Date on Master's Thesis/Doctoral Dissertation

5-2012

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Anatomical Sciences and Neurobiology

Committee Chair

Hetman, Michal

Author's Keywords

Nucleolus; Nucleolar stress; RNA Polymerase I; DNA damage; ATM; Transcription

Subject

RNA polymerases; Nucleolus

Abstract

Ribosomal biogenesis failure may contribute to neurodegenerative diseases, while its excessive activation has been shown to drive tumor growth. As ribosomal production is initiated and regulated by the Pol1-mediated transcription of rRNA genes in the nucleolus, the latter process had the attention of most researchers interested in the dysregulation of ribosomal biogenesis in disease. When this work began, regulation of Pol1-mediated transcription in neurons had been poorly characterized. The goal of this research has been to better define factors and signaling pathways that regulate neuronal Pol1 activity. The first hypothesis tested was that DNA damage induced by a DNA topoisomerase poison, etoposide, blocks neuronal Pol1. Intracarotid delivery of etoposide to adult rats resulted in Pol1 inhibition in the neocortex; however no apoptosis was found. In neonate rats that received intracerebroventricular injections of etoposide we observed inhibition of Pol1 in neurons in the neocortex and hippocampus. Neuronal apoptosis was observed in these brain structures following etoposide treatment. Therefore, these results confirm that neuronal Pol1 is sensitive to etoposide-induced DNA damage and that such a sensitivity is present in both young and mature neurons of whole rats. These results also demonstrate that Pol1 inhibition is distinct from the developmentally-restricted apoptotic response to etoposide. While working on these studies, we made the surprising finding that doses of etoposide induced Pol1-mediated transcription in cultured cortical neurons and whole rat neonate brains. Thus, low doses of etoposide that were 3 orders of magnitude lower than those inducing Pol1 block apoptosis and were sufficient to activate Poll and the DNA double strand break response, including autophosphorylation of the DNA damage signaling kinase Ataxia-telangiectasia Mutated (ATM). Therefore, it was hypothesized that ATM activated neuronal Pol1. Indeed, pharmacological inhibition of ATM with KU55933 blocked Pol1 activation in response to low concentrations of etoposide or to another topoisomerase-II inhibitor, ICRF-193. Basal levels of nucleolar transcription were also suppressed. Moreover, Pol1-driven transcription was reduced in the cerebellum of 5-week old ATM-null mice. Additionally, pharmacological inhibition of ATM reduced neuronal Pol1 activation in response to the neurotrophin, BDNF, or to increased synaptic activity. Consistent with the notion that BDNF-mediated neurite outgrowth requires Pol1, KU55933 appeared to reduce BDNF-mediated neurite outgrowth. Similar effects on ribosomal biogenesis and neuronal growth were observed with a pharmacological inhibitor of the ATM-related kinase mTOR. Finally, ATM inhibition reduced Pol1 activation in serum-stimulated human cell lines. These findings identified A TM as a novel regulator of Pol1. They further confirmed that in neurons, as in non-neuronal cells, mTOR also regulates Pol1. My results suggest that at least some consequences of ATM deficiency in humans, including neurodegeneration or impaired proliferation, result from insufficient ribosomal biogenesis.

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