Multi-species study identifies the vacuolar H+ ATPase component ATP6V0C as a human disease-associated gene

Brain. 2022 Sep 8:awac330. doi: 10.1093/brain/awac330

ATP6V0C variants impair vacuolar V-ATPase causing a neurodevelopmental disorder
often associated with epilepsy

Mattison KA, Tossing G, Mulroe F, Simmons C, Butler KM, Schreiber A, Alsadah A, Neilson DE, Naess K, Wedell A, Wredenberg A, Sorlin A, McCann E, Burghel GJ, Menendez B, Hoganson GE, Botto LD, Filloux FM, Aledo-Serrano Á, Gil-Nagel A, Tatton-Brown K, Verbeek NE, van Hirtum-Das M, Breckpot J, Hammer TB, Møller RS, Whitney A, Douglas AGL, Kharbanda M, Brunetti-Pierri N, Morleo M, Nigro V, May HJ, Tao JX, Argili E, Sherr EH, Dobyns WB, Consortium GER, Baines RA, Warwicker J, Parker JA, Banka S, Campeau PM, Escayg A

Abstract:

The vacuolar H+-ATPase (V-ATPase) is an enzymatic complex that functions in an ATP-dependent manner to pump protons across membranes and acidify organelles, thereby creating the proton/pH gradient required for membrane trafficking by several different types of transporters. We describe heterozygous point variants in ATP6V0C, encoding the c-subunit in the membrane bound integral domain of the V-ATPase, in 27 patients with neurodevelopmental abnormalities with or without epilepsy. Corpus callosum hypoplasia and cardiac abnormalities were also present in some patients. In silico modeling suggested that the patient variants interfere with the interactions between the ATP6V0C and ATP6V0A subunits during ATP hydrolysis. Consistent with decreased V-ATPase activity, functional analyses conducted in Saccharomyces cerevisiae revealed reduced LysoSensor fluorescence and reduced growth in media containing varying concentrations of CaCl2. Knockdown of ATP6V0C in Drosophila resulted in increased duration of seizure-like behavior, and the expression of selected patient variants in Caenorhabditis elegans led to reduced growth, motor dysfunction, and reduced lifespan. In summary, this study establishes ATP6V0C as an important disease gene, describes the clinical features of the associated neurodevelopmental disorder, and provides insight into disease mechanisms.

DOI: 10.1093/brain/awac330
PMID: 36074901

Drosophila used in study associating variants in UNC13B with epilepsy and/or febrile seizures

Brain. 2021 Apr 20:awab164. doi: 10.1093/brain/awab164. Online ahead of print.

UNC13B variants associated with partial epilepsy with favourable outcome.

Wang J(, Qiao JD, Liu XR, Liu DT, Chen YH, Wu Y, Sun Y, Yu J, Ren RN, Mei Z, Liu YX, Shi YW, Jiang M, Lin SM, He N, Li B, Bian WJ, Li BM(, Yi YH, Su T, Liu HK, Gu WY, Liao WP; China Epilepsy Gene 1.0 Project.

From the abstract:

"The unc-13 homolog B (UNC13B) gene encodes a presynaptic protein ... In this study we screened for novel genetic variants in a cohort of 446 unrelated cases (families) with partial epilepsy ... UNC13B variants were identified in 12 individuals affected by partial epilepsy and/or febrile seizures from eight unrelated families. ... The identified UNC13B variants included one nonsense variant, two variants at or around a splice site, one compound heterozygous missense variant, and four missense variants that cosegregated in the families. The frequency of UNC13B variants identified in the present study was significantly higher than that in a control cohort of Han Chinese and controls of the East Asian and all populations in the Genome Aggregation Database. Computational modeling, including hydrogen bond and docking analyses, suggested that the variants lead to functional impairment. In Drosophila, seizure rate and duration were increased by Unc13b knockdown compared to wild-type flies, but these effects were less pronounced than in sodium voltage-gated channel alpha subunit 1 (Scn1a) knockdown Drosophila. Electrophysiologic recordings showed that excitatory neurons in Unc13b-deficient flies exhibited increased excitability. These results suggest that UNC13B is potentially associated with epilepsy. ..."

