Research ArticleNeurodegeneration

Activation of MyD88-dependent TLR1/2 signaling by misfolded α-synuclein, a protein linked to neurodegenerative disorders

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Science Signaling  12 May 2015:
Vol. 8, Issue 376, pp. ra45
DOI: 10.1126/scisignal.2005965

Abstract

Synucleinopathies, such as Parkinson’s disease and diffuse Lewy body disease, are progressive neurodegenerative disorders characterized by selective neuronal death, abnormal accumulation of misfolded α-synuclein, and sustained microglial activation. In addition to inducing neuronal toxicity, higher-ordered oligomeric α-synuclein causes proinflammatory responses in the brain parenchyma by triggering microglial activation, which may exacerbate pathogenic processes by establishing a chronic neuroinflammatory milieu. We found that higher-ordered oligomeric α-synuclein induced a proinflammatory microglial phenotype by directly engaging the heterodimer TLR1/2 (Toll-like receptor 1 and 2) at the cell membrane, leading to the nuclear translocation of NF-κB (nuclear factor κB) and the increased production of the proinflammatory cytokines TNF-α (tumor necrosis factor–α) and IL-1β (interleukin-1β) in a MyD88-dependent manner. Blocking signaling through the TLR1/2 heterodimer with the small-molecule inhibitor CU-CPT22 reduced the nuclear translocation of NF-κB and secretion of TNF-α from cultured primary mouse microglia. Candesartan cilexetil, a drug approved for treating hypertension and that inhibits the expression of TLR2, reversed the activated proinflammatory phenotype of primary microglia exposed to oligomeric α-synuclein, supporting the possibility of repurposing this drug for synucleinopathies.

INTRODUCTION

Synucleinopathies, such as Parkinson’s disease and diffuse Lewy body disease, are progressive neurodegenerative disorders characterized by the loss of selective neurons and the accumulation of misfolded α-synuclein into hallmark pathological lesions, Lewy bodies and Lewy neurites. In addition to being the major constituent of Lewy bodies (1), α-synuclein is implicated in disease etiology because point mutations and overexpression of the gene encoding α-synuclein, SNCA, are associated with familial forms of Parkinson’s disease (26). Moreover, genome-wide association studies have linked SNCA polymorphisms with an increased risk of developing idiopathic Parkinson’s disease (79). Together, these data point to a central role for α-synuclein in disease pathophysiology.

Accumulating evidence from animal models, along with biochemical and biophysical studies, supports the hypothesis that a key event in the pathogenesis of synucleinopathies is the process by which monomeric α-synuclein misfolds and self-assembles into oligomeric α-synuclein through a nucleated polymerization mechanism (1016). Oligomeric α-synuclein has been shown to be cytotoxic, inciting neurodegeneration by disrupting proteosomal, lysosomal, and mitochondrial functions, while also increasing cell membrane conductance (1721). Evidence also demonstrates that under pathological conditions, oligomeric α-synuclein can be released from neurons through nonclassical exocytosis, enabling α-synuclein to propagate to neighboring neurons and glia, inducing inclusion body formation, neuronal death, and neuroinflammation (2233).

The present study focuses on this latter mechanism of inflammation because the role of the innate immune response in the neurodegenerative processes underlying synucleinopathies and other diseases of the central nervous system has become increasingly evident (12, 3438). Specifically, Parkinson’s disease patients demonstrate a marked increase in activated microglia (3942), with increased expression and concentration of proinflammatory cytokines such as tumor necrosis factor–α (TNF-α) and interleukin-1β (IL-1β) in the substantia nigra pars compacta (SNpc), striatum, and cerebrospinal fluid as compared to control patients (4347). In addition, α-synuclein leads to increased numbers of activated microglia in mouse models of protein overexpression before SNpc dopaminergic neuronal death and causes proinflammatory microglial activation in cell culture experiments (38, 4855). Therefore, these observations suggest a close pathophysiological relationship between disease-associated α-synuclein and microglia-mediated neuroinflammation.

As the main contributors to inflammation in the brain parenchyma, microglia can be activated by engagement of membrane-bound pattern recognition receptors, such as Toll-like receptors (TLRs), which respond to both pathogen-associated molecular patterns and danger- or damage-associated molecular patterns (DAMPs) (5662). The role of TLRs as modulators of neurological disorders has become more apparent; for example, TLR2 and TLR4 exacerbate tissue damage in animal models of stroke and mediate the extracellular clearance of amyloid β (Aβ) peptide and Aβ-induced microglial activation (6366). Linking TLRs with synucleinopathies, we previously showed that microglia exposed to misfolded α-synuclein up-regulate the expression of genes encoding TLRs and the proinflammatory molecules TNF-α and IL-1β while undergoing morphological changes indicative of classical activation (4850). Studies using cell culture and animal models have shown conflicting results regarding the requirement of TLRs in microglial activation in response to α-synuclein (55, 67). The discrepancy regarding the signaling mechanism represents the complexity of α-synuclein–mediated microglial activation, and elucidation of the intracellular molecular players involved in α-synuclein–mediated neuroinflammation enhances the probability of ameliorating disease progression.

Here, we sought to identify the molecular mechanisms involved in α-synuclein–dependent microglial activation using mouse primary microglia, and we examined the possibility of using this knowledge to treat synucleinopathies.

RESULTS

Misfolding of human α-synuclein produces different protein structures

To interrogate the molecular underpinnings of α-synuclein–mediated microglial activation, we separated misfolded human wild-type α-synuclein (SynTR) into higher-ordered oligomeric (SynO) and small oligomeric/monomeric (SynM) species using size-exclusion centrifugation. Resolution of these species using Western blot analysis under nondenaturing conditions confirmed the effective separation and enrichment of SynO and small SynM (Fig. 1A). Structural analysis of SynO and SynM using transmission electron microscopy revealed the presence of fibrils in SynO fractions, whereas fibrils were absent in SynM fractions (Fig. 1B).

Fig. 1 Higher-ordered oligomeric α-synuclein induces complex morphofunctional activation of microglia.

