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Petitgas etal. eLife 2023;12:RP88510. DOI: https://doi.org/10.7554/eLife.88510 1 of 34
Metabolic and neurobehavioral
disturbances induced by purine recycling
deficiency inDrosophila
Céline Petitgas1,2, Laurent Seugnet3, Amina Dulac1, Giorgio Matassi4,5,
Ali Mteyrek1, Rebecca Fima1, Marion Strehaiano1, Joana Dagorret1,
Baya Chérif- Zahar1, Sandrine Marie6, Irène Ceballos- Picot2, Serge Birman1*
1Genes Circuits Rhythms and Neuropathology, Brain Plasticity Unit, CNRS, ESPCI
Paris, PSL Research University, Paris, France; 2Metabolomic and Proteomic
Biochemistry Laboratory, Necker- Enfants Malades Hospital and Paris Cité University,
Paris, France; 3Integrated Physiology of the Brain Arousal Systems (WAKING), Lyon
Neuroscience Research Centre, INSERM/CNRS/UCBL1, Bron, France; 4Dipartimento
di Scienze Agroalimentari, Ambientali e Animali, University of Udine, Udine, Italy;
5UMR “Ecology and Dynamics of Anthropogenic Systems” (EDYSAN), CNRS,
Université de Picardie Jules Verne, Amiens, France; 6Laboratory of Metabolic
Diseases, Cliniques Universitaires Saint- Luc, Université catholique de Louvain,
Brussels, Belgium
Abstract Adenine phosphoribosyltransferase (APRT) and hypoxanthine- guanine phosphoribos-
yltransferase (HGPRT) are two structurally related enzymes involved in purine recycling in humans.
Inherited mutations that suppress HGPRT activity are associated with Lesch–Nyhan disease (LND),
a rare X- linked metabolic and neurological disorder in children, characterized by hyperuricemia,
dystonia, and compulsive self- injury. To date, no treatment is available for these neurological defects
and no animal model recapitulates all symptoms of LND patients. Here, we studied LND- related
mechanisms in the fruit fly. By combining enzymatic assays and phylogenetic analysis, we confirm
that no HGPRT activity is expressed in Drosophila melanogaster, making the APRT homolog (Aprt)
the only purine- recycling enzyme in this organism. Whereas APRT deficiency does not trigger
neurological defects in humans, we observed that Drosophila Aprt mutants show both metabolic
and neurobehavioral disturbances, including increased uric acid levels, locomotor impairments,
sleep alterations, seizure- like behavior, reduced lifespan, and reduction of adenosine signaling
and content. Locomotor defects could be rescued by Aprt re- expression in neurons and repro-
duced by knocking down Aprt selectively in the protocerebral anterior medial (PAM) dopaminergic
neurons, the mushroom bodies, or glia subsets. Ingestion of allopurinol rescued uric acid levels in
Aprt- deficient mutants but not neurological defects, as is the case in LND patients, while feeding
adenosine or N6- methyladenosine (m6A) during development fully rescued the epileptic behavior.
Intriguingly, pan- neuronal expression of an LND- associated mutant form of human HGPRT (I42T),
but not the wild- type enzyme, resulted in early locomotor defects and seizure in flies, similar to Aprt
deficiency. Overall, our results suggest that Drosophila could be used in different ways to better
understand LND and seek a cure for this dramatic disease.
eLife assessment
The article looks at how dysregulated purine metabolism in mutants for the Aprt gene impacts
survival, motor, and sleep behavior in the fruit fly. Interestingly, although several deficits arise from
RESEARCH ARTICLE
*For correspondence:
serge.birman@espci.fr
Competing interest: The authors
declare that no competing
interests exist.
Funding: See page 27
Sent for Review
03 June 2023
Preprint posted
23 June 2023
Reviewed preprint posted
31 July 2023
Reviewed preprint revised
21 February 2024
Version of Record published
03 May 2024
Reviewing Editor: Gaiti Hasan,
National Centre for Biological
Sciences, India
Copyright Petitgas etal. This
article is distributed under the
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Attribution License, which
permits unrestricted use and
redistribution provided that the
original author and source are
credited.
Research article Genetics and Genomics | Neuroscience
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dopaminergic neurons, dopamine levels are increased in Aprt mutants. Instead, the biochemical
change responsible for Aprt mutant neurobehavioral phenotypes appears to be a reduction in levels
of adenosine. This valuable study suggests that Drosophila Aprt mutants may serve as a model for
understanding Lesch–Nyhan disease (LND), caused by mutations in the human HPRT1 gene, and
may also potentially serve as a model to screen for drugs for the neurobehavioral deficits observed
in LND. The strength of evidence is solid.
Introduction
The purine salvage pathway is an essential component of cellular metabolism that allows the recovery
of free purine bases derived from the diet or from the degradation of nucleic acids and nucleotides,
thus avoiding the energy cost of de novo purine biosynthesis (Nyhan, 2014). Energy- intensive tissues,
such as cardiac muscle and brain cells, extensively use this pathway to maintain their purine levels
(Ipata, 2011; Johnson etal., 2019). The two main recycling enzymes involved in the salvage pathway
in mammals are hypoxanthine- guanine phosphoribosyltransferase (HGPRT), which converts hypoxan-
thine and guanine into IMP and GMP, respectively, and adenine phosphoribosyltransferase (APRT),
which converts adenine into AMP.
APRT and HGPRT deficiencies induce very different disorders in humans. Loss of APRT seems
to have only metabolic consequences, leading to the formation of 2,8- dihydroxyadenine crystals in
kidney, which can be fatal but is readily prevented by allopurinol treatment (Bollée et al., 2012;
Harambat et al., 2012). In contrast, highly inactivating mutations in HGPRT trigger Lesch–Nyhan
disease (LND), a rare neurometabolic X- linked recessive disorder with dramatic consequences for
child neurodevelopment (Lesch and Nyhan, 1964; Seegmiller et al., 1967). The metabolic conse-
quence of HGPRT deficiency is an overproduction of uric acid in the blood (hyperuricemia) that can
lead to gout and tophi, or nephrolithiasis (Sass etal., 1965; Kelley etal., 1967). Affected children
also develop severe neurological impairments, such as dystonia, choreoathetosis, spasticity, and a
dramatic compulsive self- injurious behavior (Nyhan, 1997; Jinnah etal., 2006; Torres etal., 2007a;
Schretlen etal., 2005; Madeo etal., 2019). They have a developmental delay from 3 to 6months
after birth, and most of them never walk or even sit without support. Xanthine oxidase inhibitors, such
as febuxostat or allopurinol, are given to patients after diagnosis to decrease their uric acid levels and
prevent the formation of urate crystals in kidney, which can lead to renal failure (Kelley etal., 1967;
Torres etal., 2007a; Lahaye et al., 2014). However, there is as yet no treatment to alleviate the
neurological symptoms of LND (Torres and Puig, 2007b; Jinnah etal., 2010; Madeo etal., 2019).
To date, the causes of neurobehavioral troubles in LND are still not elucidated (Jinnah etal., 2010;
Bell etal., 2016). The most favored hypothesis is a dysfunction of the brain’s basal ganglia, and partic-
ularly of its dopaminergic pathways (Baumeister and Frye, 1985; Visser etal., 2000; Nyhan, 2000;
Saito and Takashima, 2000; Egami etal., 2007). Indeed, analyses revealed a marked loss of dopa-
mine (DA) (Lloyd etal., 1981; Ernst etal., 1996) and DA transporters (Wong etal., 1996) in the basal
ganglia of LND patients. DA deficits have also been reported in HGPRT knockout rodents, but without
motor or behavioral defects (Finger etal., 1988; Dunnett etal., 1989; Jinnah etal., 1993; Jinnah
etal., 1994; Meek etal., 2016). Recent studies reported that HGPRT deficiency disrupts prolifer-
ation and migration of developing midbrain DA neurons in mouse embryos, arguing for a neurode-
velopmental syndrome (Witteveen etal., 2022). This could result from ATP depletion and impaired
energy metabolism (Bell etal., 2021) or an overactivation of de novo purine synthesis, leading to the
accumulation of potentially toxic intermediates of this pathway (López, 2008; López et al., 2020).
Pharmacological models have also been developed by injecting the neurotoxin 6- hydroxydopamine
into neonatally rat brains, which induced a self- mutilation behavior in response to DA agonist admin-
istration in adulthood. However, these models are of limited utility as they do not reproduce the basic
genetic impairment of LND (Breese etal., 1990; Knapp and Breese, 2016; Bell etal., 2016).
New animal models are therefore needed to study LND pathogenesis and find efficient therapeutic
molecules. The fruit fly Drosophila melanogaster presents many advantages for translational studies
and drug discovery (Fernández- Hernández etal., 2016; Perrimon etal., 2016; Papanikolopoulou
etal., 2019). Although the importance of this invertebrate model for studying rare human genetic
diseases is now recognized (Oriel and Lasko, 2018), a Drosophila LND model has not yet been devel-
oped to our knowledge. This is probably due to the fact that no HGPRT activity has been detected in
Research article Genetics and Genomics | Neuroscience
Petitgas etal. eLife 2023;12:RP88510. DOI: https://doi.org/10.7554/eLife.88510 3 of 34
this organism (Miller and Collins, 1973; Becker,
1974a; Becker, 1974b). However, an ortholog
of APRT is expressed in Drosophila (Johnson
et al., 1987), encoded by the Aprt gene. It is
therefore possible that part of the functions of
human HGPRT, and in particular those essential
for nervous system development and neurophysi-
ology, are endorsed by Aprt in Drosophila.
Here, we studied the effects of Aprt deficiency
on purine metabolism, lifespan, and various adult
fly behaviors, including spontaneous and startle-
induced locomotion, sleep, and seizure- like bang
sensitivity (BS). We show that Aprt is required
during development and in adult stage for many
aspects of Drosophila life, and that its activity is of
particular importance in subpopulations of brain
dopaminergic neurons and glial cells. Lack of
Aprt appears to decrease adenosinergic signaling
and induces both metabolic and neurobehavioral
symptoms in flies, as is the case with HGPRT in
humans. We also find that expression of an LND- associated mutant form of HGPRT, but not the wild-
type enzyme, in Drosophila neurons, induced neurobehavioral impairments similar to those of Aprt-
deficient flies. Such a potential toxic gain- of- function effect of mutated HGPRT had not yet been
demonstrated in an animal model .
Results
Evolution of HGPRT proteins
The pathways of purine anabolism/catabolism and recycling have been closely conserved between
Drosophila melanogaster and humans (Figure1—figure supplement 1): all genes related to purine
metabolism have homologs in both species, except for the human HPRT1 gene encoding HGPRT
(step 13 in Figure1—figure supplement 1), which has no ortholog in flies, and the lack of urate
oxidase in humans (step 20). In accordance with pioneering reports from about 50years ago (Miller
and Collins, 1973; Becker, 1974a; Becker, 1974b), we confirmed that no HGPRT enzymatic activity
can be detected in extracts of wild- type D. melanogaster (see below Table 2), using either hypoxan-
thine or guanine as substrate in the reaction medium. This intriguing observation prompted us to carry
out a more precise analysis of the evolution of HGPRT.
HGPRT proteins are ancient, for they are present in both bacteria and archaea. However, the analysis
of the phyletic distribution of HGPRT proteins revealed their striking rareness in insecta. This conclu-
sion is based on PSI- Blast sequence similarity searches on the NCBI Insecta database (taxid: 6960,
50557). Phylogenetic analysis showed that the only 11 HGPRT proteins found in Insecta cluster mainly
with bacteria, but also with fungi, apicomplexa, and acari (Figure1—figure supplement 2, red font,
see legend for details). These and further evidence support the acquisition of HGPRT in a few insecta
species by horizontal gene transfer (G. Matassi, unpublished observations). In particular, HGPRT has
no homolog in Drosophilidae, with the potential exception of a single species, Drosophila immigrans,
in which our most recent PSI- BLAST analysis detected one hit (accession KAH8256851.1, annotated
as hypothetical protein). Yet this sequence is 100% identical to the HGPRT of the Gammaproteobac-
terium Serratia marcescens. A phylogenetic analysis showed that the D. immigrans HGPRT clusters
with the Serratia genus (Figure1—figure supplement 3), suggesting either a contamination of the
sequenced sample or a very recent horizontal gene transfer event. The second scenario is more likely
since the corresponding nucleotide sequences differ by five synonymous substitutions (out of 534
positions). We also carried out structural similarity searches against the RCSB Protein Data Bank repos-
itory using the human HGPRT structure as query (PDB identifiers: 5HIA or 1Z7G). This analysis did not
identify any protein with a divergent sequence and relevant similarity with HGPRT 3D structure in D.
melanogaster, consistent with the lack of HGPRT enzymatic activity in this organism.
