Inflammatory and immune alterations are increasingly recognized as components of ALS pathology, yet whether they arise as a direct consequence of TDP-43 dysfunction or as a downstream response to neurodegeneration remains unresolved. To address this question, we profiled adult head transcriptomes of Drosophila lacking TBPH, the fly homolog of TDP-43, and identified marked overactivation of the conserved Toll/Imd/NF-κB (Relish) innate immune pathway, including increased expression of antimicrobial effector genes and inflammatory genes. We further found that TDP-43/TBPH regulates the NF-κB homolog Relish by associating with its mRNA and that its loss permits Relish-dependent immune overactivation. Genetic reduction in Relish in TDP-43-deficient flies suppressed inflammatory signaling and ameliorated neurological defects in vivo, indicating that immune dysregulation contributes to TDP-43 loss-associated phenotypes.

Transactive response DNA-binding protein 43 (TDP-43) is a key factor in motor neurons and related neurodegenerative disorders, and the presence of cytoplasmic aggregates of TDP-43 is a major hallmark of diseases such amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Nevertheless, little is known about early developmental effects or the systemic nature of TDP-43-mediated pathology. Drosophila melanogaster is acknowledged as a powerful genetic model for studying the genetic inheritance and the behavioral and developmental processes associated with human neurodegenerative diseases, including ALS. To better understand the possible roles and potential pathogenic mechanisms of TDP-43 protein in the pathogenesis of ALS, we performed a transcriptomic analysis of larvae from a Drosophila model knock-out (KO) for the TBPH gene, the fly TDP-43 ortholog. Interestingly, the Gene Ontology (GO) analysis highlighted some pathways not yet associated with this pathology and this model. We identified several genes encoding for serine proteases, a class of enzymes that in the central nervous system (CNS) play important roles in neural development, synaptic plasticity, and neurodegeneration. Our work provides insights into novel pathological mechanisms underlying the disease, thereby opening new pathways for drug discovery.

The pathological loss of nuclear TDP-43 is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leading to extensive alterations in RNA metabolism and a broad number of neuronal transcripts. However, the key effectors linking TDP-43 dysfunction to synaptic defects remain unclear. In this study, using Drosophila and human iPSC-derived motoneurons, we identify Rab4 as a direct and conserved target of TDP-43, whose expression is necessary and sufficient to recover synaptic vesicle recycling, neuromuscular junction growth, and locomotor function in TDP-43-deficient motoneurons. Moreover, Rab4 activity promotes the presynaptic recruitment of futsch/MAP1B, a microtubule-associated protein also regulated by TDP-43, which autonomously supports synaptic growth and vesicle turnover. Together, these findings define a TDP-43/Rab4/futsch/MAP1B regulatory axis that couples endosomal dynamics to cytoskeletal assembly. Furthermore, this functionally coherent module provides a mechanistic basis for understanding how synaptic vulnerability is amplified in disease and offers a framework to identify key compensatory targets capable of sustaining neuronal function in the absence of TDP-43.

Aging progressively modifies the physiological balance of the organism increasing susceptibility to both genetic and sporadic neurodegenerative diseases. These changes include epigenetic chromatin remodeling events that may modify the transcription levels of disease-causing genes affecting neuronal survival. However, how these events interconnect is not well understood. Here, we found that Su(var)3-9 causes increased methylation of histone H3K9 in the promoter region of TDP-43, the most frequently altered factor in amyotrophic lateral sclerosis (ALS), affecting the mRNA and protein expression levels of this gene through epigenetic modifications that appear to be conserved in aged Drosophila brains, mouse, and human cells. Remarkably, augmented Su(var)3-9 activity causes a decrease in TDP-43 expression followed by early defects in locomotor activities. In contrast, decreasing Su(var)3-9 action promotes higher levels of TDP-43 expression, improving motility parameters in old flies. The data uncover a novel role of this enzyme in regulating TDP-43 expression and locomotor senescence and indicate conserved epigenetic mechanisms that may play a role in the pathogenesis of ALS.

