BACKGROUND: Prenatal alcohol exposure is known to result in fetal alcohol spectrum disorders, a continuum of physiological, behavioural, and cognitive phenotypes that include increased risk for anxiety and learning-associated disorders. Prenatal alcohol exposure results in life-long disorders that may manifest in part through the induction of long-term gene expression changes, potentially maintained through epigenetic mechanisms. FINDINGS: Here we report a decrease in the expression of Canabinoid receptor 1 (Cnr1) and an increase in the expression of the regulatory microRNA miR-26b in the brains of adult mice exposed to ethanol during neurodevelopment. Furthermore, we show that miR-26b has significant complementarity to the 3’-UTR of the Cnr1 transcript, giving it the potential to bind and reduce the level of Cnr1 expression. CONCLUSIONS: These findings elucidate a mechanism through which some genes show long-term altered expression following prenatal alcohol exposure, leading to persistent alterations to cognitive function and behavioural phenotypes observed in fetal alcohol spectrum disorders

Benjamin I Laufer

Morgan L Kleiber

Randa L Stringer

Shiva M Singh

Clinical Epigenetics

PubMed

Abstract
          
            Background
            Prenatal alcohol exposure is known to result in fetal alcohol spectrum disorders, a continuum of physiological, behavioural, and cognitive phenotypes that include increased risk for anxiety and learning-associated disorders. Prenatal alcohol exposure results in life-long disorders that may manifest in part through the induction of long-term gene expression changes, potentially maintained through epigenetic mechanisms.
          
          
            Findings
            Here we report a decrease in the expression of Canabinoid receptor 1 (Cnr1) and an increase in the expression of the regulatory microRNA miR-26b in the brains of adult mice exposed to ethanol during neurodevelopment. Furthermore, we show that miR-26b has significant complementarity to the 3’-UTR of the Cnr1 transcript, giving it the potential to bind and reduce the level of Cnr1 expression.
          
          
            Conclusions
            These findings elucidate a mechanism through which some genes show long-term altered expression following prenatal alcohol exposure, leading to persistent alterations to cognitive function and behavioural phenotypes observed in fetal alcohol spectrum disorders.
          </jats:sec

Crossref

English

Reduced expression of brain cannabinoid receptor 1 (Cnr1) is coupled with an increased complementary micro-RNA (miR-26b) in a mouse model of fetal alcohol spectrum disorders

