The InteractiveFly: Drosophila as a Model for Human Diseases


Autism
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Autism spectrum disorder (ASD) is a debilitating human neurodevelopmental disorder exhibiting a complex array of symptoms such as learning deficits, hyperactivity, anxiety, and impaired social behavior and cognition. ASDs are currently estimated to occur at a frequency of 1% in the general population making them one of the most common neurodevelopmental disorders. Recent studies have demonstrated a strong genetic component with underlying genetic interactions modulating ASD characteristics. The ASD characteristics and the related phenotypes are vastly heterogeneous among individuals and frequently show incomplete penetrance. Genetic studies involving a variety of approaches including copy number variation, single gene mutations, single nucleotide polymorphism or SNP analyses, whole genome linkage, and gene association studies have implicated several hundred genes in ASD and current estimates suggest that genetic factors account for about 10-–20% of the reported ASD cases. In addition, a vast amount of data suggest that environmental and epigenetic factors contribute significantly to the etiology of the ASD phenotypes and are recognized as modulating factors (Wise, 2015 and references therein).

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Lewis, S. A., Bakhtiari, S., Forstrom, J., Bayat, A., Bilan, F., Le Guyader, G., Alkhunaizi, E., Vernon, H., Padilla-Lopez, S. R. and Kruer, M. C. (202). Abstract
AGAP1 is an Arf1 GAP that regulates endolysosomal trafficking. Damaging variants have been linked to cerebral palsy and autism. This study reports 3 new individuals with microdeletion variants in AGAP1. Affected individuals have intellectual disability (3/3), autism (3/3), dystonia with axial hypotonia (1/3), abnormalities of brain maturation (1/3), growth impairment (2/3) and facial dysmorphism (2/3). Mechanisms potentially underlying AGAP1 neurodevelopmental impairments were investigated using the Drosophila ortholog, CenG1a. Reduced axon terminal size, increased neuronal endosome abundance, and elevated autophagy were discovered at baseline. Given potential incomplete penetrance, gene-environment interactions were assessed. Basal elevation in phosphorylation of the integrated stress-response protein eIF2α and inability to further increase eIF2α-P with subsequent cytotoxic stressors was found. CenG1a -mutant flies have increased lethality from exposure to environmental insults. A model is proposed wherein disruption of AGAP1 function impairs endolysosomal trafficking, chronically activating the integrated stress response, and leaving AGAP1-deficient cells susceptible to a variety of second hit cytotoxic stressors. This model may have broader applicability beyond AGAP1 in instances where both genetic and environmental insults co-occur in individuals with neurodevelopmental disorders. This study has described 3 additional patients with heterozygous AGAP1 deletion variants and used a loss of function Drosophila model to identify defects in synaptic morphology with increased endosomal sequestration, chronic autophagy induction, basal activation of eIF2α-P, and sensitivity to environmental stressors.

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PDyson, A., Ryan, M., Garg, S., Evans, D. G. and Baines, R. A. (2022). Loss of NF1 in Drosophila Larvae Causes Tactile Hypersensitivity and Impaired Synaptic Transmission at the Neuromuscular Junction. J Neurosci 42(50): 9450-9472. PubMed ID: 36344265

Abstract
Autism spectrum disorder (ASD) is a neurodevelopmental condition in which the mechanisms underlying its core symptomatology are largely unknown. Studying animal models of monogenic syndromes associated with ASD, such as neurofibromatosis type 1 (NF1), can offer insights into its etiology. This study shows that loss of function of the Drosophila NF1 ortholog results in tactile hypersensitivity following brief mechanical stimulation in the larva (mixed sexes), paralleling the sensory abnormalities observed in individuals with ASD. Mutant larvae also exhibit synaptic transmission deficits at the glutamatergic neuromuscular junction (NMJ), with increased spontaneous but reduced evoked release. While the latter is homeostatically compensated for by a postsynaptic increase in input resistance, the former is consistent with neuronal hyperexcitability. Indeed, diminished expression of NF1 specifically within central cholinergic neurons induces both excessive neuronal firing and tactile hypersensitivity, suggesting the two may be linked. Furthermore, both impaired synaptic transmission and behavioral deficits are fully rescued via knock-down of Rasproteins. These findings validate NF1 (-/-) Drosophila as a tractable model of ASD with the potential to elucidate important pathophysiological mechanisms (Dyson, 2022).

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Palacios-Munoz, A., de Paula Moreira, D., Silva, V., Garcia, I. E., Aboitiz, F., Zarrei, M., Campos, G., Rennie, O., Howe, J. L., Anagnostou, E., Ambrozewic, P., Scherer, S. W., Passos-Bueno, M. R. and Ewer, J. (2022). Mutations in trpgamma, the homologue of TRPC6 autism candidate gene, causes autism-like behavioral deficits in Drosophila. Mol Psychiatry. PubMed ID: 35501408

Abstract
Autism Spectrum Disorder (ASD) is characterized by impaired social communication, restricted interests, and repetitive and stereotyped behaviors. The TRPC6 (transient receptor potential channel 6) represents an ASD candidate gene under an oligogenic/multifactorial model based on the initial description and cellular characterization of an individual with ASD bearing a de novo heterozygous mutation disrupting TRPC6, together with the enrichment of disruptive TRPC6 variants in ASD cases as compared to controls. This study performed a clinical re-evaluation of the initial non-verbal patient, and also present eight newly reported individuals ascertained for ASD and bearing predicted loss-of-function mutations in TRPC6. In order to understand the consequences of mutations in TRPC6 on nervous system function, the fruit fly, Drosophila melanogaster, was used to show that null mutations in transient receptor gamma (trpγ the fly gene most similar to TRPC6), cause a number of behavioral defects that mirror features seen in ASD patients, including deficits in social interactions (based on courtship behavior), impaired sleep homeostasis (without affecting the circadian control of sleep), hyperactivity in both young and old flies, and defects in learning and memory. Some defects, most notably in sleep, differed in severity between males and females and became normal with age. Interestingly, hyperforin, a TRPC6 agonist and the primary active component of the St. John's wort antidepressant, attenuated many of the deficits expressed by trpγ mutant flies. In summary, these results provide further evidence that the TRPC6 gene is a risk factor for ASD. In addition, they show that the behavioral defects caused by mutations in TRPC6 can be modeled in Drosophila, thereby establishing a paradigm to examine the impact of mutations in other candidate genes (Palacios-Munoz, 2022).

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Marcogliese, P. C., Deal, S. L., Andrews, J., ..., Marom, R., Wangler, M. F. and Yamamoto, S. (2022). Drosophila functional screening of de novo variants in autism uncovers damaging variants and facilitates discovery of rare neurodevelopmental diseases. Cell Rep 38(11): 110517. PubMed ID: 35294868

Abstract
Individuals with autism spectrum disorder (ASD) exhibit an increased burden of de novo mutations (DNMs) in a broadening range of genes. While these studies have implicated hundreds of genes in ASD pathogenesis, which DNMs cause functional consequences in vivo remains unclear.This study functionally tested the effects of ASD missense DNMs using Drosophila through "humanization" rescue and overexpression-based strategies. 79 ASD variants were examined in 74 genes identified in the Simons Simplex Collection and found 38% of them to cause functional alterations. Moreover, we identify GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in 13 previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes points to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.

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Genc, O., An, J. Y., Fetter, R. D., Kulik, Y., Zunino, G., Sanders, S. J. and Davis, G. W. (2020). Autism-like behaviors regulated by the serotonin receptor 5-HT2B in the dorsal fan-shaped body neurons of Drosophila melanogaster. Eur J Med Res 27(1): 203. PubMed ID: 36253869

Abstract

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in social interaction and repetitive stereotyped behaviors. Previous studies have reported an association of serotonin or 5-hydroxytryptamine (5-HT) with ASD, but the specific receptors and neurons by which serotonin modulates autistic behaviors have not been fully elucidated. RNAi-mediated knockdown was done to destroy the function of tryptophan hydroxylase (Trh) and all the five serotonin receptors. Given that ubiquitous knockdown of 5-HT2B showed significant defects in social behaviors, the CRISPR/Cas9 system was used to knock out the 5-HT2B receptor gene. Social space assays and grooming assays were the major methods used to understand the role of serotonin and related specific receptors in autism-like behaviors of Drosophila melanogaster. A close relationship was identified between serotonin and autism-like behaviors reflected by increased social space distance and high-frequency repetitive behavior in Drosophila. The binary expression system was further utilized to knock down all the five 5-HT receptors; the 5-HT2B receptor was observed to act as the main receptor responsible for the normal social space and repetitive behavior in Drosophila for the specific serotonin receptors underlying the regulation of these two behaviors. These data also showed that neurons in the dorsal fan-shaped body (dFB), which expressed 5-HT2B, were functionally essential for the social behaviors of Drosophila. Collectively, these data suggest that serotonin levels and the 5-HT2B receptor are closely related to the social interaction and repetitive behavior of Drosophila. Of all the 5 serotonin receptors, 5-HT2B receptor in dFB neurons is mainly responsible for serotonin-mediated regulation of autism-like behaviors (Cao, 2022).

