Cardiac stem cells (CSCs)-based therapy provides a promising avenue for the management of ischemic heart diseases. However, engrafted CSCs are subjected to acute cell apoptosis in the ischemic microenvironment. Here, stem cell antigen 1 positive (Sca-1+) CSCs proved to own therapy potential were cultured and treated with H2O2 to mimic the ischemia situation. As autophagy inhibitor, 3-methyladenine (3MA), inhibited H2O2-induced CSCs apoptosis, thus we demonstrated that H2O2 induced autophagy-dependent apoptosis in CSCs, and continued to find key proteins responsible for the crosstalk between autophagy and apoptosis. Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2), increased upon cardiomyocyte injury with unknown functions in CSCs, was increased by H2O2. NR4A2 siRNA attenuated H2O2 induced autophagy and apoptosis in CSCs, which suggested an important role of NR4A2 in CSCs survival in ischemia conditions. Reactive oxygen species (ROS) and NF- κB (P65) subunit were both increased by H2O2. Either the ROS scavenger, N-acetyl-lcysteine (NAC) or NF-κB signaling inhibitor, bay11-7082 could attenuate H2O2-induced autophagy and apoptosis in CSCs, which suggested they were involved in this process. Furthermore, NAC inhibited NF-κB activities, while bay11-7082 inhibited NR4A2 expression, which revealed a ROS/NF-κB/NR4A2 pathway responsible for H2O2- induced autophagy and apoptosis in CSCs. Our study supports a new clue enhancing the survival rate of CSCs in the infarcted myocardium for cell therapy in ischemic cardiomyopathy.

Indiana University-Purdue University Indianapolis (IUPUI) / Key regulatory decisions of protein synthesis and autophagy are controlled by the lysosomal nutrient sensing complex (LYNUS). To engage protein synthesis signaling, LYNUS requires cellular availability of amino acids, adenosine triphosphate (ATP), growth factors, and docking at the lysosomal membrane. The molecular determinants of LYNUS signaling and docking are not completely elucidated and may involve regulators of the lipid membrane structure and function of the lysosome. Since ceramides are both bioactive second messengers and determinants of lipid membrane stiffness, we investigated the role of the ceramide-producing lysosomal acid sphingomyelinase (ASM) in the homeostatic function of mammalian target of rapamycin (mTOR) signaling and autophagy. Using ASM inhibition with either imipramine or siRNA against SMPD1, in primary human lung cells or Smpd1+/- mice, we demonstrated that ASM is an endogenous inhibitor of autophagy. ASM was necessary for physiological mTOR signaling and maintenance of sphingosine levels. Whereas overstimulation of ASM has been shown to trigger autophagy with impaired flux, inhibition of ASM activity during homeostatic, non-stressed conditions triggered autophagy with degradative potential, associated with enhanced transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis genes, translocation to the nucleus and decreased sphingosine levels. These findings suggest LYNUS signaling and autophagy are partially regulated by ASM.

京都大学 / 0048 / 新制・論文博士 / 博士(人間健康科学) / 乙第13006号 / 論人健博第1号 / 新制||人健||3(附属図書館) / 32934 / (主査)教授 藤井 康友, 教授 澤本 伸克, 教授 一山 智 / 学位規則第4条第2項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20791号 / 医博第4291号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩井 一宏, 教授 松田 道行, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Introduction: Cold exposure is a challenging environmental stimulus for humans, particularly in vulnerable populations such as older adults. Decrements in cold tolerance observed in older adults may be mediated by an age-related reduction in the stress-induced cytoprotective mechanism of autophagy, which enhances cell survival by eliminating damaged cellular components. However, it is unknown how autophagy and accompanying cytoprotective pathways (i.e., heat shock proteins) respond to cold conditions in humans. Purpose: The purpose of this thesis was to evaluate the impact of aging on autophagic activity during cold exposure and assess strategies to reverse age-related autophagic dysfunction. Methods: We examined the influence of age on autophagy during acute cold exposure utilizing ex vivo, in vitro, and in vivo models in young (19-29 years) and older (54-76 years) adults. Autophagic activity in all investigations was assessed in peripheral blood mononuclear cells (i.e., immune cells) via Western blotting. Simulated hypothermic conditions (equivalent to 4-35°C core temperature) were evaluated using ex vivo whole-blood exposure. In vivo cold stress was achieved using cold-water immersions to elicit a physiologically relevant decrease in core temperature by 0.5 and 1.0°C. Techniques that potentially reversed autophagic impairments during cold exposure were assessed including, 1) an in vitro treatment of a known autophagic stimulator (rapamycin) in immune cells obtained from young and older males, and 2) an in vivo cold acclimation in young males with cold-water immersions (14°C for 60 min) repeated on 7 consecutive days. Results: Simulated hypothermia (4-35°C) induced autophagic dysfunction regardless of age or sex. Moderate cold stress (a 0.5°C reduction in core temperature) stimulated autophagy in young males. However, intense cold exposures (equivalent to ≥ 1.0°C decrease in core temperature) elicited signs of autophagic dysfunction and a shift towards apoptotic cell death. Additionally, older adults displayed evidence of autophagic dysfunction during each cold exposure, although age-related autophagic dysfunction was mitigated with acute rapamycin treatment. Further, cold acclimation robustly improved autophagic responses to cold exposure. Conclusion: Despite an observed age-related impairment in autophagic responses during cold exposure, this thesis provided the first evidence in humans that autophagic dysregulation during cold exposure can be reversed through the administration of autophagic stimulators and through cold acclimation.

