Drosophila transcription factor NF-Y suppresses transcription of the lipase 4 gene, a key gene for lipid storage.

The CCAAT motif-binding factor NF-Y consists of three different subunits, NF-YA, NF-YB, and NF-YC. Although it is suggested that NF-Y activity is essential for normal tissue homeostasis, survival, and metabolic function, its precise role in lipid metabolism is not clarified yet. In Drosophila, eye disc specific knockdown of Drosophila NF-YA (dNF-YA) induced aberrant morphology of the compound eye, the rough eye phenotype in adults and mutation of the lipase 4 (lip4) gene suppressed the rough eye phenotype. RNA-seq analyses with dNF-YA knockdown third instar larvae identified the lip4 gene as one of the genes that are up-regulated by the dNF-YA knockdown. We identified three dNF-Y-binding consensuses in the 5'flanking region of the lip4 gene, and a chromatin immunoprecipitation assay with the specific anti-dNF-YA IgG demonstrated dNF-Y binding to this genomic region. The luciferase transient expression assay with cultured Drosophila S2 cells and the lip4 promoter-luciferase fusion genes with and without mutations in the dNF-Y-binding consensuses showed that each of the three dNF-Y consensus sequences negatively regulated lip4 gene promoter activity. Consistent with these results, qRT-PCR analysis with the dNF-YA knockdown third instar larvae revealed that endogenous lip4 mRNA levels were increased by the knockdown of dNF-YA in vivo. The specific knockdown of dNF-YA in the fat body with the collagen-GAL4 driver resulted in smaller oil droplets in the fat body cells. Collectively, these results suggest that dNF-Y is involved in lipid storage through its negative regulation of lip4 gene transcription.

Association of Mutations in Replicative DNA Polymerase Genes with Human Disease: Possible Application of Drosophila Models for Studies.

Replicative DNA polymerases, such as DNA polymerase α-primase, δ and ε, are multi-subunit complexes that are responsible for the bulk of nuclear DNA replication during the S phase. Over the last decade, extensive genome-wide association studies and expression profiling studies of the replicative DNA polymerase genes in human patients have revealed a link between the replicative DNA polymerase genes and various human diseases and disorders including cancer, intellectual disability, microcephalic primordial dwarfism and immunodeficiency. These studies suggest the importance of dissecting the mechanisms involved in the functioning of replicative DNA polymerases in understanding and treating a range of human diseases. Previous studies in Drosophila have established this organism as a useful model to understand a variety of human diseases. Here, we review the studies on Drosophila that explored the link between DNA polymerases and human disease. First, we summarize the recent studies linking replicative DNA polymerases to various human diseases and disorders. We then review studies on replicative DNA polymerases in Drosophila. Finally, we suggest the possible use of Drosophila models to study human diseases and disorders associated with replicative DNA polymerases.

Role of Drosophila in Human Disease Research 3.0.

Drosophila melanogaster has become a commonly used animal model for biomedical research in a variety of areas [...].

Drosophila models to study causative genes for human rare intractable neurological diseases.

Drosophila is emerging as a convenient model for investigating human diseases. Functional homologues of almost 75% of human disease-related genes are found in Drosophila. Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease that causes defects in motoneurons. Charcot-Marie-Tooth disease (CMT) is one of the most commonly found inherited neuropathies affecting both motor and sensory neurons. No effective therapy has been established for either of these diseases. In this review, after overviewing ALS, Drosophila models targeting several ALS-causing genes, including TDP-43, FUS and Ubiquilin2, are described with their genetic interactants. Then, after overviewing CMT, examples of Drosophila models targeting several CMT-causing genes, including mitochondria-related genes and FIG 4, are also described with their genetic interactants. In addition, we introduce Sotos syndrome caused by mutations in the epigenetic regulator gene NSD1. Lastly, several genes and pathways that commonly interact with ALS- and/or CMT-causing genes are described. In the case of ALS and CMT that have many causative genes, it may be not practical to perform gene therapy for each of the many disease-causing genes. The possible uses of the common genes and pathways as novel diagnosis markers and effective therapeutic targets are discussed.

Role of Drosophila in Human Disease Research 2.0.

The fruit fly Drosophila melanogaster is a highly tractable animal model to study various human diseases [...].

