Name: sensitive detection of proteopathic seeding activity with fret flow cytometry
Uploaded: 2015-12-08T13:00:00
Description: Watch this Scientific Journal Video about Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry at JoVE.com

This work was supported by the Tau Consortium (M.I.D); National Institutes of Health Grant 1R01NS071835 (M.I.D.), a Department of Defense Grant PT110816 (to M.I.D.), 1F32NS087805 (to J.L.F.), and 1F31NS079039 (to B.B.H.).

NOTE: This protocol emphasizes the use of FRET flow cytometry for detecting seeding activity from mouse biological samples. It is also compatible with recombinant fibrils and human biological samples. Mouse euthanasia and brain harvesting was performed in accordance with IACUC-approved procedures.
1. Brain Extraction
<ol>
	<li>Following deep anesthetization with isoflurane (2%), perfuse a mouse with ice-cold PBS containing 0.03% heparin, and extract the brain following the details described in Gage et al.</e...

<ol>
	<li>Seeley, W. W., Crawford, R. K., Zhou, J., Miller, B. L., Greicius, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegenerative+diseases+target+large-scale+human+brain+networks.">Neurodegenerative diseases target large-scale human brain networks.</a> Neuron. 62 (1), 42-52 (2009).</li><li>Zhou, J., Gennatas, E. D., Efstathios, D., Kramer, J. H., Miller, B. L., Seeley, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+Regional+Neurodegeneration+from+the+Healthy+Brain+Functional+Connectome.">Predicting Regional Neurodegeneration from the Healthy Brain Functional Connectome.</a> Neuron. 73 (6), 1216-1227 (2012).</li><li>Raj, A., Kuceyeski, A., Weiner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Network+Diffusion+Model+of+Disease+Progression+in+Dementia.">A Network Diffusion Model of Disease Progression in Dementia.</a> Neuron. 73 (6), 1204-1215 (2012).</li><li>Braak, H., Braak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathological+stageing+of+Alzheimer-related+changes.">Neuropathological stageing of Alzheimer-related changes.</a> Acta Neuropathol. 82 (4), 239-259 (1991).</li><li>Braak, H., Braak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staging+of+Alzheimer's+disease-related+neurofibrillary+changes.">Staging of Alzheimer's disease-related neurofibrillary changes.</a> Neurobiol Aging. 16 (3), 271-278 (1995).</li><li>Frost, B., Diamond, M. I. <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+mechanisms+in+neurodegenerative+diseases.">Prion-like mechanisms in neurodegenerative diseases.</a> Nat Rev Neurosci. 11 (3), 155-159 (2009).</li><li>Holmes, B. B., Diamond, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+of+protein+aggregate+propagation.">Cellular mechanisms of protein aggregate propagation.</a> Current Opinion in Neurology. 25 (6), 721-726 (2012).</li><li>Kaufman, S. K., Diamond, M. I. <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+propagation+of+protein+aggregation+and+related+therapeutic+strategies.">Prion-like propagation of protein aggregation and related therapeutic strategies.</a> Neurotherapeutics. 10 (3), 371-382 (2013).</li><li>Holmes, B. B., Diamond, M. I. <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+properties+of+Tau+protein:+the+importance+of+extracellular+Tau+as+a+therapeutic+target.">Prion-like properties of Tau protein: the importance of extracellular Tau as a therapeutic target.</a> J Biol Chem. 289 (29), 19855-19861 (2014).</li><li>Guo, J. L., 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=Cell-to-cell+transmission+of+pathogenic+proteins+in+neurodegenerative+diseases.">Cell-to-cell transmission of pathogenic proteins in neurodegenerative diseases.</a> Nat Med. 20 (2), 130-138 (2014).</li><li>Frost, B., Jacks, R. L., Diamond, M. I. <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+tau+misfolding+from+the+outside+to+the+inside+of+a+cell.">Propagation of tau misfolding from the outside to the inside of a cell.</a> J Biol Chem. 284 (19), 12845-12852 (2009).