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Component 1: Interdisciplinary Research Consortium in Geroscience
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| Age-related disease is arguably the single greatest challenge for biomedicine in the 21st Century. By 2030 the national healthcare bill is predicted to be four trillion dollars, fully half of which will be required for persons aged 65 and older. Aging is the most important risk factor for human disease in developed countries, and consequently creates an enormous social and economic impact. Our ability to tackle age-related disease is undermined by our lack of understanding of the principles and mechanisms of aging. Aging is a multifaceted and profound biomedical problem that is unlikely to yield to traditional investigative techniques. We propose that only by studying the interface of normal aging and aging diseases, in an interdisciplinary fashion, can we build the knowledge that will facilitate intervention. We propose to initiate a new field (“interdiscipline”) called Geroscience that, by analogy to the creation of the field of Neuroscience, will have wide-reaching ramifications for biomedical science, healthcare, the training of future interdisciplinary researchers in Geroscience, and the national response to the many problems associated with the “graying of America.” The Buck Institute is an NIH-designated Center of Excellence, the only independent research institute in the U.S. that is focused solely on aging research, and since its very planning and inception has been devoted to interdisciplinary research; therefore, its approaches and operation to date fit extremely well with the NCRR guidelines for the current application. Based on our track record of highly collaborative, cross-discipline activity, our high density of researchers in this new field, and our designation as a Nathan Shock Center of Excellence, we believe that we are uniquely placed to form an Interdisciplinary Research Consortium in Geroscience. | |
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Component 2: mRNA Translation, TOR & Geroscience
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Principal Investigator:
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Pankaj Kapahi
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| Dietary restriction (DR) is the most robust environmental method of lifespan extension in species as diverse as yeast, worms, fruit flies and rodents. Dietary restriction (DR) has been hypothesized to influence longevity through a shift in metabolic investment away from reproduction and growth toward somatic maintenance, allowing longer survival. The TOR (target of rapamycin) pathway, conserved from yeast to humans, links nutrients in the environment to organismal growth. We have identified the TOR pathway to play a critical role in modulating lifespan upon dietary restriction in D. melanogaster (fruit fly). We have discovered a critical and novel role for mRNA translation, downstream of the TOR pathway in determining lifespan in both D. melanogaster and C. elegans. This component uses an interdisciplinary approach combining different methodologies to examine the impact of mRNA translation on lifespan and metabolism utilizing three different model systems, flies, worms and mammalian cells. The interdisciplinary aims in this component are beyond the scope of a single laboratory, therefore investigators with diverse expertise have come together to critically examine the role of mRNA translation in aging and cancer. Completion of these aims will allow integration of data from different model systems which will provide a unique perspective of mRNA translation in gerosciences. This proposal will undertake the following specific aims:
Aim 1 Examine the metabolic consequences of inhibition of the TOR pathway and DR in D. melanogaster.
Aim 2. Genome wide analysis to identify and characterize the differentially translated genes in long lived C. elegans.
Aim 3. Examine the conservation of metabolic effects of inhibiting TOR signaling in mammalian cells.
The role of the TOR pathway and translation regulation of gene expression is becoming recognized in various age related diseases including diabetes, cancer and neurodegeneration. Since a high degree of genetic similarity exists between humans and model organisms like flies and worms, we believe that taking an interdisciplinary approach by combining different approaches in various model systems will yield revealing insights into aging and age related diseases in humans.
