Size-Selective Phagocytic Clearance of Fibrillar Α-Synuclein Through

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Size-Selective Phagocytic Clearance of Fibrillar α-Synuclein through Conformational Activation of Complement Receptor 4 This information is current as of September 25, 2021. Kristian Juul-Madsen, Per Qvist, Kirstine L. Bendtsen, Annette E. Langkilde, Bente Vestergaard, Kenneth A. Howard, Martxel Dehesa-Etxebeste, Søren R. Paludan, Gregers Rom Andersen, Poul Henning Jensen, Daniel E. Otzen, Marina Romero-Ramos and Thomas Vorup-Jensen Downloaded from J Immunol published online 22 January 2020 http://www.jimmunol.org/content/early/2020/01/21/jimmun ol.1900494 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 22, 2020, doi:10.4049/jimmunol.1900494 The Journal of Immunology

Size-Selective Phagocytic Clearance of Fibrillar a-Synuclein through Conformational Activation of Complement Receptor 4

Kristian Juul-Madsen,*,† Per Qvist,†,‡,x Kirstine L. Bendtsen,{ Annette E. Langkilde,{ Bente Vestergaard,{ Kenneth A. Howard,‖ Martxel Dehesa-Etxebeste,#,** Søren R. Paludan,† Gregers Rom Andersen,†† Poul Henning Jensen,†,‡‡ Daniel E. Otzen,‖ Marina Romero-Ramos,†,‡‡,xx and Thomas Vorup-Jensen*,†,‖,xx

Aggregation of a-synuclein (aSN) is an important histological feature of Parkinson disease. Recent studies showed that the release of misfolded aSN from human and rodent neurons is relevant to the progression and spread of aSN pathology. Little is known, however, about the mechanisms responsible for clearance of extracellular aSN. This study found that human complement a a

receptor (CR) 4 selectively bound fibrillar SN, but not monomeric species. SN is an abundant protein in the CNS, which Downloaded from potentially could overwhelm clearance of cytotoxic aSN species. The selectivity of CR4 toward binding fibrillar aSN consequently adds an important aSN receptor function for maintenance of brain homeostasis. Based on the recently solved structures of aSN fibrils and the known ligand preference of CR4, we hypothesize that the parallel monomer stacking in fibrillar aSN creates a known danger-associated molecular pattern of stretches of anionic side chains strongly bound by CR4. Conformational change in the receptor regulated tightly clearance of fibrillar aSN by human monocytes. The induced change coupled concomitantly with a phagolysosome formation. Data mining of the brain transcriptome in Parkinson disease patients supported CR4 as an active SN http://www.jimmunol.org/ clearance mechanism in this disease. Our results associate an important part of the innate immune system, namely complement receptors, with the central molecular mechanisms of CNS protein aggregation in neurodegenerative disorders. The Journal of Immunology, 2020, 204: 000–000.

arkinson disease (PD), Lewy body dementia, and multiple receptor-mediated endocytosis (1). Some study ﬁndings sug- system atrophy are among the most prevalent neurode- gested that this mechanism is a means of protective clearance P generative diseases. Aggregation of the cytosolic protein (5). However, the intercellular transfer can initiate an inﬂamma- a-synuclein (aSN) into cell body inclusions (i.e., Lewy bodies) tory response in microglia and nucleate further intracellular ag-

(1), with aSN as the main component, is a shared histological gregation, which ultimately exacerbates neurodegeneration and by guest on September 25, 2021 hallmark of these diseases (2). Lewy bodies are terminal products promotes disease (1, 6). Consistent with this notion, genetic PD of the complex pathway of aSN aggregate formation; they consist risk variants are significantly enriched in gene sets functionally of fibrils of many thousands of aSN monomers. Many smaller linked to the regulation of leukocyte activity (7). Microglial cells types of aSN oligomers are also formed during aggregation. are the main resident myeloid leukocyte in the CNS. They are These oligomers may be the species responsible for cytotoxicity especially enriched in the substantia nigra, which shows the most due to their high mobility and ability to perturb the cell mem- prevalent neuronal death during PD (8). It is proposed that brane. Fibrils have been considered to be more innocuous, but microglial cells are aSN scavengers (9). Secreted aSN is cyto- study results reveal an important cell toxicity role for fibrils toxic to recipient neural cells in vitro (10) and in vitro–generated (3, 4). The aSN aggregates can be released to the extracellular oligomers of recombinant aSN are up to 17-fold more cytotoxic environment and then transfer from neuron to neuron through than monomers (11). aSN is one of the most abundant proteins in

*Biophysical Immunology Laboratory, Aarhus University, DK-8000 Aarhus C, M.R.-R. K.L.B., A.E.L., and B.V. acknowledge funding from the Lundbeck Founda- Denmark; †Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, tion Initiative BRAINSTRUC (2015-2666). Denmark; ‡iPSYCH, The Lundbeck Foundation Initiative for Integrative Psychiat- x Address correspondence and reprint requests to Prof. Thomas Vorup-Jensen, Bio- ric Research, Aarhus University, DK-8000 Aarhus C, Denmark; iSEQ, Centre for physical Immunology Laboratory, Department of Biomedicine, Aarhus University, Integrative Sequencing, Department of Biomedicine, Aarhus University, DK-8000 { The Bartholin Building (Building 1240), Wilhelm Meyers Alle, DK-8000 Aarhus C, Aarhus C, Denmark; Department of Drug Design and Pharmacology, University of ‖ Denmark. E-mail address: [email protected] Copenhagen, DK-2100 Copenhagen Ø, Denmark; Interdisciplinary Nanoscience Center, Aarhus University, DK-8000 Aarhus C, Denmark; #Neuroscience Area, Bio- The online version of this article contains supplemental material. donostia Research Institute, 20014 Donostia, San Sebastian, Spain; **CIBERNED, Abbreviations used in this article: CR, complement receptor; DAMP, danger- Instituto de Salud Carlos III, 28029 Madrid, Spain; ††Department of Molecular Biol- associated molecular pattern; Gu·HCl, guanidine hydrochloride; I, inserted; a , ogy and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark; ‡‡DANDRITE– M a-chain of CR3; a I, a I domain; MIDAS, metal ion-dependent adhesion site; Danish Research Institute of Translational Neuroscience, Aarhus University, M M xx NTA, nanoparticle tracking analysis; PD, Parkinson disease; PFF, preformed DK-8000 Aarhus C, Denmark; and NEURODIN AU IDEAS Center, Department ﬁbril; Q-dot, quantum dot; RNA-seq, RNA sequencing; RU, resonance unit; of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark aSN, a-synuclein; SPR, surface plasma resonance; tc, contact time; TEM, trans- ORCIDs: 0000-0002-5309-5221 (K.J.-M.); 0000-0002-0750-0089 (P.Q.); 0000- mission electron microscopy; ThT, thioﬂavin T; TPM, transcript per million; Wt, 0002-3976-8143 (K.L.B.); 0000-0003-2467-4205 (A.E.L.); 0000-0003-3230- wild-type; aX, a-chain of CR4; aXI, aX I domain. 9566 (M.D.-E.); 0000-0001-9180-4060 (S.R.P.); 0000-0001-6292-3319 (G.R.A.); 0000-0003-0970-578X (M.R.-R.); 0000-0002-4140-6563 (T.V.-J.). Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 Received for publication May 1, 2019. Accepted for publication December 18, 2019. This work was supported by an Aarhus University Research Foundation “NOVA” grant (AUFF-E-2015FLS-9-6) to T.V.-J. and K.J.-M. and an IDEAS Center grant to

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900494 2 a-SYNUCLEIN CLEARANCE BY COMPLEMENT RECEPTOR 4

the CNS; it accounts for 0.5–1% of all cytosolic brain protein (12). the MIDAS (32). The human aXI, especially, carries positive To limit aSN cytotoxicity during neural cell death, receptor- charges, which are involved in ligand recognition (33, 34), and mediated aSN clearance must bind aSN oligomers and fibrils almost no negative charge (Fig. 2C), which would further act to while avoiding saturation with the monomeric species. A study accommodate motifs of uninterrupted negative charge (29). The using a murine model found that predominantly aggregated aSN stretches of anionic charge, guided by the parallel organization of is an agonist of TLR-2 and stimulates microglial activation (13). the aSN monomers in the aggregates, suggest that CR4 is a strong A mouse model of multiple system atrophy revealed that TLR-4 receptor, especially for fibrillar aSN, but, to our knowledge, CR4 is associated with aSN clearance (14). It also associated with aSN has not been examined as a receptor for aSN. The roles of CR3 or activation of microglial cells by monomeric and fibrillar forms CR4 in aSN phagocytosis have also not been determined. Indi- (15). However, although TLRs are critical for the sensing of cating their importance in PD, the cerebral/cortical ITGB2 mRNA, danger-associated molecular patterns (DAMPs) (16), they are which encodes the b-chain (CD18) of CR3 and CR4, was sig- only a limited part of the physical clearance of particulates that nificantly upregulated in microglia from a small cohort of five PD carry DAMPs. The structure of DAMPs associated with aSN patients and three age-matched controls (35). However, it is un- aggregation remains unknown. known if the a-chains of these receptors, critical for ligand Studies using solid-state nuclear magnetic resonance (17) and binding, are differentially expressed in PD. cryo-electron microscopy (18–20) have revealed atomic-resolution CR3 and CR4 ligand-binding activity is tightly regulated by structures of the central core of fibrillar aSN. However, the struc- conformational changes in the receptor ectodomain (29). In the ture(s) of the toxic oligomeric state(s) is more elusive because of the ligand-binding inactive state, the a- and b-chains form bent highly dynamic nature of these species (21, 22). The aSN primary conformations, and the a-chain ligand-binding domain (i.e., the I Downloaded from structure comprises three distinct regions: the N terminus, the domain) is in proximity to the cell membrane. Alterations in nonamyloid-b-component, and the C terminus (23) (Fig. 1A). The contact between the integrin cytoplasmic tails and the adaptor lysine-rich aSN N terminus (residues 1–60) has an overall positive proteins talin and kindlin-3 enable cytoskeletal rearrangements, charge. In an aqueous solution, the monomeric form of aSN as- which permits opening of the ectodomain into the ligand-binding sumes a mostly unfolded structure. A cell membrane environment conformation (29, 36). Despite recent study findings on CD18

