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A fluorescence-coupled assay for gamma aminobutyric acid (GABA) reveals metabolic stress-induced modulation of GABA content in neuroendocrine cancer.

ABSTRACT: Pathways involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA) have been implicated in the pathogenesis of high grade neuroendocrine (NE) neoplasms as well as neoplasms from a non-NE lineage. Using The Cancer Genome Atlas, overexpression of the GABA synthetic enzyme, glutamate decarboxylase 1 (GAD1), was found to be associated with decreased disease free-survival in prostate adenocarcinoma and decreased overall survival in clear cell renal cell carcinomas. Furthermore, GAD1 was found to be expressed in castrate-resistant prostate cancer cell lines, but not androgen-responsive cell lines. Using a novel fluorescence-coupled enzymatic microplate assay for GABA mediated through reduction of resazurin in a prostate neuroendocrine carcinoma (PNEC) cell line, acid microenvironment-induced stress increased GABA levels while alkaline microenvironment-induced stress decreased GABA through modulation of GAD1 and glutamine synthetase (GLUL) activities. Moreover, glutamine but not glucose deprivation decreased GABA through modulation of GLUL. Consistent with evidence in prokaryotic and eukaryotic organisms that GABA synthesis mediated through GAD1 may play a crucial role in surviving stress, GABA may be an important mediator of stress survival in neoplasms. These findings identify GABA synthesis and metabolism as a potentially important pathway for regulating cancer cell stress response as well as a potential target for therapeutic strategies.

SUBMITTER: Ippolito JE

PROVIDER: S-EPMC3923810 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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A fluorescence-coupled assay for gamma aminobutyric acid (GABA) reveals metabolic stress-induced modulation of GABA content in neuroendocrine cancer.

Ippolito Joseph E JE Piwnica-Worms David D

PloS one 20140213 2

Pathways involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA) have been implicated in the pathogenesis of high grade neuroendocrine (NE) neoplasms as well as neoplasms from a non-NE lineage. Using The Cancer Genome Atlas, overexpression of the GABA synthetic enzyme, glutamate decarboxylase 1 (GAD1), was found to be associated with decreased disease free-survival in prostate adenocarcinoma and decreased overall survival in clear cell renal cell carcinomas. Furthermore, ...[more]

PMID: 24551133

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Project description:Background: Of the many neurotransmitters in humans, gamma-aminobutyric acid (GABA) shows potential for improving several mental health indications such as stress and anxiety. The microbiota-gut-brain axis is an important pathway for GABAergic effects, as microbially-secreted GABA within the gut can affect host mental functionhealth outcomes. Understanding the molecular characteristics of GABA production by microbes within the gut can offer insight to novel therapies for mental health. Results: Three strains of Levilactobacillus brevis with syntenous glutamate decarboxylase (GAD) operons were evaluated for overall growth, glutamate utilization, and GABA production in typical synthetic growth media supplemented with monosodium glutamate (MSG). Levilactobacillus brevis Lbr-6108 (Lbr-6108) and Levilactobacillus brevis Lbr-35 (Lbr-35) had similar growth profiles but differed significantly in GABA secretion and acid resistance. Lbr-6108 produced GABA early, within the growth phase, and produced significantly more GABA than Lbr-35 and the type strain Levilactobacillus brevis ATCC 14689 after the stationary phase. The global gene expression during GABA production was determined by RNA sequencing at several timepoints. The GAD operon, responsible for GABA production and secretion, activated in Lbr-6108 after only six hours of fermentation and continued throughout the stationary phase. Furthermore, Lbr-6108 activated many different acid resistance mechanisms concurrently, which contribute to acid tolerance and energy production. In contrast, Lbr-35, which has a genetically similar GAD operon, including two copies of the GAD gene, showed no upregulation of the GAD operon, even when cultured with MSG. Conclusions: This study is the first to evaluate whole transcriptome changes in L. brevis during GABA production over multiple timepoints. The concurrent expression of multiple acid-resistance mechanisms reveals niche-specific metabolic functionality between common human commensals and highlights the complex regulation of GABA metabolism in this important microbial species. Furthermore, the increased and rapid GABA production of Lbr-6108 highlights the strain’s potential as a therapeutic and the overall value of screening microbes for effector molecule output.

Project description:BackgroundOf the many neurotransmitters in humans, gamma-aminobutyric acid (GABA) shows potential for improving several mental health indications such as stress and anxiety. The microbiota-gut-brain axis is an important pathway for GABAergic effects, as microbially-secreted GABA within the gut can affect host mental health outcomes. Understanding the molecular characteristics of GABA production by microbes within the gut can offer insight to novel therapies for mental health.ResultsThree strains of Levilactobacillus brevis with syntenous glutamate decarboxylase (GAD) operons were evaluated for overall growth, glutamate utilization, and GABA production in typical synthetic growth media supplemented with monosodium glutamate (MSG). Levilactobacillus brevis Lbr-6108™ (Lbr-6108), formerly known as L. brevis DPC 6108, and Levilactobacillus brevis Lbr-35 ™ (Lbr-35) had similar growth profiles but differed significantly in GABA secretion and acid resistance. Lbr-6108 produced GABA early within the growth phase and produced significantly more GABA than Lbr-35 and the type strain Levilactobacillus brevis ATCC 14869 after the stationary phase. The global gene expression during GABA production at several timepoints was determined by RNA sequencing. The GAD operon, responsible for GABA production and secretion, activated in Lbr-6108 after only 6 h of fermentation and continued throughout the stationary phase. Furthermore, Lbr-6108 activated many different acid resistance mechanisms concurrently, which contribute to acid tolerance and energy production. In contrast, Lbr-35, which has a genetically similar GAD operon, including two copies of the GAD gene, showed no upregulation of the GAD operon, even when cultured with MSG.ConclusionsThis study is the first to evaluate whole transcriptome changes in Levilactobacillus brevis during GABA production in different growth phases. The concurrent expression of multiple acid-resistance mechanisms reveals niche-specific metabolic functionality between common human commensals and highlights the complex regulation of GABA metabolism in this important microbial species. Furthermore, the increased and rapid GABA production of Lbr-6108 highlights the strain's potential as a therapeutic and the overall value of screening microbes for effector molecule output.

Dataset Information

A fluorescence-coupled assay for gamma aminobutyric acid (GABA) reveals metabolic stress-induced modulation of GABA content in neuroendocrine cancer.

Publications

A fluorescence-coupled assay for gamma aminobutyric acid (GABA) reveals metabolic stress-induced modulation of GABA content in neuroendocrine cancer.

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