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Component 3: Checkpoint functions, lifespan determination and neurodegeneration
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| We have uncovered a novel genetic pathway that determines adult lifespan in the nematode Caenorhabditis elegans. This pathway encodes components of DNA damage/cell cycle checkpoints, which are known to prevent inappropriate cell division. However, because non-dividing cells comprise the C. elegans adult soma, it appears that these checkpoint proteins also control the survival of cells in a post-mitotic state. Down-regulation of checkpoint functions in adult C. elegans renders them very stress resistant and extends their lifespan. Through a whole genome RNA interference screen, we determined that many novel genes encode checkpoint functions and influence lifespan. We now propose to determine the mechanism(s) by which these genes act during aging and survival, and whether their functions are conserved in mammals. We will determine the role of lifespan-modulating checkpoint proteins in the survival of nematode neurons, which are crucial regulators of nematode lifespan, mouse cortical neurons, and dopaminergic neurons derived from human embryonic stem cells. We will modulate checkpoint functions in these post-mitotic cells and determine their resistance genotoxic and cytotoxic insults. We will identify chemical compounds that modulate survival through checkpoints, with a view to developing novel interventions into aging and neurological disease. This interdisciplinary project requires expertise in invertebrate aging, mammalian neuroscience, high throughput screening, chemical biology, molecular genetics and stem cell biology. | |
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Component 5: Stochastic Aspects of Aging
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Principal Investigator:
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Jan Vijg
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| All biological systems are subject to stochastic variation. In mammals this is obvious from the large individual variation in life span and patterns of aging-related pathology, even in genetically homogeneous animals. Stochasticity is also apparent at the molecular level. Random molecular fluctuations creating variability in gene expression within a cell population have been demonstrated in bacteria and yeast. To some extent this is inherent to the nature of the processes of information transfer, especially at small numbers of mRNA or protein products per cell. However, noise at the molecular level can also have external causes, varying from random damage to the genome to variability in regulatory signals. While sometimes advantageous, i.e., in development and evolution, increased stochasticity in aging is generally viewed as having detrimental effects on cellular function. The central hypothesis in this proposal is that oxidative stress, a likely cause of aging, increases stochastic variability of gene expression, that it does so by causing both genetic and epigenetic
changes in cells, and that cells and organisms possess a variety of genetic pathways and cellular responses to mitigate or buffer against unduly large stochastic changes. We will test this hypothesis in two specific aims. First, we will comparatively analyze four different model systems of aging, nematodes, fruit flies, mice and human cells, for mutation accumulation at a similar lacZ reporter construct. We will also investigate how such genome level stochasticity depends on genetic factors known to cause aging-related neurodegenerative disease, how it differs between human and mouse cells and how it can be modulated by genetic factors. Second, we will directly measure transcriptional noise levels in mouse neurons and neuronal stem cells during aging and in model systems for human neurodegenerative diseases. In parallel, we will study similar transcriptional noise in human and mouse fibroblasts in different genetic backgrounds and among individual nematodes during aging. We expect that the proposed study will provide a new dimension to existing paradigms in the field by defining the role of stochasticity in aging phenotypes and identifying the genetic and biochemical mechanisms that influence it. | |
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Component 6: Protein Interactions and Protein Conformation in Aging and Disease
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Principal Investigator:
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Robert Hughes
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| We propose to define and characterize protein interactions and protein conformational states relevant to aging and disease processes. A starting point for the interaction studies will be a large protein interaction network involving human orthologs of proteins known to increase longevity when mutated in models systems of aging. This scale-free network, generated using high throughput yeast two-hybrid methods, includes 2,172 human proteins interacting with 165 known longevity proteins in 3,219 highly interconnected unique binary pairs. Because of their interaction with known longevity proteins, these 2,172 are considered to be novel candidate longevity proteins. Analysis of genes encoding known and candidate longevity proteins show that they are highly enriched for genes whose expression changes during human aging (according to microarray data generated from young and old human muscle tissue). The “longevity interactome” will be mined using bioinformatic methods and novel longevity genes derived from the network will be validated using C. elegans life span assays.
A complementary approach to discovering novel proteins involved in aging will be to use mass spectrometry-based proteomic methods to discover proteins that become insoluble over time in aging and disease. We will determine the content of the SDS insoluble fraction of the proteome in aging model organisms, aging mouse tissues and brains from mouse models of neurological disease. Proteins found to partition into insoluble states during aging and/or disease will be investigated further for possible functional roles in these processes using invertebrate and mouse models.
