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 link: EntrezGene
TBPH orthologs: Biolitmine

Recent literature
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
Summary:
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
Summary:
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
Summary:
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.
Uechi, H., Kuranaga, E., Iriki, T., Takano, K., Hirayama, S., Miura, M., Hamazaki, J. and Murata, S. (2017). Ubiquitin-binding protein CG5445 suppresses aggregation and cytotoxicity of Amyotrophic Lateral Sclerosis-linked TDP-43 in Drosophila. Mol Cell Biol [Epub ahead of print]. PubMed ID: 29109084
Summary:
Ubiquitin-mediated protein degradation plays essential roles in proteostasis and is involved in the pathogenesis of neurodegenerative diseases in which ubiquitin-positive aberrant proteins accumulate. However, how such aberrant proteins are processed inside cells has not been fully explored. This study show that the product of CG5445, a previously uncharacterized Drosophila gene, prevents accumulation of aggregate-prone ubiquitinated proteins. Ubiquitin conjugates were associated with CG5445, knockdown of which caused accumulation of detergent-insoluble ubiquitinated proteins. Furthermore, CG5445 rescued eye degeneration caused by the amyotrophic lateral sclerosis (ALS)-linked mutant TAR DNA-binding protein 43-kDa (TDP-43), which often forms ubiquitin-positive aggregates in cells through the capacity of CG5445 to bind to ubiquitin chains. Biochemically, CG5445 inhibited accumulation of insoluble forms and promoted their clearance. These results demonstrate a new possible mechanism by which cells maintain ubiquitinated aggregation-prone proteins in a soluble form to decrease their cytotoxicity until they are degraded.
Manzo, E., O'Conner, A. G., Barrows, J. M., Shreiner, D. D., Birchak, G. J. and Zarnescu, D. C. (2018). Medium-chain fatty acids, beta-hydroxybutyric acid and genetic modulation of the carnitine shuttle are protective in a Drosophila model of ALS Based on TDP-43. Front Mol Neurosci 11: 182. PubMed ID: 29904341
Summary:
ALS patients exhibit dyslipidemia, hypermetabolism and weight loss; in addition, cellular energetics deficits have been detected prior to denervation. Although evidence that metabolism is altered in ALS is compelling, the mechanisms underlying metabolic dysregulation and the contribution of altered metabolic pathways to disease remain poorly understood. This study used a Drosophila model of ALS based on TDP-43 that recapitulates hallmark features of the disease including locomotor dysfunction and reduced lifespan. A global, unbiased metabolomic profiling of larvae expressing TDP-43 (wild-type, TDPWT or disease-associated mutant, TDPG298S) and identified several lipid metabolism associated alterations. Among these, a significant increase was found in carnitine conjugated long-chain fatty acids and a significant decrease in carnitine, acetyl-carnitine and beta-hydroxybutyrate, a ketone precursor. Taken together these data suggest a deficit in the function of the carnitine shuttle and reduced lipid beta oxidation. To test this possibility a combined genetic and dietary approach was used in Drosophila. The findings indicate that components of the carnitine shuttle are misexpressed in the context of TDP-43 proteinopathy and that genetic modulation of CPT1 or CPT2 expression, two core components of the carnitine shuttle, mitigates TDP-43 dependent locomotor dysfunction, in a variant dependent manner. In addition, feeding medium-chain fatty acids or beta-hydroxybutyrate improves locomotor function, consistent with the notion that bypassing the carnitine shuttle deficit is neuroprotective. Taken together, these findings highlight the potential contribution of the carnitine shuttle and lipid beta oxidation in ALS and suggest strategies for therapeutic intervention based on restoring lipid metabolism in motor neurons.
Jantrapirom, S., Piccolo, L. L., Yoshida, H. and Yamaguchi, M. (2018). Depletion of Ubiquilin induces an augmentation in soluble ubiquitinated Drosophila TDP-43 to drive neurotoxicity in the fly. Biochim Biophys Acta. PubMed ID: 29936333
Summary:
The proteostasis machinery has critical functions in metabolically active cells such as neurons. Ubiquilins (UBQLNs) may decide the fate of proteins, with its ability to bind and deliver ubiquitinated misfolded or no longer functionally required proteins to the ubiquitin-proteasome system (UPS) and/or autophagy. Missense mutations in UBQLN2 have been linked to X-linked dominant amyotrophic lateral sclerosis with frontotemporal dementia (ALS-FTD). Although aggregation-prone TAR DNA-binding protein 43 (TDP-43) has been recognized as a major component of the ubiquitin pathology, the mechanisms by which UBQLN involves in TDP-43 proteinopathy have not yet been elucidated in detail. Previous work has characterized new Drosophila Ubiquilin (dUbqn) knockdown model that produces learning/memory and locomotive deficits during the proteostasis impairment. The present study demonstrated that the depletion of dUbqn markedly affected the expression and sub-cellular localization of Drosophila TDP-43 (TBPH), resulting in a cytoplasmic ubiquitin-positive (Ub(+)) TBPH pathology. Although it was found that the knockdown of dUbqn widely altered and affected the turnover of a large number of proteins, this study showed that an augmented soluble cytoplasmic Ub(+)-TBPH is as a crucial source of neurotoxicity following the depletion of dUbqn. It was demonstrated that dUbqn knockdown-related neurotoxicity may be rescued by either restoring the proteostasis machinery or reducing the expression of TBPH. These novel results extend knowledge on the UBQLN loss-of-function pathomechanism and may contribute to the identification of new therapeutics for ALS-FTD and aging-related diseases.
Pons, M., Prieto, S., Miguel, L., Frebourg, T., Campion, D., Sune, C. and Lecourtois, M. (2018). Identification of TCERG1 as a new genetic modulator of TDP-43 production in Drosophila. Acta Neuropathol Commun 6(1): 138. PubMed ID: 30541625
Summary:
TAR DNA-binding protein-43 (TDP-43) is a ubiquitously expressed DNA-/RNA-binding protein that has been linked to numerous aspects of the mRNA life cycle. Similar to many RNA-binding proteins, TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop. Cell function and survival depend on the strict control of TDP-43 protein levels. TDP-43 has been identified as the major constituent of ubiquitin-positive inclusions in patients with Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Several observations argue for a pathogenic role of elevated TDP-43 levels in these disorders. Modulation of the cycle of TDP-43 production might therefore provide a new therapeutic strategy. Using a Drosophila model mimicking key features of the TDP-43 autoregulatory feedback loop, this study identified CG42724 as a genetic modulator of TDP-43 production in vivo. CG42724 protein was found to influence qualitatively and quantitatively the TDP-43 mRNA transcript pattern. CG42724 overexpression promotes the production of transcripts that can be efficiently released into the cytoplasm for protein translation. Importantly, TCERG1, the human homolog of the Drosophila CG42724 protein, also caused an increase of TDP-43 protein steady-state levels in mammalian cells. Therefore, these data suggest the possibility that targeting TCERG1 could be therapeutic in TDP-43 proteinopathies.
Lembke, K. M., Law, A. D., Ahrar, J. and Morton, D. B. (2018). Deletion of a specific exon in the voltage-gated calcium channel, cacophony, causes disrupted locomotion in Drosophila larvae. J Exp Biol. PubMed ID: 30397173
Summary:
Tar DNA binding protein 43 (TDP-43) is an RNA binding protein that regulates transcription, translation, and alternative splicing of mRNA. Null mutations of the Drosophila orthologue, Tar DNA-binding homologue (tbph), has been shown to cause severe locomotion defects in larvae that are mediated by a reduction in the expression of the type II voltage-gated calcium channel, cacophony (cac). TDP-43 also regulates the inclusion of alternatively spliced exons of cacophony; tbph mutants showed significantly increased expression of cacophony isoforms lacking exon 7, a particularly notable finding as only one out of the 15 predicted isoforms lacks exon 7. To investigate the function of exon 7, Drosophila mutant lines were generated with a deletion that eliminates exon 7. This deletion phenocopies many defects in tbph mutants: a reduction in Cacophony protein expression, locomotion defects in male and female third instar larvae, disrupted larval motor output, and also reduced activity levels in adult male flies. All these defects were rescued by expression of cacophony transcripts containing exon 7. By contrast, expression of a cacophony cDNA lacking exon 7 resulted in reduced Cacophony protein levels and failed to rescue larval locomotion.
Duan, Y., et al. (2019). PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins. Cell Res. PubMed ID: 30728452
Summary:
Mutations in RNA-binding proteins (RBPs) localized in ribonucleoprotein (RNP) granules, such as hnRNP A1 and TDP-43, promote aberrant protein aggregation, which is a pathological hallmark of various neurodegenerative diseases, such as myotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Protein posttranslational modifications (PTMs) are known to regulate RNP granules. This study investigated the function of poly(ADP-ribosyl)ation (PARylation), an important PTM involved in DNA damage repair and cell death, in RNP granule-related neurodegeneration. PARylation levels are a major regulator of the assembly-disassembly dynamics of RNP granules containing disease-related RBPs, hnRNP A1 and TDP-43. hnRNP A1 can both be PARylated and bind to PARylated proteins or poly(ADP-ribose) (PAR). It was further uncovered that PARylation of hnRNP A1 at K298 controls its nucleocytoplasmic transport, whereas PAR-binding via the PAR-binding motif (PBM) of hnRNP A1 regulates its association with stress granules. Moreover, it was revealed that PAR not only dramatically enhances the liquid-liquid phase separation of hnRNP A1, but also promotes the co-phase separation of hnRNP A1 and TDP-43 in vitro and their interaction in vivo. Finally, both genetic and pharmacological inhibition of PARP mitigates hnRNP A1- and TDP-43-mediated neurotoxicity in cell and Drosophila models of ALS. Together, these findings suggest a novel and crucial role for PARylation in regulating the dynamics of RNP granules, and that dysregulation in PARylation and PAR levels may contribute to ALS disease pathogenesis by promoting protein aggregation.