Cappelli et al. reported that Nitric Oxide Synthase 1 Adaptor Protein is a co-regulated transcript of the TAR DNA-binding protein 43 kDa, reduced in amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients with TAR DNA-binding protein 43 kDa pathology. Overall, their results highlight Nitric Oxide Synthase 1 Adaptor Protein as a novel druggable disease-relevant gene in TAR DNA-binding protein 43 kDa-related proteinopathies. Many lines of evidence have highlighted the role played by heterogeneous nuclear ribonucleoproteins in amyotrophic lateral sclerosis. In this study, we have aimed to identify transcripts co-regulated by TAR DNA-binding protein 43 kDa and highly conserved heterogeneous nuclear ribonucleoproteins which have been previously shown to regulate TAR DNA-binding protein 43 kDa toxicity (deleted in azoospermia-associated protein 1, heterogeneous nuclear ribonucleoprotein -Q, -D, -K and -U). Using the transcriptome analyses, we have uncovered that Nitric Oxide Synthase 1 Adaptor Protein mRNA is a direct TAR DNA-binding protein 43 kDa target, and in flies, its modulation alone can rescue TAR DNA-binding protein 43 kDa pathology. In primary mouse cortical neurons, we show that TAR DNA-binding protein 43 kDa mediated downregulation of Nitric Oxide Synthase 1 Adaptor Protein expression strongly affects the NMDA-receptor signalling pathway. In human patients, the downregulation of Nitric Oxide Synthase 1 Adaptor Protein mRNA strongly correlates with TAR DNA-binding protein 43 kDa proteinopathy as measured by cryptic Stathmin-2 and Unc-13 homolog A cryptic exon inclusion. Overall, our results demonstrate that Nitric Oxide Synthase 1 Adaptor Protein may represent a novel disease-relevant gene, potentially suitable for the development of new therapeutic strategies.

Alterations in synaptic transmission are critical early events in neuromuscular disorders. However, reliable methodologies to analyze the functional organization of the neuromuscular synapses are still needed. This manuscript provides a detailed protocol to analyze the molecular assembly of the neuromuscular synapses through immune-electrophysiology in Drosophila melanogaster. This technique allows the quantification of the molecular behavior of the neuromuscular synapses by correlating the structural configuration of the synaptic boutons with their electrical activity.

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 our previous work, we 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. In this study, we 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, we 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.

To determine the molecular and functional interactions between RNA-binding proteins (RBPs) and their targets RNAs, is of fundamental importance to understand the dynamic organization of the nervous system in health and disease. Nevertheless, this task has remained elusive due to the lack of specific protocols and experimental systems that would allow the combination of biochemical analysis with in vivo functional genetics. In this manuscript, we describe a trustworthy and detailed methodology to establish the molecular organization and intracellular function of RBPs/RNA multimeric complexes in a cell type-defined manner by using the powerful GAL4/UAS system for gene expression in Drosophila melanogaster. Graphic abstract: Immunoprecipitation for protein-RNA interaction in Drosophila.

Brain inclusions mainly composed of misfolded and aggregated TAR DNA binding protein 43 (TDP-43), are characteristic hallmarks of amyotrophic lateral sclerosis (ALS). Irrespective of the role played by the inclusions, their reduction represents an important therapeutic pathway that is worth exploring. Their removal can either lead to the recovery of TDP-43 function by removing the self-templating conformers that sequester the protein in the inclusions, and/or eliminate any potential intrinsic toxicity of the aggregates. The search for curative therapies has been hampered by the lack of ALS models for use in high-throughput screening. We adapted, optimised, and extensively characterised our previous ALS cellular model for such use. The model demonstrated efficient aggregation of endogenous TDP-43, and concomitant loss of its splicing regulation function. We provided a proof-of-principle for its eventual use in high-throughput screening using compounds of the tricyclic family and showed that recovery of TDP-43 function can be achieved by the enhanced removal of TDP-43 aggregates by these compounds. We observed that the degradation of the aggregates occurs independent of the autophagy pathway beyond autophagosome-lysosome fusion, but requires a functional proteasome pathway. The in vivo translational effect of the cellular model was tested with two of these compounds in a Drosophila model expressing a construct analogous to the cellular model, where thioridazine significantly improved the locomotive defect. Our findings have important implications as thioridazine cleared TDP-43 aggregates and recovered TDP-43 functionality. This study also highlights the importance of a two-stage, in vitro and in vivo model system to cross-check the search for small molecules that can clear TDP-43 aggregates in TDP-43 proteinopathies.

Background 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. Though alterations in the intracellular distribution of TDP-43 have been observed in skeletal muscles of patients suffering from ALS, it is not clear whether such modifications play an active role in the disease or merely represent an expression of muscle homeostatic mechanisms. Also, the molecular and metabolic pathways regulated by TDP-43 in the skeletal muscle remain largely unknown. Here, we analyze the function of TBPH, the Drosophila melanogaster ortholog of TDP-43, in skeletal muscles. Results We modulated the activity of TDP-43 in Drosophila muscles by means of RNA interference and observed that it is 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. Conclusions Our 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.

