raffaella klima - Academia.edu (original) (raw)

Papers by raffaella klima

Research paper thumbnail of Additional file 2 of TDP-43 prevents retrotransposon activation in the Drosophila motor system through regulation of Dicer-2 activity

Additional file 2 : Table S2. List of the 1408 Regulated Probesets (Fold-change ≥ 1,5; P-Value ≤ ... more Additional file 2 : Table S2. List of the 1408 Regulated Probesets (Fold-change ≥ 1,5; P-Value ≤ 0,05).

Research paper thumbnail of Additional file 3 of TDP-43 prevents retrotransposon activation in the Drosophila motor system through regulation of Dicer-2 activity

Additional file 3 : Fig. S1. a Real time quantitative PCR of blastopia, burdock and springer tran... more Additional file 3 : Fig. S1. a Real time quantitative PCR of blastopia, burdock and springer transcript levels normalized on Rpl11 (housekeeping) in w1118 - tbphΔ23,elav-GAL4/tbphΔ23; UAS-GFP/+ and tbphΔ23,elav-GAL4/tbphΔ23,UAS-TBPH. (2biological replicates, with 3 technical replicates for each), error bars SEM. b Microarray results of downregulated TEs in TBPH mutants: the fold change of TEs was reported for both tbph mutant alleles (Δ23 and Δ142) referred to w1118; TEs family and class were also indicated.

Research paper thumbnail of Additional file 6 of TDP-43 prevents retrotransposon activation in the Drosophila motor system through regulation of Dicer-2 activity

Additional file 6. : Individual data values.

Research paper thumbnail of Immunoprecipitation for Protein-Protein Interactions and for RNA Enrichment in Drosophila melanogaster

BIO-PROTOCOL, 2021

To determine the molecular and functional interactions between RNA-binding proteins (RBPs) and th... more 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.

Research paper thumbnail of ALS Sinek Modelinde Nörodejenerasyon Mekanizmasının Proteomik ve Genetik Analizi

Amac : TDP-43; RNA ba g lanma motifleri i c eren ve pre-mRNA ekleme, transkripsiyon, mRNA stabili... more Amac : TDP-43; RNA ba g lanma motifleri i c eren ve pre-mRNA ekleme, transkripsiyon, mRNA stabilitesi ve mRNA transferinde yer ald igi rapor edilen olduk c a korunmu s ve her yerde eksprese edilen n u kleer proteindir. Son zamanlarda TDP-43 yap i lan c al is malar ile Amyotrofik Lateral Skleroz (ALS) hastalar i n i n etkilenmi s beyin b o lgelerinde g o zlenen h u cre i c i inkl u zyonlar i n ana protein bile s eni olarak tan i mland i . Bu c al is mada Drosophila melanogaster ’ de TDP-43 ’ u n analo g unun (TBPH ’ in) ALS modelindeki sinekler u zerinde proteomik ve genetik mekanizmalar i i c erisindeki rol u n u n ara s t i r i lmas i ama c lanm is t i r. Materyal-Metod : WIII8,UAS GFP, GMR,GMR TBPH genotiplerinde bakire di s i sinekler ve erkekler uygun besiyeri ortam i nda c iftle s tirilerek farkl i genotip ve fenotiplerde ALS i c in etki g o sterebilen transgenik model sinekler u retildi. Transgenik sineklere TBPH entegre edildi. PCR , Jel Elektroforezi ve 2D k u tle spektrofot...

Research paper thumbnail of ResponseReticulum Stress: Signaling the Unfolded Protein

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

Neuroscience, 2015

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. The average age of onse... more 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, we created a transgenic fly 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. Our 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.

Research paper thumbnail of Additional file 1 of TDP-43 prevents retrotransposon activation in the Drosophila motor system through regulation of Dicer-2 activity

Additional file 1 : Table S1. List of the 1253 Regulated Probesets (Fold-change ≥ 1,5; P-Value ≤ ... more Additional file 1 : Table S1. List of the 1253 Regulated Probesets (Fold-change ≥ 1,5; P-Value ≤ 0,05).