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Genc, O., An, J. Y., Fetter, R. D., Kulik, Y., Zunino, G., Sanders, S. J. and Davis, G. W. (2020). Homeostatic plasticity fails at the intersection of autism-gene mutations and a novel class of common genetic modifiers, Elife 9. PubMed ID: 32609087

Abstract

This study identified a set of common phenotypic modifiers that interact with five independent autism gene orthologs [RIMS1 (Rim). CHD8 (Kismet). CHD2 (Chd1). WDFY3 (Blue cheese). ASH1L (ASH1)] causing a common failure of presynaptic homeostatic plasticity (PHP) in Drosophila. Heterozygous null mutations in each autism gene are demonstrated to have normal baseline neurotransmission and PHP. However, PHP is sensitized and rendered prone to failure. A subsequent electrophysiology-based genetic screen identifies the first known heterozygous mutations that commonly genetically interact with multiple ASD gene orthologs, causing PHP to fail. Two phenotypic modifiers identified in the screen, PDPK1 and PPP2R5D, are characterized. Finally, transcriptomic, ultrastructural and electrophysiological analyses define one mechanism by which PHP fails; an unexpected, maladaptive up-regulation of CREG, a conserved, neuronally expressed, stress response gene and a novel repressor of PHP. Thus, we define a novel genetic landscape by which diverse, unrelated autism risk genes may converge to commonly affect the robustness of synaptic transmission (Genc, 2020).

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Hahn, N., Geurten, B., Gurvich, A., Piepenbrock, D., Kästner, A., Zanini, D., Xing, .G, Xie, W., Göpfert, M.C., Ehrenreich, H. and Heinrich, R. (2013). Monogenic heritable autism gene neuroligin impacts Drosophila social behaviour. Behav Brain Res 252: 450-457. PubMed ID: PubMed ID: 23792025

Abstract
Autism spectrum disorders (ASDs) are characterized by deficits in social interactions, language development and repetitive behaviours. Multiple genes involved in the formation, specification and maintenance of synapses have been identified as risk factors for ASDs development. Among these are the neuroligin genes which code for postsynaptic cell adhesion molecules that induce the formation of presynapses, promote their maturation and modulate synaptic functions in both vertebrates and invertebrates. Neuroligin-deficient mice display abnormal social and vocal behaviours that resemble ASDs symptoms. This study shows that in the fly Drosophila melanogaster, deletion of the dnl2 gene, coding for one of four Neuroligin isoforms, impairs social interactions, alters acoustic communication signals, and affects the transition between different behaviours. dnl2-Deficient flies maintain larger distances to conspecifics and males perform less female-directed courtship and male-directed aggressive behaviours while the patterns of these behaviours and general locomotor activity are not different from wild type controls. Since tests for olfactory, visual and auditory perception reveal no sensory impairments of dnl2-deficient mutants, reduced social interactions seem to result from altered excitability in central nervous neuropils that initiate social behaviours. These results demonstrate that Neuroligins are phylogenetically conserved not only regarding their structure and direct function at the synapse but also concerning a shared implication in the regulation of social behaviours that dates back to common ancestors of humans and flies. In addition to previously described mouse models, Drosophila can thus be used to study the contribution of Neuroligins to synaptic function, social interactions and their implication in ASDs (Hahn, 2013).

Highlights

• In neuroligin2-deficient Drosophila melanogaster, social interactions are reduced and acoustic communication signals altered.
• Mutant flies have normal general activity and intact sensory perception.
• Neuroligin2-deficiency affects brain circuits that coordinate social behaviours.
• Mutant flies display the behavioural core symptoms of autism spectrum disorders.

Discussion
Postsynaptic Neuroligins contribute to the formation of bidirectional trans-synaptic signalling complexes that promote pre- and postsynaptic differentiation and regulate synaptic functions. It is believed that disturbance of Neuroligin/Neurexin signalling promotes ASDs phenotypes through impairment of synaptic development, synaptic transmission and imbalance of excitatory and inhibitory synapses in brain circuits implicated in the regulation of social behaviour. Both Neuroligins and Neurexins are evolutionary conserved and orthologs of various proteins that associate with them to form functional pre- and postsynaptic complexes in the mammalian nervous system have been identified in invertebrate species. Neuroligin/Neurexin trans-synaptic signalling has been studied in rats and mice, the nematode C. elegans, the mollusc A. californica, the honeybee A. mellifera and at the neuromuscular junction of D. melanogaster. Results of these studies suggest similar functions of Neuroligin/Neurexin signalling in the initiation, maturation, transmission and plasticity of vertebrate and invertebrate synapses (Hahn, 2013).

This study addressed the question whether impairment of Neuroligin/Neurexin trans-synaptic signalling impacts Drosophila's social behaviour and whether parallels to ASDs-like phenotypes reported in humans and mice are present. Neurexin-deficient males display severe locomotor defects and are not able to produce courtship songs, though unilateral wing extension is occasionally observed. A detailed analysis of neurexin-deficient flies was therefore not performed. In contrast, mutant lines deficient of the central nervously expressed dnl2 display no obvious motor impairments and are able to produce both types of courtship song patterns with the same accuracy as wild-type Drosophila males, suggesting that the central pattern generators for both pulse and sine song seem to function properly. Comparison of acoustic communication patterns of dnl2-deficient and wild-type flies reveals two differences, a reduced intensity of sine songs and shorter duration of inter pulse intervals. The reduced sine song intensity of dnl2KO17 mutants likely results from a weaker synaptic transmission at the neuromuscular junction causing reduced muscle activation and lower amplitudes of wing vibrations. The reduced inter pulse interval must result from altered synaptic properties in thoracic pulse song pattern generating circuits and/or differences in the intensity of their activation by descending brain neurons. The inter pulse interval is the critical parameter for species recognition and song attractiveness and deviation from a species-typical range should reduce Drosophila's courtship success and reproduction. Altered ultrasound vocalization has also been reported from mouse models for autism. While mice with impaired Neuroligin/Neurexin signalling display generally reduced calling rates, other mouse strains with ASDs-like phenotypes display abnormal spectral and temporal song patterns. Similar to the reduced rates of acoustic communication observed in mice with impaired Neuroligin/Neurexin trans-synaptic signalling, reduced courtship singing was also observed in this study with dnl2-deficient Drosophila (Hahn, 2013).

Distances between individuals of D. melanogaster have been studied in different behavioural settings which distinguish and emphasize different aspects including dispersal/exploration, intrinsic social space or group formation. It has been demonstrated that inter individual space may depend on the balance of attractive and repulsive sensory signals, previous social experience like isolation or mating and also on the type of arena and the number of flies used for the assay. The assay performed in this study excludes exploration/dispersal during the first minutes after introduction into the arena, reveals that wild type flies initially establish shorter distances to conspecifics that steadily increase between 5 and 18 min after being placed in the arena, suggesting a gradually decreasing tendency to engage in short range interactions with other individuals. In contrast, dnl2-deficient flies display this low tendency, reflected in large inter individual distances, already after 5 min in the arena without showing consistent changes with progressing time in the arena (Hahn, 2013).

Since dnl2-deficient flies are equally active as wild type flies, display no sensory and motoric impairments, and are able to produce the typical components of courtship and agonistic behaviours, their reduced social interactions and impaired transition between different behaviours (e.g. from courtship singing to subsequent behaviour; between walking and turning) appear to result from altered information processing in central nervous circuits responsible for the initiation and coordination of behaviour. Especially the mushroom bodies and the central complex, that express Dnl2, have been implicated in these functions in insects. Studies in the honeybee Apis mellifera have revealed expression of various neuroligins and neurexin in the mushroom bodies and regulation of brain neuroligin and neurexin expression by social interactions (comparison of isolated versus hive bees) during early adulthood. This suggests that Neuroligin/Neurexin signalling may also be involved in behavioural plasticity resulting from social experience, which modulates the age-related division of labour in honeybee colonies. A similar relevance of activity-dependent neuroligin- and/or neurexin-mediated synaptic plasticity in the mature brain has also been documented in mice and has been implicated in the aetiology of ASDs (Hahn, 2013).

Phenotypes very similar to those described in this study for dnl2-deficient Drosophila have also been reported in various mouse models for ASDs including neuroligin-deficient mice. Behavioural and cognitive impairments of these mice, as well as ASDs-symptoms in humans, have been linked to altered balance of excitation and inhibition in critical brain circuits that normally results from specific functions of different Neuroligin isoforms at excitatory and inhibitory synapses and altered functions of Neuroligin/Neurexin trans-synaptic signalling complexes. Similarly, differential effects on synaptic properties are also mediated by dnl1 and dnl2 at the Drosophila neuromuscular junction. Since dnl1 is expressed in muscle but not in the nervous system, it will be interesting to see, whether dnl3 or dnl4 act as “balancing counterparts” to dnl2 within the nervous system to establish proper excitation/inhibition ratios. Circuits that regulate the initiation and intensity of social behaviours including acoustic communication may be especially sensitive to disturbances of neuroligin-mediated synaptic fine tuning and this sensitivity seems to be shared by humans, mice and Drosophila and probably other social insects like honeybees. Thus, phylogenetical conservation of Neuroligins from flies to humans extends beyond their molecular structure and their direct function at the synapse and also includes their implication in the regulation of social behaviours (Hahn, 2013).

