Factors contributing to adult DRG neurotrophin-independent neuronal survival

Dodge, M. Elaine (2006) Factors contributing to adult DRG neurotrophin-independent neuronal survival. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Understanding mechanisms of cellular survival in the adult dorsal root ganglion (DRG) neuron is important in identifying targets for disease intervention and treatment in the peripheral nervous system (PNS). I have used DRG neurons to study signal transduction mechanisms involved in adult DRG neurotrophin-independent survival and response to stressful stimuli to understand mechanisms of neuroprotection and to enhance cellular survival after insult so that other processes, such as regeneration, can occur. -- Dorsal root ganglion (DRG) sensory neurons become less dependent upon neurotrophins for their survival as they mature. In Chapter 2 we show that adult DRG sensory neuronal cultures are able to survive for at least two weeks in culture in the absence of nerve growth factor (NGF). The use of pharmacological inhibitors of cellular signalling pathways confirmed the importance of the phosphoinositide kinase-3 (PI 3-K) and protein kinase C (PKC) pathways in this neurotrophin-independent neuronal survival. -- Chapter 3 shows concurrent studies investigating the role of stress-activated signalling pathways and the small heat shock protein, Hsp27, in protecting PC 12 cells from heat shock (HS) and NGF withdrawal-induced apoptosis. PC 12 cells and a stable cell line overexpressing Hsp27 (HSPC cells) were subjected to a physiological heat stress. Hsp27 associated with Akt and p38MAPK after stress and HS resulted in rapid activation of Akt followed by p38MAP kinase signalling, with phosphorylation and intracellular translocation of Hsp27 also detectable. Hsp27 protected NGF-differentiated PC12 cells against NGF-withdrawal treatment and phosphorylation of Akt was maintained in both the heat-shocked and NGF-withdrawal treated HSPC cells compared to the parental cells. -- Our studies of Hsp27 in PC 12 cells formed the basis for the experimental work outlined in Chapters 4 and 5. We investigated the possibility that Hsp27 may contribute to adult DRG neurotrophin-independent survival. Unlike adult DRG neurons in vitro, neonatal DRG neurons require NGF for survival; withdrawal of NGF results in apoptosis of a majority of neonatal neurons. Constitutive Hsp27 expression was higher in adult DRG neurons compared to neonates. After HS, Akt activation and upregulation of Hsp27 expression occurs in both adult and neonatal neurons. Increasing endogenous Hsp27 by HS in neonatal neurons was able to inhibit NGF withdrawal-induced apoptosis. Hsp27 siRNA treatment of adult neurons effected a decreased expression of Hsp27, which correlated with increased apoptosis. Hsp27 siRNA also blocked the HS-induced rescue of neonatal neurons after NGF withdrawal. These results indicate that physiologically induced upregulation of Hsp27 is sufficient to provide some degree of neuronal protection. This induction appears to be regulated by transcriptional activation of HSF-1 as shown by HSF-1 nuclear translocation and EMSA analyses of HSF-1 binding to nuclear protein. -- These results provide further evidence that adult DRG neuronal survival depends on multiple factors; one of significant importance is Hsp27. My studies show that this protein and its interactions with other signalling intermediates play important roles in neuronal survival. Further assessment of Hsp27's protein interactions and regulation will hopefully contribute to elucidating factors important for adult DRG neurotrophin-independent neuronal survival.

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