α-Synuclein fibrils recruit peripheral immune cells in the rat brain prior to neurodegeneration

Generation of α-synuclein fibrils, biosafety, and biophysical measures

Mouse α-synuclein, encoded in pRK172 was purified from BL21 (DE3) Codon Plus cells (Clontech). Bacterial growth was monitored to log-phase, IPTG added for 2 hours at 37°C, paste collected into lysis buffer consisting of 750 mM NaCl, 10 mM Tris, pH 7.6, 1 mM EDTA, 1 mM PMSF, and 1x bacterial protease inhibitor cocktail (RPI). Homogenates were sonicated and tubes placed in boiling water for 15 minutes. After centrifugation for 25 minutes at ~10,000 x g, samples were loaded into tubing (3.5 kDa MWCO, Fisher) and dialyzed into 10 mM Tris, pH 7.6 with 50 mM NaCl, 1 mM EDTA, PMSF. Supernatant were next centrifuged at 100,000 x g for 1 hour at 4 C, and concentrated using Amicon Ultra15 3.5 MWCO columns. Concentrates were separated through a HiLoad 16/600 Superdex Column, 1 x 120ml (GE Healthcare), samples with α-synuclein dialyzed and concentrated again, and separated through a HiPrep Q HP 16/10 Column, 1 x 20 mL (GE Healthcare). α-Synuclein was eluted from the column with a gradient application of high-salt buffer, and samples containing α-synuclein were dialyzed, applied to a high-capacity Endotoxin Removal spin column (Pierce), and elute concentrated a third time. Concentration of monomeric protein was determined by BCA assay (Pierce).

α-Synuclein fibrils were generated by 700 R.P.M. shaking a microcentrifuge tube containing 5 mg mL^-1 monomer α-synuclein for seven days at 37^oC. Fibrils were sheared with sonication (Fisher Scientific Sonic Dismembrator FB120110). Since sonication aerosolizes fibrils, all sonication steps were performed in a biosafety-level 2 cabinet with the operator wearing disposable wrist guards and double-gloved. Clean-up and inactivation of the fibrils on contaminated surfaces was accomplished using a 1% SDS solution as described [3, 39].

For circular dichroism, 0.1 mg mL^-1 of monomer or fibril samples were loaded onto a photospectrometer cell with a 0.2 mm path length and scanned with the CD DSM-20 system (Olis), with a wavelength from 190 to 260 nm. For dynamic light scattering, estimated molecular weight of monomers and sonicated fibrils, 0.05 mg mL^-1 of α-syn samples were measured in microcuvettes with a DynaPro NanoStar (Wyatt Technology) at 25°C, with data collected and analyzed using Dynamics software. For transmission electron microscopy, 3 μL of 0.1 mg mL^-1 samples were applied to glow-discharged 400 mesh, carbon-only, copper grids (Electron Microscopy Sciences) and negatively stained with 1% uranyl acetate (Polysciences). The grids were imaged in a FEI Tecnai F20 electron microscope (Eindhoven) operated at 200 kV with nominal magnification at 65,000x and a defocus range of -1.0 μm to -1.27 μm. Images were collected on a Gatan Ultrascan 4000 CCD camera. Endotoxin levels were determined using an LAL chromogenic endotoxin quantification kit (Pierce). Immediately prior to usage of fibrils or monomer protein in experiments, monomeric protein was subjected to ultracentrifugation 100,000 x g for one hour, and supernatants containing either monomer the prepared fibril α-synuclein protein was determined using a NanoDrop instrument with 7.45 as the extinction coefficient and 14.45 as molecular weight.

Primary microglia culture

All animal protocols were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee. Timed-pregnant C57BL/6J mice were obtained from Jackson Laboratories and P0-3 pups were isolated, meninges removed, and brain tissue dissociated and filtered (0.2 micron). Filtrate was plated into media containing 20% heat inactivated fetal bovine serum, penicillin/streptomycin, and L-Glutamine (Sigma), and 10ng mL^-1 granulocyte monocyte colony stimulating factor (PeproTech). Cells were maintained for two weeks and microglia isolated by mechanical shaking at 195 R.P.M. for 1 hour at 37C and plating to new wells.

