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CNS（脑和脊髓）及其供应毛细血管之间，为CNS和身体其他部位之间的相互作用和信息交换提供了巨大的界面。我们已经表明，BSCB不是静态的或物理被动的，而是经历动态变化，以调节从血液到CNS的细胞因子肿瘤坏死因子a（TNFa）的可用性。在所提出的研究中，我们将检验TNFa在穿过BBB/BSCB运输后可以促进脊髓损伤（SCI）后的功能恢复的假设。我们还将描述这种特定的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，脊髓摄取放射性标记的TNFe将增加特别是在一个修改后的小鼠模型的胸部半切，这种增加将不符合被动破坏的屏障（测量白蛋白的细胞旁渗透性增加）。然后，我们将通过评估运动行为、诱发电位的椎管内传导和长束再生的组织学证据来确定TNF α治疗的效果。我们预计小剂量的TNF α将促进恢复，而大剂量将恶化SCI导致的缺陷。为了鉴定p55和p75受体的作用，我们将研究在受体抗体存在下和在受体敲除小鼠中TNF α的转运。我们预测，增加脊髓摄取的TNFe SCI后将依赖于上调的受体，通过Western印迹和定量PCR分析确定。由于跨BBB/BSCB的细胞因子的转胞吞作用是一个复杂的过程，不仅涉及受体，而且还涉及其他调节蛋白，我们将通过免疫共沉淀，比较蛋白质组学，转染和运输测定来鉴定新的运输调节蛋白。我们预测转运过程将受到受体、囊泡和细胞骨架蛋白以及处于不同磷酸化状态的分子伴侣的调节。通过完成拟议的研究，我们将更好地了解TNF α在SCI中的双重作用，以及在其涉及调节蛋白的转运过程中的作用。这些转运蛋白可能是新的药物靶点，因此提供了有前途的治疗潜力。

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.