Manzo, E., Lorenzini, I., Barrameda, D., O'Conner, A. G., Barrows, J. M., Starr, A., Kovalik, T., Rabichow, B. E., Lehmkuhl, E. M., Shreiner, D. D., Joardar, A., Lievens, J. C., Bowser, R., Sattler, R. and Zarnescu, D. C. (2019). Glycolysis upregulation is neuroprotective as a compensatory mechanism in ALS. Elife 8. PubMed ID: 31180318
Summary:
Amyotrophic Lateral Sclerosis (ALS), is a fatal neurodegenerative disorder, with TDP-43 inclusions as a major pathological hallmark. Using a Drosophila model of TDP-43 proteinopathy this study found significant alterations in glucose metabolism including increased pyruvate, suggesting that modulating glycolysis may be neuroprotective. Indeed, a high sugar diet improves locomotor and lifespan defects caused by TDP-43 proteinopathy in motor neurons or glia, but not muscle, suggesting that metabolic dysregulation occurs in the nervous system. Overexpressing human glucose transporter GLUT-3 in motor neurons mitigates TDP-43 dependent defects in synaptic vesicle recycling and improves locomotion. Furthermore, PFK mRNA, a key indicator of glycolysis, is upregulated in flies and patient derived iPSC motor neurons with TDP-43 pathology. Surprisingly, PFK overexpression rescues TDP-43 induced locomotor deficits. These findings from multiple ALS models show that mechanistically, glycolysis is upregulated in degenerating motor neurons as a compensatory mechanism and suggest that increased glucose availability is protective.
Donde, A., Sun, M., Jeong, Y. H., Wen, X., Ling, J., Lin, S., Braunstein, K., Nie, S., Wang, S., Chen, L. and Wong, P. C. (2019). Upregulation of ATG7 attenuates motor neuron dysfunction associated with depletion of TARDBP/TDP-43. Autophagy. PubMed ID: 31242080
Summary:
A shared neuropathological hallmark in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is nuclear clearance and cytoplasmic aggregation of TARDBP/TDP-43 (TAR DNA binding protein). Previously work has shown that the ability of TARDBP to repress nonconserved cryptic exons was impaired in brains of patients with ALS and FTD, suggesting that its nuclear depletion contributes to neurodegeneration. However, the critical pathways impacted by the failure to repress cryptic exons that may contribute to neurodegeneration remain undefined. This report that transcriptome analysis of TARDBP-deficient neurons revealed downregulation of ATG7, a critical gene required for macroautophagy/autophagy. Mouse and Drosophila models lacking TARDBP/TBPH in motor neurons exhibiting age-dependent neurodegeneration and motor deficits showed reduction of ATG7 and accumulation of SQSTM1/p62 inclusions. Importantly, genetic upregulation of the autophagy pathway improved motor function and survival in TBPH-deficient flies. Together with the observation that ATG7 is reduced in ALS-FTD brain tissues, these findings identify the autophagy pathway as one key effector of nuclear depletion of TARDBP that contributes to neurodegeneration. It is thus suggested that the autophagy pathway is a therapeutic target for ALS-FTD and other disorders exhibiting TARDBP pathology.
Donde, A., Sun, M., Ling, J. P., Braunstein, K. E., Pang, B., Wen, X., Cheng, X., Chen, L. and Wong, P. C. (2019). Splicing repression is a major function of TDP-43 in motor neurons. Acta Neuropathol. PubMed ID: 31332509
Summary:
Nuclear depletion of TDP-43, an essential RNA binding protein, may underlie neurodegeneration in amyotrophic lateral sclerosis (ALS). As several functions have been ascribed to this protein, the critical role(s) of TDP-43 in motor neurons that may be compromised in ALS remains unknown. This study shows that TDP-43 mediated splicing repression, which serves to protect the transcriptome by preventing aberrant splicing, is central to the physiology of motor neurons. Expression in Drosophila TDP-43 knockout models of a chimeric repressor, comprised of the RNA recognition domain of TDP-43 fused to an unrelated splicing repressor, RAVER1, attenuated motor deficits and extended lifespan. Likewise, AAV9-mediated delivery of this chimeric rescue repressor to mice lacking TDP-43 in motor neurons delayed the onset, slowed the progression of motor symptoms, and markedly extended their lifespan. In treated mice lacking TDP-43 in motor neurons, aberrant splicing was significantly decreased and accompanied by amelioration of axon degeneration and motor neuron loss. This AAV9 strategy allowed long-term expression of the chimeric repressor without any adverse effects. These findings establish that splicing repression is a major function of TDP-43 in motor neurons and strongly support the idea that loss of TDP-43-mediated splicing fidelity represents a key pathogenic mechanism underlying motor neuron loss in ALS.
Cacciottolo, R., Ciantar, J., Lanfranco, M., Borg, R. M., Vassallo, N., Bordonne, R. and Cauchi, R. J. (2019). SMN complex member Gemin3 self-interacts and has a functional relationship with ALS-linked proteins TDP-43, FUS and Sod1. Sci Rep 9(1): 18666. PubMed ID: 31822699
Summary:
The predominant motor neuron disease in infants and adults is spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS), respectively. SMA is caused by insufficient levels of the Survival Motor Neuron (SMN) protein, which operates as part of the multiprotein SMN complex that includes the DEAD-box RNA helicase Gemin3/DDX20/DP103. C9orf72, SOD1, TDP-43 and FUS are ranked as the four major genes causing familial ALS. Accumulating evidence has revealed a surprising molecular overlap between SMA and ALS. This study asked the question of whether Drosophila can also be exploited to study shared pathogenic pathways. Focusing on motor behaviour, muscle mass and survival, disruption of either TBPH/TDP-43 or Caz/FUS was found to enhance defects associated with Gemin3 loss-of-function. Gemin3-associated neuromuscular junction overgrowth was however suppressed. Sod1 depletion had a modifying effect in late adulthood. This study also showed that Gemin3 self-interacts and Gem3(DeltaN), a helicase domain deletion mutant, retains the ability to interact with its wild-type counterpart. Importantly, mutant:wild-type dimers are favoured more than wild-type:wild-type dimers. In addition to reinforcing the link between SMA and ALS, further exploration of mechanistic overlaps is now possible in a genetically tractable model organism. Notably, Gemin3 can be elevated to a candidate for modifying motor neuron degeneration.
Strah, N., Romano, G., Introna, C., Klima, R., Marzullo, M., Ciapponi, L., Megighian, A., Nizzardo, M. and Feiguin, F. (2020). TDP-43 promotes the formation of neuromuscular synapses through the regulation of Disc-large expression in Drosophila skeletal muscles. BMC Biol 18(1): 34. PubMed ID: 32216790
Summary:
The ribonuclear protein TDP-43 has been implicated in the pathophysiology of amyotrophic lateral sclerosis (ALS), with genetic mutations being linked to the neurological symptoms of the disease. Alterations in the intracellular distribution of TDP-43 have been observed in skeletal muscles of patients suffering from ALS and the molecular and metabolic pathways regulated by TDP-43 in the skeletal muscle remain largely unknown. This study analyzed the function of TBPH, the Drosophila melanogaster ortholog of TDP-43, in skeletal muscles. The activity of TDP-43 in Drosophila muscles was modulated by means of RNA interference, and it was observed to be required to promote the formation and growth of neuromuscular synapses. TDP-43 regulated the expression levels of Disc-large (Dlg), and restoring Dlg expression either in skeletal muscles or in motoneurons was sufficient to suppress the locomotive and synaptic defects of TDP-43-null flies. These results were validated by the observation of a decrease in Dlg levels in human neuroblastoma cells and iPSC-differentiated motoneurons derived from ALS patients, suggesting similar mechanisms may potentially be involved in the pathophysiology of the disease. These results help to unveil the physiological role of TDP-43 in skeletal muscles as well as the mechanisms responsible for the autonomous and non-autonomous behavior of this protein concerning the organization of neuromuscular synapses.
Sanna, S., Esposito, S., Masala, A., Sini, P., Nieddu, G., Galioto, M., Fais, M., Iaccarino, C., Cestra, G. and Crosio, C. (2020). HDAC1 inhibition ameliorates TDP-43-induced cell death in vitro and in vivo. Cell Death Dis 11(5): 369. PubMed ID: 32409664
Summary:
TDP-43 pathology is a disease hallmark that characterizes both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). TDP-43 undergoes several posttranslational modifications that can change its biological activities and its aggregative propensity, which is a common hallmark of different neurodegenerative conditions. New evidence is provided by the current study pointing at TDP-43 acetylation in ALS cellular models. Using both in vitro and in vivo approaches, it was demonstrated that TDP-43 interacts with histone deacetylase 1 (HDAC1) via RRM1 and RRM2 domains, that are known to contain the two major TDP-43 acetylation sites, K142 and K192. Moreover, this study showed that TDP-43 is a direct transcriptional activator of CHOP promoter and this activity is regulated by acetylation. Finally and most importantly, it was observed both in cell culture and in Drosophila that a HDCA1 reduced level (genomic inactivation or siRNA) or treatment with pan-HDAC inhibitors exert a protective role against WT or pathological mutant TDP-43 toxicity, suggesting TDP-43 acetylation as a new potential therapeutic target. HDAC inhibition efficacy in neurodegeneration has long been debated, but future investigations are warranted in this area. Selection of more specific HDAC inhibitors is still a promising option for neuronal protection especially as HDAC1 appears as a downstream target of both TDP- 43 and FUS, another ALS-related gene.
