Triggering of Inflammasome by Aggregated α–Synuclein, an Inflammatory Response in Synucleinopathies

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. It is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta of the brain. Another feature is represented by the formation in these cells of inclusions called Lewy bodies (LB), principally constituted by fibrillar α-synuclein (αSyn). This protein is considered a key element in the aetiology of a group of neurodegenerative disorders termed synucleinopathies, which include PD, but the cellular and molecular mechanisms involved are not completely clear. It is established that the inflammatory process plays a crucial role in the pathogenesis and/or progression of PD; moreover, it is known that aggregated αSyn, released by neurons, activates microglia cells to produce pro-inflammatory mediators, such as IL-1β. IL-1β is one of the strongest pro-inflammatory cytokines; it is produced as an inactive mediator, and its maturation and activation requires inflammasome activation. In particular, the NLRP3 inflammasome is activated by a wide variety of stimuli, among which are crystallized and particulate material. In this work, we investigated the possibility that IL-1β production, induced by fibrillar αSyn, is involved the inflammasome activation. We demonstrated the competence of monomeric and fibrillar αSyn to induce synthesis of IL-1β, through TLR2 interaction; we found that the secretion of the mature cytokine was a peculiarity of the fibrillated protein. Moreover, we observed that the secretion of IL-1β involves NLRP3 inflammasome activation. The latter relies on the phagocytosis of fibrillar αSyn, followed by increased ROS production and cathepsin B release into the cytosol. Taken together, our data support the notion that fibrillar αSyn, likely released by neuronal degeneration, acts as an endogenous trigger inducing a strong inflammatory response in PD.


Introduction
Parkinson's Disease (PD) is one of the most common neurodegenerative diseases and affects more that 1% of people over the age of 60 years worldwide [1]. It is characterized by death of dopaminergic neurons of the substantia nigra pars compacta (SNpc) of the brain [2] and by the presence of intracellular aggregated inclusions containing mainly a-synuclein (aSyn), called Lewy bodies (LB) [3,4]. The disease can be divided into sporadic and early-onset familial PD; the latter is linked to three missense mutations, A53T, A30P and E46K, as well as multiple copies of the wild-type (wt) aSyn gene [5,6,7,8]. Given that aSyn is the major component of LB in both familial and sporadic PD cases, it is considered a critical factor in PD aetiology [4]. Currently, the cellular and molecular mechanisms underlying the pathological actions of aSyn are poorly understood, and the factors contributing to sporadic PD, representing about 90% of PD cases, are not known. Both in vitro and in vivo, aSyn can aggregate to form oligomeric species and ordered fibrils in LB, characterized by a cross b-sheet structure, that are morphologically similar to the amyloid fibrils found in neuritic plaques in Alzheimers disease, as well as in deposits associated with other amyloidogenic disorders [9]. Although aSyn is typically considered an intracellular protein, it has also been found in extracellular biological fluids, including human cerebrospinal fluid and blood [10,11,12,13,14]. Changes in the levels and characteristics of extracellular aSyn are associated with the disease [15]. One of the contributing factors to aSyn spread could be the membrane permeability of dying cells, but it has also been suggested that vesicle-mediated exocytosis from normal cells is probably the main source of extracellular aSyn [15]. Furthermore, extracellular aSyn has been shown to be taken up by neuronal and microglial cells in culture, although the nature of the mechanism involved is still controversial [15].
It is established that disease onset and progression are characterized by inflammation and immune abnormalities, including the activation of microglia, but the mechanism and role of this activation remains controversial. The current body of evidence points to aggregated and modified forms of aSyn as a primary cause of microglia activation [16,17,18,19,20,21,22,23]. Moreover, there is substantial evidence that microglia are activated in mouse, rat and non-human primate models of PD prior to frank neuron death. Collectively, these observations support the hypothesis that aSyn is released early in the disease and, acting as an endogenous disease-related signal, it activates microglia to release pro-inflammatory molecules, such as TNF-a and IL-1b, which are detrimental to dopamine neurons [24,25]. The progressive death of the latter is expected to be responsible for further aSyn release, thus amplifying the neurodegenerative process [26]. Although this model clearly suggests that aSyn exerts a pivotal role in promoting inflammation by acting on microglia, it remains to be firmly established which form of the protein acts as the triggering signal and through which mechanism.
IL-1b is one of the most abundant pro-inflammatory cytokines that broadly affects inflammatory processes [27]. Its production is tightly controlled: it is synthesized as an inactive pro-protein and its activation and release are controlled by the cysteine protease caspase-1, which cleaves the protein into the active form [28,29]. Caspase-1 is synthesized as an inactive zymogen (pro-caspase-1) and becomes proteolytically active only after controlled dimerization in multi-protein complexes, called inflammasomes [30,31]. Inflammasomes are a group of protein complexes that recognize a large variety of inflammation-inducing stimuli that include pathogen-associated molecular patterns (PAMPs) and dangerassociated molecular patterns (DAMPs) [32]. Different inflammasome complexes are known; among these, NLRP3 is the most studied due to the large variety of signals that activate it, including LPS, bacterial toxins, particulate materials, amyloid-b and prion protein fibrils [33,34,35,36]. Several models have been proposed to explain how all these heterogeneous signals activate the NLRP3 inflammasome; these non-exclusive mechanisms include both direct and indirect signal recognition mediated by additional accessory proteins [37,38,39].
In this work, we studied the pro-inflammatory activity exerted on monocytes by fibrillar and monomeric aSyn, and we found that both promote the expression of pro-IL-1b, following the engagement of the receptor TLR2, but only aSyn fibrils induce the release of the mature form of IL-1b, via inflammasome activation. We show that protein complex activation requires fibril phagocytosis followed by cathepsin B release into the cytosol, but it also relies on the production of ROS.
Collectively, our data provide the evidence that, among the different aSyn forms, fibrils are the main trigger of the inflammatory response, which likely precedes neurodegeneration; our observation might be useful to develop a novel therapeutic avenue for PD.

