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

突触核蛋白病性痴呆是一种与年龄相关的进行性神经退行性疾病 其特征在于富含α-突触核蛋白（αSyn）的神经元包涵体，称为Lewy小体（LB）。综合性痴呆 包括路易体痴呆症（DLB）、帕金森氏痴呆症（PDD）以及 具有LB病理的阿尔茨海默病（AD）病例占AD病例的约40%。持续 神经炎症（主要由小胶质细胞介导）和进行性α-Syn聚集是Syn-1的标志 痴呆症我们最近描述了Nod样受体蛋白-NLRP 3（NLRP 3）炎性体在人结肠癌中的激活。 小鼠突触核蛋白病模型和人PD脑中的突触核蛋白病。NLRP 3炎性小体是一种多核蛋白臂， 先天性免疫系统，当其被激活时，导致细胞因子白细胞介素-β 1 β（IL-β 1）的产生 1β），以及寡聚化的凋亡相关的斑点样蛋白，其含有半胱天冬酶募集结构域 (ASC)，其是NLRP 3炎性体的衔接蛋白。目前尚不清楚其病理作用 炎性小体激活可能在突触性痴呆的进展中起作用。我们的初步数据显示， 小胶质细胞释放的ASC可能被神经元吸收，并可能交叉种子αSyn纤维化。总体 本研究的目的是阐明小胶质细胞在神经元传递中的病理作用， 寡聚ASC促进了突触性痴呆中进行性αSyn聚集。我们还寻求 阐明小胶质细胞中导致ASC寡聚化和释放的信号机制，以及 神经元中导致其摄取的机制。在目标1中，我将进行一系列实验，包括 蛋白质错误折叠循环扩增（PMCA）和微流控室中神经元/小胶质细胞共培养的研究 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 Ted和Valina Dawson以及项目顾问Xiaobo Mao。我还将验证神经元ASC病理学作为 综合性痴呆的病理特征这将在共同研究员Juan Troncoso的实验室中完成。目标2将 涉及评估阻断小胶质细胞至神经元ASC传递对神经元αSyn病理学的影响。在 在指导阶段，我将首先抑制半胱天冬酶-β 1（Casp-β 1），这是一种从细胞中释放ASC所需的酶， 而不是其寡聚化或募集到炎性体复合物。炎性小体条件培养基 激活的WT和Casp 1-β/-β小胶质细胞将用于引发小鼠皮质神经元中的αSyn聚集。我们将 还试图确定神经元摄取机制的小胶质细胞释放ASC。这将通过利用 无偏受体筛选以鉴定和表征寡聚化ASC的新型神经元受体。这些 一组实验将在合作者毛晓波的实验室进行。在Aim-10的独立阶段， 2.我将研究通过阻断小胶质细胞释放来预防ASC传播的后果。在目标3中， 我将研究新的机制，其中E-BMP 3连接酶帕金抑制ASC寡聚化的小胶质细胞 通过泛素化炎性体受体NLRP 3并靶向其进行蛋白酶体降解。在 独立阶段，我将探讨如何成年发病敲除帕金在小胶质细胞将加速繁殖 在小鼠模型中的αSyn。在这方面，我已经生成了Parkinflx/flx/Casp 1-WT和Parkinflx/flx/Casp 1-WT。 /-我将需要这些研究的老鼠。我在痴呆生物学和衰老生物学方面的训练将得到促进 通过教学课程以及参加临床病理学研讨会、痴呆症和衰老研讨会 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。所有方面 该项目是新颖的，以前没有调查过。这个项目可能会阐明一种新的信号传导机制， 炎症小体激活直接导致神经元中αSyn聚集的管道。的培训 我承诺在约翰霍普金斯将使我能够过渡到独立和领导一个实验室，调查 神经退行性疾病病理学的分子机制。

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.