Biology subjects

Kondo, S.

Publications and source records attributed to Kondo, S..

64 records · Page 4Linked to original sources

bioRxiv2019

Patterned cell death sculpts functional and adaptive neural networks in flies

Changes to the structure and function of neural networks are thought to underlie the evolutionary adaptation of animal behaviours. Among the many developmental phenomena that generate change programmed cell death appears to play a key role. We show that cell death occurs continuously throughout insect neurogenesis and happens soon after neurons are born. Focusing on two dipterans which have lost flight during evolution we reveal that reductions in populations of flight interneurons are likely caused by increased cell death during development. Mimicking an evolutionary role for increasing cell numbers, we artificially block programmed cell death in the medial neuroblast lineage in Drosophila melanogaster, which results in the production of undead neurons with complex arborisations and distinct neurotransmitter identities. Activation of these undead neurons and recordings of neural activity in behaving animals demonstrate that they are functional. Our findings suggest that the evolutionary modulation of death-based patterning could generate novel network configurations.

neuroscience↗

bioRxiv2019

Nuclear RNA export factor variant initiates piRNA-guided co-transcriptional silencing

The PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by repressing transposable elements (TEs) in animal germ cells. Among PIWI-clade proteins in Drosophila, Piwi transcriptionally silences its targets through interactions with cofactors, including Panoramix (Panx) and forms heterochromatin characterized by H3K9me3 and H1. Here, we identified Nxf2, a nuclear RNA export factor (NXF) variant, as a protein that forms complexes with Piwi, Panx, and p15. Panx-Nxf2-p15 complex formation is necessary in the silencing by stabilizing protein levels of Nxf2 and Panx. Notably, ectopic targeting of Nxf2 initiates co-transcriptional repression of the target reporter in a manner independent of H3K9me3 marks or H1. However, continuous silencing requires HP1a and H1. In addition, Nxf2 directly interacts with target TE transcripts in a Piwi-dependent manner. These findings suggest a model in which the Nxf2-Panx-p15 complex enforces the association of Piwi with target transcripts to trigger co-transcriptional repression, prior to heterochromatin formation in the nuclear piRNA pathway.\n\nHighlightsO_LINxf2 plays an essential role in the Piwi-piRNA pathway\nC_LIO_LIFormation of Piwi-Panx-Nxf2-p15 (PPNP) complexes stabilizes both Panx and Nxf2\nC_LIO_LIThe PPNP complex triggers transcriptional silencing before heterochromatin formation\nC_LIO_LINxf2 directly binds to target transcripts in a Piwi-dependent manner\nC_LI

molecular biology↗

bioRxiv2019

Environmental light is required for maintenance of long-term memory in Drosophila

Long-term memory (LTM) is stored as functional modifications of relevant neural circuits in the brain. A large body of evidence indicates that the initial establishment of such modifications through the process known as memory consolidation requires learning-dependent transcriptional activation and de novo protein synthesis. However, it remains poorly understood how the consolidated memory is maintained for a long period in the brain, despite constant turnover of molecular substrates. Using the Drosophila courtship-conditioning assay of adult males as a memory paradigm, here, we show that in Drosophila, environmental light plays a critical role in LTM maintenance. LTM is impaired when flies are kept in constant darkness (DD) during the memory maintenance phase. Because light activates the brain neurons expressing the neuropeptide Pigment-dispersing factor (Pdf), we examined the possible involvement of Pdf neurons in LTM maintenance. Temporal activation of Pdf neurons compensated for the DD-dependent LTM impairment, whereas temporal knockdown of Pdf during the memory maintenance phase impaired LTM in light-dark cycles. Furthermore, we demonstrated that the transcription factor cAMP response element-binding protein (CREB) is required in the memory center, mushroom bodies (MBs), for LTM maintenance, and Pdf signaling regulates light-dependent transcription via CREB. Our results demonstrate for the first time that universally available environmental light plays a critical role in LTM maintenance by activating the evolutionarily conserved memory modulator CREB in MBs via the Pdf signaling pathway.\n\nSignificant StatementTemporary memory can be consolidated into long-term memory (LTM) through de novo protein synthesis and functional modifications of neuronal circuits in the brain. Once established, LTM requires continual maintenance so that it is kept for an extended period against molecular turnover and cellular reorganization that may disrupt memory traces. How is LTM maintained mechanistically? Despite its critical importance, the molecular and cellular underpinnings of LTM maintenance remain elusive. This study using Drosophila is significant because it revealed for the first time in any organism that universally available environmental light plays an essential role in LTM maintenance. Interestingly, light does so by activating the evolutionarily conserved transcription factor cAMP response element-binding protein via peptidergic signaling.

neuroscience↗

bioRxiv2019

The antimicrobial peptide Defensin cooperates with Tumour Necrosis Factor to drive tumour cell death in Drosophila

