Mutant Flies Research Articles

Drosophila tweety facilitates autophagy to regulate mitochondrial homeostasis and bioenergetics in Glia.

Mitochondria support the energetic demands of the cells. Autophagic turnover of mitochondria serves as a critical pathway for mitochondrial homeostasis. It is unclear how bioenergetics and autophagy are functionally connected. Here, we identify an endolysosomal membrane protein that facilitates autophagy to regulate ATP production in glia. We determined that Drosophila tweety (tty) is highly expressed in glia and localized to endolysosomes. Diminished fusion between autophagosomes and endolysosomes in tty-deficient glia was rescued by expressing the human Tweety Homolog 1 (TTYH1). Loss of tty in glia attenuated mitochondrial turnover, elevated mitochondrial oxidative stress, and impaired locomotor functions. The cellular and organismal defects were partially reversed by antioxidant treatment. We performed live-cell imaging of genetically encoded metabolite sensors to determine the impact of tty and autophagy deficiencies on glial bioenergetics. We found that tty-deficient glia exhibited reduced mitochondrial pyruvate consumption accompanied by a shift toward glycolysis for ATP production. Likewise, genetic inhibition of autophagy in glia resulted in a similar glycolytic shift in bioenergetics. Furthermore, the survival of mutant flies became more sensitive to starvation, underlining the significance of tty in the crosstalk between autophagy and bioenergetics. Together, our findings uncover the role for tty in mitochondrial homeostasis via facilitating autophagy, which determines bioenergetic balance in glia.

Activation of cyclic AMP signaling pathway in dopaminergic neurons rescues locomotion defects in a Drosophila larval model of Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disease showing uncontrollable motor symptoms that are primarily caused by the progressive loss of dopaminergic neurons in the brain. Currently no treatment exists to prevent PD progression. Therefore, discovery of new neuroprotective strategies still has great potential to benefit PD patients. A handful of studies show that activation of cAMP pathways is neuroprotective against PD progression. However, the neuroprotective role of this signaling cascade specifically in DA neurons has not been explored. In this study, fruit fly Drosophila melanogaster was used because of its sophisticated and powerful genetic approaches, especially with related to cAMP signaling pathway. We have investigated molecular mechanisms of neuroprotection in a fly larval model of PD by administering an environmental PD toxin rotenone. Increased cAMP signaling in the dunce mutant fly carrying defects in phosphodiesterase (PDE) gene, is neuroprotective against rotenone-induced locomotion deficits. Furthermore, the neuroprotective role of cAMP signaling specifically in DA neurons has been studied as it has not been explored. By using transgenic flies expressing designer receptors exclusively activated by designer drugs (DREADDs), we have shown that an increase of cAMP levels in DA neurons rescues rotenone-induced locomotion deficits. We also showed that this neuroprotection is mediated by activation of Gαs and PKA-C1 subunits. The results provide novel findings that expand our knowledge of neuroprotective mechanisms in DA neurons affecting PD progression, which could contribute to the development of new therapeutic treatments against PD. An important future study will explore downstream targets of cAMP-PKA signaling.

PpGpp is present in, and functions to regulate sleep of, Drosophila

Sleep is essential for animals, and receives inputs from circadian, homeostasis, and environment, yet the mechanisms of sleep regulation remain elusive. Discovery of molecules in living systems and demonstration of their functional roles are pivotal in furthering our understanding of the molecular basis of biology. Here, we report that guanosine-5′-diphosphate, 3′-diphosphate (ppGpp) is present in Drosophila, and plays an important role in regulation of sleep and starvation-induced sleep loss (SISL). ppGpp is detected in germ-free Drosophila and hydrolyzed by an enzyme encoded by the mesh1 gene in Drosophila. Nighttime sleep and SISL were defected in mesh1 mutant flies, and rescued by expression of wildtype Mesh1, but not the enzymatically defective mutant Mesh1E66A. Ectopic expression of RelA, the Escherichia coli synthetase for ppGpp, phenocopied mesh1 knockout mutants, whereas overexpression of Mesh1 resulted in the opposite phenotypes, supporting that ppGpp is both necessary and sufficient in sleep regulation. A chemo connectomic screen followed by genetic intersection experiments implicates the Dilp2 neurons in the pars intercerebralis (PI) brain region as the site of ppGpp function. Our results have thus validated the presence of ppGpp in Drosophila and revealed a physiological role of ppGpp in sleep regulation for the first time.

