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Drosophila Answers to TDP-43 Proteinopathies.
Full text linkInitially implicated in the pathogenesis of CFTR and HIV-1 transcription, nuclear factor TDP-43 was subsequently found to be involved in the origin and development of several neurodegenerative diseases. In 2006, in fact, it was reported for the first time the cytoplasmic accumulation of TDP-43 in ubiquitin-positive inclusions of ALS and FTLD patients, suggesting the presence of a shared underlying mechanism for these diseases. Today, different animal models of TDP-43 proteinopathies are available in rodents, nematodes, fishes, and flies. Although these models recapitulate several of the pathological features found in patients, the mechanisms underpinning the progressive neuronal loss observed in TDP-43 proteinopathies remain to be characterized. Compared to other models, Drosophila are appealing because they combine the presence of a sophisticated brain with the possibility to investigate quickly and massively phenotypic genetic modifiers as well as possible therapeutic strategies. At present, the development of TDP-43-related Drosophila models has further strengthened the hypothesis that both TDP-43 "loss-of-function" and "gain-of-function" mechanisms can contribute to disease. The aim of this paper is to describe and compare the results obtained in a series of transgenic and knockout flies, along with the information they have generated, towards a better understanding of the mechanisms underlying TDP-43 proteinopathies
TDP-43 regulates GAD1 mRNA splicing and GABA signaling in Drosophila CNS
No full textAlterations 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
TDP-43 Regulates Rab4 Levels to Support Synaptic Vesicle Recycling and Neuromuscular Connectivity in Drosophila and Human ALS Models
Full text linkThe 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
TBPH/TDP-43 modulates translation of Drosophila futsch mRNA through an UG-rich sequence within its 5′UTR
Full text linkNuclear factor TDP-43 is an evolutionarily conserved multifunctional RNA-binding protein associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). In recent years, Drosophila models of ALS based on TDP-43 knockdown/overexpression have allowed to find several connections with disease. Among these, we have previously described that silencing the expression of its fly ortholog (TBPH) can alter the expression of the neuronal microtubule-associated protein Futsch leading to alterations of neuromuscular junction (NMJ) organization. In particular, TBPH knocked out flies displayed a significant reduction of Futsch protein levels, although minimal variation in the futsch mRNA content was observed. These conclusions were recently validated in an independent study. Together, these observations strongly support the hypothesis that TBPH might regulate the translation of futsch mRNA. However, the mechanism of TBPH interference in futsch mRNA translation is still unknown. In this work, we use EMSA experiments coupled with RNA-protein co-immunprecipitations and luciferase assays to show that TBPH interacts with a stretch of UG within the 5′UTR of futsch mRNA and translation is positively modulated by this binding. Most importantly, this function is also conserved in human TDP-43. This result can therefore represent the first step in elucidating the relationship between TDP-43, protein translation, and eventual disease onset or progression
Chronological requirements of TDP-43 function in synaptic organization and locomotive control
No full textAbstractAlterations in TDP-43 are commonly found in patients suffering from amyotrophic lateral sclerosis (ALS) and the genetic suppression of the conserved homologue in Drosophila (TBPH) provokes alterations in the functional organization of motoneuron synaptic terminals, resulting in locomotive defects and reduced life span. To gain more insight into this pathological process, it is of fundamental importance to establish when during the fly life cycle the lack of TBPH affects motoneuron activity and whether this is a reversible phenomenon. To achieve this, we conditionally expressed the endogenous protein in TBPH minus Drosophila neurons and found that TBPH is a short lived protein permanently required for Drosophila motility and synaptic assembly through the direct modulation of vesicular proteins, such as Syntaxin 1A, indicating that synaptic transmission defects are early pathological consequences of TBPH dysfunction in vivo. Importantly, TBPH late induction is able to recover synaptogenesis and locomotion in adult flies revealing an unexpected late-stage functional and structural neuronal plasticity. These observations suggest that late therapeutic approaches based on TDP-43 functionality may also be successful for the human pathology
TDP-43 prevents retrotransposon activation in the Drosophila motor system through regulation of Dicer-2 activity
Full text linkBackground: 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
Depletion of TDP-43 affects Drosophila motoneurons terminal synapsis and locomotive behavior
No full textAbstractPathological 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 [Neumann, M. et al. (2006) Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314, 130–133, Sreedharan, J. et al. (2008) TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319, 1668–1672, Kabashi, E. et al. (2008) TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat. Genet. 40, 572–574]. However, the function of TDP-43 in vivo is unknown and a possible direct role in neurodegeneration remains speculative. Here, we report 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. Our 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
Immunoprecipitation for Protein-Protein Interactions and for RNA Enrichment in Drosophila melanogaster
Full text linkTo 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
Immuno-electrophysiology on neuromuscular junctions of drosophila third instar larva
No full textAlterations 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
TDP-43 promotes the formation of neuromuscular synapses through the regulation of Disc-large expression in Drosophila skeletal muscles
No full textBackground: 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
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