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)
Type
Research Project (R01)
Project #
2R01NS065317-06A1
Application #
8717172
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Gubitz, Amelie
Project Start
Project End
Budget Start
Budget End
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
Boeynaems, Steven; Bogaert, Elke; Michiels, Emiel et al. (2016) Drosophila screen connects nuclear transport genes to DPR pathology in c9ALS/FTD. Sci Rep 6:20877
Gitler, Aaron D; Tsuiji, Hitomi (2016) There has been an awakening: Emerging mechanisms of C9orf72 mutations in FTD/ALS. Brain Res 1647:19-29
Kramer, Nicholas J; Carlomagno, Yari; Zhang, Yong-Jie et al. (2016) Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts. Science 353:708-12
Williams, Kelly L; Topp, Simon; Yang, Shu et al. (2016) CCNF mutations in amyotrophic lateral sclerosis and frontotemporal dementia. Nat Commun 7:11253
Margulis, Neil G; Wilson, Joshua D; Bentivoglio, Christine M et al. (2016) Analysis of COPII Vesicles Indicates a Role for the Emp47-Ssp120 Complex in Transport of Cell Surface Glycoproteins. Traffic 17:191-210
Dhungel, Nripesh; Eleuteri, Simona; Li, Ling-Bo et al. (2015) Parkinson's disease genes VPS35 and EIF4G1 interact genetically and converge on α-synuclein. Neuron 85:76-87
Jovičić, Ana; Mertens, Jerome; Boeynaems, Steven et al. (2015) Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS. Nat Neurosci 18:1226-9
Coyne, Alyssa N; Yamada, Shizuka B; Siddegowda, Bhavani Bagevalu et al. (2015) Fragile X protein mitigates TDP-43 toxicity by remodeling RNA granules and restoring translation. Hum Mol Genet 24:6886-98
Figley, Matthew D; Bieri, Gregor; Kolaitis, Regina-Maria et al. (2014) Profilin 1 associates with stress granules and ALS-linked mutations alter stress granule dynamics. J Neurosci 34:8083-97
Figley, Matthew D; Thomas, Anna; Gitler, Aaron D (2014) Evaluating noncoding nucleotide repeat expansions in amyotrophic lateral sclerosis. Neurobiol Aging 35:936.e1-4

Showing the most recent 10 out of 40 publications