Ethics Statement
Peripheral blood mononuclear cells utilized in this study derived from buffy coats obtained from healthy blood donors, as anonymously provided by the Transfusion Centre of the Hospital of Padua. Written informed consent for the use of buffy coats for research purposes was obtained from blood donors by the Transfusion Centre. Data related to human samples were all analyzed anonymously. Human leukocytes, provided by the Transfusion Centre of the Hospital of Padua, were obtained not consequently to experimentation on human beings but as a consequence of voluntary and informed blood donation for transfusions: no approval of Ethics Committee is needed in such cases in our institution.

Expression and Purification of aSyn
Human aSyn and its mutant (aSynC141), suitable for fluorophore labelling via thiol chemistry, were cloned in pET-28a (Novagen, Darmstadt, Germany). The mutated form was obtained by mutagenic PCR, introducing a triplet codifying for cysteine in position 141.
Both proteins were expressed in a lipid A mutant of E. coli, BL21(DE3) [40], with strongly reduced endotoxicity (kindly provided by Prof. J.F. Gauchat). Bacteria were grown to an OD of 0.4 at 600 nm and induced with 0.1 mM IPTG for 5 h. Cells were then collected by centrifugation and recombinant proteins recovered from the periplasm by osmotic shock, as previously described [41,42].
The periplasmic homogenate was then boiled for 10 min and the soluble fraction, containing aSyn, was subjected to a two-step ammonium sulphate precipitation (corresponding, respectively to ammonium sulphate percent saturation of 35% and of 55%). The pellet was then resuspended, dialyzed against 20 mMTris-HCl pH 8.0, loaded onto a 6 ml Resource Q column (GE Healthcare, Chalfont St Giles, Buckinghamshire, UK) and eluted with a 0-500 mM linear gradient of NaCl. Protein was then dialyzed against deionized water, lyophilized and stored at 220uC.

Fluorescent Labelling of aSyn
Labelling was performed by adding a 2.5-fold molar excess of tris(2-carboxyethyl)phosphine (TCEP) to aSynC141, dissolved in PBS, pH 7.4; after 30 min of incubation at room temperature, TCEPwas added to the protein solution in a 5:1 stoichiometric ratio and the reaction was left at 45uC for 4 h.
The protein was extensively dialyzed to eliminate excess fluorophore and reducing agent; the purity of the conjugated protein was checked by reverse phase chromatography (C4 column, Phenomenex, Torrance, CA, USA).
The absorbance of rhodamine-labelled aSyn was measured at 572 nm with an UV-visible spectrophotometer (Agilent 8453, Santa Clara, CA, USA) and the protein concentration was calculated considering the molar extinction coefficient of the fluorophore (e RHOD = 95000 M 21 cm 21 ).

