Camron D. Bryant - US grants

DESCRIPTION (provided by applicant): Abuse of amphetamines is a major public health problem that warrants the identification of genetic risk factors which could lead to addiction prevention and novel treatments. Because amphetamines are also used clinically for such conditions as Attention Deficit Hyperactive Disorder, Parkinson's disease, and narcolepsy, studying the genetic basis of the biological actions of these drugs could also lead to improved pharmaceutical treatments. This application requests support for training in mouse and human genetics to test the hypothesis that polymorphisms in the candidate gene casein kinase 1-epsilon (Csnk1-e) contribute to sensitivity to the behavioral and subjective effects of amphetamines in mice and humans. In Aim 1, mice will be bred with polymorphic Csnk1-e alleles on two separate isogenic background strains with the hypothesis that Csnk1-e polymorphisms will modulate the locomotor stimulant response to methamphetamine (MA). In Aim 2, polymorphism-induced changes in Csnkle gene expression will be examined along with changes in MA-induced activation of the Darpp-32 dopamine signaling pathway and MA-induced locomotor activity in mice. The hypothesis is that an increase in Csnkle expression will result in an increase in MA-induced Darpp-32 phosphorylation and an increase in the locomotor stimulant response to MA. Furthermore, we expect these changes in Csnkle expression to result in differential sensitivity to the biochemical and behavioral effects of specific pharmacological inhibition of Csnkle. In Aim 3, parallel to the mouse studies, a human translational study will be conducted whereby association of CSNK1E polymorphisms with sensitivity to the physiological, behavioral, and subjective responses to d- amphetamine (AMPH) will be examined, including AMPH euphoria. Public Health Relevance: New insight into the genetic basis for sensitivity to amphetamines may have important implications for addiction prevention as well as the development of novel treatments for clinical conditions related to dysregulation of the dopamine system.

7. Project Summary/Abstract The nonmedical abuse of prescription opioids is a major socioeconomic problem in the United States. Drug abuse is known to have a genetic component and epidemiological studies indicate that individuals reporting a pleasurable experience are most likely to transition to drug abuse. Accordingly, this application seeks to identify the genetic basis of opioid reward and aversion. We will use a genetically informative panel of mouse strains and a behavioral model- the place conditioning assay- to screen for differences in the rewarding response to the commonly abused prescription opioid agonist oxycodone (OXY) and the aversive response to the opioid antagonist naloxone (NAL). In Aim 1, we will identify genomic regions that are responsible for differences in OXY reward and NAL aversion. In Aim 2, I will receive training in mRNA sequencing (RNA-seq) to identify differentially expressed genes and splice variants in these genomic regions as potentially causal for variation in OXY reward and NAL aversion. In Aim 3, we will generate congenic mice carrying small portions of these genomic regions to narrow the list of potential genes responsible. Within these regions, we will choose candidate genes that are differentially expressed for further analysis via molecular genetics techniques. The outcome of this proposal will aid in identifying genes contributing to opioid reward and aversion which will have implications for understanding and treating opioid abuse. My mentor, Dr. Abraham Palmer, has several years of experience with regard to all aspects of this project, including QTL mapping, transcriptome analysis, and large-scale mouse phenotyping studies. Dr. Palmer's history of expertise for transcriptome analysis has involved microarrays; thus, I have recruited Dr. Jonathan Pritchard (Co-Mentor) and Dr. Yoav Gilad (advisory committee member) to provide mentorship and training in the analytical and technical aspects of RNA-seq. Additionally, I have formed an advisory committee consisting of Dr. Palmer, Dr. Pritchard, Dr. Gilad, Dr. Harriet de Wit, and Dr. James Zacny. Dr. de Wit has extensive background and expertise in both rodent and human studies of drug abuse and is particularly interested in the translational aspect of the proposed research. Dr. de Wit conducts genetic association studies involving sensitivity to drugs of abuse, notably with subjective phenotypes such as drug reward. Thus, Dr. de Wit is well-positioned to provide expert advice on the behavioral, genetic, and pharmacological aspects of this proposal and can offer a translational context from which to apply the results. Dr. Zacny is an expert in opioid pharmacology and human phenotyping of opioid traits and has published multiple papers regarding variability in the physiological and subjective response to opioids- in particular OXY. Dr. Zacny is interested in applying the results of this proposal to translational studies regarding the genetic basis of opioid sensitivity in humans. The institutional environment for supporting and complementing this proposal is excellent. On the genetic and genomic end of the project, there are several excellent faculty in the Department of Human Genetics with whom I interact, including members of my committee. We have several seminar series affiliated with the department that I will participate in, including Work in Progress, Monthly Seminar Series, Journal Club, and Genetics of Model Organisms. There will be at least six Illumina GA2 machines in place for my RNA-seq samples to be run, three of which are located on the U of C campus. On the neuroscience end, Dr. Paul Vezina heads a NIDA-funded T32 training program, Neuropsychopharmacology Training for Drug abuse Research and seminar series that meets twice per month. Dr. de Wit and Dr. Palmer are both participating P.I.'s in this program, along with several other neuroscience faculty members who are all interested in addiction biology. My long-term objective is to establish my own academic research program at a major research university aimed at identifying genes involved with phenotypes associated with drug abuse, starting with the place conditioning assay as outlined in this proposal. In the current era of QTL mapping, transcriptome analysis is now a mainstay complementary tool for finding genes contributing to complex phenotypes such as drug sensitivity. Thus, the training I receive in RNA-seq analysis will help me to realize this career goal. There is a huge gap in the field of addiction biology regarding the genetic determinants of place preference. Because this assay is so widely used in addiction biology, this area of research will undoubtedly contribute to our understanding of the multitude of existing studies and provide novel insight into the mechanisms of drug reward and abuse. Future proposals from my laboratory will be aimed at testing genes identified in this study for pleiotropic effects on other behaviors associated with drug abuse, including withdrawal, self-administration, extinction, reconsolidation, and reinstatement. I am also interested in the intersection between reward/aversion and pain pathways and would like to further explore this area of research as a future proposal.

