Sophia A. Miracle1,2, Morgan L. Hofmeyer1,3, Ava B. Glavine1,4, Isabella C. Conti1,5, Sophia V. Pavlidis1,6 , Hala Ajjawi1 , Aleksandra G. Gorelik1,6 , Bryce Axe7,8 , Priyanka Thareja1,9 , Angelique Buton10, Kaylie R. Kaneshiro1,11 , William B. Lynch1,2 , Kelly Wingfield1,12 , Praveen P. Kulkarni8,13 , Joseph Rower14, Lili Sun14 , Stephanie G. Puig10 , Ralph Loring15 , Christopher A. Reilly14, Craig F. Ferris8,13,15, Camron D. Bryant1,2

Oxycodone (OXY; active ingredient of OxyContin®) is a major contributor to the opioid epidemic. We genetically mapped and validated loss-of-function in zinc-fingers and homeobox 2 (Zhx2) underlying increased brain oxymorphone (OMOR) in female mice. OMOR, an OXY metabolite, is a much more potent and efficacious mu opioid receptor agonist that could increase OXY addiction risk. Transcriptome analysis of Zhx2 knockout (KO) brains via bulk RNA-seq identified enrichment of extracellular matrix, endothelial cells, and cell-to-cell adhesion, suggesting Zhx2 KO compromises blood brain barrier (BBB) integrity. In support, there was a significant reduction in transcript levels of the BBB marker Claudin5 in KO females. Furthermore ex vivo analysis indicated increased permeability of sodium fluorescein but not Evans blue, specifically in hippocampus of KO females, suggesting brain region-dependent disruption of BBB. In vivo structural imaging revealed reduced water diffusion throughout the brain of KO females and enlarged ventricles. In response to OXY in awake mice, KO females showed increased OXYinduced negative bold signal in midbrain and increased OXY-induced positive bold signal in brainstem. Functional connectivity analysis identified decreased brain-wide connectivity in Zhx2 KOs. In addition to a BBB mechanism, KO females also showed increased plasma [OMOR] following systemic OXY, suggesting increased liver metabolism of OXY also contributes to increased brain [OMOR]. To summarize, multiple lines of evidence support a BBB mechanism underlying increased brain [OMOR] in Zhx2 KO females. We are currently conducting functional enzymatic assays of liver microsomes to determine whether increased liver OMOR production also contributes to the phenotype.

1Laboratory of Addiction Genetics, Department of Pharmaceutical Sciences and Center for Drug Discovery, Northeastern University, Boston, MA USA; 2Graduate Program for Neuroscience, Graduate Medical Sciences, Boston University Chobanian and Avedisian School of Medicine, Boston, MA USA; 3Undergraduate Program of Neuroscience, College of Arts and Sciences, Boston University, Boston, MA USA; 4Undergraduate program of Health Science, College of Health Sciences, Northeastern University, Boston, MA USA; 5Undergraduate Program of Behavioral Neuroscience, College of Science, Northeastern University, Boston, MA USA; 6Undergraduate Program of Biology, College of Science, Northeastern University, Boston, MA USA; 7Masters Program of Bioengineering, College of Engineering, Northeastern University, Boston, MA USA; 8Center for Translational Neuroimaging, Northeastern University, Boston, MA USA; 9Masters Program of Bioinformatics, College of Science, Northeastern University, Boston, MA USA 10Department of Psychiatry, University of Massachusetts Chan Medical School, Worcester, MA, USA; 11Undergraduate Program of Biology, College of Arts and Science, Tufts University, Boston, MA USA 12Graduate Program of Pharmacology, Boston University, Boston, MA, USA; 13Department of Psychology, Northeastern University, Boston, MA USA; 14Center for Human Toxicology, University of Utah Health, Salt Lake City, UT USA; 15Department of Pharmaceutical Sciences, Northeastern University, Boston, MA USA