Synucleinopathies are a class of neurodegenerative disease characterized by pathological lesions composed of aggregates of a-synuclein (a-syn) in select populations of neurons. Alpha-synuclein is a small, abundant lipid-binding protein of unknown function that is prone to misfolding when not bound to lipids. The accumulation of a-syn aggregates in synucleinopathies suggests that aberrations in protein homeostasis might contribute to pathogenesis in these diseases. Because protein homeostasis mechanisms are highly conserved throughout eukaryotes, yeast cells can be used as a tractable model organism to investigate factors that contribute to the pathogenesis of synucleinopathies. The Lindquist laboratory has developed a yeast model over-expressing human a-syn that recapitulates many aspects of a-syn toxicity, and has observed that sterol biosynthesis and trafficking are also perturbed. Disrupting sterol biosynthesis can potentially alter a variety of cellular pathways. In fact, sphingolipid production is responsive to flux through the sterol pathway. Interestingly, both pathways have been implicated independently in a-syn toxicity and disease progression. The contribution of lipids to a-syn toxicity remains unresolved, and understanding how lipids affect synucleinopathy pathogenesis is essential for identifying potential therapeutic targets.
The first aim of my proposal will investigate how the yeast lipidome is altered in response to a-syn expression. Lipidomic profiling has performed on yeast expressing nontoxic and toxic levels of a-syn in collaboration with Dr. Clary Clish at the Broad Institute. The lipid species identified up to this point correlate with those predicted to change upon a-syn expression. These results will then be compared to the lipid profiles of yeast co-expressing a-syn and genetic modifiers we discovered using overexpression and deletion screens. This will identify the lipids that change when toxicity is suppressed or enhanced. These targets lipids will then be manipulated in our yeast a-syn model to determine their effect on toxicity.
My second aim i s to evaluate whether the defects in cellular unesterified ergosterol distribution caused by a-syn expression lead to the mis-trafficking and accumulation of sphingolipids. Fluorescent microscopy will be used to monitor sphingolipid trafficking and localization in yeast cells. In addition, sphingolipids will be quantified using metabolic labeling. The cellular localization of unesterified ergosterol will be determined using filipin stain. Finally, my last aim will be evaluate sterol-sphingolipid regulation defects in cultured DA neurons expressing a-syn. Similar to aim 2, fluorescent microscopy will be used to monitor the trafficking and localization of unesterified cholesterol and sphingolipids, respectively. The amount of sphingolipids and sterols in a-syn expressing DA neurons will be quantified as well.

Public Health Relevance

Lipids are important macromolecules that are involved in many cellular events and have been implicated in several human diseases. Investigation of their contribution to neurodegenerative disease specifically PD, will promote further understanding of lipid homeostasis and a-syn pathobiology. The identification of lipids that might affect a-syn toxicity will be extremely useful in developing lipid-specific therapeutics reducing the pleiotropic effects associated with the general lipid drugs currently employed.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32NS066692-01A2
Application #
8003757
Study Section
Special Emphasis Panel (ZRG1-F03A-F (20))
Program Officer
Sutherland, Margaret L
Project Start
2010-09-01
Project End
2013-08-31
Budget Start
2010-09-01
Budget End
2011-08-31
Support Year
1
Fiscal Year
2010
Total Cost
$47,606
Indirect Cost
Name
Whitehead Institute for Biomedical Research
Department
Type
DUNS #
120989983
City
Cambridge
State
MA
Country
United States
Zip Code
02142