The authors would like to thank Dr. Peter Davies at Albert Einstein College of Medicine for supplying PHF-1 and CP13 antibodies and Dr. Marc Diamond at the University of Texas, Southwestern, for providing the tau constructs. This work was supported by grants from the Alzheimer&#8217;s Association (NIRG-14-322164) to S.H.Y. and from the California Institute for Regenerative Medicine (TB1-01193) to P.R.

1. Preparation of Media and Reagents
<ol>
	<li>Thaw the basement membrane matrix coating for culture plates at 4 &#176;C (do not allow the basement membrane matrix to warm up or it will solidify). Make 1 mL aliquots and store them at -20 &#176;C or -70 &#176;C.</li>
	<li>Reconstitute basic fibroblast growth factor (bFGF) in sterile phosphate-buffered saline (PBS) at 10 &#181;g/mL and make 10 &#181;L aliquots. Store them at 4 &#176;C.</li>
	<li>To a new, unopened, 500 mL bottle of DMEM/F12 with glutamine, add B27 (10 mL...

<ol>
	<li>Rojas, J. C., Boxer, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegenerative+disease+in+2015:+Targeting+tauopathies+for+therapeutic+translation.">Neurodegenerative disease in 2015: Targeting tauopathies for therapeutic translation.</a> Nature Reviews Neurology. 12 (2), 74-76 (2016).</li><li>Kadavath, 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=Tau+stabilizes+microtubules+by+binding+at+the+interface+between+tubulin+heterodimers.">Tau stabilizes microtubules by binding at the interface between tubulin heterodimers.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (24), 7501-7506 (2015).</li><li>Wang, Y., Mandelkow, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+in+physiology+and+pathology.">Tau in physiology and pathology.</a> Nature Reviews Neurology. 17 (1), 5-21 (2016).</li><li>Gomez-Ramos, 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=Characteristics+and+consequences+of+muscarinic+receptor+activation+by+tau+protein.">Characteristics and consequences of muscarinic receptor activation by tau protein.</a> European Neuropsychopharmacology. 19 (10), 708-717 (2009).</li><li>Warmus, B. 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=Tau-mediated+NMDA+receptor+impairment+underlies+dysfunction+of+a+selectively+vulnerable+network+in+a+mouse+model+of+frontotemporal+dementia.">Tau-mediated NMDA receptor impairment underlies dysfunction of a selectively vulnerable network in a mouse model of frontotemporal dementia.</a> Journal of Neuroscience. 34 (49), 16482-16495 (2014).</li><li>DeVos, S. 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=Tau+reduction+in+the+presence+of+amyloid-beta+prevents+tau+pathology+and+neuronal+death+in+vivo.">Tau reduction in the presence of amyloid-beta prevents tau pathology and neuronal death in vivo.</a> Brain. 141 (7), 2194-2212 (2018).</li><li>Cheng, J. S., 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=Tau+reduction+diminishes+spatial+learning+and+memory+deficits+after+mild+repetitive+traumatic+brain+injury+in+mice.">Tau reduction diminishes spatial learning and memory deficits after mild repetitive traumatic brain injury in mice.</a> PLOS ONE. 9 (12), e115765 (2014).</li><li>Ando, 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=Accelerated+human+mutant+tau+aggregation+by+knocking+out+murine+tau+in+a+transgenic+mouse+model.">Accelerated human mutant tau aggregation by knocking out murine tau in a transgenic mouse model.</a> The American Journal of Pathology. 178 (2), 803-816 (2011).</li><li>Reilly, 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=Novel+human+neuronal+tau+model+exhibiting+neurofibrillary+tangles+and+transcellular+propagation.">Novel human neuronal tau model exhibiting neurofibrillary tangles and transcellular propagation.</a> Neurobiology of Disease. 106, 222-234 (2017).</li><li>Kfoury, N., Holmes, B. B., Jiang, H., Holtzman, D. M., 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=Trans-cellular+Propagation+of+Tau+Aggregation+by+Fibrillar+Species.">Trans-cellular Propagation of Tau Aggregation by Fibrillar Species.</a> The Journal of Biological Chemistry. 287 (23), 19440-19451 (2012).</li><li>Yuan, S. 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=Cell-surface+marker+signatures+for+the+isolation+of+neural+stem+cells,+glia+and+neurons+derived+from+human+pluripotent+stem+cells.">Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells.</a> PLOS ONE. 6 (3), e17540 (2011).</li><li>Marchenko, S., Flanagan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passaging+human+neural+stem+cells.">Passaging human neural stem cells.</a> Journal of Visualized Experiments. (7), e263 (2007).</li><li>Lathuiliere, 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=Motifs+in+the+tau+protein+that+control+binding+to+microtubules+and+aggregation+determine+pathological+effects.">Motifs in the tau protein that control binding to microtubules and aggregation determine pathological effects.</a> Scientific Reports. 7 (1), 13556 (2017).</li><li>Asai, 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=Depletion+of+microglia+and+inhibition+of+exosome+synthesis+halt+tau+propagation.">Depletion of microglia and inhibition of exosome synthesis halt tau propagation.</a> Nature Neuroscience. 18 (11), 1584-1593 (2015).</li></ol>

