Loss of neurexin-1 in Drosophila melanogaster results in altered energy metabolism and increased seizure susceptibility.

Although autism is typically characterized by differences in language, social interaction and restrictive, repetitive behaviors, it is becoming more well known in the field that alterations in energy metabolism and mitochondrial function are comorbid disorders in autism. The synaptic cell adhesion molecule, neurexin-1 (NRXN1), has previously been implicated in autism, and here we show that in Drosophila melanogaster, the homologue of NRXN1, called Nrx-1, regulates energy metabolism and nutrient homeostasis. First, we show that Nrx-1-null flies exhibit decreased resistance to nutrient deprivation and heat stress compared to controls. Additionally, Nrx-1 mutants exhibit a significantly altered metabolic profile characterized by decreased lipid and carbohydrate stores. Nrx-1-null Drosophila also exhibit diminished levels of nicotinamide adenine dinucleotide (NAD+), an important coenzyme in major energy metabolism pathways. Moreover, loss of Nrx-1 resulted in striking abnormalities in mitochondrial morphology in the flight muscle of Nrx-1-null Drosophila and impaired flight ability in these flies. Further, following a mechanical shock Nrx-1-null flies exhibited seizure-like activity, a phenotype previously linked to defects in mitochondrial metabolism and a common symptom of patients with NRXN1 deletions. The current studies indicate a novel role for NRXN1 in the regulation of energy metabolism and uncover a clinically relevant seizure phenotype in Drosophila lacking Nrx-1.

Loss of Drosophila FMRP leads to alterations in energy metabolism and mitochondrial function.

Fragile X Syndrome (FXS), the most prevalent form of inherited intellectual disability and the foremost monogenetic cause of autism, is caused by loss of expression of the FMR1 gene . Here, we show that dfmr1 modulates the global metabolome in Drosophila. Despite our previous discovery of increased brain insulin signaling, our results indicate that dfmr1 mutants have reduced carbohydrate and lipid stores and are hypersensitive to starvation stress. The observed metabolic deficits cannot be explained by feeding behavior, as we report that dfmr1 mutants are hyperphagic. Rather, our data identify dfmr1 as a regulator of mitochondrial function. We demonstrate that under supersaturating conditions, dfmr1 mutant mitochondria have significantly increased maximum electron transport system (ETS) capacity. Moreover, electron micrographs of indirect flight muscle reveal striking morphological changes in the dfmr1 mutant mitochondria. Taken together, our results illustrate the importance of dfmr1 for proper maintenance of nutrient homeostasis and mitochondrial function.

Home-cage hypoactivity in mouse genetic models of autism spectrum disorder.

Genome-wide association and whole exome sequencing studies from Autism Spectrum Disorder (ASD) patient populations have implicated numerous risk factor genes whose mutation or deletion results in significantly increased incidence of ASD. Behavioral studies of monogenic mutant mouse models of ASD-associated genes have been useful for identifying aberrant neural circuitry. However, behavioral results often differ from lab to lab, and studies incorporating both males and females are often not performed despite the significant sex-bias of ASD. In this study, we sought to investigate the simple, passive behavior of home-cage activity monitoring across multiple 24-h days in four different monogenic mouse models of ASD: Shank3b-/-, Cntnap2-/-, Pcdh10+/-, and Fmr1 knockout mice. Relative to sex-matched wildtype (WT) littermates, we discovered significant home-cage hypoactivity, particularly in the dark (active) phase of the light/dark cycle, in male mice of all four ASD-associated transgenic models. For Cntnap2-/- and Pcdh10+/- mice, these activity alterations were sex-specific, as female mice did not exhibit home-cage activity differences relative to sex-matched WT controls. These home-cage hypoactivity alterations differ from activity findings previously reported using short-term activity measurements in a novel open field. Despite circadian problems reported in human ASD patients, none of the mouse models studied had alterations in free-running circadian period. Together, these findings highlight a shared phenotype across several monogenic mouse models of ASD, outline the importance of methodology on behavioral interpretation, and in some genetic lines parallel the male-enhanced phenotypic presentation observed in human ASDs.

Drosophila Nipped-B Mutants Model Cornelia de Lange Syndrome in Growth and Behavior.

