Restoring Iron Homeostasis to Promote Recovery after Spinal Cord Injury

描述（由申请人提供）：本提案将测试新的假设，即小胶质细胞/巨噬细胞或星形胶质细胞中的铁稳态异常会抑制脊髓损伤（SCI）后的恢复。铁是所有基本细胞功能所必需的，但过量的铁或铁代谢受损是高度毒性的。因此，哺乳动物已经进化出复杂的调节机制来维持铁稳态。脊髓损伤后，出血和细胞死亡引起慢性炎症反应，这与脊柱内铁的长期积累有关。大多数铁与活化的小胶质细胞/巨噬细胞共定位。我们的新数据表明，铁代谢和铁调节蛋白在受损脊髓中失调数周，当高度保守的巨噬细胞活化机制受损时，这种失调会加剧。具体而言，通过Toll样受体4（TLR 4）受损的信号传导加剧了SCI的恢复，并与脊柱内铁的积累增加有关。此外，两个关键蛋白，铁调素和ferroportin（FP）的表达，不成比例地调节SCI后，最显着的是在小鼠与缺陷TLR 4信号（TLR 4KO）。TLR 4KO小鼠脊髓中FP表达的显著增加有利于从活化的小胶质细胞/巨噬细胞输出螯合的铁。本实验将确定巨噬细胞和星形胶质细胞铁相关蛋白是否可以被操纵以恢复脊髓内铁稳态并促进SCI后的恢复。首先（目的1），将典型的和合成的TLR 4激动剂注射到SCI小鼠中，目的是增强小胶质细胞/巨噬细胞产生铁调素，铁调素是一种通过引起FP降解来限制铁流出的蛋白质。第二（目的2），将铁调素输注到损伤部位，从而绕过对TLR 4活化的需要。最后，在目标3中，条件性基因敲除小鼠（星形胶质细胞、小胶质细胞或单核细胞衍生的巨噬细胞中的FP敲除）将用于确定这些不同细胞亚群对SCI后过量铁释放的相对贡献;这将使未来的治疗靶向特定细胞群。与此同时，当我们评估椎管内铁的变化时，我们也将检查系统性铁调节。新的初步数据表明，铁相关蛋白在SCI后几周内在肝脏中发生了变化。由于SCI患者经常贫血（尽管脊柱内铁水平很高），因此了解全身和脊柱内铁如何受到影响非常重要。通过这样做，可以定制新的药理学或遗传干预措施，以促进有效的神经恢复，而不会引起全身病理学。

DESCRIPTION (provided by applicant): This proposal will test the novel hypothesis that aberrant iron homeostasis in microglia/macrophages or astrocytes causes inhibits recovery after spinal cord injury (SCI). Iron is essential for all basic cell functions but excess iron or impaire iron metabolism is highly toxic. Accordingly, mammals have evolved sophisticated regulatory mechanisms to maintain iron homeostasis. After SCI, hemorrhage and cell death elicit a chronic inflammatory response that is associated with prolonged accumulation of intraspinal iron. Most of this iron co- localizes with activated microglia/macrophages. Our new data show that iron metabolism and iron regulatory proteins are dysregulated in the injured spinal cord for several weeks and this dysregulation is exacerbated when highly conserved mechanisms of macrophage activation are impaired. Specifically, impaired signaling via toll-like receptor 4 (TLR4) exacerbates recovery from SCI and is associated with enhanced accumulation of intraspinal iron. Also, expression of two key proteins, hepcidin and ferroportin (FP), is disproportionately regulated after SCI, most notably in mice with deficient TLR4 signaling (TLR4KO). A significant increase in FP expression in spinal cords of TLR4KO mice favors export of sequestered iron from activated microglia/macrophages. Experiments in this proposal will determine if macrophage and astrocyte iron-related proteins can be manipulated to restore intraspinal iron homeostasis and promote recovery after SCI. First (Aim 1), canonical and synthetic TLR4 agonists will be injected into SCI mice with the goal of enhancing microglia/macrophage production of hepcidin, a protein that limits iron efflux by causing FP degradation. Second (Aim 2), hepcidin will be infused to the injury site thereby bypassing the need for TLR4 activation. Finally, in Aim 3, conditional knock-out mice (FP knockout in astrocytes, microglia or monocyte-derived macrophages) will be used to determine the relative contribution of these distinct cellular subsets to excess iron release after SCI; this will allow future therapies to be targeted to specific cell populations. In parallel, as we evaluate changes i intraspinal iron, we will also examine systemic iron regulation. Novel preliminary data show that iron-related proteins are altered for several weeks post-SCI in the liver. Since SCI patients are often anemic (despite high intraspinal iron levels), it is important to understand how systemic and intraspinal irons are affected. By doing so, new pharmacologic or genetic interventions can be customized to promote efficient neurological recovery without causing systemic pathology.