For DQ Ovalbumin measurements, microglia were treated with fibrils or monomer for 30 minutes followed by the addition of DQ Green BSA (Molecular Probes) for one hour. Anti-iNOS (Abcam) and anti-MHCII (clone M5/114.15.2, eBiosciences) were used to stain microglia as previously described (Harms 2013). Images were captured with a Lecia TCS-SP5 confocal microscope and intensities were quantified using ImageJ software. Conditioned media was collected from each experiment and supernatants analyze with the Milliplex Mouse Cytokine/Chemokine Magnetic Bead 25 Plex Kit (MD Millipore)

Injections, immunofluorescence and immunohistochemistry

Intracranial injections of fibrils or monomer control (equivalent w/v) were performed in 100 Sprague Dawley rats (Taconic Farms) aged ~8-10 weeks on a digital stereotaxic frame (David Kopf). Proteins were injected into the rats at the SNpc at 4.65 mm posterior and 2.25 rat mm lateral to Bregma, and 7.45 rat mm ventral relative to the skull). In all experiments, 4 μL of saline, or saline that included 8 μg of monomeric α-synuclein, or 8 μg of matched (same batch of protein as monomer) short-α-synuclein fibrils (concentrations determined by A₂₈₀ prior to injection via Nanodrop analysis) were injected. Rats were transcardially perfused with PBS (pH 7.4). For immunohistochemistry and imaging experiments, this was followed by freshly prepared 4% PFA buffered in PBS. Brains were removed, post-fixed for 24 hours in the 4% PFA and PBS solution, floated into 30% sucrose PBS solution for up to three days, and frozen in isopentane solution (-50 C).

Sections were cut to 40 μm on a freezing microtome and incubated in an antigen recovery solution of 10 mM sodium citrate, pH 6.0, supplemented with 0.05% tween, at 37 °C for 1 hour with gentle rocking. Sections were rinsed with tris-buffered saline (TBS, pH 7.4) and combined with blocking buffer (5% normal goat serum with 0.3% Triton X-100 in TBS). Sections were rinsed and combined with primary antibodies that include pS129-α-synuclein (clone 81A, Biolegend), NeuN (Clone A60, IgG1, EMD Millipore), tyrosine hydroxylase (rabbit polyclonal, EMD Millipore), IBA-1 (rabbit polyclonal, Wako Chemicals), CD613 (Clone ED2, Biorad), and MHCII (Clone RT1B, BD Bioscience). Sections were rinsed and combined with secondary antibodies for 24 hours at 4 °C with gentle rocking. Sections were mounted to superfrost slides and mounted to coverslips with Prolong Gold (Invitrogen). Confocal microscopy was carried out with a Leica SP5 and imaging processing with LAS X software (Leica).

For immunohistochemistry with 3,3′-diaminobenzidine (DAB) and Nissl staining, brain sections were treated as above except for a quench for 30 min at room temperature with 0.3% H₂0₂ in methanol following antigen retrieval. Secondary antibodies (Jackson Immunologic), conjugated to biotin were used and sections treated with the ABC kit (Vector) followed by development with Impact DAB reagent (Vector). Sections were dehydrated progressively in ethanol and then Histo-Clear (national Diagnostics) and mounted to coverslips with Permount reagent (Fisher). Some sections were rehydrated with immersion to water and then treated with cresyl violet solution (0.1% cresyl violet, 0.08% acetic acid) for one-minute in a microwave, and then rinsed in water and dehydrated with ethanol, and immersed back into Histo-clear and mounted onto slides. Slides were analyzed on an Olympus BX61 wide field microscope.

Mononuclear cell isolation and flow cytometry

Intracranial injections of fibrils or monomer control (equivalent w/v), or saline, were performed in 45 Sprague Dawley rats (Taconic Farms) aged ~8-10 weeks on a digital stereotaxic frame (David Kopf). Brain tissue punches from the SNpc or striatum were dissected 8 weeks after injections. Individual samples were formed by pooling (three rats each) SNpc punches, or dorsal striatum, each from different animals. In each experiment, treatment groups (PBS, monomeric alpha-synuclein, fibril alpha-synuclein) consisted of five of these independent sample pools. Mononuclear cells were isolated using enzymatic digestion with 1 mg mL^-1 Collagenase IV (Sigma) and 20 μg mL^-1 DNAseI (Sigma) diluted in RPMI 1640 with 10% heat inactivated fetal bovine serum and 1% L-glutamine from the pooled ventral midbrains or striatum of rats injected with PBS, monomeric α-synuclein, or α-synuclein fibrils. Mononuclear cells were isolated eight weeks post transduction via 30/70% percoll gradient as previously described [33]. Isolated cells were first blocked with an Fcγ receptor solution (1:500 w/v), surface stained for CD45 (OX-1), CD11b/c (OX-42), RT1D (OX-17), CD4 (W3/25), and CD8 (OX-8), all from Biolegend as previously described [33]. Stained cells were analyzed using an Attune NxT (Thermo Fischer Scientific) and data analyzed using FlowJo software. Gating scheme is provided in Additional file 1: Figure S2. In each experiment, live cells were first gated using a fixable viability dye (Vibrant Aqua eBioscience 1:1000) per the manufacturer’s instruction, followed by CD45. This gating scheme allows for selection of all immune cells first including T cells, followed by CD11b to subdivide myeloid subsets.