This protocol describes the generation of an in vitro model of human tauopathy that exhibits silver-stain-positive aggregates and thioflavin-positive neurofibrillary tangles (NFTs). Moreover, transduced cells display tau-induced pathologies such as morphological defects, reduced synaptogenesis, and an increased lysosomal volume. The main advantage of this protocol is that it provides an accessible and cost-effective model of neuronal tauopathy, which can be used for drug screening studies, as well as for the analysis of ...

Pathological aggregation of the microtubule-associated protein tau is a defining feature of many neurodegenerative diseases, including AD, frontotemporal dementia (FTD), Pick&#8217;s disease, and progressive supranuclear palsy (PSP)1. In a nondiseased state, tau binds to and stabilizes microtubule filaments in neuronal axons2. However, disease-associated hyperphosphorylation of tau promotes tau aggregation, dissociation from microtubules, and neuronal toxicity3. The toxic effects of aggregated tau may involve aberrant activation of cholinergic4 and glutamatergic receptors5 resulting in the dysregulation of intracellular calcium and, eventually, cell death. In animal models, the reduction of brain tau improves pathology in AD mice6 and in mouse models of repetitive mild traumatic brain injury7.
Mounting evidence demonstrates that the structure and binding affinity of mouse-derived tau are distinct from human-derived tau and that mouse tau is unsuitable for modeling human tauopathies8. However, human cell tauopathy models are not widely commercially available. The overall goal of this work is to describe an in vitro model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors containing mutant human tau constructs9. Tau aggregate causing lentiviral constructs encodes for the tau repeat domain harboring P301L and V337M mutations fused to a YFP reporter (Tau-RDLM-YFP) while control constructs code for the wild-type (Wt) tau repeat domain fused to a YFP reporter (Tau-Wt-YFP). Neuronal cultures transduced using this method express approximately nine times more tau than nontransduced cultures. Although the amount of tau expression overexpressed is roughly equal between Tau-RDLM-YFP- and Tau-Wt-YFP-transduced cells, only neurons transduced with Tau-RDLM-YFP display aggregates. Cultures transduced with Tau-RDLM-YFP stain positively for thioflavin and display reductions in axonal length and synaptic density. Therefore, this cellular model may be a useful tool for studying tau aggregation in vitro.

<table>
	<tbody>
		<tr>
			<td>10 cm culture dishes</td>
			<td>Thermofisher</td>
			<td>12556002</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>Biopioneer</td>
			<td>CNT-15</td>
			<td></td>
		</tr>
		<tr>
			<td>16% paraformaldehyde</td>
			<td>Thermofisher</td>
			<td>50-980-487</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well culture plates</td>
			<td>Thermofisher</td>
			<td>930186</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml tubes</td>
			<td>Biopioneer</td>
			<td>CNT-50</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol in spray bottle</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Thermofisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Basement membrane matrix (Matrigel)</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic FGF</td>
			<td>Biopioneer</td>
			<td>HRP-0011</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12 culture media with glutamine</td>
			<td>Thermofisher</td>
			<td>10565042</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (50% concentration or higher)</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Flourescently labeled secondary antibodies</td>
			<td>Various Sources, experiment dependent</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Thermofisher</td>
			<td>1254581</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Thermofisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Human neural stem cells</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentiviral vectors</td>
			<td>Various sources</td>
			<td>custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media</td>
			<td>Thermofisher</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Thermofisher</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermofisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Thermofisher</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td>Various Sources, experiment dependent</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocking or rotating platform</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cell culture hood</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioflavin S</td>
			<td>Sigma</td>
			<td>T1892-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Thermofisher</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro model for studying tau aggregation using lentiviral-mediated transduction of human neurons

