Morphine-Induced Modulation of Endolysosomal Iron Mediates Upregulation of Ferritin Heavy Chain in Cortical Neurons.

HIV-associated neurocognitive disorders (HAND) remain prevalent and are aggravated by µ-opioid use. We have previously shown that morphine and other µ-opioids may contribute to HAND by inhibiting the homeostatic and neuroprotective chemokine receptor CXCR4 in cortical neurons, and this novel mechanism depends on upregulation of the protein ferritin heavy chain (FHC). Here, we examined the cellular events and potential mechanisms involved in morphine-mediated FHC upregulation using rat cortical neurons of either sex in vitro and in vivo. Morphine dose dependently increased FHC protein levels in primary neurons through µ-opioid receptor (µOR) and Gαi-protein signaling. Cytoplasmic FHC levels were significantly elevated, but nuclear FHC levels and FHC gene expression were unchanged. Morphine-treated rats also displayed increased FHC levels in layer 2/3 neurons of the prefrontal cortex. Importantly, both in vitro and in vivo FHC upregulation was accompanied by loss of mature dendritic spines, which was also dependent on µOR and Gαi-protein signaling. Moreover, morphine upregulated ferritin light chain (FLC), a component of the ferritin iron storage complex, suggesting that morphine altered neuronal iron metabolism. Indeed, prior to FHC upregulation, morphine increased cytoplasmic labile iron levels as a function of decreased endolysosomal iron. In line with this, chelation of endolysosomal iron (but not extracellular iron) blocked morphine-induced FHC upregulation and dendritic spine reduction, whereas iron overloading mimicked the effect of morphine on FHC and dendritic spines. Overall, these data demonstrate that iron mediates morphine-induced FHC upregulation and consequent dendritic spine deficits and implicate endolysosomal iron efflux to the cytoplasm in these effects.

A Preclinical Model of Inflammatory Breast Cancer to Study the Involvement of CXCR4 and ACKR3 in the Metastatic Process.

Inflammatory breast cancer (IBC) is an aggressive and invasive tumor, accounting for 2.5% of all breast cancer cases, and characterized by rapid progression, regional and distant metastases, younger age of onset, and lower overall survival. Presently, there are no effective therapies against IBC and a paucity of model systems. Our aim was to develop a clinically relevant IBC model that would allow investigations on the role of chemokine receptors in IBC metastasis. Primary cultures of tumor cells were isolated from pleural exudates of an IBC patient and grown as spheres or monolayers. We developed a human xenograft model where patient-derived IBC cells, stably transduced with lentiviral vectors expressing fluorescent and bioluminescent markers, were inoculated directly into the left ventricle of mice. Our in vivo data show that these IBC cells (FC-IBC02A) are able to seed and proliferate into various organs, including brain, lungs, lymph nodes, and bone, closely replicating the metastatic spread observed in IBC patients. Moreover, cells were able to generate tumors when grafted in the mammary fat pad of mice. RT-PCR and microscopy studies revealed expression of both CXCR4 and ACKR3 receptors in FC-IBC02A cells. Furthermore, CXCL12 (the endogenous chemokine ligand of these receptors) induced transendothelial migration of these cells and stimulated signaling pathways involved in cell survival and migration - an effect reduced by CXCR4 or ACKR3 antagonists. This new model can be used to develop chemokine-based pharmacological approaches against the IBC metastatic process. This work also provides the first evidence of ACKR3 expression in IBC cells.