Individuals with Cornelia de Lange Syndrome (CdLS) display diverse developmental deficits, including slow growth, multiple limb and organ abnormalities, and intellectual disabilities. Severely-affected individuals most often have dominant loss-of-function mutations in the Nipped-B-Like (NIPBL) gene, and milder cases often have missense or in-frame deletion mutations in genes encoding subunits of the cohesin complex. Cohesin mediates sister chromatid cohesion to facilitate accurate chromosome segregation, and NIPBL is required for cohesin to bind to chromosomes. Individuals with CdLS, however, do not display overt cohesion or segregation defects. Rather, studies in human cells and model organisms indicate that modest decreases in NIPBL and cohesin activity alter the transcription of many genes that regulate growth and development. Sister chromatid cohesion factors, including the Nipped-B ortholog of NIPBL, are also critical for gene expression and development in Drosophila melanogaster. Here we describe how a modest reduction in Nipped-B activity alters growth and neurological function in Drosophila. These studies reveal that Nipped-B heterozygous mutant Drosophila show reduced growth, learning, and memory, and altered circadian rhythms. Importantly, the growth deficits are not caused by changes in systemic growth controls, but reductions in cell number and size attributable in part to reduced expression of myc (diminutive) and other growth control genes. The learning, memory and circadian deficits are accompanied by morphological abnormalities in brain structure. These studies confirm that Drosophila Nipped-B mutants provide a useful model for understanding CdLS, and provide new insights into the origins of birth defects.

PDE-4 inhibition rescues aberrant synaptic plasticity in Drosophila and mouse models of fragile X syndrome.

Fragile X syndrome (FXS) is the leading cause of both intellectual disability and autism resulting from a single gene mutation. Previously, we characterized cognitive impairments and brain structural defects in a Drosophila model of FXS and demonstrated that these impairments were rescued by treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium. A well-documented biochemical defect observed in fly and mouse FXS models and FXS patients is low cAMP levels. cAMP levels can be regulated by mGluR signaling. Herein, we demonstrate PDE-4 inhibition as a therapeutic strategy to ameliorate memory impairments and brain structural defects in the Drosophila model of fragile X. Furthermore, we examine the effects of PDE-4 inhibition by pharmacologic treatment in the fragile X mouse model. We demonstrate that acute inhibition of PDE-4 by pharmacologic treatment in hippocampal slices rescues the enhanced mGluR-dependent LTD phenotype observed in FXS mice. Additionally, we find that chronic treatment of FXS model mice, in adulthood, also restores the level of mGluR-dependent LTD to that observed in wild-type animals. Translating the findings of successful pharmacologic intervention from the Drosophila model into the mouse model of FXS is an important advance, in that this identifies and validates PDE-4 inhibition as potential therapeutic intervention for the treatment of individuals afflicted with FXS.

Disruption of CHTF18 causes defective meiotic recombination in male mice.

CHTF18 (chromosome transmission fidelity factor 18) is an evolutionarily conserved subunit of the Replication Factor C-like complex, CTF18-RLC. CHTF18 is necessary for the faithful passage of chromosomes from one daughter cell to the next during mitosis in yeast, and it is crucial for germline development in the fruitfly. Previously, we showed that mouse Chtf18 is expressed throughout the germline, suggesting a role for CHTF18 in mammalian gametogenesis. To determine the role of CHTF18 in mammalian germ cell development, we derived mice carrying null and conditional mutations in the Chtf18 gene. Chtf18-null males exhibit 5-fold decreased sperm concentrations compared to wild-type controls, resulting in subfertility. Loss of Chtf18 results in impaired spermatogenesis; spermatogenic cells display abnormal morphology, and the stereotypical arrangement of cells within seminiferous tubules is perturbed. Meiotic recombination is defective and homologous chromosomes separate prematurely during prophase I. Repair of DNA double-strand breaks is delayed and incomplete; both RAD51 and γH2AX persist in prophase I. In addition, MLH1 foci are decreased in pachynema. These findings demonstrate essential roles for CHTF18 in mammalian spermatogenesis and meiosis, and suggest that CHTF18 may function during the double-strand break repair pathway to promote the formation of crossovers.

PGC-1α integrates insulin signaling with mitochondrial physiology and behavior in a Drosophila model of Fragile X Syndrome.

Fragile X Syndrome (FXS) is the most prevalent monogenetic form of intellectual disability and autism. Recently, dysregulation of insulin signaling (IS) and aberrations in mitochondrial function have emerged as robust, evolutionarily conserved components of FXS pathophysiology. However, the mechanisms by which altered IS and mitochondrial dysfunction impact behavior in the context of FXS remain elusive. Here, we show that normalization of IS improves mitochondrial volume and function in flies that lack expression of dfmr1, the Drosophila homolog of the causal gene of FXS in humans. Further, we demonstrate that dysregulation of IS underlies diminished expression of the mitochondrial master regulator PGC-1α/Spargel in dfmr1 mutant flies. These results are behaviorally relevant, as we show that pan-neuronal augmentation of PGC-1α/Spargel improves circadian behavior in dfmr1 mutants. Notably, we also show that modulation of PGC-1α/Spargel expression in wild-type flies phenocopies the dfmr1 mutant circadian defect. Taken together, the results presented herein provide a mechanistic link between mitochondrial function and circadian behavior both in FXS pathogenesis as well as more broadly at the interface between metabolism and behavioral output.

