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.

<ol>
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M., Frank, L. E., Caldera-Siu, A. D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survivable+stereotaxic+surgery+in+rodents.">Survivable stereotaxic surgery in rodents.</a> Journal of Visualized Experiments. , (2008).</li><li>Tarutani, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+Fragmented+Pathogenic+alpha-Synuclein+Seeds+on+Prion-like+Propagation.">The Effect of Fragmented Pathogenic alpha-Synuclein Seeds on Prion-like Propagation.</a> Journal of Biological Chemistry. 291, 18675-18688 (2016).</li><li>Wall, N. R., De La Parra, M., Callaway, E. M., Kreitzer, A. 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Joseph Patterson, Kelvin Luk, and Caryl Sortwell are currently involved in contractual arrangements with the Michael J. Fox Foundation to quality control &#945;-syn material generated by Proteos, Inc. Coauthor Nicole Polinski, is an employee of the Michael J. Fox Foundation, which has contracted Proteos, Inc. to produce &#945;-syn monomer. Coauthors Megan Duffy, Laura Volpicelli-Daley, and Nicholas Kanaan have nothing to disclose.

Production of &#945;-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 &#945;-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 &#945;-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 &#945;-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 &#945;-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 &#181;g 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 &#945;-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.

Parkinson&#39;s disease (PD) is primarily characterized postmortem by two major pathological features: widespread and progressive alpha-synuclein (&#945;-syn) pathology, and nigrostriatal degeneration. Following injection into wildtype mice or rats, &#945;-syn preformed fibrils (PFFs) induce progressive accumulation of pathological &#945;-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 &#945;-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 &#34;seed&#34;&#160;the formation of inclusions through templating, and accumulation of endogenous &#945;-syn into phosphorylated inclusions1,7,8,9. Inclusions share similar properties to Lewy bodies: containing &#945;-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 &#945;-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 &#945;-syn inclusion formation in all cell culture models and some cultured neurons will seed better than others.
Another important feature of the in vivo &#945;-syn PFF model is the distinct sequential pathological phases that emerge over several months. In rodents, following intrastriatal injection, &#945;-syn inclusion formation generally peaks within the SNpc and many cortical regions within 1-2 months. This aggregation peak is followed by nigrostriatal degeneration &#8776;2-4 months later1,3,5. These distinct pathological stages provide researchers the platform with which to study and develop strategies that 1) decrease &#945;-syn aggregation, 2) clear already formed &#945;-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 &#945;-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 &#945;-syn pathology14. Examples of inconsistencies range from PFFs that produce little or no &#945;-syn pathology, batch to batch seeding efficiency, and even the failure of fibrils to form. Thus, the standardization of &#945;-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 &#945;-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.

<table><tbody><tr><td>1x Dulbecco&amp;rsquo;s phosphate buffered saline</td><td>Thermo Fisher (Gibco)</td><td>14190144</td><td></td></tr><tr><td>Anti-alpha-synuclein (phosphorylated at serine 129) antibody</td><td>Abcam</td><td>AB184674</td><td></td></tr><tr><td>Anti-alpha-synuclein antibody</td><td>Abcam</td><td>AB15530</td><td></td></tr><tr><td>Bicinchonic acid</td><td>Thermo Fisher (Pierce)</td><td>PI23228</td><td></td></tr><tr><td>Clear Medical Shrink Tubing (0.036&quot; inner diameter)</td><td>Nordson Medical</td><td>103-0143</td><td></td></tr><tr><td>Clear Medical Shrink Tubing (0.044&quot; inner diameter)</td><td>Nordson Medical</td><td>103-0296</td><td></td></tr><tr><td>Copper sulfate</td><td>Thermo Fisher (Pierce)</td><td>PI23224</td><td></td></tr><tr><td>Eppendorf ThermoMixer C</td><td>Eppendorf</td><td>2231000574</td><td></td></tr><tr><td>Eppendorf ThermoTop heated lid</td><td>Eppendorf</td><td>5308000003</td><td></td></tr><tr><td>Formvar/Carbon coated electron microscopy grids</td><td>Eletron Microscopy Sciences</td><td>FCF300-Cu</td><td></td></tr><tr><td>Glass capillary tube (0.53 mm outer diameter; 0.09 mm inner diameter; 54 mm length)</td><td>Drummond</td><td>22-326223</td><td></td></tr><tr><td>Glass needle puller</td><td>Narishige</td><td>PC-10</td><td></td></tr><tr><td>Hamilton syringe</td><td>Hamilton</td><td>80000</td><td></td></tr><tr><td>Human alpha-synuclein monomer to generate preformed fibrils</td><td>Proteos</td><td>RP-003</td><td></td></tr><tr><td>Mouse alpha-synuclein monomer to generate preformed fibrils</td><td>Proteos</td><td>RP-009</td><td></td></tr><tr><td>Orange Medical Shrink Tubing (0.021&quot; inner diameter)</td><td>Nordson Medical</td><td>103-0152</td><td></td></tr><tr><td>Parafilm M</td><td>Sigma-Aldrich</td><td>P7543</td><td></td></tr><tr><td>Proteinase K</td><td>Invitrogen</td><td>25530015</td><td></td></tr><tr><td>Qsonica 3.2 mm tip</td><td>Qsonica</td><td>4422</td><td></td></tr><tr><td>Qsonica Q125 sonicator</td><td>Qsonica</td><td>Q125</td><td></td></tr><tr><td>Thioflavin S</td><td>Sigma-Aldrich</td><td>T1892</td><td></td></tr><tr><td>Thioflavin T</td><td>EMD Millipore</td><td>596200</td><td></td></tr><tr><td>ToxinSensor Chromogenic LAL Endotoxin Assay Kit</td><td>Genscript</td><td>L00350C</td><td></td></tr><tr><td>Uranyl acetate</td><td>Eletron Microscopy Sciences</td><td>22400</td><td></td></tr></tbody></table>

