Browsing by Subject "Autophagy"

Tissue homeostasis is controlled by the availability of growth factors, which sustain exogenous nutrient uptake and prevent apoptosis. Cancer cells, however, can express constitutively active oncogenic kinases such as BCR-Abl that promote these processes independent of extrinsic growth factors. When cells are deprived sufficient growth signals or when oncogenic kinases are inhibited, glucose metabolism decreases and cells activate the self-digestive process of autophagy, which clears damaged organelles and provides degradation products as an alternate fuel to support mitochondrial metabolism. Importantly, loss of growth signals can also lead to apoptosis mediated through Bcl-2 family proteins, and Bcl-2 has been reported to interfere with autophagy, potentially disrupting a key nutrient source just as glucose uptake becomes limiting. Since autophagy may support survival or lead to death depending on context, the role of this pathway in apoptosis-competent growth factor deprived cells remains unclear.

In this thesis, I examine the interactions of autophagy with Bcl-2 family proteins and apoptosis upon inhibition of growth signals in hematopoietic cells. In contrast to other studies, I found autophagy was rapidly induced in growth factor deprived cells regardless of Bcl-2 or Bcl-xL expression, and this led to increased production of fatty acids and amino acids for metabolism. While these data suggested autophagy may play a key role to support metabolism of growth factor deprived cells, provision of exogenous pyruvate or lipids as alternate fuel had little affect on cell survival. Instead, I found that autophagy modulated cell stress pathways and Bcl-2 family protein expression in a context specific fashion to impact cell fate.

My results show that autophagy's effect on cell survival is dependent on its level of induction within a cell. I observed that partial suppression of autophagy protects cells from stress and induction of pro-apoptotic Bcl-2 family expression, while complete inhibition of autophagy enhances stress and is pro-apoptotic. In experiments using shRNAi to partially suppress autophagy, I found increased survival upon growth factor deprivation in several different types of cells expressing anti-apoptotic Bcl-2 or Bcl-xL, indicating that autophagy promoted cell death in these instances. Cell death was not autophagic, but apoptotic, and relied on direct Chop-dependent transcriptional induction of the pro-apoptotic Bcl-2 family protein Bim. In contrast, complete acute disruption of autophagy through conditional Cre-mediated excision of the autophagy-essential gene Atg3 led to p53 phosphorylation, upregulation of p21 and the pro-apoptotic Bcl-2 family protein Puma, and rapid cell death of cells the presence or absence of growth factor. Importantly, transformed BCR-Abl-expressing cells had low basal levels of autophagy but were highly dependent on this process. Deletion of Atg3 or treatment with chemical autophagy inhibitors led to rapid apoptosis, and BCR-Abl expressing cells were unable to form leukemia in mice in without autophagy. Together, my data demonstrate a dual role for autophagy in cell survival or cell death and suggest that the level of autophagy in a cell is critical in determining its role in apoptosis and cell fate. Ultimately, these results may help to determine future approaches to modulate autophagy in cancer therapy.

Unlabelled

Importance

Background

Materials and methods

Results

Conclusions

Cells thrive on sustaining order and balance to maintain proper homeostatic functions. However, the primary machinery involved in protein quality control including chaperones, ubiquitin proteasome system, and autophagy all decline in function and expression with age. Failures in protein quality control lead to enhanced protein misfolding and aggregation. Efficient elimination of misfolded proteins by the proteasome system is critical for cellular proteostasis. However, inadequate proteasome capacity can lead to aberrant aggregation of misfolded proteins and inclusion body formation, which is a hallmark of numerous neurodegenerative diseases. Due to the post-mitotic nature of neurons, they are more susceptible to the collapse in proteostasis correlated with age.

Here, we propose a cell based model of aggresome clearance using a reversible proteasome inhibitor, MG132, to identify the precise molecular machinery involved in proper processing of inclusions. It is known that once misfolded proteins are aggregated, the proteasome system can no longer degrade them. Furthermore, the continuous accumulation of aggregates often leads to aggresome formation, which results in amalgamated inclusion bodies that are simply too large for autophagosomes to engulf and degrade. Although, studies have shown that aggresomes can eventually be cleared by autophagy, the molecular mechanisms underlying this process remain unclear.

