Addition of exogenous α-Synuclein Pre-formed fibrils to Primary Neuronal Cultures to seed recruitment of endogenous α-Synuclein to Lewy body and Lewy Neurite-like aggregates (original) (raw)

. Author manuscript; available in PMC: 2015 Mar 25.

Published in final edited form as: Nat Protoc. 2014 Aug 14;9(9):2135–2146. doi: 10.1038/nprot.2014.143

Abstract

This protocol describes a primary neuronal model of formation of α-synuclein (α-syn) aggregates that recapitulate features of Lewy Bodies and Lewy Neurites found in Parkinson’s disease brains and other synucleinopathies. This model allows investigation of aggregate formation, their impact on neuron function, and development of therapeutics. Addition of pre-formed fibrils (PFFs) synthesized from recombinant α-syn to neurons seeds recruitment of endogenous α-syn into aggregates characterized by detergent-insolubility, and hyperphosphorylation. Aggregate formation follows a lag phase of 2–3 days, followed by formation in axons by days 4–7, spread to somatodendritic compartments by days 7–10, and neuron death around 14 days post-PFF. Here, we provide methods and highlight critical steps for PFF formation, addition to cultured hippocampal neurons, and confirmation of aggregate formation. Neurons derived from various brain regions from non-transgenic and genetically-engineered mice and rats can be used, allowing interrogation of the impact of specific genes on aggregate formation.

Introduction

Aggregates of α-syn called Lewy Bodies and Lewy neurites are a defining feature of synucleinopathies, including Parkinson’s disease, Dementia with Lewy Bodies, and the Lewy Body variant of Alzheimer’s disease. Many questions including how these aggregates form and their contribution to the etiology of PD remain to be answered. For example, early formation of these aggregates and sequestration of normal α-syn from the presynaptic terminal may contribute to neuronal dysfunction well before neurodegeneration, contributing to the prodromal phase of the disease. In addition, it is unknown whether these aggregates sequester numerous soluble toxic species providing neuroprotection, at least in the short term1–3, or represent a toxic and transmissible species, with their formation and spread directly responsible for the neurodegenerative phenotypes. Understanding how these aggregates form and their impact on neuronal function could contribute to the development of therapeutic targets to prevent the progression of these devastating neurodegenerative diseases4–7.

Modelling aggregation of α-syn

A major obstacle to elucidating the role of α-syn pathology has been the lack of model systems to study the acute effects of α-syn aggregation occurring in real-time, especially in individual neurons. First, although aggregated α-syn, as detected by either amyloid dye staining (e.g. Thioflavin S) or resistance to protease digestion, has been reported in a variety of cell lines which overexpress α-syn, few show the characteristic amyloid fibril ultrastructure seen in human Lewy pathology. In addition, transgenic mice that overexpress disease-associated mutant α-syn produce pathologic aggregates with these features, but only in mice several months of age, and aggregate formation coincides with the rapid demise of the animal, making it impossible to understand the early effects of α-syn aggregation8–10. Finally, primary neuronal cultures from these mice also do not form aggregates that recapitulate features of those found in diseased brains. By contrast, the primary neuron model described herein, combines most of the features of inclusions found in diseased brains as well as key biochemical/molecular markers of Lewy Neurites and Lewy Bodies in a genetically unmodified neuronal culture system. The defining features of these aggregates, both in diseased brains and in our model system, are that they are: insoluble in detergent, hyperphosphorylated, ubiquitinated, and filamentous ultrastructure when examined by electron microscopy11–14.

