Regulation of neurotrophin expression in the periphery

描述(申请人提供)：本研究的基本假设是周围神经元的存活受神经营养因子神经生长因子(NGF)和神经营养因子-3(NT-3)的调节。调节这些神经营养因子表达的机制还知之甚少。NGF基因产物的加工产生前神经营养因子，经过蛋白水解酶的裂解，形成成熟的NGF形式。然而，使用Western印迹分析，我们在周围神经元或靶组织中观察到非常少的成熟NGF形式。事实上，在这些组织(颈上神经节、三叉神经节和外周靶点)中表达的神经营养蛋白大多是proNGF和大分子NGF前体，每个组织都表现出特有的NGF表达模式。NT-3似乎经历了类似的过程，尽管它还没有得到很好的研究。目前项目的一个目标是使用NGF和NT-3 Western分析来将组织特异性NGF和NT-3的表达模式与使用ELISA获得的蛋白质检测相关联，并确定体内脑室内注射NGF两周的任何影响。此外，利用实时定量RT-PCR技术，我们检测到外源性NGF和NT-3在外周神经节中的转录，两者都受到外源性NGF和神经元活动的影响。本研究的主要目的是：1)研究交感、感觉神经节和周围靶组织中NGF蛋白和mRNA的表达，并确定外源性NGF在体内的作用；2)确定外源性NGF对周围神经节和靶组织中NT-3蛋白和mRNA表达的影响；3)阐明神经元活动在NGF和周围组织中NT-3表达中的作用；以及4)检测交感神经支配对周围靶组织中神经营养素表达的调节作用。该项目的结果将有助于我们理解调节神经营养素表达的复杂机制，从而有助于确定神经元在衰老和疾病中死亡的原因。

DESCRIPTION (provided by applicant): The underlying hypothesis of the present study is that the survival of peripheral neurons is regulated by the neurotrophins nerve growth factor (NGF) and neurotrophin-3 (NT-3). The mechanisms regulating the expression of these neurotrophins are poorly understood. The processing of the NGF gene product results in preproneurotrophin which, following proteolytic cleavage, results in the mature form of NGF. Yet, using Western blot analysis, we observed very little mature NGF form in peripheral neurons or target tissues. In fact, the majority of neurotrophic protein expressed in these tissues (superior cervical ganglion, trigeminal ganglion and peripheral targets) was proNGF along with large molecular weight NGF precursors, with each tissue exhibiting a characteristic NGF expression pattern. NT-3 appears to undergo similar processing though it is not well studied. One goal of the current project is to use NGF and NT-3 Western analysis to correlate tissue-specific NGF and NT-3 expression patterns with protein detection obtained using ELISA and to determine any influences of a two week in vivo intracerebroventricular infusion of NGF. Also, using real time RT-PCR, we detect NGF and NT-3 transcripts in peripheral ganglia, both of which are influenced by exogenous NGF and neuronal activity. The primary objectives of this study are to: 1) Investigate NGF protein and mRNA expression in sympathetic and sensory ganglia and peripheral target tissues and determine any effects of in vivo exogenous NGF; 2) Determine the effects of exogenous NGF on NT-3 protein and mRNA expression in peripheral ganglia and targets; 3) Delineate the role of neuronal activity in NGF and NT-3 expression in peripheral tissues; and 4) Examine the regulatory influences of sympathetic innervation on neurotrophin expression in peripheral targets. The results of this project will contribute to our understanding of the complex mechanisms regulating neurotrophin expression, and thus neuronal survival, and will help to determine the factors responsible for the neuronal death that occurs in aging and disease.