No abstract

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease, with an increasing incidence in recent years due to the aging population. Genetic mutations alone only explain <10% of PD cases, while environmental factors, including small molecules, may play a significant role in PD. In the present work, 22 plasma (11 PD, 11 control) and 19 feces samples (10 PD, 9 control) were analyzed by non-target high-resolution mass spectrometry (NT-HRMS) coupled to two liquid chromatography (LC) methods (reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC)). A cheminformatics workflow was optimized using open software (MS-DIAL and patRoon) and open databases (all public MSP-formatted spectral libraries for MS-DIAL, PubChemLite for Exposomics, and the LITMINEDNEURO list for patRoon). Furthermore, five disease-specific databases and three suspect lists (on PD and related disorders) were developed, using PubChem functionality to identifying relevant unknown chemicals. The results showed that non-target screening with the larger databases generally provided better results compared with smaller suspect lists. However, two suspect screening approaches with patRoon were also good options to study specific chemicals in PD. The combination of chromatographic methods (RP and HILIC) as well as two ionization modes (positive and negative) enhanced the coverage of chemicals in the biological samples. While most metabolomics studies in PD have focused on blood and cerebrospinal fluid, we found a higher number of relevant features in feces, such as alanine betaine or nicotinamide, which can be directly metabolized by gut microbiota. This highlights the potential role of gut dysbiosis in PD development.

Patients with Parkinson’s disease (PD) exhibit differences in their gut microbiomes compared to healthy individuals. Although differences have most commonly been described in the abundances of bacterial taxa, changes to viral and archaeal populations have also been observed. Mechanistic links between gut microbes and PD pathogenesis remain elusive but could involve molecules that promote α-synuclein aggregation. Here, we show that 2-hydroxypyridine (2-HP) represents a key molecule for the pathogenesis of PD. We observe significantly elevated 2-HP levels in faecal samples from patients with PD or its prodrome, idiopathic REM sleep behaviour disorder (iRBD), compared to healthy controls. 2-HP is correlated with the archaeal species Methanobrevibacter smithii and with genes involved in methane metabolism, and it is detectable in isolate cultures of M. smithii. We demonstrate that 2-HP is selectively toxic to transgenic α-synuclein overexpressing yeast and increases α-synuclein aggregation in a yeast model as well as in human induced pluripotent stem cell derived enteric neurons. It also exacerbates PD-related motor symptoms, α-synuclein aggregation, and striatal degeneration when injected intrastriatally in transgenic mice overexpressing human α-synuclein. Our results highlight the effect of an archaeal molecule in relation to the gut-brain axis, which is critical for the diagnosis, prognosis, and treatment of PD.

As part of the revisions of our original manuscript, we performed 13C-labelling experiments with cultures of the microorganism most strongly correlated with 2-hydroxypyridine (2-HP), i.e. the archaeal species Methanobrevibacter smithii. Although unlabelled 2-HP was detected in the cultures, the measurements by gas chromatography-mass spectrometry (GC-MS) indicated that M. smithii was not the direct source of 2-HP as labelled 2-HP was not measured. Further experiments involving a labelled solvent (deuterated pyridine) and faecal samples from our original study alongside the use of additional analytical platforms and measurements on human blood plasma and mouse brain tissues demonstrate that 2-HP is an artefact of the measurements by GC-MS. It is produced in a sample-specific manner during the derivatisation process for GC-MS by a so far unknown chemical reaction. Our correlative links between archaea (M. smithii) and 2-HP remain but, based on these most recent results, cannot be directly mechanistically linked. Apart from this central limitation of our original study, we have so far not uncovered any reasons which would draw into question the validity of our in vitro and in vivo results linking 2-HP to the observed molecular, behavioural and pathological hallmarks of Parkinson’s disease.