Embryonic microinjection of ribonucleoprotein complex (Cas9+sgRNA) of whitegene in melon fly, Zeugodacuscucurbitae (Coquillett) (Diptera: Tephritidae) produced whiteeye phenotype.

Melon fly, Zeugodacus cucurbitae (Coquillett) is a major pest of cucurbitaceous crops, and causes substantial yield losses and economic costs. CRISPR/Cas9 is a rapid and effective site-specific genome editing tool for the generation of genetic changes that are stable and heritable. The CRISPR/Cas9 tool uses synthetically designed single guide RNA (sgRNA) that is complementary to the target gene and guides the Cas9 enzyme to perform nuclease activity by making double-strand breaks in the target DNA sequences. This tool can be effectively exploited to improve traits critical for the management of insect pests by targeting specific genes encoding these traits without the need of extensive genetic information. The white gene is an important gene responsible for the transport of body pigment precursor molecules. In this study, we produced effective mutagenesis of the white gene of Z. cucurbitae using the CRISPR/Cas9 tool with double sgRNAto target multiple sites of white to increase the efficiency in the generation of frame-shift mutations resulting in the white eye phenotype in adults. This was achieved through embryonic microinjection of the ribonucleoprotein (RNP) complex in the pre-blastoderm embryo stage 1 h after embryo laying. Our success with the production of a white eye mutant fly by CRISPR/Cas9 mutagenesis is important for the research on gene function and protein-level modifications in melon fly and forms the basis for the development of new genetic control strategies such as precision guided sterile insect technique (pgSIT) for this pest of economic significance.

PINK1 and Parkin rescue motor defects and mitochondria dysfunction induced by a patient-derived HSPB3 mutant in Drosophila models

Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones with the α-crystalline domain that is critical to their chaperone activity. Within the sHSP family, three (HSPB1, HSPB3, and HSPB8) proteins are linked with inherited peripheral neuropathies, including distal hereditary motor neuropathy (dHMN) and Charco-Marie-Tooth disease (CMT). In this study, we introduced the HSPB3 Y118H (HSPB3Y118H) mutant gene identified from the CMT2 family in Drosophila. With a missense mutation on its α-crystalline domain, this human HSPB3 mutant gene induced a loss of motor activity accompanied by reduced mitochondrial membrane potential in fly neuronal tissues. Moreover, mitophagy, a critical mechanism of mitochondrial quality control, is downregulated in fly motor neurons expressing HSPB3Y118H. Surprisingly, PINK1 and Parkin, the core regulators of mitophagy, successfully rescued these motor and mitochondrial abnormalities in HSPB3 mutant flies. Results from the first animal model of HSPB3 mutations suggest that mitochondrial dysfunction plays a critical role in HSPB3-associated human pathology.

The retrograde IFT dynein is required for normal function of diverse mechanosensory cilia in Drosophila.

Cilia biogenesis relies on intraflagellar transport (IFT), a conserved transport mechanism which functions bi-directionally to bring protein complexes to the growing ciliary tip and recycle signaling and transport proteins between the cilium and cell body. In Drosophila, anterograde IFT is critical for assembly of sensory cilia in the neurons of both chordotonal (ch) organs, which have relatively long ciliary axonemes, and external sensory (es) organs, which have short axonemal segments with microtubules in distal sensory segments forming non-axonemal bundles. We previously isolated the beethoven (btv) mutant in a mutagenesis screen for auditory mutants. Although many btv mutant flies are deaf, some retain a small residual auditory function as determined both by behavior and by auditory electrophysiology. Here we molecularly characterize the btv gene and demonstrate that it encodes the IFT-associated dynein-2 heavy chain Dync2h1. We also describe morphological changes in Johnston's organ as flies age to 30 days, and we find that morphological and electrophysiological phenotypes in this ch organ of btv mutants become more severe with age. We show that NompB protein, encoding the conserved IFT88 protein, an IFT complex B component, fails to be cleared from chordotonal cilia in btv mutants, instead accumulating in the distorted cilia. In macrochaete bristles, a class of es organ, btv mutants show a 50% reduction in mechanoreceptor potentials. Thus, the btv-encoded Dync2h1 functions as the retrograde IFT motor in the assembly of long ciliary axonemes in ch organs and is also important for normal function of the short ciliary axonemes in es organs.

