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. 2010 Oct 26:5:42.
doi: 10.1186/1750-1326-5-42.

Fcγ receptors are required for NF-κB signaling, microglial activation and dopaminergic neurodegeneration in an AAV-synuclein mouse model of Parkinson's disease

Shuwen Cao  1 , Shaji Theodore, David G Standaert
Affiliations

Fcγ receptors are required for NF-κB signaling, microglial activation and dopaminergic neurodegeneration in an AAV-synuclein mouse model of Parkinson's disease

Shuwen Cao et al. Mol Neurodegener. .

Abstract

Overexpression of alpha-synuclein (α-SYN), a protein which plays an important role in the pathogenesis of Parkinson's disease (PD), triggers microglial activation and adaptive immune responses, and leads to neurodegeneration of dopaminergic (DA) neurons. We hypothesized a link between the humoral adaptive immune response and microglial activation in α-SYN induced neurodegeneration. To test this hypothesis, we employed adeno-associated virus serotype 2 (AAV2) to selectively over-express human α-SYN in the substantia nigra (SN) of wild-type mice and FcγR-/- mice, which lack high-affinity receptors for IgG. We found that in wild-type mice, α-SYN induced the expression of NF-κB p65 and pro-inflammatory molecules. In FcγR-/- mice, NF-κB activation was blocked and pro-inflammatory signaling was reduced. Microglial activation was examined using immunohistochemistry for gp91PHOX. At four weeks, microglia were strongly activated in wild-type mice, while microglial activation was attenuated in FcγR-/- mice. Dopaminergic neurodegeneration was examined using immunohistochemistry for tyrosine hydroxylase (TH) and unbiased stereology. α-SYN overexpression led to the appearance of dysmorphic neurites, and a loss of DA neurons in the SN in wild-type animals, while FcγR-/- mice did not exhibit neuritic change and were protected from α-SYN-induced neurodegeneration 24 weeks after injection. Our results suggest that the humoral adaptive immune response triggered by excess α-SYN plays a causative role in microglial activation through IgG-FcγR interaction. This involves NF-κB signaling, and leads to DA neurodegeneration. Therefore, blocking either FcγR signaling or specific intracellular signal transduction events downstream of FcγR-IgG interaction, such as NF-κB activation, may be viable therapeutic strategies in PD.

