Journal: eNeuro

Article Title: Spatiotemporal Regulation of De Novo and Salvage Purine Synthesis during Brain Development

doi: 10.1523/ENEURO.0159-23.2023

Figure Lengend Snippet: De novo and salvage purine synthesis pathways in mammals. Metabolic diagram of de novo (PAICS, FGAMS) and salvage (HGPRT) purine synthesis pathways in mammals. Enzymes involved in the de novo and salvage pathways are represented by yellow and green boxes, respectively. The route in blue represents the folate biosynthesis pathway, which is essential for substrate production of de novo purine synthesis. Red boxes indicate the inhibitors used in this study. PRPP, 5-phosphoribosyl-1-pyrophosphate; PPAT, phosphoribosyl pyrophosphate amidotransferase; 5-PRA, 5-phosphoribosylamine; GART, phosphoribosylglycinamide formyltransferase; GAR, glycinamide ribonucleotide; FGAR, formylglycinamide ribonucleotide; FGAMS, formylglycinamidine ribonucleotide synthase; FGAM, formylglycinamidine ribonucleotide; AIR, aminoimidazole ribonucleotide; PAICS, phosphoribosylaminoimidazole succinocarboxamide synthetase; CAIR, 4-carboxy-5-aminoimidazole ribonucleotide; SAICAR, 4-(N-succinylcarboxamide)−5-aminoimidazole ribonucleotide; ADSL, adenylosuccinate lyase; AICAR, aminoimidazole-4-carboxamide ribonucleotide; FAICAR, formylaminoimidazole-4-carboxamide ribonucleotide; ATIC, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase inosine monophosphate cyclohydrolase; DHF, dihydrofolate; DHFR, dihydrofolate reductase; THF, tetrahydrofolic acid; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; APRT, adenine phosphoribosyltransferase; IMP, inosine monophosphate; IMPDH, inosine monophosphate dehydrogenase; XMP, xanthosine monophosphate; GMP, guanosine monophosphate; SAMP, adenylosuccinate; AMP, adenosine monophosphate; PNP, purine nucleoside phosphorylase; XO, xanthine oxidase; MMF, mycophenolate mofetil; MTX, methotrexate; Forodesine, forodesine hydrochloride.

Article Snippet: The following drugs were used: mycophenolate mofetil (MMF; #S1501, Selleck, 100 mg/kg in vivo , 1–10 μ m in vitro , dissolved in DMSO), methotrexate (MTX; #139-13571, Fujifilm Wako, 50 mg/kg in vivo , 1–100 μ m in vitro , dissolved in 50 m m Na 2 CO 3 buffer), forodesine hydrochloride (#HY-16209, MCE, 40 mg/kg in vivo , 0.1–5 μ m in vitro , dissolved in PBS), allopurinol (#17795-21-0, Selleck, 50 mg/kg in vivo , 250 μ m in vitro , dissolved in DMSO), BrdU (#B1575, Tokyo Chemical Industry, 100 mg/kg in vivo , 10 μ m in vitro , dissolved in PBS), and MHY1485 (#S7811, Selleck, 50 mg/kg in vivo , 10 μ m in vitro , dissolved in DMSO).

Techniques:

Journal: eNeuro

Article Title: Spatiotemporal Regulation of De Novo and Salvage Purine Synthesis during Brain Development

doi: 10.1523/ENEURO.0159-23.2023

Figure Lengend Snippet: Summary of statistical analyses

Article Snippet: The following drugs were used: mycophenolate mofetil (MMF; #S1501, Selleck, 100 mg/kg in vivo , 1–10 μ m in vitro , dissolved in DMSO), methotrexate (MTX; #139-13571, Fujifilm Wako, 50 mg/kg in vivo , 1–100 μ m in vitro , dissolved in 50 m m Na 2 CO 3 buffer), forodesine hydrochloride (#HY-16209, MCE, 40 mg/kg in vivo , 0.1–5 μ m in vitro , dissolved in PBS), allopurinol (#17795-21-0, Selleck, 50 mg/kg in vivo , 250 μ m in vitro , dissolved in DMSO), BrdU (#B1575, Tokyo Chemical Industry, 100 mg/kg in vivo , 10 μ m in vitro , dissolved in PBS), and MHY1485 (#S7811, Selleck, 50 mg/kg in vivo , 10 μ m in vitro , dissolved in DMSO).

