ASC oligomerization and transmission as an initiating event for protein aggregation in Synucleinopathy Dementias

突触核变性痴呆是一种与年龄相关的进行性神经退行性疾病 其特征是富含α-突触核蛋白(αSyn)的神经元内含物，称为路易小体(Lbs)。Synn--Dementias 包括路易体痴呆症(DLB)、帕金森病痴呆症(PDD)以及 阿尔茨海默病(AD)以LB型病变为主，约占AD病例的40%。持续期 神经炎症(主要由小胶质细胞介导)和进行性α突触聚集是突触的特征 痴呆症。我们最近描述了NOD样受体蛋白-3(NLRP3)炎症体的激活。 小鼠突触核素病模型和人类帕金森病模型。NLRP3炎症体是一种多蛋白臂 先天免疫系统，当被激活时，会产生细胞因子白介素1β(IL-β 1β)，以及含有半胱氨酸天冬氨酸酶招募结构域的寡聚化的凋亡相关斑点样蛋白 (ASC)，这是NLRP3炎症体的接头蛋白。目前还不清楚是什么病理作用 炎性小体的激活可能在突发性痴呆症的进展中起作用。我们的初步数据表明 小胶质细胞释放的ASC可能被神经元摄取，并可能交叉诱导α同步纤化。最重要的是 该建议的目的是阐明小胶质细胞在神经细胞传递中的病理作用。 寡聚化的Asc促进了Syn-dementias的进行性αSYN聚集。我们还寻求 阐明小胶质细胞中导致ASC寡聚和释放的信号机制，以及 神经元中导致其摄取的机制。在目标1中，我将执行一系列实验，包括 蛋白质错折叠循环扩增(PMCA)及微流控室内神经元/小胶质细胞共培养研究 来支持我们的初步发现。这些实验将在初级导师的实验室中进行 泰德和瓦琳娜·道森以及项目顾问毛晓波。我还将验证神经元ASC病理作为一种 先天痴呆的病理特征。这将在联合导师胡安·特隆科索的实验室完成。目标2将 包括评估阻断小胶质细胞对神经元ASC传递的影响对神经元α同步病理的影响。在……里面 指导阶段，我将首先抑制Caspase-1(Casp-1)，这是一种从细胞释放Asc所必需的酶，但 而不是它的齐聚或重新聚集到炎症体复合体中。来自炎症体的条件培养液-- 激活的WT和CASP1-/-小胶质细胞将被用来诱导小鼠皮质神经元中的αSYN聚集。我们会 还试图确定小胶质细胞释放的ASC的神经元摄取机制。这将通过利用 无偏倚受体筛选以鉴定和表征寡聚ASC的新型神经元受体。这些 一系列实验将在合作者毛晓波的实验室进行。在AIM的独立阶段-- 2，我将研究通过阻止ASC的小胶质细胞释放来阻止ASC传播的后果。在《目标3》中， 我将研究E-‘3连接酶Parkin抑制小胶质细胞ASC寡聚的新机制 通过泛素化炎症体受体NLRP3并将其靶向于蛋白酶体的降解。在 独立阶段，我将探索成年后在小胶质细胞中敲除Parkin基因将如何加速其繁殖 α系统在小鼠模型中的表达。在这方面，我已经生成了Parkinflx/flx/Casp1WT和Parkinflx/flx/CASP1-- /--我将需要这些研究所需的小鼠。我在痴呆症生物学和衰老生物学方面的培训将得到促进 通过讲授课程和参加临床病理研讨会、痴呆症和老龄研讨会 在约翰霍普金斯大学医学院，并将由共同导师玛丽莲·阿尔伯特监督。各方面 这一项目是新颖的，以前从未被调查过。这个项目可能会阐明一种新的信号转导 炎性小体激活直接导致α-Syn在神经元聚集的管道。这一培训 我承诺在约翰霍普金斯大学的工作将使我能够过渡到独立，并领导一个实验室进行研究 神经退行性疾病的病理分子机制。

Synucleinopathy Dementias (Syn-­Dementias) are age related, progressive neurodegenerative diseases that are characterized by α-­Synuclein (αSyn) rich neuronal inclusions called Lewy Bodies (LBs). Syn-­Dementias include Dementia with Lewy Bodies (DLB), Parkinson’s Disease with Dementia (PDD), as well as a subset of Alzheimer’s Disease (AD) cases with LB pathology which constitute ~40% of AD cases. Sustained neuroinflammation (mediated primarily by microglia) and progressive αSyn aggregation are hallmarks of Syn-­ Dementias. We recently described the activation of the Nod Like Receptor Protein-­3 (NLRP3) inflammasome in mouse synucleinopathy models and in human PD brains. The NLRP3 inflammasome is a multi-­protein arm of the innate immune system which, when activated, results in the production of the cytokine interleukin-­1 beta (IL-­ 1β), as well as oligomerized Apoptosis-­associated Speck-­like protein containing a Caspase Recruitment Domain (ASC), which is the adaptor protein of the NLRP3 inflammasome. It is currently unknown what pathological role inflammasome activation may play in the progression of Syn-­Dementias. Our preliminary data suggests that microglia-­released ASC may be taken up by neurons and may cross-­seed αSyn fibrilization. The overarching goal of this proposal is to elucidate the pathological role of microglia to