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Figures

Figure 1
Figure 1
Effect of over-expression of human α-SYN on NF-κB activation in the mouse SN at two weeks. AAV2-SYN and AAV2-GFP (control) were injected into the right SN, and the tissue was processed for p65 and p50 staining at 2 weeks post-injection. A-D) The AAV2-SYN-injected SN displayed increased staining for p65 (red), mostly within microglia in close proximity to α-SYN-expressing neurons (green) while no induction of p65 was seen after AAV2-GFP. E) The staining for p65 (red) co-localized with the staining for CD68 (green), a marker for activated microglia, showing that NF-κB activation occurs in microglia. F) Scoring of immunostaining for p65 on a scale of 0 to 4. Compared with AAV2-GFP-injected tissues, AAV2-SYN treatment caused a significant increase in NF-κB p65. *p < 0.05 using Mann-Whitney U test (n = 6 per group). G-J) No significant difference was observed in NF-κB p50 immunoreactivity between AAV2-GFP and AAV2-SYN treated groups. Scale bars: panels A, B bar = 40 μm; panel E bar = 5 μm; panels G, H bar = 60 μm; panels C, D, I, J bar = 20 μm.
Figure 2
Figure 2
Effect of α-SYN over-expression on NF-κB activation in wild-type and FcγR-/- mice at four weeks. A-H) SN sections of wild-type and FcγR-/- mice over-expressing human α-SYN or GFP were double stained for NF-κB p65 (red) and SYN/GFP (green). Wild-type mice expressing human α-SYN revealed significantly increased immunoreactivity for NF-κB p65 while no significant enhancement of p65 was observed in FcγR-/- mice. Scale bars: panels A, B, C, D bar = 60 μm; panels E, F, G, H bar = 20 μm. I) Scoring of immunostaining for p65 using a rating scale revealed significant elevated p65 expression in wild-type mice treated with AAV2-SYN compared to AAV2-GFP group. This difference was not observed in FcγR-/- mice expressing SYN or GFP. *p < 0.05, WT-SYN vs WT-GFP, Mann-Whitney U test. J) Immunoblotting and quantification for nuclear NF-κB p65 in WT and FcγR-/- mice. In WT mice, AAV2-SYN treated mice showed significant increase in nuclear p65 level compared with AAV2-GFP controls. No difference was observed between FcγR-/- mice expressing α-SYN or GFP, but FcγR-/- mice have higher baseline levels of nuclear p65 than WT mice. *p < 0.05, WT-SYN vs WT-GFP t-test (N: untreated mice; G: AAV2-GFP mice; S: AAV2-SYN mice).
Figure 3
Figure 3
Effect of α-SYN over-expression on the transcription of NF-κB components and the pro-inflammatory mediator in wild-type and FcγR-/- mice at two weeks. There was significant increase in the mRNA level of A) NF-κB p65, B) NF-κB p50, C) intercellular adhesion molecule 1(ICAM-1) in the WT SYN group compared to the WT GFP group. **p < 0.01, *p < 0.05 WT GFP vs WT SYN; and the mRNA level of NFκB p65 was decreased in FcγR-/- SYN mice compared with FcγR-/- GFP mice. **p < 0.01 FcγR-/- SYN vs FcγR-/- GFP. One-way ANOVA with Fisher PLSD post hoc test, GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 4
Figure 4
Effect of α-SYN over-expression on microglial activation in wild-type and FcγR-/- mice. A-D) At four weeks after AAV injection, SN sections of wild-type and FcγR-/- mice over-expressing α-SYN or GFP were double-stained for gp91PHOX (red) and SYN/GFP (green). Wild-type mice expressing α-SYN revealed significantly increased immunoreactivity for gp91PHOX compared to the other three groups. gp91PHOX immunoreactivity was localized to microglia, which appear to be close to α-SYN-expressing neurons (scale bar = 50 μm). E) Scoring of microglial activation using a rating scale revealed significant microglial activation in wild-type mice expressing α-SYN compared to those expressing GFP. This difference was not observed in FcγR-/- mice expressing SYN or GFP. *p < 0.05, WT-SYN vs WT-GFP, Mann-Whitney U test.
Figure 5
Figure 5
Effect of α-SYN over-expression on dopaminergic neurodegeneration in wild-type and FcγR-/- mice. A-D) The SN of wild-type and FcγR-/- mice expressing α-SYN or GFP was stained for tyrosine hydroxylase 24 weeks after treatment. The images depict a representative sample of the injected side of the SN of the four treatment groups. E) Counts of TH positive neurons using unbiased stereology. The wild-type mice treated with AAV2-SYN revealed a significant reduction in dopamine neuron count in the injected side compared to the un-injected side. No significant loss of dopamine neurons was observed in wild-type mice treated with AAV2-GFP and FcγR-/- mice expressing GFP/SYN. *p < 0.05, one-tailed t test, ipsilateral vs contralateral in WT SYN mice. F-K) Representative neurite samples of WT AAV2-SYN and FcγR-/- AAV2-SYN mice. The α-SYN(green)-expressing neuron in WT mice has little TH (red) staining in the beaded degenerating neurite, while FcγR-/- mice are protected from neuritic change. Panel H and K are high-magnification images of the neurites in the square of panel G and J respectively. Scale bars: panels A, B, C, D bar = 200 μm; panels F, G, I, J bar = 25 μm; panels H, K bar = 6 um.
Figure 6
Figure 6
α-SYN induced neuroinflammation model. Over-abundance of α-SYN leads to the expression of a specific antigen, which induces IgG generation. The binding of IgG and FcγR on the surface of microglia results in the downstream signaling pathways, in which NF-κB is the key transcription factor, and causes microglial activation. Microglial activation results in a flooding of surrounding tissue with a variety of neurotoxic substances such as cytokines that ultimately injure neurons in the SN.
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