Animals
Mouse husbandry and procedures were performed in accordance with institutional guidelines and approved by the Stanford Administrative Panel on Animal Care (APLAC). Heterozygous human LRRK2 G2019S BAC-transgenic mice (The Jackson Laboratory, cat# 018785) were maintained on a C57BL/6 background (The Jackson Laboratory, cat# 000664). LRRK2 G2019S mice and wild-type litter mates were used for in vivo experiments. C57BL/6 mice, LRRK2 G2019S BAC mice and CAG–Cas9 transgenic mice (Jackson Laboratory cat# 024858) were used for time-mating and primary neuron cultures.
Stereotaxic injections
Stereotaxic injections were performed on 8–12 week old adult mice. Animals were placed in a stereotaxic frame and anesthetized with 2% isoflurane (2 L/min oxygen flow rate) delivered through an anesthesia nose cone. Ophthalmic eye ointment was applied to prevent desiccation of the cornea during surgery. The area around the incision was trimmed and disinfected. PFF or vehicle solutions were injected unilaterally or bilaterally into the dorsal striatum using the following coordinates (from bregma): anterior = + 0.4 mm, lateral = ± 1.85 mm from midline, depth = − 2.7 mm (from dura). Mice were injected with sonicated PFFs (5 µg/mouse) or PBS vehicle control. PFFs were sonicated prior to injection. Per hemisphere, 1 μL volume was injected at a rate of 100 nL/min using a 5 μL Hamilton syringe with a 32G needle. To limit reflux along the injection track, the needle was maintained in situ for 5 min, before being slowly retrieved. Each mouse was injected subcutaneously with analgesics and monitored during recovery.
Behavioral testing
Animals underwent a series of behavioral testing up to 6 months post injection. All behavioral assays were conducted by an investigator blinded to the genotype and treatment condition. Mice were habituated to the behavioral testing room and investigator prior to the behavioral testing. All testing equipment were cleaned with Virkon and 70% ethanol between animals.
Rotarod was used to assess motor learning and coordination. To acclimate them to the Rotarod apparatus, mice were trained on 3 consecutive days for 5 min at constant speed of 10 rpm. For the testing, the speed was gradually increased from 4 to 40 rpm over the course of 5 min. Latency to fall was measured for 3 trials/day on 3 consecutive days. The rod and chambers were wiped with 70% ethanol between trials.
Pole test was used to measure motor coordination and function. Mice were placed on a vertical pole and the latency to descend was measured for 3 trials.
Wire hang test was used to test motor function and deficit. Mice were placed on a grid that was then inverted. The latency to fall was recorded for two consecutive trials.
Open field was used to measure spontaneous activity, locomotion, and anxiety. Animals are placed in the square open field arena (76 cm × 76 cm × 50 cm) and allowed to freely move while being recorded by the automated tracking system (Ethovision) for up to 10 min. Distance travelled, number of entries and time spent in the center and periphery were automatically assessed.
Tissue processing
Mice were anesthetized with isoflurane and transcardially perfused with 0.9% saline. Brains were dissected and fixed in 4% paraformaldehyde (PFA) pH 7.4, at 4 °C for 48 h. Brains were then stored in 30% sucrose in 1× PBS at 4 °C. For unilateral injection experiments, the whole brain was fixed in PFA. For bilateral injection experiments, one hemibrain was fixed in PFA whereas the other hemibrain was sub-dissected, snap-frozen in liquid nitrogen and stored at − 80 °C for biochemical analysis. PFA-fixed brains were sectioned at 35 µm (coronal sections) with a cryo-microtome (Leica) and stored in cryoprotective medium (30% glycerol, 30% ethylene glycol) at − 20 °C.
Immunohistochemistry
Tissue processing and immunohistochemistry were performed on free-floating sections according to standard published techniques. 1:4–1:12 series coronal sections were used for all histological experiments. Sections were rinsed 3 times in TBST, pre-treated with 0.6% H2O2 and 0.1% Triton X-100 (Sigma-Aldrich) and blocked in 5% goat serum in TBST. The following primary antibodies were used: NeuN (1:1000, Millipore, Cat# MAB377), tyrosine hydroxylase (TH; 1:1000; Pel-Freez, cat# P40101-0), α-syn pSer129 (81A; 1:5000, Covance/BioLegend cat# MMS-5091); α-syn pSer129 (1:3000; Abcam cat# 51253), LRRK2 (1:1000; NeuroMAP cat# 75-253), p62 (1:1000; Proteintech cat# 18420-AP), GFAP (1:1000; Dako cat# Z0334), Iba1 (1:1000; Wako cat# 019-19741), CD68 (1:500; AbD Serotec cat# MCA1957GA), C1q (1:1000; Abcam cat# ab182451). After overnight incubation at 4 °C, the primary antibody staining was revealed using biotinylated secondary antibodies, ABC kit (Vector Laboratories) and 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich). Fluorescently labeled secondary antibodies were used for fluorescent and confocal microscopy. Sections were mounted on Superfrost Plus slides (Fisher Scientific) and coverslipped using Entellan (for DAB staining) or ProlongDiamond antifiade mountant (for fluorescent immunostaining). Imaging and unbiased stereological estimations of TH and pSer129-positive cells were performed by investigators blinded to genotype and experimental conditions. Per animal, 8–10 SNpc sections were used for TH-neuron analysis. 3–5 sections per animal and brain region were used for all other immunostainings.