Springer - Publisher Connector

Stringer et al. Clinical Epigenetics 2013, 5:14
http://www.clinicalepigeneticsjournal.com/content/5/1/14SHORT REPORT Open AccessReduced expression of brain cannabinoid receptor
1 (Cnr1) is coupled with an increased
complementary micro-RNA (miR-26b) in a mouse
model of fetal alcohol spectrum disorders
Randa L Stringer, Benjamin I Laufer, Morgan L Kleiber and Shiva M Singh*Abstract
Background: Prenatal alcohol exposure is known to result in fetal alcohol spectrum disorders, a continuum of
physiological, behavioural, and cognitive phenotypes that include increased risk for anxiety and learning-associated
disorders. Prenatal alcohol exposure results in life-long disorders that may manifest in part through the induction of
long-term gene expression changes, potentially maintained through epigenetic mechanisms.
Findings: Here we report a decrease in the expression of Canabinoid receptor 1 (Cnr1) and an increase in the
expression of the regulatory microRNA miR-26b in the brains of adult mice exposed to ethanol during
neurodevelopment. Furthermore, we show that miR-26b has significant complementarity to the 3’-UTR of the Cnr1
transcript, giving it the potential to bind and reduce the level of Cnr1 expression.
Conclusions: These findings elucidate a mechanism through which some genes show long-term altered
expression following prenatal alcohol exposure, leading to persistent alterations to cognitive function and
behavioural phenotypes observed in fetal alcohol spectrum disorders.
Keywords: Cannabinoid receptor 1, Epigenetics, Gene regulation, microRNA, Mouse, Neurodevelopment,
Prenatal alcohol exposureFindings
Fetal alcohol spectrum disorders (FASD) describe the
continuum of phenotypic effects that may result from
prenatal alcohol exposure (PAE). PAE is the most com-
mon cause of preventable neurodevelopmental disorders
in North America [1,2] and is associated with attention
deficit, impaired learning and memory, and hyperactivity
[3], as well as an increased risk for anxiety and mood
disorders [4]. These cognitive and behavioural changes
persist throughout the life of an individual following
PAE, though the mechanisms involved in maintaining
these life-long changes are not well understood. How-
ever, it has been suggested that the effects of PAE may
involve long-term changes in gene expression [5] that
may be maintained through alcohol-induced epigenetic* Correspondence: ssingh@uwo.ca
Department of Biology, Molecular Genetics Unit, Western University, London,
ON N6A 5B7, Canada
© 2013 Stringer et al.; licensee BioMed Centra
Commons Attribution License (http://creativec
reproduction in any medium, provided the orchanges. In particular, we have previously reported that
the expression of microRNAs (miRNAs) may be globally
altered in the adult mouse brain following PAE [6],
which supports recent data by other groups [7,8]. More
specifically, these changes in miRNA expression may
subsequently alter the expression of target genes, with
one miRNA having the potential to regulate many diffe-
rent genes [9]. One such gene may be cannabinoid re-
ceptor 1 (Cnr1).
We have previously shown that early neonatal ethanol
exposure in mice results in reduced Cnr1 gene ex-
pression in the adult brain [5]. Cnr1 acts within the
endocannabinoid (eCB) system, involved in modulating
neurophysiological processes controlling mood, memory,
pain sensation, and appetite [10]. Cnr1 is also thought
to be involved in the neuropharmacological effects of
alcohol [11] through inhibition of glutaminergic and
GABAergic interneurons [12]. Variations in this gene orl Ltd. This is an Open Access article distributed under the terms of the Creative
ommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
iginal work is properly cited.
Stringer et al. Clinical Epigenetics 2013, 5:14 Page 2 of 4
http://www.clinicalepigeneticsjournal.com/content/5/1/14alterations in its expression are also associated with
mood disorders, particularly fear and anxiety pheno-
types [13].
Here, we use a C57BL/6J mouse model of binge-like
exposure during the period of synaptogenesis [5] to
assess a potential relationship between Cnr1 and its
putative regulatory miRNA, miR-26b. We evaluated the
inverse expression patterns of these two transcripts, hy-
pothesizing that the up-regulation of the miRNA follo-
wing PAE may in part be responsible for the observed
reduction in transcript of a target gene in the adult
brain. In these experiments, mice were exposed to two
acute doses of alcohol (5 g/kg) at neurodevelopmental
times representing the human third trimester equivalent.
This method has been previously reported and induces a
peak blood alcohol level of over 0.3 g/dL for 4 to 5 hours
following injection, and is sufficient to induce neur-
onal apoptosis and result in FASD-related behaviour
[5,14,15]. Our results suggest that ethanol exposure during
neurodevelopment may exert its long-term effects by al-
tering the expression of regulatory miRNAs, which may
then reduce the expression of a number of target genes
that may contribute to the spectrum of phenotypes ob-
served in FASD.
Gene expression data previously was generated through
microarray analysis (GEO # GSE34539) of RNA isolated
from whole brain tissue of 60-day-old male mice exposed
to binge-like levels of alcohol during the third trimester
equivalent on postnatal days 4 and 7 (see [5] for methods).
miRNA expression array data (GEO # GSE34413) was also
generated from the same sample (see [6] for methods).Figure 1 Analysis of Gene and miRNA expression via qPCR. (A) Chang
samples normalized to control. This figure was reproduced with permission
and alcohol-treated whole brain samples normalized to control. Data are foAnalysis of these data show a reduction of Cnr1 (fold
change = −1.33, P = 6.07 x 10-5) in ethanol-treated brains
as compared to the saline controls. Also, the miRNA miR-
26b increased in ethanol-treated mice (fold change =
