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, 25 (23), 5188-5197

Genetic and Pharmacological Correction of Aberrant Dopamine Synthesis Using Patient iPSCs With BH4 Metabolism Disorders

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Genetic and Pharmacological Correction of Aberrant Dopamine Synthesis Using Patient iPSCs With BH4 Metabolism Disorders

Taizo Ishikawa et al. Hum Mol Genet.

Abstract

Dopamine (DA) is a neurotransmitter in the brain, playing a central role in several disease conditions, including tetrahydrobiopterin (BH4) metabolism disorders and Parkinson's disease (PD). BH4 metabolism disorders present a variety of clinical manifestations including motor disturbance via altered DA metabolism, since BH4 is a cofactor for tyrosine hydroxylase (TH), a rate-limiting enzyme for DA synthesis. Genetically, BH4 metabolism disorders are, in an autosomal recessive pattern, caused by a variant in genes encoding enzymes for BH4 synthesis or recycling, including 6-pyruvoyltetrahydropterin synthase (PTPS) or dihydropteridine reductase (DHPR), respectively. Although BH4 metabolism disorders and its metabolisms have been studied, it is unclear how gene variants cause aberrant DA synthesis in patient neurons. Here, we generated induced pluripotent stem cells (iPSCs) from BH4 metabolism disorder patients with PTPS or DHPR variants, corrected the gene variant in the iPSCs using the CRISPR/Cas9 system, and differentiated the BH4 metabolism disorder patient- and isogenic control iPSCs into midbrain DA neurons. We found that by the gene correction, the BH4 amount, TH protein level and extracellular DA level were restored in DA neuronal culture using PTPS deficiency iPSCs. Furthermore, the pharmacological correction by BH4 precursor sepiapterin treatment also improved the phenotypes of PTPS deficiency. These results suggest that patient iPSCs with BH4 metabolism disorders provide an opportunity for screening substances for treating aberrant DA synthesis-related disorders.

Figures

Figure 1
Figure 1
Generation and characterization of BH4 metabolism disorder iPSCs. (A) Schematic of strategy for comparing DA neuronal culture using iPSCs from BH4 metabolism disorder patients with that using genetically corrected iPSCs. (B) Phase-contrast images, immunocytochemistry for pluripotency markers NANOG and SSEA4, and karyotype analysis in PTPS deficiency iPSCs and DHPR deficiency iPSCs. Scale bars: 100 μm. (C)In vitro differentiation of PTPS deficiency iPSCs and DHPR deficiency iPSCs to three germ layers: βIII-tubulin (ectoderm), αSMA (mesoderm), and SOX17 (endoderm). Scale bars: 100 μm.
Figure 2
Figure 2
Gene correction of variant in BH4 metabolism disorder iPSCs. (A,B) Schematic of strategy used for CRISPR/Cas9-mediated gene correction of PTS c.243G>A variant (A) and QDPR c.176C>A variant (B). Red triangle denotes the gene variant. Grey triangle denotes the corrected gene variant. (C,D) Sequencing of genomic PTS locus in indicated PTPS iPSCs (C) and genomic QDPR locus in indicated DHPR iPSCs (D). The corrected gene variant is marked with an arrow. (E,F) Phase-contrast images, immunocytochemistry for pluripotency markers NANOG and SSEA4, and karyotype analysis in PTPS deficiency-corrected iPSCs (E) and DHPR deficiency-corrected iPSCs (F). Scale bars: 100 μm. (G,I) Gene expression analysis of PTPS deficiency iPSCs and PTPS deficiency-corrected iPSCs for PTPS mRNA (G) and DHPR deficiency iPSCs and DHPR deficiency-corrected iPSCs for DHPR mRNA (I). GAPDH was used as normalizer. Data represent mean ± S.D. of n =  3. *P <  0.05, **P <  0.01, one-way ANOVA with Dunnett’s tests compared to patient iPSCs. (H,J) Western blot analysis of PTPS deficiency iPSCs and PTPS deficiency-corrected iPSCs for PTPS and β-actin protein levels (H) and DHPR deficiency iPSCs and DHPR deficiency-corrected iPSCs for DHPR and β-actin protein levels (J). Asterisk indicates non-specific band.
Figure 3
Figure 3
PTPS deficiency iPSC-derived DA neuronal culture shows decreased TH protein level and extracellular DA level. (A) Gene expression analysis for key midbrain DA neuron markers. GAPDH was used as normalizer. The expression level of undifferentiated cells (day 0) was set at 1. (B) DA neuronal cultures using PTPS deficiency iPSCs and PTPS deficiency-corrected iPSCs were stained for TH, the marker for neuron βIII-tubulin and DAPI. Scale bar: 100 μm. (C–E) Percentage of βIII-tubulin-positive neurons in total cells (C), percentage of TH-positive neurons in βIII-tubulin-positive neurons (D) and the relative area of TH-positive neurons (E) in DA neuronal cultures using PTPS deficiency iPSCs and PTPS deficiency-corrected iPSCs. (F) Western blot analysis of DA neuronal cultures using PTPS deficiency iPSCs and PTPS deficiency-corrected iPSCs for TH, βIII-tubulin and β-actin protein levels. (G) Relative level of high potassium evoked-extracellular DA release in DA neuronal cultures using PTPS deficiency iPSCs and PTPS deficiency-corrected iPSCs. (H,I) Pterin metabolic profiles of DA neuronal cultures using PTPS deficiency iPSCs and PTPS deficiency-corrected iPSCs: BH4 (H), Neopterin (I). (J) Metabolic pathway of BH4 and DA synthesis in PTPS deficiency. c.243G > A variant in PTS gene causes reductions of BH4 amount, TH protein level, and extracellular DA level in DA neuronal culture. Enzymes are indicated by the following abbreviations: GTPCH, GTP cyclohydrolase I; PTPS, 6-pyruvoyltetrahydropterin synthase; SR, sepiapterin reductase; DHPR, dihydropteridine reductase; TH, tyrosine hydroxylase. Quantification represents mean ± S.D. of n = 3. *P < 0.05, **P < 0.01, Student’s t test.
Figure 4
Figure 4
Sepiapterin improves TH protein level and extracellular DA level in PTPS deficiency iPSC-derived DA neuronal culture. (A) PTPS deficiency iPSC-derived DA neuronal cultures treated with DMSO, 30 μM BH4 or 30 μM sepiapterin were stained for TH, the marker for neuron βIII-tubulin and DAPI. Scale bar: 100 μm. (B–D) Percentage of βIII-tubulin-positive neurons in total cells (B), percentage of TH-positive neurons in βIII-tubulin-positive neurons (C), and relative area of TH-positive neurons (D) in PTPS deficiency iPSC-derived DA neuronal culture treated with DMSO, 30 μM BH4 or 30 μM sepiapterin. (E) Western blot analysis of PTPS deficiency iPSC-derived DA neuronal culture treated with DMSO, 30 μM BH4 or 30 μM sepiapterin for TH, βIII-tubulin and β-actin protein levels. (F) Relative level of high potassium evoked-extracellular DA release in PTPS deficiency iPSC-derived DA neuronal culture treated with DMSO, 30 μM BH4 or 30 μM sepiapterin. Quantification represents mean ± S.D. of n =  3. *P <  0.05, ****P <  0.0001, one-way ANOVA with Dunnett’s tests compared to non-treatment control.

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