PMC:4786408 / 18697-23028 JSONTXT

{"project":"2_test","denotations":[{"id":"26812546-18056421-26883484","span":{"begin":591,"end":595},"obj":"18056421"},{"id":"26812546-16547094-26883485","span":{"begin":1992,"end":1996},"obj":"16547094"},{"id":"26812546-17360644-26883486","span":{"begin":2014,"end":2018},"obj":"17360644"},{"id":"26812546-17138868-26883487","span":{"begin":2035,"end":2039},"obj":"17138868"},{"id":"26812546-25826530-26883488","span":{"begin":2107,"end":2111},"obj":"25826530"}],"text":"ATG5E122D fails to complement the ataxic phenotype of Atg5-null flies\nTo further characterize the effect of the E122D mutation on the development of ataxia, we generated Drosophila melanogaster knockouts for Atg5 (Figure 6A), and reconstituted the Atg5-null mutant flies with transgenes expressing wild-type (WT) or E122D human ATG5 (Figure 6B-D). Unlike mouse models, Atg5-null flies are viable, although they exhibit severe mobility defects after adult eclosion as demonstrated by a negative geotaxis assay (Figure 6E and I, and Video 1), similar to Atg7 null mutant flies (Juhasz et al., 2007). These mobility defects were substantially restored by expression of ATG5WT (Figure 6F and I, and Video 2), suggesting that the molecular function of ATG5 is conserved between human and Drosophila. However, Atg5-null mutant flies expressing ATG5E122D were still defective in mobility although slightly better than Atg5-null controls (Figure 6G–I, and Videos 3 and 4), demonstrating again that ATG5 activity is compromised but not eliminated by the E122D mutation. ATG5E122D was also inferior to ATG5WT in suppressing Ref(2)P (fly p62/SQSTM1) accumulation (Figure 6J and K) and cell death (Figure 6L and M) in the brain of Atg5-null mutant flies.\n10.7554/eLife.12245.009Figure 6. Ataxic phenotype of Atg5-null flies is suppressed by human ATG5WT but not by ATG5E122D.\n(A) Genomic organization of the Atg5 locus and the Atg5-null mutant (Atg55cc5). Atg55cc5 mutants have a CRISPR-Cas9-mediated deletion in approximately 1.5 kb residues that eliminate more than 85% of Atg5-coding sequences including the translation start site. Open boxes, untranslated exons; closed boxes, protein-coding exons. Scale bar, relative length of 1 kb genomic span. (B) Schematic representation of how ATG5 transgenic flies were made. Plasmid which can express wild-type or E122D-mutated human ATG5 was inserted into an identical genomic location (the attP site) through phiC31-mediated recombination (Bateman et al., 2006; Bischof et al., 2007; Venken et al., 2006). The scheme was adapted from a previous publication (Kim and Lee, 2015). (C) Genetic scheme of how ATG5 transgenes were placed into the Atg5-null mutant flies. Atg5, UAS-ATG5 and Tub-Gal4 loci are on the X-chromosome, second chromosome and third chromosome, respectively. (D) Whole flies of indicated genotypes were analyzed by IB. (E to H) Photographs of the vials containing 2-week-old adult male flies of indicated genotypes taken at 3 sec after negative geotaxis induction: (E) Atg5-null flies exhibit severely impaired mobility. (F) Ataxic phenotype of Atg5-null flies is complemented by human ATG5WT expression. (G and H) Human ATG5E122D is less capable than human ATG5WT in suppressing the fly ataxia phenotype. (I) Quantification of the climbing speeds of 2-week-old adult male flies (n≥20) of the indicated genotype. Climbing speed is presented as mean ± standard deviation (n=5). P values were calculated using the Student’s t test (***p\u003c0.001). (J) Drosophila heads from two-weeks-old flies of the indicated genotypes were analyzed by IB. (K) Ref(2)P [p62] is an autophagy substrate. Relative protein expression was measured by densitometry and presented in a bar graph (mean ± standard error; n=4). (L) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) of Drosophila brain (middle layer of the medial compartment). (M) TUNEL-positive cells per field were quantified and presented in a bar graph (mean ± standard error; n≥5). K and M: P values were calculated using the Student’s t test (*p\u003c0.05, **p\u003c0.01, ***p\u003c0.001).\nDOI: http://dx.doi.org/10.7554/eLife.12245.009\nVideo 1. Climbing assay in 2 weeks-old wild-type flies (left) and Atg5-null flies (right).\nDOI: http://dx.doi.org/10.7554/eLife.12245.01010.7554/eLife.12245.010\nVideo 2. Climbing assay in 2 weeks-old Atg5-null flies (left) and Atg5-null flies expressing ATG5WT (right).\nDOI: http://dx.doi.org/10.7554/eLife.12245.01110.7554/eLife.12245.011\nVideo 3. Climbing assay in 2 weeks-old Atg5-null flies (left) and Atg5-null flies expressing ATG5E122D (right).\nDOI: http://dx.doi.org/10.7554/eLife.12245.01210.7554/eLife.12245.012\nVideo 4. Climbing assay in 2 weeks-old Atg5-null flies expressing ATG5WT (left) or ATG5E122D (right).\nDOI: http://dx.doi.org/10.7554/eLife.12245.01310.7554/eLife.12245.013"}