Research paper thumbnail of Additional file 5 of TDP-43 prevents retrotransposon activation in the Drosophila motor system through regulation of Dicer-2 activity

Additional file 5 : Fig. S3. Real time PCR of Dicer-2 (Dcr-2), Argonaute 2 (Ago2), Pasha, Piwi, L... more Additional file 5 : Fig. S3. Real time PCR of Dicer-2 (Dcr-2), Argonaute 2 (Ago2), Pasha, Piwi, Loquacious (Loq) and Homeless transcript levels normalized on Rpl11 (housekeeping) in adult heads of w1118, tbphΔ23/tbphΔ23 and tbphΔ142/tbphΔ142. n=2, error bars SEM.

Research paper thumbnail of Additional file 4 of TDP-43 promotes the formation of neuromuscular synapses through the regulation of Disc-large expression in Drosophila skeletal muscles

Additional file 4. Individual data values. Individual data values for: Fig. 3: pannel a; Fig. 4: ... more Additional file 4. Individual data values. Individual data values for: Fig. 3: pannel a; Fig. 4: pannels a,b,c; Fig. 5: pannels a,b,c; Additional file 1 Fig. S1.

Research paper thumbnail of Additional file 1 of TDP-43 promotes the formation of neuromuscular synapses through the regulation of Disc-large expression in Drosophila skeletal muscles

Additional file 1: Figure S1. Control of TBPH silencing. Western blot analysis on larval carcasse... more Additional file 1: Figure S1. Control of TBPH silencing. Western blot analysis on larval carcasses probed for anti-TBPH and anti-tubulin in tbphΔ23/+;Mef2-GAL4/UAS-GFP-IR and tbphΔ23/+;Mef2-GAL4/UAS-TBPH-IR. The same membrane was probed with the two antibodies and the bands of interest were cropped. n = 3 (biological replicates). Individual data values are provided in the Additional file 4. Individual Data Values.xls.

Research paper thumbnail of Additional file 2 of TDP-43 promotes the formation of neuromuscular synapses through the regulation of Disc-large expression in Drosophila skeletal muscles

Additional file 2: Figure S2. a. Western blot analysis on larval carcasses probed for anti-TBPH a... more Additional file 2: Figure S2. a. Western blot analysis on larval carcasses probed for anti-TBPH and anti-tubulin in Ctrl (w1118), tbphΔ23/tbphΔ23, tbphΔ23,UAS-TBPH/tbphΔ23;Mef2-GAL4/+, tbphΔ23/tbphΔ23;Mef2-GAL4/UAS-TBPHF/L. The same membrane was probed with two antibodies and the bands of interest were cropped. n = 3 (biological replicates). b. Western blot analysis on larval carcasses probed for anti-TDP and anti-tubulin in Ctrl (w1118) and tbphΔ23/tbphΔ23;Mef2-GAL4/UAS-TDP-43 The same membrane was probed with two antibodies and the bands of interest were cropped. n = 3 (biological replicates). c. Quantification of branches number in Ctrl, Δtb-GFP and Δtb-TBPH. n = 15. d. Quantification of boutons shape in Ctrl, Δtb-GFP and Δtb-TBPH. n = 200. e. Confocal images of third instar NMJ terminals in muscle 6/7 second segment stained with anti-HRP (in green) and anti-Dlg (in red) in Ctrl (w1118), Δtb-GFP (tbphΔ23/tbphΔ23;Mhc-GAL4/UAS-GFP), Δtb-TBPH (tbphΔ23,UAS-TBPH/tbphΔ23;Mhc-GAL4/+). f...

Research paper thumbnail of A novel fly model of TDP-43 proteinopathies : N-terminus sequences combined with the Q / N domain induce protein functional loss and locomotion defects

is ea se M o de ls & M ec ha ni sm s • D M M • A dv an ce a rt ic le © 2016. Published by The Com... more is ea se M o de ls & M ec ha ni sm s • D M M • A dv an ce a rt ic le © 2016. Published by The Company of Biologists Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

Research paper thumbnail of Thioridazine reverts the phenotype in cellular and Drosophila models of amyotrophic lateral sclerosis by enhancing TDP-43 aggregate clearance

Research paper thumbnail of TDP-43 regulates GAD1 mRNA splicing and GABA signaling in Drosophila CNS

Scientific Reports

Alterations in the function of the RNA-binding protein TDP-43 are largely associated with the pat... more 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 ...