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/Aprt5Males 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
The online version of this article includes the following
source data for table 1:
Source data 1. Source data for Table1.
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Drosophila lacking Aprt activity have a shortened lifespan
Both the phylogeny and enzymatic assays therefore strongly suggest that Aprt (Figure1—figure
supplement 1, step 12) is the only recycling enzyme of the purine salvage pathway in Drosophila.
To assess the importance of purine recycling in brain development and function in this organism, we
analyzed the phenotypes induced by a deficiency in Aprt. The Aprt5 mutant was originally gener-
ated by chemical mutagenesis followed by selection of flies resistant to purine toxicity (Johnson and
Friedman, 1981; Johnson and Friedman, 1983). Enzymatic assays confirmed a strong reduction in
Aprt activity in extracts of heterozygous Aprt5/+ mutantsand its absence in homozygous and hemizy-
gous (Aprt5/Df(3L)ED4284) mutants (Figure1—figure supplement 4 and Table1). Sequencing of the
Aprt5 cDNA (Figure1—figure supplement 5A) indicated that the Aprt5 mRNA codes for a protein
with several amino acid changes compared to D. melanogaster wild- type Aprt, modifying in partic-
ular three amino acid residues that have been conserved in the Aprt sequences from Drosophila to
humans (Figure1—figure supplement 5B). These mutations are likely to be responsible for the loss
of enzymatic activity. The homozygous Aprt5 mutants are considered viable because they develop and
reproduce normally. However, we observed that these mutants have a significantly reduced longevity,
their median lifespan being only 38d against 50d for wild- type flies (p<0.001) (Figure1A).
Uric acid levels are increased in Aprt5 mutants and rescued by
allopurinol
In humans, one of the consequences of HGPRT deficiency on metabolism is the overproduction of uric
acid (Harkness etal., 1988; Fu etal., 2015). We assayed the levels of purine metabolites by HPLC
and found that the level of uric acid was substantially increased by 37.7% on average in Drosophila
Aprt5 mutant heads (p<0.01) (Figure1B). We then tried to rescue uric acid content in flies by providing
allopurinol in the diet, as is usually done for LND patients. Allopurinol is a hypoxanthine analog and
a competitive inhibitor of xanthine oxidase, an enzyme that catalyzes the oxidation of xanthine into
uric acid (Figure1—figure supplement 1, step 19). Remarkably, the administration of 100μg/ml allo-
purinol during 5d decreased uric acid levels to a normal concentration range in Aprt5 mutant heads
(Figure1B). Therefore, quite similarly to HGPRT deficit in humans, the lack of Aprt activity in flies
increases uric acid levels and this metabolic disturbance can be prevented by allopurinol.
Aprt deficiency decreases motricity in young flies
LND patients present dramatic motor disorders that prevent them for walking at an early age. To
examine whether a deficiency in the purine salvage pathway can induce motor disturbance in young
flies, we monitored the performance of Aprt- null mutants in startle- induced negative geotaxis (SING),
a widely used paradigm to assess climbing performance and locomotor reactivity (Feany and Bender,
2000; Friggi- Grelin et al., 2003; Riemensperger et al., 2013; Sun et al., 2018). Strikingly, Aprt5
mutant flies showed a very early SING defect starting from 1day after eclosion (d a.E.) (performance
index [PI] = 0.73 vs 0.98 for wild- type flies, p<0.001) that was more pronounced at 8 d a.E. (PI = 0.51
vs 0.96 for the wild- type flies, p<0.001). The fly locomotor performance did not further decline after-
wards until 30 d a.E. (Figure1C). Df(3L)ED4284 and Df(3L)BSC365 are two partially overlapping small
genomic deficiencies that uncovers Aprt and several neighbor genes. Hemizygous Aprt5/Df(3L)ED4284
or Aprt5/Df(3L)BSC365 flies also displayed SING defects at 10 d a.E. (PI = 0.71and 0.68, respectively,
compared to 0.97 for wild- type flies, p<0.01) (Figure 1—figure supplement 6). In contrast to its
beneficial effect on uric acid levels, we observed that allopurinol treatment did not improve the loco-
motor ability of Aprt5 mutant flies, either administered 5d before the test or throughout the devel-
opment (Figure1D and E). This is comparable to the lack of effect of this drug against neurological
defects in LND patients.
To confirm the effect of Aprt deficiency on the SING behavior, we used an UAS- AprtRNAi line that
reduced Aprt activity by more than 95% when expressed in all cells with the da- Gal4 driver (Table1).
These Aprt knock- down flies also showed a strong SING defect at 10 d a.E. (PI = 0.62 against 0.97
and 0.85 for the driver and UAS- AprtRNAi alone controls, respectively, p<0.001), like the Aprt5 mutant
(Figure1F). Next, we used tub- Gal80ts, which inhibits Gal4 activity at permissive temperature (McGuire
etal., 2003), to prevent Aprt knockdown before the adult stage. Tub- Gal80ts; da- Gal4>AprtRNAi flies
raised at permissive temperature (18°C) did not show any locomotor impairment (Figure1G, white
bars). However, after being transferred for 3d (between 7 and 10 d a.E.) at a restrictive temperature
Research article Genetics and Genomics | Neuroscience
Petitgas etal. eLife 2023;12:RP88510. DOI: https://doi.org/10.7554/eLife.88510 5 of 34
Figure 1. Aprt deciency shortens lifespan and induces metabolic and neurobehavioral defects. (A) Aprt5 mutant ies have a reduced lifespan
compared to wild- type ies (median lifespan: 38 and 50d, respectively). Three independent experiments were performed on 150males per genotype
with similar results and a representative experiment is shown. Log- rank test (***p<0.001). (B) HPLCproles on head extracts revealed an increase in
uric acid levels in Aprt5 mutant ies. Administration of 100μg/ml allopurinol for 5d before the test rescued uric acid levels. Mean of three independent
Figure 1 continued on next page
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(30°C) that inactivates Gal80, the same flies demonstrated a similar SING defect as Aprt5 mutants
(PI = 0.63 compared to 0.94 and 0.83 for the driver and RNAi alone controls, respectively, p<0.001)
(Figure1G, yellow bars with dots). This demonstrates that Aprt inactivation at the adult stage only is
sufficient to alter SING performance in Drosophila, strongly suggesting that this genotype does not
result from developmental defects.
Cell specificity of Aprt requirement for startle-induced locomotion
We then tried to identify the neural cells in which Aprt is required to ensure a normal locomotor reac-
tivity in young flies by expressing AprtRNAi with selective Gal4 drivers. Expression in all neurons with
elav- Gal4 led to decreased locomotor performance in the SING test (PI = 0.68 at 10 d a.E., vs 0.90
and 0.86 for the driver and RNAi controls, respectively, p<0.05) (Figure2A), which was comparable
to the effects observed with the Aprt5 mutant or after ubiquitous expression of the RNAi. To confirm
the role of neuronal Aprt in locomotor control, we generated a UAS- Aprt line, which allowed for a
substantial increase in Aprt expression and activity (Figure2—figure supplement 1). We then found
that re- expressing Drosophila Aprt selectively in neurons in Aprt5 background partially rescued the
SING phenotype of the null mutant (PI = 0.70 vs 0.52 for driver and UAS- Aprt controls in Aprt5 back-
ground, p<0.05) (Figure2B).
Furthermore, Aprt knockdown in all glial cells with repo- Gal4, or in sub- populations of glial cells
that express the glutamate transporter Eaat1 with Eaat1- Gal4, which includes astrocyte- like glia,
cortex glia, and some subperineurial glia (Rival et al., 2004; Mazaud etal., 2019), also led to a
lower SING performance of 10- day- old flies (PI = 0.72 for repo- Gal4 vs 0.91 for both controls, p<0.05,
and PI = 0.56 for Eaat1- Gal4 vs 0.77, p<0.05, and 0.88, p<0.01, for the driver and RNAi controls,
respectively) (Figure2C and D). In contrast, MZ0709- Gal4 (Doherty etal., 2009) and NP6520- Gal4
(Awasaki et al., 2008) that selectively target the ensheathing glia did not induce any significant
locomotor defects when used to express the Aprt RNAi (Figure2—figure supplement 2). Notice-
ably, re- expressing Aprt with Eaat1- Gal4 in the Aprt5 background did not rescue the SING pheno-
type (Figure2E), in contrast to the positive effect of neuronal re- expression (Figure2B). Overall this
experiments performed on 40 ies per genotype. One- way ANOVA with Tukey’s post hoc test for multiple comparisons (*p<0.05; **p<0.01; ns: not
signicant). (C–E)Effect on climbing ability. (C)Aprt5 mutants shows early defects in the startle- induced negative geotaxis (SING) paradigm that monitors
locomotor reactivity and climbing performance. This decit was already obvious at 1 day after eclosion (d a.E.) and further decreased up to 8 d a.E., after
which it did not change signicantly up to 30 d a.E. Mean of three independent experiments performed on 50 ies per genotype. Unpaired Student’s
t- test (**p<0.01; ***p<0.001). (D, E)Administration of allopurinol does not rescue the motricity defects of Aprt- decient mutants. Feeding the Aprt5
mutants with allopurinol (100μg/ml) either in adults 5d before the test (D)or throughout all developmental stages (E)did not alter the defects observed
in SING behavior. Results of one experiment performed on 50 ies per genotype at 10 d a. E. Unpaired Student’s t- test (***p<0.001). (F)Downregulating
Aprt by RNAi in all cells (da>AprtRNAi) also led to an early impairment in climbing responses in the SING assay at 10 d a.E. compared to the driver (da/+)
and effector (AprtRNAi/+) only controls. Mean of three independent experiments performed on 50 ies per genotype. One- way ANOVA with Tukey’s
post hoc test for multiple comparisons (***p<0.001). (G)Adult- specic inactivation of Aprt (tub- Gal80ts; da- Gal4>AprtRNAi) decreased startle- induced
climbing abilities in the SING paradigm, suggesting that the locomotor impairment induced by Aprt deciency is not caused by a developmental effect.
Flies were raised at permissive temperature (18°C) in which Gal80ts suppressed Gal4- controlled AprtRNAi expression and were shifted from 18 to 30°C
for 3d before the test (between 7 and 10 d a.E.) to activate transgene expression. Mean of three independent experiments performed on 50 ies per
genotype. Two- way ANOVA with Sidak’s post hoc test for multiple comparisons (***p<0.001; ns: not signicant).
The online version of this article includes the following source data and gure supplement(s) for gure 1:
Source data 1. Source data for Figure1A–G.
Figure supplement 1. Comparison of purine metabolism pathways in Drosophila and humans.
Figure supplement 2. Urooted maximum likelihood phylogeny of hypoxanthine- guanine phosphoribosyltransferase (HGPRT) proteins (189 taxa, 130
sites).
Figure supplement 3. Urooted maximum likelihood phylogeny of HPRT proteins (20 taxa, 177 sites).
Figure supplement 4. Lack of Aprt enzymatic activity in the Aprt5 mutant.
Figure supplement 4—source data 1. Source data for Figure1—figure supplement 4.
Figure supplement 5. Alignment of wild- type and mutant Aprt cDNAs and predicted protein sequences.
Figure supplement 6. Startle- induced negative geotaxis (SING) behavior of hemizygous Aprt mutant ies.
Figure supplement 6—source data 1. Source data for Figure1—figure supplement 6.
