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Cited by 294Gut microbiota and healthAutism Spectrum Disorder ResearchTryptophan and brain disorders

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Enteric Bacterial Metabolites Propionic and Butyric Acid Modulate Gene Expression, Including CREB-Dependent Catecholaminergic Neurotransmission, in PC12 Cells - Possible Relevance to Autism Spectrum Disorders

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Nankova, Bistra B., Agarwal, Raj, MacFabe, Derrick F., La Gamma, Edmund F.·2014·PLoS ONE
DOI 10.1371/journal.pone.0103740PMC PMC4149359
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Abstract

Alterations in gut microbiome composition have an emerging role in health and disease including brain function and behavior. Short chain fatty acids (SCFA) like propionic (PPA), and butyric acid (BA), which are present in diet and are fermentation products of many gastrointestinal bacteria, are showing increasing importance in host health, but also may be environmental contributors in neurodevelopmental disorders including autism spectrum disorders (ASD). Further to this we have shown SCFA administration to rodents over a variety of routes (intracerebroventricular, subcutaneous, intraperitoneal) or developmental time periods can elicit behavioral, electrophysiological, neuropathological and biochemical effects consistent with findings in ASD patients. SCFA are capable of altering host gene expression, partly due to their histone deacetylase inhibitor activity. We have previously shown BA can regulate tyrosine hydroxylase (TH) mRNA levels in a PC12 cell model. Since monoamine concentration is known to be elevated in the brain and blood of ASD patients and in many ASD animal models, we hypothesized that SCFA may directly influence brain monoaminergic pathways. When PC12 cells were transiently transfected with plasmids having a luciferase reporter gene under the control of the TH promoter, PPA was found to induce reporter gene activity over a wide concentration range. CREB transcription factor(s) was necessary for the transcriptional activation of TH gene by PPA. At lower concentrations PPA also caused accumulation of TH mRNA and protein, indicative of increased cell capacity to produce catecholamines. PPA and BA induced broad alterations in gene expression including neurotransmitter systems, neuronal cell adhesion molecules, inflammation, oxidative stress, lipid metabolism and mitochondrial function, all of which have been implicated in ASD. In conclusion, our data are consistent with a molecular mechanism through which gut related environmental signals such as increased levels of SCFA's can epigenetically modulate cell function further supporting their role as environmental contributors to ASD.

