The CHD Protein Kismet Restricts the Synaptic Localization of Cell Adhesion Molecules at the Drosophila Neuromuscular Junction.

The appropriate expression and localization of cell surface cell adhesion molecules must be tightly regulated for optimal synaptic growth and function. How neuronal plasma membrane proteins, including cell adhesion molecules, cycle between early endosomes and the plasma membrane is poorly understood. Here we show that the Drosophila homolog of the chromatin remodeling enzymes CHD7 and CHD8, Kismet, represses the synaptic levels of several cell adhesion molecules. Neuroligins 1 and 3 and the integrins αPS2 and ßPS are increased at kismet mutant synapses but Kismet only directly regulates transcription of neuroligin 2. Kismet may therefore regulate synaptic CAMs indirectly by activating transcription of gene products that promote intracellular vesicle trafficking including endophilin B (endoB) and/or rab11. Knock down of EndoB in all tissues or neurons increases synaptic FasII while knock down of EndoB in kis mutants does not produce an additive increase in FasII. In contrast, neuronal expression of Rab11, which is deficient in kis mutants, leads to a further increase in synaptic FasII in kis mutants. These data support the hypothesis that Kis influences the synaptic localization of FasII by promoting intracellular vesicle trafficking through the early endosome.

Association of the Protein-Quality-Control Protein Ubiquilin-1 With Alzheimer's Disease Both in vitro and in vivo.

Alzheimer's disease (AD) belongs to a class of diseases characterized by progressive accumulation and aggregation of pathogenic proteins, particularly Aß proteins. Genetic analysis has identified UBQLN1 as an AD candidate gene. Ubiquilin-1 levels reduce with AD progression, suggesting a potential loss-of-function mechanism. The ubiquilin-1 protein is involved in protein quality control (PQC), which plays essential roles in cellular growth and normal cell function. Ubiquilin-1 regulates γ-secretase by increasing endoproteolysis of PS1, a key γ-secretase component. Presently, the effects of ubiquilin-1 on cellular physiology as well as Aß-related events require further investigation. Here, we investigated the effects of ubiquilin-1 on cellular growth and viability in association with APP (amyloid-ß protein precursor), APP processing-related ß-secretase (BACE1, BACE) and γ-secretase using cell and animal-based models. We showed that loss-of-function in Drosophila ubqn suppresses human APP and human BACE phenotypes in wing veins and altered cell number and tissue compartment size in the wing. Additionally, we performed cell-based studies and showed that silencing UBQLN1 reduced cell viability and increased caspase-3 activity. Overexpression of UBQLN1 significantly reduced Aß levels. Furthermore, pharmacological inhibition of γ-secretase increased ubiquilin-1 protein levels, suggesting a mechanism that regulates ubiquilin-1 levels which may associate with reduced Aß reduction by inhibiting γ-secretase. Collectively, our results support not only a loss-of-function mechanism of ubiquilin-1 in association with AD, but also support the significance of targeting ubiquilin-1-mediated PQC as a potential therapeutic strategy for AD.

Potential for Use of Erythritol as a Socially Transferrable Ingested Insecticide for Ants (Hymenoptera: Formicidae).

Ants are significant structural and agricultural pests, generating a need for human-safe and effective insecticides for ant control. Erythritol, a sugar alcohol used in many commercial food products, reduces survival in diverse insect taxa including fruit flies, termites, and mosquitos. Erythritol also decreases longevity in red imported fire ants; however, its effects on other ant species and its ability to be transferred to naïve colony members at toxic doses have not been explored. Here, we show that erythritol decreases survival in Tetramorium immigrans Santschi (Hymenoptera: Formicidae) in a concentration-dependent manner. Access to ad-libitum water reduced the toxic effects of erythritol, but worker mortality was still increased over controls with ad-lib water. Foraging T. immigrans workers transferred erythritol at lethal levels to nest mates that had not directly ingested erythritol. Similar patterns of mortality following erythritol ingestion were observed in Formica glacialis Wheeler (Hymenoptera: Formicidae), Camponotus subarbatus Emery (Hymenoptera: Formicidae), and Camponotus chromaiodes Bolton (Hymenoptera: Formicidae). These findings suggest that erythritol may be a highly effective insecticide for several genera of ants. Erythritol's potential effectiveness in social insect control is augmented by its spread at lethal levels through ant colonies via social transfer (trophallaxis) between workers.

