Damage sensing mediated by serine proteases Hayan and Persephone for Toll pathway activation in apoptosis-deficient flies.

The mechanisms by which the innate immune system senses damage have been extensively explored in multicellular organisms. In Drosophila, various types of tissue damage, including epidermal injury, tumor formation, cell competition, and apoptosis deficiency, induce sterile activation of the Toll pathway, a process that requires the use of extracellular serine protease (SP) cascades. Upon infection, the SP Spätzle (Spz)-processing enzyme (SPE) cleaves and activates the Toll ligand Spz downstream of two paralogous SPs, Hayan and Persephone (Psh). However, upon tissue damage, it is not fully understood which SPs establish Spz activation cascades nor what damage-associated molecules can activate SPs. In this study, using newly generated uncleavable spz mutant flies, we revealed that Spz cleavage is required for the sterile activation of the Toll pathway, which is induced by apoptosis-deficient damage of wing epidermal cells in adult Drosophila. Proteomic analysis of hemolymph, followed by experiments with Drosophila Schneider 2 (S2) cells, revealed that among hemolymph SPs, both SPE and Melanization Protease 1 (MP1) have high capacities to cleave Spz. Additionally, in S2 cells, MP1 acts downstream of Hayan and Psh in a similar manner to SPE. Using genetic analysis, we found that the upstream SPs Hayan and Psh contributes to the sterile activation of the Toll pathway. While SPE/MP1 double mutants show more impairment of Toll activation upon infection than SPE single mutants, Toll activation is not eliminated in these apoptosis-deficient flies. This suggests that Hayan and Psh sense necrotic damage, inducing Spz cleavage by SPs other than SPE and MP1. Furthermore, hydrogen peroxide, a representative damage-associated molecule, activates the Psh-Spz cascade in S2 cells overexpressing Psh. Considering that reactive oxygen species (ROS) were detected in apoptosis-deficient wings, our findings highlight the importance of ROS as signaling molecules that induce the activation of SPs such as Psh in response to damage.

Dendritic Eph organizes dendrodendritic segregation in discrete olfactory map formation in Drosophila.

Proper function of the neural network results from the precise connections between axons and dendrites of presynaptic and postsynaptic neurons, respectively. In the Drosophila olfactory system, the dendrites of projection neurons (PNs) stereotypically target one of ∼50 glomeruli in the antennal lobe (AL), the primary olfactory center in the brain, and form synapses with the axons of olfactory receptor neurons (ORNs). Here, we show that Eph and Ephrin, the well-known axon guidance molecules, instruct the dendrodendritic segregation during the discrete olfactory map formation. The Eph receptor tyrosine kinase is highly expressed and localized in the glomeruli related to reproductive behavior in the developing AL. In one of the pheromone-sensing glomeruli (DA1), the Eph cell-autonomously regulates its dendrites to reside in a single glomerulus by interacting with Ephrins expressed in adjacent PN dendrites. Our data demonstrate that the trans interaction between dendritic Eph and Ephrin is essential for the PN dendritic boundary formation in the DA1 olfactory circuit, potentially enabling strict segregation of odor detection between pheromones and the other odors.

Co-option of an Astacin Metalloprotease Is Associated with an Evolutionarily Novel Feeding Morphology in a Predatory Nematode.

Animals consume a wide variety of food sources to adapt to different environments. However, the genetic mechanisms underlying the acquisition of evolutionarily novel feeding morphology remain largely unknown. While the nematode Caenorhabditis elegans feeds on bacteria, the satellite species Pristionchus pacificus exhibits predatory feeding behavior toward other nematodes, which is an evolutionarily novel feeding habit. Here, we found that the astacin metalloprotease Ppa-NAS-6 is required for the predatory killing by P. pacificus. Ppa-nas-6 mutants were defective in predation-associated characteristics, specifically the tooth morphogenesis and tooth movement during predation. Comparison of expression patterns and rescue experiments of nas-6 in P. pacificus and C. elegans suggested that alteration of the spatial expression patterns of NAS-6 may be vital for acquiring predation-related traits. Reporter analysis of the Ppa-nas-6 promoter in C. elegans revealed that the alteration in expression patterns was caused by evolutionary changes in cis- and trans-regulatory elements. This study suggests that the co-option of a metalloprotease is involved in an evolutionarily novel feeding morphology.