DOI: 10.1093/brain/awab164
PMID: 33876820

Fly model related to FIG-4 associated neurological disorders uncovers potential link to epigenetic regulation

Neuroreport. 2021 May 5;32(7):562-568. doi: 10.1097/WNR.0000000000001636.

Identification of Rpd3 as a novel epigenetic regulator of Drosophila FIG 4, a
Charcot-Marie-Tooth disease-causing gene.

Muraoka Y, Nikaido A, Kowada R, Kimura H, Yamaguchi M, Yoshida H

Abstract:

Mutations in the factor-induced-gene 4 (FIG 4) gene are associated with multiple disorders, including Charcot-Marie-Tooth disease (CMT), epilepsy with polymicrogyria, Yunis-Varón syndrome and amyotrophic lateral sclerosis. The wide spectrum of disorders associated with FIG 4 may be related to the dysregulated epigenetics. Using Gene Expression Omnibus, we found that HDAC1 binds to the FIG 4 gene locus in the genome of human CD4+ T cells. Rpd3 is a well-known Drosophila homolog of human HDAC1. We previously established Drosophila models targeting Drosophila FIG 4 (dFIG 4) that exhibited defective locomotive ability, abnormal synapse morphology at neuromuscular junctions, enlarged vacuoles in the fat body and aberrant compound eye morphology. Genetic crossing experiments followed by physiological and immunocytochemical analyses revealed that Rpd3 mutations suppressed these defects induced by dFIG 4 knockdown. This demonstrated Rpd3 to be an important epigenetic regulator of dFIG 4, suggesting that the inhibition of HDAC1 represses the pathogenesis of FIG 4-associated disorders, including CMT. Defects in epigenetic regulators, such as HDAC1, may also explain the diverse symptoms of FIG 4-associated disorders.

Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.

DOI: 10.1097/WNR.0000000000001636
PMID: 33850086

Review article: "Investigating rare and ultrarare epilepsy syndromes with Drosophila models"

Fac Rev. 2021 Jan 29;10:10. doi: 10.12703/r/10-10. eCollection 2021.

Investigating rare and ultrarare epilepsy syndromes with Drosophila models.

Lasko P(1)(2), Lüthy K(1)(3).

Author information:
(1)Department of Human Genetics, Radboud University Medical Centre, Nijmegen,
Netherlands.
(2)Department of Biology, McGill University, Montréal, Québec, Canada.
(3)Donders Institute for Brain, Cognition and Behaviour, Radboud University
Medical Centre, Nijmegen, Netherlands.

Abstract:

One in three epilepsy cases is drug resistant, and seizures often begin in  infancy, when they are life-threatening and when therapeutic options are highly limited. An important tool for prioritizing and validating genes associated with epileptic conditions, which is suitable for large-scale screening, is disease  modeling in Drosophila. Approximately two-thirds of disease genes are conserved in Drosophila, and gene-specific fly models exhibit behavioral changes that are  related to symptoms of epilepsy. Models are based on behavior readouts, seizure-like attacks and paralysis following stimulation, and neuronal,  cell-biological readouts that are in the majority based on changes in nerve cell activity or morphology. In this review, we focus on behavioral phenotypes. Importantly, Drosophila modeling is independent of, and complementary to, other approaches that are computational and based on systems analysis. The large  number of known epilepsy-associated gene variants indicates a need for efficient research strategies. We will discuss the status quo of epilepsy disease modelling in Drosophila and describe promising steps towards the development of new drugs to reduce seizure rates and alleviate other epileptic symptoms.

Copyright: © 2021 Lasko P et al.

