The relationship between the gut microbiome, the kynurenine pathway and cognitive dysfunction in multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system, that causes an array of functional difficulties. Many people with MS experience some level of cognitive dysfunction, which is often attributed to focal and diffuse grey and white matter injury, cerebral atrophy, and/or disconnection of functional brain networks. Cognitive fatigue is also a common outcome of MS. A growing body of literature has implicated dysregulation of the kynurenine pathway (KP), the main catabolic pathway of tryptophan, in MS. The KP is dysregulated by chronic inflammation, which subsequently increases production of metabolites related to neuronal damage and death, at the expense of those that are neuroprotective and/or crucial for adequate cellular energy and repair. However, there is a paucity of research specifically examining the relationships between the KP and cognitive fatigue/functioning in MS, or how these may change over time. Known to also play a role in KP regulation are the trillions of microbes living in the human gut and their collective genome (the microbiome), as well as the important connection between the immune and nervous systems and the brain, known as the microbiota-gut-brain axis. Peculiarities have consistently been found in the gut microbiome of people with MS, potentially implicating the gut microbiome in the KP dysregulation and cognitive difficulties routinely observed in those with the disease. Associations between the gut, brain, KP, and cognition in MS are understudied. The therapeutic potential of altering the gut microbiome with supplements such as probiotics, to improve MS-related cognitive dysfunction via modulation of the gut microbiome and in-turn, the KP, is similarly underexplored. Based on the above, the aims of this thesis were two-fold. Firstly, to determine whether there exist relationships between cognitive dysfunction/cognitive changes over time and KP metabolites in people with MS. Secondly, to explore whether and to what extent these relationships could be altered through a probiotic intervention targeting the gut microbiome, as a known regulator of KP function. To address these aims, this thesis comprised a series of pilot studies and a review. The first study was a pilot quantitative study that specifically examined the relationships between KP metabolites and various domains of cognitive functioning in people with MS (n = 31). Whether these relationships compared to healthy control participants without MS (n = 26) was also explored. Participants completed self-report questionnaires assessing cognitive fatigue and cognitive difficulties, an objective neuropsychological test battery, and provided a blood sample for profiling of KP metabolites. This study revealed several relationships between bioenergetic and glutamatergic KP metabolites, cognitive fatigue and learning/memory performance in both groups, with several outcomes differing in the MS group compared to the control group. The results provided preliminary evidence of relationships between fatigue/cognition and KP metabolites. The second study employed a longitudinal design to further examine the relationships that might exist over time and to address the inherent limitations of the prior cross-sectional study. This study followed up 16 participants with MS from the first cross-sectional study, after an average of 4.7 years. Participants completed the same assessments and underwent the blood sample collection procedures as in the first study. The results indicated that relationships between KP metabolites and cognitive measures were different at baseline and follow-up, and baseline bioenergetic KP metabolites were found to be related to follow-up visuospatial learning and memory performance. This study lent support to the findings of the first study and provided preliminary evidence of longitudinal associations between fatigue, cognition and biological disease factors pertaining to the KP. Thirdly, in preparation for the final (intervention) study, a systematic review and meta-analysis of the literature examining the effects of prebiotic and probiotic supplementation on KP metabolites was conducted, to determine the current state of evidence in this regard. Across the prebiotic (n = 2) and probiotic (n = 12) interventions included, 11 reported a significant effect on ≥ 1 metabolite. Probiotics were found to affect kynurenine and the kynurenine:tryptophan ratio in meta-analysis. Evidence regarding prebiotics, however, was limited. Directions for future clinical trials, including several methodological considerations, were provided. Finally, a pilot, randomised, placebo-controlled clinical trial examining the efficacy of an 8-strain probiotic supplement to influence cognitive functioning and KP biomarkers in people with MS (n = 28; 14 probiotic and 14 placebo) was conducted. Participants completed the same assessments and biological procedures as in the first and second studies, both at baseline and again at the end of the 12- week supplement period. Participants also provided stool samples at both timepoints, for shotgun metagenomic sequencing. Compared to baseline, probiotic (but not placebo) supplementation was associated with compositional changes in the gut microbiome, but was not with changes in bioenergetic KP metabolites. There was tentative evidence that the probiotic-induced changes in the gut microbiome were associated with changes in cognitive outcomes. The results of this study also provided support and guidance for a larger trial, with regard to feasibility and sample size. This thesis has several strengths, including establishing relationships between the KP and cognitive fatigue/dysfunction in MS, both cross-sectionally and longitudinally, and providing a preliminary assessment of the therapeutic potential of probiotics to act on gut-brain pathways in MS. The main finding of this thesis is that microbiota-gut-brain biomarkers are associated with cognitive symptoms in MS, however, further research on their contribution to the development and progression of the disease, including cognitive symptoms, is needed. Similarly, although probiotics were found to have some capacity to alter microbiota-gut-brain pathways, additional investigation into the efficacy of probiotic therapy to directly impact cognitive and functional outcomes and to serve as an important adjunct therapy to other treatments typically given to people with MS is required.