Figure 1 continued
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Figure 2. 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 ies. (B)Pan- neuronal expression of Drosophila Aprt with the
UAS- Gal4 system partially rescued the locomotor response of Aprt5 mutants. (C, D)Downregulation of Aprt expression in all glia with repo- Gal4 (C)or
in glial cell that express the glutamate transporter Eaat1 with Eaat1- Gal4 (D)also altered SING performances. (E)Aprt re- expression in glial cells with
Eaat1- Gal4 driver did not rescue the climbing response of Aprt5 mutants. Results of three or four independent experiments performed on 50 ies per
genotype at 10 days after eclosion (d a.E.). One- way ANOVA with Tukey’s post hoc test for multiple comparisons (*p<0.05; **p<0.01; ns: not signicant).
The online version of this article includes the following source data and gure supplement(s) for gure 2:
Source data 1. Source data for Figure2A–E.
Figure supplement 1. Transgenic expression of Drosophila Aprt.
Figure supplement 1—source data 1. Source data for Figure2—figure supplement 1.
Figure supplement 2. Downregulation of Aprt expression in the ensheathing glia does not alter locomotor performances.
Figure supplement 2—source data 1. Source data for Figure2—figure supplement 2.
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suggests that Aprt is required both in neurons and glia subsets to ensure a normal SING performance
in young flies, and that neuronal but not glial Aprt re- expression is sufficient to restore a partially
functional locomotor behavior.
To identify the neuronal subpopulations in which Aprt is required to ensure proper locomotor
responses in young flies, we first tested dopaminergic drivers since we previously showed that DA
neurons play an important role in the control of the SING behavior (Riemensperger etal., 2011;
Riemensperger et al., 2013; Sun etal., 2018). Aprt knockdown targeted to these neurons with
TH- Gal4 did not induce significant impairments (Figure 3A). This driver expresses in all brain DA
neurons except a large part of the protocerebral anterior medial (PAM) cluster (Friggi- Grelin etal.,
2003; Mao and Davis, 2009; Pech etal., 2013). In contrast, downregulating Aprt with the TH- Gal4,
R58E02- Gal4 double driver, which labels all DA neurons including the PAM cluster (Sun etal., 2018),
induced a quite similar locomotor defect as did pan- neuronal Aprt knockdown (PI = 0.72 vs 0.96 and
0.93 for the driver and RNAi controls, respectively, p<0.001) (Figure3B). Besides, downregulating
Aprt in a majority of the serotonergic neurons with TRH- Gal4 (Cassar etal., 2015) did not induce a
SING defect (Figure3C).
These results suggest that some DA neurons in the PAM cluster are specifically involved in the
locomotor impairments induced by Aprt deficiency. It has been previously shown in our laboratory
that inhibiting DA synthesis in a subset of 15 PAM DA neurons cluster was able to alter markedly
SING performance in aging flies (Riemensperger etal., 2013). We and others also reported that
the degeneration or loss of PAM DA neurons is involved in the SING defects observed in several
Drosophila models of Parkinson’s disease (Riemensperger etal., 2013; Bou Dib etal., 2014; Ta s
etal., 2018; Pütz et al., 2021). Here we found that expressing AprtRNAi in the PAM cluster with
R58E02- Gal4 reproduced the same motor disturbance as pan- neuronal expression (PI = 0.74 vs
0.96, p<0.001, and 0.85, p<0.05, for the driver and RNAi controls, respectively) (Figure3D), and
this result was confirmed by using two other PAM drivers (NP6510- Gal4 – expressing only in 15
neurons – and R76F05- Gal4) (Figure3E and F). This strongly suggests that purine recycling defi-
ciency compromises the correct functioning of these neurons, leading to a defective startle- induced
locomotion.
Because PAM DA neurons innervate the horizontal lobes of the mushroom bodies (Liu etal., 2012;
Riemensperger etal., 2013), and because this structure has been shown to be involved in the control
of startle- induced climbing (Riemensperger etal., 2013; Bou Dib etal., 2014; Sun et al., 2018),
we also inactivated Aprt in mushroom body neurons with 238Y- Gal4 that strongly expresses in that
structure (Aso etal., 2009). Interestingly, this driver did induce a locomotor reactivity impairment
(PI = 0.70 vs 0.89 for both controls, p<0.01) (Figure 3G), and the same result was observed with
VT30559- Gal4, which is a very specific driver for all the mushroom body intrinsic neurons (Plaçais
etal., 2017; Figure3H). Overall, these results show that normal expression of the SING behavior
depends on Aprt expression in the PAM and mushroom body neurons in Drosophila.
Sleep disturbances induced by Aprt deficiency
Both the mushroom body and subpopulations of PAM DA neurons are known to be regulators of
sleep in Drosophila (Nall etal., 2016; Artiushin and Sehgal, 2017). The fact that Aprt deficiency in
some of these cells impaired locomotor regulation prompted us to monitor the spontaneous loco-
motion and the sleep pattern of Aprt mutants. Compared to controls, Aprt- deficient flies did not
have an altered circadian activity profile (Figure4—figure supplement 1), nor any difference in total
spontaneous locomotor activity during the day, as quantified by the number of infrared beam cuts
(events) per 30min, but they showed a 26.2% increase in total activity during the night (p<0.001)
(Figure4A). As usual, a sleep bout was defined as 5min or more of fly immobility (Huber etal., 2004),
and we checked that wild- type and Aprt mutant flies that did not move for 5min were indeed asleep
because they were less sensitive to mild mechanical stimulation (Figure4—figure supplement 2).
We found that Aprt5 mutants slept significantly less than wild- type flies and that it was the case both
during day and night (Figure4B and C). These mutants indeed showed a reduced walking speed
(Figure4D) and a smaller average sleep bout duration (Figure4E), indicating a difficulty to maintain
sleep. The reduced speed does not seem to be caused by a decreased energetic metabolism since
we could not detect different ATP levels in head and thorax of Aprt5 mutants compared to wild- type
flies (Figure4—figure supplement 3).
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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 (SING) assay. (B)In contrast, Aprt knockdown in all dopaminergic neurons
Figure 3 continued on next page
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Interestingly, RNAi- mediated downregulation of Aprt selectively in neurons (elav>AprtRNAi flies)
also significantly decreased sleep duration during day and night (Figure 4F), whereas glial- only
expression (repo>AprtRNAi) had no effect (Figure4—figure supplement 4A). Expressing the RNAi in
both neurons and glia (elav; repo>AprtRNAi) had similar effects as in neurons alone (Figure4—figure
supplement 4B). Quantification of total sleep in these experiments, and the total amount of day
and night sleep, are shown in Figure4G and Figure4—figure supplement 4C and D, respectively.
Overall, these results suggest that Aprt expression is selectively needed in neurons for a normal sleep
pattern in Drosophila.
Brain DA synthesis and levels are increased in Aprt-deficient flies
Since we found that induced locomotion and sleep, two behaviors controlled by DA neurons in
Drosophila, were altered in Aprt- deficient flies, we expected brain DA levels to be reduced in these
mutants, as is the case in the basal ganglia of LND patients. We therefore carried out comparative
immunostaining for tyrosine hydroxylase (TH), the specific enzyme for DA synthesis (Friggi- Grelin
etal., 2003; Riemensperger etal., 2011), on dissected adult brains from wild- type and Aprt5 mutant
flies. However, the global TH protein level appeared not to be decreased, but relatively increased by
17.5% in the Aprt5 mutant brain (p<0.01), in particular around the mushroom bodies, a structure that
receives dense dopaminergic projections (Figure5A and B). Moreover, DA immunostaining carried
out on whole- mount dissected brains revealed a similarly increased level of this neuromodulator in
Aprt5 flies, by 17.0% on average in the entire brain (p<0.01), but not specifically in the mushroom
bodies or another part of the brain (Figure5C and D). We also found that the transcript levels of
DTH1, encoding the TH neuronal isoform in Drosophila, were increased in Aprt5 mutants compared
to wild- type flies (Figure5E), and, conversely, decreased when Aprt was overexpressed ubiquitously
(Figure 5F). Western blot experiments further indicated that DTH1 protein levels are increased in
Aprt5 compared to controls (Figure5G and H). This indicates that disruption of the purine salvage
pathway does not impede DA synthesis and levels in the Drosophila brain, which are instead slightly
increased. We therefore searched for another neurotransmitter system that could be affected by Aprt
deficiency.
Interactions between Aprt and the adenosinergic system
Aprt catalyzes the conversion of adenine into AMP, and AMP breakdown by the enzyme 5′-nucle-
otidase produces adenosine, primarily in the extracellular space. Adenosine then acts as a wide-
spread neuromodulator in the nervous system by binding to adenosine receptors. We therefore
suspected that adenosine level could be reduced in the absence of Aprt activity, leading to alter-
ations in adenosinergic neurotransmission. Indeed, we found a significant decrease in adenosine level
either in whole flies (by 61.0% on average, p<0.01) or in heads (by 48.0%, p<0.05) in Aprt5 mutants
(Figure6A). We then examined the consequences of this reduction on molecular components of the
adenosinergic system, namely G protein- coupled adenosine receptors and equilibrative nucleoside
transporters (ENTs), which carry out nucleobase and nucleoside uptake of into cells. Only one seven-
transmembrane- domain adenosine receptor, AdoR, is present in Drosophila, which is very similar to
mammalian adenosine receptors (Dolezelova etal., 2007; Brody and Cravchik, 2000), and three
putative equilibrative nucleoside transporters (Ent1- 3) have been identified (Sankar etal., 2002) but
only one, Ent2, showed nucleoside transport activity (Machado etal., 2007). Interestingly, Ent2 has
including the PAM cluster with the TH- Gal4, R58E02- Gal4 double- driver led to a decrease in SING performance.
(C)Aprt downregulation in serotonergic neurons with TRH- Gal4 did not alter startle- induced climbing response of
the ies. (D–F) Aprt knockdown selectively in DA neurons of the PAM cluster using either R58E02- Gal4 (D),NP6510-
Gal4 (E),or R76F05- Gal4 (F)signicantly decreased climbing performance. (G, H)Aprt knockdown in all the
mushroom body intrinsic neurons (Kenyon cells) with 238Y- Gal4 (G)or VT30559- Gal4 (H)also led to a decrease
in SING performance. Results of three or four independent experiments performed on 50 ies per genotype at
10 days after eclosion (d a.E.). One- way ANOVA with Tukey’s post hoc test for multiple comparisons (*p<0.05;
**p<0.01; ***p<0.001; ns: not signicant).
The online version of this article includes the following source data for gure 3:
Source data 1. Source data for Figure3A–H.
Figure 3 continued
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Figure 4. Aprt- decient ies sleep less and walk slower than wild- type ies. (A)Quantication of total spontaneous locomotor activity during day and
night over ve light- dark (LD) cycles. Aprt5 mutants show no difference in spontaneous locomotion with wild- type ies during the day but a higher
activity at night. Three independent experiments were performed on 32 ies per genotype and mean ± SEM was plotted. Unpaired Student’s t- test
(***p<0.001; ns: not signicant). (B)Sleep pattern during a typical 24hr LD cycle showing that the total amount of sleep is smaller during day and
night in Aprt5 mutants compared to wild- type ies. (C)Quantication of day (ZT1- 12), night (ZT13- 24), and total sleep in Aprt5 mutants. ZT, zeitgeber.
(D)Locomotion speed during waking is reduced in Aprt5 mutants. (E)The average sleep bout duration is also decreased, indicating that Aprt5 mutants
have a difculty to maintain sleep. (F)Sleep pattern of elav>AprtRNAi ies, showing that knockdown of Aprt in all neurons led to sleep reduction during
the night, similarly to the mutant condition, and an even more profound sleep defect during the day. (G)Quantication of total amount of sleep when
Aprt was downregulated in all neurons (elav- Gal4), all glial cells (repo- Gal4), and both neurons and glial cells (elav- Gal4; repo- Gal4). Except for glia only,
Figure 4 continued on next page
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been shown to be enriched in the mushroom bodies and its transcript level to be highly elevated in
AdoR mutant flies (Knight etal., 2010). In Aprt5 background, we also observed a strong increase
in Ent2 mRNAs (2.3- fold higher than wild- type flies, p<0.001), but no noticeable effect on AdoR
transcript level (Figure6B). The increased expression of Ent2 could correspond to a compensatory
response to adenosine shortage in Aprt5 mutants.