Monomeric aSyn Preparation and Fibrils Growth
aSyn was resuspended in PBS, pH 7.4, and filtered to remove oligomerized protein with a 0.22 mm cut-off filter. Initial protein concentration was quantified by spectrophotometer (Agilent 8453, Santa Clara, CA, USA), measuring the absorbance at 276 nm.
Before starting the aggregation, the initial monomer concentration was adjusted to 100-200 mM and the solution was supplemented with 0.05% (w/v) sodium azide as bacteriostatic agent. The protein solution was then shaken at 1000 rpm at 37uC for 2 weeks and the fibrils were collected by centrifugation. The pellet was washed several times to remove non fibrillar aSyn and sodium azide, and resuspended in sterile PBS to a final monomer equivalent concentration of about 200 mM. The monomer equivalent concentration in the fibril pellet was calculated by difference between the starting monomer concentration and the residual monomer supernatant concentration after fibrillation. To reach the fibril amount corresponding to a concentration of 200 mM in aggregated monomers, the fibril pellet was resuspended in the proper volume of PBS. Considering the statisticalmechanical model developed to describe fibrils formation [43] and the AFM-assed average fibril length [44], we assumed an average of 5,000 to 10,000 aSyn monomers per fibril, leading to an estimated concentration of fibrils of 40 nM. Fluorescent fibrils were obtained by adding to the initial protein solution 5% of rhodamine-labelled aSynC141 and following the same procedure.
Non fluorescent and fluorescent fibrils were characterized using transmission electron microscopy, confocal microscopy and fluorescence spectroscopy.

Thioflavin T (ThT) Assay
ThT assay is typically used to detect amyloid fibrils, since the ThT dye binds to b-sheet rich structures and, when bound, it shows an enhanced fluorescence emission spectrum [45]. ThT binding assays were performed using a filtered (0.22 mm cut-off) 25 mM ThT solution in 25 mM sodium phosphate (pH 7.0). At the end of the aggregation, before dividing pellet and supernatant, an aliquot of protein samples was taken and diluted into the ThTcontaining buffer (final volume 500 ml). Fluorescence emission measurements were conducted on a fluorimeter (Ls50, Perkin Elmer, Waltham, MA, USA), at 25uC, using an excitation wavelength of 440 nm and recording the ThT fluorescence emission spectra between 450 and 600 nm. The emission maximum, at 480 nm, was used to verify the presence of b-sheet enriched protein, comparing the fluorescence intensity of the emission peak to that of the monomeric protein in solution.

TEM and Atomic Force Microscopy (AFM)
TEM fibril samples were prepared by adsorbing aSyn fibrils in PBS buffer at pH 7.4 (equivalent concentration about 25 mM) onto a carbon-coated copper grid and then performing negative staining with a 0.05% uranyl acetate solution. TEM images of aSyn fibrils were acquired to verify the morphology and dimensions of the fibrils used for the experiments ( Fig. S1A and B).
Morphological characterization of monomeric and fibrillar aSyn was also performed, by AFM imaging (Fig. S2 and Text S1).
Monocytes were fixed overnight in 0.1 M sodium cacodylate buffer at pH 7.4 containing 2.5% glutaraldehyde and then processed and embedded in LR White resin (Polysciences, Warrington, PA, USA). Ultrathin sections were then stained with 2% uranyl acetate for TEM imaging.

Monocyte Isolation and Culture
PBMCs from healthy donors were isolated by centrifugation on Ficoll-Paque solution and laid on Percoll 46% v/v solution in

Western Blot Analysis
Treated monocytes were collected, washed in ice-cold PBS, and lysed for 30 min at 4uC with ice-cold lysis buffer (20 mMTris, pH 7.15, 150 mM NaCl, 0.25% Nonidet P-40, supplemented with protease inhibitors: 1 mM PMSF, 1 mg/ml leupeptin, and 1 mg/ ml aprotinin). Cell lysates were centrifuged at 12000g for 20 min at 4uC, the supernatants (cell extracts) were kept and the protein content was quantified using the BCA assay. The entire culture supernatants (600 ml) were collected, precipitated with 10% TCA for 1 h at room temperature; protein pellets were resuspended in 15 ml of lithium dodecyl sulfate, boiled, and conserved at 220uC. Two micrograms of cell extracts and the total protein content of culture supernatants were loaded on a 4-12% SDS-PAGE and analyzed by immunoblotting. Activated caspase-1 and IL-1b were revealed by specific antibodies. b-actin and albumin were used as control for equal loading of cell lysates and cell supernatants, respectively.