Erythritol Ingestion Causes Concentration-Dependent Mortality in Eastern Subterranean Termites (Blattodea: Rhinotermitidae).

Damage from termite infestations is economically significant and control can be costly when requiring the widespread use of conventional insecticides. Erythritol, a polyalcohol sweetener that is safe for human consumption, causes increased mortality when ingested by some insects, indicating potential as a safe alternative insecticide. Here, we investigated the applicability of erythritol as a novel toxicant method of termite control. Eastern subterranean termites, Reticulitermes flavipes Kollar (Blattodea: Rhinotermitidae), were fed paper foods treated with increasing concentrations of erythritol and were assessed for mortality and bait consumption. Termite survival to 8 d (the duration of the experiment) significantly decreased as erythritol treatment concentration increased, indicating that the lethal effects of erythritol were concentration-dependent. Termites consumed erythritol-treated paper at all concentrations and did not display avoidance in choice assays, suggesting that erythritol may be practical for use as an ingestible bait. These results provide a basis for further development of erythritol as a safe alternative method of termite control.

Larval mannitol diets increase mortality, prolong development and decrease adult body sizes in fruit flies (Drosophila melanogaster).

The ability of polyols to disrupt holometabolous insect development has not been studied and identifying compounds in food that affect insect development can further our understanding of the pathways that connect growth rate, developmental timing and body size in insects. High-sugar diets prolong development and generate smaller adult body sizes in Drosophila melanogaster We tested for concentration-dependent effects on development when D. melanogaster larvae are fed mannitol, a polyalcohol sweetener. We also tested for amelioration of developmental effects if introduction to mannitol media is delayed past the third instar, as expected if there is a developmental sensitive-period for mannitol effects. Both male and female larvae had prolonged development and smaller adult body sizes when fed increasing concentrations of mannitol. Mannitol-induced increases in mortality were concentration dependent in 0 M to 0.8 M treatments with mortality effects beginning as early as 48 h post-hatching. Larval survival, pupariation and eclosion times were unaffected in 0.4 M mannitol treatments when larvae were first introduced to mannitol 72 h post-hatching (the beginning of the third instar); 72 h delay of 0.8 M mannitol introduction reduced the adverse mannitol effects. The developmental effects of a larval mannitol diet closely resemble those of high-sugar larval diets.This article has an associated First Person interview with the first author of the paper.

The CHD Protein, Kismet, is Important for the Recycling of Synaptic Vesicles during Endocytosis.

Chromatin remodeling proteins of the chromodomain DNA-binding protein family, CHD7 and CHD8, mediate early neurodevelopmental events including neural migration and differentiation. As such, mutations in either protein can lead to neurodevelopmental disorders. How chromatin remodeling proteins influence the activity of mature synapses, however, is relatively unexplored. A critical feature of mature neurons is well-regulated endocytosis, which is vital for synaptic function to recycle membrane and synaptic proteins enabling the continued release of synaptic vesicles. Here we show that Kismet, the Drosophila homolog of CHD7 and CHD8, regulates endocytosis. Kismet positively influenced transcript levels and bound to dap160 and endophilin B transcription start sites and promoters in whole nervous systems and influenced the synaptic localization of Dynamin/Shibire. In addition, kismet mutants exhibit reduced VGLUT, a synaptic vesicle marker, at stimulated but not resting synapses and reduced levels of synaptic Rab11. Endocytosis is restored at kismet mutant synapses by pharmacologically inhibiting the function of histone deacetyltransferases (HDACs). These data suggest that HDAC activity may oppose Kismet to promote synaptic vesicle endocytosis. A deeper understanding of how CHD proteins regulate the function of mature neurons will help better understand neurodevelopmental disorders.

