In vivo protein complementation demonstrates direct detection of presynaptic α-synuclein oligomerization and age-dependent accumulation of 8-16mer oligomer species

Intracellular accumulation of a-synuclein (a-syn) and formation of Lewy bodies are neuropathological characteristics of Parkinson‘s disease (PD), related a-synucleinopathies, and other neurodegenerative diseases. Recent evidence suggests that oligomerization and spreading of a-syn from neuron to neuron are key events contributing to the development of PD. To directly visualize and characterize a-syn oligomerization and spreading in vivo, we generated two independent conditional transgenic mouse models based on a-syn protein complementation assays using both split Gaussia luciferase and split Venus YFP. These inducible, neuron-specific transgenic mice allow to directly assess the quantity and subcellular distribution of a-syn oligomers in vivo. Using these innovative mouse models we demonstrate an age dependent accumulation of a specific subtype of a-syn oligomers and their synaptic localization in vivo. We provide in vivo evidence that although a-syn is found throughout neurons a-syn oligomerization takes place at the presynapse. Furthermore, our new mouse models provide strong evidence for a long distance transsynaptic cell-to-cell transfer of de novo generated a-syn oligomers in vivo. Brain total-RNA profiles of mice were generated by RNA-Seq, in 3/4 replicates, using Illumina Hiseq-2500.

α-突触核蛋白（α-synuclein, α-syn）的细胞内聚集与路易小体（Lewy bodies）的形成，是帕金森病（Parkinson’s disease, PD）、相关性α-突触核蛋白病以及其他神经退行性疾病的核心神经病理学特征。新近研究证据表明，α-syn的寡聚化过程及其在神经元间的播散，是推动帕金森病发生发展的关键事件。为实现在活体状态下直接可视化并解析α-syn的寡聚化与播散机制，我们基于分裂型高斯荧光素酶（split Gaussia luciferase）与分裂型Venus黄色荧光蛋白（split Venus YFP）构建的蛋白质互补检测体系，开发了两种独立的条件性转基因小鼠模型。该类可诱导的神经元特异性转基因小鼠，可在活体条件下直接检测α-syn寡聚体的数量与亚细胞分布特征。借助这套创新性小鼠模型，我们证实了特定亚型α-syn寡聚体的年龄依赖性聚集，及其在活体中的突触定位特征。本研究提供的活体证据表明：尽管α-syn广泛分布于神经元中，但其寡聚化过程仅特异性发生于突触前位点。此外，我们的新型小鼠模型为新生α-syn寡聚体在活体中发生的长距离跨突触细胞间转移，提供了强有力的实验依据。本研究通过Illumina Hiseq-2500平台开展RNA测序（RNA-Seq），对小鼠全脑总RNA进行了3次或4次生物学重复的转录组分析。