Thioflavin S staining
Sections were rinsed three times in PBS, mounted on Superfrost Plus slides and dried overnight. The slides with the brain sections were then incubated in 0.1% thioflavin S solution in 20% ethanol for 30 min at room temperature. The samples were rinsed twice with 20% ethanol for 2 min, followed by 2 washes in water. Following the tioflavin S staining, sections were labeled for pSer129 using standard immunohistochemistry techniques and mounted using Vectashield without DAPI (Vector Labs cat# H-1000). Fluorescence images were captured using a ZEISS LSM 800.
Western blotting
Mouse brain tissue was subdissected, snap-frozen and stored at − 80 °C until further use. In vitro, iPS cells and neurons were washed with ice-cold PBS prior to lysis. Cells or tissue was lysed on ice in 1× RIPA lysis buffer or sequentially in 1% Triton X followed by 2% SDS lysis buffer supplemented with 1× Halt protease and phosphatase Inhibitor Cocktail (Thermo Fisher Scientific cat# 78429, 78426) [59]. Crude RIPA lysates were centrifuged at 15,000 g for 10 min at 4 °C to remove cellular debris. Triton X-lysates were centrifuged at approximately 15,000 g for 10 min at 4 °C. The pellet was resuspended and sonicated in 2% SDS lysis buffer. Clarified lysates were quantified with a Pierce BCA protein assay (Thermo Fisher Scientific cat# 23225). Cell lysates were mixed with 4× NuPage LDS loading buffer or 4× Laemmli buffer and equal amounts of protein were subjected to SDS–PAGE, transferred to PVDF or nitrocellulose membranes. For the detection of total endogenously expressed α-syn (Novus Biologicals cat# NBP-92694) and α-syn pSer129 (Abcam cat# 51253), PVDF membranes were fixed with 0.4% PFA for 30 min at room temperature prior to the blocking step [37]. Membranes were blocked with 5% milk or 5% BSA in tris-buffered saline with 0.1% Tween (TBST) and immunoblotted according to standard protocols. The following additional antibodies were used: ACTB (clone C4; 1:5000; Millipore cat#), GAPDH (1:5,000, Sigma cat# G8795), total soluble α-synuclein (1:1000, Novus Biologicals cat# NBP-92694), α-synuclein pSer129 (Abcam cat# 51253, 1:5000), LRRK2 (1:1000, Abcam cat# ab133474), GBA (1:1000; Novus Biologicals cat# NBP1-32271), Synaptophysin 1 (SYSY cat# 101 002), PSD95 (1:5000; Abcam cat# ab18258), Homer 1 (SYSY cat# 160 003). Following incubation at 4 °C overnight, horseradish peroxidase-conjugated secondary antibodies and an ECL kit (GE Healthcare/Amersham Pharmacia Biotech) were used to detect protein signals. Multiple exposures were taken to select images within the dynamic range of the film (GE Healthcare Amersham Hyperfilm ECL). Protein bands were quantified using Fiji software (NIH). Actin or GAPDH bands were used for normalization.
RT-qPCR
RNA was isolated from brain tissue and cell pellets using TRIZol reagent (Thermo Fisher Scientific, Cat# 15596026) and PureLink™ RNA Mini Kit following the manufacturer’s instructions. The RNA concentration was determined via Nanodrop and RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Cat# 4368813). Real time PCR was performed on a Applied Biosystems StepOnePlus Real-Time PCR instrument using 2× TaqMan Universal Master Mix (Cat# 4440040) and gene-specific TaqMan probes against Snca (mouse, Mm01188700_m1), Lrrk2 (mouse, Mm00481934_m1), ActB (Mm02619580_g1), Ifna1 (mouse, Mm03030145_gH), Ifnb1 (mouse, Mm00439552_s1), C3 (mouse, Mm01232779_m1), C1qa (mouse, Mm00432142_m1), Il6 (mouse, Mm00446190_m1), Il1b (mouse, Mm00434228_m1), Tgfb1 (mouse, Mm01178820_m1), Il10 (mouse, Mm01288386_m1), Tnf (mouse, Mm00443258_m1), Trem2 (mouse, Mm04209424_g1), Vim (mouse, Mm01333430_m1), Cxcl10 (mouse, Mm00445235_m1), Cd44 (mouse, Mm01277161_m1), Gfap (mouse, Mm01253033_m1), SNCA (human, Hs01103383_m1), LRRK2 (human, Hs01115057_m1), GRIA1 (human, Hs00181348_m1), MAPT (human, Hs00902194_m1), MAP2 (human, Hs00258900_m1), SNAP25 (human, Hs00938957_m1). ActB was used for normalization. Each sample and primer set was run in triplicates and relative expression levels were calculated using the ΔΔCt method.