Extramacrochaetae promotes branch and bouton number via the sequestration of daughterless in the cytoplasm of neurons.

The Class I basic helix-loop-helix (bHLH) proteins are highly conserved transcription factors that are ubiquitously expressed. A wealth of literature on Class I bHLH proteins has shown that these proteins must homodimerize or heterodimerize with tissue-specific HLH proteins in order to bind DNA at E-box consensus sequences to control tissue-specific transcription. Due to its ubiquitous expression, Class I bHLH proteins are also extensively regulated posttranslationally, mostly through dimerization. Previously, we reported that in addition to its role in promoting neurogenesis, the Class I bHLH protein daughterless also functions in mature neurons to restrict axon branching and synapse number. Here, we show that part of the molecular logic that specifies how daughterless functions in neurogenesis is also conserved in neurons. We show that the Type V HLH protein extramacrochaetae (Emc) binds to and represses daughterless function by sequestering daughterless to the cytoplasm. This work provides initial insights into the mechanisms underlying the function of daughterless and Emc in neurons while providing a novel understanding of how Emc functions to restrict daughterless activity within the cell.

Mannitol ingestion causes concentration-dependent, sex-biased mortality in adults of the fruit fly (Drosophila melanogaster).

Mannitol, a sugar alcohol used in commercial food products, has been previously shown to induce sex-biased mortality in female Drosophila melanogaster when ingested at a single concentration (1 M). We hypothesized that sex differences in energy needs, related to reproductive costs, contributed to the increased mortality we observed in females compared to males. To test this, we compared the longevity of actively mating and non-mating flies fed increasing concentrations of mannitol. We also asked whether mannitol-induced mortality was concentration-dependent for both males and females, and if mannitol's sex-biased effects were consistent across concentrations. Females and males both showed concentration-dependent increases in mortality, but female mortality was consistently higher at concentrations of 0.75 M and above. Additionally, fly longevity decreased further for both sexes when housed in mixed sex vials as compared to single sex vials. This suggests that the increased energetic demands of mating and reproduction for both sexes increased the ingestion of mannitol. Finally, larvae raised on mannitol produced expected adult sex ratios, suggesting that sex-biased mortality due to the ingestion of mannitol occurs only in adults. We conclude that sex and reproductive status differences in mannitol ingestion drive sex-biased differences in adult fly mortality.

The Drosophila Chromodomain Protein Kismet Activates Steroid Hormone Receptor Transcription to Govern Axon Pruning and Memory In Vivo.

Axon pruning is critical for sculpting precise neural circuits. Although axon pruning has been described in the literature for decades, relatively little is known about the molecular and cellular mechanisms that govern axon pruning in vivo. Here, we show that the epigenetic reader Kismet (Kis) is required for developmental axon pruning in Drosophila mushroom bodies. Kis binds to cis-regulatory elements of the steroid hormone receptor ecdysone receptor (ecr) gene and is necessary for activating expression of EcR-B1. Kis promotes the active H3K36 di- and tri-methylation and H4K16 acetylation histone marks at the ecr locus. We show that transgenic EcR-B1 can rescue axon pruning and memory defects associated with loss of Kis and that the histone deacetylase inhibitor SAHA also rescues these phenotypes. EcR protein abundance is the cell-autonomous, rate-limiting step required to initiate axon pruning in Drosophila, and our data suggest this step is under the epigenetic control of Kis.

Epigenetic crosstalk: Pharmacological inhibition of HDACs can rescue defective synaptic morphology and neurotransmission phenotypes associated with loss of the chromatin reader Kismet.