Endoplasmic reticulum proteins Meigo and Gp93 govern dendrite targeting by regulating Toll-6 localization.

Neuronal target recognition is performed by numerous cell-surface transmembrane proteins. Correct folding of these proteins occurs in the endoplasmic reticulum (ER) lumen of the neuronal cells before being transported to the plasma membrane of axons or dendrites. Disturbance in this protein folding process in the ER leads to dysfunction of neuronal cell surface molecules, resulting in abnormal neuronal targeting. In this study, we report that the ER-resident protein Meigo in Drosophila, governs the dendrite targeting of olfactory projection neurons (PNs) along the mediolateral axis of the antennal lobe by regulating Toll-6 localization. Loss of Meigo causes Toll-6 mislocalization in the PNs and mediolateral dendrite targeting defects, which are suppressed by Toll-6 overexpression. Furthermore, we found that the ER-chaperone protein, Gp93, also regulates the mediolateral targeting of PN dendrites by localization of the Toll-6 protein. Gp93 overexpression in the PN homozygous for the meigo mutation, partially rescued the dendrite targeting defect, while meigo knockdown decreased Gp93 expression levels in cultured cells. These results indicate that the ER-proteins Meigo and Gp93 regulate dendrite targeting by attenuating the amount and localization of cell surface receptors, including Toll-6, implying the unexpected but active involvement of ER proteins in neural wiring.

Ubiquitin proteasome system in circadian rhythm and sleep homeostasis: Lessons from Drosophila.

Sleep is regulated by two main processes: the circadian clock and sleep homeostasis. Circadian rhythms have been well studied at the molecular level. In the Drosophila circadian clock neurons, the core clock proteins are precisely regulated by post-translational modifications and degraded via the ubiquitin-proteasome system (UPS). Sleep homeostasis, however, is less understood; nevertheless, recent reports suggest that proteasome-mediated degradation of core clock proteins or synaptic proteins contributes to the regulation of sleep amount. Here, we review the molecular mechanism of the UPS and summarize the role of protein degradation in the regulation of circadian clock and homeostatic sleep in Drosophila. Moreover, we discuss the potential interaction between circadian clock and homeostatic sleep regulation with a prime focus on E3 ubiquitin ligases.

Systemic innate immune response induces death of olfactory receptor neurons in Drosophila.

Neural functions are known to decline during normal aging and neurodegenerative diseases. However, the mechanisms of functional impairment owing to the normal aging of the brain are poorly understood. Previously, we reported that caspase-3-like protease, the protease responsible for inducing apoptosis, is activated in a subset of olfactory receptor neurons (ORNs), especially in Drosophila Or42b neurons, during normal aging. Herein, we investigated the molecular mechanism underlying age-related caspase-3-like protease activation and cell death in Or42b neurons. Gene expression profiling of young and aged fly antenna showed that the expression of antimicrobial peptides was significantly upregulated, suggesting an activated innate immune response. Consistent with this observation, inhibition or activation of the innate immune pathway caused delayed or precocious cell death, respectively, in Or42b neurons. Accordingly, autonomous cell activation of the innate immune pathway in Or42b neurons is not likely required for their age-related death, whereas the systemic innate immune response induces caspase-3-like protease activation in Or42b neurons; this indicated that the death of these neurons is regulated non-cell autonomously. We propose a possible link between the innate immune response and the death of olfactory neurons during normal aging.

Crosstalk between the mTOR and Hippo pathways.

Cell behavior changes in response to multiple stimuli, such as growth factors, nutrients, and cell density. The mechanistic target of the rapamycin (mTOR) pathway is activated by growth factors and nutrient stimuli to regulate cell growth and autophagy, whereas the Hippo pathway has negative effects on cell proliferation and tissue growth in response to cell density, DNA damage, and hormonal signals. These two signaling pathways must be precisely regulated and integrated for proper cell behavior. This integrative mechanism is not completely understood; nevertheless, recent studies have suggested that components of the mTOR and Hippo pathways interact with each other. Herein, as per contemporary knowledge, we review the molecular mechanisms of the interaction between the mTOR and Hippo pathways in mammals and Drosophila. Moreover, we discuss the advantage of this interaction in terms of tissue growth and nutrient consumption.

