w TAR DNA-binding protein-43 homolog

InteractiveFly: GeneBrief

TAR DNA-binding protein-43 homolog: Biological Overview | References

Gene name - TAR DNA-binding protein-43 homolog

Synonyms -

Cytological map position - 60A4-60A5

Function - RNA-binding protein

Keywords - regulation of synaptic efficacy and motor control, regulation of Futsch activity at the neuromuscular junction, regulation of the robustness of neuronal specification through microRNA-9a, a model for Amyotrophic lateral sclerosis (ALS), often referred to as Lou Gehrig's Disease

Symbol - TBPH

FlyBase ID: FBgn0025790

Genetic map position - chr2R:19,746,852-19,750,104

Classification - RNA recognition motif 1 in TAR DNA-binding protein 43 (TDP-43) and similar proteins

Cellular location - cytoplasmic

NCBI links: EntrezGene

Recent literature
Romano, G., Appocher, C., Scorzeto, M., Klima, R., Baralle, F. E., Megighian, A. and Feiguin, F. (2015). Glial TDP-43 regulates axon wrapping, GluRIIA clustering and fly motility by autonomous and non-autonomous mechanisms. Hum Mol Genet [Epub ahead of print]. PubMed ID: 26276811
Alterations in the glial function of TDP-43 are becoming increasingly associated with the neurological symptoms observed in Amyotrophic Lateral Sclerosis (ALS), however, the physiological role of this protein in the glia or the mechanisms that may lead to neurodegeneration are unknown. To address these issues, the expression levels of TDP-43 was modulated in Drosophila glia; the protein was found to be required to regulate the subcellular wrapping of motoneuron axons, promote synaptic growth and the formation of glutamate receptor clusters at the neuromuscular junctions. Interestingly, it was determined that the glutamate transporter EAAT1 mediates the regulatory functions of TDP-43 in the glia, and genetic or pharmacological compensations of EAAT1 activity were demonstrated to be sufficient to modulate glutamate receptor clustering and locomotive behaviors in flies. The data uncovers autonomous and non-autonomous functions of TDP-43 in glia and suggests new experimentally based therapeutic strategies in ALS.

Sreedharan, J., Neukomm, L. J., Brown, R. H., Jr. and Freeman, M. R. (2015). Age-dependent TDP-43-mediated motor neuron degeneration requires GSK3, hat-trick, and xmas-2. Curr Biol 25: 2130-2136. PubMed ID: 26234214
The RNA-processing protein TDP-43 is central to the pathogenesis of amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron (MN) disease. TDP-43 is conserved in Drosophila, where it has been the topic of considerable study, but how TDP-43 mutations lead to age-dependent neurodegeneration is unclear and most approaches have not directly examined changes in MN morphology with age. This study used a mosaic approach to study age-dependent MN loss in the adult fly leg where it is possible to resolve single motor axons, NMJs and active zones, and perform rapid forward genetic screens. Expression of the mutant protein TDP-43(Q331K) caused dying-back of NMJs and axons, which could not be suppressed by mutations that block Wallerian degeneration. Three genes were identified that suppress TDP-43 toxicity, including shaggy/GSK3, a known modifier of neurodegeneration. The two additional novel suppressors, hat-trick and xmas-2, function in chromatin modeling and RNA export, two processes recently implicated in human ALS. Loss of shaggy/GSK3, hat-trick, or xmas-2 does not suppress Wallerian degeneration, arguing TDP-43(Q331K)-induced and Wallerian degeneration are genetically distinct processes. In addition to delineating genetic factors that modify TDP-43 toxicity, these results establish the Drosophila adult leg as a valuable new tool for the in vivo study of adult MN phenotypes.

Cheng, C. W., Lin, M. J. and Shen, C. J. (2015). Rapamycin alleviates pathogenesis of a new Drosophila model of ALS-TDP. J Neurogenet: 1-47. PubMed ID: 26219309
TDP-43 is a multi-functional RNA/DNA-binding protein well-conserved among many species including mammals and Drosophila. However, it is also a major component of the pathological inclusions associated with degenerating motor neurons of amyotrophic lateral sclerosis (ALS). Further, TDP-43 is a signature protein in one subtype of frontotemporal degeneration, FTLD-U. Currently, there are no effective drugs for these neurodegenerative diseases. This study describes the generation and characterization of a new fly model of ALS-TDP with transgenic expression of the Drosophila ortholog of TDP-43, dTDP, in adult flies under the control of a temperature sensitive motor neuron-specific GAL4, thus bypassing the deleterious effect of dTDP during development. Diminished lifespan as well as impaired locomotor activities of the flies following induction of dTDP overexpression have been observed. Dissection of the T1/T2 region of the thoracic ganglia has revealed loss of these neurons. To counter the defects in this fly model of ALS-TDP, the therapeutic effects of the autophagy activator rapamycin were examined. Although harmful to the control flies, administration of 400 muM rapamycin before the induction of dTDP overexpression can significantly reduce the number of neurons bearing dTDP + aggregates as well as partially rescue the diminished lifespan and locomotive defects of the ALS-TDP flies. Furthermore, S6K, a downstream mediator of TOR pathway, was identified as one genetic modifier of dTDP. In sum, this Drosophila model of ALS-TDP under temporal and spatial control presents a useful new genetic tool for the screening and validation of therapeutic drugs for ALS. Furthermore, the data support previous findings that autophagy activators including rapamycin are potential therapeutic drugs for the progression of neurodegenerative diseases with TDP-43 proteinopathies.

Coyne, A. N., Yamada, S. B., Siddegowda, B. B., Estes, P. S., Zaepfel, B. L., Johannesmeyer, J. S., Lockwood, D. B., Pham, L. T., Hart, M. P., Cassel, J. A., Freibaum, B., Boehringer, A. V., Taylor, J. P., Reitz, A. B., Gitler, A. D. and Zarnescu, D. C. (2015). Fragile X protein mitigates TDP-43 toxicity by remodeling RNA granules and restoring translation. Hum Mol Genet. PubMed ID: 26385636
RNA dysregulation is a newly recognized disease mechanism in amyotrophic lateral sclerosis (ALS). This study identified Drosophila fragile X mental retardation protein (dFMRP) as a robust genetic modifier of TDP-43-dependent toxicity in a Drosophila model of ALS. 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. These 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.

Cragnaz, L., Klima, R., De Conti, L., Romano, G., Feiguin, F., Buratti, E., Baralle, M. and Baralle, F. E. (2015). An age-related reduction of brain TBPH/TDP-43 levels precedes the onset of locomotion defects in a Drosophila ALS model. Neuroscience 311: 415-421. PubMed ID: 26518462
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. The average age of onset of both sporadic and familial cases is 50-60 years of age. The presence of cytoplasmic inclusions of the RNA-binding protein TAR DNA-binding protein-43 (TDP-43) in the affected neurons is seen in 95% of the ALS cases, which results in TDP-43 nuclear clearance and loss of function. The Drosophila melanogaster ortholog of TDP-43 (TBPH) shares many characteristics with the human protein. Using a TDP-43 aggregation inducer previously developed in human cells, a transgenic fly was created that shows an adult locomotive defect. Phenotype onset correlates with a physiologically age-related drop of TDP-43/TBPH mRNA and protein levels, seen both in mice and flies. Artificial reduction of mRNA levels, in vivo, anticipates the locomotion defect to the larval stage. This study links, for the first time, aggregation and the age-related, evolutionary conserved reduction of TDP-43/TBPH levels with the onset of an ALS-like locomotion defect in a Drosophila model. A similar process might trigger the human disease.

Li, S., et al. (2016). Genetic interaction of hnRNPA2B1 and DNAJB6 in a Drosophila model of multisystem proteinopathy. Hum Mol Genet [Epub ahead of print]. PubMed ID: 26744327
This study sought to establish a mechanistic link between diseases caused by mutations in two genes associated with adult-onset inherited myopathies, hnRNPA2B1 and DNAJB6. Hrb98DE and mrj are the Drosophila homologs of human hnRNPA2B1 and DNAJB6, respectively. Disease-homologous mutations were introduced to Hrb98DE. Ectopic expression of the disease-associated mutant form of hnRNPA2B1 or Hrb98DE in fly muscle resulted in progressive, age-dependent cytoplasmic inclusion pathology, as observed in humans with hnRNPA2B1-related myopathy. Cytoplasmic inclusions consisted of hnRNPA2B1 or Hrb98DE protein in association with the stress granule marker ROX8 and additional endogenous RNA-binding proteins, suggesting that these pathological inclusions are related to stress granules. Notably, TDP-43 was also recruited to these cytoplasmic inclusions. Remarkably, overexpression of MRJ rescued this phenotype and suppressed the formation of cytoplasmic inclusions, whereas reduction of endogenous MRJ by a classical loss of function allele enhanced it. Moreover wild-type, but not disease-associated mutant forms of MRJ, interacted with RNA-binding proteins after heat shock and prevented their accumulation in aggregates. These results indicate both genetic and physical interaction between disease-linked RNA-binding proteins and DNAJB6/mrj, suggesting etiologic overlap between the pathogenesis of hIBM and LGMD initiated by mutations in hnRNPA2B1 and DNAJB6.

Xia, Q., Wang, H., Hao, Z., Fu, C., Hu, Q., Gao, F., Ren, H., Chen, D., Han, J., Ying, Z. and Wang, G. (2016). TDP-43 loss of function increases TFEB activity and blocks autophagosome-lysosome fusion. EMBO J 35: 121-142. PubMed ID: 26702100
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by selective loss of motor neurons in brain and spinal cord. TAR DNA-binding protein 43 (TDP-43) was identified as a major component of disease pathogenesis in ALS, frontotemporal lobar degeneration (FTLD), and other neurodegenerative disease. Despite the fact that TDP-43 is a multi-functional protein involved in RNA processing and a large number of TDP-43 RNA targets have been discovered, the initial toxic effect and the pathogenic mechanism underlying TDP-43-linked neurodegeneration remain elusive. This study found that loss of TDP-43 strongly induced a nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy, through targeting the mTORC1 key component raptor. This regulation in turn enhanced global gene expressions in the autophagy-lysosome pathway (ALP) and increased autophagosomal and lysosomal biogenesis. However, loss of TDP-43 also impaired the fusion of autophagosomes with lysosomes through dynactin 1 downregulation, leading to accumulation of immature autophagic vesicles and overwhelmed ALP function. Importantly, inhibition of mTORC1 signaling by rapamycin treatment aggravated the neurodegenerative phenotype in a TDP-43-depleted Drosophila model, whereas activation of mTORC1 signaling by PA treatment ameliorated the neurodegenerative phenotype. Taken together, these data indicate that impaired mTORC1 signaling and influenced ALP may contribute to TDP-43-mediated neurodegeneration (Xia, 2016).
Deshpande, M., Feiger, Z., Shilton, A. K., Luo, C. C., Silverman, E. and Rodal, A. A. (2016). Role of BMP receptor traffic in synaptic growth defects in an ALS model. Mol Biol Cell [Epub ahead of print]. PubMed ID: 27535427
TAR DNA-binding protein 43 (TDP-43) is genetically and functionally linked to Amyotrophic Lateral Sclerosis (ALS), and regulates transcription, splicing, and transport of thousands of RNA targets that function in diverse cellular pathways. In ALS, pathologically altered TDP-43 is thought to lead to disease by toxic gain-of-function effects on RNA metabolism, as well as by sequestering endogenous TDP-43 and causing its loss of function. However, it remains unclear which of the numerous cellular processes disrupted downstream of TDP-43 dysfunction lead to neurodegeneration. This study found that both loss- and gain-of-function of TDP-43 in Drosophila cause a reduction of synaptic-growth-promoting Bone Morphogenic Protein (BMP) signaling at the neuromuscular junction (NMJ). Further, a shift of BMP receptors from early to recycling endosomes was observed along with increased mobility of BMP receptor-containing compartments at the NMJ. Inhibition of the recycling endosome GTPase Rab11 partially rescued TDP-43-induced defects in BMP receptor dynamics and distribution, and suppressed BMP signaling, synaptic growth, and larval crawling defects. These results indicate that defects in receptor traffic lead to neuronal dysfunction downstream of TDP-43 misregulation, and that rerouting receptor traffic may be a viable strategy for rescuing neurological impairment.
Swain, A., Misulovin, Z., Pherson, M., Gause, M., Mihindukulasuriya, K., Rickels, R. A., Shilatifard, A. and Dorsett, D. (2016). Drosophila TDP-43 RNA-binding protein facilitates association of sister chromatid cohesion proteins with genes, enhancers and polycomb response elements. PLoS Genet 12: e1006331. PubMed ID: 27662615
The cohesin protein complex mediates sister chromatid cohesion and participates in transcriptional control of genes that regulate growth and development. Substantial reduction of cohesin activity alters transcription of many genes without disrupting chromosome segregation. Drosophila Nipped-B protein loads cohesin onto chromosomes, and together Nipped-B and cohesin occupy essentially all active transcriptional enhancers and a large fraction of active genes. It is unknown why some active genes bind high levels of cohesin and some do not. This study shows that the TBPH and Lark RNA-binding proteins influence association of Nipped-B and cohesin with genes and gene regulatory sequences. In vitro, TBPH and Lark proteins specifically bind RNAs produced by genes occupied by Nipped-B and cohesin. By genomic chromatin immunoprecipitation these RNA-binding proteins also bind to chromosomes at cohesin-binding genes, enhancers, and Polycomb response elements (PREs). RNAi depletion reveals that TBPH facilitates association of Nipped-B and cohesin with genes and regulatory sequences. Lark reduces binding of Nipped-B and cohesin at many promoters and aids their association with several large enhancers. Conversely, Nipped-B facilitates TBPH and Lark association with genes and regulatory sequences, and interacts with TBPH and Lark in affinity chromatography and immunoprecipitation experiments. Blocking transcription does not ablate binding of Nipped-B and the RNA-binding proteins to chromosomes, indicating transcription is not required to maintain binding once established. These findings demonstrate that RNA-binding proteins help govern association of sister chromatid cohesion proteins with genes and enhancers.
Laneve, P., Piacentini, L., Casale, A.M., Capauto, D., Gioia, U., Cappucci, U., Di Carlo, V., Bozzoni, I., Di Micco, P., Morea, V., Di Franco, C.A. and Caffarelli, E. (2017). Drosophila CG3303 is an essential endoribonuclease linked to TDP-43-mediated neurodegeneration. Sci Rep 7: 41559. PubMed ID: 28139767
Endoribonucleases participate in almost every step of eukaryotic RNA metabolism, acting either as degradative or biosynthetic enzymes. The founding member of the Eukaryotic EndoU ribonuclease family, whose components display unique biochemical features and are flexibly involved in important biological processes, such as ribosome biogenesis, tumorigenesis and viral replication, has been previously identified. This study describes the discovery of the CG3303 gene product, which was named DendoU, as a novel family member in Drosophila. Functional characterisation revealed that DendoU is essential for Drosophila viability and nervous system activity. Pan-neuronal silencing of dendoU results in immature fly phenotypes, highly reduced lifespan and dramatic motor performance defects. Neuron-subtype selective silencing shows that DendoU is particularly important in cholinergic circuits. At the molecular level, DendoU positively regulates the neurodegeneration-associated protein dTDP-43, whose downregulation recapitulates the ensemble of dendoU-dependent phenotypes. These data unveil a relevant role for DendoU in Drosophila nervous system physio-pathology and highlight that DendoU-mediated neurotoxicity is, at least in part, contributed by dTDP-43 loss-of-function.

