TREM2 regulation of macrophages in spinal cord injury and CNS endogenous repair

描述（由申请人提供）：脊髓损伤（SCI）引发神经炎症反应，可加重组织损伤（例如，神经元死亡、轴突损伤、脱髓鞘）并促进修复（例如，轴突再生、髓鞘再生、血管再生）。最近，功能不同的巨噬细胞亚群，即，已经在SCI的部位鉴定了“M1”（促炎）和“M2”（抗炎）细胞，这可能是功能二分法的基础。M1巨噬细胞具有神经毒性，并在损伤后数周内支配受损的脊髓。相比之下，M2巨噬细胞促进轴突生长，即使在抑制性分子（例如CSPG和髓磷脂）的存在下，也没有伴随的神经毒性。不幸的是，M2巨噬细胞仅在损伤部位停留几天，最终被M1巨噬细胞压倒。因此，高M1：M2比率可以解释为什么受损CNS的修复相对于外周组织缓慢且效率低下。我们预测，脊髓修复的效率和程度将通过调节巨噬细胞的表型和功能来改善。我们将使用遗传功能丧失（敲除）和功能获得（慢病毒）技术来测试这一假设，以操纵在驻留的小胶质细胞和髓样前体细胞上表达的髓样细胞-2（TREM 2）触发受体的表达，即，这些细胞产生单核细胞衍生的巨噬细胞（MDM），因为在巨噬细胞中过表达TREM 2诱导M2表型。在目标1中，我们将研究TREM 2过表达或选择性敲除小胶质细胞与MDM如何影响挫伤性脊髓损伤后的炎症、运动恢复和修复的解剖学指标。通过使用慢病毒构建体在Aim 2中的背侧脊髓半切损伤模型中在MDM上过表达TREM 2，我们将确定TREM 2操作是否影响巨噬细胞对髓鞘/轴突吞噬作用、轴突再生和轴突收缩或受损轴突的“死回”的作用。使用局灶性脊柱内脱髓鞘（溶血素）模型结合功能方案的获得，我们将确定在Aim 3中操纵巨噬细胞TREM 2是否影响脊髓内的OPC分化和髓鞘再生。目前的提案概述了原理验证实验，这些实验将促进我们对不同分子信号传导途径的理解，即，TREM 2影响SCI后CNS巨噬细胞功能的自然进程。重要的是，如果来自这些研究的数据表明，操纵TREM 2赋予解剖学或功能益处，而对CNS结构或功能的不良影响极小或没有不良影响，那么开发用于人类临床试验的类似方案应该是可行的。事实上，骨髓细胞的静脉注射（在拟议的研究中使用的主要技术）已经在SCI患者中进行了尝试，并且没有不良反应。此外，使用自体外周血单核细胞的慢病毒转导的酶替代疗法在人类受试者中显示出安全有效。

DESCRIPTION (provided by applicant): Spinal cord injury (SCI) triggers a neuroinflammatory reaction that can aggravate tissue injury (e.g., neuronal death, axonal injury, demyelination) and promote repair (e.g., axon regeneration, remyelination, revascularization). Recently, functionally distinct subsets of macrophages, i.e., "M1" (pro-inflammatory) and "M2" (anti-inflammatory) cells have been identified at sites of SCI which may underlie the functional dichotomy. M1 macrophages are neurotoxic and dominate the injured spinal cord for several weeks post-injury. In contrast, M2 macrophages promote axon growth, even in the presence of inhibitory molecules (e.g. CSPG and myelin), without concomitant neurotoxicity. Unfortunately, M2 macrophages populate the injury site for only a few days, eventually becoming overwhelmed by M1 macrophages. Thus, the high M1:M2 ratio might explain why repair of the injured CNS is slow and inefficient relative to tissues in the periphery. We predict that the efficiency and magnitude of spinal cord repair will be improved by modulating the phenotype and function of macrophages that respond to the injury. We will test this hypothesis using genetic loss-of-function (knockout) and gain-of-function (lentiviral) techniques to manipulate expression of the triggering receptor expressed on myeloid cells-2 (TREM2) on resident microglia and myeloid precursor cells, i.e., cells that give rise to monocyte-derived macrophages (MDMs), since overexpressing TREM2 in macrophages induces an M2 phenotype. In Aim 1 we will examine how TREM2 overexpression or selective knockout on microglia vs. MDMs effects inflammation, motor recovery and anatomical indices of repair after contusive spinal cord injury. By using lentiviral constructs to overexpress TREM2 on MDMs in a model of dorsal spinal hemisection injury in Aim 2, we will determine if TREM2 manipulation influences macrophage effects on myelin/axon phagocytosis, axon regeneration and axonal retraction or "die-back" of injured axons. Using a model of focal intraspinal demyelination (lysolecithin) in conjunction with the gain of function protocols, we will determine if manipulating macrophage TREM2 affects OPC differentiation and remyelination within the spinal cord in Aim 3. The current proposal outlines proof-of-principle experiments that will advance our understanding of how a distinct molecular signaling pathway, i.e., TREM2, influences the natural course of CNS macrophage function after SCI. Importantly, if data from these studies indicate that manipulating TREM2 confers anatomical or functional benefits with minimal or no adverse effects on CNS structure or function, then it should be feasible to develop similar protocols for human clinical trials. Indeed, intravenous delivery of bone marrow cells (the primary technique to be used in the proposed studies) has already been tried in SCI patients and without adverse effects. Moreover, enzyme replacement therapies, using lentiviral transduction of autologous peripheral blood mononuclear cells, were shown to be safe and effective in human subjects.