Raffaella Klima

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 [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.

TDP-43 regulates Drosophila neuromuscular junctions growth by modulating Futsch/MAP1B levels and synaptic microtubules organization.

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. Previously, we have shown that TDP-43 mutant flies presented locomotive alterations and structural defects at the neuromuscular junctions. We have now 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 we found that alterations in the organization of synaptic microtubules correlate with reduced protein levels in the microtubule associated protein futsch/MAP1B. Moreover, we observed that TDP-43 physically interacts with futsch mRNA and that its RNA binding capacity is required to prevent futsch down regulation and synaptic defects.

Characterization of the DNA-unwinding Activity of Human RECQ1, a Helicase Specifically Stimulated by Human Replication Protein A

The RecQ helicases are involved in several aspects of DNA metabolism. Five members of the RecQ family have been found in humans, but only two of them have been carefully characterized, BLM and WRN. In this work, we describe the enzymatic characterization of RECQ1. The helicase has 3′ to 5′ polarity, cannot start the unwinding from a blunt-ended terminus, and needs a 3′-single-stranded DNA tail longer than 10 nucleotides to open the substrate. However, it was also able to unwind a blunt-ended duplex DNA with a “bubble” of 25 nucleotides in the middle, as previously observed for WRN and BLM. We show that only short DNA duplexes (<30 bp) can be unwound by RECQ1 alone, but the addition of human replication protein A (hRPA) increases the processivity of the enzyme (>100 bp). Our studies done with Escherichia colisingle-strand binding protein (SSB) indicate that the helicase activity of RECQ1 is specifically stimulated by hRPA. This finding suggests that RECQ1 and hRPA may interact also in vivo and function together in DNA metabolism. Comparison of the present results with previous studies on WRN and BLM provides novel insight into the role of the N- and C-terminal domains of these helicases in determining their substrate specificity and in their interaction with hRPA.

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

SUMMARY 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, we carried out a genetic screen 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.

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

Background: TDP-43 and hnRNPA1/A2 factors are implicated in neurodegeneration. Results: The human and fruit fly TDP-43 and hnRNPA1/A2 orthologs show physical, genetic, and functional interplays. Conclusion: The functional cooperation between TBPH/Hrp38 and TDP-43/hnRNP A/B is conserved throughout evolution. Significance: TBPH/Hrp38 interplay can be critical for neurodegeneration, and Drosophila is a model suitable to study the impact of this interaction. 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, we have now characterized human TDP-43 and Drosophila TBPH similarity in terms of protein-protein interaction pathways. In this work we show 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 in TDP-43 with and without animals. These results suggest that hnRNP protein levels can play a modulatory role on TDP-43 functions.

Chronological requirements of TDP-43 function in synaptic organization and locomotive control.

Alterations 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.

A novel Drosophila model of TDP-43 proteinopathies: N-terminal sequences combined with the Q/N domain induce protein functional loss and locomotion defects

ABSTRACT 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. Summary: An engineered TDP-43 construct can be used to induce TDP-43 aggregation in Drosophila, providing a model that could be useful for characterization of pathogenetic mechanisms and drug screening.

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, we modulated the expression levels of TDP-43 in the Drosophila glia and found that the protein was required to regulate the subcellular wrapping of motoneuron axons, promote synaptic growth and the formation of glutamate receptor clusters at the neuromuscular junctions. Interestingly, we determined that the glutamate transporter EAAT1 mediated the regulatory functions of TDP-43 in the glia and demonstrated that genetic or pharmacological compensations of EAAT1 activity were sufficient to modulate glutamate receptor clustering and locomotive behaviors in flies. The data uncovers autonomous and non-autonomous functions of TDP-43 in the glia and suggests new experimentally based therapeutic strategies in ALS.

Functional screening in Drosophila reveals the conserved role of REEP1 in promoting stress resistance and preventing the formation of Tau aggregates

Pathological modifications in the microtubule-associated protein Tau is a common characteristic observed in different neurological diseases, suggesting that analogous metabolic pathways might be similarly affected during neurodegeneration. To identify these molecules and mechanisms, we utilized Drosophila models of human Tau-mediated neurodegeneration to perform an RNA interference functional screening against genes considered to be implicated in the pathogenesis of different neurodegenerative disorders. We found that the downregulation of the Drosophila REEP1 homolog protein enhanced Tau toxicity with increased formation of insoluble aggregates. On the contrary, the overexpression of either the Drosophila or the human REEP1 protein was able to revert these phenotypes and promote neuronal resistance to ER stress. These studies identify a new function for the REEP1 protein in vivo and a novel cellular mechanism to prevent Tau toxicity.

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, 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.