This work has been supported by the Italian Ministry of University and Research (MIUR) through the Career Reintegration grant scheme (RLM Program for Young Researcher) and from Scuola Normale Superiore. We thank Prof Michael Lee from University of Minnesota, USA, for providing the Prp Human A53T &#945;S Tg mice, from which aggregates are isolated.

The use of WT and Tg animals was approved and complied in full by the national and international laws for laboratory animal welfare and experimentation (EEC council directive 86/609, 12 December 1987 and Directive 2010/63/EU, 22 September 2010). All the protocols described in this paper follow the animal care guidelines of our institution.
1. Isolation of Microsomes-associated &#945;S Aggregates from Diseased A53T &#945;S Tg Mice
<ol>
	<li>Prepare the homogenization buffer which is composed of 250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 2 mM EDTA, and 1x phosphatase/protease-inhibitors. Keep the buffer on ice.</li>
	<li>Homogenize the fresh or frozen tissue in a 1:10 (w/v) volume of ice-cold homogenization buffer using a Teflon pestle homogenizer, with 10 - 15 strokes.</li>
	<li>Transfer initial homogenate (1 - 2 mL) to a microcentrifuge tube and centrifuge at 1,000 &#215; g for 10 min at 4 &#176;C using a refrigerated centrifuge in order to remove nuclei and unbroken cells in the resulting pellet (P1). Discard P1.</li>
	<li>Transfer the supernatant (S1) to a clean microcentrifuge tube and centrifuge S1 at 10,000 &#215; g for 20 min at 4 &#176;C using a refrigerated centrifuge in order to obtain the second supernatant (S10) and the pellet (P10). 
	NOTE: P10 is a crude membrane pellet that contains mitochondria and synaptosomes. Discard P10.</li>
	<li>Transfer the supernatant (S10) to a polycarbonate bottle (&#62;1 mL) and centrifuge S10 at 100,000 &#215; g, for 1 h at 4 &#176;C using an ultracentrifuge and a fixed angle rotor (90 Ti). 
	NOTE: The supernatant is the pure cytosol fraction while the pellet, P100, contains microsomes-associated &#945;S aggregates.</li>
	<li>Resuspend the P100 pellet with 500 &#181;L of the homogenization buffer.Transfer P100 to a clean microcentrifuge tube and centrifuge at 10,000 &#215; g for 20 min at 4 &#176;C in a refrigerated centrifuge.</li>
	<li>Discard the supernatant and resuspend P100 with 100 &#181;L of homogenization buffer. 
	NOTE: This fraction is the microsomes-associated &#945;S aggregates.</li>
	<li>Sonicate samples for 2 s on ice [set output power 1 watt (RMS)]. Store samples at -80 &#176;C.</li>
	<li>The day after, determine the protein amount using BCA analysis.</li>
</ol>
2. Western Blot
Note: Biochemical characterization of the microsomes-associated &#945;S aggregates is evaluated by Western Blot.
<ol>
	<li>Cast a gradient 4 - 20% Tris-glycine polyacrylamide gel on a vertical electrophoresis apparatus.</li>
	<li>Load 1 &#181;g of microsomes-associated &#945;S fractions, dissolved in the denaturing sample buffer.</li>
	<li>In a different well, load 5 &#181;L of protein standard marker.</li>
	<li>Run the gel at 100 V in a Tris/glycine/SDS running buffer until protein marker reaches the end of the gel.</li>
	<li>Transfer proteins to a nitrocellulose membrane using a basic carbonate buffer (10 mM NaCO3, 3 mM NaHCO3, 20% Methanol) O/N at 4 &#176;C, at 200 mA, constant.</li>
	<li>Block the membrane with PBS-nonionic surfactant 0.05% (PBS-T) with 5% non-fat dry milk on an orbital shaker for 30 min at RT.</li>
	<li>Briefly, rinse the membrane with PBS-T.</li>
	<li>Incubate the membrane with Syn-1 (1:5,000) or pSer129-&#945;S antibody (1:5, 000) in 2.5% non-fat dry milk in PBS-T, O/N at 4 &#176;C on an orbital shaker.</li>
	<li>Wash the membrane for 10 min at RT with PBS-T on an orbital shaker.</li>
	<li>Incubate the membrane with anti-mouse or anti-rabbit HRP-conjugated secondary antibody (1:3,000) in 2.5% non-fat dry milk in PBS-T for 1 h at RT.</li>
	<li>Wash the membrane for 10 min at RT with PBS-T on an orbital shaker. Repeat 3x.</li>
	<li>Obtain the signal through the regular chemiluminescence kits.</li>
</ol>
3. Primary Neuronal Cultures
Note: Primary neuronal cultures were prepared from the WT newborn (P0) mouse hippocampus or cortex (Line C57BL/6). The entire procedure, minus the centrifugation steps, is carried out under a cell culture hood, in sterile conditions.
<ol>
	<li>Treat coverslips (18 mm &#216;) with 65% nitric acid solution for at least 12 h at RT.</li>
	<li>Remove the nitric acid. Rinse coverslips twice with PBS 10x and several times with distilled water.</li>
	<li>Insert coverslips in 24-well dishes and coat them with poly-D-lysine (0.1 mg/mL in sterile distilled water or PBS 1x) for 1 h at 37 &#176;C.</li>