Research paper thumbnail of TDP-43 regulates GAD1 mRNA splicing and GABA signaling in Drosophila brains

ABSTRACTAlterations in the function of the RNA-binding protein TDP-43 is largely associated with ... more ABSTRACTAlterations in the function of the RNA-binding protein TDP-43 is 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. Regarding to that, we found that the expression levels of the glutamic acid decarboxylase enzyme (GAD1), responsible to convert 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 to regulate GAD1 pre-mRNA splicing and GABA levels in Drosophila brains. Interestingly, we discovered that pharmacological treatments aimed to modulate GABA neurotransmission were able to revert locomotion deficiencies in TBPH-minus flies re...

Research paper thumbnail of Immuno-electrophysiology on Neuromuscular Junctions of Drosophila Third Instar Larva

Research paper thumbnail of TDP-43 prevents retrotransposon activation in the Drosophila motor system through regulation of Dicer-2 activity

BMC Biology

Background: Mutations in the small RNA-binding protein TDP-43 lead to the formation of insoluble ... more 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.

Research paper thumbnail of TDP-43 promotes the formation of neuromuscular synapses through the regulation of Disc-large expression in Drosophila skeletal muscles

BMC Biology

Background: The ribonuclear protein TDP-43 has been implicated in the pathophysiology of amyotrop... more 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.

Research paper thumbnail of TDP-43 regulates the expression levels of Disc-large in skeletal muscles to promote the assemble of the neuromuscular synapses in Drosophila

Alterations in the intracellular distribution of TDP-43 were observed in the skeletal muscles of ... more 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. 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. Our results uncover the physiological role of TDP-43 in skeletal muscles as well as the mechanisms behind the aut...

Research paper thumbnail of Additional file 2 of TDP-43 prevents retrotransposon activation in the Drosophila motor system through regulation of Dicer-2 activity

Additional file 2 : Table S2. List of the 1408 Regulated Probesets (Fold-change ≥ 1,5; P-Value ≤ ... more Additional file 2 : Table S2. List of the 1408 Regulated Probesets (Fold-change ≥ 1,5; P-Value ≤ 0,05).

Research paper thumbnail of Additional file 3 of TDP-43 prevents retrotransposon activation in the Drosophila motor system through regulation of Dicer-2 activity

Additional file 3 : Fig. S1. a Real time quantitative PCR of blastopia, burdock and springer tran... more Additional file 3 : Fig. S1. a Real time quantitative PCR of blastopia, burdock and springer transcript levels normalized on Rpl11 (housekeeping) in w1118 - tbphΔ23,elav-GAL4/tbphΔ23; UAS-GFP/+ and tbphΔ23,elav-GAL4/tbphΔ23,UAS-TBPH. (2biological replicates, with 3 technical replicates for each), error bars SEM. b Microarray results of downregulated TEs in TBPH mutants: the fold change of TEs was reported for both tbph mutant alleles (Δ23 and Δ142) referred to w1118; TEs family and class were also indicated.

Research paper thumbnail of Additional file 6 of TDP-43 prevents retrotransposon activation in the Drosophila motor system through regulation of Dicer-2 activity

Additional file 6. : Individual data values.

Research paper thumbnail of Immunoprecipitation for Protein-Protein Interactions and for RNA Enrichment in Drosophila melanogaster

BIO-PROTOCOL, 2021

To determine the molecular and functional interactions between RNA-binding proteins (RBPs) and th... more 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.

