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: 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 T cell mediated adaptive immune cell responses in PD/DLB pathogenesis and for developing immunotherapies for a-synucleinopathies. During this period we published over 30 manuscripts mostly focusing understanding the pathogenesis of PD/DLB and developing novel pharmacological and immunotherapeutical approaches. Progress for Aim1. We recently published a review manuscript describing present progress and future directions utilizing immunotherapy in neurodegenerative disorders (Kwon et al Neurotherapeutics 2020) an expanded ou studies targeting the interactions between extracellular -synuclein aggregates and TLR2 in models of synucleinopathy by modulating synuclein transmission and neuroinflammation (Kwon et al Exp Neurobiol 2019). Other important question in the field is if there are specific antibodies targeting synuclein that will block neuron to neuron propagation and ameliorate neurodegeneration In this respect we recently reported the development (Schofield et al Neurobiology of Dis 2019) of a high affinity -synuclein antibody, MEDI1341, that can enter the brain, sequester extracellular -synuclein and attenuate -synuclein spreading in vivo. MEDI1341 binds both monomeric and aggregated forms of -synuclein. Using a novel lentiviral-based in vivo mouse model of -synuclein spreading in the brain, we show that treatment with MEDI1341 significantly reduces -synuclein accumulation and propagation along axons. MEDI1341 is now in Phase 1 human clinical trial testing for PD. Moreover, during FY20 we also investigated the role of combined immunotherapy targeting A and -synuclein in models AD/PD (Mandler et al Alz and Dem 2019). For this purpose Amyloid precursor protein/-syn tg mice were immunized with AFFITOPEs (AFF) peptides specific to A (AD02) or -syn (PD-AFF1) and the combination. Remarkably, we found that AD02 was 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. We concluded that combined active immunotherapy targeting A and/or -syn may be of potential interest for the treatment of dementia with Lewy bodies. 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. We recently published a new study (Iba et al Frontiers in Neuroscience 2020) showing that while activation of p38 has been linked to neuro-inflammation, alterations in p38 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 p38 was associated with neurons and astroglial cells, p38 localized to pre-synaptic terminals and in the brains of DLB and -syn Tg mice immunostaining in the synaptic terminals was reduced and p38 was redistributed to the neuronal cell bodies. Further, double immunolabeling showed that p38 co-localized with -syn aggregates in DLB patient and -syn Tg mice. In contrast, in the brains of DLB cases and -syn Tg mice p38 was increased in astroglial cells. These results suggest that -syn might interfere with the p38 pathway and might play a role in the mechanisms of synaptic dysfunction in DLB/PD. Next in collaboration with the Cookson group we are investigating the cross talk between TLR2 and LRRK2 and downstream signaling involving NFATC leading to neurodegeneration and inflammation in PD/DLB, the manuscript is in press in Science TM. Progress for Aim 3. In collaboration with the Sen laboratory we recently published (Iba et al J Neuroinflammation 2020) one of the first studies in the field demonstrating that neuroinflammation in synucleinopathies involves considerable T cell infiltration with potential participation of NKT cells. To understand the involvement of the peripheral immune system in PD/DLB, we evaluated T cell populations in the brains of -syn transgenic (tg) mice (e.g., Thy1 promoter line 61) and DLB patients. Our analysis showed perivascular and parenchymal infiltration by CD3+/CD4+ helper T cells, but not cytotoxic T cells (CD3+/CD8+) or B cells (CD20+), in the neocortex, hippocampus, and striatum of -syn tg mice. CD3+ cells were found in close proximity to the processes of activated astroglia, particularly in areas of the brain with significant astrogliosis, microgliosis, and expression of pro-inflammatory cytokines. A subset of CD3+ showed cytometric data consistent with NKT cells. In post-mortem DLB brains, we similarly detected increased numbers of infiltrating CD3+/CD4+ T cells in close proximity with blood vessels. These results suggest that infiltrating adaptive immune cells play an important role in neuroinflammation and neurodegeneration in synucleinopathies and that modulating peripheral T cells may be a viable therapeutic strategy for PD/DLB. Similarly we have investigated neuroinflammation in other models of synucleinopathy including MSA a neurodegenerative disorder with parkinsonism, dysautonomia and accumulation of -syn in oligodendroglial cells. This study was recently published (Hoffman et al Brain Pathol 2019) and showed increased neuroinflammation in regions with a high -syn load, A distinct gene expression profile including upregulation of cytokines important for myeloid cell attraction and proliferation was detected in -syn overexpressing oligodendrocytes. Collectively, these results imply an early crosstalk between neuroinflammation and oligodendrocytes containing -syn inclusions leading to an immune response locally restricted to white matter regions in MSA. More recently we investigated in collaboration with the Sen Laboratory the role of aging in T cell infiltration and neuroinflammation in synucleinopathies, for this purpose we utilized a novel model, for this -syn pff was injected into the striatum of wild type mice at 4 and 18 months of age and analyzed for behavioral deficits, neuropathology and immune responses at 1- and 3-month post injection periods. Following intracranial injection of -syn pff we found that in the aged mice cohort there was a significant increase in -syn pathology, infiltration of T cells, microgliosis and astrogliosis. Behavioral analysis showed increase in activity, decreased cued fear conditioning in old -syn pff mice. In conclusion, the results points to the possibility that targeting neuro-immune responses might be important in developing treatments for DLB/PD. This manuscript is under preparation for submission. 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.