Date of Award

2014-01-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Sukla Roychowdhury

Second Advisor

Siddhartha Das

Abstract

Neurodegeneration, a progressive loss of nerve cells (neurons), occurs in many neurological disorders, including Alzheimer's and Parkinson's diseases, as well as in the aging brain. Disruption of microtubules in neurons and the aggregation of proteins associated with them is the hallmark of neurodegeneration. Nevertheless, the cause of this disorder is largely unknown, and no effective drugs are available to treat the disease processes. Therefore, there is a need to understand the molecular mechanisms that drive the assembly and disassembly of microtubules during neurite outgrowth and differentiation. Evidence suggests that nerve growth factor (NGF) induces neurite outgrowth from PC12 cells by activating the receptor tyrosine kinase (TrkA) and the phosphoinositide-3-kinase (PI3K) pathway. G-protein-coupled receptors (GPCRs), as well as heterotrimeric G proteins, are also involved in regulating neurite outgrowth. However, the possible connection between these pathways and how they might ultimately converge to regulate the assembly and organization of MTs during neurite outgrowth is not well understood. In my Dissertation, I tested the hypothesis that Gβγ, an important component of the GPCR pathway, is involved in neuronal differentiation by modulating MT assembly, and that the disruption of the interaction between Gβγ and tubulin/MT in neurons will inhibit neurite outgrowth. To test this hypothesis, PC12 cells were used because they respond to NGF with growth arrest and exhibit a typical phenotype of neuronal cells sending out neurites. In addition, primary hippocampal and cerebellar neurons in culture were used for the study. It was found that NGF promoted the interaction of Gβγ with MTs and stimulated MT assembly. In further support for a role of Gβγ-MT interaction in neuronal differentiation, it was observed that overexpression of Gβγ in PC12 cells induced neurite outgrowth in the absence of added NGF. In addition, by using inhibitors of PI3K, I provide evidence that Gβγ may coordinate with PI3K signaling to regulate MT assembly and neurite outgrowth. I found that Gβγ-sequestering peptide GRK2i inhibited neurite formation, disrupted MTs, and induced axonal damage, indicating the involvement of Gβγ in this process. Because it was shown in earlier studies that prenylation and subsequent methylation/demethylation of γ subunits are required for the Gβγ-MTs interaction in vitro, in the current study, small-molecule inhibitors targeting prenylated methylated protein methyl esterase (PMPMEase) were tested, and I found that these inhibitors disrupted Gβγ and MT organization, affected cellular morphology, and inhibited neurite outgrowth. Altogether, this study demonstrates that the interaction of Gβγ with MTs could be a determining factor for MT rearrangement and the induction of neurite outgrowth, and that interfering with this process may trigger an early stage of neurodegeneration. This knowledge will be helpful for designing and developing novel therapies targeting Gβγ-MT mediated pathway that is altered during neurodegeneration.

Language

en

Provenance

Received from ProQuest

File Size

151 pages

File Format

application/pdf

Rights Holder

Jorge Anibal Sierra Fonseca

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