Journal of Neurogenetics

Misexpression of the E3 ubiquitin ligase UBE3Ais thought to contribute to a range of neurological disorders. In the context of Dup15q syndrome, excess genomic copies of UBE3A is thought to contribute to the autism, muscle tone and spontaneous seizures characteristic of the disorder. In a Drosophila model of Dup 15q syndrome, it was recently shown glial-driven expression of the UBE3A ortholog dube3a led to a "bang-sensitive" phenotype, where mechanical shock triggers convulsions, suggesting glial dube3a expression contributes to hyperexcitability in flies. Here we directly compare the consequences of glial- and neuronal-driven dube3a expression on motor coordination and neuronal excitability in Drosophila. We utilized IowaFLI tracker and developed a hidden Markov Model to classify seizure-related immobilization. Both glial and neuronal driven dube3a expression led to clear motor phenotypes. However, only glial-driven dube3a expression displayed spontaneous immobilization events, that were exacerbated at high-temperature (38 {degrees}C). Using a tethered fly preparation we monitored flight muscle activity, we found glial-driven dube3a flies display spontaneous spike discharges which were bilaterally synchronized indicative of seizure activity. Neither control flies, nor neuronal-dube3a overexpressing flies display such firing patterns. Prior drug screen indicated bang-sensitivity in glial-driven dube3a expressing flies could be suppressed by certain 5-HT modulators. Consistent with this report, we found glial-driven dube3a flies fed the serotonin reuptake inhibitor vortioxetine and the 5HT2A antagonist ketanserin displayed reduced immobilization and spike bursting. Together these findings highlight the potential for glial pathophysiology to drive Dup15q syndrome-related seizure activity.

The dauer decision in Caenorhabditis elegans is a critical developmental decision that ensures survival under harsh environmental conditions. Factors such as temperature, food availability, and pheromone strongly influence the decision to enter and exit dauer. Traditional assays that assess the dauer decision are often confounded by the influence of pheromones from the population, which are often dynamic and highly variable. To mitigate this issue, we developed a simple, single-housing assay for dauer quantification that is compatible with optogenetics. We show that insulin-like peptides (ILPs) from ASJ and other neurons strongly influence the decision to exit dauer, and that ASJ activity can be manipulated with optogenetics to influence the dauer decision in a temporally precise manner.

Drosophila is widely used to study the pathological mechanisms of human diseases in vivo, including metabolic and neurological disorders. In these models, disease-induced alterations in locomotion and stress resistance are generally monitored in comparison to healthy control flies, such the white-eyed strain w1118, used as a reference for normal physiology and behavior. Here we compared two independent w1118 lines and found that they differed strikingly in their susceptibility to oxidative stress and nutrient starvation, and less markedly in their locomotor performance. Interestingly, modulating the gut microbiome by rearing these flies under axenic conditions increased oxidative stress resistance of the more susceptible, but not the more resistant line, while it had no effect on starvation resistance for both lines. We also found that the stress-sensitive line had higher levels of Clostridiales bacteria and of the intracellular endosymbiont Wolbachia in the gut microbiota, as well as lower expression levels of immune effectors (antimicrobial peptides and lysozymes) in the head and gut. Both lines nevertheless showed similar susceptibility to pathogenic bacterial infections. In a transgenic Parkinsons disease model, the stress-resistant background strongly attenuated the progressive locomotor defects induced by pan-neuronal expression of human mutant -synuclein, but intriguingly not when -synuclein expression was restricted to a subset of brain dopaminergic neurons in the protocerebral anterior medial (PAM) cluster. These results suggest that taking into account unapparent features of the reference lines could improve the reproducibility and consistency of neurodegenerative disease models in Drosophila.