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Wise, A., Tenezaca, L., Fernandez, R.W., Schatoff, E., Flores, J., Ueda, A., Zhong, X., Wu, C.F., Simon, A.F. and Venkatesh, T. (2015). Drosophila mutants of the autism candidate gene neurobeachin (rugose) exhibit neuro-developmental disorders, aberrant synaptic properties, altered locomotion, and impaired adult social behavior and activity patterns. J Neurogenet 29: 135-143. PubMed ID: 26100104

Abstract
Autism spectrum disorder (ASD) is a neurodevelopmental disorder in humans characterized by complex behavioral deficits, including intellectual disability, impaired social interactions, and hyperactivity. ASD exhibits a strong genetic component with underlying multigene interactions. Candidate gene studies have shown that the neurobeachin (NBEA) gene is disrupted in human patients with idiopathic autism. The NBEA gene spans the common fragile site FRA 13A and encodes a signal scaffold protein. In mice, NBEA has been shown to be involved in the trafficking and function of a specific subset of synaptic vesicles. Rugose (rg) is the Drosophila homolog of the mammalian and human NBEA. Previous genetic and molecular analyses have shown that rg encodes an A kinase anchor protein (DAKAP 550), which interacts with components of the epidermal growth factor receptor or EGFR and Notch-mediated signaling pathways, facilitating cross talk between these and other pathways. This study presents functional data from studies on the larval neuromuscular junction that reveal abnormal synaptic architecture and physiology. In addition, adult rg loss-of-function mutants exhibit defective social interactions, impaired habituation, aberrant locomotion, and hyperactivity. These results demonstrate that Drosophila NBEA (rg) mutants exhibit phenotypic characteristics reminiscent of human ASD and thus could serve as a genetic model for studying ASDs (Wise, 2015).

Highlights

• rg mutants show changes in synaptic bouton number and morphology.
• Evoked responses and short-term plasticity are altered in rg mutants.
• Larval locomotion is abnormal in rg mutants.
• Adult rg mutants show altered habituation.
• Adult rg mutants exhibit hyperactivity.
• Increased social space with closest neighbor in rg mutants.

Discussion
Data presented in this study implicate Drosophila rg in synaptic development, physiology, and adult behavior. Studies on the larval NMJ show altered morphological features and physiological properties of the synapses. rg mutants show significant decreases in the number of boutons at the NMJ, altered bouton shapes, and no significant changes in the bouton size. Overall, there is a decrease in the number of boutons in all of the alleles tested. Electrophysiological studies on the larval neuromuscular junction show an increase in the size of individual EJPs, and synapses also show pair-pulse depression compared with facilitation in the CS control with repetitive stimuli. In addition, rg mutant larvae display aberrant locomotor behavior with decreases in peristaltic movement and speed, and alterations in posture. These findings are consistent with a functional role for rg at the synapse (Wise, 2015).

Rugose is the Drosophila homolog of the mammalian NBEA, a scaffolding protein implicated in neurotransmitter/endomembrane vesicle trafficking at the synapse. Other studies on NBEA lend further support to its functional role at the synapse. In mice, loss-of-function of NBEA protein completely blocks evoked synaptic transmission at neuromuscular junctions while nerve conduction, synaptic structure, and spontaneous neurotransmitter release remain normal. NBEA has also been implicated in vesicular traffic at the synapse and has been shown to be required for normal development of the synapses. Recent studies have shown that the NBEA gene is disrupted in individuals with ASD and the NBEA gene spans the common fragile site FRA 13A in humans. Individuals with fragile X syndrome and autism have reduced levels of cAMP. This has been shown to lead to a decrease in evoked synaptic potential, dendritic architecture, and actin “clumping” in areas near the post-synaptic membrane (Wise, 2015).

Results from this study are consistent with earlier studies on the effects of altered cAMP metabolism on synaptic plasticity in adult Drosophila and neurotransmission at the larval NMJ. The modulation of the cAMP signaling at the synapse by rg may be through its function as a signal scaffold for protein kinase A or A kinase anchoring protein (AKAP). Mammalian AKAPs have been shown to maintain post-synaptic scaffolds by simultaneously associating with other kinases and phosphatases. For example, AKAP79/150 has been shown to be targeted to dendritic spines by a binding motif in the N-terminus which complexes with phosphatidylinositol-4,5-bisphosphate (PIP2), F-actin, and actin-linked cadherin adhesion molecules. rg may work in a similar manner, which would allow for changes in the appearance of synaptic boutons. AKAPs are key mediators of cAMP as well as other signal transduction pathways. Presynaptically, AKAPs have been shown to regulate ion channel function, particularly that of Ca2+ channels, which are required for vesicle fusion. PKA and AKAPs together can increase the efficiency of these channels by 3-10 fold, allowing for greater current to flow. The molecular mechanism that directly links rg function to evoked synaptic transmission remains unclear. However, AKAPs have also been shown to directly interact with adenylate cyclase in neurons thereby regulating the amount of cAMP that is produced in the cell. This may provide a mechanism by which changes in presynaptic vesicular release lead to observed changes in transmission and plasticity (Wise, 2015).

In humans, disruption of the NBEA gene results in idiopathic autism and the autistic individuals typically display hyperactivity and have difficulty with social interactions. To look for similar adult behavior correlates in flies, in addition to examining rg effects in larvae, the consequences of rg mutation on the behavior of adult flies were studied. It was found that rg mutants are similarly both more active and are socially avoidant. This conclusion is based on outcomes of rg mutants’ behavior in assays of social signals. First, the ability of the flies to avoid the dSO left by agitated flies in the avoidance assay was tested. Next, flies’ response to others in social clustering, the measure of distances to their closest neighbor (their social space), in a stable undisturbed group was tested. Social space and social avoidance probably result from equilibrium between multiple attractive and repulsive cues, in addition to environmental factors. Analyzing both the response to attractive signals, as in individual space, and to repulsive signals, as in social avoidance, can help differentiate between different kinds of social deficits. Individuals who do not perform well in either assay would not recognize or care for social signals, and could be characterized as socially indifferent. However, individuals who would have a bigger individual space, but strong avoidance of stressed individuals would be efficient at recognizing social signals, and decide to avoid interactions; thus could be characterized as socially avoidant. It was found that despite proper olfaction, rg mutants unlike the CS control, tend to not avoid the stress odorant left by stressed flies. Instead, they settle further away from their neighbors. It is worth noting that the speed of flies in motion or their activity levels does not affect the distance at which flies choose to finally settle when they form immobile groups. Thus, the behavior data suggest that rg mutant flies are socially indifferent, since they are less responsive both to stressful social signals in the avoidance assay, and to their neighbor in a stable group (Wise, 2015).

The findings on several aspects of synaptic properties, from formation and development of synaptic structures to synaptic release, post-synaptic response amplitude, and behavioral output, suggest a functional role for rg at the synapse. These results are consistent with the working hypothesis that rg is important for targeting and/or sequestering various proteins of cAMP-PKA signaling pathways to specific areas in the neuron. In addition to this potential role at the synapse, it was found that rg functions in pathways involved in regulating behavior, both at the larval and adult stages, to modulate locomotion, activity levels, and response to social signals. The high degree of structural and functional similarity between rg and NBEA suggests an evolutionarily conserved functional role essential for synapse formation and transmission, in pathways of conserved function, making rg a good candidate gene for studies on autism (Wise, 2015).

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Doll, C. A., Vita, D. J. and Broadie, K. (2017). Fragile X mental retardation protein requirements in activity-dependent critical period neural circuit refinement. Curr Biol 27(15): 2318-2330.e2313. PubMed ID: 28756946

Abstract

Activity-dependent synaptic remodeling occurs during early-use critical periods, when naive juveniles experience sensory input. Fragile X mental retardation protein (FMRP) sculpts synaptic refinement in an activity sensor mechanism based on sensory cues, with FMRP loss causing the most common heritable autism spectrum disorder (ASD), fragile X syndrome (FXS). In the well-mapped Drosophila olfactory circuitry, projection neurons (PNs) relay peripheral sensory information to the central brain mushroom body (MB) learning/memory center. FMRP-null PNs reduce synaptic branching and enlarge boutons, with ultrastructural and synaptic reconstitution MB connectivity defects. Critical period activity modulation via odorant stimuli, optogenetics, and transgenic tetanus toxin neurotransmission block show that elevated PN activity phenocopies FMRP-null defects, whereas PN silencing causes opposing changes. FMRP-null PNs lose activity-dependent synaptic modulation, with impairments restricted to the critical period. It is concluded that FMRP is absolutely required for experience-dependent changes in synaptic connectivity during the developmental critical period of neural circuit optimization for sensory input (Doll, 2017).

Neural circuit remodeling during developmental critical periods requires reception of sensory experience (activity) and the responsive orchestration of synaptic refinement to optimize behavioral performance. FMRP is hypothesized to mediate these activity-dependent critical period processes in an activity sensor mechanism and as an activity-dependent translational regulator. To test these hypotheses, this study dissected FMRP requirements in the well-mapped Drosophila olfactory learning/memory circuit, focusing on projection neurons linking upstream sensory neurons to the downstream central brain mushroom body mediating learning acquisition and memory consolidation. Mushroom body KCs also associate sensory input with a valence signal from dopaminergic neurons, connecting sensory experience to the reward pathway. Null dfmr1 mutants exhibit deficits in olfactory learning and memory, KC architecture, projection neuron dendritic arborization, and activity-dependent calcium signaling. In the FXS condition, transiently altered synaptic connectivity between projection neurons and target KCs profoundly impacts establishment of specific associations between sensory input, learning/memory, and resultant behavioral output. It was predicted that the seemingly ephemeral changes have lasting impacts into maturity, when differences in synaptic architecture are minimal but strong behavior deficits persist. It is hypothesized that subtle differences in circuit connectivity, or consequent functional synaptic deficits arising from transient critical period defects, must be manifest in impairments in emergent circuit properties at maturity that result in persistent behavioral deficits (Doll, 2017).