Non-gonadal somatic piRNA pathways ensure sexual differentiation, larval growth, and wing development in silkworms.

PIWI-interacting RNAs (piRNAs) guide PIWI proteins to target transposons in germline cells, thereby suppressing transposon activity to preserve genome integrity in metazoans' gonadal tissues. Piwi, one of three Drosophila PIWI proteins, is expressed in the nucleus and suppresses transposon activity by forming heterochromatin in an RNA cleavage-independent manner. Recently, Piwi was reported to control cell metabolism in Drosophila fat body, providing an example of piRNAs acting in non-gonadal somatic tissues. However, mutant flies of the other two PIWI proteins, Aubergine (Aub) and Argonaute3 (Ago3), show no apparent phenotype except for infertility, blurring the importance of the piRNA pathway in non-gonadal somatic tissues. The silkworm, Bombyx mori, possesses two PIWI proteins, Siwi (Aub homolog) and BmAgo3 (Ago3 homolog), whereas B. mori does not have a Piwi homolog. Siwi and BmAgo3 are mainly expressed in gonadal tissues and play a role in repressing transposon activity by cleaving transposon RNA in the cytoplasm. Here, we generated Siwi and BmAgo3 loss-of-function mutants of B. mori and found that they both showed delayed larval growth and failed to become adult moths. They also exhibited defects in wing development and sexual differentiation. Transcriptome analysis revealed that loss of somatic piRNA biogenesis pathways results in abnormal expression of not only transposons but also host genes, presumably causing severe growth defects. Our results highlight the roles of non-gonadal somatic piRNAs in B. mori development.

Identifying genetic factors that increase cognitive reserve: A theoretical approach

Studies have demonstrated that some individuals display pathological hallmarks of Alzheimer's disease (AD) but are not afflicted with cognitive decline. The ability to maintain cognitive function despite the presence of pathology is referred to as cognitive reserve. This project aims to identify the molecular pathways involved in cognitive reserve using Drosophila melanogaster (Drosophila) models of AD. Specifically, a theoretical approach using experimental evolution to drive a population of AD-like Drosophila carrying a tau mutation to develop cognitive reserve is proposed. To accomplish this, a population of AD-like Drosophila will be placed in a single population cage along with wild-type flies and forced to compete for food and water. The first generation of AD-like Drosophila will be generated using random mutagenesis of the initially isogenic AD-like fly. The selected tau mutant displays a rough eye condition which allows for easy distinction between tau mutant and wild-type flies. It is hypothesised that AD-like flies with cognitive decline will be unable to survive because their limited cognitive abilities will prevent them from effectively competing for food and water. In contrast, AD-like flies with mutations that promote cognitive reserve will be better capable of survival. After 90-99% of mutant flies have died, the surviving mutant flies will be back-crossed to the P1 mutant to maintain tau mutation stability. It is expected that artificial selection will result in the creation of a generation of tau mutant flies that demonstrate cognitive abilities comparable to those of wild-type flies despite maintaining an AD-like tau mutation. This approach will monitor the successful trajectory of the evolution of increased cognitive reserve through survival curve analysis and measures of cognition. A limitation of the method is that only a dominant mutation or series of dominant mutations would be identified using this approach.

Identification and functional analysis of CG3526 in spermatogenesis of Drosophila melanogaster.