	<li>Remove poly-D-lysine and wash the coverslips three times with distilled water. Store them at 4 &#176;C until needed.</li>
	<li>Euthanize the mouse pups by decapitation and separate the heads from the bodies. Place the heads on dishes and gently dissect the skin.</li>
	<li>By using fine scissors, open the skull by making an incision at the bases of the brains. Separate the two halves of the skulls and remove them carefully.</li>
	<li>By using forceps, pinch off the brains from the bases and isolate the cortex and the hippocampus. Transfer them into two separate dishes containing Hank&#39;s Balanced Salt Solution (HBSS) medium containing 1% penicillin/streptomycin.</li>
	<li>Repeat steps 3.6 and 3.7 for each animal. Be aware that the entire process should not take more than 30 - 45 min.</li>
	<li>Mince the collected tissues and transfer them into two 50 mL conical tubes (one for hippocampus and one for cortex) with 10 mL of HBSS medium containing Trypsin 0.1%. Incubate in the water bath for 7 min at 37 &#176;C. 
	NOTE: No agitation is required.</li>
	<li>Add 1 mL of DMEM containing 10% fetal bovine serum (FBS) and 10 &#181;g/mL DNase to the homogenate to block trypsin activity.</li>
	<li>Centrifuge the resulting dissociated tissue at 200 &#215; g for 5 min at RT on a centrifuge and remove the supernatant. 
	NOTE: The pellet represents the cells dissected from the tissue.</li>
	<li>Resuspend the cells in the plating medium, containing 2% B27, 2 mM glutamine, 6 mg/mL glucose, 10% FBS, 12.5 &#181;M glutamate and 10 &#181;g/mL gentamicin.</li>
	<li>Plate dissociated neurons on poly-D-Lysine coverslips in 24 well plates according to the ratio: 1 cortex/12 wells and 1 hippocampus/6 wells, resulting in approximately 150,000/200,000 cells per well. Maintain the cells in the incubator at 37 &#176;C.</li>
	<li>The day after (day in vitro, DIV 1), replace the plating medium with the medium containing 2% B27, 10 &#181;g/mL gentamicin and 2 mM glutamine.</li>
	<li>At DIV 2, remove 1/3 of medium and add 1/3 of the fresh medium containing 2.5 &#181;M cytosine arabinoside for 48 h to reduce the glial contamination.</li>
	<li>Maintain neurons at 37 &#176;C and replace half of the medium every three days.</li>
</ol>
4. Neurons Treatment
Note: The treatment has been performed at DIV 7. All the steps are carried on under a cell culture hood in sterile conditions. An example of cortical neuronal culture density at DIV 7 is illustrated in Figure 1.
<ol>
	<li>Pool microsomes-associated &#945;S aggregates obtained from the spinal cord of three different diseased Tg mice in order to have a 1 &#181;g/&#181;L solution, using the original homogenization buffer for the dilution.</li>
	<li>Remove 1/3 of the medium and replace it gently with the fresh medium containing 2% B27, 1x gentamicin and 2 mM glutamine.</li>
	<li>Add 1 &#181;g of pooled microsomes-associated &#945;S aggregates to the cell medium. Return neurons in the incubator at 37 &#176;C.</li>
	<li>Every 3 days for 1 week, add 1/3 of fresh medium. Do not replace the medium. Just add it.</li>
	<li>After 1 week of treatment, remove 1/3 of the medium and replace it gently with the fresh medium containing 2% B27, 10 &#181;g/mL gentamicin and 2 mM glutamine. Repeat every 3 days.</li>
	<li>Fix neurons after 2 weeks of treatment (DIV 21).</li>
</ol>
5. Immunofluorescence
<ol>
	<li>Fix neurons with 2% paraformaldehyde in PBS 1x and 5% sucrose solution for 15 min at RT without shaking, under a chemical fume hood.</li>
	<li>Remove the fixing solution. Briefly, wash with PBS 1x, 3 times. 
	NOTE: Perform all the washing steps gently because primary neurons do not stick firmly to poly-lysine coverslips.</li>
	<li>Permeabilize neurons with 0.3% of nonionic surfactant in PBS 1x for 5 min at RT.</li>
	<li>Briefly, wash with PBS 1x, 3 times.</li>
	<li>Incubate neurons with 3% FBS in PBS 1x for 30 min at RT, to block unspecific binding sites, on an orbital shaker.</li>
	<li>Incubate neurons with appropriate primary antibody dissolved in 3% FBS in PBS 1x, O/N at 4 &#176;C on an orbital shaker. 
	NOTE: syn303 (1:1,000), mouse &#945;S (1:200), pser129-&#945;S (1:1,000) and Tau (1:10,000) antibodies were used.</li>
	<li>Remove the antibody solution and wash, briefly, with PBS 1x, 3 times.</li>
	<li>Incubate neurons with appropriate fluorescent secondary antibody dissolved in 3% FBS in PBS, for 1 h at RT in the dark on an orbital shaker.</li>
	<li>Remove the antibody solution and wash, briefly, with PBS 1x, 3 times.</li>
	<li>Stain neurons with DAPI solution (0.1 &#181;g/mL in PBS 1x) for 15 min at RT in the dark on an orbital shaker.</li>
	<li>Remove the antibody solution and wash, briefly, with PBS 1x, 3 times.</li>
	<li>Mount coverslips on a slide using antifade mounting medium.</li>
</ol>