Specific Aim 1. To discover and characterize novel genes relevant to longevity using an aging protein interaction network. We will mine an existing protein interaction network to identify novel protein involved in aging and longevity. Candidate proteins will be prioritized using informatic methods, compared to age-specific microarray datasets and validated experimentally in model organisms of aging. Proteins validated as having roles in aging will be studied further using MS-based proteomic methods.
Specific Aim 2. To develop proteome-scale maps of age-dependent changes in protein solubility. We will use MS-based methods to determine which proteins become insoluble in an age-dependent manner. This will be done in aging yeast, nematodes and tissues from aging and diseased mice (e.g. brain and muscle). Kinetics of changes in protein solubility will be tested in long-lived mutant yeast and nematodes and also in genetic models of late-onset neurodegeneration (e.g. AD, HD and PD mouse models). Proteins shown to be susceptible to age-dependent and disease-dependent insolubility will be functionally characterized in appropriate invertebrate, cell-based and mouse models of aging and disease. | |
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Component 7: HDACs in Neurodegeneration and Aging
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Principal Investigator:
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Lisa Ellerby
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| Age-related disease is arguably the single greatest challenge for biomedicine in the 21st Century.
Age is the largest single risk factor for panoply of diseases, including cardiovascular dysfunction, cancer, type II diabetes, osteoporosis, and neurodegeneration. Postponing (or decreasing the rate of) aging would retard the course of multiple age-related diseases and therefore substantially increase average health-span. Our ability to develop rational approaches to preventing or intervening in aging depends crucially on a thorough understanding of the basic mechanisms that cause aging, as well as the etiology of specific age related diseases. We believe that it is especially in this area of biomedicine where an interdisciplinary approach will accelerate discoveries and provide rational avenues for therapeutic intervention. In the long term this will lead to the establishment of a comprehensive new discipline, that of “Geroscience.” Despite having identified 100s of genes that determine lifespan in simple organisms, we have no true understanding of how these genes are impacting on aging and disease. This is in part that most studies are carried in isolation by either looking at the basic biology of aging or separately at the impact of these identified genes on disease. In this proposal, which is directly relevant to Specfic Aim 1 of the U54 Specific Aim 1 To establish an interdisciplinary research program on the aging-disease relationship with a focus on neurodegeneration and cancer, we will address across three neurodegenerative disease models, Huntington’s disease-Dr. Ellerby (Component 7), Dr. Hughes (Component 6), Dr. Nicholls and Gibson (Component 11), Parkinson’s disease-Dr. Andersen (Component 9) and Alzheimer’s disease-Dr. Bredesen [now Dr. Lithgow] (Component 1), and the model organism C. elegans-Dr. Kapahi (Component 2)- the role of histone deacetylase (HDACs) in neurodegeneration and aging. This represents a significant area as
researchers have made a series of important discoveries in recent years on neurodegenerative disease and in aging that these enzymes (HDACs) play a major in role in neurodegeneration or aging. However,
researchers have not yet dissected out in mammalian systems which family members are critical to target to prevent neurodegeneration and aging. A central theme of these projects is the notion that genes that are known to modulate aging are key factors in determining disease. Drugs that target the maintenance functions of these regulators or enhance their function will be important targets for proposed interventions. Our hypothesis is that compromised acetylation homoeostasis is directed coupled to neurodegeneration and identification of particular HDACs family members involved in this process will identify therapeutic targets critical to neurodegeneration and aging. | |
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Component 9: Subcellular localization of alpha-synuclein and its impact on neurodegeneration
