Final Report Summary - CELNIC (Role of gene silencing pathways in C. elegans nicotine dependence)
The aim of the CELNIC project was to use Caenorhabditis elegans (C. elegans) as a model to study nicotine dependence and the role of the microRNA (miRNA) machinery in modulating this behavior. C. elegans shows a variety of nicotine dependent behavioral responses such as acute response, adaptation, withdrawal and sensitization to addictive drugs. For instance, acute nicotine treatment stimulates locomotion and egg laying. Long-term nicotine treatment elicits tolerance in worms and these nicotine-adapted worms exhibit hyper-locomotor activity when placed in a nicotine-free environment, showing characteristics of a withdrawal response (Feng, Li et al. 2006).
The project was focused on the identification of genes and proteins that are differentially regulated upon nicotine treatment. Large-scale proteomic and genomic approaches, through microarray and mass spectrometry (MS) techniques, were adopted to identify the potential candidates. Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC), a MS-based quantitative proteomic technique, was used to identify proteins up- or downregulated after nicotine treatment. The results showed that 136 proteins were significantly regulated (alteast 2 fold) upon prolonged nicotine treatment, out of which 35 proteins were down-regulated and 101 proteins were up-regulated. The proteins identified are involved in various biological processes, such as growth, reproduction or stress responses. Interestingly, the identifications included proteins involved in small RNA interference machinery, such as MUT-7 (RNase D; involved in transposon silencing) and ERI-1 (RNase; negative regulator of RNA interference). Similarly, 373 genes were identified using microarray techniques, which were differentially regulated at transcriptional level by 1.5 fold. Those included 120 genes identified in a low-dose treatment group, and 352 genes identified in samples treated with higher doses of nicotine. There were 99 genes common between the two treatment groups (Fig. 1A, B). Comparison of the data from the MS and microarray experiments resulted in the identification of genetic factors differentially regulated both at the transcriptional and translational levels after prolonged nicotine treatment. In the future, the targets can be potential candidates to be tested for their involvement in the development of nicotine dependence in higher organisms.
The most important discovery of this project was the identification of the role of the microRNA machinery in regulating the function of acetylcholine receptors (AChRs) in the nicotine-dependent behaviors in worms. During the period of this fellowship, the applicant found that ALG-1, a key Argonaute in miRNA machinery, specifically modulates the nicotine dependent withdrawal response by regulating the expression of an acetylcholine receptor, ACR-19, through a miRNA (miR-238). This is the first time that the acr-19 gene was identified in worms as a modulator of nicotine dependent behaviors, specifically in the withdrawal response, resembling its mammalian counterpart (nACR subunit alpha-7), which is implicated in modulating a similar response (Besson, David et al. 2012). Furthermore, the nicotine mediated up-regulation of the acr-19 was suppressed in the acr-15 mutant background, indicating these two receptors were differentially regulated by nicotine. Our finding that mutants of the alg-1 gene but not of the rde-1 (an Argonaute related to the RNAi machinery) show defects in the nicotine withdrawal response suggests that modulation of the microRNA machinery plays an important role in the generation of nicotine dependent behaviors. This modulation has a very specific effect on the withdrawal response as alg-1 mutant worms exhibit a normal acute response towards nicotine. Additionally, chronic nicotine treatment also suppresses the mRNA levels of alg-1, indicating that nicotine globally regulates the expression of miRNAs to possibly control the expression of their target genes to attain a steady-state in worms. The results showed that this alg-1-mediated regulation of acr-19 occurs partially through a miRNA (miR-238), which has one binding site (best binding match) in acr-19 3’untranslated region (UTR), as mir-238 mutant worms behave similarly to the acr-19 mutant. This findings are further strengthened as transgenic acr-19 mutant worms carrying a acr-19 gene copy with a mutated miR-238 binding site in its 3’UTR can only partially rescue the nicotine withdrawal response of the mutants (Fig. 1C).
To summarize, during the course of the CELNIC project we provided evidence of the mechanisms by which the miRNA machinery regulates and is being regulated by AChRs at different levels, to generate nicotine dependent behaviors (Fig. 1D). These findings are especially relevant in understanding how the AChRs and microRNA machinery interacts to generate nicotine dependence behaviors. Additionally, it will be interesting to study in mouse models or human cell lines the role of the genes and proteins differentially regulated upon nicotine treatment, in order to find their role in the development of nicotine dependence.