Seeded formation of aggregates from α-syn endogenously expressed in primary neurons

The development of our model was based upon previous studies that utilized cell lines overexpressing mutant forms of disease-associated proteins to demonstrate that exogenous amyloid fibrils can seed transition of soluble proteins into inclusions15–18. In this model, small seeds of pre-formed α-syn fibrils (PFFs) generated from recombinant α-syn are added directly to primary neurons14. Small amounts of these PFFs are endocytosed by the neuron, without the addition of other factors to assist entry into the neuron. These seeds of PFFs induce recruitment of endogenously expressed α-syn into abnormal, phosphorylated, insoluble, ubiquitinated aggregates. Formation of these aggregates from endogenous α-syn in primary neurons derived from wild type, non-transgenic mice follows an initial lag phase of 2–4 days. By 4–7 days small, punctate insoluble, phosphorylated aggregates from in presynaptic terminals and axons. By 7–10 days post-PFF, the aggregates grow and become more elongated and serpentine in appearance, resembling Lewy Neurites. They also can be found in approximately 30% of neuronal soma and dendrites where they appear skein-like, but over time form condensed accumulations that resemble Lewy Bodies. Neuron death is negligible prior to 14 days after adding the PFFs. This permits the careful examination of α-syn aggregates from their initial early formation, to spread throughout the neuron, and ultimately neuron death, as well as how neuronal function may be perturbed at each of these stages. For example, calcium imaging experiments reveal major defects in neuronal synchronization at early time points when only small aggregates are present in axons14. In addition, one of the most predominant phenotypes is the increased expression of canonical autophagy markers19. However, although mature α-syn aggregates associate with components of autophagy machinery, they cannot be efficiently degraded by this pathway. Furthermore, mitochondrial oxidant stress contributes to PD pathogeneses and can be measured in dopaminergic neurons from the substantia nigra pars compacta by expression of mitochondrially targeted redox-sensitive green fluorescent protein (TH-mito-roGFP) expressed under control of a tyrosine hydroxylase promoter20. The use of live cell imaging of dopamine primary neurons derived from mice expressing TH-mito-roGFP, demonstrates that the presence of α-syn aggregates significantly increases mitochondrial oxidant stress, particularly in the soma and dendrites. Interestingly, the mitochondrial oxidant stress likely derives from increased lysosomal oxidase activity.

A critical aspect of this model is that none of the phenotypes described above occur when PFFs are added to primary neurons from α-syn knockout mice. For example,, the PFFs themselves are not phosphorylated, and therefore when they are added to α-syn knockout neurons, there is no p-α-syn visible by immunofluorescence or immunoblot14. Furthermore, addition of fibrils to neurons from α-syn knockout mice does not cause cell death, or changes in neuronal synchronous firing, excitation or connectivity. Thus, the pathological phenotypes are caused by “seeded” corruption of endogenous α-syn likely through both a loss of normal α-syn function and gains of toxic functions from the accumulation of the Lewy Neurite and Lewy Body-like inclusions, rather than exposure to the synthetic fibrils themselves.

Overall, this model provides researchers with the opportunity to understand how the presence of neuronal α-syn aggregates contributes to disease pathogenesis at cellular and subcellular level (Figure 1) with well-defined temporal and spatial resolution.

Figure 1.

Figure 1

Timeline of known events in PFF model of α-syn aggregation.

Applications of the Protocol

There are many other advantages afforded by this model in addition to those mentioned above. First, α-syn aggregation occurs in normal wild type neurons expressing endogenous α-syn, i.e. it does not require overexpression of wildtype or mutant α-syn14. Thus, researchers can potentially understand the impact of any gene of interest on α-syn aggregate formation utilizing knockout mice or transgenic mice. Second, α-syn aggregation occurs in primary neurons derived from hippocampus, cortex, midbrain and other brain regions and thus the impact of these aggregates on diverse neuronal populations and neuronal subtypes could be examined14, 20, 21. In addition, it provides spatial resolution to allow high resolution microscopy and/or live cell imaging, and thus future studies can elucidate of the cell biological and physiologic impact of α-syn aggregate formation. For example, transfection of fluorescent probes of localization and function of distinct organelles can be accomplished using various published protocols22–24. Furthermore, this method can potentially be used in conjunction with established methods in which neurons are compartmentalized (e.g. in microfluidic or Campenot chambers) to understand how α-syn aggregates may spread from neuron to neuron, which has recently been shown to contribute to disease progression25,26. Finally, another future potential application is the screening of putative therapeutics to prevent the formation of these aggregates.

