MhcII Regulates Transmission of α-Synuclein-Seeded Pathology in Mice

Abstract

MHCII molecules, expressed by professional antigen-presenting cells (APCs) such as T cells and B cells, are hypothesized to play a key role in the response of cellular immunity to α-synuclein (α-syn). However, the role of cellular immunity in the neuroanatomic transmission of α-syn pre-formed fibrillar (PFF) seeds is undetermined. To illuminate whether cellular immunity influences the transmission of α-syn seeds from the periphery into the CNS, we injected preformed α-syn PFFs in the hindlimb of the Line M83 transgenic mouse model of synucleinopathy lacking MhcII. We showed that a complete deficiency in MhcII accelerated the appearance of seeded α-syn pathology and shortened the lifespan of the PFF-seeded M83 mice. To characterize whether B-cell and T-cell inherent MhcII function underlies this accelerated response to PFF seeding, we next injected α-syn PFFs in Rag1-/- mice which completely lacked these mature lymphocytes. There was no alteration in the lifespan or burden of endstage α-syn pathology in the PFF-seeded, Rag1-deficient M83+/- mice. Together, these results suggested that MhcII function on immune cells other than these classical APCs is potentially involved in the propagation of α-syn in this model of experimental synucleinopathy. We focused on microglia next, finding that while microglial burden was significantly upregulated in PFF-seeded, MhcII-deficient mice relative to controls, the microglial activation marker Cd68 was reduced in these mice, suggesting that these microglia were not responsive. Additional analysis of the CNS showed the early appearance of the neurotoxic astrocyte A1 signature and the induction of the Ifnγ-inducible anti-viral response mediated by MhcI in the MhcII-deficient, PFF-seeded mice. Overall, our data suggest that the loss of MhcII function leads to a dysfunctional response in non-classical APCs and that this response could potentially play a role in determining PFF-induced pathology. Collectively, our results identify the critical role of MhcII function in synucleinopathies induced by α-syn prion seeds.

Keywords: Lewy body; MhcI; inflammation; prion; synuclein; transmission.

Figure 1

Deficiency in MhcII genes accelerates death in PFF-seeded M83 model of synucleinopathy. M83+/− mice with intact MhcII alleles (MhcII+/+) or lacking one (MhcII+/−) or both MhcII alleles (MhcII−/−) were seeded with α-syn PFFs at 2 months of age. Median survival times are indicated in months. p-values indicate comparison of all groups to MhcII+/+ mice. Gehan-Breslow-Wilcoxon test and Log rank test; ** p < 0.01. N = 10 (MhcII+/− and MhcII+/+) and 7 (MhcII−/−) mice.

Figure 2

Earlier accumulation of pathologic α-syn in PFF-seeded MhcII−/− mice. MhcII+/+ or MhcII−/− mice were seeded with α-syn PFFs at 2 months of age and were analyzed at endstage (a–d) or at a prodromal stage 2 months post PFF seeding (e,f). Representative images and magnified detail (inset) of anti-pSer129-α-syn antibody 81A stained tissue sections from spinal cord, brain stem, midbrain and thalamus shown. Scale bar, 100 µm; inset, 50 µm. n = 7–8 mice/group (a–d) and n = 5–6 mice/group (e,f). Unpaired 2-tailed t test. * p < 0.05; ** p < 0.01.

Figure 3

Reduced p62 levels in PFF-seeded MhcII−/− mice. MhcII+/+ or MhcII−/− mice were seeded with α-syn PFFs at 2 months of age and were analyzed at endstage (a–e) or at a prodromal stage 2 months post PFF seeding (f–j). Representative images and magnified detail (inset) of anti-p62 antibody-stained tissue sections from spinal cord (gray and white matter), brain stem, midbrain and thalamus shown. SpC, spinal cord. Scale bar, 100 µm; inset, 50 µm. n = 7–8 mice/group (a–e) and n = 5–6 mice/group (f–j). Unpaired 2-tailed t test. * p < 0.05.

