Metabolic and neurobehavioral disturbances induced by purine recycling deficiency in Drosophila
- Genes Circuits Rhythms and Neuropathology, Brain Plasticity Unit, CNRS, ESPCI Paris, PSL Research University, France
- Metabolomic and Proteomic Biochemistry Laboratory, Necker-Enfants Malades Hospital and Paris Cité University, France
- Integrated Physiology of the Brain Arousal Systems (WAKING), Lyon Neuroscience Research Centre, INSERM/CNRS/UCBL1, France
- Dipartimento di Scienze Agroalimentari, Ambientali e Animali, University of Udine, Italy
- UMR “Ecology and Dynamics of Anthropogenic Systems” (EDYSAN), CNRS, Université de Picardie Jules Verne, France
- Laboratory of Metabolic Diseases, Cliniques Universitaires Saint-Luc, Université catholique de Louvain, Belgium
Figures
Figure 1 with 6 supplements
Aprt deficiency shortens lifespan and induces metabolic and neurobehavioral defects.
(A) Aprt5 mutant flies have a reduced lifespan compared to wild-type flies (median lifespan: 38 and 50 d, respectively). Three independent experiments were performed on 150 males per genotype with …
-
Figure 1—source data 1
Source data for Figure 1A–G.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Comparison of purine metabolism pathways in Drosophila and humans.
(A) Schematic diagram of the purine biosynthesis and degradation pathways in Drosophila melanogaster, based on sequence homology with the human genes. (B) Percent sequence similarity between human …
Figure 1—figure supplement 2
Urooted maximum likelihood phylogeny of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) proteins (189 taxa, 130 sites).
Phylogenetic analyses show that (a) HGPRT proteins are ancient,as they are present in bacteria and archaea. (b) Two paralogs (HPRT1 and PRTFDC1) are found in human and vertebrates (red arrows). An …
Figure 1—figure supplement 3
Urooted maximum likelihood phylogeny of HPRT proteins (20 taxa, 177 sites).
The dataset comprises 17 relevant taxa used in Figure 1—figure supplement 2, together with the HGPRT proteins of Drosophila immigrans, Serratia ureilytica, and Serratia entomophila. The D. immigrans …
Figure 1—figure supplement 4
Lack of Aprt enzymatic activity in the Aprt5 mutant.
Aprt activity was assayed on whole adult fly extracts, showing that it is strongly reduced in heterozygous Aprt5/+ mutants, and absent in homozygous Aprt5 flies, compared to the wild-type. The fact …
-
Figure 1—figure supplement 4—source data 1
Source data for Figure 1—figure supplement 4.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig1-figsupp4-data1-v1.xlsx
Figure 1—figure supplement 5
Alignment of wild-type and mutant Aprt cDNAs and predicted protein sequences.
(A) Nucleotide sequence alignment of the coding regions in Drosophila Aprt and Aprt5 cDNAs. A base substitution and a deletion that are responsible for the three prominent mutations in the Aprt5 …
Figure 1—figure supplement 6
Startle-induced negative geotaxis (SING) behavior of hemizygous Aprt mutant flies.
Df(3L)ED4284 and Df(3L)BSC365 are deficiencies located in 62B4-B12 and 62B7-D3, respectively, that removes Aprt and several neighbor genes. Hemizygous Aprt5 flies, in which the mutation was placed …
-
Figure 1—figure supplement 6—source data 1
Source data for Figure 1—figure supplement 6.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig1-figsupp6-data1-v1.xlsx
Figure 2 with 2 supplements
Aprt knockdown in neurons or glial cells disrupts startle-induced locomotion in Drosophila.
(A) AprtRNAi expression in all neurons with elav-Gal4 decreased startle-induced negative geotaxis (SING) performance in 10-day-old flies. (B) Pan-neuronal expression of Drosophila Aprt with the …
-
Figure 2—source data 1
Source data for Figure 2A–E.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Transgenic expression of Drosophila Aprt.
(A) A UAS-Aprt line was generated to allow for Aprt expression in specific cells. Upon ubiquitous expression with the da-Gal4 driver (da>Aprt), Aprt mRNA level was found to be increased 18 times in …
-
Figure 2—figure supplement 1—source data 1
Source data for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
Downregulation of Aprt expression in the ensheathing glia does not alter locomotor performances.
