Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterized by progressive muscle atrophy and motor neuron death. A hallmark feature of >90% of ALS cases known to date is the mislocalization of TAR DNA Binding Protein (TDP-43) and its association with cytoplasmic aggregates in motor neurons and surrounding glia regardless of etiology. In addition, TDP-43 mutations have been found in both familial and sporadic ALS cases. Together, these findings demonstrate a central role for TDP-43 in disease. Our lab has developed a Drosophila model of ALS based on TDP-43 that exhibits age dependent neurodegeneration, locomotor defects, and decreased survival, resembling the presentation of the human disease. Using this model, we identified a robust genetic interaction between TDP-43 and Drosophila FoxO, a regulator of stress response. We found that FoxO overexpression enhances neurodegeneration caused by TDP-43 in the fly retina while reduction of FoxO expression rescued both this phenotype and the locomotor defects caused by TDP-43 expression in motor neurons. These findings led us to hypothesize that FoxO underlies at least some aspects of TDP-43 toxicity. The overarching objective of the proposed research is to determine FoxO's role in TDP-43 toxicity and begin to elucidate the mechanisms underlying this functional interaction.
In Aim 1, we will modulate FoxO expression in motor neurons and, using a battery of established assays, determine its effect on TDP-43-dependent phenotypes including abnormal NMJ morphology, altered microtubule organization, defects in distribution of synaptic markers, reduced viability and lifespan, as well as impaired locomotor function. We will determine whether FoxO affects TDP-43 aggregation by performing cellular fractionations. We will also test FoxO's effect on TDP-43's association with RNA stress granules in primary motor neurons and perform Fluorescence Recovery After Photobleaching (FRAP) analyses to determine TDP-43's mobility within neurites. Preliminary results indicate that reducing FoxO expression rescues specific TDP-43-dependent abnormalities at the NMJ and increases the solubility of TDP-43.
In Aim 2 we will use a primary motor neuron model derived from Drosophila embryos to determine the effects of FoxO on the response of TDP-43 expressing neurons to oxidative stress, as well as recovery following removal of stress. We will measure stress-induced changes in TDP-43 localization, formation of stress granules, microtubule organization, neuronal morphology, and cell death using confocal fluorescence microscopy. Live imaging and FRAP analyses will also be performed on stress-induced TDP-43 puncta to measure the kinetics of TDP-43 throughout stress response. Using this approach, we will also be able to test the emerging paradigm that TDP-43-positive stress granules may seed pathological aggregates following prolonged stress. These experiments will establish FoxO's role in TDP-43 toxicity and help determine its potential as a novel therapeutic target for ALS and related neurodegenerative diseases.

Public Health Relevance

The mechanisms underlying Amyotrophic Lateral Sclerosis (ALS) remain poorly understood, and current therapeutic options for ALS patients are at best palliative. This underscores the need for further studies in order to develop effective therapies. TDP-43 is an RNA binding protein linked to the majority of ALS cases, and recent studies including our preliminary results indicate that FoxO, a transcription factor involved in stress response, may govern some aspects of TDP-43 function. Using a Drosophila model of ALS based on TDP-43, we will determine the role of FoxO in TDP-43 dependent toxicity, which will provide novel insights into therapeutic strategies for ALS.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31NS090829-02
Application #
8987456
Study Section
Special Emphasis Panel (ZRG1-F03A-N (20))
Program Officer
Gubitz, Amelie
Project Start
2015-01-01
Project End
2017-08-31
Budget Start
2016-01-01
Budget End
2016-12-31
Support Year
2
Fiscal Year
2016
Total Cost
$40,525
Indirect Cost
Name
University of Arizona
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
806345617
City
Tucson
State
AZ
Country
United States
Zip Code
85721