Analyzing the Parkinson's Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human α-Synuclein.

Parkinson's disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, consequently, the progressive loss of control of voluntary movements. This neurodegenerative process is triggered by the deposition of protein aggregates in the brain, which are mainly constituted of α-synuclein. Several studies have indicated that neuroinflammation is required to develop the neurodegeneration associated with Parkinson's disease. Notably, the neuroinflammatory process involves microglial activation as well as the infiltration of peripheral T cells into the substantia nigra (SN). This work analyzes a mouse model of Parkinson's disease that recapitulates microglial activation, T-cell infiltration into the SN, the neurodegeneration of nigral dopaminergic neurons, and motor impairment. This mouse model of Parkinson's disease is induced by the stereotaxic delivery of adeno-associated viral vectors encoding the human wild-type α-synuclein (AAV-hαSyn) into the SN. The correct delivery of viral vectors into the SN was confirmed using control vectors encoding green fluorescent protein (GFP). Afterward, how the dose of AAV-hαSyn administered in the SN affected the extent of hαSyn expression, the loss of nigral dopaminergic neurons, and motor impairment were evaluated. Moreover, the dynamics of hαSyn expression, microglial activation, and T-cell infiltration were determined throughout the time course of disease development. Thus, this study provides critical time points that may be useful for targeting synuclein pathology and neuroinflammation in this preclinical model of Parkinson's disease.

CD4 T cells react to local increase of α-synuclein in a pathology-associated variant-dependent manner and modify brain microglia in absence of brain pathology.

We have previously shown that immunological processes in the brain during α-synuclein-induced neurodegeneration vary depending on the presence or absence of cell death. This suggests that the immune system is able to react differently to the different stages of α-synuclein pathology. However, it was unclear whether these immune changes were governed by brain processes or by a direct immune response to α-synuclein modifications. We have herein locally increased the peripheral concentration of α-synuclein or its pathology-associated variants, nitrated or fibrillar, to characterize the modulation of the CD4 T cell pool by α-synuclein and brain microglia in the absence of any α-synuclein brain pathology. We observed that α-synuclein changed the CD4:CD8 ratio by contracting the CD3+CD4+ T cell pool and reducing the pool of memory Regulatory T cells (Treg). Nitrated α-synuclein induced the expansion of both the CD3+CD4+ and CD3+CD4- T cells, while fibrils increased the percentage of Foxp3+ Treg cells and induced anti-α-synuclein antibodies. Furthermore, the activation pattern of CD3+CD4+ T cells was modulated in a variant-dependent manner; while nitrated and fibrillar α-synuclein expanded the fraction of activated Treg, all three α-synuclein variants reduced the expression levels of STAT3, CD25 and CD127 on CD3+CD4+ T cells. Additionally, while monomeric α-synuclein increased CD103 expression, the fibrils decreased it, and CCR6 expression was decreased by nitrated and fibrillar α-synuclein, indicating that α-synuclein variants affect the homing and tolerance capacities of CD3+CD4+ T cells. Indeed, this correlated with changes in brain microglia phenotype, as determined by FACS analysis, in an α-synuclein variant-specific manner and coincided in time with CD4+ T cell infiltration into brain parenchyma. We have shown that the peripheral immune system is able to sense and react specifically to changes in the local concentration and structure of α-synuclein, which results in variant-specific T cell migration into the brain. This may have a specific repercussion for brain microglia.

Dopaminergic Receptors on CD4+ T Naive and Memory Lymphocytes Correlate with Motor Impairment in Patients with Parkinson's Disease.

Parkinson's disease (PD) is characterized by loss of dopaminergic neurons in substantia nigra pars compacta, α-synuclein (α-syn)-rich intraneuronal inclusions (Lewy bodies), and microglial activation. Emerging evidence suggests that CD4+ T lymphocytes contribute to neuroinflammation in PD. Since the mainstay of PD treatment is dopaminergic substitution therapy and dopamine is an established transmitter connecting nervous and immune systems, we examined CD4+ T naive and memory lymphocytes in PD patients and in healthy subjects (HS), with specific regard to dopaminergic receptor (DR) expression. In addition, the in vitro effects of α-syn were assessed on CD4+ T naive and memory cells. Results showed extensive association between DR expression in T lymphocytes and motor dysfunction, as assessed by UPDRS Part III score. In total and CD4+ T naive cells expression of D1-like DR decrease, while in T memory cells D2-like DR increase with increasing score. In vitro, α-syn increased CD4+ T memory cells, possibly to a different extent in PD patients and in HS, and affected DR expression with cell subset-specific patterns. The present results support the involvement of peripheral adaptive immunity in PD, and may contribute to develop novel immunotherapies for PD, as well as to better use of current dopaminergic antiparkinson drugs.

