Splicing factors act as genetic modulators of TDP-43 production in a new autoregulatory TDP-43 Drosophila model.

TDP-43 is a critical RNA-binding factor associated with RNA metabolism. In the physiological state, maintaining normal TDP-43 protein levels is critical for proper physiological functions of the cells. As such, TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop. TDP-43 is a major disease-causing protein in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Several studies argue for a pathogenic role of elevated TDP-43 levels in these disorders. Modulating the cycle of TDP-43 production might therefore provide a new therapeutic strategy. In this study, we developed a new transgenic Drosophila model mimicking the TDP-43 autoregulatory feedback loop in order to identify genetic modulators of TDP-43 protein steady-state levels in vivo. First, we showed that our TDP-43_TDPBR Drosophila model recapitulates key features of the TDP-43 autoregulatory processes previously described in mammalian and cellular models, namely alternative splicing events, differential usage of polyadenylation sites, nuclear retention of the transcript and a decrease in steady-state mRNA levels. Using this new Drosophila model, we identified several splicing factors, including SF2, Rbp1 and Sf3b1, as genetic modulators of TDP-43 production. Interestingly, our data indicate that these three RNA-binding proteins regulate TDP-43 protein production, at least in part, by controlling mRNA steady-state levels.

Conjugative IncC Plasmid Entry Triggers the SOS Response and Promotes Effective Transfer of the Integrative Antibiotic Resistance Element SGI1.

The broad-host-range IncC plasmid family and the integrative mobilizable Salmonella genomic island 1 (SGI1) and its derivatives enable the spread of medically important antibiotic resistance genes among Gram-negative pathogens. Although several aspects of the complex functional interactions between IncC plasmids and SGI1 have been recently deciphered regarding their conjugative transfer and incompatibility, the biological signal resulting in the hijacking of the conjugative plasmid by the integrative mobilizable element remains unknown. Here, we demonstrate that the conjugative entry of IncC/IncA plasmids is detected at an early stage by SGI1 through the transient activation of the SOS response, which induces the expression of the SGI1 master activators SgaDC, shown to play a crucial role in the complex biology between SGI1 and IncC plasmids. Besides, we developed an original tripartite conjugation approach to directly monitor SGI1 mobilization in a time-dependent manner following conjugative entry of IncC plasmids. Finally, we propose an updated biological model of the conjugative mobilization of the chromosomal resistance element SGI1 by IncC plasmids. IMPORTANCE Antimicrobial resistance has become a major public health issue, particularly with the increase of multidrug resistance (MDR) in both animal and human pathogenic bacteria and with the emergence of resistance to medically important antibiotics. The spread between bacteria of successful mobile genetic elements, such as conjugative plasmids and integrative elements conferring multidrug resistance, is the main driving force in the dissemination of acquired antibiotic resistances among Gram-negative bacteria. Broad-host-range IncC plasmids and their integrative mobilizable SGI1 counterparts contribute to the spread of critically important resistance genes (e.g., extended-spectrum ß-lactamases [ESBLs] and carbapenemases). A better knowledge of the complex biology of these broad-host-range mobile elements will help us to understand the dissemination of antimicrobial resistance genes that occurred across Gammaproteobacteria borders.

Downregulation of Hsp90 and the antimicrobial peptide Mtk suppresses poly(GR)-induced neurotoxicity in C9ORF72-ALS/FTD.

GGGGCC repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat RNAs can be translated into dipeptide repeat proteins, including poly(GR), whose mechanisms of action remain largely unknown. In an RNA-seq analysis of poly(GR) toxicity in Drosophila, we found that several antimicrobial peptide genes, such as metchnikowin (Mtk), and heat shock protein (Hsp) genes are activated. Mtk knockdown in the fly eye or in all neurons suppresses poly(GR) neurotoxicity. These findings suggest a cell-autonomous role of Mtk in neurodegeneration. Hsp90 knockdown partially rescues both poly(GR) toxicity in flies and neurodegeneration in C9ORF72 motor neurons derived from induced pluripotent stem cells (iPSCs). Topoisomerase II (TopoII) regulates poly(GR)-induced upregulation of Hsp90 and Mtk. TopoII knockdown also suppresses poly(GR) toxicity in Drosophila and improves survival of C9ORF72 iPSC-derived motor neurons. These results suggest potential novel therapeutic targets for C9ORF72-ALS/FTD.

The enhanced association between mutant CHMP2B and spastin is a novel pathological link between frontotemporal dementia and hereditary spastic paraplegias.

Chromosome 3-linked frontotemporal dementia (FTD3) is caused by a gain-of-function mutation in CHMP2B, resulting in the production of a truncated toxic protein, CHMP2BIntron5. Loss-of-function mutations in spastin are the most common genetic cause of hereditary spastic paraplegias (HSP). How these proteins might interact with each other to drive pathology remains to be explored. Here we found that spastin binds with greater affinity to CHMP2BIntron5 than to CHMP2BWT and colocalizes with CHMP2BIntron5 in p62-positive aggregates. In cultured cells expressing CHMP2BIntron5, spastin level in the cytoplasmic soluble fraction is decreased while insoluble spastin level is increased. These pathological features of spastin are validated in brain neurons of a mouse model of FTD3. Moreover, genetic knockdown of spastin enhances CHMP2BIntron5 toxicity in a Drosophila model of FTD3, indicating the functional significance of their association. Thus, our study reveals that the enhanced association between mutant CHMP2B and spastin represents a novel potential pathological link between FTD3 and HSP.

Ik2/TBK1 and Hook/Dynein, an adaptor complex for early endosome transport, are genetic modifiers of FTD-associated mutant CHMP2B toxicity in Drosophila.

