Page 83 |
Save page Remove page | Previous | 83 of 144 | Next |
|
small (250x250 max)
medium (500x500 max)
Large (1000x1000 max)
Extra Large
large ( > 500x500)
Full Resolution
All (PDF)
|
This page
All
|
73
sequence was repeated in each of the five segments of the maze. Once the mouse entered the goal-box at the end of the final maze segment, the goal-box was exchanged with the start-box such that the mouse was again at the maze entrance. All animals received 15 massed training trials with a 1m intertrial interval (ITI) between 1300 and 1800 hours. All trials lasted a maximum of 360s. Testing was immediately halted for animals which failed to complete the maze in less than 360s on more than two maze trials.
Nissl Staining, Immunohistochemistry and Western blotting
SIRT1 wild type and KO mice were deeply anesthetized with isoflurane and intracardially perfused with cold PBS followed by 4% formaldehyde. Brains were removed, fixed for 2 hr and serially incubated in 15% and 30% sucrose for 15 hr. Then brains were snap frozen and cut into 30 μm thick coronal sections on a cryostat. Brain sections were stained with cresyl violet for Nissl staining or immunostained with corresponding antibodies. Antigen retrieval was performed with 0.1 N HCl. Half brains from the NeSTO and Nestin-Cre mice were fixed for 24-48 h in 4% paraformadehyde, paraffin embed and 5 um thick coronal sectioned. Slices were deparaffinized, rehydrated and antigen retrieval was performed by microwave irradiation. Both frozen and paraffin sections were incubated with Sirt2a 1:1000 (UPSTATE), NeuN 1:500 (Chemicon International) or GFAP 1:500 (Sigma) as primary antibodies overnight at 4°C. Alexa Fluor 488 and 568 (1:500) were used as secondary antibodies. Photographs were taken with an Olympus confocal microscope using the Kalman filter and sequential scanning mode. For immunoblotting hippocampus was finally dissected from the brain and protein
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 83 |
| Contributing entity | University of Southern California |
| Repository email | cisadmin@lib.usc.edu |
| Full text | 73 sequence was repeated in each of the five segments of the maze. Once the mouse entered the goal-box at the end of the final maze segment, the goal-box was exchanged with the start-box such that the mouse was again at the maze entrance. All animals received 15 massed training trials with a 1m intertrial interval (ITI) between 1300 and 1800 hours. All trials lasted a maximum of 360s. Testing was immediately halted for animals which failed to complete the maze in less than 360s on more than two maze trials. Nissl Staining, Immunohistochemistry and Western blotting SIRT1 wild type and KO mice were deeply anesthetized with isoflurane and intracardially perfused with cold PBS followed by 4% formaldehyde. Brains were removed, fixed for 2 hr and serially incubated in 15% and 30% sucrose for 15 hr. Then brains were snap frozen and cut into 30 μm thick coronal sections on a cryostat. Brain sections were stained with cresyl violet for Nissl staining or immunostained with corresponding antibodies. Antigen retrieval was performed with 0.1 N HCl. Half brains from the NeSTO and Nestin-Cre mice were fixed for 24-48 h in 4% paraformadehyde, paraffin embed and 5 um thick coronal sectioned. Slices were deparaffinized, rehydrated and antigen retrieval was performed by microwave irradiation. Both frozen and paraffin sections were incubated with Sirt2a 1:1000 (UPSTATE), NeuN 1:500 (Chemicon International) or GFAP 1:500 (Sigma) as primary antibodies overnight at 4°C. Alexa Fluor 488 and 568 (1:500) were used as secondary antibodies. Photographs were taken with an Olympus confocal microscope using the Kalman filter and sequential scanning mode. For immunoblotting hippocampus was finally dissected from the brain and protein |