α-Synuclein vaccination modulates regulatory T cell activation and microglia in the absence of brain pathology.

BACKGROUND: Passive and active immunization with α-synuclein has been shown to be neuroprotective in animal models of Parkinson's disease. We have previously shown that vaccination with α-synuclein, long before α-synuclein-induced brain pathology, prevents striatal degeneration by inducing regulatory T cell infiltration in parenchyma and antibody deposition on α-synuclein overexpressing neurons. However, the effect of peripheral α-synuclein on the immune system is unknown, as are the mechanistic changes induced in the CD4 T cell population during successful neuroprotective animal studies. We have studied the changes induced by vaccination with α-synuclein in the CD4 T cell pool and its impact on brain microglia to understand the immune mechanisms behind successful vaccination strategies in Parkinson's disease animal models. METHODS: Mice were immunized with WT or nitrated α-synuclein at a dose equivalent to the one used in our previous successful vaccination strategy and at a higher dose to determine potential dose-dependent effects. Animals were re-vaccinated 4 weeks after and sacrificed 5 days later. These studies were conducted in naive animals in the absence of human α-synuclein expression. RESULTS: The CD4 T cell response was modulated by α-synuclein in a dose-dependent manner, in particular the regulatory T cell population. Low-dose α-synuclein induced expansion of naive (Foxp3 + CCR6-CD127lo/neg) and dopamine receptor type D3+ regulatory T cells, as well as an increase in Stat5 protein levels. On the other hand, high dose promoted activation of regulatory T cells (Foxp3CCR6 + CD127lo/neg), which were dopamine receptor D2+D3-, and induced up-regulation of Stat5 and production of anti-α-synuclein antibodies. These effects were specific to the variant of α-synuclein used as the pathology-associated nitrated form induced distinct effects at both doses. The changes observed in the periphery after vaccination with low-dose α-synuclein correlated with an increase in CD154+, CD103+, and CD54+ microglia and the reduction of CD200R+ microglia. This resulted in the induction of a polarized tolerogenic microglia population that was CD200R-CD54CD103CD172a+ (82 % of total microglia). CONCLUSIONS: We have shown for the first time the mechanisms behind α-synuclein vaccination and, importantly, how we can modulate microglia's phenotype by regulating the CD4 T cell pool, thus shedding invaluable light on the design of neuroimmunoregulatory therapies for Parkinson's disease.

Vaccination strategies for Parkinson disease: induction of a swift attack or raising tolerance?

Parkinson disease is the second most common neurodegenerative disease in the world, but there is currently no available cure for it. Current treatments only alleviate some of the symptoms for a few years, but they become ineffective in the long run and do not stop the disease. Therefore it is of outmost importance to develop therapeutic strategies that can prevent, stop, or cure Parkinson disease. A very promising target for these therapies is the peripheral immune system due to its probable involvement in the disease and its potential as a tool to modulate neuroinflammation. But for such strategies to be successful, we need to understand the particular state of the peripheral immune system during Parkinson disease in order to avoid its weaknesses. In this review we examine the available data regarding how dopamine regulates the peripheral immune system and how this regulation is affected in Parkinson disease; the specific cytokine profiles observed during disease progression and the alterations documented to date in patients' peripheral blood mononuclear cells. We also review the different strategies used in Parkinson disease animal models to modulate the adaptive immune response to salvage dopaminergic neurons from cell death. After analyzing the evidence, we hypothesize the need to prime the immune system to restore natural tolerance against α-synuclein in Parkinson disease, including at the same time B and T cells, so that T cells can reprogram microglia activation to a beneficial pattern and B cell/IgG can help neurons cope with the pathological forms of α-synuclein.

α-Synuclein vaccination prevents the accumulation of parkinson disease-like pathologic inclusions in striatum in association with regulatory T cell recruitment in a rat model.

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.

Neuroimmunological processes in Parkinson's disease and their relation to α-synuclein: microglia as the referee between neuronal processes and peripheral immunity.

