No description available.

No description available.

Lipid droplet (LD) autophagy (lipophagy) is a recently discovered selective form of autophagy and is a pathway for LD catabolism through the lysosome or vacuole. Therefore, lipophagy has therapeutic potential in the treatment of a variety of lipid related diseases in which increased cellular LDs are associated with pathophysiologies, such as obesity or atherosclerosis. This ubiquitous process has been an ongoing area of research within the budding yeast, Saccharomyces cerevisiae. However, there remains a need to better understand the regulators of this process. I have developed and validated a lipophagy library in yeast for the assessment of novel genetic regulators of stationary phase induced lipophagy. Through the screening of my library for roles in lipophagy I have identified many genetic regulators of lipophagy which include CUE1, UBC7, LHS1, HSP31, PLN1, TFS1, LAM6, OSH3, OSH4 and OSH7, among others. My screen highlights the power of this library to identify lipophagy regulators in S. cerevisiae, which can be utilised in the future to further the understanding of lipophagy.

Autophagy, an intracellular degradation, and recycling process is essential in maintaining cellular homeostasis. Dysregulated autophagy is linked to the pathogenesis of various diseases, including inflammatory bowel disease (IBD) which consists of Crohn’s disease and ulcerative colitis. In IBD, enterochromaffin cell numbers and one of its main product serotonin (5-hydroxytryptamine; 5-HT) levels are elevated. Previously, we had shown that tryptophan hydroxylase 1 deficient (Tph1-/-) mice, with reduced gut 5-HT had decreased severity of colitis. Here, we showed that gut 5-HT plays a vital role in modulating autophagy and thus regulating gut microbial composition and susceptibility to intestinal inflammation. Tph1-/- mice, had upregulated colonic autophagy via the mammalian target of rapamycin pathway (mTOR), and decreased colitis severity. Tph1-/- mice after 5-HT replenishment, and serotonin reuptake transporter deficient (SERT-/-) mice, which have increased 5-HT levels, showed converse results. Deletion of intestinal epithelial cell-specific autophagy gene, Atg7, in Tph1-/- mice (DKO mice) abolished the protective effect of Tph1 deficiency in colitis, decreased the production of antimicrobial peptide, β-defensin 1 and promoted colitogenic microbiota. Furthermore, using cecal microbial transplantation, we found that the colitic microbiota of the DKO mice contributed to the increased severity of colitis. Supporting this pathway's translational importance, we uncovered that 5-HT treatment of peripheral blood mononuclear cells from both healthy volunteers and patients with Crohn’s disease inhibited autophagy via the mTOR pathway. Our results in this thesis emphasize the role of 5-HT-autophagymicrobiota axis in intestinal inflammation. Moreover, these findings suggest 5-HT as a novel therapeutic target in intestinal inflammatory disorders such as IBD that exhibit dysregulated autophagy. / Thesis / Doctor of Philosophy (PhD) / Approximately 0.7% of Canadians are currently affected with inflammatory bowel disease (IBD). The gut hormone serotonin, which regulates many normal functions, is elevated in gut inflammation. Reduced serotonin levels decrease the severity of inflammation. IBD pathology has been linked to a unique cell self-eating process called autophagy. Using cell lines, mice, and samples from IBD patients, we assessed the interactions between serotonin signaling and autophagy during gut inflammation. I found that an increase in serotonin levels enhances the severity of gut inflammation by inhibiting autophagy. We also established the connection between serotonin and autophagy in the intestinal epithelial cells, and how this modulates epithelial cell function. Furthermore, we demonstrated the establishment of an altered gut microbiota upon disruption of the serotonin-autophagy axis in the epithelial cells, which subsequently influenced gut inflammation severity. Thus, we identified one of the key triggers related to the pathogenesis and severity of IBD.

