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Mitochondrial oxidation of the carbohydrate fuel is required for neural precursor/stem cell function and postnatal cerebellar development

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Science Advances  10 Oct 2018:
Vol. 4, no. 10, eaat2681
DOI: 10.1126/sciadv.aat2681
  • Fig. 1 Depletion of PTPMT1 from neural precursor cells blocks postnatal cerebellar development.

    (A) Kaplan-Meier survival curves of PTPMT1fl/fl/Nestin-Cre+ (n = 18), PTPMT1+/+/Nestin-Cre+ (n = 20), and PTPMT1fl/fl/Nestin-Cre (n = 18) mice. PTPMT1+/+/Nestin-Cre+ and PTPMT1fl/fl/Nestin-Cre+ mice and brains at P12 were photographed. Representative cerebella and cerebellar sections [hematoxylin and eosin (H&E) staining] of PTPMT1+/+/Nestin-Cre+ and PTPMT1fl/fl/Nestin-Cre+ mice at P8 are shown. Cb, cerebellum; IC, inferior colliculus; CP, choroid plexus. PTPMT1 mRNA levels in freshly isolated cerebra and cerebella with the indicated genotypes (n = 3) were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). (B and C) Brain sections prepared from PTPMT1+/+/Nestin-Cre+ and PTPMT1fl/fl/Nestin-Cre+ mice at the indicated ages were processed for immunofluorescence staining with the indicated antibodies, followed by 4′,6′-diamidino-2-phenylindole (DAPI) counterstaining. (D) Cryosections of hindbrains with the indicated genotypes at E12.5, E14.5, and E17.5 were hybridized with digoxigenin (DIG)–labeled probes specific for mouse Lhx1 and Math1 mRNA. Arrows indicate Math1+ or Lhx1+ cells. (E to G) Brain sections prepared from PTPMT1+/+/Nestin-Cre+ and PTPMT1fl/fl/Nestin-Cre+ mice at the indicated ages were processed for immunofluorescence staining with the indicated antibodies, followed by DAPI counterstaining. EGL, external granule layer; PCL, Purkinje cell layer; IGL, internal granule layer; ML, molecular layer. Arrowheads in (G) indicate cleaved caspase 3+ apoptotic cells. Representative images from three mice per genotype are shown.

  • Fig. 2 Deletion of PTPMT1 from PCPs and/or GCPs generates only mild to moderate effects on cerebellar development.

    (A and B) PC-specific (PTPMT1fl/fl/PCP2-Cre+), GC-specific (PTPMT1fl/fl/Atoh1-Cre+), and PC/GC double knockout (PTPMT1fl/fl/PCP2-Cre+/Atoh1-Cre+) mice were generated. Cerebellar sections were prepared at 1 month of age for histopathological examination (H&E staining) (A) and immunofluorescence staining with the indicated antibodies (B). (C) PCs and GCs were isolated from PC-, GC-, and PC/GC double knockout mice at P4 (n = 3 per group). PTPMT1 mRNA levels in these cells were determined by qRT-PCR. (D) Footprint tests were performed to examine the gait of adult PC-specific, GC-specific, and PC/GC double knockout mice and control littermates. Dashed lines represent the direction of progression of walking. (E) PTPMT1 inducible knockout (PTPMT1fl/fl/CAG-Cre+-Esr) mice were generated and treated with tamoxifen by intraperitoneal injections (9 mg/40 g body weight, five doses over 10 days) at 6 weeks of age. Seven days after the last dose of tamoxifen administration, cerebellar sections were prepared for histopathological examination (H&E staining) and immunofluorescence staining with the indicated antibodies. (F) PTPMT1 mRNA levels in adult cerebral cortices and cerebella with the indicated genotypes (n = 4 per group) were determined by qRT-PCR. (G) Footprints of adult PTPMT1fl/fl/CAG-Cre+-Esr mice and PTPMT1+/+/CAG-Cre+-Esr littermates. Representative images from three mice per genotype are shown.

  • Fig. 3 BG development is disrupted in neural precursor/stem cell PTPMT1 knockout mice.

    (A to D) Cerebellar sections prepared from PTPMT1+/+/Nestin-Cre+ and PTPMT1fl/fl/Nestin-Cre+ mice at the indicated ages were processed for immunofluorescence staining with the indicated antibodies, followed by DAPI counterstaining. Representative images from three mice per group are shown. (E) Cerebellar cortical tissues dissected from PTPMT1+/+/Nestin-Cre+ and PTPMT1fl/fl/Nestin-Cre+ newborn pups (P0) and embryos at E17.5 were processed for microexplant culture. Seventy-two hours later, tissues and cells on the coverslips were processed for immunofluorescence staining for Nestin and GFAP, followed by DAPI counterstaining.

