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

直到最近，还没有帕金森病（PD）的动物模型充分结合了广泛的α-突触核蛋白（α-syn）病理学和持久的显着黑质纹状体变性。2012年，Luk及其同事描述了如何将合成的α-syn预形成的纤维（PFF）注射到野生型（WT）小鼠的纹状体内，接种路易体（LB）样细胞内α-syn包涵体的内源性积累和最终的黑质纹状体变性。鉴于大鼠模型系统相对于小鼠具有明显的优势（精细运动行为、更大的突触复杂性、遗传学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品收集），我们最近表征了将小鼠α-syn PFF单侧注射到大鼠纹状体中的结果。与小鼠相似，我们观察到磷酸化的α-syn神经元内积聚在几个区域，支配纹状体，最突出的是额叶和岛叶皮质，杏仁核，和黑质的丘脑部（SNpc）。α-Syn积聚与泛素、p62共定位，并且是硫磺素-S阳性和蛋白酶-k抗性。虽然α-syn包涵体内SNpc仍然同侧纹状体注射，我们观察到双边减少黑质多巴胺神经元后6个月的α-syn PFF注射。此外，注射PFF的大鼠表现出纹状体多巴胺能神经支配的减少以及纹状体多巴胺和代谢物的不足。该初步研究表明，α-syn PFF足以引发内源性α-syn的病理转化和传播，从而在大鼠中诱导α-突触核蛋白病的进行性神经退行性模型。在本IGNITE申请中，我们试图确定哪种α-syn物种（人、小鼠、大鼠）在纹状体内注射到大鼠后4个月内导致60%的黑质多巴胺神经元的持续损失（目的1）。一旦确定，我们将进行内部验证研究，以系统表征α-syn病理学的时间过程和程度、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（目标2）。最后，我们将进行PD相关的外部验证研究，包括：1）研究PFF诱导的运动缺陷是否可以用左旋多巴逆转，2）使用正电子发射断层扫描（PET）对黑质纹状体DA合成、储存和周转进行无创纵向体内成像（目的3）。我们建议在进行性大鼠α-syn PFF PD模型中进行优化、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，并促进新型疾病改善疗法的开发。

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.