Role of CNPase in TBI

说明（由申请人提供）：术语“cAMP”普遍用于指3 '，5'-环腺苷一磷酸（3 '，5'-cAMP），由Earl Southerland博士发现的著名的“第二信使”。重要的是，绝大多数研究人员使用各种市售的测定试剂盒测量3 '，5'-cAMP，我们的小组是世界上唯一一个使用高效液相色谱-串联质谱法（LC-MS/MS）常规测量3 '，5'-cAMP的小组。杰克逊博士的实验室主要关注嘌呤在肾生理学/药理学中的作用，并常规使用LC-MS/MS而不是测定试剂盒来测量3 '，5'-cAMP。在与大脑无关的实验中，杰克逊博士的实验室偶然发现，肾脏产生的2 '，3'-cAMP（3 '，5'-cAMP的位置异构体）比3 '，5'-cAMP多。Kochanek博士的实验室主要专注于创伤性脑损伤（TBI）的机制，并发现嘌呤在保护大脑免受TBI方面发挥着重要作用。由于杰克逊博士在测量嘌呤方面的专业知识和Kochanek博士对嘌呤在TBI中的作用的兴趣，这些研究人员之间形成了自然和富有成效的合作。在肾脏中偶然发现2 '，3'-cAMP后，杰克逊博士和科查内克博士决定研究大脑中是否存在2 '，3'-cAMP。在初步研究中，对来自TBI患者的44个脑脊液（CSF）样品的LC-MS/MS分析显示，2 '，3'-cAMP是来自TBI患者的CSF中的“cAMP”的主要成分。除了2 '，3'-cAMP之外，还在这些相同的样品中测量了2 '-AMP、腺苷和肌苷（腺苷的代谢物）的水平。重要的是，在人CSF中2 '，3'-cAMP和2 '-AMP、2'-AMP和腺苷以及2 '-AMP和肌苷之间存在大的和显著的相关性。这些令人惊讶的发现表明迫切需要探索2 '，3'-cAMP在TBI病理生理学中的作用，特别是关于2 '，3'-cAMP作为腺苷前体的作用。因此，该探索性项目的目的是开始通过测试2 '，3'-cAMP参与“CNS神经保护机制”的创新概念来表征2 '，3'-cAMP在TBI中的意义，其中脑损伤导致2 '，3'-cAMP从mRNA释放，并且2 '，然后3 '-cAMP被CN代谢为2'-AMP，随后2 '-AMP转化为腺苷。 公共卫生相关性：创伤性脑损伤是平民和军人死亡率、发病率和终身损伤的主要来源，治疗选择有限，结局通常令人沮丧。在TBI过程中，CNV神经保护机制在产生细胞外腺苷方面可能是极其重要的，从而提供这种保护性的“报复性”代谢物以减轻细胞损伤。通过药物操纵这一机制可以为预防和治疗TBI提供新的方法。

DESCRIPTION (provided by applicant): The term "cAMP" is used universally to refer to 3',5'-cyclic adenosine monophosphate (3',5'- cAMP), the famous "second messenger" discovered by Dr. Earl Southerland. Importantly, the vast majority of investigators measure 3',5'-cAMP using various commercially available assay kits, and our group is the ONLY group in the world that routinely measures 3',5'-cAMP using high performance liquid chromatography-tandem mass spectrometry(LC-MS/MS). Dr. Jackson's lab focuses mostly on the role of purines in renal physiology/pharmacology, and routinely employs LC-MS/MS, rather than assay kits, to measure 3',5'-cAMP. In experiments unrelated to the brain, Dr. Jackson's lab serendipitously discovered that the kidney produces more 2',3'-cAMP (a positional isomer of 3',5'- cAMP) than 3',5'-cAMP. Dr. Kochanek's lab focuses primarily on mechanisms of traumatic brain injury (TBI), and has discovered that purines play a major role in protecting the brain from TBI. Because of Dr. Jackson's expertise in measuring purines and Dr. Kochanek's interest in the role of purines in TBI, a natural and productive collaboration evolved between these investigators. After the serendipitous discovery of 2',3'-cAMP in kidneys, Drs. Jackson and Kochanek decided to investigate whether 2',3'-cAMP exists in the brain. In preliminary studies, LC-MS/MS analysis of 44 samples of cerebral spinal fluid (CSF) from TBI patients showed that 2',3'-cAMP is a major constituent of "cAMP" in CSF from patients with TBI. In addition to 2',3'-cAMP, the levels of 2'-AMP, adenosine and inosine (metabolite of adenosine) were also measured in these same samples. Importantly, there was an large and significant correlation between 2',3'-cAMP and 2'-AMP, 2'-AMP and adenosine and 2'-AMP and inosine in human CSF. These surprising discoveries suggested a critical need to explore the role of 2',3'-cAMP in TBI pathophysiology, particularly with respect to the role of 2',3'-cAMP as a precursor for adenosine. Accordingly, the purpose of this exploratory project is to begin to characterize the significance of 2',3'-cAMP in TBI by testing the innovative concept that 2',3'-cAMP is involved in a "CNPase Neuroprotection Mechanism" in which brain injury leads to release of 2',3'-cAMP from mRNA, and 2',3'-cAMP is then metabolized to 2'-AMP by CNPase followed by conversion of 2'-AMP to adenosine. PUBLIC HEALTH RELEVANCE: TBI is a major source of mortality, morbidity and life-long impairment in both the civilian and military populations, with limited treatment options and generally dismal outcomes. The CNPase Neuroprotective Mechanism could be extremely important in producing extracellular adenosine during TBI, thus providing this protective, "retaliatory" metabolite to mitigate cellular damage. Manipulation of this mechanism by drugs could provide novel approaches to prevent and treat TBI.