DESCRIPTION (provided by applicant): The addictions are highly heritable neuropsychiatric diseases; however, the responsible genetic factors remain largely unknown. Both an individual's genotype and the particular environment can powerfully influence the subjective and physiological response to drugs of abuse. Accordingly, gene x environment (G x E) interactions are hypothesized to contribute substantially to the heritability of the addictions. Mice serve as a important model organism for understanding the genetic basis of G x E interactions, permitting experimental control over genetic and environmental factors. The primary objective of this proposal is to identify the genetic basis of G x E interactions in behavioral traits that accompany the progressive stages of addiction. We observed a potent G x E interaction in the rewarding effect of the widely abused opioid oxycodone (OXY) in C57BL/6J (B6J) and C57BL/6NJ (B6NJ) substrains in the conditioned place preference (CPP) test. B6NJ showed up to a five-fold increase in OXY-CPP relative to B6J, but only when two out of four cage mates received OXY during training. In contrast, when all four mice received OXY, the effect of genotype was eliminated. Thus, the post-training social drug environment of the home cage can exert divergent effects on drug seeking behavior, depending on the genotype of the mice. We developed a multi-stage addiction assessment protocol (MSAAP) to measure a panel of addiction traits for G x E interactions in B6 substrains, including reward, analgesic tolerance, and the emotional-affective component of opioid withdrawal. In addition to substrain differences in opioid reward, preliminary studies indicate a three-fold increase in the emotional-affective component of OXY withdrawal in B6NJ versus B6J in the elevated plus maze. Because B6 substrains are nearly genetically identical, mapping the genetic basis of G x E interactions for MSAAP traits is expected to yield quantitative trait loci (QTLs) that possess very few predicted functional variants. In support, preliminary studies demonstrate that a B6J x B6NJ- F2 Reduced Complexity Cross (RCC) can be used to identify QTLs for diverse complex traits that contain fewer than 10 predicted functional variants per locus. In Aim 1, the RCC will be used to map the genetic basis of G x E interactions in MSAAP traits and select the most likely candidate genes based on the limited number of predicted functional variants that are expected to underlie the QTLs. In Aim 2, candidate genes will be validated using transcription activator-like endonucleases (TALENs) to introduce targeted null mutations and assess their role in G x E interactions in addiction traits. The identification of novel genetic factors mediating G x E interactions in multiple facets of addiction will enhance our understanding of the dynamic neurobiological mechanisms that support its progression. The results of these studies support the long-term goal of developing novel preventative and treatment strategies for addiction. An attractive notion is that clinicians will one day determine the most appropriate treatment based on both the patient's genotype and historical environment of drug abuse.