Antimicrobial peptides (AMPs) are small cationic molecules best known as mediators of the innate defence against microbial infection. While in vitro and ex vivo evidence suggest AMPs capacity to kill cancer cells, in vivo demonstration of an anti-tumour role of endogenous AMPs is lacking. Using a Drosophila model of tumourigenesis, we demonstrate a role for the AMP Defensin in the control of tumour progression. Our results reveal that Tumour Necrosis Factor mediates exposure of phosphatidylserine (PS), which makes tumour cells selectively sensitive to the action of Defensin remotely secreted from tracheal and fat tissues. Defensin binds tumour cells in PS-enriched areas, provoking cell death and tumour regression. Altogether, our results provide the first in vivo demonstration for a role of an endogenous AMP as an anti-cancer agent, as well as a mechanism that explains tumour cell sensitivity to the action of AMPs.

cancer biology↗

bioRxiv2019

ABC transporters confer multidrug resistance to Drosophila intestinal stem cells

Adult stem cells can survive a wide variety of insults from ionizing radiation to toxic chemicals1-3. To date, the multidrug resistant features of stem cells have been characterized only in vertebrates, where there is a critical need to understand how cancer stem cells thwart chemotherapy drugs4-6. These studies reveal that the ability of both normal and cancer stem cells to survive toxins hinges on their high levels of expression of ABC transporters, transmembrane pumps that efflux lipophilic compounds out of cells7,8. This has been observed across a wide spectrum of vertebrate stem cells including breast, blood, intestine, liver, and skin, suggesting that high efflux ability and multidrug resistance may be general features of stem cells that distinguish them from their differentiated daughter cells. Here we show that these previously described vertebrate stem cell features are conserved in Drosophila intestinal stem cells. Using a novel in vivo efflux assay and multiple drug challenges, we show that stem cells in the fly intestine depend on two ABC transporters--one constitutively expressed and the other induced--for efflux and multidrug resistance. These results suggest that stem cell multidrug resistance by ABC transporters is a general stem cell feature conserved over 500 million years of evolution.

cell biology↗

bioRxiv2018

Human RAD51 paralogue, SWSAP1, fosters RAD51 filament by regulating the anti-recombinase, FIGNL1 AAA+ ATPase

RAD51 assembly on single-stranded (ss)DNAs is a crucial step in the homology-dependent repair of DNA damage for genomic stability. The formation of the RAD51 filament is tightly regulated by various RAD51-interacting proteins. The familial breast cancer gene, BRCA2, as well as RAD52 and six RAD51 paralogues (SWSAP1, RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) promote RAD51 assembly on ssDNAs in humans. However, the mechanisms underlying the differential control of RAD51 filament dynamics by these factors remain largely unknown. Here, we report a role for the human RAD51 paralogue, SWSAP1, as a novel regulator of RAD51 assembly. Swsap1-deficient cells show defects in DNA damage-induced RAD51 assembly during both mitosis and meiosis. Defective RAD51 assembly in SWSAP1-depleted cells is suppressed by the depletion of FIGNL1, an AAA+ ATPase, which binds to RAD51 as well as SWSAP1. Purified FIGNL1 promotes the dissociation of RAD51 from ssDNAs. The in vitro dismantling activity of FIGNL1 does not require its ATPase but depends on RAD51-binding, suggesting a novel mechanism of RAD51 filament remodelling. Purified SWSAP1 inhibits the RAD51-dismantling activity of FIGNL1. Taken together, our data suggest that SWSAP1 protects RAD51 filaments by antagonizing the anti-recombinase, FIGNL1.

molecular biology↗

bioRxiv2018

Synergy and remarkable specificity of antimicrobial peptides in vivo using a systematic knockout approach

Antimicrobial peptides (AMPs) are host-encoded antibiotics that combat invading microorganisms. These short, cationic peptides have been implicated in many biological processes, primarily involving innate immunity. In vitro studies have shown AMPs kill bacteria and fungi at physiological concentrations, but little validation has been done in vivo. We utilised CRISPR gene editing to delete all known immune inducible AMPs of Drosophila, namely: 4 Attacins, 4 Cecropins, 2 Diptericins, Drosocin, Drosomycin, Metchnikowin and Defensin. Using individual and multiple knockouts, including flies lacking all 14 AMP genes, we characterize the in vivo function of individual and groups of AMPs against diverse bacterial and fungal pathogens. We found that Drosophila AMPs act primarily against Gram-negative bacteria and fungi, acting either additively or synergistically. We also describe remarkable specificity wherein certain AMPs contribute the bulk of microbicidal activity against specific pathogens, providing functional demonstrations of highly specific AMP-pathogen interactions in an in vivo setting.

immunology↗

bioRxiv2018

AMPK signaling linked to the schizophrenia-associated 1q21.1 deletion is required for neuronal and sleep maintenance