The AdoR receptor is highly expressed in adult fly heads and its ectopic overexpression leads to
early larval lethality (Dolezelova etal., 2007). In contrast, a null mutant of this receptor, AdoRKGex,
in which the entire coding sequence is deleted, is fully viable (Wu etal., 2009). This enabled us to
examine the consequences of a complete lack of AdoR on purine recycling in adult flies. We found that
Aprt transcripts were decreased by 29.5% on average (p<0.001) in AdoRKGex mutant heads (Figure6C,
left panel), while Aprt activity was even more decreased by 78.4% (p<0.001) compared to wild- type
flies (Figure 6C, right panel). This effect likely results from the much increased level of adenosine
uptake in the AdoR mutants (Knight et al., 2010), which would downregulate Aprt activity by a
feedback mechanism. Overall, these data indicate that extracellular adenosine levels must be strongly
decreased in Aprt mutant flies, both from the general reduction in adenosine levels and the increased
expression of the Ent2 transporter. Although AdoR expression is not affected, AdoR signaling is there-
fore expected to be significantly reduced in Aprt mutants.
Aprt mutants show epilepsy-like seizure behavior
A number of Drosophila mutants with disrupted metabolism or neural function show increased
susceptibility to seizure and paralysis following strong mechanical or electrophysiological stimulation
(Fergestad et al., 2006; Parker et al., 2011; Kroll et al., 2015). These mutants are called bang
sensitive (BS) and commonly used as models of epileptic seizure (Song and Tanouye, 2008). Here we
checked whether Aprt deficiency could trigger a BS phenotype. We observed that aged Aprt mutants
(at 30 d a.E.) recovered slowly after a strong mechanical stimulation applied by vortexing the vial for
10s at high speed. These flies took on average 17.3s to recover and get back on their legs compared
to 2.5s for wild- type flies of the same age (p<0.01) (Figure7A; see also Figure7—videos 1 and 2
). Some of the mutant flies appeared more deeply paralyzed as they did not spontaneously recover
unless the vial was stirred, so their recovery time could not be scored. Hemizygous flies of the same
age containing the Aprt5 mutation over two partially overlapping genomic deficiencies covering Aprt
also showed a BS behavior, with an average longer recovery time of 28.9s and 33.2s, respectively
(p<0.001) (Figure7B).
We then downregulated Aprt by RNAi in all cells with the da- Gal4 driver to check if this could also
induce BS behavior. As shown in Figure7C, da>AprtRNAi flies at 30 d a.E. indeed displayed seizure
after mechanical shock, quite similar to that of the Aprt5 mutants (22.7s recovery time on average
compared to 1.55s and 0.72 s for the driver and RNAi controls, respectively, p<0.05). In contrast,
inactivating Aprt selectively in neurons (elav- Gal4), glial cells (repo- Gal4), or muscles (24B- Gal4) did
not induce a BS phenotype (Figure7—figure supplement 1). This suggests that the BS phenotype
requires Aprt knockdown in other cells or in several of these cell types. Finally, in contrast to the SING
defect, we observed that adult- specific Aprt knockdown in all cells with da- Gal4 for 3d did not trigger
the resulting effect was a signicant sleep reduction. For sleep and locomotion speed, means ± SEM were plotted. Unpaired Student’s t- test (C–E)and
one- way ANOVA with Tukey’s post hoc test for multiple comparisons (G) (*p<0.05; **p<0.01; ***p<0.001; ns: not signicant).
The online version of this article includes the following source data and gure supplement(s) for gure 4:
Source data 1. Source data for Figure4A–G.
Figure supplement 1. Daily locomotor activity proles of wild- type and Aprt5 mutant ies.
Figure supplement 2. Sensitivity of resting ies to mild mechanical stimulation.
Figure supplement 2—source data 1. Source data for Figure4—figure supplement 2.
Figure supplement 3. ATP levels are not altered in head and thorax of Aprt5 ies compared to the wild- type.
Figure supplement 3—source data 1. Source data for Figure4—figure supplement 3.
Figure supplement 4. Sleep patterns of ies with cell- specic Aprt deciency.
Figure supplement 4—source data 1. Source data for Figure4—figure supplement 4.
Figure 4 continued
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Figure 5. Aprt deciency 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 ies and Aprt5 mutants. MB: mushroom body. Scale bars: 100μm. (B)Quantication of TH
immunouorescence intensity normalized to the controls in the entire brain. 4–6 brains were dissected per
Figure 5 continued on next page
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a BS behavior (Figure7D), indicating that the BS requires a longer downregulation of the gene or
might be the consequence of a developmental defect.
Administration of adenosine or N6-methyladenosine to Aprt-deficient
flies prevents seizure
Drosophila disease models are advantageously tractable for drug screening in vivo (Fernández-
Hernández etal., 2016; Perrimon et al., 2016). We thus administered several compounds related
to purine metabolism to Aprt5 flies to check if they can rescue neurobehavioral impairments (loco-
motor defects and seizure). Feeding allopurinol at the same concentration used for uric acid rescue
(100μg/ml, Figure1B), either in adults 5d before the test or throughout all developmental stages,
did not alter the BS phenotype (Figure7—figure supplement 2), as was the case for the SING assay
(Figure1D and E). Similarly in humans, it has been shown that the daily intake of allopurinol, even
from infancy, does not mitigate the neurobehavioral impairments in LND patients (Marks etal., 1968;
Torres etal., 2007a; Jinnah etal., 2010; Madeo etal., 2019).
Then, we tried to supplement Aprt mutants with various purine compounds, including adenine,
hypoxanthine, adenosine, and N6- methyladenosine (m6A), at 100 or 500 μM, either in adult stage
5d before the test or throughout larval development plus 5d before the test. None of these drugs
was able to rescue the SING defect (Figure7—figure supplement 3). In contrast and interestingly,
administration of 500μM adenosine or m6A during development rescued the BS phenotype of Aprt5
mutants (Figure7E and F). This further indicates that different mechanisms underpin SING alteration
and BS behavior in Aprt mutants and provide another evidence that the BS may be caused by a devel-
opmental defect. The results also suggest that the lower adenosine levels of Aprt mutant flies could
be at the origin of their BS.
Neuronal expression of mutant HGPRT induces early locomotor defects
and seizure behavior
In order to potentially develop another Drosophila model mimicking LND conditions, we generated
new transgenic UAS lines to express in living flies either the human wild- type HGPRT (HGPRT- WT) or
a pathogenic LND- associated mutant form of this protein (HGPRT- I42T), both isoforms being inserted
at the same genomic docking site. These lines were validated by showing that they are transcribed at
similar levels (Figure8A and B). Enzymatic assays on adult extracts of flies expressing the wild- type
form HGPRT- WT in all cells with da- GAL4 showed significant HGPRT enzyme activity, while no activity
experiment and genotype, and 6 independent experiments were performed (**p<0.01). (C)Representative
confocal projections of DA immunostaining in whole- mount adult brains of wild- type and Aprt5 mutants. Scale
bars: 100μm. (D)Quantication of DA immunouorescence intensity over the entire brain showed a slight increase
in DA content in Aprt5 mutants compared to wild- type controls. Six brains were dissected per experiment and
genotype, and three independent experiments were performed. Unpaired Student’s t- test (**p<0.01). (E) mRNA
levels of TH neuronal form DTH1 are increased in Aprt5 mutant heads compared to wild- type ies. Results of six
independent RT- qPCR experiments carried out on 3–4 different RNA extractions from 20 to 30 male heads per
genotype. Unpaired Student’s t- test (**p<0.01). (F)Conversely, overexpressing Aprt in all cells with the da- Gal4
driver (da>Aprt) reduced mRNA level of DTH1 in heads compared to the driver (da/+) and effector (Aprt/+)
controls. Mean of three independent experiments performed on three different RNA extractions from 20 to 30
male y heads. One- way ANOVA with Tukey’s post hoc test for multiple comparisons (*p<0.05, **p<0.01). (G)
Representativewestern blot of wild- type and Aprt5 mutant head extracts probed with anti- TH and anti- actin beta
antibodies. (H) Quanticationof DTH1 protein levels in adult wild- type and Aprt5 mutant heads by western blots
showed an increase in DTH1 protein level (60kDa) in Aprt5 mutants. Actin (Act5C, 42kDa) was used as a loading
control. Results are the mean of four determinations in two independent experiments. Unpaired Student’s t- test
(**p<0.01).
The online version of this article includes the following source data for gure 5:
Source data 1. Source data for Figure5A–F and H.
Source data 2. Original les for the western blot analysis of Figure5G.
Source data 3. Original les for the western blot analysis of Figure5G with relevant bands and samples labeled.
Figure 5 continued
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was detected in driver and UAS control flies, and an 80.5% lower activity was detected in Drosophila
expressing the mutant form HGPRT- I42T (Table2).
We next analyzed the consequences of human HGPRT expression on the SING and BS behav-
iors. Interestingly, the pan- neuronal expression of mutant I42T isoform specifically induced a signif-
icant early locomotor defect at 15 d a.E. (PI = 0.71 vs 0.92 and 0.90 for the driver and effector
controls, respectively, p<0.01) (Figure8C) and a relatively small but robust BS behavior at 30 d a.E.
(2.3s average recovery time vs 0.64s and 0.31s for the driver and effector only controls, p<0.001)
(Figure8D). These defective phenotypes could not be seen when HGPRT- WT was expressed. There-
fore, and remarkably, whereas wild- type HGPRT expression appears to be innocuous in Drosophila,
we observed that the neuronal expression of a pathogenic LND- associated isoform triggered neuro-
behavioral impairments comparable to those of Aprt- deficient flies.
Figure 6. Relations between Aprt and molecular components of adenosinergic signaling. (A, B) Impactsof the lack of Aprt activity on the adenosinergic
system. (A)Adenosine level was measured in whole ies or heads of Aprt5 ies by ultra performance liquid chromatography (UPLC). Compared to wild-
type ies, adenosine level was signicantly reduced in the mutants. Results of three independent experiments performed with vemales per genotype
in triplicates and two independent experiments with 30 heads per genotype in triplicates. Two- way ANOVA with Sidak’s post hoc test for multiple
comparisons (*p<0.05; **p<0.01). (B)Aprt5 mutation did not affect AdoR expression but induced a marked increase in adenosine transporter Ent2
mRNA abundance. 3–6 different RNA extractions were performed on 20–30 male heads. Results of 3–6 independent experiments. Two- way ANOVA with
Sidak’s post hoc test for multiple comparisons (***p<0.001; ns: not signicant). (C)Null AdoRKGex mutants showed decreased Aprt expression (left panel)
and a stronger decrease in Aprt activity (right panel). Four independent RNA extractions were carried out on 20–30 male heads and four independent
real- time PCR determinations were done per RNA sample. For Aprt activity, three independent determinations were performed on 20 whole ies per
genotype. Unpaired Student’s t- test (***p<0.001).
The online version of this article includes the following source data for gure 6:
Source data 1. Source data for Figure6A–C.
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Figure 7. Aprt deciency triggers a seizure- like phenotype. (A)At 30 days after eclosion (d a.E.), Aprt5 mutants need a much longer time than wild- type
ies to recover from a strong mechanical shock, showing a bang- sensitive (BS) paralysis comparable to tonic- clonic seizure. Results of three independent
experiments performed on 50 ies per genotype. Unpaired Student’s t- test; **p<0.01. (B)At 30 d a. E., hemizygous Aprt5 mutants also showed a marked
BS phenotype. Results of 2–4 independent experiments performed on 50 ies per genotype. One- way ANOVA with Dunnett’s post hoc test for multiple
Figure 7 continued on next page
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Discussion
Over the past 35years, several animal models of LND have been developed in rodents based on
HGPRT mutation in order to better understand the cause of the disease and test potential therapeutic
treatments (Finger etal., 1988; Dunnett etal., 1989; Jinnah etal., 1993; Engle etal., 1996; Meek
et al., 2016; Witteveen et al., 2022). However, none of these models recapitulated the full LND
syndrome and, particularly, the motor or neurobehavioral symptoms resulting from HGPRT deficiency.