Real-time PCR Analysis
Total RNA was extracted from 2610 6 monocytes using TRIzol solution, according to the manufacturer's instructions. RNA was reverse transcribed using SuperScript II and cDNA was amplified with the following primers: 59-AGCAACAGGGTGGTGGAC-39 and 59-GTGTGGTGGGGGACTGAG-39 for GAPDH; 59-CTGTCCTGCGTGTTGAAAGA-39 and 59-TTGGGTAATT TTTGGGATCTACA-39 for IL-1b; 59-GGAGAGACCTTTAT-GAGAAAGCAA-39 and 59-GCTGTCTTCCTGGCATATCAC A-39 for NLRP3; 59-GGAGGCCTTGGTGAAACC-39 and 59-CGATGTCACTCGGGCTATCA-39 for NLRP1. After amplification, data analysis was performed using the second derivative method algorithm by applying the 2 2DDCT method [46]. For each sample, data were normalized to an endogenous reference gene (GAPDH). Cells harvested at time zero were taken as the reference value, set to 1 AU (arbitrary unit, as shown in the figures), and the relative expression levels for treated or untreated cells were calculated and shown.

Detection of IL-1b in Culture Supernatants
Supernatants of monocyte cultures, harvested for quantification of mRNAs, were collected and stored at 280uC for subsequent quantification of cytokine content by IL-1b specific ELISA kit (eBioscience, San Diego, CA, USA), according to the manufacturer's instructions.

ROS Detection
Monocytes (2610 6 cells/well) were pre-incubated with 20 mM DPI, or left untreated, and then exposed to 40 nM aSyn fibrils (F),

Confocal Microscopy
Monocytes (2610 6 cells/well in 24-well plates) were seeded on coverslips and treated with rhodamine-labelled 8 nM aSyn in absence or presence of Baf A1. Cells were then fixed for 10 min in cold methanol, nuclei were stained with Hoechst and the coverslips were mounted on glass slides. Cells were visualized with a 636 oil immersion objective on a laser-scanning confocal microscope and images were acquired using the LAS-AF software (Leica TCS-SP5, Leica Microsystems, Wetzlar, Germany).

Statistical Analysis
Statistical analyses were performed using Student's t test preceded by ANOVA and data, reported as the mean 6 S.D., were considered significant if p-values #0.05.

Fibrillar aSyn Induces IL-1b Synthesis and Release in Human Monocytes
It is known that aSyn activates microglial cells to produce proinflammatory cytokines, such as IL-1b [17]. To investigate the mechanism of the aSyn-triggered synthesis, maturation and release of IL-1b in vitro, we incubated primary human monocytes with fibrillar aSyn (aSyn F). Monocytes were preferred to macrophages or microglial cells, particularly for the aim of verifying whether aSyn acted also as transcriptional inducer, because of the open debate on the latter two cell types. There is evidence supporting the idea that macrophages or microglia have to be primed with LPS to ensure robust induction of pro-IL-1b [33,34,35], whereas other findings claim that LPS priming is not necessary to activate these cells [20].
We found that aSyn F rapidly induced the expression of pro-IL-1b to an extent comparable to that observed following LPS stimulation: this was detectable both at the level of mRNA (Fig. 1A) and protein (Fig. 1B). It is worth mentioning that, although in a fibrillar form, aSyn induced the transcription of the pro-IL-1b gene, while other particulate structures, such as silica needle-like crystals and monosodium urate (MSU) induced the secretion of the mature cytokine from LPS-primed monocytes, but not its synthesis [35,38].
Finally, in addition to inducing pro-ILb mRNA and protein, aSyn F induced also cleavage and release of the mature cytokine. IL-1b release was measured by enzyme-linked immunosorbent assay (ELISA; Fig. 1C) and it strongly correlated with the detected value of cleaved IL-1b by immunoblot analysis (Fig. 1D), indicating that activated cells released mature IL-1b.
IL-1b Maturation Induced by aSyn F requires Caspase-1 Activation and Involves the NLRP3 Inflammasome IL-1b maturation is controlled by caspase-1 after assembly of the inflammasome, which activates pro-caspase-1. To test whether aSyn F activates caspase-1, we evaluated the release of both the active protease and IL-1b from monocytes pre-incubated with the caspase-1 specific inhibitor Ac-YVAD-cmk. Immunoblot analysis, performed with a polyclonal antibody against the active subunit p20 of caspase-1, revealed that the caspase inhibitor completely abolished the release of the mature form of the enzyme in response to the treatment with aSyn F. Moreover, the secretion of the mature cytokine was also impaired, proving that processing and secretion of IL-1b, induced by aSyn F, involves caspase-1 activation and, therefore, the inflammasome complex ( Fig. 2A).
A recent body of data revealed that needle-like particulate material (i.e., silica and MSU crystals, and fibrillar amyloid-b) activates the NLRP3 inflammasome [35,38,47]. Under resting conditions, NLRP3 is expressed but a pro-inflammatory signal is required to induce its expression to a level that leads to its activation [48]. Therefore, we decided to verify whether aSyn F increased the expression of NLRP3: in monocytes exposed to aSyn F, we measured the induction of mRNA encoding NLRP3 and compared it with that of NLRP1 (Fig. 2B). The results show that the protein induced a robust up-regulation of NLRP3, while the effect on NLRP1 expression was weaker. These data suggest that NLRP3 is the inflammasome mainly involved in aSyn F-induced IL-1b maturation.