? DESCRIPTION (provided by applicant): Methamphetamine (MA) addiction is a pervasive public health concern in the U.S. and is associated with violent crime, mental health decline, and a high rate of relapse in recovering patients. Despite decades of research, there are still no FDA-approved treatments for MA addiction. Both genetic and environmental factors contribute to the etiology of MA addiction; however, genome-wide association studies in humans are limited in their power to identify common variants associated with this devastating disease. Mouse genetics offers a powerful, complementary tool for accelerating novel gene discovery and biological mechanisms. We recently identified a 210 kb region on mouse chromosome 11 containing two protein coding genes (Hnrnph1 and Rufy1) that contributes to the locomotor stimulant response to MA. The primary objective of this proposal is to identify the novel quantitative trait gene (QTG) and the neurobiological mechanism by which this QTG regulates the stimulant and addictive properties of MA. In Aim 1, we will identify the QTG(s) that regulates MA sensitivity. We generated a 112 kb congenic mouse containing only Hnrnph1 and Rufy1, providing the unique opportunity to determine whether this region is both necessary and sufficient for MA sensitivity. Furthermore, we recently used transcriptional activator-like effecto nucleases (TALENs) to introduce null mutations in Hnrnph1 and Rufy1. Preliminary studies provide strong, replicable evidence that Hnrnph1 is a QTG for reduced MA sensitivity. To fully tackle all of the polymorphisms within the 210 kb region that could potentially contribute to behavior, we will also delete a 150 kb intergenic region using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to introduce double stranded breaks that flank the intergenic region. Finally, we will use CRISPR/Cas9 to generate knockin mice carrying SNPs from the candidate QTG. In Aim 2, we will use transcriptome analysis of the striatum and ventral midbrain via mRNA sequencing (RNA- seq) in mice carrying the congenic allele, the knockout allele of the QTG, or both alleles relative to wild-type mice to hone the molecular pathways and neurobiological mechanisms that link genetic variation with behavior. Preliminary analysis of a congenic line capturing the QTL for reduced MA sensitivity suggests perturbations in midbrain dopaminergic neuron development, glutamate signaling, and adrenergic signaling as potentially responsible for a reduction in MA-induced behavior. In Aim 3, we will use in vivo microdialysis, immunoblotting, immunohistochemistry, and operant oral MA self-administration in congenics and knockouts to functionally assess the neurochemical mechanism by which the QTG influences the stimulant and addictive properties of MA. The proposed studies employ a unique combination of genetics, genomics, and functional analyses to uncover the neurobiological mechanism by which a novel genetic factor contributes to addiction-relevant behaviors. These findings will likely have translational relevance in the genetic and molecular mechanisms that confer susceptibility/resilience to MA addiction and could inform new therapeutic avenues.

? DESCRIPTION (provided by applicant): Binge eating is a heritable trait of eating disorders that carries high physical and emotional costs on health and disease. It is defined by the uncontrolled, excessive consumption of a large amount of palatable food over a short period of time. Binge eating possesses a strong genetic component and is comorbid with mood, anxiety, and substance abuse disorders. The primary objective of this proposal is to identify novel genetic factors that contribute to binge-like eating and its motivational components in mice. In collaboration with Dr. Pietro Cottone, we developed a model of the consummatory and the conditioned rewarding properties of binge eating whereby outbred mice exhibited a nine-fold escalation in the consumption of palatable food that was tightly correlated with the degree of conditioned place preference for the palatable food-paired environment. Additionally, we observed pronounced binge eating and conditioned reward in the anxiety-prone C57BL/6NJ strain but not in the closely related C57BL/6J substrain. Because the parental substrains are nearly genetically identical, quantitative trait locus (QTL) mapping in an experimental F2 cross (Reduced Complexity Cross; RCC) will greatly facilitate the identification of novel genetic factors that underlie differences in behavior. In Aim 1, we will use the RCC to map genomic regions, or QTLs, that are causally associated with susceptibility versus resilience to binge eating, conditioned food reward, and anxiety-like behavior. We hypothesize that shared QTLs will influence the consummatory and appetitive-motivational aspects of binge-like eating and that a subset of QTLs will contribute to premorbid anxiety-like behavior which in turn, increases the risk for binge eating. In Aim 2, we will conduct transcriptome analysis via mRNA sequencing (RNA-seq) of mesocorticolimbic brain regions and the amygdala in control mice and palatable food-trained mice from the parental C57BL/6J and C57BL/6NJ substrains. The premorbid transcriptome in control mice will serve as a useful tool both in identifying candidate genes for future genome editing that are differentially expressed and underlie the behavioral QTLs as well as providing genomic insight into the neurobiological context that influences susceptibility versus resilience to binge eating. Genes that are differentially expressed as a consequence of escalated PF consumption will reveal changes in the transcriptome relevant to neural plasticity and the appetitive-motivational behaviors that support binge eating. The proposed studies will lead to the identification of novel genetic factors influencing risk versus resilience to binge eating. Furthermore, combining genetic and transcriptome analysis could lead to the identification of new downstream molecular targets for pharmacotherapeutic development.

