Among the active components of the two medicinal herbs, 20(R)-ginsenoside Rg_3, ginsenoside Re, and gypenoside ⅩⅦ exhibited the strongest protective effects on mitochondria in cardiomyocytes. Specifically, 20(R)-ginsenoside Rg_3 might upregulate expression of optic atrophy 1(OPA1) and mitofusin 2(MFN2), and ginsenoside Re and gypenoside ⅩⅦ might selectively upregulate OPA1 expression. Collectively, they promoted mitochondrial membrane fusion and mitigated mitochondrial damage, thereby exerting protective effects on cardiomyocytes. This study provides experimental support for the discovery of novel therapeutic agents for myocardial ischemia-reperfusion injury from P. ginseng and P. notoginseng and offers a novel approach for large-scale screening of bioactive compounds with cardioprotective effects from traditional Chinese medicines.

Glucose-Induced Cellular Morphological Changes Across Mammalian Species

All organisms encounter environmental fluctuations and must contend with these changes to maintain homeostasis. Some mammals tolerate a wide range of intracellular conditions while others do not. Altered glucose levels are an example of a physiological challenge that can be encountered by cells. We studied responses of dermal fibroblasts obtained from skin biopsies to altered glucose treatments in culture using a comparative approach. Specifically, we investigated responses of mammals known to tolerate a range of blood glucose levels (fruit-eating geladas and Egyptian rousettes) as well as a hibernator (ground squirrel), which exhibits a marked winter decrease in glucose metabolism. As a comparison, we investigated species that maintain fairly stable blood glucose (human and rat). Fibroblasts were cultured at baseline (8mM glucose), then subjected to hypoglycemic (2.5mM) and hyperglycemic (30mM) treatment over 24h. We assayed glycolytic rate in human and rat fibroblasts using a Seahorse XFp analyzer, and found no significance between oxygen consumption or glycolysis across glucose treatments (all p>0.05). To investigate underlying cell phenotypes, we used Cell Painting, employing fluorescent stains to mark organelles. Hoechst 34580 stained DNA, to quantify number and size of nuclei, offering insights into proliferation. MitoTracker Deep Red visualized mitochondria, providing information about energy generation. Alexa Fluor 488 Concanavalin A conjugate marked endoplasmic reticulum. F-actin was stained with Alexa Fluor 555 Phalloidin conjugate, detecting cytoskeletal organization and overall cell structure. Images were analyzed with CellProfiler software, which extracts ~1400 morphological cell features, including area, compactness, fluorescence intensity, and cell connectivity to quantify subtle changes in phenotype. Each extracted feature will be compared across treatments and within a comparative context. We predict that fibroblasts exposed to hyperglycemia exhibit mitochondrial fragmentation and redistribution (MitoTracker) and increased production of reactive oxygen species (assessed with MitoSOX), which is linked to the opening of mitochondrial permeability transition pores. Cell Profiler analysis will identify structural changes that could indicate negative outcomes, such as mitophagy. Hyperglycemia may also induce endoplasmic stress, increasing lipid droplets and altering shape and size of the endoplasmic reticulum. In contrast, hypoglycemia will cause mitochondrial degradation. In both hyper- and hypoglycemia, proliferation rates are predicted to decrease, resulting in fewer nuclei and reduced cell connectivity. Phenotypic differences across species, evaluated in the context of their organismal phenotype, will help explain the mechanisms of cell function in mammals under environmental stress. Funding by NSF #2022046. 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.

Long noncoding RNA Glis2 regulates podocyte mitochondrial dysfunction and apoptosis in diabetic nephropathy via sponging miR-328-5p.

Podocyte apoptosis exerts a crucial role in the pathogenesis of DN. Recently, long noncoding RNAs (lncRNAs) have been gradually identified to be functional in a variety of different mechanisms associated with podocyte apoptosis. This study aimed to investigate whether lncRNA Glis2 could regulate podocyte apoptosis in DN and uncover the underlying mechanism. The apoptosis rate was detected by flow cytometry. Mitochondrial membrane potential (ΔΨM) was measured using JC-1 staining. Mitochondrial morphology was detected by MitoTracker Deep Red staining. Then, the histopathological and ultrastructure changes of renal tissues in diabetic mice were observed using periodic acid-Schiff (PAS) staining and transmission electron microscopy. We found that lncRNA Glis2 was significantly downregulated in high-glucose cultured podocytes and renal tissues of db/db mice. LncRNA Glis2 overexpression was found to alleviate podocyte mitochondrial dysfunction and apoptosis. The direct interaction between lncRNA Glis2 and miR-328-5p was confirmed by dual luciferase reporter assay. Furthermore, lncRNA Glis2 overexpression alleviated podocyte apoptosis in diabetic mice. Taken together, this study demonstrated that lncRNA Glis2, acting as a competing endogenous RNA (ceRNA) of miRNA-328-5p, regulated Sirt1-mediated mitochondrial dysfunction and podocyte apoptosis in DN.