Park, J. H., Chung, C. G., Park, S. S., Lee, D., Kim, K. M., Jeong, Y., Kim, E. S., Cho, J. H., Jeon, Y. M., Shen, C. J., Kim, H. J., Hwang, D. and Lee, S. B. (2020). Cytosolic calcium regulates cytoplasmic accumulation of TDP-43 through Calpain-A and Importin alpha3. Elife 9. PubMed ID: 33305734
Summary:
Cytoplasmic accumulation of TDP-43 in motor neurons is the most prominent pathological feature in amyotrophic lateral sclerosis (ALS). A feedback cycle between nucleocytoplasmic transport (NCT) defect and TDP-43 aggregation was shown to contribute to accumulation of TDP-43 in the cytoplasm. However, little is known about cellular factors that can control the activity of NCT, thereby affecting TDP-43 accumulation in the cytoplasm. This study identified via FRAP and optogenetics cytosolic calcium as a key cellular factor controlling NCT of TDP-43. Dynamic and reversible changes in TDP-43 localization were observed in Drosophila sensory neurons during development. Genetic and immunohistochemical analyses identified the cytosolic calcium-Calpain-A-Importin α3 pathway as a regulatory mechanism underlying NCT of TDP-43. In C9orf72 ALS fly models, upregulation of the pathway activity by increasing cytosolic calcium reduced cytoplasmic accumulation of TDP-43 and mitigated behavioral defects. Together, these results suggest the calcium-Calpain-A-Importin α3 pathway as a potential therapeutic target of ALS.
Matsukawa, K., Kukharsky, M. S., Park, S. K., Park, S., Watanabe, N., Iwatsubo, T., Hashimoto, T., Liebman, S. W. and Shelkovnikova, T. A. (2021). Long non-coding RNA NEAT1_1 ameliorates TDP-43 toxicity in in vivo models of TDP-43 proteinopathy. RNA Biol: 1-9. PubMed ID: 33427561
Summary:
Pathological changes involving TDP-43 protein ('TDP-43 proteinopathy') are typical for several neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD). FTLD-TDP cases are characterized by increased binding of TDP-43 to an abundant lncRNA, NEAT1, in the cortex. However it is unclear whether enhanced TDP-43-NEAT1 interaction represents a protective mechanism. This study shows that accumulation of human TDP-43 leads to upregulation of the constitutive NEAT1 isoform, NEAT1_1, in cultured cells and in the brains of transgenic mice. Further, it was demonstrated that overexpression of NEAT1_1 ameliorates TDP-43 toxicity in Drosophila and yeast models of TDP-43 proteinopathy. Thus, NEAT1_1 upregulation may be protective in TDP-43 proteinopathies affecting the brain. Approaches to boost NEAT1_1 expression in the CNS may prove useful in the treatment of these conditions.
Baek, M., Choe, Y. J., Bannwarth, S., Kim, J., Maitra, S., Dorn, G. W., Taylor, J. P., Paquis-Flucklinger, V. and Kim, N. C. (2021). TDP-43 and PINK1 mediate CHCHD10(S59L) mutation-induced defects in Drosophila and in vitro. Nat Commun 12(1): 1924. PubMed ID: 33772006
Summary:
Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) can cause amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). However, the underlying mechanisms are unclear. This study generate CHCH10(S59L)-mutant Drosophila melanogaster and HeLa cell lines to model CHCHD10-associated ALS-FTD. The CHCHD10(S59L) mutation results in cell toxicity in several tissues and mitochondrial defects. CHCHD10(S59L) independently affects the TDP-43 and PINK1 pathways. CHCHD10(S59L) expression increases TDP-43 insolubility and mitochondrial translocation. Blocking TDP-43 mitochondrial translocation with a peptide inhibitor reduced CHCHD10(S59L)-mediated toxicity. While genetic and pharmacological modulation of PINK1 expression and activity of its substrates rescues and mitigates the CHCHD10(S59L)-induced phenotypes and mitochondrial defects, respectively, in both Drosophila and HeLa cells. These findings suggest that CHCHD10(S59L)-induced TDP-43 mitochondrial translocation and chronic activation of PINK1-mediated pathways result in dominant toxicity, providing a mechanistic insight into the CHCHD10 mutations associated with ALS-FTD.
Lehmkuhl, E. M., Loganathan, S., Alsop, E., Blythe, A. D., Kovalik, T., Mortimore, N. P., Barrameda, D., Kueth, C., Eck, R. J., Siddegowda, B. B., Joardar, A., Ball, H., Macias, M. E., Bowser, R., Van Keuren-Jensen, K. and Zarnescu, D. C. (2021). TDP-43 proteinopathy alters the ribosome association of multiple mRNAs including the glypican Dally-like protein (Dlp)/GPC6. Acta Neuropathol Commun 9(1): 52. PubMed ID: 33762006
Summary:
Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative disease in which 97% of patients exhibit cytoplasmic aggregates containing the RNA binding protein TDP-43. Using tagged ribosome affinity purifications in Drosophila models of TDP-43 proteinopathy, TDP-43 dependent translational alterations in motor neurons were identified impacting the spliceosome, pentose phosphate and oxidative phosphorylation pathways. A subset of the mRNAs with altered ribosome association are also enriched in TDP-43 complexes suggesting that they may be direct targets. Among these, dlp mRNA, which encodes the glypican Dally like protein (Dlp)/GPC6, a wingless (Wg/Wnt) signaling regulator is insolubilized both in flies and patient tissues with TDP-43 pathology. While Dlp/GPC6 forms puncta in the Drosophila neuropil and ALS spinal cords, it is reduced at the neuromuscular synapse in flies suggesting compartment specific effects of TDP-43 proteinopathy. These findings together with genetic interaction data show that Dlp/GPC6 is a novel, physiologically relevant target of TDP-43 proteinopathy.
Zhao, M. J., Yao, X., Wei, P., Zhao, C., Cheng, M., Zhang, D., Xue, W., He, W. T., Xue, W., Zuo, X., Jiang, L. L., Luo, Z., Song, J., Shu, W. J., Yuan, H. Y., Liang, Y., Sun, H., Zhou, Y., Zhou, Y., Zheng, L., Hu, H. Y., Wang, J. and Du, H. N. (2021). O-GlcNAcylation of TDP-43 suppresses proteinopathies and promotes TDP-43's mRNA splicing activity. EMBO Rep: e51649. PubMed ID: 33855783
Summary:
Pathological TDP-43 aggregation is characteristic of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP); however, how TDP-43 aggregation and function are regulated remain poorly understood. This study shows that O-GlcNAc transferase OGT-mediated O-GlcNAcylation of TDP-43 suppresses ALS-associated proteinopathies and promotes TDP-43's splicing function. Biochemical and cell-based assays indicate that OGT's catalytic activity suppresses TDP-43 aggregation and hyperphosphorylation, whereas abolishment of TDP-43 O-GlcNAcylation impairs its RNA splicing activity. This study further showed that TDP-43 mutations in the O-GlcNAcylation sites improve locomotion defects of larvae and adult flies and extend adult life spans, following TDP-43 overexpression in Drosophila motor neurons. This study demonstrates that O-GlcNAcylation of TDP-43 promotes proper splicing of many mRNAs, including STMN2, which is required for normal axonal outgrowth and regeneration. These findings suggest that O-GlcNAcylation might be a target for the treatment of TDP-43-linked pathogenesis.
Loganathan, S., Ball, H. E., Manzo, E. and Zarnescu, D. C. (2021). Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy. J Vis Exp(174). PubMed ID: 34424253
Summary:
Als is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy broad ranging metabolic deficits have been identified. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700μδ6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700μδ6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43(G298S), an ALS causing variant. This study shows how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700μδ6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.
Wall, J. M., Basu, A., Zunica, E. R. M., Dubuisson, O. S., Pergola, K., Broussard, J. P., Kirwan, J. P., Axelrod, C. L. and Johnson, A. E. (2021). CRISPR/Cas9-engineered Drosophila knock-in models to study VCP diseases. Dis Model Mech 14(7). PubMed ID: 34160014
Summary:
Mutations in Valosin Containing Protein (VCP) are associated with several degenerative diseases, including multisystem proteinopathy (MSP-1) and amyotrophic lateral sclerosis. However, patients with VCP mutations vary widely in their pathology and clinical penetrance, making it difficult to devise effective treatment strategies. A deeper understanding of how each mutation affects VCP function could enhance the prediction of clinical outcomes and design of personalized treatment options. The power of a genetically tractable model organism coupled with well-established in vivo assays and a relatively short life cycle make Drosophila an attractive system to study VCP disease pathogenesis. Using CRISPR/Cas9, this study generated individual Drosophila knock-in mutants that include nine hereditary VCP disease mutations. The models display many hallmarks of VCP-mediated degeneration, including progressive decline in mobility, protein aggregate accumulation and defects in lysosomal and mitochondrial function. Some novel and unexpected findings were made, including nuclear morphology defects and sex-specific phenotypic differences in several mutants. Taken together, the Drosophila VCP disease models generated in this study will be useful for studying the etiology of individual VCP patient mutations and testing potential genetic and/or pharmacological therapies.