</li><li>Guo, J. L., 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=Seeding+of+normal+tau+by+pathological+tau+conformers+drives+pathogenesis+of+Alzheimer-like+tangles.">Seeding of normal tau by pathological tau conformers drives pathogenesis of Alzheimer-like tangles.</a> Journal of Biological Chemistry. , (2011).</li><li>Holmes, B. 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=Heparan+sulfate+proteoglycans+mediate+internalization+and+propagation+of+specific+proteopathic+seeds.">Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds.</a> Proc Natl Acad Sci U S A. 110 (33), E3138-E3147 (2013).</li><li>de Calignon, 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+Tau+Pathology+in+a+Model+of+Early+Alzheimer's+Disease.">Propagation of Tau Pathology in a Model of Early Alzheimer's Disease.</a> Neuron. 73 (4), 685-697 (2012).</li><li>Liu, 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=Trans-Synaptic+Spread+of+Tau+Pathology+In+Vivo.">Trans-Synaptic Spread of Tau Pathology In Vivo.</a> PLoS One. 7 (2), e31302 (2012).</li><li>Holmes, B. 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=Proteopathic+tau+seeding+predicts+tauopathy+in+vivo.">Proteopathic tau seeding predicts tauopathy in vivo.</a> Proc Natl Acad Sci U S A. 111 (41), E4376-E4385 .</li><li>Yoshiyama, 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=Synapse+loss+and+microglial+activation+precede+tangles+in+a+P301S+tauopathy+mouse+model.">Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model.</a> Neuron. 53 (3), 337-351 (2007).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J Vis Exp. 65 (65), (2012).</li><li>Hagihara, H., Toyama, K., Yamasaki, N., Miyakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+hippocampal+dentate+gyrus+from+adult+mouse.">Dissection of hippocampal dentate gyrus from adult mouse.</a> J Vis Exp. (33), (2009).</li><li>Yan, Z. X., Stitz, L., Heeg, P., Pfaff, E., Roth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectivity+of+prion+protein+bound+to+stainless+steel+wires:+a+model+for+testing+decontamination+procedures+for+transmissible+spongiform+encephalopathies.">Infectivity of prion protein bound to stainless steel wires: a model for testing decontamination procedures for transmissible spongiform encephalopathies.</a> Infect Control Hosp Epidemiol. 25 (4), 280-283 (2004).</li><li>McDonnell, G., 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=Cleaning,+disinfection+and+sterilization+of+surface+prion+contamination.">Cleaning, disinfection and sterilization of surface prion contamination.</a> J Hosp Infect. 85 (4), 268-273 (2013).</li><li>Banning, 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=A+flow+cytometry-based+FRET+assay+to+identify+and+analyse+protein-protein+interactions+in+living+cells.">A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells.</a> PLoS One. 5 (2), e9344 (2010).</li><li>Morozova, O. A., March, Z. M., Robinson, A. S., Colby, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+Features+of+Tau+Fibrils+from+Alzheimer's+Disease+Brain+Are+Faithfully+Propagated+by+Unmodified+Recombinant+Protein.">Conformational Features of Tau Fibrils from Alzheimer's Disease Brain Are Faithfully Propagated by Unmodified Recombinant Protein.</a> Biochemistry. , (2013).</li><li>Sui, D., Liu, M., Kuo, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+aggregation+assays+using+hyperphosphorylated+tau+protein.">In vitro aggregation assays using hyperphosphorylated tau protein.</a> J Vis Exp. (95), e51537 (2015).</li></ol>

The FRET flow cytometry system described here is a powerful tool for quickly and quantitatively assessing tau seeding activity. It requires only moderate cell culture experience and a working knowledge of FRET and flow cytometry. Other seeding assays, such as Thioflavin T - which exhibits enhanced fluorescence when bound to beta sheet structure - are laborious and require a pure, recombinant protein substrate. Additionally, in vitro seeding assays for tau are only semi-quantitative and generally insensitive to s...