(A) Representative Western blot analysis of misfolded human α-synuclein under nondenaturing conditions. Purified recombinant α-synuclein was misfolded (SynTR) and subsequently separated to isolate higher-ordered oligomeric conformers (SynO; bracket) from the monomeric and dimeric structures (SynM; arrowhead). (B) Representative transmission electron microscopy of SynO and SynM at ×75,000 magnification. Fibrils are present in the SynO fraction (black arrows). Scale bars, 100 nm. (C) Immunohistochemistry for Iba-1 in microglia after a 24-hour exposure to SynM or SynO (80.0 ng/ml). DAPI, 4′,6-diamidino-2-phenylindole. Scale bars, 20 μm. (D) Quantitative real-time polymerase chain reaction (qRT-PCR) for TNF-α and IL-1β expression in microglia after exposure to SynM or SynO (80.0 ng/ml). (E) Enzyme-linked immunosorbent assay (ELISA) for the concentrations of TNF-α and IL-1β in the conditioned medium from microglia cultured for 2 and 24 hours with SynO or SynM (80.0 ng/ml). (F) qRT-PCR analysis for ARG-1 (arginase 1) and IL-10 gene expression in microglia cultured with SynO or SynM (0.95 μg/ml) for 24 hours. Black dashed line, gene expression in vehicle control. (G) ELISA for the concentration of IL-10 in the supernatant of microglia cultured with either SynM or SynO (80 ng/ml) for 2 and 24 hours. All data are means ± SEM (n = 3 experiments). *P = 0.01, ***P < 0.0001. In (E), $$$P < 0.0001, comparing 2- and 24-hour SynO data. Data were analyzed by unpaired Student’s t test (D) or one-way analysis of variance (ANOVA) with Bonferroni post hoc test (E to G). N.D., not detectable.

Oligomeric α-synuclein induces a complex activation phenotype in microglia

To explore the differential effects of α-synuclein conformers on microglial activation, we exposed primary microglia to either SynM or SynO. We first characterized microglial morphology after exposure to these α-synuclein species by immunocytochemical staining for Iba-1, a microglia-specific calcium-binding protein. Microglia exposed to SynM retained a bipolar morphology, whereas microglia exposed to higher-ordered oligomers of α-synuclein (SynO) undertook an amoeboid, phagocytic morphology, indicative of classical activation (Fig. 1C). Furthermore, this SynO-induced morphological change was concurrent with a biochemical activation profile, such that these microglia had increased mRNA abundance and protein secretion of the proinflammatory cytokines TNF-α and IL-1β (Fig. 1, D and E). In contrast, the monomeric form of α-synuclein had no effect on these proinflammatory molecules. We next probed for changes in the expression of genes encoding the immunoresolution or alternative activation molecules arginase 1 and IL-10. The gene expression of these molecules was unchanged in the SynM-exposed microglia but was increased upon exposure to higher-ordered oligomeric α-synuclein (SynO; Fig. 1F). Furthermore, an ELISA specific for IL-10 showed that SynO-exposed microglia had a significant increase in the secretion of this cytokine (Fig. 1G). However, unlike the robust up-regulation of the proinflammatory cytokine TNF-α, which was detectable after 2- and 24-hour exposures to SynO (Fig. 1E), IL-10 was detectable only after the longer exposure time. These data suggest a temporally distinct activation profile in response to SynO, consisting of an initial proinflammatory phase followed by an up-regulation of anti-inflammatory factors.

Because the expression of genes encoding TNF-α, IL-1β, arginase 1, and IL-10 is regulated in part by the transcription factor nuclear factor κB (NF-κB), we next interrogated whether SynO exposure causes nuclear translocation of the active p65 subunit of this sequence-specific DNA binding protein. From our initial exploratory assays (fig. S1), we focused on the 2-hour time point to assess NF-κB translocation. Microglia exposed to SynO demonstrated a robust nuclear translocation of the NF-κB p65 subunit compared to cells exposed to SynM (Fig. 2A). Quantification of nuclear NF-κB p65 signal intensity by immunostaining demonstrated a significant translocation of p65 in the presence of SynO compared with SynM (Fig. 2B). We next used an ELISA-based activity assay to determine whether this nuclear p65 binds to its consensus DNA sequence, indicative of activity. Nuclear extracts from microglia exposed to SynO exhibited an increase in nuclear NF-κB activity compared with SynM-exposed cells (Fig. 2C). Together, these data suggest that the higher-ordered oligomeric form of α-synuclein incites a complex inflammatory activation profile that may be mediated through the NF-κB transcription pathway.

Fig. 2 Higher-ordered oligomeric α-synuclein induces nuclear translocation of active NF-κB.

(A) Immunocytochemistry for the p65 subunit of NF-κB in primary microglia after a 2-hour incubation with α-synuclein (80.0 ng/ml). Arrows represent nuclear localization of p65 whereas arrowheads indicate unappreciable nuclear localization. Scale bars, 20 μm. (B) Quantification of p65 nuclear signal intensity in (A). Data are means ± SEM from three independent replicates of 33 nuclei per condition. A.U., arbitrary units. (C) Quantification of active nuclear p65 NF-κB. SynO exposure increases active nuclear p65 NF-κB in comparison to SynM. Data are means ± SEM (n = 3 experiments). ***P ≤ 0.0001, comparing SynO to SynM, analyzed by unpaired Student’s t test.

Oligomeric α-synuclein localizes to the microglial cell surface and signals through a MyD88-dependent pathway

Having established that higher-ordered oligomeric α-synuclein induces inflammatory activity in microglia and NF-κB nuclear translocation, we next probed the signaling mechanism mediating the observed proinflammatory response. Our data thus far supported a possible role for NF-κB–mediated transcriptional regulation, and it is well established that this DNA binding protein is activated through microglial surface receptor signaling. Further linking surface receptors to α-synuclein–specific microglial activation, we previously reported that microglia stimulated with misfolded α-synuclein containing higher-ordered oligomeric, small oligomeric, and monomeric conformers increased the expression of genes encoding TLRs and the TLR adaptor protein MyD88 (encoded by myeloid differentiation primary response gene 88) (50). Together, these data suggest that α-synuclein may initiate microglial activation at the cell surface by engaging TLRs.