To date, the causes of the neurobehavioral impairments in LND are not yet clearly elucidated and
the disease is still incurable (Fu etal., 2014; Bell etal., 2016; López etal., 2020; Bell etal., 2021;
Witteveen etal., 2022). Here we used two different strategies to develop new models of this disease
in Drosophila, a useful organism to conduct genetic and pharmacological studies. First, we show that
Aprt deficiency induces both metabolic and neurobehavioral disturbances in Drosophila, similar to the
loss of HGPRT, but not APRT, activity in humans. Secondly, we expressed an LND- associated mutant
form of human HGPRT in Drosophila neurons, which also yielded behavioral symptoms. Our results
suggest that the fruit fly can be used to study the consequences of defective purine recycling pathway
and HGPRT mutation in the nervous system.
Aprt-deficient flies replicate lifespan and metabolic defects caused by
HGPRT deficiency
Flies that carry a homozygous null- mutation in Aprt develop normally and live until the adult stage
(Johnson and Friedman, 1983). While a previous study reported that heterozygous Aprt/+ flies have
an extended lifespan (Stenesen etal., 2013), we observed that homozygous Aprt5 mutants have in
contrast a significantly reduced longevity. The lack of HGPRT activity in LND also reduces lifespan
expectancy, generally under 40years of age for properly cared patients. Stenesen et al., 2013
showed that, in Drosophila, dietary supplementation with adenine, the Aprt substrate, prevented
the longevity extension conferred either by dietary restriction or heterozygous mutations of AMP
biosynthetic enzymes. This suggests that lifespan depends on accurate adenine level regulation. It
is possible that adenine could accumulate to toxic levels during aging in homozygous Aprt mutants,
explaining their shorter lifespan. Alternatively, since AMP is the Aprt product, AMP- activated protein
kinase (AMPK), an enzyme that protects cells from stresses inducing ATP depletion, could be less acti-
vated in Aprt mutants. Multiple publications explored the role of AMPK in lifespan regulation (Sinnett
and Brenman, 2016) and downregulating AMPK by RNAi in adult fat body or muscles (Stenesen
comparisons (***p<0.001). (C)RNAi- mediated downregulation of Aprt in all cells (da>AprtRNAi) also led to BS phenotype in adults at 30 d a.E., but not
with the driver and effector controls. Results of three independent experiments performed on 50 ies per genotype. One- way ANOVA with Tukey’s post
hoc test for multiple comparisons (*p<0.05). (D)Aprt knockdown by RNAi during the adult stage for 3d before the test was not sufcient to induce bang
sensitivity, suggesting that this phenotype could be caused by a developmental defect or a longer downregulation of Aprt. Results of two independent
experiments performed on 50 ies per genotype. Two- way ANOVA with Sidak’s post hoc test for multiple comparisons; ns: not signicant. (E, F)The BS
phenotype of 30- day- old Aprt5 mutants was rescued by feeding either 500µM adenosine (ado) (E)or 500µM N6- methyladenosine (m6A) (F)during all
developmental stages plus 5d before the test. Results of four or six independent experiments performed on 50 ies per genotype. One- way ANOVA
with Tukey’s post hoc test for multiple comparisons (***p<0.001, ns: not signicant).
The online version of this article includes the following video, source data, and gure supplement(s) for gure 7:
Source data 1. Source data for Figure7A–F.
Figure supplement 1. Aprt knockdown selectively in neurons, glia, or muscle cells did not induce bang sensitivity.
Figure supplement 1—source data 1. Source data for Figure7—figure supplement 1.
Figure supplement 2. Administration of allopurinol does not rescue the bang sensitivity phenotype of Aprt- decient mutants.
Figure supplement 2—source data 1. Source data for Figure7—figure supplement 2.
Figure supplement 3. Administration of various purine compounds does not rescue the motricity defects of Aprt- decient mutants.
Figure supplement 3—source data 1. Source data for Figure7—figure supplement 3.
Figure 7—video 1. Bang sensitivity phenotype of Aprt- decient ies: wild- type ies.
https://elifesciences.org/articles/88510/gures#g7video1
Figure 7—video 2. Bang sensitivity phenotype of Aprt- decient ies: Aprt5 mutant ies.
https://elifesciences.org/articles/88510/gures#g7video2
Figure 7 continued
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et al., 2013), as well as its ubiquitous inactivation under starvation (Johnson et al., 2010), both
reduced fly lifespan.
In humans, HGPRT deficiency induces hypoxanthine and guanine accumulation, resulting from lack
of recycling, and increased de novo purine synthesis (Harkness etal., 1988; Fu etal., 2015; Ceballos-
Picot etal., 2015). This in turn leads to uric acid overproduction that increases the risk for nephroli-
thiasis, renal failure, and gouty arthritis if not properly treated. In insects, the end product of purine
catabolism is not uric acid but allantoin (Figure1—figure supplement 1, step 20). However, urate
oxidase, the enzyme that converts uric acid into allantoin, is specifically expressed in the Malpighi
da/+
da>HPRT1-WT
da>HPRT1-I42T
HPRT1-WT/+
HPRT1-I42T/+
0
2
4
6
8
10
HPRT1/rp49 transcript level
elav/+
elav>HPRT1-WT
HPRT1-WT/+
elav>HPRT1-I42T
HPRT1-I42T/+
0.0
0.5
1.0
Performance Index (PI)
ns
**
elav/+
elav>HPRT1-WT
HPRT1-WT/+
elav>HPRT1-I42T
HPRT1-I42T/+
0
1
2
3
Average recovery time (sec)
******
ns
Figure 8. Expression of a pathogenic mutant isoform of human hypoxanthine- guanine phosphoribosyltransferase (HGPRT) induces neurobehavioral
defects in ies. (A, B) Ubiquitousexpression of human HPRT1with da- Gal4. (A)Amplication of human HPRT1 transcripts detected by RT- PCR in head
extracts of da>HPRT1WT and da>HPRT1- I42T ies. A band with lower intensity was also detected in the effector controls (UAS- HPRT1- WT/+ and
UAS- HPRT1- I42T/+), and not in the driver control (da/+), which indicates a small amount of driver- independent transgene expression. (B)Quantication
of the previous experiment. (C, D)Expression of the Lesch–Nyhan disease (LND)- associated I42T isoform in all neurons (elav>HPRT1- I42T), but not of
the wild- type form (elav>HPRT1- WT), induced an early SING defect at 15 d a.E. (C)and a BS phenotype at 30 d a.E. (D),compared to the driver (elav/+)
and effector (UAS- HPRT1- I42T/+) only controls. Results of three independent experiments performed on 50 ies per genotype. One- way ANOVA with
Tukey’s post hoc test for multiple comparisons (**p<0.01; ***p<0.001; ns: not signicant).
The online version of this article includes the following source data for gure 8:
Source data 1. Original le for the DNA gel electrophoresis of Figure8A.
Source data 2. Original le for the DNA gel electrophoresis of Figure8A with relevant bands and samples labeled.
Source data 3. Source data for Figure8B–D.
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tubules, an excretory organ producing pre- urine
(Wallrath et al., 1990). Uric acid could there-
fore accumulate in fly tissues and hemolymph if
purine salvage pathway is impaired. Accordingly,
we observed an increase in uric acid levels in
Drosophila Aprt mutant heads, which could be
reduced to normal levels by providing allopu-
rinol, a xanthine oxidase inhibitor used to protect
against renal failure in LND patients.
Aprt is required in dopaminergic
and mushroom body neurons for
young fly motricity
We found that Aprt- null adult flies show reduced
performance in the SING test, which monitors
locomotor reactivity to startle and climbing
ability. This phenotype appears at an early age,
starting from 1 d a.E. The performance continued
to decrease until 8 d a.E. and then appeared to
stabilize up to 30 d a.E. This defect is quite different from the locomotor impairments described in
Drosophila Parkinson’s disease models, in which SING performance starts declining at around 25 d
a.E. (Feany and Bender, 2000; Riemensperger etal., 2013; Rahmani etal., 2022). This phenotype
of Aprt- deficient flies could be reminiscent of the early onset of motor symptoms in LND patients,
which appear most often between 3 and 6months of age (Jinnah etal., 2006). Interestingly, knocking
down Aprt during 3d only in adult flies also induced SING impairment, which argues against a devel-
opmental flaw. Although Aprt mutants walked slower than wild- type flies, they were no less active
and their ATP levels were not different compared to controls, excluding a major failure in energy
metabolism.
Downregulating Aprt in all neurons reproduced the locomotor defect of the Aprt5 mutant, and
Aprt mutant locomotion could be partially rescued by neuronal Aprt re- expression. The fact that
rescue was not complete could be due to a dominant negative effect of the mutation, as suggested
by enzymatic assays on extracts of heterozygous Aprt5 mutants (Figure1—figure supplement 4).
Previous work from our and other laboratories showed that DA neurons control the SING behavior
in Drosophila (Feany and Bender, 2000; Friggi- Grelin etal., 2003; Riemensperger et al., 2011;
Vaccaro etal., 2017; Sun etal., 2018) and that PAM DA neurondegeneration induces SING defects
in various Parkinson’s disease models (Riemensperger etal., 2013; Bou Dib etal., 2014; Tas etal.,
2018; Pütz etal., 2021). Here, we found that knocking down Aprt either in all DA neurons or only
in the PAM DA neurons was sufficient to induce early SING defects. In contrast, knocking down Aprt
with TH- Gal4 that labels all DA neurons except for a major part of the PAM cluster did not induce
SING defects, indicating that Aprt expression in subsets of PAM neurons is critical for this locomotor
behavior.
Inactivating Aprt in all mushroom body neurons also induced a lower performance in the SING
assay. This important brain structure receives connections from DA neurons, including the PAM, and
is enriched in DA receptors (Waddell, 2010). We recently reported that activation of MB- afferent
DA neurons decreased the SING response, an effect that requires signalization by the DA receptor
dDA1/Dop1R1 in mushroom body neurons (Sun etal., 2018). We also observed a SING defect after
knocking down Aprt either in all glial cells or more selectively in glial subpopulations expressing
the glutamate transporter Eaat1, but not in the ensheathing glia. Astrocyte- like glial cells expressing
Eaat1 extend processes forming a thick mesh- like network around and inside the entire mushroom
body neuropil (Sinakevitch et al., 2010). It could be hypothesized that SING also requires Aprt
expression in these MB- associated astrocytes. However, re- expressing Aprt with Eaat1- Gal4 did not
lead to SING rescue in Aprt mutant background. This suggests that the presence of Aprt in neurons
can somehow compensate for Aprt deficiency in glia, but the reverse is not true. In conclusion, proper
startle- induced locomotion in young flies depends on Aprt activity in PAM DA and mushroom body
neurons, and in Eaat1- expressing glial cells.
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
The online version of this article includes the following
source data for table 2:
Source data 1. Source data for Table2.
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Neuronal Aprt regulates spontaneous activity and sleep
Because sleep is regulated by the mushroom body as well as DA neurons in flies (Artiushin and
Sehgal, 2017), we monitored spontaneous locomotor activity and sleep pattern of Aprt5 mutants.
Their activity profile appeared normal, with unaltered morning and evening anticipation, indicating
that the circadian rhythms are surprisingly maintained in light- dark (LD) conditions in the absence of
a functional purine recycling pathway (Figure4—figure supplement 1). This experiment also high-
lighted that Aprt- deficient flies are hyperactive during the night, which suggests that they sleep less
than wild- type flies. This could be confirmed by measuring their sleep pattern on video recordings.
Aprt mutants show a reduced walking speed, sleep less during both day and night, and have diffi-
culty in maintaining sleep. Downregulating Aprt selectively in neurons reproduced the sleep defect,
whereas doing it in glial cells only had no effect. We have not attempted here to identify further the
neuronal cells involved in this phenotype. Like for the SING behavior defect, it could involve PAM DA
neurons as subpopulations of this cluster have been shown to regulate sleep in Drosophila (Nall etal.,
2016). Therefore, the lack of purine recycling markedly disrupts sleep in Aprt- deficient flies, making
them more active at night. This is strikingly comparable to young LND patients who have a much
disturbed sleep time during the night (Mizuno etal., 1979).