Ishiguro, T., et al. (2017). Regulatory role of RNA chaperone TDP-43 for RNA misfolding and repeat-associated translation in SCA31. Neuron 94(1): 108-124.e107. PubMed ID: 28343865
Microsatellite expansion disorders are pathologically characterized by RNA foci formation and repeat-associated non-AUG (RAN) translation. However, their underlying pathomechanisms and regulation of RAN translation remain unknown. This study reports that expression of expanded UGGAA (UGGAAexp) repeats, responsible for spinocerebellar ataxia type 31 (SCA31) in Drosophila, causes neurodegeneration accompanied by accumulation of UGGAAexp RNA foci and translation of repeat-associated pentapeptide repeat (PPR) proteins, consistent with observations in SCA31 patient brains. Motor-neuron disease (MND)-linked RNA-binding proteins (RBPs), TDP-43, FUS, and hnRNPA2B1, bind to and induce structural alteration of UGGAAexp. These RBPs suppress UGGAAexp-mediated toxicity in Drosophila by functioning as RNA chaperones for proper UGGAAexp folding and regulation of PPR translation. Furthermore, nontoxic short UGGAA repeat RNA suppressed mutated RBP aggregation and toxicity in MND Drosophila models. Thus, functional crosstalk of the RNA/RBP network regulates their own quality and balance, suggesting convergence of pathomechanisms in microsatellite expansion disorders and RBP proteinopathies.
Appocher, C., Mohagheghi, F., Cappelli, S., Stuani, C., Romano, M., Feiguin, F. and Buratti, E. (2017). Major hnRNP proteins act as general TDP-43 functional modifiers both in Drosophila and human neuronal cells. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 28575377
Nuclear factor TDP-43 is known to play an important role in several neurodegenerative pathologies. In general, TDP-43 is an abundant protein within the eukaryotic nucleus that binds to many coding and non-coding RNAs and influence their processing. Using Drosophila, a functional screening was performed to establish the ability of major hnRNP proteins to affect TDP-43 overexpression/depletion phenotypes. Interestingly, lowering hnRNP and TDP-43 expression has a generally harmful effect on flies locomotor abilities. In parallel, this study has also identified a distinct set of hnRNPs that is capable of powerfully rescuing TDP-43 toxicity in the fly eye (Hrb27c, CG42458, Glo and Syp). Most importantly, removing the human orthologs of Hrb27c (DAZAP1) in human neuronal cell lines can correct several pre-mRNA splicing events altered by TDP-43 depletion. Moreover, using RNA sequencing analysis, DAZAP1 and TDP-43 were shown too co-regulate an extensive number of biological processes and molecular functions potentially important for the neuron/motor neuron pathophysiology. These results suggest that changes in hnRNP expression levels can significantly modulate TDP-43 functions and affect pathological outcomes.
Lembke, K. M., Scudder, C. and Morton, D. B. (2017). Restoration of motor defects caused by loss of Drosophila TDP-43 by expression of the voltage-gated calcium channel, Cacophony, in central neurons. J Neurosci 37(39): 9486-9497. PubMed ID: 28847811
Defects in the RNA-binding protein, TDP-43, are known to cause a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar dementia. A variety of experimental systems have shown that neurons are sensitive to TDP-43 expression levels, yet the specific functional defects resulting from TDP-43 dysregulation have not been well described. Using the Drosophila TDP-43 ortholog TBPH, it has been shown that TBPH-null animals display locomotion defects as third instar larvae. Furthermore, loss of TBPH caused a reduction in cacophony, a Type II voltage-gated calcium channel, expression and that genetically restoring cacophony in motor neurons in TBPH mutant animals was sufficient to rescue the locomotion defects. The present study examined the relative contributions of neuromuscular junction physiology and the motor program to the locomotion defects and identified subsets of neurons that require cacophony expression to rescue the defects. At the neuromuscular junction, it was shown that mEPP amplitudes and frequency require TBPH. Cacophony expression in motor neurons rescued mEPP frequency but not mEPP amplitude. It was also shown that TBPH mutants displayed reduced motor neuron bursting and coordination during crawling and restoring cacophony selectively in two pairs of cells located in the brain, the AVM001b/2b neurons, also rescued the locomotion and motor defects, but not the defects in neuromuscular junction physiology. These results suggest that the behavioral defects associated with loss of TBPH throughout the nervous system can be associated with defects in a small number of genes in a limited number of central neurons, rather than peripheral defects.


TDP-43 is an evolutionarily conserved RNA binding protein recently associated with the pathogenesis of different neurological diseases. At the moment, neither its physiological role in vivo nor the mechanisms that may lead to neurodegeneration are well known. Previous studies have shown that TDP-43 mutant flies presented locomotive alterations and structural defects at the neuromuscular junctions (Feiguin, 2009). This study investigated the functional mechanism leading to these phenotypes by screening several factors known to be important for synaptic growth or bouton formation. As a result it was found that alterations in the organization of synaptic microtubules correlate with reduced protein levels in the microtubule associated protein Futsch/MAP1B. Moreover, TDP-43 was found to physically interact with futsch mRNA and that its RNA binding capacity is required to prevent futsch down-regulation and synaptic defects (Godena, 2011).

TDP-43 is an RNA binding protein of 43 kDa that belongs to the hnRNP family and plays numerous roles in mRNA metabolism such as transcription, pre-mRNA splicing, mRNA stability, microRNA biogenesis, transport and translation. TDP-43 is very well conserved during the evolution, especially with regards to the two RNA-recognition motifs (RRMs), the first (RRM1) being responsible for the binding of TDP-43 with UG rich RNA. In consonance with these described functions, TDP-43 prevalently resides in the cell nucleus where it co-localizes with other members of the RNA processing machinery. Nevertheless, in pathological conditions such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), TDP-43 appears in the form of large insoluble protein aggregates redistributed within the cytoplasm. At the moment, however, it is not clear how these alterations may lead to neurodegeneration. In theory, the cytosolic accumulation of TDP-43 may induce a toxic, gain of function effect on motoneurons while the exclusion of TDP-43 from the cell nucleus may lead to neurodegeneration due to a loss of function mechanism. At present, several lines of evidence mainly from different cellular and animal models support either view suggesting that both models may be acting to lead the disease condition. Recently, to determine the physiological role of TDP-43 in vivo it has been reported that the flies which lack the TDP-43 homologue (TBPH) closely reproduce many of the phenotypes observed in ALS patients, such as progressive defects in the animal locomotion and reduced life span. Moreover, it was observed that loss of TDP-43 function in Drosophila results in reduced number of motoneurons terminal branches and synaptic boutons at neuromuscular junctions (NMJs), indicating TDP-43 may regulate the assembly and organization of these structures. In coincidence with that, it should be noted that overexpression of TDP-43 in Drosophila has been reported to increase dendritic branching (Lu, 2009), lead to motor dysfunction and reduced life span, axon loss and neuronal death, is generally toxic regardless of inclusion formations, and at least in part is the cause behind the degeneration associated with TER94 mutations which is the Drosophila homologue of the VCP protein. Taken together, and in consideration that Drosophila TDP-43 (TBPH) can functionally substitute for human TDP-43 in functional splicing assays, these reports confirm that Drosophila may represent a highly suitable animal model to investigate TDP-43 functions both in normal and disease conditions (Godena, 2011).

Drosophila larval NMJ is a well-characterized system to analyze the cellular and molecular events that are involved in synapse development and plasticity. Synaptic growth during larval development is expanded according to muscle size and is accomplished by the addition of new boutons to the existing presynaptic terminals. Typically, defects in synapse formation and synaptic growth are linked to cytoskeleton abnormalities, since the synaptic boutons and the newly formed buds require the underlying presynaptic microtubules to maintain their structural organization and plasticity inside the innervated muscles. Thus, to determine the physiological role of TDP-43 in vivo and the pathological consequences of its altered function, the molecular organization of Drosophila NMJs during larval development was examined in TDP-43 minus flies (Godena, 2011).

This work, shows that in TBPH minus Drosophila model the changes observed at the level of NMJs and synaptic boutons formation can be explained by defects at the cytoskeleton level, which in turn are mediated by a down-regulation of the futsch protein (but not mRNA). These results provide additional insight with regards to potential disease mechanisms mediated by TDP-43 and considerably extend knowledge with regards to defining the basic molecular functions of this protein. Future work will be aimed at better characterizing more in depth the functional mechanism through which TBPH regulates futsch protein levels and how these results can be extended to the human disease model (Godena, 2011).

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. This study provides evidence that Drosophila 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 higher levels of transcript were found 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. futsch overexpression was shown to be 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 these observations in Drosophila motor neurons. Together, these results suggest a microtubule-dependent mechanism in motor neuron disease caused by TDP-43-dependent alterations in futsch mRNA localization and translation in vivo (Coyne, 2014).

TDP-43, an RNA-binding protein linked to a significant fraction of ALS cases, associates with futsch mRNA in a complex in vivo and regulates its localization and translation in Drosophila motor neurons. Using polysome fractionations, wild-type and disease-associated mutant TDP-43 was shown to cofractionate with both the untranslated fractions, namely RNPs and ribosomal subunits, and actively translating polyribosomes. These results add translation regulation to TDP-43's plethora of known roles in RNA processing, such as transcription, splicing, and mRNA transport, and suggest that TDP-43 contributes to the pathophysiology of ALS via multiple RNA-based mechanisms (Coyne, 2014).

These data provide the first in vivo demonstration that TDP-43 associates with polysomes and regulates the translation of futsch mRNA. These findings are consistent with previous reports that futsch mRNA coimmunoprecipitates with Drosophila TBPH, and the mammalian homolog, MAP1B, is a candidate target of TDP-43 in mouse. The results showing a decrease in Futsch levels at the NMJ and an increase in Futsch levels in motor neuron cell bodies suggest a model whereby futsch/MAP1B mRNA may not be properly transported into axons. This is substantiated by qPCR from ventral ganglia where futsch mRNA is found at higher levels than at the NMJ compared with controls. Although it cannot be excluded that TDP-43 regulates futschmRNA stability, given the more pronounced reduction in protein versus transcript levels at the NMJ compared with cell bodies and the shift to untranslated fractions in polysomes, these data suggest TDP-43-dependent defects in futsch/MAP1B mRNA transport and protein expression at the NMJ. Although the underlying cause remains unknown, Futsch protein levels are also reduced at the NMJ in the absence of TBPH (the Drosophila homolog of TDP-43), suggesting a loss-of-function mechanism for the disease. Interestingly, there is a marked increased in futsch mRNA in motor neuron cell bodies in ventral ganglia relative to Futsch protein levels. This suggests that TDP-43 also regulates the expression of Futsch in motor neuron cell bodies possibly through sequestration into RNP complexes. Although cytoplasmic TDP-43 puncta were not seen in vivo, a plausible scenario is that TDP-43-containing complexes are below the limit of resolution of the confocal microscope, as suggested by cellular fractionation experiments indicating the presence of cytoplasmic TDP-43. Since futsch is the Drosophila homolog of MAP1B and MAP1B mRNA has been identified in TDP-43-containing RNP complexes in mouse models, these results predict that MAP1B and microtubule-based processes may also be affected in ALS patient tissues. Indeed, similar to the current results in the fly, immunohistochemistry experiments reveal a significant accumulation of MAP1B in motor neuron cell bodies in ALS spinal cords compared with controls but not in the hippocampus. Although these alterations may be the result of ongoing neurodegeneration, the remarkable similarities with the fly model suggest that comparable defects in transport and translation processes may occur in the human disease. It is interesting to note that spinal cord motor neurons in the patient with the C9ORF72 repeat expansion failed to contain MAP1B immunostaining in the cell body, suggesting altered pathogenic mechanisms from sporadic ALS patients, though further studies with an increased number of patients within this repeat expansion are required to confirm this finding (Coyne, 2014).

Interestingly, Futsch protein expression is similarly inhibited by wild-type or mutant TDP-43, supporting a scenario in which MAP1B dysregulation may be a shared feature of ALS cases with TDP-43-positive pathology, regardless of etiology. It is possible that other targets, which remain to be identified, are regulated in a variant-dependent manner. Indeed, RNA sequencing experiments from wild-type and mutant polysome fractions indicate several distinct, up- and down-translated mRNAs. Perhaps TDP-43's role in translation is not surprising as it has previously been shown to associate with stress granules (SGs), which are known to sequester mRNAs and inhibit their translation during environmental stress. Although TDP-43 does not seem to be required for SG formation, ALS-linked mutations in TDP-43 were shown to alter the dynamics of SG assembly and disassembly. This is consistent with previous findings that the molecular mobility of wild-type TDP-43 differs from that of the mutant variants in primary motor neurons. Furthermore, TDP-43-containing cytoplasmic aggregates can 'evolve' from paraquat-induced SG. Together, these findings suggest a scenario whereby, in response to stress, possibly caused by aging or environmental factors, TDP-43 localizes to cytoplasmic SGs that in turn lead to altered ribostasis, including abnormal futsch mRNA expression as demonstrated by these experiments (Coyne, 2014).