Effect of maternal age on ATP content and distribution of mitochondria in bovine oocytes.

Our objective was to understand how maternal age influences the mitochondrial population and ATP content of in vivo matured bovine oocytes. We hypothesized that in vivo matured oocytes from older cows would have altered mitochondrial number and distribution patterns and lower cytoplasmic ATP content compared to the oocytes obtained from younger cows. Follicles ≥5mm were ablated in old cows (13 to 22 yrs, Old Group, n = 7) and their younger daughters (4 to 10 years old, Young Group; n = 7) to induce the emergence of a new follicular wave. Cows were treated twice daily with eight doses of FSH starting 24 hr after ablation (Day 0, day of wave emergence). Prostaglandin F2alpha (PGF) was given on Days 3 and 3.5, LH on Day 4.5, and cumulus-oocyte-complexes were collected 18-20 hours post-LH by ultrasound-guided follicular aspiration. Oocytes were either processed for staining with MitoTracker Deep Red FM or for ATP assay. Stained oocytes were imaged with a Zeiss LSM 710 confocal microscope, and mitochondria were segmented in the oocyte volume sets using Imaris Pro 7.4. In vivo matured oocytes obtained from old cows were similar in morphological grades to those from young cows. However, the oocytes of COC from older cows had 23% less intracellular ATP (27.4±1.9 vs 35.7±2.2 pmol per oocyte, P = 0.01) than those of young cows. Furthermore, the average volume of individual mitochondria, indicated by the number of image voxels, was greater (P<0.05) in oocytes from older cows than in those from younger cows. Oocytes from older cows also tended to have a greater number of mitochondrial clusters (P = 0.06) and an increased number of clusters in the central region of the oocytes (P = 0.04) compared to those from younger cows. In conclusion, our study demonstrated that maternal age was associated with a decrease in the cytoplasmic ATP content of in vivo mature oocytes and an altered distribution of mitochondrial structures. These findings suggest that maternal age may negatively influence the developmental competence of oocytes from older cows.

Performance of TMRM and Mitotrackers in mitochondrial morphofunctional analysis of primary human skin fibroblasts

Mitochondrial membrane potential (Δψ) and morphology are considered key readouts of mitochondrial functional state. This morphofunction can be studied using fluorescent dyes (“probes”) like tetramethylrhodamine methyl ester (TMRM) and Mitotrackers (MTs). Although these dyes are broadly used, information comparing their performance in mitochondrial morphology quantification and Δψ-sensitivity in the same cell model is still scarce. Here we applied epifluorescence microscopy of primary human skin fibroblasts to evaluate TMRM, Mitotracker Red CMXros (CMXros), Mitotracker Red CMH2Xros (CMH2Xros), Mitotracker Green FM (MG) and Mitotracker Deep Red FM (MDR). All probes were suited for automated quantification of mitochondrial morphology parameters when Δψ was normal, although they did not deliver quantitatively identical results. The mitochondrial localization of TMRM and MTs was differentially sensitive to carbonyl cyanide-4-phenylhydrazone (FCCP)-induced Δψ depolarization, decreasing in the order: TMRM ≫ CHM2Xros = CMXros = MDR > MG. To study the effect of reversible Δψ changes, the impact of photo-induced Δψ “flickering” was studied in cells co-stained with TMRM and MG. During a flickering event, individual mitochondria displayed subsequent TMRM release and uptake, whereas this phenomenon was not observed for MG. Spatiotemporal and computational analysis of the flickering event provided evidence that TMRM redistributes between adjacent mitochondria by a mechanism dependent on Δψ and TMRM concentration. In summary, this study demonstrates that: (1) TMRM and MTs are suited for automated mitochondrial morphology quantification, (2) numerical data obtained with different probes is not identical, and (3) all probes are sensitive to FCCP-induced Δψ depolarization, with TMRM and MG displaying the highest and lowest sensitivity, respectively. We conclude that TMRM is better suited for integrated analysis of Δψ and mitochondrial morphology than the tested MTs under conditions that Δψ is not substantially depolarized.

