Author response: Pathway-specific effects of ADSL deficiency on neurodevelopment
2021-12-22

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Appendix 2 Data availability References Decision letter Author response Article and author information Metrics Abstract 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. Editor's evaluation This article attempts to elucidate adenylosuccinate lyase (ADSL) deficiency in neurodevelopment using a variety of approaches. It provides a better understanding of the underlying mechanisms that lead to microcephaly in some patients affected by ADSL deficiency. https://doi.org/10.7554/eLife.70518.sa0 Decision letter eLife's review process Introduction Adenylosuccinate lyase (ADSL) is a conserved homotetrameric enzyme that catalyzes the eighth reaction in the de novo purine synthesis (DNPS) pathway and also participates in the purine nucleotide cycle (Figure 1—figure supplement 1; Daignan-Fornier and Pinson, 2019). Mutations in ADSL cause adenylosuccinate lyase deficiency (ADSLD), an autosomal recessive disorder characterized by defects in purine metabolism and heterogeneous neurological phenotypes that include lack of eye-to-eye contact, auto-aggressive behavior, speech impairment, mild psychomotor delay, transient contact defects, autism spectrum disorder, epilepsy, and in some cases, microcephaly, encephalopathy, ataxia, or coma vigil (Jaeken and Van den Berghe, 1984; Jurecka et al., 2015). While the incidence of ADSLD has not been fully established, over 100 patients have been diagnosed to date and subcategorized based on their symptoms that range from premature death to milder developmental and behavioral disorders (Jurecka et al., 2015; http://www.adenylosuccinatelyasedeficiency.com/). ADSLD can be diagnosed by detecting elevated levels of the substrates of ADSL, SAICAR (also referred to as SZMP), and S-AMP, as well as their dephosphorylated riboside forms, SAICAr and S-Ado, in bodily fluids (Jaeken and Van den Berghe, 1984). As normal levels of purine nucleotides were detected in serum from ADSLD patients, the accumulation of S-Ado, and particularly SAICAr, was proposed to play a role in the disease pathology (Jaeken and Van den Berghe, 1984; Jurecka et al., 2015; Stone et al., 1998). In yeast, ADSL (Ade13) loss provokes genomic instability and is lethal (Daignan-Fornier and Pinson, 2019; Giaever et al., 2002; Pinson et al., 2009; Chen et al., 2016). Lethality in yeast can be rescued by deletion of a number of DNPS enzymes upstream of ADSL, or the transcription factors that regulate the pathway, indicating that the accumulation of metabolic intermediates, rather than impaired DNPS, underlies the toxicity (Daignan-Fornier and Pinson, 2019; Chen et al., 2016). In Caenorhabditis elegans, ADSL loss caused delayed growth, infertility, reduced life span, and locomotion defects. In some studies, growth, life span, and locomotion could be linked to the accumulation of SAICAR (Chen et al., 2016; Marsac et al., 2019; Fenton et al., 2017). Perfusion of rat brains with SAICAr caused cellular attrition in the hippocampus, leading to the proposition that SAICAr accumulation is neurotoxic, although the potential mechanism remains unknown (Stone et al., 1998). In glucose-deprived cancer cells, SAICAR accumulation was shown to activate PKM2, and a number of other kinases, to promote cancer survival in glucose-limiting conditions, suggesting that purine metabolite accumulation could have distinct signaling outcomes that impact on cell behavior and fate during development (Keller et al., 2012; Keller et al., 2014; Yan et al., 2016). However, despite extensive enzymology and structural information, the underlying mechanisms by which neuropathology arises in ADSLD remain unknown. To address the potential roles of ADSL deficiency in neurodevelopment, we systematically examined the consequences of ADSL depletion in diploid human cells and in vivo. We found that ADSL depletion in human epithelial cells impaired cell cycle progression, induced DNA damage signaling, and impaired primary ciliogenesis. Deletion of p53 or supplementation with nucleosides rescued cell cycle progression and DNA damage signaling, respectively. In contrast, ciliogenesis defects were unaffected by p53 status or nucleoside supplementation and