First, to interrogate whether SynM and SynO localized to the microglial cell surface, microglia were exposed to the two α-synuclein species followed by biotinylation of cell surface proteins. Biotinylated proteins were subsequently isolated using an avidin-binding column and subjected to Western blot analyses under nondenaturing conditions. Successful isolation of cell membrane proteins was confirmed by probing for the highly expressed microglial surface receptors TLR1 and TLR2 in all treatment groups (Fig. 3, A and B). Probing for α-synuclein revealed that only the higher-ordered oligomeric form of α-synuclein interacted with the microglial membrane (Fig. 3C).

Fig. 3 Higher-ordered oligomeric α-synuclein localizes to the surface of microglia and mediates MyD88-dependent signaling.

(A to C) Nondenaturing Western blot analyses of biotinylated surface TLR1 (A) and TLR2 (B) (arrowheads) and human α-synuclein (C) (bracket) from microglia exposed to SynM or SynO (0.8 μg/ml; 30 min). Blots are representative of three experiments. (D) Peptide inhibition of MyD88 homodimerization. SynO-induced TNF-α and IL-1β expression in microglia treated with either control peptide (CTLpep) or inhibitory MyD88 peptide (MyD88pep) for 30 min before being exposed to SynO (80.0 ng/ml; 2 hours). (E) ELISA for the concentration of TNF-α in the conditioned medium from (D). Data are means ± SEM (n = 3 experiments). **P ≤ 0.005 by unpaired Student’s t test.

Our data support that higher-ordered oligomeric α-synuclein acts at the microglial cell surface and possibly in an NF-κB–dependent pathway, suggestive of TLR engagement. Given that a subset of TLRs requires the homodimerization of MyD88 to promote nuclear translocation of NF-κB, we used a MyD88 peptide inhibitor to determine whether MyD88-dependent pathways are relevant to SynO-mediated microglial activation. We pretreated microglia with a control peptide (CTLpep) or an inhibitory peptide against MyD88 homodimerization (MyD88pep) and subsequently exposed the cells to SynO. Using qRT-PCR, we found a significant reduction in TNF-α and IL-1β gene expression in cells treated with the MyD88 inhibitor compared to those treated with the control peptide (Fig. 3D). Moreover, in parallel with reduced proinflammatory gene expression, inhibition of MyD88 signaling decreased the amount of TNF-α released from microglia in response to stimulation with SynO (Fig. 3E). These data suggest that the higher-ordered oligomeric α-synuclein–induced proinflammatory response in microglia acts at the cell membrane through a MyD88-dependent mechanism.

Oligomeric α-synuclein interacts with TLR1/2

Thus far, we have established that α-synuclein–induced microglial activation is dependent on both the higher-ordered oligomeric structure of α-synuclein and the homodimerization of MyD88, a critical adaptor protein for pattern recognition receptor signaling. Because our previous studies found that microglia-like murine cells (BV2 cells) and primary microglia have increased gene expression of TLR1, TLR2, and TLR3 upon exposure to misfolded α-synuclein (48, 50), we next inquired whether our isolated forms of α-synuclein induced differential expression of these pattern recognition receptors. We exposed microglia to oligomeric or monomeric α-synuclein for 24 hours and quantified the expression of these pattern recognition receptors. There was a significant increase in TLR1, TLR2, and TLR3 gene expression in microglia exposed to SynO compared to that in either SynM or vehicle conditions, with TLR1 demonstrating the greatest increase in expression (Fig. 4A). There were no significant differences between SynM- and vehicle-treated conditions for all gene expressions measured.

Fig. 4 SynO increases microglial TLR expression and interacts with hTLR2 and TLR1.

(A) qRT-PCR for TLR expression after a 24-hour exposure to SynO (80.0 ng/ml). Dashed line indicates vehicle gene expression level. Data are means ± SEM (n = 2 experiments). (B) SEAP activity in HEK-hTLR cells. HEK-hTLR cells were exposed to vehicle, the TLR1/2 agonist Pam (1 μg/ml), the TLR3 agonist poly I:C [P(I:C); 1 μg/ml], SynM, or SynO (80.0 ng/ml per α-synuclein treatment) for 20 hours followed by SEAP quantification. Data are means ± SEM (n = 3 experiments). (C) Representative PLA images of microglia after exposure to vehicle, SynM, or SynO (0.8 μg/ml) for 10 min with probes against human α-synuclein (LB509) and TLR1 antibodies (white dots indicate interaction). Nuclei were counterstained with DAPI. Insets represent enlarged image of one cell. Scale bars, 20 μm. (D) Quantification of SynO/TLR1 interaction. Dashed line represents vehicle signal intensity; normalized signal intensity was determined from four random fields of view across two separate experiments. (A) **P = 0.008, ***P < 0.0001, comparing SynM and SynO; $$P = 0.008, $$$P < 0.0001, comparing vehicle and SynO; (B) ***P < 0.0001; (D) ***P < 0.0001, comparing SynM and SynO; $$$P < 0.0001, comparing vehicle and SynO by one-way ANOVA with Bonferroni post hoc test. For all data, there was no significant difference between SynM and vehicle control.

Given this robust increase in TLR1 expression and that TLR1 heterodimerizes with TLR2 to initiate MyD88-dependent intracellular signaling, we used a secreted embryonic alkaline phosphatase (SEAP) reporter system in human embryonic kidney (HEK) 293 cells that overexpresses human TLR2 and expresses endogenous amounts of TLR1 (HEK-hTLR2) to determine whether α-synuclein directly interacted with these pattern recognition receptors. We first established that HEK-hTLR2 reporter cells responded to a specific TLR1/2 agonist, Pam3CSK4 (Pam). HEK-hTLR2 cells exposed to Pam showed a robust increase in SEAP reporter activity (Fig. 4B), indicating that these cells are capable of forming and signaling through the TLR1/2 receptor heterodimer. We then used this system to test whether α-synuclein signaled through this same pathway. SynO exposure induced TLR2-SEAP activity, whereas SynM had no detectable effect (Fig. 4B). Furthermore, because TLR3 mRNA abundance was also increased in microglia upon exposure to SynO, we used a similar SEAP reporter system, but with cells overexpressing human TLR3 (HEK-hTLR3), to investigate whether SynO engaged TLR3 homodimers. Only the TLR3-specific agonist polyinosinic:polycytidylic acid (poly I:C) increased SEAP activity (Fig. 4B), indicating that neither form of α-synuclein directly interacted with TLR3, a MyD88-independent receptor.

