The regulation of the cytoskeleton in promoting axon growth

Williams, Kristy L. (2009) The regulation of the cytoskeleton in promoting axon growth. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

The overall objective of this study was to investigate the role that the small heat shock protein Hsp27 plays in neurite initiation and growth, through its regulation of the neuronal cytoskeleton. The present investigation was carried out using adult rat dorsal root ganglion (DRG) neurons to study the behavior of neurite initiation and extension. -- The extracellular environment triggers the production and extension of a neurite via extracellular cues. To trigger morphological changes in the neuron these cues must be transformed into signals that converge on the cytoskeleton. The cytoskeletal components, such as actin, tubulin and neurofilament light chain (NF-L), can be modified though interaction with other cellular proteins, including Hsp27. Hsp27 is regulated through intracellular signaling cascades, and has been demonstrated to interact with cytoskeletal components. This positioning of Hsp27 as a possible transducer of extracellular signals to the cytoskeleton formed the basis for my hypothesis that Hsp27 plays a role in neurite growth. -- In Chapter 2 I showed that Hsp27 was present and colocalized with actin and tubulin in lamellipodia, filopodia, focal contacts, neurite shafts, branch points and growth cones during the stages of neurite formation and growth. The use of upstream p38 MAPK pharmacological inhibitors attenuated Hsp27 phosphorylation resulting in aberrant neurite growth. Further study of the effects of the p38 MAPK inhibitors in Chapter 5 indicated they increased F-actin levels in the neuron, suggesting a link between Hsp27 phosphorylation and actin dynamics in neurite growth. -- Chapter 3 confirmed the importance of the presence, and protein level of Hsp27 in neurite growth. Small interfering RNA (siRNA) was used to knock down endogenous Hsp27 protein levels in the DRG neurons, resulting in decreased neurite growth and altered actin localization. Alternatively, overexpression of Hsp27, resulted in increased neurite growth. -- An attempt was made to clarify the role of Hsp27 phosphorylation in Chapter 4; this was done using rodent hamster Hsp27 constructs with mutations in their serine 15, and serine 90 phosphorylation sites to mimic either constitutively nonphosphorylated or constitutively phosphorylated Hsp27. Results suggested that the phosphorylation state of Hsp27 plays a role in neurite growth; specifically, constitutive phosphorylation of either site was inhibitory to neurite growth. -- These results support my hypothesis that Hsp27 is involved in neurite growth via regulation of the actin cytoskeleton, although the underlying mechanisms have not been fully elucidated in neurons.

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