  • Fig. 4 Induced deletion of PTPMT1 from neural precursor cells at E13.5, but not E18.5, blocks cerebellar development.

    PTPMT1fl/+/Nestin-Cre+-Esr1 mice were generated and used to cross PTPMT1fl/+ mice. (A to C) Timed-pregnant female mice at E13.5 were administered tamoxifen by an intraperitoneal injection [2 mg dissolved in corn oil/ethanol (9:1) mixture]. After giving birth, lactating mothers were treated with tamoxifen again (83.5 mg/kg body weight, daily for 5 days). (A) Kaplan-Meier survival curves of PTPMT1fl/fl/Nestin-Cre+-Esr1 (n = 8) and PTPMT1+/+/ Nestin-Cre+-Esr1 (n = 24) pups. Representative cerebellar and cerebral sections (H&E staining) of PTPMT1fl/fl/Nestin-Cre+-Esr1 and PTPMT1+/+/ Nestin-Cre+-Esr1 pups at P13 are shown. Cx, cortex; Hp, hippocampus. Cerebellar sections were also processed for immunofluorescence staining with the indicated antibodies, followed by DAPI counterstaining. (B) Representative images. (C) PTPMT1 mRNA levels in freshly isolated cerebral cortices and cerebella with the indicated genotypes (n = 3 mice per group) were determined by qRT-PCR. (D and E) Timed-pregnant female mice at E18.5 were administered tamoxifen as above. Cerebellar sections (H&E staining) of PTPMT1fl/fl/Nestin-Cre+-Esr1 and PTPMT1+/+/ Nestin-Cre+-Esr1 pups at P13 are shown (D). PTPMT1 mRNA levels in freshly isolated cerebral cortices and cerebella with the indicated genotypes (n = 3 mice per group) were determined by qRT-PCR (E).

  • Fig. 5 PTPMT1 depletion causes bioenergetic stress and cell cycle arrest in cerebellar NSPCs, leading to diminished self-renewal and senescence.

    (A to C) Prominin+Lin cells were sorted from individual PTPMT1fl/fl/Nestin-Cre+ and PTPMT1+/+/Nestin-Cre+ cerebella at P4 (n = 3 mice per group). These cells were assessed by the neurosphere assay. Neurospheres derived were counted after 7 days of culture. Representative neurospheres are shown on the left. Primary (1°) neurospheres were harvested and assayed by the neurosphere culture again. Secondary (2°) neurospheres derived were counted 7 days later. (A) Total cell numbers were determined at the indicated time points. Primary neurosphere cells were assayed by apoptosis analyses. (B) Annexin V+ apoptotic cells were quantified by fluorescence-activated cell sorting (FACS). (C) Cell cycle profiles of primary neurosphere cells were determined by Ki67 and Hoechst 33342 staining, followed by FACS analyses. (D) mRNA levels of the indicated cell cycle regulatory genes in primary neurosphere cells were determined by qRT-PCR (n = 6 mice per group). (E) Total DNA was extracted from neurospheres. Mitochondrial content was estimated by comparing mitochondrial DNA (mtDNA; cytochrome B) levels to genomic DNA levels by qPCR (n = 3 mice per group). NS, not significant. (F and G) Total cellular ATP and ROS levels in neurosphere cells were determined. (H) Prominin-1+Lin cells were sorted from individual cerebella dissected from PTPMT1fl/fl/Nestin-Cre+ (n = 5) and PTPMT1+/+/Nestin-Cre+ (n = 3) pups at P4. Whole-cell lysates were prepared and examined by immunoblotting with the indicated antibodies. Representative results are shown. ACC, acetyl–coenzyme A (CoA) carboxylase. (I) Neurospheres generated from the cortices of PTPMT1fl/fl/Nestin-Cre+ and PTPMT1+/+/Nestin-Cre+ mice (n = 3 per group) were lysed and examined by immunoblotting with the indicated antibodies. Representative results are shown.

  • Fig. 6 PTPMT1 depletion from the mitochondria reprograms cellular metabolism by limiting mitochondrial pyruvate utilization.

