Drosophila assay developed to test variants of human PTEN, a gene associate with cancers and ASD

A scalable Drosophila assay for clinical interpretation of human PTEN variants in suppression of PI3K/AKT induced cellular proliferation

Payel Ganguly, Landiso Madonsela, Jesse T. Chao, Christopher J. R. Loewen, Timothy P. O’Connor, Esther M. Verheyen, Douglas W. Allan

Abstract: "Gene variant discovery is becoming routine, but it remains difficult to usefully interpret the functional consequence or disease relevance of most variants. ... Drosophila melanogaster offers great potential as an assay platform, but was untested for high numbers of human variants adherent to these guidelines. Here, we wished to test the utility of Drosophila as a platform for scalable well-established assays. We took a genetic interaction approach to test the function of ~100 human PTEN variants in cancer-relevant suppression of PI3K/AKT signaling in cellular growth and proliferation. We validated the assay using biochemically characterized PTEN mutants as well as 23 total known pathogenic and benign PTEN variants, all of which the assay correctly assigned into predicted functional categories. ... Overall, we demonstrate that Drosophila offers a powerful assay platform for clinical variant interpretation, that can be used in conjunction with other well-established assays, to increase confidence in the accurate assessment of variant function and pathogenicity."

Access a Science in Vancouver feature on this article here.

Preprint: Results of a Drosophila study of ASD-associated gene variants impacts understanding of neurological diseases

Drosophila functional screening of de novo variants in autism uncovers deleterious variants and facilitates discovery of rare neurodevelopmental diseases

Marcogliese et al.

BioRxiv pre-print:
https://www.biorxiv.org/content/10.1101/2020.12.30.424813v1

Abstract: Individuals with autism spectrum disorders (ASD) exhibit an increased burden of de novo variants in a broadening range of genes. We functionally tested the effects of ASD missense variants using Drosophila through ‘humanization’ rescue and overexpression-based strategies. We studied 79 ASD variants in 74 genes identified in the Simons Simplex Collection and found 38% of them caused functional alterations. Moreover, we identified GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in eight previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes point to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.

Drosophila studies help identify and understand role of the CSDE1 gene in an autism-related syndrome

Guo H, Li Y, Shen L, Wang T, Jia X, Liu L, Xu T, Ou M, Hoekzema K, Wu H, et al. Disruptive variants of CSDE1 associate with autism and interfere with neuronal development and synaptic transmission. Sci Adv. 2019 Sep 25;5(9):eaax2166. PubMed PMID: 31579823; PubMed Central PMCID: PMC6760934.

Abstract: "RNA binding proteins are key players in posttranscriptional regulation and have been implicated in neurodevelopmental and neuropsychiatric disorders. Here, we report a significant burden of heterozygous, likely gene-disrupting variants in CSDE1 (encoding a highly constrained RNA binding protein) among patients with autism and related neurodevelopmental disabilities. Analysis of 17 patients identifies common phenotypes including autism, intellectual disability, language and motor delay, seizures, macrocephaly, and variable ocular abnormalities. HITS-CLIP revealed that Csde1-binding targets are enriched in autism-associated gene sets, especially FMRP targets, and in neuronal development and synaptic plasticity-related pathways. Csde1 knockdown in primary mouse cortical neurons leads to an overgrowth of the neurites and abnormal dendritic spine morphology/synapse formation and impaired synaptic transmission, whereas mutant and knockdown experiments in Drosophila result in defects in synapse growth and synaptic transmission. Our study defines a new autism-related syndrome and highlights the functional role of CSDE1 in synapse development and synaptic transmission."

Flies used to test drug activity predicted using dynamic combinatorial chemistry approach

Canal-Martín A, Sastre J, Sánchez-Barrena MJ, Canales A, Baldominos S, Pascual N, Martínez-González L, Molero D, Fernández-Valle ME, Sáez E, Blanco-Gabella P, Gómez-Rubio E, Martín-Santamaría S, Sáiz A, Mansilla A, Cañada FJ, Jiménez-Barbero J, Martínez A, Pérez-Fernández R. Insights into real-time chemical processes in a calcium sensor protein-directed dynamic library. Nat Commun. 2019 Jun 26;10(1):2798. PMID: 31243268; PMCID: PMC6595003.

Abstract: "Dynamic combinatorial chemistry (DCC) has proven its potential in drug discovery speeding the identification of modulators of biological targets. However, the exchange chemistries typically take place under specific reaction conditions, with limited tools capable of operating under physiological parameters. Here we report a catalyzed protein-directed DCC working at low temperatures that allows the calcium sensor NCS-1 to find the best ligands in situ. Ultrafast NMR identifies the reaction intermediates of the acylhydrazone exchange, tracing the molecular assemblies and getting a real-time insight into the essence of DCC processes at physiological pH. Additionally, NMR, X-ray crystallography and computational methods are employed to elucidate structural and mechanistic aspects of the molecular recognition event. The DCC approach leads us to the identification of a compound stabilizing the NCS-1/Ric8a complex and whose therapeutic potential is proven in a Drosophila model of disease with synaptic alterations."