could be reproduced by SAICAr supplementation or attenuated by the inhibition of phosphoribosylaminoimidazole carboxylase (PAICS), which is required to generate SAICAR/r upstream of ADSL. Depletion of ADSL in chicken or zebrafish embryos impaired neurogenesis and caused developmental defects. Both chicken and zebrafish embryos exhibited microcephaly, which is observed in some ADSLD patients, and increased DNA damage was evident in zebrafish. In addition, fish embryos displayed ciliopathy-related phenotypes and interventions to limit SAICAR accumulation-rescued impaired neurogenesis. Together, our results indicate that ADSL depletion causes context-dependent phenotypes associated with its role in both the purine nucleotide cycle and DNPS, which together impact neurodevelopment. Results ADSL depletion causes p53-dependent proliferation defects To investigate the impact of ADSL on cellular homeostasis, we depleted ADSL with a pool of four siRNAs in hTERT-immortalized human retinal epithelial cells (hTERT-RPE-1, referred to henceforth as RPE-1). Depletion of ADSL was effective as we observed 80% depletion of the mRNA and a clear reduction in protein levels (Figure 1A and B). This was accompanied by reduced levels of AMP and GMP, as well as accumulation of S-Ado (Figure 1C, Figure 1—figure supplement 2A and B). SAICAr levels were at the limit of detection in RPE-1 cells and differences could not be assessed (Figure 1—figure supplement 2B), but elevated SAICAr could be readily observed following ADSL depletion in HeLa, potentially due to elevated levels of DNPS enzymes that have been observed in cancer cells (Figure 1—figure supplement 2C; Zurlo et al., 2019; Agarwal et al., 2020; Yamauchi et al., 2022; Taha-Mehlitz et al., 2021). We deconvolved the siRNA pool and identified an effective single siRNA (#2) to further validate results from the siRNA pool (Figure 1—figure supplement 2D). Figure 1 with 2 supplements see all Download asset Open asset Adenylosuccinate lyase (ADSL) depletion causes p53-dependent proliferation defects. (A) Reduced mRNA levels of ADSL confirmed by qRT-PCR experiments. hTERT-RPE-1 were silenced with smart pool RNAi for 96 hr before harvesting. Two independent experiments in triplicate are shown in the panel (n = 2, two-tailed t-test, ****p<0.0001) (B) Western blot of RPE-1 cell extracts treated as in (A). One experiment is shown as representative of four independent experiments. Actin was used as a loading control. Quantifications of ADSL intensity in four different experiments were performed by ImageJ software and normalized to actin first and then to the relative controls (n = 4, two-tailed t-test, ****p<0.0001). Ponceau is shown as an additional loading and transfer control. (C) S-Ado levels in RPE-1 cells silenced with a single control or ADSL siRNA (n = 4, two-tailed t-test, *p<0.05). (D) Cell proliferation rates of RPE-1 cells quantified every 3 days after treatment with a single control or ADSL siRNA in medium with serum (n = 3, two-tailed t-test ****p<0.0001). ΔPDL represent the difference in population doubling levels quantified through the formula described in Materials and methods. (E) Ki67-positive cells (indicated by arrows) upon 96 hr of silencing with control or ADSL smart pool siRNAs. Scale bar 10 μm (n = 4, scored 767 cells for siC, 694 cells for siADSL conditions, ****p<0.0001). (F) The percentage of p53-positive cells (indicated by arrows) following treatment with control or ADSL smart pool siRNAs was quantified in three independent experiments (n = 3, scored 835 cells for siC and 1126 cells for siADSL, *p<0.05). (G) Cell proliferation rates in RPE-1 wt and p53 knockout KO cells as in (D) were counted for 6 days (n = 3, two-tailed t-test, **p<0.01, *p<0.05). (H) RPE-1 cells stably expressing EGFP or ADSL*-EGFP were transfected with a single control or ADSL siRNAs for 96 hr and immunostained with anti-Ki67 antibody. Scale bar = 20 μm. Quantification of Ki67-positive cells (n = 3, scored 278 cells for EGFP siC, 264 for EGFP siADSL, 266 for ADSL* siC, 232 cells for ADSL* siADSL conditions, ns not significant, ****p<0.0001). (I) Quantification of RPE-1 transfected with a single control or ADSL siRNA for 96 hr in the presence or absence of 60 μM nucleosides. Cells were fixed and immunostained with