PFF preparation
The expression and purification of mouse or human wild-type α-syn was performed as previously described [24]. α-Syn fibril formation was induced by incubation in 50 mM Tris–HCl, pH 7.5, 150 mM KCl buffer at 37 °C under continuous shaking in an Eppendorf Thermomixer at 600 rpm. α-Syn fibrils were centrifuged twice at 15,000 g for 10 min and resuspended in PBS. For in vitro uptake and transport experiments, the fibrils were labeled with Alexa-488 or Alexa-555 (Life Technology, # A-20009) NHS fluorophore following the manufacturer’s instructions using a protein:dye ratio of 1:2. The labeling reactions were arrested by addition of 1 mM Tris pH 7.5. Free fluorophores were removed by two cycles or centrifugations at 15,000 g for 10 min and resuspension of the pelleted fibrils in PBS. All fibrils were fragmented prior to in vivo or in vitro use by sonication for 20 min in 2-mL Eppendorf tubes in a Vial Tweeter powered by an ultrasonic processor UIS250v (250 W, 2.4 kHz; Hielscher Ultrasonic, Teltow, Germany). For human α-syn PFFs, 6.5 µg/mL (0.5 µM) was used for aggregation experiments and 13 µg/mL (1 µM) was used for internalization experiments with human-induced neurons. For mouse α-syn PFFs, 5 µg of fibrils/mouse was used for in vivo mouse experiments. 2.6 µg/mL (0.2 µM) was used for aggregation experiments and 6.5 µg/mL (0.5 µM) was used for internalization experiments in primary mouse neurons.
Virus production
HEK293T (ATCC) cells were cultured under standard conditions (DMEM, 10% FBS, penicillin–streptomycin) and used to package lentiviral particles according to standard protocols with third-generation packaging plasmids (pMDlg-pRRE, pRSV-ReV, pMD2.0; Addgene). Lentiviral backbone and packaging plasmids were transfected into HEK293T cells using Lipofectamine 2000 and Lentivirus-containing medium was harvested after 48 h. Media was centrifuged at 300 g for 5 min to remove cellular debris, and concentrated using ultracentrifugation (26,000 rpm/2 h/4 °C) or Lenti-X concentrator (Clontech) before being added to cell cultures. ShRNAs were expressed from H1 promoters (pGreenPuro backbone, SystemBio), gRNAs were expressed from U6 promoters. For overexpression experiments, ORFs from the human ORFeome collection were cloned into the pLEX307 destination plasmid (Addgene cat# 41392) using the standard LR clonase protocol (Thermo Fischer Scientific).
Primary neuron cultures
Primary mouse hippocampal and cortical neurons were dissociated into single-cell suspensions from E16.5 mouse brains with a papain dissociation system (Worthington, Cat# LK003153). Neurons were seeded onto poly-L-lysine-coated plates [0.1% (wt/vol)] and grown in Neurobasal medium (Gibco) supplemented with B-27 serum-free supplement (Gibco), GlutaMAX, and penicillin–streptomycin (Gibco) in a humidified incubator at 37 °C, with 5% CO2. Half-media changes were performed every 4–5 days, or as required. Neurons were plated on 12 mm glass coverslips (Carolina Biological Supplies cat# 633009) in 24-well plates (100,000 cells/well), in 12-well plates for biochemical studies (500,000 cells/well), or in 96-well plates for automated imaging and luminescence-based survival assays (25,000–35,000 cells/well). For the targeted shRNA-based screens, neurons were grown in clear bottom, black wall plates (Sigma cat# CLS3340-50EA), transduced on 1–3 day in vitro (DIV). For gene-knock out experiments in Cas9 cortical neurons, lentiviruses encoding sgRNAs expressed from a U6 promoter were transduced on 1 DIV. Sonicated mouse PFFs were added to neurons on 10 DIV. 2.6 µg/mL PFFs was used for shRNA-based screens and 6.5 µg/mL for internalization experiments. Neurons were fixed or lysed on 17-21 DIV.
