IntroductionWith 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.MethodsThis 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.ResultsWe 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.DiscussionThis 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.

IntroductionIt has been established that UBR4 encodes E3 ubiquitin ligase, which determines the specificity of substrate binding during protein ubiquitination and has been associated with various functions of the nervous system but not the reproductive system. Herein, we explored the role of UBR4 on fertility with a Drosophila model.MethodsDifferent Ubr4 knockdown flies were established using the UAS/GAL4 activating sequence system. Fertility, hatchability, and testis morphology were studied, and bioinformatics analyses were conducted. Our results indicated that UBR4 deficiency could induce male sterility and influent egg hatchability in Drosophila.ResultsWe found that Ubr4 deficiency affected the testis during morphological analysis. Proteomics analysis indicated 188 upregulated proteins and 175 downregulated proteins in the testis of Ubr4 knockdown flies. Gene Ontology analysis revealed significant upregulation of CG11598 and Sfp65A, and downregulation of Pelota in Ubr4 knockdown flies. These proteins were involved in the biometabolic or reproductive process in Drosophila. These regulated proteins are important in testis generation and sperm storage promotion. Bioinformatics analysis verified that UBR4 was low expressed in cryptorchidism patients, which further supported the important role of UBR4 in male fertility.DiscussionOverall, our findings suggest that UBR4 deficiency could promote male infertility and may be involved in the protein modification of UBR4 by upregulating Sfp65A and CG11598, whereas downregulating Pelota protein expression.

The vast majority of medically intractable epilepsy cases can be traced to polygenic backgrounds, and pathogenic variants contribute to genetic risk in the complex inheritance of this common disorder. We generated monogenic and digenic drosophila models by knock-down of two epilepsy-associated genes, CACNA1A and CELSR2, with impacts on intersynaptic calcium activities. Monogenic knockdown of CACNA1A or CELSR2 could induce seizure-like behavior in flies. Interestingly, seizure behavior tests showed that CACNA1A deficiency could attenuate the seizure-like activities of the CACNA1A-CELSR2 digenic knockdown flies. The protective effect of CACNA1A deficiency was quantitatively fitted with multiple linear regression equation based on the seizure rates. In calcium imaging results, CACNA1A monogenic knockdown flies showed higher frequency of large spikes calcium activity than that of wild-type flies. That would be the neural base of the seizure in CACNA1A deficiency flies. However, the area under curve of calcium activity showed no difference between CACNA1A monogenic knockdown flies and wild-type flies. The calcium imaging results could partially illustrate the mechanism of the protective effect of CACNA1A deficiency. Furthermore, our study could be an ideal strategy to study polygenic effect on epilepsy by using animal model.

It has been established that UBR4 encodes E3 ubiquitin ligase, which determines the specificity of substrate binding during protein ubiquitination and has been associated with various functions of the nervous system but not the reproductive system. Herein, we explored the role of UBR4 on fertility and development with a drosophila model. Different UBR4 knockdown flies were established using the GAL4/upstream activating sequence system. Fertility, hatchability and testis morphology were studied, and bioinformatics analyses were conducted. Our results indicated that UBR4 deficiency could promote larvae developmental delay and male drosophila fertility. We found that UBR4 deficiency affected the testis during morphological analysis. Bioinformatics analysis indicated 188 upregulated proteins and 175 downregulated proteins in the testis of UBR4 knockdown flies. Gene Ontology analysis revealed that significant upregulation of Sfp65A and downregulation of Pelota were involved in the reproductive process in drosophila, important in testis generation and sperm storage promotion. Overall, our findings suggest that UBR4 deficiency could promote male infertility in drosophila and may be involved in the protein modification of UBR4 by upregulating Sfp65A anddownregulating Pelota protein expression.