stimulates a-helical folding with a resulting structure that is re- integrins in neurodegenerative diseases (37), the roles of confor- http://www.jimmunol.org/ markably similar to the neural protein, myelin basic protein (24). mational activation of CD18 integrins in neuroinflammatory dis- The aSN C terminus (residues 96–140) carries an overall negative ease remain unknown. charge and remains unfolded, even when in contact with cell In this study, we found that fibrillar aSN was a strong ligand for membranes. In the fibrillar state, a central b-sheeted core as- CR4, but the monomeric form was not. Our results were also sembles from parallel-stacked monomers (Fig. 1B). It then further consistent with those of a previous study of CR3 as a receptor for assembles into a dimer of cores, still exposing some of the surface presumably unaggregated aSN (38). Unlike CR4, CR3 showed no of the original b-sheeted core (17–20). In the available structures major difference in the recognition of monomeric and aggre- (17–20), the N and C termini are not visible. This characteristic gated forms of aSN. Conformational activation of CD18 integrins may be due to fibril heterogeneity and terminus flexibility. The strongly enhanced phagocytosis concomitantly with the intracel- by guest on September 25, 2021 segments protrude away from the longitudinal axis of the fibril as lular formation of lysosomal vesicles. Conformationally activated highly charged, brush-like appendages to the core (17). On- CD18 integrins efficiently cleared fibrillar aSN but not mono- pathway oligomers (21), or oligomers that coexist with the fibril meric aSN. Guided by these results, we reanalyzed published PD form (25), are likely to have a related structure. b-Sheeted aSN brain transcriptome data and found a previously unappreciated forms are the most toxic (26). The parallel-type of stacking of the upregulation of expression of all components for CR3 and CR4 in fibril and related oligomers creates stretches, sometimes referred patients with PD compared with age-matched controls. This up- to as ladders (27), of repeated side chains that are most noticeable regulation correlated with the gene expression of the conformation- in the b-sheet core (Fig. 1C), but they may also occur at least in regulating proteins kindlin-3 and talin. Our data now indicate an patches of the unfolded regions. It remains unclear if this pattern important role in PD of CR4 ligand selectivity and conformational or other structural patterns affect clearance of these toxic forms. activation in clearance of fibrillar aSN. Complement receptor 3 (CR3; also named Mac-1, integrin a b a b M 2, or CD11b/CD18) and CR4 (p150,95, integrin X 2,or Materials and Methods CD11c/CD18) mediate microglial phagocytosis (28). Both re- a-Synuclein ceptors bind proteolytic cleavage products of C3, which is part of Human wild-type (Wt) aSN and aSND2–11 were prepared using recombinant the innate immune system. Human CR4 has a strong preference expression in Escherichia coli as a protein source (39). for molecules with motifs of uninterrupted negative charge, such For the cellular experiments with fibrillar aSN, monomeric aSN as the proteins polyglutamate and osteopontin, and the glucos- (346 mM) was assembled into preformed fibrils (PFF) using incubation amine glycan heparin (29). By contrast, CR3 appears to interact under sterile conditions at 37˚C in PBS (pH 7.4) (Life Technologies) with continuous shaking at 1050 rpm (Eppendorf ThermoTop) for 7 d. Aggre- with less homogeneous motifs containing positively charged gation was monitored by removing samples for thioflavin S fluorescence protein side chains (24, 30, 31). These differences in ligand rec- spectroscopy. The final incubation product was sedimented using centri- ognition are explainable from properties of the major ligand fugation at 15,600 3 g for 20 min to isolate the insoluble PFF from the soluble aSN. After aSN sedimentation, the PFFs were diluted to 2 mg/ml binding site in the a-chain of CR3 (aM) and a-chain of CR4 (aX), in sterile PBS (pH 7.4) (Life Technologies) and subjected to ultrasound usually referred to as the inserted (I) domain. Both the aM and aX a a breakage for 20 min using a Branson-Emerson 250 Analog Sonifier I domain ( MI and XI, respectively) take the Rossmann fold with equipped with a water jacket cooling system to avoid sample heating. The seven amphipathic a helices surrounding a hydrophobic b-sheet settings were a 30% duty cycle and an output control of 3. The size- core (29). Opposite the connection to the main body of the re- distribution profiles of the PFFs in suspension were measured using dy- ceptors, the I domains chelate an Mg2+ ion in the metal ion- namic light scattering (DynaPro NanoStar instrument; Wyatt) at 25˚C. The data analysis of the PFFs sample showed a homogeneous monodisperse dependent adhesion site (MIDAS) (Fig. 2A, 2B). Despite high population of 44-nm hydrodynamic-radius PFFs. a a overall sequence and structural similarity, the human MI and XI For transmission electron microscopy (TEM) experiments, lyophilized differ with regard to the presentation of electrostatic charge near powder of human Wt aSN (40) was dissolved in PBS buffer and filtrated The Journal of Immunology 3 using 0.22-mm filters. Thioflavin T (ThT) dye was added to a final con- surfaces were coupled with Wt aSN or aSN with an N-terminal deletion centration of 20 mM and 6 mg/ml aSN. aSN was fibrillated in a 96-well missing residues 2–11 (aSND2–11) using the amine-coupling chemistry plate (Thermo Fisher Scientific) by incubation and shaking at 37˚C for 7 d. method as described (43). A reference surface was prepared by coupling The fibrillation was done in triplicates of 150 ml with a 3-mm glass the surfaces with ethanolamine rather than protein. bead added in each well for agitation. ThT fluorescence was followed The aMI and aXI were stabilized in the activation conformation by in a Fluostar Optima plate reader (BMG Labtech) by the emission at mutation of a C-terminal Ile residue to glycine and were prepared as de- 480 6 5nmuponexcitationat4506 5 nm to ensure full fibrillation. scribed earlier (31). The I domains were diluted in 150 mM NaCl, 1 mM For buffer change and separation from soluble aSN fractions, the fibrils MgCl2, 5.0 mM, HEPES [pH 7.4] (running buffer) to concentrations of were sedimented, washed, and finally resuspended in Tris buffer (150 mM 156, 325, 625, 1250, and 2500 nM. The prepared solutions were then in- NaCl, 20 mM Tris [pH 7.4]). The fibrils were subsequently sonicated with jected over the surfaces; the contact time (tc) was 245 s and the following 10 s pulsed sonication using a Sonopuls mini20 (Bandelin). dissociation phase time was 255 s. The surfaces were then regenerated in 50 mM EDTA, 1.5 M NaCl, and 0.1 M HEPES [pH 7.4]. The data col- Cells sources lection rate was one data point per 0.4 s. The sensorgrams were manually aligned using BIAevaluation soft- Primary human monocytes were isolated from buffy coats obtained with an ware (GE Healthcare), and the signals from the reference surfaces were established collaboration with the Aarhus University Hospital Blood Bank subtracted from the signals from the ligand-coated surfaces. The according to ethically approved protocols (Protocol No. 77). The monocytes resulting a I sensorgrams were analyzed using an algorithm for were isolated from the buffy coat, by initial erythrocyte depletion by density M combined affinity and rate constant distributions of ligand populations gradient centrifugation (no. 17-1440-02, Ficoll-Paque PLUS; GE Health- from experimental surface binding kinetics and equilibria using the care) followed by negative selection using Dynabeads Untouched Human fitting tool “EVILFIT” (44) implemented in MATLAB 2012a (Math- Monocytes kit (no. 11350D; Invitrogen). Purified cells were stored at 2135˚C works). The injection start was 0 s and the injection end was 240 s; the in RPMI 1640 with L-glutamine, 20% (v/v) heat-inactivated FCS (Life dissociation start was 250 s and the dissociation end was 450 s. The Technologies), and 10% (v/v) DMSO. The cells were thawed immediately operator-set boundaries for the distributions were uniformly set to limit Downloaded from before use. Cell population viability was .90% for all experiments. 210 22 the KD values in the 10 –10 M interval and the dissociation Immortalized myelogenous K562 cell lines with recombinant expression 2 2 2 k values in the 10 4–10 1 s 1 interval. In the case of the a I, a more of CR3 or CR4 were made and cultivated as described (41) together with the d X limited analysis was made using the BIAevaluation software parental K562 cell line. Briefly, the cells were cultured at 37˚C and 5% (GE Healthcare, Norwalk, CT). k was calculated from the sensorgram CO in RPMI 1640 with NaHCO and 10 mM HEPES [pH 7.2], 10% (v/v) d 2 3 by 1:1 Langmuir binding isotherm for the dissociation phase defined by FCS, and penicillin and streptomycin. Selection for recombinant expres- the k =1/S 3 dS/dt,whereS is the SPR signal and with local fitting sion was maintained by adding 4 mg/ml puromycin dihydrochloride to the d applied. CR3/K562 culture medium and 16 mg/ml hygromycin B to the CR4/K562 http://www.jimmunol.org/ For preparation of EM grids, the sonicated aSN fibrils and the a I medium. X weremixedina1:1Mratio(12mM, based on the aSN monomer Cell adhesion assays with human monocytes and recombinant concentrations) with added 2 mM MgCl2 or 2 mM EDTA, respectively. The mixtures were then incubated for 30 min at room temperature. Five- K562 cells microliter samples were then allowed to absorb onto glow-discharged Cell adhesion was tested using a centrifugation-based assay as described Formvar/carbon-coated 200 mesh Cu grids (Electron Microscopy Sci- m (42). In brief, polystyrene 96-well microtiter plates with v-shaped bottoms ences) for 1 min before blotting and washing with 5 lMQH2Oina (no. 3896; Costar) were coated for 1 h at 37˚C with Wt aSN diluted in 1 min incubation. The absorbed material was negatively stained using a 150 mM NaCl and 20 mM Tris [pH 9.4] (coating buffer) at concentrations 1 min incubation with 5 ml 2% (w/v) uranyl formate. The TEM was of 0.940, 1.88, 3.75, 7.5, 15, or 30 mg/ml or left uncoated for reference. performed using a CM100 TWIN Transmission Electron Microscope Each concentration was prepared in triplicate. To remove oligomeric aSN (Philips). by guest on September 25, 2021 species, some plates were emptied, and 100 ml 6 M guanidine hydrochloride (Gu·HCl) was added. The samples were then dissolved in coating aSN/quantum dot phagocytosis quantified using imaging flow buffer, followed by incubation for 1.5 h at room temperature. All plates cytometry and nanoparticle tracking analysis were then washed in 200 ml PBS with 0.05% (v/v) Tween 20 and further blocked in this buffer for 1 h at 37˚C. For establishing assays to characterize cellular uptake and size selec- a Monocytes were thawed and added to PBS supplemented with 20% (v/v) tivity in phagocytosis of SN, monocytes from two donors were pu- FCS, collected using centrifugation, and resuspended in RPMI 1640 sup- rified and used separately in six independent experiments as described 2 plemented with 2% (v/v) FCS. The monocytes were fluorescently labeled in Fig. 3. The monocytes were retrieved from 135˚C storage on the using incubation with 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein day of experiment and kept on dry ice until use (Fig. 3A). Biotinylated a m acetoxymethyl ester (no. 14562; Sigma-Aldrich) at 37˚C and 5% (v/v) Wt and fibrillar SN samples were preincubated with 12.5 lofa 40-nM streptavidin/quantum dot (Q-dot) solution (no. Q10123MP; CO2 for 15 min, washed twice, and resuspended in 150 mM NaCl, 5 mM KCl, 1 mM MgCl , 1.8 mM CaCl , 10 mM HEPES [pH 7.4], with Molecular Probes) for 30 min at 37˚C; the final concentrations of 2 2 m 5 mM glucose and 2.5% (v/v) FCS (binding buffer) to a final cell con- proteins were 10 or 20 g/ml. The cells were thawed and resuspended 3 6 centration of 6–10 3 105 cells/ml. The cells were centrifuged for 5 min at in 1 ml RPMI to a concentration of 40 10 cells/ml. Twenty-five a 230 3 g, washed twice, and resuspended in binding buffer with 5 mg/ml microliters of cell suspension was added to the SN/Q-dot solutions or m CD18 integrin-activating Ab KIM127 (CRL-2838). To test the contribution Q-dots without protein (control) together with 5 g/ml CD18 integrin- of CR4 to adhesion, 1 mg/ml predialyzed function-blocking Ab was added activating Ab KIM127 (CRL-2838; ATCC), followed by incubation for to the a chain (clone “3.9”, MA1-46052; Thermo Fisher Scientific). 30 min at 37˚C with 5% (v/v) CO2. All samples were then washed X 3 Mouse IgG1 (M7894; Dako) was added to obtain an isotypic control. The twice with PBS. After centrifugation at 230 g for 5 min, the su- microtiter plates were emptied and kept at 37˚C in a heating block, a pernatant was saved for nanoparticle tracking analysis (NTA) (de- 100-ml cell suspension was then transferred to each well using an auto- scribed below) (Fig. 3B). Five microliters CD14 Ab conjugated with matic multichannel pipette, and the plates were incubated at 37˚C with Brilliant Violet 421 (no. 301829; BioLegend) was then added to each sample, and the samples were incubated for 25 min at 37˚C with CO2 for 10 min. The plates were then centrifuged at 50 3 g for 5 min, and the fluorescence count was read in the nadir of the wells using a Victor3 5% (v/v) CO2. Ten microliters of a 1:200 dilution in PBS of 1 mM Lyso 1420 multilabel counter (485-nm excitation wavelength, 535-nm emission Tracker stain Green DND 26 (no. L7526; Thermo Fisher Scientific) wavelength; Wallac). was then added to each sample, followed by an additional 5 min in- The K562 cell lines were treated as for the human monocytes, except cubation. The samples were then washed twice in PBS and resuspended in 50 ml PBS. Five microliters of 100 mM DNA nucleus stain that integrin activation was achieved by addition of 1 mM MnCl2 rather than Ab. Appropriate centrifugation force was achieved using centrifu- CyTRAK Orange (CO50050; Biostatus) was then added, and all tubes gation at 10 3 g for5minandagainat503 g for 5 min. The fluo- were incubated for 15 min. All samples were then kept on ice until rescence count was recorded at each step. imaging flow cytometry analysis. Flow cytometry was performed us- inganAmnisImageStreamXMKII(Amnis, Seattle, WA); the sensi- Binding of the CR3 and CR4 I domains to aSN, analyzed using tivity was set to high with a 603 image magnification. Images from surface plasma resonance and TEM 40,000–60,000 cells were recorded for all samples. Membrane and intracellular masks were determined from the locations of the CD14 The surface plasma resonance (SPR) assays were performed in CM-4 chips staining. The data were analyzed using the IDEAS software package and run in the BIAcore 3000 instrument (GE Health Care). The chip (Amnis) (Fig. 3C). 4 a-SYNUCLEIN CLEARANCE BY COMPLEMENT RECEPTOR 4

