“Satisfaction lies in the effort, not in the attainment. Full effort is full victory”.

-Mahatma Gandhi

The keys to success:
collaboration, mentoring, independence

Brandon Johnson ‘03 helps start the lab 2002 and begins the budding yeast model!

Samantha England ‘04 joins the lab in 2003 and a love for research begins that leads to her biology PhD in 2011 from the University of Rochester

Samantha England ‘04 helps pioneer the fission yeast model

Nijee Sharma ‘04 mentors Sara Herrera ‘05 in the learning fluorescence microscopy. They help bring GFP live cell imaging into lab.

Samantha England ‘04 mentors Richter Scholar Katrina Brandis ‘06 in the intricacies of mastering yeast growth assays

Dr. D and Katrina Brandis ‘06 meeet and discuss research progress regularly

Michael White ‘07 teaches future PhD student Lokesh Kukreja ‘08 how to do a Western blot

Alexandra Ayala ‘09 teaches Richter Scholar Alina Konnikova ‘11 how to prepare yeast cultures

Collaborating and teaching during research is replete with lighter moments! This early camaraderie can lead to lifelong friendships.

Alexandra Ayala ‘09 enjoys teaching Jaime Perez ‘10. No wonder she is now a K-12 teacher with Teach for America!

Why reinvent the wheel? What you learn from those before you, you teach to others.What Jaime Perez ‘10 learned from Alexandra Ayala ‘09, he now teaches Madhavi Senagolage ‘12.

Here, Madhavi Senagolage ‘12 guides and trains Richter Scholar Rida Khan ‘14. Research mentoring pipelines like this one sustains project productivity, even as students graduate and move on.

Mentoring often leads to productive partnerships. Michael Fiske ‘10 and Keith Solvang ‘11 form one such effective team that contributes to two publcations!

Lab graduate Keith Solvang ’11 stays the summer after graduation serving as Lab Manager and training Richter Scholar Katrina Campbell ‘14

Here is Keith Solvang ‘11 again, this time training another first year research scholar Ryan Vlaar ‘14

Maintaining regular, accurate, and complete records is a pre-requisite to a scientific career. Michael Fiske ‘10 ends his daily routine by logging his entries in his lab notebook.

Analyzing and interpreting data is one of the best ways to sharpen critical thinking skills and develop keener insight. Dr.D discuss  GFP live cell imaging with Ray Choi ‘09

All work and no play is hardly how it shapes out in D-Lab.  Even during times of intense focus, shared humor is not far.

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 are 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.

Publication Outcomes

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.

Publication Outcomes

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.
doi:10.5402/2011/521847
#: co-first authors.

2011 Senagolage, M*#, Perez, J*#, Ayala, A*, Vahedi, M*, Price, J*, Fiske, M*, and DebBurman, SK. Complex regulation of alpha-synuclein pathotoxic properties in yeasts by endocytosis genes.  #: co-first authors. In prep.

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.
WebID: http://www.jyi.org/articletools/print.php?id=1350

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.

Publication Outcome:

This work has been presented at several national conferences since 2009, including Society for Neuroscience and American society for Cell Biology meetings.

2011-12 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 (2012-2015): Alpha-synuclein Degradation, Nitration, & Combination Mutant analysis

Grant proposal currently submitted to NIH for 2012-2015

This second grant renewal application seeks 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, and they may crosstalk with the proteasome, and be modulated by nitrative stress and features intrinsic to alpha-synuclein itself.

We propose five related aims to be tested in both yeast models that will specifically examine these hypotheses.

1. Endocytosis is a route for alpha-synuclein degradation
2. Autophagy is a route for alpha-synuclein degradation
3.The proteasome and two lysosomal pathways (autophagy and endocytosis) degrade alpha-synuclein by also regulating each other
4. Nitration regulates alpha-synuclein pathologic properties
5. Individual amino acid determinants within alpha-synuclein regulate pathologic properties in a combinatorial manner