Oxidative stress is a hallmark of several aging and trauma related neurological disorders, but the precise details of how altered neuronal activity elicits subcellular redox changes have remained difficult to resolve. Current redox sensitive dyes and fluorescent proteins can quantify spatially distinct changes in reactive oxygen species levels, but multicolor probes are needed to accurately analyze compartment-specific redox dynamics in single cells that can be masked by population averaging. Our lab previously engineered a genetically-encoded red-shifted redox-sensitive fluorescent protein sensors using a Förster resonance energy transfer relay strategy. Here, we developed a second-generation excitation ratiometric sensor called rogRFP2 with improved red emission for quantitative live-cell imaging. Using this sensor to measure activity-dependent redox changes in individual cultured neurons, we observed an anticorrelation in which mitochondrial oxidation was accompanied by a concurrent reduction in the cytosol. This behavior was dependent on the activity of Complex I of the mitochondrial electron transport chain and could be modulated by the presence of co-cultured astrocytes. We also demonstrated that the red fluorescent rogRFP2 facilitates ratiometric redox imaging in Drosophila retinas. The proof-of-concept studies reported here demonstrate that this new rogRFP2 redox sensor can be a powerful tool for understanding redox biology both in vitro and in vivo across model organisms. In addition, we have used these tools that monitor cellular redox, to study oxidative stress and ROS changes in Parkinson’s disease models. Here, we have established cellular models for studying Parkinson’s disease causing LRRK2 mutations to create a platform for future work to explore the relationship between PD associated LRRK2 variants and oxidative stress.

No / Here, we studied how epithelial energy metabolism impacts overall skin development by selectively deleting intraepithelial mtDNA in mice by ablating a key maintenance factor (TfamEKO), which induces loss of function of the electron transport chain (ETC). Quantitative (immuno)histomorphometry demonstrated that TfamEKO mice showed significantly reduced hair follicle (HF) density and morphogenesis, fewer intrafollicular keratin15+ epithelial progenitor cells, increased apoptosis, and reduced proliferation. TfamEKO mice also displayed premature entry into (aborted) HF cycling by apoptosis-driven HF regression (catagen). Ultrastructurally, TfamEKO mice exhibited severe HF dystrophy, pigmentary abnormalities, and telogen-like condensed dermal papillae. Epithelial HF progenitor cell differentiation (Plet1, Lrig1 Lef1, and β-catenin), sebaceous gland development (adipophilin, Scd1, and oil red), and key mediators/markers of epithelial–mesenchymal interactions during skin morphogenesis (NCAM, versican, and alkaline phosphatase) were all severely altered in TfamEKO mice. Moreover, the number of mast cells, major histocompatibility complex class II+, or CD11b+ immunocytes in the skin mesenchyme was increased, and essentially no subcutis developed. Therefore, in contrast to their epidermal counterparts, pilosebaceous unit stem cells depend on a functional ETC. Most importantly, our findings point toward a frontier in skin biology: the coupling of HF keratinocyte mitochondrial function with the epithelial–mesenchymal interactions that drive overall development of the skin and its appendages.

Myocardial and skeletal muscle high energy phosphate metabolism is abnormal in heart failure, but the pathophysiology is not understood. Plasma non-esterified fatty acids (NEFA) increase in heart failure due to increased sympathetic drive, and regulate the transcription of mitochondrial uncoupling protein-3 (UCP3), through peroxisome proliferator-activated receptor-α. The aim of the work in this thesis was to determine whether cardiac PCr/ATP ratios and skeletal muscle PCr kinetics during exercise were related to cardiac and skeletal muscle UCP3 levels respectively, thus providing a mechanism for the apparent mitochondrial dysfunction observed in heart failure. Patients having cardiac surgery underwent pre-operative testing, including cardiac and gastrocnemius 31P magnetic resonance spectroscopy. Intra-operatively, ventricular, atrial and skeletal muscle biopsies were taken for measurement of mitochondrial protein levels by immunoblotting, along with mitochondrial function by tissue respiration rates. Fasting plasma NEFA concentrations increased in patients with ventricular dysfunction and with New York Heart Association (NYHA) class. Ventricular UCP3 levels increased and cardiac PCr/ATP decreased with NYHA class, however, demonstrated no relationship to each other. In skeletal muscle, maximal rates of oxidative ATP synthesis (Qmax) related to functional capacity. Skeletal muscle UCP3 levels increased with NYHA class but were unrelated to skeletal muscle Qmax. Tissue respiration experiments revealed no relationship between ventricular function and indices of mitochondrial coupling, furthermore, indices of mitochondrial coupling were unrelated to tissue UCP3 levels. No evidence was found to support mitochondrial uncoupling, mediated through UCP3, as a cause of the abnormalities in cardiac and skeletal muscle high energy phosphate metabolism.

