June 18, 2019
Introduction: Our country is experiencing an emergent clinical and epidemiological crisis of opiate addition and opiate overdose deaths. While there are many arguments advanced about the “nature-nurture” origins of this crisis, all agree that opiate addiction is a chronic brain disease, is difficult to manage, and that there is no single effective solution.
Opiate drugs are miraculous pain relievers that have revolutionized pain management, including chronic pain due to injury or cancer. However, opiates that interact primarily with the mu family of opiate receptors (MOR) both relieve pain and mediate addiction. Withdrawal from MOR drugs, either by cessation of intake or acute administration of OR antagonists such as naloxone, can precipitate drug craving and a physiological withdrawal syndrome in humans and experimental animals. Such MOR withdrawal itself can be lethal.
Molecular mechanisms and therapiesto blunt opiate withdrawal symptoms are sorely needed. At the moment, treatment of those with opiate abuse disorder consists of administration of MOR agonists (such as methadone) or time-release administration of a mixed MOR agonist-antagonist (such as buprenorphine). No therapies address underlying molecular mechanisms of addiction.
We propose to develop a human neural cell model of opiate addiction and acute withdrawal by studying what happens to human neural cells (SH-SY5Y) exposed for 72 hrs to a potent MOR agonist (fentanyl), followed by 24 hrs of an OR antagonist (naloxone).
Results: We found that SH-SY5Y cells expressed MOR but not kappa opiate receptors (KOR) or delta opiate receptors (DOR). Figure 1 shows the effects on mRNA gene expression of 24 hrs of exposure to the naturally occurring MOR agonists enkephalin (DAMGO, a hydrolysis-resistant enkephalin analogue) and beta-endorphin peptide. Note that the gene expression in the presence of naloxone has been subtracted from total expression. The difference is due nearly completely to MOR activation.
Figure 2 shows the results of the same experiment analyzed for microRNA (miRNA) expression. Note the excellent correlation.
Finally, Figure 3 shows the result of mRNA gene expression in total cell RNA (top) and exosome RNA (bottom) in SY5Y cells treated with fentanyl for 72 hrs followed by naloxone for 24 hrs (or PBS as vehicle control). Exosomes are small vesicles of pinched off cell material that cross the blood-brain barrier and can be assayed in blood plasma. Both of these experiments were repeated once (ie, done twice). Note that naloxone-precipitated mRNA gene expression is notably less in exosomes compared to whole cells.
We feel that the human SH-SY5Y neural cell has excellent potential to be developed as a model for opiate “addiction” and “withdrawal”. Particularly if the exosome findings repeat, then it may be possible to define which persons with opiate abuse disorder could benefit from miRNA expression manipulation, so as to blunt withdrawal symptoms in these persons.