Spermatogenesis is a critical part of reproduction in insects; however, its molecular mechanism is still largely unknown. In this study, we identified a testis-specific gene CG3526 in Drosophila melanogaster. Bioinformatics analysis showed that CG3526 contains a zinc binding domain and 2 C2 H2 type zinc fingers, and it is clustered to the vertebrate really interesting new gene (RING) family E3 ubiquitin-protein ligases. When CG3526 was knocked down by RNA interference (RNAi), the testis became much smaller in size, and the apical tip exhibited a sharp and thin end instead of the blunt and round shape in the control testis. More importantly, compared to the control flies, only a few mature sperm were present in the seminal vesicle of C587-Gal4>CG3526 RNAi flies. Immunofluorescence staining of the testis from CG3526 RNAi flies showed that the homeostasis of testis stem cell niche was disrupted, cell distribution in the apical tip was scattered, and the process of spermatogenesis was not completed. Furthermore, we found that the phenotype of CG3526 RNAi flies' testis was similar to that of testis of Stat92E RNAi flies, the expression level of CG3526 was significantly downregulated in the Stat92EF06346 mutant flies, and the promoter activity of CG3526 was upregulated by STAT92E. Taken together, our results indicated that CG3526 is a downstream effector gene in the JAK-STAT signaling pathway that plays a key role in the spermatogenesis of Drosophila.

Loss of STING in parkin mutant flies suppresses muscle defects and mitochondria damage.

The early pathogenesis and underlying molecular causes of motor neuron degeneration in Parkinson's Disease (PD) remains unresolved. In the model organism Drosophila melanogaster, loss of the early-onset PD gene parkin (the ortholog of human PRKN) results in impaired climbing ability, damage to the indirect flight muscles, and mitochondrial fragmentation with swelling. These stressed mitochondria have been proposed to activate innate immune pathways through release of damage associated molecular patterns (DAMPs). Parkin-mediated mitophagy is hypothesized to suppress mitochondrial damage and subsequent activation of the cGAS/STING innate immunity pathway, but the relevance of this interaction in the fly remains unresolved. Using a combination of genetics, immunoassays, and RNA sequencing, we investigated a potential role for STING in the onset of parkin-null phenotypes. Our findings demonstrate that loss of Drosophila STING in flies rescues the thorax muscle defects and the climbing ability of parkin-/- mutants. Loss of STING also suppresses the disrupted mitochondrial morphology in parkin-/- flight muscles, suggesting unexpected feedback of STING on mitochondria integrity or activation of a compensatory mitochondrial pathway. In the animals lacking both parkin and sting, PINK1 is activated and cell death pathways are suppressed. These findings support a unique, non-canonical role for Drosophila STING in the cellular and organismal response to mitochondria stress.

New mutant alleles for Spargel/dPGC-1 highlights the function of Spargel RRM domain in oogenesis and expands the role of Spargel in embryogenesis and intracellular transport.

Energy metabolism in vertebrates is controlled by three members of the PGC-1 (PPAR γ- coactivator 1) family, transcriptional coactivators that shape responses to physiological stimuli by interacting with the nuclear receptors and other transcription factors. Multiple evidence now supports that Spargel protein found in insects and ascidians is the ancestral form of vertebrate PGC-1's. Here, we undertook functional analysis of srl gene in Drosophila, asking about the requirement of Spargel per se during embryogenesis and its RNA binding domains. CRISPR- engineered srl gene deletion turned out to be an amorphic allele that is late embryonic/early larval lethal and Spargel protein missing its RNA binding domain (SrlΔRRM) negatively affects female fertility. Overexpression of wild-type Spargel in transgenic flies expedited the growth of egg chambers. On the other hand, oogenesis is blocked in a dominant-negative fashion in the presence of excess Spargel lacking its RRM domains. Finally, we observed aggregation of Notch proteins in egg chambers of srl mutant flies, suggesting that Spargel is involved in intracellular transport of Notch proteins. Taken together, we claim that these new mutant alleles of spargel are emerging powerful tools for revealing new biological functions for Spargel, an essential transcription coactivator in both Drosophila and mammals.

Damage sensing mediated by serine proteases Hayan and Persephone for Toll pathway activation in apoptosis-deficient flies.

