Micro-NMR elucidates altered metabolites in the Parkinson’s disease-related catp-6 genotype of Caenorhabditis elegans

Abstract

A severe form of Parkinson’s disease (PD) is the Kufor-Rakeb syndrome. Here mutations in the ATP13A2 (PARK9) gene lead to an early juvenile-onset Parkinsonism often accompanied by dementia. ATP13A2 encodes a lysosomal P-type ATPase. Its ortholog in Caenorhabditis elegans is the catp-6 gene where phenotypes with mutations in the alleles ok3473 and tm3190 show high mortality and low reproduction. Since PD is difficult to study in humans we wanted to investigate the potential to use C. elegans as model for the Kufor-Rakeb syndrome. As it is difficult to obtain enough catp-6 mutant worms for standard NMR metabolic profiling, we explored focused ultrasonication extraction and miniaturized NMR as techniques to overcome this limitation. One- and two-dimensional NMR experiments (1 H, JRES, TOCSY) were performed with a commercial high-resolution magic angle spinning (HR-MAS) probe (25 µL sample volume). Significant features were identified through analysis of variance (ANOVA, p 1.5), PCA, and PLS-DA. Assignment of statistically relevant peaks resulted in the identification of twenty altered metabolites. Previous studies on catp-6 mutants identified strong morphological and functional changes in their mitochondria. Our findings of altered TCA metabolites (fumarate, succinate), branched-chain amino acids (leucine, isoleucine and valine) and nucleotides (AMP, ATP and GTP), formate and hypoxanthine appear to support these findings. Highest fold changes (< −5) in wildtype relative to both catp-6 strains were found for GTP. Formic acid is known to inhibit the mitochondrial respiratory chain complex IV and high hypoxanthine in catp-6 indicates an increased nucleotide salvage pathway. Alterations in most of the remaining metabolites may be the result of the recently discovered activation of AMPK (AMP-activated protein kinase) and inhibition of mTOR (mechanistic target of rapamycin) pathways together with a catabolic response to recover energy production. If the effect of the catp-6 mutation in C. elegans at the level of metabolites is correlated to the metabolic dysfunction in the human PARK9 ortholog, then it may be possible to uncover the molecular mechanism behind Parkinsonism and the Kufor-Rakeb syndrome.