The role of neuroinflammation and the adaptive immune system in PD (Parkinson's disease) has been the subject of intense investigation in recent years, both in animal models of parkinsonism and in post-mortem PD brains. However, how these processes relate to and modulate α-syn (α-synuclein) pathology and microglia activation is still poorly understood. Specifically, how the peripheral immune system interacts, regulates and/or is induced by neuroinflammatory processes taking place during PD is still undetermined. We present herein a comprehensive review of the features and impact that neuroinflamation has on neurodegeneration in different animal models of nigral cell death, how this neuroinflammation relates to microglia activation and the way microglia respond to α-syn in vivo. We also discuss a possible role for the peripheral immune system in animal models of parkinsonism, how these findings relate to the state of microglia activation observed in these animal models and how these findings compare with what has been observed in humans with PD. Together, the available data points to the need for development of dual therapeutic strategies that modulate microglia activation to change not only the way microglia interact with the peripheral immune system, but also to modulate the manner in which microglia respond to encounters with α-syn. Lastly, we discuss the immune-modulatory strategies currently under investigation in animal models of parkinsonism and the degree to which one might expect their outcomes to translate faithfully to a clinical setting.

Chronic intranasal deferoxamine ameliorates motor defects and pathology in the α-synuclein rAAV Parkinson's model.

Parkinson's disease is characterized by neuronal death in the substantia nigra and the presence of intracellular inclusions of α-synuclein in the Lewy bodies. Several lines of data support a role for iron in Parkinson's disease: iron is present in Lewy bodies, iron accumulates in the dopaminergic neurons in the substantia nigra, and Parkinson's disease is correlated with polymorphisms of several genes implicated in iron metabolism. Furthermore, iron can compromise the solubility of α-synuclein through direct interaction and can induce neurotoxicity in vitro. Here, we investigate the possible neuroprotective effect of the iron chelator deferoxamine in vivo to elucidate whether iron chelation can provide meaningful therapy for Parkinson's disease. Hence, we used a Parkinson's disease animal model based on unilateral injection of a recombinant adeno-associated viral vector encoding α-synuclein in the rat midbrain. Rats were treated with a novel deferoxamine delivery approach: 6 mg of the compound was administered intranasally three times a week for 3 or 7 weeks. The behavior of the animals and histopathological changes in the brain were analyzed. Our data show that although intranasal administration of deferoxamine in rats did not protect them from dopaminergic cell death, it did decrease the number of the pathological α-synuclein formations at the terminal level. In addition, this treatment resulted in changes in the immune response and an overall partial improvement in motor behavior. Taken together, our data show that in vivo iron chelation can modulate α-synuclein-induced pathology in the central nervous system. Our data suggest that chronic administration of intranasal deferoxamine may be a valid approach to limiting the mishandling of α-synuclein in the central nervous system observed in Parkinson's disease and slowing disease progression.

Microglia acquire distinct activation profiles depending on the degree of alpha-synuclein neuropathology in a rAAV based model of Parkinson's disease.

Post-mortem analysis of brains from Parkinson's disease (PD) patients strongly supports microglia activation and adaptive immunity as factors contributing to disease progression. Such responses may be triggered by alpha-synuclein (alpha-syn), which is known to be the main constituent of the aggregated proteins found in Lewy bodies in the brains of PD patients. To investigate this we used a recombinant viral vector to express human alpha-syn in rat midbrain at levels that induced neuronal pathology either in the absence or the presence of dopaminergic cell death, thereby mimicking early or late stages of the disease. Microglia activation was assessed by stereological quantification of Mac1+ cells, as well as the expression patterns of CD68 and MCH II. In our study, when alpha-syn induced neuronal pathology but not cell death, a fast transient increase in microglia cell numbers resulted in the long-term induction of MHC II+ microglia, denoting antigen-presenting ability. On the other hand, when alpha-syn induced both neuronal pathology and cell death, there was a delayed increase in microglia cell numbers, which correlated with long-lasting CD68 expression and a morphology reminiscent of peripheral macrophages. In addition T-lymphocyte infiltration, as judged by the presence of CD4+ and CD8+ cells, showed distinct kinetics depending on the degree of neurodegeneration, and was significantly higher when cell death occurred. We have thus for the first time shown that the microglial response differs depending on whether alpha-syn expression results on cell death or not, suggesting that microglia may play different roles during disease progression. Furthermore, our data suggest that the microglial response is modulated by early events related to alpha-syn expression in substantia nigra and persists at the long term.

