Date of Award

1-1-2014

Document Type

Dissertation

Degree Name

Ph.D.

Organizational Unit

College of Natual Science and Mathematics

First Advisor

Daniel A. Linseman, Ph.D.

Second Advisor

Joseph Angleson

Third Advisor

Todd Blankenship

Fourth Advisor

James Fogleman

Fifth Advisor

Kingshuk Ghosh

Sixth Advisor

David Patterson

Keywords

Apoptosis, Motor neurons, Neurons, Rac GTPase, Rho GTPase

Abstract

Several studies have identified Rho family GTPases (i.e. Rho, Rac, Cdc42) as mediators of diverse critical cellular processes, such as actin cytoskeleton remodeling, gene transcription, cell-cell adhesion, and cell cycle progression. However, more recent data highlight an essential role for Rho GTPases as regulators of neuronal morphology and neuronal survival. In particular, Rac GTPase generally induces neurite outgrowth and promotes neuronal survival while Rho GTPase typically provokes neurite retraction and induces neuronal apoptosis. However, the precise signaling pathways that regulate neuronal survival downstream of Rho GTPases and the potential involvement of dysregulated activity of Rho GTPases as a causative factor in the progression of neurodegenerative diseases remains to be elucidated.

Consistent with a pro-survival function for Rac in neurons, inhibition of Rac with Clostridium difficle toxin B (ToxB) or expression of a dominant negative Rac1 mutant significantly induces activation of the "death executioner" caspase-3 and subsequent apoptosis in primary cerebellar granule neuron (CGN) cultures. We have previously shown that Rac inhibition evokes apoptosis via inactivation of a pro-survival mitogen activated protein kinase kinase (MEK)1/2/ extracellular signal regulated-kinase (ERK)1/2 cascade which functions in healthy CGNs to promote the degradation of the pro-apoptotic Bcl-2 homology-3 domain (BH3)-only protein Bim while also repressing a pro-apoptotic Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) signaling cascade. Here, we reveal that ToxB induces the JAK-dependent phosphorylation (activation) of STAT5, which is sufficient to induce apoptosis through transcriptional repression of pro-survival Bcl-xL. Collectively, these data implicate Rac GTPase as a critical mediator of CGN survival.

Given that ToxB inhibits Rho, Rac, and Cdc42, we aimed to elucidate the effects of more targeted Rac inhibition in CGNs. NSC23766 specifically inhibits Rac GTPase activation by the Rac-specific GEFs Tiam1 and Trio. Targeted inhibition of Rac GTPase via NSC23766 treatment in CGNs evoked apoptosis via repression of unique MEK5/ERK5, p90Rsk, and Akt-dependent pro-survival pathways. Furthermore, selective inactivation of Rac induced the activation and translocation of the pro-apoptotic BH3-only protein Bad to the mitochondria, where it has been demonstrated to induce apoptosis. Intriguingly, adenoviral expression of a constitutively active MEK5 protected CGNs from apoptosis induced by NSC23766, but not ToxB. These data demonstrate that selective inhibition of Rac GTPase with NSC23766 versus global inhibition of Rho GTPases with ToxB induces cell death via repression of distinct MAP kinase signaling pathways.

We also report that treatment with either ToxB or Clostridium sordelli lethal toxin, which also inhibits Rac GTPase, induces caspase-dependent downregulation of the pro-survival transcriptional corepressor, C-terminal binding proteins (CtBPs), in CGNs. Interestingly, we demonstrate that downregulation of CtBPs undergoing apoptosis does not appear to occur via enhanced degradation or reduced transcription. Instead, our data suggest that caspases indirectly regulate the expression of CtBPs in CGNs undergoing apoptosis in a manner that occurs post-transcriptionally and is dependent on intact miRNA machinery. Finally, inhibition of CtBPs with 4-methylthio-2-oxobutyric acid (MTOB) provoked CGN apoptosis that was associated with induction of the CtBP target Noxa. These data indicate a novel function for CtBPs in regulating survival of primary CGNs.

Finally, we examined the involvement of Rho GTPases in regulating the survival of motor neurons derived from embryonic stem cells (ESCs) as selective degeneration of motor neurons in the spinal cord and motor cortex underlies the pathology of amyotrophic lateral sclerosis (ALS). Consistent with our previous findings in CGNs, inhibition of Rac GTPase via NSC23766 treatment resulted in apoptotic cell death of ESC-derived motor neurons that was coincident with deactivation of pro-survival ERK5 and Akt signaling pathways. Surprisingly, we report that the BH3-only protein Bad localized exclusively to the nucleus in ESC-derived motor neurons treated with the Rac inhibitor. In agreement with an antagonistic relationship exerted between Rac GTPase and Rho GTPase as a determinant of neuronal survival, we also report that constitutive activation of Rho GTPase via CN03 treatment induced Rho kinase (ROCK)-dependent apoptosis of ESC-derived motor neurons. Next, we investigated Rac and Rho GTPase activitity and localization in motor neurons in vivo . In the G93A mutant Cu, Zn superoxide dismutase (mSOD1) transgenic mouse model of ALS, we demonstrate that active Rac1-GTP is reduced within spinal cord motor neurons when compared to age-matched wild type (WT) littermates. Furthermore, we report that total RhoB is expressed in the soma and nucleus of WT motor neurons; however, RhoB shows a marked redistribution to neuronal processes in end-stage mice harboring the G93A SOD1 mutation. These data underscore the importance of Rho family GTPases in maintaining motor neuronal survival and suggest that diminished Rac GTPase activity or enhanced Rho GTPase activity may each contribute to selective loss of motor neurons during the progression of ALS.

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Rights Holder

Trisha Stankiewicz

Provenance

Received from ProQuest

File Format

application/pdf

Language

en

File Size

250 p.

Discipline

Neurosciences, Biology, Biochemistry

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