2,3 cAMP in Traumatic Brain Injury

描述（由申请人提供）：术语“cAMP”通常是指第二信使3',5'-环腺苷单磷酸。我们偶然发现器官系统可以产生（从mRNA降解）并向细胞外室输出3',5'-cAMP的位置异构体，即2',3'-cAMP。我们发现器官系统将细胞外的2',3'-cAMP转化为2'-AMP + 3'-AMP，并能将2'-AMP和3'-AMP代谢为腺苷。我们把这个途径称为“2',3'- camp -腺苷途径”。我们还发现细胞外2',3'-cAMP可显著增加啮齿动物和人类脑外伤后损伤（TBI）；当通路受损时，啮齿动物的创伤性脑损伤结果会恶化。细胞内2',3'-cAMP打开线粒体通透性过渡孔，而细胞外腺苷具有神经保护作用。因此，“2',3'- camp -腺苷途径”可能在TBI中很重要，因为它消除细胞内神经毒素（2',3'- camp的输出）并产生细胞外神经保护剂（2',3'- camp转化为腺苷）。我们还发现了神秘的髓磷脂蛋白2',3'-环核苷酸3'-磷酸二酯酶（CNPase）是将细胞外2',3'-cAMP代谢为2'-AMP的主要酶（转化为腺苷的关键步骤）。缺乏CNPase的KO小鼠在tbi后产生较少的细胞外腺苷，更容易受到损伤并随着年龄的增长发生轴突变性，尽管没有明显的髓鞘异常。假设：“2’，3’- camp -腺苷途径”是脑外伤后内源性细胞保护机制。我们将阐明哪些CNS细胞类型产生2',3'-cAMP，哪些损伤触发2',3'-cAMP的产生，2',3'-cAMP如何被运输出细胞，下游amp如何转化为腺苷，以及操纵2',3'-cAMP-腺苷途径是否会改变继发性损伤。特异性目的1：确定损伤后哪些CNS细胞类型产生2',3'-cAMP。由于体内TBI增加了细胞外2',3'-cAMP，因此确定哪些CNS细胞产生2',3'-cAMP以及这种作用是否依赖于损伤类型是很重要的。目的1将确定代谢应激、缺氧或机械损伤是否会增强星形胶质细胞、小胶质细胞、神经元或少突胶质细胞产生2',3'-cAMP。特异性目的2：确定MRP4是否介导2',3'-cAMP的表达。由于2',3'-cAMP是一种细胞内毒素，阐明2',3'-cAMP是如何从中枢神经系统细胞中挤出的至关重要。目的2将检验MRP4输出2',3'-cAMP的假设。特异性目的3：确定组织碱性磷酸酶（Tissue Alkaline Phosphatase， TAP）是否参与2'-AMP和3'- AMP （2',3'- camp的下游代谢物）向腺苷的胞外代谢。由于细胞外腺苷具有神经保护作用，因此了解细胞外2'-AMP和3'-AMP如何转化为细胞外腺苷是至关重要的。具体目的4：验证2',3'- camp -腺苷途径是脑外伤后内源性保护机制的假设。Aim 4将通过确定抑制或增强2',3'- camp -腺苷途径对TBI结果的影响，进一步验证2',3'- camp -腺苷途径具有细胞保护作用的假设。

DESCRIPTION (provided by applicant): The term "cAMP" usually refers to the second messenger 3',5'-cyclic adenosine monophosphate. We serendipitously discovered that organ systems can produce (from mRNA degradation) and export to the extracellular compartment a positional isomer of 3',5'-cAMP, namely 2',3'-cAMP. We showed that organ systems convert extracellular 2',3'-cAMP to 2'-AMP + 3'-AMP and can metabolize 2'-AMP and 3'-AMP to adenosine. We refer to this pathway as the "2',3'-cAMP-adenosine pathway." We also showed that extracellular 2',3'-cAMP increases greatly post-traumatic brain injury (TBI) in brain in rodents and humans; and that when the pathway is impaired, TBI outcomes worsen in rodents. Intracellular 2',3'-cAMP opens mitochondrial permeability transition pores while extracellular adenosine is neuroprotective. Thus the "2',3'- cAMP-adenosine pathway" may be important in TBI because it eliminates an intracellular neurotoxin (export of 2',3'-cAMP) and generates an extracellular neuroprotectant (conversion of 2',3'-cAMP to adenosine). We also identified the enigmatic myelin protein 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) to be the major enzyme that metabolizes extracellular 2',3'-cAMP to 2'-AMP (a key step toward conversion into adenosine). KO mice lacking CNPase produce less extracellular adenosine post-TBI, are more susceptible to injury and develop axonal degeneration with age despite no gross myelin abnormalities. Hypothesis: the "2',3'-cAMP- adenosine pathway" is an endogenous cytoprotective mechanism after TBI. We will elucidate which CNS cell types produce 2',3'-cAMP, what kinds of injury trigger 2',3'-cAMP production, how 2',3'-cAMP is transported out of cells, how downstream AMPs are converted to adenosine, and if manipulating the 2',3'-cAMP-adenosine pathway alters secondary damage. Specific Aim 1: To determine which CNS cell types produce 2',3'-cAMP after injury. Because in vivo TBI increases extracellular 2',3'-cAMP, it is important to determine which CNS cells produce 2',3'-cAMP and whether the effect is injury-type dependent. Aim 1 will determine if metabolic stress, hypoxia or mechanical injury enhances 2',3'-cAMP production by astrocytes, microglia, neurons or oligodendrocytes. Specific Aim 2: To determine whether Multidrug Resistance Protein 4 (MRP4) mediates egress of 2',3'-cAMP. Because 2',3'-cAMP is an intracellular toxin, it is critical to elucidate how 2',3'-cAMP is extrude from CNS cells. Aim 2 will test the hypothesis that MRP4 exports 2',3'-cAMP. Specific Aim 3: To determine if Tissue Alkaline Phosphatase (TAP) participates in the extracellular metabolism of 2'-AMP and 3'- AMP (downstream metabolites of 2',3'-cAMP) to adenosine. Because extracellular adenosine is neuroprotective it is essential to understand how extracellular 2'-AMP and 3'-AMP are converted to extracellular adenosine. Specific Aim 4: To test the hypothesis that the 2',3'-cAMP-adenosine pathway is an endogenous protective mechanism post-TBI. Aim 4 will further test the hypothesis that the 2',3'-cAMP- adenosine pathway is cytoprotective by determining the effect of inhibiting or augmenting it on TBI outcomes.