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 synucleinopathies of the aging population. We propose 3 Aims, the first investigating the role of innate immune responses and immunotherapy targeting Toll-like receptors in PD/DLB; the second assessing downstream pro-inflammatory signaling pathways including MAPK-p38 and the third evaluating the role of T cell mediated adaptive immune cell responses in PD/DLB models. During this period we published over 10 manuscripts mostly focusing on innate immune responses in PD/DLB and novel immunotherapeutical approaches. Progress for Aim1. Immunotherapy targeting Toll-like receptor 2 alleviates neurodegeneration in models of synucleinopathy by modulating a-synuclein transmission and neuroinflammation. TLR2 a pattern recognition immune receptor, has been implicated in the pathogenesis of synucleinopathies because TLR2 is elevated in the brains of patients with DLB/PD and TLR2 is a mediator of the neurotoxic and pro-inflammatory effects of extracellular a-synuclein (a-syn) aggregates. Therefore, blocking TLR2 might alleviate a-syn pathological and functional effects. For this purpose, herein, we targeted TLR2 using a functional inhibitory antibody (anti-TLR2). Two different human a-syn overexpressing tg mice were used in this study. a-syn low expresser mouse (a-syn-tg, under the PDGFb promoter, D line) was stereotaxically injected with TLR2 overexpressing lentivirus to demonstrate that increment of TLR2 expression triggers neurotoxicity and neuroinflammation. The a-syn high expresser mouse (a-syn-tg; under mThy1 promoter, Line 61) was administrated with anti-TLR2 to examine that functional inhibition of TLR2 ameliorates neuropathology and behavioral defect in the synucleinopathy animal model. In vitro a-syn transmission live cell monitoring system was used to evaluate the role of TLR2 in a-syn cell-to-cell transmission. We demonstrated that administration of anti-TLR2 alleviated a-syn accumulation in neuronal and astroglial cells, neuroinflammation, neurodegeneration, and behavioral deficits in an a-syn tg mouse model of PD/DLB. Moreover, in vitro studies with neuronal and astroglial cells showed that the neuroprotective effects of anti-TLR2 antibody were mediated by blocking the neuron-to-neuron and neuron-to-astrocyte a-synuclein transmission which otherwise promotes NFkB dependent pro-inflammatory responses. This study proposes TLR2 immunotherapy as a novel therapeutic strategy for synucleinopathies of the aging population. This study was recently completed and published (Kim et al Molecular Neurodegeneration 2018), we are now investigating in collaboration with the laboratory of Drs. Cookson and Singleton the interactions between extracellular a-syn, TLR2 and LRRK2 and their role in microglial activation and inflammation. We are also actively investigating immunomodulatory compounds targeting the interphase between TLR2 and LRRK2 as novel therapeutics for synucleinopathies. Progress for Aim 2. Role of alterations in protein kinase p38 gamma in the pathogenesis of synucleinopathies of the aging population. We have previously shown a role of extracellular a-syn at activating microglia through TLR2 signaling via NFB and probably p38 MAPK. For this project we wanted to expand studies of the role of p38 on inflammation and neurodegeneration in PD/DLB models. Although p38 has been studied in AD less is known as to the role of MAPK p38 in DLB/PD and other synucleinopathies. For this purpose, we investigated the expression of the p38 family in a-syn overexpressing tg mice (Thy1 promoter line 61) as well as brains from patients with DLB/PD. Immunohistochemical analysis revealed that while in healthy control human and non-tg mice p38g localized to synaptic terminals and p38a in glial cells, in DLB and a-syn tg mice immunostaining in the synaptic terminals was reduced and p38 was redistributed to the neuronal cell bodies; while p38a in glial cells was increased. Further, double immunolabeling showed that p38g co-localized with a-syn aggregates in DLB patient and a-syn tg mice; whilst p38a co-localized with astroglia and microglial cells. Further biochemical and mRNA analysis confirmed a tendency toward increased p38 in DLB and a-syn tg mice. One p38g target in the synapses is syntrophin, in human controls and non-tg mice this substrate co-localizes with p38g and a-syn in the synaptic terminals but not in the neuronal cell body, in contrast in a-syn tg mice and DLB patients syntrophin co-localized with a-syn in the intra-neuronal inclusions. In agreement with these findings, in vitro studies showed that a-syn co-immunoprecipitates with p38g (but not p38a). These results were presented at the 2017 SFN meeting in Wash DC and a manuscript is in preparation. Next we will expand our studies of p38a in immune innate immune responses and test compounds that block this pathway in combination with immunotherapy. Progress for Aim 3. Neuro-inflammation is associated with T cell infiltration in an alpha-synuclein tg models of Lewy body disease. In addition to activation of the innate immune system (eg; TLR2 as shown in the previous section), recent evidence suggests that alterations of the adaptive immune system including T cell activation might play a role. To better understand the underlying alterations of the immune system in DLB/PD, in collaboration with the LMBI of Drs. Sen, we evaluated 10-12 months old a-syn tg mouse model which overexpress human wild type a-syn (Thy1 promoter line 61) comparing its littermates non-transgenic mice (non-tg). Interestingly, flow cytometric analysis of immune cells revealed that a-syn tg showed significant decrease of natural killer T (NKT) cells in spleen, liver and lymph node, although the level of NKT cells in the brain significantly increased in a-syn tg. Immunohistochemical analysis of brain sections revealed significant increase of CD3 and CD4 (T cells), but not CD20 (B cells) in cortex, hippocampus and striatum of a-syn tg compare to non-Tg mouse. A subset of the CD3 positive cells colocalized with the NKT cell marker IFNg and tetramer. Macrophage marker CD68 was also increased in the same brain regions in a-syn tg as well as microglia and astrocytes. These results will be 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 a 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). 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.