AAV MEDIATED NEUROTROPHIC FACTOR GENE DELIVERY TO BRAIN

这项修订后的拨款的目标仍然是检验几个关于神经营养因子体细胞基因转移到隔海马区系统的假说。这些研究使用腺相关病毒(AAV)重组载体(含有神经元特异性烯醇化酶(NSE)启动子)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)在转导后的隔区和海马区有较强的表达，现已扩展到对这一途径中营养因子的表达、胆碱能标志物的水平和Trk受体密度的观察。在所研究的每个区域中，表达都是神经元特有的。神经元亚群的某种程度的趋向性在海马区也是明显的，尽管在隔区不是这样。我们的研究建议测试：1)NSE驱动的GFP在不同的海马神经元和隔区神经元中是否存在剂量和时间依赖性的表达：2)在完整的隔区和海马区异位表达BDNF和NGF是否在不改变GABA能活性的情况下提高胆碱能和Trk标记物；3)BDNF和NGF基因转移到隔区是否以一种类似的基因转移或GABA能神经元所未见的方式优先保护切断的胆碱能神经元：4)海马和隔区营养因子基因转移是否能改善部分穹隆损伤动物的记忆相关行为；5)神经生长因子或脑源性神经营养因子基因转移能否克服与年龄相关的记忆相关行为和乙酰胆碱释放缺陷；6)衰老对转基因神经生长因子表达的干扰是否大于脑源性神经营养因子对衰老过程中内源性营养因子调节的影响。这些研究可能最终导致与隔-海马胆碱能缺陷相关的疾病的基因治疗试验，如阿尔茨海默病。

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.