The Rat Pre-Formed Alpha-Synuclein Fibril Model of Parkinson's Disease

直到最近，还没有一种帕金森病（PD）的动物模型充分结合了广泛的α-突触核蛋白（α-syn）病理和长期显著的黑质纹状体变性。2012年，Luk及其同事描述了将合成α-syn预形成原纤维（pff）注射到野生型（WT）小鼠的纹状体内，如何导致内源性路易体（LB）样细胞内α-syn包涵体的积累和最终的黑质纹状体变性。鉴于大鼠模型系统比小鼠具有明显的优势（精细的运动行为，更大的突触复杂性，遗传和药代动力学更类似于人类，更大的大脑和身体尺寸更适合神经外科干预和样本收集），我们最近描述了将小鼠α-syn pff单侧注射到大鼠纹状体中的结果。与小鼠相似，我们在纹状体的几个神经支配区域观察到磷酸化的α-syn在神经元内的积累，最突出的是额叶和岛叶皮层、杏仁核和黑质致密部（SNpc）。α-Syn积累与泛素p62共定位，并呈硫黄素- s阳性和蛋白酶k抗性。尽管纹状体注射后SNpc内的α-syn包涵体仍在同侧，但我们观察到注射α-syn PFF 6个月后，双侧黑质多巴胺神经元减少。此外，注射PFF的大鼠纹状体多巴胺能神经支配减少，纹状体多巴胺及其代谢物缺失。本初步研究表明，α-syn pff足以诱导内源性α-syn的病理转化和繁殖，从而诱导大鼠α-突触核蛋白病的进行性、神经退行性模型。在目前的IGNITE应用程序中，我们试图确定哪种α-syn物种（人类、小鼠、大鼠）在大鼠纹状体内注射后4个月内导致60%的黑质多巴胺神经元持续损失（目的1）。一旦确定，我们将进行内部验证研究，以系统地表征α-syn病理、纹状体多巴胺和代谢物损失、纹状体多巴胺能神经支配（酪氨酸羟化酶、囊泡单胺转运蛋白和多巴胺转运蛋白）水平和运动行为缺陷的时间过程和程度（目的2）。最后，我们将进行pd相关的外部验证研究，包括：1)调查pff诱导的运动缺陷是否可以通过左旋多巴逆转；2)利用正电子发射断层扫描（PET）对黑质纹状体DA合成、储存和转换进行无创的体内纵向成像（Aim 3）。我们建议对进行性大鼠α-syn PFF PD模型进行优化、内部验证和外部验证研究，以便与PD患者的临床指标进行直接比较，并促进新型疾病修饰疗法的开发。

Until recently, no animal model of Parkinson's disease (PD) has adequately incorporated both widespread alpha-synuclein (α-syn) pathology and protracted significant nigrostriatal degeneration. In 2012, Luk and colleagues described how intrastriatal injection of synthetic α-syn preformed fibrils (PFFs) into wildtype (WT) mice seeded endogenous accumulation of Lewy Body (LB)-like intracellular α-syn inclusions and ultimate nigrostriatal degeneration. In light of the fact that the rat model system offers distinct advantages over mice (fine motor behaviors, greater synaptic complexity, genetics and pharmacokinetics more similar to humans, larger brain and body size more amenable to neurosurgical interventions and sample collection), we recently characterized the results of unilateral injection of mouse α-syn PFFs into the striatum of rats. Similar to the mouse, we observed phosphorylated α-syn intraneuronal accumulations in several areas that innervate the striatum, most prominently the frontal and insular cortices, the amygdala, and the substantia nigra pars compacta (SNpc). α-Syn accumulations co-localized with ubiquitin, p62, and were thioflavin-S-positive and proteinase-k resistant. Although α-syn inclusions within the SNpc remained ipsilateral to striatal injection, we observed bilateral reductions in nigral dopamine neurons 6 months following α-syn PFF injection. Further, PFF injected rats exhibited reductions in striatal dopaminergic innervation as well as deficits in striatal dopamine and metabolites. This initial study demonstrates that α-syn PFFs are sufficient to seed the pathological conversion and propagation of endogenous α-syn to induce a progressive, neurodegenerative model of α-synucleinopathy in rats. In the present IGNITE application we seek to identify which α-syn species (human, mouse, rat) results in consistent loss of 60% nigral dopamine neurons over the course of 4 months after intrastriatal injection into rats (Aim 1). Once identified, we will conduct internal validation studies to systematically characterize the time course and magnitude of α-syn pathology, striatal dopamine and metabolite loss, levels of striatal dopaminergic innervation (tyrosine hydroxylase, vesicular monoamine transporter, and dopamine transporter) and deficits in motor behaviors (Aim 2). Lastly, we will conduct PD-relevant external validation studies including: 1) investigating whether PFF-induced motor deficits are reversible with levodopa and 2) conduct non-invasive longitudinal in vivo imaging of nigrostriatal DA synthesis, storage and turnover using positron emission tomography (PET) (Aim 3). We propose that optimization, internal validation and external validation studies in the progressive rat α-syn PFF PD model will allow for direct comparison to clinical metrics in PD patients and facilitate the development of novel disease modifying therapies.