Effects of CAG/Qn Expansions on KGDHC and Other Enzymes

解释阿尔茨海默病（AD）中α -酮戊二酸脱氢酶复合物缺乏的意义的一个困难是，能量/氧化代谢受损也发生在其他神经退行性疾病中，包括CAGn/Qn疾病。该项目将测试一些可能在CAGn/Qn疾病中损害线粒体的机制，包括KGDHC。我们已经证明，组织转谷氨酰胺酶（tTGase；来自豚鼠肝脏）可以将Qn扩增物共价附着在KGDHC和甘油醛-3-磷酸脱氢酶上，使它们失活。组蛋白的tTGase作用使它们聚集。许多其他蛋白质都不是底物。线粒体损伤可能会损害钙的调节，从而激活（钙依赖的）谷氨酰胺转氨酶，导致恶性循环，即使在线粒体中没有发现在CAGn/Qn疾病中积累的谷氨酰胺转氨酶和聚集体。将进行六个系列的实验。(1)人脑转谷氨酰胺酶与广泛使用的豚鼠肝酶不同，将被纯化、表征、克隆和测序。（ii）由于转谷氨酰胺酶活性几乎不存在于人成纤维细胞中，其活性将通过提高胞质Ca2+、地塞米松诱导和可能通过转染而增加。细胞中异常的存在和时间将根据转谷氨酰胺酶在时间和酶诱导量方面的增加而确定。这些研究将澄清线粒体损伤（包括KGDHC）是否发生在该模型中，以及它是在其他细胞损伤证据之前还是之后。（iii）体外作为转谷氨酰胺酶底物的蛋白质（包括KGDHC）将在转基因CAGn/Qn小鼠的大脑中进行检测（与项目5一起）。这些研究将确定能量代谢成分（包括KGDHC）的变化是否发生在这些模型中，以及它们是在聚集体积累之前还是之后。（iv）合理设计的转谷氨酰胺酶抑制剂将在纯化蛋白、培养细胞和转基因动物中进行测试。(v)将在人体组织以及动物和培养细胞模型中测试epsilon-（γ -谷氨酰）赖氨酸作为CAGn/Qn紊乱标记物的效用。如果这些研究表明，CAGn/Qn疾病中的线粒体损伤是细胞内聚集物积累的结果，那么未来的研究将关注线粒体（包括KGDHC）对AD和其他神经退行性疾病中积聚的聚集物的影响。

A difficulty in interpreting the significance of the well-documented deficiency of the alpha-ketoglutarate dehydrogenase complex in Alzheimer's Disease (AD) is that impaired energy/oxidative metabolism also occurs in other neurodegenerative disorders including the CAGn/Qn disorders. This project will test a number of mechanisms that might damage mitochondria, including KGDHC, in the CAGn/Qn disorders. We have shown that the enzyme tissue transglutaminase (tTGase; from guinea pig liver) can covalently attach Qn expansions to KGDHC and to glyceraldehyde-3-phosphate dehydrogenase, inactivating them. tTGase action of histone aggregates them. A variety of other proteins were not substrates. Mitochondrial damage might impair calcium regulation and thereby activate (calcium-dependent) transglutaminase, leading to a vicious spiral, even though transglutaminase and the aggregates which accumulate in the CAGn/Qn disorder are not found in mitochondria. Six series of experiments will be done. (I) The human brain transglutaminase(s), which appear to differ from the widely used guinea pig liver enzyme, will be purified, characterized, cloned, and sequenced. (ii) Since transglutaminase activity is almost absent from human fibroblasts, its activity will be increased by raising cytosolic Ca2+, by dexamethasone induction, and possibly by transfection. The presence and timing of abnormalities in the cells will be determined in relation to the increases in transglutaminase in terms of both time and quantity of enzyme induced. These studies will clarify whether damage to mitochondria (including to KGDHC) occurs in this model and whether it precedes or follows other evidence of cell damage. (iii) Proteins which are transglutaminase substrates in vitro (including KGDHC) will be examined in the brains of transgenic CAGn/Qn mice (with Project 5). These studies will determine whether changes in constituents of energy metabolism (including KGDHC) occur in these models and whether they precede or follow accumulation of aggregates. (iv) Rationally designed transglutaminase inhibitors will be tested with purified proteins, cultured cells, and in transgenic animals. (v) The utility of epsilon-(gamma- glutamyl)lysine as a marker for CAGn/Qn disorders will be tested, in human tissues as well as in animal and cultured cell models. If these studies indicate that mitochondrial damage in the CAGn/Qn disorders is a consequence of the accumulation of intracellular aggregates, future studies will focus on the effects of mitochondria (including KGDHC) of the aggregates that accumulate in AD as well as those in other neurodegeneration.