Limitations of the protocol

In cultures from non-transgenic mice, there is a 2–4 day lag phase before insoluble, phosphorylated aggregates appear. Thus, any compounds tested during this lag phase using this model must not be detrimental to neuron health when used over this time course. Although, it may be possible to reduce the concentrations of test compounds such that they remain effective but do not lead to neuron death. Furthermore, this lag phase can be reduced by overexpression of α-syn.

Experimental design

Primary neuron culture

While we provide some information here regarding culturing of primary neurons, these methods have been presented in detail elsewhere and we reference these protocols for full details23, 27, 28. Other labs have reported strong PFF-seeding using their own culturing methods20, 21. Recommended plating densities are provided for culturing neurons as we have found that the efficiency of aggregate formation depends on synaptic densities; the higher the density of the cultures, the more aggregates are produced. The procedure describes culture of hippocampal neurons from 1 pregnant C57BL/6 mouse with approximately 6–8 E16–E17 embryos. We generally perform experiments using hippocampal neurons from non-transgenic mice because: α-syn aggregates are found in the hippocampus in diseased brains and correlate with cognitive impairments29–31 ; the morphological and physiological properties of these neurons in culture have been well defined32; and we can obtain enough neurons for both imaging and biochemical studies. Seeded α-syn inclusions also develop in primary neurons from cortex and tyrosine hydroxylase-positive neurons cultured midbrain14, 20. However, the presence of inclusions may depend on the subtype of neuron, for example, GAD-positive inhibitory neurons do not form inclusions because of low to no expression of α-syn in these neurons21.

PFFs

Storage of the PFFs is another crucial factor in the effectiveness of the protocol. Once generated, we recommend dividing the PFFs into aliquots and storing them at −80°C because we and others33 have observed that storage at 4°C results in loss of activity. Sonication with a probe tip sonicator is another critical step in this protocol to generate small seeds (Video 1, Figure 2). We recommend adding PFFs at DIV 5–10 because α-syn is only expressed at mature synapses34. PFFs can also be added at later time points and pathology will proceed more quickly because higher levels of α-syn are expressed at presynaptic terminals. However, because neurons in culture typically only last approximately 21–31 days in culture in general, we do not recommend adding PFFs at too late a time point because the neurons (including control neurons) may begin to die before the aggregates spread throughout the neuron, thus adding a confounding factor. Also, we provide our recommended concentrations of PFFs to add to neurons. However, we suggest testing a range of concentrations to determine which concentration is best for each researcher’s particular assay.

Figure 2.

Figure 2

Electron micrographs of PFFs before (A) and after (B) sonication. Scale bars= 500 nm42.

Choice of controls

Here, we describe using PBS as a control. Other controls may include treatment of the neurons with α-syn monomer which does not produce α-syn aggregates. However, given the propensity for α-syn to aggregate, it is important to centrifuge the monomer after thawing (benchtop ultracentrifuge, 100,000 × g, 1hr) to remove any small fibrils that may have formed, and use only the resulting supernatant. As mentioned above, cultures from α-syn knockout mice can be used as a control to confirm that phenotypes result from aggregates formed from endogenously expressed α-syn and not from addition of PFFs.

MATERIALS

Reagents

Box 1: Purification of α-syn.

The methods for purifying α-syn have been published in detail in several publications35–39. Here, we describe our protocol for purifying human full length α-syn (NM_000345) or mouse full length α-syn (NM_001042451). The final goal is to obtain a high concentration of untagged α-synuclein in a final buffer composed of Tris and NaCl.

