Peptide synthesis and fibril formation
Lyophilized peptides and proteins were commercially purchased: amyloid-β 40 (KE Biochem), amyloid-β 42 (Celtek Peptides), α-synuclein (rPeptide). The lyophilized powder was resuspended in PBS (Thermo Fisher) at concentrations of 2 or 5 mg/mL for amyloid-β 40, amyloid-β 42, and α-synuclein. Fibril formation was induced by agitation at 1,500 × rpm for 5–10 days at 37°C using an Eppendorf Thermomixer. EF-C (PTD-A) peptide was produced by standard Fmoc solid-phase synthesis, purified by preparative RP HPLC, and analyzed by HPLC and MS at the Core Facility for Functional Peptidomics Ulm. The lyophilized powder was solubilized in dimethyl sulfoxide (Merck) at a concentration of 10 mg/mL. Fibrils formed instantly upon 10-fold dilution of the peptide stock in PBS, as described previously33. EF-C seeds were generated by the sonication of fibrils for 60 sec, amplitude 50%, with pulses 1 sec on, 1 sec off. Fibril formation was verified by Thioflavin T (ThT) binding assay and electron microscopy.
ATTO NHS ester labeling of fibrils
ATTO labeling was performed as previously reported74. In brief, 1 mg of ATTO NHS dye was mixed with the peptide or protein stock solution and incubated for 1 h at room temperature (RT). Labeled monomer solutions were formed by mixing 1 µL ATTO 647N, or ATTO 495 labeled monomers with 99 µL unlabeled monomers. Cross-seeded fibrils were formed by mixing 10% v/v 1% labeled and sonicated EF-C fibrils with 90% v/v 1% labeled for amyloid-β 40, or α-synuclein monomers. Fibril formation was initiated by agitation in the dark at 1,500 × rpm for 5–10 days at 37°C using an Eppendorf Thermomixer. To remove unbound dye, labeled fibrils were washed by centrifugation at 14,000 × rpm for 15 min, and resuspended in equal amount of PBS. Fluorescence microscopy was performed using a Leica Dmi8 confocal microscope.
ProteoStat and Thioflavin T labeling
Labeling with Proteostat was performed according to the Proteostat amyloid plaque detection kit (Enzo LifeSciences). Fibrils were diluted to a final concentration of 200 µg/mL in the ProteoStat staining solution and incubated in the dark for 15 min at RT. For ThT binding, a 2.5 stock solution of ThT in PBS was prepared and sterile-filtered. 10 µL of fibril solution (1 mg/mL) were mixed with 1 µL of the ThT stock solution, 89 µL PBS were added and samples were incubated for 10 min in the dark at RT. Fluorescence intensity scans were performed by excitation at 450 nm and an emission of 450–650 nm for spectral scanning, or 482 nm for single wavelength analysis. To monitor the fibril formation kinetics, samples (1 mg/mL) were incubated with 25 mM ThT. The mix was pipetted into a Corning 3575 plate and two glass beads with a diameter of 1–2 mm were added in each well. For the cross-seeding experiments, samples were mixed with the indicated amounts of seeds, or sucrose-purified HIV-1. The plate was sealed and incubated at 37°C, under continuous orbital shaking (1 mm) at a frequency of 800 cmp. Fluorescence was measured at an excitation wavelength of 450 nm and an emission endpoint of 490 nm. Measurements were performed using a Synergy H1 hybrid multi-mode reader (BioTek).
Nanoparticle tracking analysis
To measure the concentration, size distribution, or surface charge (zeta potential), samples were diluted in 1 mL of ddH2O and measured three times using a ZetaView TWIN (Particle Metrix). The zeta potential of fibrils was calculated based on the electrophoretic mobility of the fibril samples.
Transmission electron microscopy
To visualize fibrils by electron microscopy, the samples were diluted to 1 mg/mL in PBS. 5 µL of the samples were incubated for 5 min on carbon-coated formvar film, glow discharged 300 mesh copper grids. Afterwards, three washing steps with a series of three drops of double-distilled water (ddH2O), and one staining step with 2% uranyl acetate in H2O were performed. The dye excess was removed using filter paper. Samples were air-dried for 1–2 h and visualized using a Jeol 1400 transmission electron microscope (Jeol) operated at 120 kV.
