Exploring the role of divalent metal transporter 1 in morphine-mediated upregulation of neuronal ferritin heavy chain
Year: 2020
Type: dissertation
Abstract: Clinical studies indicate that HIV-associated neurocognitive disorders (HAND) are exacerbated by opioid use, but the underlying mechanisms are controversial. Our previous work demonstrated that morphine triggers iron release from endolysosomes and upregulates the iron storage protein ferritin heavy chain in cortical neurons, thus reducing dendritic spine density. Given the role of dendritic spines in cognitive processes and the reported spine loss in HAND, this pathway could be involved in HIV/opioid-induced neuropathology. Here, we hypothesized that morphine elicits endolysosomal iron efflux through the common endolysosomal iron transporter divalent metal transporter 1 (DMT1). To start testing this hypothesis, the first aim of this study was to examine expression of DMT1 and its isoforms in cultured cells, including cortical neurons. Since DMT1 bands were not clearly evident using standard western blot protocols/reagents, we tested different sample preparation methods using cells overexpressing DMT1. We found that endogenous DMT1±IRE isoforms were visible in unboiled cell lysates. We also optimized conditions to recognize DMT1 isoforms by immunocytochemistry, and most neurons showed staining in the soma. Next, we performed a dual stain for DMT1 and the endolysosomal marker LAMP1, which suggested that DMT1 was present in neuronal endolysosomes. Our second aim was to test DMT1's involvement in the induction of FHC by morphine, and we found that pretreatment of cortical neurons with the DMT1 inhibitor ebselen prevented morphine- mediated FHC upregulation. Overall, these studies have provided a foundation to further investigate the role of DMT1 and intracellular iron changes in morphine-mediated FHC regulation in cortical neurons.
DOI: 10.17918/00000264
Authors: Ipek Gozen Eralp, Olimpia Meucci, Seena Ajit
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
OpenAlex ID: W4205742219