    (A and B) Neurospheres derived from PTPMT1+/+/Nestin-Cre+ and PTPMT1fl/fl/Nestin-Cre+ mice (n = 3 mice per group) were dissociated into single cells. OCR (A) and ECAR (B) of these live cells in the medium containing all metabolic substrates were measured. Representative results are shown. (C to F) Mitochondria were isolated from adult brains dissected from PTPMT1fl/fl/CAG-Cre+-Esr mice and PTPMT1+/+/CAG-Cre+-Esr littermates (n = 3 mice per group) 10 days following tamoxifen administration. Oxygen consumption of the mitochondria was measured in the presence of pyruvate/malate (C), palmitoyl-CoA/carnitine/malate (D), glutamate/malate (E), or succinate (F), following the addition of ADP, oligomycin, FCCP, and rotenone. Representative results are shown. (G to I) Pyruvate and α-ketoglutarate levels in the lysates of the neurosphere cells derived from PTPMT1+/+/Nestin-Cre+ and PTPMT1fl/fl/Nestin-Cre+ mice (n = 3 mice per group) or in the lysates of the mitochondria isolated from the cerebella of adult PTPMT1fl/fl/CAG-Cre+-Esr mice and PTPMT1+/+/CAG-Cre+-Esr littermates (n = 3 mice per group) 10 days following tamoxifen administration were measured. (J and K) Mitochondria were isolated from the cerebella freshly dissected from adult PTPMT1fl/fl/CAG-Cre-Esr+ and PTPMT1+/+/CAG-Cre-Esr+ mice (n = 3 mice per group) as above. After being washed three times in mitochondrial assay solution (MAS) buffer, mitochondria were incubated with pyruvate/malate (5 mM) and ADP (4 mM) in a non-CO2 incubator. Five minutes later, mitochondria were collected, washed, and lysed. (J) α-Ketoglutarate levels in the mitochondrial lysates were measured. (K) PDH activities in the mitochondrial lysates were determined with a PDH assay kit following the manufacturer’s instructions. (L) Neurospheres derived from PTPMT1fl/fl/Nestin-Cre+ and PTPMT1+/+/Nestin-Cre+ mice (n = 3 mice per group) were dissociated into single cells. These cells were treated with the MPC inhibitor UK5099 (25 nM) or vehicle for 20 min. OCRs of the cells in the presence of all metabolic substrates were then measured as described above. Statistical analysis results between UK5099-treated and vehicle-treated PTPMT1+/+/Nestin-Cre+ cells are shown. (M to O) Cerebral cortices dissected from PTPMT1fl/fl/Nestin-Cre+ and PTPMT1+/+/Nestin-Cre+ mice (n = 3 per group) were assessed by the neurosphere assay in the presence of the MPC inhibitor UK5099 at the indicated concentrations (M), methyl pyruvate (20 mM), dimethyl α-ketoglutarate (6 mM) (N), or the PIKfyve inhibitor (YM201636) at the indicated concentrations (O). Neurospheres derived were counted after 7 days of culture.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/10/eaat2681/DC1

    Fig. S1. PTPMT1 depletion from neural precursor cells compromises cerebral development.

    Fig. S2. Active proliferation of GCPs and increased apoptosis in postnatal PTPMT1 knockout cerebella.

    Fig. S3. Correctly localized BG with radial fibers are missing in PTPMT1 knockout cerebella.

    Fig. S4. Decreased cell proliferation and increased apoptosis in PTPMT1 knockout cerebra.

    Fig. S5. Loss of PTPMT1 causes cell cycle arrest in cerebral NSPCs, leading to defective self-renewal and senescence.

    Fig. S6. Deletion of p53 does not rescue PTPMT1 knockout mice or NSPC self-renewal.

    Fig. S7. Loss of PTPMT1 causes bioenergetic stress in cerebral NSPCs.

    Fig. S8. The entire cerebral cortex cell population is less affected by PTPMT1 ablation.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. PTPMT1 depletion from neural precursor cells compromises cerebral development.
    • Fig. S2. Active proliferation of GCPs and increased apoptosis in postnatal PTPMT1 knockout cerebella.
    • Fig. S3. Correctly localized BG with radial fibers are missing in PTPMT1 knockout cerebella.
    • Fig. S4. Decreased cell proliferation and increased apoptosis in PTPMT1 knockout cerebra.
    • Fig. S5. Loss of PTPMT1 causes cell cycle arrest in cerebral NSPCs, leading to defective self-renewal and senescence.
    • Fig. S6. Deletion of p53 does not rescue PTPMT1 knockout mice or NSPC self-renewal.
    • Fig. S7. Loss of PTPMT1 causes bioenergetic stress in cerebral NSPCs.
    • Fig. S8. The entire cerebral cortex cell population is less affected by PTPMT1 ablation.

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