Generation and Characterization of the Drosophila melanogaster paralytic Gene Knock-Out as a Model for Dravet Syndrome.
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ABSTRACT:
SUBMITTER: Tapia A
PROVIDER: S-EPMC8619338 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Dravet syndrome (DS) is an epileptic syndrome caused by mutations in the Scn1a gene encoding the α1 subunit of the sodium channel Nav1.1, which is associated with febrile seizures that progress to severe tonic-clonic seizures and associated comorbidities. Treatment with cannabidiol has been approved to reduce seizures in DS, but it may also be active against these comorbidities. The aim of this study was to validate a new mouse model of DS having lower mortality than previous models, which may serve to further evaluate therapies for the long-term comorbidities. This new model consists of heterozygous conditional knock-in mice carrying a missense mutation (A1783V) in Scn1a gene expressed exclusively in neurons of the CNS (Syn-Cre/Scn1aWT/A1783V). These mice have been used here to determine the extent and persistence of the behavioral deterioration in different postnatal days (PND), as well as to investigate the alterations that the disease produces in the endocannabinoid system and the contribution of inflammatory events and impaired neurogenesis in the pathology. Syn-Cre/Scn1aWT/A1783V mice showed a strong reduction in hindlimb grasp reflex at PND10, whereas at PND25, they presented spontaneous convulsions and a greater susceptibility to pentylenetetrazole-induced seizures, marked hyperactivity, deficient spatial working memory, lower levels of anxiety, and altered social interaction behavior. These differences disappeared at PND40 and PND60, except the changes in social interaction and anxiety. The analysis of CNS structures associated with these behavioral alterations revealed an elevated glial reactivity in the prefrontal cortex and the dentate gyrus. This was associated in the dentate gyrus with a greater cell proliferation detected with Ki67 immunostaining, whereas double-labeling analyses identified that proliferating cells were GFAP-positive suggesting failed neurogenesis but astrocyte proliferation. The analysis of the endocannabinoid system of Syn-Cre/Scn1aWT/A1783V mice confirmed reductions in CB1 receptors and MAGL and FAAH enzymes, mainly in the cerebellum but also in other areas, whereas CB2 receptors became upregulated in the hippocampus. In conclusion, Syn-Cre/Scn1aWT/A1783V mice showed seizuring susceptibility and several comorbidities (hyperactivity, memory impairment, less anxiety, and altered social behavior), which exhibited a pattern of age expression similar to DS patients. Syn-Cre/Scn1aWT/A1783V mice also exhibited greater glial reactivity and a reactive response in the neurogenic niche, and regional changes in the status of the endocannabinoid signaling, events that could contribute in behavioral impairment.
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Project description:Hundreds of mutations in the SCN1A sodium channel gene confer a wide spectrum of epileptic disorders, requiring efficient model systems to study cellular mechanisms and identify potential therapeutic targets. We recently demonstrated that Drosophila knock-in flies carrying the K1270T SCN1A mutation known to cause a form of genetic epilepsy with febrile seizures plus (GEFS+) exhibit a heat-induced increase in sodium current activity and seizure phenotype. To determine whether different SCN1A mutations cause distinct phenotypes in Drosophila as they do in humans, this study focuses on a knock-in line carrying a mutation that causes a more severe seizure disorder termed Dravet syndrome (DS). Introduction of the DS SCN1A mutation (S1231R) into the Drosophila sodium channel gene para results in flies that exhibit spontaneous and heat-induced seizures with distinct characteristics and lower onset temperature than the GEFS+ flies. Electrophysiological studies of GABAergic interneurons in the brains of adult DS flies reveal, for the first time in an in vivo model system, that a missense DS mutation causes a constitutive and conditional reduction in sodium current activity and repetitive firing. In addition, feeding with the serotonin precursor 5-HTP suppresses heat-induced seizures in DS but not GEFS+ flies. The distinct alterations of sodium currents in DS and GEFS+ GABAergic interneurons demonstrate that both loss- and gain-of-function alterations in sodium currents are capable of causing reduced repetitive firing and seizure phenotypes. The mutation-specific effects of 5-HTP on heat-induced seizures suggest the serotonin pathway as a potential therapeutic target for DS.