<ol>
	<li>Goedert, M., Spillantini, M. G., Del Tredici, K., Braak, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=100+years+of+Lewy+pathology.">100 years of Lewy pathology.</a> Nat. Rev. Neurol. 9, 13-24 (2012).</li><li>Visanji, N. P., 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=&#945;-Synuclein-Based+Animal+Models+of+Parkinson's+Disease:+Challenges+and+Opportunities+in+a+New+Era.">&#945;-Synuclein-Based Animal Models of Parkinson's Disease: Challenges and Opportunities in a New Era.</a> Trends Neurosci. 39, 750-762 (2016).</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=Pathological+&#945;-Synuclein+Transmission+Initiates+Parkinson-like+Neurodegeneration+in+Nontransgenic+Mice.">Pathological &#945;-Synuclein Transmission Initiates Parkinson-like Neurodegeneration in Nontransgenic Mice.</a> Science. 338, 949-953 (2012).</li><li>Rey, N. L., Petit, G. H., Bousset, L., Melki, R., Brundin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+human+&#945;-synuclein+from+the+olfactory+bulb+to+interconnected+brain+regions+in+mice.">Transfer of human &#945;-synuclein from the olfactory bulb to interconnected brain regions in mice.</a> Acta Neuropathol. (Berl). 126, 555-573 (2013).</li><li>Guo, J. 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=Distinct+&#945;-Synuclein+Strains+Differentially+Promote+Tau+Inclusions+in+Neurons.">Distinct &#945;-Synuclein Strains Differentially Promote Tau Inclusions in Neurons.</a> Cell. 154, 103-117 (2013).</li><li>Masuda-Suzukake, 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=Prion-like+spreading+of+pathological+&#945;-synuclein+in+brain.">Prion-like spreading of pathological &#945;-synuclein in brain.</a> Brain. 136, 1128-1138 (2013).</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+&#945;-Synuclein+Fibrils+Induce+Lewy+Body+Pathology+Leading+to+Synaptic+Dysfunction+and+Neuron+Death.">Exogenous &#945;-Synuclein Fibrils Induce Lewy Body Pathology Leading to Synaptic Dysfunction and Neuron Death.</a> Neuron. 72, 57-71 (2011).</li><li>Peelaerts, W., 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=&#945;-Synuclein+strains+cause+distinct+synucleinopathies+after+local+and+systemic+administration.">&#945;-Synuclein strains cause distinct synucleinopathies after local and systemic administration.</a> Nature. 522, 340-344 (2015).</li><li>L&#225;zaro, D. F., Pavlou, M. A. S., Outeiro, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+models+as+tools+for+the+study+of+the+role+of+alpha-synuclein+in+Parkinson's+disease.">Cellular models as tools for the study of the role of alpha-synuclein in Parkinson's disease.</a> Exp. Neurol. 298, 162-171 (2017).</li><li>Lee, H. -. J., Patel, S., Lee, S. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravesicular+localization+and+exocytosis+of+alpha-synuclein+and+its+aggregates.">Intravesicular localization and exocytosis of alpha-synuclein and its aggregates.</a> J. Neurosci. Off. J. Soc. Neurosci. 25, 6016-6024 (2005).</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=Intracerebral+inoculation+of+pathological+&#945;-synuclein+initiates+a+rapidly+progressive+neurodegenerative+&#945;-synucleinopathy+in+mice.">Intracerebral inoculation of pathological &#945;-synuclein initiates a rapidly progressive neurodegenerative &#945;-synucleinopathy in mice.</a> J. Exp. Med. 209, 975-986 (2012).</li><li>Mougenot, A. -. 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=Prion-like+acceleration+of+a+synucleinopathy+in+a+transgenic+mouse+model.">Prion-like acceleration of a synucleinopathy in a transgenic mouse model.</a> Neurobiol. Aging. 33, 2225-2228 (2012).</li><li>Recasens, 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=Lewy+body+extracts+from+Parkinson+disease+brains+trigger+&#945;-synuclein+pathology+and+neurodegeneration+in+mice+and+monkeys:+LB-Induced+Pathology.">Lewy body extracts from Parkinson disease brains trigger &#945;-synuclein pathology and neurodegeneration in mice and monkeys: LB-Induced Pathology.