Domain-sensory processing approach--new fly model for study of genetic basis of autism

Vilidaite G, Norcia AM, West RJH, Elliott CJH, Pei F, Wade AR, Baker DH. Autism sensory dysfunction in an evolutionarily conserved system. Proc Biol Sci. 2018 Dec 19;285(1893):20182255. PMID: 30963913.

Abstract: "There is increasing evidence for a strong genetic basis for autism, with many genetic models being developed in an attempt to replicate autistic symptoms in animals. However, current animal behaviour paradigms rarely match the social and cognitive behaviours exhibited by autistic individuals. Here, we instead assay another functional domain-sensory processing-known to be affected in autism to test a novel genetic autism model in Drosophila melanogaster. We show similar visual response alterations and a similar development trajectory in Nhe3 mutant flies (total n = 72) and in autistic human participants (total n = 154). We report a dissociation between first- and second-order electrophysiological visual responses to steady-state stimulation in adult mutant fruit flies that is strikingly similar to the response pattern in human adults with ASD as well as that of a large sample of neurotypical individuals with high numbers of autistic traits. We explain this as a genetically driven, selective signalling alteration in transient visual dynamics. In contrast to adults, autistic children show a decrease in the first-order response that is matched by the fruit fly model, suggesting that a compensatory change in processing occurs during development. Our results provide the first animal model of autism comprising a differential developmental phenotype in visual processing."

Preprint: New fly model of SeSAME syndrome

Posted December 2018 on the preprint server:

Ravi K Nadella, Anirudh Chellappa, Anand G Subramaniam, View ORCID ProfileAman Aggarwal, Ravi Prabhakar More, Mahalakshmi Dhana sekar, Srividya Shetty, Suriya Prakash, Nikhil Ratna, Vandana VP, Meera Purushotham, Jitender Saini, Biju Viswanath, Bindu PS, Madhu Nagappa, Bhupesh Mehta, Sanjeev Jain, View ORCID ProfileRamakrishnan Kannan

Novel KCNJ10 mutation identified in a SeSAME family compromise channel function and impairs Drosophila locomotor behavior
https://www.biorxiv.org/content/10.1101/506949v1

From the preprint abstract: "Deficits in the inwardly rectifying K channel, Kir4.1, cause SeSAME syndrome, autism spectrum disorders with seizures, multiple sclerosis, Huntington disease and Rett syndrome. ... We used whole exome sequencing (WES), channel physiology in patient-specific lymphoblastoid cells (LCLs) and established a Drosophila model to examine the functional effects of a KCNJ10 variant identified in SeSAME like family. ... Drosophila irk2 mutant, a human homolog of Kir4.1, exhibited impairment locomotion, shortened life span and age dependent degeneration of dopaminergic neurons in the adult brain. ... neural specific expression of either Kir4.1 or Irk2 alleviate irk2 mutant phenotypes, while the Kir4.1(T290A) and Irk2(T290A) mutant proteins failed to do so. These results imply the functional conservation of Kir4.1 across species thereby elevate the potential of using Drosophila model to improve our understanding of the SeSAME syndrome. ..."

Neurons vs. Glia: "Paradigm shift" in understanding a disease mechanism suggested by results of fly research

Hope KA, LeDoux MS, Reiter LT. Glial overexpression of Dube3a causes seizures and synaptic impairments in Drosophila concomitant with down regulation of the Na(+)/K(+) pump ATPα. Neurobiol Dis. 2017 Dec;108:238-248. PMID: 28888970; PMCID: PMC5675773.

Abstract: "Duplication 15q syndrome (Dup15q) is an autism-associated disorder co-incident with high rates of pediatric epilepsy. Additional copies of the E3 ubiquitin ligase UBE3A are thought to cause Dup15q phenotypes, yet models overexpressing UBE3A in neurons have not recapitulated the epilepsy phenotype. We show that Drosophila endogenously expresses Dube3a (fly UBE3A homolog) in glial cells and neurons, prompting an investigation into the consequences of glial Dube3a overexpression. Here we expand on previous work showing that the Na+/K+ pump ATPα is a direct ubiquitin ligase substrate of Dube3a. A robust seizure-like phenotype was observed in flies overexpressing Dube3a in glial cells, but not neurons. Glial-specific knockdown of ATPα also produced seizure-like behavior, and this phenotype was rescued by simultaneously overexpressing ATPα and Dube3a in glia. Our data provides the basis of a paradigm shift in Dup15q research given that clinical phenotypes have long been assumed to be due to neuronal UBE3A overexpression."