We are beginning to appreciate the complex mechanisms by which epigenetic proteins control chromatin dynamics to tightly regulate normal development. However, the interaction between these proteins, particularly in the context of neuronal function, remains poorly understood. Here, we demonstrate that the activity of histone deacetylases (HDACs) opposes that of a chromatin remodeling enzyme at the Drosophila neuromuscular junction (NMJ). Pharmacological inhibition of HDAC function reverses loss of function phenotypes associated with Kismet, a chromodomain helicase DNA-binding (CHD) protein. Inhibition of HDACs suppresses motor deficits, overgrowth of the NMJ, and defective neurotransmission associated with loss of Kismet. We hypothesize that Kismet and HDACs may converge on a similar set of target genes in the nervous system. Our results provide further understanding into the complex interactions between epigenetic protein function in vivo.

Non-Nutritive Polyol Sweeteners Differ in Insecticidal Activity When Ingested by Adult Drosophila melanogaster (Diptera: Drosophilidae).

Previous work showed the non-nutritive polyol sweetener Erythritol was toxic when ingested by Drosophila melanogaster (Meigen, 1930). This study assessed whether insect toxicity is a general property of polyols. Among tested compounds, toxicity was highest for erythritol. Adult fruit flies (D. melanogaster) fed erythritol had reduced longevity relative to controls. Other polyols did not reduce longevity; the only exception was a weaker but significant reduction of female (but not male) longevity when flies were fed D-mannitol. We conclude at least some non-nutritive polyols are not toxic to adult D. melanogaster when ingested for 17 days. The longer time course (relative to erythritol) and female specificity of D-mannitol mortality suggests different mechanisms for D-mannitol and erythritol toxicity to D. melanogaster.

Type I bHLH Proteins Daughterless and Tcf4 Restrict Neurite Branching and Synapse Formation by Repressing Neurexin in Postmitotic Neurons.

Proneural proteins of the class I/II basic-helix-loop-helix (bHLH) family are highly conserved transcription factors. Class I bHLH proteins are expressed in a broad number of tissues during development, whereas class II bHLH protein expression is more tissue restricted. Our understanding of the function of class I/II bHLH transcription factors in both invertebrate and vertebrate neurobiology is largely focused on their function as regulators of neurogenesis. Here, we show that the class I bHLH proteins Daughterless and Tcf4 are expressed in postmitotic neurons in Drosophila melanogaster and mice, respectively, where they function to restrict neurite branching and synapse formation. Our data indicate that Daughterless performs this function in part by restricting the expression of the cell adhesion molecule Neurexin. This suggests a role for these proteins outside of their established roles in neurogenesis.

The Drosophila ortholog of the Zc3h14 RNA binding protein acts within neurons to pattern axon projection in the developing brain.

The dNab2 polyadenosine RNA binding protein is the D. melanogaster ortholog of the vertebrate ZC3H14 protein, which is lost in a form of inherited intellectual disability (ID). Human ZC3H14 can rescue D. melanogaster dNab2 mutant phenotypes when expressed in all neurons of the developing nervous system, suggesting that dNab2/ZC3H14 performs well-conserved roles in neurons. However, the cellular and molecular requirements for dNab2/ZC3H14 in the developing nervous system have not been defined in any organism. Here we show that dNab2 is autonomously required within neurons to pattern axon projection from Kenyon neurons into the mushroom bodies, which are required for associative olfactory learning and memory in insects. Mushroom body axons lacking dNab2 project aberrantly across the brain midline and also show evidence of defective branching. Coupled with the prior finding that ZC3H14 is highly expressed in rodent hippocampal neurons, this requirement for dNab2 in mushroom body neurons suggests that dNab2/ZC3H14 has a conserved role in supporting axon projection and branching. Consistent with this idea, loss of dNab2 impairs short-term memory in a courtship conditioning assay. Taken together these results reveal a cell-autonomous requirement for the dNab2 RNA binding protein in mushroom body development and provide a window into potential neurodevelopmental functions of the human ZC3H14 protein.

Vertebrate Fidgetin Restrains Axonal Growth by Severing Labile Domains of Microtubules.