Non-apoptotic function of Drosophila caspase activation in epithelial thorax closure and wound healing.

Non-apoptotic caspase activation involves multiple cellular events. However, the link between visible non-apoptotic caspase activation and its function in living organisms has not yet been revealed. Here, we visualized sub-lethal activation of apoptotic signaling with the combination of a sensitive indicator for caspase 3 activation and in vivo live-imaging analysis of Drosophila During thorax closure in pupal development, caspase 3 activation was specifically observed at the leading edge cells, with no signs of apoptosis. Inhibition of caspase activation led to an increase in thorax closing speed, which suggests a role of non-apoptotic caspase activity in cell motility. Importantly, sub-lethal activation of caspase 3 was also observed during wound closure at the fusion sites at which thorax closure had previously taken place. Further genetic analysis revealed that the activation of the initiator caspase Dronc is coupled with the generation of reactive oxygen species. The activation of Dronc also regulates myosin levels and delays wound healing. Our findings suggest a possible function for non-apoptotic caspase activation in the fine-tuning of cell migratory behavior during epithelial closure.

Amyotrophic lateral sclerosis-associated Vap33 is required for maintaining neuronal dendrite morphology and organelle distribution in Drosophila.

VAMP-associated protein (VAP) is an endoplasmic reticulum (ER) membrane protein that functions as a tethering protein at the membrane contact sites between the ER and various intracellular organelles. Mutations such as P56S in human VAPB cause neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, VAP functions in neurons are poorly understood. Here, we utilized Drosophila olfactory projection neurons with a mosaic analysis with a repressible cell marker (MARCM) to analyze the neuronal function of Vap33, a Drosophila ortholog of human VAPB. In vap33 null mutant clones, the dendrites of projection neurons exhibited defects in the maintenance of their morphology. The subcellular localization of the Golgi apparatus and mitochondria were also abnormal. These results indicate that Vap33 is required for neuronal morphology and organelle distribution. Additionally, to examine the impact of ALS-associated mutations in neurons, we overexpressed human VAPB-P56S in vap33 null mutant clones (mosaic rescue experiments) and found that, in aged flies, human VAPB-P56S expression caused mislocalization of Bruchpilot, a presynaptic protein. These results implied that synaptic protein localization and ER quality control may be affected by disease mutations. We provide insights into the physiological and pathological functions of VAP in neurons.

Dronc-independent basal executioner caspase activity sustains Drosophila imaginal tissue growth.

Caspase is best known as an enzyme involved in programmed cell death, which is conserved among multicellular organisms. In addition to its role in cell death, caspase is emerging as an indispensable enzyme in a wide range of cellular functions, which have recently been termed caspase-dependent nonlethal cellular processes (CDPs). In this study, we examined the involvement of cell death signaling in tissue-size determination using Drosophila wing as a model. We found that the Drosophila executioner caspases Dcp-1 and Decay, but not Drice, promoted wing growth independently of apoptosis. Most of the reports on CDPs argue the importance of the spatiotemporal regulation of the initiator caspase, Dronc; however, this sublethal caspase function was independent of Dronc, suggesting a more diverse array of CDP regulatory mechanisms. Tagging of TurboID, an improved promiscuous biotin ligase that biotinylates neighboring proteins, to the C terminus of caspases revealed the differences among the neighbors of executioner caspases. Furthermore, we found that the cleavage of Acinus, a substrate of the executioner caspase, was important in promoting wing growth. These results demonstrate the importance of executioner caspase-mediated basal proteolytic cleavage of substrates in sustaining tissue growth. Given the existence of caspase-like DEVDase activity in a unicellular alga, our results likely highlight the original function of caspase-not cell death, but basal proteolytic cleavages for cell vigor.

Efficient visual screening of CRISPR/Cas9 genome editing in the nematode Pristionchus pacificus.