Using genetic interaction approaches, this study shows that futsch is a physiologically significant RNA target of TDP-43 and can alleviate locomotor dysfunction and increase life span. At the structural level, Futsch protein shares homology with mammalian MAP1B as well as neurofilaments, which do not exist per se in Drosophila. Interestingly, TDP-43 was shown to bind, transport, and sequester neurofilament light (NEFL) mRNA into SG, which contributes to ALS-like phenotypes in motor neurons. Given Futsch's known requirement in axonal and dendritic development and the organization of microtubules at the synapse, the current findings suggest that these processes may be involved in the pathophysiology of ALS. Indeed, recent studies in SOD1- and TDP-43-based models of ALS demonstrate an impairment in microtubule-based axonal transport (Coyne, 2014).

Consistent with previous studies in which tubulin acetylation has been shown to rescue transport defects in neurodegeneration, these data show that TDP-43 leads to reduced levels of acetylated tubulin, and this is rescued by futsch overexpression. Other TDP-43 targets such as HDAC6, which is regulated by TDP-43 at the level of transcription, are also linked to microtubule stability, providing additional support to the notion that microtubule stability is an important factor mediating TDP-43 toxicity. It is possible that microtubule stability is regulated locally by an interplay between Futsch and HDAC6 at the NMJ. Additionally, microtubule integrity and stress granules have been intricately linked. It has been demonstrated that microtubule integrity is important for the transportation and disassembly of stress granules. Also, SGs can be cleared by autophagy, and microtubules are used to transport autophagosomes to the microtubule organizing center where they fuse with lysosomes. Notably, microtubule stability can promote transport by association of motors that preferentially bind acetylated microtubules. Futsch is often associated with the stabilization of microtubules at the synapse through the formation of loop structures as this study has seen in the context of TDP-43. Therefore, restoration of Futsch levels and microtubule stability may mitigate TDP-43 toxicity through multiple mechanisms, including restoring transport of target mRNAs essential for synaptic stability and transport, disassembly, and/or clearance of stress granules, which in turn may interfere with aggregate formation as suggested by the observed decrease in insoluble TDP-43. In conclusion, futsch has been identified as a disease-relevant and functionally significant post-transcriptional target of TDP-43. Given the role of futsch/MAP1B in microtubule and synaptic stabilization, the current findings point to microtubule-based processes as targets for the development of therapeutic strategies for TDP-43 proteinopathies (Coyne, 2014).

Reduced TDP-43 expression improves neuronal activities in a Drosophila model of Perry syndrome

Parkinsonian Perry syndrome, involving mutations in the dynein motor component dynactin or p150Glued, is characterized by TDP-43 pathology in affected brain regions, including the substantia nigra. However, the molecular relationship between p150Glued and TDP-43 is largely unknown. This study reports that a reduction in TDP-43 protein levels alleviates the synaptic defects of neurons expressing the Perry mutant p150G50R in Drosophila. Dopaminergic expression of p150G50R, which decreases dopamine release, disrupts motor ability and reduces the lifespan of Drosophila. p150G50R expression also causes aggregation of dense core vesicles (DCVs), which contain monoamines and neuropeptides, and disrupts the axonal flow of DCVs, thus decreasing synaptic strength. The above phenotypes associated with Perry syndrome are improved by the removal of a copy of Drosophila TDP-43, TBPH, thus suggesting that the stagnation of axonal transport by dynactin mutations promotes TDP-43 aggregation and interferes with the dynamics of DCVs and synaptic activities (Hosaka, 2017)

Perry syndrome (PS) is an autosomal dominant disorder characterized by parkinsonism with depression, sleep disturbance, weight loss, and central hypoventilation. Genome-wide linkage analysis has identified disease-segregating missense mutations located in the dynactin (DCTN1) gene. The gene product of dynactin, p150Glued, forms a complex with dynein, the microtubule-dependent retrograde motor. Disease-associated missense mutations (G71R, G71E, G71A, T72P, Q74P) are located in the cytoskeleton-associated protein Gly-rich (CAP-Gly) domain of p150Glued, which has been implicated in binding to microtubules recruiting dynein and stabilizing the plus-end of microtubules. A glycine to serine substitution at residue 59 (G59S), which causes distal hereditary motor neuropathy 7B (HMN7B), appears to affect the structure of the CAP-Gly domain and produces severe synaptic phenotypes, including p150Glued aggregation, dynein accumulation at nerve terminals and disruption of axonal transport. Mutations associated with PS show milder synaptic phenotypes but cause impaired retrograde flux (Hosaka, 2017)

The TAR DNA-binding protein of 43 kDa (TDP-43) is a highly conserved heterogeneous ribonucleoprotein (hnRNP) involved in the transcription, splicing, stabilization and transport of specific mRNAs. TDP-43 has been identified as the key component of intracellular ubiquitin-positive inclusions observed in affected brain areas of patients with amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and Alzheimer's disease. A pathological feature of PS is the accumulation of TDP-43 in affected areas. Increased TDP-43 is toxic to neurons, possibly because of its proneness to aggregation and conversion to an abnormal protein structure similar to that of prion and α-Synuclein. On the other hand, TDP-43 is indispensable to mouse development and Drosophila survival , thus suggesting that the control of appropriate protein levels is critical for TDP-43 function (Hosaka, 2017)

The molecular relationship between p150Glued and TDP-43 is largely unknown. To determine whether TDP-43 contributes to PS phenotypes, TDP-43 protein levels wetr manipulated in a Drosophila PS model, and reduced TDP-43 was found to improve defects in axonal transport and the synaptic activity of central dopaminergic neurons, as well as motor neurons, caused by the neuron-specific expression of a PS-associated p150Glued mutation (Hosaka, 2017)

Whereas mutations of p150Glued cause PS, in which TDP-43 pathology has been reported in the basal ganglia, including the substantia nigra, whether TDP-43 contributes to neurodegeneration in PS remained unknown. This study analyzed the molecular relationship between p150Glued and TDP-43 in a Drosophila PS model (Hosaka, 2017)

Neuronal expression of the p150G50R mutation, located in the CAP-Gly domain, led to swelling of distal boutons and the prominent aggregation of DCVs but not mitochondria in axons and synapses. DCVs travel between proximal axons and terminal boutons via axonal transport, which efficiently delivers neurotransmitters to synaptic boutons. Although p150G50R disrupts both the anterograde and retrograde flow of DCVs, the anterograde/retrograde ratios of moving DCVs suggested that the retrograde flow is especially affected by p150G50R, thus leading to accumulation of organelles and materials at nerve terminals. Consistently with the morphological abnormalities in synaptic phenotypes, synaptic strength, dopamine release and motor ability were decreased by p150G50R expression without detectable neuron loss. Although p150WT expression also affected the retrograde flow of DCVs and the DCV distribution at the terminal boutons, the phenotypes were milder than those of p150G50R. Given that p150WT protein was expressed at higher levels than p150G50R in transgenic flies, in which both p150WT and p150G50R transgenes were inserted in the same genomic locus and both transcript levels were similar, p150G50R exerts highly deleterious effects on neuronal activity despite its unstable expression. Thus, the data obtained with p150G50R expression would reflect synaptic dysfunction as an early neurodegeneration event in PS (Hosaka, 2017)

Altered mitochondrial distribution at the boutons by p150G50R was not significantly rescued by TBPH reduction, thus suggesting that the improvement of DCV phenotypes markedly contributes to the rescue effects in neuronal functions and survival. However, it cannot be excluded that mitochondrial functions may be affected in p150G50R flies, because the fluorescence intensity of mitoGFP (cytochrome C oxidase subunit VIII-GFP fusion protein), which inserts in the mitochondrial inner membrane in a membrane potential-dependent manner, was somewhat reduced by p150G50R. Ultrastructural analysis of synaptic mitochondria also suggested that mitochondrial cristae were partly damaged in p150G50R flies. Because mitochondrial shuttling between cell bodies and nerve terminals in neurons would be important to maintain mitochondrial proteins derived from the nuclear genome, which include the respiratory complex I, III, IV and V subunits, the effects of PS mutations on mitochondrial functions at nerve terminals should be examined in future studies (Hosaka, 2017)

TDP-43 accumulation in affected regions is a prominent feature of PS pathology. TDP-43 forms cytoplasmic messenger ribonucleoprotein (mRNP) granules, which also move via axonal transport to synaptic boutons to deliver mRNA for synaptic activities. The discovery in Drosophila that the ablation of a copy of the TBPH gene improves the axonal aggregation of DCVs and synaptic defects provides three possible molecular mechanisms: First, a reduced concentration of TBPH in axons and nerve terminals improves axonal flow, suppressing the aggregation of TBPH. Second, TBPH reduction alters the expression of proteins regulated by TBPH at transcript levels, thus alleviating synaptic dysfunctions. Third, the above two mechanisms contribute to rescue effects. The third mechanism is preferred for the reasons listed below (Hosaka, 2017)

The DCV aggregates in axons and synapses were suppressed by TBPH reduction without improving the velocity of axonal transport, thus suggesting that aggregation-prone TBPH promotes DCV aggregation when the axonal flow stagnates. However, the numbers of DCVs and SVs were increased in the synaptic boutons of TBPH+/− flies, thus suggesting that TBPH negatively regulates DCV and SV production. A variety of TBPH target mRNAs have been reported in Drosophila, and further studies may reveal a target(s) to regulate DCVs and SVs. Regarding synaptic stabilization, microtubule-associated MAP1B/Futsch is an evolutionarily conserved target of TDP-43/TBPH, which negatively or positively regulates synaptic MAP1B/Futsch expression and might maintain axonal transport through regulating microtubule dynamics. Although no obvious changes were detected in the levels of Futsch protein in TBPH+/− flies, the downregulation of Futsch/MAP1B may explain the observation that some phenotypes in TBPH+/− flies were reversed by p150G50R expression. Because p150Glued stabilizes the plus ends of microtubules, the ectopic expression of p150G50R may partially alleviate the microtubule destabilization caused by Futsch/MAP1B downregulation. Alternatively, the decreased axonal flow resulting from p150G50R expression may enable efficient synaptic capture of TDP-43-mRNP granules, which regulate local protein translation for synaptic activity including Futsch/MAP1B (Hosaka, 2017).

Autoregulation of TDP-43 mRNA has been demonstrated in mammals and has been suggested in Drosophila. Consistently with these reports, the decrease in TBPH protein was at most 30% in TBPH+/− flies. Although the genetic ablation of a copy of the TBPH gene in itself produced some synaptic phenotypes and decreased the lifespan in flies, the transient knockdown of TDP-43 would be a suitable strategy for therapeutic intervention in PS. It has been demonstrated that the transient inhibition of a truncated N-terminal huntingtin with an abnormal polyQ stretch improves the neuropathology and the motor phenotype in a Huntington's disease mouse model. Thus, disease phenotypes caused by TDP-43 proteinopathies may be reversible under appropriate levels of TDP-43 control (Hosaka, 2017).

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). This study identified Drosophila Fragile X Mental Retardation Protein (dFMRP) as a robust genetic modifier of TDP-43 dependent toxicity in a Drosophila model of ALS. dFMRP overexpression 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 (PABP) 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 overexpression also relieves the translation inhibition of futsch mRNA, a TDP-43 target mRNA, which regulates neuromuscular synapse architecture. Restoration of futsch translation by dFMRP overexpression mitigates Futsch dependent morphological phenotypes at the neuromuscular junction including synaptic size and presence of satellite boutons. These data suggest a model whereby dFMRP is neuroprotective by remodeling TDP-43 containing RNA granules, reducing aggregation and restoring the translation of specific mRNAs (Coyne, 2015).

This study used a combination of genetic and molecular approaches to uncover a novel functional interaction between dFMRP and TDP-43. Taken together, the results support a model whereby dFMRP, a well established translational regulator, can modulate the neurotoxicity caused by TDP-43 overexpression. When overexpressed, dFMRP decreases the association of TDP-43 with the aggregate-like fraction. Together with immunoprecipitation and binding experiments, these findings support a model whereby dFMRP promotes the remodeling of the RNP by 'extracting' TDP-43 and freeing the sequestered mRNA from the protein-RNA complex. This in turn may alleviate the negative impact that TDP-43 exerts on its mRNA targets as is the case for futsch mRNA. Indeed, dFMRP OE in the context of TDP-43 restores the expression of futsch, which is a translation target of TDP-43. While the change in Futsch expression is slight in magnitude, it is statistically significant. These findings suggest a scenario whereby the robust synaptic phenotypes observed in ALS may result from the combinatorial effect of decreased expression for multiple TDP-43 targets at the NMJ. In future studies it will be interesting to determine additional synaptic targets of TDP-43 whose expression is restored upon dFMRP OE. While futsch mRNA can be translationally controlled by both dFMRP and TDP-43, in the context of TDP-43 RNA granules, dFMRP appears to favor an association with TDP-43 protein over its translation target, leaving futsch mRNA available for protein synthesis, which explains the translation restoration observed in the context of dFMRP overexpression. Given the wide repertoire of RNA binding protein partners of TDP-43, it will be interesting in the future to determine whether others can also confer neuroprotection to TDP-43 dependent toxicity and whether they do so by a similar molecular mechanism. This would be expected given that Futsch expression is significantly increased but not fully restored by dFMRP OE at the NMJ (Coyne, 2015).

Previous studies have shown that TDPWT and disease linked mutations, although expressed at comparable levels, confer differential toxicity in various phenotypic assays. This study provides evidence that TDPWT and TDPG298S also interact differentially with protein partners. TDPG298S colocalizes with PABP to a lesser extent than TDPWT. Further evidence is provided that TDPWT and TDPG298S exhibit distinct molecular mobilities within neurites, which is consistent with previous reports that although wild-type and disease linked variants both associate with stress granules, their dynamics, persistence and size differ dramatically (Coyne, 2015).

Taken together, these findingssuggest that ALS may be a consequence of chronic translation inhibition. This could result from dysregulation of RNA granule physiology in the context of excess cellular stress as previously suggested. This scenario is consistent with previous findings that inhibition of SG is neuroprotective and provides a plausible mechanism for how TDP-43 mutations lead to disease. Additionally, it can explain the association of wild-type TDP-43 with cytoplasmic aggregates in the majority of ALS cases, regardless of etiology. One possibility is that, in the context of aging related or other cellular stress, wild-type TDP-43 enters the RNA stress granule cycle, contributing to translation inhibition and disease pathophysiology (Coyne, 2015).