The accumulation of intracellular tau amyloids defines tauopathies such as Alzheimer&#39;s disease. In early disease stages, pathology is generally localized to discrete regions of the brain, but with disease progression, pathology invariably spreads along distinct neural networks1-5. Accumulating evidence suggests transcellular propagation of toxic protein aggregates underlies this pathology (reviewed in 6-10). In this model, proteopathic seeds (e.g., tau) are released from donor cells and enter neighboring cells, transforming native tau protein into the misfolded form via templated conformational change11-15. The assay described here was developed to sensitively detect such seeding activity. It is compatible with recombinant protein and biological samples and enables quantification of minute levels of proteopathic seeding activity16.
HEK 293T cells that stably express tau repeat domain (RD) containing the disease-associated P301S mutation fused to either CFP or YFP (hereafter referred to as tau-RD-CFP/YFP cells) serve as a stable biosensor of seeding activity. In the absence of proteopathic seeds, the cells maintain tau as a soluble monomer, and have no appreciable background FRET. Spontaneous uptake or liposome-mediated transduction of tau seeds into cells, however, results in RD-CFP and RD-YFP aggregation, which produces a FRET signal that is measured within single cells via flow cytometry.
Numerous components of this assay were engineered to enhance sensitivity and reduce variability. A monoclonal cell line with a 1:1 RD-CFP/YFP expression ratio was selected, as it provides optimal signal:noise. To increase sensitivity, phospholipids are used to introduce seeds directly into cells (although to study biological mechanisms of uptake, this can be omitted). Finally, flow cytometry monitors FRET at a population level and a single cell level, unlike other protein aggregation assays. The final outcome measure, integrated FRET density, is highly quantitative and accounts both for the number of cells with aggregation, and the degree to which aggregation has occurred within each cell. All of these optimized parameters enhance sensitivity and ensure reproducibility.
This system was recently employed in a comprehensive study in transgenic P301S tauopathy mice17 that evaluated the temporal onset and progression of tau seeding activity relative to other commonly used tau pathological markers (e.g., MC1, AT8, PG5, and ThioflavinS). Seeding activity is by far the earliest and most robust marker of tau pathology evaluated, preceding histological detection by at least 6 weeks. Seeding activity appears at 1.5 months and increases progressively with age, suggesting a causal role of proteopathic seeds in the onset and/or progression of neurodegeneration16.
Precise quantitation of minute levels of seed material from biological samples can facilitate studies that monitor early disease progression. By shortening trial duration and enabling use of younger animals, this could increase the efficiency and accuracy of preclinical animal trials. For example, in the P301S mouse previously described, lead compounds could be delivered as early as 4-6 weeks (immediately prior to, or at onset of seeding activity), and monitored for efficacy 2-4 weeks later. The assay should accurately quantify any reductions in seeding activity. FRET flow cytometry has in vitro screening applications as well. For example, anti-tau reagents (e.g., antibodies, small molecules, etc.) can be tested rapidly for their capacity to block seeding induction directly in culture, using either recombinant tau aggregates or brain-derived lysates as a seed source (Figure 5). With this setup, once seed material is prepared, an experiment takes just three days to complete, including data analysis. The rapid quantitation of proteopathic seeding activity can thus facilitate many studies of neurodegeneration.