BACKGROUND:Mutations in the small RNA-binding protein TDP-43 lead to the formation of insoluble cytoplasmic aggregates that have been associated with the onset and progression of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder affecting homeostasis of the motor system which is also characterized by aberrant expression of retrotransposable elements (RTEs). Although the TDP-43 function was shown to be required in the neurons and glia to maintain the organization of neuromuscular synapses and prevent denervation of the skeletal muscles, the molecular mechanisms involved in physiological dysregulation remain elusive. Here, we address this issue using a null mutation of the TDP-43 Drosophila homolog, TBPH.RESULTS:Using genome-wide gene expression profiles, we detected a strong upregulation of RTE expression in TBPH-null Drosophila heads, while the genetic rescue of the TDP-43 function reverted these modifications. Furthermore, we found that TBPH modulates the small interfering RNA (siRNA) silencing machinery responsible for RTE repression. Molecularly, we observed that TBPH regulates the expression levels of Dicer-2 by direct protein-mRNA interactions in vivo. Accordingly, the genetic or pharmacological recovery of Dicer-2 activity was sufficient to repress retrotransposon activation and promote motoneuron axonal wrapping and synaptic growth in TBPH-null Drosophila.CONCLUSIONS:We identified an upregulation of RTE expression in TBPH-null Drosophila heads and demonstrate that defects in the siRNA pathway lead to RTE upregulation and motoneuron degeneration. Our results describe a novel physiological role of endogenous TDP-43 in the prevention of RTE-induced neurological alterations through the modulation of Dicer-2 activity and the siRNA pathway.

The aberrant expression of retrotransposable elements (RTEs) was observed in different neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), a terminal disorder characterized by functional alterations in the small RNA-binding protein TDP-43, suggesting that these events might be connected. Using genome wide gene expression profiles, we detected RTEs highly upregulated in TDP-43-null Drosophila heads while, the genetic rescue of TDP-43 function reverted these modifications. Furthermore, we found that TDP-43 modulates the small interfering RNA (siRNA) silencing machinery responsible for RTEs repression. Molecularly, we observed that TDP-43 regulates the expression levels of Dicer-2 by direct protein mRNA interactions in vivo. Accordingly, the genetic or pharmacological recovery of Dicer-2 activity was sufficient to repress retrotransposons activation and revert the neurodegeneration in TDP-43-null Drosophila motoneurons. Our results, describe a novel physiological role of endogenous TDP-43 in the prevention of RTEs-induced neurodegeneration through the modulation of Dicer-2 activity and the siRNA pathway.

Background Alterations in the intracellular distribution of TDP-43 were observed in the skeletal muscles of patients suffering from ALS. However, it is not clear whether these modifications play an active role in the disease or represent a physiological adaptation to muscles homeostasis. Result To answer these questions, we modulated the activity of this protein in Drosophila muscles and observed that TDP-43 was required in these tissues to promote the formation and growth of the neuromuscular synapses. Moreover, we identified that TDP-43 regulates the expression levels of Disc-large (Dlg) and demonstrated that the modulation of Dlg activity, in skeletal muscles or motoneurons, was sufficient to recover the TDP-43-null locomotive and synaptic defects in flies. Additionally, we found that similar mechanisms are conserved in human cell lines and present in tissues derived from ALS patients. Conclusions Our results uncover the physiological role of TDP-43 in skeletal muscles as well as the mechanisms behind the autonomous and non-autonomous behavior of this protein in the organization of the neuromuscular synapses.

TDP-43 is an ubiquitous and highly conserved ribonucleoprotein involved in several cellular processes including pre-mRNA splicing, transcription, mRNA stability and transport. Notwithstanding the evidence of TDP-43 involvement in the pathogenesis of different neurodegenerative disorders (i.e. ALS and FTLD), the underlying mechanisms are still unclear. Given the high degree of functional similarity between the human and fly orthologs of TDP-43, Drosophila melanogaster is a simple and useful model to study the pathophysiological role of this protein in vivo. It has been demonstrated that the depletion of the TDP-43 fly ortholog (tbph) induces deficient locomotive behaviors and reduces life span and anatomical defects at the neuromuscular junction. In this study, using the known binding specificity of TDP-43/tbph for (UG) repeated sequences, we performed a bioinformatic screening for fly genes with at least 6 (TG) repeats in a row within the 3'-UTR regions in order to identify the genes that might be regulated by this factor. Among these genes, we were able to identify RhoGAPp190 as a potential target of the tbph-mediated neurodegeneration. RhoGAPp190 is a negative regulator of Drosophila RhoA, a GTPase protein implicated in the fine modulation of critical cellular processes including axon branch stability and motor axon defasciculation at muscle level and cognitive processes. We were able to demonstrate that the RhoGAPp190 expression is upregulated in a tbph-null fly model, providing evidence that this deregulation is associated to tbph silencing. Our results introduce RhoGAPp190 as a novel potential mediator in the complex scenario of events resulting from in vivo tbph loss-of-function.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that affects the motor system, comprised of motoneurons and associated glia. Accordingly, neuronal or glial defects in TDP-43 function provoke paralysis due to the degeneration of the neuromuscular synapses in Drosophila. To identify the responsible molecules and mechanisms, we performed a genome wide proteomic analysis to determine differences in protein expression between wild-type and TDP-43-minus fly heads. The data established that mutant insects presented reduced levels of the enzyme glutamic acid decarboxylase (Gad1) and increased concentrations of extracellular glutamate. Genetic rescue of Gad1 activity in neurons or glia was sufficient to recuperate flies locomotion, synaptic organization and glutamate levels. Analogous recovery was obtained by treating TDP-43-null flies with glutamate receptor antagonists demonstrating that Gad1 promotes synapses formation and prevents excitotoxicity. Similar suppression of TDP-43 provoked the downregulation of GAD67, the Gad1 homolog protein in human neuroblastoma cell lines and analogous modifications were observed in iPSC-derived motoneurons from patients carrying mutations in TDP-43, uncovering conserved pathological mechanisms behind the disease.