The mechanisms by which the innate immune system senses damage have been extensively explored in multicellular organisms. In Drosophila, various types of tissue damage, including epidermal injury, tumor formation, cell competition, and apoptosis deficiency, induce sterile activation of the Toll pathway, a process that requires the use of extracellular serine protease (SP) cascades. Upon infection, the SP Spätzle (Spz)-processing enzyme (SPE) cleaves and activates the Toll ligand Spz downstream of two paralogous SPs, Hayan and Persephone (Psh). However, upon tissue damage, it is not fully understood which SPs establish Spz activation cascades nor what damage-associated molecules can activate SPs. In this study, using newly generated uncleavable spz mutant flies, we revealed that Spz cleavage is required for the sterile activation of the Toll pathway, which is induced by apoptosis-deficient damage of wing epidermal cells in adult Drosophila. Proteomic analysis of hemolymph, followed by experiments with Drosophila Schneider 2 (S2) cells, revealed that among hemolymph SPs, both SPE and Melanization Protease 1 (MP1) have high capacities to cleave Spz. Additionally, in S2 cells, MP1 acts downstream of Hayan and Psh in a similar manner to SPE. Using genetic analysis, we found that the upstream SPs Hayan and Psh contributes to the sterile activation of the Toll pathway. While SPE/MP1 double mutants show more impairment of Toll activation upon infection than SPE single mutants, Toll activation is not eliminated in these apoptosis-deficient flies. This suggests that Hayan and Psh sense necrotic damage, inducing Spz cleavage by SPs other than SPE and MP1. Furthermore, hydrogen peroxide, a representative damage-associated molecule, activates the Psh-Spz cascade in S2 cells overexpressing Psh. Considering that reactive oxygen species (ROS) were detected in apoptosis-deficient wings, our findings highlight the importance of ROS as signaling molecules that induce the activation of SPs such as Psh in response to damage.

Disease-Modifying Effects of Vincamine Supplementation in Drosophila and Human Cell Models of Parkinson's Disease Based on DJ-1 Deficiency.

Parkinson's disease (PD) is an incurable neurodegenerative disorder caused by the selective loss of dopaminergic neurons in the substantia nigra pars compacta. Current therapies are only symptomatic and are not able to stop or delay its progression. In order to search for new and more effective therapies, our group carried out a high-throughput screening assay, identifying several candidate compounds that are able to improve locomotor ability in DJ-1β mutant flies (a Drosophila model of familial PD) and reduce oxidative stress (OS)-induced lethality in DJ-1-deficient SH-SY5Y human cells. One of them was vincamine (VIN), a natural alkaloid obtained from the leaves of Vinca minor. Our results showed that VIN is able to suppress PD-related phenotypes in both Drosophila and human cell PD models. Specifically, VIN reduced OS levels in PD model flies. Besides, VIN diminished OS-induced lethality by decreasing apoptosis, increased mitochondrial viability, and reduced OS levels in DJ-1-deficient human cells. In addition, our results show that VIN might be exerting its beneficial role, at least partially, by the inhibition of voltage-gated sodium channels. Therefore, we propose that these channels might be a promising target in the search for new compounds to treat PD and that VIN represents a potential therapeutic treatment for the disease.

Сerebrolysin® influences in Sod- and sws-dependent neurodegenerative models of Drosophila melano­gaster

Background. The incidence of human neurodegenerative disorders increases continuously as the human population ages. To date, these diseases remain incurable and require complex experimental approaches using tractable models to study the degeneration mechanisms and potential drug intervention regimens. In the current work, we assessed the impact of the neuroprotective drug Сerebrolysin on these neurodegenerative processes in Drosophila Sod1 and swiss cheese (sws) mutants. Materials and Methods. The experiments were conducted using a D. melano­gaster Sod1- and sws-dependent neurodegeneration model. Сerebrolysin (3 μL/mL) was added for larvae feeding. In order to evaluate Сerebrolysin influence, several tests were performed: locomotor activity assay, lifespan, size of brain tissue degeneration zones and sensitivity to prooxidant exposion. Results. Dietary supplementation with Сerebrolysin extended the lifespan of all flies under normal circumstances. The drug treatment also reduced the sensitivity of mutant flies to pro-oxidant effects. Moreover, treatment with Сerebrolysin partially diminished the size of degeneration zones in the brain tissue of sws1 mutant flies, without any notable effects on locomotor ability. Conclusions. The data obtained confirm the moderate neuroprotective and/or antioxidant action of Сerebrolysinagainst neurodegenerative processes under different genetic backgrounds.