The genetic tractability, well-mapped circuitry, and diverse behavioral repertoire of the nematode C. elegans make it an ideal model for physiological and behavioral studies. A wide range of methods has been developed for analyzing C. elegans behaviors, evolving with advances in technology such as videography and computer-assisted analysis. However, unlike organisms with distinct body features such as limbs and wings, the contour of C. elegans is rather uniform, posing unique challenges for automated analyses of C. elegans behavior. Here, we introduce LabGym--an open-source, artificial intelligence (AI)-based platform we recently developed--to the C. elegans research community. We trained deep learning models in LabGym capable of automatically categorizing and quantifying multiple user-defined parameters of worm locomotion behavior in multi-worm videos with high accuracy. Furthermore, we demonstrated their efficacy in quantifying locomotion changes in aging worms. Our work offers a cost-effective, user-accessible, and comprehensive approach to behavioral analysis in C. elegans.

Animals balance staying in a favorable environment with exploring new ones. In C. elegans chemotaxis, the process by which worms migrate toward an attractant has been extensively studied. However, what happens after they reach it remains largely unexplored, partly because conventional assays immobilize worms at the point of arrival. Here, we quantitatively analyzed chemotactic behavior upon reaching an attractive odor source using an immobilization-free chemotaxis assay. We observed that 62% animals left the isoamyl alcohol region after initially approaching it, a behavior we termed "leaving behavior." Quantitative analysis revealed that leaving behavior represents a distinct locomotor state compared with free-moving, high-concentration odor avoidance, and approach behavior. To test whether leaving behavior is related to olfactory adaptation, we analyzed mutants in adaptation-related genes. The proportion of leaving behavior was significantly increased in egl-4 loss-of-function mutants compared with wild-type animals, whereas arr-1 mutants showed no significant difference. These results suggest that egl-4 negatively regulates leaving behavior, suggesting a role for this kinase in stabilizing post-arrival behavioral states beyond its known function in olfactory adaptation. Our findings indicate that chemotaxis involves dynamic behavioral transitions even after reaching an attractant, consistent with an exploration-exploitation trade-off framework.

Understanding how diverse cell types come together to form a functioning brain relies on the ability to specifically target these cells. This is often done using genetic tools such as the GAL4/UAS system in Drosophila melanogaster. Surprisingly, despite its extensive usage during studies of the ageing brain, detailed spatio-temporal characterisation of GAL4 driver lines in adult flies has been lacking. Here we show that three commonly used neuronal drivers (elav[C155]-GAL4, nSyb[R57C10]-GAL4 and ChAT-GAL4) and the commonly used glial driver repo-GAL4 all show rapid and pronounced decreases in activity over the first 1.5 weeks of adult life, with activity becoming undetectable in some regions after 30 days. In addition to an overall decrease in GAL4 activity over time, we found notable differences in spatial patterns, mostly occurring soon after eclosion. Although all lines showed these changes, the nSyb-GAL4 line exhibited the most consistent and stable expression patterns over ageing. Our findings suggest that gene transcription of key loci decreases in the aged brain, a finding broadly similar to previous work in mammalian brains. Our results also raise questions over past work on long-term expression of disease models in the brain, and stress the need to find better genetic tools for ageing studies.

Genetic defects in AP2M1, which encodes the -subunit of the adaptor protein complex 2 (AP-2) essential for clathrin-mediated endocytosis (CME), cause a rare form of developmental and epileptic encephalopathy (DEE). In this study, we modeled AP2M1-DEE in Drosophila melanogaster to gain deeper insights into the underlying disease mechanisms. Pan-neuronal knock-down of the Drosophila AP2M1 ortholog, AP-2{micro}, resulted in a consistent heat-sensitive paralysis phenotype and altered morphology in class IV dendritic arborization (c4da) neurons. Unexpectedly, affected flies were resistant to antiseizure medications and exhibited increased resistance to electrically induced seizures. A CRISPR-engineered fly line carrying the recurrent human disease variant p.Arg170Trp displayed a milder seizure resistance phenotype. While these findings contrast with the human phenotype, they align with previous studies on other CME-related genes in Drosophila. Our results suggest that hyperexcitability and seizures in AP2M1-DEE may stem from broader defects in neuronal development rather than direct synaptic dysfunction.

