Morphine Increases Brain Levels of Ferritin Heavy Chain Leading to Inhibition of CXCR4-Mediated Survival Signaling in Neurons

Abstract

This study focuses on the effect of -opioid receptor agonists on CXCR4 signaling in neurons and the mechanisms involved in regulation of neuronal CXCR4 by opiates. The data show that CXCR4 is negatively modulated by long-term morphine treatments both in vitro and in vivo; CXCR4 inhibition is caused by direct stimulation of -opioid receptors in neurons, leading to alterations of ligand-induced CXCR4 phosphorylation and upregulation of protein ferritin heavy chain (FHC), a negative intracellular regulator of CXCR4. Reduced coupling of CXCR4 to G-proteins was found in the brain of morphine-treated rats, primarily cortex and hippocampus. CXCR4-induced G␣ i /G␤␥ activities were suppressed after 24 h treatment of cortical neurons with morphine or the selective -opioid agonist DAMGO (D-Ala2-NMe-Phe 4 -glycol 5 -enkephalin), as shown by analysis of downstream targets of CXCR4 (i.e., cAMP, Akt, and ERK1/2). These agonists also prevented CXCL12-induced phosphorylation of CXCR4, indicating a deficit of CXCR4 activation in these conditions. Indeed, morphine (or DAMGO) inhibited prosurvival signaling in neurons. These effects are not attributable to a reduction in CXCR4 expression or surface levels but rather to upregulation of FHC by opioids. The crucial role of FHC in inhibition of neuronal CXCR4 was confirmed by in vitro and in vivo RNA interference studies. Overall, these findings suggest that opiates interfere with normal CXCR4 function in the brain. By this mechanism, opiates could reduce the neuroprotective functions of CXCR4 and exacerbate neuropathology in opiate abusers who are affected by neuroinflammatory/infectious disorders, including neuroAIDS.

Biology of ferritin in mammals: an update on iron storage, oxidative damage and neurodegeneration

“…The increased capacity to sequester iron is an efficient protective mechanism against cellular stress, as shown by the block of the TNF-/JNK mediated apoptosis obtained through the NF-kb-dependent activation of ferritin transcription (Pham et al 2004). Other pathways of regulation suggest the possible association of ferritin with alternative, cell specific functions (see below); adiponectin can increase ferritin expression in primary skeletal muscle cells by recruitment of NF-kb (Ikegami et al 2009) while in neurons a transcription-dependent increase of ferritin H is observed upon the activation of -opiod receptors (Sengupta et al 2009). A strong post-transcriptional regulation of ferritin is superimposed to the transcriptional one to guarantee rapid adaptations to changes in iron levels and oxidative stress.…”

“…Stimulation of CXCR4 by its ligand CXCL12 induces FtH phosphorilation at serine 178 and subsequent translocation to the nucleus. The discovery that opioids induce new synthesis of FtH can explain the heterologous desensitization observed between -opioid receptors (MOR) and CXCR4 in neurons (Sengupta et al 2009), a possible mechanism underlining the neurological dysfunction and cerebral damage determined by opiates. This activity is independent of iron binding since it is maintained by a ferroxidase deficient FtH (Pitcher et al 2014).…”

Neuronal ferritin heavy chain and drug abuse affect HIV-associated cognitive dysfunction

“…Recently, our group has shown that morphine (and selective μ-opioid receptor [MOR] agonists, such as DAMGO) specifically impairs CXCL12/CXCR4 signaling in neurons, both in vitro and in vivo, by increasing protein levels of ferritin heavy chain (FHC), a recently described CXCR4 regulator (25,(27)(28)(29). Morphine increases the level of FHC and its association with CXCR4, thereby inhibiting CXCR4 activation and downstream prosurvival signaling pathways (27).…”

“…Recently, our group has shown that morphine (and selective μ-opioid receptor [MOR] agonists, such as DAMGO) specifically impairs CXCL12/CXCR4 signaling in neurons, both in vitro and in vivo, by increasing protein levels of ferritin heavy chain (FHC), a recently described CXCR4 regulator (25,(27)(28)(29). Morphine increases the level of FHC and its association with CXCR4, thereby inhibiting CXCR4 activation and downstream prosurvival signaling pathways (27). This novel CXCR4-regulatory function of FHC occurs seemingly independent from the protein's primary role as a critical regulator of intracellular iron levels; in this context, FHC functions by binding, oxidizing, and sequestering free reactive iron, which is stably stored within the ferritin complex formed by FHC and its partner subunit, ferritin light chain (FLC).…”

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

“…At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods [1][2][3] . Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology 4 , cellular and molecular biology 5-8 , biochemistry 5 , imaging and microscopy 4,6,7,9,10 .…”