To confirm that higher-ordered oligomeric α-synuclein interacts with TLR1 in primary microglia, we performed an in situ proximity ligation assay (PLA) using an antibody specific for human synuclein (LB509) in conjunction with a TLR1 antibody after microglial exposure to vehicle, SynM, or SynO. Confocal microscopy of microglia after in situ PLA revealed numerous punctate signals after SynO exposure, indicative of a robust interaction between SynO and TLR1 (Fig. 4C). In contrast, vehicle- and SynM-treated conditions elicited no appreciable interaction between α-synuclein and TLR1 (Fig. 4C). Quantification of the PLA further supported a significant interaction between the higher-ordered oligomeric form and TLR1 (Fig. 4D). Together, these data show that α-synuclein–mediated microglial activation is specific to the higher-ordered oligomeric form of α-synuclein, which can directly signal through TLR1/2 heterodimers in a MyD88-dependent mechanism.

Pharmacological inhibition of TLR1/2 attenuates oligomeric α-synuclein–mediated effects

Because SynO directly interacted with the TLR1/2 receptor complex, we next asked whether blocking this interaction diminishes the higher-ordered oligomeric α-synuclein–mediated proinflammatory response. We previously developed a novel small-molecule antagonist of the TLR1/2 complex, CU-CPT22 (68). To test the effectiveness of this compound in blocking the SynO-TLR1/2 interaction, we used the HEK-hTLR2 reporter cells, which (as shown above) respond to the TLR1/2 activator Pam and SynO. Incubation of these reporter cells with CU-CPT22 and either Pam or SynO caused a significant decrease in reporter activity (Fig. 5A), further supporting the hypothesis that SynO can signal through the TLR1/2 complex.

Fig. 5 CU-CPT22 attenuates SynO-mediated signaling and activation.

(A) SEAP activity of HEK-hTLR2 cells (n = 3) after simultaneous exposure to Pam (1 ng/ml) or SynO (8.0 ng/ml) and CU-CPT22 or vehicle. Data are means ± SEM (n = 3). ***P < 0.0001, comparing CU-CPT22 condition to vehicle condition for Pam and SynO analyzed by unpaired Student’s t test. (B) Nuclear NF-κB translocation after treatment of microglia with SynO (8.0 ng/ml) and vehicle (white arrows) or CU-CPT22 (white arrow heads). Scale bars, 20 μm. (C) Quantification of the change in nuclear NF-κB p65 shown in (B). (D) ELISA for the concentration of TNF-α in conditioned medium from microglia simultaneously treated with SynO (80.0 ng/ml; 72 hours) and vehicle or CU-CPT22 (10 μM). Data are means ± SEM (n = 3 experiments). *P < 0.01 by one-way ANOVA with Bonferroni post hoc test.

We next sought to translate these results to our primary cell culture model by exposing microglia to SynO while simultaneously inhibiting TLR1/2. To isolate the effect of TLR1/2 signaling on nuclear NF-κB translocation after SynO exposure, we treated primary microglia with CU-CPT22, the TLR1/2 antagonist, and simultaneously exposed the cells to SynO. Immunocytochemical analysis of NF-κB confirmed that SynO enhanced the nuclear translocation of p65 (Fig. 5B) but that blocking TLR1/2 with CU-CPT22 significantly decreased this effect (Fig. 5, B and C). Treatment with CU-CPT22 also significantly decreased the SynO-mediated release of TNF-α (Fig. 5D). Together, these data support that TLR1/2 engagement is important for oligomeric synuclein–induced microglial activation.

We tested a second pharmacological agent, candesartan cilexetil (candesartan), for its ability to attenuate synuclein-mediated activation of microglia. Candesartan is an angiotensin II receptor blocker (ARB) that also inhibits TLR2 receptor expression and activity in both in vitro and in vivo models of inflammation (69). Given its reported effect on the expression of the gene encoding TLR2, and because of the ability of higher-ordered oligomeric α-synuclein to activate the TLR1/2 complex, we reasoned that candesartan might prove effective in dampening SynO-mediated microglial activation. To test this hypothesis, we pretreated microglia with increasing concentrations of candesartan and subsequently exposed these cells to higher-ordered oligomeric α-synuclein. We observed a marked morphological and functional change in candesartan-treated microglia (Fig. 6). Specifically, increasing concentrations of candesartan more potently reversed the prototypical amoeboid morphology indicative of classically activated microglia (Fig. 6A) and significantly decreased SynO-induced TNF-α secretion (Fig. 6B). These data show that this U.S. Food and Drug Administration (FDA)–approved drug attenuates the SynO-induced microglial proinflammatory phenotype and that TLR1/2 is a potential target to suppress synuclein-mediated glial activation.

Fig. 6 Candesartan diminishes SynO-mediated morphofunctional responses in microglia.

(A) Iba-1 immunocytochemistry (red) of microglia pretreated with 10, 20, or 30 μM candesartan cilexetil (CD) or vehicle (equivalent to 30 μM CD) for 2 hours and subsequently exposed to SynO (8.0 ng/ml) or buffer for 12 hours. Cell nuclei were stained with DAPI (blue). Scale bars, 20 μm. (B) ELISA for the concentration of TNF-α in conditioned medium of microglia treated as in (A). Data are means ± SEM (n = 3). *P = 0.01, **P = 0.001, comparing 10, 20, or 30 μM conditions to vehicle + SynO by one-way ANOVA with Bonferroni post hoc test.

DISCUSSION

We previously showed that higher-ordered oligomeric α-synuclein causes a complex morphofunctional response in microglia (4850). This response is conformation-specific because monomeric or smaller oligomeric species of α-synuclein do not incite a similar activation profile. Here, we identified the mechanism by which higher-ordered oligomeric α-synuclein induces microglial activation through a MyD88-dependent TLR1/2 pathway. Together, our findings suggest that this pattern recognition receptor complex is a druggable target to treat neuroinflammation in synucleinopathies.