Recent research evidence demonstrates that the inability to orient oneself and navigate space is an early indicator of Alzheimers Disease. The video game Sea Hero Quest (SHQ) was designed to assess the players navigation ability, and several research works analysed the SHQ data using simple metrics such as length and time of navigation paths. Expanding these analyses, we propose new performance metrics that capture the geometry of paths, and analyse datasets of more than 60,000 navigators. The metrics identify players who failed the navigation task, the dementia patients, and carriers of the at-risk allele of the Apolipoprotein-E [APOE]. Furthermore the metrics detect weak navigation ability when only a fraction of navigation paths are used, with superior performance to baseline methods. Our findings demonstrate that the proposed performance metrics pave the way to a comprehensive pre-clinical screening toolbox for Alzheimers Disease. TEASERWe propose geometric methods to capture decline in navigation ability from dementia.

Animals sleep more when they are sick. In C. elegans, stress-induced sleep (SIS) follows cellular injury such as exposure to ultraviolet (UV) light. The genetic regulators of SIS remain incompletely defined. Using a worm-picking robot, multi-well WorMotel imaging, and association analysis we performed a semi-automated screen of 941 whole-genome-sequenced Million Mutation Project (MMP) strains. We quantified behavioral activity and quiescence before and after ultraviolet (UV) radiation. We applied the Sequence Kernel Association Test (SKAT) to this behavioral data to prioritize 6,663 genes and observed significant enrichment of known SIS genetic regulators. Based on these results, we conducted a candidate validation screen for additional genes regulating SIS. We identified three genes (strd-1, egl-8, cla-1), mutations in which reproducibly influence SIS. Further exploration of these genes holds potential for enhancing our understanding of the molecular basis of SIS. These findings establish a pipeline for automated behavioral phenotyping coupled with gene-based association to accelerate studies of C. elegans neurogenetics.

While astrocytes are known to be important for development and nourishment of the nervous system, the field is just beginning to explore how astrocytes respond to environmental stimuli. Using Caenorhabditis elegans and their astrocyte-equivalent, CEPsh, we asked whether astrocyte-like glia respond to odor exposure. We found one day-old adult C. elegans decrease hlh-17 promoter (CEPsh glia marker) mediated fluorescent expression when trained with an innately attractive odor butanone. Moreover, the olfactory training paradigm itself affects phlh-17 expression, but in a different way. This suggests astrocyte-like CEPsh glia can integrate environmental information to respond to changes in the environment, which enhances survival.

Parkinsons disease is a complex and multifactorial disorder, but how its biological and clinical complexity emerge from molecular to macroscopic brain interactions remains poorly understood. Here, we use a personalized multiscale generative brain model to characterize direct spatiotemporal links between genes and multimodal neuroimaging-derived biological factors in PD. We identified a set of genes modulating PD-associated longitudinal changes in dopamine transporter level, neuronal activity, dendrite density and tissue atrophy. Inter-individual heterogeneity in the gene-mediated biological mechanisms is associated with five distinct configurations of PD motor and non-motor symptoms. Although characterized by distinctive biological pathways, all the symptom configurations are associated with cell cycle processes. Notably, the protein-protein interaction networks underlying these configurations revealed distinct hub genes including MYC, CCNA2, CCDK1, SRC, STAT3 and PSMD4. We also uncovered the biological mechanisms associated with physical activities performance in PD, and observed that leisure and work activities are principally related to neurotypical cholesterol homeostasis and inflammatory response processes, respectively. Finally, patient-tailored in silico gene perturbations revealed a set of putative disease-modifying drugs with potential to effectively treat PD, most of which are associated with dopamine reuptake and anti-inflammation. Our study constitutes the first self-contained multiscale approach providing comprehensive insights into the complex multifactorial pathogenesis of PD, unravelling key biological modulators of physical and clinical deterioration, and serving as a blueprint for optimum drug selection at personalized level.