References
Besson, M., V. David, et al. (2012). "Alpha7-nicotinic receptors modulate nicotine-
induced reinforcement and extracellular dopamine outflow in the mesolimbic system in mice." Psychopharmacology 220(1): 1-14.
Feng, Z., W. Li, et al. (2006). "A C. elegans model of nicotine-dependent behavior: regulation by TRP-family channels." Cell 127(3): 621-633.
The project was focused on the identification of genes and proteins that are differentially regulated upon nicotine treatment. Large-scale proteomic and genomic approaches, through microarray and mass spectrometry (MS) techniques, were adopted to identify the potential candidates. Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC), a MS-based quantitative proteomic technique, was used to identify proteins up- or downregulated after nicotine treatment. The results showed that 136 proteins were significantly regulated (alteast 2 fold) upon prolonged nicotine treatment, out of which 35 proteins were down-regulated and 101 proteins were up-regulated. The proteins identified are involved in various biological processes, such as growth, reproduction or stress responses. Interestingly, the identifications included proteins involved in small RNA interference machinery, such as MUT-7 (RNase D; involved in transposon silencing) and ERI-1 (RNase; negative regulator of RNA interference). Similarly, 373 genes were identified using microarray techniques, which were differentially regulated at transcriptional level by 1.5 fold. Those included 120 genes identified in a low-dose treatment group, and 352 genes identified in samples treated with higher doses of nicotine. There were 99 genes common between the two treatment groups (Fig. 1A, B). Comparison of the data from the MS and microarray experiments resulted in the identification of genetic factors differentially regulated both at the transcriptional and translational levels after prolonged nicotine treatment. In the future, the targets can be potential candidates to be tested for their involvement in the development of nicotine dependence in higher organisms.
The most important discovery of this project was the identification of the role of the microRNA machinery in regulating the function of acetylcholine receptors (AChRs) in the nicotine-dependent behaviors in worms. During the period of this fellowship, the applicant found that ALG-1, a key Argonaute in miRNA machinery, specifically modulates the nicotine dependent withdrawal response by regulating the expression of an acetylcholine receptor, ACR-19, through a miRNA (miR-238). This is the first time that the acr-19 gene was identified in worms as a modulator of nicotine dependent behaviors, specifically in the withdrawal response, resembling its mammalian counterpart (nACR subunit alpha-7), which is implicated in modulating a similar response (Besson, David et al. 2012). Furthermore, the nicotine mediated up-regulation of the acr-19 was suppressed in the acr-15 mutant background, indicating these two receptors were differentially regulated by nicotine. Our finding that mutants of the alg-1 gene but not of the rde-1 (an Argonaute related to the RNAi machinery) show defects in the nicotine withdrawal response suggests that modulation of the microRNA machinery plays an important role in the generation of nicotine dependent behaviors. This modulation has a very specific effect on the withdrawal response as alg-1 mutant worms exhibit a normal acute response towards nicotine. Additionally, chronic nicotine treatment also suppresses the mRNA levels of alg-1, indicating that nicotine globally regulates the expression of miRNAs to possibly control the expression of their target genes to attain a steady-state in worms. The results showed that this alg-1-mediated regulation of acr-19 occurs partially through a miRNA (miR-238), which has one binding site (best binding match) in acr-19 3’untranslated region (UTR), as mir-238 mutant worms behave similarly to the acr-19 mutant. This findings are further strengthened as transgenic acr-19 mutant worms carrying a acr-19 gene copy with a mutated miR-238 binding site in its 3’UTR can only partially rescue the nicotine withdrawal response of the mutants (Fig. 1C).
To summarize, during the course of the CELNIC project we provided evidence of the mechanisms by which the miRNA machinery regulates and is being regulated by AChRs at different levels, to generate nicotine dependent behaviors (Fig. 1D). These findings are especially relevant in understanding how the AChRs and microRNA machinery interacts to generate nicotine dependence behaviors. Additionally, it will be interesting to study in mouse models or human cell lines the role of the genes and proteins differentially regulated upon nicotine treatment, in order to find their role in the development of nicotine dependence.
References
Besson, M., V. David, et al. (2012). "Alpha7-nicotinic receptors modulate nicotine-
induced reinforcement and extracellular dopamine outflow in the mesolimbic system in mice." Psychopharmacology 220(1): 1-14.
Feng, Z., W. Li, et al. (2006). "A C. elegans model of nicotine-dependent behavior: regulation by TRP-family channels." Cell 127(3): 621-633.