The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.
Use of the in vivo alpha-synuclein preformed fibril (α-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson's disease synucleinopathy and nigrostriatal degeneration. The standardization of α-syn PFF generation and in vivo application is critical in order to ensure consistent, robust α-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric α-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of α-syn PFFs into rats or mice. Starting with monomeric α-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed α-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated α-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.
Parkinson's disease (PD) is primarily characterized postmortem by two major pathological features: widespread and progressive alpha-synuclein (α-syn) pathology, and nigrostriatal degeneration. Following injection into wildtype mice or rats, α-syn preformed fibrils (PFFs) induce progressive accumulation of pathological α-syn, which can result in protracted degeneration of substantia nigra pars compacta (SNpc) dopamine neurons over the course of many months, as well as sensorimotor deficits1,2,3,4,5,6. Neurons are exposed to α-syn fibrils, either via direct intracerebral injection or added to the media of cultured neurons. When the PFFs are taken into the neurons, the PFFs act to "seed" the formation of inclusions through templating, and accumulation of endogenous α-syn into phosphorylated inclusions1,7,8,9. Inclusions share similar properties to Lewy bodies: containing α-syn phosphorylated at serine 129 (pSyn), ubiquitin, and p62; possess amyloid quaternary structures as shown with positive thioflavin staining; and are resistant to proteinase K digestion1,3,5,7,8,9,10,11,12. PFF exposure leads to α-syn inclusion formation in primary and some immortalized neurons in culture, as well as mice, rats, and non-human primates in vivo1,2,3,4,5,6,7,8,9,13. It is important to note that PFFs will not lead to α-syn inclusion formation in all cell culture models and some cultured neurons will seed better than others.
Another important feature of the in vivo α-syn PFF model is the distinct sequential pathological phases that emerge over several months. In rodents, following intrastriatal injection, α-syn inclusion formation generally peaks within the SNpc and many cortical regions within 1-2 months. This aggregation peak is followed by nigrostriatal degeneration ≈2-4 months later1,3,5. These distinct pathological stages provide researchers the platform with which to study and develop strategies that 1) decrease α-syn aggregation, 2) clear already formed α-syn inclusions, and/or 3) prevent subsequent neurodegeneration. The PFF model offers distinct advantages and disadvantages as compared to neurotoxicant, transgenic, and viral vector mediated α-syn overexpression models as previously reviewed6. The choice of which model or approach to take should be determined by which model best suits the question the investigators are asking.
Although the PFF model has been successfully utilized by many labs, there are still groups that have experienced inconsistencies with generating fibrils and producing consistent α-syn pathology14. Examples of inconsistencies range from PFFs that produce little or no α-syn pathology, batch to batch seeding efficiency, and even the failure of fibrils to form. Thus, the standardization of α-syn PFF generation and in vivo application is critical in order to allow for accurate interpretations regarding the impact of novel therapeutic interventions. The following protocol outlines the steps required for the generation of PFFs from α-syn monomers, the in vitro quality control of the PFFs once formed, the sonication and measurement of PFFs prior to use, and suggestions to facilitate successful in vivo injection of PFFs into rats or mice.
Production of α-syn PFFs capable of seeding neurons and leading to Lewy body-like inclusions is dependent on multiple factors and steps. A critical factor is that the monomers used for generating fibrils need to be specifically formulated for fibrilization4,9,14,15. If the monomers are not formulated for fibrilization, fibrils may not form or the fibrils that do form may not produce α-syn pathology. Likewise, the buffer that the monomers are in also influence fibrilization. As such, for the best results, the salt concentration should be approximately 100 mM NaCl and pH between 7.0 and 7.3. An initial step that introduces variability is the method whereby initial protein content is determined, with measurement at A280 likely to produce more accurate results and therefore is the preferred method. The discrepancy in protein concentration can decrease the efficacy of the fibrilization process, as well as alter the assumed PFF concentration used in experiments. Both could lead to a decrease in seeding efficiency and batch variation between experiments.
Initial quality control steps will confirm critical features of the PFFs, specifically that they have a fibrillary conformation (electron microscopy), contain amyloid structures (thioflavin T assay), and are pelletable (sedimentation assay). It is important to note that the results of the thioflavin T assay will fluctuate with time and are not a direct measure of the amount of amyloid structures present, rather, the thioflavin T assay should be used only as an indicator of amyloid structures presence within the sample. Thioflavin T is typically used in in vitro assays, such as the aforementioned assay to show the fibrils contain amyloid structures. Alternatively, thioflavin S is used in tissue to detect amyloid structures, as shown in Figure 7. In regards to the sedimentation assay, the results show only that the PFFs are found predominantly in the pelletable fraction. As the samples are run in denaturing conditions, a single prominent band of approximately 14 kDa, the size of α-syn monomers, is present on the gels. This is unlike the multiple bands present at higher molecular weights that would be expected with PFFs if a native or non-denaturing gel was used. Lastly, successful passing of all these initial quality control steps does not guarantee α-syn inclusion seeding activity. For this reason, cell culture experiments or a small cohort of surgically-injected animals should be used to test the efficacy of the PFFs before use in larger experiments.
