Modeling and Pathological Study of Sporadic Parkinson's Disease

1.LRRK2与Tau在帕金森病发病中的相互作用。 为了研究LRRK2和tau在神经退行性变中的潜在致病相互作用，我们将CaMKII-TTA/Teto-G2019S LRRK2双转基因小鼠与tau转基因小鼠品系PRNP-tau B6D2-TG(PRNP-MAPT)43Vle/J杂交，产生CaMKII-TTA/Teto-G2019S/PRNP-tau三重转基因小鼠，在大脑皮层、纹状体和海马区的神经元中共表达人LRRK2和tau。我们发现，人G2019S LRRK2和野生型tau的共同表达进一步降低了脑匀浆中游离微管蛋白的水平。同时，抗tau抗体免疫染色显示，CaMKII-TTA/Teto-G2019S/PRNP-tau三重转基因小鼠皮质中tau阳性聚集体的形成增加。这些初步数据表明，LRRK2可能调节tau的功能和聚集，并且LRRK2和tau之间的协同作用可能进一步损害神经元的微管动力学。为了进一步研究LRRK2和tau在中脑多巴胺能神经元中是否有协同作用，我们建立了一组PITX3-TTA/Teto-LRRK2WT/Teto-Tau WT转基因小鼠，其中野生型人LRRK2和tau由中脑DA神经元选择性表达。在下一财年，我们将对这些小鼠进行行为、病理和神经化学研究。同时，我们还将研究受影响的DA神经元的亚细胞表型，特别是轴突运输和多巴胺的释放。 2.α-突触核蛋白与LRRK2在帕金森病发病中的相互作用。 我们先前已经证明，LRRK2的过度表达加剧了帕金森病相关的A53Tα-突触核蛋白介导的前脑区神经变性；而抑制LRRK2的表达则改善了α-突触核蛋白诱导的神经元丢失。然而，目前尚不清楚LRRK2和α-突触核蛋白在中脑DA神经元中的作用方式是否相同。为了解决这一问题，我们建立了PITX3-TTA/Teto-LRRK2/Teto-α-突触核蛋白三重转基因小鼠，以允许在中脑DA神经元中选择性表达人类野生型和PD相关突变体LRRK2(G2019)和α-突触核蛋白(A53T)。此外，我们还在LRRK2基因敲除背景下培育了PITX3/A53T小鼠。我们系统地检测了α-突触核蛋白和LRRK2突变小鼠在1个月和18个月龄时中脑DA神经元的数量。我们的初步数据表明，野生型和G2019S LRRK2的过度表达加速了1月龄小鼠A53Tα-突触核蛋白介导的DA神经元的丢失。相反，LRRK2的缺失改善了A53Tα-突触核蛋白诱导的DA神经变性。在下一个财政年度，我们将产生一个新的野生型LRRK2和α-突触核蛋白转基因小鼠队列，以研究野生型α-突触核蛋白和LRRK2的上调是否足以导致DA神经元丢失，以及潜在的致病机制。本研究为研究散发性帕金森病的病因和治疗提供了一种有用的动物模型。 3.α-突触核蛋白与tau在帕金森病发病中的相互作用。 已有研究表明，α-突触核蛋白和tau的过度表达极大地加速了它们在体外的聚集。我们决定评估α-突触核蛋白和tau相互作用在中脑DA神经元中的致病后果。与先林博士的实验室一起，我们将建立PITX3/α-突触核蛋白/tau三重转基因小鼠，以研究α-突触核蛋白和LRRK2之间潜在的致病相互作用，方法与上面概述的相同。 4.野生型α-突触核蛋白、LRRK2和tau在帕金森病发病中的相互作用。 最后，我们计划建立PITX3/α-突触核蛋白/LRRK2/tau复合转基因小鼠，以探讨这三种遗传危险因素在中脑DA神经元变性中的作用。本研究为研究散发性帕金森病的病因和治疗提供了一种有用的动物模型。

1. Pathogenic Interplay between LRRK2 and Tau in PD. To examine a potential pathogenic interplay between LRRK2 and tau in neurodegeneration, we crossbred CaMKII-tTA/tetO-G2019S LRRK2 double transgenic mice with a line of tau transgenic mice Prnp-tau B6D2-Tg(Prnp-MAPT)43Vle/J to generate CaMKII-tTA/tetO-G2019S/Prnp-tau triple transgenic mice that co-expressed human LRRK2 and tau in neurons of forebrain regions such as cerebral cortex, striatum and hippocampus. We found that co-expression of human G2019S LRRK2 and wild-type tau further decreased the level of free tubulin in the brain homogenate. Meanwhile, the formation of tau-positive aggregates was increased in the cortex of CaMKII-tTA/tetO-G2019S/Prnp-tau triple transgenic mice as revealed by immunostaining with antibodies against tau. These preliminary data indicate that LRRK2 may regulate the function and aggregation of tau and a synergistic interaction between LRRK2 and tau may further impair the microtubule dynamics in neurons. To further investigate whether LRRK2 and tau act synergistically in the midbrain dopaminergic (DA) neurons, we have generated a cohort of PITX3-tTA/tetO-LRRK2 WT/tetO-Tau WT transgenic mice in which wild-type human LRRK2 and tau are selectively expressed by the midbrain DA neurons. In the coming fiscal year, we will carry out behavioral, pathological, neurochemical studies of these mice. Meanwhile, we will also examine the subcellular phenotype in the affected DA neurons, particularly on the axonal transport and dopamine release. 