Figure 4

Reduced CD3+ T cell in endstage PFF-seeded MhcII−/− mice. MhcII+/+ or MhcII−/− mice were seeded with α-syn PFFs at 2 months of age and were analyzed at endstage (CD3, (a–d); CD4, (e–h)). Representative images and magnified detail of anti-CD3 and anti-CD4 antibody-stained tissue sections from spinal cord, brain stem, midbrain and thalamus shown. Scale bar, 100 µm; inset, 50 µm. n = 6–8 mice/group. Unpaired 2-tailed t test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

Rag1 deficiency does not influence lifespan in PFF-seeded M83 model of synucleinopathy. M83+/− mice with intact Rag1 (Rag1+/+) or lacking both Rag1 alleles (Rag1−/−) were seeded with α-syn PFFs at 2 months of age. Survival curve and median survival times are indicated in months (a). p-values indicate comparison of all groups to Rag1+/+ mice. Gehan-Breslow-Wilcoxon test and Log rank test N = 10 (Rag1+/+) and 9 (Rag1−/−) mice. Representative images and of anti-pSer129-α-syn antibody 81A stained tissue sections from spinal cord, brain stem, midbrain and thalamus of PFF-seeded mice at endstage (b–e). Scale bar, 100 µm; inset, 50 µm. n = 8–10 mice/genotype. Unpaired 2-tailed t test.

Figure 6

Increased microgliosis in PFF-seeded MhcII−/− mice at endstage. MhcII+/+ or MhcII−/− mice were seeded with α-syn PFFs at 2 months of age and were analyzed at endstage (a–e) or at a prodromal stage 2 months post PFF seeding (f–j). Representative images and magnified detail (inset) of anti-Iba-1 antibody-stained tissue sections from spinal cord (gray and white matter), brain stem, midbrain and thalamus shown. SpC, spinal cord. Scale bar, 100 µm; inset, 50 µm. n = 7–8 mice/group (a–e) and n = 5–6 mice/group (f–j). Unpaired 2-tailed t test. * p < 0.05; ** p < 0.01.

Figure 7

Reduced CD68 levels in PFF-seeded MhcII−/− mice at endstage. MhcII+/+ or MhcII−/− mice were seeded with α-syn PFFs at 2 months of age and were analyzed at endstage (a–e). Representative images and magnified detail (inset) of anti-CD68 antibody-stained tissue sections from spinal cord, brain stem, midbrain and thalamus shown. SpC, spinal cord. Scale bar, 100 µm; inset, 50 µm. n = 7–8 mice/group. Unpaired 2-tailed t test. * p < 0.05; ** p < 0.01.

Figure 8

Astrocytosis is blunted in PFF-seeded MhcII−/− mice at endstage. MhcII+/+ or MhcII−/− mice were seeded with α-syn PFFs at 2 months of age and were analyzed at endstage (a–e) or at a prodromal stage 2 months post PFF seeding (f–j). Representative images and magnified detail (inset) of anti-GFAP antibody-stained tissue sections from spinal cord (gray and white matter), brain stem, midbrain and thalamus shown. SpC, spinal cord. Scale bar, 100 µm; inset, 50 µm. n = 7–8 mice/group (a–e) and n = 5–6 mice/group (f–j). Unpaired 2-tailed t test. ** p < 0.01.

Figure 9

Focused transcriptomic analysis reveals altered immune function in PFF-seeded MhcII−/− mice. Differential gene expression was analyzed using NanoString Glial Profiling Panel in α-syn PFF-seeded MhcII+/+ or MhcII−/− mice. Mice were analyzed at endstage (paralysis) or at a prodromal stage (2 months post PFF seeding). Volcano plot and top 10 altered genes shown for prodromal cohort (a,b) and endstage cohort (c,d). Orange dots, upregulated genes; blue dots, downregulated genes. FC = fold change; padj = p-value adjusted for multiple comparison. The list of differentially expressed genes was used to impute enrichment of functional pathways (Gene Ontogeny Molecular Function, GO MF) among the upregulated genes (e) and downregulated genes (f) in bubble plots. Over-represented pathways with p-value ≤ 0.01, the number of module genes within the pathway > 5 and an enrichment score > 2.0 are depicted. p-value is indicated by the color score and the enrichment score by the dot size. Differential gene expression patterns of cell-type-specific markers in different cell types were imputed (g–k). Disease-associated glial signatures were identified in the prodromal and endstage mice (l–n). One-way Anova with Tukey’s correction. *** p < 0.001; ** p < 0.01; * p < 0.05. N = 5–6 mice/group.

Figure 10

Schematic depiction of immune-mediated pathways influencing peripheral to central progression of synucleinopathy. Schematic summary of the main outcomes of our data pointing to a potential key role of MhcII activity in regulating disease progression of synucleinopathies. Increased astrocytosis and microgliosis accompanied worsened lifespan in α-Syn PFF-seeded mice constitutively lacking MhcII but not in mice lacking functional T cells or B cells, supporting a key role for non-lymphocytic antigen presentation activity in this process. Increased expression of MhcI locus genes in α-Syn PFF-seeded MhcII−/− mice also indicates its potential involvement in α-Syn pathogenesis.