Expression in e.coli
Cell lysis and preclearing
Purification

Equipment

Reagent setup

PROCEDURE

CAUTION: Appropriate personal protective equipment should be used at all times. WEAR GLOVES, LAB COAT, FACE MASK (such as: VWR, 414004-670), AND PROTECTIVE GOGGLES and PERFORM SONICATION IN A CLASS II BIOSAFETY CABINET TO AVOID INHALING OR GETTING AEROSOLIZED PFFS IN EYES (Video 1). Although studies have not performed on the safety of handling of PFFs, we recommend referring to the prion literature for the utmost safety precautions40, 41. Decontaminate PFFs or any spills with a final concentration of 1 N NaOH or 20,000 ppm bleach (two parts 5.25% household bleach to three parts water) for 1 hour. Make NaOH or bleach solutions fresh daily. Use disposable, absorbent pads whenever possible.

Preparation of PFFs

Timing: 7 days

Sedimentation assay for confirmation of pelletable, active PFFs

Figure 3. Typical results seen at step 12 following the sedimentation assay of PFFs.

Figure 3

PFFs were prepared as described in steps 1–12 and the supernatant and pellet resolved by SDS-PAGE and stained with Coomassie Blue. This batch of PFFs had approximately equivalent amounts of α-syn in the supernatant and pellet was efficient at seeding aggregates from endogenous α-syn in primary neurons.

Culturing neurons

Timing: About 8 hours

Adding PFFs to neurons

Timing: about 60 min

CRITICAL Perform 5–10 days after plating neurons

Confirmation of PFF transduction and seeding

Takes 2–3 days. CRITICAL Perform 7–23 days after transduction

PFF transduction and seeding can be confirmed by immunofluorescence (option A) or sequential extraction and immunoblotting (option B). Abnormal α-syn derived from endogenous α-syn can be detected via immunofluorescence with a p-α-syn specific antibody. These phosphorylated inclusions will not be visible when PFFs are added to α-syn knockout neurons (Figure 4). Alternatively, the neurons can be fixed with 4%paraformaldehyde/4%sucrose/1% Tx-100 and stained with an antibody to total α-syn. The normal, “synaptic” α-syn will be extracted and will not be visible, but the PFF-induced α-syn inclusions will not be extracted and will be visible by immunofluorescence (Figure 5).To distinguish exogenously added human PFFs from inclusions formed from endogenous α-syn, neurons can be co-stained using an antibody that is specific for human α-syn (LB509 or Syn204; both raised in mouse) and an antibody for p-α-syn (MJFR1, raised in rabbit) (Figure 6).

Figure 4. Visualization of PFF-induced formation of α-syn aggregates using an antibody to p-α-syn.

Figure 4

A. Fourteen days following the addition of PFFs to nontransgenic primary neurons, p-α-syn inclusions, visualized by immunofluorescence using mAB81A, were abundant throughout neurites and soma (top panels)14. These inclusions were also insoluble as determined by fixation in paraformaldehyde/4% sucrose/1% Tx (top, right). p-α-Syn inclusions were not apparent in PBS or PFF treated (14 days post-treatment) in primary neurons from α-syn knockout mice (bottom panels). Scale bar = 50 µm. B. PFFs (top panels) or monomeric, non-fibrillar α-syn (bottom panels) were added to primary neurons. Immunofluorescent imaging was performed using antibodies MJFR1, which recognizes p-α-syn, and NeuN, to visualize neuronal soma. Inclusions positive for p-α-syn (green) were abundant in PFF-treated neurons, but not neurons treated with soluble, monomeric α-syn. Scale bar = 20 µm.

Figure 5. Immunofluorescence of α -syn aggregates.

