Autophagy induction by tetrahydrobiopterin deficiency

Abstract

Tetrahydrobiopterin (BH₄) deficiency is a genetic disorder associated with a variety of metabolic syndromes such as phenylketonuria (PKU). In this article, the signaling pathway by which BH₄ deficiency inactivates mTORC1 leading to the activation of the autophagic pathway was studied utilizing BH₄-deficient Spr(-/-) mice generated by the knockout of the gene encoding sepiapterin reductase (SR) catalyzing BH₄ synthesis. We found that mTORC1 signaling was inactivated and autophagic pathway was activated in tissues from Spr(-/-) mice. This study demonstrates that tyrosine deficiency causes mTORC1 inactivation and subsequent activation of autophagic pathway in Spr(-/-) mice. Therapeutic tyrosine diet completely rescued dwarfism and mTORC1 inhibition but inactivated autophagic pathway in Spr(-/-) mice. Tyrosine-dependent inactivation of mTORC1 was further supported by mTORC1 inactivation in Pah(enu2) mouse model lacking phenylalanine hydroxylase (Pah). NIH3T3 cells grown under the condition of tyrosine restriction exhibited autophagy induction. However, mTORC1 activation by RhebQ64L, a positive regulator of mTORC1, inactivated autophagic pathway in NIH3T3 cells under tyrosine-deficient conditions. In addition, this study first documents mTORC1 inactivation and autophagy induction in PKU patients with BH₄ deficiency.

Figure 1

Autophagy induction in BH₄-deficient Spr^−/− mice. (A) Inactivation of mTORC1 and induction of autophagy in Spr^−/− mice. Tissue homogenates from 25-d-old Spr^+/+ or Spr^−/− mice fed a normal diet were analyzed by protein gel blotting to examine the phosphorylation status of S6K and the conversion of LC3-I into LC3-II. (B) Relative band intensities in protein gel blot in (A) were quantified by using ImageJ software and the ratio of phosphorylated S6K (p-S6K) to S6K or LC3-II to LC3-I was presented. The ratio of band intensities of p-S6K/S6K in each tissue from Spr^+/+ mice was individually set to 1.0 (upper). The ratio of band intensities of LC-II/LC3-I in tissues from Spr^+/+ mice was set to 1.0 (bottom). Values were means ± SD (n = 3 experiments). Statistical significance was determined by Student's t-test, *p < 0.05, compared with Spr^+/+ mice. (C) Representative electron micrographs show the formation of autophagic vesicles in liver cells from 25-d-old Spr^−/− mouse. Scale bars represent 2 µm. (D) Representative example of liver cells from Spr^+/+ or Spr^−/− mice indicating reduced cell size in Spr^−/− mice. Liver sections from 25-d-old Spr^+/+ (left) or Spr^−/− (right) mice were stained with hematoxylin and eosine (H&E). Scale bars represent 20 µm. (E) Average number of cells in 10 randomly selected areas (100 µm × 100 µm) was presented. Statistical significance was determined by Student's t-test. Values represent means ± SD **p < 0.01, compared with Spr^+/+ mice. (F) Dispensable role of BH₄ deficiency on the activity of Akt or AMPK. Levels of Akt, AMPK, and their phosphorylated forms in livers and muscles from 25-d-old Spr^−/− mice fed a normal diet were compared with those in Spr^+/+ mice. The activation of Akt or AMPK was evaluated by examining the phosphorylation status of Akt at Ser473 or AMPK at Thr172 by protein gel blotting. (G) Relative band intensities in protein gel blot in (F) were quantified and ratios of p-Akt/Akt and p-AMPK/AMPK in the liver or muscle from Spr^+/+ or Spr^−/− mice were determined. The ratio of band intensities of p-Akt/Akt or p-AMPK/AMPK in tissues from Spr^+/+ mice was respectively set to 1.0. Values represent means ± SD (n = 3 experiments). #, 0.1 < p < 1.0.

Figure 2

Tyrosine deficiency causes autophagy induction following mTORC1 inactivation in Spr^−/− mice. (A) Concentrations of liver phenylalanine and tyrosine in 25-d-old Spr^+/+ or Spr^−/− mice (n = 5 for each genotype). Data represent means ± SD, **p < 0.01, compared with Spr^+/+ mice. (B) Weight gain of 35-d-old experimental mice. Newborn Spr^−/− mice were pretreated with ‘high dose of BH₄’ as described in Materials and Methods. For a control experiment, Spr^−/− mice were fed a normal diet (ND) with or without BH₄ solution (+BH₄) for an additional 10 d. For replacement therapy, therapeutic tyrosine diet (+Tyr) or leucine diet (+Leu) was prepared as described in Materials and Methods. Spr^−/− mice were fed a tyrosine or leucine supplemented diet for 10 d under ad libitum conditions. (C) Effects of tyrosine replacement therapy on weight gain of Spr^−/− mice (n = 5 for each genotype per experimental group). Each experimental mouse was weighed before and after replacement therapy for 10 d. Data represent means ± SD, **p < 0.01. ***p < 0.001, compared with body weight of Spr^+/+ mice fed a normal diet, one-way analysis of variance (ANOVA), #, 0.1 < p < 1.0. (D) Dietary tyrosine supplementation increases mTORC1 activity with subsequent inactivation of autophagic pathway in Spr^−/− mice. Tissue homogenates from 35-d-old Spr^+/+ or Spr^−/− mice fed a normal diet, therapeutic tyrosine or leucine diet were analyzed by protein gel blotting to examine the phosphorylation of S6K and the conversion of LC3-I into LC3-II. (E) Band intensities of protein gel blot in (D) were quantified and ratios of p-S6K/S6K and LC3-II/LC3-I were determined (n = 3 experiments). The ratio of band intensities of p-S6K/S6K or LC3-II/LC3-I in liver or muscle tissue from Spr^+/+ mice fed a normal diet was respectively set to 1.0. Values represent means ± SD, *p < 0.05.

