Novel Pre-clinical PD Model (Line 61-PFF). We previously showed that the JAK1/2 inhibitor AZD1480 prevents neuroinflammation and protects against dopaminergic neuron loss in response to AAV-human-α-Syn overexpression in rats (31). To better mimic idiopathic PD, we tested mouse PFFs and found that injection into mouse striatum activated microglia but did not induce the accumulation of MHC Class II-expressing immune cells (data not shown) that occur in human PD brains (49). We therefore tested whether injecting human α-Syn PFFs into the striatum of Line 61 mice, which use the Thy1 promoter to express human α-Syn (32), would induce more robust inflammatory phenotypes. Using an antibody to α-Syn phosphorylated at serine 129 (p-α-Syn), we identified α-Syn inclusions in the SNpc four weeks after injecting Line 61 mice with human PFFs (Figs. 1A, B). We also observed abundant MHC Class II-positive immune cells in the SNpc (Figs. 1A, 1C). Injecting monomeric α-Syn into Line 61 mice did not produce p-α-Syn inclusions or MHC Class II-positive cells (Fig. 1A).
We tested the JAK1/2 inhibitor AZD1480 in the Line 61-PFF model as described (31). Two weeks after PFF injections, AZD1480 or VH was administered for two weeks, then mice were sacrificed for immunofluorescence studies. Figures 1A-C show that AZD1480 treatment significantly reduced α-Syn inclusions and the number of MHC Class II-positive cells in the SNpc.
Sections from the SNpc were stained for TH to determine the number of dopaminergic neurons in mice injected with monomer or PFFs and treated with VH or AZD1480. Line 61-PFF mice did not exhibit a loss of TH-positive neurons compared to monomer-injected mice at a 12-week time point, and AZD1480 treatment produced no significant differences (Supplemental Figs. 1A, B).
Neuroinflammation in Line 61-PFF Mice. Injecting human PFFs into the striatum of Line 61 mice increased MHC Class II positive immune cells in the SNpc, and AZD1480 treatment reduced the abundance of these cells (Fig. 1). Immune cell phenotypes in the midbrain were further characterized by flow cytometry (Figs. 2A, B). In Line 61-PFF mice, total immune cells in the midbrain were significantly higher than in monomer α-Syn-injected mice (Fig. 2C). We identified microglia as CD45MidCD11b+, macrophages as CD45HiCD11b+, dendritic cells (DCs) as CD45HiCD11b+CD11c+, and lymphocytes as CD45+CD11b−. In Line 61-PFF mice, macrophages, DCs, and lymphocytes showed significant increases compared to monomer-injected mice, but the total number of microglia did not increase (Fig. 2C).
We assessed the activation status of innate immune cell subsets by determination of MHC Class II expression. Absolute numbers of MHC Class II positive microglia, macrophages, and DCs were significantly higher in the midbrain of Line 61-PFF mice than in the monomer group (Fig. 2D).
Since adaptive immune cells are also implicated in PD (3, 21–24), we determined how pathologic α-Syn affects infiltration of CD4+ T-cells, CD8+ T-cells, and CD19+ B-cells. We found a significant increase in CD19+ B-cell but not CD4+ or CD8+ T-cell infiltration in Line 61-PFF mice compared to the monomer group, although CD4+ T-cell infiltration trended towards significance (Fig. 2E).
We next determined how AZD1480 treatment influenced specific subsets of immune cells (Figs. 2A, B). AZD1480 or VH was administered two weeks after PFF injections, and treatment continued for four weeks, when mice were sacrificed for analysis. Compared to VH, AZD1480 significantly suppressed absolute numbers of immune cells (microglia, macrophages, DCs, and lymphocytes), the numbers of MHC Class II positive microglia, macrophages and DCs, and the number of CD4+ T-cells, CD8+ T-cells, and CD19+ B-cells in the midbrain of Line 61-PFF mice (Figs. 2C-E). These data show that inhibiting the JAK/STAT pathway suppresses the infiltration and activation of innate and adaptive immune cells.