Agonist-driven development of CD4+CD25+Foxp3+ regulatory T cells requires a second signal mediated by Stat6.

The factors that induce Foxp3 expression and regulatory T (Treg) cell development remain unknown. In this study, we investigated the role of STAT4 and STAT6 in agonist-driven generation of Ag-specific Foxp3-expressing Treg cells. Our findings indicate that fully efficient induction of Foxp3 expression and development of Ag-specific Treg cells requires the synergistic action of two signals: a TCR-mediated signal and a second signal mediated by STAT6. Indeed, by comparing the development of wild-type and STAT4- and STAT6-deficient hemagglutinin-specific T cells in the presence of hemagglutinin Ag, we found that the absence of STAT6 impaired the generation of Ag-specific CD4+CD25+Foxp3+ cells. Moreover, in transgenic mice expressing a constitutively active form of STAT6, we found that the fraction of CD4+Foxp3+ cells exceeds that of control wild-type littermates. Overall these findings support a role for the STAT6 pathway in Treg cell development and maintenance.

CD8 T cell sensory adaptation dependent on TCR avidity for self-antigens.

Adaptation of the T cell activation threshold may be one mechanism to control autoreactivity. To investigate its occurrence in vivo, we engineered a transgenic mouse model with increased TCR-dependent excitability by expressing a Zap70 gain-of-function mutant (ZAP-YEEI) in postselection CD8 thymocytes and T cells. Increased basal phosphorylation of the Zap70 substrate linker for activation of T cells was detected in ZAP-YEEI-bearing CD8 T cells. However, these cells were not activated, but had reduced levels of TCR and CD5. Moreover, they produced lower cytokine amounts and showed faster dephosphorylation of linker for activation of T cells and ERK upon activation. Normal TCR levels and cytokine production were restored by culturing cells in the absence of TCR/spMHC interaction, demonstrating dynamic tuning of peripheral T cell responses. The effect of avidity for self-ligand(s) on this sensory adaptation was studied by expressing ZAP-YEEI in P14 or HY TCR transgenic backgrounds. Unexpectedly, double-transgenic animals expressed ZAP-YEEI prematurely in double-positive thymocytes, but no overt alteration of selection processes was observed. Instead, modifications of TCR and CD5 expression due to ZAP-YEEI suggested that signal tuning occurred during thymic maturation. Importantly, although P14 x ZAP-YEEI peripheral CD8 T cells were reduced in number and showed lower Ag-induced cytokine production and limited lymphopenia-driven proliferation, the peripheral survival/expansion and Ag responsiveness of HY x ZAP-YEEI cells were enhanced. Our data provide support for central and peripheral sensory T cell adaptation induced as a function of TCR avidity for self-ligands and signaling level. This may contribute to buffer excessive autoreactivity while optimizing TCR repertoire usage.

Different competitive capacities of Stat4- and Stat6-deficient CD4+ T cells during lymphophenia-driven proliferation.

The outcome of an immune response relies on the competitive capacities acquired through differentiation of CD4(+) T cells into Th1 or Th2 effector cells. Because Stat4 and Stat6 proteins are implicated in the Th1 vs Th2 generation and maintenance, respectively, we compare in this study the kinetics of Stat4(-/-) and Stat6(-/-) CD4(+) T cells during competitive bone marrow reconstitution and lymphopenia-driven proliferation. After bone marrow transplantation, both populations reconstitute the peripheral T cell pools equally well. After transfer into lymphopenic hosts, wild-type and Stat6(-/-) CD4(+) T cells show a proliferation advantage, which is early associated with the expression of an active phospho-Stat4 and the down-regulation of Stat6. Despite these differences, Stat4- and Stat6-deficient T cells reach similar steady state numbers. However, when both Stat4(-/-) and Stat6(-/-) CD4(+) T cells are coinjected into the same hosts, the Stat6(-/-) cells become dominant and out-compete Stat4(-/-) cells. These findings suggest that cell activation, through the Stat4 pathway and the down-regulation of Stat6, confers to pro-Th1 T cells a slight proliferation advantage that in a competitive situation has major late repercussions, because it modifies the final homeostatic equilibrium of the populations and favors the establishment of Th1 CD4(+) T cell dominance.