</a> Ann. Neurol. 75, 351-362 (2014).</li><li>Woerman, A. 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=Propagation+of+prions+causing+synucleinopathies+in+cultured+cells.">Propagation of prions causing synucleinopathies in cultured cells.</a> Proc. Natl. Acad. Sci. 112, 4949-4958 (2015).</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> Proc. Natl. Acad. Sci. U. S. A. 106, 20051-20056 (2009).</li><li>Sacino, A. N., 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=Intramuscular+injection+of+&#945;-synuclein+induces+CNS+&#945;-synuclein+pathology+and+a+rapid-onset+motor+phenotype+in+transgenic+mice.">Intramuscular injection of &#945;-synuclein induces CNS &#945;-synuclein pathology and a rapid-onset motor phenotype in transgenic mice.</a> Proc. Natl. Acad. Sci. U. S. A. 111, 10732-10737 (2014).</li><li>Lee, M. 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=Human+alpha-synuclein-harboring+familial+Parkinson's+disease-linked+Ala-53+-->+Thr+mutation+causes+neurodegenerative+disease+with+alpha-synuclein+aggregation+in+transgenic+mice.">Human alpha-synuclein-harboring familial Parkinson's disease-linked Ala-53 --> Thr mutation causes neurodegenerative disease with alpha-synuclein aggregation in transgenic mice.</a> Proc. Natl. Acad. Sci. U. S. A. 99, 8968-8973 (2002).</li><li>Colla, E., 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=Endoplasmic+Reticulum+Stress+Is+Important+for+the+Manifestations+of+-Synucleinopathy+In+Vivo.">Endoplasmic Reticulum Stress Is Important for the Manifestations of -Synucleinopathy In Vivo.</a> J. Neurosci. 32, 3306-3320 (2012).</li><li>Fujiwara, 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+is+phosphorylated+in+synucleinopathy+lesions.">alpha-Synuclein is phosphorylated in synucleinopathy lesions.</a> Nat. Cell Biol. 4, 160-164 (2002).</li><li>Colla, E., 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=Toxic+properties+of+microsome-associated+alpha-synuclein+species+in+mouse+primary+neurons.">Toxic properties of microsome-associated alpha-synuclein species in mouse primary neurons.</a> Neurobiol. Dis. 111, 36-47 (2018).</li><li>Colla, E., 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=Accumulation+of+Toxic+-Synuclein+Oligomer+within+Endoplasmic+Reticulum+Occurs+in+-Synucleinopathy+In+Vivo.">Accumulation of Toxic -Synuclein Oligomer within Endoplasmic Reticulum Occurs in -Synucleinopathy In Vivo.</a> J. Neurosci. 32, 3301-3305 (2012).</li><li>Volpicelli-Daley, L. A., Luk, K. C., Lee, V. M. -. Y. <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+&#945;-synuclein+preformed+fibrils+to+primary+neuronal+cultures+to+seed+recruitment+of+endogenous+&#945;-synuclein+to+Lewy+body+and+Lewy+neurite-like+aggregates.">Addition of exogenous &#945;-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous &#945;-synuclein to Lewy body and Lewy neurite-like aggregates.</a> Nat. Protoc. 9, 2135-2146 (2014).</li><li>Li, W., 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=Aggregation+promoting+C-terminal+truncation+of+alpha-synuclein+is+a+normal+cellular+process+and+is+enhanced+by+the+familial+Parkinson's+disease-linked+mutations.">Aggregation promoting C-terminal truncation of alpha-synuclein is a normal cellular process and is enhanced by the familial Parkinson's disease-linked mutations.</a> Proc. Natl. Acad. Sci. U. S. A. 102, 2162-2167 (2005).</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 Rep. 16, 3373-3387 (2016).</li><li>Volpicelli-Daley, L. A., Kirik, D., Stoyka, L. E., Standaert, D. G., Harms, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+can+rAAV-&#945;-synuclein+and+the+fibril+&#945;-synuclein+models+advance+our+understanding+of+Parkinson's+disease.">How can rAAV-&#945;-synuclein and the fibril &#945;-synuclein models advance our understanding of Parkinson's disease.</a> J. Neurochem. 139, 131-155 (2016).</li></ol>

