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Molecular Basis of Parkinson’s Disease
The D-lab is fascinated by how cells manipulate protein shapes. Our research explores why some proteins cause incurable diseases when they change their normal shape. Cells contain a myriad of proteins that, when in their proper shapes, perform tasks essential for life. Within the intensely crowded environment of cells, most proteins require cooperation of chaperones, which help the proteins fold into proper shapes. If the proteins still misfold, they are targeted for destruction. Sometimes, however, proteins acquire wrong shapes that escape the chaperone assistance and quality control; their buildup can kill cells, especially in the brain, and cause neurological disease. Our goal is to characterize molecular mechanisms and identify proteins that can regulate the toxicity that is linked to alpha-synuclein, the protein that kills nerve cells in Parkinson’s disease (PD). Misfolding and aggregation of this protein, oxidative damage, and impairment of protein degradation and protein folding pathways are all hypotheses for the molecular cause of the selective neurotoxicity. To evaluate these hypotheses, we combine molecular genetics, biochemistry, and cell biology, and employ two yeasts as model systems.
The history of our work on PD can be divided in three phases of past (2001-2007) present (2007-2012), and future (2012-2015).
Phase I (2001-2007): Developing Yeast Models to Study Neurodegenerative Disease Mechanisms
Budding Yeast (S. cerevisiae) has emerged as a powerful model system for understanding molecular aspects of many human diseases. PD is one of the most common NDDs, where the misfolding of a specific protein (alpha-synuclein) is thought to cause selective neuronal death. A S. cerevisiae expression system for studying alpha-synuclein has recently been developed in our lab. Preliminary evidence supports that both wildtype and disease-associated mutants are aggregating within yeast cells and upon purification. Three goals were proposed.
Goals (supported by NIH AREA R15 grant 2004-2007)
1) Misfolding properties between wildtype and mutant versions of both proteins will be investigated in vivo (immunofluorescence and GFP-based localization and assessment of protein half-life) and in vitro (by measuring protease sensitivity and differential solubility).
2) Influences of chaperones and ubiquitin-proteasomal pathway proteins on folding and degradation of these proteins will be assessed in strains compromised for chaperone/proteasomal function, or those that overexpress chaperones, and by co-immunoprecipitation assessment. 3) A fission yeast (S. pombe) expression model for alpha-synuclein and atrophin properties (as in Aim 1) will be developed and compared with the S. cerevisiae model; NDD models have not been reported in S. pombe.
2006 Sharma, N*, Brandis, K*, Herrera, SK*, Johnson, BE*, Vaidya, T*, and DebBurman, SK. ALPHA-SYNUCLEIN BUDDING YEAST MODEL: Toxicity Enhanced By Impaired Proteasome and Oxidative Stress. Journal of Molecular Neuroscience 28, 171-178.
2006 Brandis, K*, Holmes, I*, England, S*, Sharma, N*, Kukreja, L*, and DebBurman, SK. ALPHA-SYNUCLEIN FISSION YEAST MODEL: Concentration-Dependent Aggregation Without Membrane Localization or Toxicity. Journal of Molecular Neuroscience 28, 179-192.
2006 Paul, AG* (Faculty Advisor: DebBurman, SK.) Evaluation of STP2-dependent alpha-synuclein toxicity in a budding yeast model: Is GAPDH involved? Impulse: Impulse: An Undergraduate Journal in Neuroscience.
Phase IIa (2007-2012): Phospholipid binding, E46K familial mutant, & Endocytosis regulation
By continuing comparative analysis using both yeast models and by employing genetic manipulation in living cells, the following specific questions that all centrally focus on the molecular determinants within alpha-synuclein and cellular pathways that regulate its misfolding, lipid binding, degradation, and toxicity can be examined. What is the significance of the newly discovered familial PD mutation E46K in vivo? Does alpha-synuclein membrane localization in vivo involve specific phospholipids and is membrane interaction required for in vivo toxicity? Does alpha-synuclein contain domains that confer plasma membrane localization and aggregation in vivo? Can cytoplasmic oxidative stress also cause alpha-synuclein-mediated lethality or is lethality limited to mitochondrial stress? Lastly, does the lysosome also degrade alpha-synuclein and by what mechanism?
Goals (supported by NIH AREA R15 Grant 2007-2012)
1. To test the hypothesis that alpha-synuclein mutant E46K is significantly toxic to cells and binds phospholipids in vivo.
2. To test the hypothesis that specific phospholipid composition and total phospholipid content is critical to alpha-synuclein membrane association and toxicity.
3. To test the hypothesis that specific aggregation domains and lipid-binding domains mediate alpha-synuclein properties.
4. To test the hypothesis that cytoplasmic oxidative stress also contributes to alpha-synuclein-mediated toxicity.
5. To test the hypothesis that the lysosome pathway also degrades alpha-synuclein.
Senagolage, M*, Perez, J*, Ayala, A*, Vahedi, M*, Price, J*, Fiske, M*, Khan, R*, Tembo, M*, and DebBurman, SK. Complex regulation of alpha-synuclein pathotoxic properties in yeasts by endocytosis genes. #: co-first authors. In prep.
