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Background: Parkinson's disease (PD) is characterized by alpha-synuclein (α-Syn) pathology, neurodegeneration and neuroinflammation. Human leukocyte antigen (HLA) variants associated with PD and α-Syn specific CD4+ T lymphocytes in PD patients highlight the importance of antigen presentation in PD etiology. The class II transactivator (CIITA) regulates major histocompatibility complex class II (MHCII) expression. Reduced Ciita levels significantly increase α-Syn pathology, nigrostriatal neurodegeneration and behavioral deficits in α-Syn-induced rat PD models., Objective: Characterize immune profiles associated with enhanced PD-like pathology observed in rats expressing lower Ciita levels (DA.VRA4) compared to the background strain (DA)., Methods: To model PD, we combined rAAV-mediated α-Syn overexpression in the substantia nigra with striatal injection of α-Syn preformed fibrils. Immune profiles in brain and blood were analyzed by flow cytometry and multiplexed ELISA in naïve rats, 4- and 8 weeks post rAAV injection., Results: Flow cytometry showed Ciita-dependent regulation of MHCII on microglia, brain macrophages and circulating myeloid cells. The MHCII-dependent microglial response was highest at 4 weeks post rAAV injection, whereas the MHCII levels in circulating myeloid cells was highest at 8 weeks. There was no major infiltration of macrophages or T lymphocytes into the CNS in response to α-Syn and only subtle Ciita- and/or α-Syn-dependent changes in the T lymphocyte compartment. Lower Ciita levels were consistently associated with higher TNF levels in serum., Conclusions: Ciita regulates susceptibility to PD-like pathology through minor but detectable changes in resident and peripheral immune cells and TNF levels, indicating that mild immunomodulatory therapies could have therapeutic effects in PD.

Parkinson's disease (PD) is characterized by the accumulation of alpha-synuclein (α-syn) inclusions, the major component of Lewy bodies. Extracellular α-syn aggregates act as a damage-associated molecular pattern (DAMP) and the presence of autoantibodies against α-syn species in the cerebrospinal fluid and the serum of PD patients implicate the involvement of innate and adaptive immune responses. In non-transgenic (Tg) mice, intrastriatal injection of preformed fibril (PFF) α-syn results in widespread pathologic α-syn inclusions in the CNS. While the PFF model has been broadly utilized to study the mechanistic relationship between α-syn transmission and other neuropathological phenotypes, the immune phenotypes in this model are not clearly demonstrated. This study aimed to characterize the immune phenotypes during pathologic α-syn propagation by utilizing PFF α-syn-injected non-tg mice. Here, we showed that pathologic α-syn inclusions are prevalent in various brain regions and the gut at 5 months post injection (p.i.), preceding the degeneration of dopaminergic neurons in substantia nigra (SN). We discovered a distinct inflammatory response involving both activation of microglia and astrocytes and infiltration of B, CD4+ T, CD8+ T, and natural killer cells in the brain at 5 months p.i. Moreover, PFF α-syn-injected mice display significant alterations in the frequency and number of leukocyte subsets in the spleen and lymph nodes with minimum alterations in the blood. Our data provide primary evidence that intracerebral-initiated synucleinopathies in non-tg mice alter immune cell profiles both in the CNS and peripheral lymphoid organs. Furthermore, our data provides support for utilizing this mouse model to assess the mechanistic connection between immune responses and synuclein pathology.

Human leukocyte antigen-DR induction and lymphocyte infiltrates in the brains of patients with Parkinson disease (PD) and the presence in serum of α-synuclein (α-syn)-specific antibodies suggest that the peripheral immune system may have an active role in the progression of PD. We designed a vaccination strategy to attempt to control these processes and mediate protection against disease progression in a rat PD model. Using a recombinant adeno-associated viral vector, we unilaterally overexpressed human α-syn in the rat substantia nigra to induce a progressive neuropathologic process. Prior to stereotactic delivery of the viral vector, animals were vaccinated with recombinant α-syn (asyn). This resulted in a high-titer anti-α-syn antibody response on α-syn overexpression; the accumulation of CD4-positive, MHC II-positive ramified microglia in the substantia nigra; long-lasting infiltration of CD4-positive, Foxp3-positive cells throughout the nigrostriatal system; and fewer pathologic aggregates in the striatum versus control animals that had received a mock vaccine. A long-term increase in GDNF levels in the striatum and IgG deposition in α-syn-overexpressing cells and neurites in the substantia nigra were also observed. Together, these results suggest that a protective vaccination strategy results in induction of regulatory T cells and distinctly activated microglia, and that this can induce immune tolerance against α-syn.