Kanamycin treatment in the pre-symptomatic stage of a Drosophila PD model prevents the onset of non-motor alterations

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor alterations, which is preceded by a prodromal stage where non-motor symptoms are observed. Over recent years, it has become evident that this disorder involves other organs that communicate with the brain like the gut. Importantly, the microbial community that lives in the gut plays a key role in this communication, the so-called microbiota-gut-brain axis. Alterations in this axis have been associated to several disorders including PD.Here we proposed that the gut microbiota is different in the presymptomatic stage of a Drosophila model for PD, the Pink1B9 mutant fly, as compared to that observed in control animals. Our results show this is the case: there is basal dysbiosis in mutant animals evidenced by substantial difference in the composition of midgut microbiota in 8–9 days old Pink1B9 mutant flies as compared with control animals. Further, we fed young adult control and mutant flies kanamycin and analyzed motor and non-motor behavioral parameters in these animals. Data show that kanamycin treatment induces the recovery of some of the non-motor parameters altered in the pre-motor stage of the PD fly model, while there is no substantial change in locomotor parameters recorded at this stage. On the other hand, our results show that feeding young animals the antibiotic, results in a long-lasting improvement of locomotion in control flies.Our data support that manipulations of gut microbiota in young animals could have beneficial effects on PD progression and age-dependent motor impairments.This article is part of the Special Issue on “Microbiome & the Brain: Mechanisms & Maladies”.

Curcumin-mediated gene expression changes in Drosophila melanogaster

Background: Curcumin has been suggested to be useful in the treatment of numerous diseases, including autoinflammation, that has been implicated in some pathological conditions. Experimentally, autoinflammatory phenotypes were observed in the short-lived Peptidoglycan recognition protein LB (PGRP-LB) mutant of Drosophila melanogaster. Objective: This study aimed to determine the effect of curcumin on the expression of ageing-related genes in the autoinflammatory model of D. melanogaster. Method: This study was performed using five test groups including untreated control, solvent control, and three groups given a series of curcumin concentrations: 10 µM, 50 µM, and 250 µM, separately. Survival assay and gene expression studies were carried out on these test groups. Result: The results revealed that the lifespan of the curcumin-treated groups was significantly improved in comparison to the control groups. Such phenotype was accompanied by the increased expression of srl and hsp22 genes in most, if not all, of the curcumin-treated groups and elevated expression of tom40, pepck, and cat genes was specifically detectable only in groups treated with 250 µM curcumin. On the contrary, the expression of indy was significantly reduced upon the administration of curcumin at all given concentrations. Conclusion: Based on these results, it can be inferred that supplementation of curcumin can improve the lifespan of the PGRP-LB mutant flies and this might be related to the changes in the expression of ageing-related genes.

Juan Modolell (1937-2023).

Juan Modolell (1937-2023).

Understanding how GBA mutations influence Parkinson’s disease progression (S42.003)