2. Pathogenic Interplay between alpha-synuclein and LRRK2 in PD. We have previously shown that over-expression of LRRK2 exacerbates PD-related A53T alpha-synuclein-mediated neurodegeneration in the forebrain regions; while inhibition of LRRK2 expression ameliorates the alpha-synuclein-induced neuronal loss. However, it remains unclear whether LRRK2 and alpha-synuclein act the same way in the midbrain DA neurons. To address this question, we generate PITX3-tTA/tetO-LRRK2/tetO-alpha-synuclein triple transgenic mice to allow a selective expression of human wild-type as well as PD-related mutant LRRK2 (G2019) and alpha-synuclein (A53T) in the midbrain DA neurons. In addition, we also bred PITX3/A53T mice in the LRRK2 knockout background. We have systematically examined the numbers of midbrain DA neurons in the alpha-synuclein and LRRK2 mutant mice at one and eighteen months of age. Our preliminary data indicate that over-expression of both wild-type and G2019S LRRK2 accelerated A53T alpha-synuclein-mediated DA neuron loss in 1-month old mice. By contrast, loss of LRRK2 ameliorated A53T alpha-synuclein-induced DA neurodegeneration. In the coming fiscal year, we will generate a new cohort of wild-type LRRK2 and alpha-synuclein transgenic mice in order to investigate if the up-regulation of both wild-type alpha-synuclein and LRRK2 is sufficient to cause DA neuronal loss, and the underlying pathogenic mechanisms. This study may provide a useful animal model for studying the cause and treatment of sporadic PD. 3. Pathogenic Interplay between alpha-synuclein and tau in PD. It has been shown previously that over-expression of alpha-synuclein and tau greatly accelerate their aggregation in vitro. We decided to evaluate the pathogenic consequence of alpha-synuclein and tau interaction in the midbrain DA neurons. Together with Dr. Xian Lin's lab, we will generate PITX3/alpha-synuclein/tau triple transgenic mice to study the potential pathogenic interplay between alpha-synuclein and LRRK2 using the same strategy as we outlined the above. 4. Pathogenic Interplay between Wild-type alpha-synuclein, LRRK2, and tau in PD. Finally, we plan to develop PITX3/alpha-synuclein/LRRK2/tau compound transgenic mice to investigate the contribution of these three genetic risk factors in the degeneration of midbrain DA neurons. This study may provide a useful animal model for studying the cause and treatment of sporadic PD.