Research paper thumbnail of ALS Sinek Modelinde Nörodejenerasyon Mekanizmasının Proteomik ve Genetik Analizi

Amac : TDP-43; RNA ba g lanma motifleri i c eren ve pre-mRNA ekleme, transkripsiyon, mRNA stabili... more Amac : TDP-43; RNA ba g lanma motifleri i c eren ve pre-mRNA ekleme, transkripsiyon, mRNA stabilitesi ve mRNA transferinde yer ald igi rapor edilen olduk c a korunmu s ve her yerde eksprese edilen n u kleer proteindir. Son zamanlarda TDP-43 yap i lan c al is malar ile Amyotrofik Lateral Skleroz (ALS) hastalar i n i n etkilenmi s beyin b o lgelerinde g o zlenen h u cre i c i inkl u zyonlar i n ana protein bile s eni olarak tan i mland i . Bu c al is mada Drosophila melanogaster ’ de TDP-43 ’ u n analo g unun (TBPH ’ in) ALS modelindeki sinekler u zerinde proteomik ve genetik mekanizmalar i i c erisindeki rol u n u n ara s t i r i lmas i ama c lanm is t i r. Materyal-Metod : WIII8,UAS GFP, GMR,GMR TBPH genotiplerinde bakire di s i sinekler ve erkekler uygun besiyeri ortam i nda c iftle s tirilerek farkl i genotip ve fenotiplerde ALS i c in etki g o sterebilen transgenik model sinekler u retildi. Transgenik sineklere TBPH entegre edildi. PCR , Jel Elektroforezi ve 2D k u tle spektrofot...

Research paper thumbnail of ResponseReticulum Stress: Signaling the Unfolded Protein

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

Neuroscience, 2015

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. The average age of onse... more 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, we created a transgenic fly 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. Our 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.

Research paper thumbnail of Additional file 1 of TDP-43 prevents retrotransposon activation in the Drosophila motor system through regulation of Dicer-2 activity

Additional file 1 : Table S1. List of the 1253 Regulated Probesets (Fold-change ≥ 1,5; P-Value ≤ ... more Additional file 1 : Table S1. List of the 1253 Regulated Probesets (Fold-change ≥ 1,5; P-Value ≤ 0,05).

Research paper thumbnail of Additional file 5 of TDP-43 prevents retrotransposon activation in the Drosophila motor system through regulation of Dicer-2 activity

Additional file 5 : Fig. S3. Real time PCR of Dicer-2 (Dcr-2), Argonaute 2 (Ago2), Pasha, Piwi, L... more Additional file 5 : Fig. S3. Real time PCR of Dicer-2 (Dcr-2), Argonaute 2 (Ago2), Pasha, Piwi, Loquacious (Loq) and Homeless transcript levels normalized on Rpl11 (housekeeping) in adult heads of w1118, tbphΔ23/tbphΔ23 and tbphΔ142/tbphΔ142. n=2, error bars SEM.

Research paper thumbnail of Additional file 4 of TDP-43 promotes the formation of neuromuscular synapses through the regulation of Disc-large expression in Drosophila skeletal muscles

Additional file 4. Individual data values. Individual data values for: Fig. 3: pannel a; Fig. 4: ... more Additional file 4. Individual data values. Individual data values for: Fig. 3: pannel a; Fig. 4: pannels a,b,c; Fig. 5: pannels a,b,c; Additional file 1 Fig. S1.

Research paper thumbnail of Additional file 1 of TDP-43 promotes the formation of neuromuscular synapses through the regulation of Disc-large expression in Drosophila skeletal muscles

Additional file 1: Figure S1. Control of TBPH silencing. Western blot analysis on larval carcasse... more Additional file 1: Figure S1. Control of TBPH silencing. Western blot analysis on larval carcasses probed for anti-TBPH and anti-tubulin in tbphΔ23/+;Mef2-GAL4/UAS-GFP-IR and tbphΔ23/+;Mef2-GAL4/UAS-TBPH-IR. The same membrane was probed with the two antibodies and the bands of interest were cropped. n = 3 (biological replicates). Individual data values are provided in the Additional file 4. Individual Data Values.xls.