Gene expression of PLAT and ATS3 proteins increases plant resistance to insects.

MAIN CONCLUSION: Genes of the PLAT protein family, including PLAT and ATS3 subfamilies of higher plants and homologs of liverwort, are involved in plant defense against insects. Laticifer cells in plants contain large amounts of anti-microbe or anti-insect proteins and are involved in plant defense against biotic stresses. We previously found that PLAT proteins accumulate in laticifers of fig tree (Ficus carica) at comparable levels to those of chitinases, and the transcript level of ATS3, another PLAT domain-containing protein, is highest in the transcriptome of laticifers of Euphorbia tirucalli. In this study, we investigated whether the PLAT domain-containing proteins are involved in defense against insects. Larvae of the lepidopteran Spodoptera litura showed retarded growth when fed with Nicotiana benthamiana leaves expressing F. carica PLAT or E. tirucalli ATS3 genes, introduced by agroinfiltration using expression vector pBYR2HS. Transcriptome analysis of these leaves indicated that ethylene and jasmonate signaling were activated, leading to increased expression of genes for PR-1, ß-1,3-glucanase, PR5 and trypsin inhibitors, suggesting an indirect mechanism of PLAT- and ATS3-induced resistance in the host plant. Direct cytotoxicity of PLAT and ATS3 to insects was also possible because heterologous expression of the corresponding genes in Drosophila melanogaster caused apoptosis-mediated cell death in this insect. Larval growth retardation of S. litura occurred when they were fed radish sprouts, a good host for agroinfiltration, expressing any of nine homologous genes of dicotyledon Arabidopsis thaliana, monocotyledon Brachypodium distachyon, conifer Picea sitchensis and liverwort Marchantia polymorpha. Of these nine genes, the heterologous expression of A. thaliana AT5G62200 and AT5G62210 caused significant increases in larval death. These results indicated that the PLAT protein family has largely conserved anti-insect activity in the plant kingdom (249 words).

Novel genetic link between the ATP-binding cassette subfamily A gene and hippo gene in Drosophila.

The pan-neuron-specific knockdown of dABCA, a Drosophila homologue of the human ATP binding cassette subfamily A member 13 gene, increases social space without affecting climbing ability and induces the early onset of evening activity in adult flies followed by relatively high activity throughout the day. Satellite bouton numbers in the presynaptic terminals of motor neurons are increased in dABCA knockdown flies. In the present study, we further characterized pan-neuron-specific dABCA knockdown flies and found that active zones in the presynaptic terminals of motor neurons increased, whereas learning abilities decreased in larvae. Genetic crossing experiments revealed that the hippo mutation enhanced the hyperactivity phenotype of adults, but suppressed the increased satellite bouton phenotype induced by the dABCA knockdown. Drosophila ABCA is predicted to transport lipid molecules and impair the asymmetric distribution of phospholipids across the plasma membrane, and these local changes are considered to be important for various cellular functions. The disruption of lipid homeostasis in central and peripheral nervous systems by the dABCA knockdown may affect the Hippo-related signaling pathway in order to induce the observed phenotypes.

Identification of CR43467 encoding a long non-coding RNA as a novel genetic interactant with dFIG4, a CMT-causing gene.

The eye imaginal disc-specific knockdown of dFIG4, a Drosophila homolog of FIG4 that is one of the Charcot-Marie-Tooth disease (CMT)-causing genes, induces an aberrant adult compound eye morphology, the so-called rough eye phenotype. We previously performed modifier screening on the dFIG4 knockdown-induced rough eye phenotype and identified several genes, including CR18854, encoding a long non-coding RNA (lncRNA) as genetic interactants with dFIG4. In the present study, in more extensive genetic screening, we found that the deletion of a gene locus encoding both Odorant rector 46a (Or46a) and lncRNA CR43467 effectively suppressed the rough eye phenotype induced by the knockdown of dFIG4. Both genes were located on the same locus, but oriented in opposite directions. In order to identify which of these genes is responsible for the suppression of the rough eye phenotype, we established a CR43467-specific knockdown line using the CRISPR-dCas9 system. By using this system, we demonstrated that the CR43467 gene, but not the Or46a gene, genetically interacted with the dFIG4 gene. The knockdown of CR43467 rescued the reductions in the length of synaptic branches and number of boutons at neuromuscular junctions induced by the knockdown of dFIG4. The vacuole enlargement phenotype induced by the fat body-specific dFIG4 knockdown was also effectively suppressed by the knockdown of CR43467. The knockdown of CR43467 also suppressed the rough eye phenotype induced by other peripheral neuropathy-related genes, such as dCOA7, dHADHB, and dPDHB. We herein identified another gene encoding lncRNA, CR43467 as a genetic interactant with the CMT-causing gene.

