Program Project on Reactive Oxygen Species and Aging
Mission
Phases I and II
Research into the molecular, genetic, and cellular aspects of aging has greatly expanded our understanding of the basic processes that contribute to both longevity and age-related diseases. The primary focus of the Program was the assertion that age-dependent increases in oxidative stress and alterations in cellular handling of calcium converge to produce several of the well-known manifestations of aging, such as progressive muscle weakness, compromised cognitive performance, and declining immune function. Oxidative stress has been invoked as an important mechanism for cell degeneration in many human diseases, including conditions such as Alzheimer’s and Parkinson’s disease. Formation of high levels of oxidants in cells in the body is also linked to loss of control of calcium levels inside cells. We have shown such loss of control of calcium levels with advancing age, in particular in brain nerve cells. In addition to the loss of control of calcium, we have found clear evidence of increased oxidative stress with aging. During the period of funding of this Program Project, we have developed unique capabilities in the analysis of protein markers of oxidative stress and characterized the effects of oxidation and aging on critical calcium-regulating proteins.
Phase III
During the previous decade of funding of the Program, enormous shifts have occurred in the overall research enterprise, primarily due to the sequencing of the human genome and development of remarkable technologies for high throughput analyses of genomic, proteomic, and signaling events occurring in cells and tissues. As a result, the strategies used in the last stage of this project was to study the aging process changed to take advantage of the latest research advances in these fields. In the last phase of this Program, we continued the investigations into aging, oxidative stress and calcium regulation by focusing our activities on three projects and two scientific cores. We concentrated on the molecular causes of loss of muscle mass and strength and of nerve cell degeneration and loss of memory as a result of advancing age.
Scientific Cores
Protein Complex Identification, Microcharacterization & Lipid Profiling
Personnel
Core Leader: Todd Williams
Core Advisor: Christian Schöneich
Co-Investigator: John Stobaugh
Informatics Specialist: Jianwen Fang Mass.
Spec. Specialist: Nadezhda Galeva
Goals
The goals for this core were to:
- Use proteomics based detection strategies to identify proteins isolated in the form of protein complexes isolation experiments
- Use MALDI TOF and TOF/TOF to interrogate subcelluar preparations or pull down samples to rapidly identify proteins and guide sample preparations.
- Use LC/MS/MS on the hybrid tandem mass spectrometer composed of a quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance (FT) analyzers (or LTQ-FT) to improve protein identification coverage. Capillary HPLC (5µl/min on 320µm inner diameter (ID) columns) will be used for large protein loads and nanoUPLC (100nl/min on 75µm ID columns) for low abundance samples.
- Improve information content gleaned from LC/MS/MS experiments on the LTQ-FT or quadrupole-time of flight hybrid tandem (QTOF) instruments using MALDI search engines on LC/MS1 "survey" data.
- Develop an alternative protein ID validation strategy.
- Quantify stoichiometry and detect post translational modifications in protein complexes
- Use LC/MS/MS on LTQ-FT or QTOF to extend protein sequence coverage and find modified peptides.
- Use LCMS on LTQ-FT or QTOF to measure relative ions signal from peptides derived from Stable Isotope Labeling with Amino acids in Cell culture (SILAC), 16O/18O labeling, or spiked internal standard experiments on protein complex samples.
- Improve data handling strategies to harvest intensity information from LC/MS data.
- Profile lipids in raft or membrane subdomain preparations
- Profile sphingo- and phospholipids using head group specific MS/MS scans.
- Improve the quantitation and data handling in sphingolipid profiling.
- Determine levels of 7-keto-cholesterol in raft lipid preparations.
Animal Models, Electron Microscopy, Cell Culture and Molecular Biology
Personnel
Core Leader: Mary L. Michaelis
Co-Leader: Dianne Durham
Mol. Biol./Transgenic Mice Specialist: Dongwei Hui
Informatics Specialist: Jianwen Fang
Goals
The goals for this core were to:
- Maintain a web-accessible relational database that integrates information about the animals used in the projects and continuously updates all data derived from experiments with each animal and from the cell culture models.
- Coordinate activities involving animals, including the ordering, harvesting tissues cross breeding and genotyping Tg mice, maintaining special diets, monitoring longevity, and maintaining fully documented records for the database.