Research paper thumbnail of Additional file 2 of TDP-43 promotes the formation of neuromuscular synapses through the regulation of Disc-large expression in Drosophila skeletal muscles

Additional file 2: Figure S2. a. Western blot analysis on larval carcasses probed for anti-TBPH a... more Additional file 2: Figure S2. a. Western blot analysis on larval carcasses probed for anti-TBPH and anti-tubulin in Ctrl (w1118), tbphΔ23/tbphΔ23, tbphΔ23,UAS-TBPH/tbphΔ23;Mef2-GAL4/+, tbphΔ23/tbphΔ23;Mef2-GAL4/UAS-TBPHF/L. The same membrane was probed with two antibodies and the bands of interest were cropped. n = 3 (biological replicates). b. Western blot analysis on larval carcasses probed for anti-TDP and anti-tubulin in Ctrl (w1118) and tbphΔ23/tbphΔ23;Mef2-GAL4/UAS-TDP-43 The same membrane was probed with two antibodies and the bands of interest were cropped. n = 3 (biological replicates). c. Quantification of branches number in Ctrl, Δtb-GFP and Δtb-TBPH. n = 15. d. Quantification of boutons shape in Ctrl, Δtb-GFP and Δtb-TBPH. n = 200. e. Confocal images of third instar NMJ terminals in muscle 6/7 second segment stained with anti-HRP (in green) and anti-Dlg (in red) in Ctrl (w1118), Δtb-GFP (tbphΔ23/tbphΔ23;Mhc-GAL4/UAS-GFP), Δtb-TBPH (tbphΔ23,UAS-TBPH/tbphΔ23;Mhc-GAL4/+). f...

Research paper thumbnail of A novel fly model of TDP-43 proteinopathies : N-terminus sequences combined with the Q / N domain induce protein functional loss and locomotion defects

is ea se M o de ls & M ec ha ni sm s • D M M • A dv an ce a rt ic le © 2016. Published by The Com... more is ea se M o de ls & M ec ha ni sm s • D M M • A dv an ce a rt ic le © 2016. Published by The Company of Biologists Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

Research paper thumbnail of Thioridazine reverts the phenotype in cellular and Drosophila models of amyotrophic lateral sclerosis by enhancing TDP-43 aggregate clearance

Research paper thumbnail of TDP-43 regulates GAD1 mRNA splicing and GABA signaling in Drosophila CNS

Scientific Reports

Alterations in the function of the RNA-binding protein TDP-43 are largely associated with the pat... more 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 ...

Research paper thumbnail of TDP-43 regulates GAD1 mRNA splicing and GABA signaling in Drosophila brains

ABSTRACTAlterations in the function of the RNA-binding protein TDP-43 is largely associated with ... more ABSTRACTAlterations in the function of the RNA-binding protein TDP-43 is 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. Regarding to that, we found that the expression levels of the glutamic acid decarboxylase enzyme (GAD1), responsible to convert 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 to regulate GAD1 pre-mRNA splicing and GABA levels in Drosophila brains. Interestingly, we discovered that pharmacological treatments aimed to modulate GABA neurotransmission were able to revert locomotion deficiencies in TBPH-minus flies re...

Research paper thumbnail of Immuno-electrophysiology on Neuromuscular Junctions of Drosophila Third Instar Larva

Research paper thumbnail of TDP-43 prevents retrotransposon activation in the Drosophila motor system through regulation of Dicer-2 activity

BMC Biology

Background: Mutations in the small RNA-binding protein TDP-43 lead to the formation of insoluble ... more 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.

Research paper thumbnail of TDP-43 promotes the formation of neuromuscular synapses through the regulation of Disc-large expression in Drosophila skeletal muscles

BMC Biology

Background: The ribonuclear protein TDP-43 has been implicated in the pathophysiology of amyotrop... more 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.

Research paper thumbnail of TDP-43 regulates the expression levels of Disc-large in skeletal muscles to promote the assemble of the neuromuscular synapses in Drosophila

Alterations in the intracellular distribution of TDP-43 were observed in the skeletal muscles of ... more 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. 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. Our results uncover the physiological role of TDP-43 in skeletal muscles as well as the mechanisms behind the aut...