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

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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.