Epigenetic Regulation of ALS and CMT: A Lesson from Drosophila Models.

Amyotrophic lateral sclerosis (ALS) is the third most common neurodegenerative disorder and is sometimes associated with frontotemporal dementia. Charcot-Marie-Tooth disease (CMT) is one of the most commonly inherited peripheral neuropathies causing the slow progression of sensory and distal muscle defects. Of note, the severity and progression of CMT symptoms markedly vary. The phenotypic heterogeneity of ALS and CMT suggests the existence of modifiers that determine disease characteristics. Epigenetic regulation of biological functions via gene expression without alterations in the DNA sequence may be an important factor. The methylation of DNA, noncoding RNA, and post-translational modification of histones are the major epigenetic mechanisms. Currently, Drosophila is emerging as a useful ALS and CMT model. In this review, we summarize recent studies linking ALS and CMT to epigenetic regulation with a strong emphasis on approaches using Drosophila models.

Overexpression of H3K36 methyltransferase NSD in glial cells affects brain development in Drosophila.

NSD1 is a histone methyltransferase that methylates the lysine 36 at histone H3. NSD duplication is associated with short stature, microcephaly, intellectual disability, and behavioral defects in humans. Ectopic overexpression of NSD, an NSD1 homolog in Drosophila, was shown to induce developmental abnormalities via apoptosis. In this study, to investigate the effects of NSD overexpression on Drosophila brain development, we first examined the typical NSD expression pattern in larval brains and found that endogenous NSD promoter activity was detected only in subsets of glial cells. Pan-glial, but not pan-neuronal, NSD overexpression induced apoptosis in larval brain cells. However, pan-glial NSD overexpression also induced caspase-3 cleavage in neuronal cells. Among the various glial cell types, NSD overexpression in only astrocytic glia induced apoptosis and abnormal learning defects in the larval stage. Furthermore, NSD overexpression downregulated the expression of various astrocyte-specific genes, including draper (drpr), possibly owing to an indirect effect of NSD overexpression-induced astrocytic apoptosis. Since drpr plays a role in axon pruning during mushroom body (MB) formation in Drosophila astrocytes, we examined the effect of astrocytic NSD overexpression on this process and found that it disrupted the clearance of γ-neurons in the MB, subsequently inducing arrhythmic locomotor activity of the fly. Thus, these results suggest that aberrant NSD overexpression may cause neurodevelopmental disorders by interfering with crucial functions of astrocytes in the brain, underlining the importance of the tightly controlled astrocytic NSD expression for proper neurodevelopment.

The human Bcl-2 family member Bcl-rambo and voltage-dependent anion channels manifest a genetic interaction in Drosophila and cooperatively promote the activation of effector caspases in human cultured cells.

We previously reported that the Bcl-2 family member human Bcl-rambo, also known as BCL2L13, induces apoptosis in human embryonic kidney 293T cells. Mouse Bcl-rambo has recently been reported to mediate mitochondrial fragmentation and mitophagy. In the present study, we showed that the transfection of human Bcl-rambo and its microtubule-associated protein light chain 3-interacting region motif mutant (W276A/I279A) caused mitochondrial fragmentation and the perinuclear accumulation of fragmented mitochondria in human lung adenocarcinoma A549 cells. In comprehensive screening using the Drosophila model in which human Bcl-rambo was ectopically expressed in eye imaginal discs, voltage-dependent anion channels (VDAC), also known as mitochondrial porin, were found to manifest a genetic interaction with human Bcl-rambo. In addition to human adenine nucleotide translocase (ANT) 1 and ANT2, the human Bcl-rambo protein bound to human VDAC1, albeit to a lesser extent than ANT2. Moreover, human VDAC1 and human VDAC2 in particular promoted the activation of effector caspases only when they were co-expressed with human Bcl-rambo in 293T cells. Bcl-rambo induced the perinuclear accumulation of fragmented mitochondria by the knockdown of VDAC1, VDAC2, and VDAC3 in A549 cells. Thus, the present study revealed that human Bcl-rambo and VDAC cooperatively promote the activation of effector caspases in human cultured cells.