2011 Fiske, M*#, Valtierra, S*,# Solvang, K*, Zorniak, M*, White, M.*, Herrera, S*, Brezinsky, R*, Konnikova, A* and DebBurman, SK. Contribution of serine phosphorylation and alanine-76 to alpha-synuclein membrane association and aggregation in yeast models. Parkinson’s Disease Volume 2011, 12 pages, Article ID 392180; doi:10.4061/2011/392180
#: co-first authors.
2011 Fiske, M*#, White M*#, Valtierra, S*, Herrera, S*, Solvang, K*, Konnikova, A* and DebBurman, SK. E46K Alpha-Synuclein Familial Mutant Binds Membranes, Aggregates, and Induces Strain-Selective Toxicity in Yeast Models. ISRN Neurology, vol. 2011, Article ID 521847, 14 pages, 2011.
#: co-first authors.
2009 Kukreja, L., and DebBurman, SK. Oxidants Induce alpha-Synuclein-Independent Toxicity in a Fission Yeast Model for Parkinson’s Disease. Journal of Young Investigators. Volume 19, Issue 17 on 03 November. (http://www.jyi.org/research/re.php?id=3529)
2008 Kukreja, L* (Faculty Advisor: DebBurman, SK.) Evaluating alpha-synuclein’s interaction with cellular phospholipids and potential toxicity in yeast models for Parkinson’s disease. AJUR: The Journal for undergraduate research in the pure and applied sciences, Vol 7, 9-24.
2007 Zorniak, M* & Brandis, K* (Faculty Advisor: DebBurman, SK.) Can Oxidative Stress and Mitochondrial Dysfunction Enhance alpha-synuclein Toxicity in a Yeast Model of Parkinson’s Disease?
Journal of Young Investigators Vol 17, issue 6.
Phase IIb (2008-2011): Autophagic regulation
Supported by the American Parkinson Disease Association 2008-2010
PD is caused by the degeneration of midbrain dopaminergic neurons, which accumulate misfolded, aggregated, and toxic forms of alpha-synuclein. An attractive hypothesis is that increasing the degradation of alpha-synuclein will reduce its toxicity. Mounting evidence points to the proteasome and lysosome as cellular sites for alpha-synuclein degradation. Multiple pathways, including autophagy, target diverse proteins to the lysosome, but the specificity with which alpha-synuclein utilizes these pathways is unclear. In this proposal, we will test the hypothesis that autophagy regulates alpha-synuclein-dependent toxicity. In a yeast model, we will measure autophagy status in alpha-synuclein expressing cells that exhibit varying levels of toxicity. We will also evaluate toxicity, and alpha-synuclein aggregation and its turnover in cells induced or repressed for autophagy. The discovery of specific autophagy genes that regulate alpha-synuclein toxicity holds substantial therapeutic promise.
This work has been presented at several national conferences since 2009, including Society for Neuroscience and American society for Cell Biology meetings.
Konnikova, A*, Choi, R*, Sanchez, D*, Ahlstrand, K*, Sullivan, P*, Perez, J*, and DebBurman, SK. Autophagic regulation of alpha-synuclein membrane association, aggregation and toxicity in budding yeast. Manuscript in prep.
Phase IIb (2012-2017): Alpha-synuclein Degradation, Sumoylation, & Variant analysis
Grant proposal in preparation
This current grant renewal application will seek to extend support for some of the determinants identified above by testing the overall hypothesis that two forms of clearance–endocytosis and autophagy—may both be involved in degrading alpha-synuclein, and they may crosstalk with the proteasome. These processes are modulated by sumoylation, nitrosylation, c-terminal truncations, and splice variants of alpha-synuclein, and its new disease mutants (H50Q, G51D, and A53E).
We propose the following aims to be tested in both yeast models that will specifically examine these hypotheses:
1. The proteasome and two lysosomal pathways (autophagy and endocytosis) degrade alpha-synuclein by also regulating each other
2. Splice variants regulate alpha-synuclein pathologic properties
3. C-terminal truncation variants contribute to PD pathology
4. Sumoylation reduces alpha-synuclein mediated toxicity
5. New PD alpha-synuclein mutant (H50Q, G51D, and A53E) have altered membrane binding and aggregation properties and increase toxicity.
Current projects in progress
Solvang, K*, Fiske, M*, DebBurman, SK. Alpha-synuclein nitrosylation regulates aggregation and cellular toxicity in budding yeast.
Kukulka, N*, Vlaar, R*, Senagolage, M*, DebBurman, SK. Combination mutant analysis of familial Parkinson’s disease alpha-synuclein mutants in two yeast models: Not all mutants are equal.
Campbell, K*, Lipkin, G*, Solvang, K*, Senagolage, M*, DebBurman, SK. Amino acid determinants of alpha-synuclein membrane association, aggregation and toxicity.
Lipkin, G*, Hamid, K*, Campbell, K*, DebBurman, SK. Evaluation of alpha-synuclein sumoylation in yeasts.
Tembo, M*, Senagolage, M*, DebBurman, SK. Evaluation of alpha-synuclein threonine phosphorylation in yeast.
Kukulka, N*, DebBurman, SK. Evaluation of alpha-synuclein splice variants in yeast.
Alvarado, C*, Chiren, S*, James, J*, Roman, A*, Munoz, M*, Campbell, K*, and DebBurman, SK. Evaluation of alpha-synuclein C-terminal truncations in yeast.