A splicing variant of Charlatan, a Drosophila REST-like molecule, preferentially localizes to axons.

Neuron-restrictive silencing factor (NRSF), also known as RE-1 silencing transcription factor (REST), has pivotal functions in many neuron-specific genes. Previous studies revealed that neuron-specific alternative splicing (AS) of REST produces divergent forms of REST variants and provides regulatory complexity in the nervous system. However, the biological significance of these variants in the regulation of neuronal activities remains to be clarified. Here, we revealed that Charlatan (Chn), a Drosophila REST-like molecule, is also regulated by neuron-specific AS. Neuron-specific AS produced six divergent variants of Chn proteins, one of which preferentially localized to axons. A small sequence of this variant was especially important for the axonal localization. Our data suggest that some variants have roles beyond the transcriptional regulation of neuronal activities.

Induction of ganglioside synthesis in Drosophila brain accelerates assembly of amyloid ß protein.

The assembly and deposition of amyloid ß protein (Aß) 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 Aß, which facilitates the assembly of soluble Aßs; however, the molecular mechanisms underlying the binding of Aß 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 Aß assembly in vivo. Our results support the hypothesis that gangliosides are responsible for Aß assembly in vivo and also provide an opportunity to develop a valuable model for basic research as well as a therapeutic strategy for AD.

Pyroglutamate-amyloid-ß peptide expression in Drosophila leads to caspase-dependent and endoplasmic reticulum stress-related progressive neurodegeneration.

Alzheimer's disease (AD) is the most common neurodegenerative disorder among the elderly. During the progression of AD, massive neuronal degeneration occurs in the late stage of the disease; however, the molecular mechanisms responsible for this neuronal loss remain unknown. AßpE3-42 (an N-terminal-truncated amyloid-ß peptide that begins with pyroglutamate at the third position) is produced during late-stage AD. It also aggregates more rapidly in vitro and exhibits greater toxicity in neurons than full-length Aß1-42. In the present study, we established a Drosophila melanogaster model that expresses Aß3-42E3Q, which effectively produces AßpE3-42, and investigated the function of AßpE3-42 using the photoreceptor neurons of Drosophila. AßpE3-42 induced caspase-dependent apoptosis and caused progressive degeneration in photoreceptor neurons. Mutations in ER stress response genes or the administration of an inhibitor of the ER stress response markedly suppressed the degeneration phenotype, suggesting that the ER stress response plays an important role in neurodegeneration caused by AßpE3-42. We also confirmed that human Tau-dependent apoptotic induction was strongly enhanced by AßpE3-42. Thus, AßpE3-42 expression system in the fly may be a promising new tool for studying late-onset neurodegeneration in AD.

Expression of amyloid-ß in mouse cochlear hair cells causes an early-onset auditory defect in high-frequency sound perception.

Increasing evidence indicates that defects in the sensory system are highly correlated with age-related neurodegenerative diseases, including Alzheimer's disease (AD). This raises the possibility that sensory cells possess some commonalities with neurons and may provide a tool for studying AD. The sensory system, especially the auditory system, has the advantage that depression in function over time can easily be measured with electrophysiological methods. To establish a new mouse AD model that takes advantage of this benefit, we produced transgenic mice expressing amyloid-ß (Aß), a causative element for AD, in their auditory hair cells. Electrophysiological assessment indicated that these mice had hearing impairment, specifically in high-frequency sound perception (>32 kHz), at 4 months after birth. Furthermore, loss of hair cells in the basal region of the cochlea, which is known to be associated with age-related hearing loss, appeared to be involved in this hearing defect. Interestingly, overexpression of human microtubule-associated protein tau, another factor in AD development, synergistically enhanced the Aß-induced hearing defects. These results suggest that our new system reflects some, if not all, aspects of AD progression and, therefore, could complement the traditional AD mouse model to monitor Aß-induced neuronal dysfunction quantitatively over time.

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.

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-ß-like 1 and transducin-ß-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.