Virus stocks
Infectious molecular clones of HIV-1 Bal and NL4-3 (X4) strains were obtained from NIH AIDS Reagent Program. HIV-1 AD8 was provided by Kathleen Collins (University of Michigan, Michigan, USA), and HIV-1 JRCSF by Beatrice Hahn (University of Pennsylvania School of Medicine, Philadelphia, USA), while HIV-1 NL4-3 R5 and HIV-1 NL4-3 R5-IRES-G-Luc were generated in house. To generate virus stocks, 9 × 105 HEK293T cells were seeded 24 h before transfection in 6-well tissue culture plates (Sarstedt). Cells were transfected with 2.5 µg per well of plasmid DNA using TransIT®-LT1 (Mirus) according to the manufacturer. Virus stocks were harvested 48 h post-infection and stored at -80°C. For the fibril-forming experiment, HIV-1 was purified using 20% sucrose by layering 4 parts of freshly produced virus on 1-part sucrose, and centrifugation for 3 h, 14,000 × rpm, at 4°C. The virus pellet was resuspended in PBS, particle concentration measured by nanoparticle tracking, adjusted to 3.5 × 109, and fixed in 4% PFA.
Cell lines and culture
Human Embryonic Kidney (HEK) 293T, H4 neuroglioma cells (H4), and Microglia Clone 3 (HMC-3) cells were purchased from American Type Culture Collection (ATCC). CEM-M7, U373-MAGI, and TZM-bl cells were obtained from the NIH AIDS Reagent Program. HEK 293T, U373-MAGI, and TZM-bl cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Gibco), 2 mM L-glutamine, 100 µg/mL streptomycin, and 100 U/mL penicillin (all PANBiotech). H4 cells were cultivated in DMEM (Gibco) supplemented with 10% (v/v) heat-inactivated FBS and 4.5 g/L D-glucose and L-glutamine (Bio & SELL). HMC-3 cells were cultivated in Eagle's Minimum Essential Medium (EMEM, ATCC) supplemented with 10% (v/v) heat-inactivated FBS. CEM-M7 cells were cultivated in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) supplemented with 10% (v/v) heat-inactivated FBS, 2 mM L-glutamine, 100 mg/mL streptomycin, and 100 U/mL penicillin. All cells were cultured at 37°C, 90% humidity, and 5% CO2. Adherent cell lines were passaged with 0.05% Trypsin/EDTA (PANBiotech) and used for experiments when they reached 70–80% confluence.
Isolation of primary blood cells
Buffy coats from healthy donors were obtained from the DRK blood bank (Ulm). To obtain CD4 + T cells, they were diluted 1:2 with PBS and incubated with 50 µL/mL CD4 + T cells enrichment cocktail (RosetteSep) for 20 min. The mixture was further diluted to 1:3 with PBS, layered on human Pancoll (PAN-Biotech), and centrifuged at 1,200 × g for 30 min with the brakes off. The white interface layer containing leukocytes and platelets was collected and washed with PBS. Red blood cells were lysed with ammonium-chloride-potassium lysing buffer (TheraPEAK) for 5 min. After washing with PBS, 1 × 106 cells/mL were cultured in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated FBS, 2 mM L-glutamine, 100 mg/mL streptomycin, 100 U/mL penicillin, 10 ng/mL IL-2 (Miltenyi Biotec), and anti-CD3/CD28 dynabeads (bead-to-cell ratio of 1:1, Thermo Fisher Scientific) for three days. PBMCs)were isolated from buffy coats using human Pancoll (PAN-Biotech). To generate human monocyte-derived macrophages (MDMs), 1 × 106 isolated blood cells/mL were cultured in 48-well tissue culture plates (Sarstedt) pre-coated with poly-L-lysine (Sigma-Aldrich), using DMEM supplemented with 10% (v/v) FBS, antibiotics, 10% (v/v) human serum (PeproTech), and 15 ng/mL M-CSF (PeproTech). To generate human monocyte-derived microglia (MMG), 1 × 106 isolated cells/mL were cultured in 48-well tissue culture plates pre-coated with poly-L-lysine, using RPMI 1640 supplemented with 1% (v/v) FBS, antibiotics, 100 ng/mL IL-34 (PeproTech), 100 ng/mL MCP-1 (PeproTech), 10 ng/mL M-CSF, 10 ng/mL GM-CSF (PeproTech), and 10 ng/mL β-nerve growth factor (PeproTech). Three days later, non-adherent cells (consisting mainly of T-lymphocytes) were removed by washing with the respective cell medium. To induce the differentiation of MDMs and MMG, cells were cultured in the respective supplemented FBS-free cell medium for up to 14 days.
