The authors from the United States conducted a targeted metabolomic analysis of plasma and cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) and healthy controls. The results demonstrated a presence of potentially neurotoxic group of metabolites (indoxyl sulphate, p-cresol sulphate, and N-phenylacetylglutamine). The researchers suggested that these three metabolites are microbially derived, and represent possible candidates for ‘toxic’ gut–brain communication in patients with multiple sclerosis.
Recent studies have supported the concept of microbial dysbiosis in the pathogenesis of several neurological disorders. There are differences in the composition of the gut microbiota in patients with MS compared to healthy controls. It has been proposed that certain metabolites or bacterial toxins produced in the gut may potentially enter the bloodstream and subsequently be transferred to the CSF, where they may exert a toxic effect and influence the progression of disease.
The authors of this study speculated that an imbalance between bacteria with beneficial effects and bacteria with detrimental effects, as detected in patients with MS, may lead to the depletion of neuroprotective metabolites and the potential accumulation of neurotoxic compounds. Therefore, they conducted an in-depth analysis of the metabolites obtained in CSF samples from patients with relapsing remitting multiple sclerosis (RRMS) before and after treatment with dimethyl fumarate (DMF). DMF has various immunomodulatory, epigenetic and gut microbiome-related effects. The same research group previously demonstrated that DMF has a significant impact on the composition of the gut microbiota.
About the study
The study included two cohorts. The first cohort included 16 patients with RRMS, 17 patients with secondary progressive multiple sclerosis (SPMS), and 20 healthy controls. The second cohort included patients who received a treatment: 11 additional patients with RRMS who were treated with DMF and 8 patients with RRMS who were treated with ocrelizumab. The study included patients with diagnosis of multiple sclerosis according to McDonald criteria. Exclusion criteria were current smoking status and treatment with steroids within 30 days from enrollment.
Participants underwent lumbar puncture and venipuncture to obtain CSF and plasma, respectively, at baseline and six months after treatment. The enzyme-linked immunosorbent assay was used to measure the CSF levels of neurofilament light chain (NFL), which is a biomarker for neurodegeneration. Regarding the second cohort, which consisted of patients treated with DMF or ocrelizumab, plasma samples were collected at baseline and after 6 months of treatment. These samples were analyzed the same way as the first group. The magnetic resonance imaging (MRI) scans were performed at baseline and during follow-up visits.
To assess neurotoxicity, cultured hippocampal and cortical neurons isolated from Sprague–Dawley rat embryos were exposed to CSF samples from MS patients before and after DMF treatment. A metabolomic analysis of plasma and CSF samples was done to identify metabolites whose abundance correlated with the neurotoxic potential of the CSF.
Results
The CSF samples obtained from patients with RRMS or SPMS had a significant impact and induced signs of axonal damage in cultured hippocampal and cortical rat neurons. Nonetheless, the CSF samples obtaimed from patients with RRMS who were treated with DMF exhibited a reduction in neurotoxicity, whereas CSF samples obtained from patients with SPMS did not show this effect. The CSF samples from healthy individuals did not show any effect on cultured rat neurons.
The authors then performed an in-depth analysis of the metabolites present in plasma and CSF samples from patients with RRMS. The investigation revealed a specific group of three metabolites from catabolism of two essential amino acids, tryptophan and phenylalanine. The ‘red module’ group included indoxyl sulphate (IS), an indole-derivative from the tryptophan metabolism, and two phenol-derivatives from the tyrosine and phenylalanine metabolism, p-cresol sulphate (pCS), and N-phenylacetylglutamine (PAG). The relative concentrations of these three metabolites were highly correlated between plasma and CSF, and their levels as a ‘group’ were higher in the CSF of RRMS patients than in controls.
Chronic exposure of cultured neurons to increasing concentrations of three “red module” metabolites produced a dose-dependent neurotoxic effect that directly resulted in axonal damage and neuronal dysfunction, and decreased spontaneous neuronal activity, such as average firing rate, number of spikes per second and number of network bursts. The independent electrophysiological testing of each metabolite belonging to the “red module” in cultured neurons showed significant and distinct effect for each individual metabolite. However, their overall impact on the mean firing rate and the number of spikes per second was synergistic.
Importantly, mitochondrial function did not show a difference in oxygen consumption rate or respiratory capacity, and oxidative stress did not differ between neurons that were treated with increasing concentrations of “red module” metabolites and neurons that were not treated. Researchers concluded that the neurotoxic effect observed in cultured neurons was independent of mitochondrial dysfunction and oxidative stress.
A specific group of “red module” metabolites was found to correlate with the MS treatment. The concentrations of pCS, IS and PAG were significantly decreased in both CSF and plasma from patients with RRMS who were treated with DMF for 6 months. The authors noted that they have previously demonstrated that DMF treatment depleted the gut flora. Also, the relative levels of NFL, which is a biomarker for neurodegeneration, were correlated with reduced levels of pCS, IS and PAG in samples from patients with RRMS treated with DMF. In contrast, the levels of the same metabolites remained unchanged in a cohort of age-related RRMS patients treated with anti-CD20 therapy. This treatment has disease-modifying properties that are independent of gut alterations.
Interestingly, the levels of ‘red module’ metabolites in the CSF samples from RRMS patients after 12 months of DMF treatment showed an inverse correlation with MRI metrics of cortical volume. There was no correlation with the volume of deep gray matter structure. The superficial brain layers are in the closer contact with CSF and are exposed to neurotoxic microbial metabolites, whereas the deeper structures of the gray matter are more protected from the influence of CSF composition.
Researchers noted that, under physiological conditions, the healthy gut-microbiota processes tryptophan, to generate serotonin or metabolites such as kynurenate (which can be further converted into nicotinamide). However, in pathological conditions, the levels of serotonin and kynurenic acid are often decreased, as tryptophan is converted into an excess of indole-derivatives, such as indole acetate and indoxyl sulphate. The authors believe that these results support a pathological ‘metabolic shunt’ from healthy to toxic catabolites in patients with RRMS because lower levels of kynurenic acid and higher levels of indoxyl sulphate were found in patients with RRMS compared to healthy controls.
Researchers suggested that phenol and indole derivatives, products of the catabolism of tryptophan and phenylalanine, two essential amino acids, are microbially derived metabolites. These metabolites, after being produced by microbial species in the gut, enter the bloodstream and reach the CNS via CSF. This way, they come into contact and interact with neurons in the superficial cortical layers. This mechanism identifies these “red module” metabolites in the CSF of RRMS patients as potential candidates for a ‘toxic’ gut–brain communication in patients with multiple sclerosis. Future studies are needed to accurately decipher the selective contribution of each of these metabolites to CSF toxicity and to thoroughly examine their precise mechanism of action.
This article was published in Brain.
Journal Reference
Ntranos A et al. Bacterial neurotoxic metabolites in multiple sclerosis cerebrospinal fluid and plasma. Brain 2022: 145; 569–583. https://doi.org/10.1093/brain/awab320