The authors have nothing to disclose.

We described a method to obtain formation of &#945;S inclusions in brain-derived primary neuronal cultures from WT mice, through the addition of purified microsomes-associated &#945;S aggregates isolated from &#945;S Tg animal models.
Critical steps of this protocol are the following: the ratio of &#181;g of microsomes-associated &#945;S aggregates/neurons and the source of &#945;S aggregates. As shown in the results session, it is crucial to optimize the ratio of &#181;g of microsomes-associated &#945;S aggregates/number of neurons since working in suboptimal conditions can lead to the premature cell death or too scarce intracellular aggregation (see the Representative Results section). Because of this, it is very important to assess the density of the neuronal culture at DIV 7 (shown in Figure 1) before starting the treatment. Additionally, &#945;S aggregates have to be purified from diseased &#945;S Tg mice, i.e. from animal models that accumulate LB-like inclusions characterized by phosphorylated and detergent insoluble &#945;S HMW fibrils.
Possible modifications to this protocol regard the tissue, frozen or fresh, from which microsomes can be isolated and the starting amount. While we used SpC of Tg mice because of the high content of &#945;S insoluble aggregates, the protocol is suitable to isolate microsomes-associated aggregates from any tissues, provided that the area has a high content in &#945;S aggregates. Frozen samples can also be used since freezing does not affect the purification steps of microsomes-associated aggregates or the aggregates per se. While the recommended starting weight for tissues is about 100 - 150 mg, this protocol is suitable for obtaining microsomes from as low as 50 mg of raw material (no maximum weight limit). In the case of the amount lower than 100 mg, however, the appropriate homogenization ratio will be 1:20 (w/v) in order to have at least 1 mL of supernatant S10 to load on the polycarbonate bottle for the ultracentrifuge precipitation. In fact, loading volumes smaller than 1 mL may result in the collapse of the tube and sample loss. Increasing the homogenization volume will lead to a more diluted supernatant but the concentration of the microsomal pellet will remain unaffected.
A limitation of this protocol concerns the inefficient cross-seeding in the formation of &#945;S inclusions that have been recently reported in the case administration of &#945;S PFFs of human origin to murine neuronal cultures as opposed to mouse &#945;S PFFs24. Since increasing the amount of exogenous &#945;S fibrils given to murine cultures can bypass this issue, we recommend to finely tune the amount of microsomes-associated aggregates in the case of administration of fibrils obtained from other &#945;S variants or from different species to mouse neuronal cultures than what we described.
Exogenous &#945;S aggregates added to the culture media can be from different sources. In vitro&#160;&#945;S PFFs have been previously used as seeding template of intracellular &#945;S aggregates in cell cultures, primary neurons, and animal models3,7,8,15. Compared to our method where microsomes-associated &#945;S aggregates can be isolated in few hours, the formation of PFFs is lengthy and laborious, requiring multiple steps of purifications, followed by additional assays to check &#945;S aggregates confomations25. In addition, PFFs being obtained from bacterial-expressed human or mouse &#945;S, i.e. lacking posttranslational modifications typical of eukaryotes, can present different conformations, with selective seeding and pathogenic properties, according to the nucleation protocols followed (i.e. ribbons vs fibrils)5,8 leading to different results and conclusions. Instead, single administration of in vivo purified &#945;S aggregates guarantees the transmission of more authentic pathogenic templates, mimicking closely the process of formation of &#945;S inclusions in animal models and PD patients.
As a future application of this technique, we believe that this protocol can be successfully used to isolate &#945;S pathogenic seeds from the brain of PD patients or other &#945;S Tg animal models, provided that diseased area from which microsomes are isolated are rich in &#945;S inclusions.
To our knowledge, this is the first method that allows the purification of native toxic species of &#945;S from in vivo PD models to be used as seeding template for obtaining formation of &#945;S inclusions in primary neurons.
We believe that this method is extremely versatile and can provide an exceptional cell-based model to study the different aspects of &#945;S aggregation and its influence on the cell pathophysiology. Because the formation of &#945;S inclusions represent a complex process that has been difficult to replicate in cultured cells, we are hopeful that this model will provide great insights in acute pathogenic mechanisms, hard to identify in chronic and more elaborate systems such as are animal models.

Accumulation of alpha-synuclein (&#945;S) proteinaceous inclusions is a prominent and important feature of Parkinson&#39;s Disease (PD) and alpha-synulceinopathies1. Unfortunately, while animal models are able to provide a sufficient cellular and biochemical environment to induce the aggregation steps necessary for the formation of protein fibrils2, replicating formation of complex Lewy Body (LB)-like aggregates in cell cultures is difficult and challenging.
Here we describe a method to induce the formation of &#945;S inclusions, similar to protein aggregates obtained in animal models and PD patients, in cultured cells, using brain isolated mouse primary neurons. Our protocol is based on the exogenous administration of microsomes-associated &#945;S aggregates isolated from &#945;S symptomatic transgenic (Tg) mice to mouse hippocampal or cortical primary neurons. This method takes advantage of the spreading and propagation ability of &#945;S toxic species which, once added to the culture medium, are able to become internalized, and induce the formation of mature &#945;S-positive aggregates3,4,5,6,7,8.
Originally, standard methods to obtain formation of &#945;S fibrils in cell cultures were based on the overexpression of the corresponding &#945;S cDNA through the regular transfection protocols or viral-mediated infection9. While in the first case obtaining LB-like &#945;S aggregates were fortuitous, showed low efficiency, and depended on the cell type, the second protocol led to the formation of insoluble fibrils, including high molecular weight (HMW) species in 24 - 48 h from infection10. In these methods, the formation of aggregates was probably due to an excessive and unbalanced in &#945;S protein amount that becomes insoluble rather than a pathological conversion of the &#945;S conformation that dictates aggregation. Instead, the technique that we are presenting here does not alter the &#945;S expression level but induces widespread protein aggregation due to the internalization of exogenous fibrils. Moreover, the formation of &#945;S aggregates through the administration of exogenous fibrils is a lengthy process that requires days or weeks to become exhaustive allowing us to study early and intermediate stages of &#945;S inclusions formation in a time-lapse fashion and to correlate it with the cellular biochemical changes. Thus, our method is a valuable application to create cellular models of &#945;S aggregation that are helpful to study &#945;S fibril formation microscopically in relation to cellular pathophysiology.
In addition although administration of raw brain extracts from diseased &#945;S Transgenic (Tg) mice11,12 or human PD brains6,13 is able to induce &#945;S deposition in Tg or wild-type (WT) animals, application of the same procedure to cell cultures has not proven to be as successful, possibly because of the low amount of aggregates in the samples used and the lack of a standard procedure to isolate native &#945;S toxic species14. Because of this, in vitro preformed fibrils (PFFs) of &#945;S have been the aggregates source of choice until now for the induction of &#945;S inclusions in cells and animal models3,4,6,7,15,16. With our protocol, however, we show that microsomes-associated &#945;S aggregated species isolated from &#945;S Tg mice can efficiently induce accumulation of intracellular LB-like &#945;S inclusions in primary neurons.
In our lab, microsomes-associated &#945;S aggregated species are isolated from the spinal cord (SpC) tissue of the diseased Tg mice expressing human A53T &#945;S gene under the control of the mouse prion protein (PrP) promoter [Prp Human A53T &#945;S Tg mice, Line G2-317]. These mice show an age-dependent neurodegenerative phenotype that includes robust motor dysfunction and formation of inclusions in the central nervous system made of phosphorylated, ubiquitinated, and insoluble &#945;S, starting after 9 months of age. Once motor dysfunction appears, the phenotype rapidly evolves into paralysis, starting from posterior limbs, that leads to the death in 2 - 3 weeks. Accumulation of &#945;S aggregates parallels disease manifestation. Mice sacrificed at the onset of the motor dysfunction show a robust degree of &#945;S aggregation in the SpC, brain stem and cerebellum. There is no need to wait until paralysis sets to sacrifice the mouse. Presymptomatic mice are taken at 9 months-old animals that do not display motor dysfunction.