Techniques: Control

Journal: eNeuro

Article Title: Spatiotemporal Regulation of De Novo and Salvage Purine Synthesis during Brain Development

doi: 10.1523/ENEURO.0159-23.2023

Figure Lengend Snippet: Inhibition of de novo purine pathway disturbs NSPC proliferation in vitro . A–D , Primary cultured NSPCs from the E12.5 telencephalon were treated for 48 h with PBS or DMSO as control ( A ), forodesine (0.1, 1, 5 μ m ; B ), or mycophenolate mofetil (MMF; 1, 5 μ m ; C ), followed by BrdU labeling for 24 h. NSPCs were immunostained with anti-Nestin (green) and anti-BrdU (red) antibodies. D , Quantified comparison of the number of the BrdU + Nestin + cells to total Nestin + cells. ns, not significant; *** p < 0.001, χ 2 tests with Holm–Bonferroni correction. control PBS, n = 326; control DMSO, n = 346; forodesine 0.1 μ m , n = 435; forodesine 1 μ m , n = 283; forodesine 5 μ m , n = 310; MMF 1 μ m , n = 114; MMF 5 μ m , n = 72. E–G , Primary cultured NSPCs were electroporated with control shRNA ( E ) or PAICS shRNAs (#1 or #3; F ) together with EGFP-expression plasmid (green), followed by labeling with BrdU (red) for 24 h. Arrowheads indicate GFP + BrdU + proliferating NSPCs. The arrow indicates EGFP + but BrdU – cells. ( G ) The numbers of GFP + BrdU + cells were compared with the total GFP + cells using the χ 2 tests with Holm–Bonferroni correction. ns, not significant; *** p < 0.001. control shRNA, n = 100; PAICS shRNA#1, n = 100; PAICS shRNA#3, n = 100. Scale bars, 50 μm. Data supporting that inhibition of de novo purine synthesis disturbs the proliferation of cerebellar NSPCs is provided in Extended Data .

Article Snippet: The following drugs were used: mycophenolate mofetil (MMF; #S1501, Selleck, 100 mg/kg in vivo , 1–10 μ m in vitro , dissolved in DMSO), methotrexate (MTX; #139-13571, Fujifilm Wako, 50 mg/kg in vivo , 1–100 μ m in vitro , dissolved in 50 m m Na 2 CO 3 buffer), forodesine hydrochloride (#HY-16209, MCE, 40 mg/kg in vivo , 0.1–5 μ m in vitro , dissolved in PBS), allopurinol (#17795-21-0, Selleck, 50 mg/kg in vivo , 250 μ m in vitro , dissolved in DMSO), BrdU (#B1575, Tokyo Chemical Industry, 100 mg/kg in vivo , 10 μ m in vitro , dissolved in PBS), and MHY1485 (#S7811, Selleck, 50 mg/kg in vivo , 10 μ m in vitro , dissolved in DMSO).

Techniques: Inhibition, In Vitro, Cell Culture, Control, Labeling, Comparison, shRNA, Expressing, Plasmid Preparation

Journal: eNeuro

Article Title: Spatiotemporal Regulation of De Novo and Salvage Purine Synthesis during Brain Development

doi: 10.1523/ENEURO.0159-23.2023

Figure Lengend Snippet: Embryonic brain malformations induced by inhibiting de novo purine pathway. Horizontal frozen sections of E12.5 brains. Control DMSO ( A ), MMF ( B ), MHY1485 ( C ), MMF and MHY1485 ( D ), forodesine ( E ), allopurinol ( F ), MTX ( G ), or MTX and allopurinol ( H ) were successively administered during E9.5–E11.5 and brains were harvested at E12.5. Sections were stained with Hoechst dye (cyan). A’–F’ , Magnified views of the forebrain of A–H . lv, lateral ventricles; 3v, third ventricle. Scale bars, 500 μm ( A–H ), 100 μm ( A’–H’ ). Data supporting the anatomic changes in the embryonic brain caused by each inhibitor is shown in Extended Data .

Article Snippet: The following drugs were used: mycophenolate mofetil (MMF; #S1501, Selleck, 100 mg/kg in vivo , 1–10 μ m in vitro , dissolved in DMSO), methotrexate (MTX; #139-13571, Fujifilm Wako, 50 mg/kg in vivo , 1–100 μ m in vitro , dissolved in 50 m m Na 2 CO 3 buffer), forodesine hydrochloride (#HY-16209, MCE, 40 mg/kg in vivo , 0.1–5 μ m in vitro , dissolved in PBS), allopurinol (#17795-21-0, Selleck, 50 mg/kg in vivo , 250 μ m in vitro , dissolved in DMSO), BrdU (#B1575, Tokyo Chemical Industry, 100 mg/kg in vivo , 10 μ m in vitro , dissolved in PBS), and MHY1485 (#S7811, Selleck, 50 mg/kg in vivo , 10 μ m in vitro , dissolved in DMSO).