The human 1q21.1 deletion of ten genes is associated with increased risk schizophrenia. The deletion involves the {beta}-subunit of the AMP-activated protein kinase (AMPK) complex, a key energy sensor in the cell. Although neurons have a high demand for energy and low capacity to store nutrients, the role of AMPK in neuronal physiology is poorly defined. Here we show that AMPK is important in the nervous system for maintaining neuronal integrity and for stress survival and longevity in Drosophila. To understand its impact on behavior and potential contribution to the 1q21.1 deletion syndrome, we focused on sleep, as its main role is proposed to be to reestablish neuronal energy levels that are diminished during energy-demanding wakefulness. Sleep disturbances are one of the most common problems affecting individuals with psychiatric disorders. We show that AMPK is required to maintain proper sleep architecture and for sleep recovery following sleep deprivation. Neuronal AMPK{beta} loss specifically leads to sleep fragmentation and causes dysregulation of genes believed to play a role in sleep homeostasis. Our data also suggest that AMPK{beta} loss may contribute to the increased risk of developing mental disorders and sleep disturbances associated with the human 1q21.1 deletion. Sleep is regulated by circadian and homeostatic processes. While the circadian clock is well studied, the molecular mechanism underlying homeostasis remains largely unknown. Our data suggest that AMPK is required for sleep maintenance and may be involved in the homeostatic process of sleep regulation.\n\nAuthor SummaryThe human 1q21.1 chromosomal deletion is associated with increased risk of schizophrenia. Because this deletion affects only a small number of genes, it provides a unique opportunity to identify the specific disease-causing gene(s) using animal models. Here, we report the use of a Drosophila model to identify the potential contribution of one gene affected by the 1q21.1 deletion - PRKAB2 - to the pathology of the 1q21.1 deletion syndrome. PRKAB2 encodes a subunit of the AMP-activated protein kinase (AMPK) complex, the main cellular energy sensor. We show that AMPK deficiency reduces lifespan and causes structural abnormalities in neuronal dendritic structures, a phenotype which has been linked to schizophrenia. Furthermore, cognitive impairment and altered sleep patterning are some of the most common symptoms of schizophrenia. Therefore, to understand the potential contribution of PRKAB2 to the 1q21.1 syndrome, we tested whether AMPK alterations would cause defects in learning and sleep. Our study shows that lack of PRKAB2 and AMPK-complex activity, in the nervous system leads to reduced learning and to dramatic sleep disturbances. Thus, our data links a single 1q21.1-related gene with phenotypes that resemble common symptoms of schizophrenia, suggesting that this gene, PRKAB2, may contribute to the risk of developing schizophrenia.

neuroscience↗

bioRxiv2018

More than black and white: complex relationships involving serine proteases regulate the Toll pathway and the melanization response in Drosophila

The melanization response is a rapid and important defense mechanism in arthropods. Melanin is produced around wound sites and invading microorganisms by phenoloxidases (POs), which need to be activated by the sequential activation of an extracellular serine protease (SP) cascade. Drosophila melanogaster has been a useful genetic model for dissecting insect immune signaling, but understanding these proteolytic cascades has been complicated by the large number of SP genes, possibly with redundant function. Taking advantage of recently-generated null and compound mutants, we re-investigated the role of SPs involved in the melanization response in D. melanogaster and discovered phenotypes previously concealed in single mutant analysis. We found that two of them, Hayan and Sp7, can activate the melanization response in two different manners: Hayan is required in the local blackening of wound sites, while Sp7 regulates an alternate melanization reaction responsible for the clearance of septic infections with Staphylococcus aureus. We present evidence that both Sp7 and Hayan regulate the Toll NF-kB pathway. Sp7 is regulated by canonical Toll signaling downstream of PGRP-SA, ModSP, and Grass, leading to control of septic infections via a Sp7-dependent melanization response. Additionally, we found that Hayan and the Toll-regulating SP Psh are the result of a recent gene duplication. Using genetic manipulations, we reveal the hidden role for Hayan, alongside Psh, in propagating Toll signaling downstream of pattern recognition receptors. Thus, we describe the existence of two pathways leading to the melanization response and reveal previously unknown dynamics in the activation of the Toll pathway.

immunology↗

bioRxiv2018

Data-driven analysis of motor activity implicated 5-HT2A neurons in backward locomotion of larval Drosophila

Rhythmic animal behaviors are regulated in part by neural circuits called the central pattern generators (CPGs). Classifying neural population activities correlated with body movements and identifying the associated component neurons are critical steps in understanding CPGs. Previous methods that classify neural dynamics obtained by dimension reduction algorithms often require manual optimization which could be laborious and preparation-specific. Here, we present a simpler and more flexible method that is based on the pre-trained convolutional neural network model VGG-16 and unsupervised learning, and successfully classifies the fictive motor patterns in Drosophila larvae under various imaging conditions. We also used voxel-wise correlation mapping to identify neurons associated with motor patterns. By applying these methods to neurons targeted by 5-HT2A-GAL4, which we generated by the CRISPR/Cas9-system, we identified two classes of interneurons, termed Seta and Leta, which are specifically active during backward but not forward fictive locomotion. Optogenetic activation of Seta and Leta neurons increased backward locomotion. Conversely, thermogenetic inhibition of 5-HT2A-GAL4 neurons or application of a 5-HT2 antagonist decreased backward locomotion induced by noxious light stimuli. This study establishes an accelerated pipeline for activity profiling and cell identification in larval Drosophila and implicates the serotonergic system in the modulation of backward locomotion.

neuroscience↗