Abstract 213: High-Throughput Analysis of Mitochondrial Morphology in Neurons Post-Mitochondrial Transplantation

Background: Mitochondrial transplantation (MTx) improves neurological and survival outcomes in rats after cardiac arrest. However, the impact of transferred mitochondria on native mitochondrial features in neurons remains poorly assessed. Evaluating mitochondrial morphology provides insights into post-MTx mitochondrial dynamics in recipient neurons, aiding the understanding of cellular processes and targeted therapy development. Aim: To investigate whether increased uptake of donor mitochondria influences the morphology of native mitochondrial networks in neurons. Methods: We conducted in vitro MTx using the HT-22 neuron cell line. Native mitochondria in neurons were pre-labeled with MitoTracker Green. Extracellular mitochondria were isolated from healthy adult rat muscle tissue, labeled with MitoTracker Deep Red, and co-cultured with neurons. Confocal microscopy captured 2D fluorescence images. MitoHacker, a computational program, facilitated high-throughput analysis of 27 mitochondrial morphometric markers. Results: We analyzed 62 images of cells ( Figure ). MitoHacker successfully identified features of native and transferred donor mitochondria. Linear regression analysis revealed that total donor mitochondrial area had positive correlations with native mitochondrial counts, total area, minor axis length of mitochondrion, mitochondrial averaged density, and mitochondrial branch length, and a negative correlation with distance between mitochondrial location and nucleus (all P&lt;0.05). These findings suggest that increased amounts of transferred mitochondria can positively influence native mitochondrial biogenesis, proliferation, accumulation, and activated mitochondrial dynamics and networking in recipient neurons. Conclusion: Using the deep learning-based mitochondrial morphology analysis, our study provides evidence that MTx significantly impacts mitochondrial morphology and dynamics in recipient cells.

Abstract 165: Immediate Internalization of Transplanted Mitochondria in Circulatory Immune Cells After Cardiac Arrest Resuscitation in Rats

Background: Mitochondrial transplantation (MTx) is an innovative technology that has the potential effect for attenuating ischemia reperfusion injury. We previously demonstrated that MTx significantly improves survival and neurological outcomes after cardiac arrest (CA) and resuscitation in rats. However, the underlying mechanism by which MTx enhances CA outcomes remains unclear. Aim: To investigate the hypothesis that injected donor mitochondria are rapidly internalized by circulatory immune cells, particularly monocytes/macrophages, which play crucial roles in post-CA inflammatory signaling. Methods and Results: Adult male Sprague-Dawley rats underwent sham operation or 10 minutes of asphyxial CA followed by resuscitation with CPR. Either a respiration buffer as a vehicle or donor mitochondria labeled with MitoTracker Deep Red (MTDR) was intravenously injected to animals of both groups. In the CA groups, the injection was performed upon achieving spontaneous circulation. Serial blood samples were collected at 30, 75, and 150 seconds after injection. Flow cytometry analysis revealed that a marked increase of MTDR-positive ratios, peaking at 30 seconds, in CD45 + leukocytes, CD3 + T lymphocytes, and CD11b/c + monocytes/macrophages in the MTx-treated CA group compared to the other groups (Figure A). Notably, in sham-operated rats with donor mitochondrial injection, only minimal donor mitochondria were found in the circulating immune cells at those time points. Brain and spleen 1 h post-CA with MTx showed enhanced signals of MTDR measured using an in vivo imaging system, indicating successful transfer of injected mitochondria to organs (Figure B). Conclusion: Our findings demonstrate rapid internalization of injected mitochondria by circulatory immune cells after MTx in post-CA rats, suggesting a pivotal role for circulating immune cells in the mechanism underlying the beneficial effects of MTx in CA resuscitation.