Mitochondrial dynamics includes the processes of fusion, fission, and motility. These processes form interdependent adaptive mechanisms that, together with autophagy, maintain mitochondrial function to meet cellular needs. Mitochondrial dynamics control function directly by inducing bioenergetic remodeling or indirectly by promoting turnover of mitochondria via autophagy. Importantly, mitochondrial dysfunction has been implicated in beta-cell failure during type 2 diabetes. This thesis will investigate the role of dynamics and autophagy in regulating mitochondrial and pancreatic beta-cell function during chronic exposure to excess glucose and fatty acids, termed glucolipotoxicity (GLT). It remains ill-defined what role fusion and motility play in determining mitochondrial turnover, as current methodologies to assess turnover lack subcellular resolution. To address this need we developed the use of MitoTimer, a mitochondrial fluorescent probe that undergoes a time-dependent green-to-red transition. Turnover was revealed by the integrated proportions of young (green) and old (red) MitoTimer protein. The results demonstrate that mitochondrial fusion and motility regulate turnover by promoting the distribution of newer protein to subsets of mitochondria in the network. GLT inhibits mitochondrial fusion and networking in pancreatic beta-cells. Since fusion is dependent on motility we tested the hypothesis that GLT impairs fusion by affecting motility. We determined that GLT arrests motility, which may contribute to mitochondrial and beta-cell dysfunction. We show that excess nutrients increase O-linked β-N-acetyl glucosamine (O-GlcNAc) modification of mitochondrial motor adaptor Milton1, which decreases its activity and results in arrest of motility and increased fission. Thus Milton1 O-GlcNAc modification acts as a nutrient-sensor linking fusion, fission, and motility to nutrient supply in the beta-cell. Finally, GLT inhibits autophagic flux with concurrent lysosomal pH increase in beta-cells. To address the hypothesis that impaired lysosomal acidification is a causative event inhibiting autophagic flux and beta-cell function, we developed lysosome-localizing nanoparticles that expand and acidify upon UV photo-activation. Increasing lysosomal acidity with the nanoparticles increased autophagic flux and restored beta-cell function under GLT, establishing lysosomal pH as a key mediator of nutrient-induced beta-cell dysfunction. In summary the work elucidates the interdependence and specific roles of mitochondrial fusion, fission, motility, and autophagy in dictating beta-cell responses to excess nutrient environment. / 2017-06-15T00:00:00Z

The maintenance of protein homeostasis is vital for all cells, but it is of utmost importance in post-mitotic cells, such as neurons that cannot dilute aggregates by cell division. Dysregulation of the proteostasis network can lead to neurodegenerative disorders such as Parkinson’s disease (PD), Alzheimer’s disease, Huntington’s disease, Amyotrophic Lateral Sclerosis (ALS), and prion diseases. The small molecule Sephin1 is a promising lead against proteostasis disruption, but its mechanism of action is uncertain. We assessed the therapeutic efficacy of Sephin1 in an established PD mouse model. Our laboratory has recently characterized a mouse expressing via bacterial artificial chromosome (BAC) the human LRRK2 G2019S protein, a variant linked to PD. Our data show that Sephin1 treatment rescues the motor deficit observed in BAC human-G2019S mice. Our experimental evidence shows that Sephin1 binds the monomeric globular actin (G-actin) in cell-free assays. By combining PAL chemistry to MS/MS analysis we identified the putative Sephin1 binding site on actin. In vitro, Sephin1 drives actin misfolding, and eventually, its precipitation. Upon Sephin1 treatment in HeLa cells, we visualized actin clusters localized to the lysosomes. This event at the lysosome impairs the normal autophagic flux. At the same time, Sephin1 induces the inactivation of the mammalian target of rapamycin (mTORC1), thus allowing the nuclear translocation of the transcription Factor EB (TFEB) and the expression of TFEB-direct target genes, on the longer term. In parallel, Sephin1 elicits the phosphorylation of the α subunit of the Eukaryotic Initiation Factor 2 (eIF2) and the ER-stress independent expression of the C/EBP homologous protein (CHOP). CHOP is a transcription factor that contributes to the integrated stress response as well as to autophagy. As such, Sephin1 triggers the activation of two main players in the autophagic response, TFEB and CHOP. Accordingly, we reported that, after the initial impairment, Sephin1 stimulates autophagy. Taken together, our results reveal a novel Sephin1 molecular mechanism in which lysosomal stress may regulate autophagy via mTORC1-TFEB complemented with the eIF2α signalling pathway. Although several questions remain to be answered, Sephin1, which successfully completed the phase I clinical trial for ALS and Charcot–Marie–Tooth disease, represents a promising therapeutic strategy that targets autophagy to regulate the homeostatic balance of proteins in neurodegenerative diseases.