anti-Ki67 antibody (n = 3, at least 100 cells per conditions were counted in each experiment, ns not significant, ****p<0.0001). (J) Quantification of RPE-1 in the same conditions of (I) and immunostained with anti-p53 antibody (n = 3, at least 100 cells were counted for each condition per experiment, ns not significant, ****p<0.0001). (K) Quantification of p53-positive cells in ADSL-depleted cells in the presence or absence of the phosphoribosylaminoimidazole carboxylase (PAICS) inhibitor MRT00252040 (n = 3, scored 223 cells for siC, 248 cells for siADSL, 336 cells for siC+ MRT00252040, 365 cells for siADSL + MRT00252040, ns not significant, ****p<0.001, *p<0.05). (L) Quantification of Ki67-positive cells in ADSL-depleted cells in the presence or absence of MRT00252040 (n = 5, more than 60 cells were counted in each condition for each experiment, ns not significant, ***p<0.001). Positive cells are indicated with arrows in siADSL panels. All graphs depict means ± SD with individual values shown in circles. Figure 1—source data 1 Related to Figure 1A and C–L. GraphPad Prism file. https://cdn.elifesciences.org/articles/70518/elife-70518-fig1-data1-v4.zip Download elife-70518-fig1-data1-v4.zip Figure 1—source data 2 Related to Figure 1B. jpg file. https://cdn.elifesciences.org/articles/70518/elife-70518-fig1-data2-v4.zip Download elife-70518-fig1-data2-v4.zip As ADSL is critical for DNPS, we examined cell growth following ADSL depletion and found reduced levels of proliferation in ADSL-depleted cells compared to controls (Figure 1D). ADSL-depleted cells frequently lacked Ki67 expression, indicating that some cells were exiting the cell cycle, had increased levels of p53, and showed accumulation in G1 phase of the cell cycle (Figure 1E and F, Figure 1—figure supplement 2E). Trypan Blue and β-galactosidase assays indicated that cell death was not increased (Figure 1—figure supplement 2F) and that the Ki67-negative cells were likely quiescent and not senescent (Figure 1—figure supplement 2G). Deletion of TP53 rescued proliferation, prevented G1 arrest, and restored the number of Ki67-positive cells (Figure 1G, Figure 1—figure supplement 2H and I). The reduction in Ki67-positive cells could be rescued by stable expression of an siRNA-resistant allele of ADSL (ADSL*) (Figure 1H, Figure 1—figure supplement 2J). We also checked whether ADSL-depleted RPE-1 cells underwent differentiation by staining with vimentin, a marker of undifferentiated cells (Tamiya et al., 2010), and cytokeratin 20 (CK20), a marker of differentiation, upon ADSL depletion. We did not observe any CK20 signal or a reduction in vimentin-positive cells in the population upon ADSL silencing compared to the controls, arguing against premature differentiation (Figure 1—figure supplement 2K). To identify the cause of cell cycle exit in ADSL-depleted cells, we performed specific treatments. To restore purine levels, we supplemented cells with a mixture of nucleosides or with adenosine alone. To reduce potential accumulation of SAICAr that is generated by DNPS, we treated with MRT00252040, a small-molecule inhibitor of PAICS, the enzyme required to generate SAICAr upstream of ADSL (Figure 1—figure supplement 1; Hoxhaj et al., 2017). Supplementation of ADSL-depleted RPE-1 cells with nucleosides or adenosine did not prevent p53 induction or cell cycle exit (Figure 1I and J). Similarly, treatment with MRT00252040 did not influence p53 induction or loss of Ki67 (Figure 1K and L). This demonstrated that ADSL depletion in non-transformed human epithelial cells leads to a partial p53-dependent cell cycle exit/arrest that is not rescued by complementing the defects of ADSL depletion with nucleoside supplementation or inhibiting PAICS to prevent SAICAR/r production upstream of ADSL. ADSL depletion causes elevated DNA damage signaling Reduced levels of purine nucleotides in ADSL-depleted cells may cause replication stress and DNA damage (Kim et al., 2015; Benedict et al., 2018; Gottifredi et al., 2001). To address this, we examined the levels of chromatin-bound RPA, a surrogate marker of ssDNA accumulation and indicator of replication stress. In ADSL-depleted cells, chromatin-bound RPA levels were increased, indicative of replication stress, although the effect was mild (Figure 2A). Additionally, we observed an increased number of cells with more than five 53BP1 foci per cell, indicative of DNA double-strand break (DSBs) accumulation (Figure 2B). 