Lack of Aprt reduces adenosine signaling leading to DA neuron
overactivation
We observed that Aprt deficiency did not decrease DA levels in the Drosophila brain. This prompted
us to study another neuromodulator, adenosine, which is indirectly a product of Aprt enzymatic
activity. The purine nucleoside adenosine is one of the building blocks of RNA and the precursor of
ATP and cAMP, but is also the endogenous ligand of adenosine receptors that modulate a wide range
of physiological functions. In brain, adenosine regulates motor and cognitive processes, such as the
sleep- wake cycle, anxiety, depression, epilepsy, memory, and drug addiction (Soliman etal., 2018).
In normal metabolic conditions, adenosine is present at low concentrations in the extracellular space
and its level is highly regulated, either taken up by cells and incorporated into ATP stores or deami-
nated by adenosine deaminase into inosine. In mammals, several nucleoside transporters mediate the
uptake of adenosine and other nucleosides into cells, named equilibrative and concentrative nucleo-
side transporters, respectively (Gray etal., 2004; Boswell- Casteel and Hays, 2017; Pastor- Anglada
and Pérez- Torras, 2018).
We observed a marked reduction in adenosine levels in Aprt mutant flies. While we did not observe
any alteration in the transcript levels of AdoR, the gene coding for the only adenosine receptor in
Drosophila, transcript levels of an adenosine transporter, Ent2, were increased more than twofold in
Aprt mutants. Interestingly, one paper reported a similar strong increase in Ent2 expression in AdoR
mutant flies (Knight etal., 2010). These results suggest that AdoR signaling is less activated in Aprt-
deficient flies compared to controls. We also observed that the lack of AdoR decreased Aprt expres-
sion and activity in Drosophila, possibly from increased Ent2 expression and so higher adenosine influx
which could downregulate Aprt by a feedback mechanism.
Adenosine and DA receptors are known to interact closely in mammals (Franco etal., 2000; Kim
and Palmiter, 2008). A previous study performed in Drosophila larvae showed that an increase in
astrocytic Ca2+ signaling can silence DA neurons through AdoR stimulation by a mechanism potentially
involving the breakdown of released astrocytic ATP into adenosine (Nall etal., 2016). We previously
showed that DA neuron activation can decrease fly performance in the SING test (Sun etal., 2018).
Fly nocturnal hyperactivity can also be caused by an increase in DA signaling (Kumar etal., 2012;
Lee etal., 2013), in accordance with our observation that Aprt- deficient flies sleep less and are more
active during the night. Therefore, both the locomotor and sleep defects induced by the lack of Aprt
activity could be explained by DA neuron overactivation that would result from reduced adenosinergic
signaling.
Adenosine has been proposed before to be involved in neurological consequences of LND (Visser
et al., 2000). Adenosine transport is decreased in peripheral blood lymphocytes of LND patients
(Torres etal., 2004), as well as A2A adenosine receptor mRNA and protein levels (García etal., 2009;
García etal., 2012). In a murine LND model, adenosine A1 receptor expression was found to be
strongly increased and that of A2A slightly decreased, while A2B expression was not affected (Bertelli
et al., 2006). Chronic administration of high doses of caffeine, an adenosine receptor antagonist,
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caused self- injurious behavior in rats (Miñana etal., 1984; Jinnah etal., 1990). Moreover, central
injection of an adenosine agonist is sufficient to prevent self- mutilation induced by dopaminergic
agonist administration in neonatally 6- OHDA lesioned rats (Criswell etal., 1988).
Adenosine or N6-methyladenosine supplementation rescues the
epilepsy behavior of Aprt mutants
LND is characterized by severe behavioral troubles, including dystonia, spasticity, and involuntary
movements (choreoathetosis). Some patients can also show an epileptic disorder (Jinnah etal., 2006;
Madeo etal., 2019). In flies, the BS test is often used to model epileptic seizures (Song and Tanouye,
2008; Parker et al., 2011). Here, we observed that 30- day- old Aprt mutant flies show a transient
seizure- like phenotype after a strong mechanical shock. Although seizure duration appeared shorter
in Aprt5 than in typical BS mutants such as bss, at least one other BS mutant, porin, was reported
to have similar short recovery times as Aprt- deficient flies (Graham et al., 2010). Previous works
demonstrated that BS is linked to neuronal dysfunction in Drosophila (Parker etal., 2011; Kroll and
Tanouye, 2013; Kroll etal., 2015; Saras and Tanouye, 2016). Knocking down Aprt in specific cells
such as neurons, glia, or muscles did not trigger this phenotype, suggesting that Aprt must be inacti-
vated in several cell types to induce seizure.
Interestingly, knocking down Aprt by RNAi in all cells during development also induced the BS
behavior, but not for 3d only in adult flies, at variance with the SING phenotype. We have fed the
mutants with a diet supplemented with various compounds involved in purine metabolism, including
allopurinol, adenine, hypoxanthine, adenosine, or its analog N6- methyladenosine (m6A) either
throughout larval development or in adult stage. Only adenosine and m6A, ingested during devel-
opment, rescued the BS behavior. This suggests that loss of Aprt induces a lack of adenosine in the
developing nervous system, as we observed in adult flies (Figure6), which may alter neural circuits
controlling BS behavior in adults. The adenosine analog m6A cannot be incorporated into nucleic acids
and is excreted in the urine (Schram, 1998; Batista, 2017). The rescuing effect we observed with
m6A suggests thereby that both this compound and adenosine are required as adenosine receptor
agonists or allosteric regulators during development, rather than nucleotide precursors.
Adenosine can strongly inhibit cerebral activity and its role as endogenous anticonvulsant and
seizure terminator is well established in humans (Boison, 2005; Masino etal., 2014; Weltha etal.,
2019). Conversely, deficiencies in the adenosine- based neuromodulatory system can contribute
to epileptogenesis. For instance, increased expression of astroglial adenosine kinase (ADK), which
converts adenosine into AMP, leads to a reduction in brain adenosine level that plays a major role in
epileptogenesis (Weltha etal., 2019). Hence, therapeutic adenosine increase is a rational approach
for seizure control. Our observation that feeding adenosine or its derivative m6A rescued the seizure-
like phenotype of Aprt mutant flies further suggests that adenosinergic signaling has partly similar
functions in the fly and mammalian brains. In addition, the decrease in adenosine levels we observed
in Aprt mutants could result from enhanced ADK activity that would compensate for the lack of Aprt-
produced AMP.
We and others recently observed that m6A and related compounds sharing an adenosine moiety
are able to rescue the viability of LND fibroblasts and neural stem cells derived from induced plurip-
otent stem cells (iPSCs) of LND patients cultured in the presence of azaserine, a potent blocker of de
novo purine synthesis (Petitgas and Ceballos- Picot, unpublished results; Ruillier etal., 2020). Like in
flies again, allopurinol was not capable of rescuing the cell viability in this in vitro model. The similarity
of these results increases confidence that Aprt- deficient Drosophila could be used as an animal model
of LND.
Expression of mutant HGPRT triggers locomotor and seizure
phenotypes
How HGPRT deficiency can cause such dramatic neurobehavioral troubles in LND patients still remains
a crucial question. To date, cellular (Smith and Friedmann, 2000; Torres etal., 2004; Ceballos- Picot
etal., 2009; Cristini etal., 2010; Guibinga etal., 2012; Sutcliffe etal., 2021) and rodent (Finger
etal., 1988; Dunnett etal., 1989; Jinnah etal., 1993; Meek etal., 2016; Witteveen etal., 2022)
models only focused on the consequences of HGPRT deficiency to phenocopy the disease. Such an
approach was justified by the fact that the lower the residual activity of mutant HGPRT, the more
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severe the symptoms are in patients (Fu and Jinnah, 2012. Fu et al., 2014). However, it could be
conceivable that part of these symptoms result from compensatory physiological mechanisms or a
deleterious gain- of- function conferred to the HGPRT protein by the pathogenic mutations. Here, we
observed that neuronal expression of mutant human HGPRT- I42T, which expresses a low enzymatic
activity, but not the wild- type HGPRT protein, induced early locomotor defects in young flies and BS in
older flies, similarly to the defects induced by Aprt deficiency. This suggests a potential neurotoxicity
of the pathological mutant form of HGPRT, which could be related to disturbances in purine metab-
olism or other signaling pathways. The human mutant form might not be properly degraded and
accumulate as aggregates, potentially exerting neuronal toxicity. Such an approach opens interesting
perspectives to better understand the endogenous function of HGPRT and its pathogenic forms.
Indeed, the identification of a potential inherent neurotoxicity of defective forms of human HGPRT is
a new element, which could be explored in further work in the fly and also in rodent models.
A new model of LND in an invertebrate organism?
LND, a rare X- linked metabolic disorder due to mutations of the HPRT1 gene, has dramatic neuro-
logical consequences for affected children. To date, no treatment is available to abrogate these trou-
bles, and no fully satisfactory in vivo models exist to progress in the understanding and cure of this
disease. HGPRT knockout rodents do not show comparable motor and behavioral troubles, which
makes these models problematic for testing new therapeutic treatments. Drosophila does not express
HGPRT- like activity and our phylogenetic analysis established that no HGPRT homolog is present in
D. melanogaster (Figure1—figure supplement 2 and Figure1—figure supplement 3), confirming
that Aprt is the only enzyme of the purine salvage pathway in this organism. APRT and HGPRT are
known to be functionally and structurally related. Both human APRT and HGPRT belong to the type I
PRTases family identified by a conserved phosphoribosyl pyrophosphate (PRPP) binding motif, which
is used as a substrate to transfer phosphoribosyl to purines. This binding motif is only found in PRTases
from the nucleotide synthesis and salvage pathways (Sinha and Smith, 2001). Moreover, the purine
substrates adenine, hypoxanthine, and guanine share the same chemical skeleton and APRT can bind
hypoxanthine, indicating that APRT and HGPRT also share similarities in their substrate binding sites
(Ozeir etal., 2019).
Here, we find that Aprt mutant flies show symptoms partly comparable to the lack of HGPRT in
humans, including increase in uric acid levels, reduced longevity, and various neurobehavioral defects
such as early locomotor decline, sleep disorders, and epilepsy behavior. This animal model therefore
recapitulates both salvage pathway disruption and motor symptoms, as observed in LND patients.
Moreover, our results highlight that Aprt deficiency in Drosophila has more similarities with HGPRT
than APRT deficiency in humans. Aprt mutant flies also show a disruption of adenosine signaling, and
we found that adenosine itself or a derivative compound can relieve their epileptic symptoms. Finally,
neuronal expression of a mutant form of human HGPRT that causes LND also triggers abnormalities
in fly locomotion and seizure- like behavior, which has not been documented to date in other models.
The use of Drosophila to study LND could therefore prove valuable to better understand the link
between purine recycling deficiency and brain functioning and carry out drug screening in a living
organism, paving the way toward new improvements in curing this dramatic disease.
Materials and methods
Drosophila culture and strains
Flies were raised at 25°C on standard cornmeal- yeast- agar medium supplemented with methyl hydroxy-
benzoate as an antifungal under a 12hr:12hr LD cycle. The Drosophila mutant lines were obtained
either from the Bloomington Drosophila Stock Center (BDSC), the Vienna Drosophila Resource Center
(VDRC) or our own collection: Aprt5 (Johnson and Friedman, 1983) (BDSC #6882), Df(3L)ED4284
(BDSC #8056), Df(3L)BSC365 (BDSC #24389), UAS- AprtRNAi (VDRC #106836), AdoRKG03964ex (Wu etal.,
2009) here named AdoRKGex (BDSC #30868), and the following Gal4 drivers: 238Y- Gal4, 24B- Gal4, da-
Gal4, Eaat1- Gal4 (Rival etal., 2004), elav- Gal4 (elavC155), MZ0709- Gal4, NP6510- Gal4, NP6520- Gal4,
R58E02- Gal4 (Liu etal., 2012), R76F05- Gal4, repo- Gal4, TH- Gal4 (Friggi- Grelin etal., 2003), TRH-
Gal4 (Cassar et al., 2015), tub- Gal80ts , and VT30559- Gal4. The Canton- S line was used as wild-
type control. The simplified driver>effector convention was used to indicate genotypes in figures, for
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example, elav>AprtRNAi for elav- Gal4; UAS- AprtRNAi. In some experiments, to restrict RNAi- mediated
Aprt inactivation at the adult stage, we have used the TARGET system (McGuire etal., 2003). Flies
were raised at 18°C (permissive temperature) where Gal4 transcriptional activity was inhibited by
tub- Gal80ts, and shifted to 30°C (restrictive temperature) for 3d before the test to enable Gal4- driven
AprtRNAi expression.