<h3>Objective:</h3> To investigate mechanisms underlying the clinical observation that GBA mutations are associated with increased risk of developing PD, along with faster progression of motor and cognitive symptoms. <h3>Background:</h3> Mutations in the gene <i>glucosidase, beta acid 1</i> (<i>GBA</i>) are the strongest genetic risk factor for Parkinson’s Disease (PD) and accelerate disease progression. Our work using a <i>Drosophila GBA</i> deficient model revealed altered exosomes may act as vehicles to accelerate protein aggregate spread. We are further investigating how <i>GBA</i> affects neuronal endolysosomal trafficking and exosome biogenesis. <h3>Design/Methods:</h3> We developed a <i>Drosophila</i> model of <i>GBA</i> deficiency (<i>GBA</i>^<i>del</i>) by deleting the <i>Drosophila</i> homolog of <i>GBA</i>. Human induced pluripotent stem cells (iPSCs) were generated from an individual with PD carrying the IVS2+1G&gt;A <i>GBA</i> mutation (<i>GBA</i>^<i>IVS</i> PD). Neurons were differentiated from <i>GBA</i>^<i>IVS</i> PD, isogenic <i>GBA</i>^<i>WT</i> PD, and age- and sex-matched healthy control iPSCs using StemCell Technologies reagents and protocols. Confirmation for differentiation was performed by IHC. Neuronal EVs are isolated by size exclusion chromatography from conditioned media. <h3>Results:</h3> Exosomes isolated from <i>GBA</i>^<i>del</i> mutant flies have altered protein cargo, including increased levels of exosome-intrinsic proteins Rab11 and Rab7, and increased oligomerized Ref(2)p, the Drosophila ortholog for p62. Expression of wildtype d<i>GBA1b</i> in flight muscle or glia of <i>GBA</i>^<i>del</i> mutant flies rescued protein aggregation in the brain, and also rescued levels of exosomal Rab11, Rab 7 and Ref(2)p. <h3>Conclusions:</h3> Our Drosophila model supports the hypothesis that <i>GBA</i> deficiency alters exosomes, which may act as a vehicle to accelerate the spread of Lewy pathology. We are now examining how <i>GBA</i> alters endolysosomal trafficking leading to exosome biogenesis in our iPSC model, and how altered exosomes can be a vehicle for Lewy pathology propagation. This could elucidate mechanisms to halt or slow down the spread of pathogenic protein aggregation in PD. <b>Disclosure:</b> Mr. Khera has nothing to disclose. Ms. Fish has nothing to disclose. Ms. Estes has nothing to disclose. Selina Yu has nothing to disclose. Leo Pallanck has nothing to disclose. The institution of Dr. Young has received research support from NIH. The institution of Dr. Young has received research support from BrightFocus Foundation. The institution of Dr. Young has received research support from Biogen. The institution of Dr. Davis has received research support from NIH NINDS. The institution of Dr. Davis has received research support from University of Washington Institute for Stem Cell and Regenerative Medicine. The institution of Dr. Davis has received research support from VA BLRD. Dr. Davis has received personal compensation in the range of $500-$4,999 for serving as a study section grant reviewer with NIH. Dr. Davis has received personal compensation in the range of $500-$4,999 for serving as a Grant reviewer with Parkinson’s Foundation.

A role of cryptochrome for magnetic field-dependent improvement of sleep quality, lifespan, and motor function in Drosophila.

Understanding the molecular genetic basis of animal magnet reception has been one of the big challenges in molecular biology. Recently it was discovered that the magnetic sense of Drosophila melanogaster is mediated by the ultraviolet (UV)-A/blue light photoreceptor cryptochrome (Cry). Here, using the fruit fly as a magnet-receptive model organism, we show that the magnetic field exposure (0.4-0.6 mT) extended lifespan under starvation, but not in cryptochrome mutant flies (cryb ). The magnetic field exposure increases motor function in wild type and neurodegenerative disease model flies. Furthermore, the magnetic field exposure improved sleep quality at night-time specific manner, but not in cryb . We also showed that repeated AC magnetic field exposure increased climbing activity in wild-type Drosophila, but not in cryb . The data suggests that magnetic field-dependent improvement of lifespan, sleep quality, and motor function is mediated through a cry-dependent pathway in Drosophila.

Efficient strategies based on behavioral and electrophysiological methods for epilepsy-related gene screening in the Drosophila model.

With the advent of trio-based whole-exome sequencing, the identification of epilepsy candidate genes has become easier, resulting in a large number of potential genes that need to be validated in a whole-organism context. However, conducting animal experiments systematically and efficiently remains a challenge due to their laborious and time-consuming nature. This study aims to develop optimized strategies for validating epilepsy candidate genes using the Drosophila model. This study incorporate behavior, morphology, and electrophysiology for genetic manipulation and phenotypic examination. We utilized the Gal4/UAS system in combination with RNAi techniques to generate loss-of-function models. We performed a range of behavioral tests, including two previously unreported seizure phenotypes, to evaluate the seizure behavior of mutant and wild-type flies. We used Gal4/UAS-mGFP flies to observe the morphological alterations in the brain under a confocal microscope. We also implemented patch-clamp recordings, including a novel electrophysiological method for studying synapse function and improved methods for recording action potential currents and spontaneous EPSCs on targeted neurons. We applied different techniques or methods mentioned above to investigate four epilepsy-associated genes, namely Tango14, Klp3A, Cac, and Sbf, based on their genotype-phenotype correlation. Our findings showcase the feasibility and efficiency of our screening system for confirming epilepsy candidate genes in the Drosophila model. This efficient screening system holds the potential to significantly accelerate and optimize the process of identifying epilepsy candidate genes, particularly in conjunction with trio-based whole-exome sequencing.