1.284, P = 0.0364) compared to controls.
The potential interaction of the genes and miRNAs
identified as differentially expressed by the array studies
were analysed using Ingenuity’s® Micro-RNA Target Filter.
This analysis identified miR-26b as a high-confidence pre-
dicted regulator of Cnr1 expression.
The reduction of Cnr1 transcript was confirmed by
real time RT-PCR [5], showing a 1.14-fold decrease in
expression in ethanol-treated male brains as compared
to matched controls (P = 0.004; Figure 1A). Further, we
demonstrated a significant increase in the level of miR-
26b miRNA in ethanol-treated samples (fold change =
3.71, P = 0.012) compared to matched controls (see
[6] for methods) (Figure 1B). This inverse relationship
within the same sample set suggests that the two ob-
servations may be biologically related. This potential
interaction was further analysed using the TargetScan®
Human 6.2 predictor for miRNA targets [16], which
shows that the seed region of miR-26b possesses com-
plementarity to the 3’-UTR of the Cnr1 transcript and
has a significant potential to bind this region (Figure 2).
The probability of conserved targeting (PCT) analyses
the preferential conservation of binding sites [16]. It
has the advantage of identifying targeting interactions
that are not only more likely to be effective but also
those that are more likely to be consequential for the
animal, given the evolutionary conservation. The analysise in Cnr1 mRNA levels in male control and alcohol-treated whole brain
from the authors [5]. (B) Change in miR-26b levels in male control
ld change ± SEM. Control n = 5, alcohol n = 5. *P <0.01, **P <0.05.
Figure 2 TargetScan® analysis of miR-26b-Cnr1 binding sites provides a model for Cnr1 and miR-26b interaction. The seed region of
miR-26b has the potential to bind to the 3’-UTR of the Cnr1 transcript.
Stringer et al. Clinical Epigenetics 2013, 5:14 Page 3 of 4
http://www.clinicalepigeneticsjournal.com/content/5/1/14calculated a PCT score of 0.84, which indicates a significant
degree of confidence in the predicted interaction. Next,
we evaluated expression of Cnr1 and miR-26b to confirm
their relative expression levels.
miR-26b is encoded from an intron of small C-
terminal domain phosphatase [17]. Interestingly, it is
involved in neuronal differentiation as its transcrip-
tion results in a negative feedback loop that is absent
in neural stem cells [18]. miR-26b has also been shown to
regulate the expression of brain-derived neurotrophic fac-
tor (BDNF), a gene strongly implicated in neurodeve-
lopment and related disorders (i.e., schizophrenia) [19],
including the effects of PAE [5].
This altered expression of miR-26b may have the abil-
ity to affect downstream gene expression by binding to
the mRNA transcripts of its target genes. We have de-
monstrated that miR-26b shows complementarity to a
region of the 3’-UTR of the Cnr1 transcript (Figure 2),
which gives it the potential to regulate the expression of
Cnr1. This regulation by miRNAs generally occurs
through blocking of translation and/or promoting deg-
radation of the target transcript [9]. The up-regulation
of miR-26b correlates with the reduced Cnr1 transcript
observed in the adult brain of mice neurodevelop-
mentally exposed to alcohol. [7] Our results suggest
that this regulatory mechanism also occurs in vivo,
and that the stable alteration of miRNA as a result
of neurodevelopmental teratogenesis may affect long-
term gene expression of its target transcript(s) long
after exposure.
It is possible that relationships such as these may have
the ability to influence the aberrant behavioural pheno-
types seen in FASD. The eCB system, for instance, plays
a strong role in anxiety-related behaviour [20], whichhas been shown to increase in adult mice following PAE
[21]. Previous studies evaluating Cnr1 knockout mice
have demonstrated increased anxiety-like phenotypes
[13]. This suggests that the observed reduction in Cnr1
expression demonstrated here may contribute to our ob-
servation of anxiety-like behaviour following PAE.
Ultimately, these findings provide a mechanism by
which the long-term change in Cnr1 expression is main-
tained following PAE. They also suggest that the alter-
ation of neurodevelopmentally-important miRNAs can
influence the long-term function of biological pathways
that influence cognition and behaviour. Epigenetic re-
gulators of gene expression may then be affected by
PAE, subsequently exerting pleiotropic effects on nu-
merous gene targets that then contribute to the long-
term and variable neurobehavioural effects associated
with FASD.
Abbreviations
Cnr1: Cannabinoid receptor 1; eCB: Endocannabinoid; FASD: Fetal alcohol
spectrum disorders; miRNA: microRNA; PAE: Prenatal alcohol exposure.
Competing interests
The authors declare no competing financial interests.
Authors’ contributions
This project was developed by RLS, BIL, MLK, and SMS. MLK raised the mice,
performed the experimental interventions, and extracted the RNA. RLS
performed the qPCR reactions. BIL and RLS performed the bioinformatic
analysis. RLS, BIL, MLK, and SMS wrote the manuscript. All authors read and
approved the final manuscript.
Acknowledgements
The authors would like to thank David Carter from the London Regional
Genomics Centre (LRGC) for hybridizing the expression array and aiding in
the analysis. The authors would also like to thank Eric Diehl for providing a
proofreading of the manuscript.
Stringer et al. Clinical Epigenetics 2013, 5:14 Page 4 of 4
http://www.clinicalepigeneticsjournal.com/content/5/1/14Received: 7 May 2013 Accepted: 28 June 2013
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doi:10.1186/1868-7083-5-14
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Reduced expression of brain cannabinoid receptor 1 (Cnr1) is coupled with an increased complementary micro-RNA (miR-26b) in a mouse model of fetal alcohol spectrum disorders

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