This web page was produced as an assignment for Genetics 564, an undergraduate course at UW-Madison .
Although impulsivity is a multifactorial trait with many variations as to how it is defined, it is generally referred to as as 'rapid, unplanned reactions towards internal and external stimuli [1]'. Rash impulsiveness is a symptom in a number of behavioral disorders including substance abuse, conditions like ADHD, to mood or brain disorders like mania and schizophrenia [2]. It can be debilitating due to the inability of patients to control their behavior appropriately, thus leading to altered quality of life.
It has been thought that misregulation of dopamine signalling can lead to impulsive behaviour. One component of dopaminergic pathways is the dopamine receptor: In humans, there are five known dopamine receptors; DRD2 being one of them. DRD2 is highly expressed in certain parts of the brain like the striatum, nucleus accumbens, and the hypothalamus [3]. It is involved in G-protein receptor signalling, is localised to the cell membrane of neural synapses, and is well-conserved across vertebrates.
There are several studies linking DRD2 to impulsivity-related habits: Some report polymorphisms associated with habits like drug abuse [4] and smoking [5]; others report low DRD2 levels in obese subjects [6]. Behavioral tests also been performed on mice and rhesus monkeys: Those studies report that DRD2 knockout and knockdown animals have a higher rate of substance self-administration compared to wild-type animals [7].
DRD2 mRNA is can also be alternatively spliced to give rise to different DRD2 isoforms. Although there have not been studies specifically addressing how regulation of these isoforms may lead to impulsive behavior, it is possible that they may play a role since the long isoform (D2L)--which is the predominantly expressed isoform--acts at postsynaptic sites, while the short isoform (D2S) is truncated in its amino acid sequence and acts at presynaptic sites. Both isoforms have been reported to have different functions [3]. In addition, a third isoform (D2(Longer)) has been described in patients who died with psychosis [8].
It should be noted that there are also several studies countering claims of associations between DRD2 and impulsive behavior. One explanation as to why conflicting studies on associations between impulsivity and DRD2 alleles exist are the complex regulatory mechanisms of DRD2 and the protein and gene level. I therefore hypothesize that regulation of the expression of DRD2 isoforms and polymorphisms is critical for impulse control and responsiveness to antipsychotic drug treatment.
It has been thought that misregulation of dopamine signalling can lead to impulsive behaviour. One component of dopaminergic pathways is the dopamine receptor: In humans, there are five known dopamine receptors; DRD2 being one of them. DRD2 is highly expressed in certain parts of the brain like the striatum, nucleus accumbens, and the hypothalamus [3]. It is involved in G-protein receptor signalling, is localised to the cell membrane of neural synapses, and is well-conserved across vertebrates.
There are several studies linking DRD2 to impulsivity-related habits: Some report polymorphisms associated with habits like drug abuse [4] and smoking [5]; others report low DRD2 levels in obese subjects [6]. Behavioral tests also been performed on mice and rhesus monkeys: Those studies report that DRD2 knockout and knockdown animals have a higher rate of substance self-administration compared to wild-type animals [7].
DRD2 mRNA is can also be alternatively spliced to give rise to different DRD2 isoforms. Although there have not been studies specifically addressing how regulation of these isoforms may lead to impulsive behavior, it is possible that they may play a role since the long isoform (D2L)--which is the predominantly expressed isoform--acts at postsynaptic sites, while the short isoform (D2S) is truncated in its amino acid sequence and acts at presynaptic sites. Both isoforms have been reported to have different functions [3]. In addition, a third isoform (D2(Longer)) has been described in patients who died with psychosis [8].
It should be noted that there are also several studies countering claims of associations between DRD2 and impulsive behavior. One explanation as to why conflicting studies on associations between impulsivity and DRD2 alleles exist are the complex regulatory mechanisms of DRD2 and the protein and gene level. I therefore hypothesize that regulation of the expression of DRD2 isoforms and polymorphisms is critical for impulse control and responsiveness to antipsychotic drug treatment.
Aim 1: To study the prevalence of the third DRD2 variant.
To do this, mRNA samples from post-mortem brain tissue of individuals would be obtained and placed into three groups: One control group, one group of subjects with history of substance abuse, and one group of subjects with psychiatric illnesses. The tissue would be from key areas of the brain in which DRD2 is known to be expressed. Then, RNA-Seq will be performed to quantify the abundance of the third DRD2 mRNA variant. The graph shows an speculated example of what results may look like in one part of the brain--the striatum, in this case (Fig. 1). |
Figure 1. Example of what results for mRNA expression may look like in tissue obtained from brain striata.
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Based on studies in animals and humans reporting low D2L receptor levels in subjects with impulsive habits, I would expect D2L expression to be lower in substance abusers and probably even more so in patients of psychiatric illnesses. Due to misregulation of alternative splicing, it is also possible that D2(Longer) may be more highly expressed in people exhibiting certain degrees of impulsive behavior.
Figure 2. A protein interaction network for D2L as visualized by STRING. The proteins are all involved in signal transduction.
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Specific Aim 2: To identify protein interactions with different DRD2 isoforms.