The primary treatment options for head and neck cancer are radiation therapy or surgery, or both combined; chemotherapy is often used as an additional, or adjuvant, treatment. Patients treated with radiotherapy are exposed to a high cumulative dose of radiation over a period of time and there is a 17-33% chance of recurrence. High cumulative doses of radiation, a long time course of treatment, side effects and the possibility of recurrence provide the rationale for developing approaches for radiation sensitization, which could be helpful to patients in decreasing the dose, duration of radiation, side effects, or the chance of recurrence. Radiation induces autophagy, which is a catabolic process involving the degradation of the cell’s own components to generate energy under conditions of stress. Autophagy can be cytoprotective helping the cell to survive during stress such as nutrient deprivation or it can be cytotoxic, leading the cell toward death. We investigated whether blocking autophagy by the use of the antimalarial drug, chloroquine, could sensitize head and neck cancer cells to radiation. Studies were performed using the HN30 human head and neck cancer line (p53 wild type) derived from the pharynx as well as HN6 human cells (p53 mutant) derived from the base of the tongue. Cell viability was determined by cell counting and clonogenic survival assays, autophagy was monitored based on acridine orange staining accompanied by flow cytometry, while western blotting, DAPI and TUNEL staining and PI/annexin/FACS were utilized for determination and quantification of apoptosis. Senescence was monitored by beta-galactosidase staining/ FACS analysis. Radiation alone produced a transient growth arrest followed by proliferative recovery in both the HN30 and HN6 cancer cells. Radiation also promoted autophagy in both cell lines. The combination of chloroquine with radiation inhibited autophagy and promoted apoptotic cell death and suppression of proliferative recovery for the HN30 cells, but had little effect on sensitivity to radiation and proliferative recovery in the HN6 cells. The data suggest that autophagy induced by radiation serves a protective function in the HN30 cells and that a blockade to autophagy by chloroquine drives the cell toward apoptosis and death. In contrast, autophagy in HN6 cells appears to be non-protective as a pharmacological blockade did not sensitize the HN6 cells to radiation. These studies support the premise that autophagy induction by radiation need not necessarily have a cytoprotective function and further indicates that caution should be exercised in efforts to sensitize head and neck cancer to radiation through the clinical suppression of autophagy.