CRISPR/Cas9 genome editing has been applied to a wide variety of organisms, including nematodes such as Caenorhabditis elegans and Pristionchus pacificus. In these nematodes, genome editing is achieved by microinjection of Cas9 protein and guide RNA into the hermaphrodite gonads. However, P. pacificus is less efficient in CRISPR/Cas9 genome editing and exogenous gene expression. Therefore, it takes considerable time and effort to screen for target mutants if there are no visual markers that indicate successful injection. To overcome this problem, co-injection markers (gRNA for Ppa-prl-1, which induces the roller phenotype, and Ppa-egl-20p::turboRFP, a plasmid expressing a fluorescent protein) have been developed in P. pacificus. By selecting worms with the roller phenotype or turboRFP expression, screening efficiency is substantially increased to obtain worms with desired mutations. Here, we describe a step-by-step protocol for the visual screening system for CRISPR/Cas9 genome editing in P. pacificus. We also describe technical tips for microinjection, which is difficult for beginners. This protocol will facilitate genome editing in P. pacificus and may be applied to other nematode species.

Screening for CRISPR/Cas9-induced mutations using a co-injection marker in the nematode Pristionchus pacificus.

CRISPR/Cas9 genome-editing methods are used to reveal functions of genes and molecular mechanisms underlying biological processes in many species, including nematodes. In evolutionary biology, the nematode Pristionchus pacificus is a satellite model and has been used to understand interesting phenomena such as phenotypic plasticity and self-recognition. In P. pacificus, CRISPR/Cas9-mediated mutations are induced by microinjecting a guide RNA (gRNA) and Cas9 protein into the gonads. However, mutant screening is laborious and time-consuming due to the absence of visual markers. In this study, we established a Co-CRISPR strategy by using a dominant roller marker in P. pacificus. We found that heterozygous mutations in Ppa-prl-1 induced the roller phenotype, which can be used as an injection marker. After the co-injection of Ppa-prl-1 gRNA, target gRNA, and the Cas9 protein, roller progeny and their siblings were examined using the heteroduplex mobility assay and DNA sequencing. We found that some of the roller and non-roller siblings had mutations at the target site. We used varying Cas9 concentrations and found that a higher concentration of Cas9 did not increase genome-editing events. The Co-CRISPR strategy promotes the screening for genome-editing events and will facilitate the development of new genome-editing methods in P. pacificus.

Impairment of cytokinesis by cancer-associated DAPK3 mutations.

Death-associated protein kinase 3 (DAPK3), a member of the DAPK family, contributes to cytokinesis by phosphorylating myosin II regulatory light chain (MRLC). Missense mutations in DAPK3, T112M, D161N, and P216S, were observed in the lung, colon, and cervical cancers, respectively, but the effects of these mutations on cytokinesis remain unclear. Here, we show that cells expressing EGFP-DAPK3-T112M, -D161N, or -P216S exhibited reduced rates of cytokinesis, with an increased ratio of multinucleated cells. In addition, these cells exhibited reduced levels of phosphorylated MRLC at the contractile ring. Collectively, our data demonstrates that cancer-associated DAPK3 mutations impair cytokinesis by reducing phosphorylated MRLC.

Biallelic TBCD Mutations Cause Early-Onset Neurodegenerative Encephalopathy.

We describe four families with affected siblings showing unique clinical features: early-onset (before 1 year of age) progressive diffuse brain atrophy with regression, postnatal microcephaly, postnatal growth retardation, muscle weakness/atrophy, and respiratory failure. By whole-exome sequencing, we identified biallelic TBCD mutations in eight affected individuals from the four families. TBCD encodes TBCD (tubulin folding co-factor D), which is one of five tubulin-specific chaperones playing a pivotal role in microtubule assembly in all cells. A total of seven mutations were found: five missense mutations, one nonsense, and one splice site mutation resulting in a frameshift. In vitro cell experiments revealed the impaired binding between most mutant TBCD proteins and ARL2, TBCE, and ß-tubulin. The in vivo experiments using olfactory projection neurons in Drosophila melanogaster indicated that the TBCD mutations caused loss of function. The wide range of clinical severity seen in this neurodegenerative encephalopathy may result from the residual function of mutant TBCD proteins. Furthermore, the autopsied brain from one deceased individual showed characteristic neurodegenerative findings: cactus and somatic sprout formations in the residual Purkinje cells in the cerebellum, which are also seen in some diseases associated with mitochondrial impairment. Defects of microtubule formation caused by TBCD mutations may underlie the pathomechanism of this neurodegenerative encephalopathy.