<table>
	<tbody>
		<tr>
			<td>TBS</td>
			<td>Sigma</td>
			<td>T5912</td>
		</tr>
		<tr>
			<td>cOmplete Protease Inhibitors (EDTA-free)</td>
			<td>Roche</td>
			<td>4693159001</td>
		</tr>
		<tr>
			<td>Cryo-vials</td>
			<td>Sarstedt</td>
			<td>72.694.006</td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>Mettler Toledo</td>
			<td>XSE 105DU</td>
		</tr>
		<tr>
			<td>Weighing Boats</td>
			<td>Fisher Scientific</td>
			<td>13-735-743</td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>USA Scientific</td>
			<td>1475-0501</td>
		</tr>
		<tr>
			<td>Omni Sonic Ruptor Ultrasonic Homogenizer</td>
			<td>Omni International</td>
			<td>18-000-115</td>
		</tr>
		<tr>
			<td>Micro-Tip for Ultrasonic Homogenizer</td>
			<td>Omni International</td>
			<td>OR-T-156</td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Fisher Scientific</td>
			<td>A451</td>
		</tr>
		<tr>
			<td>Noise Cancelling Ear Muffs</td>
			<td>Fisher Scientific</td>
			<td>19-145-412</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Fisher Scientific</td>
			<td>S47299</td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>USA Scientific</td>
			<td><a href="http://www.usascientific.com/Seal-Rite-1.5-ml-tube-sterile.aspx">1615-5510</a></td>
		</tr>
		<tr>
			<td>Microcentrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5424 000.215</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Life Technologies</td>
			<td>14190-136</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11965-084</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>HyClone</td>
			<td>SH30071.03</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
		</tr>
		<tr>
			<td>50 mL Conical Tubes</td>
			<td>Phenix Research</td>
			<td>SS-PH15</td>
		</tr>
		<tr>
			<td>25 mL reagent resevoirs</td>
			<td>VWR</td>
			<td>41428-954</td>
		</tr>
		<tr>
			<td>Multi channel pipet</td>
			<td>Fisher Scientific</td>
			<td>TI13-690-049</td>
		</tr>
		<tr>
			<td>96 well flat bottom plates</td>
			<td>Corning</td>
			<td>3603</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668019</td>
		</tr>
		<tr>
			<td>96 well round bottom plates</td>
			<td>Corning</td>
			<td>3788</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>RT 15710</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>P5493</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>ED2SS</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Life Technologies</td>
			<td>14185-052</td>
		</tr>
		<tr>
			<td>Sorvall ST 40 Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004509</td>
		</tr>
		<tr>
			<td>BIOLiner Swinging Bucket Rotor</td>
			<td>Thermo Scientific</td>
			<td>75003796</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>VWR</td>
			<td>15170-172</td>
		</tr>
		<tr>
			<td>MACSQuant VYB Flow Cytomter</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-116</td>
		</tr>
		<tr>
			<td>Chill 96 Rack</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-459</td>
		</tr>
		<tr>
			<td>Flow Jo analysis software</td>
			<td>Flow Jo</td>
			<td></td>
		</tr>
		<tr>
			<td>20 uL pipet tips</td>
			<td>Rainin</td>
			<td>GPS-L10</td>
		</tr>
		<tr>
			<td>200 uL pipet tips</td>
			<td>Rainin</td>
			<td>GPS-250</td>
		</tr>
		<tr>
			<td>1 mL pipet tips</td>
			<td>Rainin</td>
			<td>GPS-1000</td>
		</tr>
		<tr>
			<td>200 uL pipet tips</td>
			<td>USA Scientific</td>
			<td>1111-1800</td>
		</tr>
		<tr>
			<td>5 mL serological pipett</td>
			<td>Phenix Research</td>
			<td>SPG-606180</td>
		</tr>
		<tr>
			<td>10 mL serological pipett</td>
			<td>Phenix Research</td>
			<td>SPG-607180</td>
		</tr>
		<tr>
			<td>25 mL Serological pipett</td>
			<td>Phenix Research</td>
			<td>SPG-760180</td>
		</tr>
	</tbody>
</table>

sensitive detection of proteopathic seeding activity with fret flow cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

FRET flow cytometry enables sensitive, quantitative, and rapid detection of seeding activity from recombinant or biological samples. Assay setup is facile: monoclonal-derived stable cell lines expressing tau-RD-CFP/YFP are transduced with seed material, incubated for 24-48 hr, and subjected to flow cytometry analysis (Figure 1A). In the absence of seeds, biosensor cells maintain tau in a soluble, monomeric form (Figure 1B). In the presence of seeds, however, biosensor cells convert tau i...

Watch this Scientific Journal Video about Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry at JoVE.com

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

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