Aprt downregulation targeted to the ensheathing glial cells using MZ0709-Gal4 (A) or NP6520-Gal4 (B) did not impair startle-induced climbing of the flies at 10 days after eclosion (d a.E.). Results …
-
Figure 2—figure supplement 2—source data 1
Source data for Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig2-figsupp2-data1-v1.xlsx
Figure 3
Aprt downregulation in dopamine (DA) neurons of the protocerebral anterior medial (PAM) cluster and in mushroom body neurons impairs startle-induced locomotion.
(A) RNAi-mediated Aprt inactivation in brain DA neurons except a large part of the PAM cluster with the TH-Gal4 driver did not lead to locomotor defects in the startle-induced negative geotaxis …
-
Figure 3—source data 1
Source data for Figure 3A–H.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig3-data1-v1.xlsx
Figure 4 with 4 supplements
Aprt-deficient flies sleep less and walk slower than wild-type flies.
(A) Quantification of total spontaneous locomotor activity during day and night over five light-dark (LD) cycles. Aprt5 mutants show no difference in spontaneous locomotion with wild-type flies …
-
Figure 4—source data 1
Source data for Figure 4A–G.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Daily locomotor activity profiles of wild-type and Aprt5 mutant flies.
Actograms showing activity profiles during a 12 hr:12 hr light-dark (LD) cycles from wild-type (A) and Aprt5 (B) male flies at 8 days after eclosion (d a.E.). Aprt deficiency does not seem to alter …
Figure 4—figure supplement 2
Sensitivity of resting flies to mild mechanical stimulation.
Wild-type and Aprt5 mutant flies display similarly decreasing responses to stimulations following periods of immobility exceeding 5 min, confirming the standard criteria for sleep. Flies were …
-
Figure 4—figure supplement 2—source data 1
Source data for Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig4-figsupp2-data1-v1.xlsx
Figure 4—figure supplement 3
ATP levels are not altered in head and thorax of Aprt5 flies compared to the wild-type.
ATP was measured by a bioluminescence assay. Mean of three or four independent experiments performed on 30 heads or five thoraces per genotype. Unpaired Student’s t-test,ns: not significant.
-
Figure 4—figure supplement 3—source data 1
Source data for Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig4-figsupp3-data1-v1.xlsx
Figure 4—figure supplement 4
Sleep patterns of flies with cell-specific Aprt deficiency.
(A) Sleep pattern of repo>AprtRNAi flies, showing that knockdown of Aprt in all glial cells did not induce any sleep defect. (B) In contrast, knocking down Aprt in all neurons and glial cells (elav; …
-
Figure 4—figure supplement 4—source data 1
Source data for Figure 4—figure supplement 4.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig4-figsupp4-data1-v1.xlsx
Figure 5
Aprt deficiency increases dopamine (DA) synthesis and content in the Drosophila brain.
(A) Representative confocal projections of tyrosine hydroxylase (TH)-immunostained whole-mount adult brains from wild-type flies and Aprt5 mutants. MB: mushroom body. Scale bars: 100 μm. (B) …
-
Figure 5—source data 1
Source data for Figure 5A–F and H.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig5-data1-v1.xlsx
-
Figure 5—source data 2
Original files for the western blot analysis of Figure 5G.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig5-data2-v1.zip
-
Figure 5—source data 3
Original files for the western blot analysis of Figure 5G with relevant bands and samples labeled.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig5-data3-v1.zip
Figure 6
Relations between Aprt and molecular components of adenosinergic signaling.
(A, B) Impacts of the lack of Aprt activity on the adenosinergic system. (A) Adenosine level was measured in whole flies or heads of Aprt5 flies by ultra performance liquid chromatography (UPLC). …
-
Figure 6—source data 1
Source data for Figure 6A–C.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig6-data1-v1.xlsx
Figure 7 with 5 supplements
Aprt deficiency triggers a seizure-like phenotype.
(A) At 30 days after eclosion (d a.E.), Aprt5 mutants need a much longer time than wild-type flies to recover from a strong mechanical shock, showing a bang-sensitive (BS) paralysis comparable to …
-
Figure 7—source data 1
Source data for Figure 7A–F.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
Aprt knockdown selectively in neurons, glia, or muscle cells did not induce bang sensitivity.