Sonication is a crucial step in the process and parameters will differ depending on the model of sonicator used. Sonication parameters must be applied and verified to show that short PFF fragments have been produced. Fibril size has bearing on the seeding, with shorter fibrils seeding more efficiently. Though shorter fibrils seed more efficiently, this effect plateaus and the optimal PFF length is approximately 50 nm4,18. It is also important not to over-sonicate the samples and expose the PFFs to excessive heat, as this may decrease seeding efficiency. These sonicated PFFs should be tested for efficacy in small cell culture or in vivo experiments prior to use in larger scale experiments. As different sonication sessions have the potential to introduce variability, experimental treatment groups should be planned accordingly.
When delivering PFFs in vivo, the localization of the injection site(s) and the total amount PFFs used can affect the number of neurons that will develop inclusions as well as the extent of neurodegeneration7,14. The coordinates in the protocol provide a place to start, but should be tested within the lab to ensure the desired target region develops α-syn pathology prior to use in large scale experiments. If desired, tracking dye or fluorescent beads can be used as a way to test regional targeting before using PFFs. The amount of PFFs used in vivo varies between groups, with most groups using a total between 5 to 20 µg of PFFs at one or divided between two injection sites1,2,3,4,5,6,7,14. As the number of injection sites, location of injection sites, and amount of PFFs injected can effect results and progression of the synucleinopathy, the downstream outcomes of the parameters used should be characterized prior to using the model to test potential interventions or examining temporal features of the model.
When selecting a model to use for testing therapeutics or studying disease progression, the model used should be selected to best answer the question asked. Not all models will possess certain disease features of PD, or offer the timeframe needed to test potential interventions. The PFF model recapitulates key features of PD, such as α-syn pathology and neurodegeneration, and can lead to modest motor impairments. The model offers a predictable and protracted time-course, where inclusions form months before neurodegeneration. This allows researchers to examine and exploit the different phases throughout the protracted progression of the synucleinopathy. The current and future use of the model overall is expected to be beneficial in the study of disease progression and development of novel therapies.
The authors have nothing to disclose.
This research was supported by grants from the Michael J. Fox Foundation, the National Institute of Neurological Disorders and Stroke (NS099416) and the Weston Brain Institute.
1x Dulbecco’s phosphate buffered saline | Thermo Fisher (Gibco) | 14190144 | |
Anti-alpha-synuclein (phosphorylated at serine 129) antibody | Abcam | AB184674 | |
Anti-alpha-synuclein antibody | Abcam | AB15530 | |
Bicinchonic acid | Thermo Fisher (Pierce) | PI23228 | |
Clear Medical Shrink Tubing (0.036″ inner diameter) | Nordson Medical | 103-0143 | |
Clear Medical Shrink Tubing (0.044″ inner diameter) | Nordson Medical | 103-0296 | |
Copper sulfate | Thermo Fisher (Pierce) | PI23224 | |
Eppendorf ThermoMixer C | Eppendorf | 2231000574 | |
Eppendorf ThermoTop heated lid | Eppendorf | 5308000003 | |
Formvar/Carbon coated electron microscopy grids | Eletron Microscopy Sciences | FCF300-Cu | |
Glass capillary tube (0.53 mm outer diameter; 0.09 mm inner diameter; 54 mm length) | Drummond | 22-326223 | |
Glass needle puller | Narishige | PC-10 | |
Hamilton syringe | Hamilton | 80000 | |
Human alpha-synuclein monomer to generate preformed fibrils | Proteos | RP-003 | |
Mouse alpha-synuclein monomer to generate preformed fibrils | Proteos | RP-009 | |
Orange Medical Shrink Tubing (0.021″ inner diameter) | Nordson Medical | 103-0152 | |
Parafilm M | Sigma-Aldrich | P7543 | |
Proteinase K | Invitrogen | 25530015 | |
Qsonica 3.2 mm tip | Qsonica | 4422 | |
Qsonica Q125 sonicator | Qsonica | Q125 | |
Thioflavin S | Sigma-Aldrich | T1892 | |
Thioflavin T | EMD Millipore | 596200 | |
ToxinSensor Chromogenic LAL Endotoxin Assay Kit | Genscript | L00350C | |
Uranyl acetate | Eletron Microscopy Sciences | 22400 |