Synaptic connectivity investigations show two primary defects in FMRP-deficient mPN2 neurons: (1) truncated synaptic branches in the posterior mushroom body calyx and (2) enlarged synaptic boutons on postsynaptic KCs. Importantly, both defects manifest only during the early-use critical period and are not detectably present at maturity, after FMRP expression has precipitously declined. Milder, persistent synaptic architecture defects are detected in some cases, dependent on the genetic background. Null dfmr1 mutant boutons also display a critical-period-restricted reduction in presynaptic active zone scaffold Brp (Drosophila ELKS protein) only during the critical period, showing that FMRP regulates a core organizing component of presynaptic maturation selectively during this transient time window. Using transgenic GFP reconstitution to test synapse connectivity, this study found that FMRP-deficient mPN2 neurons develop impaired synaptic partner interactions with reduced mPN2-KC contacts. GRASP synaptic defects likewise are restricted to the early-use critical period. Electron microscopy during the critical period reveals greatly enlarged synaptic boutons with reduced active zone density in dfmr1-null mutants compared to age-matched controls. These ultrastructural results are consistent with the light microscopy findings, revealing expanded synaptic bouton area coupled with reduced synaptic density during the critical period. Taken together, these combined approaches reveal compromised synaptic connectivity in the Drosophila disease model, consistent with defects in the mouse FXS model, which transiently occur only during the early-use critical period (Doll, 2017).

Next, activity-dependent FMRP roles were explored in the critical period. Critical period exposure to sensory olfactory experience causes dramatic changes in mPN2 mushroom body synaptic connectivity, reminiscent of odorant-induced critical period changes in antennal lobe synaptic glomeruli. Synaptic remodeling is FMRP dependent, and critical period activity phenocopies dfmr1-null defects. Induced changes are specific to the pyrrolidine-sensitive VL1-mPN2 glomerulus, as other odorants (i.e., ethyl acetate) do not alter mPN2 synapses. Importantly, olfactory experience at maturity has no effect on wild-type mPN2s but does cause minor changes in dfmr1-null mPN2s, which supports the 'shifted critical period' Autism spectrum disorder (ASD) hypothesis. FMRP and activity may function in parallel pathways, but the fact that FMRP is activity regulated and mediates activity-dependent processes strongly suggests a direct activity-dependent FMRP mechanism for critical period synaptic refinement. mPN2-targeted optogenetic stimulation during the critical period phenocopies FXS model synaptic defects, with reduced branching and enlarged synaptic boutons, reminiscent of defects in downstream KCs. Similar cell-autonomous optogenetic stimulation causes erroneous axonpathfinding and diminished axon outgrowth. Importantly, both sensory stimulation via peripheral odorant exposure and direct mPN2 stimulation via channelrhodopsin optogenetics phenocopy FXS model defects. All activity-dependent changes require FMRP and are tightly restricted to the early-use critical period. Together, these results support the FXS hyperexcitation theory and highlight a critical period deficit in the suppression of excitatory synapses (Doll, 2017).

In contrast to stimulation paradigms, cell-targeted halorhodopsinsuppression of neuronal activity causes increased mPN2 synaptic branching in the MB calyx. This result demonstrates bidirectional capacity for mPN2 to manifest activity-dependent changes in synaptic connectivity during the early-use critical period. This phenotype is comparable to the overgrown axonal projections that result from developmental application of the GABA antagonist picrotoxin, suggesting that activity normally limits synaptic connectivity. Surprisingly, hyperpolarization of wild-type mPN2 neurons also caused increased synaptic bouton size at maturity, albeit not during the critical period. It is therefore clear that neuronal hyperpolarization impacts synaptic connectivity and architecture in a distinct mechanism compared to excess excitation. However, it is not clear what role the FMRP activity sensor plays when neuronal activity is dampened. Indeed, it was surprising that halorhodopsin hyperpolarization influences dfmr1-null mPN2 synaptic bouton area, suggesting that neurons lacking FMRP retain some capacity to function in activity-dependent synaptic bouton refinement during critical period development. There is evidence that FXS disease model dysfunction can be alleviated through increased activation of the inhibitory neural circuitry: for example, pharmacological enhancement of GABAergic signaling is sufficient to rescue some FXS hyperexcitation and can rescue biochemical, morphological, and behavioral phenotypes in the Drosophila FXS disease model. Thus, excitation/inhibition balance appears important for sculpting synaptic circuit connectivity during the critical period (Doll, 2017).

The blockade of mPN2 neurotransmission by conditional, targeted expression of the tetanus neurotoxin (TNT) leads to striking synaptic overgrowth in wild-type neurons that represents an opposite extreme in comparison to dfmr1-null phenotypes. Suppressed circuit activity (via both halorhodopsin and tetanus toxin manipulations) may spur increased process exploration or connectivity with potential synaptic targets in the mushroom body calyx, further suggesting that reduced branching in FMRP-deficient mPN2 neurons may stem from excess excitation during critical period development. TNT neurotransmission blockade similarly causes aberrant competition for glomerular space during olfactory circuit targeting and enlarged downstream postsynaptic terminals within motor circuits. In dfmr1-null mutants, neurotransmission blockade has little impact on mPN2 presynaptic architecture, demonstrating yet another level of activity-dependent FMRP requirement. As tools are not yet available to assay mPN2 postsynaptic partners, no insight was gained into postsynaptic KC differentiation downstream of the TNT neurotransmission blockade. Planned future work to manipulate neuronal excitability and neurotransmission strength should provide more precise understanding of FMRP function in limiting excitatory synapse connectivity in the developing brain circuitry. The clear requirement for FMRP in activity-dependent synaptic refinement during the early-use critical period, evidence of temporally shifted critical periods in the FXS condition, and the promise of new paradigms to rebalance excitatory/inhibitory synaptic connectivity all hold tremendous future therapeutic potential for combatting the FXS disease state (Doll, 2017).

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Shilpa, O., Anupama, K. P., Antony, A. and Gurushankara, H. P. (2021). Lead (Pb)-induced oxidative stress mediates sex-specific autistic-like behaviour in Drosophila melanogaster. Mol Neurobiol. PubMed ID: 34528217

Abstract

Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder characterised by three main behavioural symptoms: abnormal social interaction, verbal and non-verbal communication impairments, and repetitive and restricted activities or interests. Even though the exact aetiology of ASD remains unknown, studies have shown a link between genetics and environmental pollutants. Heavy metal lead (Pb), the environmental pollutant, is associated with ASD. Pb may also exhibit sex-specific ASD behaviour, as has been demonstrated in the global human populations. Drosophila melanogaster as a model has been used in the present study to understand the involvement of Pb-induced oxidative stress in developing ASD behaviour. The larval feeding technique has been employed to administer different Pb concentrations (0.2-0.8 mM) to Oregon-R (ORR), superoxide dismutase (Sod), or catalase (Cat) antioxidants overexpressed or knockdown flies. Adult Drosophila (5-day old) were used for Pb content, biochemical, and behavioural analysis. Pb accumulated in the Drosophila brain induces oxidative stress and exhibited a human autistic-like behaviour such as reduced climbing, increased grooming, increased social spacing, and decreased learning and memory in a sex-specific manner. Pb-induced autistic-like behaviour was intensified in Sod or Cat-knockdown flies, whereas Sod or Cat-overexpressed flies overcome that behavioural alterations. These results unequivocally proved that Pb-induced oxidative stress causes ASD behaviour of humans in Drosophila. Thus, Drosophila is used as a model organism to analyse ASD-like human behaviour and underlines the importance of using antioxidant therapy in alleviating ASD symptoms in children (Shilpa, 2021).

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Matta, S. M., Moore, Z., Walker, F. R., Hill-Yardin, E. L. and Crack, P. J. (2020). An altered glial phenotype in the NL3(R451C) mouse model of autism. Sci Rep 10(1): 14492. PubMed ID: 32879325

Abstract

Autism Spectrum Disorder (ASD; autism) is a neurodevelopmental disorder characterised by deficits in social communication, and restricted and/or repetitive behaviours. Increasing evidence supports a role for dysregulated neuroinflammation in the brain with potential effects on synapse function. Characteristics of microglia and astrocytes were studied in the Neuroligin-3 (NL3(R451C); see Drosophila Nlg) mouse model of autism. Increased microglial density was observed in the dentate gyrus (DG) of NL3(R451C) mice without morphological differences. In contrast, WT and NL3(R451C) mice had similar astrocyte density but astrocyte branch length, the number of branch points, as well as cell radius and area were reduced in the DG of NL3(R451C) mice. Because retraction of astrocytic processes has been linked to altered synaptic transmission and dendrite formation, regional changes in pre- and postsynaptic protein expression were assessed in the cortex, striatum and cerebellum in NL3(R451C) mice. NL3(R451C) mice showed increased striatal postsynaptic density 95 (PSD-95; see Drosophila Discs large) protein levels and decreased cortical expression of synaptosomal-associated protein 25 (SNAP-25; see Drosophila Snap-25). These changes could contribute to dysregulated neurotransmission and cognition deficits previously reported in these mice (Matta, 2020).