Construction of transgenic lines
To generate a UAS- Aprt strain, a 549bp Aprt insert containing the coding sequence was PCR amplified
from the ORF clone BS15201 (Drosophila Genomics Resource Center, Bloomington, IN) using primers
with added restriction sites (in bold type): forward 5′- AGGG AATT GG GAAT TC GTTA TCAG TCGA
CATG AGCC C, reverse 5′- ACAA AGAT CC TCTA GA TCTA GAAA GCTT TCAG TACT TAAT G. After diges-
tion with EcoRI and XbaI, the Aprt cDNA was subcloned into the pUASTattB vector (Bischof etal.,
2007) using the In- Fusion HD Cloning Kit (Takara Bio, Kyoto, Japan) according to the manufacturer’s
instructions, and the insertion verified by sequencing (Eurofins Genomics, Ebersberg, Germany). The
construction was sent to BestGene (Chino Hills, CA, USA) for Drosophila germline transformation into
the attP14 docking site on the 2d chromosome. The UAS- Aprt line yielding the strongest expression
was selected and used in the experiments.
A clone containing the human wild- type HPRT1 cDNA was kindly provided to us by Prof. Hyder
A. Jinnah (Emory University, GA). We constructed the HPRT1- I42T cDNA from this clone by site-
directed mutagenesis using the QuikChange II Site- Directed Mutagenesis Kit (Agilent Technologies,
Santa Clara, CA). Primers sequences used to create the mutation were: forward 5'- CAGT CCTG TCCA
TA G TTAG TCCA TGAG GAAT AAAC ACCC T and reverse 5'- AGGG TGTT TATT CCTC ATGG ACTA A C TATG
GACA GGAC TG (the bases modified to create the mutation are in bold type). The cDNA obtained
was verified by sequencing. Then, the 657bp HPRT1- WT and HPRT1- I42T inserts were PCR amplified
using primers with added restriction sites (in bold type) and Drosophila translation start consensus
sequence: forward 5′- AGGG AATT GG GAAT TC AAGA AGGA GAT ACAA A ATGG C and reverse 5′- ACAA
AGAT CC TCTA GA GCTC GGAT CCTT ATCA TTAC (the bases modified to match the Drosophila transla-
tion initiation consensus sequence are underlined). They were subcloned into pUASTattB and verified
by sequencing. The transgenes were sent to BestGene for Drosophila transformation and inserted
into the attP40 docking site on the 2d chromosome.
Sequencing of Aprt5
For sequencing of the Aprt5 cDNA, total RNA was isolated by standard procedure from 20 to 30 heads
of homozygous Aprt5 flies and reverse transcribed using oligo d(T) primers (PrimeScript RT Reagent
Kit, Takara Bio). At least 750ng of the first- strand cDNA was amplified by PCR in 20μl of reaction
mixture using PrimeStar Max DNA polymerase (Takara Bio) with a Techne Prime Thermal Cycler appa-
ratus (Bibby Scientific, Burlington, NJ). The program cycles included 10s denaturation at 98°C, 10s
annealing at 55°C, and 10s elongation at 72°C, repeated 35 times. 1μl of the PCR product was
amplified again with the same program, in 30μl of reaction mixture. After elution on 1% agarose gel,
DNA were purified using the Wizard SV Gel and PCR Clean- Up System protocol (Promega, Madison,
WI) according to the manufacturer’s instructions. Finally, 7.5μl of purified DNA were sent with 2.5μl
of primers (forward and reverse in separate tubes) for sequencing (Eurofins Genomics).
Phylogenetic analyses
HGPRT homologs were identified by BlastP searching the NCBI GenBank non- redundant protein
database (last October 2019 version). A subset of interest was selected for phylogenetic analyses. For
Figure1—figure supplement 2, multiple sequence alignment was performed using MAFFT (- ensi)
(Katoh and Standley, 2013). The confidence of aligned residues was assessed using the TCSindex
(Chang etal., 2014) only columns with TCS index 7–9 (on a 0–9 scale) were retained. ProtTest v3.2
(Darriba etal., 2011) was used to assess the best model fitting of the data. Maximum likelihood tree
was inferred in IQ- TREE (Trifinopoulos etal., 2016), under the LG + R6 model. Bayesian inference
was carried out in PhyloBayes v. 3.3 (Lartillot etal., 2009), under the LG + Γ4 model. For Figure1—
figure supplement 3, the whole analysis was performed in SeaView 5.0.4 (Gouy et al., 2010).
Multiple sequence alignment was performed using MUSCLE (Edgar, 2004). Maximum likelihood tree
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was inferred using PhyML 3.0 (Guindon etal., 2010) (under the LG + Γ4 model; best of NNI and SPR
tree searching option; invariable sites optimized).
Lifespan assay
Longevity study was performed as previously described (Riemensperger etal., 2011). Drosophila
adult males were collected within 24hr of emergence and maintained on standard medium at 25°C
under a 12:12hr LD cycle. They were transferred into fresh bottles every 2–3d, and the number or
surviving flies was scored. Also, 50 flies per bottle in triplicate were tested for each genotype and the
experiment was done three times.
Startle-induced negative geotaxis (SING)
SING assays were performed as previously described (Rival etal., 2004; Riemensperger etal., 2011;
Sun etal., 2018). For each genotype, 50 adult males divided into five groups of 10 flies were placed in
a vertical column (25cm long, 1.5cm diameter) with a conic bottom end and left for about 30min for
habituation. Then, columns were tested individually by gently tapping them down (startle), to which
flies normally responded by climbing up. After 1min, flies having reached at least once the top of the
column (above 22cm) and flies that never left the bottom (below 4cm) were counted. Each fly group
was assayed three times at 15min intervals. The PI for each column is defined as ½[1 + (ntop- nbot)/ntot],
where ntot is the total number of flies in the column, ntop the number of flies at the top, and nbot the
number of flies at the bottom after 1min. SING was performed at 1, 8, 10, and 30d a.E.
Spontaneous locomotion and sleep monitoring
Spontaneous locomotor activity was recorded as previously described (Vaccaro etal., 2017) using
Drosophila activity infrared beam monitors (DAM, TriKinetics Inc, Waltham, MA) placed in incubators
at 25°C equipped with standard white light. Eight- day- old male flies were maintained individually for
5–6d under 12:12hr LD cycle in 5 × 65mm glass tubes containing 5% sucrose, 1.5% agar medium.
Data analysis was performed with the FaasX software (Klarsfeld etal., 2003). Histograms represent
the distribution of the activity through 24hr in 30min bins, averaged for 32 flies per genotype over
4–5 cycles.
For sleep monitoring, 2–4- day- old virgin female flies were transferred individually into 5 × 65mm
glass tubes containing standard food and their movements were recorded for up to 5d using DAM
infrared beam monitors (TriKinetics Inc) or the Drosophila ARousal Tracking (DART) video system
(Faville etal., 2015), in a 12hr:12hr LD cycle with 50–60%humidity. Control and experimental flies
were recorded simultaneously. Each experiment included at least 14 flies for each condition and was
repeated 2–3 times with independent groups of flies. Fly sleep, defined as periods of immobility
lasting more than 5min (Huber et al., 2004), was computed with a Microsoft Excel macro for the
infrared beam data and with the DART software for the video data. Distribution and homogeneity as
well as statistical group comparisons were tested using Microsoft Excel plugin software StatEL. The
p- value shown is the highest obtained among post hoc comparisons and means ± SEM were plotted.
Immunohistochemistry
Brains of 8–10- day- old adult flies were dissected in ice- cold Ca2+- free Drosophila Ringer’s solu-
tion and processed for whole- mount anti- TH or anti- DA immunostaining as previously described
(Riemensperger etal., 2011; Cichewicz et al., 2017). The primary antibodies used were mouse
monoclonal anti- TH (1:1000, Cat# 22941, ImmunoStar, Hudson, WI) and mouse monoclonal anti- DA
(1:100, Cat# AM001, GemacBio, Saint- Jean- d'Illac, France). The secondary antibody was goat anti-
mouse conjugated to Alexa Fluor 488 (1:1000, Thermo Fisher Scientific, Waltham, MA). Brains were
mounted with antifade reagent, either Prolong Gold (Thermo Fisher Scientific) or, alternatively, 65%
2,2′-thiodiethanol (Sigma- Aldrich, St. Louis, MI) (Cichewicz et al., 2017) for DA staining. Images
were acquired on a Nikon A1R confocal microscope with identical laser, filter, and gain settings
for all samples in each experiment. Immunofluorescence intensity levels were quantified using the
Fiji software. Experiments were repeated independently at least three times on 4–6 brains per
genotype.
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Bang sensitivity test
Bang sensitivity assays were performed as previously described (Howlett etal., 2013). 30- day- old
males were divided into five groups of 10 flies under CO2 and allowed to recover overnight. The
following day, each group was placed in a vial without food and after 20min of habituation, the vials
were stimulated vigorously with a vortex mixer for 10s at 2500rpm. The recovery time was measured
for each fly, from the end of the stimulation until they reached a normal standing position. Results are
the mean of the recovery time for at least 50 flies per genotype.
Drug administration
Allopurinol (A8003, Sigma- Aldrich) was diluted in standard medium at 100μg/ml and flies were placed
for 5d on this medium before metabolite extraction. Adenine, adenosine, and hypoxanthine (A2786,
A9251, and H9377, Sigma- Aldrich) or N6- methyladenosine (m6A) (QB- 1055, Combi- Blocks, San Diego,
CA) were diluted in fly food medium at 500μM. Parents were allowed to lay eggs on this medium in
order to have exposition to the drug throughout all larval development of the progeny. Adults were
collected and placed in normal medium until 5d before the test, when they were placed again in food
supplemented with adenosine or m6A at the same concentrations.
Uric acid quantification
For purine metabolite extraction, 40 heads from 8- day- old flies were ground in 80% ethanol, heated
for 3min at 80°C, and evaporated in a Speedvac apparatus. Dried residues were resuspended in MilliQ
water and total protein content of each homogenate was measured with the Pierce BCA Protein Assay
Kit (Thermo Fisher Scientific). 20µl of each sample was injected into a 25cm × 4.6mm C18 Nucleosil
column with 5 µm particles (Interchim, Montluçon, France). Purine metabolites were detected by
an Agilent 1290 Infinity HPLC system coupled with a diode array detector as recommended by the
ERDNIM advisory document. Seven wavelengths were used for detection (230, 240, 250, 260, 266,
270, and 280nm) in order to have the spectrum of each compound for identification in addition to the
retention time. The mobile phases contained 0.05M monopotassium phosphate pH 5 and 65% (v/v)
acetonitrile. The flux was fixed at 1ml/min. For analysis, the maximum height of the compound was
normalized to protein content and compared to the control genotype.
ATP assay
ATP level was measured by bioluminescence using the ATP Determination Kit (A22066, Thermo Fisher
Scientific) on a TriStar 2 Spectrum LB942S microplate reader (Berthold Technologies, Bad Wildbad,
Germany) according to the manufacturer’s instructions. Samples were prepared as described previ-
ously (Fergestad etal., 2006). Briefly, 30 heads or 5 thoraces from 8- day- old flies were homogenized
in 200µl of 6M guanidine- HCl to inhibit ATPases, boiled directly 5min at 95°C, and then centrifuged
for 5min at 13,000× g and 4°C. Total protein content of each supernatant was measured with the
BCA Protein Assay Kit. Each supernatant was then diluted at 1:500 in TE buffer pH 8 and 10µl were
placed in a 96- well white- bottom plate. The luminescent reaction solution (containing d- luciferine,
recombinant firefly luciferase, dithiothreitol, and reaction buffer) was added to each well with an
injector and high- gain 1 s exposure glow reads were obtained at 15 min after reaction initiation.