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| Alpha-synuclein is a major protein component of Lewy bodies, a cardinal feature of the degenerating Parkinsonian brain. Alpha-synuclein has been demonstrated to intercalate into lipid membranes via formation of an alpha helical structure in its N-terminal end. We recently demonstrated that either incubation of recombinant A53T mutant alpha-synuclein protein with mitochondria isolated from immortalized midbrain-derived dopaminergic neurons or following A53T expression within dopaminergic cells either in culture or in transgenic animals, the protein localizes to the inner mitochondrial membrane (IMM) in the form of oligomers. Mitochondrial localization may be due to A53T’s inability to undergo serine 129 (ser-129) phosphorylation as this event, which can be induced by oxidative stress, drives the wildtype protein towards cytoplasmic localization and oligomerization. Localization of A53T to the IMM is accompanied by decreased mitochondrial membrane potential (MMP) and increased mitochondrial autophagy (mitophagy). Decreases in MMP has been shown by others to influence the mitochondrial fission-fusion rate, driving mitochondria towards mitophagy. In aim #1 of this grant application, we propose to: (1) Assess the ability of alpha-synuclein ser-129 phosphorylation to determine its subcellular localization and oligomerization (self-interaction) state with the assistance of Drs. Gibson and Hughes of Components 11 and 6, respectively, (2) Examine the impact of mitochondrial localization on mitochondrial fission-fusion ratios and mitophagy with the assistance of Dr. Nicholls of Component 11, and (3) Examine the effects of alterations in TOR activity on associated mitophagy with the assistance of Dr. Kapahi (Component 2 and co-PI, Component 9).
Nuclear translocation of alpha-synuclein selectively into dopaminergic midbrain neurons has been demonstrated following either its overexpression or increased oxidative stress. Nuclear translocation may also be dependent upon the protein’s ser-129 phosphorylation state and/or its cleavage as well as its ability to associate with dopaminergic cytoplasmic factors. Nuclear localization appears to result in neurotoxicity via alpha-synuclein’s ability to bind histones within the nucleus reducing their acetylation. Reduced histone acetylation could impact on gene transcription. In aim 2 of the application, we propose to assess alpha-synuclein for post-translational modifications and interactions with dopaminergic cytosolic factors associated with its nuclear translocation with the assistance of Drs. Gibson and Hughes of Components 11 and 6, respectively. With Dr. Ellerby as part of Component 7, we will also examine the ability of HDAC inhibitors or specific HDAC siRNAs to protect against these effects and with Drs. Vijg and Melov as part of Component 5 the impact of nuclear localization of alpha synuclein on gene transcription. | |
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Component 8: Neuronal Stem Cells and Aging
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| Aging is associated with increased susceptibility to a variety of diseases and diminished capacity for
tissue repair. Although many factors are likely to be involved, one proposed explanation for the less
complete recovery from injury or disease that is often observed in aged individuals is impairment in the
number or function of adult (tissue) stem cells. These cells persist throughout life in many tissues, where they may proliferate and differentiate in response to physiological cues and pathogenic insults. We hypothesize that although basal levels of neurogenesis decline with aging, the neurogenesis response to injury can be restored toward youthful levels for therapeutic purposes. Further, we anticipate that this is the case for both endogenous neurogenesis and neurogenesis from transplanted neuronal precursor cells (NPCs). Finally, we propose that the mechanisms responsible for the age-related decline in adult neurogenesis can be localized to one of two compartments: the NPCs themselves or the vascular niche in which they arise. We will test these hypotheses with the following specific aims: (1) Determine how aging alters injury-induced neurogenesis in the adult mouse brain; 2) Examine whether age-related defects in injury-induced endogenous neurogenesis are imposed by neuronal precursor cells (NPCs) themselves or by their tissue environment; (3) Evaluate the extent to which the age of a recipient mouse determines the transplantation efficacy of human embryonic stem cell (hESC)-derived NPCs after injury; and (4) Identify candidate mediators of the age-induced decline in injury-induced endogenous neurogenesis by screening for changes in the proteome of endogenous NPCs and DG or SVZ endothelial cells. | |
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Component 10: Postdoctoral Research Training and Education in Geroscience