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Vissers, L., Kalvakuri, S., ..., Bodmer, R. and de Brouwer, A. P. M. (2020). De Novo Variants in CNOT1, a Central Component of the CCR4-NOT Complex Involved in Gene Expression and RNA and Protein Stability, Cause Neurodevelopmental Delay. Am J Hum Genet 107(1): 164-172. PubMed ID: 32553196

Abstract

CNOT1 is a member of the CCR4-NOT complex, which is a master regulator, orchestrating gene expression, RNA deadenylation, and protein ubiquitination. This study reports on 39 individuals with heterozygous de novo CNOT1 variants, including missense, splice site, and nonsense variants, who present with a clinical spectrum of intellectual disability, motor delay, speech delay, seizures, hypotonia, and behavioral problems. To link CNOT1 dysfunction to the neurodevelopmental phenotype observed, variant-specific Drosophila models were generated that showed learning and memory defects upon CNOT1 knockdown. Introduction of human wild-type CNOT1 was able to rescue this phenotype, whereas mutants could not or only partially, supporting the hypothesis that CNOT1 impairment results in neurodevelopmental delay. Furthermore, the genetic interaction with autism-spectrum genes, such as ASH1L, DYRK1A, MED13, and SHANK3, was impaired in our Drosophila models. Molecular characterization of CNOT1 variants revealed normal CNOT1 expression levels, with both mutant and wild-type alleles expressed at similar levels. Analysis of protein-protein interactions with other members indicated that the CCR4-NOT complex remained intact. An integrated omics approach of patient-derived genomics and transcriptomics data suggested only minimal effects on endonucleolytic nonsense-mediated mRNA decay components, suggesting that de novo CNOT1 variants are likely haploinsufficient hypomorph or neomorph, rather than dominant negative. In summary, this study provides strong evidence that de novo CNOT1 variants cause neurodevelopmental delay with a wide range of additional co-morbidities. Whereas the underlying pathophysiological mechanism warrants further analysis, the data demonstrate an essential and central role of the CCR4-NOT complex in human brain development (Vissers, 2020).

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Hope, K. A., McGinn, A. and Reiter, L. T. (2019). A genome-wide enhancer/suppressor screen for Dube3a interacting genes in Drosophila melanogaster. Sci Rep 9(1): 2382. PubMed ID: 30787400

Abstract

The genetics underlying autism spectrum disorder (ASD) are complex. Approximately 3-5% of ASD cases arise from maternally inherited duplications of 15q11.2-q13.1, termed Duplication 15q syndrome (Dup15q). 15q11.2-q13.1 includes the gene UBE3A which is believed to underlie ASD observed in Dup15q syndrome. UBE3A is an E3 ubiquitin ligase that targets proteins for degradation and trafficking, so finding UBE3A substrates and interacting partners is critical to understanding Dup15q ASD. This study took an unbiased genetics approach to identify genes that genetically interact with Dube3a, the Drosophila melanogaster homolog of UBE3A. An enhancer/suppressor screen was conducted using a rough eye phenotype produced by Dube3a overexpression with GMR-GAL4. Using the DrosDel deficiency kit, 3 out of 346 deficiency lines were identified that enhanced rough eyes when crossed to two separate Dube3a overexpression lines, and subsequently IA2, GABA-B-R3, and lola were identified as single genes responsible for rough eye enhancement. Using the FlyLight GAL4 lines to express uas-Dube3a + uas-GFP in the endogenous lola pattern, an increase was observed in the GFP signal compared to uas-GFP alone, suggesting a transcriptional co-activation effect of Dube3a on the lola promoter region. These findings extend the role of Dube3a/UBE3A as a transcriptional co-activator, and reveal new Dube3a interacting genes (Hope, 2019).

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Fenckova, M., Blok, L. E. R., Asztalos, L., Goodman, D. P., Cizek, P., Singgih, E. L., Glennon, J. C., IntHout, J., Zweier, C., Eichler, E. E., von Reyn, C. R., Bernier, R. A., Asztalos, Z. and Schenck, A. (2019). Habituation learning is a widely affected mechanism in Drosophila models of intellectual disability and autism spectrum disorders. Biol Psychiatry 86(4): 294-305. PubMed ID: 31272685

Abstract

Although habituation is one of the most ancient and fundamental forms of learning, its regulators and its relevance for human disease are poorly understood. This study manipulated the orthologs of 286 genes implicated in intellectual disability (ID) with or without comorbid autism spectrum disorder (ASD) specifically in Drosophila neurons, and these models were tested in light-off jump habituation. Neuronal substrates underlying the identified habituation deficits were dissected and genotype-phenotype annotations, gene ontologies, and interaction networks were integrated to determine the clinical features and molecular processes that are associated with habituation deficits. >100 genes required for habituation learning were identified. For 93 of these genes, a role in habituation learning was previously unknown. These genes characterize ID disorders with macrocephaly and/or overgrowth and comorbid ASD. Moreover, individuals with ASD from the Simons Simplex Collection carrying damaging de novo mutations in these genes exhibit increased aberrant behaviors associated with inappropriate, stereotypic speech. At the molecular level, ID genes required for normal habituation are enriched in synaptic function and converge on Ras/mitogen-activated protein kinase (Ras/MAPK) signaling. Both increased Ras/MAPK signaling in gamma-aminobutyric acidergic (GABAergic) neurons and decreased Ras/MAPK signaling in cholinergic neurons specifically inhibit the adaptive habituation response. This work supports the relevance of habituation learning to ASD, identifies an unprecedented number of novel habituation players, supports an emerging role for inhibitory neurons in habituation, and reveals an opposing, circuit-level-based mechanism for Ras/MAPK signaling. These findings establish habituation as a possible, widely applicable functional readout and target for pharmacologic intervention in ID/ASD (Fenckova, 2019).

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Hope, K. A., Flatten, D., Cavitch, P., May, B., Sutcliffe, J. S., O'Donnell, J. and Reiter, L. T. (2019). The Drosophila gene Sulfateless modulates autism-like behaviors. Front Genet 10: 574. PubMed ID: 31316544

Abstract

Major challenges to identifying genes that contribute to autism spectrum disorder (ASD) risk include the availability of large ASD cohorts, the contribution of many genes overall, and small effect sizes attributable to common gene variants. An alternative approach is to use a model organism to detect alleles that impact ASD-relevant behaviors and ask whether homologous human genes infer ASD risk. This study used the Drosophila genetic reference panel (DGRP) as a tool to probe for perturbation in naturally occurring behaviors in Drosophila melanogaster that are analogous to three behavior domains: impaired social communication, social reciprocity and repetitive behaviors or restricted interests. Using 40 of the available DGRP lines, single nucleotide polymorphisms (SNPs) were identified in or near genes controlling these behavior domains, including ASD gene orthologs (neurexin 4 and neuroligin 2), an intellectual disability (ID) gene homolog (kirre), and a gene encoding a heparan sulfate (HS) modifying enzyme called sulfateless (sfl). SNPs in sfl were associated with all three ASD-like behaviors. Using RNAi knock-down of neuronal sfl expression, significant changes were observed in expressive and receptive communication during mating, decreased grooming behavior, and increased social spacing. These results suggest a role for HS proteoglycan synthesis and/or modification in normal social communication, repetitive behavior, and social interaction in flies. Finally, using the DGRP to directly identify genetic effects relevant to a neuropsychiatric disorder further demonstrates the utility of the Drosophila system in the discovery of genes relevant to human disease (Hope, 2019).

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Lodge, W., Zavortink, M., Golenkina, S., Froldi, F., Dark, C., Cheung, S., Parker, B. L., Blazev, R., Bakopoulos, D., Christie, E. L., Wimmer, V. C., Duckworth, B. C., Richardson, H. E. and Cheng, L. Y. (2021). Tumor-derived MMPs regulate cachexia in a Drosophila cancer model. Dev Cell. PubMed ID: 34473940

Abstract
Cachexia, the wasting syndrome commonly observed in advanced cancer patients, accounts for up to one-third of cancer-related mortalities. A Drosophila larval model of organ wasting was established whereby epithelial overgrowth in eye-antennal discs leads to wasting of the adipose tissue and muscles. The wasting is associated with fat-body remodeling and muscle detachment and is dependent on tumor-secreted matrix metalloproteinase 1 (Mmp1). Mmp1 can both modulate TGFβ signaling in the fat body and disrupt basement membrane (BM)/extracellular matrix (ECM) protein localization in both the fat body and the muscle. Inhibition of TGFβ signaling or Mmps in the fat body/muscle using a QF2-QUAS binary expression system rescues muscle wasting in the presence of tumor. Altogether, this study proposes that tumor-derived Mmps are central mediators of organ wasting in cancer cachexia.