In several neurodegenerative diseases including Alzheimers disease (AD), tau, a microtubule-associated protein (MAP) enriched in the axon, becomes hyperphosphorylated, detaches from microtubules, redistributes to the somato-dendritic compartment and self-aggregates. The mechanisms leading to neuronal dysfunction and death by tau pathology remain to be fully elucidated. C. elegans has been successfully used by several groups including ours to identify mechanisms involved in neurodegeneration. We generated three strains, one overexpressing wild-type human tau (WT Tau), one a tau mutant mimicking hyperphosphorylation (hyperP Tau) and one preventing phosphorylation (hypoP Tau) in GABA motor neurons. A significant reduction of body size and egg laying was noted in these tau strains. Starting at day 1, we found that the worms overexpressing hyperP Tau were smaller than the N2 control strain and the worms either overexpressing WT Tau or hypoP Tau. Starting at day 5, the worms overexpressing WT Tau were smaller than control and the worms overexpressing hypoP Tau. Egg laying was reduced in both hyperP Tau and WT Tau worms. Survival was only decreased in WT Tau worms. Motility deficits were also observed. For age-dependent paralysis, a difference was noted between control and hyperP Tau. Swimming activity and speed were increased in hypoP Tau and decreased in hyperP Tau strains. Axonal integrity was altered in all tau strains. In the case of synaptic activity, at day 1, it was increased in the hypoP Tau strain and decreased in the hyperP Tau one. Collectively, our data revealed that overexpression of tau exerted neuronal and peripheral defects indicating that tau dysfunction could affect cell-cell communication.

When exposed to noxious thermal or mechanical stimuli, Drosophila melanogaster larvae will aZempt to escape by performing a stereotypical nocifensive rolling behavior. Here, we report the identification of a mutation in Drosophila that specifically alters the thermal nocifensive escape behaviors of larvae. We provide genetic, molecular, and histological evidence that this mutation maps to the FucTA gene, which encodes an -1,3-fucosyltransferase. Our results suggest that fucosylation is important for the thermal nociception rolling behavior of Drosophila larvae.

Molecular tool development in traditionally non-tractable animals opens new avenues to study gene functions in the relevant ecological context. Entomopathogenic nematodes (EPN) Steinernema and their symbiotic bacteria of Xenorhabdus spp are a valuable experimental system in the laboratory and are applicable in the field to promote agricultural productivity. The infective juvenile (IJ) stage of the nematode packages mutualistic symbiotic bacteria in the intestinal pocket and invades insects that are agricultural pests. The lack of consistent and heritable genetics tools in EPN targeted mutagenesis severely restricted the study of molecular mechanisms underlying both parasitic and mutualistic interactions. Here, I report a protocol for CRISPR-Cas9 based genome-editing that is successful in two EPN species, S. carpocapsae and S. hermaphroditum. I adapted a gonadal microinjection technique in S. carpocapsae, which created on-target modifications of a homologue Sc-dpy-10 (cuticular collagen) by homology-directed repair. A similar delivery approach was used to introduce various alleles in S. hermaphroditum including Sh-dpy-10 and Sh-unc-22 (a muscle gene), resulting in visible and heritable phenotypes of dumpy and twitching, respectively. Using conditionally dominant alleles of Sh-unc-22 as a co-CRISPR marker, I successfully modified a second locus encoding Sh-Daf-22 (a homologue of human sterol carrier protein SCPx), predicted to function as a core enzyme in the biosynthesis of nematode pheromone that is required for IJ development. As a proof of concept, Sh-daf-22 null mutant showed IJ developmental defects in vivo (in insecta). This research demonstrates that Steinernema spp are highly tractable for targeted mutagenesis and has great potential in the study of gene functions under controlled laboratory conditions within the relevant context of its ecological niche.