The idea that higher-ordered α-synuclein acts as a DAMP promoting neuroinflammation has implications for disease pathogenesis. In support of this, studies show that oligomeric α-synuclein is released from neurons and subsequently propagates to neighboring neurons and glia (26, 2833, 70). Human pathological studies also support the concept that oligomeric α-synuclein spreads throughout the nervous system, with evidence for α-synuclein pathology in the gut, olfactory system, brain stem, midbrain, and cortical regions; however, there has been debate about the exact mechanism of this “prion-like” pathology (7178). Injection of preformed recombinant human α-synuclein fibrils into mice results in a spreading of Lewy body–like pathology, lending further support to the propagation theory for synucleinopathies (7984). Likewise, the injection of Lewy body extracts from the brains of Parkinson’s disease patients into those of either mice or rhesus monkeys demonstrates the ability of this misfolded protein to incite disease pathology (85). Collectively, these studies support a role for oligomeric α-synuclein in Parkinson’s disease pathogenesis.

The present work supports a role for higher-ordered oligomeric α-synuclein in the initiation of neuroinflammation, a common feature of synucleinopathies (8689). Although we are unable to pinpoint the exact molecular structure of the inflammation-mediating α-synuclein, we have demonstrated that only the higher-ordered oligomeric fibrillar conformers (>720 kD) cause a robust inflammatory response; the smaller oligomeric and monomeric forms of this protein do not incite a measurable response in microglia. It would be useful in future studies to further classify the oligomeric α-synuclein subspecies because this information could be used for the development of additional drug therapies. Furthermore, work by others suggests that not all oligomeric species are toxic to cells because only higher-ordered oligomers cause a microglial response [reviewed in (16)], and here we support this hypothesis.

As the innate immune cells of the central nervous system, microglia work to maintain homeostasis, responding to changes in the microenvironment with complex and variable immunofunctional profiles. However, activation states are not simply categorical because microglial cells can adopt a mixed phenotype within the spectrum of “resting” to “fully activated.” In fact, here, we demonstrated that in a nearly pure population of cultured microglia, there is evidence for both “classically” activated (such as TNF-α and IL-1β abundance and release) and “immune resolution” or “alternatively” activated phenotypes [such as IL-10 and arginase 1 abundance; reviewed in (90)]. Previous studies using recombinant human α-synuclein report detectable mRNA expression of proinflammatory molecules in microglia 45 min after exposure (52). Here, we captured early and late mRNA expression changes and found that proinflammatory TNF-α was detectable as early as 2 hours after exposure to higher-ordered oligomeric α-synuclein, whereas proinflammatory IL-1β as well as anti-inflammatory IL-10 was not evident until 24 hours after exposure. Previous studies show that microglial IL-10 abundance is up-regulated during proinflammatory events and subsequently inhibits the expression of genes encoding cytokines (such as TNF-α) and cytokine receptors (91). Thus, our results suggest that IL-10 expression is increased in response to microglial activation in an attempt to maintain homeostasis.

The signaling pathway leading to these varied activation states are controlled by the engagement of surface receptors by specific ligands (59, 60, 9296). Our findings suggest that higher-ordered oligomeric α-synuclein is a DAMP that interacts with a specific TLR complex. Although our work substantiates a role for TLR1/2 in Parkinson’s disease neuroinflammation, we recognize that other pattern recognition and surface receptors also play a role in synucleinopathies. Previous work suggests that CD36 is important for α-synuclein–mediated microglial activation (52). However, that study did not isolate different α-synuclein structures, and primary microglia from CD36 knockout mice did not completely abrogate the synuclein-mediated effect, suggesting that multiple receptors could be important for microglial activation or that the response is α-synuclein structure–specific. Others have implicated TLR4 in Parkinson’s disease: Fellner et al. (55) demonstrated that microglial phagocytosis, NF-κB nuclear translocation, release of proinflammatory molecules, and nitric oxide production all decrease after α-synuclein exposure in microglial cells isolated from TLR4 knockout mice. In a mouse model of α-synuclein overexpression (Thy1-aSyn mice), the expression of TLR1, TLR4, and TLR8 in the substantia nigra are up-regulated at about 6 months of age, whereas only TLR2 is increased at 14 months of age (97), suggesting a temporally specific TLR effect. In cell culture, oligomeric α-synuclein released from SH-SY5Y cells activates microglia, and this effect is reduced in microglia derived from TLR2 knockout mice (67). Other studies also support an involvement of TLR2 in synuclein-mediated inflammation (98, 99). Overall, there is strong support for TLR signaling in α-synuclein–mediated activation of microglia converging on TLR2 and TLR4. Other DAMPs, including fibrillar β-amyloid, signal through both TLR2 and TLR4, suggesting that these receptors are promiscuous [reviewed in (100) and (64, 101, 102)].

One outstanding question is whether TLRs and subsequently activated microglia are the cause or consequence of neuronal death in synucleinopathies. Work by Doorn et al. (103) shows that TLR2 colocalizes with microglia and is increased in postmortem brains of incidental Lewy body disease cases as well as those from Parkinson’s disease patients. Furthermore, these findings support that α-synuclein is the trigger for microglial activation in synucleinopathies because activated microglia were only present in brain regions displaying α-synuclein deposits. Doorn et al. (103) also demonstrated that neuronal death was not required for microglial activation because activated glia were evident in areas that had no measurable neuronal death. This pathologically based study suggests that activated microglia are dynamic contributors to pathogenesis rather than simply harbingers of neuronal death.

Our identification of a TLR1/2-dependent mechanism in a Lewy body disease–relevant inflammatory pathway provides us the opportunity to target this pathway with new or repurposed therapies (103, 105107), such as with CU-CPT22, a benzotropolone analog with specificity and potency for TLR1/2, or candesartan cilexetil, an FDA-approved drug that is already used to treat cerebrovascular disorders, hypertension, and chronic heart failure [reviewed in (108)]. The group of drugs to which candesartan belongs (called sartans) are also potent antiatherosclerotic and neuroprotective agents that decrease vascular inflammation, autoimmune neuroinflammation, dopaminergic neuronal death, and α-synuclein aggregation (104, 109119). Evidence suggests that the antiatherosclerotic activity of sartans is mediated through activation of the peroxisome proliferator–activated receptor-γ (PPARγ) (120122). However, candesartan is not a potent agonist of PPARγ (123), suggesting that the effect of this drug on α-synuclein–induced microglial activation is not mediated through PPARγ. Candesartan can decrease the abundance of TLR2 and the signaling activity of the TLR1/2 receptor in monocytes (69). Future studies are needed to decipher the exact mechanism of candesartan’s effect on α-synuclein–induced microglial activation (and presumably neuroinflammation), but we hypothesize that it does so by suppressing overall TLR1/2 signaling in the microglia. Thus, our work suggests that inhibitors of TLR1/2 may suppress neuroinflammation and improve disease pathology in patients with synucleinopathies.

