Wild-type and A315T mutant TDP-43 exert differential neurotoxicity in a Drosophila model of ALS
ABSTRACT The RNA-binding protein TDP-43 has been linked to amyotrophic lateral sclerosis (ALS) both as a causative locus and as a marker of pathology. With several missense mutations being identified within TDP-43, efforts have been directed towards generating animal models of ALS in mouse, zebrafish, Drosophila and worms. Previous loss of function and overexpression studies have shown that alterations in TDP-43 dosage recapitulate hallmark features of ALS pathology, including neuronal loss and locomotor dysfunction. Here we report a direct in vivo comparison between wild-type and A315T mutant TDP-43 overexpression in Drosophila neurons. We found that when expressed at comparable levels, wild-type TDP-43 exerts more severe effects on neuromuscular junction architecture, viability and motor neuron loss compared with the A315T allele. A subset of these differences can be compensated by higher levels of A315T expression, indicating a direct correlation between dosage and neurotoxic phenotypes. Interestingly, larval locomotion is the sole parameter that is more affected by the A315T allele than wild-type TDP-43. RNA interference and genetic interaction experiments indicate that TDP-43 overexpression mimics a loss-of-function phenotype and suggest a dominant-negative effect. Furthermore, we show that neuronal apoptosis does not require the cytoplasmic localization of TDP-43 and that its neurotoxicity is modulated by the proteasome, the HSP70 chaperone and the apoptosis pathway. Taken together, our findings provide novel insights into the phenotypic consequences of the A315T TDP-43 missense mutation and suggest that studies of individual mutations are critical for elucidating the molecular mechanisms of ALS and related neurodegenerative disorders.
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ABSTRACT: Dysfunction of the RNA-binding protein, TDP-43, is strongly implicated as a causative event in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). TDP-43 is normally found in the nucleus and pathological hallmarks of ALS include the presence of cytoplasmic protein aggregates containing TDP-43 and an associated loss of TDP-43 from the nucleus. Loss of nuclear TDP-43 likely contributes to neurodegeneration. Using Drosophila melanogaster to model TDP-43 loss of function, we show that reduced levels of the voltage-gated calcium channel, cacophony, mediate some of the physiological effects of TDP-43 loss. Null mutations in the Drosophila orthologue of TDP-43, named TBPH, resulted in defective larval locomotion and reduced levels of cacophony protein in whole animals and at the neuromuscular junction. Restoring the levels of cacophony in all neurons or selectively in motor neurons rescued these locomotion defects. Using TBPH immunoprecipitation, we showed that TBPH associates with cacophony transcript, indicating that it is likely to be a direct target for TBPH. Loss of TBPH leads to reduced levels of cacophony transcript, possibly due to increased degradation. In addition, TBPH also appears to regulate the inclusion of some alternatively spliced exons of cacophony. If similar effects of cacophony or related calcium channels are found in human ALS patients, these could be targets for the development of pharmacological therapies for ALS.Brain research 11/2013; 1584. DOI:10.1016/j.brainres.2013.11.019 · 2.83 Impact Factor
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ABSTRACT: Amyotrophic Lateral Sclerosis (ALS) is a progressive neuromuscular disease for which there is no cure. We have previously developed a Drosophila model of ALS based on TDP-43 that recapitulates several aspects of disease pathophysiology. Using this model, we designed a drug screening strategy based on the pupal lethality phenotype induced by TDP-43 when expressed in motor neurons. In screening 1,200 FDA approved compounds, we identified the PPARγ agonist pioglitazone as neuroprotective in Drosophila. Here we show that pioglitazone can rescue TDP-43 dependent locomotor dysfunction in motor neurons and glia but not in muscles. Testing additional models of ALS we find that pioglitazone is also neuroprotective when FUS, but not SOD1, is expressed in motor neurons. Interestingly, survival analyses of TDP or FUS models show no increase in lifespan, which is consistent with recent clinical trials. Using a pharmacogenetic approach, we show that the predicted Drosophila PPARγ homologs, E75 and E78 are in vivo targets of pioglitazone. Finally, using a global metabolomic approach, we identify a set of metabolites that pioglitazone can restore in the context of TDP-43 expression in motor neurons. Taken together, our data provide evidence that modulating PPARγ activity, although not effective at improving lifespan, provides a molecular target for mitigating locomotor dysfunction in TDP-43 and FUS but not SOD1 models of ALS in Drosophila. Furthermore, our data also identifies several “biomarkers” of the disease that may be useful in developing therapeutics and in future clinical trials.Human Molecular Genetics 11/2014; · 6.68 Impact Factor
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ABSTRACT: The human TAR DNA binding protein 43 (TDP-43), encoded by the gene TARDBP, plays a central role in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. TDP-43 inclusions are also found in up to approximately 60% of Alzheimer's disease (AD) brains. Although ALS-causing TARDBP mutations cluster in the C-terminal glycine-rich region of the protein, the pathogenic nature of the atypical missense variants p.A90V (located between the bipartite nuclear localization signal) and p.D169G (located in the first RNA-binding domain) is unclear. In addition, whether causal ALS mutations represent gain or loss-of-function alleles remains unknown. We recently reported that loss-of-function of the highly conserved TARDBP ortholog in Drosophila (called TBPH) leads to death of bursicon neurons resulting in adult maturation and wing expansion defects. Here, we compared wild-type TARDBP, 2 typical ALS-causing mutations (p.G287S and p.A315T) and 2 atypical variants (p.A90V and p.D169G), for their ability to complement neuronal TBPH loss-of-function. Although p.D169G rescued organismal pupal lethality and neuronal loss to a similar extent as wild-type TARDBP, p.A90V, p.G287S, and p.A315T were less efficient. Accordingly, p.A90V, p.G287S, and p.A315T but not p.D169G or wild-type protein promoted a shift of TDP-43 from the nucleus to the cytoplasm in approximately 12%-14% of bursicon neurons. Finally, we found that the carrier frequency of rare variant p.A90V was higher in French-Belgian AD cases (5/1714, 0.29%) than in controls of European descent (5/9436, 0.05%) (odds ratio = 5.5; 95% confidence interval, 1.6-19.0; p = 0.009). We propose that pathogenic TARDBP mutations have partial loss-of-function properties and that TARDBP p.A90V may increase AD risk by the same mechanism. Copyright © 2014 Elsevier Inc. All rights reserved.Neurobiology of aging 12/2014; 36(2). DOI:10.1016/j.neurobiolaging.2014.09.001 · 4.85 Impact Factor