Ex vivo proton spectroscopy (1 H-NMR) analysis of inborn errors of metabolism: Automatic and computer-assisted analyses.

There are about 1500 genetic metabolic diseases. A small number of treatable diseases are diagnosed by newborn screening programs, which are continually being developed. However, most diseases can only be diagnosed based on clinical symptoms or metabolic findings. The main biological fluids used are urine, plasma and, in special situations, cerebrospinal fluid. In contrast to commonly used methods such as gas chromatography and high performance liquid chromatography mass spectrometry, ex vivo proton spectroscopy (1 H-NMR) is not yet used in routine clinical practice, although it has been recommended for more than 30 years. Automatic analysis and improved NMR technology have also expanded the applications used for the diagnosis of inborn errors of metabolism. We provide a mini-overview of typical applications, especially in urine but also in plasma, used to diagnose common but also rare genetic metabolic diseases with 1 H-NMR. The use of computer-assisted diagnostic suggestions can facilitate interpretation of the profiles. In a proof of principle, to date, 182 reports of 59 different diseases and 500 reports of healthy children are stored. The percentage of correct automatic diagnoses was 74%. Using the same 1 H-NMR profile-targeted analysis, it is possible to apply an untargeted approach that distinguishes profile differences from healthy individuals. Thus, additional conditions such as lysosomal storage diseases or drug interferences are detectable. Furthermore, because 1 H-NMR is highly reproducible and can detect a variety of different substance categories, the metabolomic approach is suitable for monitoring patient treatment and revealing additional factors such as nutrition and microbiome metabolism. Besides the progress in analytical techniques, a multiomics approach is most effective to combine metabolomics with, for example, whole exome sequencing, to also diagnose patients with nondetectable metabolic abnormalities in biological fluids. In this mini review we also provide our own data to demonstrate the role of NMR in a multiomics platform in the field of inborn errors of metabolism.

Human Dopaminergic Neurons Lacking PINK1 Exhibit Disrupted Dopamine Metabolism Related to Vitamin B6 Co-Factors.

PINK1 loss-of-function mutations cause early onset Parkinson disease. PINK1-Parkin mediated mitophagy has been well studied, but the relevance of the endogenous process in the brain is debated. Here, the absence of PINK1 in human dopaminergic neurons inhibits ionophore-induced mitophagy and reduces mitochondrial membrane potential. Compensatory, mitochondrial renewal maintains mitochondrial morphology and protects the respiratory chain. This is paralleled by metabolic changes, including inhibition of the TCA cycle enzyme mAconitase, accumulation of NAD+, and metabolite depletion. Loss of PINK1 disrupts dopamine metabolism by critically affecting its synthesis and uptake. The mechanism involves steering of key amino acids toward energy production rather than neurotransmitter metabolism and involves cofactors related to the vitamin B6 salvage pathway identified using unbiased multi-omics approaches. We propose that reduction of mitochondrial membrane potential that cannot be controlled by PINK1 signaling initiates metabolic compensation that has neurometabolic consequences relevant to Parkinson disease.

Generation of iPSCs carrying a common LRRK2 risk allele for in vitro modeling of idiopathic Parkinson's disease.

Induced pluripotent stem cells (iPSCs) have recapitulated several aspects of Parkinson's disease (PD), but most iPSCs are derived from familial cases, which account for only about 15% of patients. Thus, while the emphasis has justifiably been on using iPSCs to model rare familial cases, models for the most common forms of PD are critically lacking. Here, we report the generation of an iPSC-based model of idiopathic PD (iPD) with or without RS1491923, which is a common risk variant in the LRRK2 locus. Consistent with GWA studies, we found large variability in our datasets. However, iPSC-derived neurons carrying the risk allele emerged for displaying subtle disturbances of cellular degradative systems, in line with familial PD models. We also observed that treatment with the LRRK2 inhibitor CZC-25146 slightly reduced a marker of aSYN pathology in all iPD lines. Future iPSC-based studies may need to be structured similarly to large GWA studies in order to obtain relevant statistical power. However, results from this pilot study suggest that iPSC-based modeling represents an attractive way to investigate idiopathic diseases.

Metformin reverses TRAP1 mutation-associated alterations in mitochondrial function in Parkinson's disease.

The mitochondrial proteins TRAP1 and HTRA2 have previously been shown to be phosphorylated in the presence of the Parkinson's disease kinase PINK1 but the downstream signalling is unknown. HTRA2 and PINK1 loss of function causes parkinsonism in humans and animals. Here, we identified TRAP1 as an interactor of HTRA2 using an unbiased mass spectrometry approach. In our human cell models, TRAP1 overexpression is protective, rescuing HTRA2 and PINK1-associated mitochondrial dysfunction and suggesting that TRAP1 acts downstream of HTRA2 and PINK1. HTRA2 regulates TRAP1 protein levels, but TRAP1 is not a direct target of HTRA2 protease activity. Following genetic screening of Parkinson's disease patients and healthy controls, we also report the first TRAP1 mutation leading to complete loss of functional protein in a patient with late onset Parkinson's disease. Analysis of fibroblasts derived from the patient reveal that oxygen consumption, ATP output and reactive oxygen species are increased compared to healthy individuals. This is coupled with an increased pool of free NADH, increased mitochondrial biogenesis, triggering of the mitochondrial unfolded protein response, loss of mitochondrial membrane potential and sensitivity to mitochondrial removal and apoptosis. These data highlight the role of TRAP1 in the regulation of energy metabolism and mitochondrial quality control. Interestingly, the diabetes drug metformin reverses mutation-associated alterations on energy metabolism, mitochondrial biogenesis and restores mitochondrial membrane potential. In summary, our data show that TRAP1 acts downstream of PINK1 and HTRA2 for mitochondrial fine tuning, whereas TRAP1 loss of function leads to reduced control of energy metabolism, ultimately impacting mitochondrial membrane potential. These findings offer new insight into mitochondrial pathologies in Parkinson's disease and provide new prospects for targeted therapies.

Generation of induced pluripotent stem cells derived from a 77-year-old healthy woman as control for age related diseases.

Induced pluripotent stem cells (iPSCs) hold great promise to model diseases, where the disease affected cell type is difficult to access. A major obstacle for the development of disease models is the lack of well characterized control iPSCs from old people not affected by such a disease. Furthermore, gene-editing approaches often require iPSCs from healthy donors, where pathogenic mutations can be inserted if patient material is not available. Here, we report the generation of an iPSC line (16423 #6) from a 77-year-old woman, who did not display any disease symptoms at the time, when the skin biopsy was taken.