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Figure 1 PPA activates the transcription of TH gene in CREB-dependant manner. Plasmid constructs with wild type (WT) rat TH promoter (−773/+27 bp) driving the expression of luciferase reporter gene or having a G-to A transition mutation in the CRE promoter element (mCRE) were electroporated in PC12 cells pre-treated for one day with increasing concentrations of PPA (0.1 to 10 mM, Fig. 1A)). The same doses of PPA were given immediately after electroporation for additional 24 hours, when changes in reporter gene activity were determined. In the second set of experiments (Figure 1B), PC12 cells were co-transfected with combination of wt TH promoter construct and plasmids expressing wild type or dominant negative CREB mutants (binding-defective K-CREB or phosphorylation-defective 133CREB). To half of the cultures, 10 mM PPA was given 1 day before and immediately after electroporation for additional 24 h (black bars). Controls received vehicle 24 hrs before and immediately after electroporation (open bars). The values shown are means ± SEM from three experiments. The luciferase activities (RLU/s/µg protein) are expressed as fold stimulation relative to control cells co-transfected with the same plasmids but not treated with PPA. *P<0.05 (PPA vs. vehicle treated; **P<0.002 (wt vs. mutant constructs).
Figure 1 PPA activates the transcription of TH gene in CREB-dependant manner. Plasmid constructs with wild type (WT) rat TH promoter (−773/+27 bp) driving the expression of luciferase reporter gene or having a G-to A transition mutation in the CRE promoter element (mCRE) were electroporated in PC12 cells pre-treated for one day with increasing concentrations of PPA (0.1 to 10 mM, Fig. 1A)). The same doses of PPA were given immediately after electroporation for additional 24 hours, when changes in reporter gene activity were determined. In the second set of experiments (Figure 1B), PC12 cells were co-transfected with combination of wt TH promoter construct and plasmids expressing wild type or dominant negative CREB mutants (binding-defective K-CREB or phosphorylation-defective 133CREB). To half of the cultures, 10 mM PPA was given 1 day before and immediately after electroporation for additional 24 h (black bars). Controls received vehicle 24 hrs before and immediately after electroporation (open bars). The values shown are means ± SEM from three experiments. The luciferase activities (RLU/s/µg protein) are expressed as fold stimulation relative to control cells co-transfected with the same plasmids but not treated with PPA. *P<0.05 (PPA vs. vehicle treated; **P<0.002 (wt vs. mutant constructs).CC BY · PubMed Central
Figure 2 PPA induces accumulation of TH mRNA and TH protein in vitro. PC12 cells were treated with SCFA: propionic acid (PPA, 1 mM), butyric acid (BA, 1 mM) or valproic acid (VPA, 0.1 mM). At indicated times (48 hrs) total RNA or total cell lysates (0, 24, 48 and 72 hrs) were prepared and subjected to Northern (A) or Western blot analyses (B, PPA and BA). The data on Fig. 2A are presented as mean ± SEM value relative to the mRNA levels in control, vehicle treated cells. 18S rRNA was used as a house keeping gene for normalization of data. *P<0.05 SCFA vs. vehicle treated. For Western blotting (2B) cells were lysed after indicated incubation times and equal amounts of proteins were subjected to analyses using TH-specific antibody as described in Experimental procedures. Membranes were re-probed with β-actin specific antibody as loading control. The autoradiographs were analyzed by densitometry. Values are presented as the ratio of TH to that of beta-actin as fold difference from control, vehicle- treated cells. N = 6–8 independent samples per group, and the experiment was repeated twice with similar results [63].
Figure 2 PPA induces accumulation of TH mRNA and TH protein in vitro. PC12 cells were treated with SCFA: propionic acid (PPA, 1 mM), butyric acid (BA, 1 mM) or valproic acid (VPA, 0.1 mM). At indicated times (48 hrs) total RNA or total cell lysates (0, 24, 48 and 72 hrs) were prepared and subjected to Northern (A) or Western blot analyses (B, PPA and BA). The data on Fig. 2A are presented as mean ± SEM value relative to the mRNA levels in control, vehicle treated cells. 18S rRNA was used as a house keeping gene for normalization of data. *P<0.05 SCFA vs. vehicle treated. For Western blotting (2B) cells were lysed after indicated incubation times and equal amounts of proteins were subjected to analyses using TH-specific antibody as described in Experimental procedures. Membranes were re-probed with β-actin specific antibody as loading control. The autoradiographs were analyzed by densitometry. Values are presented as the ratio of TH to that of beta-actin as fold difference from control, vehicle- treated cells. N = 6–8 independent samples per group, and the experiment was repeated twice with similar results [63].CC BY · PubMed Central
Figure 3 Differential gene expression profiles evoked by SCFA in PC12 cells. Total RNA was isolated from PC12 cells following 48 hrs exposures to BA (6 mM), PPA (10 mM) or vehicle and subjected to microarray analysis. Genes that changed two-fold or greater in SCFA-treated groups (*p<0.05) compared to the control (vehicle treated PC12 cells) group were imported into MetaCore GeneGo for comparison analyses. Differentially expressed unique (1599 for BA and 355 for PPA experimental group) and common for both groups genes (1010) were identified. Fisher's exact test p-value was <2.2e-16.
Figure 3 Differential gene expression profiles evoked by SCFA in PC12 cells. Total RNA was isolated from PC12 cells following 48 hrs exposures to BA (6 mM), PPA (10 mM) or vehicle and subjected to microarray analysis. Genes that changed two-fold or greater in SCFA-treated groups (*p<0.05) compared to the control (vehicle treated PC12 cells) group were imported into MetaCore GeneGo for comparison analyses. Differentially expressed unique (1599 for BA and 355 for PPA experimental group) and common for both groups genes (1010) were identified. Fisher's exact test p-value was <2.2e-16.CC BY · PubMed Central
Figure 4 Enrichment analysis of differentially expressed genes: distribution by gene ontology (GO) processes. Differentially expressed genes in BA and PPA groups (t-test compared to control group p<0.01) were subjected to enrichment analysis (which consists of matching gene IDs for the common, similar and unique sets of the uploaded files with gene IDs in functional ontologies in MetaCore). The figure illustrates the distribution by GO processes. The gene content is aligned between all uploaded files. The set of common gene IDs is marked as blue and white stripes. The unique genes for the files are marked as colored bars (BA - orange; PPA- blue). The sorting was done by common gene IDs; p-value was set for 0.05; both signals (induced and repressed) were included. The data shown are for sorting method “similarity by”. The degree of “relevance” to different categories for the uploaded datasets is defined by p values, so that the lower p-value gets higher priority. The top 10 processes are listed based on their −log (p-value).
Figure 4 Enrichment analysis of differentially expressed genes: distribution by gene ontology (GO) processes. Differentially expressed genes in BA and PPA groups (t-test compared to control group p<0.01) were subjected to enrichment analysis (which consists of matching gene IDs for the common, similar and unique sets of the uploaded files with gene IDs in functional ontologies in MetaCore). The figure illustrates the distribution by GO processes. The gene content is aligned between all uploaded files. The set of common gene IDs is marked as blue and white stripes. The unique genes for the files are marked as colored bars (BA - orange; PPA- blue). The sorting was done by common gene IDs; p-value was set for 0.05; both signals (induced and repressed) were included. The data shown are for sorting method “similarity by”. The degree of “relevance” to different categories for the uploaded datasets is defined by p values, so that the lower p-value gets higher priority. The top 10 processes are listed based on their −log (p-value).CC BY · PubMed Central