Figure 5

A. Neurons were fixed in 4% paraformaldehyde/4% sucrose and immunofluorescence was performed using an antibody to total α-syn (Syn1) and an antibody which recognizes the presynaptic marker, VAMP2

In control PBS-treated neurons, α-syn colocalized extensively with VAMP2 at presynaptic terminals. Fourteen days following PFF treatment, α-syn no longer localized to the presynaptic terminal, but was found in longer serpentine aggregates in axons and skein-like filaments in the soma. Scale bar = 10 µm. B. Neurons were fixed in 4% paraformaldehyde/4% sucrose/1% Tx-100 and immunofluorescence was performed using an antibody to total α-syn and an antibody which recognizes the neuron specific marker, NeuN. In control neurons, α-syn was completely extractable but in PFF treated neurons (here, 10 days post), α-syn was insoluble and thus visible throughout the culture. Scale bar = 50 µm. C. 4% paraformaldehyde/4% sucrose/1% Tx-100 and co-stained using an antibody to total α-syn and NeuN. Scale bar = 10 µm. D. Seven days following PFF treatment, neurons were fixed with 4% paraformaldehyde/4% sucrose and co-stained with a p- α-syn (red) and tau (green) antibody to label axons.Scale bar = 10 µm.

Figure 6. Visualization of exogenously added PFFs and p-α-syn aggregates formed from endogenous α-syn.

Figure 6

Neurons were fixed 14 days after treatment with PFFs and double immunofluorescence was performed using a rabbit antibody to p-α-syn generated in the Lee lab (green)14, and a mouse antibody, Syn204, that specifically recognizes the exogenously added human PFFs (red). There was minimal colocalization between the exogenous PFFs and the p-α-syn inclusions derived from endogenous α-syn. Scale bar = 50 µm.