Our research reveals that regardless of impaired proteolysis, proteasomes can still stimulate autophagy-dependent aggresome clearance by producing unanchored lysine (K)63-linked ubiquitin chains via the deubiquitinating enzyme Poh1. Unanchored ubiquitin chains activate ubiquitin-binding histone deacetylase 6, which mediates actin-dependent disassembly of aggresomes. This crucial de-aggregation of aggresomes allows autophagosomes to efficiently engulf and eliminate the protein aggregates. Interestingly, the canonical function of Poh1 involves the cleavage of ubiquitin chains en bloc from proteasomal substrates prior to their degradation by the 20S core, which requires intact 26S proteasomes. In contrast, here we present evidence that during aggresome clearance, 20S proteasomes dissociate from protein aggregates, while Poh1 and selective subunits of 19S proteasomes are retained as an efficient K63 deubiquitinating enzyme complex. The dissociation of 20S proteasome components requires the molecular chaperone Hsp90. Hsp90 inhibition suppresses 26S proteasome remodeling, unanchored ubiquitin chain production, and aggresome clearance. Ultimately, we hope to apply these molecular markers of inclusion body processing to identify the underlying lesion in aggregate prone neurodegenerative disease.

Tuberculosis is a devastating disease that has been plaguing humankind for millennia. Co-evolution of humans with Mycobacterium tuberculosis, the causative agent of the disease, has allowed for the pathogen to possess an abundance of survival mechanisms. The outcome of this is the ability of the bacterium to create an intracellular niche lifestyle inside host cells where it can successfully evade the host immune system. While there is a vaccine available, named the BCG vaccine, it confers little protection to adults in the pulmonary form of the disease. The lack of an effective vaccine and the rise of Multidrug-Resistant (MDR) and Extensively Drug-Resistant (XDR) tuberculosis highlight the need for more research into combating Mycobacterium tuberculosis. The purpose of this work is to enhance the field of knowledge of how mycobacterial virulence factors affect host cellular pathways so that the interactions can be exploited for novel therapeutics and vaccine development.

One of the hypotheses for the poor efficacy of the BCG vaccine is that it fails to elicit a strong CD8+ T cell response during infection. Studies have found that vaccinating mice with apoptotic bodies containing mycobacterial antigens were able to protect mice to a greater degree than BCG and that this is dependent on CD8+ T cell activation. Thus, we hypothesized that a pro-apoptotic mutant of M. tuberculosis could be utilized as a novel vaccine candidate. Through screening a library of M. tuberculosis transposon mutants, we identified an Enhanced Cell Death mutant (ECD19) that functions through caspase 3 mediated apoptosis. Sequencing revealed that the mutant has a transposon insertion in Rv2456c, a probable integral membrane transport protein. Immunogenicity testing via Enzyme-Linked ImmunoSpot (ELISPOT) and Intracellular Cytokine Staining (ICS) assays demonstrated that ECD19 induced an altered immune response when compared to the parental strain M. tuberculosis H37Rv. Additionally, ECD19 has reduced survival in an in vitro THP-1 cell model and in an in vivo mouse model. Taken together, our data suggest that Rv2456c is important to the survival of H37Rv in host cells and that deletion of the gene may enhance the immunogenicity of the bacterium.

Inappropriate dosing and poor adherence to antibiotics in the treatment of tuberculosis has led to MDR and XDR, the highest incidences of which can be found in the KwaZulu-Natal (KZN) province of South Africa. Little is known about the virulence of these strains, but it is hypothesized that the drug resistance mechanisms come at a cost to the bacteria. In an in vitro assay, we have found that clinical isolates from the KZN region induce higher levels of necrosis than virulent laboratory strains of M. tuberculosis. Additionally, our in vivo studies show that the drug-resistant isolates do not disseminate as well as susceptible strains, and in both immunocompetent and immunocompromised mouse models, mice infected with the drug-resistant strains are able to live longer than mice infected with drug-sensitive strains. As all strains are highly related on a genetic level, we can say that the drug-resistant mechanisms acquired by the strains come at a cost of reduced virulence. Thus, it is likely that higher prevalence of the MDR and XDR in the KZN province is due to the high rate of HIV+, immunocompromised individuals living in the region.