{"project":"MyTest","denotations":[{"id":"26812546-18056421-26883484","span":{"begin":591,"end":595},"obj":"18056421"},{"id":"26812546-16547094-26883485","span":{"begin":1992,"end":1996},"obj":"16547094"},{"id":"26812546-17360644-26883486","span":{"begin":2014,"end":2018},"obj":"17360644"},{"id":"26812546-17138868-26883487","span":{"begin":2035,"end":2039},"obj":"17138868"},{"id":"26812546-25826530-26883488","span":{"begin":2107,"end":2111},"obj":"25826530"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"text":"ATG5E122D fails to complement the ataxic phenotype of Atg5-null flies\nTo further characterize the effect of the E122D mutation on the development of ataxia, we generated Drosophila melanogaster knockouts for Atg5 (Figure 6A), and reconstituted the Atg5-null mutant flies with transgenes expressing wild-type (WT) or E122D human ATG5 (Figure 6B-D). Unlike mouse models, Atg5-null flies are viable, although they exhibit severe mobility defects after adult eclosion as demonstrated by a negative geotaxis assay (Figure 6E and I, and Video 1), similar to Atg7 null mutant flies (Juhasz et al., 2007). These mobility defects were substantially restored by expression of ATG5WT (Figure 6F and I, and Video 2), suggesting that the molecular function of ATG5 is conserved between human and Drosophila. However, Atg5-null mutant flies expressing ATG5E122D were still defective in mobility although slightly better than Atg5-null controls (Figure 6G–I, and Videos 3 and 4), demonstrating again that ATG5 activity is compromised but not eliminated by the E122D mutation. ATG5E122D was also inferior to ATG5WT in suppressing Ref(2)P (fly p62/SQSTM1) accumulation (Figure 6J and K) and cell death (Figure 6L and M) in the brain of Atg5-null mutant flies.\n10.7554/eLife.12245.009Figure 6. Ataxic phenotype of Atg5-null flies is suppressed by human ATG5WT but not by ATG5E122D.\n(A) Genomic organization of the Atg5 locus and the Atg5-null mutant (Atg55cc5). Atg55cc5 mutants have a CRISPR-Cas9-mediated deletion in approximately 1.5 kb residues that eliminate more than 85% of Atg5-coding sequences including the translation start site. Open boxes, untranslated exons; closed boxes, protein-coding exons. Scale bar, relative length of 1 kb genomic span. (B) Schematic representation of how ATG5 transgenic flies were made. Plasmid which can express wild-type or E122D-mutated human ATG5 was inserted into an identical genomic location (the attP site) through phiC31-mediated recombination (Bateman et al., 2006; Bischof et al., 2007; Venken et al., 2006). The scheme was adapted from a previous publication (Kim and Lee, 2015). (C) Genetic scheme of how ATG5 transgenes were placed into the Atg5-null mutant flies. Atg5, UAS-ATG5 and Tub-Gal4 loci are on the X-chromosome, second chromosome and third chromosome, respectively. (D) Whole flies of indicated genotypes were analyzed by IB. (E to H) Photographs of the vials containing 2-week-old adult male flies of indicated genotypes taken at 3 sec after negative geotaxis induction: (E) Atg5-null flies exhibit severely impaired mobility. (F) Ataxic phenotype of Atg5-null flies is complemented by human ATG5WT expression. (G and H) Human ATG5E122D is less capable than human ATG5WT in suppressing the fly ataxia phenotype. (I) Quantification of the climbing speeds of 2-week-old adult male flies (n≥20) of the indicated genotype. Climbing speed is presented as mean ± standard deviation (n=5). P values were calculated using the Student’s t test (***p\u003c0.001). (J) Drosophila heads from two-weeks-old flies of the indicated genotypes were analyzed by IB. (K) Ref(2)P [p62] is an autophagy substrate. Relative protein expression was measured by densitometry and presented in a bar graph (mean ± standard error; n=4). (L) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) of Drosophila brain (middle layer of the medial compartment). (M) TUNEL-positive cells per field were quantified and presented in a bar graph (mean ± standard error; n≥5). K and M: P values were calculated using the Student’s t test (*p\u003c0.05, **p\u003c0.01, ***p\u003c0.001).\nDOI: http://dx.doi.org/10.7554/eLife.12245.009\nVideo 1. Climbing assay in 2 weeks-old wild-type flies (left) and Atg5-null flies (right).\nDOI: http://dx.doi.org/10.7554/eLife.12245.01010.7554/eLife.12245.010\nVideo 2. Climbing assay in 2 weeks-old Atg5-null flies (left) and Atg5-null flies expressing ATG5WT (right).\nDOI: http://dx.doi.org/10.7554/eLife.12245.01110.7554/eLife.12245.011\nVideo 3. Climbing assay in 2 weeks-old Atg5-null flies (left) and Atg5-null flies expressing ATG5E122D (right).\nDOI: http://dx.doi.org/10.7554/eLife.12245.01210.7554/eLife.12245.012\nVideo 4. Climbing assay in 2 weeks-old Atg5-null flies expressing ATG5WT (left) or ATG5E122D (right).\nDOI: http://dx.doi.org/10.7554/eLife.12245.01310.7554/eLife.12245.013"}