Exploring cellular stress responses: HspB1 and autophagy

Clarke, Joseph-Patrick William Edward (2016) Exploring cellular stress responses: HspB1 and autophagy. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Through an understanding of the innate mechanisms that neurons undertake when they undergo episodes of stress, we can develop effective strategies and treatments that ultimately prevent neuron death and promote neuron survival and neurite growth. In this study, the overall objectives were to investigate how the innate cellular mechanisms of both the heat stress response and autophagy promote survival in neurons during episodes of cell stress. Chapter 2 focuses upon the interaction dynamics between heat shock protein B1 (HspB1) and filamentous actin (F-actin) in stressed cells. This study demonstrates that under non-stressed conditions HspB1 interacts with F-actin as a non-phosphorylated protein, but after heat stress, both phosphorylated and non-phosphorylated forms of HspB1 interact with F-actin. Furthermore, by inhibiting HspB1 phosphorylation during heat stress, there is attenuation in the interaction between phosphorylated HspB1 and F-actin. This study was the first to show the interaction differences between the phosphorylated and non-phosphorylated forms of HspB1 with F-actin using an endogenous HspB1 expressing model. Chapter 3 explores the interaction between HspB1 and death-associated protein 6 (Daxx) in dorsal root ganglion (DRG) neurons, and how this interaction could influence autophagic activity. This study demonstrates that high glucose cell stress in DRG neurons affects the mRNA expression and the cellular localization of Daxx, HspB1 and B-cell lymphoma 2 protein (Bcl-2). Subsequent changes in autophagic activity associated with the changes found in Daxx, HspB1 or Bcl-2 expression, however, were not detected. Overall, the experimental results observed in this study can be used as a basis for further research into this topic. Chapter 4, 5 and 6 focus on how the process of autophagy affects DRG neuron survival and neurite growth. These studies demonstrate that adult rat DRG neurons activate autophagy when exposed to the stressful conditions of nutrient starvation and hydrogen peroxide. Furthermore, treatment with 3-methyladenine (3-MA) inhibits both endogenous and stress activated autophagy, while treatment with rapamycin does not further activate autophagy past levels obtained by stressful conditions. Additionally, inhibition of autophagy with 3-MA resulted in a reduction in cell survival, neurite growth, branching and initiation from the cell soma. Overall, these results suggest that autophagic activity plays a significant role in DRG survival and neurite growth.

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