Drosophila melanogaster is a valuable model for studying various aspects of human cancer, including proliferative capacity, invasiveness and metabolic adaptation typical of tumour cells to support cell growth. One of the major players in this process is Myc, which can promote tumorigenesis by triggering a metabolic reprogramming that allows cells to produce macromolecules, by modulating glycolytic flux, glutaminolysis, lipidogenesis, and autophagy. The process by which hyperproliferative cells undergo metabolic reprogramming to sustain growth can be recapitulated in the epithelial cells from Drosophila imaginal discs, where different levels of Myc induce cell competition. This process is a mechanism for selection of cells expressing higher level of Myc that acquire a super-competitor condition, with the ability to non-autonomously kill the neighbouring slow-growing cells. The direct connection between Myc, glutamine metabolism and autophagy and their role in competitive events between cancerous cells and wild type cells have not been clearly explained; therefore, the main purpose of this project is to determine a plausible link between Myc and autophagy, by examining the dependency of Myc-induced autophagy on glutaminase and major regulators of autophagy, such as TOR, Atg1, Atg5 and ammonia, a by-product of glutamine catabolism, by dissecting these mechanisms both in normal epithelial clones and hyperproliferating RasV12 -expressing cells. Our results show that Myc promotes the transcription of glutamine-related genes and the production of ammonia, and that glutaminase is necessary for Myc-induced autophagy in epithelial cells of clones of the wing imaginal discs, with a mechanism independent from TOR and Atg1. Conversely, the effect of Myc on autophagy induction is mediated by Atg5. We then investigated the contribute of Myc in autophagy in RasV12-transformed cells, that upregulate Myc to sustain growth and hyperproliferation. Intriguingly, our data report that autophagy is increased non-autonomously in neighbouring wild type cells, and that this non-autonomous RasV12-driven autophagic flux depends on Myc activity. Moreover, downregulation of glutaminase in RasV12-expressing cells significantly reduces non-autonomous autophagy. Collectively, our results give new insights on how glutamine metabolism can contribute to Myc-induced autophagy and how this enhances cancerous cell fitness.

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24206号 / 薬科博第159号 / 新制||薬科||17(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 伊藤 貴浩, 教授 中山 和久, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

No description available.

p97 is a member of the AAA family of proteins (ATPase Associated with various cellular Activities). It is a highly conserved and abundant protein and functions in numerous ubiquitin-mediated processes including ERAD. Endoplasmic Reticulum Associated Degradation is the process by which misfolded/ubiquitinated proteins translocate out of the ER and migrate to the proteasome for degradation. p97 maintains substrate misfolding and mediates its exit from the ER and trafficking to the 26S proteasome. It also plays important roles in protein trafficking, the cell-cycle, apoptosis and homeotypic Golgi Apparatus and Endoplasmic Reticulum membrane fusion after mitosis. In addition, p97 plays a role in the aggresome-autophagy degradation pathway, which handles the ubiquitin-mediated destruction of aggregate-prone, misfolded, cytosolic proteins. p97 mutation is the causative alteration in the disorder, IBMPFD, which is marked by defects in autophagy. This broad diversity of function is mediated through p97's interaction with a large group of adaptor proteins. Many of these adaptors harbor both p97 interaction motifs and ubiquitin association domains. However, more than half of known p97 adaptors do not. Their function is largely unknown. UBXD1 is one known adaptor for p97 that does not have a ubiquitin association domain (UBA), and has been shown to have decreased interaction with IBMPFD mutant p97R155H and p97A232E. Recently, it has been suggested to perform a role in protein trafficking, specifically in monoubiquitinated caveolin-1 internalization and trafficking to the endosome. A novel high abundance UBXD1 interacting partner has been identified via solution-based mass spectrometric analyses. ERGIC-53, the namesake of the ER-Golgi Intermediate Compartment, has been shown to be involved in bi-directional trafficking between the ER and Golgi. The association between UBXD1 and ERGIC-53 is unique among UBX family members. Deletional analysis has shown that unlike p97, the ERGIC-53-UBXD1 interaction takes place in the extreme amino terminus of UBXD1, (within the first 10 amino acids) which is predicted by computer modeling to form a hydrophobic binding pocket. Further site-directed mutagenesis work has clearly shown four amino acids (3 highly hydrophobic) are crucial for maintaining this interaction. They have been modeled to form a conserved alpha-helix. ßCOPI, a primary member of the COPI coatomer complex which is involved in protectively coating ERGIC-53 positive vesicles, is also thought to be involved with the ERGIC-53-UBXD1-p97 pathway. ßCOPI has been identified as a UBXD1-independent interactor with p97. Modest UBXD1 over- expression using a ponasterone inducible system has shown that UBXD1 modulates ERGIC-53 localization. Additionally, a functional link between UBXD1, p97 and ERGIC-53 in autophagy has been discovered through the use of a highly efficient, miR30-based, inducible knockdown system. Upon individual knockdown of UBXD1, p97 and ERGIC-53, autophagic markers p62 and LC3-II accumulate at relatively high levels in normal culture conditions, strongly suggesting a role in mediating basal autophagy. However, when placed under starvation conditions, autophagy progresses and p62 is degraded. It is speculated from these studies that a p97/UBXD1 complex plays a role in regulating the trafficking of ERGIC-53 positive vesicles and this activity plays an important role in autophagy. / Molecular Biology and Genetics