Hughes, M. P., Goldschmidt, L. and Eisenberg, D. S. (2021). Prevalence and species distribution of the low-complexity, amyloid-like, reversible, kinked segment (LARKS) structural motif in amyloid-like fibrils. J Biol Chem: 101194. PubMed ID: 34537246
Summary:
Membraneless Organelles (MLOs) are vital and dynamic reaction centers in cells that compartmentalize the cytoplasm in the absence of a membrane. Multivalent interactions between protein Low-Complexity Domains (LCDs) contribute to MLO organization. Previously, This study used computational methods to identify structural motifs termed Low-complexity Amyloid-like Reversible Kinked Segments (LARKS) that promote phase-transition to form hydrogels and that are common in human proteins that participate in MLOs. This study searched for LARKS in the proteomes of six model organisms: Homo sapiens, Drosophila melanogaster, Plasmodium falciparum, Saccharomyces cerevisiae, Mycobacterium tuberculosis, and Escherichia coli to gain an understanding of the distribution of LARKS in the proteomes of various species. This study found that LARKS are abundant in M. tuberculosis, D. melanogaster, and H. sapiens, but not in S. cerevisiae or P. falciparum. LARKS have high glycine content, which enables kinks to form as exemplified by the known LARKS-rich amyloidogenic structures of TDP43, FUS, and hnRNPA2, three proteins that are known to participate in MLOs. These results support the idea of LARKS as an evolved structural motif. Based on these results, this study also established the LARKSdb webserver, which permits users to search for LARKS in their protein sequences of interest (Hughes, 2021).
Romano, G., Holodkov, N., Klima, R. and Feiguin, F. (2021). TDP-43 regulates GAD1 mRNA splicing and GABA signaling in Drosophila CNS. Sci Rep 11(1): 18761. PubMed ID: 34548578
Summary:
Alterations in the function of the RNA-binding protein TDP-43 are largely associated with the pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating disease of the human motor system that leads to motoneurons degeneration and reduced life expectancy by molecular mechanisms not well known. In previous work, it was found found that the expression levels of the glutamic acid decarboxylase enzyme (GAD1), responsible for converting glutamate to γ-aminobutyric acid (GABA), were downregulated in TBPH-null flies and motoneurons derived from ALS patients carrying mutations in TDP-43, suggesting that defects in the regulation of GAD1 may lead to neurodegeneration by affecting neurotransmitter balance. This study observed that TBPH was required for the regulation of GAD1 pre-mRNA splicing and the levels of GABA in the Drosophila central nervous system (CNS). Interestingly, this study discovered that pharmacological treatments aimed to potentiate GABA neurotransmission were able to revert locomotion deficiencies in TBPH-minus flies, revealing novel mechanisms and therapeutic strategies in ALS.
Azpurua, J., El-Karim, E. G., Tranquille, M. and Dubnau, J. (2021). A behavioral screen for mediators of age-dependent TDP-43 neurodegeneration identifies SF2/SRSF1 among a group of potent suppressors in both neurons and glia. PLoS Genet 17(11): e1009882. PubMed ID: 34723963
Summary:
Cytoplasmic aggregation of Tar-DNA/RNA binding protein 43 (TDP-43) occurs in 97 percent of amyotrophic lateral sclerosis (ALS), ~40% of frontotemporal dementia (FTD) and in many cases of Alzheimer's disease (AD). Cytoplasmic TDP-43 inclusions are seen in both sporadic and familial forms of these disorders, including those cases that are caused by repeat expansion mutations in the C9orf72 gene. To identify downstream mediators of TDP-43 toxicity, This study expressed human TDP-43 in a subset of Drosophila motor neurons. Such expression causes age-dependent deficits in negative geotaxis behavior. Using this behavioral readout of locomotion, this study conducted an shRNA suppressor screen and identified 32 transcripts whose knockdown was sufficient to ameliorate the neurological phenotype. The majority of these suppressors also substantially suppressed the negative effects on lifespan seen with glial TDP-43 expression. In addition to identification of a number of genes whose roles in neurodegeneration were not previously known, this screen also yielded genes involved in chromatin regulation and nuclear/import export - pathways that were previously identified in the context of cell based or neurodevelopmental suppressor screens. A notable example is SF2, a conserved orthologue of mammalian SRSF1, an RNA binding protein with roles in splicing and nuclear export. The identification SF2/SRSF1 as a potent suppressor of both neuronal and glial TDP-43 toxicity also provides a convergence with C9orf72 expansion repeat mediated neurodegeneration, where this gene also acts as a downstream mediator (Azpurua, 2021).
Jeon, Y. M., Kwon, Y., Lee, S., Kim, S., Jo, M., Lee, S., Kim, S. R., Kim, K. and Kim, H. J. (2021). Vitamin B12 Reduces TDP-43 Toxicity by Alleviating Oxidative Stress and Mitochondrial Dysfunction. Antioxidants (Basel) 11(1). PubMed ID: 35052586
Summary:
TAR DNA-binding protein 43 (TDP-43) is a member of an evolutionarily conserved family of heterogeneous nuclear ribonucleoproteins that modulate multiple steps in RNA metabolic processes. Cytoplasmic aggregation of TDP-43 in affected neurons is a pathological hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer's disease (AD), and limbic predominant age-related TDP-43 encephalopathy (LATE). Mislocalized and accumulated TDP-43 in the cytoplasm induces mitochondrial dysfunction and reactive oxidative species (ROS) production. This study shows that TDP-43- and rotenone-induced neurotoxicity in the human neuronal cell line SH-SY5Y were attenuated by hydroxocobalamin (Hb, vitamin B(12) analog) treatment. Although Hb did not affect the cytoplasmic accumulation of TDP-43, Hb attenuated TDP-43-induced toxicity by reducing oxidative stress and mitochondrial dysfunction. Moreover, a shortened lifespan and motility defects in TDP-43-expressing Drosophila were significantly mitigated by dietary treatment with hydroxocobalamin. Taken together, these findings suggest that oral intake of hydroxocobalamin may be a potential therapeutic intervention for TDP-43-associated proteinopathies.
Lee, S., Jo, M., Lee, H. E., Jeon, Y. M., Kim, S., Kwon, Y., Woo, J., Han, S., Mun, J. Y. and Kim, H. J. (2021). HEXA-018, a Novel Inducer of Autophagy, Rescues TDP-43 Toxicity in Neuronal Cells. Front Pharmacol 12: 747975. PubMed ID: 34925009
Summary:
The autophagy-lysosomal pathway is an essential cellular mechanism that degrades aggregated proteins and damaged cellular components to maintain cellular homeostasis. This study identified HEXA-018, a novel compound containing a catechol derivative structure, as a novel inducer of autophagy. HEXA-018 increased the LC3-I/II ratio, which indicates activation of autophagy. Consistent with this result, HEXA-018 effectively increased the numbers of autophagosomes and autolysosomes in neuronal cells. This study also found that the activation of autophagy by HEXA-018 is mediated by the AMPK-ULK1 pathway in an mTOR-independent manner. It was further shown that ubiquitin proteasome system impairment- or oxidative stress-induced neurotoxicity was significantly reduced by HEXA-018 treatment. Moreover, oxidative stress-induced mitochondrial dysfunction was strongly ameliorated by HEXA-018 treatment. In addition, the efficacy of HEXA-018 in models of TDP-43 proteinopathy was investigated. HEXA-018 treatment mitigated TDP-43 toxicity in cultured neuronal cell lines and Drosophila. These data indicate that HEXA-018 could be a new drug candidate for TDP-43-associated neurodegenerative diseases.
Farrugia, M., Vassallo, N. and Cauchi, R. J. (2022). Disruption of Survival Motor Neuron in Glia Impacts Survival but has no Effect on Neuromuscular Function in Drosophila. Neuroscience 491: 32-42. PubMed ID: 35314252
Summary:
Increasing evidence points to the involvement of cell types other than motor neurons in both amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), the predominant motor neuron disease in adults and infants, respectively. The contribution of glia to ALS pathophysiology is well documented. Studies have since focused on evaluating the contribution of glia in SMA. This study made use of the Drosophila model to ask whether the survival motor neuron (Smn) protein, the causative factor for SMA, is required selectively in glia. The specific loss of Smn function in glia during development reduced survival to adulthood but did not affect motoric performance or neuromuscular junction (NMJ) morphology in flies. In contrast, gain rather than loss of ALS-linked TDP-43, FUS or C9orf72 function in glia induced significant defects in motor behaviour in addition to reduced survival. Furthermore, glia-specific gain of TDP-43 function caused both NMJ defects and muscle atrophy. Smn together with Gemins 2-8 and Unrip, form the Smn complex which is indispensable for the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). This study shows that glial-selective perturbation of Smn complex components or disruption of key snRNP biogenesis factors pICln and Tgs1, induce deleterious effects on adult fly viability but, similar to Smn reduction, had no negative effect on neuromuscular function. These findings suggest that the role of Smn in snRNP biogenesis as part of the Smn complex is required in glia for the survival of the organism, underscoring the importance of glial cells in SMA disease formation.
Loganathan, S., Wilson, B. A., Carey, S. B., Manzo, E., Joardar, A., Ugur, B. and Zarnescu, D. C. (2022). TDP-43 Proteinopathy Causes Broad Metabolic Alterations including TCA Cycle Intermediates and Dopamine Levels in Drosophila Models of ALS. Metabolites 12(2). PubMed ID: 35208176
Summary:
