Amyotrophic lateral sclerosis (ALS) is a devastating adult-onset neurodegenerative disease that wreaks havoc on motor neurons. A progressive and fatal muscle paralysis ensues, causing death within 2 to 5 years. Recently, a central role for RNA binding proteins and RNA metabolism pathways has emerged. The protein TDP-43 was recently identified as the major disease protein in pathological inclusions in both ALS and frontal temporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Moreover, mutations in the TDP-43 gene have now been identified in sporadic and familial ALS patients. Pathology and genetics both converge on TDP-43 being central to the pathogenesis of these diseases. In the first five years of this research project, we have generated in vitro and in vivo TDP-43 proteinopathy models to explore TDP-43. We have harnessed the simple yeast model system to study TDP-43's properties and the effect of ALS-linked mutations. Our preliminary data demonstrate: 1) a critical role for the RNA recognition motif and carboxy-terminal region of TDP-43 in mediating aggregation and cellular toxicity, 2) increased aggregation and toxicity caused by a disease-linked TDP-43 mutation, and 3) genetic screens identified multiple RNA binding proteins as potent toxicity modifiers. One of these, Pbp1, is the yeast homolog of human ataxin 2 and we identified polyglutamine expansions in ataxin 2 as a major genetic risk factor for ALS in humans. We also discovered that deletion of the Dbr1 gene potently suppresses TDP-43 toxicity. The identification of a major genetic risk factor for ALS in humans and a novel and unexpected therapeutic target for ALS starting from the simple yeast model, illustrates the power of this approach. We have also pursued studies beyond TDP-43, focusing on additional RNA-binding proteins, such as FUS, TAF15, EWSR1, and several more. We have discovered a prion-like domain in TDP-43 and FUS and have used this domain to link additional RNA-binding proteins to a class of proteins with similar structural and functional properties. For the next five years of this project, with the goal to define TDP-43 disease mechanisms from multiple angles we propose three Specific Aims: 1) Continuing to characterize hits from our yeast TDP-43 toxicity modifier screens to elucidate additional mechanisms of TDP- 43 toxicity and to perform an additional yeast screen of ~1,000 essential genes;2) Defining the mechanism by which Dbr1 inhibition suppresses TDP-43 toxicity and extending these studies to mammalian cells and animal models;3) Testing the hypothesis that aggregation-prone RNA-binding proteins contribute broadly to ALS using next generation sequencing approaches.

Public Health Relevance

As our population continues to age, neurodegenerative disease will increase in prevalence and thus pose a daunting challenge to public health worldwide. The protein TDP-43 has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontal temporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), but very little is known about the mechanisms by which TDP-43 causes neurodegeneration. Our project harnesses multiple in vitro and in vivo models and techniques to gain mechanistic insight into how TDP-43 contributes to human disease, which will suggest novel avenues for therapeutic intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
2R01NS065317-06A1
Application #
8717172
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Gubitz, Amelie
Project Start
2009-03-01
Project End
2019-02-28
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
6
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Stanford University
Department
Genetics
Type
Schools of Medicine
DUNS #
City
Stanford
State
CA
Country
United States
Zip Code
94304
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