Name: generation of alpha-synuclein preformed fibrils from monomers and use in vivo
Uploaded: 2019-06-02T15:00:00
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Description: Watch this Scientific Journal Video about Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo at JoVE.com

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>
	<li>Luk, K. C., 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=Pathological+alpha-synuclein+transmission+initiates+Parkinson-like+neurodegeneration+in+nontransgenic+mice.">Pathological alpha-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice.</a> Science. 338, 949-953 (2012).</li><li>Luk, K. C., 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=Molecular+and+Biological+Compatibility+with+Host+Alpha-Synuclein+Influences+Fibril+Pathogenicity.">Molecular and Biological Compatibility with Host Alpha-Synuclein Influences Fibril Pathogenicity.</a> Cell Reports. 16, 3373-3387 (2016).</li><li>Paumier, K. L., 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=Intrastriatal+injection+of+pre-formed+mouse+alpha-synuclein+fibrils+into+rats+triggers+alpha-synuclein+pathology+and+bilateral+nigrostriatal+degeneration.">Intrastriatal injection of pre-formed mouse alpha-synuclein fibrils into rats triggers alpha-synuclein pathology and bilateral nigrostriatal degeneration.</a> Neurobiology of Disease. 82, 185-199 (2015).</li><li>Abdelmotilib, H., 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=alpha-Synuclein+fibril-induced+inclusion+spread+in+rats+and+mice+correlates+with+dopaminergic+Neurodegeneration.">alpha-Synuclein fibril-induced inclusion spread in rats and mice correlates with dopaminergic Neurodegeneration.</a> Neurobiology of Disease. 105, 84-98 (2017).</li><li>Duffy, M. F., 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=Lewy+body-like+alpha-synuclein+inclusions+trigger+reactive+microgliosis+prior+to+nigral+degeneration.">Lewy body-like alpha-synuclein inclusions trigger reactive microgliosis prior to nigral degeneration.</a> Journal of Neuroinflammation. 15, 129 (2018).</li><li>Duffy, M. F., 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=Quality+Over+Quantity:+Advantages+of+Using+Alpha-Synuclein+Preformed+Fibril+Triggered+Synucleinopathy+to+Model+Idiopathic+Parkinson's+Disease.">Quality Over Quantity: Advantages of Using Alpha-Synuclein Preformed Fibril Triggered Synucleinopathy to Model Idiopathic Parkinson's Disease.</a> Frontiers in Neuroscience. 12, 621 (2018).</li><li>Luk, K. C., 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=Exogenous+alpha-synuclein+fibrils+seed+the+formation+of+Lewy+body-like+intracellular+inclusions+in+cultured+cells.">Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 20051-20056 (2009).</li><li>Volpicelli-Daley, L. 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=Exogenous+alpha-synuclein+fibrils+induce+Lewy+body+pathology+leading+to+synaptic+dysfunction+and+neuron+death.">Exogenous alpha-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death.</a> Neuron. 72, 57-71 (2011).</li><li>Volpicelli-Daley, L. A., Luk, K. C., Lee, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addition+of+exogenous+alpha-synuclein+preformed+fibrils+to+primary+neuronal+cultures+to+seed+recruitment+of+endogenous+alpha-synuclein+to+Lewy+body+and+Lewy+neurite-like+aggregates.">Addition of exogenous alpha-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous alpha-synuclein to Lewy body and Lewy neurite-like aggregates.</a> Nature Protocols. 9, 2135-2146 (2014).</li><li>Kuusisto, E., Salminen, A., Alafuzoff, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin-binding+protein+p62+is+present+in+neuronal+and+glial+inclusions+in+human+tauopathies+and+synucleinopathies.">Ubiquitin-binding protein p62 is present in neuronal and glial inclusions in human tauopathies and synucleinopathies.</a> Neuroreport. 12, 2085-2090 (2001).</li><li>Neumann, M., 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=Misfolded+proteinase+K-resistant+hyperphosphorylated+alpha-synuclein+in+aged+transgenic+mice+with+locomotor+deterioration+and+in+human+alpha-synucleinopathies.">Misfolded proteinase K-resistant hyperphosphorylated alpha-synuclein in aged transgenic mice with locomotor deterioration and in human alpha-synucleinopathies.</a> The Journal of Clinical Investigation. 110, 1429-1439 (2002).</li><li>Li, J. Y., 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=Characterization+of+Lewy+body+pathology+in+12-+and+16-year-old+intrastriatal+mesencephalic+grafts+surviving+in+a+patient+with+Parkinson's+disease.">Characterization of Lewy body pathology in 12- and 16-year-old intrastriatal mesencephalic grafts surviving in a patient with Parkinson's disease.</a> Movement disorders : official journal of the Movement Disorder Society. 25, 1091-1096 (2010).</li><li>Shimozawa, 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=Propagation+of+pathological+alpha-synuclein+in+marmoset+brain.">Propagation of pathological alpha-synuclein in marmoset brain.</a> Acta Neuropathologica Communications. 5, 12 (2017).</li><li>Polinski, N. K., 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=Best+Practices+for+Generating+and+Using+Alpha-Synuclein+Pre-Formed+Fibrils+to+Model+Parkinson's+Disease+in+Rodents.">Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson's Disease in Rodents.</a> Journal of Parkinson's Disease. 8, 303-322 (2018).</li><li>Fares, M. B., 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=Induction+of+de+novo+alpha-synuclein+fibrillization+in+a+neuronal+model+for+Parkinson's+disease.">Induction of de novo alpha-synuclein fibrillization in a neuronal model for Parkinson's disease.</a> Proceedings of the National Academy of Sciences of the United States of America. 113, 912-921 (2016).</li><li>Fenyi, A., Coens, A., Bellande, T., Melki, R., Bousset, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+efficacy+of+different+procedures+that+remove+and+disassemble+alpha-synuclein,+tau+and+A-beta+fibrils+from+laboratory+material+and+surfaces.">Assessment of the efficacy of different procedures that remove and disassemble alpha-synuclein, tau and A-beta fibrils from laboratory material and surfaces.</a> Scientific Reports. 8, 10788 (2018).</li><li>Geiger, B. 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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+innervation+of+direct-+and+indirect-pathway+striatal+projection+neurons.">Differential innervation of direct- and indirect-pathway striatal projection neurons.</a> Neuron. 79, 347-360 (2013).</li></ol>

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'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" inner diameter)</td>
			<td >Nordson Medical</td>
			<td >103-0143</td>
			<td></td>
		</tr>
		<tr >
			<td >Clear Medical Shrink Tubing (0.044" 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" 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 ...

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