The supernatants obtained in the experiments described above (Fig. 3B) from the Gene Expression Omnibus data repository (https://www.ncbi. were diluted in PBS (1:1000) to obtain a particle concentration suitable nlm.nih.gov/geo/; accession code no. GSE68719). The reads were for analysis. The particles present in the samples were analyzed using quality filtered and mapped to the Ensembl Homo Sapiens. GRCh38.94 a NanoSight LM10 system (Malvern Instruments, Malvern, United human reference genome using HISAT2 (version 2.1.0) (46). Gene ex- Kingdom) (Fig. 3C). The system was configured with a 405-nm laser and pression quantification was performed using StringTie version 1.3.4 (47), a high-sensitivity scientific complementary metal–oxide–semiconductor the Ensemble Homo Sapiens (GRCh38.94 annotation gtf file), and the camera (OrcaFlash2.8, Hamamatsu C11440; Malvern Instruments). The alignment BAM files. Gene abundances were reported as transcripts per sample chamber was washed twice with PBS before each measurement. million (TPM). All samples were thoroughly mixed before measurement and were then For the heatmaps, the rows were centered, and unit variance scaling was injected into the sample chamber using 1-ml syringes. The measure- applied to the rows. The rows and columns were clustered using correlation ments were initialized within 10 s of injection into the chamber. Ap- distance and average linkage. Lists of glia specifically expressed genes were proximately 20–70 particles were in the field of view, corresponding to obtained from https://web.stanford.edu/group/barres_lab/brain_rnaseq. 2 3 108–1.3 3 109 particles/ml. The videos were collected and ana- html with a low count threshold of 1 fragment per kilobase per million. lyzed using NTA software (version 2.3, build 0025). The automatic Only genes with TPM .1 in the StringTie count files were considered for settings were used for the minimal expected particle size, minimum inclusion. track length, and blur setting. To enable recording of the movements of To assess expression of integrin-related genes in monocytes in PD small particles, the camera sensitivity was set to maximum (level 16) patients and controls, TPM values based on RNA-seq data reported in and the detection threshold was set close to minimum (level 3). A 650- Schlachetzki et al. (48) were obtained from Gene Expression Omnibus nm long-pass filter was used for all recordings. The temperature (range, data repository (https://www.ncbi.nlm.nih.gov/geo/; accession code no. 23–25˚C) was recorded manually. Three 60-s-duration videos were GSE88888). For comparison of transcriptomic profiles between microglia recorded for each sample (i.e., three replicates for each measurement). and circulating monocytes, normalized gene expression matrices were To analyze the biological variation in the size-selective phagocytosis, a obtained from RNA-seq data [https://science.sciencemag.org/content/356/ further seven donors were subjected to the NTA-based analysis following 6344/eaal3222.long (49)]. Downloaded from the procedures described in Fig. 3A–C, however, without technical replicates and only using the highest concentration of Wt or fibrillar aSN Statistical analysis and reproducibility m (10.0 g/ml; or blank as control). All statistical analyses were performed using Prism software (GraphPad Analysis of PD transcriptome Software, San Diego, CA). A p value ,0.05 was considered to be statistically significant. All information on experimental replicates, The RNA sequencing (RNA-seq) data from prefrontal cortex Brodmann number of donors tested, and applied statistical tests are stated in figure area 9 from 29 patients with PD and 44 controls (45) were downloaded legends. http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. Structural models of monomeric and filament aSN. Monomer organization of human Wt (Wt) aSN. (A) An illustrative model of human Wt aSN, generated using an ensemble optimization method based on SAXS data (77), where this model is one of many plausible conformations. The N-terminal domain (residues 1–60) is orange, the nonamyloid-b-component (NAC) region (residues 61–95) is green, and the C-terminal (residues 96–140) is teal. The positive-charge lysine residues are blue (there are no arginine residues in the sequence), and the negative-charge residues (glutamate and aspartate) are red. (B) Organization of human single protofilament/fibrillar aSN based on Protein Data Bank entry 2N0A (17). The b-sheet core, which resembles a “Greek key,” is shown with charged residues colored as for the monomeric aSN. (C) Zoom-in on part of the filament aSN in the b-sheet region with anionic stretches generated by the aligned Glu residues. The measurements also indicate the distances between the Cas of Glu46 in the strands. The Journal of Immunology 5

Results compared with conditions without integrin activation. As noted Conformational activation of CR3 and CR4 strongly enhances earlier (39), the applied Wt aSN contains a small amount of ag- cell adhesion to aSN gregated aSN (Supplemental Fig. 1). To test the effect of aggregates on cell adhesion, surfaces were treated with Gu·HCl. Primary human monocytes coexpress CR3 and CR4 similarly to This exposure caused a significant reduction in the adhesion in human microglial cells (50). We used these cells as a robust CR4/K562 cells, whereas the adhesion in CR3/K562 cells was model system for investigating the ability of human CD18 unchanged. integrins to bind in cell adhesion experiments with titrations of a the SN coating concentration (Figs. 1–3). The cell adhesion in Binding of aMI and aXItoaSN each titration point was determined from independent experi- To better understand on a quantitative basis how CR3 and CR4 ments with monocytes from three donors. The titration experi- recognize aSN, we followed a strategy used in previous studies ments were analyzed in a one-way ANOVA involving all to design a SPR assay (43, 51). a Ianda I were injected over titration points. When the CD18 integrin-activating Ab KIM127 M X surfaces covalently coupled with Wt aSN. The well-established was added, monocytes adhered robustly to microtiter wells heterogeneous a I interactions with ligands (29) were analyzed coated with recombinant Wt aSN and were significantly stronger M by resolving the combined set of association and dissociation than for conditions without KIM127 (Fig. 4A). When adding a phases (Fig. 5A, 5C, 5E, 5G) into an ensemble of 1:1 interac- function-blocking Ab to the aXI of CR4, the adhesion of the tions; each was typified by their ka and kd rates (44, 52). From the monocytes was significantly reduced compared with conditions simple relationship KD = kd/ka, the distribution in ligand binding in which an isotypic control Ab was added. The peak in differ- kinetics for the ensemble was shown in three-dimensional plots Downloaded from ence between the titration curves for the function-blocking and with axes of k , the equilibrium constant K , and the SPR signal, a m d D controlAbswasata SN coating concentration of 15 g/ml. To R, in arbitrary resonance units (RU) with contours to indicate the test if this amounted to a significant difference in a single ti- volume of each type of 1:1 interaction (Fig. 5B, 5D, 5F, 5H). For tration point, we increased the number of independent experi- aMI, the model was clearly consistent with the experimental data ments for this condition with monocytes from a total of eight as shown by the residuals in panels below the sensorgrams and donors permitting the use of a nonparametric (paired) Wilcoxon small root-mean square deviations not exceeding 2% of the http://www.jimmunol.org/ test. For all donors, the addition of function-blocking Ab reduced maximum SPR signal in either of the experiments (Fig. 5A, 5C, the adhesion. The variation between donors in inhibition relative 5E, 5G). The ensemble binding to native Wt aSN (Fig. 5B) was to control Ab-treated cells ranged from 60 to 0.4%, with a mean easily divided by eye into four bins (I–IV); for each bin, the value at 16% (Fig. 4B); the difference was highly significant in weighted mean KD and kd were calculated with results listed in testing of the absolute adhesion for function-blocking versus Table I. For native Wt aSN, the ensembles comprised interac- , 24 26 control Ab-treated monocytes with p 0.0078. This result tions with KD values in the orders of ∼10 and 10 M (bins clearly indicated that CR4 participated in the adhesion to aSN as I–III). A population that was minor (bin IV), yet distinct and with a 28 expressed in the monocyte cell membrane. good total signal, had a KD of 10 M (Fig. 5B). Treatment of These findings were further supported by the use of K562 cells the surface with Gu·HCl reduced the SPR signals (Fig. 5E) by guest on September 25, 2021 with recombinant expression of CR4 and CR3 (Fig. 4C). Mn2+ roughly proportional to the loss of dry-mass level (38%) of activation of the integrins again induced strong adhesion to aSN immobilized aSN (Fig. 5A, 5E). Although some features were in CR4/K562 and, albeit more attenuated, also in CR3/K562 cells missing because of the total lower signal, the ensemble binding

FIGURE 2. Homology modeling and calculation of electrostatic surface potentials for human and rodent aXIandaMI. (A and B) Models of the mouse and rat aXIandaMI were created using the SWISS-MODEL server (58), with the primary structures for Mus musculus and Rattus norvegicus taken from Bajic et al. (31) and based on the Protein Data Bank entries for open-conformation human aXI 4NEN (78) and aMI 1IDO (79) as templates. 2+ (A) Structural alignment of human (purple), mouse (teal), and rat (orange) aXI. The location of the Mg ion in the MIDAS is shown as a green sphere. 2 2 (B) Same analysis and coloring with the human, mouse, and rat aMI. (C) Electrostatic surface potential representation from 25kT/e (red) to 5 kT/e (blue) for human and rodent aXIs and aMIs. All surfaces were scaled and oriented according to the structures in (A)and(B). The electrostatic surface potential was calculated using Adaptive Poisson-Boltzmann Solver using default parameters (80). Because of the difﬁculties in calculating the electrostatic potential of coordinated divalent metal ions (81), structures were modeled without such, which makes the electrostatic charge of the unoc- cupied MIDAS negative (red). 6 a-SYNUCLEIN CLEARANCE BY COMPLEMENT RECEPTOR 4