Different DRD2 protein isoforms, especially D2S and D2(Longer) wold first be isolated from the brain striata of post-mortem human brain tissue samples. This would be done by tandem affinity purification and co-immunoprecipitation (Ideally, the use of one method would confirm the results of the other, thus enabling higher accuracy). Tandem mass spectrometry can then be used to identify proteins interacting with the DRD2 proteins. The proteins identified can be compared to STRING to find out what proteins interact or do not interact with the different isoforms (STRING presents information about D2L protein interactions but not for the other DRD2 isoforms). Due to its truncated sequence, I hypothesize that fewer proteins will interact with D2S compared to D2L. But for both D2S and D2(longer), there may possibly be interacting proteins different than those listed on STRING. |
Specific Aim 3: To study the relationship between gene polymorphisms and DRD2 protein affinity to specific compounds.
First, genotyping would be performed on individuals with different DRD2 gene polymorphisms with sequencing. Based on the sequences obtained from the individuals, human cell lines expressing the different polymorphisms can then be made by editing their genomes with CRISPR-Cas9. Next, the DRD2 proteins expressed by the cell lines can be isolated and their their affinity for compounds known to interact with DRD2 can be tested. The compounds chosen would be based on databases like PubChem or antipsychotic drugs known to block DRD2 like haloperidol, clozapine and remoxipride. Based on screens, there are about 12000 compounds listed on PubChem that actively interact with DRD2. Testing the affinity of such a large number of compounds would therefore be a large project to undertake. |
Figure 3. Examples of compounds known to interact with DRD2. a) Clozapine; b) Remoxiprode; c) Haloperidol
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Future Directions
Although DRD2 is only a small part of the larger dopaminergic network, studying it will give insight to impulse-related behavior. The long-term goal would be to use all of the different findings and try developing therapies to manage impulse control dysregulation that is characteristic of psychiatric disorders.
Some future research that could be carried out include elucidating the function and cellular localization of D2(Longer). One problem with studying this longer isoform is that it is not very highly expressed [8]; therefore it may be difficult to obtain samples. But with the development of more sophisticated techniques for editing gene expression, it may be possible to create cell lines or model organisms expressing higher levels of specific protein isoforms. Also, it would probably be useful to conduct more association studies in order to better characterize the link between DRD2 and impulsive behavior.
Although DRD2 is only a small part of the larger dopaminergic network, studying it will give insight to impulse-related behavior. The long-term goal would be to use all of the different findings and try developing therapies to manage impulse control dysregulation that is characteristic of psychiatric disorders.
Some future research that could be carried out include elucidating the function and cellular localization of D2(Longer). One problem with studying this longer isoform is that it is not very highly expressed [8]; therefore it may be difficult to obtain samples. But with the development of more sophisticated techniques for editing gene expression, it may be possible to create cell lines or model organisms expressing higher levels of specific protein isoforms. Also, it would probably be useful to conduct more association studies in order to better characterize the link between DRD2 and impulsive behavior.
References
(1) Moeller FG, Barratt ES, Dougherty DM, Schmitz JM, Swann AC. Psychiatric aspects of impulsivity. Am J Psychiatry. 2001;158:1783-1793
(2) Dalley, J. W., & Roiser, J. P. (2012). Dopamine, serotonin and impulsivity. Neuroscience, 215, 42-58
(3) Beaulieu, J. M., & Gainetdinov, R. R. (2011). The physiology, signaling, and pharmacology of dopamine receptors. Pharmacological reviews, 63(1), 182-217.
(4) Najafabadi, M. S., et al. (2005). Association between the DRD2 A1 allele and opium addiction in the Iranian population. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics, 134(1), 39-41
(5) Comings, D. E., et al. (1996). The dopamine D2 receptor (DRD2) gene: a genetic risk factor in smoking. Pharmacogenetics and Genomics, 6(1), 73-79.
(6) Volkow, N. D., et al. (2008). Low dopamine striatal D2 receptors are associated with prefrontal metabolism in obese subjects: possible contributing factors.
(7) Dalley, J. W., et al. (2007). Nucleus accumbens D2/3 receptors predict trait impulsivity and cocaine reinforcement. science, 315(5816), 1267-1270
(8) Seeman, P., Nam, D., Ulpian, C., Liu, I. S., & Tallerico, T. (2000). New dopamine receptor, D2 Longer, with unique TG splice site, in human brain. Molecular brain research, 76(1), 132-141
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(1) Moeller FG, Barratt ES, Dougherty DM, Schmitz JM, Swann AC. Psychiatric aspects of impulsivity. Am J Psychiatry. 2001;158:1783-1793
(2) Dalley, J. W., & Roiser, J. P. (2012). Dopamine, serotonin and impulsivity. Neuroscience, 215, 42-58
(3) Beaulieu, J. M., & Gainetdinov, R. R. (2011). The physiology, signaling, and pharmacology of dopamine receptors. Pharmacological reviews, 63(1), 182-217.
(4) Najafabadi, M. S., et al. (2005). Association between the DRD2 A1 allele and opium addiction in the Iranian population. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics, 134(1), 39-41
(5) Comings, D. E., et al. (1996). The dopamine D2 receptor (DRD2) gene: a genetic risk factor in smoking. Pharmacogenetics and Genomics, 6(1), 73-79.
(6) Volkow, N. D., et al. (2008). Low dopamine striatal D2 receptors are associated with prefrontal metabolism in obese subjects: possible contributing factors.
(7) Dalley, J. W., et al. (2007). Nucleus accumbens D2/3 receptors predict trait impulsivity and cocaine reinforcement. science, 315(5816), 1267-1270
(8) Seeman, P., Nam, D., Ulpian, C., Liu, I. S., & Tallerico, T. (2000). New dopamine receptor, D2 Longer, with unique TG splice site, in human brain. Molecular brain research, 76(1), 132-141
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