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      Discovery and inhibition of an interspecies gut bacterial pathway for Levodopa metabolism

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          Abstract

          The human gut microbiota metabolizes the Parkinson’s disease medication Levodopa ( L-dopa), potentially reducing drug availability and causing side effects. However, the organisms, genes, and enzymes responsible for this activity in patients and their susceptibility to inhibition by host-targeted drugs are unknown. Here, we describe an interspecies pathway for gut bacterial L-dopa metabolism. Conversion of L-dopa to dopamine by a pyridoxal phosphate-dependent tyrosine decarboxylase from Enterococcus faecalis is followed by transformation of dopamine to m-tyramine by a molybdenum-dependent dehydroxylase from Eggerthella lenta. These enzymes predict drug metabolism in complex human gut microbiotas. Although a drug that targets host aromatic amino acid decarboxylase does not prevent gut microbial L-dopa decarboxylation, we identified a compound that inhibits this activity in Parkinson’s patient microbiotas and increases L-dopa bioavailability in mice.

          INTRODUCTION

          Parkinson’s disease is a debilitating neurological condition affecting more than 1% of the global population aged 60 and above. The primary medication used to treat Parkinson’s disease is levodopa ( L-dopa). To be effective, L-dopa must enter the brain and be converted to the neurotransmitter dopamine by the human enzyme aromatic amino acid decarboxylase (AADC). However, the gastro-intestinal tract is also a major site for L-dopa decarboxylation, and this metabolism is problematic because dopamine generated in the periphery cannot cross the blood-brain barrier and causes unwanted side effects. Thus, L-dopa is coadministered with drugs that block pe-ripheral metabolism, including the AADC in-hibitor carbidopa. Even with these drugs, up to 56% of L-dopa fails to reach the brain. Moreover, the efficacy and side effects of L-dopa treatment are extremely heterogeneous across Parkinson’s patients, and this variability cannot be completely explained by differences in host metabolism

          RATIONALE

          Previous studies in humans and animal models have demonstrated that the gut microbiota can metabolize L-dopa. The major proposed pathway involves an initial decarboxylation of L-dopa to dopamine, followed by conversion of dopamine to m-tyramine by means of a distinctly microbial dehydroxylation reaction. Although these metabolic activities were shown to occur in complex gut microbiota samples, the specific organisms, gene, and enzymes responsible were unknown. The effects of host-targeted inhibitors such as carbidopa on gut microbial L-dopa metabolism were also unclear. As a first step toward understanding the gut microbiota’s effect on Parkinson’s disease therapy, we sought to elucidate the molecular basis for gut microbial L-dopa and dopamine metabolism.

          RESULTS

          Hypothesizing that L-dopa decarboxylation would require a pyridoxal phosphate (PLP)–dependent enzyme, we searched gut bacterial genomes for candidates and identified a conserved tyrosine decarboxylase (TyrDC) in Enterococcus faecalis. Genetic and biochemical experiments revealed that TyrDC simultaneously decarboxylates both L-dopa and its preferred substrate, tyrosine. Next, we used enrichment culturing to isolate a dopamine dehydroxylating strain of Eggerthella lenta, a species previously implicated in drug metabolism. Transcriptomics linked this activity to a molybdenum cofactor–dependent dopamine dehydroxylase (Dadh) enzyme. Unexpectedly, the presence of this enzyme in gut bacterial genomes did not correlate with dopamine metabolism; instead, we identified a single-nucleotide polymorphism (SNP) in the dadh gene that predicts activity. The abundance of E. faecalis, tyrDC, and the individual SNPs of dadh correlated with L-dopa and dopamine metabolism in complex gut microbiotas from Parkinson’s patients, indicating that these organisms, genes, enzymes, and even nucleotides are relevant in this setting.

          We then tested whether the host-targeted AADC inhibitor carbidopa affected L-dopa decarboxylation by E. faecalis TyrDC. Carbidopa displayed greatly reduced potency toward bacteria and was completely ineffective in complex gut microbiotas from Parkinson’s patients, suggesting that this drug likely does not prevent microbial L-dopa metabolism in vivo. To identify a selective inhibitor of gut bacterial L-dopa decarboxylation, we leveraged our molecular understanding of gut microbial L-dopa metabolism. Given TyrDC’s preference for tyrosine, we examined tyrosine mimics and found that (S)-α-fluoromethyltyrosine (AFMT) prevented L-dopa decarboxylation by TyrDC and E. faecalis as well as complex gut microbiota samples from Parkinson’s patients. Coadministering AFMT with L-dopa and carbidopa to mice colonized with E. faecalis also increased the peak serum concentration of L-dopa. This observation is consistent with inhibition of gut microbial L-dopa metabolism in vivo.

          CONCLUSION

          We have characterized an interspecies pathway for gut bacterial L-dopa metabolism and demonstrated its relevance in human gut microbiotas. Variations in these microbial activities could possibly contribute to the heterogeneous responses to L-dopa observed among patients, including decreased efficacy and harmful side effects. Our findings will enable efforts to elucidate the gut microbiota’s contribution to treatment outcomes and highlight the promise of developing therapies that target both host and gut microbial drug metabolism.

