The main objective of the Molecular Neuropathology unit (MNU) at Laboratory of Neurogenetics (LNG) is to harness the immune system to better understand the mechanisms of neurodegeneration and to develop therapies for synucleinopathies of the aging population such as DLB, PD and AD. We propose 3 Aims, the first investigating the role of innate immune responses and combinatorial immunotherapy targeting LRRK2, Toll-like receptors, p38, NFAT and the protein aggregates (eg: a-synuclein, 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 aging in T cell mediated adaptive immune cell responses in PD/DLB pathogenesis and for developing immunotherapies for synucleinopathies. During this period, we published 9 manuscripts mostly focusing understanding the pathogenesis of PD/DLB and developing novel pharmacological and immunotherapeutical approaches. Progress for Aim1. In previous studies we identified several immune receptors that mediate neuroinflammation in synucleinopathies, including Toll-like receptor 2 (TLR2) and have shown which species of a-synuclein bind TLR2 and mediates neuroinflammation. These results suggest that cell type-specific modulation of TLR2 may be a multifaceted and promising therapeutic strategy for synucleinopathies; inhibition of neuronal and astroglial TLR2 decreases pathogenic a-syn transmission, but activation of microglial TLR2 enhances microglial extracellular a-syn clearance. Along these lines we recently published a manuscript (Torres et al Neurobiol of Dis 2022) characterizing in vivo in our a-synuclein transgenic model the relationship between degeneration of interneurons and neuroinflammation in the amygdala. As an extension and given the impact of the COVID19 pandemic and potential role of SARS-CoV2 in AD/ADRD we investigated (in collaboration with the Nath laboratory) inflammation in the brains of COVID19 patients with evidence of neurological involvement, we found increased a-synuclein accumulation in axons and glial cells as well as astrocytic activation and T cell trafficking in affected CNS areas. Moreover, preliminary studies indicate that the SARS-CoV2 envelope protein might interact with TLR2 and play a role in inflammation and a-synuclein driven pathology, we published a manuscript addressing this possibility (Szabo et al Exp Mol Med 2022). Finally, this year in collaborative projects with academia we made considerable progress developing novel ADRD immunotherapies that either target 3RTau (Spencer et al Methods Mol Biol 2022) or simultaneously target multiple epitopes of a-synuclein. For Tau we developed a novel brain penetrating single chain antibody packed in a viral vector that can be applied peripherally. For a-synuclein, we used the MultiTEP based DNA vaccine technology which is an approach especially useful for the elderly with immunosenescence. All vaccines induced high titers of antibodies specific to a-synuclein that significantly reduced PD/DLB-like pathology in mice. The most significant effects were observed in mice vaccinated with PV-1949D and PV-1950D in a sex-dependent manner. We published two recent studies (Kim et al Vaccines 2022 and Zagorski et al Int J Mol Scie 2022). Progress for Aim 2. In collaboration with the Cookson group (Kim et al Science TM 2020) showed that LRRK2 plays a critical role in the activation of microglia by extracellular a-synuclein. We determined that LRRK2 promoted a neuroinflammatory cascade by selectively phosphorylating and inducing nuclear translocation of the immune transcription factor nuclear factor of activated T cells, cytoplasmic 2 (NFATc2). Our results suggest that modulation of LRRK2 and its downstream signaling mediator NFATc2 might be therapeutic targets for treating synucleinopathies. Next, we investigated the hypothesis that blocking NFATc2 translocation might ameliorate a-synuclein-mediated microglial neurotoxicity. Preliminary studies identified a novel peptide compound (11r VIVIT) that when administered peripherally into -syn tg mice reduced neuroinflammation, restores NFATc2 cytoplasmic localization and ameliorates behavioral deficits. We are currently expanding these studies to include detailed characterization of microglial transcriptomic, phenotypic profiling (M1 vs M2), neuropathological and functional examination. Our results suggest that modulation of LRRK2 and its downstream signaling mediator NFATc2 might be therapeutic targets for treating synucleinopathies. This paper is currently under preparation for submission. The next step is to investigate the immunomodulatory role of p38 inhibitors in models of synucleinopathy. We have previously shown that the synaptic MAPK p38-gamma plays a role synaptotoxicity in DLB/PD, while p38-alpha plays a role in neuroinflammation. We treated our animal models of DLB/PD with a p38 inhibitor that crosses the blood brain barrier and evaluated the effects on synaptic function and neuroinflammation. The results of this study were presented at the intramural NIA retreat and at SfN and a manuscript was recently submitted to Science TM. Progress for Aim 3. In collaboration with the Sen laboratory we showed that T cell infiltration with potential participation of NKT cells play an important role in DLB/PD. Next, we explored the role of aging in the pathogenesis of PD/DLB by analyzing, behavior, neuropathology and transcriptomics and immune responses in young and aged wildtype mice that received intrastriatal injections with a-synuclein preformed fibrils (pff). We found that aged mice showed more extensive accumulation of a-synuclein and behavioral deficits that was associated with greater infiltration of T cells and microgliosis. Distinct inflammatory patterns of gene expression in microglia showed that a-synuclein pff-induced genes networks in young mice (eg: CSF2, TNF, IL1b, IL6) that overlaps with genes differentially expressed in microglia in the aged mice. These results indicate that the aged and more inflamed brain micro-environment directly influences the disease outcome of pathological accumulation of a-syn in age-related chronic diseases such as DLB and PD. This manuscript was recently re-submitted to Molecular Neurodegeneration. Other experiments underway includes investigating the effects of aging and deleting microglia with the PLX3387 (a compound that targets CSF1R) on synucleinopathies in DLB/PD models.We are also investigating the effects of aging and inflammation in models where T cells and microglia are deleted or where CSF1 and CSF2 are modulated, manuscripts for all these studies are under preparation. Other collaborations at LNG are with Drs. Cookson, Singleton, Traynor and Scholz on the genetic architecture of FTD, DLB and MSA by providing expert neuropathological assessment, animal models and human postmorten brain tissues. In addition to the collaborations with LNG, we have been collaborating with the laboratory of Dr. Avi Nath at NINDS investigating the neuropathology of COVID19 that resulted on a recent publication on the microvascular alterations in NeuroCOVID (Nath et al NEJM 2021) and with Dr. Robert Tycho on modeling mechanisms of a-syn protein aggregation.