FIGURE 3. Schematic representation of interdonor and interassay variation testing and total set of experiments for measuring Q-dot internalization using ImageStream flow cytometry and NTA. (A) Buffy coats from two donors were used for purification of monocytes. For each donor, approximately $9 3 106 cells were distributed into each of three vials. (B) The vials were treated with KIM127 Ab to activate CD18 integrins or were untreated. As indicated with colored lines, the KIM127 treatments Downloaded from were distributed to enable an indication of the interdonor and interassay variation, both with and without KIM127 treatment. Sam- ples from each of the six experiments were further mixed with 2.5–10 mg/ml Wt or fibrillar (Fibril) aSN with Q-dots, or treated as controls. (C) Following incubation, the http://www.jimmunol.org/ supernatants were collected and stored at 220˚C for later NTA profiling. The cell fraction was kept on ice for immediate imaging flow cytometry analysis. by guest on September 25, 2021

was largely similar to the untreated surfaces (Fig. 5B, 5F). We the dissociation phases for the injections of 156–1250 nM aXI also performed experiments with an N-terminal truncated aSN that were analyzed as a 1:1 interaction. Generally, the experimental 2 11 contained a deletion of aSN residues Asp to Ala (aSND2–11) data matched this approach with only small residuals (Fig. 5I). On (Fig. 5C). We used this construct to represent the minimal engi- average, based on measurements for two experiments with four neered intervention sufficient to reduce formation of aSN aggre- aXI concentration, the kd (mean 6 SD) was approximately equal 24 21 gates (39). As indicated from the SPR signal, the aSND2–11 was a to (8.66 6 1.39) 3 10 s . This almost equaled the slowest somewhat poorer ligand for aMI compared with Wt aSN (Fig. 5A, dissociation rates determined for the aMI binding of Wt aSN 5C). Shown by the contour plots, aSND2–11 differed because it had (Table I). When surfaces were treated with Gu·HCl, the aXI SPR almost 10-fold less (∼5 RU) of the high affinity interactions in bin signal vanished (Fig. 5K), unlike what was found for aMI. Similar IV (Fig. 5B, 5D) compared with the Wt construct (∼45 RU), to the cell adhesion experiments with the CR4/K562 cells whereas interactions in the other bins were only reduced ∼2-fold. (Fig. 4C), these findings indicated a critical role of aggregated As in the case of Wt aSN, Gu·HCl removed noncovalently bound aSN, which was further supported by the aSND2–11 experiments. material (Fig. 5C, 5G), which caused a drop in total signal but did Compared with Wt aSN, aXI had much more limited interaction not change the KD distribution. with aSND2–11 (Fig. 5J). This result occurred even though the The binding of aXItoWtaSN produced a more multifaceted surfaces were coupled with the same amounts of protein. Gu·HCl result than for aMI, especially because the sensorgrams were not treatment again ablated the signal (Fig. 5K, 5L). Taken together, analyzable as ensembles of 1:1 interactions. At the start of the these findings indicated that CR4 aXI strongly discriminated be- association phase (ti), a sharp peak appeared, followed by a slow tween monomeric and aggregated aSN, whereas CR3 aMI did not. raise in the signal toward the end of the tc. The dissociation phase The interaction between aXI and sonicated fibrillar aSN was had a remarkably slow progression (Fig. 5I). Altogether, the aXI investigated using negative-stain TEM. Similar to the results of sensorgrams shared features similar to those published earlier for other EM studies (19, 54), the slender fibrils appeared with the binding to heparin (53). As in the case of the binding to well-defined perimeters, often with dark rims (Fig. 5M, heparin, analysis by a simple 1:1 model, or even as an ensemble Supplemental Fig. 2A). When aXI was added in the presence of 2+ of 1:1 interactions used for aMI above (Fig. 5B, 5D, 5F, 5H), Mg , the perimeters appeared more ruffled, which reduced the failed to fit the experimental data. Instead, to obtain at least some rim contrast (Supplemental Fig. 2B). In some locations, there quantitative information on the aXI binding of aSN, exclusively were also high-contrast aXI-like features associated with the The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 4. Cell adhesion to aSN-coated surfaces. (A) Monocyte adhesion was studied in four conditions using a centrifugation-based assay in the presence or absence of CD18 integrin-activating Ab KIM127. The contribution of CR4 was tested using murine function-blocking Ab 3.9 to CD11c with murine isotypic IgG1 Ab as a control. For each condition, the mean and SEM are indicated from independent experiments using monocytes from by guest on September 25, 2021 three donors applied to each condition. The statistical analyses were performed using two-way ANOVAs and Bonferroni correction for multiple comparisons. (B) From the peak in difference between conditions with and without function-blocking Ab at a coating concentration of 15 mg/ml Wt aSNshownin(A), the adhesion at this coating concentration was analyzed using monocytes from eight donors. Adhesion was made with function- blocking or isotypic control Ab added. Paired results for each donor, indicated with color label, are shown with connecting, hatched lines. The mean values with and without function-blocking Ab are indicated with the ends of a curly bracket and the 6 SEM with connected black bars. The relative inhibition by the 3.9 Ab, shown next to the bracket, was calculated from normalization to the adhesion in the presence of the control Ab and shown as the mean value 6 SEM. The statistical comparison was made with a (paired) Wilcoxon test. (C) Adhesion of K562 cells with a recombinant expression of CR3 and CR4 and parental K562 cells as a control repeated in three independent experiments. Integrins were activated by the addition of MnCl2.The effect of ﬁbrillar aSN on CR3 and CR4 binding was tested using preincubation of the coated surfaces with Gu·HCl. For each condition, the error bars indicate the mean and SEM values from the independent experiments. The statistical analyses were performed using two-way ANOVAs and Bonferroni correction for multiple comparisons as in (A).

fibrils (Fig. 5N). The dark rims were restored when the EDTA- The preparations of Wt or fibrillar aSN were biotinylated and containing buffer that prevented aXI ligand interactions was conjugated with streptavidin-coupled Q-dots (Fig. 6A, 6B). The used (29) (Supplemental Fig. 2C), and the associated aXI-like cells were stained for CD14 expression to locate the membrane, features appeared in only a few positions (Fig. 5O). DNA to locate the nucleus, and for the intracellular phagolysosomes. Quantification of uptake was performed based on a Size-selective phagocytosis of SN is regulated by images of the entire cell. Image masks were made to further CD18 integrin activation distinguish fully internalized Q-dots from those associated with The results of the experiments above suggested that aggregated the membrane (Fig. 6C). The analyses were performed with cells aSN is a ligand for CR3 and CR4 and that CR4 has a specific role incubated with the CD18 integrin-activating Ab KIM127 (distri- in recognition of the aggregated forms. We designed an assay for butions indicated with a black line) or without such activation direct quantification of the CD18 integrin-mediated phagocytosis (gray lines). The results for the differences in median Q-dot of aggregated aSN analyzing the need for CD18 integrin activa- fluorescence (DIM) and for the Kolmogorov–Smirnov variable D tion found in the experiments described above. Initially, mono- (measures the maximum difference between the normalized cytes from two donors were analyzed also with replicates curves) (inserts, Fig. 6D–F) indicated that CD18-integrin activa- (Fig. 3B) to overall assess the technical stability with regard to tion increased the uptake of fibrillar aSN only (Fig. 6E). This intra-assay variation. The experiments focused on microscopically finding was also clear when comparing the variation between evaluating the phagocytic process by image stream flow cytometry donors and in the technical repeats (Supplemental Fig. 3A–F). and quantification of clearance with regard to the size of the The conformation-changing KIM127 Ab strongly induced the phagocytosed particles by use of NTA. stain for phagolysosomes independent of the provided substrates 8 a-SYNUCLEIN CLEARANCE BY COMPLEMENT RECEPTOR 4 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. Interactions of the CR3 (aMI) and CR4 (aXI) ligand-binding domains to aSN-coupled surfaces. (A–L) SPR surfaces were coupled with Wt aSN (A, E, and I) or truncated aSN (aSND2-11)(C, G, and J), applied to the experiments in their native states (A, C, I, and J) or following treatment with Gu·HCl (E, G, K, and L). Sensorgrams for a titration of I domain concentration are shown with indications of the start of the injection (ti) and the end of the tc and with the residuals between the experimental data (in colored lines) and the model (in broad gray lines) shown in panels below the sensorgram. The root-mean square deviation (RMSD) between the experimental data and the model applied was also calculated and stated for each set of sensorgrams (A, C, E, G, and I). For the data in (J)–(L), no modeling was made. The amount of immobilized protein for each of the surfaces is stated in picomoles per square millimeter. SPR signals were analyzed as ensembles of 1:1 interactions, each interaction typified by its equilibrium dissociation constant (KD) and dissociation rate (kd) (29, 43, 52, 82). Results are presented on two-dimensional grids with log10 (KD) on the abscissa and log10 (kd) on the ordinate axes, and contours (in RU, with 10-RU stepping) indicating the amount of the interaction. (A–D) Binding of the aMI to native Wt aSN and aSND2-11.In(B), (D), (F), and (H) the four bins, numbered I–IV, are shown for quantifying the KD and kd values listed in Table I. (E–H) Same analyses as in (A)–(D) for surfaces treated with Gu·HCl with 2-RU stepping between contours in (F) and (H). (I–L) Binding of the aXI to either native (I and J) or Gu·HCl-treated (K and L) Wt aSN and aSND2-11. SPR experiments were repeated twice on the same chip with decreased signal but overall similar binding kinetics in the second run of the same chip and again twice with a fresh chip and a similar decrease in signal for the second run. Shown sensograms are representative of the runs from the fresh chip. (M–O) TEM imaging of the aXI binding to sonicated aSN fibrils. Images were made either with sonicated fibrils alone (M), with sonicated 2+ fibrils and aXIinMg -containing buffer permitting binding of the I domain (N), or, as a control, with sonicated fibrils and aXI in EDTA-containing buffer not permitting such binding (O). In (N) and (O), aXI-like features in proximity of the fibrils are highlighted using a yellow circle next to the feature. The diameter of the circle was equivalent to ∼4.5 nm; this result was consistent with a previous study of negative-stained TEMs of aXI using class averaging (83). A scale bar (200 nm) is below the micrographs. The Journal of Immunology 9

Table I. Binding kinetic parameters for the CR3 aMI

Wt aSN aSND2–11

21 21 KD (M) kd (s ) KD (M) kd (s ) Native structured proteina Bin I (4.6 6 0.8) 3 1025 (3.4 6 1.7) 3 1023 (5.3 6 0.3) 3 1025 (5.4 6 0.1) 3 1023 Bin II (9.6 6 8.5) 3 1025 (1.2 6 2.2) 3 1022 (3.5 6 2.3) 3 1025 (1.2 6 5.0) 3 1023 Bin III (5.5 6 0.4) 3 1027 (1.9 6 0.6) 3 1022 (7.9 6 5.1) 3 1027 (2.2 6 0.4) 3 1022 Bin IV (0.8 6 0.8) 3 1028 (3.2 6 3.2) 3 1023 (0.8 6 0.8) 3 1028 (3.3 6 3.3) 3 1023 Gu·HCl-treated (denatured) proteina Bin Ib N/D N/D N/D N/D Bin II (8.5 6 6.6) 3 1025 (1.1 6 0.6) 3 1021 (2.4 6 2.2) 3 1025 (1.4 6 0.7) 3 1021 Bin III (6.5 6 1.9) 3 1027 (5.1 6 1.5) 3 1022 (1.0 6 0.4) 3 1026 (6.3 6 1.5) 3 1022 Bin IV (1.5 6 1.4) 3 1029 (4.9 6 3.2) 3 1023 (1.4 6 1.1) 3 1029 (4.4 6 1.7) 3 1023 aBinding kinetic parameters extracted from the analysis made in Fig. 5B, 5D, 5F, and 5H. For each bin, the value was calculated as a mean weighted by the volume of the interactions in the bin. All tabulated values are the mean of two experiments 6 SD. b Bin 1 collects weak interactions, which were N/D for the surface with denatured protein at the applied aMI concentrations. N/D, not detected.

