2,3 cAMP in Traumatic Brain Injury

说明书(申请人提供)：术语“cAMP”通常指第二信使3‘，5’-环状腺苷一磷酸。我们偶然发现，器官系统可以产生3‘，5’-cAMP的位置异构体，即2‘，3’-cAMP，并将其输出到细胞外隔室。我们发现，器官系统将胞外2‘，3’-cAMP转化为2‘-AMP 3’-AMP，并能将2‘-AMP和3’-AMP代谢为腺苷。我们将这一途径称为“2‘，3’-cAMP-腺苷途径”。我们还发现，在啮齿动物和人类的脑创伤后，细胞外2‘，3’-cAMP显著增加；当该通路受损时，啮齿动物的脑创伤结局恶化。细胞内2‘，3’-cAMP开放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”在脑损伤中可能是重要的，因为它消除了细胞内的神经毒素(2‘，3’-cAMP的输出)，并产生了细胞外的神经保护剂(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(CNPase)是将胞外2’，3‘-cAMP代谢成2’-AMP的主要酶(这是转化为腺苷的关键步骤)。缺乏CNPase的KO小鼠在脑损伤后产生的细胞外腺苷较少，更容易受到损伤，并随着年龄的增长而发展为轴突变性，尽管没有明显的髓鞘异常。假设：“2‘，3’-cAMP-腺苷途径”是脑外伤后的一种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，什么样的损伤触发2‘，3’-cAMP的产生，2‘，3’-cAMP是如何被转运出细胞的，下游的AMP是如何转化为腺苷的，以及操纵2‘，3’-cAMP-腺苷途径是否会改变二次损伤。具体目的1：确定哪些中枢神经系统细胞在损伤后产生2‘，3’-cAMP。由于在活体脑损伤后细胞外2‘，3’-cAMP水平升高，因此确定哪些中枢神经系统细胞产生2‘，3’-cAMP以及这种作用是否与损伤类型有关是很重要的。目的1确定代谢应激、低氧或机械损伤是否促进星形胶质细胞、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：确定多药耐药蛋白4(MRP4)是否介导2‘，3’-cAMP的外流。由于2‘，3’-cAMP是一种细胞内毒素，因此阐明2‘，3’-cAMP是如何从中枢神经系统细胞中排出的至关重要。目标2将检验MRP4输出2‘，3’-cAMP的假设。具体目的3：确定组织碱性磷酸酶(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。由于胞外腺苷具有神经保护作用，因此了解胞外2‘-AMP和3’-AMP如何转化为胞外腺苷是非常必要的。具体目的4：验证2‘，3’-cAMP-腺苷途径是脑创伤后内源性保护机制的假说。目的4将通过确定抑制或增强2‘，3’-cAMP-腺苷途径对脑损伤结局的影响来进一步检验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.