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Project description:Here we report the molecular characterization of Out-cold (Ocd) mutants of Drosophila melanogaster, which produce a dominant, X-linked, cold-sensitive paralytic phenotype. From its initial 1.5-Mb cytological location within 13F1-16A2, P-element and SNP mapping reduced the Ocd critical region to <100 kb and to six candidate genes: hangover, CG9947, CG4420, eIF2a, Rbp2, and paralytic (para). Complementation testing with para null mutations strongly suggests Ocd and para are allelic, as does gene rescue of Ocd semilethality with a wild-type para transgene. Pesticide resistance and electrophysiological phenotypes of Ocd mutants support this conclusion. The para gene encodes a voltage-gated sodium channel. Sequencing the Ocd lines revealed mutations within highly conserved regions of the para coding sequence, in the transmembrane segment S6 of domain III (I1545M and T1551I), and in the linker between domains III and IV (G1571R), the location of the channel inactivation gate. The G1571R mutation is of particular interest as mutations of the orthologous residue (G1306) in the human skeletal muscle sodium channel gene SCN4A are associated with cases of periodic paralysis and myotonia, including the human cold-sensitive disorder paramyotonia congenita. The mechanisms by which sodium channel mutations cause cold sensitivity are not well understood. Therefore, in the absence of suitable vertebrate models, Ocd provides a system in which genetic, molecular, physiological, and behavioral tools can be exploited to determine mechanisms underlying sodium channel periodic paralyses.
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Project description:microRNA-142 (miR-142) is an important regulator of many biological processes and associated signaling pathways during embryonic development, homeostasis and disease. The miR-142 hairpin gives rise to the "guide strand" miR-142-3p and the sister "passenger" strand miR-142-5p. miR-142-3p has been shown to play critical, non-redundant functions in the development of the hematopoietic lineage. We have recently reported that miR-142-3p is critical for the control of Wnt signaling in the mesenchyme of the developing lung. miR-142-5p has been proposed to control adaptive growth in cardiomyocytes postnatally and its increase is associated with extensive apoptosis and cardiac dysfunction in a murine heart failure model. Using homologous recombination, we now report the generation and validation of miR-142-null mice. miR-142-null mice show a significant decrease in th expression levels of both the 3p and 5p isoforms. The expression of Bzrap1, a gene immediately flanking miR-142 is not altered while the expression of a long non-coding RNA embedded within the miR-142 gene is decreased. miR-142-null newborn pups appear normal and are normally represented indicating absence of embryonic lethality. At embryonic day 18.5, miR-142-null lungs display increased Wnt signaling associated with the up-regulation of Apc and p300, two previously reported targets of miR-142-3p and -5p, respectively. Adult miR-142-null animals display impaired hematopoietic lineage formation identical to previously reported miR-142 gene trap knockdown mice. We report, for the first time, the homologous recombination-based miR-142-null mice that will be useful for the scientific community working on the diverse biological functions of miR-142.
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Project description:Spinocerebellar ataxia 3 (SCA3) is a genetic disorder resulting from the expansion of the CAG repeats in the ATXN3 gene. The pathogenesis of SCA3 is based on the toxic function of the mutant ataxin-3 protein, but the exact mechanism of the disease remains elusive. Various types of transgenic mouse models explore different aspects of SCA3 pathogenesis, but a knock-in humanized mouse has not yet been created. The initial aim of this study was to generate an ataxin-3 humanized mouse model using a knock-in strategy. The human cDNA for ataxin-3 containing 69 CAG repeats was cloned from SCA3 patient and introduced into the mouse ataxin-3 locus at exon 2, deleting it along with exon 3 and intron 2. Although the human transgene was inserted correctly, the resulting mice acquired the knock-out properties and did not express ataxin-3 protein in any analyzed tissues, as confirmed by western blot and immunohistochemistry. Analyses of RNA expression revealed that the entire locus consisting of human and mouse exons was expressed and alternatively spliced. We detected mRNA isoforms composed of exon 1 spliced with mouse exon 4 or with human exon 7. After applying 37 PCR cycles, we also detected a very low level of the correct exon 1/exon 2 isoform. Additionally, we confirmed by bioinformatic analysis that the structure and power of the splicing site between mouse intron 1 and human exon 2 (the targeted locus) was not changed compared with the native mouse locus. We hypothesized that these splicing aberrations result from the deletion of further splicing sites and the presence of a strong splicing site in exon 4, which was confirmed by bioinformatic analysis. In summary, we created a functional ataxin-3 knock-out mouse model that is viable and fertile and does not present a reduced life span. Our work provides new insights into the splicing characteristics of the Atxn3 gene and provides useful information for future attempts to create knock-in SCA3 models.