Do metabolic deficits contribute to sleep disruption in monogenic intellectual disability syndromes?

Intellectual disability is defined as limitations in cognitive and adaptive behavior that often arise during development. Disordered sleep is common in intellectual disability and, given the importance of sleep for cognitive function, it may contribute to other behavioral phenotypes. Animal models of intellectual disability, in particular of monogenic intellectual disability syndromes (MIDS), recapitulate many disease phenotypes and have been invaluable for linking some of these phenotypes to specific molecular pathways. An emerging feature of MIDS, in both animal models and humans, is the prevalence of metabolic abnormalities, which could be relevant for behavior. Focusing on specific MIDS that have been molecularly characterized, we review sleep, circadian, and metabolic phenotypes in animal models and humans and propose that altered metabolic state contributes to the abnormal sleep/circadian phenotypes in MIDS.

Using Drosophila as a tool to identify pharmacological therapies for fragile X syndrome.

Despite obvious differences such as the ability to fly, the fruit fly Drosophila melanogaster is similar to humans at many different levels of complexity. Studies of development, cell growth and division, metabolism and even cognition, have borne out these similarities. For example, Drosophila bearing mutations in the fly gene homologue of the known human disease fragile X are affected in fundamentally similar ways as affected humans. The ramification of this degree of similarity is that Drosophila, as a model organism, is a rich resource for learning about human cells, development and even human cognition and behavior. Drosophila has a short generation time of ten days, is cheap to propagate and maintain and has a vast array of genetic tools available to it; making Drosophila an extremely attractive organism for the study of human disease. Here, we summarize research from our lab and others using Drosophila to understand the human neurological disease, called fragile X. We focus on the Drosophila model of fragile X, its characterization, and use as a tool to identify potential drugs for the treatment of fragile X. Several clinical trials are in progress now that were motivated by this research.

Arginine methylation of Aubergine mediates Tudor binding and germ plasm localization.

Piwi proteins such as Drosophila Aubergine (Aub) and mouse Miwi are essential for germline development and for primordial germ cell (PGC) specification. They bind piRNAs and contain symmetrically dimethylated arginines (sDMAs), catalyzed by dPRMT5. PGC specification in Drosophila requires maternal inheritance of cytoplasmic factors, including Aub, dPRMT5, and Tudor (Tud), that are concentrated in the germ plasm at the posterior end of the oocyte. Here we show that Miwi binds to Tdrd6 and Aub binds to Tudor, in an sDMA-dependent manner, demonstrating that binding of sDMA-modified Piwi proteins with Tudor-domain proteins is an evolutionarily conserved interaction in germ cells. We report that in Drosophila tud(1) mutants, the piRNA pathway is intact and most transposons are not de-repressed. However, the localization of Aub in the germ plasm is severely reduced. These findings indicate that germ plasm assembly requires sDMA modification of Aub by dPRMT5, which, in turn, is required for binding to Tudor. Our study also suggests that the function of the piRNA pathway in PGC specification may be independent of its role in transposon control.

Using Drosophila as a tool to identify Pharmacological Therapies for Fragile X Syndrome.

Despite obvious differences such as the ability to fly, the fruit fly Drosophila melanogaster is similar to humans at many different levels of complexity. Studies of development, cell growth and division, metabolism, and even cognition, have borne out these similarities. For example, Drosophila bearing mutations in the fly gene homologue of the known human disease Fragile X, are affected in fundamentally similar ways as affected humans. The ramification of this degree of similarity is that Drosophila, as a model organism, is a rich resource for learning about human cells, development and even human cognition and behavior. Drosophila has a short generation time of ten days, is cheap to propagate and maintain and has a vast array of genetic tools available to it; making Drosophila an extremely attractive organism for the study of human disease. Here, we summarize research from our lab and others using Drosophila to understand the human neurological disease, called Fragile X. We focus on the Drosophila model of fragile X, its characterization, and use as a tool to identify potential drugs for the treatment of Fragile X. Several clinical trials are in progress now that were motivated by this research.

Pharmacological and genetic reversal of age-dependent cognitive deficits attributable to decreased presenilin function.

