Leo Tsuda

An NRSF/REST‐like repressor downstream of Ebi/SMRTER/Su(H) regulates eye development in Drosophila

The corepressor complex that includes Ebi and SMRTER is a target of epidermal growth factor (EGF) and Notch signaling pathways and regulates Delta (Dl)‐mediated induction of support cells adjacent to photoreceptor neurons of the Drosophila eye. We describe a mechanism by which the Ebi/SMRTER corepressor complex maintains Dl expression. We identified a gene, charlatan (chn), which encodes a C2H2‐type zinc‐finger protein resembling human neuronal restricted silencing factor/repressor element RE‐1 silencing transcription factor (NRSF/REST). The Ebi/SMRTER corepressor complex represses chn transcription by competing with the activation complex that includes the Notch intracellular domain (NICD). Chn represses Dl expression and is critical for the initiation of eye development. Thus, under EGF signaling, double negative regulation mediated by the Ebi/SMRTER corepressor complex and an NRSF/REST‐like factor, Chn, maintains inductive activity in developing photoreceptor cells by promoting Dl expression.

Involvement of the Mitochondrial Protein Translocator Component Tim50 in Growth, Cell Proliferation and the Modulation of Respiration in Drosophila

Allelic mutants exhibiting growth defects in Drosophila were isolated. Molecular cloning identified the responsible gene as a budding yeast Tim50 ortholog, and thus it was named tiny tim 50 (ttm50). The weak allele (ttm50Gp99) produced small flies due to reduced cell size and number, and growth terminated at the larval stage in the strong alleles (ttm50IE1 and ttm50IE2). Twin-spot analysis showed fewer cells in ttm50Gp99 clones, whereas ttm50IE1 clones did not proliferate, suggesting that the gene has an essential cellular function. Tim50 is known to maintain mitochondrial membrane potential (MMP) while facilitating inner-membrane protein transport. We found that tagged Ttm50 also localized to mitochondria and that mitochondrial morphology and MMP were affected in mutants, indicating that mitochondrial dysfunction causes the developmental phenotype. Conversely, ttm50 overexpression increased MMP and apoptosis. Co-expression of p35 suppressed this apoptosis, resulting in cell overproliferation. Interestingly, ttm50 transcription was tissue specific, corresponding to elevated MMP in the larval midgut, which was decreased in the mutant. The correlation of ttm50 expression levels with differences in MMP match its proposed role in mitochondrial permeability barrier maintenance. Thus a mitochondrial protein translocase component can play active roles in regulating metabolic levels, possibly for modulation of physiological function or growth in development.

Ebi/AP-1 suppresses pro-apoptotic genes expression and permits long-term survival of Drosophila sensory neurons.

Sensory organs are constantly exposed to physical and chemical stresses that collectively threaten the survival of sensory neurons. Failure to protect stressed neurons leads to age-related loss of neurons and sensory dysfunction in organs in which the supply of new sensory neurons is limited, such as the human auditory system. Transducin β-like protein 1 (TBL1) is a candidate gene for ocular albinism with late-onset sensorineural deafness, a form of X-linked age-related hearing loss. TBL1 encodes an evolutionarily conserved F-box–like and WD40 repeats–containing subunit of the nuclear receptor co-repressor/silencing mediator for retinoid and thyroid hormone receptor and other transcriptional co-repressor complexes. Here we report that a Drosophila homologue of TBL1, Ebi, is required for maintenance of photoreceptor neurons. Loss of ebi function caused late-onset neuronal apoptosis in the retina and increased sensitivity to oxidative stress. Ebi formed a complex with activator protein 1 (AP-1) and was required for repression of Drosophila pro-apoptotic and anti-apoptotic genes expression. These results suggest that Ebi/AP-1 suppresses basal transcription levels of apoptotic genes and thereby protects sensory neurons from degeneration.

fat facets induces polyubiquitination of Imd and inhibits the innate immune response in Drosophila

The IMD pathway is one of the major regulators of the innate immune response in Drosophila. Although extensive analysis of the IMD pathway has been carried out, precise mechanisms for how each target gene of the pathway is down‐regulated remain to be clarified. Here, we carried out genetic screening and found that fat facets (faf), which encodes a deubiquitinating enzyme, inhibited the expression of the target genes of the IMD pathway. Overexpression of faf suppressed the infection‐induced expression of Diptericin and increased susceptibility to bacterial infection in flies, whereas faf loss‐of‐function mutants decreased susceptibility. Time course analysis revealed that specific subsets of the target genes of the IMD pathway were affected by faf. Biochemical analysis showed that Faf made a complex with Imd, and both Faf and Imd were polyubiquitinated when they were co‐overexpressed. Given that faf‐dependent Imd polyubiquitination did not seem to cause protein degradation of Imd, Faf might inhibit the IMD pathway by modulating the state of Imd ubiquitination and/or stability.

Robust specification of sensory neurons by dual functions of charlatan, a Drosophila NRSF/REST‐like repressor of extramacrochaetae and hairy

Sensory bristle formation in Drosophila is a well‐characterized system for studying sensory organ development at the molecular level. The master proneural genes of the achaete‐scute (ac‐sc) complex, which encode basic‐helix‐loop‐helix (bHLH) transcription factors, are necessary and sufficient for sensory bristle formation. charlatan (chn) was originally identified as a transcriptional activator of ac‐sc gene expression through interaction with its enhancer, an activity that promotes sensory bristle development. In contrast, Chn was also identified as a functional homologue of mammalian neuron‐restrictive silencing factor or RE1 silencing transcription factor (NRSF/REST), an important transcriptional repressor during vertebrate neurogenesis and stem cell development that acts through epigenetic gene silencing. Here, we report that Chn acts as a repressor of extramacrochaetae (emc) and hairy, molecules that inhibit ac‐sc expression. This double‐negative mechanism, together with direct activation via the achaete enhancer, increases expression of achaete and ensures robust development of sensory neurons. A mutation in the C‐terminal repressor motif of Chn, which causes Chn to lose its repression activity, converted Chn to an activator of emc and hairy, suggesting that Chn is a dual functional regulator of transcription. Because chn‐like sequences are found among arthropods, regulation of neuronal development by Chn‐like molecules may be widely conserved.

