Indiana University-Purdue University Indianapolis (IUPUI) / Type 1 diabetes (T1D) is a multifactorial disease involving genetic and environmental factors. One of the factors implicated in disease pathogenesis is early life viral infection. A typical immune response to viral infection includes production of type 1 interferons (IFN), such as IFN-α, which can induce stress in the pancreatic β-cells. Reactive oxygen species (ROS) accumulation occurs after exposure to other inflammatory cytokines, causing oxidative stress that may be linked to T1D pathogenesis. Therefore, we hypothesized that IFN-α may also elicit β-cell ROS accumulation. Our in vivo and in vitro experiments with human islets showed rapid and heterogenous ROS accumulation with IFN-α. Although T1D is characterized by autoimmune destruction of β-cells, some cells survive this persistent attack. We hypothesized that survival/ death of β-cells could be attributed to the ability to effectively mitigate ROS accumulation. One mechanism to mitigate ROS is autophagy, which degrades and recycles cellular components to promote cellular homeostasis. We observed an impairment in autophagy in β-cells of donors with T1D as well as in islets of diabetic non-obese diabetic (NOD) mouse model of autoimmune diabetes. Autophagic flux was also impaired in diabetic NOD mouse islets, further confirming impairment of autophagy. Interestingly, we observed an induction of autophagy after acute treatment with IFN-α both in vitro and in vivo, suggesting compensatory upregulation of autophagy to restore homeostasis. Similarly, we observed an increase in autophagosomes and telolysosomes in β-cells of normoglycemic autoantibody positive organ donors compared to nondiabetic organ donors. Together, these data implicate a defect in the final degradation step of autophagy involving lysosomes. Therefore, we analyzed the activity and expression of lysosomal cysteine protease Cathepsin H (CTSH, a T1D susceptibility locus), and found both to be increased in islets of pre-diabetic NOD mice. Together, these data support compensatory hyperactivation of lysosomal enzymes prior to overt diabetes, potentially to rid the cell of ROS and degradation-resistant oxidized proteins and lipids. We also observed that C57Bl/6J mice lacking a key autophagy enzyme, ATG7, in their β-cells, spontaneously developed hyperglycemia. Collectively, these data highlight the importance of -phagic degradation process in the pathogenesis of T1D. / 2022-12-28

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20566号 / 医博第4251号 / 新制||医||1022(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 竹内 理, 教授 小柳 義夫, 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

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21799号 / 工博第4616号 / 新制||工||1719(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 浜地 格, 教授 跡見 晴幸, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Iron homeostasis is a vital process that balances access to free iron to fuel physiological processes with iron storage to reduce the deleterious aspects of excess free iron. Dysfunctional iron homeostasis can lead to iron overload, in which excess free iron can promote cellular injury, ferroptosis, and eventual organ damage as a result. The cellular consequences of long-term iron overload including the cellular pathways that lead to pathological changes or those that may provide protection against damage are incompletely defined. Here, we use dietary and genetically engineered mouse models of iron overload combined with mass spectrometry-based quantitative proteomics to determine the iron overload liver proteome. We identify and quantify over 8,000 proteins representing the most in-depth iron overload proteome evaluation to date. Using bioinformatics, we identify conserved upregulated pathways including ‘response to oxidative stress’ and downregulated pathways including ‘steroid homeostasis.’ Furthermore, we identify an unexpected role for NCOA4, an autophagy adaptor that targets ferritin for autophagic degradation during iron deprivation, during iron overload. This work highlights the importance of further investigation into NCOA4 and its role in diseases of iron overload. / 2025-01-30T00:00:00Z

<p>Lysosomal storage diseases (LSDs) are devastating neurological disorders caused by mutations in lysosomal hydrolases that result in accumulations of hydrolase substrates. Tay-Sachs disease (TSD) is an LSD that specifically results in the accumulation of GM2 gangliosides causing the activation of inflammatory signaling pathways, and leading to microglial activation and apoptotic cell death. The detailed mechanisms through which cell death occurs have not been completely elucidated, however, excitotoxicity is thought to play a major role. Here, we investigated the role of hypoxia-inducible factor-1α (HIF- 1α) and its effector microRNA, miR-210, and the impact they have on the expression of important molecules involved in excitotoxicity, namely neuronal pentraxin 1 (NPTX1) and potassium channel KCNK2 (KCNK2). We discovered that TSD neuroglia are inefficient at stabilizing HIF-1α in hypoxic conditions. Furthermore, miR-210 expression is significantly higher in TSD neuroglia compared to normal neuroglia at baseline and during hypoxia. In addition, TSD neuroglia expressed <em>NPTX1</em>, <em>NPTX2 </em>and <em>KCNK2 </em>at higher levels, and neuronal pentraxin receptor at lower levels than normal neuroglia, implicating excitotoxicity in disease pathogenesis. We also confirmed that miR-210 binds to the 3’ UTR of <em>NPTX1 </em>to repress its expression in TSD neuroglia. The presence of reverse hypoxia response elements in the promoter of KCNK2 and the repression of <em>KCNK2 </em>expression by HIF-1α stabilization suggest that KCNK2 is directly regulated by HIF-1α. Moreover, the glucosylceramide synthase inhibitor, NBDNJ, which is used to reduce ganglioside synthesis, caused expression of <em>NPTX1 </em>to decrease but <em>KCNK2 </em>expression to increase, indicating this drug can modify multiple parameters of disease. This study identifies major gene expression changes between normal and TSD neuroglia that affect the excitability and therefore the viability of TSD cells. This information provides new insight into the mechanisms of neurodegeneration experienced by TSD neuroglia.</p> / Master of Science (MSc)

