The MNU at the 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: syn, 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 syn bind TLR2 and mediates neuroinflammation. We were interested at better characterizing the downstream pathways mediating neurodegeneration by using multiomics approaches. For this purpose, we collaborated with Dr. Jung laboratory in South Korea. First, we establish transcriptomic and epigenomic landscapes of the substantia nigra by profiling 113,207 nuclei obtained from healthy controls and patients with PD. Our multiomics data integration provides cell type annotation of 128,724 cis-regulatory elements (cREs) and uncovers cell type-specific dysregulations in cREs with a strong transcriptional influence on genes implicated in PD. The establishment of high-resolution three-dimensional chromatin contact maps identifies 656 target genes of dysregulated cREs and genetic risk loci, uncovering both potential and known PD risk genes. Notably, these candidate genes exhibit modular gene expression patterns with unique molecular signatures in distinct cell types, highlighting altered molecular mechanisms in immune cells like microglia as well as in oligodendroglial cells (Lee et al, Sci Adv 2023). In addition, mice treated with LV-shRNA α-syn had amelioration of abnormal microglial activation and astrocytosis in AD mice. This suggests a link between Aβ and α-syn in pathology pointing to a possible therapeutic angle for AD targeting α-syn. This paper was published in JAD (Leitao et al JAD 2023). In other studies, in collaboration with the Cookson lab we studied the distribution of p-a-synuclein in control and PD brains using the PLA method (Arlinghaus et al J Park Dis 2023) and more recently spatial transcriptomics studies in the brains of a-synuclein tg mice and DLB brains (Horan-Portelance et sl BioRvix 2024). Progress for Aim 2. We previously showed that once extracellular -syn binds TLR2, (Kim et al Science TM 2020) 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 in collaboration with the Gerez lab in Germany we investigated the propagation effects of syn in a novel reporter model. Expression of the secreted syn in the mouse brain was under the control of a novel hybrid promoter in combination with adeno-associated virus serotype 9 (AAV9). The secreted syn is aggregation-prone and amyloidogenic, and when expressed in the brain of wild-type non-transgenic mice, it induces a Parkinson's disease-like phenotype that includes a robust syn pathology in the substantia nigra, neuronal loss, neuroinflammation, and motor deficits, all the key features of experimental animal models of DLB/PD ( Gerez et al NPJ Parkinson’s Disease 2024). Finally, we investigated the role of p38 MAPK as a potential immunomodulatory pathway. We hypothesized that p38α might be associated with neuronal p38γ distribution and synaptic dysfunction in these diseases. We found that inhibition of p38α reduced neuroinflammation and ameliorated synaptic, neurodegenerative, and motor behavioral deficits in transgenic mice overexpressing human α-syn. Moreover, treatment with SKF-86002 promoted the redistribution of p38γ to synapses and reduced the accumulation of α-syn in mice overexpressing human α-syn. Supporting the potential value of targeting p38 in DLB/PD, we found that SKF-86002 promoted the redistribution of p38γ in neurons differentiated from iPS cells derived from patients with familial PD (carrying the A53T α-syn mutation) and healthy controls. These findings provide a mechanistic connection between p38α and p38γ as well as a rationale for targeting this pathway in DLB/PD. This study was recently published in Science Translational Medicine (Iba et al Science TM 2023). 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. To evaluate the potential of targeting NKT cells to modulate neuroinflammation, we treated α-syn transgenic (tg) mice (e.g.: Thy1 promoter line 61) with an antibody against CD1d, which is a glycoprotein expressed in antigen presenting cells (APCs). CD1d-presented lipid antigens activate NKT cells through the interaction with T cell receptor in NKTs, resulting in the production of cytokines. Thus, we hypothesized that blocking the APC-NKT interaction with an anti-CD1d antibody might reduce neuroinflammation and neurodegeneration in models of DLB/PD. Treatment with the anti-CD1d antibody did not have effects on CD3 (T cells), slightly decreased CD4 and increased CD8 lymphocytes in the mice. Moreover, double labeling studies showed that compared to control (IgG) treated α-syn tg mice, treatment with anti-CD1d decreased numbers of CD3/interferon γ (IFN γ)-positive cells, consistent with NKTs. Further double labeling studies showed that CD1d-positive cells co-localized with the astrocytes marker GFAP and that anti-CD1d antibody reduced this effect. While in control α-syn tg mice CD3 positive cells were near astrocytes, this was modified by the treatment with the CD1d antibody. By qPCR, levels of IFN γ, CCL4, and interleukin-6 were increased in the IgG treated α-syn tg mice. Treatment with CD1d antibody blunted this cytokine response that was associated with reduced astrocytosis and microgliosis in the CNS of the α-syn tg mice treated with CD1d antibody. These results suggest that reducing infiltration of NKT cells with an antibody against CD1d might be a potential therapeutical approach for DLB/PD (Iba et al J Neuroinflammation 2024). 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. Microglial depletion also ameliorated neuronal and synaptic degeneration in -syn-tg mice, thereby resulted partially improving the motor behavioral deficit in syn-tg mice. We suggest that microglia play a critical role in the pathogenesis of synucleinopathies, and that modulation of microglial state might be an effective therapeutic strategy for synucleinopathies. 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 tissues.