NMR STUDIES OF TRANSGENIC MOUSE MODELS OF HUNTINGTONS DISEASE & ALS

该项目需要测量代谢物水平(‘H和 “c)在转基因小鼠的脑提取液中 肌萎缩侧索硬化症(ALS)和亨廷顿病(HD)。 这些代谢物水平然后被用来在体内补充 使用夫人测定许多相同的化学物质我们的目标是 确定TCA循环代谢是否存在缺陷 这些小鼠的神经元-神经胶质细胞循环。谷氨酸水平升高 通常与肌萎缩侧索硬化症有关，NAA水平的大幅下降通常是 以高清模式显示。我们用体内MRS(4.7T)对小鼠进行了纵向研究 在MGH)，然后在不同的时间间隔提取大脑 500 MHz磁体的体外核磁共振波谱研究 实验室。从这些提取物中，我们确定了绝对的 用一维和二维质子光谱测定代谢物的浓度。我们 还利用直接和间接观测的碳-13光谱来 测定小鼠体内三氯乙酸循环代谢产物的分级富集度 在献祭前注入标记的葡萄糖。这些也是 与在体内获得的动态碳-13谱相比， 局部的大脑区域。然后将核磁共振数据与高效液相色谱进行关联。 对一些相同的化合物进行测量以确定其准确性 并提供对结果的绝对量化 代谢物水平。此外，我们还进行了组织化学和 对相同动物进行组织学检查，以确定 生化缺陷伴发其他神经病变。结果是 到目前为止，我们已经得到了NAA的深刻干扰 HD动物神经元的代谢，尽管事实是 组织学检查显示神经元丢失很少。此外，还有一个 这些小鼠体内谷氨酸-谷氨酰胺循环严重失衡。这个 NAA扰动随着时间的推移呈指数级增长。在ALS小鼠身上， 相比之下，我们发现NAA代谢的变化很小，而 发现谷氨酸含量大幅增加。这些结果清楚地表明 核磁共振波谱有可能产生有价值的见解 这些疾病的基本生化病因学。

This project entails measurement of metabolite levels (both 'H and "C) in brain extracts of transgenic mice that are models for amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD). These metabolite levels are then used to complement in vivo determination of many of the same chemicals using MRS. Our goal is to determine if there are defects in TCA cycle metabolism and neuronal-glial cycling in these mice. Elevated glutamate levels have often been implicated in ALS and profound decreases in NAA are often seen in HD. We study the mice longitudinally using in vivo MRS (4.7T at MGH) and then take extracts of brains at various time intervals for study using in vitro NMR spectroscopy at 500 MHz at the Magnet Laboratory. From these extracts, we determine the absolute concentrations of metabolites using 1D and 2D proton spectroscopy. We also utilize direct and indirect observe carbon-13 spectroscopy to determine fractional enrichements of TCA cycle metabolites from mice infused with labeled glucose prior to sacrifice. These are also compared to the in vivo dynamic carbon-13 spectroscopy obtained in localized brain regions. The NMR data is then correlated with HPLC measurements of some of the same compounds to ascertain the accuracy of the results and to provide absolute quantification of the metabolite levels. In addition, we have performed histochemistry and histology on the same animals to determine the correlation of the biochemical defects with other neuropathological changes. The results we have obtained so far indicate a profound disturbance of NAA metabolism in the neurons of the HD animals in spite of the fact that histology shows very little neuronal loss. In addition, there is a profound imbalance in glutamate-glutamine cycling in these mice. The NAA disturbance progresses exponentially over time. In the ALS mice, in contrast, we have found very small changes in NAA metabolism while finding large increases in glutamate. These results clearly indicate that NMR spectroscopy has the potential to yield valuable insight intoi the basic biochemical etiology of these diseases.