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| The proposed program in research training and education is designed to prepare NRSA and non-NRSA postdoctoral trainees for careers in the emerging field of “geroscience” — the integrated, interdisciplinarystudy of aging and age-related diseases. The Buck Institute for Age Research is uniquely positioned toprovide broad-based training and education in this field, and thereby ready its participants to make importantcontributions to our understanding of the biology of aging and its attendant health consequences. Some keyassets of the Buck Institute in this regard are its strong institutional focus on aging research, highlyinterdisciplinary approach to research problems, advanced technologic infrastructure, outstanding facultywith diverse scientific and clinical expertise and prior teaching experience, and the unsurpassed regionalresearch environment and facilities of the San Francisco Bay Area. The Buck Institute has devoted majoreffort and resources to the training of junior scientists, especially postdoctoral fellows, in aging research. Inaddition to working in the laboratories of Buck Institute faculty members, these fellows have access to didactic courses designed to better equip them for independent careers in aging research in academic or biotechnology settings, as well as practical instruction in career-development skills including research ethics, scientific presentations, grant writing and teaching. This postdoctoral training program’s Specific Aims are to: AIM 1. Provide postdoctoral trainees with interdisciplinary laboratory experience in geroscience — the integrated study of aging and age-related diseases. AIM 2. Provide postdoctoral trainees with didactic teaching in disciplines inherent to geroscience and practical instruction that will assist in their scientific career development. | |
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Component 11: Mass Spectrometry and Imaging Technologies in Geroscience
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Principal Investigator:
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Brad Gibson
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| The ‘Mass Spectrometry and Imaging Technologies in Geroscience’ P30 Core will consist of an integrated suite of mass spectrometry and microscopic imaging technologies coupled to specific protein and small molecule drug and metabolite analysis assays and bioenergetic analysis. The aims of this P30 Core are to integrate the unique instrumentation facilities at the Buck Institute in imaging and mass spectrometry to target the needs of the 10 components of the U54 in structural and functional proteomics, and for the analysis of drugs and targeted metabolites that constitute this Geroscience initiative. Specifically, the imaging and mass spectrometry technologies will encompass; (i) functional and fixed-cell imaging using protein-specific fluorophores and antibodies, (ii) functional analysis of intact neurons and other cells including bioenergetic and redox measurements, (iii) development and implementation of protein chemistries, (iv) mass spectrometry-based analysis of proteins, including their identification, quantitation, interactions and posttranslational modifications, (v) mass spectrometry imaging and profiling studies, and (vi) drug and metabolite analyses. These studies will be carried out on a range of aging and aging-related disease models that constitute the components of this U54. The P30 core will consist of two main but integrated parts: ‘Functional and Structural Imaging Technologies’ directed by Dr. Nicholls and ‘Mass Spectrometry Technologies' directed by the principal investigator, Dr. Bradford Gibson. We propose to apply and integrate these instrument-based technologies at the very earliest stages of experimental design and work closely with the other investigators to develop, optimize, and implement these mass spectrometry and imaging technologies. | |
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Fly-Through of A Four Day Old Worm Nuclei
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2009
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| Bell R, Hubbard A, Chettier R, Chen D, Miller JP, Kapahi P, Tarnopolosky M, Saharsabuhde S, Melov S, Hughes RE. A human protein interaction network shows conservation of aging processes between human invertebrate species” (2009 PLos Genet ): 5(3). PMID 19293945
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| Chen D, Thomas EL, Kapahi P. HIF-1 Modulates dietary Restriction-mediated Lifespan Extension via IRE-1 in C. elegans. Plos Genet 2009 5(5):e1000486. PMCID: PMC 2676694.