53BP1 foci were reduced by treatment with a small-molecule inhibitor for ATM (Figure 2C), indicating an active DNA damage response. Supplementation of cells with nucleosides suppressed the appearance of DNA DSBs detected by 53BP1 following ADSL depletion or treatment with the ribonucleotide reductase inhibitor hydroxyurea (HU) that depletes dNTP pools (Figure 2D and E). A similar reduction in 53BP1 foci was also observed with adenosine supplementation of ADSL-depleted cells (Figure 2F). Nucleoside supplementation also reduced the increase in γH2AX, the phosphorylated form of the histone variant H2AX, which is also a marker of DSBs (Figure 2G). In contrast to nucleoside supplementation, the PAICS inhibitor MRT00252040 did not rescue the increased levels of DSBs (Figure 2H). These data indicate that ADSL depletion in cultured cells induces mild levels of DNA damage signaling that can be suppressed by nucleoside supplementation, but not by the inhibition of PAICS. This implicates defects in the purine nucleotide cycle, rather than impaired DNPS or specific metabolite accumulation resulting from ADSL depletion, in replication stress and DSB formation. In addition, it indicates that the partial p53-dependent cell cycle exit is not solely a consequence of DNA damage signaling or purine metabolite accumulation, as it was not rescued by either intervention. Figure 2 Download asset Open asset Adenylosuccinate lyase (ADSL) depletion caused elevated DNA damage signaling. (A) RPE-1 were silenced for 96 hr with a smart pool of ADSL siRNAs, chromatin extracted, and immunostained with an anti-RPA2 antibody. Nuclear intensity of cells was measured- Quantification of one representative experiment of four that showed similar results is shown; SD and average are indicated in black. After normalization to the average of the control (siC), two-tailed t-test was used for statistical analysis of n = 4 independent experiments: *p<0.05 was observed for siADSL to siC. (B) RPE-1 were silenced for 96 hr with a smart pool of ADSL siRNAs, fixed, and immunostained with anti-53BP1 antibody. Scale bar = 10 μm. Quantification of positive cells that have more than five foci per cell (n = 5 scored 1134 cells for siC, 1144 cells for siADSL, ***p<0.001). (C) RPE-1 were silenced with a single control or ADSL siRNA with or without 5 mM ATM inhibitor (KU5593) (n = 4, scored 359 cells for siC, 322 cells for siADSL, 307 cells for siC + KU5593, 279 cells for siADSL + KU5593, ****p<0.0001). (D) Cells were silenced for 96 hr, treated or not with 60 μM (1×) nucleosides, and stained for 53BP1. Scale bar = 10 μm (n = 5, scored 717 cells for siC, 608 cells for siADSL, 576 cells for siC + nucl, 512 cells for siADSL + nucl, ****p<0.0001). (E) RPE-1 cells were treated with 2 mM hydroxyurea (HU) for 6 hr and treated or not with 1× nucleosides and stained for 53BP1. Cells with more than five foci per nucleus were counted as positive. Three independent experiments were performed (n = 3, at least 100 cells per experiment per condition were counted, ****p<0.0001). (F) Cells were silenced for 96 hr, treated or not with 80 μg/ml adenosine, and stained for 53BP1 (n = 3, scored 544 cells for siC, 428 cells for siADSL, 485 cells for siC+ adenosine, 411 cells for siADSL + adenosine, ****p<0.0001). (G) RPE-1 treated as in (A) were fixed and stained for γH2AX (H2AX phosphorylated on Ser-139). Scale bar = 10 μm. 5 Gy X-ray irradiation (IR) was used as positive control. Quantification of one representative experiment of two that showed similar results is shown; median is indicated in black. After normalization to the average of the control (siC), one-tailed t-test was used for statistical analysis of n = 3 independent experiments: *p<0.05 was observed for siADSL (to siC), and for siADSL relative to siADSL + nucl. There is no statistical difference between siC and siC + nucl. (H) RPE-1 were silenced in the presence or absence of 4 μM MRT00252040, fixed and stained for 53BP1 (n = 4, scored 367 cells for siC, 313 cells for siADSL, 294 cells for siC + MRT00252040, 241 cells for siADSL + MRT00252040, ns not significant, ****p<0.0001). All bar graphs