Amyotrophic lateral sclerosis (ALS) is a fatal, complex neurodegenerative disorder that causes selective degeneration of motor neurons. ALS patients exhibit symptoms consistent with altered cellular energetics such as hypermetabolism, weight loss, dyslipidemia, insulin resistance, and altered glucose tolerance. Although evidence supports metabolic changes in ALS patients, metabolic alterations at a cellular level remain poorly understood. This study used a Drosophila model of ALS based on TDP-43 expression in motor neurons that recapitulates hallmark features of motor neuron disease including TDP-43 aggregation, locomotor dysfunction, and reduced lifespan. To gain insights into metabolic changes caused by TDP-43, global metabolomic profiling was performed in larvae expressing TDP-43 (WT or ALS associated mutant variant, G298S) and identified significant alterations in several metabolic pathways. This study reports alterations in multiple metabolic pathways and highlight upregulation of Tricarboxylic acid (TCA) cycle metabolites and defects in neurotransmitter levels. It was also shown that modulating TCA cycle flux either genetically or by dietary intervention mitigates TDP-43-dependent locomotor defects. In addition, dopamine levels are significantly reduced in the context of TDP-43(G298S), and it was found that treatment with pramipexole, a dopamine agonist, improves locomotor function in vivo in Drosophila models of TDP-43 proteinopathy.
François-Moutal, L., Scott, D. D., Ambrose, A. J., Zerio, C. J., Rodriguez-Sanchez, M., Dissanayake, K., May, D. G., Carlson, J. M., Barbieri, E., Moutal, A., Roux, K. J., Shorter, J., Khanna, R., Barmada, S. J., McGurk, L. and Khanna, M. (2022). Heat shock protein Grp78/BiP/HspA5 binds directly to TDP-43 and mitigates toxicity associated with disease pathology. Sci Rep 12(1): 8140. PubMed ID: 35581326
Summary:
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure or effective treatment in which TAR DNA Binding Protein of 43 kDa (TDP-43) abnormally accumulates into misfolded protein aggregates in affected neurons. It is widely accepted that protein misfolding and aggregation promotes proteotoxic stress. The molecular chaperones are a primary line of defense against proteotoxic stress, and there has been long-standing interest in understanding the relationship between chaperones and aggregated protein in ALS. Of particular interest are the heat shock protein of 70 kDa (Hsp70) family of chaperones. However, defining which of the 13 human Hsp70 isoforms is critical for ALS has presented many challenges. To gain insight into the specific Hsp70 that modulates TDP-43, this study investigated the relationship between TDP-43 and the Hsp70s using proximity-dependent biotin identification (BioID) and discovered several Hsp70 isoforms associated with TDP-43 in the nucleus, raising the possibility of an interaction with native TDP-43. It was further found that HspA5 bound specifically to the RNA-binding domain of TDP-43 using recombinantly expressed proteins. Moreover, in a Drosophila strain that mimics ALS upon TDP-43 expression, the mRNA levels of the HspA5 homologue (Hsc70.3) were significantly increased. Similarly this study observed upregulation of HspA5 in prefrontal cortex neurons from human ALS patients. Finally, overexpression of HspA5 in Drosophila rescued TDP-43-induced toxicity, suggesting that upregulation of HspA5 may have a compensatory role in ALS pathobiology.
Pereira, P. E., ..., Dermaut, B. (2023). C-terminal frameshift variant of TDP-43 with pronounced aggregation-propensity causes rimmed vacuole myopathy but not ALS/FTD. Acta Neuropathol. PubMed ID: 37000196
Summary:
Neuronal TDP-43-positive inclusions are neuropathological hallmark lesions in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Pathogenic missense variants in TARDBP, the gene encoding TDP-43, can cause ALS and cluster in the C-terminal prion-like domain (PrLD), where they modulate the liquid condensation and aggregation properties of the protein. TDP-43-positive inclusions are also found in rimmed vacuole myopathies, including sporadic inclusion body myositis, but myopathy-causing TDP-43 variants have not been reported. This study identified a conclusively linked frameshift mutation in TDP-43 producing a C-terminally altered PrLD (TDP-43(p.Trp385IlefsTer10)) (maximum multipoint LOD-score 3.61). Patient-derived muscle biopsies showed TDP-43-positive sarcoplasmic inclusions, accumulation of autophagosomes and transcriptomes with abnormally spliced sarcomeric genes (including TTN and NEB) and increased expression of muscle regeneration genes. In vitro phase separation assays demonstrated that TDP-43(Trp385IlefsTer10) does not form liquid-like condensates and readily forms solid-like fibrils indicating increased aggregation propensity compared to wild-type TDP-43. In Drosophila TDP-43(p.Trp385IlefsTer10) behaved as a partial loss-of-function allele as it was able to rescue the TBPH (fly ortholog of TARDBP) neurodevelopmental lethal null phenotype while showing strongly reduced toxic gain-of-function properties upon overexpression. Accordingly, TDP-43<(p.Trp385IlefsTer10) showed reduced toxicity in a primary rat neuron disease model. Together, these genetic, pathological, in vitro and in vivo results demonstrate that TDP-43(p.Trp385IlefsTer10) is an aggregation-prone partial loss-of-function variant that causes autosomal dominant vacuolar myopathy but not ALS/FTD. This study genetically links TDP-43 proteinopathy to myodegeneration, and reveals a tissue-specific role of the PrLD in directing pathology.
Chang, Y. H. and Dubnau, J. (2023). Endogenous retroviruses and TDP-43 proteinopathy form a sustaining feedback driving intercellular spread of Drosophila neurodegeneration. Nat Commun 14(1): 966. PubMed ID: 36810738
Summary:
Inter-cellular movement of "prion-like" proteins is thought to explain propagation of neurodegeneration between cells. For example, propagation of abnormally phosphorylated cytoplasmic inclusions of TAR-DNA-Binding protein (TDP-43) is proposed to underlie progression of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). But unlike transmissible prion diseases, ALS and FTD are not infectious and injection of aggregated TDP-43 is not sufficient to cause disease. This suggests a missing component of a positive feedback necessary to sustain disease progression. This study demonstrated that endogenous retrovirus (ERV) expression and TDP-43 proteinopathy are mutually reinforcing. Expression of either Drosophila mdg4-ERV (gypsy) or the human ERV, HERV-K (HML-2) are each sufficient to stimulate cytoplasmic aggregation of human TDP-43. Viral ERV transmission also triggers TDP-43 pathology in recipient cells that express physiological levels of TDP-43, whether they are in contact or at a distance. This mechanism potentially underlies the TDP-43 proteinopathy-caused neurodegenerative propagation through neuronal tissue.
Chang, Y. H. and Dubnau, J. (2023). Endogenous retroviruses and TDP-43 proteinopathy form a sustaining feedback driving intercellular spread of Drosophila neurodegeneration. Nat Commun 14(1): 966. PubMed ID: 36810738
Summary:
Inter-cellular movement of "prion-like" proteins is thought to explain propagation of neurodegeneration between cells. For example, propagation of abnormally phosphorylated cytoplasmic inclusions of TAR-DNA-Binding protein (TDP-43) is proposed to underlie progression of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). But unlike transmissible prion diseases, ALS and FTD are not infectious and injection of aggregated TDP-43 is not sufficient to cause disease. This suggests a missing component of a positive feedback necessary to sustain disease progression. This study demonstrates that endogenous retrovirus (ERV) expression and TDP-43 proteinopathy are mutually reinforcing. Expression of either Drosophila mdg4-ERV (gypsy) or the human ERV, HERV-K (HML-2) are each sufficient to stimulate cytoplasmic aggregation of human TDP-43. Viral ERV transmission also triggers TDP-43 pathology in recipient cells that express physiological levels of TDP-43, whether they are in contact or at a distance. This mechanism potentially underlies the TDP-43 proteinopathy-caused neurodegenerative propagation through neuronal tissue.
Huber, R. G., Pandey, S., Chhangani, D., Rincon-Limas, D. E., Staff, N. P. and Yeo, C. J. J. (2022). Identification of potential pathways and biomarkers linked to progression in ALS. Ann Clin Transl Neurol. PubMed ID: 36533811
Summary:
To identify potential diagnostic and prognostic biomarkers for clinical management and clinical trials in amyotrophic lateral sclerosis. Proteomics data of ALS patient-induced pluripotent stem cell-derived motor neurons available through the AnswerALS consortium were analyzed. After stratifying patients using clinical ALSFRS-R and ALS-CBS scales, differentially expressed proteins indicative of ALS disease severity and progression rate were identified as candidate ALS-related and prognostic biomarkers. Pathway analysis for identified proteins was performed using STITCH. Protein sets were correlated with the effects of drugs using the Connectivity Map tool to identify compounds likely to affect similar pathways. RNAi screening was performed in a Drosophila TDP-43 ALS model to validate pathological relevance. A statistical classification machine learning model was constructed using ridge regression that uses proteomics data to differentiate ALS patients from controls. This study identified 76, 21, 71 and 1 candidate ALS-related biomarkers and 22, 41, 27 and 64 candidate prognostic biomarkers from patients stratified by ALSFRS-R baseline, ALSFRS-R progression slope, ALS-CBS baseline and ALS-CBS progression slope, respectively. Nineteen proteins enhanced or suppressed pathogenic eye phenotypes in the ALS fly model. Nutraceuticals, dopamine pathway modulators, statins, anti-inflammatories and antimicrobials were predicted starting points for drug repurposing using the connectivity map tool. Ten diagnostic biomarker proteins were predicted by machine learning to identify ALS patients with high accuracy and sensitivity. This study showcases the powerful approach of iPSC-motor neuron proteomics combined with machine learning and biological confirmation in the prediction of novel mechanisms and diagnostic and predictive biomarkers in ALS.
