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
© Stringer et al.; licensee BioMed Central Ltd. 2013
Received: 7 May 2013
Accepted: 28 June 2013
Published: 2 August 2013
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.
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.
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.
KeywordsCannabinoid receptor 1 Epigenetics Gene regulation microRNA Mouse Neurodevelopment Prenatal alcohol exposure
Fetal alcohol spectrum disorders (FASD) describe the continuum of phenotypic effects that may result from prenatal alcohol exposure (PAE). PAE is the most common cause of preventable neurodevelopmental disorders in North America[1, 2] and is associated with attention deficit, impaired learning and memory, and hyperactivity, as well as an increased risk for anxiety and mood disorders. 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. However, it has been suggested that the effects of PAE may involve long-term changes in gene expression that may be maintained through alcohol-induced epigenetic changes. In particular, we have previously reported that the expression of microRNAs (miRNAs) may be globally altered in the adult mouse brain following PAE, 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 different genes. One such gene may be cannabinoid receptor 1 (Cnr1).
We have previously shown that early neonatal ethanol exposure in mice results in reduced Cnr1 gene expression in the adult brain. Cnr1 acts within the endocannabinoid (eCB) system, involved in modulating neurophysiological processes controlling mood, memory, pain sensation, and appetite. Cnr1 is also thought to be involved in the neuropharmacological effects of alcohol through inhibition of glutaminergic and GABAergic interneurons. Variations in this gene or alterations in its expression are also associated with mood disorders, particularly fear and anxiety phenotypes.
Here, we use a C57BL/6J mouse model of binge-like exposure during the period of synaptogenesis to assess a potential relationship between Cnr1 and its putative regulatory miRNA, miR-26b. We evaluated the inverse expression patterns of these two transcripts, hypothesizing that the up-regulation of the miRNA following 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 neuronal 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 altering 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 observed 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 for methods). miRNA expression array data (GEO # GSE34413) was also generated from the same sample (see for methods). Analysis 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 predicted regulator of Cnr1 expression.
miR-26b is encoded from an intron of small C-terminal domain phosphatase. Interestingly, it is involved in neuronal differentiation as its transcription results in a negative feedback loop that is absent in neural stem cells. miR-26b has also been shown to regulate the expression of brain-derived neurotrophic factor (BDNF), a gene strongly implicated in neurodevelopment and related disorders (i.e., schizophrenia), including the effects of PAE.
This altered expression of miR-26b may have the ability to affect downstream gene expression by binding to the mRNA transcripts of its target genes. We have demonstrated 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 degradation of the target transcript. The up-regulation of miR-26b correlates with the reduced Cnr1 transcript observed in the adult brain of mice neurodevelopmentally exposed to alcohol. 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 phenotypes seen in FASD. The eCB system, for instance, plays a strong role in anxiety-related behaviour, which has been shown to increase in adult mice following PAE. Previous studies evaluating Cnr1 knockout mice have demonstrated increased anxiety-like phenotypes. This suggests that the observed reduction in Cnr1 expression demonstrated here may contribute to our observation of anxiety-like behaviour following PAE.
Ultimately, these findings provide a mechanism by which the long-term change in Cnr1 expression is maintained following PAE. They also suggest that the alteration of neurodevelopmentally-important miRNAs can influence the long-term function of biological pathways that influence cognition and behaviour. Epigenetic regulators of gene expression may then be affected by PAE, subsequently exerting pleiotropic effects on numerous gene targets that then contribute to the long-term and variable neurobehavioural effects associated with FASD.
Cannabinoid receptor 1
Fetal alcohol spectrum disorders
Prenatal alcohol exposure.
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.
- Chudley AE, Conry J, Cook JL, Loock C, Rosales T, LeBlanc N: Public Health Agency of Canada's National Advisory Committee on Fetal Alcohol Spectrum Disorder. Fetal alcohol spectrum disorder: Canadian guidelines for diagnosis. CMAJ. 2005, 172 (5 Suppl): S1-S21.PubMed CentralView ArticlePubMedGoogle Scholar
- May PA, Gossage JP, Kalberg WO, Robinson LK, Buckley D, Manning M, Hoyme HE: Prevalence and epidemiologic characteristics of FASD from various research methods with an emphasis on recent in-school studies. Dev Disabil Res Rev. 2009, 15 (3): 176-192. 10.1002/ddrr.68.View ArticlePubMedGoogle Scholar