show means ± SD with individual values in circles. Figure 2—source data 1 Related to Figure 2. GraphPad Prism file. https://cdn.elifesciences.org/articles/70518/elife-70518-fig2-data1-v4.zip Download elife-70518-fig2-data1-v4.zip ADSL depletion impairs neurogenesis in the developing chicken neural tube Given the effects of ADSL depletion on cell growth and proliferation, we sought to examine the consequences of its loss in vivo. To this end, we used the chicken embryo system to examine the influence of ADSL depletion on nervous system development. We electroporated one side of the neural tube with plasmid expressing GFP as a transfection marker in combination with either control or ADSL shRNA vectors. After confirming efficient ADSL depletion (Figure 3A), we evaluated neurogenesis by staining with markers for proliferating neural progenitors (SOX2 positive) and post-mitotic neurons (ELAVL3/4 positive). We found that in the ADSL-depleted side both cell populations were reduced when compared to the non-transfected side (Figure 3B) and that the size of the tissue was smaller, suggesting reduced growth and/or increased cell death. Staining for the apoptotic marker cleaved caspase-3 (CC3) revealed no notable differences, suggesting that this was not due to increased cell death (Figure 3—figure supplement 1A). Figure 3 with 1 supplement see all Download asset Open asset Adenylosuccinate lyase (ADSL) depletion causes neurodevelopmental delay in the chicken neural tube. (A) mRNA levels of ADSL and GAPDH were measured by qRT-PCR in chicken embryonic fibroblasts (CEFs) transfected for 24 hr with shCONTROL or shADSL to confirm knockdown efficiency (n = 3, two-tailed t-test, **p<0.01). (B) Transverse sections of HH12 chicken neural tubes 48 hr post electroporation (hpe) with shCONTROL or shADSL plasmids and stained with antibodies against SOX2 (green) and ELAVL3/4 (magenta). Transfection was detected by GFP (blue). Scale bar = 50 μm. Average ratio of neural stem cells (NSCs, SOX2+) 48 hpe with shCONTROL or shADSL obtained by comparing the mean number of SOX2+ cells on the electroporated and non-electroporated side (n = 7 embryos, two-tailed t-test, **p<0.01). Average ratio of cells differentiated into neurons (ELAVL3/4) at 48 hpe with shCONTROL or shADSL obtained by comparing the mean number of ELAVL3/4-positive cells on the electroporated and the non-electroporated side (n = 7 embryos, two-tailed t-test, ***p<0.001). (C) Percentage of electroporated cells identified as NSCs (SOX2) or neurons (ELAVL3/4) 48 hpe with shCONTROL or shADSL (n = 11 embryos, two-tailed t-test, ****p<0.0001). (D) The cell cycle profiles of NSCs (GFP+/ELAVL3/4-) obtained by fluorescence-associated cell sorting (FACS) 48 hpe with shCONTROL or shADSL into HH12 chicken neural tubes. The mean of two independent experiments is shown in the left panel. 6–8 embryos per condition were used for each experiment. Two-tailed t-test was used for statistical analysis of n = 2 independent experiments, ns not significant, Cell cycle profiles of a representative experiment are shown in the panels. (E) Transverse sections of HH12 chicken neural tubes 48 hpe with shCONTROL or shADSL and stained with (green) and an antibody against (magenta). Transfection was detected by GFP (blue). Scale bar = 50 μm. indicated in are in the in black. in show Percentage of transfected cells identified as 48 hpe with shCONTROL or shADSL (n = embryos and embryos two-tailed t-test, ****p<0.0001). Percentage of the cell population 48 hpe with shCONTROL or shADSL (n = embryos, two-tailed t-test, ns not significant, **p<0.01, ****p<0.0001). Average ratio of and cells 48 hpe of shCONTROL or shADSL obtained by comparing the mean number of cells on the electroporated and the non-electroporated side n = 11 embryos embryos two-tailed t-test, n = embryos embryos two-tailed t-test, ns not (F) Percentage of and the cell population 48 hpe of shCONTROL or shADSL plasmids (n = embryos, two-tailed t-test, ns not significant, ****p<0.0001). graphs show means ± Figure data 1 Related to Figure GraphPad Prism file. Download We then SOX2 and ELAVL3/4 staining the transfected cells and found that ADSL depletion increased the percentage of progenitors relative to ELAVL3/4-positive neurons (Figure Figure 3—figure supplement This that