The gram-negative endotoxin, lipopolysaccharide (LPS), has been extensively researched in high doses (10-200ng/ml) and is well-documented in the literature for its ability to result in devastating effects such as multi-organ failure, sepsis, and septic shock. In high doses, LPS signals through Toll-like-receptor 4 (TLR4) and triggers a cascade of events culminating in the release of pro- and anti-inflammatory cytokines and the activation of NF-κB. In contrast, super-low doses of LPS (1-100pg/ml) are able to trigger the persistent release of pro-inflammatory mediators while evading the compensatory activation of NF-κB. This mild yet persistent induction of inflammation may lie at the heart of numerous inflammatory diseases and disorders and warrants studies such as this to elucidate the novel mechanisms. In this study, we explored the novel mechanisms utilized by super-low dose LPS in cellular stress and low-grade inflammation. In the first study, the molecular mechanisms governing the role of super-low dose LPS on cellular stress and necroptosis were examined. We show that in the presence of super-low dose LPS (50pg/ml), the key regulators of mitochondrial fission and fusion, Drp1 and Mfn1 respectively, are inversely regulated. An increase in mitochondrial fragmentation and cell death which was not dependent on caspase activation was observed. In addition, super-low dose LPS was able to activate RIP3, a kinase responsible for inducing the inflammatory cell death, necroptosis. These mechanisms were regulated in an Interleukin-1 receptor-associated kinase 1 (IRAK-1) dependent manner. In the second study, the molecular mechanisms governing the role of super-low dose LPS on cellular stress and endosome/lysosome fusion were examined. In the presence of low-dose LPS (50pg/ml), endosomal-lysosomal fusion is inhibited and a loss of endosomal acidification required for the successful clearance of cellular debris and resolution of inflammation was observed. Additionally, super-low dose LPS induced the accumulation of p62 indicative of the suppression of autophagy. Tollip and Interleukin-1 receptor-associated kinase 3 (IRAK-M) appear to be critical regulators in this process. Collectively, these studies show that low-dose endotoxemia is capable of causing persistent cellular stress, not observed in the presence of high-dose LPS (10-200ng/ml), and that it promotes necroptotic cell death while suppressing mechanisms necessary for the resolution of inflammation such as endosome-lysosome fusion. This research reveals novel mechanisms utilized by low-dose endotoxemia which could aid future efforts to develop prevention and treatment for various debilitating inflammatory diseases. / Ph. D.

Le lupus érythémateux disséminé est une maladie autoimmune systémique provoquant des lésions tissulaires graves. Notre laboratoire a découvert un peptide phosphorylé, appelé P140, présentant des propriétés thérapeutiques pour le traitement du lupus. Le mode d’action du peptide P140 dans le traitement du lupus repose sur son interaction avec la protéine de choc thermique HSPA8/HSC70 et l’objectif de mon projet de thèse a été de consolider et compléter ce mécanisme. Nous avons démontré qu’après internalisation par endocytose dépendante des clathrines, le peptide P140 se localise rapidement au sein du lysosome des lymphocytes B. Dans cet organelle, il réduit l’import de substrat cytosolique par autophagie dépendante des chaperonnes en ciblant et réduisant l’activité de HSPA8 intralysosomale. Nous avons également entrepris une analyse comparative du répertoire des lymphocytes T et B des souris lupiques par rapport aux souris saines. Nos résultats révèlent un changement dans la fréquence de certains réarrangements du TCR entre les souris lupiques et les souris saines et un effet bénéfique du peptide P140 sur certains réarrangements associés au lupus. / Systemic lupus erythematosus is a multi-organ autoimmune disease provoking tissue damages. Our laboratory has discovered a phosphorylated peptide, named P140, with therapeutic activities in lupus. The mode of action used by P140 peptide relies on its interaction with the heat shock protein HSPA8/HSC70. The aim of my thesis project was to consolidate and complete this HSPA8-dependent mechanism. We have demonstrated that, upon internalization by clathrin-mediated endocytosis, P140 peptide homes rapidly into B cell lysosome. In this organelle, the peptide reduces chaperone-mediated autophagy by interacting and inhibiting intralysosomal HSPA8 activity. We also performed a comparative analysis of T cell and B cell repertoire on lupic mice compared to healthy mice Our results show a modification in the frequency of certain TCR rearrangements between lupus-prone mice and healthy mice and a beneficial effect of P140 peptide on certain lupus-associated rearrangements.