Phagocytosis of aSyn F is Required for IL-1b Release
We next explored the mechanism by which aSyn F activates NLRP3 inflammasome. It is established that, in case of particulate material, inflammasome activation occurs following its phagocytosis [38,47,49]. Therefore, we first evaluated whether monocytes internalized the fibrils. Transmission electron microscopy (TEM) analyses of monocytes exposed to aSyn F for 6 h (a time considered sufficient to induce detectable IL-1b release) revealed that aSyn F accumulated in the cells in enlarged and swollen compartments, probably phagosomes or phago-lysosomes; this observation, together with the fact that no similar structures were appreciable in monocytes treated with saline (vehicle), demonstrated that aSyn F was phagocytosed by monocytes and that phago-lysosome swelling, and possible dysfunction, occurs during internalization (Fig. 3A). It is worth noting that, in TEM images, aSyn F can be clearly discriminated within these swollen compartments. We therefore investigated whether phagocytosis was required for aSyn F-induced release of IL-1b. We incubated monocytes, before and during stimulation with aSyn F, with Baf A1: this macrolide antibiotic neutralizes acidic organelles, such as endosomes, and inhibits the endocytotic pathway from early endosomes [50]. As expected, Baf A1 did not affect IL-1b synthesis induced by aSyn F (Fig. S3); however, it dramatically reduced aSyn F-mediated IL-1b release, as well as caspase-1 activation, as revealed by both ELISA and immunoblot analyses (Fig. 3B), and maintained confined aSyn F just below the plasma membrane (Fig. 3C). Collectively, these data show that aSyn Fis phagocytosed and that it accumulates in phagosomal compartments that become swollen, suggesting that a destabilization occurs; most importantly, internalization of the protein is essential for aSyn F-induced IL-1b release.