(Fig. 6G–J). All experiments found an associated increased analyzed in two bins: one ranging from 100 to 500 nm and another Downloaded from phagolysosomal response with KIM127 Ab-induced conforma- from 500 to 1000 nm. In each bin, the normalized cumulative size tional change in the CD18 integrins. An experiment using distribution was calculated for each of the nine donors, either in high preactivation had the same result (Fig. 6I, Supplemental the presence or absence of KIM127. From these curves, median Fig. 3G–L). size distributions were calculated and compared for each bin and The experiments described above served to identify the size- for each of the three types of particle. To make a statistical

selective phagocytosis of aSN via CD18-integrin. A related, but evaluation, the total number of particles in each bin and in the http://www.jimmunol.org/ not identical, question involves the ability of these processes to presence or absence of KIM127 was also calculated and compared clear aSN from the extracellular environment to limit inflammatory in inserts in the panels (Fig. 7C, 7D). As already noted above, responses to this material (55). Supernatants from the phagocytosis there was a clear removal from the supernatants of particles with experiments above were analyzed using laser-equipped NTA fibrillar aSN in the size interval 500–1000 nm when KIM127 was equipment to track and size-determine Q-dot/aSN conjugates based added (Fig. 7D), but almost never in the size interval 100–500 nm on hydrodynamic-radius measurement in the complex medium, as (Fig. 7C). A closer inspection of the cumulative distributions permitted by the nonbleaching Q-dots. The size distributions ob- revealed that the most pronounced induction of clearance by tained from phagocytosis samples were analyzed using a particle KIM127 occurred for particles with a size of ∼700 nm (Fig. 7D). diameter cut-off ,100 nm to exclude all unconjugated Q-dot The total number of particles in the 500–1000 nm bin showed a by guest on September 25, 2021 particles. With the presence of 5–10 streptavidin molecules per highly significant (p = 0.0024) 38% lowering when KIM127 was Q-dot, the aSN conjugation capacity enabled good size separation added to cells incubated with fibrillar aSN conjugates (Fig. 7D). from the unconjugated 20-nm Q-dots (Fig. 7A, 7B). Results from As expected, for Wt aSN conjugates, the number of particles in the NTA analysis were presented as the raw file with the particle the 500–1000 nm bin was low, only permitting a description of the concentration versus size and scattering intensity (insert) and as full cumulative distribution in the absence of KIM127 (Fig. 7D), plots with particle concentration versus size for the experiments but again confirming the efficient clearance of the larger particles. with 10 mg/ml aSN (Fig. 7A, 7B). Similar to the analysis using The number of smaller particles in the 100- to 500-nm bin were image stream flow cytometry, all interdonor and interassay rep- not affected by KIM127 addition (Fig. 7C). Unconjugated parti- licates (Fig. 3B) were used to calculate a single median distri- cles behaved opposite the aSN conjugate with no evidence of bution (Fig. 7A, 7B). Addition of KIM127 reduced the median efficient phagocytosis (Fig. 7C, 7D). size of the Q-dot/fibrillar aSN from 417 to 108 nm (Fig. 7A, 7B). In contrast, there was essentially no change in the size distri- Microglial integrin gene expression in PD bution of Q-dot/Wt aSN conjugate (Fig. 7A, 7B). The median PD has a strong genetic component (56); 44 risk loci have been values were 132 nm without KIM127 addition and 160 nm with found (57). Pathway-based analysis of associated variants has KIM127 addition. Both values were close to median sizes of the provided support for immune-related genetic susceptibility to PD Q-dot/fibrillar aSN left behind in the experiment with KIM127. with, in particular, enrichment of genes implicated with leukocyte This result also supported the finding that naked Q-dots had only function (7, 48). Findings using PD cortical tissue indicate that a minor change in size distribution and that the change was microglial genes are generally upregulated, including the CD18 opposite from the Q-dots/fibrillar aSN result. The same results encoding gene, ITGB2 (35). Guided by the in vitro results, we werealsofoundwhenalowerconcentration(5mg/ml) of aSN assessed the expression of an integrin-related panel of genes using was used (Supplemental Fig. 4). Taken together, the experiments a dataset from a recent, large RNA-seq study involving cortical (Fig. 7A, 7B) established a separation at ∼500 nm between the samples from 29 patients with PD and 44 aged-matched controls easily phagocytozed aSN-conjugated particles and particles (45) (Fig. 8A). The hierarchical clustering result generally strati- smaller than this value, which were essentially not phagocytozed fied samples based on disease state. Most (60%) of the PD cases with the involvement of CD18 integrin activation. were in a distinct cluster of 94% PD cases; there were also two To further test the biological validity of our findings concerning clusters consisting of 86 and 70% controls (Fig. 8A). The PD- size-selective aSN phagocytosis, we used the NTA for investi- enriched cluster was characterized by a marked upregulation of gating the response with monocytes from nine different donors genes in a cluster containing microglia-specific genes implicated (Fig. 7C, 7D). The size distribution of particles conjugated and with leukocyte-specific function in particular (Fig. 8A). These incubated as for the results shown in Fig. 7A and 7B were CD18 integrin-related genes included ITGB2 and ITGAL, ITGAM, 10 a-SYNUCLEIN CLEARANCE BY COMPLEMENT RECEPTOR 4 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. Image flow cytometry of CD18 integrin-mediated aSN phagocytosis. (A and B) In preparation for the phagocytosis experiment, either Wt (A) or fibrillar aSN (B) were biotinylated and mixed with PEG and streptavidin-coated 20-nm ZnS/CdSe Q-dots. The particles and the aSN species are drawn to scale. (C) Two representative events from the population of Q-dot-positive monocytes (events 882 and 1111) are presented with masks placed to collect Q-dot fluorescence in the entire cell or in the intracellular or membrane compartments. The masks were based on staining of the monocyte cell membrane through binding of CD14 Ab, indicated in purple. The masks are indicated in dark gray. The Q-dots are red, further highlighted with white arrowheads in the mask covering the entire Q-dot positive cell. (D–F) Image-based calculations on Q-dot uptake. The distributions of Q-dot fluorescence intensities in the entire cell and in the intracellular and membrane compartments, as defined in (C), were calculated from three independent experiments with purified monocytes from two donors. A combined total of 22,000 image events for all three experiments without the addition of KIM127 Ab, and 60,000 events for all three experiments with KIM127 Ab addition, were analyzed. The distributions were compared using the differences in median levels (DIM), and using the Kolmogorov–Smirnov statistic D, the maximum difference between the normalized cumulative distributions (shown in inserts) of the Q-dot intensity for the KIM127 untreated and treated cells. Analyses were performed with naked Q-dots (D), or 10 mg/ml fibrillar aSN and Q-dots (E), or 10 mg/ml Wt aSN and Q-dots (F). Except for the comparison of Wt aSN-coupled Q-dots in the membrane compartment (F), all other comparisons were statistically significant (Kolmogorov–Smirnov test, p , 0.002). (G–J) Stain (Figure legend continues) The Journal of Immunology 11 Downloaded from

FIGURE 7. Concentrations of Q-dots and in monocyte culture supernatants following incubation without and with KIM127 Ab. (A and B) NTA analysis of the sizes and concentrations of aSN-coupled or naked Q-dots in supernatants from monocyte cultures, either treated with KIM127 (B) or left as untreated http://www.jimmunol.org/ (A). Interdonor and intra-assay replicates are described in Fig. 3B. Because of large numbers of unbound Q-dots, the curves display the particle distributions with a lower cut-off at 100 nm. From the raw files (inserts) containing information on particle concentration, size, and scatter intensity, two-dimensional plots of particle size versus concentration were made for each condition. For each particle size, the median concentration calculated from the three experiments (Fig. 3B) is indicated with a solid black line, and the SEM result in gray. For each condition (Q-dots with no aSN, Wt aSN, or fibrillar aSN), the median value (M) was calculated. Black, hatched lines separates particle sizes below and above 500 nm. (C and D) Analysis of biological variation (interdonor variation) of phagocytic clearance of aSN-coupled or naked Q-dots. Monocytes from nine donors were incubated with Q-dots with no aSN, Wt aSN, or fibrillar aSN either in the presence of KIM127 (eight donors) or absence of KIM127 (nine donors). For each donor, a two-dimensional plot of particle size versus concentration was established as in Fig. 7A, 7B for the size intervals 100–500 nm and 500–1000 nm and further used to calculate the cumulative distribution for each interval. The median cumulative curve (read in % on left axis) was produced for conditions with (black color) and without

(gray color) addition of KIM127 from the eight and nine experiments, respectively. The two-dimensional plots were also used to calculate the total number by guest on September 25, 2021 of particles with GraphPad Prism’s area-under-curve function. For each type of particle and application of KIM127, the mean particle concentration (shown as bars in inserts, read on the right axis in 108 particles ml, and with color coding as for the curves) and confidence interval (error bars showing upper-half interval) was calculated. Based on the mean value, confidence interval and number of donors tested, the statistical significance were calculated in an unequal variance t test (Welch test). For all calculations made, the p values are stated in the inserts. and ITGAX, which encode LFA-1, CR3, and CR4, respectively. healthy tissue ex vivo from 19 individual with no diagnosis of PD TLN1 and FERMT3, which encode talin and kindlin-3, respec- (49). Although the expression pattern contains several differences tively, important for CD18 integrin conformation regulation, were overall, the expression of the CD18 integrin chains and their also included. The results were similar for C3, which upon com- conformational regulators was highly correlated, and, in conse- plement activation and proteolytic regulation is converted to iC3b, quence, had a ranking in expression largely similar for the two cell a major ligand for CR3 and CR4 (58). Among the CD18 integrin types, again showing a higher expression of ITGAM and, more a-chain genes, ITGAM and ITGAX mRNA were notably more moderately, ITGAX than ITGAL. The C3 expression was not fol- abundant than ITGAL mRNA (Fig. 8B). To assess whether the lowing this pattern with a notably higher expression in microglia increased mRNA level of microglial integrin-related genes in than in monocytes (Fig. 9C). To investigate if changes in the CD18 cortical tissue from PD patients was due to a general upregula- integrin-related expression also could be found in peripheral tion of microglial genes reflecting multiplying microglial cells monocytes from PD patients, we used the recent RNA-seq data (59) or monocytic invasion (60), we examined the expression of (48) on peripheral monocytes from a smaller cohort of PD patients microglia-specific genes in the dataset (Fig. 9A). Although the and aged-matched controls (Fig. 8C). In this case, no striking majority of microglial genes were upregulated in PD, as reported differences in expression were observed between patients and by others (35), we found that approximately one fourth were controls. downregulated (Fig. 9A). In the cortical tissue, there was a notable Taken together, the results of the analysis revealed the strong correlation of expression of genes for CR4 and for kindlin-3 linkage between CD18 integrin-related gene expression and PD. (Fig. 9B). We also examined the similarity in gene expression Also, the similarity in CD18 integrin-related gene expression between human peripheral monocytes and microglia isolated from between microglial cell and monocytes supports the use of the latter

for lysosomes. Four representative events were selected, including two events with low (167 and 700) and two events with high (411 and 869) lysosomal staining (G). For all events described above, the lysosomal stain in the presence or absence of KIM127 Ab was compared for naked Q-dots (H), or 10 mg/ml ﬁbrillar aSN and Q-dots (I), or 10 mg/ml Wt aSN and Q-dots (J). 12 a-SYNUCLEIN CLEARANCE BY COMPLEMENT RECEPTOR 4 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 8. Expression of integrins and related molecules in cortical tissue and monocytes. (A and B) Expression in the prefrontal cortex (Brodmann area 9) in postmortem samples obtained from patients with PD (n = 29) and healthy controls (n = 44) reported in (45). (A) Heat map of row normalized mRNA expression values (TPM) for expression of genes encoding all core components of integrins and integrin inside out signaling, including all integrin a-chains (ITGAs), all integrin b-chains (ITGBs), select integrin ligands (C3, ICAM1, MADCAM-1, VCAM1,andITGB1BP1), and intracellular regulators of CD18 integrin conformation through inside-out signaling (TLN1 and FERMT3). The genes were clustered based on their correlation among patients (horizontal clades) and cell expression (vertical clades). Tight correlation between integrin a-chains and conformational regulators is indicated using braces. The clade collecting the most patients with PD is indicated with an asterisk. (B) Normalized mRNA expression values (TPM) for the CD18 integrin genes, regulators of their conformation, and C3. (C) Normalized mRNA expression in TPM for the same gene as in (B) from peripheral monocytes obtained from early-stage PD patients (n = 10) and age-matched controls (n = 10) (48). The Journal of Immunology 13 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 9. Gene expression of microglial-specific genes in cortical tissue and correlation of expression in microglia and monocytes. (A and B) Gene expression and correlation of expression of microglia-specific genes in cortical tissuefrom data (45). (A) Altered microglial gene expression in patients with PD versus controls. The 500 most-expressed genes in microglial cells were analyzed for changes in patients with PD versus controls, as indicated by colors. (B) Correlation coefficients for gene expression of CD18 integrin loci and function-related genes. (C) Correlation between gene expression in microglial cells and peripheral monocytes from data reported in Gosselin et al. (49). Microglia were isolated from brain tissue resected for treatment of epilepsy, brain tumors, or acute ischemia in 19 individuals. Leukocyte-specific gene expressions in the microglia and monocytes were correlated with CD18 integrin genes, regulators of their conformation, and C3 indicated in red in the plot. cell type as a model for CD18 integrin function in microglial cells (61). Microglia clears aggregated aSN from the extracellular (Fig. 10). milieu, but the molecular mechanisms responsible for this process are not well understood (9). We found that the strong Discussion binding of microglial-expressed receptor CR4 to aSN selected PD is an important disease of the CNS with an urgent, unmet the fibrillary forms over the monomeric species. aSN phagocy- need for effective therapy. However, understanding of the tosis was dependent on CD18-integrin conformational regulation molecular-level mechanisms associated with PD pathogenesis and also activated a phagolysosomal response. Our findings are remains incomplete. This deficiency impedes the progress in directly connected to the recently characterized structures of fi- designing appropriate pharmacological treatments. Recently, brillar or similarly-folded aSN aggregates (17–20); they define the intercellular transmission of aggregated aSN has received a novel role for the CR4 receptor in aSN-related diseases attention as especially relevant for disease etiology and progression (Fig. 10). 14 a-SYNUCLEIN CLEARANCE BY COMPLEMENT RECEPTOR 4 Downloaded from http://www.jimmunol.org/