- Prepare primary neuronal and muscle cell cultures and to maintain the C2C12 myocyte and SH-SY5Y human neuroblastoma cell lines.
- Assist investigators in the design and use of new molecular biology reagents for overexpression, suppression or mutation of targeted genes, including the performance of transfections.
- Prepare animals for light and electron microscopy studies (perfusion fixation) and conduct the ultrastructure analyses of brain and muscle.
Projects
Sarcopenia and Apoptosis: Role of SERCA and Bcl-2
Project Leader
Christian Schöneich
Goals
The goals for this project were to:
- Characterize (a) the complex between Bcl-2 and SERCA in specific muscle types in vitro and in vivo, (b) the effect of aging and Bcl-2 phosphorylation on SERCA/Bcl-2 complex formation and SERCA translocation from CRDs in isolated SR, and (c) the physiologic importance of Bcl-2/SERCA interaction
- Characterize the effects of the pro-apototic proteins Bak and Bad on the SERCA/Bcl-2 interaction in vitro.
- Determine in vivo (muscle and cell culture) the role of Hsp70 on SERCA/Bcl-2 co-localization and functional interaction, the stoichiometry of SERCA/Bcl-2 complexes, and the age-dependent interaction of Bcl-2 with the IP3R.
- Determine in vitro (isolated SR) the role of Hsp70 and other SERCA-binding proteins on SERCA/Bcl-2 functional interaction, the stoichiometry of SERCA/Bcl-2 complexes, and the interaction of Bcl-2 with the IP3R.
Age-Dependent Changes in Synaptic Raft Domains and Plasma Membrane Ca2+ - ATPase
Project Leader
Mary Lou Michaelis
Goals
The goals for this project were to:
- Characterize synaptic membrane rafts from 5, 22 and 34-mos old rats in terms of protein oxidation, lipid peroxidation, sphingolipid composition, and effects of in vitro oxidative stress on the distribution of PMCA in raft vs non-raft domains.
- Use pharmacological and genetic manipulations to elucidate the role of the raft lipids in the membrane localization and the kinetic properties of PMCA.
- Determine the role of protein interactions in PMCA localization in rafts by identifying proteins that form complexes with PMCA in the raft domains, determining their levels in membranes from 5, 22, and 34- mos old rats, and altering expression of the major partners in cell models to confirm effects on PMCA.
Glutamate Neurotransmission, Aging, Longevity and Neurite Remodeling
Project Leader
Elias K. Michaelis
Genomics
Xinkun Wang
Goals
The goals for this project were to:
- Assess longevity and protein and DNA oxidation levels in brain and other tissues of wt and Tg mice across three ages.
- Determine the effects of neuronal GLUD1 overexpression and of aging on structural, metabolic and gene expression changes in vulnerable and resistant brain neurons.
- Determine the age-dependent changes in expression, composition and activity of a Ca2+-sensitive, dendrite-growth controlling complex in GLUD1 and wt mice.
Participants
| Name | Assignment |
|---|---|
| Elias K. Michaelis | Program Director Co-Leader, Administrative Core Project Leader, Project 3, Phases III |
| Diana Bigelow | Project Leader, Project 3, Phases I & II |
| Xue-Wen Chen | Scientific Advisor |
| Rick T. Dobrowsky | Scientific Advisor |
| Elena Dremina | Project 1 |
| Dianne Durham | Co-Leader, Core 2 |
| Jainwen Fang | Informatics Leader, Core 1 |
| Nadezhda A. Galeva | Mass Spec. Specialist, Core 1 |
| Dongwei Hui | Molecular Biology/Transgenic Mice Specialist, Core 2 |
| Lei Jiang | Project 2 |
| Mary L. Michaelis | Core Leader, Core 2 Project Leader, Project 2, Phase III |
| Ranu Pal | Core 2 and Project 3 |
| Christian Schöneich | Project Leader, Project 1, Phases I, II, and III Core Advisor, Core 1 |
| Tom Squire | Project Leader, Project 2, Phases I & II |
| John F. Stobaugh | Co-Investigator, Core 1 |
| Xinkun Wang | Genomics Leader, Project 3 |
| Todd D. Williams | Core Leader, Core 1 |