The results indicate that FMRP remodels TDP-43 RNP granules and this restores futsch translation and expression at the NMJ. This in turn, can alleviate phenotypes associated with microtubule instability such as the presence of satellite boutons. Altered microtubule stability is emerging as a prominent pathological mechanism underlying the progression of ALS and may provide a useful avenue for the development of therapeutics. In addition, altered ribostasis has emerged as a major hypothesis for explaining the progression from RNA stress granules to aggregates seen in disease. This model suggests altered translational regulation as a molecular mechanism underlying disease progression. The current results support this model and provide evidence that mitigating translational repression can suppress disease phenotypes (Coyne, 2015).

In future studies it will be important to establish whether blanket approaches such as RNA SG inhibition or translation restoration offer more promise than targeted strategies based on specific targets. Two recent studies have shown that TDP-43 suppresses toxicity in CGG repeat expansion models of Fragile X associated tremor/ataxia syndrome (FXTAS). Removing a portion of the C-terminus of TDP- 43 in which interactions with hnRNP A2/B1 typically occur, abolishes the ability of TDP-43 to suppress toxicity. These results suggest that TDP-43 may work to mitigate CGG RNA toxicity via interactions with its protein partners by preventing them from sequestration into toxic RNA foci. Thus, in the case of CGG repeat disorders, TDP-43 may alter RNP complexes similar to how dFMRP overexpression alters RNP complexes in the TDP-43 model of ALS. Together with these studies, the current results provide evidence for common mechanisms underlying neurodegenerative diseases and repeat expansion disorders. In both cases, remodeling of RNP granules and the 'freeing' of RNA binding proteins or mRNA targets mitigates toxicity (Coyne, 2015).

In conclusion, this study identified a novel strategy for mitigating TDP-43 dependent phenotypes in vivo, based on FMRP mediated remodeling of RNA granules, which provides relief to chronic translation inhibition for specific mRNA targets such as futsch. The results suggest that targeting RNP remodeling or translation restoration may prove useful as therapeutic strategies. Future experiments are aimed at identifying additional translational targets of TDP-43 in vivo that will broaden the repertoire of therapeutic strategies for ALS and related neurodegenerative diseases (Coyne, 2015).

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. A Drosophila model has been developed of ALS based on TDP-43 that recapitulates several aspects of disease pathophysiology. Using this model, a drug screening strategy was designed based on the pupal lethality phenotype induced by TDP-43 when expressed in motor neurons. In screening 1,200 FDA approved compounds, the PPARgamma agonist pioglitazone was found to be neuroprotective in Drosophila. This study shows that pioglitazone can rescue TDP-43 dependent locomotor dysfunction in motor neurons and glia but not in muscles. Testing additional models of ALS it was found 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, this study showed that the predicted Drosophila PPARγ homologs, E75 and E78 are in vivo targets of pioglitazone. Finally, using a global metabolomic approach, a set of metabolites was identified that pioglitazone can restore in the context of TDP-43 expression in motor neurons. Taken together, these 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, these data also identifies several 'biomarkers' of the disease that may be useful in developing therapeutics and in future clinical trials (Joardar, 2014).

ALS is the third most common form of neurodegeneration following Alzheimer's and Parkinson's disease. Although Riluzole is approved for ALS patients, its benefits are marginal, and at this time, there are no known effective treatments for the disease. There have been several efforts to design therapeutics using the SOD1 mouse, the most commonly used animal model of ALS. However, despite promising preclinical results, these candidate drugs have been disappointing in humans. To address this significant issue, efforts are being made to develop other animal models of ALS that can be used not only to identify phenotypes and 'early biomarkers' of the disease but also will be useful in drug screens for therapeutic purposes. Previously work has generated a Drosophila model of ALS based on TDP-43, which recapitulates several aspects of the human disease including locomotor dysfunction and reduced lifespan Using this model, this study shows that the antidiabetic drug pioglitazone acts as a neuroprotectant for aspects of TDP-43 proteinopathy by activating the putative Drosophila PPARγ homologs E75 and E78. It was also shown that pioglitazone mitigates FUS but not SOD1-dependent toxicity in Drosophila, consistent with previous published work showing that distinct mechanisms are likely at work in the context of these different models of ALS. Interestingly, pioglitazone did not improve, and in some cases worsened, the lifespan of TDP-43-expressing flies, when administered either during development, or after 'disease onset', which is consistent with results from recent clinical trials. This apparent disconnect is consistent with the effects of pioglitazone on cellular metabolism. As described earlier, while pioglitazone treatment restored some metabolites altered owing to TDP-43 overexpression in motor neurons, others were unchanged or even worsened. This provides a potential explanation for why some phenotypes but not others are rescued by pioglitazone. Aside from the possibility that different drug concentrations may be needed, it remains unclear why pioglitazone is protective in mouse but not fly SOD1 models and, in retrospect, given the similarities between the effect of pioglitazone in Drosophila models of ALS and humans, the fly appears to be a more accurate predictor of clinical trial outcomes (Joardar, 2014).

It is tempting to speculate that the predictive power of the Drosophila model may lie in the tools that enable motor neuronal versus glial versus muscle-specific expression of the toxic TDP-43 protein. The current results show that pioglitazone mitigates neuronal and glial TDP-43-dependent toxicity but has no effect on the locomotor dysfunction caused by muscle-specific expression of TDP-43. This type of knowledge is easily obtainable in the fly model and can provide helpful information about cell autonomous versus non-autonomous effects as well as the efficacy of candidate drugs in different tissues of interest. While it was shown that pioglitazone reduces inflammation in the glia, its effects in neurons or muscles have not been studied in the mouse prior to human trial. The current results indicate that the protective effects of pioglitazone are specific to the nervous system and were not observed in muscles, at least within the limits of the experimental conditions (i.e., tissue-specific levels of expression and drug concentration). These findings suggest that future preclinical studies may benefit from testing candidate therapies in multiple disease models in which tissue specificity and several phenotypic outcomes are easily ascertained (Joardar, 2014).

Pioglitazone has been originally developed for the treatment of type 2 diabetes as PPARγ activation in the liver improves glucose metabolism systemically. In the nervous system, activation of the nuclear hormone receptor PPARγ has been shown to have anti-inflammatory and neuroprotective effects. In the current model, pioglitazone restored a rather limited set of metabolites altered in a TDP-43-dependent manner. Evidence was found evidence of altered glutamine/glutamate metabolism in TDPWT flies, as displayed by elevated levels of N-acetylglutamine, which is restored by pioglitazone. Excessive levels of extracellular glutamate in the central nervous system cause hyperexcitability of neurons, ultimately leading to their death. The glutamate transporter GLT1/EAAT2 plays a major role in maintaining extracellular glutamate levels below the excitotoxic concentrations by efficiently transporting this metabolite. Interestingly, astrocytic GLT1/EAAT2 gene is a target of PPARγ, leading to neuroprotection by increasing glutamate uptake. Furthermore, pyruvate, which is significantly high in both TDPWT and TDPG298S, shows a trend toward reduction upon pioglitazone treatment for TDPWT. Pyruvate is a central metabolite that lies at the junction of several intersecting cellular pathways including glucose and fatty acid metabolism. It is converted to oxaloacetate by the enzyme pyruvate carboxylase, which is a key step in lipogenesis. Interestingly, PPARγ, the target of pioglitazone, is a direct transcriptional modulator of the pyruvate carboxylase gene. Given the fact that ALS patients suffer from massive weight loss, this provides a possible explanation for the potential protective effects of pioglitazone through increased lipogenesis. Taken together, this metabolomics approach provides useful insights for understanding the molecular mechanisms underlying ALS pathophysiology. Interestingly, altered cellular metabolism has previously been implicated in ALS pathophysiology with patients exhibiting signs of hypermetabolism. Notably, the fly model also showed signs of hypermetabolism including an increase in pyruvate, a key metabolite linking glucose metabolism to the TCA cycle. Additionally, the ketone body GHB is reduced in the context of TDPWT, consistent with a clinical study showing that a ketogenic diet slowed ALS disease progression. Given the similarities between the metabolic profile of the Drosophila model and human samples, it will be interesting in the future, to design therapeutic approaches aimed at restoring these common metabolic changes using nutritional supplementation (Joardar, 2014).

In summary, these data show the potential of using the fly model of ALS as a rapid and efficacious system for drug screening in vivo. The results using FUS and SOD1 fly models of ALS indicate that pioglitazone is effective in mitigating some, but not all forms of the disease, which suggests that stratification of patient populations should be considered in future clinical trials. The primary endpoint tested in prior clinical trials, namely lifespan, was not improved by pioglitazone, which is consistent with the data in Drosophila. Although the results from the clinical trials have not shown much promise in ALS patients, the use of pioglitazone as a tool to dissect molecular mechanisms of the disease remains attractive. The metabolomic profiling with and without pioglitazone pinpoints pathways that could be targeted either by drugs or by diet modifications. Also, it is possible that chemical modifications of pioglitazone, which was optimized for adipose tissue, skeletal muscle and liver, are needed for increased efficacy in the nervous system. The protective effect of pioglitazone opens up avenues for designing small molecules with modifications around the basic structure of the drug, and testing their potential in vivo, in the fly model. Furthermore, clinical trials have not been stratified for TDP-43 pathology or mutations; thus, significant results may have been missed. The developmental and adult feeding experiments clearly demonstrate that locomotor function is improved by pioglitazone suggesting that despite its lack of effect on lifespan, PPARγ remains a molecular target with therapeutic potential, perhaps in combination with other strategies based on restoring the metabolic alterations caused by TDP-43 in the nervous system (Joardar, 2014).

The FTD/ALS-associated RNA-binding protein TDP-43 regulates the robustness of neuronal specification through microRNA-9a in Drosophila

TDP-43 is an evolutionarily conserved RNA-binding protein currently under intense investigation for its involvement in the molecular pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 is normally localized in the nucleus, but translocated to the cytoplasm in diseased neurons. The endogenous functions of TDP-43 in the nervous system remain poorly understood. This study shows that the loss of Drosophila TDP-43 (dTDP-43) results in an increased production of sensory bristles and sensory organ precursor (SOP) cells on the notum of some but not all flies. The location of ectopic SOPs varies among mutant flies. The penetrance of this novel phenotype is dependent on the gender and sensitive to environmental influences. A similar SOP phenotype was also observed on the wing and in the embryos. Overexpression of dTDP-43 causes both loss and ectopic production of SOPs. Ectopic expression of ALS-associated mutant human TDP-43 (hTDP-43(M337V) and hTDP-43(Q331K)) produces a less severe SOP phenotype than hTDP-43(WT), indicating a partial loss of function of mutant hTDP-43. In dTDP-43 mutants, miR-9a expression is significantly reduced. Genetic interaction studies further support the notion that dTDP-43 acts through miR-9a to control the precision of SOP specification. These findings reveal a novel role for endogenous TDP-43 in neuronal specification and suggest that the FTD/ALS-associated RNA-binding protein TDP-43 functions to ensure the robustness of genetic control programs (Li, 2013).

This study identified a novel function for the evolutionarily conserved RNA-binding protein TDP-43 in neural specification in Drosophila. Unlike several well-studied transcription factors in the Notch-Delta lateral inhibition pathway, TDP-43 seems to function as a 'robustness' factor in a manner similar to that of miR-9a. In the absence of TDP-43 activity, ectopic bristles appear at one of several locations and SOP specification becomes more sensitive to environmental influences. Thus, TDP-43 seems to serve as a 'gatekeeper' to ensure the reproducible execution of genetic control programs and to canalize developmental phenotypes (Li, 2013).

Since mRNA synthesis can fluctuate, regulation of mRNA metabolism plays a central role in gene expression. miRNAs are a key class of regulatory molecules that ensure the robustness of developmental programs and may play a key role in canalization, which is partly due to the modest effect of each miRNA on expression levels of multiple mRNA targets. Hundreds of RNA-binding proteins are encoded by the Drosophila genome or in other species, and many simultaneously regulate multiple mRNAs. It is likely some other RNA-binding proteins also serve as robustness factors through direct interactions with target mRNAs (Li, 2013).

The current findings show that the 'robustness' function of TDP-43 is mediated in part by miR-9a in one specific neural developmental process. The molecular functions of TDP-43 in development as revealed in this study may provide a new perspective on its endogenous role in neurodegeneration. Although the loss of TDP-43 leads to an early lethal phenotype in mouse embryos, loss of nuclear TDP-43 does not necessarily result in rapid neuronal cell death. Gene expression in individual cells is tightly regulated and also varies significantly, in part due to stochastic biochemical events. Chronic loss of nuclear TDP-43 or compromise of its buffering function by genetic mutations may cause an imbalance in protein homeostasis in human neurons before eventual neurodegeneration, during which miRNAs may play an essential role downstream of TDP-43. Both TDP-43 and miR-9 are highly conserved through evolution and their interaction may occur in mammalian cells as well. It is interesting to note that miR-9 is significantly downregulated in Huntington's disease and a mouse model of spinal motor neuron disease. Thus, downregulation of miR-9 and possibly other miRNAs as well in stressed neurons in which TDP-43 has been depleted from the nucleus may be a common contributing factor in different neurodegenerative disorders (Li, 2013).

HDAC6 is a Bruchpilot deacetylase that facilitates neurotransmitter release

Presynaptic densities are specialized structures involved in synaptic vesicle tethering and neurotransmission; however, the mechanisms regulating their function remain understudied. In Drosophila, Bruchpilot is a major constituent of the presynaptic density that tethers vesicles. This study shows that HDAC6 is necessary and sufficient for deacetylation of Bruchpilot. HDAC6 expression is also controlled by TDP-43, an RNA-binding protein deregulated in amyotrophic lateral sclerosis (ALS). Animals expressing TDP-43 harboring pathogenic mutations show increased HDAC6 expression, decreased Bruchpilot acetylation, larger vesicle-tethering sites, and increased neurotransmission, defects similar to those seen upon expression of HDAC6 and opposite to hdac6 null mutants. Consequently, reduced levels of HDAC6 or increased levels of ELP3, a Bruchpilot acetyltransferase, rescue the presynaptic density defects in TDP-43-expressing flies as well as the decreased adult locomotion. This work identifies HDAC6 as a Bruchpilot deacetylase and indicates that regulating acetylation of a presynaptic release-site protein is critical for maintaining normal neurotransmission (Miskiewicz, 2014).