Nontoxic singlet oxygen generator as a therapeutic candidate for treating tauopathies

Methylene blue (MB) inhibits the aggregation of tau, a main constituent of neurofibrillary tangles. However, MB’s mode of action in vivo is not fully understood. MB treatment reduced the amount of sarkosyl-insoluble tau in Drosophila that express human wild-type tau. MB concurrently ameliorated the climbing deficits of transgenic tau flies to a limited extent and diminished the climbing activity of wild-type flies. MB also decreased the survival rate of wild-type flies. Based on its photosensitive efficacies, we surmised that singlet oxygen generated through MB under light might contribute to both the beneficial and toxic effects of MB in vivo. We identified rose bengal (RB) that suppressed tau accumulation and ameliorated the behavioral deficits to a lesser extent than MB. Unlike MB, RB did not reduce the survival rate of flies. Our findings indicate that singlet oxygen generators with little toxicity may be suitable drug candidates for treating tauopathies.

Regulatory system for the G1‐arrest during neuronal development in Drosophila

Neuronal network consists of many types of neuron and glial cells. This diversity is guaranteed by the constant cell proliferation of neuronal stem cells following stop cell cycle re‐entry, which leads to differentiation during development. Neuronal differentiation occurs mainly at the specific cell cycle phase, the G1 phase. Therefore, cell cycle exit at the G1 phase is quite an important issue in understanding the process of neuronal cell development. Recent studies have revealed that aberrant S phase re‐entry from the G1 phase often links cellular survival. In this review we discuss the different types of G1 arrest on the process of neuronal development in Drosophila. We also describe the issue that aberrant S phase entry often causes apoptosis, and the same mechanism might contribute to sensory organ defects, such as deafness.

Ebi alleviates excessive growth signaling through multiple epigenetic functions in Drosophila.

As multicellular organisms develop, many cells permanently stop dividing and undergo terminal differentiation. The G1 phase of the cell cycle is thought to be the critical decision point for differentiation. Many growth factors, such as epidermal growth factor, are involved in regulating the G1 to S phase transition, and aberrant activation of growth factor signaling is one of the critical causes of tumor formation. Therefore, each cell must have proper mechanisms to suppress inappropriate/excessive activation of growth factor signaling, but the underlying molecular mechanisms remain undefined. Here, we found that ebi, a Drosophila homologue of genes encoding transducin‐β‐like 1 and transducin‐β‐like 1‐related protein, mitigated excess growth stimulation by taking advantage of its distinct epigenetic functions. Ebi acted as a corepressor of transcription by forming a complex with retinoblastoma family protein (RBF), a Drosophila homologue of retinoblastoma, and regulating the expression of specific target genes of the Rbf/E2F pathway. Furthermore, ebi also sustained expression of certain genes, including Rbf, encoding factors that inhibit progression out of G1. Our genetic studies suggest that the antagonistic function of ebi against the Polycomb group silencing complex plays a role in the G1/S phase transition.

Cellular Defense and Sensory Cell Survival Require Distinct Functions of ebi in Drosophila

The innate immune response and stress-induced apoptosis are well-established signaling pathways related to cellular defense. NF-κB and AP-1 are redox-sensitive transcription factors that play important roles in those pathways. Here we show that Ebi, a Drosophila homolog of the mammalian co-repressor molecule transducin β-like 1 (TBL1), variously regulates the expression of specific genes that are targets of redox-sensitive transcription factors. In response to different stimuli, Ebi activated gene expression to support the acute immune response in fat bodies, whereas Ebi repressed genes that are involved in apoptosis in photoreceptor cells. Thus, Ebi seems to act as a regulatory switch for genes that are activated or repressed in response to different external stimuli. Our results offer clear in vivo evidence that the Ebi-containing co-repressor complex acts in a distinct manner to regulate transcription that is required for modulating the output of various processes during Drosophila development.

Induction of ganglioside synthesis in Drosophila brain accelerates assembly of amyloid β protein

The assembly and deposition of amyloid β protein (Aβ) is a fundamental event during the early stages of Alzheimer’s disease (AD) and cerebral amyloid angiopathy. A growing body of evidence indicates that gangliosides form a pathological platform for the generation of ganglioside-bound Aβ, which facilitates the assembly of soluble Aβs; however, the molecular mechanisms underlying the binding of Aβ to gangliosides in the brain remain unclear due to the lack of an in vivo system that may address this issue. In insects, including the fruit fly Drosophila melanogaster, gangliosides are not intrinsically present at a detectable level. We herein demonstrate that ganglioside expression is inducible in Drosophila via the expression of transgenes of ganglioside synthesis enzymes and the feeding of exogenous sialic acid, and also that the induction of ganglioside synthesis significantly accelerates Aβ assembly in vivo. Our results support the hypothesis that gangliosides are responsible for Aβ assembly in vivo and also provide an opportunity to develop a valuable model for basic research as well as a therapeutic strategy for AD.