Lysosomal Destabilization and ROS Production Trigger aSyn F-induced Inflammasome Activation
It is established that inflammasome activation, which follows phagocytosis of particulate substances, requires the release of active cathepsin B from lysosomes into the cytosol [35]. In order to prove that aSyn F destabilizes the phagosomal membrane, the pH-dependent fluorescence probe acridine orange was trapped within the phago-lysosomes and its leakage into the cytosol was monitored as fluorescence change. Cytofluorimetric analysis revealed that the monomeric protein was not competent to induce leakage of the probe, while aSyn F destabilized the phagosomal membrane already after a 2 h-treatment (Fig. 4A).
To investigate whether the activity of cathepsin B, after stimulation with aSyn F, was functionally linked to inflammasome activation, we measured the effect of the inhibition of cathepsin B on the release of IL-1b from monocytes. We found that the specific inhibitor of cathepsin B, CA-074-Me, strongly inhibited IL-1b release. Moreover, the fact that, in the presence of the inhibitor, the activation of caspase-1 was also impaired, demonstrated that the cathepsin B-induced release of IL-1b occurred through activation of caspase-1 and not independently (Fig. 4B).
Collectively, these data indicate that phagocytosis of aSyn F by monocytes leads to an enlargement of phago-lysosomes that is associated with a loss of their membrane integrity: the release of specific proteases, such as cathepsin B, into the cytosol is causally related to inflammasome activation.
Next, we assessed the requirement of ROS generation, which has been implicated in inflammasome activation in response to the majority of known NLRP3 agonists [51]. The exposure of monocytes to aSyn F resulted in a time-dependent increase of ROS production (Fig. 4C): notably, levels of ROS induced by aSyn F were comparable to those elicited by LPS, which is a known trigger of ROS production in immune cells [52]. Pretreatment of monocytes with (DPI), a chemical inhibitor of ROS generation, diminished the amount of ROS (Fig. 4C) as well as inflammasome-dependent caspase-1 activation and IL-1b secretion in response to aSyn F (Fig. 4D).
Collectively, our data demonstrate that aSyn F-induced inflammasome activation requires phagocytosis, cathepsin-like activity and ROS production. The Activation of the Inflammasome is a Peculiarity of aSyn F, not Shared by its Monomeric Form As mentioned above, aSyn exists in a monomeric form but, under particular conditions, it assembles into aggregates which are thought to have a pathogenic role in PD due to their proinflammatory activity [17,53]. Considering that we could not exclude that also monomers were endowed with pro-inflammatory properties, we addressed the role of monomeric aSyn (aSyn M) in promoting the synthesis of pro-IL-1b and in activating the inflammasome. The concentration of monomer we adopted for the experiments was 1 mM, while the monomer equivalent molar concentration for aSyn fibrils was about 5 mM. However, the concentration of monomers is likely to be much higher than the actual concentration of aSyn F used in the experiments described above, because the formation of an individual aSyn fibrils is likely to recruit thousands of monomers. Therefore, the actual concentration of aSyn F used is expected to be in the range of nM, much lower than the concentration of aSyn M (1 mM).
We first compared the ability of the two forms of protein to induce ROS production; interestingly, both aSyn M and aSyn F induced ROS production in monocytes to a similar extent and with a similar time dependence. Moreover, in both cases, preincubation of cells with DPI strongly reduced ROS production (Fig. 5A).
Analysis of pro-IL-1b mRNA expression revealed that it was similarly induced by aSyn M and aSyn F (Fig. 5B); on the contrary, analysis of culture supernatants revealed that the monomeric aSyn was unable to induce the release of mature IL-1b (Fig. 5C). These results suggest that ROS are necessary but not sufficient for aSyn-induced inflammasome activation and that lysosome destabilization, induced only by fibrils, is a required factor for IL-1b maturation.

TLR2 is Involved in IL-1b Expression Induced by aSyn
Microglia cells express on their surface Toll-like receptors (TLRs); these receptors recognize conserved molecular motifs associated to pathogens (PAMPs) or endogenous danger signals (DAMPs). The engagement of a TLR triggers a downstream molecular pathway ending with the translocation of the transcription factor NFkB to the nucleus and the subsequent up-regulation of the expression of pro-inflammatory molecules [54]. It has been recently reported that aSyn activates this pathway and also that it modulates TLR expression: in particular, it up-regulates the expression of TLR2 [16]. Therefore, the possibility is there that HSyn binds to TLR2. To address such a possibility, monocytes were pre-incubated with a TLR2-blocking antibody before stimulation with aSyn F or aSyn M. Real-time PCR analysis revealed that, in presence of the blocking antibody, the expression of pro-IL-1b was strongly reduced, independently of the form of the protein (Fig. 6A). These data show that both fibrillar and monomeric aSyn are able to activate an inflammatory response and demonstrate that the latter involves the interaction with TLR2. As a consequence of the reduced synthesis of IL-1b, also the level of the mature cytokine decreased. The effect of the blocking antibody on the release of the mature cytokine induced by aSyn M was negligible: this is expected since it was also negligible the amount of cytokine released from monocytes exposed to the monomeric form in absence of any antibody ( Fig. 5 and Fig. 6B).