(A–C) Downregulation of Aprt by RNAi either in all neurons (A), all glial cells (B), or all muscles (C) did not induce a seizure phenotype, in contrast to the effects of ubiquitous downregulation. …
-
Figure 7—figure supplement 1—source data 1
Source data for Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig7-figsupp1-data1-v1.xlsx
Figure 7—figure supplement 2
Administration of allopurinol does not rescue the bang sensitivity phenotype of Aprt-deficient mutants.
Feeding the Aprt5 mutants with allopurinol at the same concentration used for uric acid normalization (100 μg/ml) either in adults 5 d before the test (A) or throughout all developmental stages (B) …
-
Figure 7—figure supplement 2—source data 1
Source data for Figure 7—figure supplement 2.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig7-figsupp2-data1-v1.xlsx
Figure 7—figure supplement 3
Administration of various purine compounds does not rescue the motricity defects of Aprt-deficient mutants.
(A–D) Feeding the Aprt5 mutant flies with adenine (A, B) or hypoxanthine (C, D) at 100 μM, either in adults 5 d before the test (A, C) or throughout all developmental stages plus 5 d before the test …
-
Figure 7—figure supplement 3—source data 1
Source data for Figure 7—figure supplement 3.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig7-figsupp3-data1-v1.xlsx
Figure 7—video 1
Bang sensitivity phenotype of Aprt-deficient flies: wild-type flies.
Wild-type Canton S flies (CS) are not paralyzed and recover rapidly after 10 s vortexing.
Figure 7—video 2
Bang sensitivity phenotype of Aprt-deficient flies: Aprt5 mutant flies.
In contrast, Aprt5 mutant flies (APRT) show seizure-like behavior and paralysis, and they recover slowly after such mechanical stimulation.
Figure 8
Expression of a pathogenic mutant isoform of human hypoxanthine-guanine phosphoribosyltransferase (HGPRT) induces neurobehavioral defects in flies.
(A, B) Ubiquitous expression of human HPRT1with da-Gal4. (A) Amplification of human HPRT1 transcripts detected by RT-PCR in head extracts of da>HPRT1 WT and da>HPRT1-I42T flies. A band with lower …
-
Figure 8—source data 1
Original file for the DNA gel electrophoresis of Figure 8A.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig8-data1-v1.zip
-
Figure 8—source data 2
Original file for the DNA gel electrophoresis of Figure 8A with relevant bands and samples labeled.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig8-data2-v1.zip
-
Figure 8—source data 3
Source data for Figure 8B–D.
- https://cdn.elifesciences.org/articles/88510/elife-88510-fig8-data3-v1.xlsx
Author response image 1
Tables
Table 1
Aprt activity in wild-type and Aprt-deficient flies.
| Genotypes | Sex | Aprt activity (nmol/min/mg prot) |
|---|---|---|
| Wild type | Males | 1.32 ± 0.17 |
| Females | 2.77 ± 0.27 | |
| Aprt5/Aprt5 | Males and females | 0.04 ± 0.02 |
| Aprt5/Df(3L)ED4284 | Males | 0.02 ± 0.01 |
| da/+ | Males | 2.78 ± 0.41 |
| da>AprtRNAi | Males | 0.10 ± 0.01 |
| AprtRNAi/+ | Males | 2.16 ± 0.37 |
-
Table 1—source data 1
Source data for Table 1.
- https://cdn.elifesciences.org/articles/88510/elife-88510-table1-data1-v1.xlsx
Table 2
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity in transgenic flies expressing the wild-type or a Lesch–Nyhan disease (LND)-associated mutant form of human HPRT1.
| Genotypes | HGPRT activity (nmol/min/mg) |
|---|---|
| da/+ | 0 |
| da>HPRT1-WT | 13.88 ± 3.75 |
| da>HPRT1-I42T | 2.70 ± 1.44 |
| HPRT1-WT/+ | 0 |
| HPRT1-I42T/+ | 0 |
-
Table 2—source data 1
Source data for Table 2.
- https://cdn.elifesciences.org/articles/88510/elife-88510-table2-data1-v1.xlsx