Figure 3

Inactivation of mTORC1 and autophagy induction in Pah^enu2 mice. (A and B) The growth of 25-d-old Pah^enu2 mice was compared with that of 25-d-old wild-type mice (n = 6). Data represent means ± SD **p < 0.01, compared with the control group. (C) Inactivation of mTORC1 and induction of autophagy in Pah^enu2 mice. Liver and muscle homogenates from 25-d-old wild-type or Pah^enu2 mice fed a normal diet were analyzed by protein gel blotting to examine the phosphorylation of S6K and the conversion of LC3-I into LC3-II. (D) Band intensities of protein gel blot in (C) were quantified and ratios of p-S6K/S6K and LC3-II/LC3-I were presented. Values represent means ± SD (n = 3 experiments). *p < 0.05, compared with wild-type mice.

Figure 4

Induction of autophagy by tyrosine deficiency, but not by phenylalanine excess in NIH 3T3 cells. (A) NIH 3T3 cells were grown for 24 h in DMEM, tyrosine-restricted media (-Tyr), DMEM containing an excess amount of phenylalanine (4.0 mM) or tyrosine-restricted media containing 4.0 mM phenylalanine. Cells were examined for the phosphorylation of S6K and the conversion of LC3-I into LC3-II. (B) Band intensities of protein gel blot were quantified and the ratio of p-S6K/S6K or LC3-II/LC3-I was shown. The ratio of band intensities of p-S6K/S6K or LC-II/LC3-I in cells grown in complete DMEM was respectively set to 1.0. Values represent means ± SD (n = 3 experiments). *p < 0.05. (C) Electron micrographs demonstrate the induction of autophagy in NIH 3T3 cells grown in tyrosine-deprived DMEM for 24 h. Scale bars represent 2 µm. (D and E) NIH 3T3-GFP-LC3 cells stably expressing GFP-LC3 were incubated in DMEM, tyrosine-restricted media, DMEM containing excess amount of phenylalanine (4.0 mM) or tyrosine-restricted media containing 4.0 mM phenylalanine for 24 h. Accumulation of GFP-LC3 was visualized by fluorescent microscope and quantified. Combined results from three independent experiments were shown as means ± SD of the percentage of GFP-LC3-positive cells with punctate dots. **p < 0.01, compared with cells incubated in complete DMEM.

Figure 5

Inactivation of autophagic pathway by constitutively active Rheb in NIH 3T3 cells grown under tyrosine deficiency. (A) NIH 3T3 cells (1 × 10⁶) grown in complete media were transiently cotransfected with Flag-RhebQ64L (0.8 µg), GFP-LC3 (0.1 µg) and HA-S6K (0.1 µg). At 16 h after transfection, culture media were replaced with fresh DMEM or tyrosine-deficient media (-Tyr) and incubated for additional 24 h. The phosphorylation of ectopically expressed S6K (HA-S6K) and conversion of GFP-LC3-I into GFP-LC3-II were examined by protein gel blotting. (B) Band intensities were quantified and the ratio of p-S6K/S6K or LC3-II/LC3-I was shown. (n = 3 experiments). Values represent means ± SD, *p < 0.05.

Figure 6

Downregulation of mTORC1 activates autophagic pathway in BH₄-responsive PKU patients. (A) Levels of phosphorylated S6K and LC3-II in lymphocytes from BH₄-responsive PKU patients with BH₄ deficiency left untreated (Patient #1 and #3) or treated with 400 mg/day BH₄ (Patient #2 and #4) were examined by protein gel blotting. Patient #1, #2 and #4: PTPS deficiency, Patient #3: DHPR deficiency. (B) Band intensities were quantified and ratios of p-S6K/S6K and LC3-II/actin were determined. Values represent means ± SD (n = 3 experiments). *p < 0.05. #, 0.1 < p < 1.0. (C) Levels of S6K and phosphorylated S6K in 50 µg of lymphocyte lysate protein from a healthy child (Normal #1) were compared with those in 250 µg of lymphocyte lysate protein from BH₄-responsive PKU patient (Patient #1). (D) Band intensities were quantified and the ratio of p-S6K/S6K or LC3-II/LC3-I in PKU patient was compared with that in normal control. Values represent means ± SD (n = 3 experiments). *p < 0.05.