FIGURE 10. Schematic overview of the relationship between CR3 and CR4 conformational activation and aSN clearance. (A) The endosomal transport in neurons of aSN (in a mixture of oligomeric or fibrillar forms) to the extracellular environment enables microglial phagocytosis of the material. The conformational regulation of CR3 and CR4 involves the adaptor proteins talin and kindling-3 under physiologic conditions. Part of the conformational regulation can be mimicked by the KIM127 Ab by stabilizing CR3 and CR4 in their active conformations. (B) If CR4 is appropriately activated, the oligomeric or fibrillar forms of aSN are phagocytosed. Once inside the microglial cells they are loaded into more phagolysosomes, which enables deg- radation. (C) If CR4 is not activated, the oligomeric or fibrillar forms of aSN remain in the extracellular environment. by guest on September 25, 2021

Study results suggest that CR3 and CR4 are functionally dif- (53), produced a more orderly response in keeping with inability ferent with regard to ligand recognition (29, 62). This difference is of aMI to bind well polyanions (51). Probably in consequence of also relevant for recognition of aSN, with surprising conse- the complexity of the binding scheme, we were not able to model quences for the clearance of potentially toxic aggregates. We comprehensively the binding of aXItoWtaSN. However, an confirmed (38) that CR3 is a receptor for aSN. Our study now also estimate of the dissociation rate at ∼1023 s21 seemed to account reveals that CR4 binds aSN. Expressed in monocytes and pre- well for most of the dissociation phase, suggesting that such sented to a surface coated with Wt aSN, which is mostly non- slowly releasing bonds constituted most of the interaction be- fibrillar, CR4 contributed ∼16% of adhesion as judged from the tween aXI and Wt aSN. Again, this was unlike aMI, which had use of function-blocking Ab. In experiments with a recombinant several interactions with considerable faster dissociation rates at expression of CR4 in K562 cells, the adhesion was strong but ∼1022 s21. Removal of aggregated aSN by Gu·HCl treatment attenuated by treatment with Gu·HCl, which was unlike the im- ablated the binding by the CR4 aXI. Compared with the Wt aSN, provement in CR4 binding when albumin and fibrinogen was binding to the aSND2–11 construct was much reduced; the treated with denaturing agents as reported earlier (51, 63).These aSND2–11 construct contains smaller amounts of aggregated aSN observations prompted us to investigate if aggregated aSN played than Wt aSN according to ThT spectrosopy (39). This type of a special role as ligand for CR4. We immobilized preparations of aggregate quantification is directly linked with the formation of aSN in SPR flow cells to study the binding of aXI. The binding to side chain ladders (27). Wt aSN was strong, with fast association and slow dissociation Why is CR4 a receptor selective for aggregated aSN? CR4 binds rates, but also with a striking feature in the early association phase, well to uninterrupted stretches of anionic moieties, such as those where the SPR signal peaked initially followed by a slow increase found in polyglutamate, osteopontin, and heparin (29). This in signal toward the end of the tc. The results resemble those of a preference is consistent with the electrostatic charge distribution previous study of binding to heparin (53). Both fibrillar aSN and on ligand binding interface of human aXI as also noted elsewhere heparin share the exposure of dense negatively charged carbox- (29, 32, 51). Other studies found these mostly positive charges as ylates, known to be good ligands for aXI (64). The electrically important in aXI ligand recognition (33, 34), which agrees with charged groups engage in a hydrated layer, which is at least our data, although we did not map the specific interactions temporarily distorted by the aXI binding, producing the sharp between aXIandtheaSN aggregates. Although monomeric peak in SPR signal. This similarity in SPR response is conse- aSN contains an acidic C-terminal domain, uninterrupted quently pointing to the densely packed carboxylates in both ma- stretches of negative charge in the primary structure are not terials as involved in the aXI binding. By contrast, the binding of abundant, at least not compared with other CR4 ligands (29). the CR3 aMItoWtaSN, and as previously reported to heparin The recent structural description of fibrillar aSN indicates how The Journal of Immunology 15 these stretches, nevertheless, are formed by this material (17–20). recently be successfully used for understanding the contribution of The C-terminal acidic appendages with high flexibility offer an CR3 to human neuropathology (72), whereas, to our knowledge, anionic environment similar to the glutamate-coupled matrix no similar progress was made in the case of CR4. From our found to efficiently pull down aXI (51). The parallel stacking of structural comparison of the aMI and aXI (Fig. 2), it seems likely the monomeric aSN within the fibrils is even more striking. This that CR3 ligand binding is conserved between rodents and human, structure creates long stretches, or ladders, of uninterrupted, an- whereas the same is not expected to be the case for CR4. This ionic side chains perpendicular to the peptide backbone, with an supports the relevance of the cross-species studies in the case of inter-Glu-Ca distance of 4.9 A˚ (Fig. 1C). This distance is within CR3 functions, whereas it is unlikely that a direct comparison of the range of the 3.7-A˚ distance of the peptide backbone in poly- human and rodent CR4 would be equally helpful in understanding glutamate and the 5-A˚ distance of the anionic monosaccharide the role of CR4 in clearing aSN aggregates. Of course, this would units in heparin; both are confirmed ligands of CR4 (51, 53). The be true for analyzing cellular systems of rodent origin in vitro as highlighted stretches (Fig. 1C) would be equally accessible in the well. Finally, several lines of evidence now relate changes in the double-stranded models of fibrillar aSN (18–20). Because of its peripheral immune system to PD as recently demonstrated for flexibility, the distance between Glu residues could be even lower blood monocytes (73). The experimental observation that enteric in the acidic C-terminal domain. The TEM imaging indicated that aSN pathology may spread to the brain points to tissues outside the aXI domains unevenly decorated the perimeter of the aSN the brain as possible sites for initiation of PD (74). As shown by fibrils under conditions permitting such binding. Consequently, us, RNA-seq data (48) do not suggest changes in expression of CR4 binding motifs previously found to represent endogenous CD18 integrins or their conformational regulators in monocytes

DAMPs (51) are critically linked with the parallel stacking of at from early-stage PD patients compared with controls. This is Downloaded from least some types of aSN aggregates. The strong toxicity of these unlike what seems to be the case for microglia in brain tissue (45). oligomers was recently revealed (26), further emphasizing the Even with the reservations in such a comparison, which come necessity for rapid and selective clearance. from the differences between these studies in mode of tissue ex- The CR4 binding of aSN aggregates was repeated in the traction and study cohorts, this clearly ﬁts an understanding of the downstream process of phagocytosis, although now with direct brain as the major site of disease-related inﬂammatory processes

