Genetic and pharmacological correction of aberrant dopamine synthesis using patient iPSCs with BH4 metabolism disorders

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.

Oral berberine improves brain dopa/dopamine levels to ameliorate Parkinson’s disease by regulating gut microbiota

“… 11 A deficiency of BH 4 or the reductase will directly inhibit the hydroxylation of Phe to Tyr and/or Tyr to dopa, which will block the Phe–Tyr–dopa–dopamine metabolism pathway and lead to nervous system diseases. 50 At present, the main clinical treatment strategy for BH 4 deficiency is to increase the intake of synthetic BH 4 (trade name Kuvan). 51 Here, BBR might be the first agonist of Enterococcus bacteria that leads to the production of l -dopa by triggering BH 4 formation in the gut bacteria.…”

The functional genomics laboratory: functional validation of genetic variants

“…Rescue: introduction by lentiviral transduction of wild type LYRM7 cDNA in fibroblasts from a patient with a defect in LYRM7 results in normalization of mitochondrial Rieske Fe-S protein (Hempel et al 2017 ). CRISPR/Cas9: Absence of thymidine hydroxylase (TH) staining in iPSC-derived dopaminergic nerve cells from a patient with a defect in PTPS , and normalization of TH expression after CRISPR/Cas9-mediated correction of the PTPS gene (Ishikawa et al 2016 ). Biomarkers: detail of a 1H NMR spectrum of a CSF sample from a NANS patient, showing the presence of alpha and beta forms of N -acetylmannosamine (van Karnebeek et al 2016 ).…”

Stem Cells, Genome Editing, and the Path to Translational Medicine

“…Engineering of isogenic hPSCs by either correcting the disease-associated mutation in patient-derived hiPSCs or insertion into wild-type hPSCs has become standard to distinguish disease-associated effects from background variation. Examples include Parkinson’s and Alzheimer’s disease (Paquet et al, 2016; Reinhardt et al, 2013; Schöndorf et al, 2014), hereditary motor and sensory neuropathy (Murakami et al, 2017), frontotemporal lobar degeneration (Imamura et al, 2016), Huntington disease (Xu et al, 2017), tetrahydrobiopterin metabolism disorder (Ishikawa et al, 2016), fragile X syndrom (Xie et al, 2016) and the functional analysis of neurorexin-1 in neuropsychiatric diseases (Pak et al, 2015). Limitations to this approach are off-target cleavage of site-specific nucleases and undesired genetic alterations in cell survival pathways such as P53 mutations as a consequence of genome engineering associated genotoxic stress and isolating single cell clones (Haapaniemi et al, 2018; Ihry et al, 2018; Merkle et al, 2017).…”