A New Tool for Targeted Antisense Knockdown in Brain

描述（由申请人提供）： 该项目的目标是开发一种新的工具，用于下调成年大脑中选择性靶向细胞中特定蛋白质的表达。该方法将使用抗体靶向的反义PNA（肽核酸）来选择性地敲低基底前脑中靶向的p75 NTR表达细胞内的神经生长因子受体TrkA的表达。我们的长期目标是使用该方法来检验以下假设：TrkA的持续减少，特别是内侧隔和基底核大细胞胆碱能神经元内的减少，是基底前脑胆碱能功能和认知能力与年龄相关的下降的原因。一旦开发出来，这种工具可以用来选择性地敲低这些胆碱能神经元内的各种蛋白质（例如，神经递质受体、囊泡转运蛋白、离子通道等），以及在大脑和外周的其他部分内的p75 NTR表达神经元中，以评估它们在神经变性疾病和与年龄相关的认知衰退中的功能作用。类似的方法可以用于调节其他（非p75 NTR表达）细胞类型中的蛋白质，这些细胞类型适合于抗体靶向。研究将使用已建立的方法将荧光标记的PNA与抗小鼠p75 NTR（小鼠-192-IgG）的抗体偶联。结合物将通过TOF质谱法和体外结合试验进行表征。PNA将通过脑室直接递送到大鼠脑中。将通过荧光显微镜结合胆碱乙酰转移酶（ChAT）、p75 NTR和TrkA蛋白的免疫组织化学检测来评价基底前脑中胆碱能神经元对PNA的摄取。然后，研究将使用标准Nissl染色以及神经胶质细胞酸性蛋白的免疫组织化学检测和小神经胶质细胞浸润的组织化学检测来评估抗体-PNA缀合物对细胞活力的影响。通过Western印迹、125 I-NGF从海马到内侧隔的逆行转运以及测量隔和海马中的ChAT活性，评价对TrkA表达、NGF结合和胆碱能功能的影响。这将是第一次开发出靶向反义敲低方法，并用于选择性地调节成人大脑中已鉴定的神经元亚群内的基因表达。

DESCRIPTION (provided by applicant): The goal of this project is to develop a new tool for down regulating the expression of specific proteins in selectively targeted cells in the adult brain. The approach will use antibody-targeted antisense PNA (peptide nucleic acid) to selectively knock down expression of the nerve growth factor receptor TrkA within targeted p75NTR-expressing cells in the basal forebrain. Our long-term goal is to use the method to test the hypothesis that sustained reductions in TrkA, specifically within cholinergic neurons in the medial septum and nucleus basalis magnocellularis, are responsible for age-related declines in basal forebrain cholinergic function and cognitive performance. Once developed, this tool could be used to selectively knock down a wide range of proteins within these cholinergic neurons (e.g., neurotransmitter receptors, vesicular transporters, ion channels, etc...), as well as in p75NTR-expressing neurons within other parts of the brain and periphery, in order to assess their functional role in neurodegenerative diseases and age-related cognitive decline. Similar methods could be used to regulate proteins in other (non-p75NTR-expressing) cell types that are amenable to antibody targeting. Studies will use established methods to conjugate fluorescently-tagged PNA to an antibody raised against mouse p75NTR (mouse-192-IgG). Conjugates will be characterized by TOF mass spectrometry and by in vitro binding assays. PNAs will be delivered intraventricularly directly into the rat brain. Uptake of the PNA by cholinergic neurons in the basal forebrain will be evaluated by fluorescence microscopy, combined with immunohistochemical detection of choline acetyltransferase (ChAT), p75NTR, and TrkA protein. Studies will then evaluate the effects of antibody-PNA conjugates on cell viability using standard Nissl stain, as well as immunohistochemical detection of glial fibrillary acidic protein, and histochemical detection of microglial infiltration. Effects on TrkA expression, NGF binding, and cholinergic function will be evaluated by Western blot, by retrograde transport of 125I-NGF from the hippocampus to the medial septum, and by measuring ChAT activity in the septum and hippocampus. This will be the first time that a targeted antisense knockdown approach will have been developed and used to selectively regulate gene expression within an identified subpopulation of neurons in the adult brain.