Discussion
The neurodegenerative process in PD and related synucleinopathies is accompanied by the presence of a neuro-inflammatory response, which has been proposed to contribute to disease progression [55,56]. Several studies lead to the definition of a role for aSyn in the initiation and maintenance of inflammation in PD, through the activation of microglia cells [57,58]. Once activated, these cells would release the pro-inflammatory TNF-a and IL-1b cytokines [17,56], thus leading to the accumulation of reactive oxygen species and generating an adverse environment for adjacent neurons, responsible for their damage and death [24,59]. This is particularly true for dopaminergic neurons, in which the redox chemistry of dopamine present in the cytoplasm could be enhanced by ROS, leading to the formation of toxic dopamine-derived quinones [60].
Although it is established that aSyn may activate microglia, the mechanism by which this activation occurs remained an elusive issue. Within this framework, since aSyn is a natively unstructured protein that can present itself in several conformations, the definition of the form of aSyn which mainly exerts a proinflammatory activity becomes a germane question.
Here we demonstrated that insoluble aSyn fibrils induced monocytes to release IL-1b following the activation of NLRP3 inflammasome. In contrast to all previous studies, aimed at evaluating the pro-inflammatory activity of aSyn and using microglia as a cell model [16,56], we made the choice to work with monocytes. Our choice is based on two reasons. First, several reports have demonstrated that macrophage-like cells, such as microglia, have to be primed with LPS to ensure robust induction of pro-IL-1b [33,34,35]; this experimental approach not only hampers the possibility of verifying whether an agonist promotes by itself the expression of the cytokine but, most importantly, it does not replicate the in vivo situation of PD patients, in whose brains LPS is not present. The second reason relied on the evidence that blood circulating monocytes are recruited during inflammatory events occurring in the central nervous system and they are responsible for microglial cell replenishment [61,62]; therefore, they may reasonably represent a target of aSyn in vivo. When we exposed monocytes to fibrillar aSyn, we found that not only it stimulated the release of the mature cytokine IL-1b but, notably, it also enhanced its gene expression. This result differentiates aSyn from other particulate structures that induce the secretion of IL-1b activating inflammasome, without inducing its synthesis [38,47].
Our data support the view that IL-1b synthesis occurs following the engagement of TLR2, which mediates a downstream pathway that results in the translocation of NFkB to the nucleus. Interestingly, aSyn engaged TLR2 and activated the downstream pathway also as monomer. Although this observation sounds peculiar considering that monomers and fibrillar aSyn are structurally very different, it must be considered that TLR2 has the capability to recognize a wide range of structurally unrelated PAMPs and DAMPs [63]. However, since the C-terminal domain is unstructured and solvent-exposed in both monomeric and fibrillar aSyn, the possibility exists that the interaction between TLR2 and aSyn occurs within this region [64].
The similarities between aSyn F and aSyn M are restricted to their ability to induce the expression of pro-IL-1b. It is only the former that is capable of inducing the secretion of the mature cytokine, following the activation of inflammasome. We showed by confocal microscopy and TEM that monocytes phagocytosed aSyn fibrils that, accumulating in phago-lysosomes, led to their swelling. Phagocyotosis of aSyn F is crucial for its ability to activate the inflammasome, as demonstrated by the fact that once phagocytosis was hampered with Baf A1 in monocytes, the secretion of IL-1b induced by aSyn F was markedly blocked.
The leakage of the lysosomal cysteine protease cathepsin B into the cytosol has been associated with the activation of the NLRP3 inflammasome by silica and cholesterol crystals [35,65] and it reflects a destabilization of the membrane of phago-lysosomes as a consequence of their swelling. Similarly, also for aSyn the cytosolic activity of cathepsin B seemed to be an essential intermediate step for inflammasome activation, indicating that fibrils-induced swelling of phago-lysosomal compartments resulted in an alteration of their membrane integrity and in the leakage of the enzyme into the cytosol.
Finally, in accordance with the fact that phagocytosis triggers the assembly of the NADPH oxidase complex on the plasma membrane and on the phagosomal membrane, we found that aSyn induces the production of oxygen radicals, which are also crucial for inflammasome activation. Fitting together all these data, allows us to propose a model outlining how aSyn may exert its pro-inflammatory activity.
Moreover, the characterization of the steps and molecules involved opens the possibility of reducing aSyn-triggered inflammation by blocking the activity of cytokine IL-1b, the final product of inflammasome activation. Albeit for a different disease, this blockage has already been pursued effectively: since 2002, one of the approaches for treatment of rheumatoid arthritis is the administration of Anakinra, an IL-1b receptor antagonist. It may therefore be worthwhile to explore the possibility of using an already FDA-approved drug as a new therapeutic avenue in PD, at least in managing the progression of the disease where neurodegeneration leads to the release of aggregated aSyn [15]. However, this potential approach has to be balanced with the possibility that turning off inflammation may enhance the neuronto-neuron progression of the disease [66] because of the expected accumulation of extracellular aSyn F. Figure S1 (TIF) Figure S2 (TIF) Figure S3 (TIF) Text S1