Minimizing sperm oxidative stress using nanotechnology for breeding programs in rams

BackgroundArtificial insemination (AI) is a routine breeding technology in animal reproduction. Nevertheless, the temperature-sensitive nature and short fertile lifespan of ram sperm samples hamper its use in AI. In this sense, nanotechnology is an interesting tool to improve sperm protection due to the development of nanomaterials for AI, which could be used as delivery vehicles. In this work, we explored the feasibility of vitamin E nanoemulsion (NE) for improving sperm quality during transport.ResultsWith the aim of evaluating this proposal, ejaculates of 7 mature rams of Manchega breed were collected by artificial vagina and extended to 60 × 106 spz/mL in Andromed®. Samples containing control and NE (12 mmol/L) with and without exogenous oxidative stress (100 µmol/L Fe2+/ascorbate) were stored at 22 and 15 ºC and motility (CASA), viability (YO-PRO/PI), acrosomal integrity (PNA-FITC/PI), mitochondrial membrane potential (Mitotracker Deep Red 633), lipoperoxidation (C11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (SCSA®) monitored during 96 h. Our results show that NE could be used to maintain ram spermatozoa during transport at 15 and 22 ºC for up to 96 h, with no appreciable loss of kinematic and physiological characteristics of freshly collected samples.ConclusionsThe storage of ram spermatozoa in liquid form for 2–5 d with vitamin E nanoemulsions may lead more flexibility to breeders in AI programs. In view of the potential and high versatility of these nanodevices, further studies are being carried out to assess the proposed sperm preservation medium on fertility after artificial insemination.Graphical

An Fgr kinase inhibitor attenuates sepsis-associated encephalopathy by ameliorating mitochondrial dysfunction, oxidative stress, and neuroinflammation via the SIRT1/PGC-1α signaling pathway

BackgroundSepsis-associated encephalopathy (SAE) is characterized by diffuse brain dysfunction, long-term cognitive impairment, and increased morbidity and mortality. The current treatment for SAE is mainly symptomatic; the lack of specific treatment options and a poor understanding of the underlying mechanism of disease are responsible for poor patient outcomes. Fgr is a member of the Src family of tyrosine kinases and is involved in the innate immune response, hematologic cancer, diet-induced obesity, and hemorrhage-induced thalamic pain. This study investigated the protection provided by an Fgr kinase inhibitor in SAE and the underlying mechanism(s) of action.MethodsA cecal ligation and puncture (CLP)-induced mouse sepsis model was established. Mice were treated with or without an Fgr inhibitor and a PGC-1α inhibitor/activator. An open field test, a novel object recognition test, and an elevated plus maze were used to assess neurobehavioral changes in the mice. Western blotting and immunofluorescence were used to measure protein expression, and mRNA levels were measured using quantitative PCR (qPCR). An enzyme-linked immunosorbent assay was performed to quantify inflammatory cytokines. Mitochondrial membrane potential and morphology were measured by JC-1, electron microscopy, and the MitoTracker Deep Red probe. Oxidative stress and mitochondrial dysfunction were analyzed. In addition, the regulatory effect of Fgr on sirtuin 1 (SIRT1) was assessed.ResultsCLP-induced sepsis increased the expression of Fgr in the hippocampal neurons. Pharmacological inhibition of Fgr attenuated CLP-induced neuroinflammation, the survival rate, cognitive and emotional dysfunction, oxidative stress, and mitochondrial dysfunction. Moreover, Fgr interacted with SIRT1 and reduced its activity and expression. In addition, activation of SIRT1/PGC-1α promoted the protective effects of the Fgr inhibitor on CLP-induced brain dysfunction, while inactivation of SIRT1/PGC-1α counteracted the benefits of the Fgr inhibitor.ConclusionsTo our knowledge, this is the first report of Fgr kinase inhibition markedly ameliorating SAE through activation of the SIRT1/PGC-1α pathway, and this may be a promising therapeutic target for SAE.Graphical

Cryopreservation effect on sperm viability, mitochondrial membrane potential, acrosome integrity and sperm capacitation of alpaca spermatozoa detected by imaging flow cytometry.

Eighty-five sperm samples were cryopreserved and SYBR14/PI, MitoTracker Deep Red FM, FITC-PSA/PI and chlortetracycline were used for imaging flow cytometry evaluation of sperm viability, mitochondrial membrane potential (MMP), acrosome integrity and sperm capacitation, respectively. Sperm motility was also registered. Sperm motility (46.1 ± 7.7 vs. 24.1% ± 6.5%), sperm viability (49.8 ± 11.5 vs. 32.3% ± 9.6%) and high MMP (49.8% ± 12.4% vs. 34.9% ± 9.9%) decreased significantly (p < .05) during cryopreservation process, in contrast to acrosome-reacted in viable spermatozoa (1.0% ± 1.6% vs. 1.0% ± 1.0%) and sperm capacitation (10.0 ± 9.8 vs. 8.2% ± 12.4%) that were similar (p > .05) before and after cryopreservation. Positive correlations were found between sperm motility versus high MMP (r=.63), sperm motility versus sperm viability (r=.67) and sperm viability versus high MMP (r=.88). In conclusion, cryopreservation of alpaca spermatozoa is related to a decrease in sperm motility, sperm viability and high MMP, meanwhile acrosome integrity and sperm capacitation are not affected.

