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 ...

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

Research

JoVE Journal

Neuroscience

21.0K Views.  Michigan State University. 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.

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

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

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 ...