- Bhatara V, Loudenberg R, Ellis R: Association of attention deficit hyperactivity disorder and gestational alcohol exposure: an exploratory study. J Atten Disord. 2006, 9 (3): 515-522. 10.1177/1087054705283880.View ArticlePubMedGoogle Scholar
- Hellemans KG, Verma P, Yoon E, Yu W, Weinberg J: Prenatal alcohol exposure increases vulnerability to stress and anxiety-like disorders in adulthood. Ann N Y Acad Sci. 2008, 1144: 154-175. 10.1196/annals.1418.016.View ArticlePubMedGoogle Scholar
- Kleiber ML, Mantha K, Stringer RL, Singh SM: Neurodevelopmental alcohol exposure elicits long-term changes to gene expression that alter distinct molecular pathways dependent on timing of exposure. J Neurodev Disord. 2013, 5 (1): 6-10.1186/1866-1955-5-6.PubMed CentralView ArticlePubMedGoogle Scholar
- Laufer BI, Mantha K, Kleiber ML, Diehl EJ, Addison SM, Singh SM: Long lasting alterations to DNA methylation and ncRNAs may underlie the effects of fetal alcohol exposure in mice. Dis Model Mech. 2013, 6 (4): 977-992. 10.1242/dmm.010975.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang LL, Zhang Z, Li Q, Yang R, Pei X, Xu Y, Wang J, Zhou SF, Li Y: Ethanol exposure induces differential microRNA and target gene expression and teratogenic effects which can be suppressed by folic acid supplementation. Hum Reprod. 2009, 24 (3): 562-579.View ArticlePubMedGoogle Scholar
- Soares AR, Pereira PM, Ferreira V, Reverendo M, Simoes J, Bezerra AR, Moura GR, Santos MA: Ethanol exposure induces upregulation of specific microRNAs in zebrafish embryos. Toxicol Sci. 2012, 127 (1): 18-28. 10.1093/toxsci/kfs068.View ArticlePubMedGoogle Scholar
- Mukherji S, Ebert MS, Zheng GX, Tsang JS, Sharp PA, van Oudenaarden A: MicroRNAs can generate thresholds in target gene expression. Nat Genet. 2011, 43 (9): 854-859. 10.1038/ng.905.PubMed CentralView ArticlePubMedGoogle Scholar
- Pertwee RG: The pharmacology of cannabinoid receptors and their ligands: an overview. Int J Obes (Lond). 2006, 30 (Suppl 1): S13-S18.View ArticleGoogle Scholar
- Adermark L, Jonsson S, Ericson M, Soderpalm B: Intermittent ethanol consumption depresses endocannabinoid-signaling in the dorsolateral striatum of rat. Neuropharmacology. 2011, 61 (7): 1160-1165. 10.1016/j.neuropharm.2011.01.014.View ArticlePubMedGoogle Scholar
- Elphick MR, Egertova M: The neurobiology and evolution of cannabinoid signalling. Philos Trans R Soc Lond B Biol Sci. 2001, 356 (1407): 381-408. 10.1098/rstb.2000.0787.PubMed CentralView ArticlePubMedGoogle Scholar
- Dubreucq S, Kambire S, Conforzi M, Metna-Laurent M, Cannich A, Soria-Gomez E, Richard E, Marsicano G, Chaouloff F: Cannabinoid type 1 receptors located on single-minded 1-expressing neurons control emotional behaviors. Neuroscience. 2012, 204: 230-244.View ArticlePubMedGoogle Scholar
- Ikonomidou C, Bittigau P, Ishimaru MJ, Wozniak DF, Koch C, Genz K, Price MT, Stefovska V, Horster F, Tenkova T, Dikranian K, Olney JW: Ethanol-induced apoptotic neurodegeneration and fetal alcohol syndrome. Science. 2000, 287 (5455): 1056-1060. 10.1126/science.287.5455.1056.View ArticlePubMedGoogle Scholar
- Wozniak DF, Hartman RE, Boyle MP, Vogt SK, Brooks AR, Tenkova T, Young C, Olney JW, Muglia LJ: Apoptotic neurodegeneration induced by ethanol in neonatal mice is associated with profound learning/memory deficits in juveniles followed by progressive functional recovery in adults. Neurobiol Dis. 2004, 17 (3): 403-414. 10.1016/j.nbd.2004.08.006.View ArticlePubMedGoogle Scholar
- Friedman RC, Farh KK, Burge CB, Bartel DP: Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 2009, 19 (1): 92-105.PubMed CentralView ArticlePubMedGoogle Scholar
- Sowa N, Horie T, Kuwabara Y, Baba O, Watanabe S, Nishi H, Kinoshita M, Takanabe-Mori R, Wada H, Shimatsu A, Hasegawa K, Kimura T, Ono K: MicroRNA 26b encoded by the intron of small CTD phosphatase (SCP) 1 has an antagonistic effect on its host gene. J Cell Biochem. 2012, 113 (11): 3455-3465. 10.1002/jcb.24222.View ArticlePubMedGoogle Scholar
- Dill H, Linder B, Fehr A, Fischer U: Intronic miR-26b controls neuronal differentiation by repressing its host transcript, ctdsp2. Genes Dev. 2012, 26 (1): 25-30. 10.1101/gad.177774.111.PubMed CentralView ArticlePubMedGoogle Scholar
- Caputo V, Sinibaldi L, Fiorentino A, Parisi C, Catalanotto C, Pasini A, Cogoni C, Pizzuti A: Brain derived neurotrophic factor (BDNF) expression is regulated by microRNAs miR-26a and miR-26b allele-specific binding. PLoS One. 2011, 6 (12): e28656-10.1371/journal.pone.0028656.PubMed CentralView ArticlePubMedGoogle Scholar
- Ashton CH, Moore PB: Endocannabinoid system dysfunction in mood and related disorders. Acta Psychiatr Scand. 2011, 124 (4): 250-261. 10.1111/j.1600-0447.2011.01687.x.View ArticlePubMedGoogle Scholar
- Mantha K, Kleiber M, Singh S: Neurodevelopmental timing of ethanol exposure may contribute to observed heterogeneity of behavioral deficits in a mouse model of fetal alcohol spectrum disorder (FASD). J Behav Brain Sci. 2013, 3: 85-99. 10.4236/jbbs.2013.31009.View ArticleGoogle Scholar
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