demonstration of the CD18 integrin-mediated binding to fibrillar in PD, at least with regard to those involving CD18 integrins. http://www.jimmunol.org/ forms. Conformational activation of CD18 integrins (i.e., CR3 and Nevertheless, CD18 integrin-mediated aSN phagocytosis by pe- CR4) with the Ab KIM127 promoted uptake of fibrillar aSN but ripheral monocytes may still be speculated to be triggered as an not Wt aSN or unconjugated Q-dots. The results of the image adjunct consequence of non–PD-related inflammatory responses, stream flow cytometry analysis confirmed the presence of the including infection or autoinflammatory responses. particles in the intracellular compartment. KIM127 Ab stimu- Among the integrins, roles for especially CR3 and CR4 in PD are lation also induced robust formation of phagolysosomes. The supported by transcriptional analysis of samples from the CNS of conformation-regulated CD18 integrin signaling associated with patients. Microarray analysis of human brain microglia revealed formation of lysosomes is a novel aspect of the structural biology PD-associated alterations in ITGB2 expression (35). Our data of integrin conformation; it is, of course, a logical coupling to mining of a larger data set provided (45) was prompted by the by guest on September 25, 2021 the phagocytic function of CR3 and CR4. results of the biochemical and cellular studies; this research ap- The choice of monocytes as our experimental model system was proach enabled a guided inquiry into the PD brain transcriptome made as they are primary leukocytes with a physiologic regulation of microglia. The analysis revealed a high expression of the genes of integrin ligand binding activity and a similar phagocytic capacity encoding the CR3 and CR4 a-chains that was further increased in of microglial cells through the shared expression of both CR3 and PD. In contrast, LFA-1 a-chain expression was almost 10-fold less CR4 (65). From the data presented in our study, we conclude that than that of the CR4 a-chain and was not strongly changed in PD. CD18 integrin activation is required for aSN clearance by primary Consistently, LFA-1 has little or no role in phagocytosis, whereas leukocytes. Notwithstanding the challenges in getting access to this is the major function of CR3 and CR4 (29). This observation, human brain tissue with viable microglial cells, if integrin acti- which to our knowledge is novel, strongly supports CR3 and CR4 vation studies were to be made on cells extracted from resected as parts of an aSN clearance mechanism differentially regulated in brain tissue as used by others for genetic analyses (49), it would the CNS and hence as participants in PD molecular pathogenesis. be difficult to ensure that their native integrin conformation The correlation in gene expression between the adaptor proteins remained unperturbed. Hence, the role of integrin activation would talin and kindlin-3 versus CR3 and CR4 represents even stronger be complex to ascertain. A recent development suggests that evidence. There were pairwise correlations of TLN1 with ITGAM microglia-like cells can be derived from differentiation of human and FERMT3 with ITGAX. This result was not expected during the monocytes in vitro, especially permitting investigations on neu- design of the genetic analysis, as these proteins also associate with rologic disease-associated alleles (66). It is not clear, however, other integrins included in the analysis (36, 75). Nevertheless, it how this model functions with regard to CD18 integrins, and more seems to be consistent with the role of CR3 and CR4 conforma- characterization is required to appropriately apply it for the studies tional regulation in aSN clearance revealed by this study. The presented in our report. The uncertainties with regard to using genetic analyses also lend support to our use of monocytes as primary microglia or microglial-like cells are in contrast to models of CD18 integrin-mediated functions in microglia. From monocytes extracted from blood by a negative selection protocol, broad transcriptomic analyses, it is clear that the human microglial which routinely has provided a source of leukocytes suitable for cells share several expression patterns relevant to integrin func- studying CD18 integrin activation and phagocytosis (24, 67–69). tion with human monocytes across neurodegenerative diseases Human microglial cell lines are available through immortalization (35, 50). In recently obtained data (49), we found that the ex- by viral transduction with oncogenes (70). As a model for pression of CD18 integrin genes, together with genes encoding the studying integrin function, concerns arrive from the cellular at- molecules regulating integrin conformation, are highly correlated tenuation of integrin-mediated functions by the transduced onco- between human microglia and monocytes. Although select parts genes (71), although the specific consequences still need to be of the phagocytic capabilities of microglia (i.e., those involving elucidated for CD18 integrins. Rodent in vivo models have CD33) may be subject to transcriptional regulations different from 16 a-SYNUCLEIN CLEARANCE BY COMPLEMENT RECEPTOR 4 monocytes (66), the evidence presented in our study suggests that 13. Kim, C., D.-H. Ho, J.-E. Suk, S. You, S. Michael, J. Kang, S. Joong Lee, E. Masliah, D. Hwang, H.-J. Lee, and S.-J. Lee. 2013. Neuron-released oligo- CD18 integrin-related functions are conserved between the two meric a-synuclein is an endogenous agonist of TLR2 for paracrine activation of cell types. microglia. Nat. Commun. 4: 1562. Taken together, our study now identifies human CR4 as a 14. Stefanova, N., L. Fellner, M. Reindl, E. Masliah, W. Poewe, and G. K. Wenning. a 2011. Toll-like receptor 4 promotes a-synuclein clearance and survival of nigral prominent part of SN clearance in its aggregated form. The dopaminergic neurons. Am. J. Pathol. 179: 954–963. structural biology responsible for the aggregate recognition is, on 15. Fellner, L., R. Irschick, K. Schanda, M. Reindl, L. Klimaschewski, W. Poewe, one hand, entirely consistent with what has been found for several G. K. Wenning, and N. Stefanova. 2013. Toll-like receptor 4 is required for a-synuclein dependent activation of microglia and astroglia. Glia 61: 349–360. other CR4 ligands, and, on the other hand, apparently the conse- 16. Blander, J. M., and R. Medzhitov. 2004. Regulation of phagosome maturation by quence of a previously unappreciated evolutionary specialization signals from toll-like receptors. Science 304: 1014–1018. of the a I compared both to its rodent homologs and the a I. The 17. Tuttle, M. D., G. Comellas, A. J. Nieuwkoop, D. J. Covell, D. A. Berthold, X M K. D. Kloepper, J. M. Courtney, J. K. Kim, A. M. Barclay, A. Kendall, et al. ligand recognition by the rodent homologs are not extensively 2016. Solid-state NMR structure of a pathogenic fibril of full-length human tested. Hence, the functional differences between human and ro- a-synuclein. Nat. Struct. Mol. Biol. 23: 409–415. dent CR4 remain speculative at this stage and would seem to 18. Li, Y., C. Zhao, F. Luo, Z. Liu, X. Gui, Z. Luo, X. Zhang, D. Li, C. Liu, and X. Li. 2018. Amyloid fibril structure of a-synuclein determined by cryo-electron deserve more attention. Nevertheless, with ageing as the most microscopy. Cell Res. 28: 897–903. important risk factor for development of pathological protein ag- 19. Li, B., P. Ge, K. A. Murray, P. Sheth, M. Zhang, G. Nair, M. R. Sawaya, W. S. Shin, D. R. Boyer, S. Ye, et al. 2018. Cryo-EM of full-length a-synuclein gregation (76), the associated diseases are especially challenging reveals fibril polymorphs with a common structural kernel. Nat. Commun. 9: to long-lived humans relative to the shorter-lived rodents. In this 3609. perspective, human CR4’s ability to convey aggregate clearance 20. Guerrero-Ferreira, R., N. M. Taylor, D. Mona, P. Ringler, M. E. Lauer, R. Riek, M. Britschgi, and H. Stahlberg. 2018. Cryo-EM structure of alpha-synuclein Downloaded from is well in accordance with a needed mechanism of maintaining fibrils. Elife 7: e36402. tissue homeostasis. 21. Giehm, L., D. I. Svergun, D. E. Otzen, and B. Vestergaard. 2011. Low-resolution structure of a vesicle disrupting α-synuclein oligomer that accumulates during fibrillation. Proc. Natl. Acad. Sci. USA 108: 3246–3251. Acknowledgments 22. Pieri, L., K. Madiona, L. Bousset, and R. Melki. 2012. Fibrillar a-synuclein and We thank Bettina W. Grumsen and Kirsten S. Petersen for excellent tech- huntingtin exon 1 assemblies are toxic to the cells. Biophys. J. 102: 2894–2905. 23. Hashimoto, M., and E. Masliah. 1999. Alpha-synuclein in Lewy body disease nical assistance. We acknowledge the kind help of the Core Facility for In- and Alzheimer’s disease. Brain Pathol. 9: 707–720. tegrated Microscopy, Faculty of Health and Medical Sciences, University 24. Stapulionis, R., C. L. Oliveira, M. C. Gjelstrup, J. S. Pedersen, M. E. Hokland, http://www.jimmunol.org/ of Copenhagen and the FACS core facility in Department of Biomedicine, S. V. Hoffmann, K. Poulsen, C. Jacobsen, and T. Vorup-Jensen. 2008. Structural Aarhus University. insight into the function of myelin basic protein as a ligand for integrin alpha M beta 2. J. Immunol. 180: 3946–3956. 25. Skamris, T., C. Marasini, K. L. Madsen, V. Fodera`, and B. Vestergaard. 2019. Disclosures Early stage alpha-synuclein amyloid fibrils are reservoirs of membrane-binding species. Sci. Rep. 9: 1733. The authors have no financial conflicts of interest. 26. Fusco, G., S. W. Chen, P. T. F. Williamson, R. Cascella, M. Perni, J. A. Jarvis, C. Cecchi, M. Vendruscolo, F. Chiti, N. Cremades, et al. 2017. Structural basis of membrane disruption and cellular toxicity by a-synuclein oligomers. Science References 358: 1440–1443. 1. Lashuel, H. A., C. R. Overk, A. Oueslati, and E. Masliah. 2013. The many faces 27. Riek, R., and D. S. Eisenberg. 2016. The activities of amyloids from a structural by guest on September 25, 2021 of a-synuclein: from structure and toxicity to therapeutic target. Nat. Rev. perspective. Nature 539: 227–235. Neurosci. 14: 38–48. 28. Akiyama, H., and P. L. McGeer. 1990. Brain microglia constitutively express 2. Spillantini, M. G., R. A. Crowther, R. Jakes, M. Hasegawa, and M. Goedert. beta-2 integrins. J. Neuroimmunol. 30: 81–93. 1998. alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkin- 29. Vorup-Jensen, T., and R. K. Jensen. 2018. Structural immunology of complement son’s disease and dementia with lewy bodies. Proc. Natl. Acad. Sci. USA 95: receptors 3 and 4. Front. Immunol. 9: 2716. 6469–6473. 30. Podolnikova, N. P., A. V. Podolnikov, T. A. Haas, V. K. Lishko, and a b 3. Peelaerts, W., L. Bousset, A. Van der Perren, A. Moskalyuk, R. Pulizzi, T. P. Ugarova. 2015. Ligand recognition specificity of leukocyte integrin M 2 M. Giugliano, C. Van den Haute, R. Melki, and V. Baekelandt. 2015. a-Synu- (Mac-1, CD11b/CD18) and its functional consequences. Biochemistry 54: 1408– clein strains cause distinct synucleinopathies after local and systemic adminis- 1420. tration. Nature 522: 340–344. 31. Bajic, G., L. Yatime, R. B. Sim, T. Vorup-Jensen, and G. R. Andersen. 2013. 4. Peelaerts, W., L. Bousset, V. Baekelandt, and R. Melki. 2018. ɑ-Synuclein Structural insight on the recognition of surface-bound opsonins by the integrin I strains and seeding in Parkinson’s disease, incidental Lewy body disease, de- domain of complement receptor 3. Proc. Natl. Acad. Sci. USA 110: 16426– mentia with Lewy bodies and multiple system atrophy: similarities and differ- 16431. ences. Cell Tissue Res. 373: 195–212. 32. Vorup-Jensen, T., C. Ostermeier, M. Shimaoka, U. Hommel, and T. A. Springer. 5. Lee, H. J., J. E. Suk, E. J. Bae, J. H. Lee, S. R. Paik, and S. J. Lee. 2008. 2003. Structure and allosteric regulation of the alpha X beta 2 integrin I domain. Assembly-dependent endocytosis and clearance of extracellular alpha-synuclein. Proc. Natl. Acad. Sci. USA 100: 1873–1878. Int. J. Biochem. Cell Biol. 40: 1835–1849. 33. Lee, J. H., J. Choi, and S. U. Nham. 2007. Critical residues of alphaX I-domain 6. Ferreira, S. A., and M. Romero-Ramos. 2018. Microglia response during Par- recognizing fibrinogen central domain. Biochem. Biophys. Res. Commun. 355: kinson’s disease: alpha-synuclein intervention. Front. Cell. Neurosci. 12: 247. 1058–1063. 7. Holmans, P., V. Moskvina, L. Jones, M. Sharma, A. Vedernikov, F. Buchel, 34. Gang, J., J. Choi, J. H. Lee, and S. U. Nham. 2007. Identification of critical M. Saad, J. M. Bras, F. Bettella, N. Nicolaou, et al; International Parkinson’s residues for plasminogen binding by the alphaX I-domain of the beta2 integrin, Disease Genomics Consortium. 2013. A pathway-based analysis provides ad- alphaXbeta2. Mol. Cells 24: 240–246. ditional support for an immune-related genetic susceptibility to Parkinson’s 35. Itoh, Y., and R. R. Voskuhl. 2017. Cell specificity dictates similarities in gene disease. [Published erratum appears in 2014 Hum. Mol. Genet. 23: 562.] Hum. expression in multiple sclerosis, Parkinson’s disease, and Alzheimer’s disease. Mol. Genet. 22: 1039–1049. PLoS One 12: e0181349. 8. Perry, V. H. 2012. Innate inflammation in Parkinson’s disease. Cold Spring Harb. 36. Hogg, N., I. Patzak, and F. Willenbrock. 2011. The insider’s guide to leukocyte Perspect. Med. 2: a009373. integrin signalling and function. Nat. Rev. Immunol. 11: 416–426. 9. Lee, H. J., J. E. Suk, E. J. Bae, and S. J. Lee. 2008. Clearance and deposition of 37.Shi,Q.,S.Chowdhury,R.Ma,K.X.Le,S.Hong,B.J.Caldarone,B.Stevens, extracellular alpha-synuclein aggregates in microglia. Biochem. Biophys. Res. and C. A. Lemere. 2017. Complement C3 deficiency protects against neuro- Commun. 372: 423–428. degeneration in aged plaque-rich APP/PS1 mice. Sci. Transl. Med. 9: 10. Emmanouilidou, E., K. Melachroinou, T. Roumeliotis, S. D. Garbis, M. Ntzouni, eaaf6295. L. H. Margaritis, L. Stefanis, and K. Vekrellis. 2010. Cell-produced alpha- 38. Hou, L., X. Bao, C. Zang, H. Yang, F. Sun, Y. Che, X. Wu, S. Li, D. Zhang, and synuclein is secreted in a calcium-dependent manner by exosomes and im- Q. Wang. 2018. Integrin CD11b mediates a-synuclein-induced activation of pacts neuronal survival. J. Neurosci. 30: 6838–6851. NADPH oxidase through a Rho-dependent pathway. Redox Biol. 14: 600–608. 11. Lorenzen, N., S. B. Nielsen, A. K. Buell, J. D. Kaspersen, P. Arosio, B. S. Vad, 39. Lorenzen, N., L. Lemminger, J. N. Pedersen, S. B. Nielsen, and D. E. Otzen. W. Paslawski, G. Christiansen, Z. Valnickova-Hansen, M. Andreasen, et al. 2014. The N-terminus of a-synuclein is essential for both monomeric and 2014. The role of stable a-synuclein oligomers in the molecular events under- oligomeric interactions with membranes. FEBS Lett. 588: 497–502. lying amyloid formation. J. Am. Chem. Soc. 136: 3859–3868. 40. van Maarschalkerweerd, A., V. Vetri, A. E. Langkilde, V. Fodera`,and 12. Iwai, A., E. Masliah, M. Yoshimoto, N. Ge, L. Flanagan, H. A. de Silva, B. Vestergaard. 2014. Protein/lipid coaggregates are formed during a-synuclein- A. Kittel, and T. Saitoh. 1995. The precursor protein of non-A beta component of induced disruption of lipid bilayers. Biomacromolecules 15: 3643–3654. Alzheimer’s disease amyloid is a presynaptic protein of the central nervous 41. Petruzzelli, L., J. Luk, and T. A. Springer. 1995. Adhesion structure subpanel 5, system. Neuron 14: 467–475. leukocyte integrins: CD11a, CD11b, CD11c, CD18. In Leucocyte Typing V: The Journal of Immunology 17