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			<td height="20" style="height:20px;width:400px;">cOmplete Mini&#160;</td>
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			<td height="21" style="height:21px;width:400px;">BCA Protein Assay Kit</td>
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			<td height="21" style="height:21px;width:400px;">Criterion TGX 4-20% Stain Free, 18 wells</td>
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			<td>5678094</td>
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			<td height="21" style="height:21px;width:400px;">Supported Nitrocellulose membrane&#160;</td>
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			<td>1620097</td>
			<td>0.2 &#956;m</td>
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			<td height="21" style="height:21px;width:400px;">Blotting-Grade Blocker&#160;</td>
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			<td>1706404</td>
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			<td height="21" style="height:21px;width:400px;">SuperSignal West Pico Chemiluminescent Substrate</td>
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			<td>1004411000</td>
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			<td>1014355118NR1</td>
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			<td style="width:119px;">14170-500 mL</td>
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			<td>10,000 U/mL, 100 mL</td>
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			<td height="21" style="height:21px;width:400px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium&#160;</td>
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			<td style="width:119px;">D5796-500 mL</td>
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			<td height="21" style="height:21px;width:400px;">Trypsin-EDTA&#160;</td>
			<td style="width:245px;">Termo Fisher Scientific</td>
			<td>15400054</td>
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			<td>17504044</td>
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			<td height="21" style="height:21px;">90 Ti rotor</td>
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			<td style="width:119px;">N/A</td>
			<td>Ultracentrifuge rotor</td>
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			<td height="21" style="height:21px;">Optima L-90K Ultracentrifuge</td>
			<td style="width:245px;">Beckman</td>
			<td style="width:119px;">N/A</td>
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			<td height="21" style="height:21px;width:400px;">Syn-1 antibody, clone 42&#160;</td>
			<td style="width:245px;">BD Biosciences</td>
			<td>610786</td>
			<td>anti-mouse WB: 1:5000</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Syn303 antibody&#160;</td>
			<td style="width:245px;">BioLegend</td>
			<td style="width:119px;">824301</td>
			<td>anti-mouse IF: 1:1000</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Tau antibody&#160;</td>
			<td style="width:245px;">Synaptic Systems</td>
			<td>314&#160;002</td>
			<td>anti-rabbit IF: 1:10,000</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:400px;">pser129-&#945;S antibody&#160;</td>
			<td style="width:245px;">A gift from Fujiwara et al, reference 19</td>
			<td style="width:119px;"></td>
			<td>anti-rabbit WB: 1:5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">pser129-&#945;S antibody</td>
			<td style="width:245px;">Abcam</td>
			<td style="width:119px;">ab51253</td>
			<td>anti-rabbit&#160; IF: 1:1000</td>
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			<td height="21" style="height:21px;width:400px;">Mouse &#945;S (D37A6) XP&#160;</td>
			<td style="width:245px;">Cell Signaling</td>
			<td style="width:119px;">4179</td>
			<td>anti-rabbit IF 1:200</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Alexa fluor 555-conjugated anti-rabbit antibody&#160;</td>
			<td style="width:245px;">Termo Fisher Scientific</td>
			<td style="width:119px;">A27039</td>
			<td></td>
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			<td height="21" style="height:21px;width:400px;">Alexa fluor 488-conjugated anti-mouse antibody&#160;</td>
			<td style="width:245px;">Termo Fisher Scientific</td>
			<td>A-11029</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Microson XL-2000&#160;</td>
			<td style="width:245px;">Misonix</td>
			<td style="width:119px;"></td>
			<td>Sonicator&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ultra Bottles (Oakridge Bottles), PCB, 16x76mm, Assembly, Noryl Cap, Beckman-type</td>
			<td style="width:245px;">Science Service EU</td>
			<td style="width:119px;">S4484</td>
			<td>Ultracentrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AXIO Observer Inverted Light Microscope&#160;</td>
			<td style="width:245px;">Zeiss</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TCS SP2 laser scanning confocal microscope</td>
			<td style="width:245px;">Leica</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted epi-fluorescence microscope&#160;</td>
			<td style="width:245px;">Nikon</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:400px;">Triton x-100</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td>X100-500ML</td>
			<td>Nonionic surfactant&#160;</td>
		</tr>
	</tbody>
</table>

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.