Les lysosomes sont importants pour la survie et la fonction des cellules du système nerveux central et en particulier des neurones. Le mécanisme de la reformation des lysosomes est crucial pour maintenir une quantité adéquate de lysosomes fonctionnels dans les cellules. La spatacsine, qui joue un rôle dans le ce mécanisme est impliquée dans la paraplégie spastique de type SPG11 ; une maladie caractérisée par des troubles moteurs et cognitifs sévères. L’utilisation de modèles cellulaires de cette pathologie permet d’étudier les mécanismes physiopathologiques à l’origine d’altérations de la reformation des lysosomes. J’ai montré que la perte de fonction de la spatacsine est responsable de l’accumulation de lipides dans les lysosomes. Ces accumulations sont constituées de gangliosides et de cholestérol et sont présentes dans les autolysosomes perturbant leur recyclage en lysosomes, notamment en empêchant le recrutement de protéines impliquées dans le mécanisme. Les accumulations de gangliosides rendent les neurones à l’exposition au glutamate ce qui suggère que ces altérations pourraient avoir un rôle dans la neurodégénérescence. J’ai aussi montré que l’absence de spatacsine provoque une dérégulation de l’import de Ca2+ extracellulaire par le « store-operated calcium entry » ce qui conduit à altération de l’homéostasie calcique. L’inhibition de l’import de calcium par le SOCE permet de réduire les accumulations de lipides et de rétablir partiellement le recyclage des lysosomes. Ainsi, l’absence de spatacsine induit une altération de l’homéostasie calcique qui participe à l’accumulation de lipides dans le système lysosomal ce qui est délétère pour la survie des neurones. / Lysosomal dysfunctions are involved in a large number of neurodegenerative diseases highlightingthe crucial function of lysosomes in neuron survival and function. The mechanism of lysosomereformation from autolysosomes allow cells to maintain the ool of functional lysosomes.Disruptions of this rocess are involved in athologies affecting the central nervous system. Inparticular, spatacsin that lays a role in lysosome recycling is implicated in hereditary spasticparaplegia type SPG11, a severe disease characterized by motors and cognitive alterations. Thispathology is caused by loss of function mutations in SPG11, encoding spatacsin. The study ofSPG11 cellular models gives the opportunity to decipher the hysiopathological mechanismsunderlying lysosomal reformation disruptions.During my thesis, I showed that loss of spatacsin function induces lipid accumulation in lysosomesand articularly in autolysosomes both in fibroblasts and neurons from Spg11-/- mice. Gangliosidesand cholesterol are among lipids that accumulate in autolysosomes impairing lysosomal membranerecycling by disrupting the recruitment of keys roteins. Neurons with ganglioside accumulationsare more sensitive to glutamate induced neuronal death, suggesting that these accumulations areinvolved in neurodegeneration. These results could be of great importance since accumulations ofgangliosides in lysosomes arise in many diseases.I also showed that loss of spatacsin disrupts extracellular calcium import by the store-operatedcalcium entry (SOCE) leading to an increase in cytosolic calcium levels. Lysosomal calcium contentis also increased in Spg11-/- cells and calcium release from lysosome by TRPML1 is reduced.Inhibiting SOCE and stimulating lysosomal calcium release by TRPML1 reduced lipidsaccumulations in lysosomes and artially restored lysosome reformation.Our data suggest that absence of spatacsin is responsible for a disruption of calcium homeostasisthat contributes to lipid accumulation in autolysosomes, disturbing reformation of lysosomes fromautolysosomes. Inhibiting gangliosides synthesis could be used as a therapeutic strategy. However,understanding how loss of function of spatacsin alters these cellular athways will allow thedevelopment of targeted therapeutic approaches.