White Cell Differentiation Antigens. S. F. Schlossman, L. Boumsell, W. Gilks, 63. Davis, G. E. 1992. The Mac-1 and p150,95 beta 2 integrins bind denatured J. Harlan, T. Kishimoto, T. Morimoto, J. Ritz, S. Shaw, R. Silverstein, and proteins to mediate leukocyte cell-substrate adhesion. Exp. Cell Res. 200: 242– T. A. Springer, eds. Oxford University Press, New York, p. 1581. 252. 42. Weetall, M., R. Hugo, C. Friedman, S. Maida, S. West, S. Wattanasin, R. Bouhel, 64. Kla¨ning, E., B. Christensen, G. Bajic, S. V. Hoffmann, N. C. Jones, M. M. Callesen, G. Weitz-Schmidt, and P. Lake. 2001. A homogeneous fluorometric assay for G. R. Andersen, E. S. Sørensen, and T. Vorup-Jensen. 2015. Multiple low-affinity measuring cell adhesion to immobilized ligand using V-well microtiter plates. interactions support binding of human osteopontin to integrin aXb2. Biochim. Anal. Biochem. 293: 277–287. Biophys. Acta 1854: 930–938. 43. Vorup-Jensen, T. 2012. Surface plasmon resonance biosensing in studies of the 65. Griffiths, M. R., P. Gasque, and J. W. Neal. 2009. The multiple roles of the innate binding between b2 integrin I domains and their ligands. Methods Mol. Biol. immune system in the regulation of apoptosis and inflammation in the brain. 757: 55–71. J. Neuropathol. Exp. Neurol. 68: 217–226. 44. Gorshkova, I. I., J. Svitel, F. Razjouyan, and P. Schuck. 2008. Bayesian analysis 66. Ryan, K. J., C. C. White, K. Patel, J. Xu, M. Olah, J. M. Replogle, M. Frangieh, of heterogeneity in the distribution of binding properties of immobilized surface M. Cimpean, P. Winn, A. McHenry, et al. 2017. A human microglia-like cellular sites. Langmuir 24: 11577–11586. model for assessing the effects of neurodegenerative disease gene variants. Sci. 45. Dumitriu, A., J. Golji, A. T. Labadorf, B. Gao, T. G. Beach, R. H. Myers, Transl. Med. 9: eaai7635. K. A. Longo, and J. C. Latourelle. 2016. Integrative analyses of proteomics and 67. Støy, S., T. D. Sandahl, A. L. Hansen, B. Deleuran, T. Vorup-Jensen, RNA transcriptomics implicate mitochondrial processes, protein folding path- H. Vilstrup, and T. W. Kragstrup. 2018. Decreased monocyte shedding of the ways and GWAS loci in Parkinson disease. BMC Med. Genomics 9: 5. migration inhibitor soluble CD18 in alcoholic hepatitis. [Published erratum 46. Kim, D., B. Langmead, and S. L. Salzberg. 2015. HISAT: a fast spliced aligner appears in 2018 Clin. Transl. Gastroenterol. 9: 171.] Clin. Transl. Gastroenterol. with low memory requirements. Nat. Methods 12: 357–360. 9: 160. 47. Pertea, M., G. M. Pertea, C. M. Antonescu, T. C. Chang, J. T. Mendell, and 68. Jensen, M. R., G. Bajic, X. Zhang, A. K. Laustsen, H. Koldsø, K. K. Skeby, S. L. Salzberg. 2015. StringTie enables improved reconstruction of a tran- B. Schiøtt, G. R. Andersen, and T. Vorup-Jensen. 2016. Structural basis for scriptome from RNA-seq reads. Nat. Biotechnol. 33: 290–295. simvastatin competitive antagonism of complement receptor 3. J. Biol. Chem. 48. Schlachetzki, J. C. M., I. Prots, J. Tao, H. B. Chun, K. Saijo, D. Gosselin, 291: 16963–16976. B. Winner, C. K. Glass, and J. Winkler. 2018. A monocyte gene expression 69. Zhang, X., G. Bajic, G. R. Andersen, S. H. Christiansen, and T. Vorup-Jensen. signature in the early clinical course of Parkinson’s disease. Sci. Rep. 8: 10757. 2016. The cationic peptide LL-37 binds Mac-1 (CD11b/CD18) with a low dis- 49. Gosselin, D., D. Skola, N. G. Coufal, I. R. Holtman, J. C. M. Schlachetzki, sociation rate and promotes phagocytosis. Biochim. Biophys. Acta 1864: 471– Downloaded from E. Sajti, B. N. Jaeger, C. O’Connor, C. Fitzpatrick, M. P. Pasillas, et al. 2017. An 478. environment-dependent transcriptional network specifies human microglia 70. Timmerman, R., S. M. Burm, and J. J. Bajramovic. 2018. An overview of in vitro identity. Science 356: eaal3222. methods to study microglia. Front. Cell. Neurosci. 12: 242. 50. Raj, T., K. Rothamel, S. Mostafavi, C. Ye, M. N. Lee, J. M. Replogle, T. Feng, 71. Cheng, A., G. S. Bal, B. P. Kennedy, and M. L. Tremblay. 2001. Attenuation M. Lee, N. Asinovski, I. Frohlich, et al. 2014. Polarization of the effects of of adhesion-dependent signaling and cell spreading in transformed fibroblasts autoimmune and neurodegenerative risk alleles in leukocytes. Science 344: 519– lacking protein tyrosine phosphatase-1B. J. Biol. Chem. 276: 25848–25855. 523. 72. Hong, S., V. F. Beja-Glasser, B. M. Nfonoyim, A. Frouin, S. Li, S. Ramakrishnan,

51. Vorup-Jensen, T., C. V. Carman, M. Shimaoka, P. Schuck, J. Svitel, and K. M. Merry, Q. Shi, A. Rosenthal, B. A. Barres, et al. 2016. Complement and http://www.jimmunol.org/ T. A. Springer. 2005. Exposure of acidic residues as a danger signal for recog- microglia mediate early synapse loss in Alzheimer mouse models. Science 352: nition of fibrinogen and other macromolecules by integrin alphaXbeta2. Proc. 712–716. Natl. Acad. Sci. USA 102: 1614–1619. 73. Nissen, S. K., K. Shrivastava, C. Schulte, D. E. Otzen, D. Goldeck, D. Berg, 52. Svitel, J., A. Balbo, R. A. Mariuzza, N. R. Gonzales, and P. Schuck. 2003. H. J. Møller, W. Maetzler, and M. Romero-Ramos. 2019. Alterations in blood Combined affinity and rate constant distributions of ligand populations from monocyte functions in Parkinson’s disease. Mov. Disord. 34: 1711–1721. experimental surface binding kinetics and equilibria. Biophys. J. 84: 4062–4077. 74. Van Den Berge, N., N. Ferreira, H. Gram, T. W. Mikkelsen, A. K. O. Alstrup, 53. Vorup-Jensen, T., L. Chi, L. C. Gjelstrup, U. B. Jensen, C. A. Jewett, C. Xie, N. Casadei, P. Tsung-Pin, O. Riess, J. R. Nyengaard, G. Tamgu¨ney, et al. 2019. M. Shimaoka, R. J. Linhardt, and T. A. Springer. 2007. Binding between the integrin Evidence for bidirectional and trans-synaptic parasympathetic and sympathetic alphaXbeta2 (CD11c/CD18) and heparin. J. Biol. Chem. 282: 30869–30877. propagation of alpha-synuclein in rats. Acta Neuropathol. 138: 535–550. 54. Vilar, M., H. T. Chou, T. Lu¨hrs, S. K. Maji, D. Riek-Loher, R. Verel, 75. Sun, Z., M. Costell, and R. Fa¨ssler. 2019. Integrin activation by talin, kindlin and G. Manning, H. Stahlberg, and R. Riek. 2008. The fold of alpha-synuclein fibrils. mechanical forces. Nat. Cell Biol. 21: 25–31.

Proc. Natl. Acad. Sci. USA 105: 8637–8642. 76. Groh, N., A. Bu¨hler, C. Huang, K. W. Li, P. van Nierop, A. B. Smit, M. Fa¨ndrich, by guest on September 25, 2021 55. Allen Reish, H. E., and D. G. Standaert. 2015. Role of a-synuclein in inducing F. Baumann, and D. C. David. 2017. Age-dependent protein aggregation initiates innate and adaptive immunity in Parkinson disease. J. Parkinsons Dis. 5: 1–19. amyloid-b aggregation. Front. Aging Neurosci. 9: 138. 56. Thacker, E. L., and A. Ascherio. 2008. Familial aggregation of Parkinson’s 77. Tria, G., H. D. Mertens, M. Kachala, and D. I. Svergun. 2015. Advanced en- disease: a meta-analysis. Mov. Disord. 23: 1174–1183. semble modelling of flexible macromolecules using X-ray solution scattering. 57. Chang, D., M. A. Nalls, I. B. Hallgrı´msdo´ttir, J. Hunkapiller, M. van der Brug, IUCrJ 2: 207–217. F. Cai, G. A. Kerchner, G. Ayalon, B. Bingol, M. Sheng, et al; International 78. Sen, M., K. Yuki, and T. A. Springer. 2013. An internal ligand-bound, metastable Parkinson’s Disease Genomics Consortium; 23andMe Research Team. 2017. A state of a leukocyte integrin, aXb2. J. Cell Biol. 203: 629–642. meta-analysis of genome-wide association studies identifies 17 new Parkinson’s 79. Lee, J. O., P. Rieu, M. A. Arnaout, and R. Liddington. 1995. Crystal structure of disease risk loci. Nat. Genet. 49: 1511–1516. the A domain from the alpha subunit of integrin CR3 (CD11b/CD18). Cell 80: 58. Waterhouse, A., M. Bertoni, S. Bienert, G. Studer, G. Tauriello, R. Gumienny, 631–638. F. T. Heer, T. A. P. de Beer, C. Rempfer, L. Bordoli, et al. 2018. SWISS- 80. Baker, N. A., D. Sept, S. Joseph, M. J. Holst, and J. A. McCammon. 2001. MODEL: homology modelling of protein structures and complexes. Nucleic Electrostatics of nanosystems: application to microtubules and the ribosome. Acids Res. 46(W1): W296–W303. Proc. Natl. Acad. Sci. USA 98: 10037–10041. 59. Perry, V. H., and C. Holmes. 2014. Microglial priming in neurodegenerative 81. San Sebastian, E., J. M. Mercero, R. H. Stote, A. Dejaegere, F. P. Cossıó, and disease. Nat. Rev. Neurol. 10: 217–224. X. Lopez. 2006. On the affinity regulation of the metal-ion-dependent adhesion 60. Harms, A. S., and D. G. Standaert. 2014. Monocytes and Parkinson’s disease: sites in integrins. J. Am. Chem. Soc. 128: 3554–3563. invaders from outside? Mov. Disord. 29: 1242. 82. Zhao, H., I. I. Gorshkova, G. L. Fu, and P. Schuck. 2013. A comparison of 61. Brundin, P., and R. Melki. 2017. Prying into the prion hypothesis for Parkinson’s binding surfaces for SPR biosensing using an antibody-antigen system and af- disease. J. Neurosci. 37: 9808–9818. finity distribution analysis. Methods 59: 328–335. 62. Erdei, A., S. Lukaćsi, B. Maćsik-Valent, Z. Nagy-Balo´, I. Kurucz, and Z. Bajtay. 83. Xu, S., J. Wang, J. H. Wang, and T. A. Springer. 2017. Distinct recognition of 2019. Non-identical twins: different faces of CR3 and CR4 in myeloid and complement iC3b by integrins aXb2 and aMb2. Proc. Natl. Acad. Sci. USA 114: lymphoid cells of mice and men. Semin. Cell Dev. Biol. 85: 110–121. 3403–3408.