Preprint describes contribution of Drosophila double-knockdown assay to understanding 16p11.2 deletion syndrome

Pervasive epistasis in cell proliferation pathways modulates neurodevelopmental defects of autism-associated 16p11.2 deletion

Janani Iyer, Mayanglambam Dhruba Singh, Matthew Jensen, Payal Patel, Lucilla Pizzo, Emily Huber, Haley Koerselman, Alexis T. Weiner, Paola Lepanto, Komal Vadodaria, Alexis Kubina, Qingyu Wang, Abigail Talbert, Sneha Yennawar, Jose Badano, J. Robert Manak, Melissa M. Rolls, Arjun Krishnan, Santhosh Girirajan

https://www.biorxiv.org/content/early/2017/09/20/185355

From the abstract: "We used RNA interference in Drosophila melanogaster to evaluate the phenotype, function, and interactions of conserved 16p11.2 homologs ... Leveraging the Drosophila eye for studying gene interactions, we performed 561 pairwise knockdowns of gene expression, and identified 24 interactions between 16p11.2 homologs ... and 62 interactions with other neurodevelopmental genes ... Overall, these results point towards a new model for pathogenicity of rare CNVs, where CNV genes interact with each other in conserved pathways to modulate expression of the neurodevelopmental phenotype."

Fly study suggests link between bisphenol A (BPA), found in some plastics, and ADHD and autism

The results suggest BPA is a risk factor for the behavioral impairments that are the basis of autism and ADHD https://t.co/xzhUxws8ql

— Thomas Brody (@interactivefly) February 15, 2016

Review of Drosophila a model for neuropsychopharmacology-related research

Narayanan AS, Rothenfluh A. I Believe I Can Fly!: Use of Drosophila as a Model Organism in Neuropsychopharmacology Research. Neuropsychopharmacology. 2015 Oct 30. PMID: 26576740.

From the abstract:  "... Here, we outline why we study an invertebrate organism in the context of neuropsychiatric disorders, and we discuss how we can gain insight from studies in Drosophila. ... Highlighting some translational examples, we underline the fact that their brains works more like ours than one would have anticipated."

Their Fig. 1 provides a nice graphical summary of translational approaches.

Review looks at contributions of Drosophila research related to Fragile X syndrome and discusses discordance

Weisz ED, Monyak RE, Jongens TA. Deciphering Discord: How Drosophila research has enhanced our understanding of the importance of FMRP in different spatial and temporal contexts. Exp Neurol. 2015 May 27. pii: S0014-4886(15)30001-7. PMID: 26026973.

From the abstract: "Fragile X Syndrome (FXS) is the most common heritable form of intellectual impairment as well as the leading monogenetic cause of autism. ... Research efforts in Drosophila melanogaster have revealed key insights into the mechanistic underpinnings of FXS. While much remains unknown, it is increasingly apparent that FXS involves a myriad of spatial and temporally specific alterations in cellular function. Consequently, the literature is filled with numerous discordant findings. Researchers and clinicians alike must be cognizant of this dissonance, as it will likely be important for the design of preclinical studies to assess the efficacy of therapeutic strategies to improve the lives of FXS patients."

Drosophila study contributes to understanding of autism spectrum disorders-associated gene variants

Grice SJ, Liu JL, Webber C. Synergistic Interactions between Drosophila Orthologues of Genes Spanned by De Novo Human CNVs Support Multiple-Hit Models of Autism. PLoS Genet. 2015 Mar 27;11(3):e1004998. PMID: 25816101.

Fig. 1 from Grice et al. 2015 PLoS Genet.

From the abstract: "Autism spectrum disorders (ASDs) are highly heritable ... 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, we observe numerous synergistic interactions between pairs of informatically-identified candidate genes whose orthologues are jointly affected by large de novo copy number variants (CNVs). ... We first demonstrate 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. We then considered pairwise dosage changes within the set of orthologues of candidate genes that were affected by the same single human de novo CNV. ... Our study illustrates mechanisms through which synergistic effects resulting from large structural variation can contribute to human disease."

Impaired GABAergic circuit structure and function reported for fly model of Fragile-X syndrome

Gatto CL, Pereira D, Broadie K. GABAergic circuit dysfunction in the Drosophila Fragile X syndrome model. Neurobiol Dis. 2014 May;65:142-59. PMID: 24423648; PMCID: PMC3988906.

Reveiw--model systems in the study of autism spectrum disorders

Doll CA, Broadie K. Impaired activity-dependent neural circuit assembly and refinement in autism spectrum disorder genetic models. Front Cell Neurosci. 2014 Feb 7;8:30. eCollection 2014. Review. PMID: 24570656; PMCID: PMC3916725.