Reduction of Rpd3 suppresses defects in locomotive ability and neuronal morphology induced by the knockdown of Drosophila SLC25A46 via an epigenetic pathway.

Mitochondrial dysfunction causes various diseases. Mutations in the SLC25A46 gene have been identified in mitochondrial diseases that are sometimes classified as Charcot-Marie-Tooth disease type 2, optic atrophy, and Leigh syndrome. A homolog of SLC25A46 was identified in Drosophila and designated as dSLC25A46 (CG5755). We previously established mitochondrial disease model targeting of dSLC25A46, which causes locomotive dysfunction and morphological defects at neuromuscular junctions, such as reduced synaptic branch lengths and decreased numbers of boutons. The diverse symptoms of mitochondrial diseases carrying mutations in SLC25A46 may be associated with the dysregulation of some epigenetic regulators. To investigate the involvement of epigenetic regulators in mitochondrial diseases, we examined candidate epigenetic regulators that interact with human SLC25A46 by searching Gene Expression Omnibus (GEO). We discovered that HDAC1 binds to several SLC25A46 genomic regions in human cultured CD4 (+) cells, and attempted to prove this in an in vivo Drosophila model. By demonstrating that Rpd3, Drosophila HDAC1, regulates the histone H4K8 acetylation state in dSLC25A46 genomic regions, we confirmed that Rpd3 is a novel epigenetic regulator modifying the phenotypes observed with the mitochondrial disease model targeting of dSLC25A46. The functional reduction of Rpd3 rescued the deficient locomotive ability and aberrant morphology of motoneurons at presynaptic terminals induced by the dSLC25A46 knockdown. The present results suggest that the inhibition of HDAC1 suppresses the pathogenic processes that lead to the degeneration of motoneurons in mitochondrial diseases.

Neuron-specific knockdown of Drosophila HADHB induces a shortened lifespan, deficient locomotive ability, abnormal motor neuron terminal morphology and learning disability.

Mutations in the HADHB gene induce dysfunctions in the beta-oxidation of fatty acids and result in a MTP deficiency, which is characterized by clinical heterogeneity, such as cardiomyopathy and recurrent Leigh-like encephalopathy. In contrast, milder forms of HADHB mutations cause the later onset of progressive axonal peripheral neuropathy (approximately 50-80%) and myopathy with or without episodic myoglobinuria. The mechanisms linking neuronal defects in these diseases to the loss of HADHB function currently remain unclear. Drosophila has the CG4581 (dHADHB) gene as a single human HADHB homologue. We herein established pan-neuron-specific dHADHB knockdown flies and examined their phenotypes. The knockdown of dHADHB shortened the lifespan of flies, reduced locomotor ability and also limited learning abilities. These phenotypes were accompanied by an abnormal synapse morphology at neuromuscular junctions (NMJ) and reduction in both ATP and ROS levels in central nervous system (CNS). The Drosophila NMJ synapses are glutamatergic that is similar to those in the vertebrate CNS. The present results reveal a critical role for dHADHB in the morphogenesis and function of glutamatergic neurons including peripheral neurons. The dHADHB knockdown flies established herein provide a useful model for investigating the pathological mechanisms underlying neuropathies caused by a HADHB deficiency.

Investigating Developmental and Epileptic Encephalopathy Using Drosophila melanogaster.