La macroautophagie assure l'homéostasie cellulaire en recyclant du matériel cytosolique obsolète ou délétère et sa dérégulation est associée à plusieurs pathologies. Elle constitue aussi un mécanisme de défense car elle peut éliminer des pathogènes intracellulaires. L'étape cruciale de l'autophagie est la maturation lors de laquelle la vésicule renfermant des substrats cytosoliques, l'autophagosome, fusionne avec des lysosomes et la dégradation a lieu. Nous nous intéressons à la régulation de l'autophagie et aux conséquences de sa perturbation lors des infections, notamment par le virus de la rougeole (VR). Les données de l'équipe montrent qu'il induit et utilise toutes les étapes de l'autophagie, afin de se répliquer efficacement. Mes travaux montrent que des protéines du virus peuvent interagir avec au moins deux protéines cellulaires NDP52 et T6BP qui sont des récepteurs autophagiques (protéines cytosoliques ayant un domaine de liaison aux autophagosomes et un domaine de liaison au substrat à dégrader, par exemple des pathogènes). J'ai alors étudié le rôle des récepteurs autophagiques T6BP, NDP52 et Optineurine dans la réplication virale. J'ai aussi participé à une étude décrivant que NDP52 et Optineurine régulent en plus la maturation. Mes travaux de thèse démontrent un tel double rôle pour T6BP. Cependant, seuls T6BP et NDP52 sont nécessaires à la réplication du VR bien qu'elle requiert la maturation autophagique. Ainsi mes résultats suggèrent d'une part que les trois récepteurs puissent réguler la maturation d'autophagosomes distincts.D'autre part, le VR pourrait exploiter individuellement les autophagosomes dont la maturation dépend de T6BP et NDP52 pour se répliquer / Macroautophagy ensures cell homeostasis through the recycling of obsolete or deleterious cytosolic components and its deregulation is associated with several pathologies. It is also a defense mechanism as it allows the elimination of intracellular pathogens. The most important autophagic step is maturation, during which the cytosolic substrate-containing vesicle, the autophagosome, fuses with lysosomes and the degradation occurs. We study autophagy regulation and the consequences of its disruption during infections and in particular by measles virus (MeV). Our team has shown that MeV induces and exploits all steps of autophagy, to replicate more efficiently. My results indicate that viral proteins can interact with at least two cellular proteins, NDP52 and T6BP, which are autophagy receptors (cytosolic proteins that carry an autophagosome-binding domain and a domain binding substrates that would be degraded, such as intracellular pathogens). I then studied the role of autophagic receptors T6BP, NDP52 and OPTINEURIN in viral replication. I also took part in a study describing NDP52 and OPTINEURIN as autophagosome maturation regulators. My work depicts the same dual role for T6BP. However, only T6BP and NDP52 are necessary for MeV replication even though it requires autophagosome maturation. Thus, my results suggest that the three autophagy receptors might regulate distinct autophagosome maturation on one hand. On the other, MeV could individually exploit autophagosomes, the maturation of which is regulated by T6BP or NDP2 to replicate efficiently

In general, ionizing radiation promotes cytoprotective autophagy in a majority of breast tumor cells. Previous studies from our laboratory indicated that radiation (5x2 Gy) induces cytoprotective autophagy in MCF-7 cells. In the current work, inhibition of autophagy by silencing of Beclin-1 in MCF-7 cells resulted in an increase in sensitivity to radiation based both on cell number and clonogenic survival; however, there was no increase in apoptosis and the basis for this sensitization is currently under investigation. Unexpectedly, enhancement of autophagy by silencing of Bcl-2 also led to an increase in sensitivity to radiation, possibly through the conversion of cytoprotective to cytostatic autophagy. In contrast to the MCF-7 cells, radiation (5x2 Gy) induces non-protective autophagy in Hs578t cells. Interference with autophagy through silencing of Beclin-1 or induction of Bcl-2 did not alter radiation sensitivity in the Hs578t cells. Since the induction of cytoprotective autophagy can represent an impediment to radiation therapy, it is important to understand the types of autophagy that occur in response to radiation in specific cellular settings and whether interference with autophagy can increase sensitivity to different forms of cancer treatment.

Autophagy, senescence and apoptosis are tightly linked processes which together determine the fate of cells in response to various stresses. There is ample evidence supporting the notion that senescent cells are highly dependent on autophagy and this process is here much more intensive than in nonsenescent cells. Autophagy may to some extent compensate increased energetic and metabolic demands of senescent cells and also helps with removal of toxic products such as oxidized proteins, protein aggregates and damaged organelles resulting from an overloaded metabolism of some senescent cells. In addition, some studies reported the need of autophagy for the adoption of senescent phenotype. However, there are also studies with seemingly contradictory results claiming that increased autophagy prevents or delays cellular senescence. Relationship of autophagy to apoptosis is similarly ambivalent. Whereas intact autophagy is necessary for the cell, while slightly increased autophagy still has a rather positive impact, excessive autophagy may lead to degradation of critical components necessary for cell function and survival and can trigger one of the modes of programmed cell death. In the first part of this work, we focused on the analysis of autophagic response in senescent and proliferating pancreatic...