ES/iPS and iN culture conditions
ES and iPS cells were cultured using feeder-free conditions on Matrigel (Fisher Scientific cat# CB-40230) using mTeSR1 media (Stemcell Technologies, cat# 85850). ROCK inhibitor Y-27632 was added to the culture media during passaging. Cells were transduced with a Tet-On inducible system allowing for the expression of the transcription factor Ngn2 upon addition of doxycycline to the media. Cells were differentiated as previously described in DMEM/F12 media supplemented with 1× N2 (ThermoFisherScientific) and 1 ug/mL doxycycline [66]. Cells were dissociated on day 3–5 of differentiation and replated on poly-L-lysine-coated tissue culture plates, glass coverslips or microfluidics chambers (Xona microfluidics SND450). iNs were transitioned to Neurobasal media with 1× B27 and doxycycline starting on day 6. Half-media changes were performed every 3–4 days, or as required.
Internalization of labelled alpha-synuclein fibrils
The internalization experiments were performed as previously described [10]. Primary mouse neurons or induced neurons were grown in 8-well Lab Tek II chamber slides (ThermoFisherScientific cat# 155409). For induced human neurons, sonicated Alexa-488-labeled α-synuclein fibrils were added at a final concentration of 13 µg/mL (1 μM) after 14–21 days of differentiation. For primary neurons, labeled mouse fibrils were added at a final concentration of 6.5 µg/mL (0.5 μM). Internalization was measured at 1, 2, 4, 6, 24 h after the addition of fibrils to the culture media. At each time point, the medium was removed, the cells were washed gently 2–3 times with PBS. 0.1% Trypan blue (ThermoFischerScientific, T10282) was added to each well to quench the non-internalized fluorescent signal and the cells were imaged using a Leica DMI6000B inverted fluorescence microscope with a 20× objective. Control wells without fibrils were used to measure the background fluorescence. 20 images were acquired per well and the percentage of neurons containing fluorescent puncta as well as the average intensity per neuron was measured using ImageJ. More than 100 neurons were assessed for each well.
Immunocytochemistry
Cells were grown on poly-L-lysine-coated glass coverslips [0.1% (wt/vol)] in standard multi-well cell culture plates and were stained through standard immunocytochemistry techniques. Briefly, cells were fixed with 4% formaldehyde for 15 min at room temperature, rinsed with PBS, permeabilized with 0.1% Triton X-100, blocked with 5% normal goat serum in PBS. The following primary antibodies were used for labeling: NeuN (1:1000, Millipore cat# MAB377), α-syn pSer129 (1:5000; Covance cat# MMS-5091), α-syn pSer129 (1:3000, Abcam ab51253), α-syn (1:1000, Abcam cat# ab138501), p62 (1:1000; Proteintech cat# 18420-AP), ubiquitin (1:500, Millipore cat# MAB1510), Map2 (1:1000; SYSY cat# 188 004), Map2A/B (1:1000; Millipore cat# MAB378), βIII-tubulin (1:1000; SYSY cat# 303 304). After overnight primary antibody incubation, cells were rinsed 3× with PBS. Cells were incubated with fluorescently labeled secondary antibodies for 1 h at room temperature. Coverslips were mounted with Prolong Diamond Antifade mountant with DAPI (Thermo Fisher Scientific). Images were acquired with a Leica DMI6000B inverted fluorescence microscope and a confocal Zeiss LSM710 microscope. Automated imaging and analysis were performed with the IncuCyte life cell analysis imaging system and Molecular Devices ImageXpress Micro and MetaXpress 5. The percentage area of MAP2, βIII-tubulin and pSer129 immunofluorescence staining was quantified in ImageJ or MetaXpress 5. The number of NeuN and DAPI labeled-cells was counted using MetaXpress 5 and the analyze particle function in Fiji. The experimentors were blinded to the treatment conditions. For the targeted shRNA-based screen, neurons were grown in clear bottom, black wall plates optimized for automated imaging. Images were acquired in a 4 × 3 grid pattern for each well (12–24 wells/shRNA/screen). The area covered by pSer129 and the number of NeuN-positive nuclei were measured for each image using MetaXpress. Scramble shRNA and untreated control wells were included on each plate. The pSer129 aggregation score was calculated using the pSer129 intensity and area and normalized to the NeuN counts. Scramble controls were included in all plates and used as comparison between plates.
Statistics
Statistical tests were performed with GraphPad Prism 7. Differences between treatment conditions were established using a unpaired Student’s t test (for two conditions). For experiments with > 2 groups, one-way ANOVA with a Tukey’s post-test for multiple comparisons or two-way ANOVA with Bonferroni correction was performed. Pearson r was calculated for correlation analysis. p < 0.05 was considered statistically significant. Statistic details are indicated in the respective figure legends.