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Project description:Modern molecular genetics studies necessitate the manipulation of genes in their endogenous locus, but most of the current methodologies require an inefficient donor-dependent homologous recombination step to locally modify the genome. Here we describe a methodology to efficiently generate Drosophila knock-in alleles by capitalizing on the availability of numerous genomic MiMIC transposon insertions carrying recombinogenic attP sites. Our methodology entails the efficient PhiC31-mediated integration of a recombination cassette flanked by unique I-SceI and/or I-CreI restriction enzyme sites into an attP-site. These restriction enzyme sites allow for double-strand break-mediated removal of unwanted flanking transposon sequences, while leaving the desired genomic modifications or recombination cassettes. As a proof-of-principle, we mutated LRRK, tau, and sky by using different MiMIC elements. We replaced 6 kb of genomic DNA encompassing the tau locus and 35 kb encompassing the sky locus with a recombination cassette that permits easy integration of DNA at these loci and we also generated a functional LRRK(HA) knock in allele. Given that ~92% of the Drosophila genes are located within the vicinity (<35 kb) of a MiMIC element, our methodology enables the efficient manipulation of nearly every locus in the fruit fly genome without the need for inefficient donor-dependent homologous recombination events.
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Project description:Background: Inactivation of microRNA-100 (miR-100) is involved in hepatocellular carcinoma (HCC) and miR-100 behaves as a tumor suppressor. To understand miR-100 function in HCC genesis and development in vivo, we developed hepatocyte-specific miR-100 deficient mice. Methods: Mice homozygous for floxed miR-100 allele that carried the Alb-Cre transgene (miR-100flox/floxAlb -Cre+) were developed by mating miR-100flox/flox mice with Alb-Cre+/+mice. The mice tails DNA were genotyped using the primers for LoxP sites and Cre recombinase, respectively. The specific deletion of miR-100 in the livers was verified by quantitative Real-time PCR (qRT-PCR). HE-staining was performed for histology analysis. Liver function was assessed by transaminase activity. The metabolic profiles of the hepatocytes were detected using a Seahorse XFe24 extracellular flux analyzer. The direct targets of miR-100 (such as IGF1R-β, mTOR and CDC25A) and HCC related protein (SHP-2) were detected by qRT-PCR and Western blot in liver tissues. Results: The resultant homozygous knockout mice with genotype of miR-100flox/flox-Alb-Cre+ showed an 80% decrease in hepatic miR-100 expression. In adult mice, miR-100 knockout has no effect on the liver function and morphology. In aged mice, HE staining showed that miR-100 knockout caused infiltration of inflammatory cells and expansion of hepatocellular nuclei. Consistently, liver function was impaired in miR-100 knockout aged mice as indicated by increased serum AST and ALT levels. The metabolic analysis demonstrated that the miR-100 knockout hepatocytes tend to adopt glycolysis. The expressions of the miR-100 target genes, such as IGF1R-β, CDC25A and mTOR, were increased. In addition, the known HCC related protein, SHP-2 also was up-regulated in the knockout livers. Conclusions: We successfully generated a miR-100 hepatocyte-specific knock-out mouse model. The malignant transformation related to HCC were observed in aged mice. Therefore, this model is suitable for investigating the mechanism of miR-100 inactivation contributing to HCC genesis in vivo.
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