Background: Activated microglia are a feature of the host response to neurodegeneration in Parkinson's disease (PD) and are thought to contribute to disease progression. Recent evidence suggests that extracellular α-synuclein (eSNCA) may play an important role in the pathogenesis of PD and that this may be mediated by a microglial response., Methods: We wished to discover whether the host response to eSNCA would be sufficient to induce significant cytokine production. In vitro cultured BV-2 microglia were used to determine the basic inflammatory response to eSNCA. In vivo, 8-week old Biozzi mice were subjected to a single intranigral injection of either 3 μg SNCA, lipopolysaccharide (LPS) or serum protein (BSA) and allowed to recover for 24 hours. A second cohort of animals were peripherally challenged with LPS (0.5 mg/kg) 6 hours prior to tissue collection. Inflammation was studied by quantitative real-time PCR for a number of pro-inflammatory genes and immunohistochemistry for microglial activation, endothelial activation and cell death., Results: In vitro data showed a robust microglial response to SNCA, including a positive NFĸB response and the production of pro-inflammatory cytokines. Direct injection of SNCA into the substantia nigra resulted in the upregulation of mRNA expression of proinflammatory cytokines, the expression of endothelial markers of inflammation and microglial activation. However, these results were significantly different to those obtained after direct injection of LPS. By contrast, when the animals were injected intracerebrally with SNCA and subsequently challenged with systemic LPS, the level of production of IL-1β in the substantia nigra became comparable to that induced by the direct injection of LPS into the brain. The injection of albumin into the nigra with a peripheral LPS challenge did not provoke the production of a significant inflammatory response. Direct injection of LPS into the substantia nigra also induces cell death in a more robust manner than direct injection of either SNCA or BSA., Conclusion: These results suggest that the presence of eSNCA protein 'primes' microglia, making them susceptible to environmental proinflammatory challenge. For this reason, we hypothesise that where 'inflammation' contributes to the disease progression in PD, it does so in a punctuate manner (on-off) as a result of systemic events.

Background: Mechanisms whereby gene-environment interactions mediate chronic, progressive neurodegenerative processes in Parkinson's disease (PD)-the second most common neurodegenerative disease-remain elusive., Objective: We created a two-hit [neuroinflammation and mutant α-synuclein (α-syn) overexpression] animal model to investigate mechanisms through which mutant α-syn and inflammation work in concert to mediate chronic PD neurodegeneration., Methods: We used an intraperitoneal injection of the inflammogen lipopolysaccharide (LPS; 3 × 106 EU/kg) to initiate systemic and brain inflammation in wild-type (WT) mice and transgenic (Tg) mice overexpressing human A53T mutant α-syn. We then evaluated nigral dopaminergic neurodegeneration, α-syn pathology, and neuroinflammation., Results: After LPS injection, both WT and Tg mice initially displayed indistinguishable acute neuroinflammation; however, only Tg mice developed persistent neuroinflammation, chronic progressive degeneration of the nigrostriatal dopamine pathway, accumulation of aggregated, nitrated α-syn, and formation of Lewy body-like inclusions in nigral neurons. Further mechanistic studies indicated that 4-week infusion of two inhibitors of inducible nitric oxide synthase and NADPH oxidase, major free radical-generating enzymes in activated microglia, blocked nigral α-syn pathology and neurodegeneration in LPS-injected Tg mice., Conclusions: Microglia-derived oxidative stress bridged neuroinflammation and α-syn pathogenic alteration in mediating chronic PD progression. Our two-hit animal model involving both a genetic lesion and an environmental trigger reproduced key features of PD and demonstrated synergistic effects of genetic predisposition and environmental exposures in the development of PD. The chronic progressive nature of dopaminergic neurodegeneration, which is absent in most existing PD models, makes this new model invaluable for the study of mechanisms of PD progression.

The Lewy body is a pathological hallmark of Parkinson's disease. It has been revealed that the Lewy body contains nitrated alpha-synuclein which is prone to oligomerization. We tested the hypothesis that aging may enhance nitration of alpha-synuclein due to an exaggerated neuroinflammatory reaction such as an excessive induction of the inducible nitric oxide synthase, which occurs post-intrapallidal lipopolysaccharide (LPS) injection. Here, we show microglia activation and proinflammatory cytokine expression are more evident in the substantia nigra of elderly rats following intrapallidal LPS. In addition, greater nitration of proteins like alpha-synuclein occurs in the substantia nigra of 16-month-old rats versus 3-month-old rats, which is accompanied by a higher expression level of inducible nitric oxide synthase. These results imply that an exaggerated neuroinflammatory response that occurs with aging might be involved in the increase in prevalence of neurodegenerative diseases like Parkinson's disease., (Copyright 2008 Elsevier Inc. All rights reserved.)

Microglial activation and adaptive immunity have been implicated in the neurodegenerative processes in Parkinson disease. It has been proposed that these responses may be triggered by modified forms of alpha-synuclein (alpha-SYN), particularly nitrated species, which are released as a consequence of dopaminergic neurodegeneration. To examine the relationship between alpha-SYN, microglial activation, and adaptive immunity, we used a mouse model of Parkinson disease in which human alpha-SYN is overexpressed by a recombinant adeno-associated virus vector, serotype 2 (AAV2-SYN); this overexpression leads to slow degeneration of dopaminergic neurons. Microglial activation and components of the adaptive immune response were assessed using immunohistochemistry; quantitative polymerase chain reaction was used to examine cytokine expression. Four weeks after injection, there was a marked increase in CD68-positive microglia and greater infiltration of B and T lymphocytes in the substantia nigra pars compacta of the AAV2-SYN group than in controls. At 12 weeks, CD68 staining declined, but B- and T-cell infiltration persisted. Expression of proinflammatory cytokines was enhanced, whereas markers of alternative activation (i.e. arginase I and interleukins 4 and 13) were not altered. Increased immunoreactivity for mouse immunoglobulin was detected at all time points in the AAV2-SYN animals. These data show that overexpression of alpha-SYN alone, in the absence of overt neurodegeneration, is sufficient to trigger neuroinflammation with both microglial activation and stimulation of adaptive immunity.