This study finds that HDAC6 controls vesicle tethering and synaptic transmission by regulating BRP deacetylation, thereby antagonizing ELP3, a BRP acetyltransferase (Miśkiewicz, 2011). This work defines BRP as a deacetylation target of HDAC6. Acetylation of the C-terminal end of BRP results in more condensed T-bars, while deacetylation leads the protein to send excessive tentacles into the cytoplasm to contact more synaptic vesicles. Similar to chromatin structure being regulated by electrostatic mechanisms at the level of histone acetylation, it is proposed that electrostatic interactions between acetylated and deacetylated lysines in individual BRP strands regulate presynaptic density structure and function (Miskiewicz, 2014).

While many HDAC-like proteins are present in the nucleus to deacetylate histones, HDAC6 predominantly locates to the cytoplasm, where it has been implicated in the modification of different proteins, including α-tubulin, contractin, and HSP90. In neurons, HDAC6-dependent α-tubulin deacetylation may affect axonal transport by promoting kinesin-1 and dynein binding to microtubules. However, hdac6 null mutant flies did not show overt changes in synaptic features other than T-bar morphology as gauged by electron microscopy, suggesting that axonal transport as a consequence of tubulin defects was not massively affected, although more subtle transport defects cannot be excluded (Miskiewicz, 2014).

BRP is a presynaptic density structural component important to cluster calcium channels at release sites while tethering synaptic vesicles at its C-terminal end. The regulation of BRP by HDAC6-dependent deacetylation indicates the BRP C-terminal end is important to sustain neurotransmitter release during intense (60 Hz) stimulation by orchestrating vesicle tethering. Corroborating these results, mutations in the BRP C-terminal end (brpnude) cause defects in vesicle tethering and the maintenance of release during intense 60 Hz stimulation (Hallermann et al., 2010a). Similarly brp-isoform mutations that leave calcium channel clustering intact but result in a much more condensed T-bar top show a smaller readily releasable vesicle pool, very similar to the defects when BRP is excessively acetylated. The brpnude mutation shows somewhat less severe defects to maintain synaptic transmission, possibly because more vesicles still manage to tether in these mutants during stimulation compared to the conditions that result in strong shrinking of the T-bar top. Nonetheless, the data indicate that in flies, BRP orchestrates efficient synaptic transmission during intense activity (Miskiewicz, 2014).

In the model presented in this study, ELP3 and HDAC6 antagonistically control presynaptic function. TDP-43, a gene mutated in ALS, positively regulates HDAC6 expression, and in flies, increased HDAC6 activity or expression of pathogenic TDP-43 results in the deacetylation of active zone material and increased synaptic release. Remarkably, the presence of an ALS risk-associated ELP3 allele in humans correlates with reduced ELP3 expression in ALS patient spinal cords. In flies, elp3 mutants also cause active zone deacetylation and more synaptic release. Together with genetic interactions in fruit flies, the data suggest that decreased HDAC6 function and increased ELP3 function act antagonistically, both in flies and humans. However, the target(s) on which these enzymes converge in humans remains to be discovered. In flies, the data are consistent with ELP3-dependent acetylation to occur at the C-terminal tail of the BRP protein. However, the mammalian BRP counterpart, ELKS/CAST, that resides in the presynaptic density, does not contain a long C-terminal tail. ELKS/CAST in mammals has been found to be associated with filamentous structures, and the activity to concentrate synaptic vesicles near release sites may thus be executed by binding partners of ELKS/CAST such as Picollo or Bassoon. Hence, it will be interesting to test if ELP3 and HDAC6 regulate acetylation at the much shorter ELKS/CAST tail or whether ELKS/CAST binding partners are acetylated also in the context of ALS. It is in this perspective interesting to note that another active zone-associated protein, UNC13A, is implicated in ALS as well, but the pathomechanism of how UNC13A is implicated remains to be elucidated (Miskiewicz, 2014).

Drosophila Valosin-containing protein is required for dendrite pruning through a regulatory role in mRNA metabolism

The dendritic arbors of the larval Drosophila peripheral class IV dendritic arborization neurons degenerate during metamorphosis in an ecdysone-dependent manner. This process-also known as dendrite pruning-depends on the ubiquitin-proteasome system (UPS), but the specific processes regulated by the UPS during pruning have been largely elusive. This study shows that mutation or inhibition of Valosin-Containing Protein (VCP; termed TER94 by FlyBase), a ubiquitin-dependent ATPase whose human homolog is linked to neurodegenerative disease, leads to specific defects in mRNA metabolism and that this role of VCP is linked to dendrite pruning. Specifically, it was found that VCP inhibition causes an altered splicing pattern of the large pruning gene Molecule interacting with CasL and mislocalization of the Drosophila homolog of the human RNA-binding protein TAR-DNA-binding protein of 43 kilo-Dalton (TDP-43). These data suggest that VCP inactivation might lead to specific gain-of-function of TDP-43 and other RNA-binding proteins. A similar combination of defects is also seen in a mutant in the ubiquitin-conjugating enzyme ubcD1 (Effete) and a mutant in the 19S regulatory particle of the proteasome, but not in a 20S proteasome mutant. Thus, these results highlight a proteolysis-independent function of the UPS during class IV dendritic arborization neuron dendrite pruning and link the UPS to the control of mRNA metabolism (Rumpf, 2014).

To achieve specific connections during development, neurons need to refine their axonal and dendritic arbors. This often involves the elimination of neuronal processes by regulated retraction or degeneration, processes known collectively as pruning. In the Drosophila, large-scale neuronal remodeling and pruning occur during metamorphosis. For example, the peripheral class IV dendritic arborization (da) neurons specifically prune their extensive larval dendritic arbors, whereas another class of da neurons, the class III da neurons, undergo ecdysone- and caspase-dependent cell death. Class IV da neuron dendrite pruning requires the steroid hormone ecdysone and its target gene SOX14, encoding an HMG box transcription factor. Class IV da neuron dendrites are first severed proximally from the soma by the action of enzymes like Katanin-p60L and Mical that sever microtubules and actin cables, respectively. Later, caspases are required for the fragmentation and phagocytic engulfment of the severed dendrite remnants. Another signaling cascade known to be required for pruning is the ubiquitin-proteasome system (UPS). Covalent modification with the small protein ubiquitin occurs by a thioester cascade involving the ubiquitin-activating enzyme Uba1 (E1), and subsequent transfer to ubiquitin-conjugating enzymes (E2s) and the specificity-determining E3 enzymes. Ubiquitylation of a protein usually leads to the degradation of the modified protein by the proteasome, a large cylindrical protease that consists of two large subunits, the 19S regulatory particle and the proteolytic 20S core particle. Several basal components of the ubiquitylation cascade-Uba1 and the E2 enzyme ubcD1-as well as several components of the 19S subunit of the proteasome have been shown to be required for pruning, as well as the ATPase associated with diverse cellular activities (AAA) ATPase Valosin-Containing Protein (VCP) (CDC48 in yeast, p97 in vertebrates, also known as TER94 in Drosophila), which acts as a chaperone for ubiquitylated proteins. Interestingly, autosomal dominant mutations in the human VCP gene cause hereditary forms of ubiquitin-positive frontotemporal dementia (FTLD-U) and amyotrophic lateral sclerosis (ALS). A hallmark of these diseases is the occurrence of both cytosolic and nuclear ubiquitin-positive neuronal aggregates that often contain the RNA-binding protein TAR-DNA-binding protein of 43 kilo-Dalton (TDP-43). It has been proposed that ubcD1 and VCP promote the activation of caspases during dendrite pruning via degradation of the caspase inhibitor DIAP1. However, mutation of ubcD1 or VCP inhibit the severing of class IV da neuron dendrites from the cell body, whereas in caspase mutants, dendrites are still severed from the cell body, but clearance of the severed fragments is affected. This indicates that the UPS must have additional, as yet unidentified, functions during pruning (Rumpf, 2014).

This study further investigated the role of UPS mutants in dendrite pruning. vcp mutation was shown to lead to a specific defect in ecdysone-dependent gene expression, as VCP is required for the functional expression and splicing of the large ecdysone target gene molecule interacting with CasL (MICAL). Concomitantly, mislocalization of Drosophila TDP-43 and up-regulation of other RNA-binding proteins were observed, and genetic evidence suggests that these alterations contribute to the observed pruning defects in VCP mutants. Defects in MICAL expression and TDP-43 localization are also induced by mutations in ubcD1 and in the 19S regulatory particle of the proteasome, but not by a mutation in the 20S core particle, despite the fact that proteasomal proteolysis is required for dendrite pruning, indicating the requirement for multiple UPS pathways during class IV da neuron dendrite pruning (Rumpf, 2014).

Class IV da neurons have long and branched dendrites at the third instar larval stage. In wild-type animals, these dendrites are completely pruned at 16-18 h after puparium formation (h APF). VCP mutant class IV da neurons were generated by the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique for clonal analysis. Mutant vcp26-8 class IV da neurons displayed strong pruning defects and retained long dendrites at 16 h APF. Expression of an ATPase-deficient dominant-negative VCP protein (VCP QQ) under the class IV da neuron-specific driver ppk-GAL4 recapitulated the pruning phenotype and also led to the retention of long and branched dendrites at 16 h APF. VCP inhibition also causes defects in class III da neuron apoptosis. This combination of defects in both pruning and apoptosis is reminiscent of the phenotypes caused by defects in ecdysone-dependent gene expression. Indeed, overexpression of the transcription factor Sox14, which induces pruning genes, led to a nearly complete suppression of the pruning phenotype caused by VCP QQ. This genetic interaction suggested that VCP might be required for the expression of one or several ecdysone target genes during pruning (Rumpf, 2014).

How could VCP be linked to Sox14? The suppression of the vcp mutant phenotype by Sox14 overexpression could be achieved in one of several ways. Sox14 could be epistatic to VCP-that is, VCP could be required for functional Sox14 expression-and this effect would be mitigated by Sox14 overexpression. However, VCP could also be required for the expression of one or several Sox14 target genes, and enhanced Sox14 expression could overcome this requirement either via enhanced induction of one or several particular targets or via enhanced induction of other pruning genes, in which case Sox14 would be a bypass suppressor of VCP QQ. To distinguish between these possibilities, the effects were assessed of VCP inhibition on the expression of known genes in the ecdysone cascade required for pruning in class IV da neurons. Class IV da neuron pruning is governed by the Ecdysone Receptor B1 (EcR-B1) isoform, which in turn directly activates the transcription of Sox14 and Headcase (Hdc), a pruning factor of unknown function. Sox14, on the other hand, activates the transcription of the MICAL gene encoding an actin-severing enzyme. In immunostaining experiments, VCP QQ did not affect the expression of EcR-B1, Sox14, or Hdc at the onset of the pupal phase. However, the expression of Mical was selectively abrogated in class IV da neurons expressing VCP QQ, or in vcp26-8 class IV da neuron MARCM clones . These data indicated that VCP might affect dendrite pruning by regulating the expression of the Sox14 target gene Mical, indicating that Sox14 might act as a bypass suppressor of VCP QQ (Rumpf, 2014).

How could VCP inhibition suppress Mical expression? To answer this question, whether Mical mRNA could still be detected in class IV da neurons expressing VCP QQ was assessed. To this end, enzymatic tissue digestion and FACS sorting were used to isolate class IV da neurons from early pupae (1-5 h APF). Total RNA was then extracted from the isolated neurons, and the presence of Mical mRNA expression was assessed by RT-PCR, using control samples or samples from animals expressing VCP QQ under ppk-GAL4. The Mical gene is large (~40 kb) and spans multiple exons that are transcribed to yield a ∼15 kb mRNA. To detect Mical cDNA, primer pairs spanning several exons were used for two different regions of Mical mRNA, exons 14-16 and exons 8-12. [MICAL is on the (-) strand, but the exon numbering denoted by Flybase follows the direction of the (+) strand. Therefore, exons 14-16 are upstream of exons 8-12, and the latter are closer to the 3' end of MICAL mRNA.] MICAL mRNA was detectable upon VCP inhibition in these extracts with a primer pair spanning exons 14-16. The second primer pair spanning exons 8-12 also detected MICAL mRNA in both samples, but the RT-PCR product from the VCP QQ-expressing class IV da neurons had a larger molecular weight. Sequencing of the PCR products indicated that MICAL mRNA from VCP QQ-expressing class IV da neurons contained exon 11, which was not present in Mical mRNA from the control sample. Exon 11 is absent from all predicted MICAL splice isoforms except for a weakly supported isoform designated 'Mical-RM.' It introduces a stop codon into MICAL mRNA that would lead to the truncation of the C-terminal 1,611 amino acids from Mical protein. This portion of Mical protein contains several predicted protein interaction domains such as a proline-rich region, a coiled-coil region with similarity to Ezrin/Radixin/Moesin (ERM) domains, and a C-terminal PDZ-binding motif, and is required for the interaction between Mical and PlexinA. In addition, the truncated region contains the epitope for the antibody used in the immunofluorescence experiments, thus explaining the observed lack of Mical expression upon VCP inhibition. Given that a mutant of Mical with a smaller C-terminal truncation (compared with the one induced by VCP inhibition) was not sufficient to rescue the class IV da neuron dendrite pruning defect of mical mutants, disruption of VCP function likely results in expression of a truncated Mical protein without pruning activity. Taken together, these data suggest that the observed defect in MICAL mRNA splicing contributes significantly to the pruning defects of VCP mutants (Rumpf, 2014).