Les G-quadruplexes (G4) sont des structures non canoniques des acides nucléiques qui peuvent être formés dans des régions d’ADN ou d’ARN riches en guanines. Les ligands G4 (LG4), sont des molécules capables d’interagir et de stabiliser les structures G4, qui présentent de nombreuses propriétés anti-cancéreuses. Nous avons travaillé avec le LG4 20A, appartenant à la famille des triarylpyridines, qui stabilise efficacement les structures G4 in vitro. Les objectifs de ce travail ont été de déterminer les mécanismes moléculaires et cellulaires responsables des effets anti-prolifératifs du 20A dans des cellules cancéreuses. Dans cette étude, nous avons montré que le 20A induit un arrêt de la croissance cellulaire de cellules en culture et dans un modèle de xénogreffe tumorale, grâce à l’induction de la sénescence et de la mort cellulaire par apoptose. Ces réponses sont associées à l’activation de la voie des réponses aux dommages à l’ADN (DDR) via la kinase ATM, qui favorise l’autophagie (un processus catabolique) et la sénescence, tout en protégeant les cellules de l’apoptose. De plus, nous avons observé que le 20A induit un échec de la cytokinèse, conduisant à l’accumulation de cellules binucléées qui présentent une résistance à la mort cellulaire. De façon inattendue, nous avons trouvé que le 20A s’accumule dans les lysosomes, induisant une augmentation de la taille de ces derniers. La combinaison du 20A et de l’agent lysomotropique chloroquine, potentialise de façon importante la perméabilisation de la membrane lysosomale (LMP) et la mort cellulaire. En particulier, cette combinaison sensibilise de façon notable ces cellules binucléées à la mort cellulaire. L’ensemble de ces résultats révèle une relation entre les processus de mort cellulaire et de sénescence induits par le LG4 20A, et les voies de DDR et lysosomales. Ces régulations devraient être prises en considération lors de l’utilisation d’agents antiprolifératifs susceptibles d’interférer avec les fonctions lysosomales. / G-quadruplexes (G4) are unusual nucleic acid structures that can be formed by guanine-rich DNA and RNA. Through their ability to stabilize G4 structures, G4 ligands (G4L) have been described to display potent anticancer properties. Here, we studied the G4L 20A belonging to the triarylpyridine family of compounds that have the ability to efficiently bind to and stabilize G4 structures in vitro. The objectives of this work were to determine the molecular and cellular mechanisms responsible for the anti-proliferative effects of 20A in cancer cells. In this study, we showed that 20A causes cancer cell growth arrest in cell culture and a mice tumour xenograft model, through induction of senescence and apoptotic cell death. These cellular responses are associated with the induction of the DNA damage response pathway (DDR), in particular ATM activation, which promotes the induction of both autophagy (a lysosomal catabolic pathway) and senescence, while protecting cells against apoptosis. Furthermore, we found that 20A induces failure of cytokinesis which results in the accumulation of binucleated cells that display marked resistance to 20A-induced cell death. Unexpectedly, we found that 20A accumulates in the lysosomal compartment and causes lysosome enlargement. The combination of a lysosomotropic agent, chloroquine, and 20A promotes a significant induction of lysosomal membrane permeabilization (LMP) and a robust cell death. In particular, this combination significantly sensitizes binucleated cells to cell death. Altogether, our results uncover the relationship of the DDR and lysosomal pathways to cell death and senescence induced by the G4L 20A. Such regulation should also be taken into account when using antiproliferative drugs susceptible to interfere with the lysosomal functions.

Lysosomes are acidic, hydrolase-rich vesicles capable of degrading most biological macromolecules. During the past several decades, much has been learned about different aspects of lysosome biogenesis. The selective phosphorylation of mannose residues on lysosomal enzymes, in conjunction with specific receptors for the mannose-6-phosphate recognition marker, has been found to be largely responsible for the targeting of newly synthesized lysosomal enzymes to lyzosomes. It is known that lysosomes receive input from both the endocytotic and biosynthetic pathways. Nevertheless the exact molecular mechanisms responsible for sorting of the biosynthetic imput involved in the lysosome biogenesis is still a matter of debate. Because osteoclast precursors do not secrete their lysosomal enzymes and osteoclasts do, the observation of modifications occuring during osteoclastogenesis is a good model to observe mechanisms responsible for lysosomal enzymes traffic. Osteoclasts are bone-degrading cells. To perform this specific task they have to reorganise the sorting of their lysosomal enzymes to be able to target them toward the bone surface in mature cells. Since few years, the differentiation of osteoclasts in vitro did help to study these cells. Osteoclast morphology has been therefore already well studied, and the nature of their specific membrane domains is now established. Sensing the proximity of a bone-like surface the cell reorganises its cytoskeleton, and creates specific membrane domains: an actin-rich ring-like zone (named actin ring) surrounded by highly ruffled membrane (named the ruffled border) where enzymes are secreted, while subsequent bone degradation products are endocytosed. Endocytosed material is then transported through the cell inside transcytotic vesicles and released at the top of the cell in an area named the functional secretory domain. Several molecular machineries are thought to control these different phenomena. The main purpose of this thesis was to identify the major regulators of lysosomal enzymes secretion and therefore to identify the molecular switches responsible for such a membrane traffic re-organisation.