Pathway-specific effects of ADSL deficiency on neurodevelopment

Adenylosuccinate lyase (ADSL) functions in de novo purine synthesis (DNPS) and the purine nucleotide cycle. ADSL deficiency (ADSLD) causes numerous neurodevelopmental pathologies, including microcephaly and autism spectrum disorder. ADSLD patients have normal serum purine nucleotide levels but exhibit accumulation of dephosphorylated ADSL substrates, S-Ado, and SAICAr, the latter being implicated in neurotoxic effects through unknown mechanisms. We examined the phenotypic effects of ADSL depletion in human cells and their relation to phenotypic outcomes. Using specific interventions to compensate for reduced purine levels or modulate SAICAr accumulation, we found that diminished AMP levels resulted in increased DNA damage signaling and cell cycle delays, while primary ciliogenesis was impaired specifically by loss of ADSL or administration of SAICAr. ADSL-deficient chicken and zebrafish embryos displayed impaired neurogenesis and microcephaly. Neuroprogenitor attrition in zebrafish embryos was rescued by pharmacological inhibition of DNPS, but not increased nucleotide concentration. Zebrafish also displayed phenotypes commonly linked to ciliopathies. Our results suggest that both reduced purine levels and impaired DNPS contribute to neurodevelopmental pathology in ADSLD and that defective ciliogenesis may influence the ADSLD phenotypic spectrum.ID was funded by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 754510, THS, JL, and SP were funded by the Ministry of Science, Innovation and Universities (MCIU; PGC2018-095616-B-I00 to THS, PGC2018-099562-B-I00 to JL, and BFU2017-83562-P to SP), the 2017 SGR 1089 (AGAUR), FEDER, the Centres of Excellence Severo Ochoa award, and the CERCA Programme. THS was supported by the NIH Intramural Research Program, National Cancer Institute, Center for Cancer Research. MP was funded by grants from the Deutsche Forschungsgemeinschaft (DFG PH144/4-1 and PH144/6-1). MZ, OS, and VS were supported by Charles University, program PROGRES Q26/LF1. We would like to thank Biocev, First Faculty of Medicine, Charles University, for the opportunity to use their department’s equipment

Improved diagnostics of purine and pyrimidine metabolism disorders using LC-MS/MS and its clinical application

Objectives: To develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify 41 different purine and pyrimidine (PuPy) metabolites in human urine to allow detection of most known disorders in this metabolic pathway and to determine reference intervals. Methods: Urine samples were diluted with an aqueous buffer to minimize ion suppression. For detection and quantification, liquid chromatography was combined with electrospray ionization, tandem mass spectrometry and multiple reaction monitoring. Transitions and instrument settings were established to quantify 41 analytes and nine stable-isotope-labeled internal standards (IS). Results: The established method is precise (intra-day CV: 1.4–6.3%; inter-day CV: 1.3–15.2%), accurate (95.2% external quality control results within ±2 SD and 99.0% within ±3 SD; analyte recoveries: 61–121%), sensitive and has a broad dynamic range to quantify normal and pathological metabolite concentrations within one run. All analytes except aminoimidazole ribonucleoside (AIr) are stable before, during and after sample preparation. Moreover, analytes are not affected by five cycles of freeze-thawing (variation: −5.6 to 7.4%), are stable in thymol (variation: −8.4 to 12.9%) and the lithogenic metabolites also in HCl conserved urine. Age-dependent reference intervals from 3,368 urine samples were determined and used to diagnose 11 new patients within 7 years (total performed tests: 4,206). Conclusions: The presented method and reference intervals enable the quantification of 41 metabolites and the potential diagnosis of up to 25 disorders of PuPy metabolism

Source data supporting Figure 4 of "Pathway specific effects of ADSL deficiency on neurodevelopment" [Dataset]

Source microscopy data for Fig4C, D, E, G, S1A, S1C, S1D, S1F and S1G (.lif files) in a single zip file.Resources available on the publisher's site: https://doi.org/10.25452/figshare.plus.18321611.v1Marie Skłodowska-Curie grant agreement No. 754510 Ministry of Science, Innovation and Universities PGC2018-095616-B-I00 Ministry of Science, Innovation and Universities PGC2018-099562-B-I00Peer reviewe

Source data supporting Figure 8 of "Pathway specific effects of ADSL deficiency on neurodevelopment" [Dataset]

Source microscopy data for Fig8A, B, C, D and S1 (.lif and .jpg file) in a single zip file.Resources available on the publisher's site: https://doi.org/10.25452/figshare.plus.18312410.v1Deutsche Forschungsgemeinschaft DFG PH144/4-1 and PH144/6-1Peer reviewe

Source data supporting Figure 7 of "Pathway specific effects of ADSL deficiency on neurodevelopment" [Dataset]

Raw microscopy data for Fig7A-B, C and D-G (.lif and .jpg files) in a single zip file.Resources available on the publisher's site: https://doi.org/10.25452/figshare.plus.18316226.v1Deutsche Forschungsgemeinschaft DFG PH144/4-1 and PH144/6-1Peer reviewe

Source data supporting Figure 5 of "Pathway specific effects of ADSL deficiency on neurodevelopment" [Dataset]

Raw microscopy data for Fig5E (.lif files) in a single zip file.Resources available on the publisher's site: https://doi.org/10.25452/figshare.plus.18348926.v1Marie Skłodowska-Curie grant agreement No. 754510 Ministry of Science, Innovation and Universities PGC2018-095616-B-I00 Ministry of Science, Innovation and Universities PGC2018-099562-B-I00Peer reviewe

Source data supporting Figure 3 of "Pathway specific effects of ADSL deficiency on neurodevelopment" [Dataset]

Raw microscopy data for Fig3B, 3E and 3S1 (.lif format) in a single zip file.Resources available on the publisher's site: https://doi.org/10.25452/figshare.plus.18309440.v1Ministry of Science, Innovation and Universities BFU2017-83562-PPeer reviewe

Source data supporting Figure 2 of "Pathway specific effects of ADSL deficiency on neurodevelopment" [Dataset]

Raw microscopy data for Fig2A and G in .lif format in a single zip file.Resources available on the publisher's site: https://doi.org/10.25452/figshare.plus.19064324.v1Marie Skłodowska-Curie grant agreement No. 754510 Ministry of Science, Innovation and Universities PGC2018-095616-B-I00 Ministry of Science, Innovation and Universities PGC2018-099562-B-I00 NIH Intramural Funding, National Cancer InstitutePeer reviewe

Source data supporting Figure 6 of "Pathway specific effects of ADSL deficiency on neurodevelopment" [Dataset]

Raw microscopy data for Fig6A, F, G, S1 and S2 (.lif or .jpg format) in a single zip file.Resources available on the publisher's site: https://doi.org/10.25452/figshare.plus.18310361.v1Deutsche Forschungsgemeinschaft DFG PH144/4-1 and PH144/6-1Peer reviewe