Tissue nonautonomous effects of fat body methionine metabolism on imaginal disc repair in Drosophila.

Regulatory mechanisms for tissue repair and regeneration within damaged tissue have been extensively studied. However, the systemic regulation of tissue repair remains poorly understood. To elucidate tissue nonautonomous control of repair process, it is essential to induce local damage, independent of genetic manipulations in uninjured parts of the body. Herein, we develop a system in Drosophila for spatiotemporal tissue injury using a temperature-sensitive form of diphtheria toxin A domain driven by the Q system to study factors contributing to imaginal disc repair. Using this technique, we demonstrate that methionine metabolism in the fat body, a counterpart of mammalian liver and adipose tissue, supports the repair processes of wing discs. Local injury to wing discs decreases methionine and S-adenosylmethionine, whereas it increases S-adenosylhomocysteine in the fat body. Fat body-specific genetic manipulation of methionine metabolism results in defective disc repair but does not affect normal wing development. Our data indicate the contribution of tissue interactions to tissue repair in Drosophila, as local damage to wing discs influences fat body metabolism, and proper control of methionine metabolism in the fat body, in turn, affects wing regeneration.

Familial Amyotrophic Lateral Sclerosis-linked Mutations in Profilin 1 Exacerbate TDP-43-induced Degeneration in the Retina of Drosophila melanogaster through an Increase in the Cytoplasmic Localization of TDP-43.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive and selective loss of motor neurons. Causative genes for familial ALS (fALS), e.g. TARDBP or FUS/TLS, have been found, among which mutations within the profilin 1 (PFN1) gene have recently been identified in ALS18. To elucidate the mechanism whereby PFN1 mutations lead to neuronal death, we generated transgenic Drosophila melanogaster overexpressing human PFN1 in the retinal photoreceptor neurons. Overexpression of wild-type or fALS mutant PFN1 caused no degenerative phenotypes in the retina. Double overexpression of fALS mutant PFN1 and human TDP-43 markedly exacerbated the TDP-43-induced retinal degeneration, i.e. vacuolation and thinning of the retina, whereas co-expression of wild-type PFN1 did not aggravate the degenerative phenotype. Notably, co-expression of TDP-43 with fALS mutant PFN1 increased the cytoplasmic localization of TDP-43, the latter remaining in nuclei upon co-expression with wild-type PFN1, whereas co-expression of TDP-43 lacking the nuclear localization signal with the fALS mutant PFN1 did not aggravate the retinal degeneration. Knockdown of endogenous Drosophila PFN1 did not alter the degenerative phenotypes of the retina in flies overexpressing wild-type TDP-43 These data suggest that ALS-linked PFN1 mutations exacerbate TDP-43-induced neurodegeneration in a gain-of-function manner, possibly by shifting the localization of TDP-43 from nuclei to cytoplasm.

Hierarchical axon targeting of Drosophila olfactory receptor neurons specified by the proneural transcription factors Atonal and Amos.

Sensory information is spatially represented in the brain to form a neural map. It has been suggested that axon-axon interactions are important for neural map formation; however, the underlying mechanisms are not fully understood. We used the Drosophila antennal lobe, the first olfactory center in the brain, as a model for studying neural map formation. Olfactory receptor neurons (ORNs) expressing the same odorant receptor target their axons to a single glomerulus out of approximately 50 glomeruli in the antennal lobe. Previous studies have showed that the axons of Atonal ORNs, specified by Atonal, a basic helix-loop-helix (bHLH) transcription factor, pioneer antennal lobe formation; however, the details remain to be elucidated. Here, we show that genetic ablation of Atonal ORNs affects antennal lobe structure and axon targeting of Amos ORNs, another type of ORN specified by the bHLH transcription factor Amos. During development, Atonal ORNs reach the antennal lobe and form the axon commissure before Amos ORNs. We also found that N-cadherin knockdown specifically in Atonal ORNs disrupts the glomerular boundary in the whole antennal lobe. Our results suggest that Atonal ORNs function as pioneer axons. Thus, correct axon targeting of Atonal ORNs is essential for formation of the whole antennal lobe.