Drosophila adult and larvae exhibit sophisticated behaviors that are widely used in development, synaptic transmission, sensory physiology, and learning and memory research. Many of these behaviors depend on locomotion, the ability of an animal to move. However, the statistical analysis of locomotion is not trivial. Here we use an open-source Fiji plugin TrackMate to track the locomotion of Drosophila adults and larvae. We build optimal experimental setups to rapidly process recordings by Fiji and analyze by TrackMate. We also provide tips for analyzing non-optimal recordings. TrackMate extracts the X and Y positions of an animal on each frame of an image sequence or a video. This information allows for generating moving trajectories, calculating moving distances, and determining preference indices in two-choice assays. Notably, this free-cost analysis method does not require programming skills. Summary statementThis study uses an open-source Fiji plugin TrackMate to computationally analyze Drosophila adult and larval behavioral assays, which does not require programming skills.

Epilepsy affects approximately 30% of individuals with autism spectrum disorder (ASD). Consistent with these observations, while PRICKLE mutations are primarily linked with epilepsy, there is an enrichment of pathogenic DNA sequence variants in PRICKLE genes carried by individuals with ASD. Nonetheless, a connection between PRICKLE function and ASD warrants further investigation. Here, we show that a seizure-prone Drosophila prickle mutant (prickle-spiny-legs, or pksple) exhibits learning and memory deficits, increased pain sensitivity, both communication and social interaction difficulties, and restrictive repetitive grooming behaviors, all of which are strongly correlated with ASD, while a non-seizure prone prickle mutant (prickle-prickle, or pkpk) does not, thereby providing a direct genetic connection between epilepsy and ASD through prickle. Comparing headed versus headless pksple mutants, we also show that the excessive grooming requires higher level cognitive processing from the brain. Finally, both pksple and pkpk mutants exhibit circadian rhythm defects, another feature correlated with ASD, as well as distinct yet overlapping neurological anomalies in processes that include innate immune response, oxidative stress response, neuronal cell death, neurodegeneration, motor dysfunction and reduced lifespan, likely reflecting the unique isoform expression patterns observed in the developing CNS. Collectively, this study highlights the broadscale effects of PRICKLE mutations that extend beyond the primary clinical features of epilepsy to include several of the core features of ASD.

Calcium (Ca2+) homeostasis is fundamental to neuronal physiology, including in the regulation of membrane excitability and synaptic transmission. Disruptions in the ion transporters regulating Ca2+ influx and efflux are clearly linked to seizure disorders and age-related neurodegenerative disease. Yet, the specific contributions of variants in genes encoding these transporters to neurological disease remain to be fully understood. Drosophila melanogaster has proven to be a powerful genetic model for uncovering such mechanisms, particularly through studies of mutants that display temperature-sensitive (TS) behavioral phenotypes. In a forward genetic screen, we identified a mutant line that exhibited TS convulsions along with progressive, age-dependent neurodegeneration. We mapped the mutation to Nckx30c, specifically within the transmembrane ion-binding region of this K+-dependent Na+/Ca2+ exchanger. Characterization of this mutant, together with a second Nckx30c allele, revealed TS convulsions, impaired locomotion, a markedly shortened lifespan, neurodegeneration with age, along with structural defects at larval and adult neuromuscular junctions (NMJs). Nckx30c mutants also displayed altered neural motor circuit performance. Gene expression analysis confirmed that Nckx30c levels were reduced in heads of Nckx30c loss-of-function mutants. Tissue-specific manipulation revealed that knockdown of Nckx30c in neurons recapitulated the TS convulsions, locomotor defects, and shortened lifespan phenotypes. Drosophila Nckx30c is highly conserved and shares homology with mammalian SLC24A2, a solute carrier family 24 member whose neurological role is not yet fully elucidated. Our work establishes Nckx30c as an essential regulator of neuronal health and provides an in vivo framework for investigating the contribution of SLC24A2 to neuronal Ca2+ homeostasis, seizures and age-related neurodegeneration.

