2,3 cAMP in Traumatic Brain Injury

说明（由申请人提供）：术语“cAMP”通常是指第二信使3 '，5'-环腺苷一磷酸。我们偶然发现，器官系统可以产生（从mRNA降解）并输出到细胞外室的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，并且可以将2 '-AMP和3'-AMP代谢为腺苷。我们将该途径称为“2 '，3'-cAMP-腺苷途径。“我们还表明，细胞外2 '，3'-cAMP大大增加了啮齿动物和人类大脑中的创伤后脑损伤（TBI）;当该途径受损时，啮齿动物的TBI结果会恶化。细胞内2 '，3'-cAMP打开线粒体通透性转换孔，而细胞外腺苷具有神经保护作用。因此，“2 '，3'-cAMP-腺苷途径”在TBI中可能是重要的，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）。我们还确定了神秘的髓鞘蛋白2 '，3'-环核苷酸3 '-磷酸二酯酶（CNE 3）是将细胞外2'，3 '-cAMP代谢为2'-AMP（转化为腺苷的关键步骤）的主要酶。敲除小鼠缺乏CNclase产生更少的细胞外腺苷后TBI，更容易受到损伤，并发展轴突变性与年龄，尽管没有严重的髓鞘异常。假设：“2 '，3'-cAMP-腺苷途径”是TBI后的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，什么类型的损伤触发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。具体目的1：确定哪些CNS细胞类型在损伤后产生2 '，3'-cAMP。因为体内TBI增加细胞外2 '，3'-cAMP，所以重要的是确定哪些CNS细胞产生2 '，3'-cAMP以及该作用是否是损伤类型依赖性的。目的1将确定代谢应激、缺氧或机械损伤是否增强星形胶质细胞、小胶质细胞、神经元或少突胶质细胞的2 '，3'-cAMP产生。具体目的2：确定多药耐药蛋白4（MRP 4）是否介导2 '，3'-cAMP的排出。由于2 '，3'-cAMP是一种细胞内毒素，因此阐明2 '，3'-cAMP是如何从CNS细胞中挤出的至关重要。目的2将检验MRP 4输出2 '，3'-cAMP的假设。具体目标3：确定组织碱性磷酸酶（TAP）是否参与细胞外2 '-AMP和3'-AMP（2 '，3'-cAMP的下游代谢产物）代谢为腺苷。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至关重要。具体目的4：检验2 '，3'-cAMP-腺苷途径是TBI后内源性保护机制的假设。目的4将通过确定抑制或增强2 '，3'-cAMP-腺苷途径对TBI结果的影响来进一步检验2 '，3'-cAMP-腺苷途径具有细胞保护性的假设。

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.