Following the protocol described above and summarized in Figure 2, we purified microsomes-associated &#945;S aggregates from three diseased A53T &#945;S Tg mice (Figure 3). Microsomes are crude membrane pellet fractions that contain the endoplasmic reticulum, Golgi, and small synaptic vesicles. The degree of purity of the microsomal pellet compared to other fractions was previously evaluated using specific organelle markers18.
Once isolated, biochemical characterization of &#945;S aggregates is assessed through denaturing SDS-Page, followed by incubation with Syn-1 or phosphorylated &#945;S at Serine 129 (pSer129-&#945;S) antibody. Compared to presymptomatic (PreS) and age-matched non-Tg (nTg) mice, microsomes isolated from sick mice co-precipitate with &#945;S aggregates. Microsomes-associated &#945;S species show the typical features of &#945;S aggregates, such as accumulation of HMW detergent-resistant species, phosphorylation at serine 12919 and C-and N-terminal truncated fragments (for a full characterization of microsomes-associated &#945;S aggregates see reference18,20,21). These are fundamental requirements since monomeric &#945;S does not get efficiently internalized and does not induce &#945;S deposition7,15,22. It is important not to use any detergent (ionic or non-ionic) to resuspend P100 pellets since it can be harmful to cells. Also, in order to avoid sample variability, microsomes-associated &#945;S aggregates from three different diseased mice will be pooled for neuronal treatment.
Administration of 1 &#181;g of pooled microsomes-associated &#945;S aggregates from diseased A53T mice to the culture medium of cortical or hippocampal neurons induces a time-dependent formation of &#945;S inclusions, positive for aggregates-specific &#945;S antibodies such as Syn303 (Figure 4, 5) or pser129-&#945;S (Figure 5). After two days (2d) of treatment these aggregates appear as small, scattered puncta that will become more abundant at later time points. After two weeks, &#945;S inclusions resemble long and mature beads-like structures, heavily spread throughout the neuronal cultures, following a neurite pattern and partially co-localizing with presynaptic and neurites markers (Figure 5). Occasionally, newly formed &#945;S inclusions can be seen to co-localize and stain the cell soma or cover the entire process, resembling necrotic neurites.
Although microsomes-associated &#945;S aggregates fractions can efficiently spread in neuronal cultures, their amount has to be finely tuned to the number of neurons plated (Figure 1). In fact, exceeding the recommended ratio, &#181;g of microsomes-associated aggregates: number of neurons, will induce premature cell death within a few days (Figure 6), while an insufficient amount of microsomes-associated &#945;S aggregates will lead to a scarce and reduced number of inclusions after two weeks of treatment, similar to what was obtained at earlier time points (Figure 4A).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57884/57884fig1.jpg" /> 
Figure 1. Cortical neuronal cultures. Representative image showing density at DIV 7 of cortical neuronal cultures. Images were taken with an inverted light microscope, 10X objective. Scale bar = 100 &#181;m. <a href="/files/ftp_upload/57884/57884fig1large.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/57884/57884fig2.jpg" /> 
Figure 2. Isolation of microsomes-associated &#945;S aggregates from mouse SpC. Flowchart of the purification protocol of microsomes-associated &#945;S aggregates from SpC of diseased mice. <a href="/files/ftp_upload/57884/57884fig2large.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/57884/57884fig3.jpg" /> 
Figure 3. Isolation of microsomes fractions from diseased, presymptomatic A53T &#945;S Tg and nTg mice. Western blot analysis showing purified microsomes-associated &#945;S aggregates isolated from three diseased (Sick), presymptomatic (PreS) and aged-matched nTg mice. Diseased mice are A53T &#945;S Tg mice that show the motor and neurological dysfunction, including the accumulation of &#945;S inclusions, while presymptomatic animals are healthy A53T &#945;S Tgs of 9 months of age that do not show yet any &#945;S pathology related phenotype. nTg mice are littermates of diseased mice which do not carry the &#945;S transgene and therefore do not develop &#945;S induced pathology. 1 &#181;g of each purified fractions were run on a denaturing SDS-Page, transferred on a nitrocellulose membrane and blotted with Syn-1 or pSer129-&#945;S antibody. Only microsomes fractions isolated from sick mice contained HMW detergent-resistant &#945;S aggregates that were phosphorylated at serine 129 and showed C-and N-terminal truncation. This figure has been adapted from Colla et al.18. <a href="/files/ftp_upload/57884/57884fig3large.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/57884/57884fig4.jpg" /> 
Figure 4. Time-dependent induction of &#945;S deposition after administration of microsomes-associated &#945;S aggregates. (A) Immunofluorescence of primary hippocampal neurons treated with 1 &#181;g of microsomes-associated &#945;S aggregates fractions pooled from three different diseased mice. Neurons were fixed at 2 days (2d), 1 week (1w) or 2 weeks (2w) of treatment and immunostained with syn303 (S303, 1:1000), an antibody specific for oxidized and aggregated &#945;S. Cells were counterstained with DAPI. Confocal images were taken using a laser scanning confocal microscope, 63X objective. Scale bar = 50 &#181;m. (B) Quantitative analysis of total &#64258;uorescence, after background subtraction, was done using the particles count plugin of the Image J software. Values were normalized for the number of nuclei per field (DAPI count) and expressed as the percentage of the S303 fluorescence signal at 2D. Values are given as the mean &#177; SD (n = 5). **p &#60;0.001, ****p &#60;0.00001, One-way ANOVA, followed by Fisher&#39;s LSD post-hoc test. <a href="/files/ftp_upload/57884/57884fig4large.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/57884/57884fig5.jpg" /> 
Figure 5. Intracellular &#945;S inclusions colocalize with cortical neurites network. Representative confocal images of cortical neurons treated with microsomes-associated &#945;S aggregates obtained from diseased Tg mice. After 2 weeks of treatment neurons were fixed and double stained with aggregates-specific antibodies [S303 (A, B) or pser129-&#945;S, 1:1,000 (C)] and neurite markers [mouse &#945;S, 1:200 (A,B) or Tau, 1:10,000 (C)]. Co-labelling of the fluorescent signals demonstrated partial co-localization of newly formed &#945;S bead-like structures with the neurites network. Occasionally &#945;S inclusions accumulate within the neuronal soma (A, B, arrowheads). Stacked images were acquired with a laser scanning confocal microscope, 63X&#160;objective. Scale bar = 50 &#181;m. This figure has been modified from Colla et al.20. <a href="/files/ftp_upload/57884/57884fig5large.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/57884/57884fig6.jpg" /> 
Figure 6. Addition of suboptimal amount of microsomes-associated &#945;S aggregates is toxic to neurons. DAPI staining of neurons treated with increasing concentration of microsomes-associated &#945;S aggregates leads to cell death. (A) Cortical neurons were treated with 1, 2 &#181;g of microsomes-associated &#945;S aggregates extracted from diseased mice or only buffer that does not contain aggregates (B) and stained with DAPI. Fluorescent images were acquired with an epi-fluorescence microscope using a 20X objective. Scale bar = 100 &#181;m.(B) DAPI-positive cells were counted using the Image J software. The graph shows a reduction in the number of nuclei with increasing concentration of microsomes-associated &#945;S aggregates added to the neuronal media. Values are expressed as % of b and are given as the mean &#177; SD (n = 5), ***p &#60;0.0001, One-way ANOVA, followed by Fisher&#39;s LSD post-hoc test. <a href="/files/ftp_upload/57884/57884fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation at JoVE.com

