NeuroHIV and other neurologic disorders present with altered iron metabolism in central nervous system neurons. Many people with HIV also use opioids, which can worsen neuroHIV symptoms by further dysregulating neuronal iron metabolism. Our previous work demonstrated that the μ-opioid agonist morphine causes neuronal endolysosomes to release their iron stores, and neurons respond by upregulating ferritin heavy chain (FHC), an iron storage protein associated with cognitive impairment in neuroHIV. Here, we investigated if this process required divalent metal transporter 1 (DMT1), a well-known iron transporter expressed on endolysosomes. We first optimized conditions to detect DMT1 isoforms (DMT1 1B ± iron responsive element) using fluorescently labeled rat DMT1 constructs expressed in HEK-293 cells. We also expressed these constructs in primary rat cortical neurons to compare their expression and subcellular distribution with endogenous DMT1 isoforms. We found endogenous DMT1 isoforms in the cytoplasm that colocalized with lysosomal-associated protein 1 (LAMP1), a marker of endolysosomes. Next, we blocked endogenous DMT1 isoforms using ebselen, a potent pharmacological inhibitor of DMT1 iron transport. Ebselen pre-treatment blocked morphine’s ability to upregulate FHC protein, suggesting this pathway requires DMT1 iron transport from endolysosomes. This was further validated using viral-mediated genetic silencing of DMT1±IRE in cortical neurons, which also blocked FHC upregulation in the presence of morphine. Overall, our work demonstrates that the μ-opioid agonist morphine utilizes the endolysosomal iron transporter DMT1 to modulate neuronal cellular iron metabolism, upregulate FHC protein, and contribute to cognitive decline in neuroHIV.

Graphical Abstract

Morphine requires DMT1 to upregulate neuronal FHC. Cortical neurons treated with morphine release their endolysosomal iron stores to the cytoplasm and upregulate FHC, an iron storage protein associated with dendritic spine deficits and cognitive impairment in neuroHIV. This pathway requires the endolysosomal iron transporter DMT1, as pharmacological and genetic inhibitors of the transporter completely block morphine’s ability to upregulate FHC. Created with BioRender.com.

中文翻译：

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吗啡调节神经元铁蛋白重链需要内溶酶体转运蛋白 DMT1

NeuroHIV 和其他神经系统疾病表现为中枢神经系统神经元铁代谢的改变。许多艾滋病毒感染者还使用阿片类药物，这会通过进一步失调神经元铁代谢来恶化神经艾滋病毒症状。我们之前的工作表明，μ-阿片受体激动剂吗啡会导致神经元内溶酶体释放铁储备，神经元通过上调铁蛋白重链（FHC）做出反应，铁蛋白重链是一种与神经艾滋病毒认知障碍相关的铁储备蛋白。在这里，我们研究了这个过程是否需要二价金属转运蛋白 1 (DMT1)，这是一种在内溶酶体上表达的众所周知的铁转运蛋白。我们首先使用 HEK-293 细胞中表达的荧光标记的大鼠 DMT1 构建体优化检测 DMT1 亚型（DMT1 1B ± 铁反应元件）的条件。我们还在原代大鼠皮质神经元中表达了这些构建体，以将它们的表达和亚细胞分布与内源性 DMT1 亚型进行比较。我们在细胞质中发现了内源性 DMT1 亚型，与溶酶体相关蛋白 1 (LAMP1)（内溶酶体的标志物）共定位。接下来，我们使用依布硒啉 (ebselen) 阻断内源性 DMT1 亚型，依布硒啉是 DMT1 铁转运的有效药理学抑制剂。Ebselen 预处理阻断吗啡上调 FHC 蛋白的能力，表明该途径需要从内溶酶体转运 DMT1 铁。使用皮质神经元中病毒介导的 DMT1±IRE 基因沉默进一步验证了这一点，这也阻止了吗啡存在下 FHC 的上调。总体而言，我们的工作表明，μ-阿片受体激动剂吗啡利用内溶酶体铁转运蛋白 DMT1 调节神经元细胞铁代谢，上调 FHC 蛋白，并导致神经 HIV 认知能力下降。

图形概要

吗啡需要 DMT1 上调神经元 FHC。用吗啡处理的皮层神经元将其内溶酶体铁储存释放到细胞质中，并上调 FHC（一种与神经艾滋病毒树突棘缺陷和认知障碍相关的铁储存蛋白）。该途径需要内溶酶体铁转运蛋白 DMT1，因为转运蛋白的药理学和遗传抑制剂完全阻断吗啡上调 FHC 的能力。使用 BioRender.com 创建。