Meso pyridinium BODIPY-based long wavelength infrared mitochondria-targeting fluorescent probe with high photostability.

Mito-tracker deep red (MTDR) as a commercially available mitochondria-targeting probe was easily bleached upon imaging. We designed and synthesized a family of meso-pyridinium BODIPY and introduced lipophilic methyl or benzyl as the head moiety to develop a mitochondria-targeting deep red probe. Moreover, we changed the substitution of the 3,5-phenyl moieties with the methoxy or methoxyethoxyethyl group to balance hydrophilicity. The designed BODIPY dyes possessed long absorption and good fluorescence emission. Among them, meso ortho-pyridinium BODIPYs with benzyl head and glycol substitution on phenyl moiety (3h) with favorable Stokes shift were found to have the best mitochondrial targeting performance. 3h was easily uptaken by cells and was less toxic and more photostable than MTDR. An immobilizable probe (3i) was further developed, and nice mitochondria targeting properties under the damaging condition of mitochondria membrane potential were maintained. BODIPY 3h or 3i may become alternative long-wavelength mitochondria targeting probes apart from MTDR and be suitable for long-term mitochondrial tracking studies.

Abstract 15006: Imaging the Mitochondrial Nucleoid Body in Pulmonary Arterial Hypertension Reveals Changes in Nucleoid Body Distribution and Composition in Human Pulmonary Artery Smooth Muscle Cells

Introduction: Pulmonary arterial hypertension (PAH) exhibits excessive proliferation of pulmonary artery smooth muscle cells (PASMC). This is related to mitochondrial abnormalities, including a metabolic shift to aerobic glycolysis, and increased mitochondrial fragmentation. Mitochondrial DNA (mtDNA) encodes 13 proteins critical to mitochondrial function and is supported by multi-protein complexes called nucleoid bodies (NB) comprised of key mtDNA maintenance proteins. Previous data shows increased mtDNA damage and increased NB protein levels in PAH vs control PASMC. This seems to suggest an attempted compensatory response to mtDNA damage in PAH. We aim to identify NB composition and distribution changes in PAH. We hypothesize that NB structure and distribution throughout the mitochondrial network are dysregulated in PAH. Methods: NBs were visualized in control and PAH PASMC (n=15 per group). Cells were loaded with MitoTracker Deep Red (mitochondrial matrix dye) and visualized using stimulated emission depletion (STED) microscopy. We simultaneously imaged mtDNA and NB proteins TFAM, POLG and SSBP. NB protein distribution was quantified by comparing the rates of mtDNA-protein interactions per mitochondrial area. Results: On visualization of PASMC, mitochondrial fragmentation was evident in PAH vs control. Qualitative findings in PAH PASMC included increased heterogeneity of NB size (diameter) and spatial distribution in PAH compared to control. IF quantitation showed decreased mtDNA/cell (193±20 vs 326±28 mtDNA/cell, p&lt;0.05) and increased SSBP (0.91±0.07 vs 0.59±0.37 SSBP/mtDNA, p&lt;0.05) in PAH cells. These results are consistent with previous findings. Conclusion: These findings suggest a compensatory remodeling of NB in PASMC and is an important step in characterizing the NB shape, distribution, and composition in PAH. These findings will direct experiments analyzing gene and protein expression to further elucidate the role of NB in PAH.

Vitamin E Lipid-Based Nanodevices as a Tool for Ovine Sperm Protection against Oxidative Stress: Impact on Sperm Motility.

