PPAR gamma activation is neuroprotective in a Drosophila model of ALS based on TDP-43.

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 1200 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 identify several 'biomarkers' of the disease that may be useful in developing therapeutics and in future clinical trials.

Fragile X protein mitigates TDP-43 toxicity by remodeling RNA granules and restoring translation.

RNA dysregulation is a newly recognized disease mechanism in amyotrophic lateral sclerosis (ALS). Here we identify Drosophila fragile X mental retardation protein (dFMRP) as a robust genetic modifier of TDP-43-dependent toxicity in a Drosophila model of ALS. We find that dFMRP overexpression (dFMRP OE) mitigates TDP-43 dependent locomotor defects and reduced lifespan in Drosophila. TDP-43 and FMRP form a complex in flies and human cells. In motor neurons, TDP-43 expression increases the association of dFMRP with stress granules and colocalizes with polyA binding protein in a variant-dependent manner. Furthermore, dFMRP dosage modulates TDP-43 solubility and molecular mobility with overexpression of dFMRP resulting in a significant reduction of TDP-43 in the aggregate fraction. Polysome fractionation experiments indicate that dFMRP OE also relieves the translation inhibition of futsch mRNA, a TDP-43 target mRNA, which regulates neuromuscular synapse architecture. Restoration of futsch translation by dFMRP OE mitigates Futsch-dependent morphological phenotypes at the neuromuscular junction including synaptic size and presence of satellite boutons. Our data suggest a model whereby dFMRP is neuroprotective by remodeling TDP-43 containing RNA granules, reducing aggregation and restoring the translation of specific mRNAs in motor neurons.

Futsch/MAP1B mRNA is a translational target of TDP-43 and is neuroprotective in a Drosophila model of amyotrophic lateral sclerosis.

TDP-43 is an RNA-binding protein linked to amyotrophic lateral sclerosis (ALS) that is known to regulate the splicing, transport, and storage of specific mRNAs into stress granules. Although TDP-43 has been shown to interact with translation factors, its role in protein synthesis remains unclear, and no in vivo translation targets have been reported to date. Here we provide evidence that TDP-43 associates with futsch mRNA in a complex and regulates its expression at the neuromuscular junction (NMJ) in Drosophila. In the context of TDP-43-induced proteinopathy, there is a significant reduction of futsch mRNA at the NMJ compared with motor neuron cell bodies where we find higher levels of transcript compared with controls. TDP-43 also leads to a significant reduction in Futsch protein expression at the NMJ. Polysome fractionations coupled with quantitative PCR experiments indicate that TDP-43 leads to a futsch mRNA shift from actively translating polysomes to nontranslating ribonuclear protein particles, suggesting that in addition to its effect on localization, TDP-43 also regulates the translation of futsch mRNA. We also show that futsch overexpression is neuroprotective by extending life span, reducing TDP-43 aggregation, and suppressing ALS-like locomotor dysfunction as well as NMJ abnormalities linked to microtubule and synaptic stabilization. Furthermore, the localization of MAP1B, the mammalian homolog of Futsch, is altered in ALS spinal cords in a manner similar to our observations in Drosophila motor neurons. Together, our results suggest a microtubule-dependent mechanism in motor neuron disease caused by TDP-43-dependent alterations in futsch mRNA localization and translation in vivo.

Wild-type and A315T mutant TDP-43 exert differential neurotoxicity in a Drosophila model of ALS.

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.

Staufen- and FMRP-containing neuronal RNPs are structurally and functionally related to somatic P bodies.

Local control of mRNA translation modulates neuronal development, synaptic plasticity, and memory formation. A poorly understood aspect of this control is the role and composition of ribonucleoprotein (RNP) particles that mediate transport and translation of neuronal RNAs. Here, we show that staufen- and FMRP-containing RNPs in Drosophila neurons contain proteins also present in somatic "P bodies," including the RNA-degradative enzymes Dcp1p and Xrn1p/Pacman and crucial components of miRNA (argonaute), NMD (Upf1p), and general translational repression (Dhh1p/Me31B) pathways. Drosophila Me31B is shown to participate (1) with an FMRP-associated, P body protein (Scd6p/trailer hitch) in FMRP-driven, argonaute-dependent translational repression in developing eye imaginal discs; (2) in dendritic elaboration of larval sensory neurons; and (3) in bantam miRNA-mediated translational repression in wing imaginal discs. These results argue for a conserved mechanism of translational control critical to neuronal function and open up new experimental avenues for understanding the regulation of mRNA function within neurons.

Fragile X protein controls the efficacy of mRNA transport in Drosophila neurons.

Fragile X syndrome, the most common form of inherited mental retardation is caused by mutations in the FMR1 gene. FMR1 encodes an RNA-binding protein thought to control the transport and translation of target mRNAs. While the function of FMRP in translational control has been clearly demonstrated, its role in mRNA transport and localization in neurons remains elusive. Using a genetically encoded mRNA imaging system in Drosophila we provide the first demonstration that FMRP controls mRNA transport. Live imaging of FMRP associated mRNAs show that mRNA granules are less motile and exhibit decreased directional movement in dFmr1 mutant neurons. Furthermore, Fluorescence Recovery After Photobleaching experiments show that the mobile fraction of mRNA molecules within neurites is dependent on FMRP dosage. These data support a model whereby FMRP regulates transport efficacy, by regulating the association between mRNA cargo and microtubules and suggest a new mechanism for the disease.

miR-9a mediates the role of Lethal giant larvae as an epithelial growth inhibitor in Drosophila.