Thermotolerance limit in aquatic organism is set by the ability to sustain aerobic scope to sudden temperature shifts. This study tested the genetic and plastic differences in thermotolerance of Daphnia that can be explained by the differences in the ability to retain mitochondrial integrity at high temperatures. Five genotypes with different biogeographic origins were acclimated to 18ᵒC and 25ᵒC. We developed a rhodamine 123 in-vivo assay to measure mitochondrial membrane potential and observed higher fluorescent in heat damaged tissues as the disruption of the mitochondrial membrane potential. Significant effects on temperature tolerance were observed with CCCP and DNP but not with NaN3. Effects of toxins were significant in temperature sensitive genotype and high concentration of lactate was observed in 18ᵒC acclimated genotype only. We conclude that genetic and physiological differences are intricately linked to the ability of sustaining aerobic respiration at high temperatures which sets limit to the thermotolerance.

Mitochondrial dysfunction is a phenomenon observed in models of Traumatic Brain Injury (TBI). Loss of mitochondrial bioenergetics can result in diminished cellular homeostasis leading to cellular dysfunction and possible cellular death. Consequently, the resultant tissue damage can manifest as functional deficits and/or disease states. Therapeutic strategies to target this mitochondrial dysfunction have been investigated for models TBI and have shown promising effects. For this project, we tested the hypothesis that mitoNEET, a novel mitochondrial membrane protein, is a target for pioglitazone mediated neuroprotection. To test this, we used a severe Controlled Cortical Impact (CCI) injury model in mitoNEET null and wild-type mice. We then dosed these animals with pioglitazone or NL-1, which is a compound that has a similar structure to pioglitazone allowing us to hone in one the importance of mitoNEET binding. Wild-type animals treated with the mitoNEET ligands, both pioglitazone and NL-1, had improved mitochondrial function, tissue sparing and functional recovery, compared to mitoNEET null animals. In addition to this specific hypothesis tested, our experiments provided insight casting doubt on the central dogma that mitochondrial dysfunction following TBI is the result of vast oxidative damage and consequential irreversible mitochondrial loss. The data from these studies show that when mitoNEET is targeted with pioglitazone at 12 hours’ post-injury, mitochondrial dysfunction can be reversed. Additionally, when bypassing proteins upstream of Complex I with an alternative biofuel, such as beta-hydroxybuterate (BHB), TBI related mitochondrial dysfunction is once again reversed. This leads to novel hypothesis for future work which posits mitoNEET as a redox sensitive switch; when mitoNEET senses changes in redox, as seen in TBI, it inhibits mitochondrial respiration. When targeted with an agonist/ligand or bypassed with a biofuel TBI mitochondrial dysfunction can be reversed. These studies support the role of mitoNEET in the neuropathological sequelae of brain injury, supporting mitoNEET as a crucial target for pioglitazone mediated neuroprotection following TBI. Lastly, these studies propose a mechanism of TBI related mitochondrial dysfunction which can reversed with pharmacological agents.

The discovery of biomarkers for Parkinson's disease (PD) is challenging due to the heterogeneous nature of this disorder, and a poor correlation between the underlying pathology and the clinically expressed phenotype. An ideal biomarker would inform on PD-relevant pathological changes via an easily assayed biological characteristic, which reliably tracks clinical symptoms. Human dermal (skin) fibroblasts are accessible peripheral cells that constitute a patient-specific system, which potentially recapitulates the PD chronological and epigenetic aging history. Here, we compared primary skin fibroblasts obtained from individuals diagnosed with late-onset sporadic PD, and healthy age-matched controls. These fibroblasts were studied from fundamental viewpoints of growth and morphology, as well as redox, mitochondrial, and autophagic function. It was observed that fibroblasts from PD subjects had higher growth rates, and appeared distinctly different in terms of morphology and spatial organization in culture, compared to control cells. It was also found that the PD fibroblasts exhibited significantly compromised mitochondrial structure and function when assessed via morphological and oxidative phosphorylation assays. Additionally, a striking increase in baseline macroautophagy levels was seen in cells from PD subjects. Exposure of the skin fibroblasts to physiologically relevant stress, specifically ultraviolet irradiation (UVA), further exaggerated the autophagic dysfunction in the PD cells. Moreover, the PD fibroblasts accumulated higher levels of reactive oxygen species (ROS) coupled with lower cell viability upon UVA treatment. In essence, these studies highlight primary skin fibroblasts as a patient-relevant model that captures fundamental PD molecular mechanisms, and supports their potential utility to develop diagnostic and prognostic biomarkers for the disease.