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

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 ...

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Universidad de Cartagena UDEC

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Neuroscience

7.5K Views.  Scuola Normale Superiore. 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.

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

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, ...

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Antibodies provide the ability to gain novel insight into various events taking place in living systems. The ability to produce highly specific antibodies to target proteins has allowed for very precise biological questions to be addressed. Importantly, antibodies have been implicated in the pathogenesis of a number of human diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multiple sclerosis (MS) and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP) 1-9. How antibodies cause disease is an area of ongoing investigation, and data suggests that interactions between antibodies and various intracellular molecules results in inflammation, altered cellular messaging, and apoptosis 10. It has been shown that patients with MS and HAM/TSP produce autoantibodies to the intracellular RNA binding protein heterogeneous ribonuclear protein A1 (hnRNP A1) 3, 5-7, 9, 11. Recent data indicate that antibodies to both intra-neuronal and surface antigens are pathogenic 3, 5-9, 11. Thus, a procedure that allows for the study of intracellular antibody:protein interactions would lend great insight into disease pathogenesis.
Genes are commonly transfected into primary cells and cell lines in culture, however transfection of antibodies into cells has been hindered by alteration of antibody structure or poor transfection efficiency 12. Other methods of transfection include antibody transfection based on cationic liposomes (consisting of DOTAP/DOPE) and polyethylenimines (PEI); both of which resulted in a ten-fold decrease in antibody transfection compared to controls 12. The method performed in our study is similar to cationic lipid-mediated methods and uses a lipid-based mechanism to form non-covalent complexes with the antibodies through electrostatic and hydrophobic interactions 13. We utilized Ab-DeliverIN reagent, which is a lipid formulation capable of capturing antibodies through non-covalent electrostatic and hydrophobic interactions and delivering them inside cells. Thus chemical and genetic couplings are not necessary for delivery of functional antibodies into living cells. This method has enabled us to perform various antibody tracing and protein localization experiments, as well as the analyses of the molecular consequences of intracellular antibody:protein interactions 9.
In this protocol, we will show how to transfect antibodies into neurons rapidly, reproducibly and with a high degree of transfection efficiency. As an example, we will use anti-hnRNP A1 and anti-IgG antibodies. For easy quantification of transfection efficiency we used anti-hnRNP A1 antibodies labelled with Atto-550-NHS and FITC-labeled IgG. Atto550 NHS is a new label with high molecular absorbtion and quantum yield. Excitation source and fluorescent filters for Atto550 are similar to Cy3 (Ex. 556 Em. 578). In addition, Atto550 has high photostability. FITC-labeled IgG were used as a control to show that this method is versatile and not dye dependent. This approach and the data that is generated will assist in understanding of the role that antibodies to intracellular target antigens might play in the pathogenesis of human diseases.

A rapid approach to investigate interactions and effects on molecular mechanisms related to the presence of antibodies in an intracellular environment is described. The method involves transfection of antibodies into live cells using a non-covalent complex formation based on a lipid formulation. The technique is adaptable to immortalized cell lines and primary cells.

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

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, ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

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 ...

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...

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;

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 ...

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

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Chapters in this video

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

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Calcium Phosphate Transfection of Primary Hippocampal Neurons

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

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

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

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

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