Multiple sclerosis (MS) is one of the most important neurological diseases, characterized by multiple areas of inflammation and demyelination in the white matter of the central nervous system (CNS). Demyelinating lesions of the CNS, MS plaques, are characterized by infiltration of mononuclear cells, the proliferation of macrophages, and loss of oligodendrocytes, myelin-producing cells. There are currently four recognized courses of MS: clinically isolated syndrome (CIS), relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and secondary progressive MS (SPMS). The most common MS phenotype is RRMS, which affects approximately 85% of patients. Previous data support the hypothesis that certain metabolites or bacterial toxins produced in the gut may enter the bloodstream, be transferred to the cerebrospinal fluid (CSF), exert a toxic effect on CNS, and influence disease progression. Therefore, this metabolomic analysis of plasma and CSF samples collected from patients diagnosed with RRMS before and after dimethyl fumarate (DMF) treatment, investigated “toxic” gut-brain communication in MS patients. The authors also assessed the neurotoxicity of CSF samples collected from patients with RRMS or SPMS before and after DMF treatment in cultured hippocampal and cortical rat neurons.
The same research group from the United States demonstrated before that DMF treatment significantly affects gut microbiota composition in MS patients.
About the study
The authors analyzed the metabolites in the CSF samples from patients diagnosed with RRMS before and after treatment with DMF. MS diagnosis was established according to McDonald’s criteria. The exclusion criteria were current smoking status and treatment with steroids within 30 days from enrollment. Participants underwent lumbar puncture and venipuncture at baseline and six months after the treatment. The magnetic resonance imaging (MRI) scans were performed at baseline and during follow-up visits. The enzyme-linked immunosorbent assay was used to assess concentrations of neurofilament light chain (NfL), a neurodegeneration biomarker.
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.
Results
Metabolomic analysis of plasma and CSF samples of RRMS patients before and after DMF treatment
The analysis of the metabolites in plasma and CSF samples of patients diagnosed with RRMS demonstrated a specific group of three metabolites called the ‘red module’ group which included indoxyl sulfate (IS), an indole-derivative from the tryptophan metabolism, and two phenol-derivatives from the tyrosine and phenylalanine metabolism, p-cresol sulfate (pCS), and N-phenylacetylglutamine (PAG). The concentrations of three metabolites as a ‘group’ were higher in the CSF of RRMS patients than in healthy controls.
After 6 months of DMF treatment, patients diagnosed with RRMS had decreased concentrations of pCS, IS, and PAG in the CSF and plasma. In contrast, the levels of the same metabolites remained unchanged in age-related RRMS patients treated with anti-CD20 therapy. Additionally, concentrations of NfL, a neurodegeneration biomarker, correlated with reduced levels of pCS, IS, and PAG in CSF and plasma of RRMS patients treated with DMF.
After 12 months of DMF treatment, the concentrations of ‘red module’ metabolite in the CSF samples of RRMS patients showed an inverse correlation with MRI metrics of cortical volume. There was no correlation with the volume of deep gray matter. The authors emphasized that phenol and indole derivatives after being produced by microbial species in the gut, enter the bloodstream and reach the CNS via CSF. They come into contact and interact with neurons in the superficial cortical layers which are in closer contact with CSF and are exposed to neurotoxic microbial metabolites. Consequently, the deeper structures of the gray matter are more protected from the CSF and neurotoxic microbial metabolites.
A culture of hippocampal and cortical rat neurons exposed to CSF from MS patients
CSF samples from healthy individuals did not affect cultured hippocampal and cortical rat neurons. In contrast, cultured hippocampal and cortical rat neurons exposed to CSF samples from patients with RRMS or SPMS showed signs of axonal damage. After DMF treatment, CSF samples of patients with RRMS exhibited reduced neurotoxicity, but this effect was not observed with CSF samples of patients with SPMS.
Chronic exposure of cultured hippocampal and cortical rat neurons to increasing concentrations of three “red module” metabolites resulted in a dose-dependent neurotoxic effect, axonal damage, neuronal dysfunction, and reduction of spontaneous neuronal activity (average firing rate, number of spikes per second, and number of network bursts) compared to neurons that were not treated. There was no difference in mitochondrial function, oxygen consumption rate, or respiratory capacity. The authors concluded that the neurotoxic effect observed in cultured neurons treated with increasing concentrations of “red module” metabolites was independent of mitochondrial dysfunction and oxidative stress.
The electrophysiological testing of cultured neurons demonstrated a significant and distinct effect of each metabolite from the “red module”, but also their synergistic effect on the mean firing rate and the number of spikes per second.
Conclusion
This study demonstrated the presence of a potentially neurotoxic group of metabolites (indoxyl sulfate, p-cresol sulfate, and N-phenylacetylglutamine). According to the authors, these three metabolites are microbially derived and represent candidates for “toxic” gut-brain communication in MS patients. These results support a pathological ‘metabolic shunt’ from healthy to toxic catabolites in patients diagnosed with RRMS.
The authors noted that the healthy gut microbiota under physiological conditions processes tryptophan, to generate serotonin or metabolites such as kynurenate (which can be further converted into nicotinamide). In pathological conditions, serotonin and kynurenic acid levels decrease, as tryptophan is converted into an excess of indole-derivatives, such as indole acetate and indoxyl sulphate. An imbalance between bacteria with beneficial effects and bacteria with detrimental effects, as detected in patients with MS, may lead to the potential accumulation of neurotoxic compounds.
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