Doxorubicin (DOX)-induced cardiotoxicity is thought to be mediated by the generation of superoxide anion radicals (superoxide) from redox cycling of DOX in cardiomyocyte mitochondria. Reduction of superoxide generates H2O2, which diffuses throughout the cell and potentially contributes to oxidant-mediated cardiac injury. The mitochondrial and cytosolic glutathione peroxidase 1 (Gpx1) primarily functions to eradicate H2O2. In this study, we hypothesize that Gpx1 plays a pivotal role in the clearance of H2O2 generated by DOX. To test this hypothesis, we compared DOX-induced cardiac dysfunction, mitochondrial injury, protein nitration, and apoptosis in Gpx1-deficient and wild type mouse hearts. The Gpx1-deficient hearts showed increased susceptibility to DOX-induced acute functional derangements than wild type hearts, including impaired contractility and diastolic properties, decreased coronary flow rate, and reduced heart rate. In addition, DOX treatment impaired the mitochondrial function of Gpx1-deficient hearts. Specifically, Gpx1-deficient hearts treated with DOX demonstrated an increased rate of NAD-linked state 4 respiration and a decline in the P/O ratio relative to wild type hearts, suggesting that DOX uncouples the electron transfer chain and oxidative phosphorylation in Gpx1-deficient hearts. Finally, apoptosis and protein nitration were significantly increased in Gpx1-deficient mouse hearts compared to wild type hearts. These studies suggest that Gpx1 plays significant roles in protecting DOX-induced mitochondrial impairment and cardiac dysfunction in the acute phase.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19175号 / 医博第4017号 / 新制||医||1010(附属図書館) / 32167 / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邊 直樹, 教授 松原 和夫, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The thermotolerance, an adaptive phenomenon that is accompanied by the phenotypic plasticity which is the adjustment of physiology, biochemistry and metabolism of every cellular function by the hidden mechanism. Mitochondrion, the powerhouse of the cell that determines the functional integrity of every cellular homeostasis and functional phycological processes should provide its association in regulating the thermotolerance as well. This study assessed the mitochondrial function in regulating and determining the limit of thermo tolerance in the Daphnia magna of different geographical regions of the world, mainly sub grouped as temperature tolerant clones (IL) and temperature sensitive clones (GB). The acclimation effects or the adjustment of the preexisting biological properties help the organism adjust its biological processes to the changing habitat to maintain the cellular functional integrity. The clonal divergence as well as the acclimation show a clear pattern in limiting the thermotolerance and the prediction is the temperature tolerant clones should show higher adjustment of the mitochondrial function than temperature sensitive ones. We hypothesize that the damage in the mitochondrial membrane integrity by different mito-toxins should decrease the heat tolerance by decreasing the membrane potential and fluidity. The integrated mitochondrial function was assessed in acclimated clones by using the molecular studies as well as observation of behavioral and phenotypic plasticity. Due to the specific effects of each mito-toxins (CCCP, NaN3 and DNP) on different complexes (I-IV and ATP synthase) in ETC, we determined the mitochondrial membrane integrity by the Rhodamine 123 alongside with the lactate assay for measuring the mitochondrial integrity. Among all these three mito-toxins, CCCP show significant effect on limiting the heat tolerance. The lower lactate accumulation was observed in the temperature-tolerant clones acclimated in cold temperatures (18°C) which indicates the higher mitochondrial adjustment than the temperature sensitive clones. The concluding remark is that thermal tolerance is determined by the adjustment of mitochondrial function which accompanied with the adjustment to the mitochondrial respiration as well as the adjustment to membrane potential and fluidity.

Cellular stress is central to the understanding of pathological mechanisms and the development of new therapeutic strategies and serves as a biomarker for disease progression in neurodegeneration, diabetes, cancer, cardiovascular and other chronic diseases. The common cellular stress assay (CSA) based on Seahorse technology in peripheral blood mononuclear cells (PBMCs) shows inconsistent results, which prevents its use as a biomarker for the progression of chronic diseases. Therefore, the aim of this study was to investigate potential factors that affect the CSA in PBMCs. We measured the CSA parameters in PBMCs from study participants and compared the results according to the potential factors, namely, the PBMC isolation method, age, seasonal variation and the gender of the study participants. PBMCs were isolated by OptiPrep® and RobosepTM-S methods. PBMCs isolated with the OptiPrep method showed much higher extracellular acidification and higher respiration compared to Robosep-isolated cells. Moreover, OptiPrep-isolated cells showed a higher number of outliers for the proton production rate (PPR) and a high respiratory quotient, indicating impurities with other cells, such as platelets, and technical inconsistencies. PBMCs from older individuals showed higher maximal respiration, spare capacity and extracellular acidification than younger participants. Additionally, in winter, maximal respiration and spare capacity decreased. From spring until early autumn, spare capacity and maximal respiration continuously increased. Elderly males also showed higher basal respiration, spare capacity and extracellular acidification than females. In conclusion, the findings of this study clearly demonstrate that the results of CSA parameters measured in PBMCs are influenced by the PBMC isolation method, age, seasonal variation and gender. Therefore, we recommend that researchers and physicians properly interpret the results of CSA parameters in PBMCs by considering these factors. It is important to use separate CSA evaluation standards based on the isolation method, age, gender and season-dependent factors. To assess the cellular stress situation in PBMCs, both extracellular acidification and mitochondrial respiration should be taken into account. Further study of additional factors, such as mitochondrial mass, should be conducted to improve the measurement of CSA parameters for the assessment of the real mitochondrial fitness.