DOI: 10.12703/r/10-10
PMCID: PMC7894260
PMID: 33659928

Experiments in Drosophila contribute to study of human gene variants associated with an early onset epileptic encephalopathy

Suzuki H, Yoshida T, Morisada N, Uehara T, Kosaki K, Sato K, Matsubara K, Takano-Shimizu T, Takenouchi T. De novo NSF mutations cause early infantile epileptic encephalopathy. Ann Clin Transl Neurol. 2019 Nov 1. doi: 10.1002/acn3.50917. PubMed PMID: 31675180.

Abstract: "N-ethylmaleimide-sensitive factor (NSF) plays a critical role in intracellular vesicle transport, which is essential for neurotransmitter release. Herein, we, for the first time, document human monogenic disease phenotype of de novo pathogenic variants in NSF, that is, epileptic encephalopathy of early infantile onset. When expressed in the developing eye of Drosophila, the mutant NSF severely affected eye development, while the wild-type allele had no detectable effect under the same conditions. Our findings suggest that the two pathogenic variants exert a dominant negative effect. De novo heterozygous mutations in the NSF gene cause early infantile epileptic encephalopathy."

Review highlights Drosophila research contribution to understanding mTOR and tuberous sclerosis

Franz DN, Krueger DA. mTOR inhibitor therapy as a disease modifying therapy for tuberous sclerosis complex. Am J Med Genet C Semin Med Genet. 2018 Sep;178(3):365-373. doi: 10.1002/ajmg.c.31655. Review. PubMed PMID: 30307123.

Abstract: "Between 1993 and 2003, through experiments involving Drosophila sp., cancer biologists identified the protein kinase known as the mammalian target of rapamycin, its pathway, and its relationship to the genes responsible for tuberous sclerosis. Thereafter, clinical research has resulted in regulatory approval of mTOR inhibitors for four distinct manifestations of the disease: giant cell astrocytoma, angiomyolipoma, lymphangioleiomyomatosis, and epilepsy. These developments are summarized and the practical use of mTOR inhibitors to improve the lives of patients with tuberous sclerosis reviewed."

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."

FlyRNAi: Drosophila cell-based RNAi screen related to treat...

FlyRNAi: Drosophila cell-based RNAi screen related to treat...: Lin WH, He M, Fan YN, Baines RA. An RNAi-mediated screen identifies novel targets for next-generation antiepileptic drugs based on increased...

Crystal structure of fly protein reveals new functional information relevant to epilepsy and DOOR syndrome

Fischer B, Lüthy K, Paesmans J, De Koninck C, Maes I, Swerts J, Kuenen S, Uytterhoeven V, Verstreken P, Versées W. Skywalker-TBC1D24 has a lipid-binding pocket mutated in epilepsy and required for synaptic function. Nat Struct Mol Biol. 2016 Nov;23(11):965-973. PMID: 27669036.

From the abstract: "Mutations in TBC1D24 cause severe epilepsy and DOORS syndrome, but the molecular mechanisms underlying these pathologies are unresolved. We solved the crystal structure of the TBC domain of the Drosophila ortholog Skywalker, revealing an unanticipated cationic pocket conserved among TBC1D24 homologs. ... The most prevalent patient mutations affect the phosphoinositide-binding pocket and inhibit lipid binding. ... Hence, we discovered that a TBC domain affected by clinical mutations directly binds phosphoinositides through a cationic pocket and that phosphoinositide binding is critical for presynaptic function."

Exploring a linke between Parkinson's and epilepsy through study of the Drosophila mutant easily shocked

Witt SN. Lipid disequilibrium in biological membranes, a possible pathway to neurodegeneration. Commun Integr Biol. 2015 Jan 8;7(6):e993266. PMID: 26480301; PMCID: PMC4594524.

From the abstract: "... Here we highlight a Drosophila mutant called easily shocked-thought to be a model of epilepsy-that cannot use ethanolamine to synthesize PE. ... We propose that disruptions in lipid homeostasis (synthesis and degradation) may be responsible for some cases of PD and epilepsy."