Unbiased approaches, stereology, and statistics

Unbiased stereological estimations of the total number of TH+ (Nissl counterstain) in the SNpc was performed using an optical fractionator probe (Stereologer software, Stereology Resource Center) by an investigator blinded to the experiment identity. Sections used for counting covered the entire SNpc and were equally spaced 120 μm apart, with random frame placement through all counting areas. Guard zone height was 2 μm to avoid artifacts on the cut surface of sections. Sampling grid size (e.g., 200 μm) was adjusted as needed, due to the heterogeneity of lesion size, to allow up to five objects on average counted at each of 100 to 200 x-y locations (minimum 100 objects counted for each observation, based on mean section thickness). Cells that were large (~30-50 μm) and Nissl+ but not TH positive within the borders of the SNpc were also counted (less than 1% of the total counts in each experimental group reported herein). For counting soma inclusions through the SNpc or striatum, a rare-object counting probe was utilized. Only somatic perinuclear (e.g., adjacent to Nissl bodies) phosphorylated α-synuclein inclusions were counted.

Statistical analysis and graphs were performed and created with Graphpad Prism 5.0 software. Two-tailed one-way ANOVA tests were used to calculate p values, where less than 0.05 was considered significant, and for those groups with significant differences, a post-hoc test (Tukey’s) was applied to each possible combination. Linear regression lines are shown for some plots.

α-Synuclein fibril exposure promotes antigen processing, presentation and activation in primary microglia

Aggregated forms of α-synuclein may activate primary cultured microglia through toll-like receptor signaling pathways [11, 37]. Very short α-synuclein fibrils have been identified as conformers that can seed the formation of new inclusions in neurons, and eventually cause progressive dopaminergic neurodegeneration in rats [1, 29]. We wondered whether the concentrations and compositions of short-fibrils we and others have used in primary neurons to induce inclusions might cause microglia activation phenotypes similar to responses observed with applying large α-synuclein aggregates and fibrils to microglia [14, 20, 21].

To generate short fibrils and matched monomer preparations (protein from the same catch of starting recombinant protein), we purified recombinant α-synuclein from bacteria and depleted endotoxin content with reduced poly-lysine columns. Next, we verified preparations of either monomeric (depleted of aggregates via ultracentrifugation) or sonicated short fibrils using dynamic light scattering (Fig. 1a). The fibril preparation had an average length of ~30 nm compared to monomeric protein (average length of ~2 nm). Larger α-synuclein conformers greater than 2 nm in the monomeric preparation could not be detected by light scattering. Based on A₂₈₀ measurements of protein concentration, the amount of α-synuclein protein did not vary in the monomeric or short-fibril preparations. Endotoxin as measured through Limulus Amebocyte Lysate testing revealed a concentration of 14.9 endotoxin units (EU) per milligram (i.e., 0.015 EU μg^-1) of protein common to the preparations of monomer, unsonicated fibrils, and short-fibrils. Circular dichroism easily distinguished the monomer protein from the fibrils based on β-sheet conformations inherent to α-synuclein protein fibrils (Fig. 1b). Electron microscopy of the fibril preparation corroborated the light scattering-predicted length of the fibrils (Fig. 1c).

To determine whether monomeric or short α-synuclein fibrils might provoke differential effects in microglia, we treated mouse primary microglia with monomer or fibrils using matched amounts of α-synuclein protein (based on A₂₈₀) with amounts of fibrils that can result in inclusions in primary neurons (~1 nM fibrils, see [1]). After four hours, we measured MHCII and iNOS expression that are both canonical markers of pro-inflammatory activation and found that the fibrils induced these markers three to six-fold (respectively) over monomer α-synuclein exposures (Fig. 1 d, e). To determine whether the fibrils could increase antigen processing and presentation, DQ ovalbumin was added to the primary microglia cultures for one hour. Following the addition of α-synuclein fibrils to primary microglia for thirty minutes, a marked induction of processing was observed via fluorescence emitted from cleaved DQ ovalbumin (Fig. 1f,g), consistent with MHCII upregulation.

