Role of the Src Family Kinases in Traumatic Brain Injury

 描述(申请人提供)：创伤性脑损伤(TBI)与血管和出血有关。原发性颅脑损伤可引起凝血酶、氧合血红蛋白(OxHb)、细胞因子(ROS)等释放。这些分子通过多条途径介导继发性损伤。由于许多分子和信号通路参与了脑损伤后的损伤和行为缺陷，许多分子，包括活性氧，阻断单一介质或单一途径在临床上可能并不有效。这导致这一提案的调查人员考虑了一种将阻塞多条路径的方法。SRC家族激酶(SFK)是一类非受体酪氨酸激酶，包括9个家族成员：C-Src、Fyn、Lyn、Yrk、Fgr、Yes、HCK、Blk和Lck。SFK可被多种跨膜受体激活，如黏附受体、酪氨酸激酶受体、G蛋白偶联受体、细胞因子受体等。SFK的这一独特功能使其成为许多有毒分子(如凝血酶、氧合Hb、ROS、细胞因子等)的汇合点，这些分子被释放并介导脑损伤后的继发性损伤。此外，激活的SFK还启动了许多神经毒性下游信号通路，包括RhoA-Rho-kinase1(ROCK1)、Jun N-末端激酶(JNK)、P38和Erk丝裂原激活的蛋白激酶(MAPKs)。因此，研究人员认为，阻断SFK可能通过阻断许多SFK上游有毒分子(如凝血酶、氧合Hb、ROS、细胞因子等)以及许多SFK下游效应因子(如ROCK1、JNK、P38、ERK等)的多条有害途径，为脑外伤的治疗提供一种新的有效途径。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的重点放在一条线性途径上，以证明SFK在脑外伤中发挥重要作用的原理：脑创伤→出血→凝血酶→SFKs→ROCK1→继发性损伤。研究人员推测，脑损伤后急性阻断SFK及其下游靶点ROCK1将防止海马细胞丢失，并改善脑损伤后数周的认知结果。在这一提议中，利用啮齿动物中度侧向流体冲击(LFP)模型，研究人员表明：(1)脑损伤后脑脊液(CSF)中凝血酶活性增加，而凝血酶单独通过激活SFK活性引起认知障碍；(2)使用非特异性SFK抑制剂PP2或体内基于纳米颗粒的小干扰RNA(SiRNAs)急性抑制SFK亚型(Fyn和c-Src)，保护海马神经元，改善脑损伤后的认知功能；(3)PP2阻断脑损伤后ROCK1的表达，ROCK1抑制剂Y27632可改善脑损伤后海马神经元的存活和记忆功能。该项目有可能被翻译到人类身上，因为SFK拮抗剂已经被安全地给予人类，体内纳米颗粒的释放方法也被FDA批准用于人类。

 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.