Role of the Src Family Kinases in Traumatic Brain Injury

 描述（由申请人提供）：创伤性脑损伤（TBI）与血管和出血有关。原发性TBI损伤引发凝血酶、氧合血红蛋白（oxyHb）、细胞因子、ROS等的释放。这些分子通过多种途径介导继发性损伤。由于许多分子和信号传导途径涉及破裂后的损伤和行为缺陷，因此许多分子（包括活性氧物质）阻断单一介体或单一途径在人TBI中可能不具有临床有效性。这导致这项提议的研究人员考虑一种可以阻断多种途径的方法。Src家族激酶（SFKs）是一类非受体酪氨酸激酶，包括c-Src、Fyn、林恩、Yrk、Fgr、Yes、Hck、Blk和Lck等9个家族成员。SFK可被许多跨膜受体激活，如粘附受体、酪氨酸激酶受体、G蛋白偶联受体、细胞因子受体等。SFK的这种独特特征使其成为许多毒性分子（例如凝血酶、oxyHb、ROS、细胞因子等）的汇聚点，这些毒性分子在TBI后释放并介导继发性损伤。此外，激活的SFK启动许多神经毒性下游信号通路，包括RhoA-Rho-激酶1（ROCK 1）、Jun N-末端激酶（JNK）、P38和Erk丝裂原活化蛋白激酶（MAPK）。 因此，研究人员提出，阻断SFK可能提供一种新的和强大的方法，用于通过阻断多种有害途径来治疗TBI，其中许多SFK上游毒性分子（例如，凝血酶、oxyHb、ROS、细胞因子等）以及许多SFK下游效应物（例如ROCK 1、JNK、P38、Erk等）都涉及。然而，研究所有这些途径超出了单一提案的范围。因此，研究人员将把这项研究集中在一个线性途径上，以证明SFKs在TBI中发挥重要作用的原理：TBI →出血→凝血酶→ SFKs → ROCK 1 →继发性损伤。研究人员假设，TBI后急性阻断SFK和急性阻断SFK下游靶点ROCK 1将防止海马细胞丢失并改善TBI后数周的认知结果。 在本研究中，研究者使用啮齿类动物中度侧向液压冲击（LFP）模型，显示（1）TBI后脑脊液（CSF）中凝血酶增加和SFK活性增加，并且凝血酶单独通过激活SFK引起认知缺陷;（2）使用非特异性SFK抑制剂PP 2对SFK的急性抑制，或基于纳米颗粒的体内小干扰RNA（siRNA）（Fyn和c-Src），保护海马神经元，改善TBI后的认知功能;（3）PP 2阻断了TBI后ROCK 1的表达，ROCK 1抑制剂Y27632可改善TBI后海马神经元的存活和记忆功能。该项目有可能转化为人类，因为SFK拮抗剂已安全地给予人类，并且体内纳米颗粒递送方法已被FDA批准用于人类使用。

 DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is associated with blood vessels and hemorrhage. The primary TBI injury initiates release of thrombin, oxyhemoglobin (oxyHb), cytokines, ROS), and others. These molecules mediate secondary injury through multiple pathways. Since a number of molecules and signaling pathways are implicated in the injury and behavioral deficits post- ruptured many molecules, including reactive oxygen species, blocking a single mediator or single pathway may not be as clinically effective in human TBI. This led the investigators of this proposal to consider an approach that would block multiple pathways. Src family kinases (SFKs) are non-receptor tyrosine kinases, including nine family members: c-Src, Fyn, Lyn, Yrk, Fgr, Yes, Hck, Blk and Lck. SFKs can be activated by many trans-membrane receptors, such as adhesion receptors, tyrosine kinase receptors, G protein-coupled receptors, cytokine receptors, and others. This unique feature of SFKs makes them a point of convergence for many toxic molecules (e.g. thrombin, oxyHb, ROS, cytokines, and others) that are released and mediate secondary injury after TBI. Moreover, the activated SFKs initiate many neurotoxic down-stream signaling pathways including RhoA- Rho- kinase1 (ROCK1), Jun N-terminal kinase (JNK), P38 and Erk mitogen-activated protein kinases (MAPKs). Therefore, the investigators propose that blocking SFKs may provide a novel and powerful approach for treating TBI via blocking multiple detrimental pathways in which many SFK upstream toxic molecules (e.g., thrombin, oxyHb, ROS, cytokines and others) and as well as many SFK downstream effectors (e.g. ROCK1, JNK, P38, Erk, and others) are implicated. However, studying all of these pathways is beyond the scope of a single proposal. Thus, the investigators will focus this study on a linear pathway to show proof of principle that SFKs play an important role in TBI: TBI → bleeding → thrombin → SFKs → ROCK1 → secondary injury. The investigators hypothesize that acutely blocking SFKs and acutely blocking SFK down-stream target ROCK1 post TBI will prevent hippocampal cell loss and improve cognitive outcomes many weeks after TBI. Using a rodent moderate lateral fluid percussion (LFP) model in this proposal, the investigators have shown (1) thrombin increases in cerebrospinal fluid (CSF) and SFK activity increases after TBI , and thrombin alone causes cognitive deficits through activation of SFKs; (2) Acute inhibition of SFKs using a non-specific SFK inhibitor, PP2, or nanoparticle-based in vivo small interfering RNAs (siRNAs) to SFK subtypes (Fyn and c-Src), protects hippocampal neurons and improves cognitive function after TBI; (3) PP2 blocks ROCK1 expression after TBI, and a ROCK 1 inhibitor, Y27632, improves hippocampal neuron survival and memory function after TBI. The project has the potential to be translated to humans, since SFK antagonists have been safely given to humans, and the in vivo nanoparticle delivery methods are FDA approved for human use.