Khalil, B., Chhangani, D., .., Rincon-Limas, D. E. and Rossoll, W. (2022). Nuclear import receptors are recruited by FG-nucleoporins to rescue hallmarks of TDP-43 proteinopathy. Mol Neurodegener 17(1): 80. PubMed ID: 36482422
Summary:
Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) disease spectrum, causing both nuclear loss-of-function and cytoplasmic toxic gain-of-function phenotypes. While TDP-43 proteinopathy has been associated with defects in nucleocytoplasmic transport, this process is still poorly understood. This study examined the role of karyopherin-β1 (KPNB1) and other nuclear import receptors in regulating TDP-43 pathology. Several members of the nuclear import receptor protein family were able to reduce the formation of pathological TDP-43 aggregates. Using KPNB1 as a model, its activity was found to depend on the prion-like C-terminal region of TDP-43, which mediates the co-aggregation with phenylalanine and glycine-rich nucleoporins (FG-Nups) such as Nup62. KPNB1 is recruited into these co-aggregates where it acts as a molecular chaperone that reverses aberrant phase transition of Nup62 and TDP-43. These findings are supported by the discovery that Nup62 and KPNB1 are also sequestered into pathological TDP-43 aggregates in ALS/FTD postmortem CNS tissue, and by the identification of the fly ortholog of KPNB1 as a strong protective modifier in Drosophila models of TDP-43 proteinopathy. These results show that KPNB1 can rescue all hallmarks of TDP-43 pathology, by restoring its solubility and nuclear localization, and reducing neurodegeneration in cellular and animal models of ALS/FTD. hese findings suggest a novel NLS-independent mechanism where, analogous to its canonical role in dissolving the diffusion barrier formed by FG-Nups in the nuclear pore, KPNB1 is recruited into TDP-43/FG-Nup co-aggregates present in TDP-43 proteinopathies and therapeutically reverses their deleterious phase transition and mislocalization, mitigating neurodegeneration.
Yusuff, T., Chang, Y. C., Sang, T. K., Jackson, G. R. and Chatterjee, S. (2023). Codon-optimized TDP-43 mediates neurodegeneration in a Drosophila model of ALS/FTLD. Front Genet 14: 881638. PubMed ID: 36968586
Summary:
Transactive response DNA binding protein-43 (TDP-43) is known to mediate neurodegeneration associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The exact mechanism by which TDP-43 exerts toxicity in the brains, spinal cord, and lower motor neurons of affected patients remains unclear. In a novel Drosophila melanogaster model, this study reports gain-of-function phenotypes due to misexpression of insect codon-optimized version of human wild-type TDP-43 (CO-TDP-43) using both the binary GAL4/UAS system and direct promoter fusion constructs. The CO-TDP-43 model showed robust tissue specific phenotypes in the adult eye, wing, and bristles in the notum. Compared to non-codon optimized transgenic flies, the CO-TDP-43 flies produced increased amount of high molecular weight protein, exhibited pathogenic phenotypes, and showed cytoplasmic aggregation with both nuclear and cytoplasmic expression of TDP-43. Further characterization of the adult retina showed a disruption in the morphology and function of the photoreceptor neurons with the presence of acidic vacuoles that are characteristic of autophagy. Based on these observations, it is proposed that TDP-43 has the propensity to form toxic protein aggregates via a gain-of-function mechanism, and such toxic overload leads to activation of protein degradation pathways such as autophagy. The novel codon optimized TDP-43 model is an excellent resource that could be used in genetic screens to identify and better understand the exact disease mechanism of TDP-43 proteinopathies and find potential therapeutic targets.
Lo Piccolo, L., Umegawachi, T., Yeewa, R., Potikanond, S., Nimlamool, W., Prachayasittikul, V., Gotoh, Y., Yoshida, H., Yamaguchi, M. and Jantrapirom, S. (2023). A Novel Drosophila-based Drug Repurposing Platform Identified Fingolimod As a Potential Therapeutic for TDP-43 Proteinopathy. Neurotherapeutics. PubMed ID: 37493896
Summary:
Pathogenic changes to TAR DNA-binding protein 43 (TDP-43) leading to alteration of its homeostasis are a common feature shared by several progressive neurodegenerative diseases for which there is no effective therapy. Here, we developed Drosophila lines expressing either wild type TDP-43 (WT) or that carrying an Amyotrophic Lateral Sclerosis/Frontotemporal Lobar Degeneration-associating G384C mutation that recapitulate several aspects of the TDP-43 pathology. To identify potential therapeutics for TDP-43-related diseases, a drug repurposing strategy was utilized that involved three consecutive steps. Firstly, the improvement of eclosion rate was evaluated, followed by the assessment of locomotive functions at early and late developmental stages. Through this approach, fingolimod, was successfully identified as a promising candidate for modulating TDP-43 toxicity. Fingolimod exhibited several beneficial effects in both WT and mutant models of TDP-43 pathology, including post-transcriptional reduction of TDP-43 levels, rescue of pupal lethality, and improvement of locomotor dysfunctions. These findings provide compelling evidence for the therapeutic potential of fingolimod in addressing TDP-43 pathology, thereby strengthening the rationale for further investigation and consideration of clinical trials. Furthermore, our study demonstrates the utility of our Drosophila-based screening pipeline in identifying novel therapeutics for TDP-43-related diseases. These findings encourage further scale-up screening endeavors using this platform to discover additional compounds with therapeutic potential for TDP-43 pathology.
Krupp, S., Tam, O., Gale Hammell, M. and Dubnau, J. (2023). TDP-43 pathology in Drosophila induces glial-cell type specific toxicity that can be ameliorated by knock-down of SF2/SRSF1. bioRxiv. PubMed ID: 37205372
Summary:
Accumulation of cytoplasmic inclusions of TAR-DNA binding protein 43 (TDP-43) is seen in both neurons and glia in a range of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Alzheimer's disease (AD). Disease progression involves non-cell autonomous interactions among multiple cell types, including neurons, microglia and astrocytes. This study investigated the effects in Drosophila of inducible, glial cell type-specific TDP-43 overexpression, a model that causes TDP-43 protein pathology including loss of nuclear TDP-43 and accumulation of cytoplasmic inclusions. TDP-43 pathology in Drosophila is sufficient to cause progressive loss of each of the 5 glial sub-types. But the effects on organismal survival were most pronounced when TDP-43 pathology was induced in the perineural glia (PNG) or astrocytes. In the case of PNG, this effect is not attributable to loss of the glial population, because ablation of these glia by expression of pro-apoptotic reaper expression has relatively little impact on survival. To uncover underlying mechanisms, cell-type-specific nuclear RNA sequencing was used to characterize the transcriptional changes were identified that were induced by pathological TDP-43 expression. Numerous glial cell-type specific transcriptional changes. Notably,SF2/SRSF1 levels were found to be decreased in both PNG and in astrocytes. Further knockdown of SF2/SRSF1 in either PNG or astrocytes lessens the detrimental effects of TDP-43 pathology on lifespan, but extends survival of the glial cells. Thus TDP-43 pathology in astrocytes or PNG causes systemic effects that shorten lifespan and SF2/SRSF1 knockdown rescues the loss of these glia, and also reduces their systemic toxicity to the organism.
Yue, W., Deng, X., Wang, Z., Jiang, M., Hu, R., Duan, Y., Wang, Q., Cui, J. and Fang, Y. (2023). Inhibition of the MEK/ERK pathway suppresses immune overactivation and mitigates TDP-43 toxicity in a Drosophila model of ALS. Immun Ageing 20(1): 27. PubMed ID: 37340309 TDP-43 is an important DNA/RNA-binding protein that is associated with age-related neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD); however, its pathomechanism is not fully understood. In a transgenic RNAi screen using Drosophila as a model, this study uncovered that knockdown (KD) of Dsor1 (the Drosophila MAPK kinase dMEK) suppressed TDP-43 toxicity without altering TDP-43 phosphorylation or protein levels. Further investigation revealed that the Dsor1 downstream gene rl (dERK) was abnormally upregulated in TDP-43 flies, and neuronal overexpression of dERK induced profound upregulation of antimicrobial peptides (AMPs). A robust immune overactivation was pbserved in TDP-43 flies, which could be suppressed by downregulation of the MEK/ERK pathway in TDP-43 fly neurons. Furthermore, neuronal KD of abnormally increased AMPs improved the motor function of TDP-43 flies. On the other hand, neuronal KD of Dnr1, a negative regulator of the Drosophila immune deficiency (IMD) pathway, activated the innate immunity and boosted AMP expression independent of the regulation by the MEK/ERK pathway, which diminished the mitigating effect of RNAi-dMEK on TDP-43 toxicity. Finally, this study showed that an FDA-approved MEK inhibitor trametinib markedly suppressed immune overactivation, alleviated motor deficits and prolonged the lifespan of TDP-43 flies, but did not exhibit a lifespan-extending effect in Alzheimer disease (AD) or spinocerebellar ataxia type 3 (SCA3) fly models. Together, these findings suggest an important role of abnormal elevation of the MEK/ERK signaling and innate immunity in TDP-43 pathogenesis and propose trametinib as a potential therapeutic agent for ALS and other TDP-43-related diseases (Yue, 2023).