Individual microtubules (MTs) in the axon consist of a stable domain that is highly acetylated and a labile domain that is not. Traditional MT-severing proteins preferentially cut the MT in the stable domain. In Drosophila, fidgetin behaves in this fashion, with targeted knockdown resulting in neurons with a higher fraction of acetylated (stable) MT mass in their axons. Conversely, in a fidgetin knockout mouse, the fraction of MT mass that is acetylated is lower than in the control animal. When fidgetin is depleted from cultured rodent neurons, there is a 62% increase in axonal MT mass, all of which is labile. Concomitantly, there are more minor processes and a longer axon. Together with experimental data showing that vertebrate fidgetin targets unacetylated tubulin, these results indicate that vertebrate fidgetin (unlike its fly ortholog) regulates neuronal development by tamping back the expansion of the labile domains of MTs.

Kismet positively regulates glutamate receptor localization and synaptic transmission at the Drosophila neuromuscular junction.

The Drosophila neuromuscular junction (NMJ) is a glutamatergic synapse that is structurally and functionally similar to mammalian glutamatergic synapses. These synapses can, as a result of changes in activity, alter the strength of their connections via processes that require chromatin remodeling and changes in gene expression. The chromodomain helicase DNA binding (CHD) protein, Kismet (Kis), is expressed in both motor neuron nuclei and postsynaptic muscle nuclei of the Drosophila larvae. Here, we show that Kis is important for motor neuron synaptic morphology, the localization and clustering of postsynaptic glutamate receptors, larval motor behavior, and synaptic transmission. Our data suggest that Kis is part of the machinery that modulates the development and function of the NMJ. Kis is the homolog to human CHD7, which is mutated in CHARGE syndrome. Thus, our data suggest novel avenues of investigation for synaptic defects associated with CHARGE syndrome.

Cyclopamine modulates γ-secretase-mediated cleavage of amyloid precursor protein by altering its subcellular trafficking and lysosomal degradation.

Alzheimer disease (AD) is a progressive neurodegenerative disease leading to memory loss. Numerous lines of evidence suggest that amyloid-ß (Aß), a neurotoxic peptide, initiates a cascade that results in synaptic dysfunction, neuronal death, and eventually cognitive deficits. Aß is generated by the proteolytic processing of the amyloid precursor protein (APP), and alterations to this processing can result in Alzheimer disease. Using in vitro and in vivo models, we identified cyclopamine as a novel regulator of γ-secretase-mediated cleavage of APP. We demonstrate that cyclopamine decreases Aß generation by altering APP retrograde trafficking. Specifically, cyclopamine treatment reduced APP-C-terminal fragment (CTF) delivery to the trans-Golgi network where γ-secretase cleavage occurs. Instead, cyclopamine redirects APP-CTFs to the lysosome. These data demonstrate that cyclopamine treatment decreases γ-secretase-mediated cleavage of APP. In addition, cyclopamine treatment decreases the rate of APP-CTF degradation. Together, our data demonstrate that cyclopamine alters APP processing and Aß generation by inducing changes in APP subcellular trafficking and APP-CTF degradation.

Development and characterization of an aged onset model of Alzheimer's disease in Drosophila melanogaster.

The biggest risk factor for developing Alzheimer's disease (AD) is age. Depending on the age of onset, AD is clinically categorized into either the early-onset form (before age 60years old), or the late-onset form (after age 65years old), with the vast majority of AD diagnosed as late onset (LOAD). LOAD is a progressive neurodegenerative disorder that involves the accumulation of ß-amyloid (Aß) plaques and neurofibrillary tangles in the brains of elderly patients. Affected individuals often experience symptoms including memory loss, confusion, and behavioral changes. Though many animal models of AD exist, very few are capable of analyzing the effect of older age on AD pathology. In an attempt to better model LOAD, we developed a novel "aged AD" model using Drosophila melanogaster. In our model, we express low levels of the human AD proteins APP (amyloid precursor protein) and BACE1 (ß-site APP cleaving enzyme BACE) specifically in the fly's central nervous system. Advantages of our model include the onset of behavioral and neuropathological symptoms later in the fly's lifespan due to a gradual accrual of Aß within the central nervous system (CNS), making age the key factor in the behavioral and neuroanatomical phenotypes that we observe in this model.

