The main objective of the Molecular Neuropathology section (MNS) at Laboratory of Neurogenetics (LNG) is to harness the immune system to better understand the mechanisms of neurodegeneration and to develop therapies for a-synucleinopathies of the aging population. We propose 3 Aims, the first investigating the role of innate immune responses and combinatorial immunotherapy targeting LRRK2, Toll-like receptors and the protein aggregates (eg: a-synuclei,n Abeta, tau) in PD/DLB; the second assessing downstream pro-inflammatory signaling pathways including MAPK-p38, NFAT and NFkB and the third evaluating the role of T cell mediated adaptive immune cell responses in PD/DLB pathogenesis and for developing immunotherapies for a-synucleinopathies. During this period we published over 24 manuscripts mostly focusing understanding the pathogenesis of PD/DLB and developing novel pharmacological and immunotherapeutical approaches. Progress for Aim1. We recently published the results of our studies demonstrating that blocking the interactions between extracellular a-synuclein aggregates and TLR2 with neutralizing Immunotherapy targeting Toll-like receptor 2 alleviates neurodegeneration in models of synucleinopathy by modulating a-synuclein transmission and neuroinflammation (Kim et al Molecular Neurodegeneration 2018). During FY19 we also investigated other single and combined siRNA (Spencer et al, Neurobiol of Dis 2019) pharmacological (Hassan et al, and Snow et al, Sci Reports 2018) and immunotherapeutical approaches (mandler et al Alz and Dem 2019). We know that Immunotherapeutic approaches targeting amyloid (A) protein and tau in Alzheimer's disease and -synuclein (-syn) in Parkinson's disease are being developed for treating dementia with Lewy bodies. However, it is unknown if single or combined immunotherapies targeting A and/or -syn may be effective. Amyloid precursor protein/-syn tg mice were immunized with AFFITOPEs (AFF) peptides specific to A (AD02) or -syn (PD-AFF1) and the combination. AD02 more effectively reduced A and pTau burden; however, the combination exhibited some additive effects. Both AD02 and PD-AFF1 effectively reduced -syn, ameliorated degeneration of pyramidal neurons, and reduced neuroinflammation. PD-AFF1 more effectively ameliorated cholinergic and dopaminergic fiber loss; the combined immunization displayed additive effects. AD02 more effectively improved buried pellet test behavior, whereas PD-AFF1 more effectively improved horizontal beam test; the combined immunization displayed additive effects. We conclude that combined active immunotherapy targeting A and -syn may be of potential interest for the treatment of dementia with Lewy bodies. We are now investigating in collaboration with the laboratory of Drs. Cookson and Singleton the interactions between extracellular a-synuclein, TLR2 and LRRK2 and their role in microglial activation and inflammation. This work was recemtly presented at the SFN meeting in San Diego Nov 2018. Progress for Aim 2. Alterations in kinase pathways including mitogen activated protein kinase (MAPK) p38 has been proposed to play a in PD/DLB and AD. While activation of p38a has been linked to neuro-inflammation, alterations in p38g has been associated with Tau phosphorylation in AD. Although p38 has been studied in AD less is known as to the role of MAPK p38 in DLB/PD and other synucleinopathies. While in healthy control human and Non-Tg mice p38a was associated with neurons and astroglial cells, p38g localized to pre-synaptic terminals and in the brains of DLB and a-syn Tg mice immunostaining in the synaptic terminals was reduced and p38g was redistributed to the neuronal cell bodies. Further, double immunolabeling showed that p38g co-localized with a-syn aggregates in DLB patient and a-syn Tg mice. In contrast, in the brains of DLB cases and a-syn Tg mice p38a was increased in astroglial cells. These results suggest that a-syn might interfere with the p38 pathway and might play a role in the mechanisms of inflammation and synaptic dysfunction in DLB/PD. This study was presented at the intra-mural retreat in 2019 and the manuscript will be submitted to Acta Neuropath Comm. In addition and in collaboration with Drs. Cookson and Singleton labs we are investigating the pro-inflammatory signaling pathways downstream of LRRK2 in microglia, specifically we found that LRRK2 promoted this neuroinflammatory cascade by selectively phosphorylating and inducing nucleus translocation of the immune transcription factor, nuclear factor of activated T-cells, cytoplasmic 2 (NFATc2). Likewise, activation of NFATc2 cascade was identified in the brains of synucleinopathies patients including Parkinsons disease (PD) and dementia with Lewy bodies (DLB) and in animal models of synucleinopathy. Therefore, we propose the modulation of LRRK2 and its downstream signaling mediator NFATc2 as a novel therapeutic strategy for synucleinopathies. The results of this study were presented at SFN in san Diego Nov 2018 and at the intramural retreat in 2019. The revised manuscript is under consideration by Science TM. Progress for Aim 3. As a first step to understand the involvement of the peripheral immune system in DLB/PD, we evaluated T cell populations in -syn transgenic (tg) mice (eg: Thy1 promoter line 61). FACS and neuropathologica analysis showed increase of CD3+ T cells but not CD20 (B cells) in the brains -syn tg mice. Furthermore, CD3+ cells co-expressed interferon and where found in close proximity of astroglial cells processes in areas of the brain displaying increased neuro-inflammatory cells (eg: astrogliosis, microgliosis) and pro-inflammatory cytokines. These results suggest that infiltrating adaptive immune responses play an important role in neuro-inflammation and neurodegeneration in synucleinopathies and that modulating peripheral NKT and T cells might be a viable therapeutic strategy for DLB/PD. These results were presented at the 2018 SFN meeting in San Diego. We are in the process of preparing the manuscript for publication as well as comparing NKT in the brains of a-syn tg younger (3-4 m/o) and older mice (18-24 m/o). As a follow up to these studies and as part of an inter-lab project we are investigating T cell responses in an a-syn prion-like seeding models of PD/DLB at various ages and by deleting T cell populations (in collaborations with Drs. Jyoti and Ranjan Sen). We hypothesize that neurodegeneration in the -syn pff model might be associated with an age dependent increase in cytotoxic T cell responses. Following intracranial injection of -syn pff into striatum there was extensive accumulation of -syn that extended to the amygdala, hippocampus and neocortex with greater pathology when comparing the 1 vs the 3 months post injection of both mice cohorts, but aged mice cohort showed significantly increased -syn pathology compare to young mice cohort that was accompanied by infiltration of T cells, microgliosis and astrogliosis. Interestingly we observed greater increase of T cell numbers detected by CD3 antibody in 1 month post injection of aged mice cohort compared to young mice cohort. Additional studies of analyzing immune cell distributions with flow cytometry and microglia RNA sequence are underway to better understand immune response of intracranial injection of -syn pff. These results will be presented at the SFN meeting in Chicago in 2019. Other collaborations at LNG are with Drs. Andy Singleton, Bryan Traynor and Sonja Scholz on the genetic architecture of FTD, DLB and MSA by providing expert neuropathological assessment, animal models and human postmorten brain tissues.