Drosophila lethal giant larvae (lgl) encodes a conserved tumor suppressor with established roles in cell polarity, asymmetric division, and proliferation control. Lgl's human orthologs, HUGL1 and HUGL2, are altered in human cancers, however, its mechanistic role as a tumor suppressor remains poorly understood. Based on a previously established connection between Lgl and Fragile X protein (FMRP), a miRNA-associated translational regulator, we hypothesized that Lgl may exert its role as a tumor suppressor by interacting with the miRNA pathway. Consistent with this model, we found that lgl is a dominant modifier of Argonaute1 overexpression in the eye neuroepithelium. Using microarray profiling we identified a core set of ten miRNAs that are altered throughout tumorigenesis in Drosophila lgl mutants. Among these are several miRNAs previously linked to human cancers including miR-9a, which we found to be downregulated in lgl neuroepithelial tissues. To determine whether miR-9a can act as an effector of Lgl in vivo, we overexpressed it in the context of lgl knock-down by RNAi and found it able to reduce the overgrowth phenotype caused by Lgl loss in epithelia. Furthermore, cross-comparisons between miRNA and mRNA profiling in lgl mutant tissues and human breast cancer cells identified thrombospondin (tsp) as a common factor altered in both fly and human breast cancer tumorigenesis models. Our work provides the first evidence of a functional connection between Lgl and the miRNA pathway, demonstrates that miR-9a mediates Lgl's role in restricting epithelial proliferation, and provides novel insights into pathways controlled by Lgl during tumor progression.

Functional dissection of a eukaryotic dicistronic gene: transgenic stonedB, but not stonedA, restores normal synaptic properties to Drosophila stoned mutants.

The dicistronic Drosophila stoned mRNA produces two proteins, stonedA and stonedB, that are localized at nerve terminals. While the stoned locus is required for synaptic-vesicle cycling in neurons, distinct or overlapping synaptic functions of stonedA and stonedB have not been clearly identified. Potential functions of stoned products in nonneuronal cells remain entirely unexplored in vivo. Transgene-based analyses presented here demonstrate that exclusively neuronal expression of a dicistronic stoned cDNA is sufficient for rescue of defects observed in lethal and viable stoned mutants. Significantly, expression of a monocistronic stonedB trangene is sufficient for rescuing various phenotypic deficits of stoned mutants, including those in organismal viability, evoked transmitter release, and synaptotagmin retrieval from the plasma membrane. In contrast, a stonedA transgene does not alleviate any stoned mutant phenotype. Novel phenotypic analyses demonstrate that, in addition to regulation of presynaptic function, stoned is required for regulating normal growth and morphology of the motor terminal; however, this developmental function is also provided by a stonedB transgene. Our data, although most consistent with a hypothesis in which stonedA is a dispensable protein, are limited by the absence of a true null allele for stoned due to partial restoration of presynaptic stonedA by transgenically provided stonedB. Careful analysis of the effects of the monocistronic transgenes together and in isolation clearly reveals that the presence of presynaptic stonedA is dependent on stonedB. Together, our findings improve understanding of the functional relationship between stonedA and stonedB and elaborate significantly on the in vivo functions of stonins, recently discovered phylogenetically conserved stonedB homologs that represent a new family of "orphan" medium (mu) chains of adaptor complexes involved in vesicle formation. Data presented here also provide new insight into potential mechanisms that underlie translation and evolution of the dicistronic stoned mRNA.

Motor neurons and glia exhibit specific individualized responses to TDP-43 expression in a Drosophila model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by complex neuronal and glial phenotypes. Recently, RNA-based mechanisms have been linked to ALS via RNA-binding proteins such as TDP-43, which has been studied in vivo using models ranging from yeast to rodents. We have developed a Drosophila model of ALS based on TDP-43 that recapitulates several aspects of pathology, including motor neuron loss, locomotor dysfunction and reduced survival. Here we report the phenotypic consequences of expressing wild-type and four different ALS-linked TDP-43 mutations in neurons and glia. We show that TDP-43-driven neurodegeneration phenotypes are dose- and age-dependent. In motor neurons, TDP-43 appears restricted to nuclei, which are significantly misshapen due to mutant but not wild-type protein expression. In glia and in the developing neuroepithelium, TDP-43 associates with cytoplasmic puncta. TDP-43-containing RNA granules are motile in cultured motor neurons, although wild-type and mutant variants exhibit different kinetic properties. At the neuromuscular junction, the expression of TDP-43 in motor neurons versus glia leads to seemingly opposite synaptic phenotypes that, surprisingly, translate into comparable locomotor defects. Finally, we explore sleep as a behavioral readout of TDP-43 expression and find evidence of sleep fragmentation consistent with hyperexcitability, a suggested mechanism in ALS. These findings support the notion that although motor neurons and glia are both involved in ALS pathology, at the cellular level they can exhibit different responses to TDP-43. In addition, our data suggest that individual TDP-43 alleles utilize distinct molecular mechanisms, which will be important for developing therapeutic strategies.