α-Synuclein fibril exposure in the rat SNpc induces a rapid MHCII response

To determine whether α-synuclein fibril exposures could provoke rapid inflammation phenotypes in the brain as suggested by our in vitro studies with primary microglia (Fig. 1), we injected 4 μL of saline, or saline that included 8 μg of monomeric α-synuclein, or 8 μg of matched (same batch of protein as monomer, both with ~0.12 total EU included) α-synuclein fibrils into the rat SNpc. In serial sections across the midbrain, the injected fibrils could be detected by immunohistochemistry and rapidly spread across the entirety of the SNpc as observed twenty-four hours after injection (Fig. 2a). By 72 hours post-injection, the fibril material could not be detected in the SNpc. To determine microglial activation profiles in the SNpc in the presence of the injected fibrils or monomer protein at 24 hours, as well as inflammation caused by the surgery and saline injections alone, we immunostained sections of midbrain with MHCII to probe pro-inflammatory responses, IBA-1 to identify resident microglia, microgliosis (expansion of microglia, and/or reduction of turnover), and tyrosine hydroxylase (TH) for dopaminergic neuron identification. IBA-1 expression appeared upregulated 24-hours after injections in all conditions when compared to the contralateral side, consistent with effects due to the surgery alone (Fig. 2c-e). The saline injections failed to induce any MHCII expression. In contrast, both monomer and fibril α-synuclein upregulated MHCII in many of the ramified IBA-1 cells (Fig. 2c-e). We noticed that fibril injected rat sections often harbored a proportion of MHCII-positive cells that were much weaker in IBA-1 expression and had amoeboid morphology distinct from typical microglia (Fig. 2e). CD163 is a scavenger receptor demonstrated to demarcate monocytes and macrophages from the periphery [31], and these cells poorly express IBA-1 but can robustly express MHCII. We could not detect CD163-positive cells in the SNpc of saline or monomer α-synuclein injected rats, but all the fibril injected rats demonstrated robust CD163 staining (Fig. 2h). These results demonstrate that all conditions including surgery alone active the innate immune response to some extent, but α-synuclein fibrils were unique in recruiting MHCII-positive IBA-1 weak and CD163-positive cells within the fibril injection field.

α-Synuclein fibrils but not monomer result in a sustained MHCII response in the SNpc

In vitro experiments with primary microglia suggest monomeric α-synuclein poorly induces pro-inflammatory responses compared to fibrillar α-synuclein. We further probed the monomer and fibril injected rats by evaluating the SNpc injection site one, three, and six months later. In contrast to fibril-injected rats, monomer exposures did not lead to a sustained MHCII expression in the SNpc (or saline-only) injections (Fig. 3a-f). CD163-positive cells in the monomer-injected SNpc could be detected only in the vasculature, indicative of the normal presence of perivascular macrophages (Fig. 3g-i). In contrast, CD163-positive cells were detected in the SNpc distant from any vasculature in fibril-injected rats at 1, 3 and 6 months post-injection (Fig. 3j-l). Efforts to further probe the surface markers of these MHCII expressing cells using antibodies to TMEM119 and CX3CR1 were unsuccessful due to a lack of antibody efficacy and specificity in rats compared to mice where the antibodies work well (Additional file 1: Figure S1). Overall, these results indicate that the inflammatory response observed at twenty-four hours with monomeric α-synuclein protein had resolved, whereas α-synuclein fibrils cause a more persistent MHCII expression that may involve peripheral immune cells.

α-Synuclein fibrils promote microglial activation (but not microgliosis) with peripheral immune cell infiltration in the SNpc

Our confocal analyses have identified two features that distinguish fibril-α-synuclein effects from monomer effects: recruitment of CD163-positive monocytes and macrophages and sustained MHCII responses in fibril, but not monomer, injected rats. To quantify these processes, we developed a protocol for mononuclear cell isolation from microdissected rat brain tissue and subsequent flow cytometry analysis from matched saline, monomer, and fibril injected rats, two-months post-injection. Cells were first gated for CD45 and CD11b expression to select myeloid lineages (gating strategy presented in Additional file 1: Figure S2). Measurements of CD11b expressing CD45^hi cells that mark infiltrating monocytes and macrophages [5] revealed significant increases with fibril exposures as compared to monomer or saline injections (Fig. 4). Monomer and saline controls were similar in all our measures, showing that the effects of the surgical procedure and monomeric α-synuclein protein (and co-contaminants inherent to the α-synuclein protein preparation) resolved and could not be distinguished two-months after injections (Fig. 4a).

