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MOLECULAR BASIS OF NEURODEGENERATION The DebBurman lab is fascinated by how cells manipulate protein shapes. My 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. 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 biology, biochemistry, and genetics, and employ two yeasts as model systems. Since 1999, our research national and private foundations have funded our research, including the NIH, NSF, Parkinson Disease Foundation, Michigan Parkinson Foundation, and the Campbell Foundation. Training of Undergraduates as Scholars: Undergraduate research is a powerful form of teaching. Since 1999, the DebBurman lab environment has provided rigorous research experiences to thirty undergraduates to date. The lab's shared philosophy is to "work hard, enjoy science, collaborate, be ethical, and respect diversity." Nine are already co-authors in published manuscripts and several others will become authors as current research gets completed. Many students have successfully receiving nationally competitive grants to support their training. Several have gone on to receiving regional and national recognition for their scholarship. All graduates who have trained in the lab have pursued post-graduate studies (including MD, PhD, and MD/PhD degrees). Click here to learn who these students are and where they are headed. Phase I: Yeast Models to Study Neurodegenerative Disease Mechanisms In 2004, we received an NIH AREA grant to conduct the following project: Budding Yeast (S. cerevisiae) has emerged as a powerful model system for understanding molecular aspects of many human diseases. Protein misfolding linked to certain neurodegenerative diseases (NDDs) like Huntington Disease, Lou Gehrig's disease, and prion diseases have been succesfully recapitulated in S. cerevisiae and led to identification of therapeutically relevant regulators of misfolding. No S. cerevisiae models for Parkinson Disease (PD) or dentatorubral pallidoluysian atrophy (DRPLA) have been reported. PD is one of the most common NDDs, while DRPLA is a rare inherited NDD of the triplet repeat disease family. In both diseases, misfolding of a specific protein (alpha- for PD and atrophin for DRPLA) is thought to cause selective neuronal death. Unlike the well-characterized huntingtin protein in Huntington Disease (which shares many similarities to DRPLA), less is known about the misfolding of mutant atrophin in DRPLA. 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. A similar effort to establish atrophin-1 expression in yeast is underway. To extend initial observations with alpha-synuclein in yeast and fully develop a yeast model for atrophin, three goals are proposed. 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. These studies may further clarify the molecular bases for misfolding and degradation of PD- and DRPLA-linked proteins and extend the usefulness of yeast models. Importantly, the scientific training of many undergraduates will be supported, strengthening their cell biology and molecular genetics skills and appreciation for model organisms. In published findings from our lab with budding yeast (Sharma et al. 2006) and fission yeast (Brandis et al. 2006) models for alpha-synuclein misfolding and toxicity, we provided genetic and live cell support for these hypotheses, while uncovering unexpected yeast-specific alpha-synuclein property differences. In budding yeast, expression of alpha-synuclein alone does not cause toxicity, but the addition of either proteasomal dysfunction or mitochondrial oxidative stress is synthetic lethal. This lethality does not correlate well with alpha-synuclein aggregation, instead, alpha-synuclein localizes primarily to the yeast plasma membrane even when toxic. In fission yeast, alpha-synuclein misfolds and aggregates within the cytoplasm in an exquisitely time and concentration-dependent manner, providing crucial live cell evidence for a mechanism that follows the nucleation-polymerization model. Despite the extensive aggregation, alpha-synuclein is not toxic. Even at low concentrations, it does not localize to the plasma membrane. Citations: Sharma, N*, Brandis, K*, Herrera, SK*, Johnson, BE*, Vaidya, T*, and DebBurman, SK. (2006) ALPHA-SYNUCLEIN BUDDING YEAST MODEL: Toxicity Enhanced By Impaired Proteasome and Oxidative Stress. Journal of Molecular Neuroscience 28, 171-178. Brandis, K*, Holmes, I*, England, S*, Sharma, N*, Kukreja, L*, and DebBurman, SK. (2006) ALPHA-SYNUCLEIN FISSION YEAST MODEL: Concentration-Dependent Aggregation Without Membrane Localization or Toxicity. Journal of Molecular Neuroscience 28, 179-192. Phase II: Molecular Regulation of alpha-Synuclein in Yeast Models In 2007, we renewed our NIH grant to further both yeast models, as described below: 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? Our current grant aims are:
Pilot undergraduate projects underway have already provided initial evidence to support some of these hypotheses. Once completed, these related studies, intentionally diverse in scope and designed to attract a diverse and large set of undergraduates to my lab, will together clarify the molecular bases for the regulation of the normal biology and pathotoxicity of alpha-synuclein. They will also expand the usefulness of multiple yeast models to study diverse protein misfolding diseases. |