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| Edman U, Garcia, AM, Busuttil RA, Sorensen D, Lundell M, Kapahi P, Vijg J. “Lifespan extension by dietary restriction is not linked to protection against somatic DNA damage in Drosophila melanogaster.” Aging Cell (2009) 2009 8(3): 331-338. PMCID: NIHMSID 154550
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| Zid BM, Rogers A, Katewa SD, Au Lu T, Benzer S, Kapahi P. 4E-BP modulates lifespan and mitochondrial translation upon dietary restriction in Drosophila. Cell 2009 139(1):149-160. PMCID: PMC2759400
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| Mallajoysyula JK, Chinta SJ, Rajagoppalan S, Nicholls DG, and Andersen JK (2009). Metabolic control analysis in a cellular model of elevated MAO-B: relevance to Parkinson’s disease. Neurotox Res. 16: 186-193. PMCID: 2727365
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| Chinta SJ, Rane A, Yadava N., Andersen JK, Nicholls DG, and Polster BM (2009). Reactive oxygen species regulation by AIF- and complex I-depleted brain mitochondria.
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| Chinta SJ, Poksay KS, Kaundinya G, Hart M, Bredesen DE, Andersen JK, and Rao RV (2009). Endoplasmic reticulum stress-induced cell death in dopaminergic cells: Effect of resveratrol. J. Mol. Neurosci. 39: 157-168. No PMCID
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| Mallajoysyula JK, Chinta SJ, Rajagoppalan S, Nicholls DG, and Andersen JK (2009). Metabolic control analysis in a cellular model of elevated MAO-B: relevance to Parkinson’s disease. Neurotox Res. 16: 186-193. PMCID: 2727365
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| Danielson S, Held J, Schilling B, Oo M, Gibson B, and Andersen, JK (2009). Preferentially increased nitration of alpha-synuclein at tyrosine-39 in a cellular oxidative model of Parkinson’s disease. Anal. Chem. 81: 7823-7828 No PMCID
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2008
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| Bhaumik D, Scott GK, Schokrpur S, Patil CK, Campisi J, Benz CC (2008) Expression of microRNA 146 suppresses NF-kappa activity with reduction of metastatic potential in breast cancer cells. Oncogene 27 (42): 5643-5647. PMCID 2811234
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| Peng J, Xie L, Jin K, Greenberg D.A. Andersen JK (2008) Fibroblast growth factor 2 enhances striatal and nigral neurogenesis in the acute 1-methyl -4-phenyl-1,2,3,6-tetrahydrophyridine model of parkinson’s disease. Neuroscience 153(3): 664-670. No PMCID
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| Chinta S, Rane A, Poksay KS, Bredesen DE, Andersen JK, and Rammohan R (2008). Coupling endoplasmic reticulum stress to the cell death program in dopaminergic cells: effects of paraquat. NeuroMol. Med. 10: 333-342. No PMCID
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| Vali S, Chinta S, Peng J, Sultana Z, Singh N, Sharma P, Sharada S, Andersen JK, and Bharath MMS (2008). Insights into the effects of alpha-synuclein expression and proteasome inhibition on glutathione metabolism through a dynamic in silico model of Parkinson’s disease: validation by cell culture data FRBM 45: 1290-1301. No PMCID
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2007
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| Young JE, Gouw L, Propp S, Sopher BL, Taylor J, Lin A, Hermel E, Logvinova A, Chen SF, Chen S, Bredesen D, Truant R, Ptacek LJ, La Spada AR, Ellerby LM (2007). Proteolytic Cleavage of Ataxin-7 by Caspase-7 Modulates Cellular Toxicity and Transcriptional Dysregulation. J. Biological Chemsitry: 282(41): 30150-30165).
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| Mallajosyula JK, Kaur D, Chinta SJ, Rajagopalan S, Rane A, Nicholls,DG, DiMonte D, Maccarthur H, Andersen JK. MAO-B elevation in mouse brain astrocytes results in Parkinson’s pathology. Plos One 3(2): e1616 No PMCID
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