Alzheimer's disease (AD) is the leading cause of cognitive loss and neurodegeneration in the developed world. Although its genetic and environmental causes are not generally known, familial forms of the disease (FAD) are attributable to mutations in a single copy of the Presenilin (PS) and amyloid precursor protein genes. The dominant inheritance pattern of FAD indicates that it may be attributable to gain or change of function mutations. Studies of FAD-linked forms of presenilin (psn) in model organisms, however, indicate that they are loss of function, leading to the possibility that a reduction in PS activity might contribute to FAD and that proper psn levels are important for maintaining normal cognition throughout life. To explore this issue further, we have tested the effect of reducing psn activity during aging in Drosophila melanogaster males. We have found that flies in which the dosage of psn function is reduced by 50% display age-onset impairments in learning and memory. Treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium during the aging process prevented the onset of these deficits, and treatment of aged flies reversed the age-dependent deficits. Genetic reduction of Drosophila metabotropic glutamate receptor (DmGluRA), the inositol trisphosphate receptor (InsP(3)R), or inositol polyphosphate 1-phosphatase also prevented these age-onset cognitive deficits. These findings suggest that reduced psn activity may contribute to the age-onset cognitive loss observed with FAD. They also indicate that enhanced mGluR signaling and calcium release regulated by InsP(3)R as underlying causes of the age-dependent cognitive phenotypes observed when psn activity is reduced.

Arginine methylation of vasa protein is conserved across phyla.

Recent studies have uncovered an unexpected relationship between factors that are essential for germline development in Drosophila melanogaster: the arginine protein methyltransferase 5 (dPRMT5/Csul/Dart5) and its cofactor Valois, methylate the Piwi family protein Aub, enabling it to bind Tudor. The RNA helicase Vasa is another essential protein in germline development. Here, we report that mouse (mouse Vasa homolog), Xenopus laevis, and D. melanogaster Vasa proteins contain both symmetrical and asymmetrical dimethylarginines. We find that dPRMT5 is required for the production of sDMAs of Vasa in vivo. Furthermore, we find that the mouse Vasa homolog associates with Tudor domain-containing proteins, Tdrd1 and Tdrd6, as well as the Piwi proteins, Mili and Miwi. Arginine methylation is thus emerging as a conserved and pivotal post-translational modification of proteins that is essential for germline development.

Mitochondrial dynamics: Shaping and remodeling an organelle network.

Mitochondria form networks that continually remodel and adapt to carry out their cellular function. The mitochondrial network is remodeled through changes in mitochondrial morphology, number, and distribution within the cell. Mitochondrial dynamics depend directly on fission, fusion, shape transition, and transport or tethering along the cytoskeleton. Over the past several years, many of the mechanisms underlying these processes have been uncovered. It has become clear that each process is precisely and contextually regulated within the cell. Here, we discuss the mechanisms regulating each aspect of mitochondrial dynamics, which together shape the network as a whole.

Mitochondrial adaptor TRAK2 activates and functionally links opposing kinesin and dynein motors.

Mitochondria are transported along microtubules by opposing kinesin and dynein motors. Kinesin-1 and dynein-dynactin are linked to mitochondria by TRAK proteins, but it is unclear how TRAKs coordinate these motors. We used single-molecule imaging of cell lysates to show that TRAK2 robustly activates kinesin-1 for transport toward the microtubule plus-end. TRAK2 is also a novel dynein activating adaptor that utilizes a conserved coiled-coil motif to interact with dynein to promote motility toward the microtubule minus-end. However, dynein-mediated TRAK2 transport is minimal unless the dynein-binding protein LIS1 is present at a sufficient level. Using co-immunoprecipitation and co-localization experiments, we demonstrate that TRAK2 forms a complex containing both kinesin-1 and dynein-dynactin. These motors are functionally linked by TRAK2 as knockdown of either kinesin-1 or dynein-dynactin reduces the initiation of TRAK2 transport toward either microtubule end. We propose that TRAK2 coordinates kinesin-1 and dynein-dynactin as an interdependent motor complex, providing integrated control of opposing motors for the proper transport of mitochondria.

The Drosophila fragile X protein functions as a negative regulator in the orb autoregulatory pathway.

Translational regulation of maternal mRNAs in distinct temporal and spatial patterns underlies many key decisions in developing eggs and embryos. In Drosophila, Orb is responsible for mediating the translational activation of mRNAs localized within the developing oocyte. Orb is a germline-specific RNA binding protein and is one of the founding members of the CPEB family of translational regulators. Here we show that Orb associates with the Drosophila Fragile X Mental Retardation (dFMR1) protein as part of a ribonucleoprotein complex that controls the localized translation of mRNAs in developing egg chambers. One of the key orb regulatory targets is orb mRNA, and this autoregulatory activity is critical for ensuring that Orb protein is expressed at high levels in the oocyte. We show that dFMR1 functions as a negative regulator in the orb autoregulatory circuit, downregulating orb mRNA translation.