generation of alpha-synuclein preformed fibrils from monomers and use in vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-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 &#945;-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 &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

Generation of fibrils from &#945;-syn monomers begins with determining the concentration of the monomers. Both the BCA assay and measurement of absorbance at 280 nm (A280) can be used to measure protein content; the BCA assay results, however, suggested a higher concentration than the A280 method. PFFs derived from mouse &#945;-syn monomer had a BCA value of 14.05 &#177; 0.22 and a A280 of 8.05 &#177; 0.03 &#181;g/&#181;L (Figure 1). Likewise, PFFs derived from human &#945;-syn monomer also appeared to be at a higher concentration, with a BCA value of 12.95 &#177; 0.38 and a A280 of 7.83 &#177; 0.05 &#181;g/&#181;L (Figure 1). The A280 measurements are specific to &#945;-syn based on the inclusion of the extinction coefficients and these results were used to dilute the monomers prior to 7-day incubation.
Prior to incubation, the liquid containing the &#945;-syn monomers was clear, but should appear turbid after fibril formation. Examination with transmission electron microscopy confirmed the presence of long fibrils, measuring 10-20 nm wide (Figure 4). In comparison, &#945;-syn monomers were barely visible with no discernible shape apparent (Figure 4). With visual confirmation of fibrillar structures, amyloid conformation of the fibrils is the next feature of PFFs that should confirmed using a thioflavin T assay. Thioflavin T exhibits enhanced fluorescence when binding to amyloid; thus, increased fluorescent signal from the samples indicates presence of amyloid. As an example, thioflavin in dPBS produced a signal of 3,287 &#177; 580 relative fluorescent units (RFU), mouse &#945;-syn monomer produced a signal of 4,174 &#177; 158 RFU, and mouse PFFs produced a signal of 59,754 &#177; 6,224 RFU (Figure 5). In comparison, human &#945;-syn monomer produced a similar signal of 4,158 &#177; 105 RFU to mouse monomer, and human PFFs produced a higher signal of 1,235,967 &#177; 113,747 RFU as compared to mouse PFFs (Figure 5). To assess the presence of pelletable fibrils, a sedimentation assay was performed. Fibrils will pellet with centrifugation. In both the mouse and human PFF samples, the supernatant fraction should have more protein in the pellet than the supernatant (Figure 6). In contrast, the majority of the protein from the mouse and human monomers was present in the supernatant, with little present in the pellet (Figure 6). With the PFFs visibly present by electron microscopy, amyloid structures present, and fibrils pelletable, the PFFs passed all in vitro quality control steps.
Both mouse and human PFFs were sonicated to produce PFFs of appropriate lengths for seeding &#945;-syn inclusions4,18. PFFs were diluted to the desired concentration of 4 &#181;g/&#181;L and sonicated. Immediately prior to surgery, 25 representative fibrils were imaged by electron microscopy and measured to spot check the fibril size. The sonicated mouse PFFs measured 48.8 &#177; 3.1 nm, whereas the human PFFs measured 52.1 &#177; 4.4 nm in length; PFFs of both species were therefore the appropriate length (50 nm or less) to induce seeding activity. More comprehensive examination of approximately 500 fibrils revealed the average length and length distribution of the sonicated mouse fibrils. The average length was 44.4 &#177; 0.6 nm, with 86.6% of the PFFs measuring 60 nm or less (Figure 4). In comparison, human PFFs averaged 55.9 &#177; 1.1 nm with 69.6% of the PFFs measuring 60 nm or less (Figure 4).
Following intrastriatal injection of sonicated mouse PFFs into rats as previously described3,5, a series of tissue sections were processed at 2 months post-surgery, when the number of inclusion containing neurons is known to peak in the SNpc, for the confirmation of phosphorylated &#945;-syn inclusions5. Inclusion bearing neurons, as indicated by immunohistochemical staining for pSyn (antibody in Table of Materials) are present within the SNpc (Figure 7), as well as other regions throughout the brain which innervate the striatum (anterior olfactory nucleus, motor, cingulate, piriform, prelimbic, somatosensory, entorhinal, and insular cortices, amygdala, striatum)1,3,4,5,19. These inclusions share similar properties with Lewy bodies, such as binding thioflavin S, and resistance of total &#945;-syn to proteinase K (Figure 7), as shown by immunohistochemical staining (antibody and proteinase K in Table of Materials). Confirmation of seeding within the brain indicates the in vivo quality control has been passed, and aliquots of PFFs previously frozen and saved from the same batch may be sonicated under identical parameters, with lengths validated, in larger experiments.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59758/59758fig1.jpg" /> 