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Liu, Y., Shen, L., Zhang, Y., Zhao, R., Liu, C., Luo, S., Chen, J., Xia, L., Li, T., Peng, Y. and Xia, K. (2021). Rare NRXN1 missense variants identified in autism interfered protein degradation and Drosophila sleeping. J Psychiatr Res 143: 113-122. PubMed ID: 34487988

Abstract
NRXN1 is involved in synaptogenesis and has been implicated in Autism spectrum disorders (ASDs). However, many rare inherited missense variants of NRXN1 have not been thoroughly evaluated. In this study, functional analyses in vitro and in Drosophila of three NRXN1 missense mutations, Y282H, L893V, and I1135V identified in ASD patients in a previous study were performed. The results showed these three mutations interfered protein degradation compared with NRXN1-WT protein. Expressing human NRXN1 in Drosophila could lead to abnormal circadian rhythm and sleep behavior, and three mutated proteins caused milder phenotypes, indicating the mutations may change the function of NRXN1 slightly. These findings highlight the functional role of rare NRXN1 missense variants identified in autism patients, and provide clues for a better understanding of the pathogenesis of abnormal circadian rhythm and sleep behavior of other organisms, including humans.

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Campbell, N. G., et al. (2019). Structural, functional, and behavioral insights of dopamine dysfunction revealed by a deletion in SLC6A3. Proc Natl Acad Sci U S A 116(9): 3853-3862. PubMed ID: 30755521

Abstract

The human dopamine (DA) transporter (hDAT) mediates clearance of DA. Genetic variants in hDAT have been associated with DA dysfunction, a complication associated with several brain disorders, including autism spectrum disorder (ASD). This study investigated the structural and behavioral bases of an ASD-associated in-frame deletion in hDAT at N336 (N336). The deletion promoted a previously unobserved conformation of the intracellular gate of the transporter, likely representing the rate-limiting step of the transport process. It is defined by a "half-open and inward-facing" state (HOIF) of the intracellular gate that is stabilized by a network of interactions conserved phylogenetically, as was demonstrated in hDAT by Rosetta molecular modeling and fine-grained simulations, as well as in its bacterial homolog leucine transporter by electron paramagnetic resonance analysis and X-ray crystallography. The stabilization of the HOIF state is associated both with DA dysfunctions demonstrated in isolated brains of Drosophila melanogaster expressing hDAT N336 and with abnormal behaviors observed at high-time resolution. These flies display increased fear, impaired social interactions, and locomotion traits that are associated with DA dysfunction and the HOIF state. Together, these results describe how a genetic variation causes DA dysfunction and abnormal behaviors by stabilizing a HOIF state of the transporter (Campbell, 2019).

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Park, S.M., Park, H.R. and Lee, J.H. (2017). MAPK3 at the autism-linked human 16p11.2 locus influences precise synaptic target selection at Drosophila larval neuromuscular junctions. Mol Cells [Epub ahead of print]. PubMed ID: 28196412

Abstract

Proper synaptic function in neural circuits requires precise pairings between correct pre- and post-synaptic partners. Errors in this process may underlie development of neuropsychiatric disorders, such as autism spectrum disorder (ASD). Development of ASD can be influenced by genetic factors, including copy number variations (CNVs). This study focused on a CNV occurring at the 16p11.2 locus in the human genome and investigated potential defects in synaptic connectivity caused by reduced activities of genes located in this region at Drosophila larval neuromuscular junctions, a well-established model synapse with stereotypic synaptic structures. A mutation of rolled, the Drosophila homolog of human mitogen-activated protein kinase 3 (MAPK3) at the 16p11.2 locus, causes ectopic innervation of axonal branches and their abnormal defasciculation. The specificity of these phenotypes was confirmed by expression of wild-type rolled in the mutant background. Albeit to a lesser extent, ectopic innervation patterns were also observed in mutants defective in Cdk2, Gaq, and Gp93, all of which are expected to interact with Rolled MAPK3. Further genetic analysis in double heterozygous combinations reveals a synergistic interaction between rolled and Gp93. In addition, results from RT-qPCR analyses indicate consistently reduced rolled mRNA levels in Cdk2, Gaq, and Gp93 mutants. Taken together, these data suggest a central role of MAPK3 in regulating the precise targeting of presynaptic axons to proper postsynaptic targets, a critical step that may be altered significantly in ASD (Park, 2017).

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Park, S.M., Littleton, J.T., Park, H.R. and Lee, J.H. (2016). Drosophila homolog of human KIF22 at the autism-linked 16p11.2 loci influences synaptic connectivity at larval neuromuscular junctions. Exp Neurobiol 25: 33-39. PubMed ID: 26924931

Abstract
Copy number variations at multiple chromosomal loci, including 16p11.2, have been implicated in the pathogenesis of autism spectrum disorder (ASD), a neurodevelopmental disease that affects 1~3% of children worldwide. This study investigated the roles of human genes at the 16p11.2 loci in synaptic development using Drosophila larval neuromuscular junctions (NMJ), a well-established model synapse with stereotypic innervation patterns. A preliminary genetic screen based on RNA interference was conducted in combination with the GAL4-UAS system, followed by mutational analyses. Data indicate that disruption of klp68D, a gene closely related to human KIF22, causes ectopic innervations of axon branches forming type III boutons in muscle 13, along with less frequent re-routing of other axon branches. In addition, mutations in klp64D, of which gene product forms Kinesin-2 complex with KLP68D, leads to similar targeting errors of type III axons. Mutant phenotypes are at least partially reproduced by knockdown of each gene via RNA interference. Taken together, these data suggest the roles of Kinesin-2 proteins, including KLP68D and KLP64D, in ensuring proper synaptic wiring (Park, 2016).

Highlights

• Aberrant synaptic targeting of presynaptic axons upon knockdown of Klp68D.
• Ectopic innervation of type III axons in muscle 13 in Klp68D mutants.
• Ectopic innervation patterns are reproduced by disruption in KLP64D, another Kinesin-2 complex protein assembled with KLP68D.

Discussion
Recent clinical studies on ASD have revealed gross alterations in the structure of nervous system. For instance, total brain size and the rate of neuronal proliferation in the prefrontal cortex are significantly increased in ASD patients. Such structural changes may reflect altered neuronal connectivity between specific brain regions. Moreover, the proposed candidate genes responsible for ASD include various synaptic proteins that play important roles in neurite outgrowth, axonal guidance, axonal targeting and synaptogenesis, suggesting structural abnormalities at a synaptic level responsible for expression of ASD phenotypes. Based on these findings, it was hypothesized that genetic perturbation at the 16p11.2 loci would lead to aberrant synaptic connectivity, thus underlying functional disturbances that lead to ASD. Results demonstrate significant axon targeting errors caused by defects in KLP68D, a Drosophila Kinesin-2 protein closely related to human KIF22 at the autism-linked 16p11.2 loci (Park, 2016).

Hetero-trimeric Kinesin-2 complex in Drosophila, consisting of KLP68D, KLP64D and DmKAP, has been implicated in microtubule organization and axonal transport of synaptic proteins such as choline acetyltransferase. However, experimental evidence is missing to support the idea that Kinesin-2 complex may participate in delivering molecules important for axon targeting. Potential cargos of KLP68D and KLP64D motors have been estimated to include Unc-51/ATG1, Fasciclin II, EB1, Armadillo, Bazooka, and DE-cadherin, most of whom have been well characterized for their roles in synaptogenesis. It will be important to investigate whether disruptions of any of these potential cargos lead to aberrant axon targeting phenotypes observed in Klp68D and Klp64D mutants (Park, 2016).

It should be noted that Drosophila Nod ("no distributive disjunction"), mostly involved in chromosomal segregation has been recognized as a homolog for human KIF22. However, similar levels of sequence homology to KIF22 were found in both Nod and KLP68D. In fact, a blast analysis results in higher sequence identity between KIF22 and KLP68D than Nod (41% vs. 33%). The specificity of motor protein cargos is often predicted to depend on the amino acid composition of motor proteins outside their core motor domain. Therefore, relatively lower level of homology between human KIF22 and Drosophila KLP68D may correspond to their distinct molecular functions. In contrast to KLP68D, the role of KIF22 in the mammalian nervous system has not been extensively investigated, but only limited to chromosomal segregation and genomic stability. Whether Drosophila KLP68D can be functionally replaced by human KIF22 in transgenic animals awaits further investigations (Park, 2016).

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Dong, T., He, J., Wang, S., Wang, L., Cheng, Y. and Zhong, Y. (2016). Inability to activate Rac1-dependent forgetting contributes to behavioral inflexibility in mutants of multiple autism-risk genes. Proc Natl Acad Sci U S A 113: 7644-7649. Exp Neurobiol 25: 33-39. PubMed ID: 27335463

Abstract
The etiology of autism is so complicated because it involves the effects of variants of several hundred risk genes along with the contribution of environmental factors. Therefore, it has been challenging to identify the causal paths that lead to the core autistic symptoms such as social deficit, repetitive behaviors, and behavioral inflexibility. As an alternative approach, extensive efforts have been devoted to identifying the convergence of the targets and functions of the autism-risk genes to facilitate mapping out causal paths. This study used a reversal-learning task to measure behavioral flexibility in Drosophila and determined the effects of loss-of-function mutations in multiple autism-risk gene homologs in flies. Mutations of five autism-risk genes with diversified molecular functions all lead to a similar phenotype of behavioral inflexibility indicated by impaired reversal-learning. These reversal-learning defects result from the inability to forget or rather, specifically, to activate Rac1 (Ras-related C3 botulinum toxin substrate 1)-dependent forgetting. Thus, behavior-evoked activation of Rac1-dependent forgetting has a converging function for autism-risk genes (Dong, 2016).