Techniques: Control, Staining

Journal: eNeuro

Article Title: Spatiotemporal Regulation of De Novo and Salvage Purine Synthesis during Brain Development

doi: 10.1523/ENEURO.0159-23.2023

Figure Lengend Snippet: De novo pathway inhibition causes forebrain-specific malformation. A–AE , Control DMSO ( A , F , K , P , U , Z , AC ), MMF ( B , G , L , Q , V , AA , AD ), MMF and MHY1485 ( C , H , M , R , W , AB , AE ), forodesine ( D , I , N , S , X ), or allopurinol ( E , J , O , T , Y ) was administered to pregnant mice between E9.5–11.5 and analyzed at E12.5. Horizontal frozen sections were immunostained with antibodies to cleaved-caspase3 (red; A–E ), Pax6 (green) and DCX (red; F–J ), Tbr2 (green; K–O ), Ki67 (red; P–T ), pH3 (green; U–Y ), Nestin (green; Z–AB ), or GSH2 (green; AC–AE ). The forebrain region treated with MMF was divided by dotted lines into the rostral (Pax6 – GSH2 + ) and caudal area (Pax6 + GSH2 – ). The asterisk in G denotes the expanded cortical layer filled with DCX + immature neurons. AF , AG , Quantification of cleaved-caspase3 + ( AF ) or pH3 + ( AG ) cells in the cerebral cortex (number of cells per 500-μm width of the cortex). In embryos treated with MMF, cells were counted individually in the rostral and caudal areas. ns, not significant, * p < 0.05, ** p < 0.01, Welch’s t test followed by Holm–Bonferroni correction. Scale bars, 100 μm ( A–AE ). AH , AI , Expression of cleaved-caspase3. Embryos were treated with MTX or MTX and MHY1485 during E9.5–E11.5, and the brains were subjected to Western blot analysis at E12.5. AH , Representative Western blotting illustrating the accelerated apoptosis in MTX-treated brains and the rescue effect of MHY1485. The blot was reprobed with α-Tubulin antibody (bottom) to examine quantitative protein loading. AI , Quantified comparison of cleaved-caspase3 expression. Data are presented as the mean ± SEM. AJ , Schematic model of brain abnormalities caused by de novo pathway inhibition. Control (left upper panel) or MMF-treated brains (left lower panel) were cut in the horizontal (middle panels) or sagittal plane (right panels). Horizontal sections were sliced through the orange line shown in the 3D brain (left). MMF-treated brains show a loss or hypoplasia of the rostral neocortex and a dorsal expansion of striatal GE (blue), resulting in abnormal forebrain structures. In MMF-treated embryos, the rostral neocortex containing Pax6 + VZ disappeared; instead, the GE containing GSH2 + VZ ectopically appeared on the dorsal surface. lv, lateral ventricles; 3v, third ventricle; 4v, fourth ventricle; GE, ganglionic eminence. Data demonstrating that administration of MHY1485 alone does not affect embryonic brain development is shown in Extended Data .

Article Snippet: The following drugs were used: mycophenolate mofetil (MMF; #S1501, Selleck, 100 mg/kg in vivo , 1–10 μ m in vitro , dissolved in DMSO), methotrexate (MTX; #139-13571, Fujifilm Wako, 50 mg/kg in vivo , 1–100 μ m in vitro , dissolved in 50 m m Na 2 CO 3 buffer), forodesine hydrochloride (#HY-16209, MCE, 40 mg/kg in vivo , 0.1–5 μ m in vitro , dissolved in PBS), allopurinol (#17795-21-0, Selleck, 50 mg/kg in vivo , 250 μ m in vitro , dissolved in DMSO), BrdU (#B1575, Tokyo Chemical Industry, 100 mg/kg in vivo , 10 μ m in vitro , dissolved in PBS), and MHY1485 (#S7811, Selleck, 50 mg/kg in vivo , 10 μ m in vitro , dissolved in DMSO).

Techniques: Inhibition, Control, Expressing, Western Blot, Comparison