Caspase inhibition in select olfactory neurons restores innate attraction behavior in aged Drosophila.

Sensory and cognitive performance decline with age. Neural dysfunction caused by nerve death in senile dementia and neurodegenerative disease has been intensively studied; however, functional changes in neural circuits during the normal aging process are not well understood. Caspases are key regulators of cell death, a hallmark of age-related neurodegeneration. Using a genetic probe for caspase-3-like activity (DEVDase activity), we have mapped age-dependent neuronal changes in the adult brain throughout the lifespan of Drosophila. Spatio-temporally restricted caspase activation was observed in the antennal lobe and ellipsoid body, brain structures required for olfaction and visual place memory, respectively. We also found that caspase was activated in an age-dependent manner in specific subsets of Drosophila olfactory receptor neurons (ORNs), Or42b and Or92a neurons. These neurons are essential for mediating innate attraction to food-related odors. Furthermore, age-induced impairments of neural transmission and attraction behavior could be reversed by specific inhibition of caspase in these ORNs, indicating that caspase activation in Or42b and Or92a neurons is responsible for altering animal behavior during normal aging.

Linking cell surface receptors to microtubules: tubulin folding cofactor D mediates Dscam functions during neuronal morphogenesis.

Formation of functional neural networks requires the coordination of cell surface receptors and downstream signaling cascades, which eventually leads to dynamic remodeling of the cytoskeleton. Although a number of guidance receptors affecting actin cytoskeleton remodeling have been identified, it is relatively unknown how microtubule dynamics are regulated by guidance receptors. We used Drosophila olfactory projection neurons to study the molecular mechanisms of neuronal morphogenesis. Dendrites of each projection neuron target a single glomerulus of ∼50 glomeruli in the antennal lobe, and the axons show stereotypical pattern of terminal arborization. In the course of genetic analysis of the dachsous mutant allele (ds(UAO71)), we identified a mutation in the tubulin folding cofactor D gene (TBCD) as a background mutation. TBCD is one of five tubulin-folding cofactors required for the formation of α- and ß-tubulin heterodimers. Single-cell clones of projection neurons homozygous for the TBCD mutation displayed disruption of microtubules, resulting in ectopic arborization of dendrites, and axon degeneration. Interestingly, overexpression of TBCD also resulted in microtubule disruption and ectopic dendrite arborization, suggesting that an optimum level of TBCD is crucial for in vivo neuronal morphogenesis. We further found that TBCD physically interacts with the intracellular domain of Down syndrome cell adhesion molecule (Dscam), which is important for neural development and has been implicated in Down syndrome. Genetic analyses revealed that TBCD cooperates with Dscam in vivo. Our study may offer new insights into the molecular mechanism underlying the altered neural networks in cognitive disabilities of Down syndrome.

RNA binding mediates neurotoxicity in the transgenic Drosophila model of TDP-43 proteinopathy.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive and selective loss of motor neurons. The discovery of mutations in the gene encoding an RNA-binding protein, TAR DNA-binding protein of 43 kD (TDP-43), in familial ALS, strongly implicated abnormalities in RNA processing in the pathogenesis of ALS, although the mechanisms whereby TDP-43 leads to neurodegeneration remain elusive. To clarify the mechanism of degeneration caused by TDP-43, we generated transgenic Drosophila melanogaster expressing a series of systematically modified human TDP-43 genes in the retinal photoreceptor neurons. Overexpression of wild-type TDP-43 resulted in vacuolar degeneration of the photoreceptor neurons associated with thinning of the retina, which was significantly exacerbated by mutations of TDP-43 linked to familial ALS or disrupting its nuclear localization signal (NLS). Remarkably, these degenerative phenotypes were completely normalized by addition of a mutation or deletion of the RNA recognition motif that abolishes the RNA binding ability of TDP-43. Altogether, our results suggest that RNA binding is key to the neurodegeneration caused by overexpression of TDP-43, and that abnormalities in RNA processing may be crucial to the pathogenesis of TDP-43 proteinopathy.