WAC is an autism-associated gene involved in neurodevelopment. However, the effects of reduced WAC function on behavior and synaptic regulation in vivo remain unclear. Taking cues from the previous studies on the wac gene and the C. elegans model of ASD, we elucidated the effects of wac gene deletion on food-leaving behavior, a known parameter linked to ASD associated genes along with the cholinergic pathway. wac-deficient worms exhibited curtailed food-leaving behavior. Notably, observed phenotype was similar to that exhibited by nematodes with mutation in ASD related gene, neuroligin. In addition, wac-deficient worms showed impaired growth, reduced pharyngeal pumping, and lifespan. To examine potential synaptic mechanisms, we analyzed expression of genes related to cholinergic signaling across all developmental stages (L1-L4) through young adult (YA). Stage-specific transcriptional changes were observed, with increased expression of ace-1 and acr-3 at L1, acr-3 at L3, and acr-3, cha-1, lev-1, and lev-10 at L4. The transcriptomic alteration was most prominent at YA stage, exhibiting upregulation of ace-1, cha-1, cho-1, lev-1, lev-10, unc-17, unc-29, unc-38, and unc-50. To identify specific suppressor of upmodulated Ach signaling, RNAi of the upregulated genes was performed. cho-1 was identified as a specific suppressor of elevated Ach signaling. cho-1 encodes a high-affinity choline transporter responsible for choline uptake in the pre-synapse. These studies identify the molecular mechanisms pertaining to up-modulation of cholinergic signaling in wac mutant worms. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/719318v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@13b9510org.highwire.dtl.DTLVardef@b74e11org.highwire.dtl.DTLVardef@6676e0org.highwire.dtl.DTLVardef@1068f35_HPS_FORMAT_FIGEXP M_FIG C_FIG

Accurate and scalable sleep assessment is crucial for diagnosing sleep disorders and advancing personalized medicine. However, current approaches heavily rely on manual scoring of polysomnography (PSG) recordings, which is labor-intensive, costly, and difficult to scale. Here, we introduce SleepGPT, the first sleep-specific language model trained on over 5.8 million sleep stage annotations from 5,793 whole-night recordings to model the sequential dynamics of sleep macrostructure. We demonstrate that SleepGPT consistently enhances the performance of multiple state-of-the-art sleep staging models across diverse independent datasets (total N=1,320,654), and enables high staging accuracy from low-density wearable EEG recordings (N=120,095). We further show that SleepGPT supports robust diagnosis of sleep disorders when embedded into a sequence-level Transformer framework. The model accurately identifies abnormal sleep stage dynamics and Type-1 narcolepsy across multiple clinical datasets (total N=685). Attention-based visualization of model outputs revealed interpretable macrostructural patterns associated with diagnostic labels. Together, these results demonstrate that SleepGPT serves as a general-purpose foundation model for sleep sequence modeling, offering a scalable and clinically translatable tool for automated sleep staging and sleep disorder diagnosis.

Lowe syndrome (LS) is a rare X-linked monogenic disorder resulting from mutations in the OCRL1 gene that encodes a phosphatidylinositol 4,5-bisphosphate 5 phosphatase enzyme. Patients with LS exhibit a range of neurological symptoms, including neurodevelopmental delays, hypotonia, febrile seizures, and behavioural abnormalities; however, the cellular and developmental origins of LS remain poorly understood. The Drosophila genome encodes a single homolog of OCRL (docrl). Here, we report that a germline null allele of docrl (docrlKO) shows heat induced seizures reminiscent of the febrile seizures in LS patients. Cell type specific deletion of docrl in neurons was sufficient to recapitulate the heat induced seizures seen in docrlKO indicating a cell autonomous requirement of docrl in neurons to prevent seizures. Temporally controlled deletion of docrl showed that heat induced seizure in adults were predetermined by a requirement of docrl in neural stem cells during embryonic neurogenesis. Collectively, our findings demonstrate the developmental origin of the neurological manifestations of LS highlighting the need to target potential therapeutic interventions during this developmental time window.