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          Most cited references70

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          Gut Microbiota Regulate Motor Deficits and Neuroinflammation in a Model of Parkinson's Disease.

          The intestinal microbiota influence neurodevelopment, modulate behavior, and contribute to neurological disorders. However, a functional link between gut bacteria and neurodegenerative diseases remains unexplored. Synucleinopathies are characterized by aggregation of the protein α-synuclein (αSyn), often resulting in motor dysfunction as exemplified by Parkinson's disease (PD). Using mice that overexpress αSyn, we report herein that gut microbiota are required for motor deficits, microglia activation, and αSyn pathology. Antibiotic treatment ameliorates, while microbial re-colonization promotes, pathophysiology in adult animals, suggesting that postnatal signaling between the gut and the brain modulates disease. Indeed, oral administration of specific microbial metabolites to germ-free mice promotes neuroinflammation and motor symptoms. Remarkably, colonization of αSyn-overexpressing mice with microbiota from PD-affected patients enhances physical impairments compared to microbiota transplants from healthy human donors. These findings reveal that gut bacteria regulate movement disorders in mice and suggest that alterations in the human microbiome represent a risk factor for PD.
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            Gut microbiota are related to Parkinson's disease and clinical phenotype.

            In the course of Parkinson's disease (PD), the enteric nervous system (ENS) and parasympathetic nerves are amongst the structures earliest and most frequently affected by alpha-synuclein pathology. Accordingly, gastrointestinal dysfunction, in particular constipation, is an important non-motor symptom in PD and often precedes the onset of motor symptoms by years. Recent research has shown that intestinal microbiota interact with the autonomic and central nervous system via diverse pathways including the ENS and vagal nerve. The gut microbiome in PD has not been previously investigated. We compared the fecal microbiomes of 72 PD patients and 72 control subjects by pyrosequencing the V1-V3 regions of the bacterial 16S ribosomal RNA gene. Associations between clinical parameters and microbiota were analyzed using generalized linear models, taking into account potential confounders. On average, the abundance of Prevotellaceae in feces of PD patients was reduced by 77.6% as compared with controls. Relative abundance of Prevotellaceae of 6.5% or less had 86.1% sensitivity and 38.9% specificity for PD. A logistic regression classifier based on the abundance of four bacterial families and the severity of constipation identified PD patients with 66.7% sensitivity and 90.3% specificity. The relative abundance of Enterobacteriaceae was positively associated with the severity of postural instability and gait difficulty. These findings suggest that the intestinal microbiome is altered in PD and is related to motor phenotype. Further studies are warranted to elucidate the temporal and causal relationships between gut microbiota and PD and the suitability of the microbiome as a biomarker.
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              The Central Nervous System and the Gut Microbiome.

              Neurodevelopment is a complex process governed by both intrinsic and extrinsic signals. While historically studied by researching the brain, inputs from the periphery impact many neurological conditions. Indeed, emerging data suggest communication between the gut and the brain in anxiety, depression, cognition, and autism spectrum disorder (ASD). The development of a healthy, functional brain depends on key pre- and post-natal events that integrate environmental cues, such as molecular signals from the gut. These cues largely originate from the microbiome, the consortium of symbiotic bacteria that reside within all animals. Research over the past few years reveals that the gut microbiome plays a role in basic neurogenerative processes such as the formation of the blood-brain barrier, myelination, neurogenesis, and microglia maturation and also modulates many aspects of animal behavior. Herein, we discuss the biological intersection of neurodevelopment and the microbiome and explore the hypothesis that gut bacteria are integral contributors to development and function of the nervous system and to the balance between mental health and disease.
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                Author and article information

                Journal
                Science
                Science
                SCIENCE
                Science (New York, N.y.)
                American Association for the Advancement of Science
                0036-8075
                1095-9203
                14 June 2019
                2019
                : 364
                : 6445
                : eaau6323
                Affiliations
                [1 ]Department of Chemistry abmknd Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA
                [2 ]Department of Microbiology and Immunology, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA
                [3 ]Department of Chemistry, University of California, Irvine, 1102 Natural Sciences 2, Irvine, CA 92617, USA
                [4 ]Department of Molecular Biology and Biochemistry, University of California, Irvine, 1102 Natural Sciences 2, Irvine, CA 92617, USA
                [5 ]Chan Zuckerberg Biohub, San Francisco, CA 94158, USA
                Author notes
                Corresponding author. Email: balskus@ 123456chemistry.harvard.edu (E.P.B.); peter.turnbaugh@ 123456ucsf.edu (P.J.T.)
                Article
                SCIENCE-364-6445-aau6323
                10.1126/science.aau6323
                7745125
                31196984
                ed046bc7-3dc6-49a9-b2d4-218d14774c18
                © 2019 American Association for the Advancement of Science

                This is an open access article distributed under the terms of the Creative Commons Non-Commercial, No Derivatives (CC BY-NC-ND) license ( http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 29 June 2018
                : 18 April 2019
                : 02 May 2019
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