How is VCP linked to alternative splicing of MICAL mRNA? A plausible mechanism for the control of an alternative splicing event would be the modulation of specific (pre)mRNA-binding proteins. VCP has recently been linked to several RNA-binding proteins: human autosomal dominant VCP mutations cause frontotemporal dementia or ALS with inclusion bodies that contain aggregated human TDP-43; a genetic screen in Drosophila identified the RNA-binding proteins Drosophila TDP-43, HRP48, and x16 as weak genetic interactors of the dominant effects of VCP disease mutants; and HuR (a human homolog of the neuronal Drosophila RNA-binding protein elav) was recently shown to bind human VCP. Of these, TDP-43 and also elav have been linked to alternative splicing in various model systems, including Drosophila. Therefore this study used available specific antibodies to assess the levels and distribution of Drosophila TDP-43 (hereafter referred to as TDP-43) and elav. TDP-43 has previously been shown to localize to the nucleus in Drosophila motoneurons and mushroom body Kenyon cells. Surprisingly, TDP-43 was largely localized to the cytoplasm in class IV da neurons, where it was enriched in a punctate pattern around the nucleus, with only a small fraction also detectable in the nucleus, a localization pattern that could be reproduced with transgenic N-terminally HA-tagged TDP-43. Elav is a known nuclear marker for Drosophila neurons; in class IV da neurons, it was somewhat enriched in nuclear punctae. The effects of VCP inhibition on these two RNA-binding proteins was assessed. Elav localization did not change notably upon VCP QQ expression. Strikingly, TDP-43 became depleted from the cytoplasm of class IV da neurons and relocalized to the nucleus upon VCP QQ expression. Closer inspection revealed that TDP-43 in VCP-inhibited neurons was now enriched in nuclear dots that often also exhibited increased elav staining. The relocalization of TDP-43 from the cytoplasm to the nucleus was also observed in vcp26-8 mutant class IV da neuron MARCM clones. Importantly, quantification and normalization of TDP-43 levels showed that VCP inhibition did not alter the absolute levels of TDP-43, suggesting that the observed effects were not a consequence of a defect in TDP-43 degradation. In fact, the only manipulation that resulted in a mild but significant increase in TDP-43 levels-but without a change in localization-was the expression of an RNAi directed against the autophagy factor ATG7, perhaps reflecting the degradation of cytoplasmic RNA granules through the autophagy pathway (Rumpf, 2014).

It was next asked whether manipulation of TDP-43 would affect class IV da neuron dendrite pruning. A previously characterized TDP-43 mutant, TDP-43 Q367X, did not display pruning defects, but overexpression of TDP-43 led to strong dendrite pruning defects at 16 h APF. In support of the hypothesis that TDP-43 acts in the same or a similar pathway as VCP during dendrite pruning, it was also found that a more weakly expressed TDP-43 transgene (UAS-TDP-43weak) and VCP A229E, a weakly dominant-active VCP allele corresponding to a human VCP disease mutation, exhibited a synergistic inhibition of pruning when coexpressed. Interestingly, manipulation of elav gave very similar results as with TDP-43: elav down-regulation by RNAi did not affect pruning, but elav overexpression led to highly penetrant pruning defects (Rumpf, 2014).

To exclude the possibility that the pruning defects induced by TDP-43 or elav overexpression were due to long-term expression and aggregation of RNA-binding proteins, TDP-43 and elav overexpression was also induced acutely (24 h before the onset of pupariation). Pruning was still inhibited in these cases. Also, overexpression of several other RNA-binding proteins did not cause pruning defects, with two exceptions: a UAS-carrying P-element in the promotor of the adjacent x16 and HRP48 genes caused a strong pruning defect when expression was induced in class IV da neurons, and levels of a GFP protein trap insertion into the x16 gene were also markedly increased in class IV da neurons expressing VCP QQ, possibly indicating a role for VCP in x16 degradation. In further support of an involvement of VCP with RNA-binding proteins during neuronal pruning processes, it was also found that VCP is required for mushroom body γ neuron axon pruning and induces the accumulation of Boule, an RNA-binding protein that had previously been shown to inhibit γ neuron axon pruning when overexpressed. Thus, the data suggest that VCP regulates a specific subset of RNA-binding proteins and that this regulatory role of VCP is associated with its role in pruning (Rumpf, 2014).

As VCP is an integral component of the UPS, it was next asked whether the role of VCP in MICAL regulation and TDP-43 localization was also dependent on ubiquitylation and/or the proteasome. To address this question, Mical levels and TDP-43 distribution was assessed in UPS mutants with known pruning defects. An ubiquitylation enzyme known to be required for pruning is the E2 enzyme ubcD1. When TDP-43 localization was assessed in larval ubcD1D73 mutant class IV da neurons, TDP-43 was again localized to the nucleus in these cells. Furthermore, a pronounced reduction of Mical expression in ubcD1D73 mutant class IV da neurons was noted during the early pupal stage, indicating that ubiquitylation through ubcD1 is involved in the regulation of TDP-43 localization and Mical expression (Rumpf, 2014).

TDP-43 localization and Mical expression were assessed in proteasome mutants. A previously characterized mutant in the Mov34 gene encoding the 19S subunit Rpn8 was used. TDP-43 was again relocalized to the nucleus in Mov34 mutant class IV da neurons, and Mical expression was absent from Mov34 mutant class IV da neurons at 2 h APF. To rigorously address whether proteasomal proteolysis was also required for TDP-43 localization and Mical expression, the effect was assessed of Pros261, a previously characterized mutation in the 20S core particle subunit Prosβ6. In contrast to Mov34 mutant class IV da neurons, Pros261 mutant class IV da neurons displayed cytoplasmic TDP-43 localization, and robust Mical expression was detected in these neurons at 2 h APF. Thus, although ubiquitylation and the 19S proteasome are both required for Mical expression and normal TDP-43 localization, proteolysis through the 20S core particle of the proteasome is not. Importantly, Pros261 MARCM class IV da neurons showed strong dendrite pruning defects at 16 h APF, as did expression of RNAi constructs directed against subunits of the 20S core particle (Rumpf, 2014).

These data indicate that there must be several ubiquitin- and proteasome-dependent pathways that are required for dendrite pruning: one pathway requires ubcD1, VCP, and the 19S regulatory particle of the proteasome, but not the 20S core particle. This pathway regulates MICAL expression. A second UPS pruning pathway does depend on proteolysis through the 20S core. In an E3 ubiquitin ligase candidate screen, cul-1/lin19 was identified as a pruning mutant. Cul-1 encodes cullin-1, a core component of a class of multisubunit ubiquitin ligases known as SCF (for Skp1/Cullin/F-box) ligases. Class IV da neurons mutant for cul-1 or class IV da neurons expressing an RNAi construct directed against cul-1 had not pruned their dendrites at 16 h APF. However, unlike with VCP, ubcD1, and Mov34, cul-1 mutation did not affect Mical expression at 2 h APF, indicating that cullin-1 is not a component of the VCP-dependent UPS pathway involved in splicing and might thus be a component of a proteolytic UPS pathway. In support of this notion, a recent report independently identified cul-1 as a pruning mutant and associated it with protein degradation (Rumpf, 2014).

It has been proposed that the E2 enzyme ubcD1 and VCP would act to activate caspases during pruning. However, the dendrite pruning defects caused by those UPS mutants are much stronger than the phenotypes caused by caspase inactivation, which mostly causes a delay in the phagocytic uptake of severed dendrites by the epidermis. Although it cannot be excluded that ubcD1 and VCP contribute to caspase activation during pruning, the new mechanism proposed in this study - control of RNA-binding proteins and MICAL expression - likely makes a much stronger contribution to the drastic pruning phenotypes of UPS mutants (Rumpf, 2014).

How precisely do VCP, ubcD1, and the 19S proteasome contribute to MICAL expression? The data indicate that VCP inhibition causes missplicing of MICAL mRNA that likely leads to the expression of an inactive Mical protein variant. At the same time, VCP inhibition leads to the mislocalization of TDP-43, and possibly the dysregulation of a number of other RNA-binding proteins. The fact that these phenotypes correlate in the vcp, ubcD1, and Mov34 mutants gives a strong indication that they are related. TDP-43 had previously been identified as a suppressor of the toxicity induced by a weak VCP disease allele in the Drosophila eye. In class IV da neurons, reducing the amounts of TDP-43 (with a deficiency) or elav (by RNAi) did not ameliorate the pruning defect induced by VCP inhibition. Therefore, the possibility cannot be excluded that the two proteins act in parallel rather than in an epistatic fashion. As VCP has been shown to remodel protein complexes that contain ubiquitylated proteins and is structurally similar to the 19S cap, it is interesting to speculate that VCP and the 19S cap might alter the subunit composition of ubiquitylated TDP-43-containing complexes of RNA-binding proteins, and that this activity-rather than a direct action on TDP-43 (or maybe also elav) alone-might lead to both MICAL missplicing and TDP-43 mislocalization (Rumpf, 2014).

Interestingly, autosomal dominant mutations in human VCP cause frontotemporal dementia and ALS, a hallmark of which is the formation of aggregates that contain TDP-43. Most of these aggregates are cytoplasmic (and contain TDP-43 that has relocalized from the nucleus to the cytoplasm), but VCP mutations also induce TDP-43 aggregation in the nucleus, a situation that might be similar to the situation caused by VCP inhibition in class IV da neurons. Although human VCP disease mutations have been proposed to act as dominant-active versions of VCP with enhanced ATPase activity, both the disease allele and the dominant-negative ATPase-dead VCP QQ mutant cause class IV da neuron pruning defects and TDP-43 relocalization to the nucleus of class IV da neurons and therefore act in the same direction. It is thought that VCP can only bind substrates when bound to ATP, and will release bound substrates upon ATP hydrolysis. Thus, it is conceivable that the phenotypic outcome of inhibiting the ATPase (no substrate release) should be similar to that of ATPase overactivation (reduced substrate binding or premature substrate release): in both cases, a substrate protein complex would not be properly remodeled (Rumpf, 2014).

Taken together, these results indicate the existence of a nonproteolytic function of VCP and the UPS in RNA metabolism and highlight its importance during neuronal development (Rumpf, 2014).

Drosophila lines with mutant and wild type human TDP-43 replacing the endogenous gene reveals phosphorylation and ubiquitination in mutant lines in the absence of viability or lifespan defects

Mutations in TDP-43 (see Drosophila TDPH) are associated with proteinaceous inclusions in neurons and are believed to be causative in neurodegenerative diseases such as frontotemporal dementia or amyotrophic lateral sclerosis. This study describes a Drosophila system where the genome was engineered to replace the endogenous TDP-43 orthologue with wild type or mutant human TDP-43(hTDP-43). In contrast to other models, these flies express both mutant and wild type hTDP-43 at similar levels to those of the endogenous gene and importantly, no age-related TDP-43 accumulation was observed among all the transgenic fly lines. Immunoprecipitation of TDP-43 showed that flies with hTDP-43 mutations had increased levels of ubiquitination and phosphorylation of the hTDP-43 protein. Furthermore, histologically, flies expressing hTDP-43 M337V showed global, robust neuronal staining for phospho-TDP. All three lines: wild type hTDP-43, -G294A and -M337V were homozygous viable, with no defects in development, life span or behaviors observed. The primary behavioral defect was that flies expressing either hTDP-43 G294A or M337V showed a faster decline with age in negative geotaxis. Together, these observations implied that neurons could handle these TDP-43 mutations by phosphorylation- and ubiquitin-dependent proteasome systems, even in a background without the wild type TDP-43. These findings suggest that these two specific TDP-43 mutations are not inherently toxic, but may require additional environmental or genetic factors to affect longevity or survival (Chang, 2017).

Therapeutic modulation of eIF2alpha phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models

Amyotrophic lateral sclerosis (ALS) is a fatal, late-onset neurodegenerative disease primarily affecting motor neurons. A unifying feature of many proteins associated with ALS, including TDP-43 and ataxin-2 (see Drosophila Ataxin-2), is that they localize to stress granules. Unexpectedly, this study found that genes that modulate stress granules are strong modifiers of TDP-43 toxicity in Saccharomyces cerevisiae and Drosophila melanogaster. eIF2alpha phosphorylation is upregulated by TDP-43 toxicity in flies, and TDP-43 interacts with a central stress granule component, polyA-binding protein (PABP). In human ALS spinal cord neurons, PABP accumulates abnormally, suggesting that prolonged stress granule dysfunction may contribute to pathogenesis. The efficacy of a small molecule inhibitor of eIF2alpha phosphorylation was investigated in ALS models. Treatment with this inhibitor mitigated TDP-43 toxicity in flies and mammalian neurons. These findings indicate that the dysfunction induced by prolonged stress granule formation might contribute directly to ALS and that compounds that mitigate this process may represent a novel therapeutic approach (Kim, 2014)

The RNA-binding protein TDP-43 selectively disrupts microRNA-1/206 incorporation into the RNA-induced silencing complex

MicroRNA (miRNA) maturation is regulated by interaction of particular miRNA precursors with specific RNA-binding proteins. Following their biogenesis, mature miRNAs are incorporated into the RNA-induced silencing complex (RISC) where they interact with mRNAs to negatively regulate protein production. However, little is known about how mature miRNAs are regulated at the level of their activity. To address this, a screen was performed for proteins differentially bound to the mature form of the miR-1 or miR-133 miRNA families. These muscle-enriched, co-transcribed miRNA pairs cooperate to suppress smooth muscle gene expression in the heart. However, they also have opposing roles, with the miR-1 family, composed of miR-1 and miR-206, promoting myogenic differentiation, whereas miR-133 maintains the progenitor state. This study describes a physical interaction between TDP-43, an RNA-binding protein that forms aggregates in the neuromuscular disease, amyotrophic lateral sclerosis, and the miR-1, but not miR-133, family. Deficiency of the TDP-43 Drosophila ortholog enhanced Drosophila miR-1 activity in vivo. In mammalian cells, TDP-43 limited the activity of both miR-1 and miR-206, but not the miR-133 family, by disrupting their RISC association. Consistent with TDP-43 dampening miR-1/206 activity, protein levels of the miR-1/206 targets, IGF-1 and HDAC4, were elevated in TDP-43 transgenic mouse muscle. This occurred without corresponding Igf-1 or Hdac4 mRNA increases and despite higher miR-1 and miR-206 expression. These findings reveal that TDP-43 negatively regulates the activity of the miR-1 family of miRNAs by limiting their bioavailability for RISC loading and suggest a processing-independent mechanism for differential regulation of miRNA activity (King, 2014).

Evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins functionally interact with human and Drosophila TAR DNA-binding protein 43 (TDP-43)

Human TDP-43 represents the main component of neuronal inclusions found in patients with neurodegenerative diseases, especially frontotemporal lobar degeneration and amyotrophic lateral sclerosis. In vitro and in vivo studies have shown that the TAR DNA-binding protein 43 (TDP-43) Drosophila ortholog (TBPH) can biochemically and functionally overlap the properties of the human factor. The recent direct implication of the human heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1, known TDP-43 partners, in the pathogenesis of multisystem proteinopathy and amyotrophic lateral sclerosis supports the hypothesis that the physical and functional interplay between TDP-43 and hnRNP A/B orthologs might play a crucial role in the pathogenesis of neurodegenerative diseases. To test this hypothesis and further validate the fly system as a useful model to study this type of diseases, this study characterized human TDP-43 and Drosophila TBPH similarity in terms of protein-protein interaction pathways. This work shows that TDP-43 and TBPH share the ability to associate in vitro with Hrp38/Hrb98DE/CG9983, the fruit fly ortholog of the human hnRNP A1/A2 factors. Interestingly, the protein regions of TDP-43 and Hrp38 responsible for reciprocal interactions are conserved through evolution. Functionally, experiments in HeLa cells demonstrate that TDP-43 is necessary for the inhibitory activity of Hrp38 on splicing. Finally, Drosophila in vivo studies show that Hrp38 deficiency produces locomotive defects and life span shortening. These results suggest that hnRNP protein levels can play a modulatory role on TDP-43 functions (Romano, 2014).

Loss and gain of Drosophila TDP-43 impair synaptic efficacy and motor control leading to age-related neurodegeneration by loss-of-function phenotypes

Cytoplasmic accumulation and nuclear clearance of TDP-43 characterize familial and sporadic forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, suggesting that either loss or gain of TDP-43 function, or both, cause disease formation. This study has systematically compared loss- and gain-of-function of Drosophila TDP-43, TAR DNA Binding Protein Homolog (TBPH), in synaptic function and morphology, motor control, and age-related neuronal survival. Both loss and gain of TBPH severely affect development and result in premature lethality. TBPH dysfunction caused impaired synaptic transmission at the larval neuromuscular junction (NMJ) and in the adult. Tissue-specific knockdown together with electrophysiological recordings at the larval NMJ also revealed that alterations of TBPH function predominantly affect pre-synaptic efficacy, suggesting that impaired pre-synaptic transmission is one of the earliest events in TDP-43-related pathogenesis. Prolonged loss and gain of TBPH in adults resulted in synaptic defects and age-related, progressive degeneration of neurons involved in motor control. Toxic gain of TBPH did not downregulate or mislocalize its own expression, indicating that a dominant-negative effect leads to progressive neurodegeneration also seen with mutational inactivation of TBPH. Together these data suggest that dysfunction of Drosophila TDP-43 triggers a cascade of events leading to loss-of-function phenotypes whereby impaired synaptic transmission results in defective motor behavior and progressive deconstruction of neuronal connections, ultimately causing age-related neurodegeneration (Diaper, 2013a).

Drosophila TDP-43 dysfunction in glia and muscle cells cause cytological and behavioural phenotypes that characterize ALS and FTLD

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are neurodegenerative disorders that are characterized by cytoplasmic aggregates and nuclear clearance of TAR DNA-binding protein 43 (TDP-43). Studies in Drosophila, zebrafish and mouse demonstrate that the neuronal dysfunction of TDP-43 is causally related to disease formation. However, TDP-43 aggregates are also observed in glia and muscle cells, which are equally affected in ALS and FTLD; yet, it is unclear whether glia- or muscle-specific dysfunction of TDP-43 contributes to pathogenesis. This study shows that similar to its human homologue, Drosophila TDP-43, Tar DNA-binding protein homologue (TBPH), is expressed in glia and muscle cells. Muscle-specific knockdown of TBPH causes age-related motor abnormalities, whereas muscle-specific gain of function leads to sarcoplasmic aggregates and nuclear TBPH depletion, which is accompanied by behavioural deficits and premature lethality. TBPH dysfunction in glia cells causes age-related motor deficits and premature lethality. In addition, both loss and gain of Drosophila TDP-43 alter mRNA expression levels of the glutamate transporters Excitatory amino acid transporter 1 (EAAT1) and EAAT2. Taken together, these results demonstrate that both loss and gain of TDP-43 function in muscle and glial cells can lead to cytological and behavioural phenotypes in Drosophila that also characterize ALS and FTLD and identify the glutamate transporters EAAT1/2 as potential direct targets of TDP-43 function. These findings suggest that together with neuronal pathology, glial- and muscle-specific TDP-43 dysfunction may directly contribute to the aetiology and progression of TDP-43-related ALS and FTLD (Diaper, 2013b).

Motor neuron expression of the voltage-gated calcium channel cacophony restores locomotion defects in a Drosophila, TDP-43 loss of function model of ALS

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, this study shows 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 rescues these locomotion defects. Using TBPH immunoprecipitation, TBPH was shown to associate 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 (Chang, 2013).

Muscleblind, BSF and TBPH are mislocalized in the muscle sarcomere of a Drosophila myotonic dystrophy model

Myotonic dystrophy type 1 (DM1) is a genetic disease caused by the pathological expansion of a CTG trinucleotide repeat in the 3' UTR of the DMPK gene. In the DMPK transcripts, the CUG expansions sequester RNA-binding proteins into nuclear foci, including transcription factors and alternative splicing regulators such as MBNL1. MBNL1 sequestration has been associated with key features of DM1. However, the basis behind a number of molecular and histological alterations in DM1 remain unclear. To help identify new pathogenic components of the disease, a genetic screen was carried using a Drosophila model of DM1 that expresses 480 interrupted CTG repeats, i(CTG)480, and a collection of 1215 transgenic RNA interference (RNAi) fly lines. Of the 34 modifiers identified, two RNA-binding proteins, TBPH (homolog of human TAR DNA-binding protein 43 or TDP-43) and BSF (Bicoid stability factor; homolog of human LRPPRC), were of particular interest. These factors modified i(CTG)480 phenotypes in the fly eye and wing, and TBPH silencing also suppressed CTG-induced defects in the flight muscles. In Drosophila flight muscle, TBPH, BSF and the fly ortholog of MBNL1, Muscleblind (Mbl), were detected in sarcomeric bands. Expression of i(CTG)480 resulted in changes in the sarcomeric patterns of these proteins, which could be restored by coexpression with human MBNL1. Epistasis studies showed that Mbl silencing was sufficient to induce a subcellular redistribution of TBPH and BSF proteins in the muscle, which mimicked the effect of i(CTG)480 expression. These results provide the first description of TBPH and BSF as targets of Mbl-mediated CTG toxicity, and they suggest an important role of these proteins in DM1 muscle pathology (Llamusi, 2013).

Identification of genetic modifiers of TDP-43 neurotoxicity in Drosophila

Cytosolic aggregation of the nuclear RNA-binding protein TDP-43 is a histopathologic signature of degenerating neurons in amyotrophic lateral sclerosis (ALS), and mutations in the TARDBP gene encoding TDP-43 cause dominantly inherited forms of this condition. To understand the relationship between TDP-43 misregulation and neurotoxicity, Drosophila has been used as a model system, in which overexpression of either wild-type TDP-43 or its ALS-associated mutants in neurons is sufficient to induce neurotoxicity, paralysis, and early death. Using microarrays, this study examined gene expression patterns that accompany TDP-43-induced neurotoxicity in the fly system. Constitutive expression of TDP-43 in the Drosophila compound eye elicited widespread gene expression changes, with strong upregulation of cell cycle regulatory genes and genes functioning in the Notch intercellular communication pathway. Inducible expression of TDP-43 specifically in neurons elicited significant expression differences in a more restricted set of genes. Genes that were upregulated in both paradigms included SpindleB and the Notch target Hey, which appeared to be a direct TDP-43 target. Mutations that diminished activity of Notch or disrupted the function of downstream Notch target genes extended the lifespan of TDP-43 transgenic flies, suggesting that Notch activation was deleterious in this model. Finally, mutation of the nucleoporin Nup50 increased the lifespan of TDP-43 transgenic flies, suggesting that nuclear events contribute to TDP-43-dependent neurotoxicity. The combined findings identified pathways whose deregulation might contribute to TDP-43-induced neurotoxicity in Drosophila (Zhan, 2013; PubMed).

TDP-43 loss-of-function causes neuronal loss due to defective steroid receptor-mediated gene program switching in Drosophila

TDP-43 proteinopathy is strongly implicated in the pathogenesis of amyotrophic lateral sclerosis and related neurodegenerative disorders. Whether TDP-43 neurotoxicity is caused by a novel toxic gain-of-function mechanism of the aggregates or by a loss of its normal function is unknown. This study increased and decreased expression of TDP-43 (dTDP-43) in Drosophila. Although upregulation of dTDP-43 induced neuronal ubiquitin and dTDP-43-positive inclusions, both up- and downregulated dTDP-43 resulted in selective apoptosis of bursicon neurons and highly similar transcriptome alterations at the pupal-adult transition. Gene network analysis and genetic validation showed that both up- and downregulated dTDP-43 directly and dramatically increased the expression of the neuronal microtubule-associated protein Map205, resulting in cytoplasmic accumulations of the ecdysteroid receptor (EcR) and a failure to switch EcR-dependent gene programs from a pupal to adult pattern. It is proposed that dTDP-43 neurotoxicity is caused by a loss of its normal function (Vanden Broeck, 2013; PubMed).

Comparison of parallel high-throughput RNA sequencing between knockout of TDP-43 and its overexpression reveals primarily nonreciprocal and nonoverlapping gene expression changes in the central nervous system of Drosophila

The human Tar-DNA binding protein, TDP-43, is associated with amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. TDP-43 contains two conserved RNA-binding motifs and has documented roles in RNA metabolism, including pre-mRNA splicing and repression of transcription. Using Drosophila melanogaster as a model, this study generated loss-of-function and overexpression genotypes of Tar-DNA binding protein homolog (TBPH) to study their effect on the transcriptome of the central nervous system (CNS). By using massively parallel sequencing methods (RNA-seq) to profile the CNS, it was found that loss of TBPH results in widespread gene activation and altered splicing, much of which are reversed by rescue of TBPH expression. Conversely, TBPH overexpression results in decreased gene expression. Although previous studies implicated both absence and mis-expression of TDP-43 in ALS, these data exhibit little overlap in the gene expression between them, suggesting that the bulk of genes affected by TBPH loss-of-function and overexpression are different. In combination with computational approaches to identify likely TBPH targets and orthologs of previously identified vertebrate TDP-43 targets, this study provides a comprehensive analysis of enriched gene ontologies. The data suggest that TDP-43 plays a role in synaptic transmission, synaptic release, and endocytosis. A potential novel regulation was uncovered of the Wnt and BMP pathways, many of whose targets appear to be conserved (Hazelett, 2012).

Neuronal function and dysfunction of Drosophila dTDP

TDP-43 is an RNA- and DNA-binding protein well conserved in animals including the mammals, Drosophila, and C. elegans. In mammals, the multi-function TDP-43 encoded by the TARDBP gene is a signature protein of the ubiquitin-positive inclusions (UBIs) in the diseased neuronal/glial cells of a range of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-U). This paper reports a study of the function and dysfunction of the Drosophila ortholog of the mammalian TARDBP gene, dTDP, by genetic, behavioral, molecular, and cytological analyses. It was found that depletion of dTDP expression caused locomotion defect accompanied with an increase of the number of boutons at the neuromuscular junctions (NMJ). These phenotypes could be rescued by overexpression of Drosophila dTDP in the motor neurons. In contrast, overexpression of dTDP in the motor neurons also resulted in reduced larval and adult locomotor activities, but this was accompanied by a decrease of the number of boutons and axon branches at NMJ. Significantly, constitutive overexpression of dTDP in the mushroom bodies caused smaller axonal lobes as well as severe learning deficiency. In contrast, constitutive mushroom body-specific knockdown of dTDP expression did not affect the structure of the mushroom bodies, but it impaired the learning ability of the flies, albeit moderately. Overexpression of dTDP also led to the formation of cytosolic dTDP (+) aggregates. These data together demonstrate the neuronal functions of dTDP, and by implication the mammalian TDP-43, in learning and locomotion. The effects of mis-expression of dTDP on Drosophila NMJ suggest that eukaryotic TDP-43 guards against over development of the synapses. The conservation of the regulatory pathways of functions and dysfunctions of Drosophila dTDP and mammalian TDP-43 also shows the feasibility of using the flies as a model system for studying the normal TDP-43 function and TDP-43 proteinopathies in the vertebrates including human (Lin, 2011).

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. This study reports a direct in vivo comparison between wild-type and A315T mutant TDP-43 overexpression in Drosophila neurons. 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, it was shown 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, these 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 (Estes, 2011).

The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span

The fatal adult motor neuron disease amyotrophic lateral sclerosis (ALS) shares some clinical and pathological overlap with frontotemporal dementia (FTD), an early-onset neurodegenerative disorder. The mammalian RNA/DNA-binding proteins fused in sarcoma (FUS; also known as TLS) and TAR DNA binding protein-43 (TDP-43) have recently been shown to be genetically and pathologically associated with familial forms of ALS and FTD. It is currently unknown whether perturbation of these proteins results in disease through mechanisms that are independent of normal protein function or via the pathophysiological disruption of molecular processes in which they are both critical. This paper reports that Drosophila mutants in which the homolog of FUS is disrupted exhibit decreased adult viability, diminished locomotor speed, and reduced life span compared with controls. These phenotypes were fully rescued by wild-type human FUS, but not ALS-associated mutant FUS proteins. A mutant of the Drosophila homolog of TDP-43 had similar, but more severe, deficits. Through cross-rescue analysis, it was demonstrated that FUS acted together with and downstream of TDP-43 in a common genetic pathway in neurons. Furthermore, it was found that these proteins associated with each other in an RNA-dependent complex. These results establish that FUS and TDP-43 function together in vivo and suggest that molecular pathways requiring the combined activities of both of these proteins may be disrupted in ALS and FTD (Wang, 2011).