Ethics
The use of human PBMCs was approved by the Ethics Committee of the Ulm UniversityMedical Center (Approval 93/21-FSt/TR). All donors were anonymized prior to the experiments and randomly chosen from a pool of healthy donors. Informed written consent was given and no compensation provided. Sex and/or gender were not considered for the study design and were determined based on self-report. Analyses of brain samples received ethical approval by the Ulm University Ethics Committee (Ulm/Germany; Decision-No. 342/14) and by the UZ Leuven ethical committee (Leuven/Belgium; Decision-No. S-59295). In accordance with the Declaration of Helsinki an informed consent for autopsy and scientific use of autopsy tissue with clinical information was granted. All methods have been performed in accordance with the relevant guidelines and regulations.
Verification of macrophage and microglia markers
To analyze the expression of markers on macrophage and microglia cells by flow cytometry, cells were detached using Versene (Gibco) and washed using FACS buffer (PBS with 1% (v/v) FBS). For membrane markers, cells were stained with FITC-anti-human P2RY12 Ab (BioLegend), FITC- anti-human CD45 Ab (BioLegend), Alexa Fluor® 647 anti-human IBA1 Ab (Abcam), Brilliant Violet 605™ anti-human CD4 Ab (BioLegend), APC anti-human CCR5 Ab (BD Biosciences), or PE anti-human CXCR4 Ab (BD Biosciences), for 1 h in a wet chamber at RT. Then, cells were washed three times in FACS buffer and fixed in 2% (v/v) PFA. For intracellular markers, cells were permeabilized in 0.2% Triton-X in PBS for 10 min at RT before th addition of antibodies. Samples were acquired on a CytoFLEX flow cytometer equipped with CytExpert software. Acquired data were analyzed using Flowjo 10.9.0 software. To analyze the expression of markers on macrophage and microglia cells by confocal microscopy, cells were blocked in 1% BSA (v/v) in TBS-T for 30 min at RT, followed by antibody staining as indicated above. Cell nuclei were stained with Hoechst for 30 min at RT. Then, the cells were washed three times in PBS and fixed in 4% (v/v) PFA. For intracellular markers, before the addition of antibody, cells were permeabilized in 0.2% Triton-X in PBS for 10 min at RT.
Infection assays
To evaluate the HIV-1 infection in the reporter cell line TZM-bl75, 1 × 104 cells were seeded in 96-well tissue culture plates (Starstedt). The next day, HIV-1 was pretreated with different compound dilutions at a ratio of 1:1, for 3–5 min at 37°C. Then, 20 µL of the mix was added to 80 µL medium in triplicates. Three days post-infection, the medium was discarded and 40 µL of 1:4 diluted GalScreen® substrate (Applied Biosystems) in PBS containing 0.214% Triton X (Sigma-Aldrich) was added to the cells. After 45 min of incubation in the dark at RT, 35 µL were transferred to Nunc™ MicroWell™ 96-well plate (Thermo Fisher Scientific) and β-galactosidase activities were quantified as relative light units per second (RLU/s) using the Orion II microplate luminometer (Tiertek-Berthold). Values were corrected for the background signal derived from the uninfected cells. CEM-M7 cells were seeded and infected as described above for TZM-bl cells. Three days post-infection, cells were washed with PBS and fixed in 4% (v/v) PFA. In all the experiments, GFP + gates were set based on the uninfected cells treated in parallel. Samples were acquired on a CytoFLEX flow cytometer equipped with CytExpert software. Acquired data were analyzed using CytExpert 2.3.0.84. U373-MAGI cells were seeded at a density of 1 × 104 cells/well in 96-well tissue culture plates one day before infection, and infected as described above for TZM-bl cells. Two hours post-infection, the inoculum was washed off and fresh medium was added. Supernatants were collected immediately and further every three days, frozen at -80°C. For infection of primary cells, 1 × 105 CD4 + T cells were seeded in 96-U-well plates (Sarstedt), and MDM and MMG cells, differentiated in 48-well plates as described above, were infected with a ratio of 1:4 (v/v) HIV-1 treated with different compound dilutions (1:1), for 3–5 min at 37°C. To determine the infectivity of virions produced in primary blood cells, ~ 50% (v/v) of supernatants of primary CD4 + T cells and ~ 30% (v/v) of supernatants of MDMs and MMG cultures at the indicated time points were collected and frozen at -80°C. TZM-bl cells were seeded in 96-well plates (Sarstedt) at a density of 1 × 104 cells in 100 µL per well and infected with 25 µL of the supernatants collected from primary CD4 + T cells, MDMs, or MMG. Three days post-infection, viral infectivity was determined using the β-galactosidase screen kit.
