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localized in the cytosol (Fig. 13, middle panel), implicating that SIRT1 can shuttle into cytosol in response to oxidative stress. We also treated neurons with NMDA, an agonist for NMDA-subtype glutamate receptor. At excitotoxic concentrations NMDA (100 μM, 1 hr; neurons were fixed right after treatment) showed similar effects to that of H2O2 (SIRT1 was localized in the cytosol in 47 among 426 SIRT1-Flag positive neurons counted), while it showed no effect at physiological concentrations (5 μM, 5 min treatment; neurons were fixed one hour after treatment) (Fig. 13, lower panel). These data suggest that SIRT1 is mainly localized in the cytosol in the brain and it can shuttle between the cytosol and the nucleus upon oxidative stress or other environmental insults.
Figure 13. Nucleus-cytosol shuttling of SIRT1 in cultured neurons
Cultured rat cortical neurons (10 DIV) were transfected with SIRT1-Flag and treated with H2O2 (200 μM, 1 hr, middle panel), NMDA (100 μM, 1 hr, lower panel), or vehicle (upper panel) 48 hrs after transfection. The neurons were then fixed and immunostained with MAP2 and FLAG and counterstained with DAPI. In cultured neurons exogenously expressed SIRT1 was exclusively localized in the nucleus, whereas it shuttled into the cytosol upon insults including H2O2 or NMDA in ~10% of neurons. 42
Object Description
| Title | Roles of SIRT1 in neuronal oxidative damage and brain function |
| Author | Li, Ying |
| Author email | lying@usc.edu; yingraceli@yahoo.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Neuroscience |
| School | College of Letters, Arts and Sciences |
| Date defended/completed | 2008-09-12 |
| Date submitted | 2008 |
| Restricted until | Unrestricted |
| Date published | 2008-10-30 |
| Advisor (committee chair) | Longo, Valter D. |
| Advisor (committee member) |
Baudry, Michel Pike, Christian J. Madigan, Stephen A. |
| Abstract | Aging is a common phenomenon of multiple organisms. In humans aging is frequently accompanied by cognitive decline and occurrence of neurodegenerative diseases which reduce the quality of life and impose financial stress on society. Delaying the aging process, extending life span and decreasing the occurrence of age-related brain function deficit have always been aspirations of human kind. Extensive research has advanced our understanding of the mechanisms underlying aging, among which is the ability of calorie restriction to increase longevity, and the pivotal regulatory roles of insulin/IGF-1 signaling pathway. Some recent studies identified silent information regulator 2 (Sir2; SIRT1 is the mammalian homolog) as a key mediator of the beneficial effects of calorie restriction and this prompted development of SIRT1 activators for human consumption to delay aging and accompanying cognitive decline. However, our laboratory previously showed in yeast that Sir2 can increase stress sensitivity and limit life span extension under certain conditions, calling for more detailed characterization of SIRT1. In the research described in this dissertation I extended this study to the mammalian system and focused on the role of SIRT1 on the health of neurons and brain functions, especially learning and memory.; This dissertation consists of three chapters. In chapter 1 I briefly review some recent progress on aging, oxidative stress, insulin/IGF-1 signaling pathway and learning and memory with emphasis on the involvement of SIRT1 in these processes. In chapter 2 I focused on the role of SIRT1 in oxidative stress in neurons and its mechanisms. I found that SIRT1 inhibition increased resistance to oxidative damage and this effect is partially mediated by a reduction in IGF-I/IRS-2/Ras/ERK1/2 signaling. In chapter 3 I studied the functions of SIRT1 in learning and memory. The experiments showed that deletion of SIRT1 impairs a certain form of synaptic plasticity and reduce performance in several different learning and memory tasks while overexpressing SIRT1 did not substantially affect learning and memory.; Together, my studies reveal that SIRT1 exacerbates neuronal oxidative damage but is essential in learning and memory, indicating that SIRT1 plays multiple roles in aging and brain functions and that caution should be exercised in designing anti-aging or therapeutic approaches that involve targeting SIRT1. |
| Keyword | SIRT1; neurons; brain; oxidative damage; learning and memory |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m1723 |
| Contributing entity | University of Southern California |
| Rights | Li, Ying |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | cisadmin@lib.usc.edu |
| Filename | etd-LI-2405 |
| Archival file | uscthesesreloadpub_Volume44/etd-LI-2405.pdf |
Description
| Title | Page 52 |
| Contributing entity | University of Southern California |
| Repository email | cisadmin@lib.usc.edu |
| Full text | localized in the cytosol (Fig. 13, middle panel), implicating that SIRT1 can shuttle into cytosol in response to oxidative stress. We also treated neurons with NMDA, an agonist for NMDA-subtype glutamate receptor. At excitotoxic concentrations NMDA (100 μM, 1 hr; neurons were fixed right after treatment) showed similar effects to that of H2O2 (SIRT1 was localized in the cytosol in 47 among 426 SIRT1-Flag positive neurons counted), while it showed no effect at physiological concentrations (5 μM, 5 min treatment; neurons were fixed one hour after treatment) (Fig. 13, lower panel). These data suggest that SIRT1 is mainly localized in the cytosol in the brain and it can shuttle between the cytosol and the nucleus upon oxidative stress or other environmental insults. Figure 13. Nucleus-cytosol shuttling of SIRT1 in cultured neurons Cultured rat cortical neurons (10 DIV) were transfected with SIRT1-Flag and treated with H2O2 (200 μM, 1 hr, middle panel), NMDA (100 μM, 1 hr, lower panel), or vehicle (upper panel) 48 hrs after transfection. The neurons were then fixed and immunostained with MAP2 and FLAG and counterstained with DAPI. In cultured neurons exogenously expressed SIRT1 was exclusively localized in the nucleus, whereas it shuttled into the cytosol upon insults including H2O2 or NMDA in ~10% of neurons. 42 |