Mutations in CHMP2B, encoding a protein in the endosomal sorting complexes required for transport (ESCRT) machinery, causes frontotemporal dementia linked to chromosome 3 (FTD3). FTD, the second most common form of pre-senile dementia, can also be caused by genetic mutations in other genes, including TANK-binding kinase 1 (TBK1). How FTD-causing disease genes interact is largely unknown. We found that partial loss function of Ik2, the fly homologue of TBK1 also known as I-kappaB kinase ε (IKKε), enhanced the toxicity of mutant CHMP2B in the fly eye and that Ik2 overexpression suppressed the effect of mutant CHMP2B in neurons. Partial loss of function of Spn-F, a downstream phosphorylation target of Ik2, greatly enhanced the mutant CHMP2B phenotype. An interactome analysis to understand cellular processes regulated by Spn-F identified a network of interacting proteins including Spn-F, Ik2, dynein light chain, and Hook, an adaptor protein in early endosome transport. Partial loss of function of dynein light chain or Hook also enhanced mutant CHMP2B toxicity. These findings identify several evolutionarily conserved genes, including ik2/TBK1, cut up (encoding dynein light chain) and hook, as genetic modifiers of FTD3-associated mutant CHMP2B toxicity and implicate early endosome transport as a potential contributing pathway in FTD.

Intracellular Positioning Systems Limit the Entropic Eviction of Secondary Replicons Toward the Nucleoid Edges in Bacterial Cells.

Bacterial genomes, organized intracellularly as nucleoids, are composed of the main chromosome coexisting with different types of secondary replicons. Secondary replicons are major drivers of bacterial adaptation by gene exchange. They are highly diverse in type and size, ranging from less than 2 to more than 1000 kb, and must integrate with bacterial physiology, including to the nucleoid dynamics, to limit detrimental costs leading to their counter-selection. We show that large DNA circles, whether from a natural plasmid or excised from the chromosome tend to localize in a dynamic manner in a zone separating the nucleoid from the cytoplasm at the edge of the nucleoid. This localization is in good agreement with silico simulations of DNA circles in the nucleoid volume. Subcellular positioning systems counteract this tendency, allowing replicons to enter the nucleoid space. In enterobacteria, these systems are found in replicons above 25 kb, defining the limit with small randomly segregated plasmids. Larger replicons carry at least one of the three described family of systems, ParAB, ParRM, and StbA. Replicons above 180 kb all carry a ParAB system, suggesting this system is specifically required in the cases of large replicons. Simulations demonstrated that replicon size profoundly affects localization, compaction, and dynamics of DNA circles in the nucleoid volume. The present work suggests that presence of partition systems on the larger plasmids or chromids is not only due to selection for accurate segregation but also to counteract their unmixing with the chromosome and consequent exclusion from the nucleoid.

FTLD/ALS-linked TDP-43 mutations do not alter TDP-43's ability to self-regulate its expression in Drosophila.

TDP-43 is a major disease-causing protein in amyotrophic lateral sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Today, >50 missense mutations in the TARDBP/TDP-43 gene have been described in patients with FTLD/ALS. However, the functional consequences of FTLD/ALS-linked TDP-43 mutations are not fully elucidated. In the physiological state, TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop. Maintaining normal TDP-43 protein levels is critical for proper physiological functions of the cells. In the present study, we investigated whether the FTLD/ALS-associated mutations could interfere with TDP-43 protein's capacity to modulate its own protein levels using Drosophila as an experimental model. Our data show that FTLD/ALS-associated mutant proteins regulate TDP-43 production with the same efficiency as the wild-type form of the protein. Thus, FTLD/ALS-linked TDP-43 mutations do not alter TDP-43's ability to self-regulate its expression and consequently of the homeostasis of TDP-43 protein levels.

Identification of TCERG1 as a new genetic modulator of TDP-43 production in Drosophila.

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, we identified CG42724 as a genetic modulator of TDP-43 production in vivo. We found that CG42724 protein influences 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, we showed that TCERG1, the human homolog of the Drosophila CG42724 protein, also caused an increase of TDP-43 protein steady-state levels in mammalian cells. Therefore, our data suggest the possibility that targeting TCERG1 could be therapeutic in TDP-43 proteinopathies.

A New Behavioral Test and Associated Genetic Tools Highlight the Function of Ventral Abdominal Muscles in Adult Drosophila.

The function of the nervous system in complex animals is reflected by the achievement of specific behaviors. For years in Drosophila, both simple and complex behaviors have been studied and their genetic bases have emerged. The neuromuscular junction is maybe one of the prototypal simplest examples. A motor neuron establishes synaptic connections on its muscle cell target and elicits behavior: the muscle contraction. Different muscles in adult fly are related to specific behaviors. For example, the thoracic muscles are associated with flight and the leg muscles are associated with locomotion. However, specific tools are still lacking for the study of cellular physiology in distinct motor neuron subpopulations. Here we decided to use the abdominal muscles and in particular the ventral abdominal muscles (VAMs) in adult Drosophila as new model to link a precise behavior to specific motor neurons. Hence, we developed a new behavioral test based on the folding movement of the adult abdomen. Further, we performed a genetic screen and identify two specific Gal4 lines with restricted expression patterns to the adult motor neurons innervating the VAMs or their precursor cells. Using these genetic tools, we showed that the lack of the VAMs or the loss of the synaptic transmission in their innervating motor neurons lead to a significant impairment of the abdomen folding behavior. Altogether, our results allow establishing a direct link between specific motor neurons and muscles for the realization of particular behavior: the folding behavior of the abdomen in Drosophila.