Figure 1. Methods for the generation of &#945;-syn fibrils. Outline of the steps required to produce fibrils from &#945;-syn monomers. Monomers are centrifuged for 10 min (15,000 x g, at 4 &#176;C). Supernatant is transferred to a clean tube and the protein concentration is determined by either the absorbance at 280 nm, or a BCA assay. Graph shows concentrations from human and mouse &#945;-syn monomers. Columns indicate the group means, error bars represent &#177;1 standard error of the mean. After protein concentration is determined, &#945;-syn monomers are diluted, briefly vortexed and incubated for 37 &#176;C for 7 days, while shaking on an orbital mixer set at 1,000 RPM. <a href="https://www.jove.com/files/ftp_upload/59758/59758fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59758/59758fig2.jpg" /> 
Figure 2. Staining methods for transmission electron microscopy. Diagram of negative staining for electron microscopy. A) Images depicting electron microscopy specimen grids. The grid has a dull or light side and a shiny or dark side. The shiny/dark side is coated with a formvar/carbon support film. B) Illustration of staining procedure. The grid is floated shiny/dark side down on the first drop of ddH2O for 1 min and the excess is wicked away with filter paper. The process is repeated with the second drop of ddH2O, diluted PFFs or monomers, two drops of uranyl acetate, and two additional drops of ddH2O. Grids may be stored in a grid box until imaged. Scale bar = 3 mm. <a href="https://www.jove.com/files/ftp_upload/59758/59758fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59758/59758fig3.jpg" /> 
Figure 3. Assembly of custom glass needle syringes. Diagram of the steps required to assemble glass needle attached syringes. Siliconized glass capillary tubes are pulled and cut in the middle to produce glass needles. Shrink wrap tubing is used to prepare the metal needle and form an inner seal when the glass needle is slid onto the metal needle. Two additional layers of shrink-wrap tubing that overlap the base of the glass needle and the metal needle are consecutively added and heat applied to secure the glass needle and form a water-tight seal. <a href="https://www.jove.com/files/ftp_upload/59758/59758fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59758/59758fig4.jpg" /> 
Figure 4. Visualization of &#945;-syn monomers and &#945;-syn fibrils via transmission electron microscopy. Representative micrographs of &#945;-syn monomers and fibrils. Top panels: Mouse and human &#945;-syn monomer. Middle panels: Full length mouse and human &#945;-syn PFFs. Bottom panels: Mouse and human &#945;-syn PFFs after sonication. Bottom graph: Distribution of sonicated mouse and human &#945;-syn PFF lengths. Scale bar = 500 nm. <a href="https://www.jove.com/files/ftp_upload/59758/59758fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59758/59758fig5.jpg" /> 
Figure 5. Confirmation of amyloid structures by thioflavin T assay. Measurement of fluorescent signal from mouse and human &#945;-syn monomer and PFF samples. Left: Results from mouse &#945;-syn monomers and &#945;-syn PFFs. Right: Results from human &#945;-syn monomers and &#945;-syn PFFs. A dPBS negative control is shown in each graph. All measurements are expressed as relative fluorescent units (RFU). Columns indicate the group means, error bars represent &#177;1 standard error of the mean. <a href="https://www.jove.com/files/ftp_upload/59758/59758fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59758/59758fig6.jpg" /> 
Figure 6. Sedimentation assay for pelletable &#945;-syn. Images from Coomassie stained gels. Bands shown are at approximately 14 kDa based on the protein ladder. Left: Mouse monomer and PFFs. Right: Human monomers and PFFs. For all monomer and PFF samples, the resuspended pellet (P) and supernatant (S) are shown. <a href="https://www.jove.com/files/ftp_upload/59758/59758fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59758/59758fig7.jpg" /> 
Figure 7. Features of inclusions in the rat model confirming &#945;-syn pathology. Representative micrographs from the substantia nigra pars compacta at 2 months post-injection. Left: Neurons containing pSyn and counterstained with cresyl violet. Middle: Thioflavin S positive neurons. Right: &#945;-syn-containing inclusions resistant to proteinase K. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/59758/59758fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo at JoVE.com