neuron transmission of oligomerized ASC in contributing to progressive αSyn aggregation in Syn-­Dementias. We also seek to elucidate the signaling mechanisms in microglia that lead to the oligomerization and release of ASC, and the mechanisms in neurons that lead to its uptake. In Aim 1, I will perform a series of experiments including Protein misfolding cyclic amplification (PMCA) and neuron/microglia co-­culture studies in microfluidic chambers to support our preliminary findings. These experiments will be performed in the laboratories of primary mentors Ted and Valina Dawson and project consultant Xiaobo Mao. I will also validate neuronal ASC pathology as a pathological hallmark of Syn-­Dementias. This will be done in the lab of co-­mentor Juan Troncoso. Aim 2 will involve assessing the effects of blocking microglia to neuron ASC transmission on neuronal αSyn pathology. In the mentored phase, I will first inhibit Caspase-­1 (Casp-­1), an enzyme required for ASC release from cells but not its oligomerization or recruitment to inflammasome complexes. Conditioned medium from inflammasome-­ activated WT and Casp1-­/-­ microglia will be used to elicit αSyn aggregation in mouse cortical neurons. We will also seek to identify a neuronal uptake mechanism for microglia-­released ASC. This will be done by utilizing unbiased receptor screening to identify and characterize novel neuronal receptors for oligomerized ASC. These set of experiments will be performed in collaborator Xiaobo Mao’s laboratory. In the independent phase of Aim-­ 2, I will investigate the ramifications of preventing ASC transmission by blocking its microglial release. In Aim 3, I will investigate the novel mechanism by which the E-­3 ligase Parkin inhibits ASC oligomerization in microglia by ubiquitinating the inflammasome receptor NLRP3 and targeting it for proteasomal degradation. In the independent phase, I will explore how adult onset knockout of Parkin in microglia will hasten the propagation of αSyn in mouse models. In this regard, I have already generated the Parkinflx/flx/Casp1WT and Parkinflx/flx/Casp1-­ /-­ mice that I will need for these studies. My training in the biology of dementia and aging biology will be facilitated by didactic courses as well as by participation in Clinical Pathological Seminars, Dementia and Aging Symposia at the Johns Hopkins University School of Medicine and will be overseen by co-­mentor Marilyn Albert. All aspects of this project are novel and have not been investigated before. This project may elucidate a novel signaling conduit through which inflammasome activation directly leads to αSyn aggregation in neurons. The training that I undertake at Johns Hopkins will enable me to transition to independence and lead a laboratory that investigates the molecular mechanisms of pathology in neurodegenerative diseases.