Zhx2

We have multiple experiments being conducted regarding Zhx2.

1. Validating the transcription factor gene Zhx2 as a quantitative trait gene regulating oxycodone metabolism and oxycodone addiction behaviors. While prescription opioids continually substantially to the worsening opioid epidemic in the United States, understanding of how their metabolism contributes to their addictive profile is incomplete. Previous studies in the lab have yielded the hypothesis that an active metabolite of oxycodone, oxymorphone, contributes substantially to oxycodone addiction model behaviors. Genetic mapping identified Zhx2 as a candidate gene underlying oxymorphone content, hypothesized to regulate the expression of Cytochrome P450 2D (CYP2D) enzymes in the brain and liver, to influence the metabolism of oxycodone to oxymorphone. While humans have known genetic polymorphisms of CYP2D which alter opioid metabolism and underlie opioids’ analgesic efficacy, how these differential metabolism rates across individuals alter opioids’ addictive profiles between humans is unclear. We are utilizing gene manipulation methods like CRISPR-Cas9 and targeted adenoassociated viral injections combined with addiction behavioral tests like open-field locomotion and conditioned place preference as well as molecular quantification techniques like quantitative PCR, liquid chromatography mass-spectrometry, and CUT&RUN sequencing to validate Zhx2 in regulating oxycodone metabolite generation and addiction behaviors, uncovering the intermediate mechanisms in the process. This work could have important translational value to humans seeking additional insight and treatments to combat opioid use disorder.
2. Zhx2 Brain Overexpression: Oxycodone (OXY) is a semi-synthetic opioid that, when metabolized, produces a more potent metabolite, oxymorphone (OMOR). The presence of OMOR is hypothesized to increase addiction-like phenotypes. In addition, previous eQTL analysis has identified the transcriptional repressor protein, Zhx2, as having potential implications in the metabolism of OXY to OMOR. The lab uses behavioral (conditioned place preference, locomotion) and molecular assays (proteomics, western blot, immunohistochemistry, qPCR) to better understand the role of Zhx2 in opioid use disorder.
3. Withdrawal Machine Learning: Opioid withdrawal severity is one of the leading drivers of relapse. Because of the symptoms associated with opioid withdrawal, users are more likely to continue using to prevent the onset of the withdrawal phenotypes. We hypothesize that with an increased concentration of oxymorphone (OMOR), there is an increase in addiction-like phenotypes. In contrast, with an increase of Zhx2 expression, there is a decrease in OMOR concentration and decrease in addiction-like phenotypes, which could subsequently attenuate the opioid-induced withdrawal. We will implement machine learning using a 3-dimensional recording system to identify behavioral nuances during spontaneous and naloxone-precipitated withdrawal paradigms. This will allow us to better understand the complexities of opioid-induced withdrawal and to further explore the relationship between Zhx2 and opioid-use disorder.

Rat Genetics

From our recent paper: Forward genetic mapping of F2 crosses between closely related substrains of inbred rodents – referred to as a reduced complexity cross (RCC) – is a relatively new strategy for accelerating the pace of gene discovery for complex traits, such as drug addiction. RCCs to date were generated in mice, but rats are thought to be optimal for addiction genetic studies. Based on past literature, one inbred Spontaneously Hypertensive Rat substrain, SHR/NCrl, is predicted to exhibit a distinct behavioral profile as it relates to cocaine self-administration traits relative to another substrain, SHR/NHsd. Direct substrain comparisons are a necessary first step before implementing an RCC. We evaluated model traits for cocaine addiction risk and cocaine self-administration behaviors using a longitudinal within-subjects design. Impulsive-like and compulsive-like traits were greater in SHR/NCrl than SHR/NHsd, as were reactivity to sucrose reward, sensitivity to acute psychostimulant effects of cocaine, and cocaine use studied under fixed-ratio and tandem schedules of cocaine self-administration. Compulsive-like behavior correlated with the acute psychostimulant effects of cocaine, which in turn correlated with cocaine taking under the tandem schedule. Compulsive-like behavior also was the best predictor of cocaine seeking responses. Heritability estimates indicated that 22 %–40 % of the variances for the above phenotypes can be explained by additive genetic factors, providing sufficient genetic variance to conduct genetic mapping in F2 crosses of SHR/NCrl and SHR/NHsd. These results provide compelling support for using an RCC approach in SHR substrains to uncover candidate genes and variants that are of relevance to cocaine use disorders.