Fragile X protein functions with lgl and the par complex in flies and mice.

Fragile X syndrome, the most common form of inherited mental retardation, is caused by loss of function for the Fragile X Mental Retardation 1 gene (FMR1). FMR1 protein (FMRP) has specific mRNA targets and is thought to be involved in their transport to subsynaptic sites as well as translation regulation. We report a saturating genetic screen of the Drosophila autosomal genome to identify functional partners of dFmr1. We recovered 19 mutations in the tumor suppressor lethal (2) giant larvae (dlgl) gene and 90 mutations at other loci. dlgl encodes a cytoskeletal protein involved in cellular polarity and cytoplasmic transport and is regulated by the PAR complex through phosphorylation. We provide direct evidence for a Fmrp/Lgl/mRNA complex, which functions in neural development in flies and is developmentally regulated in mice. Our data suggest that Lgl may regulate Fmrp/mRNA sorting, transport, and anchoring via the PAR complex.

Age-dependent cognitive impairment in a Drosophila fragile X model and its pharmacological rescue.

Fragile X syndrome afflicts 1 in 2,500 individuals and is the leading heritable cause of mental retardation worldwide. The overriding clinical manifestation of this disease is mild to severe cognitive impairment. Age-dependent cognitive decline has been identified in Fragile X patients, although it has not been fully characterized nor examined in animal models. A Drosophila model of this disease has been shown to display phenotypes bearing similarity to Fragile X symptoms. Most notably, we previously identified naive courtship and memory deficits in young adults with this model that appear to be due to enhanced metabotropic glutamate receptor (mGluR) signaling. Herein we have examined age-related cognitive decline in the Drosophila Fragile X model and found an age-dependent loss of learning during training. We demonstrate that treatment with mGluR antagonists or lithium can prevent this age-dependent cognitive impairment. We also show that treatment with mGluR antagonists or lithium during development alone displays differential efficacy in its ability to rescue naive courtship, learning during training and memory in aged flies. Furthermore, we show that continuous treatment during aging effectively rescues all of these phenotypes. These results indicate that the Drosophila model recapitulates the age-dependent cognitive decline observed in humans. This places Fragile X in a category with several other diseases that result in age-dependent cognitive decline. This demonstrates a role for the Drosophila Fragile X Mental Retardation Protein (dFMR1) in neuronal physiology with regard to cognition during the aging process. Our results indicate that misregulation of mGluR activity may be causative of this age onset decline and strengthens the possibility that mGluR antagonists and lithium may be potential pharmacologic compounds for counteracting several Fragile X symptoms.

Pharmacological rescue of synaptic plasticity, courtship behavior, and mushroom body defects in a Drosophila model of fragile X syndrome.

Fragile X syndrome is a leading heritable cause of mental retardation that results from the loss of FMR1 gene function. A Drosophila model for Fragile X syndrome, based on the loss of dfmr1 activity, exhibits phenotypes that bear similarity to Fragile X-related symptoms. Herein, we demonstrate that treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium can rescue courtship and mushroom body defects observed in these flies. Furthermore, we demonstrate that dfmr1 mutants display cognitive deficits in experience-dependent modification of courtship behavior, and treatment with mGluR antagonists or lithium restores these memory defects. These findings implicate enhanced mGluR signaling as the underlying cause of the cognitive, as well as some of the behavioral and neuronal, phenotypes observed in the Drosophila Fragile X model. They also raise the possibility that compounds having similar effects on metabotropic glutamate receptors may ameliorate cognitive and behavioral defects observed in Fragile X patients.

Drosophila lacking dfmr1 activity show defects in circadian output and fail to maintain courtship interest.

Fragile X mental retardation is a prominent genetic disorder caused by the lack of the FMR1 gene product, a known RNA binding protein. Specific physiologic pathways regulated by FMR1 function have yet to be identified. Adult dfmr1 (also called dfxr) mutant flies display arrhythmic circadian activity and have erratic patterns of locomotor activity, whereas overexpression of dFMR1 leads to a lengthened period. dfmr1 mutant males also display reduced courtship activity which appears to result from their inability to maintain courtship interest. Molecular analysis fails to reveal any defects in the expression of clock components; however, the CREB output is affected. Morphological analysis of neurons required for normal circadian behavior reveals subtle abnormalities, suggesting that defects in axonal pathfinding or synapse formation may cause the observed behavioral defects.