Developmental and epileptic encephalopathies (DEEs) are the spectrum of severe epilepsies characterized by early-onset, refractory seizures occurring in the context of developmental regression or plateauing. Early infantile epileptic encephalopathy (EIEE) is one of the earliest forms of DEE, manifesting as frequent epileptic spasms and characteristic electroencephalogram findings in early infancy. In recent years, next-generation sequencing approaches have identified a number of monogenic determinants underlying DEE. In the case of EIEE, 85 genes have been registered in Online Mendelian Inheritance in Man as causative genes. Model organisms are indispensable tools for understanding the in vivo roles of the newly identified causative genes. In this review, we first present an overview of epilepsy and its genetic etiology, especially focusing on EIEE and then briefly summarize epilepsy research using animal and patient-derived induced pluripotent stem cell (iPSC) models. The Drosophila model, which is characterized by easy gene manipulation, a short generation time, low cost and fewer ethical restrictions when designing experiments, is optimal for understanding the genetics of DEE. We therefore highlight studies with Drosophila models for EIEE and discuss the future development of their practical use.

Correction: Ueoka, I., et al. Autism Spectrum Disorder-Related Syndromes: Modeling with Drosophila and Rodents. Int. J. Mol. Sci. 2019, 20, 4071.

The author wishes to make the following correction to this paper [...].

Bacopa monnieri (L.) Wettst. Extract Improves Memory Performance via Promotion of Neurogenesis in the Hippocampal Dentate Gyrus of Adolescent Mice.

Bacopa monnieri L. Wettst. (BM) is a botanical component of Ayurvedic medicines and of dietary supplements used worldwide for cognitive health and function. We previously reported that administration of BM alcoholic extract (BME) prevents trimethyltin (TMT)-induced cognitive deficits and hippocampal cell damage and promotes TMT-induced hippocampal neurogenesis. In this study, we demonstrate that administration of BME improves spatial working memory in adolescent (5-week- old) healthy mice but not adult (8-week-old) mice. Moreover, improved spatial working memory was retained even at 4 weeks after terminating 1-week treatment of adolescent mice. One-week BME treatment of adolescent mice significantly enhanced hippocampal BrdU incorporation and expression of genes involved in neurogenesis determined by RNAseq analysis. Cell death, as detected by histochemistry, appeared not to be significant. A significant increase in neurogenesis was observed in the dentate gyrus region 4 weeks after terminating 1-week treatment of adolescent mice with BME. Bacopaside I, an active component of BME, promoted the proliferation of neural progenitor cells in vitro in a concentration-dependent manner via the facilitation of the Akt and ERK1/2 signaling. These results suggest that BME enhances spatial working memory in healthy adolescent mice by promoting hippocampal neurogenesis and that the effects of BME are due, in significant amounts, to bacopaside I.

Mutations in COA7 cause spinocerebellar ataxia with axonal neuropathy.

Several genes related to mitochondrial functions have been identified as causative genes of neuropathy or ataxia. Cytochrome c oxidase assembly factor 7 (COA7) may have a role in assembling mitochondrial respiratory chain complexes that function in oxidative phosphorylation. Here we identified four unrelated patients with recessive mutations in COA7 among a Japanese case series of 1396 patients with Charcot-Marie-Tooth disease (CMT) or other inherited peripheral neuropathies, including complex forms of CMT. We also found that all four patients had characteristic neurological features of peripheral neuropathy and ataxia with cerebellar atrophy, and some patients showed leukoencephalopathy or spinal cord atrophy on MRI scans. Validated mutations were located at highly conserved residues among different species and segregated with the disease in each family. Nerve conduction studies showed axonal sensorimotor neuropathy. Sural nerve biopsies showed chronic axonal degeneration with a marked loss of large and medium myelinated fibres. An immunohistochemical assay with an anti-COA7 antibody in the sural nerve from the control patient showed the positive expression of COA7 in the cytoplasm of Schwann cells. We also observed mildly elevated serum creatine kinase levels in all patients and the presence of a few ragged-red fibres and some cytochrome c oxidase-negative fibres in a muscle biopsy obtained from one patient, which was suggestive of subclinical mitochondrial myopathy. Mitochondrial respiratory chain enzyme assay in skin fibroblasts from the three patients showed a definitive decrease in complex I or complex IV. Immunocytochemical analysis of subcellular localization in HeLa cells indicated that mutant COA7 proteins as well as wild-type COA7 were localized in mitochondria, which suggests that mutant COA7 does not affect the mitochondrial recruitment and may affect the stability or localization of COA7 interaction partners in the mitochondria. In addition, Drosophila COA7 (dCOA7) knockdown models showed rough eye phenotype, reduced lifespan, impaired locomotive ability and shortened synaptic branches of motor neurons. Our results suggest that loss-of-function COA7 mutation is responsible for the phenotype of the presented patients, and this new entity of disease would be referred to as spinocerebellar ataxia with axonal neuropathy type 3.