Characterization of sleep in a fly model of epilepsy

Petruccelli E, Lansdon P, Kitamoto T. Exaggerated Nighttime Sleep and Defective Sleep Homeostasis in a Drosophila Knock-In Model of Human Epilepsy. PLoS One. 2015 Sep 11;10(9):e0137758. PMID: 26361221; PMCID: PMC4567262.

From the abstract: "Despite an established link between epilepsy and sleep behavior, it remains unclear how specific epileptogenic mutations affect sleep and subsequently influence seizure susceptibility. ... Here, we show that at room temperature the GEFS+ mutation dominantly modifies sleep, with mutants exhibiting rapid sleep onset at dusk and increased nighttime sleep as compared to controls.  ... Additionally, analyses under other light conditions suggested that the GEFS+ mutation led to reduced buffering of behavioral responses to light on and off stimuli, which contributed to characteristic GEFS+ sleep phenotypes. ... Our study has revealed the sleep architecture of a Drosophila VGSC mutant that harbors a human GEFS+ mutation, and provided unique insight into the relationship between sleep and epilepsy."

Using the fly system to explore the role of glia in neurological diseases (review)

Zwarts L, Van Eijs F, Callaerts P. Glia in Drosophila behavior. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 2015 Sep;201(9):879-93. PMID: 25336160.

From the abstract: "Glial cells constitute about 10% of the Drosophila nervous system. The development of genetic and molecular tools has helped greatly in defining different types of glia. ... We here summarize recent work describing the role of glia in normal behavior and in Drosophila models for neurological and behavioral disorders."

FlyRNAi: in vivo RNAi used to validate at gene level hits i...

FlyRNAi: in vivo RNAi used to validate at gene level hits i...: Talsma AD, Chaves JF, LaMonaca A, Wieczorek ED, Palladino MJ. Genome-wide screen for modifiers of Na (+) /K (+) ATPase alleles identifies cr...

Sleep-deprived and feeling it: new use of fly as a model of sleep deprivation-related seizures

Lucey BP, Leahy A, Rosas R, Shaw PJ. A new model to study sleep deprivation-induced seizure. Free online at Sleep. 2015 May 1;38(5):777-85. PMID: 25515102.

Fly study related to neurodegeneration, epilepsy and DOOR syndrome

Fernandes AC, Uytterhoeven V, Kuenen S, Wang YC, Slabbaert JR, Swerts J, Kasprowicz J, Aerts S, Verstreken P. Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration. J Cell Biol. 2014 Nov 24;207(4):453-62. PMID: 25422373; PMCID: PMC4242831.

From the abstract: “Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. ... we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. ... Our findings ... identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans.”

Review written for clinicians and mammalian researchers about non-mammalian models of epilepsy

Cunliffe VT, Baines RA, Giachello CN, Lin WH, Morgan A, Reuber M, Russell C, Walker MC, Williams RS. Epilepsy research methods update: Understanding the causes of epileptic seizures and identifying new treatments using non-mammalian model organisms. Seizure. 2014 Oct 13. PMID: 25457452.

From the abstract:  "This narrative review is intended to introduce clinicians treating epilepsy and researchers familiar with mammalian models of epilepsy to experimentally tractable, non-mammalian research models used in epilepsy research, ranging from unicellular eukaryotes to more complex multicellular organisms. The review focuses on four model organisms: the social amoeba Dictyostelium discoideum, the roundworm Caenorhabditis elegans, the fruit fly Drosophila melanogaster and the zebrafish Danio rerio. ..."

Neurodegenerative models--catching up

Catching up on recent papers related to Drosophila as a model for neurodegenerative diseases.

Fernandes AC, Uytterhoeven V, Kuenen S, Wang YC, Slabbaert JR, Swerts J, Kasprowicz J, Aerts S, Verstreken P. Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration. J Cell Biol. 2014 Nov 24;207(4):453-62. PMID: 25422373; PMCID: PMC4242831. From the abstract: "In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, ... Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average ... Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking ... rescued the neurotransmission and neurodegeneration defects in sky mutants. ... Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans. "

Vanden Broeck L, Kleinberger G, Chapuis J, Gistelinck M, Amouyel P, Van Broeckhoven C, Lambert JC, Callaerts P, Dermaut B. Functional complementation in Drosophila to predict the pathogenicity of TARDBP variants: evidence for a loss-of-function mechanism. Neurobiol Aging. 2014 Sep 28. pii: S0197-4580(14)00596-X. PMID: 25442115.