To determine if the population of monocytes and macrophages from the periphery were in-part responsible for the enhanced MHCII expression in the SNpc we could observe by confocal analysis, we gated CD11b expressing and CD45^hi cells for MHCII expression using the same MHCII antibody clone used for confocal analysis. Fibril exposures approximately doubled the number of CD45^hi and CD11b-expressing cells, and those that expressed the highest MHCII levels (Fig. 4b, c). The total number of microglia did not change compared to saline or monomer exposures, suggesting a lack of microglia proliferation or changes in turn-over (Fig. 4d). The overall increase in MHCII positive cells was therefore due in-part to infiltrating monocytes and macrophages.

Finally, we analyzed the SNpc cell isolate for CD4 positive T-cells of the adaptive immune system and again detected a robust increase in peripheral immune cells in fibril-injected rats as compared to monomer or saline-only injections (Fig. 4e). While T-cells lack MHCII expression, they corroborate the exacerbated and sustained pro-inflammatory response defined by MHCII expression and peripheral cell recruitment caused by α-synuclein fibril exposures.

α-Synuclein fibril exposures in the SNpc promote pSer129-α-synuclein inclusions, progressive loss of tyrosine-hydroxylase (TH) expression, and striatal degeneration

Confocal analyses revealed sparse or no phosphorylated α-synuclein inclusions one-month post fibril injection, whereas robust inclusions in TH-expressing cells in the SNpc could be detected three and six-months post-fibril injection (Fig. 3a-f). To quantify inclusion formation and loss of TH-expression in the context of persistent neuroinflammation, serial sections of the rat SNpc processed for MHCII and CD163 staining were also analyzed via unbiased stereology for TH-expressing neurons at one, three, and six months’ post-injection. We found a significant loss of TH-positive cells three and six-months post injection (Fig. 5a). No significant change in the numbers of TH-positive neurons were detected in the monomer injected SNpc between 1, 3, and 6 months (average 8779±323 neurons, n=25). In contrast, the number of TH-positive neurons in the fibril-injected SNpc reduced from 1 to 3 to 6 months (average 6697±341, 6213±1303, and 4426±688, respectively, n=34). The number of TH-positive neurons in the un-injected SNpc was similar between all groups (cumulative average 8129±257, n=59).

The number of inclusions in the SNpc peaked at three-months with thousands of inclusions detected in some rats, whereas hundreds of inclusions were detected across the SNpc at one and six-months post fibril injection (Fig. 5b). pSer129-α-synuclein inclusions were never detected in any of the monomer-injected rats. To determine the viability of dopaminergic nigro/striatal projections at these time-points, striatal TH fiber density in the dorsal striatum was measured via LiCOR tissue imaging and decreased densities could be detected three and six months’ post α-syn fibril injections (Fig. 5c). Decreased TH fiber density correlated well (r=0.93) with decreases in the numbers of TH-expressing neurons in the SNpc (Fig. 5d). The timing of neurodegeneration and inclusion distribution with SNpc fibril injections is therefore different than previous reports of fibril injections in the dorsal striatum in rats [29].

α-Synuclein fibril exposure in the SNpc induces progressive inflammation and inclusion formation in the dorsal striatum

A main feature of fibril injections in the rat SNpc is that inclusions eventually spread to spiny projection neurons in the dorsal striatum and this correlates with dopaminergic neurodegeneration [1]. To determine whether α-synuclein fibrils introduced in the SNpc induce inflammation in the dorsal striatum, we analyzed tissue dissected from the striatum via flow cytometry. Two-months post fibril injection, we detected an increase in the number of monocytes and macrophages in the striatum, without an increase in MHCII expression, in fibril-injected rats (Fig. 6a, Additional file 1: Figure S3). The number of microglia in the striatum 2-months post-fibril injection were unchanged in all conditions (Fig. 6b, c).

Stereological counts of inclusions through the striatum revealed that inclusion spread from the SNpc to the striatum was a slow process where inclusion loads could only be detected at 6-months post-injection (Fig. 6d). Concomitant with the formation of new α-synuclein inclusions in the dorsal striatum, MHCII expression and CD163-cell recruitment in the striatum became robust six-months post-fibril injection in the SNpc in comparison to monomer-injected rats (Fig. 6e-j). These results demonstrate a marked pro-inflammatory response that spreads from the initial site of α-synuclein fibril injection in the SNpc to the striatum, with full MHCII expression and peripheral cell recruitment that may depend on the availability of local α-synuclein inclusions and fibrils (Fig. 6i, j).