Highlights

• Mutation of Fmr1 leads to impaired reversal-learning through an inability to activate Rac1-dependent forgetting.
• Acute down-regulation of Fmr1 expression leads to behavioral inflexibility.
• Disruption of Ube3a impairs Rac1-dependent forgetting.
• Impairment of Rac1-dependent active forgetting in three other autism-susceptibility gene mutants.

Discussion
This study investigates whether forgetting-dependent behavioral inflexibility is a common phenotype for autism-risk genes and whether behavior-dependent activation of Rac1 is the convergent molecular target for these genes. To minimize genetic and behavioral variations, flies for all genotypes had the same isogenic background, and behavioral assays were performed using the well-established balanced protocol. Aversive conditioning and its associated reversal-learning task was used to investigate the functions of five autism-susceptibility genes: Fmr1, Ube3a, Nrx-1, Nlg4, and Tsc1. Four major findings emerge from the current study. First, mutation and RNAi-induced knockdown of these five autism-risk genes result in strong reversal-learning defects independent of learning ability. Second, these reversal-learning phenotypes are all caused by the inability to forget the old memory. Third, all the autism-risk gene mutants studied failed to trigger the behavior-dependent activation of Rac1, a reported regulator of forgetting. Fourth, the effects of these genes are confined within the mushroom body, in which Rac1-dependent forgetting is involved. These results suggest that the inability to activate Rac1-dependent forgetting is a converging mechanism for multiple autism-risk genes (Dong, 2016).

It is interesting that all five of the autism-risk genes with diverse functions funnel to a deficit in Rac1 activation. These five autism-risk genes have been reported to be involved in the Rac1 signaling pathway. The cytoplasmic FMRP-interacting protein (CYFIP) directly links Rac1 and FMRP to modulate cytoskeleton remodeling; Tsc1 functionally regulates Rac1 activity; Ube3a promotes Rho-GEF Pbl degradation via ubiquitination to affect Rac1 activation; and upon synaptic activation Rho-GEF Kal-7 disassembles from the Nrx-1/Nlg4/DISC1 complex to modulate the Rac1 pathway. Several other autism-risk genes, such as Nlg1, Nrx-4, P-Rex1, and Shank-3, have also been reported to participate in the Rac1-signaling pathway. In addition, when the gene–environment interactions of 122 genes and 191 factors in the autistic context were analyzed by systems biology, Rac1 was predicted to be a converging node that genetically links to the neurobiology of autism. Taken together, these findings indicate that Rac1 is a functional converging site for autism-risk genes (Dong, 2016).

Although autism is considered to be a developmental disorder, emerging evidence points to the postdevelopmental effects of autism-risk genes in adults. In this study, acute down-regulation of these five autism-risk genes at the adult stage lead to impaired behavioral flexibility with reduced reversal-learning and resistant old memory. Thus, all these five autism-risk genes are physiologically involved in regulating behavioral flexibility (Dong, 2016).

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Grice, S.J., Liu, J.L. and Webber, C. (2015). Synergistic interactions between Drosophila orthologues of genes spanned by de novo human CNVs support multiple-hit models of autism. PLoS Genet 11: e1004998. PubMed ID: 25816101

Abstract
Autism spectrum disorders (ASDs) are highly heritable and characterised by deficits in social interaction and communication, as well as restricted and repetitive behaviours. Although a number of highly penetrant ASD gene variants have been identified, there is growing evidence to support a causal role for combinatorial effects arising from the contributions of multiple loci. By examining synaptic and circadian neurological phenotypes resulting from the dosage variants of unique human:fly orthologues in Drosophila, this study observed numerous synergistic interactions between pairs of informatically-identified candidate genes whose orthologues are jointly affected by large de novo copy number variants (CNVs). These CNVs were found in the genomes of individuals with autism, including a patient carrying a 22q11.2 deletion. It was demonstrated that dosage alterations of the unique Drosophila orthologues of candidate genes from de novo CNVs that harbour only a single candidate gene display neurological defects similar to those previously reported in Drosophila models of ASD-associated variants. Next, pairwise dosage changes within the set of orthologues of candidate genes that were affected by the same single human de novo CNV were analyzed. For three of four CNVs with complete orthologous relationships, significant synergistic effects were found following the simultaneous dosage change of gene pairs drawn from a single CNV. The phenotypic variation observed at the Drosophila synapse that results from these interacting genetic variants supports a concordant phenotypic outcome across all interacting gene pairs following the direction of human gene copy number change. Specificity as well as transitivity was observed between interactors, both within and between CNV candidate gene sets, supporting shared and distinct genetic aetiologies. It was also shown that different interactions affect divergent synaptic processes, demonstrating distinct molecular aetiologies. Overall, the data illustrate mechanisms through which synergistic effects resulting from large structural variation can contribute to human disease (Grice, 2015).

Highlights

• Modelling ASD genes in the fly with NMJ and circadian phenotypes.
• Drosophila models of monogenic forms of ASD yield neurological phenotypes.
• Drosophila models of polygenic causes of ASD are driven by genetic interactions.
• A Drosophila model of a human gain CNVs supports convergent aetiologies following copy number change in ASD.
• Specific subsets of the candidates modify the Neurxin IV background.
• Genetic interaction subsets cause differential synaptic defects.

Discussion
This study developed an in vivo model system in Drosophila to determine how genes can synergistically interact within ASD associated de novo CNVs. Specifically, it was shown that (i) of the 4 human CNVs containing 2, 2, 5 and 6 network-identified candidate genes respectively (from a combined total of 114 copy-changed protein-coding genes), pairwise interactions between Drosophila orthologues yielding changes in the neuromuscular junction (NMJ) bouton number and circadian rhythms are observed for 3 CNVs; (ii) that the interactions observed are synergistic, as opposed to additive, in nature, and (iii) that the synaptic bouton counts observed following the simultaneous dosage change of all 5 pairs of interacting CNV candidate genes’ orthologues within Drosophila support a convergent phenotypic outcome arising from these genes’ dosage change for the individuals with ASD within whom they were identified. The combinations of genes drawn from these CNVs that interact are specific, both within a CNV and between CNVs, supporting distinct aetiologies underlying ASD. Finally, these specific interactions act through different molecular aetiologies, supporting the role of distinct molecular aetiologies in ASD (Grice, 2015).

The synergistic, as opposed to additive, nature of the pairwise genetic interactions that are observed in Drosophila has important consequences for identifying the genetic causes of ASD, and (i) the conserved orthology of the interactors, (ii) the human orthologues’ participation in an ASD-relevant network constructed from known mammalian interactions, and (iii) the concordance between the direction of dosage change and phenotype all support the inter-species relevance of our findings. Although there are over 100 ASD candidate genes currently identified, at least 70% of the genetic causes remain to be explained. The presence of multiple genetic variants in many patients suggests that inherited variants might lead to ASD through the combinatorial effects of distinct deleterious variants which affect a shared biological pathway. Where variants that act additively to cause ASD in a proband are inherited from each parent, those variants individually may cause detectable ASD-relevant traits in the parents (Grice, 2015).

However, if combinations of variants act only synergistically to cause ASD, there would be no expectation of ASD-relevant traits in either parent. Importantly, if sub-threshold ASD traits affect fecundity then variants that are only deleterious in combination may rise to a higher frequency in the population. Results in Drosophila show that only particular combinations of dosage variants act together to yield an abnormal phenotype. Identifying those variants that contribute to ASD only in combination with other specific variants, amongst a background of large amounts of non-contributing genetic variation, will be challenging because the variety of gene variant combinations is extremely large, and allele frequencies are likely very rare (Grice, 2015).

The genes participating in the pairwise genetic interactions identified by the screen are discs large (dlg: human orthologue (h.o) DLG1), p21-activated kinase (pak: h.o. PAK2), p20 catenin (p120ctn: h.o. CTNND2), Notch (N: h.o Notch 1), shibire/dynamin (shi/dynamin: h.o. DNM1), alpha-Spectrin (α-spec: h.o. SPTAN1), optomotor-blind-related-gene-1 (org-1: h.o. TBX1), partner of drosha (pasha: h.o. DGCR8) and Septin 4 (Sep4: h.o. SEPT5). An examination of CNVs listed in the Database of Genomic Variants (DGV) reveals that most of these genes are found to be individually dosage changed in the same direction in apparently healthy individuals (DLG1, 7 CNVs; PAK2, 1 CNV; DNM1, 1 CNV; SPTAN, 1 CNV; SEPT5, 2 CNVs; TBX1, 9 CNVs; DGCR8 5 CNVs). However, only one of these CNVs might simultaneously change two genes that were found to genetically-interact in the fly (variant nsv828939) and CNVs strongly implicated in ASD have previously been reported in apparently healthy individuals (Grice, 2015).