BIOLOGICAL OVERVIEW

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).

Loss of ISWI function in Drosophila nuclear bodies drives cytoplasmic redistribution of Drosophila TDP-43

Over the past decade, evidence has identified a link between protein aggregation, RNA biology, and a subset of degenerative diseases. An important feature of these disorders is the cytoplasmic or nuclear aggregation of RNA-binding proteins (RBPs). Redistribution of RBPs, such as the human TAR DNA-binding 43 protein (TDP-43) from the nucleus to cytoplasmic inclusions is a pathological feature of several diseases. Indeed, sporadic and familial forms of amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration share as hallmarks ubiquitin-positive inclusions. Recently, the wide spectrum of neurodegenerative diseases characterized by RBPs functions' alteration and loss was collectively named proteinopathies. This study shows that TBPH (TAR DNA-binding protein-43 homolog), the Drosophila ortholog of human TDP-43 TAR DNA-binding protein-43, interacts with the 'architectural RNA' (arcRNA) hsromega and with hsromega-associated hnRNPs. Additionally, it was found that the loss of the omega speckles remodeler ISWI (Imitation SWI) changes the TBPH sub-cellular localization to drive a TBPH cytoplasmic accumulation. These results, hence, identify TBPH as a new component of omega speckles and highlight a role of chromatin remodelers in hnRNPs nuclear compartmentalization (Lo Piccolo, 2018).

To confirm these interactions, a co-immunoprecipitation assay was conducted using anti-TBPH antibody starting from wild-type (WT) fresh larval nuclear extracts. Squid and Hrb87F are enriched in TBPH pulled-down fractions (Lo Piccolo, 2018).

Several in vitro experiments through proteomic studies and co-immunoprecipitation assay in HEK293 cells showed that in human cells TDP-43 interacts with the Drosophila orthologs of Squid and Hrb87F hnRNPs. This study confirmed these results in vivo, showing that in Drosophila tissue TBPH also interacts with Squid and Hrb87F hnRNP (Lo Piccolo, 2018).

Considering the co-localization between TBPH and Squid/Hrb87F hnRNPs, it was asked whether TBPH and hsrω could physically interact as well. To answer this question, an immunofluorescence and fluorescence RNA in situ hybridization was conducted. A physical interaction was observed between TBPH and the architectural RNA (arcRNA) hsrω in Malpighian tubules (MT) in vivo (Lo Piccolo, 2018).

As hnRNPs are known to shuttle between nucleus and cytoplasm, Western blot analysis was performed using a previously described method to produce in a single experiment nuclear and cytoplasmic protein fractions (NF and CF). These experiments were performed using MT and brain cells (BCs). Omega speckles are present in all the larval and adult Drosophila cell-type tissues but cells from Malpighian tubules were used for their large nuclear size, which allow a better understanding of nuclear bodies' distribution, as well as the eventual hsrω-interacting protein subcellular localization. BCs were characterized, as TBPH is largely expressed and has fundamental roles in the brain (Lo Piccolo, 2018).

The localization of TBPH in BCs is predominantly localized in the nucleus, but in BCs there is also a fraction of TBPH protein in the cytoplasm (Lo Piccolo, 2018b).

To rule out that the physical association observed between TBPH and hsrω was due to fortuitous interactions occurring during nuclear extract preparation, a cross-linking RNA-immuno-precipitation (CLIP-RIP) biochemical assay was performed using the anti-TBPH antibody on fixed larval nuclear extracts from brain cells. The CLIP-RIP data confirmed the specific interaction between TBPH and hsrω in the nuclear extract from the brain cells (+3.05-fold), compared to Rox1 (+0.77-fold) and U4 (+1.1-fold), two other abundant nuclear non-coding RNAs (Lo Piccolo, 2018).

It was thus demonstrated that TBPH, as previously reported for Hrb87F and Squid, is able to bind the hsrω RNA in vitro. Furthermore, using a gel shift assay employing an hsrω-n repeat unit (280b) transcribed in vitro and a full-length recombinant TBPH, it was shown that TBPH effectively retards hsrω RNA gel mobility. Finally, as seen for Hrb87F and Squid hnRNPs, the addition of ISWI protein in the reaction is a strong modulator of the interaction between TBPH-hsrω, changing the gel shift delay (Lo Piccolo, 2018).

In conclusion, these experiments confirmed the interaction of TBPH with hsrω arcRNA, Squid and Hrb87F hnRNPs in the omega speckles context. These results strongly suggest that, like Hrb87F and Squid, TBPH is another hnRNP belonging to the omega speckles complexes. Moreover, as shown for Squid and Hrb87F hnRNPs, ISWI function is essential for the modulation of TBPH/hsrω interaction (Lo Piccolo, 2018).

The chromatin remodeler ISWI is essential for a correct organization of the nucleoplasmic omega speckles (Oranati, 2011). Indeed, the organization and distribution of omega speckles are profoundly altered in ISWI null mutants when compared to wild-type cells. Omega speckles lose their dot shape and assume a trail shape distribution in the nucleus, suggesting a severe defect in their maturation or organization. Squid and Hrb87F hnRNPs also form trail-like structures in the nucleus of ISWI null mutants, showing that not only the distribution of the hsrω arcRNA, but also that of omega speckle-associated hnRNPs is compromised (Lo Piccolo, 2018).