  1. Immunofluorescence
    1. Aspirate media from coverslips (aspirate from only a few coverslips at a time)
    2. Add either 4%paraformaldehyde/4%sucrose (0.5mL for 24 well plate) or 4%paraformaldehyde/4%sucrose/1% Tx-100 (0.5mL for 24 well plate), called “Tx-100 extracted neurons”.
    3. Incubate room temperature for 15 minutes. CRITICAL STEP Unless otherwise noted, this and all subsequent steps should be performed at room temperature.
    4. Rinse 5X with 0.5 mL PBS/well
    5. Permeabilize and block neurons with 3%BSA/0.1% Tx-100 for 15 minutes.
    6. Rinse 3X with PBS
    7. Dilute antibody in blocking buffer. We prefer to perform double staining immunofluorescence using 2 antibodies such as LB509/p-α-syn (as described above) or an α-syn antibody with MAP2 or tau to help resolve the spatial relationship of inclusions within dendrites or axons, respectively, or combined with an antibody to any other marker of interest. Incubate neurons in primary antibody for 2 hours at room temperature or 4°C overnight.
    8. Rinse 5X with PBS
    9. Dilute goat anti-mouse Alexafluor conjugated secondary antibody and/or goat anti-rabbit Alexafluor conjugated in blocking buffer 1:500 (Life Technologies). (The choice of Alexafluor depends on the microscope filters or lasers, if using confocal microscopy). Incubate neurons in secondary antibody for 1 hour.
    10. Rinse 5X with PBS
    11. Mount coverslips onto glass slides with Prolong Gold mounting media. Visualize pathology with epifluorescent microscope.
  2. Sequential Extraction and Immunoblotting
    1. Rinse neurons 2X with PBS
    2. Place dish on ice. Working one well at a time, completely aspirate PBS and add following volumes of ice cold 1%Tx-100/TBS with protease and phosphatase inhibitors:
      6 well: 250 µL per well
      6 cm dish: 500 µL
    3. Use cell scraper to thoroughly scrape all neurons from well
    4. Place in polyallomar tube for table top ultracentrifuge, keep on ice
    5. Sonicate 10X, 0.5 sec pulse, power at 10%
    6. Incubate on ice for 30 minutes.
    7. Centrifuge at 100,000 × g at 4°C for 30 minutes.
    8. Add 4X Laemmli to about 150–200 µL of Tx-100 supernatant (some is usually lost during steps iii-viii, we recommend measuring remaining supernatant before determining how much to remove for laemmli buffer). Save about 20 µL of supernatant for protein assay. Retain on ice or in −20°C freezer.
    9. To the pellet, add the same volume of ice cold 1% Tx-100 with protease and phosphatase inhibitors
      6 well: 250 µL
      6 cm dish: 500 µL
    10. Sonicate 10X, 0.5 sec pulse, power 10%. Keep tip of probe toward bottom of tube to prevent frothing. Make sure that pellet is completely dispersed.
    11. Centrifuge at 100,000 × g at 4°C for 30 minutes.
    12. Discard supernatant.
    13. Add 2% SDS/TBS to pellet with protease and phosphatases inhibitors. To a 6 well plate add 125 µL. To a 6 cm dish add 250 µL.
    14. Sonicate 15X, 0.5 sec pulse, power 10%. Keep tip of probe toward bottom of tube. Make sure that pellet is completely dispersed.
    15. Remove supernatant and place into new microcentrifuge tube.
    16. Perform BCA/protein assay on Tx-100 supernatant and SDS extract. Typically a 1:5 dilution for the BCA assay is sufficient.
    17. Dilute 2% SDS extract from step Xv into Laemmli buffer to 2X volume for corresponding Tx-100 fraction (regardless of protein concentration of SDS fraction). For example, if you have 180 µL Tx-100 extract (from step viii) at 1 mg/mL and 90 µL of SDS extract, add 60 µL of 4X Laemmli to the Tx-100 extract and 30 µL of 4X Laemmli to the SDS extract. Load 12.5 µL of both the Tx-100 extract (10 µg) and SDS extract. We suggest 2X volume because it makes the insoluble α-syn species more abundant and thus easier to visualize and quantify by immunoblot.
    18. Load samples on 4–20% gel and run according to manufacturer’s directions. We use 85V constant voltage until the dye front runs off the gel. Be sure not to let the 10 kDa marker to run off the gel.
    19. Transfer following manufacturers instructions at 100 V for 75 minutes or overnight at 40V.
    20. Block membrane for 1 hour with TBS/5% milk
    21. Dilute primary antibodies in TBS/5% milk and incubate overnight at 4°C with shaking.
    22. Rinse 3X with TBS/T, 10 minutes each rinse.
    23. Incubate with HRP conjugated secondary antibodies for 1 hour at room temperature.
    24. Rinse 4X with TBS/T, 10 minutes each rinse.
    25. Develop with ECL

Anticipated results

Immunofluorescence

Significant α-syn pathology is detectable 4–10 days following addition of PFFs to primary hippocampal neurons from non-transgenic, wildtype mice using this protocol. When immunofluorescence is performed with an antibody to total α-syn, α-syn will localize to presynaptic puncta in control neurons, and to longer serpentine aggregates that do not colocalize with presynaptic markers in PFF-treated neurons (Figure 4,5). Note that some presynaptic a-syn that has not converted to aggregates will remain, especially at early time points after PFF addition. This is why we recommend also performing Tx-100 extraction. In neurons fixed with 4% paraformaldehyde/4% sucrose/1% Tx-100, there will be no staining for α-syn in the control neurons, but the aggregates will remain in Tx-100 extracted PFF-treated neurons. When immunofluorescence is performed using antibodies to p-α-syn, the control neurons will show light, diffuse staining. However the staining in the PFF-treated neurons will be substantially more intense and discrete puncta and longer serpentine and skein-like aggregates will be apparent.