PFF Induction of STAT3 Activation. Activation of the JAK/STAT pathway results in phosphorylation of STAT proteins (28). We assessed activation in Line 61-PFF mice by measuring tyrosine phosphorylation of STATs, particularly STAT1 and STAT3 (30, 50). Immunoblotting was performed to measure protein expression levels of total STAT1, phosphorylated STAT-1 (p-STAT1), total STAT3, and p-STAT3 in mononuclear cells isolated from the midbrain. We observed a significant increase in p-STAT3 in Line 61-PFF mice treated with VH compared to monomer, which was inhibited by AZD1480 treatment (Figs. 3A, B). There were no statistical differences in total STAT3, total STAT1, and p-STAT1 expression (Figs. 3A-C). Immunohistochemistry confirmed the significant increase in p-STAT3+ cells in Line 61-PFF mice treated with VH compared to monomer treatment, and suppression after AZD1480 treatment (Fig. 3D). These data indicate that PFF injection activates the JAK/STAT pathway as demonstrated by phosphorylated STAT3, and AZD1480 inhibits STAT3 activation in mononuclear cells.
scRNA-Seq Characterization of Immune Cell Clusters in Line 61-PFF Mice. Several scRNA-Seq studies point to the heterogeneity of cells involved in PD pathogenesis (51–54). To identify cell-specific contributions in the Line 61-PFF model, we performed scRNA-Seq on sorted CD45+ leukocytes obtained from Line 61 mice injected with PBS, monomer, PFF plus VH, or PFF plus AZD1480 (Supplemental Fig. 2). Cell clusters were annotated with the top differentially expressed biomarkers and canonical markers for microglia (MG), monocytes/macrophages (MM), T-cells (T), B-cells (B) and neutrophils (Neu) (Supplemental Fig. 3A). We observed no difference in the percentage of each cluster or total cell numbers between the PBS and monomer groups, indicating that monomer injection did not affect the immune cell subsets found in PBS-treated Line 61 mice (Supplemental Figs. 3B, C). Most cells (> 80%) were identified as MG and separated into 9 clusters (MG1-9; Supplemental Fig. 3A). Upon PFF injection, either the percentage or absolute numbers of MG2, MG4, and MG5 clusters were higher than those in PBS- and monomer-injected mice (Supplemental Figs. 3B, C). Percentages and cell numbers of MM clusters and the T-cell cluster were higher in PFF injected mice than in PBS- and monomer-injected mice (Supplemental Figs. 3B, C). Neu and B-cell clusters were not examined due to low (< 1%) numbers.
scRNA-Seq Reveals an PFF-induced MM Inflammatory Cluster Which is Modulated by AZD1480 Treatment. We found no differences in MM cell cluster percentage or numbers between the PBS and monomer groups (Supplemental Figs. 3B, C). To further characterize MM clusters, we used the original annotated dataset to recluster MM1 and MM2 as MM1- MM5 (Supplemental Fig. 3D). MM1, MM2, and MM3 were the major clusters identified in the monomer group (Figs. 4A, B). Examining their transcriptional profiles, Gng10 and Ldhb were highly expressed in the MM1 cluster, while genes related to antigen presentation, H2-Eb1, H2-Aa, H2-Ab1, and Cd74 (55, 56), were enriched in MM2 and MM4 (Supplemental Figs. 4A, B). Genes related to macrophage function and signal transduction, including F13a1, Cd163, Mrc1, Ms4a7, and Pf4 (57) were differentially expressed in MM3, while Nav2, Nav3, Ldlrad4, Dennd4a, and Csmd3, genes associated with Alzheimer’s Disease, Autism Spectrum Disorders (ASD), aging, axon guidance and signal transduction (58–63) were elevated in MM4. Only MM5 expressed Nfib, Dnm3, and Fermt2 (Supplemental Figs. 4A, B). GSEA revealed that the MM2 and MM4 clusters were enriched in IFN-α and IFN-γ responses, IL-6-JAK-STAT3 signaling, TNF-α signaling via NF-κB, Hedgehog signaling, inflammatory responses and hypoxia (Supplemental Fig. 4C).
We examined whether the MM clusters changed with PFF injection. MM1, MM2, MM3, and MM4 cell numbers increased in Line 61-PFF mice compared to monomer, with the MM4 cluster showing the greatest increase (Figs. 4A, B), and DEG analysis revealed upregulated expression of MM4 genes (Fig. 4C).
AZD1480 treatment reduced MM2, MM3, and MM4 cell numbers, almost eliminating the MM4 cluster, and increased MM1 cell numbers (Fig. 4B). H2-Eb1, H2-Aa, H2-Ab1, and Cd74 genes were suppressed in the MM2 and MM4 clusters (Figs. 4C, D) and Nav2, Csmd3, Nav3, Ldlrad4, and Dennd4a in the MM4 cluster (Fig. 4C). Collectively, these results suggest that MM4 is a potentially pathogenic cluster that emerges after PFF injection, and AZD1480 treatment abrogates the appearance of the MM4 cluster.