Paclitaxel is a cytoskeletal drug commonly used for the treatment of breast, lung, and ovarian cancer. Peripheral neuropathic pain (CIPN) is one of the most common and serious adverse effects experienced by cancer patients treated with paclitaxel. CIPN can be a dose-limiting factor for chemotherapy, leading to premature termination of treatment, thereby influencing survival and quality of life. Currently, no therapies have been identified that address the underlying pathogenic mechanisms such as neurodegeneration; in fact, the current symptomatic therapies are frequently ineffective in mitigating the painful symptoms of CIPN in the majority of patients. Therefore, the identification of alternative forms of therapy is a crucial medical need. The primary objective of this proposal is to identify novel genetic factors that contribute to paclitaxel- induced neuropathy in mice. We observed pronounced paclitaxel-induced CIPN in C57BL/6NJ strain but not in the closely related C57BL/6J substrain. Because the parental substrains are nearly genetically identical, quantitative trait locus (QTL) mapping in an experimental F2 cross (Reduced Complexity Cross; RCC) will greatly facilitate the identification of novel genetic factors that underlie differences in CIPN behaviors. In Aim 1, we will use the RCC to map genomic regions, or QTLs, that are causally associated with susceptibility versus resilience to multiple measures of CIPN. In Aim 2, we will conduct transcriptome analysis via mRNA sequencing (RNA-seq) of spinal and peripheral neuronal regions in control mice and paclitaxel-treated mice from the parental male and female C57BL/6J and C57BL/6NJ substrains. The transcriptome in control mice will aid in identifying differentially expressed, candidate CIPN susceptibility genes underlying the QTLs . Genes that are differentially expressed as a consequence of paclitaxel will reveal changes in the transcriptome relevant to central and peripheral neuronal plasticity and the behaviors/changes that support the long-term establishment of CIPN that may be important for treatment reversal. In Aim 3, we will validate candidate quantitative trait genes and functional variants that influence susceptibility to and establishment of CIPN. These studies will provide rapid genetic and neurobiological insight into CIPN. Future studies will test for translational potential in human genetics, human experimental model systems (e.g., hIPSCs), and new potential therapeutics to combat the debilitating side effects of CIPN in cancer patients.

PROJECT SUMMARY Substance use disorders (SUDs) are heritable psychiatric disorders with a significant genetic component. Opioid dependence, one of the most heritable SUDS, has reached epidemic proportions in the United States. Human genome-wide association studies (GWAS) are statistically underpowered to detect the majority of common genetic variation that contributes to opioid dependence. Discovery-based genetics in mammalian model organisms is a powerful complement to human GWAS and can uncover novel genetic factors, biological pathways, and gene networks underlying addiction traits. Mouse models are advantageous because they enable collection of the relevant brain tissue at the appropriate time points under controlled opioid dosing. Furthermore, gene editing permits the validation of functional variants in vivo within the same species on a controlled, genetic background. Reduced Complexity Crosses (RCCs) are genetic crosses between inbred mouse substrains that are nearly genetically identical and can vastly improve the speed at which causal genetic factors can be identified. Our primary objective is to use an RCC between BALB/c substrains to discover the genetic and molecular basis of opioid addiction-relevant traits at two stages of opioid dependence following repeated administration of the mu opioid receptor agonist oxycodone (OXY; the active ingredient of Oxycontin®). We found robust differences between BALB/c substrains in opioid adaptive behaviors, including state-dependent learning of OXY-induced locomotor stimulation and reward following limited, low-dose administration (1.25 mg/kg, IP) as well as the emotional-affective component of opioid withdrawal and weight loss following repeated high-dose administration (40 mg/kg, IP). In Aim 1, we will map quantitative trait loci (QTLs) underlying these OXY phenotypes in an RCC F2 cross. In Aim 2, we will map QTLs controlling gene expression (eQTLs) in the relevant brain tissues of control F2 mice and in OXY-trained F2 mice. We will then nominate candidate causal genes and nucleotides underlying behavior by integrating eQTL with behavioral QTL analysis. To increase precision in assigning candidate variants with the regulation of gene expression and behavior and to identify biological pathways and opioid-adaptive gene networks in specific cell types, we will use single nucleus RNA- seq (snRNA-seq) of brain tissue following limited, low-dose OXY and repeated high-dose OXY. In Aim 3, we will validate candidate functional variants underlying OXY phenotypes using CRISPR/Cas9 gene editing of each of the two alternate alleles onto each reciprocal substrain background. This approach will allow us to demonstrate both necessity and sufficiency of the quantitative trait nucleotides. The proposed studies will identify the genetic basis of unique opioid phenotypes across two stages of opioid dependence. Independent from gene discovery, these studies have broader application in revealing novel, actionable insight toward cellular adaptations at progressive stages of the opioid addiction process and potentially improving behavioral outcomes.