HIV-1 infection in trans
To examine HIV-1 in trans-infection, 5 × 103 HMC-3 cells were seeded one day prior to infection in 96-well plates (Sarstedt). The next day, 20 µL of a 1:1 fibril-virus mix was added in a volume of 80 µL medium. After 4 h of incubation, the cells were washed 3 × with PBS, and 5 × 103 TZM-bl cells were added in 200 µL of medium. As a Gaussia luciferase reporter virus was used, supernatants were collected prior to the addition of TZM-bl, representing the washing control, further collected every three days, and frozen at -80°C.
Gaussia Luciferase assay
To measure the infection of Gaussia luciferase reporter HIV-1, coelenterazine substrate (PJK GmbH) was prepared by dissolving 1 mg in acidified methanol (1 drop concentrated HCl to 10 mL of methanol), and stored at -80°C. For the measurements, 100 µL of the substrate diluted 1:120 in PBS was added to 20 µL of supernatant, and activity was quantified as RLU/s using the Orion II microplate luminometer (Tiertek-Berthold). For the bioluminescence complementation assay with ASYN-hGLuc1 (S1) and ASYN-hGLuc2 (S2) constructs, cells were transfected as described above and conditioned media collected 48 hours post-transfection. Luciferase activity from protein complementation was measured using 100 µL conditioned media in an automated plate reader (Victor X3 microplate reader, Perkin-Elmer) at 480 nm with a signal integration time of 1 second after injection of 100 µL coelenterazine (1 mg/mL, PJK GmbH).
Virion fusion
HIV-1 BlaM-Vpr particles were generated by cotransfection of HEK293T cells with HIV-1 AD8 proviral DNA, pCMV-BlaM-Vpr, and pAdVAntage vectors, as previously described49. HIV-1 virion fusion was measured based on the incorporation of a β-lactamase Vpr (BlaM-Vpr) fusions into the virions and its subsequent transfer into a target cell where CCF2, the fluorescent dye substrate of β-lactamase is cleaved. Cleavage triggers a fluorescence shift of CCF2 from green (520 nm) to blue (447 nm), after excitation at 405 nm. To quantify HIV-1 fusion in PBMC-derived macrophages and microglia, 2.5 × 105 freshly isolated PBMCs were seeded in 8-well Ibidi slides (Ibidi) pre-coated with poly-L-lysine (Sigma-aldrich). At day 14 of differentiation, cells were infected with 100 µL of 1:1 fibril-virus mix in 100 µL medium for 6 h at 37°C. Then, the cells were washed 3 × with PBS, and loaded with CCF2 dye and the reagents from the Beta-lactamase Loading Solutions kit (Invitrogen) (0.3 µL CCF dye, 1.2 µL solution B, and 1.5 µL solution D in 150 µL CO2-independent medium/sample) and incubated overnight at RT in a wet chamber. At the next day, cells were washed 3 × with PBS, fixed in 4% paraformaldehyde (PFA, ChemCruz), and imaged by fluorescence microscopy using a Leica Dmi8 confocal microscope (Leica). The mean fluorescence of the green and blue channels was quantified using Fiji ImageJ.