Tetrahydrocurcumin extends life span and inhibits the oxidative stress response by regulating the FOXO forkhead transcription factor.

The O-type forkhead domain transcription factor (FOXO) is involved in many biological processes such as aging, the oxidative stress response, and growth regulation. FOXO activity is tightly controlled within cells. In particular, growth factor signaling pathways and the oxidative stress response can both stimulate nuclear translocation of this transcription factor. Here, we show that tetrahydrocurcumin (THC), a curcumin metabolite, regulates the oxidative stress response and aging via FOXO. In NIH3T3 cells, THC induced nuclear accumulation of FOXO4, a member of the FOXO family of transcription factors, by inhibiting phosphorylation of protein kinase B (PKB)/Akt. In Drosophila melanogaster, THC attenuated the oxidative stress response, an effect that was blocked in a foxo mutant background. THC also extended the life span of Drosophila under normal conditions, and loss of either foxo or Sir2 activity eliminated this effect. Based on these results, THC may regulate the aging process via an evolutionarily conserved signaling pathway that includes both foxo and Sir2.

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.

Negative modulation of bone morphogenetic protein signaling by Dullard during wing vein formation in Drosophila.

Studies in Xenopus have shown that the C-terminal domain phosphatase-like domain (CPD) phosphatase Dullard is essential for proper neural development via inhibition of bone morphogenetic protein (BMP) signaling receptors. In contrast, the orthologous budding yeast Nem1 and human Dullard have been shown to dephosphorylate the phosphatidate phosphatases yeast Smp2/Pah1 and human Lipin, and the relationship between phospholipid metabolism and BMP signaling remain unsolved. Here we report evidence that the Dullard-Lipin phosphatase cascade in Drosophila can regulate BMP signaling, most likely by affecting the function of the nuclear envelope. Manipulating expression levels of either the Drosophila Dullard gene, d-dullard (ddd) or the Lipin gene, DmLpin affected wing vein formation in a manner suggesting a negative effect on BMP signaling. Furthermore, both genes exhibit genetic interaction with BMP signaling pathway components, and can affect the levels of phosphorylated-Mothers against dpp (p-Mad). Although changing ddd expression levels did not have an obvious effect on overall nuclear envelope morphology as has been shown for yeast nem1, the nuclear import machinery components Importin-ß and RanGAP were mislocalized and membrane lipid staining was altered in cells overexpressing ddd. Considering the known genetic interaction between Nup84 complex nucleoporins and nem1 in yeast, and the recently reported requirement for components from the orthologous nucleoporin complex in the nuclear translocation of Drosophila Mad (Chen & Xu 2010), it is likely that the role of Drosophila Dullard in regulating membrane lipid homeostasis is conserved and is critical for normal BMP signaling.

Mi-2 chromatin remodeling factor functions in sensory organ development through proneural gene repression in Drosophila.

Mi-2, the central component of the nucleosome remodeling and histone deacetylation (NuRD) complex, is known as an SNF2-type ATP-dependent nucleosome remodeling factor. No morphological mutant phenotype of Drosophila Mi-2 (dMi-2) had been reported previously; however, we found that rare escapers develop into adult flies showing an extra bristle phenotype. The dMi-2 enhanced the phenotype of ac(Hw49c), which is a dominant gain-of-function allele of achaete (ac) and produces extra bristles. Consistent with these observations, the ac-expressing proneural clusters were expanded, and extra sensory organ precursors (SOP) were formed in the dMi-2 mutant wing discs. Immunostaining of polytene chromosomes showed that dMi-2 binds to the ac locus, and dMi-2 and acetylated hisotones distribute on polytene chromosomes in a mutually exclusive manner. The chromatin immunoprecipitation assay of the wing imaginal disc also demonstrated a binding of dMi-2 on the ac locus. These results suggest that the Drosophila Mi-2/NuRD complex functions in neuronal differentiation through the repression of proneural gene expression by chromatin remodeling and histone deacetylation.