Jäckel S, Summerer AK, Thömmes CM, Pan X, Voigt A, Schulz JB, Rasse TM, Dormann D, Haass C, Kahle PJ. Nuclear import factor transportin and arginine methyltransferase 1 modify FUS neurotoxicity in Drosophila. Neurobiol Dis. 2014 Nov 8;74C:76-88. PMID: 25447237. From the abstract: "... To investigate the requirements for the nuclear import of FUS in an in vivo model, we generated different transgenic Drosophila lines expressing human FUS wild type (hFUS wt) and two disease-related variants P525L and R495X, in which the NLS is mutated or completely absent, respectively. ..."

Pogson JH, Ivatt RM, Sanchez-Martinez A, Tufi R, Wilson E, Mortiboys H, Whitworth AJ. The Complex I Subunit NDUFA10 Selectively Rescues Drosophila pink1 Mutants through a Mechanism Independent of Mitophagy. PLoS Genet. 2014 Nov 20;10(11):e1004815. doi: 10.1371/journal.pgen.1004815. PMID: 25412178; PMCID: PMC4238976.

Jahromi SR, Haddadi M, Shivanandappa T, Ramesh SR. Modulatory effect of Decalepis hamiltonii on ethanol-induced toxicity in transgenic Drosophila model of Parkinson's disease. Neurochem Int. 2014 Nov 5;80C:1-6. doi: PMID: 25451756.

Siddique YH, Faisal M, Naz F, Jyoti S, Rahul. Role of Ocimum sanctum leaf extract on dietary supplementation in the transgenic Drosophila model of Parkinson's disease. Chin J Nat Med. 2014 Oct;12(10):777-81. PMID: 25443371.

Fly study suggests role for cation-chloride cotransporters in glia in seizure disorders

Rusan ZM, Kingsford OA, Tanouye MA. Modeling Glial Contributions to Seizures and Epileptogenesis: Cation-Chloride Cotransporters in Drosophila melanogaster. PLoS One. 2014 Jun 27;9(6):e101117. PMID: 24971529; PMCID: PMC4074161.

From the abstract: "Flies carrying a kcc loss-of-function mutation are more seizure-susceptible than wild-type flies. ... Here, we examined the spatial and temporal requirements for kcc loss-of-function to modify seizure-susceptibility in flies. Targeted RNA interference (RNAi) of kcc in various sets of neurons was sufficient to induce severe seizure-sensitivity. Interestingly, kcc RNAi in glia was particularly effective in causing seizure-sensitivity. ... The findings presented are the first attributing a causative role for glial cation-chloride cotransporters in seizures and epileptogenesis. The importance of elucidating glial cell contributions to seizure disorders and the utility of Drosophila models is discussed."

New fly model of epilepsy. Sodium channels. Recent report.

Howlett IC, Rusan ZM, Parker L, Tanouye MA. Drosophila as a Model for Intractable Epilepsy: gilgamesh Suppresses Seizures in parabss1 Heterozygote Flies. G3 (Bethesda). 2013 Jun 24. PMID: 23797108.

New approach for study of learning and memory in flies. Relevance to disease-related study. Recent report.

The authors indicate relevance to "neurological pathologies such as Parkinson's disease, Alzheimer's disease, addiction, epilepsy and autism spectrum disorders."

Mejia M, Heghinian MD, Marí F, Godenschwege TA. New Tools for Targeted Disruption of Cholinergic Synaptic Transmission in Drosophila melanogaster. PLoS One. 2013 May 30;8(5):e64685. PMID: 23737994; PMCID: PMC3667824.