Immunoblots

When probed using an antibody against total α-syn, PBS-treated control neurons will show a band slightly above the 15 kDa band corresponding to monomeric α-syn exclusively in the Tx-100 soluble fraction (Figure 7). Following 14 days treatment with PFFs, neuronal lysates will show reduced levels of α-syn in the Tx-100 soluble fraction relative to control neuronal lysates, and an appearance of α-syn in the SDS-soluble fraction. These bands typically appear as a series of bands within the SDS fraction, likely corresponding to α-syn oligomers. PBS-treated control neurons will not show p-α-syn immunoreactivity in the Tx-100 or the SDS fraction. PFF-treated neurons will show pathologic p-α-syn immunoreactivity predominately in the SDS soluble fraction that again will appear as a series of bands.

Figure 7. Typical immunoblot results seen following sequential extraction of neurons in 1% Tx-100 followed by SDS.

Figure 7

A. Immunoblotting was performed using Syn1, an antibody that recognizes total α-syn. In control neurons, α-syn was completely extractable in Tx-100. In PFF treated neurons, there was a decrease in Tx-100 soluble α-syn, and increase in SDS-soluble α-syn. B. Immunoblots of SDS extracts (Tx-100 insoluble) using mAB81a that recognizes p-α-syn. There was no p-α-syn in control neurons but a substantial amount of p-α-syn in PFF treated neurons.

Troubleshooting

Preparation of PFFs

Preparation of PFFs from recombinant, purified protein without tags is the most effective. Generating PFFs from α-syn purchased from various suppliers has not been successful in our hands, likely because many suppliers utilize a tag to assist in purification. The composition of the buffers used to purify α-syn and generate PFFs, including salt concentrations and pH, is also crucial.

Inclusion formation

The density of the neurons has been optimized for efficient inclusion formation. Using lower neuronal densities decreases the abundance of inclusions. Also, as mentioned throughout the protocol, sonication with a probe tip sonicator is critical for this protocol. Investigators who have tried bath sonicators for PFF sonication have not been as successful in generating abundant inclusions. Note that PFFs may lose activity over multiple freeze thaw cycles. Verify that PFFs were diluted and stored correctly prior to addition to neurons. In addition, our system has been optimized for primary hippocampal neurons from C57Bl/6 mice or CD1 mice. Some neuronal populations may be more or less sensitive to the effects of the PFFs and therefore it may be necessary to perform a time course and a concentration curve after PFF addition to determine the time point and concentration at which pathology is maximal.

Immunoblots

p-α-Syn can sometimes be difficult to detect by immunoblot. If this is difficult, we recommend loading as much protein as possible (≥10µg). Using BSA in the blocking buffer instead of milk may also be helpful. Using lysates from α-syn knockout mice is also recommended as a control to distinguish specific bands.

Supplementary Material

Video. Supplementary Video 1.

This video demonstrates sonication PFFs using a probe tip sonicator, a critical step for the success of this protocol.

Summary.

Lewy bodies and Lewy neurites are found in the brains of patients with Parkinson’s disease and other synucleinopathies. This protocol describes how to induce α-synuclein aggregates in a primary neuronal culture system, creating a cellular model of these synucleinopathies.

Acknowledgements

We would like to thank Andrew B. West for allowing us to create of video of him demonstrating the sonication of the fibrils. We also thank the reviewers whose careful attention to details and suggestions greatly improved this protocol. This study was supported by NIH grant, P50 NS053488 to V.M.-Y.L.

Footnotes

Author Contributions: L.V.D. carried out the experiments that formed the basis of the protocol. K.C.L. provided the electron microscopy images of the sonicated fibrils. V.M.-Y.L. supervised the project. L.V.D., K.L. and V.M.-Y.L. provided intellectual input that contributed to the development of the protocol. L.V.D. wrote the paper, and K.L. and V.M.-Y.L. provided valuable editorial input.

Competing Financial Interests: The authors report no competing financial interests

Contributor Information

Kelvin C. Luk, Email: kelvincl@mail.med.upenn.edu.

Virginia M.-Y. Lee, Email: vmylee@upenn.edu.

References

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Video. Supplementary Video 1.

This video demonstrates sonication PFFs using a probe tip sonicator, a critical step for the success of this protocol.