MATERIALS AND METHODS

Preparation and misfolding of α-synuclein

Human α-synuclein was prepared as previously described (124, 125), except that after purification, the α-synuclein was lyophilized and stored at −80°C until use. To misfold α-synuclein, the lyophilized protein was resuspended in TEN buffer [10 mM tris-HCl (pH 7.5), 1 mM EDTA, 20 mM NaCl] to a final concentration of 1 mg/ml and sonicated at 20 Hz (two 10-s bursts with a 10-s rest in between bursts). α-Synuclein was subsequently incubated for 5 days at 37°C (T) with rotation at 1000 rpm (R), resulting in the SynTR fraction (ThermoMixer, Eppendorf).

SynTR was then separated by size-exclusion centrifugation to isolate high–molecular weight oligomers from the monomeric conformer (48). Specifically, SynTR (0.3 mg) was placed onto a 150-kD molecular weight cutoff concentrator (Thermo Scientific) and centrifuged at 2000g for 30 min. The concentrate (SynO) was resuspended in TEN buffer (100 μl) and used for subsequent analyses. The flow-through was processed using a 20-kD concentrator (Thermo Scientific) and centrifuged at 3000g for 30 min; the concentrate (SynM) was resuspended in TEN buffer (100 μl) and used for subsequent analyses.

This method of isolation resulted in recombinant synuclein fractions with an endotoxin content that is below the detection limit of the E-TOXATE test kit [0.03 endotoxin units (EU)/ml; Sigma-Aldrich, cat. no. ET0200; 1 EU/ml ≈ 0.1 to 0.2 ng endotoxin/ml], consistent with our previous reports (48, 52).

Western blot

SynTR, SynO, and SynM fractions (0.1 μg) were subjected to native polyacrylamide gradient (3 to 12% bis-tris) gel electrophoresis using the NativePAGE Novex Bis-Tris Gel System (Life Technologies). Proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane and probed for α-synuclein using the Syn211 clone antibody (1:1000; Thermo Scientific). Immune complexes were visualized on film (Amersham Hyperfilm ECL, General Electric Healthcare) after incubation with horseradish peroxidase (HRP)–conjugated goat anti-mouse secondary antibody (1:20,000; Chemicon) and SuperSignal West Femto chemiluminescent substrate (Thermo Scientific).

To probe for α-synuclein in the cell surface isolation experiment, microglia were treated and cell surface proteins were isolated as described below. Isolated biotinylated proteins were subjected to native polyacrylamide gradient (3 to 12% bis-tris) gel electrophoresis using the NativePAGE Novex Bis-Tris Gel System. Proteins were transferred onto PVDF membranes, and the membranes were blocked for 1 hour at room temperature in TBST/NFDM [20 mM tris-HCl (pH 7.5), 150 mM NaCl, 0.1% (v/v) Tween, 5% (w/v) nonfat dry milk (NFDM)], followed by incubation with primary antibody (Syn211; 1:1000). Immune complexes were visualized on film after incubation with HRP-conjugated goat anti-mouse secondary antibody (1:5000; Chemicon) and Amersham chemiluminescent substrate (General Electric Healthcare). The membranes were reprobed using rabbit anti-TLR2 (1:1000; LifeSpan Biosciences, cat. no. LS-C98268) and rabbit anti-TLR1 (1:250; Imgenex, cat. no. IMG-5012), which served as controls for successful isolation of surface proteins.

Electron microscopy

SynO or SynM (1.0 μg) was placed on a carbon/formvar-coated 300-mesh copper grid (Electron Microscopy Sciences). Samples were allowed to adhere to each grid for 20 min, followed by staining with 2% uranyl acetate for 2 min. Grids were imaged using Hitachi H-7600 filament transmission electron microscope with a Hammamatsu ORCA-HR charge-coupled device camera in collaboration with A. Popratiloff at The George Washington University.

Preparation and treatment of primary microglia

Primary microglia cultures derived from C57/Bl6 mouse cortices were prepared as previously described (126). Microglia were plated at a density of 1 × 105 cells per well (24-well plates) in 0.5 ml of microglia growth medium [MGM; Earle’s minimum essential medium supplemented with 1 mM sodium pyruvate, 0.6% (v/v) d-(+)-glucose, 1 mM l-glutamine, penicillin/streptomycin (100 μg/ml), and 5% (v/v) fetal bovine serum]. For immunocytochemistry experiments, microglia were plated on sterile glass coverslips using the same procedure. For isolation of surface proteins experiments, microglia were plated at 6 × 105 cells per well (six-well plates) in 2.0 ml of MGM. For in situ PLA experiments, microglia were plated at 2 × 104 cells per well in 16-well chamber slides (Nunc Lab-Tek, Thermo Scientific, cat. no. 178599). For quantification of nuclear NF-κB activity, microglia were plated at 8.5 × 105 cells per well (six-well plates). Microglia were subsequently treated with SynO, SynM, or 20 mM TEN buffer (vehicle) in MGM as indicated in the figure legends.

Isolation of surface proteins

Microglia were plated as described above and treated with SynO or SynM (0.8 μg/ml) for 30 min at 4°C. Medium was subsequently removed, microglia were washed with ice-cold phosphate-buffered saline (PBS; 1×), and surface proteins were biotinylated and isolated according to the manufacturer’s instructions (Thermo Scientific, cat. no. 89881). This procedure was performed in three separate experiments with three biological replicates.

