RESUMO
Parkinson's disease (PD) is characterized by neuroinflammation, progressive loss of dopaminergic neurons, and accumulation of a-synuclein (a-Syn) into insoluble aggregates called Lewy pathology. The Line 61 a-Syn mouse is an established preclinical model of PD; Thy-1 is used to promote human a-Syn expression, and features of sporadic PD develop at 9-18 months of age. To accelerate the PD phenotypes, we injected sonicated human a-Syn preformed fibrils (PFFs) into the striatum, which produced phospho-Syn (p-a-Syn) inclusions in the substantia nigra pars compacta and significantly increased MHC Class II-positive immune cells. Additionally, there was enhanced infiltration and activation of innate and adaptive immune cells in the midbrain. We then used this new model, Line 61-PFF, to investigate the effect of inhibiting the JAK/STAT signaling pathway, which is critical for regulation of innate and adaptive immune responses. After administration of the JAK1/2 inhibitor AZD1480, immunofluorescence staining showed a significant decrease in p-a-Syn inclusions and MHC Class II expression. Flow cytometry showed reduced infiltration of CD4+ T-cells, CD8+ T-cells, CD19+ B-cells, dendritic cells, macrophages, and endogenous microglia into the midbrain. Importantly, single-cell RNA-Sequencing analysis of CD45+ cells from the midbrain identified 9 microglia clusters, 5 monocyte/macrophage (MM) clusters, and 5 T-cell (T) clusters, in which potentially pathogenic MM4 and T3 clusters were associated with neuroinflammatory responses in Line 61-PFF mice. AZD1480 treatment reduced cell numbers and cluster-specific expression of the antigen-presentation genes H2-Eb1, H2-Aa, H2-Ab1, and Cd74 in the MM4 cluster and proinflammatory genes such as Tnf, Il1b, C1qa, and C1qc in the T3 cluster. Together, these results indicate that inhibiting the JAK/STAT pathway suppresses the activation and infiltration of innate and adaptive cells, reducing neuroinflammation in the Line 61-PFF mouse model.
RESUMO
Autosomal dominant pathogenic mutations in Leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease (PD). The most common mutation, G2019S-LRRK2, increases the kinase activity of LRRK2 causing hyper-phosphorylation of its substrates. One of these substrates, Rab10, is phosphorylated at a conserved Thr73 residue (pRab10), and is one of the most abundant LRRK2 Rab GTPases expressed in various tissues. The involvement of Rab10 in neurodegenerative disease, including both PD and Alzheimer's disease makes pinpointing the cellular and subcellular localization of Rab10 and pRab10 in the brain an important step in understanding its functional role, and how post-translational modifications could impact function. To establish the specificity of antibodies to the phosphorylated form of Rab10 (pRab10), Rab10 specific antisense oligonucleotides were intraventricularly injected into the brains of mice. Further, Rab10 knock out induced neurons, differentiated from human induced pluripotent stem cells were used to test the pRab10 antibody specificity. To amplify the weak immunofluorescence signal of pRab10, tyramide signal amplification was utilized. Rab10 and pRab10 were expressed in the cortex, striatum and the substantia nigra pars compacta. Immunofluorescence for pRab10 was increased in G2019S-LRRK2 knockin mice. Neurons, astrocytes, microglia and oligodendrocytes all showed Rab10 and pRab10 expression. While Rab10 colocalized with endoplasmic reticulum, lysosome and trans-Golgi network markers, pRab10 did not localize to these organelles. However, pRab10, did overlap with markers of the presynaptic terminal in both mouse and human cortex, including α-synuclein. Results from this study suggest Rab10 and pRab10 are expressed in all brain areas and cell types tested in this study, but pRab10 is enriched at the presynaptic terminal. As Rab10 is a LRRK2 kinase substrate, increased kinase activity of G2019S-LRRK2 in PD may affect Rab10 mediated membrane trafficking at the presynaptic terminal in neurons in disease.