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

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 (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...

generation-alpha-synuclein-preformed-fibrils-from-monomers-use

Research

JoVE Journal

Neuroscience

21.2K Views.  Michigan State University. Het Alpha-Synuclein Preformed Fibril model wordt door laboratoria wereldwijd gebruikt om synucleinopathieën te bestuderen. Hoewel het model met succes is gebruikt door vele laboratoria, hebben sommige groepen inconsistenties ervaren die fibrils genereren en consistente alfa-synuclein pathologie produceren. We hopen dat dit protocol, dat de generatie fibrils van alfa-synuclein monomeren en het gebruik van voorgevormde fibrils in vivo beschrijft, vragen kan beantwoorden die onderzoekers hebben...

Video: Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

Medicine

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Biology

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.

We describe how to successfully inject solutions into specific brain areas of rodents using a stereotaxic frame. This survival surgery is a well-established method used to mimic various aspects of Parkinson's disease.

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in ...

&#945;-Synuclein Propagation: Intranasal Administration in Parkinson&#39;s Disease

Technique for Intranasal Administration of &#945;-Synuclein Aggregates

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of Lewy bodies, which are aggregates of &#945;-synuclein (&#945;-Syn). Recently, the disease was proposed to develop and progress through the prion-like propagation of &#945;-Syn aggregates from the olfactory bulb (OB) or dorsal nucleus of the vagus nerve. Although the origin of &#945;-Syn aggregates in the OB remains unclear, their propagation from the olfactory mucosa has been recently suggested. We previously showed that intranasal administration of &#945;-Syn aggregates in a mouse model induced &#945;-Syn pathology in the OB of mice. In this study, we present a method of intranasal administration of &#945;-Syn aggregates that induced &#945;-Syn pathology in the OB of mice. Intranasal administration of &#945;-Syn aggregates is a very simple and straightforward method, and we believe it will be a useful tool in the research for elucidating the origin of &#945;-Syn pathology in the OB and the pathway of &#945;-Syn propagation through the olfactory system.

The present protocol describes a method for intranasal administration of &#945;-synuclein aggregates. This method provides insights into &#945;-synuclein propagation from the olfactory mucosa to the olfactory bulb in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of Lewy bodies, which are aggregates of &#945;-synuclein ...

A Method to Study &#945;-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy bodies containing misfolded and aggregated &#945;-synuclein. &#945;-synuclein is an abundant presynaptic protein that regulates synaptic vesicle trafficking, but the accumulation of its proteinaceous inclusions results in neurotoxicity. Recent studies have revealed that various genetic factors, including bacterial chaperones, could reduce the formation of &#945;-synuclein aggregates in vitro. However, it is also important to monitor the anti-aggregation effect in the cell to apply this as a potential treatment for the patients. It would be ideal to use neuronal cells, but these cells are difficult to handle and take a long time to exhibit the anti-aggregation phenotype. Therefore, a quick and effective in vivo tool is required for the further evaluation of in vivo anti-aggregation activity. The method described here was used to monitor and analyze the anti-aggregation phenotype in the humanized yeast Saccharomyces cerevisiae, which expressed human &#945;-synuclein. This protocol demonstrates in vivo tools that could be used for monitoring &#945;-synuclein-induced cellular toxicity, as well as the formation of &#945;-synuclein aggregates in cells.

An in vivo physiological model of &#945;-synuclein is required to study and understand the pathogenesis of Parkinson's disease. We describe a method to monitor the cytotoxicity and aggregate formation of &#945;-synuclein using a humanized yeast model.

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy ...

Induction of Neuroinflammation in Mice via Injection of Alpha-Synuclein Fibrils

Source: Breid, S. et al. <a href="https://app.jove.com/v/55503">Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils.</a> J. Vis. Exp. (2017).
This video demonstrates the injection of alpha-synuclein fibrils into a mouse to induce neuroinflammation. The fibrils are injected intraperitoneally into a transgenic mouse expressing mutated alpha-synuclein proteins. Upon reaching the central nervous system, the fibrils enter neurons and induce aggregation of the mutated proteins. The aggregates spread to neighboring neurons, microglia, and astrocytes. Recognition of the aggregates triggers microglia to induce neuroinflammation, damaging the neurons.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160; ...

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

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