AZD1480 Treatment Reduces MG Cluster Cell Numbers but Does Not Influence Transcriptional Profiles in Line 61-PFF Mice. Microglia play a critical role in PD pathogenesis (64–66), so we assessed their transcriptional profiles in Line 61 mice. MG1, MG3, MG4, and MG6 were the major MG clusters found in the PBS and monomer groups, with no differences in the percentage or cell numbers of the 9 MG clusters (Supplemental Figs. 3B, C). All MG clusters expressed the canonical gene markers P2ry12 and Cx3cr1. MG1 strongly expressed Klf2 and Egr3, MG3 expressed Maf and Slc2a5, and MG5 expressed Spp1 and Apoe. Although MG2 primarily upregulated Gm42418 and Cmss1, and MG4, Gng10 and Alox5ap, neither does so exclusively. MG6 upregulated two genes, Snx29 and Anks1, and downregulated Maf and Slc2a5. The MG7 cluster upregulated Stat3 and Cd83 and downregulated Klf2 and Egr3. MG8 expressed Top2a and Mki67, and MG9 uniquely expressed Ifit2, Ifit3, Oasl, and Irf7 (Supplemental Figs. 5A, B). GSEA revealed that the MG2 and MG9 clusters were enriched in IFN-α and IFN-γ response pathways, and MG2 was also enriched in TGF-β and PI3K-AKT-MTOR signaling (Supplemental Fig. 5C).
PFF injection increased MG2, MG4, MG5, MG8, and MG9 cell numbers compared to monomer injection (Figs. 5A, 5B), with MG2 showing the greatest increase. However, PFF injection did not change transcriptional profiles to any great extent (Fig. 5C). While AZD1480 treatment reduced cell numbers of MG1, MG4, MG5, MG8, and MG9 clusters overall and MG2 cell numbers to the low levels seen in monomer mice (Fig. 5B), DEG analysis identified only subtle changes in Gm42418, Cmss1, Apoe, and Actb (Figs. 5C, D).
scRNA-Seq Reveals an PFF-induced T-cell Inflammatory Cluster Which is Modulated by AZD1480 Treatment. We identified five T-cell clusters (T1 to T5; Fig. 6A and Supplemental Figs. 3A, E). Most of the T-cells in monomer-treated mice were in cluster T1, which strongly expressed Cd3e, Cd3d, Cd3g, and Trac, genes encoding TCR (67). The T2 cluster expressed Ncr1, Xcl1, Klrb1b, and Klrb1c; T4, Gzma and Klri2; and T5, Gata3, a gene related to Th2 differentiation (68, 69) (Supplemental Figs, 6A, B). Strikingly, PFF injection uniquely induced a new cluster, T3, which most strongly expressed numerous proinflammatory genes, including Cst3, Csmd3, C1qc, C1qa, Cd83, Tnf, and Il1b (Figs. 6A, B; Supplemental Figs. 6A-C). GSEA revealed that the T2 and T4 clusters were enriched in the IFN-γ response pathway and IL-2-STAT5 pathway, while PI3K-AKT-MOTR, IL-6-JAK-STAT3, TNF-α signaling via NF-κB and Notch signaling were enhanced in the T5 cluster. The T3 cluster showed enriched Myc-target signaling (Supplemental Fig. 6E). Cell numbers in the T1 and T2 clusters were increased by PFF injection, and as mentioned previously, the T3 cluster emerged only after PFF injection (Fig. 6B).
AZD1480 treatment reduced T1 and T2 cell numbers, with an almost complete abrogation of the T3 cluster (Figs. 6A, B). Tnf, Il1b, C1qa, and C1qc gene expression was also reduced upon AZD1480 treatment (Figs. 6C, D). Interestingly, AZD1480 restored expression of the naïve T-cell-related genes Cd3d and Cd3e (70, 71) in the T1 cluster as well as the Th2-related genes Gata3 and Il4 (72) in the T5 cluster (Figs. 6C, E).
Collectively, these results reveal that a novel inflammatory T-cell cluster, T3, is induced in response to PFF injection, and is associated with neuroinflammatory responses. AZD1480 treatment significantly decreased T3 cell numbers and suppressed proinflammatory gene expression. AZD1480 also restored T1 and T5 transcriptional profiles, suggesting that the JAK/STAT pathway affects T-cells in the Line 61-PFF model of PD.