Immunocytochemistry

Microglia were plated on glass coverslips (12 mm; Deckglaser), treated as described, and subsequently processed for immunocytochemistry. More specifically, after treatment, cells were washed with PBS for 5 min, fixed with PBS containing 4% (w/v) paraformaldehyde and 4% (w/v) sucrose (pH 7.4) at room temperature for 15 min, permeabilized in PBS containing 0.1% (v/v) Triton X-100 for 5 min, and blocked for 1 hour with PBS containing 10% (v/v) goat serum. Cells were subsequently incubated overnight at 4°C with either rabbit Iba-1 antibody (anti–Iba-1; 1:750; Wako) or rabbit anti–NF-κB (1:1000; α-p65; Abcam) in blocking buffer. Antibody/antigen complexes were visualized after incubation with Alexa Fluor 594–conjugated goat anti-rabbit immunoglobulin G secondary antibody (1:1000) in PBS containing 0.1% (v/v) Triton X-100 and 1% goat serum. Unbound secondary antibodies were removed by washing with PBS containing 0.1% (v/v) Triton X-100. Cells were counterstained with DAPI (13.0 ng/μl) in PBS for 5 min followed by two washes with PBS. Coverslips were mounted with CitiFluor (Ted Pella) and sealed with nail polish. Cells were imaged using a Zeiss Axioskop fluorescence microscope (Carl Zeiss), and images were captured using an AxioCam HRm camera (Carl Zeiss).

RNA extraction

After treatment, RNA was harvested from microglia using an RNeasy Mini Kit with on-column deoxyribonuclease I digestion according to the manufacturer’s instructions (Qiagen). RNA concentrations were measured using a NanoDrop 1000 spectrophotometer (Thermo Scientific).

Quantitative RT-PCR

RNA (1 μg) was reverse-transcribed in a 20-μl reaction volume using a High-Capacity cDNA Archive Kit (Life Technologies). β-Actin RT-PCR was subsequently performed to verify the quality of the complementary DNA (cDNA). Gene expression was quantified by qRT-PCR in a 96-well format. Specifically, cDNA (2.5 μl) was combined with master mix (17.5 μl) containing the appropriate primer/probe pairs and TaqMan Universal PCR Master Mix. qRT-PCR reactions were performed using the ABI Prism 7900HT Sequence Detection System (Life Technologies). Data were analyzed using the relative quantification ΔΔCt method, normalizing target gene expression to a GAPDH (glyceraldehyde phosphate dehydrogenase) endogenous control, followed by normalization to either SynM or TEN buffer–treated controls. The primers/probes used were as follows: ARG-1 Mm00833903_m1, IL-10 Mm0434228_m1, TNF-α Mm00443258_m1, IL-1β Mm0434228_m1, TLR1 Mm0120884_m1, TLR2 Mm00442346_m1, TLR3 Mm00446577_g1, and GAPDH 4352339E. Gene expression changes are represented as fold change (2−ΔΔCt). Statistical analyses were performed on ΔCt values as specified in the figure legends, and the significance threshold was set at P < 0.05. All measurements were performed in three separate experiments with three biological replicates and duplicate technical replicates, unless otherwise noted in the figure legends.

Enzyme-linked immunosorbent assay

TNF-α, IL-1β, and IL-10 protein concentrations in cell culture supernatants were quantified using an ELISA according to the manufacturer’s instructions (R&D Systems). All measurements were performed in three separate experiments with three biological replicates and duplicate technical replicates.

MyD88 homodimerization inhibition

Primary microglia were plated as described above. Cells were then pretreated with 200 μM of control peptide or MyD88 inhibitory peptide (Imgenex) for 30 min before a 2-hour exposure to SynO (80.0 ng/ml). RNA was then harvested from the treated cells, and the gene expression of TNF-α and IL-1β was quantified using qRT-PCR as described above. All measurements were performed in three separate experiments with three biological replicates and duplicate technical replicates.

In situ PLA

Microglia were plated as described above in 16-well chamber slides. Microglia were exposed to vehicle, SynM, or SynO (0.8 μg/ml) for 10 min at 37°C. After the exposure time, medium was removed, and cells were washed twice with ice-cold PBS (1×) and fixed with PBS containing 4% (w/v) paraformaldehyde and 4% (w/v) sucrose (pH 7.4) at room temperature for 15 min. PLA was performed according to the manufacturer’s instructions (Duolink, Sigma-Aldrich). In brief, microglia were permeabilized in PBS containing 0.1% (v/v) Triton X-100 for 5 min and blocked for 1 hour with PBS containing 10% (v/v) normal goat serum. Cells were subsequently incubated in a dropwise reaction for 1 hour in a humidified chamber at 37°C with mouse synuclein antibody (anti-synuclein LB509; 1:1000; Sigma-Aldrich) and rabbit anti-TLR1 (IMG-5012; 1:250; Imgenex) in blocking buffer. Removal of unbound primary antibodies was performed by washing in PBS containing 0.1% (v/v) Triton X-100 and 0.1% (v/v) normal goat serum. Donkey anti-rabbit (+) PLA probe (DUO92002; Sigma-Aldrich) and donkey anti-mouse (−) PLA probe (DUO92004) were diluted (1:5) in blocking buffer and applied to cells in a dropwise reaction. Cells were incubated with PLA probes for 1 hour in a humidified chamber at 37°C. Cells were subsequently washed in wash buffer; PLA probes were ligated in 1× ligation buffer with ligase (DUO92013) for 30 min, followed by rolling circular amplification in 1× amplification buffer with polymerase (DUO92013); and amplified signal was visualized with far-red fluorescent detection molecule (DUO92013). Cells were mounted with an in situ mounting medium containing DAPI (DUO82040). In situ PLA signals were imaged using an LSM510 confocal microscope. Images are representative Z-stack projections (7-μm stack; 1 μm per section). This procedure was repeated in three separate experiments with three biological replicates.

HEK-Blue TLR reporter assay

HEK293 reporter cells stably expressing human TLR2 (HEK-hTLR2) and human TLR3 (HEK-hTLR3) were used according to the manufacturer’s instructions (InvivoGen). HEK-hTLR2 cells were exposed to either Pam (1.0 μg/ml) or the different forms of α-synuclein (80.0 ng/ml) and allowed to incubate for 20 hours. Similarly, HEK-hTLR3 cells were exposed to either poly I:C (1.0 μg/ml) or the different forms of α-synuclein (80.0 ng/ml) and allowed to incubate for 20 hours. All measurements were performed in two separate experiments in three biological replicates with duplicate technical replicates.

