Transport of TNFalpha across the BBB

描述（由申请人提供）：血脑和血脊髓屏障（BBB/BSCB）位于中枢神经系统（脑和脊髓）及其供应毛细血管之间，为中枢神经系统和身体其他部分之间的相互作用和信息交换提供了巨大的接口。我们已经证明，BSCB不是静态的或物理被动的，而是经历动态变化，以调节细胞因子肿瘤坏死因子a （TNFa）从血液到中枢神经系统的可用性。在拟议的研究中，我们将验证TNFa在通过血脑屏障/BSCB运输后可以促进脊髓损伤（SCI）后功能恢复的假设。我们还将描述这种特定运输可以被调节的机制。为了确定TNFe转运的功能意义，我们将首先表明，在改良的小鼠胸椎半切模型中，脊髓对放射性标记的TNFe的摄取会特异性增加，并且这种增加不会与被动破坏屏障（通过增加白蛋白的细胞旁通透性来测量）相一致。然后，我们将通过评估运动行为、脊髓内诱发电位传导和长束再生的组织学证据来确定TNFa治疗的效果。我们预计小剂量的TNFa将促进恢复，而大剂量会加重脊髓损伤造成的缺陷。为了确定p55-和p75受体的作用，我们将研究受体抗体存在和受体敲除小鼠中TNFa的转运。我们预测脊髓损伤后脊髓对TNFe摄取的增加将取决于受体的上调，这是通过Western blot和定量PCR分析确定的。由于细胞因子跨血脑屏障/BSCB的胞饮作用是一个复杂的过程，不仅涉及受体，还涉及其他调节蛋白，因此我们将通过共免疫沉淀、比较蛋白质组学、转染和运输试验来鉴定新的运输调节蛋白。我们预测转运过程将受到不同磷酸化状态的受体、囊泡蛋白和细胞骨架蛋白以及伴侣蛋白的调控。通过完成本研究，我们将更好地了解TNFa在SCI中的双重作用及其涉及调节蛋白的转运过程。这些转运蛋白可能是新的药物靶点，因此具有良好的治疗潜力。

DESCRIPTION (provided by applicant): The blood-brain and blood-spinal cord barriers (BBB/BSCB), lying between the CNS (brain and spinal cord) and its supplying capillary blood vessels, provide an immense interface for interaction and exchange of information between the CNS and the rest of the body. We have shown that the BSCB is not static or physically passive, but undergoes dynamic changes, to regulate the availability of the cytokine tumor necrosis factor a (TNFa) from blood to the CNS. In the proposed studies, we will test the hypothesis that TNFa, after being transported across the BBB/BSCB, can facilitate functional recovery after spinal cord injury (SCI). We will also characterize the mechanisms by which this specific transport can be regulated. To determine the functional implications of TNFe transport, we will first show that spinal cord uptake of radioactively labeled TNFe will increase specifically in a modified mouse model of thoracic hemisection, and that this increase will not coincide with passive disruption of the barrier (measured by increased paracellular permeability of albumin). We will then determine the effects of TNFa treatment by evaluating locomotor behavior, intraspinal conduction of evoked potentials, and histological evidence of long tract regeneration. We expect that small doses of TNFa will facilitate recovery whereas large doses will worsen the deficits resulting from SCI. To identify the roles of p55- and p75-receptors, we will study TNFa transport in the presence of receptor antibodies and in the receptor knockout mice. We predict that increased spinal cord uptake of TNFe after SCI will be dependent upon upregulation of the receptors, as determined by Western blot and quantitative PCR analysis. Since transcytosis of a cytokine across the BBB/BSCB is a complicated process involving not only the receptors but also other regulatory proteins, we will identify novel transport regulatory proteins by co-immunoprecipitation, comparative proteomics, transfection and transport assays. We predict that the transport process will be regulated by the receptors, vesicular and cytoskeletal proteins, and chaperons in different phosphorylation states. By completing the proposed study, we will better understand the dual roles of TNFa in SCI and in its transport process involving the regulatory proteins. These transporters could be novel drug targets and therefore provide promising therapeutic potential.