The advent of nanotechnology in the field of animal reproduction has led to the development of safer and more efficient therapies. The use of nanotechnology allows us to avoid the detrimental effects of certain traditional antioxidants, such as Vitamin E. Its hydrophobic nature makes mandatory the use of organic solvents, which are toxic to sperm cells. This study aims to evaluate the efficiency of vitamin E nanoemulsions (NE) on ram (Ovis aries) spermatozoa. For this purpose, the effect of three NE concentrations (6, 12, and 24 mM) were assessed on sperm of 10 mature rams of the Manchega breed. Sperm samples were collected by artificial vagina, pooled, and diluted in Bovine Gamete Medium. The samples were stored at 37 °C and assessed at 0, 4, 8, and 24 h under oxidative stress conditions (100 µM Fe2+/ascorbate). Motility (CASA), viability (YO-PRO/IP), acrosomal integrity (PNA-FITC/IP), mitochondrial membrane potential (Mitotracker Deep Red 633), lipoperoxidation (C11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (SCSA®®) were assessed. A linear mixed-effects models were used to analyze the effects of time, NE, and oxidant (fixed factors) on sperm parameters, and a random effect on the male was also included in the model with Tukey’s post hoc test. Protection of ram spermatozoa with NE resulted in a more vigorous motility under oxidative stress conditions with respect Control and Free vitamin E, while preventing the deleterious effects of oxidative stress coming from the production of free radicals and lipid peroxidation. These results ascertain the high relevance of the use of delivery systems for sperm physiology preservation in the context of assisted reproduction techniques.

Alcohol Dysregulates CD4 T Cell Mitochondrial Homeostasis

CD4 T cell differentiation to pro‐inflammatory and immunosuppressive subsets requires distinct metabolic pathways. Pro‐inflammatory CD4 subsets rely on glycolysis, while immunosuppressive (Treg cells) subsets, require functional mitochondria for their differentiation and function. Previous studies have shown that binge alcohol (ethanol, EtOH) administration increased Tbet‐expressing (Th1) and decreased FOXP3‐expressing (Treg) CD4 T cells in the colons of mice. We tested the hypothesis that EtOH dysregulates normal CD4 T cell differentiation, after stimulation, by impairing mitochondrial homeostasis. Human naïve CD4 T cells were isolated from buffy coats from blood bank donors (N = 6) using MACS sorting. Cells were stimulated using anti‐CD3‐coated dishes in the presence of anti‐CD28 and IL‐12, and exposed to EtOH (0 and 50 mM) for 3 days. Mitochondrial content was measured with Mitotracker Deep Red. Gene expression indicative of: autophagosome formation (ATG5, ATG7, ATG13, MAP1LC3B, BECN1, BNIP3L, ULK1), mitophagy (PINK1, PRKN),mitochondrial fusion (MFN1, MFN2, OPA1), mitochondrial fission (MFFand FIS1), and mitochondrial biogenesis (PPARC1A, PPARC1B, TFAM) was determined by RT2 profiler arrays. EtOH‐treated CD4 T cells had increased mitochondrial content (p = 0.0008) with Tregs accounting for the greatest increase in mitochondria (p = 0.04). There was a main effect of stimulation (p &lt; 0.05) to increase ATG5, ATG13, MAP1LC3B, BECN1, BNIP3L, ULK1, MFF, PPARC1B, and TFAM, and a main effect of EtOH (p &lt; 0.05) to increase PINK1 and decrease ATG7. There was a main effect of both EtOH and stimulation (p &lt; 0.05) to increase MFN2, and OPA1.Taken together, these results indicate that EtOH increases mitochondrial content in Treg cells and dysregulates mitochondrial gene expression important for mitochondrial repair and mitophagy. These EtOH‐mediated alterations in gene expression could result in an inability of CD4 T cells to maintain mitochondrial homeostasis and remove damaged mitochondria that is required for normal differentiation and function of anti‐inflammatory Treg cells.

Dysfunction of metabolic activity of bone marrow mesenchymal stem cells in aged mice.

ObjectivesEvidences have suggested that the metabolic function is the key regulator to the fate of MSCs, but its function in senescence of MSC and the underlying mechanism is unclear. Therefore, the purpose of this study was to investigate the metabolic activity of MSCs and its possible mechanism during aging.Materials and MethodsWe used the Seahorse XF24 Analyzer to understand OCR and ECAR in BMSCs and used RT‐PCR to analyze the gene expression of mitochondrial biogenesis and key enzymes in glycolysis. We analyzed BMSC mitochondrial activity by MitoTracker Deep Red and JC‐1 staining, and detected NAD+/NADH ratio and ATP levels in BMSCs. Microarray and proteomic analyses were performed to detect differentially expressed genes and proteins in BMSCs. The impact of aging on BMSCs through mitochondrial electron transport chain (ETC) was evaluated by Rotenone and Coenzyme Q10.ResultsOur results demonstrated that the oxidative phosphorylation and glycolytic activity of BMSCs in aged mice were significantly decreased when compared with young mice. BMSCs in aged mice had lower mitochondrial membrane potential, NAD+/NADH ratio, and ATP production than young mice. FABP4 may play a key role in BMSC senescence caused by fatty acid metabolism disorders.ConclusionsTaken together, our results indicated the dysfunction of the metabolic activity of BMSCs in aged mice, which would play the important role in the impaired biological properties. Therefore, the regulation of metabolic activity may be a potential therapeutic target for enhancing the regenerative functions of BMSCs.