reduced tissue growth was not due to premature differentiation but due to a proliferation in the To cell cycle progression in neural stem cells, we performed fluorescence-associated cell sorting (FACS) analysis of cells following electroporation of control or ADSL We found that was a increase in the population after ADSL depletion (Figure analysis of stained tissue sections showed that ADSL depletion caused a reduction in the of cells that and an increase in the of cells positive for the marker phosphorylated histone (Figure We the cells into two cells, identified by and cells, This revealed that the of cells was increased by ADSL depletion, indicating that ADSL depletion caused a specific delay in phase in the SOX2+ rather than during (Figure Together, our in data indicate that ADSL depletion leads to a mild induction of DNA damage signaling and impaired cell cycle In this as reduced in the developing without a clear induction of cell death or defects following ADSL depletion As are cells in the and ADSL depletion caused cell cycle exit in RPE-1 cells, a condition frequently accompanied by we the of control and ADSL-depleted RPE-1 cells to treatment with cells were for 48 hr and by Ki67 staining confirmed that of the cells in both conditions the cell cycle (Figure We examined ciliogenesis by staining for the marker and the marker cells treated with the ADSL siRNA pool had and the that were were when compared to controls (Figure We also observed upon depletion with single siRNAs for ADSL (Figure supplement 1A). To the that ciliogenesis was we quantified the number of cells hr after serum and observed a similar (Figure supplement ciliogenesis was rescued by expression of an siRNA-resistant but not by nucleoside supplementation (Figure and of the DNPS pathway with which impairs in DNPS and of ADSL (Figure 1—figure supplement had no effect on ciliogenesis in control cells (Figure supplement but rescued both the number of cells and in ADSL-depleted RPE-1 cells (Figure supplement 1D). Figure 4 with 1 supplement see all Download asset Open asset ciliogenesis defects following adenylosuccinate lyase (ADSL) depletion. (A) RPE-1 were transfected with control or ADSL smart pool siRNAs. After 96 hr, cells were serum for 48 hr to ciliogenesis by staining against Ki67 and cells quantified (n = 2, two-tailed t-test, ns not (B) cells silenced as in (A) were stained for and Scale bar = 10 μm. show of the show of cells and indicates (n = 3, scored cells for siC, cells for siADSL, ****p<0.0001). (C) cells stably expressing EGFP or ADSL*-EGFP were silenced for 96 hr with control or a single ADSL serum for 48 hr, fixed, and stained for Scale bar = 5 μm. three experiments ns not significant, **p<0.01, *p<0.05). (D) RPE-1 cells silenced with a single ADSL siRNA for 96 hr in the absence or presence of 1× nucleosides. and were mean ± SD of n = 3 siC and siADSL n = 5 for siC and siADSL with nucleosides, ns not significant, (E) RPE-1 cells were treated or not with MRT00252040 and serum and then immunostained for and and were quantified (n = 3, scored cells for siC, cells for siADSL, cells for siC + cells for siADSL + MRT00252040, **p<0.01, ****p<0.0001). Scale bar = 10 μm. (F) Quantification of the in control and cells (n = 4, scored cells for cells for ****p<0.0001). (G) of cells treated as in (F) (n = 5, two-tailed t-test, *p<0.05). Figure data 1 Related to Figure 4 and Figure supplement GraphPad Prism file. Download defective ciliogenesis caused by ADSL depletion was rescued by but not by nucleoside supplementation, we examined whether specific inhibition of PAICS was to the ADSL depletion, we treated cells with the PAICS inhibitor MRT00252040 et al., 2017). to this rescued ciliogenesis as the number and of were similar in control and ADSL-depleted cells (Figure As inhibition of PAICS impairs DNPS upstream of SAICAR we treated cells with SAICAr and observed that this the ciliogenesis observed in ADSL-depleted cells (Figure and SAICAr treatment did not activate which has been shown to ciliogenesis due to serum (Figure supplement et al., 2019). To additional effects as a cause for the reduction in the number of cells, as cell cycle progression defects caused by DNA p53 or defective from cells, we the experiment in p53 KO cells and examined following the of