CU-CPT22 treatment

A previously described TLR1/2 inhibitor, CU-CPT22 (68), was used to block signaling in primary microglia and HEK-hTLR2 cells. HEK-hTLR2 cells were simultaneously treated with 100 μM CU-CPT22 and either SynM (8.0 ng/ml), SynO (8.0 ng/ml), or the TLR1/2 agonist Pam (1 μg/ml) for 20 hours, followed by SEAP reporter activity quantification according to the manufacturer’s instructions (InvivoGen). Primary microglia were simultaneously treated with 400 μM CU-CPT22 and SynO (8.0 ng/ml) for 10 or 20 min, and then NF-κB nuclear translocation was quantified as described below (see “Quantification of nuclear NF-κB activity”). For experiments quantifying TNF-α release, primary microglia were simultaneously treated with 10 μM CU-CPT22 and SynO (80.0 ng/ml) for 72 hours. TNF-α was quantified as described above (see “Enzyme-linked immunosorbent assay”). All measurements were performed in two separate experiments in duplicate biological replicates with duplicate technical replicates.

Microglial nuclear extraction

Microglial cells were plated as described above and exposed to either SynM (80.0 ng/ml) or SynO (80.0 ng/ml) for 2 hours. Preparation of nuclear extracts was subsequently performed according to the manufacturer’s instructions (Nuclear Extraction Kit, Active Motif, cat. no. 40010). In brief, cell culture supernatant was removed, and cells were washed with 2 ml of PBS/phosphatase inhibitor solution (125 mM NaF, 250 mM β-glycerophosphate, 250 mM p-nitrophenyl phosphate, 25 mM NaVO3). Cells were subsequently harvested in 1 ml of PBS/phosphatase inhibitor solution by cell scraping, and cell suspension was centrifuged for 5 min at 200g in a microcentrifuge precooled at 4°C. The supernatant was discarded, and the cell pellet was resuspended in 125 μl of 1× hypotonic buffer [20 mM Hepes (pH 7.5), 5 mM sodium fluoride, 10 μM sodium molybdate, 0.1 mM EDTA] and incubated for 15 min on ice to allow cells to swell. To release cell nuclei, cells were lysed by adding detergent (5 μl of 10% NP-40) and vortexed for 10 s. This suspension was centrifuged for 30 s at 14,000g in a microcentrifuge precooled at 4°C to pellet cell nuclei. The supernatant (cytoplasmic fraction) was collected and stored at −80°C. Nuclear pellets were resuspended in 50 μl of complete lysis buffer [Lysis Buffer AM1 supplemented with 1 mM dithiothreitol and 1.0% (v/v) protease inhibitor cocktail] and vortexed for 10 s. The suspensions were incubated for 30 min on ice with rotary shaking, vortexed for 30 s, and subsequently centrifuged for 10 min at 14,000g in a microcentrifuge precooled at 4°C. The nuclear fractions were stored at −80°C until used for quantification of nuclear NF-κB activity.

Quantification of nuclear NF-κB activity

Activation of nuclear NF-κB was measured in nuclear protein extracts using the TransAM p65 NF-κB Kit (Active Motif, cat. no. 40596), an ELISA-based method designed to quantify NF-κB p65 subunit activation, according to the manufacturer’s instructions. Absorbances were measured at 450 nm using a microplate absorbance reader model 680 (Bio-Rad). All measurements were performed in two separate experiments in triplicate biological replicates with duplicate technical replicates.

Nuclear NF-κB translocation quantification

Cells were processed for NF-κB immunocytochemistry as described above. Images of NF-κB and DAPI staining were captured as described above from 10 distinct, randomly selected regions of each treatment condition by a blinded observer. Quantification of nuclear NF-κB intensity was completed as previously described (127, 128) using ImageJ software [National Institutes of Health (NIH)]. A total of 52 cells were analyzed for each condition. The percent change in nuclear NF-κB was calculated by normalizing the raw data obtained from microglia treated with inhibitor to their respective vehicle control. All measurements were performed in two separate experiments in duplicate biological replicates. Percent decrease from both experiments was plotted as means ± SEM.

Candesartan treatment

Primary microglia were plated as described above on glass coverslips followed by pretreatment with 10, 20, or 30 μM candesartan cilexetil (SML0245, Sigma-Aldrich) or vehicle (dimethyl sulfoxide) for 2 hours. Pretreated cells were subsequently exposed to SynO (8.0 ng/ml) for 12 hours and processed for Iba-1 immunocytochemistry as described above. All measurements were performed in triplicate in three separate biological replicates.

Statistical analyses

All statistical analyses were performed using GraphPad Prism 5 (GraphPad Software Inc.). For qRT-PCR, all statistical analyses were performed on ΔCt values. One-way ANOVA was performed followed by an appropriate post hoc test, as specified in the figure legends. All data are reported as means ± SEM, and significance was set at P ≤ 0.05; all P values for each statistical analysis are reported in the appropriate figure legend.

SUPPLEMENTARY MATERIALS

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Fig. S1. Preliminary time course of p65 staining in microglia.

REFERENCES AND NOTES

Acknowledgments: We thank A. Edwards for technical assistance and A. Popratiloff for assistance with electron microscopy. Funding: This work was supported in part by Parkinson’s Disease Foundation Summer Student Fellowship PDF-SFW-1350 (C.D.), NIH/National Institute of Neurological Disorders and Stroke T32NS041218 (D.B.), NIH R01GM101279 (H.Y.), NIH/National Institute of Environmental Health Sciences R01ES014470 (K.A.M.-Z.), and a Parkinson’s Movement Disorder Foundation grant (K.A.M.-Z.). Author contributions: S.G.D. and K.A.M.-Z. designed the research, analyzed the data, and wrote the paper; S.G.D. performed most of the experiments; D.B. performed the electron microscopy and qRT-PCR for TLR1 to TLR3; C.D. performed the HEK-hTLR experiments; S.G.D., D.B., C.D., and K.A.M.-Z. prepared and analyzed the synuclein; and K.C. and H.Y. prepared the CU-CPT22 and advised on the use of this compound. All authors edited the paper. Competing interests: The authors declare that they have no competing interests. Data and materials availability: Patents existing related to this work: “Antagonists of the Toll-like receptor 1/2 complex”, Yin, H.; Cheng, K.; PCT Int. Appl. 2013, US2013/52517; USA Appl. 2014, 14/417,676; EU Appl. 2015, 13826030.2.
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