Knockdown of transactive response DNA-binding protein (TDP-43) downregulates histone deacetylase 6

TDP-43 is an RNA/DNA-binding protein implicated in transcriptional repression and mRNA processing. Inclusions of TDP-43 are hallmarks of frontotemporal dementia and amyotrophic lateral sclerosis. Besides aggregation of TDP-43, loss of nuclear localization is observed in disease. To identify relevant targets of TDP-43, expression profiling was performed. Thereby, histone deacetylase 6 (HDAC6) downregulation was discovered on TDP-43 silencing, and this was confirmed at the mRNA and protein level in human embryonic kidney HEK293E and neuronal SH-SY5Y cells. This was accompanied by accumulation of the major HDAC6 substrate, acetyl-tubulin. HDAC6 levels were restored by re-expression of TDP-43, dependent on RNA binding and the C-terminal protein interaction domains. Moreover, TDP-43 bound specifically to HDAC6 mRNA arguing for a direct functional interaction. Importantly, in vivo validation in TDP-43 knockout Drosophila melanogaster confirmed the specific downregulation of HDAC6. HDAC6 is necessary for protein aggregate formation and degradation. Indeed, HDAC6-dependent reduction of cellular aggregate formation and increased cytotoxicity of polyQ-expanded ataxin-3 were found in TDP-43 silenced cells. In conclusion, loss of functional TDP-43 causes HDAC6 downregulation and might thereby contribute to pathogenesis (Fiesel, 2010).

A Drosophila model for TDP-43 proteinopathy

Neuropathology involving TAR DNA binding protein-43 (TDP-43) has been identified in a wide spectrum of neurodegenerative diseases collectively named as TDP-43 proteinopathy, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). To test whether increased expression of wide-type human TDP-43 (hTDP-43) may cause neurotoxicity in vivo, transgenic flies were generated expressing hTDP-43 in various neuronal subpopulations. Expression in the fly eyes of the full-length hTDP-43, but not a mutant lacking its amino-terminal domain, led to progressive loss of ommatidia with remarkable signs of neurodegeneration. Expressing hTDP-43 in mushroom bodies (MBs) resulted in dramatic axon losses and neuronal death. Furthermore, hTDP-43 expression in motor neurons led to axon swelling, reduction in axon branches and bouton numbers, and motor neuron loss together with functional deficits. Thus, transgenic flies expressing hTDP-43 recapitulate important neuropathological and clinical features of human TDP-43 proteinopathy, providing a powerful animal model for this group of devastating diseases. This study indicates that simply increasing hTDP-43 expression is sufficient to cause neurotoxicity in vivo, suggesting that aberrant regulation of TDP-43 expression or decreased clearance of hTDP-43 may contribute to the pathogenesis of TDP-43 proteinopathy (Li, 2010).

TDP-43 mediates degeneration in a novel Drosophila model of disease caused by mutations in VCP/p97

Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD) is a dominantly inherited degenerative disorder caused by mutations in the valosin-containing protein (VCP7) gene. VCP (p97 in mouse, TER94 in Drosophila melanogaster, and CDC48 in Saccharomyces cerevisiae) is a highly conserved AAA(+) (ATPases associated with multiple cellular activities) ATPase that regulates a wide array of cellular processes. The mechanism of IBMPFD pathogenesis is unknown. To elucidate the pathogenic mechanism, a Drosophila model of IBMPFD (mutant-VCP-related degeneration) was developed and characterized. Based on genetic screening of this model, three RNA-binding proteins were identified that dominantly suppressed degeneration; one of these was TBPH, the Drosophila homolog of TAR (trans-activating response region) DNA-binding protein 43 (TDP-43). VCP and TDP-43 interact genetically, and disease-causing mutations in VCP lead to redistribution of TDP-43 to the cytoplasm in vitro and in vivo, replicating the major pathology observed in IBMPFD and other TDP-43 proteinopathies. It was also demonstrated that TDP-43 redistribution from the nucleus to the cytoplasm is sufficient to induce cytotoxicity. Furthermore, it was determined that a pathogenic mutation in TDP-43 promotes redistribution to the cytoplasm and enhances the genetic interaction with VCP. Together, these results show that degeneration associated with VCP mutations is mediated in part by toxic gain of function of TDP-43 in the cytoplasm. It is suggested that these findings are likely relevant to the pathogenic mechanism of a broad array of TDP-43 proteinopathies, including frontotemporal lobar degeneration and amyotrophic lateral sclerosis (Ritson, 2010).

Depletion of TDP-43 affects Drosophila motoneurons terminal synapsis and locomotive behavior

Pathological modifications in the highly conserved and ubiquitously expressed heterogeneous ribonucleoprotein TDP-43 were recently associated to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), a late-onset disorder that affects predominantly motoneurons. However, the function of TDP-43 in vivo is unknown and a possible direct role in neurodegeneration remains speculative. This study reports that flies lacking Drosophila TDP-43 appeared externally normal but presented deficient locomotive behaviors, reduced life span and anatomical defects at the neuromuscular junctions. These phenotypes were rescued by expression of the human protein in a restricted group of neurons including motoneurons. These results demonstrate the role of this protein in vivo and suggest an alternative explanation to ALS pathogenesis that may be more due to the lack of TDP 43 function than to the toxicity of the aggregates (Feiguin, 2009).


Search PubMed for articles about Drosophila Tbp-43

Chang, J. C., Hazelett, D. J., Stewart, J. A. and Morton, D. B. (2013). Motor neuron expression of the voltage-gated calcium channel cacophony restores locomotion defects in a Drosophila, TDP-43 loss of function model of ALS. Brain Res. PubMed ID: 24275199

Chang, J. C. and Morton, D. B. (2017). Drosophila lines with mutant and wild type human TDP-43 replacing the endogenous gene reveals phosphorylation and ubiquitination in mutant lines in the absence of viability or lifespan defects. PLoS One 12(7): e0180828. PubMed ID: 28686708

Coyne, A. N., Siddegowda, B. B., Estes, P. S., Johannesmeyer, J., Kovalik, T., Daniel, S. G., Pearson, A., Bowser, R. and Zarnescu, D. C. (2014). Futsch/MAP1B mRNA is a translational target of TDP-43 and is neuroprotective in a Drosophila model of Amyotrophic Lateral Sclerosis J Neurosci 34: 15962-15974. PubMed ID: 25429138

Coyne, A. N., Yamada, S. B., Siddegowda, B. B., Estes, P. S., Zaepfel, B. L., Johannesmeyer, J. S., Lockwood, D. B., Pham, L. T., Hart, M. P., Cassel, J. A., Freibaum, B., Boehringer, A. V., Taylor, J. P., Reitz, A. B., Gitler, A. D. and Zarnescu, D. C. (2015). Fragile X protein mitigates TDP-43 toxicity by remodeling RNA granules and restoring translation. Hum Mol Genet [Epub ahead of print]. PubMed ID: 26385636

Diaper, D. C., Adachi, Y., Sutcliffe, B., Humphrey, D. M., Elliott, C. J., Stepto, A., Ludlow, Z. N., Vanden Broeck, L., Callaerts, P., Dermaut, B., Al-Chalabi, A., Shaw, C. E., Robinson, I. M. and Hirth, F. (2013a). Loss and gain of Drosophila TDP-43 impair synaptic efficacy and motor control leading to age-related neurodegeneration by loss-of-function phenotypes. Hum Mol Genet 22: 1539-1557. PubMed ID: 23307927

Diaper, D. C., Adachi, Y., Lazarou, L., Greenstein, M., Simoes, F. A., Di Domenico, A., Solomon, D. A., Lowe, S., Alsubaie, R., Cheng, D., Buckley, S., Humphrey, D. M., Shaw, C. E. and Hirth, F. (2013b). Drosophila TDP-43 dysfunction in glia and muscle cells cause cytological and behavioural phenotypes that characterize ALS and FTLD. Hum Mol Genet 22: 3883-3893. PubMed ID: 23727833

Estes, P. S., Boehringer, A., Zwick, R., Tang, J. E., Grigsby, B. and Zarnescu, D. C. (2011). Wild-type and A315T mutant TDP-43 exert differential neurotoxicity in a Drosophila model of ALS. Hum Mol Genet 20: 2308-2321. PubMed ID: 21441568

Feiguin, F., Godena, V. K., Romano, G., D'Ambrogio, A., Klima, R. and Baralle, F. E. (2009). Depletion of TDP-43 affects Drosophila motoneurons terminal synapsis and locomotive behavior. FEBS Lett 583: 1586-1592. PubMed ID: 19379745

Fiesel, F. C., Voigt, A., Weber, S. S., Van den Haute, C., Waldenmaier, A., Gorner, K., Walter, M., Anderson, M. L., Kern, J. V., Rasse, T. M., Schmidt, T., Springer, W., Kirchner, R., Bonin, M., Neumann, M., Baekelandt, V., Alunni-Fabbroni, M., Schulz, J. B. and Kahle, P. J. (2010). Knockdown of transactive response DNA-binding protein (TDP-43) downregulates histone deacetylase 6. EMBO J 29: 209-221. PubMed ID: 19910924

Godena, V. K., Romano, G., Romano, M., Appocher, C., Klima, R., Buratti, E., Baralle, F. E. and Feiguin, F. (2011). TDP-43 regulates Drosophila neuromuscular junctions growth by modulating Futsch/MAP1B levels and synaptic microtubules organization. PLoS One 6: e17808. PubMed ID: 21412434

Hazelett, D. J., Chang, J. C., Lakeland, D. L. and Morton, D. B. (2012). Comparison of parallel high-throughput RNA sequencing between knockout of TDP-43 and its overexpression reveals primarily nonreciprocal and nonoverlapping gene expression changes in the central nervous system of Drosophila. G3 (Bethesda) 2: 789-802. PubMed ID: 22870402

Hosaka, Y., Inoshita, T., Shiba-Fukushima, K., Cui, C., Arano, T., Imai, Y. and Hattori, N. (2017). Reduced TDP-43 expression improves neuronal activities in a Drosophila model of Perry syndrome. EBioMedicine [Epub ahead of print]. PubMed ID: 28625517

Joardar, A., Menzl, J., Podolsky, T. C., Manzo, E., Estes, P. S., Ashford, S. and Zarnescu, D. C. (2014). PPAR gamma activation is neuroprotective in a Drosophila model of ALS based on TDP-43. Hum Mol Genet 24(6): 1741-54. PubMed ID: 25432537

Kim, H. J., Raphael, A. R., Ladow, E. S., McGurk, L., Weber, R. A., Trojanowski, J. Q., Lee, V. M., Finkbeiner, S., Gitler, A. D. and Bonini, N. M. (2014). Therapeutic modulation of eIF2alpha phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models. Nat Genet 46: 152-160. PubMed ID: 24336168

King, I. N., Yartseva, V., Salas, D., Kumar, A., Heidersbach, A., Ando, D. M., Stallings, N. R., Elliott, J. L., Srivastava, D. and Ivey, K. N. (2014). The RNA-binding protein TDP-43 selectively disrupts microRNA-1/206 incorporation into the RNA-induced silencing complex. J Biol Chem 289: 14263-14271. PubMed ID: 24719334

Li, Y., Ray, P., Rao, E. J., Shi, C., Guo, W., Chen, X., Woodruff, E. A., Fushimi, K. and Wu, J. Y. (2010). A Drosophila model for TDP-43 proteinopathy. Proc Natl Acad Sci U S A 107: 3169-3174. PubMed ID: 20133767

Li, Z., Lu, Y., Xu, X. L. and Gao, F. B. (2013). The FTD/ALS-associated RNA-binding protein TDP-43 regulates the robustness of neuronal specification through microRNA-9a in Drosophila. Hum Mol Genet 22: 218-225. PubMed ID: 23042786

Llamusi, B., Bargiela, A., Fernandez-Costa, J. M., Garcia-Lopez, A., Klima, R., Feiguin, F. and Artero, R. (2013). Muscleblind, BSF and TBPH are mislocalized in the muscle sarcomere of a Drosophila myotonic dystrophy model. Dis Model Mech 6: 184-196. PubMed ID: 23118342

Lin, M. J., Cheng, C. W. and Shen, C. K. (2011). Neuronal function and dysfunction of Drosophila dTDP. PLoS One 6: e20371. PubMed ID: 21673800

Miskiewicz, K., Jose, L. E., Yeshaw, W. M., Valadas, J. S., Swerts, J., Munck, S., Feiguin, F., Dermaut, B. and Verstreken, P. (2014). HDAC6 is a Bruchpilot deacetylase that facilitates neurotransmitter release. Cell Rep 8: 94-102. PubMed ID: 24981865

Ritson, G. P., Custer, S. K., Freibaum, B. D., Guinto, J. B., Geffel, D., Moore, J., Tang, W., Winton, M. J., Neumann, M., Trojanowski, J. Q., Lee, V. M., Forman, M. S. and Taylor, J. P. (2010). TDP-43 mediates degeneration in a novel Drosophila model of disease caused by mutations in VCP/p97. J Neurosci 30: 7729-7739. PubMed ID: 20519548

Romano, M., Buratti, E., Romano, G., Klima, R., Del Bel Belluz, L., Stuani, C., Baralle, F. and Feiguin, F. (2014). Evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins functionally interact with human and Drosophila TAR DNA-binding protein 43 (TDP-43). J Biol Chem 289: 7121-7130. PubMed ID: 24492607

Rumpf, S., Bagley, J. A., Thompson-Peer, K. L., Zhu, S., Gorczyca, D., Beckstead, R. B., Jan, L. Y. and Jan, Y. N. (2014), Drosophila Valosin-Containing Protein is required for dendrite pruning through a regulatory role in mRNA metabolism. Proc Natl Acad Sci U S A 111(20):7331-6. PubMed ID: 24799714

Vanden Broeck, L., Naval-Sanchez, M., Adachi, Y., Diaper, D., Dourlen, P., Chapuis, J., Kleinberger, G., Gistelinck, M., Van Broeckhoven, C., Lambert, J. C., Hirth, F., Aerts, S., Callaerts, P. and Dermaut, B. (2013). TDP-43 loss-of-function causes neuronal loss due to defective steroid receptor-mediated gene program switching in Drosophila. Cell Rep 3: 160-172. PubMed ID: 23333275

Wang, J. W., Brent, J. R., Tomlinson, A., Shneider, N. A. and McCabe, B. D. (2011). The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span. J Clin Invest 121: 4118-4126. PubMed ID: 21881207

Zhan, L., Hanson, K. A., Kim, S. H., Tare, A. and Tibbetts, R. S. (2013). Identification of genetic modifiers of TDP-43 neurotoxicity in Drosophila. PLoS One 8: e57214. PubMed ID: 23468938

Biological Overview

date revised: 12 November 2017

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