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Metadata

Authors
Nankova, B. B., Agarwal, R., MacFabe, D. F., & La Gamma, E. F.
Affiliations
  • New York Medical College (US)
  • Western University (CA)
Corresponding author
Nankova, Bistra B.
ORCID
Nankova, Bistra B.
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2014-08-29
Volume
9
Issue
8
Pages
e103740
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0103740
PMC
PMC4149359
OpenAlex
W2028901643
Cited by
294
References
136
Citations by year
2026: 24 · 2025: 26 · 2024: 30 · 2023: 29 · 2022: 43 · 2021: 34 · 2020: 37 · 2019: 19 · 2018: 21 · 2017: 8 · 2016: 14 · 2015: 8 · 2014: 1
FWCI
4.23
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Touro Scholar (Touro College) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Figshare (submittedVersion, cc-by)
Funders
Autism Research Institute
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
  • Life Sciences › Neuroscience › Cognitive Neuroscience › Autism Spectrum Disorder Research
  • Life Sciences › Neuroscience › Biological Psychiatry › Tryptophan and brain disorders
Keywords
propionic acid, butyrate, short-chain fatty acids, gut microbiota, autism spectrum disorder, catecholaminergic neurotransmission, tyrosine hydroxylase, CREB transcription factor, PC12 cells, histone deacetylase inhibition, epigenetic regulation, monoamines, neurodevelopmental disorders, gut-brain axis
MeSH terms
Animals, Child Development Disorders, Pervasive, Enterobacteriaceae, Gene Expression Regulation, Synaptic Transmission, Propionates, Tyrosine 3-Monooxygenase, Transcriptional Activation, PC12 Cells, Cyclic AMP Response Element-Binding Protein, Butyric Acid, Rats, Gene Regulatory Networks, Host-Pathogen Interactions

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