A new Drosophila model of Ubiquilin knockdown shows the effect of impaired proteostasis on locomotive and learning abilities.

Ubiquilin (UBQLN) plays a crucial role in cellular proteostasis through its involvement in the ubiquitin proteasome system and autophagy. Mutations in the UBQLN2 gene have been implicated in amyotrophic lateral sclerosis (ALS) and ALS with frontotemporal lobar dementia (ALS/FTLD). Previous studies reported a key role for UBQLN in Alzheimer's disease (AD); however, the mechanistic involvement of UBQLN in other neurodegenerative diseases remains unclear. The genome of Drosophila contains a single UBQLN homolog (dUbqn) that shows high similarity to UBQLN1 and UBQLN2; therefore, the fly is a useful model for characterizing the role of UBQLN in vivo in neurological disorders affecting locomotion and learning abilities. We herein performed a phenotypic and molecular characterization of diverse dUbqn RNAi lines. We found that the depletion of dUbqn induced the accumulation of polyubiquitinated proteins and caused morphological defects in various tissues. Our results showed that structural defects in larval neuromuscular junctions, abdominal neuromeres, and mushroom bodies correlated with limited abilities in locomotion, learning, and memory. These results contribute to our understanding of the impact of impaired proteostasis in neurodegenerative diseases and provide a useful Drosophila model for the development of promising therapies for ALS and FTLD.

Neuron-specific knockdown of Drosophila PDHB induces reduction of lifespan, deficient locomotive ability, abnormal morphology of motor neuron terminals and photoreceptor axon targeting.

Pyruvate dehydrogenase complex deficiency (PDCD) is a common primary cause of defects in mitochondrial function and also can lead to peripheral neuropathy. Pyruvate dehydrogenase E1 component subunit beta (PDHB) is a subunit of pyruvate dehydrogenase E1, which is a well-known component of PDC. In Drosophila melanogaster, the CG11876 (dPDHB) gene is a homolog of human PDHB. In this study, we established a Drosophila model with neuron-specific knockdown of dPDHB to investigate its role in neuropathy pathogenesis. Knockdown of dPDHB in pan-neurons induced locomotor defects in both larval and adult stages, which were consistent with abnormal morphology of the motor neuron terminals at neuromuscular junctions and mitochondrial fragmentation in brains. Moreover, neuron-specific knockdown of dPDHB also shortened the lifespan of adult flies. In addition, flies with knockdown of dPDHB manifested a rough eye phenotype and aberrant photoreceptor axon targeting. These results with the Drosophila model suggest the involvement of PDHB in peripheral neuropathy.

Hippo, Drosophila MST, is a novel modifier of motor neuron degeneration induced by knockdown of Caz, Drosophila FUS.

Mutations in the Fused in Sarcoma (FUS) gene have been identified in familial ALS in human. Drosophila contains a single ortholog of human FUS called Cabeza (Caz). We previously established Drosophila models of ALS targeted to Caz, which developed the locomotive dysfunction and caused anatomical defects in presynaptic terminals of motoneurons. Accumulating evidence suggests that ALS and cancer share defects in many cellular processes. The Hippo pathway was originally discovered in Drosophila and plays a role as a tumor suppressor in mammals. We aimed to determine whether Hippo pathway genes modify the ALS phenotype using Caz knockdown flies. We found a genetic link between Caz and Hippo (hpo), the Drosophila ortholog of human Mammalian sterile 20-like kinase (MST) 1 and 2. Loss-of-function mutations of hpo rescued Caz knockdown-induced eye- and neuron-specific defects. The decreased Caz levels in nuclei induced by Caz knockdown were also rescued by loss of function mutations of hpo. Moreover, hpo mRNA level was dramatically increased in Caz knockdown larvae, indicating that Caz negatively regulated hpo. Our results demonstrate that hpo, Drosophila MST, is a novel modifier of Drosophila FUS. Therapeutic targets that inhibit the function of MST could modify the pathogenic processes of ALS.