AAV MEDIATED NEUROTROPHIC FACTOR GENE DELIVERY TO BRAIN

这项修订的拨款的目标是继续测试一些关于神经营养因子体细胞基因转移到中隔海马系统的假设。这些研究使用含有神经元特异性烯醇化酶启动子的腺相关病毒（AAV）重组载体来驱动神经生长因子（NGF）或脑源性神经营养因子（BDNF）的表达。最初的研究表明，标志基因绿色荧光蛋白（GFP）在转导后的中隔和海马中有较长时间的强劲表达，现在已经扩展到观察这一途径中营养因子的表达、胆碱能标记物的水平和trk受体密度。在研究的每个区域中，表达是神经特异性的。神经元亚群的一定程度的趋向性在海马体中也很明显，尽管在隔膜中不如此。本项目拟检测：1)nse驱动的GFP在不同海马和间隔神经元中是否存在剂量和时间依赖性表达；2)完整间隔和海马中BDNF和NGF的异位表达是否会升高胆碱能和trk标志物，但不会改变gaba能活性；3) BDNF和NGF基因转移到中隔是否优先保护轴突化的胆碱能神经元，这在海马或gaba能神经元中都没有发现；4)海马和中隔营养因子基因转移是否能改善部分穹穴病变动物的记忆相关行为；5)是否可以通过中隔NGF或BDNF基因转移来克服年龄相关的记忆相关行为和乙酰胆碱释放缺陷；6)衰老对转基因NGF表达的干扰是否大于对BDNF表达的干扰，如衰老过程中内源性营养因子调节的研究所预测的那样。这些研究可能最终导致与中隔海马胆碱能缺陷相关的疾病（如阿尔茨海默病）的基因治疗试验。

The goal of this revised grant continues to be testing several hypotheses about neurotropic factor somatic gene transfer into the septohippocampal system. These studies use adeno-associated virus (AAV) recombinant vectors containing the neuron specific enolase (NSE) promoter to drive expression of nerve growth factor (NGF) or brain derived neurotrophic factor (BDNF). Initial studies demonstrating robust expression of the marker gene green fluorescent protein (GFP) in septum and hippocampus for extended intervals after transduction have been extended to observations about trophic factor expression, levels of cholinergic markers and trk-receptor density in this pathway. Expression is neuro-specific in each region studied. Some degree of tropism for subpopulations o neurons is also apparent in hippocampus, though not so in septum. Our grant proposes to test: 1) whether there are dose- and time-dependent expressions of NSE-driven GFP expression in different hippocampal and septal neurons: 2) if ectopic BDNF and NGF expression in intact septum and hippocampus elevates cholinergic and trk markers without altering GABAergic activity; 3) if BDNF and NGF gene transfer into septum preferentially protects axotomized cholinergic neurons in a manner not seen with either similar gene transfer in hippocampus or with GABAergic neurons: 4) whether both hippocampal and septal trophic factor gene transfer can improve memory-related behaviors in animals receiving partial fornix lesions; 5) whether age-related deficits in memory-related behaviors and acetylcholine release can be overcome with septal NGF or BDNF gene transfer; and 6) if aging interferes with the expression of transgenic NGF more than that of BDNF, as predicted from studies of endogenous trophic factor regulation during senescence. These studies may lead eventually to gene therapy trials for conditions associated with septohippocampal cholinergic deficits such as Alzheimer's disease.