Oridonin Alleviates LPS-Induced Depression by Inhibiting NLRP3 Inflammasome via Activation of Autophagy.

Objective: Oridonin (Ori) is a diterpene compound that has multiple biological properties. Here, our study was conducted to observe the therapeutic effect of Ori on depression as well as to uncover the mechanism.Methods: Lipopolysaccharide (LPS)-induced depression models were established both in C57BL/6 mice and primary astrocytes, which were treated with Ori, autophagy agonist Rapamycin (Rap) and autophagy inhibitor 3-Methyladenine (3-MA). The depressive-like behaviors were assessed with behavioral tests. Autophagy was evaluated in the hippocampus and astrocytes by investigating autophagosomes under transmission electron microscope (TEM) and detecting LC3II/I, Beclin1 and P62 through western blotting. Astrocyte marker glial fibrillary acidic protein (GFAP) was investigated by immunofluorescence. NLRP3 inflammasome activation was evaluated by detecting IL-1β, NLRP3, ASC and Caspase-1 expression and reactive oxygen species (ROS) accumulation was quantified via DCFH-DA probe. Autolysosomes, autophagosomes and mitophagy were separately observed through mTag-Wasabi-LC3 plasmid, MitoTracker Deep Red staining, and TEM.Results: Our results showed that Ori administration alleviated LPS-induced depressive-like behaviors and increased GFAP expression in the hippocampus. Furthermore, Ori treatment promoted autophagy activation and cell viability as well as weakened NLRP3 inflammasome activation and ROS accumulation both in LPS-induced mice and astrocytes. Ori promoted the autophagic flux unblocked through enhancing fusion of autophagosomes with lysosomes as well as enhanced mitophagy in LPS-treated astrocytes. The therapeutic effect of Ori was enhanced by Rap and weakened by 3-MA.Conclusion: Collectively, our findings provided a promising antidepressant drug and uncovered that Ori alleviated LPS-induced depression by inhibiting NLRP3 inflammasome through activation of autophagy.

Assessment of sperm mitochondrial activity by flow cytometry and fluorescent microscopy: a comparative study of mitochondrial fluorescent probes in bovine spermatozoa.

While conventional semen analysis is a simple, time-saving, and economical means to evaluate sperm quality, it leaves biochemical and metabolic characteristics of spermatozoa aside. To address this issue, the use of fluorescent probes assessing functional sperm parameters, such as JC-1, DiOC6 (3) and MitoTracker, has increased over the last decades. Apparently contradictory observations have nevertheless fostered an ongoing debate on their sensitivity and ability to evaluate the mitochondrial membrane potential (MMP) of sperm cells, thus warranting a re-examination of these probes. The present study aims to elucidate the suitability and sensitivity of each probe to evaluate the MMP of bovine spermatozoa by flow cytometry. Cryopreserved spermatozoa from ten bulls were thawed, stained with JC-1/SYTOXRed, DiOC6 (3)/propidium iodide (PI) or MitoTracker Deep Red (MTDR)/PI, and evaluated with flow cytometry and fluorescence microscopy. DiOC6 (3), JC-1 and MTDR can be simultaneously co-stained with a viability marker. The results of the present study support the ability of DiOC6 (3)/PI and JC-1/SYTOXRed, but not that of MTDR/PI, to monitor the MMP of spermatozoa. JC-1/SYTOXRed assessed by flow cytometry was found to be the most sensitive and robust fluorescent probe to assess MMP. Moreover, DiOC6 (3)/PI could be a suitable alternative when the flow cytometer is not equipped with a red laser and/or an adequate optical filter. Both DiOC6 (3) and JC-1, but not MTDR, could be used as probes to assess the mitochondrial membrane potential of bovine spermatozoa.