Vanesa Sanchez-Guajardo

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 Parkinsons disease (PD) patients strongly supports microglia activation and adaptive immunity as factors contributing to disease progression. Such responses may be triggered by α-synuclein (α-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 α-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 α-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 α-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 α-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 α-syn expression in substantia nigra and persists at the long term.

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 (Parkinsons 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.

The relation between α-synuclein and microglia in Parkinson’s disease: Recent developments

Recent research suggests a complex role for microglia not only in Parkinsons disease but in other disorders involving alpha-synuclein aggregation, such as multiple system atrophy. In these neurodegenerative processes, the activation of microglia is a common pathological finding, which disturbs the homeostasis of the neuronal environment otherwise maintained, among others, by microglia. The term activation comprises any deviation from what otherwise is considered normal microglia status, including cellular abundance, morphology or protein expression. The microglial response during disease will sustain survival or otherwise promote cell degeneration. The novel concepts of alpha-synuclein being released and uptaken by neighboring cells, and their importance in disease progression, positions microglia as the main cell that can clear and handle alpha-synuclein efficiently. Microglias behavior will therefore be a determinant on the diseases progression. For this reason we believe that the better understanding of microglias response to alpha-synuclein pathological accumulation across brain areas and disease stages is essential to develop novel therapeutic tools for Parkinsons disease and other alpha-synucleinopathies. In this review we will revise the most recent findings and developments with regard to alpha-synuclein and microglia in Parkinsons disease.

Long-term polarization of microglia upon α-synuclein overexpression in nonhuman primates.

We have previously shown that persistent α-synuclein overexpression in ventral midbrain of marmoset leads to a distinctive neurodegenerative process and motor defects. The neurodegeneration was confined to caudate putamen dopaminergic fibers in animals overexpressing wild-type (wt) α-synuclein. However, A53T α-synuclein overexpression induced neurodegeneration that resulted in nigral dopaminergic cell death. Here, we analyze the microglia population in the midbrain of these animals by stereological quantification of Iba1+ cells. Our data here show that monkeys overexpressing A53T α-synuclein showed a long-term increase in microglia presenting macrophagic morphology. However, wt α-synuclein overexpression, despite the absence of dopaminergic cell death, resulted in a permanent robust increase of the microglia population characterized by a range of distinct morphological types that persisted after 1 year. These results confirm that the microglial response differs depending on the type of α-synuclein (wt/A53T) and/or whether α-synuclein expression results in cell death or not, suggesting that microglia may play different roles during disease progression. Furthermore, the microglial response is modulated by events related to α-synuclein expression in substantia nigra and persists in the long term. The data presented here is in agreement with that previously observed in a recombinant adeno-associated virus (rAAV) α-synuclein rat model, thereby validating both the findings and the model, and highlighting the translational potential of the rodent model to higher species closer to humans.

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

Parkinsons 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 Parkinsons disease: iron is present in Lewy bodies, iron accumulates in the dopaminergic neurons in the substantia nigra, and Parkinsons 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 Parkinsons disease. Hence, we used a Parkinsons 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 Parkinsons disease and slowing disease progression.

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.

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.

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.

Neuroprotective role of Regulatory T cells in Parkinson's disease: Effect of COP-1/alpha-synuclein vaccination on pathology progression

Since costimulation is crucial for T cell activation, attempts to block those pathways have been effective in preventing unwanted immune reactions. In particular, the CTLA-4Ig fusion protein (blocking CD28/CTLA4/B7 signaling) is highly effective in preventing T cell mediated diseases in animal models, and is in clinical use to treat rheumatoid arthritis (abatacept) and rejection after renal transplantation (belatacept). In order to examine the potential use of CTLA-4Ig (abatacept) as a treatment for multiple sclerosis (MS), we used amousemodel of myelin oligodendrocyte glycoprotein (MOG) induced experimental autoimmuneencephalomyelitis (EAE). As an effective treatment forMS patients needs to be able to interfere with an ongoing inflammatory reaction, we applied CTLA-4Ig at day 7 and 9 after immunization, when myelinreactive T cells are already activated and start to migrate towards the central nervous system (CNS). Surprisingly, we found that CTLA-4Ig treatment at day 7 and 9 after immunization exacerbated clinical EAE symptoms (peak score on day 21: 2.35 vs. 1.45) and resulted in more severe inflammation and demyelination within the CNS. Similar observations were made when the CD28/CTLA-4/B7 interaction was blocked with anti-B7 antibodies (peak score on day 21: 2.17 vs. 1.06). The exacerbated disease was associated with an increased production of the inflammatory cytokines IL-17 in the CNS (day 21: 6.75% vs. 20.89% CD4IL-17) and IFN-gamma in the periphery (day 21: 4.38% vs. 10.64% CD4IFN-gamma). The proportion of Foxp3 regulatory T (Treg) cells was reduced in peripheral lymphoid organs immediately after treatment (day 10: 9.3% vs. 7.9% CD4Foxp3). Altogether these data suggest that blocking the B7 receptors after T cell priming does not interfere with CD28 mediated T cells activation, but rather inhibits regulatory processes that control the disease. Therefore, we hypothesize that CTLA-4Ig treatment at this late stage rather interferes with the CTLA-4/B7 interaction and blocks CTLA-4 mediated cell-intrinsic negative regulation of Teff cells and/ or interferes with Treg cell function or induction.

Alpha-synuclein immunization, in the absence of alpha-synuclein over-expression or neural pathology, modifies microglia activation patterns and T cells' activation profiles

Dendritic cells (DCs) are responsible for the differentiation of naive CD4 T cells into T helper (Th) cells, which orchestrate the consequent adaptive immune response. Distinct Th subsets with different functions have been described, such as Th1, Th2, Th9, Th17 and Th22. Autoimmune diseases and, particularly, multiple sclerosis (MS), are characterized by persistent inflammation generated by Th1 and Th17 cells. However, the role of human DCs in shaping adaptive Th cell response in MS has not been investigated. In this study, we compared the production of Th cytokines by naive T cells polarizedwithmyeloid and plasmacytoid dendritic cells (mDCs and pDCs) in healthy donors (HD) and relapsing–remitting (RR)MS patients. We found that after stimulation mDCs are able to activate a Th17 immune response, while pDCs induce IL-10 production. Surprisingly, we found that stimulated pDCs from MS patients are also involved in the polarization of Th9 cells, which produce IL-9 and are known to be involved in allergic diseases. We investigated the potential role of pDCmediated IL-9 production inMS.We found that IL-9 activates STAT1 and STAT5 phosphorylation in Th17 cells and interferes with IL-17 production and IRF-4 expression by Th17-polarized cells. We measured IL-9 levels in the cerebrospinalfluid (CSF) ofMSpatients and found them to be inversely correlatedwith the severity ofMS andwith levels of IL-17 in the CSF. Moreover, we analysed CSF contents of IL-9 and we found that IL-9 is inversely correlated with indexes of inflammatory activity, neurodegeneration and disability progression of MS. Furthermore, high levels of IL-9 are associated with the absence of IL-17 in the CSF of RR-MS patients. In conclusion, these results suggest an important immunoregulatory role exerted by pDCs in driving the Th9 profile, which attenuate the exaggerated Th17 inflammatory response in MS.