Results were compared to a standard curve generated with known ATP concentrations, and final
values were normalized to the protein content.
Enzyme activity assay
HGPRT and APRT activities were assessed in Drosophila by adapting the methods previously estab-
lished for human cells (Cartier and Hamet, 1968; Ceballos- Picot et al., 2009). Twenty whole male
flies were homogenized in 250µl of 110 mM Tris, 10mM MgCl2 pH 7.4 (Tris- MgCl2 buffer), imme-
diately frozen and kept at least one night at –80°C before the assay. After 5min of centrifugation
at 13,000× g, total protein content of each supernatant was measured with the BCA Protein Assay
Kit to normalize activity level. Kinetics of [14C]-hypoxanthine conversion to IMP (or in some cases
[14C]-guanine conversion to GMP), and [14C]-adenine conversion to AMP were assessed for HGPRT
and Aprt assays, respectively. Compositions of reaction mediums were 25µl of a radioactive solution
made with 38µl of [14C]-hypoxanthine (25 µCi/ml) diluted in 1 ml of 1.2mM cold hypoxanthine (or
in some cases 38µl of [14C]-guanine [25µCi/ml] diluted in 1ml of 1.2mM cold guanine), for HGPRT
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assay, or 25µl of a radioactive solution made with 75µl of [14C]-adenine (50µCi/ml) diluted in 1ml of
1.2mM cold adenine for Aprt assay, and a volume of fly extract equivalent to 200µg protein diluted
in Tris- MgCl2 buffer to 150µl. Reactions were monitored at 37°C and started by adding 25µl of the
co- factor 5- phosphoribosyl- 1- pyrophosphate (PRPP) at 10mM. After 6, 12, 24, and 36min, 40µl of
each pool was placed in a tube containing either 25µl of HIE (3mM hypoxanthine, 6mM IMP, 200mM
EDTA) or AAE (3mM adenine, 6mM AMP, 200mM EDTA) solutions and incubated for 3min at 95°C
to stop the reaction. The different radioactive compounds were separated by paper chromatography
on Whatman 3MM strips using 28% NH4OH, 50mM EDTA as solvent for about 1 hr 30 min. Then, the
substrates and products were visualized under a UV lamp at 254nm and placed separately in vials in
2ml of Scintran (VWR, Radnor, PA). The radioactivity in disintegrations per minute was measured in
a Packard Tri- Carb 1600 TR liquid scintillation analyzer (PerkinElmer, Waltham, MA). The percentage
of substrate transformation as a function of time was converted in nmol/min/mg protein and finally
normalized to wild- type control values.
Protein extraction and western blots
Thirty heads of 8–10- day- old males per genotype were homogenized in 30 μl Laemmli buffer
containing protease inhibitor (cOmplete mini Protease Inhibitor Cocktail, Roche Diagnostics) using a
Minilys apparatus (Bertin Instruments, Montigny- le- Bretonneux, France). The lysates were incubated
on ice for 30min and centrifuged at 8000× g for 10min at 4°C. The extracted proteins were heated
at 95°C for 10min. Western blots were performed as previously described (Issa etal., 2018). Briefly,
proteins were separated in 4–12% Novex NuPAGE Bis- Tris precast polyacrylamide gels (Life Tech-
nologies) following the manufacturer’s protocol in a MOPS- SDS running buffer. A semi- dry transfer
was done onto polyvinylidene difluoride membranes (Amersham Hybond P 0.45μm) using a Hoefer
TE77 apparatus. Membranes were probed overnight at 4°C with mouse monoclonal anti- TH (1:5000,
Cat# 22941, ImmunoStar) and mouse monoclonal anti- actin beta (1:5000, Cat# ab20272, Abcam,
Cambridge, UK) that cross- reacts with Drosophila Actin 5C (Act5C). After incubation with horseradish
peroxidase (HRP)- conjugated anti- mouse (1:5000, Cat# 115- 035- 146, Jackson ImmunoResearch) as
secondary antibody, immunolabeled bands were revealed by chemiluminescence staining using ECL
RevelBlOt Intense (Ozyme, Saint- Cyr- l'École, France, Cat# OZYB002- 1000) and then digitally acquired
with the ImageQuant TL software (GE Healthcare Life Science). Densitometry measures were made
with the Fiji software and normalized to Act5C values as internal controls.
Reverse transcription-PCR and quantitative PCR
Total RNA was isolated by standard procedure from 20 to 30 heads of 8- day- old males collected on
ice and lysed in 600µl QIAzol Reagent (QIAGEN, Venlo, Netherlands). 1μg of total RNA was treated
by DNase (DNase I, RNase- free, Thermo Fisher Scientific) according to the manufacturer’s instructions.
5μl of treated RNA was reverse transcribed using oligo d(T) primers (PrimeScriptRT Reagent Kit,
Takara Bio). Then, at least 750ng of the first- strand cDNA was amplified in 20μl of reaction mixture
using PrimeStar Max DNA polymerase (Takara Bio) with a Techne Prime Thermal Cycler apparatus
(Bibby Scientific). The program cycles included 10s denaturation at 98°C, 10s annealing at 55°C, and
10s elongation at 72°C, repeated 30 times. PCR product levels were measured after electrophoresis
by densitometry analysis with the Fiji software. Data were normalized to amplification level of the
ribosomal protein rp49/RpL32 transcripts as internal control. Sequences of the primers used were
for HPRT: forward 5′- GAGA TACA AAAT GGCG ACCC GCAG CCCT , reverse, 5′- GCTC GGAT CCTT ATCA
TTAC TAGG CTTT G (amplicon 686bp); and for rp49: forward 5′- GACG CTTC AAGG GACA GTAT C and
reverse rp49, 5′AAAC GCGG TTCT GCAT GAG (amplicon 144bp).
For RT- qPCR, approximately 40 ng of the first- strand cDNA was amplified in 10 μl of reaction
mixture using the LightCycler 480 SYBR Green I Master reaction mix (Roche Applied Science,
Mannheim, Germany) and a LightCycler 480 Instrument (Roche Applied Science). The program
cycles included a 10min preincubation step at 95°C, 40 cycles of amplification (10s denaturation
at 95°C, 10 s annealing at 55°C, 20 s elongation at 72°C), followed by a melting curves analysis
for PCR product identification. Data were normalized to amplification level of the ribosomal protein
rp49/RpL32 transcripts as internal control. The genes analyzed and primer sequences used for qPCR
are AdoR, forward 5′- GGAG AAAT TGCG ATCG GATG ACAC , reverse 5′- TCTT CAGC GAAC TCCG AGTG
AATG ; Aprt, forward 5′- AATC AGCG CGGA AGAC AAGC TA, reverse, 5′- CCAC CTTG CCGA TGAG TTCA
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GT; DTH1, forward 5′- GGAT CGAA AGCC AACC AAGT G, reverse 5′- CTTG GGGA CCAA CTGC GCTT TA;
Ent2, forward 5′- ACGG CAAG GGAT CAAC GTC, reverse 5′- CCGT GCAG CAGG AATA TAAA GA; rp49,
forward 5′- GACG CTTC AAGG GACA GTAT C; reverse 5′- AAAC GCGG TTCT GCAT GAG.
Adenosine assay
Adenosine was determined by ultra performance liquid cChromatography (UPLC). Here, 5 whole
flies or 30 heads were homogenized in 120 µl 0.9% (w/v) NaCl using a Minilys apparatus (Bertin
Instruments) and frozen at –80°C. After unfreezing and 5min of microcentrifugation, 20 µl of 10%
perchloric acid were added to 70µl of the supernatant and the mixture was left for 5min on ice. After
a new centrifugation, 20µl of a neutralization solution (made by mixing 3 volumes of 3M K2CO3 with
1volume of 6.4mM NaOH containing 0.4mg/ml bromothymol blue) were added and the mixture was
centrifuged again before injection (5µl). Samples were analyzed with a UV diode- array detector on an
Acquity UPLC HSS T3 column (1,8µm, 2.1 × 150mm) (Waters Corporation, Milford, MA). The mobile
phases consisted of Buffer A (30mM ammonium acetate, pH 4.0 with 1:10,000 heptafluorobutyric
acid [HPFA]) and Buffer B (acetonitrile with 1:10,000 HPFA) using a flow rate of 0.3ml/min. Chromato-
graphic conditions were 3.5min 100% Buffer A, 16.5min up to 6.3% Buffer B, 2min up to 100% Buffer
B, and 1min 100% Buffer B. The gradient was then returned over 5min to 100% Buffer A, restoring
the initial conditions. Results were normalized to protein levels for each sample.
Statistics
Statistical significance was determined using the Prism 6 software (GraphPad Software, La Jolla,
CA). Survival curves for longevity experiments were analyzed using the log- rank test. Student’s t- test
was used to compare two genotypes or conditions, and one- way or two- way ANOVA with Tukey’s,
Dunnett’s, or Sidak’s post hoc multiple comparison tests for three or more conditions. Results are
presented as mean ± SEM. Probability values in all figures: *p<0.05, **p<0.01, ***p<0.001.
Acknowledgements
We are grateful to Pr. Hyder A Jinnah for the gift of HPRT cDNAs and to Dr Thomas Riemensperger
for helpful discussions. Part of the phylogenetic analyses were performed using the computing facil-
ities of the PRABI- AMSB bioinformatics platform, Laboratory of Biometry and Evolutionary Biology,
Lyon, France. This work was supported by funding from CNRS and ESPCI Paris to SB and Association
Malaury to ICP. CP is a recipient of PhD fellowships from the Association Lesch- Nyhan Action (LNA)
and Labex MemoLife.
Additional information
Funding
Funder Grant reference number Author
Centre National de la
Recherche Scientique Laboratory funding Serge Birman
École Supérieure de
Physique et de Chimie
Industrielles de la Ville de
Paris
Laboratory funding Serge Birman
Association Malaury Laboratory funding Irène Ceballos-Picot
Association Lesch-Nyhan
Action Graduate Student
Fellowship Céline Petitgas
Labex MemoLife Graduate Student
Fellowship Céline Petitgas
The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication.
Research article Genetics and Genomics | Neuroscience
Petitgas etal. eLife 2023;12:RP88510. DOI: https://doi.org/10.7554/eLife.88510 28 of 34
Author contributions
Céline Petitgas, Conceptualization, Formal analysis, Validation, Investigation, Methodology, Writing
– original draft, Writing – review and editing; Laurent Seugnet, Sandrine Marie, Conceptualization,
Formal analysis, Investigation, Methodology; Amina Dulac, Rebecca Fima, Marion Strehaiano, Joana
Dagorret, Formal analysis, Investigation; Giorgio Matassi, Formal analysis, Investigation, Method-
ology; Ali Mteyrek, Baya Chérif- Zahar, Methodology; Irène Ceballos- Picot, Conceptualization, Super-
vision, Funding acquisition, Validation, Writing – original draft, Writing – review and editing; Serge
Birman, Conceptualization, Supervision, Funding acquisition, Validation, Writing – original draft,
Project administration, Writing – review and editing
Author ORCIDs
Laurent Seugnet
http://orcid.org/0000-0003-1617-5721
Giorgio Matassi
http://orcid.org/0000-0003-4923-226X
Serge Birman
http://orcid.org/0000-0002-4278-454X
Peer review material
Reviewer #1 (Public review): https://doi.org/10.7554/eLife.88510.3.sa1
Reviewer #2 (Public review): https://doi.org/10.7554/eLife.88510.3.sa2
Reviewer #3 (Public review): https://doi.org/10.7554/eLife.88510.3.sa3
Author response https://doi.org/10.7554/eLife.88510.3.sa4
Additional files
Supplementary files
• MDAR checklist
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files;
source data files have been provided for all the figures and linked figure supplements.
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