Virus-fibril interaction
ProteoStat stained fibrils were pre-incubated with diluted MLV GAG YFP virus-like particles (VLPs) for 3–5 min at 37°C, and 30 µL of the mixture were transferred to an Ibidi µ-slide chamber (Ibidi) for visualization. To detect the interaction of the fibril-virus mix with cells, 3 × 104 TZM-bl cells were seeded in 8-well slides (Ibidi) one day before the addition of amyloids, viral particles, or mixtures thereof. For cell staining, the CellTrace Violet Cell Proliferation Kit (Invitrogen) was used. Fluorescence microscopy was performed using an LSM 710 confocal microscope (Zeiss).
Fibril-virus pull-down assay
To determine the binding between fibril and virus, HIV-1 was incubated with different compound dilutions for 3–5 min at 37°C, then the mixture was pelleted by centrifugation at 3,041 × g for 10 min. 20 µL of supernatant was carefully collected, the pellet was resuspended with 20 µL of medium, and the supernatant and resuspended pellet were added to TZM-bl cells. Three days post-infection, β-galactosidase assay described above was performed.
Generation of α-synuclein supernatants
H4 cells were cultivated as described above and seeded in 6-well plates (Sarstedt) 48 hours before transfection. The ASYN-hGLuc1 (S1) and ASYN-hGLuc2 (S2) fusion constructs (wildtype and mutants) were generated as previously described76. The Gaussia luciferase is split into two parts and each part is fused to either wild-type α-synuclein or one of the mutant constructs (A30P, E46K, A53T). Upon α-synuclein oligomerization, the reporter parts form a functional reconstituted bioluminescent active luciferase. H4 cells were transfected with equal amounts of split ASYN constructs using PolyFect Transfection Reagent (Qiagen) according to the manufacturer’s instructions. Opti-MEM (1X) reduced serum medium supplemented with L-glutamine and HEPES (Gibco) was used as the growth medium. Cells were further cultured for 48 hours before media were collected for HIV-1 infection assays. For the depletion of α-synuclein in the supernatant of wild-type ASYN transfected cells, 150 µL protein G Mag Sepharose Xtra beads (GE Healthcare) were supplemented with 1 mL Opti-MEM (1X) reduced serum medium supplemented with L-glutamine and HEPES (Gibco) and rotated at 4°C for 10 min. The beads were centrifuged at maximum speed for 1 min and placed in a magnet (Invitrogen). The supernatant was discarded and the washing step was repeated a total of three times. In a pre-clearing step, 50 µL of washed beads were incubated with 700 µL of conditioned media for 1 hour at 4°C on a rotator (Stuart). The beads were centrifuged at maximum speed for 10 min and placed in a magnet to transfer the supernatant in a new tube. 5 µg SYN-1 antibody (BD Bioscience) was added to the conditioned medium and rotated for at least 3 hours at 4°C. The remaining 100 µL of washed G beads were then added and rotated overnight at 4°C. The next day, the sample was centrifuged at maximum speed for 10 min and the supernatant was used as α-synuclein depleted media.
Processing of brain lysates
Human brain lysates were obtained from post-mortem tissue of freshly frozen human occipital (Brodmann areas 17–19) and temporal cortex (Brodmann areas 35 and 36). Two donors were Alzheimer`s patients, two donors were diagnosed with Lewy body disease, whereas the remaining donors represented age-matched control individuals without neurological disorders (Supplementary Table 1). Samples were homogenized by sonication, and the insoluble fraction was separated by centrifugation at 14,000 × g, for 2 h at RT, and discarded. The supernatants containing the soluble fraction were kept and normalized by protein concentration. Then, TZM-bl cells seeded one day prior at a density of 1 × 104 cells/well, were infected with a 1:1 mix of titrated brain extract and HIV-1. The inoculum was washed after 3 hours, and two days post-infection the β-galactosidase assay described above was performed.
Statistical Analysis
All experiments were independently repeated three times unless specified otherwise. Where statistics were calculated using unpaired t-test analysis at least three replicates were measured in each experiment. Calculations were made using the Prism 7.05 software (GraphPad, La Jolla, CA, USA). Graphical data presentations show mean values and SEM; asterisks indicate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Significances for pattern analysis were determined using unpaired t-test analysis.