Erythritol, a non-nutritive sugar alcohol sweetener and the main component of truvia®, is a palatable ingested insecticide.

Insecticides have a variety of commercial applications including urban pest control, agricultural use to increase crop yields, and prevention of proliferation of insect-borne diseases. Many pesticides in current use are synthetic molecules such as organochlorine and organophosphate compounds. Some synthetic insecticides suffer drawbacks including high production costs, concern over environmental sustainability, harmful effects on human health, targeting non-intended insect species, and the evolution of resistance among insect populations. Thus, there is a large worldwide need and demand for environmentally safe and effective insecticides. Here we show that Erythritol, a non-nutritive sugar alcohol, was toxic to the fruit fly Drosophila melanogaster. Ingested erythritol decreased fruit fly longevity in a dose-dependent manner, and erythritol was ingested by flies that had free access to control (sucrose) foods in choice and CAFE studies. Erythritol was US FDA approved in 2001 and is used as a food additive in the United States. Our results demonstrate, for the first time, that erythritol may be used as a novel, environmentally sustainable and human safe approach for insect pest control.

Synaptic abnormalities in a Drosophila model of Alzheimer's disease.

Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by memory loss and decreased synaptic function. Advances in transgenic animal models of AD have facilitated our understanding of this disorder, and have aided in the development, speed and efficiency of testing potential therapeutics. Recently, we have described the characterization of a novel model of AD in the fruit fly, Drosophila melanogaster, where we expressed the human AD-associated proteins APP and BACE in the central nervous system of the fly. Here we describe synaptic defects in the larval neuromuscular junction (NMJ) in this model. Our results indicate that expression of human APP and BACE at the larval NMJ leads to defective larval locomotion behavior, decreased presynaptic connections, altered mitochondrial localization in presynaptic motor neurons and decreased postsynaptic protein levels. Treating larvae expressing APP and BACE with the γ-secretase inhibitor L-685,458 suppresses the behavioral defects as well as the pre- and postsynaptic defects. We suggest that this model will be useful to assess and model the synaptic dysfunction normally associated with AD, and will also serve as a powerful in vivo tool for rapid testing of potential therapeutics for AD.

Pathogenic mutation of spastin has gain-of-function effects on microtubule dynamics.

Mutations to the SPG4 gene encoding the microtubule-severing protein spastin are the most common cause of hereditary spastic paraplegia. Haploinsufficiency, the prevalent model for the disease, cannot readily explain many of its key aspects, such as its adult onset or its specificity for the corticospinal tracts. Treatment strategies based solely on haploinsufficiency are therefore likely to fail. Toward developing effective therapies, here we investigated potential gain-of-function effects of mutant spastins. The full-length human spastin isoform called M1 or a slightly shorter isoform called M87, both carrying the same pathogenic mutation C448Y, were expressed in three model systems: primary rat cortical neurons, fibroblasts, and transgenic Drosophila. Although both isoforms had ill effects on motor function in transgenic flies and decreased neurite outgrowth from primary cortical neurons, mutant M1 was notably more toxic than mutant M87. The observed phenotypes did not result from dominant-negative effects of mutated spastins. Studies in cultured cells revealed that microtubules can be heavily decorated by mutant M1 but not mutant M87. Microtubule-bound mutant M1 decreased microtubule dynamics, whereas unbound M1 or M87 mutant spastins increased microtubule dynamics. The alterations in microtubule dynamics observed in the presence of mutated spastins are not consistent with haploinsufficiency and are better explained by a gain-of-function mechanism. Our results fortify a model wherein toxicity of mutant spastin proteins, especially mutant M1, contributes to axonal degeneration in the corticospinal tracts. Furthermore, our results provide details on the mechanism of the toxicity that may chart a course toward more effective treatment regimens.