Lastly, we are interested in building on the knowledge that avirulent mycobacteria are able to induce autophagy in a murine macrophage cell line. Through the use of Mammalian Target of Rapamycin (mTOR) inhibitors and autophagy-deficient macrophages, we were able to show that Mycobacterium smegmatis is able to induce both mTOR and autophagy during infection. Additionally, we found that mycobacterial killing occurs in the absence of autophagy when mTOR is inhibited. This effect is not due to a bactericidal effect of the mTOR inhibitors. From these data, we show that there is an underappreciated role in the induction of mTOR after mycobacterial infection. By studying the interplay of mTOR and autophagy, therapies targeted to favoring host defenses could be developed.

In summary, the insights from this work enhance the knowledge of how mycobacteria are able to be successful pathogens. This data may be useful in the creation of novel vaccine candidates or the identification of potential drug targets to bolster the therapeutic options in treating those afflicted with tuberculosis.

Neurons, with their enormous membrane contents, depend heavily on regulated membrane trafficking processes to maintain their morphology and function. The phosphatidylinositol 3-kinase class III, or PIK3C3, plays a critical role in various membrane trafficking processes including both the endocytic and autophagic pathways. The functions of PIK3C3 in the nervous system in vivo are un-characterized. We reasoned that studying PIK3C3 in neurons would provide us an entry point into understanding the regulations and functions of the neuronal membrane trafficking processes and their roles in neuronal morphogenesis and homeostasis.

We generated a conditional allele of Pik3c3 and first deleted it specifically in the peripheral sensory neurons. Mutant large-diameter myelinated sensory neurons accumulated numerous enlarged vacuoles and ubiquitin-positive aggregates and underwent rapid degeneration. By contrast, Pik3c3-deficient small-diameter unmyelinated neurons accumulated excessive numbers of lysosome-like organelles and degenerated slower than large-diameter neurons. These differential degenerative phenotypes are unlikely caused by a disruption of the autophagy pathway, because inhibiting autophagy alone by conditional deletion of Atg7 results in a completely distinct subcellular phenotypes and very slow degenerations of all sensory neurons. More surprisingly, a noncanonical PIK3C3-independent LC3-positive autophagosome formation pathway was activated in Pik3c3-deficient small-diameter neurons. This work uncovered unexpected differences of the endo-lysosomal systems in different types of neurons and discovered a novel autophagy initiation pathway in vivo in neurons.

To examine the role of PIK3C3 in the central nervous system (CNS), we next deleted Pik3c3 in CNS neural progenitor cells using the Nestin-Cre transgenic line. The resulting conditional knockout mice displayed a severe cortical lamination abnormality caused by defective cortical neuron migration. This finding uncovered a previously under-appreciated role of endocytic trafficking in neural migration, which was further confirmed by electron microscopic analyses of the developing cortex. Moreover, overexpressing the dominant negative forms of Dynamin2 or Rab5, two regulators of endocytosis, caused similar migration defects as Pik3c3-deletion. Mechanistically, Pik3c3-deficient cortical neurons drastically reduced surface Reelin binding sites, and showed significantly decreased levels of Dab1 phosphorylation, despite expressing normal total amount of Reelin receptor ApoER2. This work suggests endocytosis and recycling of Reelin receptors are likely to play an important role in cortical migration regulated by the Reelin signaling pathway.

These studies represent the first in vivo characterization of PIK3C3 functions in mammals, and provide insight into the complexity and functional importance of neuronal endo-lysosomal and autophagic pathways.