Many of the interacting genes have known functions in the nervous system. For example the localisation of the septate junction and neuronal adhesion protein Dlg at the NMJ has been shown to be regulated by Pak serine/threonine-protein kinase activity. In addition, it is interesting to point out that p21-activated kinase (PAK) has been shown to interact with the protein SHANK3 in rat, whose disruption can also cause ASD, with mutant Shank3 altering actin dynamics driven by PAK signalling. Destabilisation of the actin filaments at the NMJ leads to defective NMDAR-mediated synaptic current in neurons. PAK inhibitors have also been shown to rescue fragile X syndrome phenotypes in Fmr1 KO mice, suggesting an important role for Pak serine/threonine-protein kinase activity in ASD and ID. The gene alpha-spectrin, which was shown to genetically interact with the dynamin protein shabire, is known to cross link actin, and has been shown to be important for the localisation of Dlg at the synapse. The phenotypes resulting from the combination of these genes’ variants suggests an important role for the control of synapse integrity via actin stabilisation in ASD. This again is supported up by a particular enrichment for genes directly and indirectly associated with both cell adhesion and cytoskeletal associated cell membrane proteins in interacting genes (5 out of 9; discs large, p120 catenin, Notch, alpha-spectrin, pak), several of which have been identified to have properties in the neuron (Grice, 2015).

Many studies have linked neurodevelopmental disorders, including ASD, to mutations in synaptic adhesion proteins, including the neurexins and neuroligins, and mutations in these in Drosophila have yielded both behavioural and larval NMJ defects. This study found specific interactions between P120ctn, dlg and pak with Drosophila neurexin IV, which has been shown to be involved in the maturation of the Drosophila NMJ. Notably, the ASD-network orthologues (namely org-1, pasha and sep4) that contribute to the interactions modelling the CNV 12239_chr22_loss_17249508_l that covers the 22q11.2 microdeletion critical region, do not yield phenotypes in the sensitised NrxIV background suggesting that these intracellular genes may be exerting phenotypic effects through an alternative process. While other (non-ASD network) genes in this 22q11.2 critical region have received interest in effecting the many associated phenotypes, data from this study suggest that interactions between the human genes TBX1, DGCR8 and SEPT5 may play a significant causal role (Grice, 2015).

Alterations in active zone structures have been connoted in ASD. Moreover, neuron specific knockdown of the Drosophila orthologues of the ASD genes CNTNAP2 and NRXN1, NrxIX and Nrx-1 (dnrx), have been shown to alter the levels of the active zone protein BRP. BRP shows both sequence and functional homology with the mammalian ELKS/CAST proteins that are structural components of the vertebrate active zone. It was shown that dosage changes created by transheretozygotes between NrxIV, dlg and pak lead to a reduction in BRP foci. Dlg is a postsynaptic anchoring protein which is required for the development and stability of the postsynaptic subsynaptic reticulum (SSR), whilst Pak is known to phosphorylate Dlg and control its abundance at the synapse. NrxIV is predominantly presynaptic, but is required for the cell-cell contacts that influence synaptic development, and govern the interconnectivity between both neurons, glial cells and the pre- and postsynapse. Dosage alterations in NrxIV with Dlg, Pak and p120 catenin may lead to alterations in adhesion protein interactions, causing the destabilisation of the synaptic architecture in both the pre- and postsynapse, ultimately leading to defective synaptic maturation (Grice, 2015).

In the null mutant of the Drosophila orthologue of NRXN1, Nrx-1 (dnrx), GluRIIA subunit fluorescence and BRP active zone density are increased, although bouton numbers still remain reduced. It has been suggested that interactions between Drosophila neurexins and neuroligins may synchronise GluRIIA, and presynaptic active zone neurexin and neuroligin may be involved in the link between GluRIIA expression and presynaptic active zone dynamics. The interactions observed between P120ctn, NrxIV, dlg and pak also result in synaptic maturation defects. Null mutants in pak and dlg have also been shown to lead to alterations in glutamate receptor subunits (GluRIIA), however, a significant interaction between the dlg/pak transheterozygotes, or the interactions with NrxIV was not observed. GluRIIA levels are affected in the pasha/Sep4 cross. Reductions in GluRIIA have been found to lead to a compensatory increase in active zone size. Changes in active zone puncta in the pasha/Sep4 cross were not observed, suggesting that these compensatory mechanisms may be compromised in this case (Grice, 2015).

It is also worth noting that, through changes in the mammalian target of rapamycin mTOR, altered eIF4E-dependent translation results in ASD-relevant phenotypes in mouse and altered regulation of the synthesis of neuroligins. Mutations in Drosophila TOR and eIF4E alter levels of GluRIIA but do not alter the active zones. Interestingly, the fragile X syndrome associated protein FMRP (fragile X syndrome has 30% co-morbidity with ASD) and the miRNA pathway are known to mechanistically interact (Pasha, is part of the miRNA microprocessor complex), while the mRNA of the Sept4 human orthologue (SEPT5) is an FMRP target. Both FMRP, which is known to pause ribosomal translocation, and Pasha are involved in translational repression. In addition, both mutations in FMRP and the microRNA processing machinery affect the ratios of GluR subunits. It may be that pasha/Sep4 deficit leads to the suboptimal translation of Sep4, which functions in complexes that associate with cellular membranes and actin filaments. This may lead to inefficient synaptic anchoring. Further analysis of this process, and those arising from the gene-gene interactions in this study, can now be performed. In summary, the in vivo model system described in this study may be well suited to rapidly evaluate how combinations of genes may contribute synergistically to the neurological defects that, in turn, may contribute to ASD (Grice, 2015).

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Valdez, C., Scroggs, R., Chassen, R. and Reiter, L.T. (2015). Variation in Dube3a expression affects neurotransmission at the Drosophila neuromuscular junction. Biol Open 4: 776-782. PubMed ID: 25948754

Abstract
Changes in UBE3A expression levels in neurons can cause neurogenetic disorders ranging from Angelman syndrome (AS) (decreased levels) to autism (increased levels). This study investigated the effects on neuronal function of varying UBE3A levels using the Drosophila neuromuscular junction as a model for both of these neurogenetic disorders. Stimulations that evoke excitatory junction potentials (EJPs) at 1 Hz intermittently fail to evoke EJPs at 15 Hz in a significantly higher proportion of Dube3a over-expressors using the pan neuronal GAL4 driver C155-GAL4 (C155-GAL4>UAS-Dube3a) relative to controls (C155>+ alone). However, in the Dube3a over-expressing larval neurons with no failures, there is no difference in EJP amplitude at the beginning of the train, or the rate of decrease in EJP amplitude over the course of the train compared to controls. In the absence of tetrodotoxin (TTX), spontaneous EJPs are observed in significantly more C155-GAL4>UAS-Dube3a larva compared to controls. In the presence of TTX, spontaneous and evoked EJPs are completely blocked and mEJP amplitude and frequency does not differ among genotypes. These data suggest that over-expression of wild type Dube3a, but not a ubiquitination defective Dube3a-C/A protein, compromises the ability of motor neuron axons to support closely spaced trains of action potentials, while at the same time increasing excitability. EJPs evoked at 15 Hz in the absence of Dube3a (Dube3a15b homozygous mutant larvae) decay more rapidly over the course of 30 stimulations compared to w1118 controls, and Dube3a15b larval muscles have significantly more negative resting membrane potentials (RMP). However, these results could not be recapitulated using RNAi knockdown of Dube3a in muscle or neurons alone, suggesting more global developmental defects contribute to this phenotype. These data suggest that reduced UBE3A expression levels may cause global changes that affect RMP and neurotransmitter release from motorneurons at the neuromuscular junction. Similar affects of under- and over-expression of UBE3A on membrane potential and synaptic transmission may underlie the synaptic plasticity defects observed in both AS and autism (Valdez, 2015).

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Guven-Ozkan, T., Busto, G.U., Schutte, S.S., Cervantes-Sandoval, I., O'Dowd, D.K. and Davis, R.L. (2016). miR-980 is a memory suppressor microRNA that regulates the autism-susceptibility gene A2bp1. Cell Rep 14: 1698-1709. PubMed ID: 26876166

Abstract
MicroRNAs have been associated with many different biological functions but little is known about their roles in conditioned behavior. This study demonstrates that Drosophila miR-980 is a memory suppressor gene functioning in multiple regions of the adult brain. Memory acquisition and stability are both increased by miR-980 inhibition. Whole cell recordings and functional imaging experiments indicate that miR-980 regulates neuronal excitability. The autism susceptibility gene, A2bp1, was identified as an mRNA target for miR-980. A2bp1 levels vary inversely with miR-980 expression; memory performance is directly related to A2bp1 levels. In addition, A2bp1 knockdown reverses the memory gains produced by miR-980 inhibition, consistent with A2bp1 being a downstream target of miR-980 responsible for the memory phenotypes. These results indicate that miR-980 represses A2bp1 expression to tune the excitable state of neurons, and the overall state of excitability translates to memory impairment or improvement (Guven-Ozkan, 2016).

Highlights

• miR-980 inhibition enhances olfactory learning and memory stability.
• miR-980 enhances memory when inhibited during adulthood in multiple areas of the CNS.
• A2bp1 is expressed in the nuclei of most brain neurons and miR-980 represses A2bp1 protein expression.
• Overexpression of A2bp1 in the adult mushroom bodies enhances memory.

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Reviews

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More in IF

Ube3a, the E3 ubiquitin ligase causing Angelman syndrome and linked to autism, regulates protein homeostasis through the proteasomal shuttle Rpn10

Neuroligin 2 is required for synapse development and function at the Drosophila neuromuscular junction

Neurexin-1 is required for synapse formation and larvae associative learning in Drosophila

Mechanisms of TSC-mediated control of synapse assembly and axon guidance

Back to Drosophila as a Model for Human Diseases

Date revised: 02 Feb 2017

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