Astrocyte dysfunction in EAE

描述(申请人提供)：星形胶质细胞是维持中枢神经系统(CNS)内稳态所必需的。这些细胞产生凝血酶原蛋白(TSPs)，这是一种大的寡聚基质细胞蛋白，在细胞附着、细胞迁移、细胞骨架动力学和血管生成中发挥重要作用。TSP-1和TSP-2也被认为是星形胶质细胞在体外和体内发育过程中促进兴奋性突触形成的主要因子。这种活性已经被映射到它们的表皮生长因子样重复序列，通过与神经元a2d-1电压门控钙通道结合，该通道也是药物加巴喷丁(GBP)的受体。GBP在体外和体内发育过程中都能有效地抑制TSP介导的突触形成。虽然TSP水平在成年中枢神经系统中普遍下降，但我们在正常断奶小鼠的脊髓中发现了可测量的数量的TSP-1。此外，在复发性实验性自身免疫性脑脊髓炎(EAE)期间，脊髓TSP-1水平迅速下降，然后随着后肢瘫痪的出现和消失而恢复。EAE是一种已建立的人类脱髓鞘疾病多发性硬化症(MS)的啮齿动物模型。我们和其他人已经证明，在复发性EAE小鼠的腰椎脊髓中可以发现可逆的突触病理，我们发现，当脊髓TSP-1水平处于最低点并刚刚开始恢复时，对EAE动物在高峰瘫痪时开始全身给予GBP，显著延迟了临床改善和腰椎灰质中运动突触的重建。综上所述，这些发现使我们推测，星形胶质细胞在成年脊髓中产生TSP-1，以维持运动通路中的兴奋性突触，但在复发的EAE过程中，局部炎症环境导致这一介质变得失调，导致一过性突触改变和可逆性后肢瘫痪。如果得到证实，这将使星形胶质细胞成为典型的动物中枢神经系统脱髓鞘疾病的可逆神经功能障碍的主要贡献者，进而成为人类治疗干预的新靶点。在此，我们提出以下目标：1)证实星形胶质细胞来源的TSP-1/TSP-2信号通过神经元a2d-1电压门控钙通道作用于脊髓运动通路的兴奋性突触，在介导复发性EAE可逆性瘫痪中的重要性；2)研究在体外和体内导致星形胶质细胞下调TSP-1表达的机制，并确定导致EAE缓解时脊髓TSP-1表达恢复和临床恢复的因素。这些研究将阐明在已建立的中枢神经系统脱髓鞘疾病动物模型中存在可逆性神经缺陷的新的细胞和分子机制。由于临床复发会导致人类多发性硬化症的慢性疾病进展，因此更好地了解这些可逆事件并开发新的方法来阻止它们是至关重要的。目前的MS治疗针对的是炎症反应，但目前还没有直接干预神经元或神经胶质细胞水平的药物。

DESCRIPTION (provided by applicant): Astrocytes are essential for the maintenance of homeostasis within the central nervous system (CNS). These cells produce thrombospondins (TSPs), large oligomeric matricellular proteins with important roles in cell attachment, cell migration, cytoskeletal dynamics and angiogenesis. TSP-1 and TSP-2 have also recently been identified as the main astrocyte-derived factors that promote excitatory synapse formation in vitro and during development in vivo. This activity has been mapped to their epidermal growth factor-like repeats, via binding to the neuronal a2d-1 voltage-gated calcium channel that is also a receptor for the drug, gabapentin (GBP). GBP potently inhibits TSP-mediated synapse formation in vitro and during development in vivo. While TSP levels generally decline in the adult CNS, we find measurable amounts of TSP-1 in the spinal cords of normal weanling mice. Furthermore, spinal cord TSP-1 levels fall rapidly and then recover in parallel to the appearance and disappearance of hind limb paralysis during relapsing experimental autoimmune encephalomyelitis (EAE), an established rodent model of the human demyelinating disease, multiple sclerosis (MS). We and others have shown that reversible synaptic pathology can be found in the lumbar spinal cords of mice with relapsing EAE, and we find that systemic administration of GBP to animals with EAE starting at peak paralysis, when spinal cord TSP-1 levels are at their nadir and just beginning to recover, significantly delays both clinical improvement and the reestablishment of motor synapses in lumbar spinal grey matter. Taken together, these findings lead us to hypothesize that astrocytes produce TSP-1 in the adult spinal cord to maintain excitatory synapses in the motor pathway, but the local inflammatory milieu during relapsing EAE causes this mediator to become dysregulated resulting in transient synaptic changes and reversible hind limb paralysis. If confirmed, this would make astrocytes central contributors to the reversible neurological deficits that typify animal CNS demyelinating diseases, and by extension, novel targets for therapeutic intervention in humans. Here, the following aims are proposed: 1) To confirm the importance of astrocyte-derived TSP- 1/TSP-2 signaling through neuronal a2d-1 voltage-gated calcium channels in mediating the reversible paralysis of relapsing EAE by acting on excitatory synapses in motor pathways of the spinal cord, and 2) To characterize the mechanisms that cause astrocytes to down-regulate TSP-1 expression in vitro and in the spinal cord during EAE in vivo, and determine the factors responsible for the return of spinal cord TSP-1 expression and clinical recovery as EAE remission occurs. These studies will clarify novel cellular and molecular mechanisms underlying reversible neurological deficits in an established animal model of CNS demyelinating disease. Since clinical relapses beget chronic disease progression in human MS, it is essential to better understand these reversible events and to develop novel approaches that block them. Current MS therapies target the inflammatory response, but none as of yet directly intervene at either the neuronal or the glial cell level.