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. Here, we show that TBPH (TAR DNA-binding protein-43 homolog), the Drosophila ortholog of human TDP-43 TAR DNA-binding protein-43, interacts with the arcRNA hsrω and with hsrω-associated hnRNPs. Additionally, we found that the loss of the omega speckles remodeler ISWI (Imitation SWI) changes the TBPH sub-cellular localization to drive a TBPH cytoplasmic accumulation. Our results, hence, identify TBPH as a new component of omega speckles and highlight a role of chromatin remodelers in hnRNPs nuclear compartmentalization.

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, we have performed a functional screening to establish the ability of major hnRNP proteins to affect TDP-43 overexpression/depletion phenotypes. Interestingly, we observed that lowering hnRNP and TDP-43 expression has a generally harmful effect on flies locomotor abilities. In parallel, our 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 we show that DAZAP1 and TDP-43 can co-regulate an extensive number of biological processes and molecular functions potentially important for the neuron/motor neuron pathophysiology. Our results suggest that changes in hnRNP expression levels can significantly modulate TDP-43 functions and affect pathological outcomes.

SMN (Survival Motor Neuron) deficiency is the predominant cause of spinal muscular atrophy (SMA), a severe neurodegenerative disorder that can lead to progressive paralysis and death. Although SMN is required in every cell for proper RNA metabolism, the reason why its loss is especially critical in the motor system is still unclear. SMA genetic models have been employed to identify several modifiers that can ameliorate the deficits induced by SMN depletion. Here we focus on WDR79/TCAB1, a protein important for the biogenesis of several RNA species that has been shown to physically interact with SMN in human cells. We show that WDR79 depletion results in locomotion defects in both Drosophila and Caenorhabditis elegans similar to those elicited by SMN depletion. Consistent with this observation, we find that SMN overexpression rescues the WDR79 loss-of-function phenotype in flies. Most importantly, we also found that WDR79 overexpression ameliorates the locomotion defects induced by SMN depletion in both flies and worms. Our results collectively suggest that WDR79 and SMN play evolutionarily conserved cooperative functions in the nervous system and suggest that WDR79/TCAB1 may have the potential to modify SMA pathogenesis.

Transactive response DNA-binding protein 43 kDa (TDP-43, also known as TBPH in Drosophila melanogaster and TARDBP in mammals) is the main protein component of the pathological inclusions observed in neurons of patients affected by different neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD). The number of studies investigating the molecular mechanisms underlying neurodegeneration is constantly growing; however, the role played by TDP-43 in disease onset and progression is still unclear. A fundamental shortcoming that hampers progress is the lack of animal models showing aggregation of TDP-43 without overexpression. In this manuscript, we have extended our cellular model of aggregation to a transgenic Drosophila line. Our fly model is not based on the overexpression of a wild-type TDP-43 transgene. By contrast, we engineered a construct that includes only the specific TDP-43 amino acid sequences necessary to trigger aggregate formation and capable of trapping endogenous Drosophila TDP-43 into a non-functional insoluble form. Importantly, the resulting recombinant product lacks functional RNA recognition motifs (RRMs) and, thus, does not have specific TDP-43-physiological functions (i.e. splicing regulation ability) that might affect the animal phenotype per se. This novel Drosophila model exhibits an evident degenerative phenotype with reduced lifespan and early locomotion defects. Additionally, we show that important proteins involved in neuromuscular junction function, such as syntaxin (SYX), decrease their levels as a consequence of TDP-43 loss of function implying that the degenerative phenotype is a consequence of TDP-43 sequestration into the aggregates. Our data lend further support to the role of TDP-43 loss-of-function in the pathogenesis of neurodegenerative disorders. The novel transgenic Drosophila model presented in this study will help to gain further insight into the molecular mechanisms underlying neurodegeneration and will provide a valuable system to test potential therapeutic agents to counteract disease.