Restoring Iron Homeostasis to Promote Recovery after Spinal Cord Injury

描述（由申请人提供）：本提案将验证小胶质细胞/巨噬细胞或星形胶质细胞中异常铁稳态导致脊髓损伤（SCI）后抑制恢复的新假设。铁对所有基本细胞功能都是必需的，但过量的铁或铁代谢受损是剧毒的。因此，哺乳动物已经进化出复杂的调节机制来维持铁的体内平衡。脊髓损伤后，出血和细胞死亡引发慢性炎症反应，这与脊髓内铁的长期积累有关。大部分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损伤脊髓中出现数周的失调，当巨噬细胞激活的高度保守机制受损时，这种失调会加剧。具体来说，通过toll样受体4 （TLR4）的信号传导受损会加剧脊髓损伤的恢复，并与脊髓内铁的积累增加有关。此外，两种关键蛋白hepcidin和铁转运蛋白（FP）的表达在脊髓损伤后不成比例地受到调节，尤其是在TLR4信号传导缺陷（TLR4KO）的小鼠中。TLR4KO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/巨噬细胞中输出隔离铁。本实验将确定是否可以操纵巨噬细胞和星形胶质细胞铁相关蛋白来恢复脊髓损伤后的脊髓内铁稳态并促进恢复。首先（目的1），将标准的和合成的TLR4激动剂注射到SCI小鼠中，目的是增强小胶质细胞/巨噬细胞hepcidin的产生，hepcidin是一种通过引起FP降解来限制铁外排的蛋白质。其次（目的2），hepcidin将被灌注到损伤部位，从而绕过TLR4激活的需要。最后，在Aim 3中，将使用条件敲除小鼠（星形胶质细胞、小胶质细胞或单核细胞来源的巨噬细胞中的FP敲除）来确定这些不同的细胞亚群对脊髓损伤后过量铁释放的相对贡献；这将使未来的治疗能够针对特定的细胞群。同时，当我们评估椎管内铁的变化时，我们也将检查全身铁调节。新的初步数据显示，肝脏中铁相关蛋白在脊髓损伤后数周内发生改变。由于脊髓损伤患者通常是贫血的（尽管脊髓内铁含量很高），了解全身铁和脊髓内铁是如何受到影响的是很重要的。通过这样做，新的药理学或遗传干预可以定制，以促进有效的神经恢复，而不会引起全身病理。

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.