Therefore, the distribution of TBPH protein was analyzed in ISWI null mutants to check if loss of ISWI could influence TBPH organization in omega speckles NBs as for Hrb87F and Squid hnRNPs. Remarkably, it was found that compared to wild-type cells, loss of ISWI function changes TBPH distribution in the context of omega speckles, inducing a dramatic alteration of TBPH sub-cellular localization. While in WT MT TBPH immunoreactive spots are nucleus limited, in ISWI null mutants' MT cytoplasmic TBPH-positive spots and trails were detected. Of note, these cytoplasmic spots show to be organized in different shapes, as indicated by arrows and arrowheads (Lo Piccolo, 2018).

These data were confirmed in vitro by Western blot of nuclear and cytoplasmic fractions in WT versus ISWI null mutant MT and BCs. In detail, this study showed that in ISWI null mutant the TBPH protein in MT disappears from the nucleus while moving to the cytoplasm, where the TBPH abundance increased compared to the WT NF (+2.37-fold). It was also observed a similar phenomenon in BCs where it was found that in ISWI null mutant TBPH disappears from the NF while its cytoplasmic fraction (CF) is not significantly changed compared to WT (Lo Piccolo, 2018).

Analyzing in detail the ventral ganglion of WT and ISWI null larvae, it was observed that the mean intensity of TBPH in motoneuron nuclei of ISWI null mutants is reduced compared to WT (Lo Piccolo, 2018).

Interestingly, loss of Squid in the Squid- null mutant also affects the cellular distribution of TBPH and causes its aberrant cytoplasmic localization in MT. While in WT MT TBPH immunoreactive spots are nucleus-limited, TBPH-positive cytoplasmic spots and trails were detected in MT of Squid null mutants. These data were confirmed in vitro by performing Western blots of nuclear and cytoplasmic fractions in WT versus Squid null mutant MT and BCs. In detail, it was shown that in Squid null mutant the TBPH protein in MT disappears from the nucleus while moving to the cytoplasm, where the TBPH abundance increased compared to the WT nuclear fraction (Lo Piccolo, 2018).

The human orthologs of Squid and Hrb87F proteins interact with TDP-43 to function cooperatively in RNA metabolism regulation. This study addressed whether TBPH interacts with Hrb87F and Squid in Drosophila cells as well. Double-immunofluorescence was conducted for TBPH/Hrb87F and TBPH/Squid and it was found that TBPH co-localizes in Malpighian tubules with Squid and Hrb87F hnRNPs in vivo (Lo Piccolo, 2018).

A similar phenomenon was observed in BCs where it was found that in Squid null mutant TBPH disappear from the NF while its cytoplasmic fraction is quite similar compared to the WT (Lo Piccolo, 2018).

Unlike Squid, Hrb87F does not affect TBPH subcellular distribution. To explain this result, the existence of a hierarchical order was hypothesized in omega speckles assembling, and it was speculated that Squid together with ISWI could be master regulators in the formation of physiologically functional hnRNP-hsrω complexes. In this case it could be hypothesized that the loss of Squid protein forces TBPH protein to escape the nucleus as a consequence of incorrect interaction among all omega speckle-associated hnRNPs (Lo Piccolo, 2018).

All these data collectively suggest that, in the Drosophila cells, the disorganization of omega speckles' compartments caused by loss of ISWI's role lead to a redistribution of TBPH protein from the nucleus to the cytoplasm. This could be a very important observation, considering that intracellular deposition of aggregated and ubiquitinated proteins are a prominent cyto-pathological feature of most neurodegenerative disorders frequently correlated with neural cell death (Lo Piccolo, 2018).

To explain all the results presented, it is hypothesized that loss of ISWI's function may indirectly affect TBPH distribution as a consequence of incorrect interaction among the omega speckle-associated hnRNPs and hsrω arcRNA. Indeed, while Squid and Hrb87F in ISWI null mutants are disorganized in their structure, but remain in the nucleus, TBPH seems to be more affected and to escape from the nucleus to the cytoplasm (Lo Piccolo, 2018).

For instance, the data reinforce the role of the chromatin remodeler ISWI in the modulation of the cellular localization of aggregation-prone proteins and show that the correct nuclear compartmentalization of TBPH hnRNP is dependent on nuclear body maintenance regulated by the chromatin remodeler. Finally, the data are in line with the recent findings showing that TDP-43-dependent reduction of the chromatin remodeler Chd1's recruitment to chromatin affects the induction of several key stress genes necessary to protect from diseases like ALS and FTD (Frontotemporal Dementia) (Lo Piccolo, 2018).

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).

Glial TDP-43 regulates axon wrapping, GluRIIA clustering and fly motility by autonomous and non-autonomous mechanisms

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 (Romano, 2015).

Age-dependent TDP-43-mediated motor neuron degeneration requires GSK3, hat-trick

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 (Sreedharan, 2015).

Rapamycin alleviates pathogenesis of a new Drosophila model of ALS-TDP

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 (Cheng, 2015).

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 (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 (Coyne, 2015).

An age-related reduction of brain TBPH/TDP-43 levels precedes the onset of locomotion defects in a Drosophila ALS model

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 (Cragnaz, 2015).

Genetic interaction of hnRNPA2B1 and DNAJB6 in a Drosophila model of multisystem proteinopathy

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 (Lee, 2016).

TDP-43 loss of function increases TFEB activity and blocks autophagosome-lysosome fusion

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).

Role of BMP receptor traffic in synaptic growth defects in an ALS model

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 (Deshpande, 2016).

Drosophila TDP-43 RNA-binding protein facilitates association of sister chromatid cohesion proteins with genes, enhancers and polycomb response elements

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 (Swain, 2016).

Drosophila CG3303 is an essential endoribonuclease linked to TDP-43-mediated neurodegeneration

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 (Laneve, 2017).

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).

Overexpression of ter94, Drosophila VCP, improves motor neuron degeneration induced by knockdown of TBPH, Drosophila TDP-43

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease characterized by the motor neuron degeneration that eventually leads to complete paralysis and death within 2-5 years after disease onset. One of the major pathological hallmark of ALS is abnormal accumulation of inclusions containing TAR DNA-binding protein-43 (TDP-43). TDP-43 is normally found in the nucleus, but in ALS, it localizes in the cytoplasm as inclusions as well as in the nucleus. Loss of nuclear TDP-43 functions likely contributes to neurodegeneration. TBPH is the Drosophila ortholog of human TDP-43. This study confirmed that Drosophila models harboring TBPH knockdown develop locomotive deficits and degeneration of motoneurons (MNs) due to loss of its nuclear functions, recapitulating the human ALS phenotypes. Previous work has suggested that ter94, the Drosophila ortholog of human Valosin-containing protein (VCP), is a modulator of degeneration in MNs induced by knockdown of Caz, the Drosophila ortholog of human FUS. In this study, to determine the effects of VCP on TDP-43-associated ALS pathogenic processes, genetic interactions were examined between TBPH and ter94. Overexpression of ter94 suppressed the compound eye degeneration caused by TBPH knockdown and suppressed the morbid phenotypes caused by neuron-specific TBPH knockdown, such as locomotive dysfunction and degeneration of MN terminals. Further immunocytochemical analyses revealed that the suppression is caused by restoring the cytoplasmically mislocalized TBPH back to the nucleus. Consistent with these observations, a loss-of-function mutation of ter94 enhanced the compound eye degeneration caused by TBPH knockdown and partially enhanced the locomotive dysfunction caused by TBPH knockdown. The data demonstrated that expression levels of ter94 influenced the phenotypes caused by TBPH knockdown, and indicate that reagents that up-regulate the function of human VCP could modify MN degeneration in ALS caused by TDP-43 mislocalization (Kushimura, 2018).

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).


REFERENCES

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

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

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

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

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

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 27(19):2898-910. PubMed ID: 27535427

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

Kushimura, Y., Tokuda, T., Azuma, Y., Yamamoto, I., Mizuta, I., Mizuno, T., Nakagawa, M., Ueyama, M., Nagai, Y., Yoshida, H. and Yamaguchi, M. (2018). Overexpression of ter94, Drosophila VCP, improves motor neuron degeneration induced by knockdown of TBPH, Drosophila TDP-43. Am J Neurodegener Dis 7(1): 11-31. PubMed ID: 29531866

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<

Li, S., et al. (2016). Genetic interaction of hnRNPA2B1 and DNAJB6 in a Drosophila model of multisystem proteinopathy. Hum Mol Genet 25(5):936-50. PubMed ID: 26744327

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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

Lo Piccolo, L., Bonaccorso, R., Attardi, A., Li Greci, L., Romano, G., Sollazzo, M., Giurato, G., Ingrassia, A. M. R., Feiguin, F., Corona, D. F. V. and Onorati, M. C. (2018). Loss of ISWI function in Drosophila nuclear bodies drives cytoplasmic redistribution of Drosophila TDP-43. Int J Mol Sci 19(4). PubMed ID: 29617352

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, 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

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Biological Overview

date revised: 18 February 2024

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