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). Although mouse models of tauopathy have provided a valuable resource for investigating the neurotoxic mechanisms of aggregated tau, it is becoming increasingly apparent that, due to interspecies differences in neurophysiology, the mouse brain is unsuitable for modeling the human condition. Advances in cell culture methods have made human neuronal cultures accessible for experimental use in vitro and have aided in the development of neurotherapeutics. However, despite the adaptation of human neuronal cell cultures, in vitro models of human tauopathy are not yet widely available. This protocol describes a cellular model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors that code for pathogenically mutated tau fused to a yellow fluorescent protein (YFP) reporter. Transduced cultures produce tau aggregates that stain positively for thioflavin and display markers of neurotoxicity, such as decreased axonal length and increased lysosomal volume. This procedure may be a useful and cost-effective model for studying human tauopathies.

Tau-RDLM-YFP-transduced neurons were fluorescently tagged with YFP, and RDLM-transduced cultures displayed aggregates after transduction. These inclusions stained positive for thioflavin (Figure 1). As Figure 1 demonstrates, this protocol produces neuronal cultures that display thioflavin-positive tau aggregates. For initial experiments, it is recommended that neuronal differentiation is confirmed by immunolabeling the neuron-specific marker &#946;-tubulin III i...

Watch this Scientific Journal Video about An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons at JoVE.com

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

In this protocol we detail an in vitro model of human tauopathy. This method fills a materials need which may be useful for drug screening and disease mechanism studies. The main advantage of this technique is that the cell culture paradigm allows for a more rapid assessment of tau toxicity and potential tau therapeutics compared to animal studies To begin this procedure thaw the basement membrane matrix coating for culture plates at four degrees Celsius.Make one-milliliter aliquots and store them at minus 20 or minus 70 degrees Celsius. Next, reconstitute the basic fibroblast growth factor in sterile PBS at a concentration of 10 micrograms per milliliter. Make 10-microliter aliquots and store them at four degrees Celsius.Then set out a new unopened 500-milliliter bottle of DMEM-F12 with glutamine. Add 10 milliliters of B27, five milliliters of N2, and five milliliters of penicillin-streptomycin to prepare neural stem cell media. Place 50 milliliters of the NSC media into a conical tube, and add 10 microliters of the prepared bFGF.Store this NSC plus bFGF media at four degrees Celsius. First, remove an aliquot of frozen basement membrane matrix coating for cell culture plates from the freezer and it allow to thaw at four degrees Celsius. Add 385 microliters of the thawed basement membrane matrix coating to five milliliters of DMEM-F12 media containing penicillin-streptomycin.If cells are being thawed from frozen NSC stocks, take a vial of frozen cells from the freezer and warm it in a water bath heated to 37 degrees Celsius by moving the vial back and forth in the water. After this, spray the vial with 70%ethanol. Then under a cell culture hood, transfer the cells to approximately 10 milliliters of DMEM-F12 media containing penicillin-streptomycin.Centrifuge at room temperature at 1, 000 times g for five minutes. Then aspirate the media and resuspend the cells in NSC media. Dilute the cells in NSC media to obtain the appropriate seeding density for the cell culture vessel that is being used.Add enough cells suspended in NSC media to seed the basement membrane matrix coated culture dishes. Grow the cells at 37 degrees Celsius with 5%carbon dioxide until they are 75 to 80%confluent, making sure to change the media every other day. To transduce neurons with lentivirus, use a titer count of 340, 000 transducible units per cell.Dilute the transducible units in cell culture media to the necessary concentration, and add them to the cells. Two days after adding the lentivirus, wash the cells once with fresh media that does not contain bFGF. Continue culturing the cells at 37 degrees Celsius with 5%carbon dioxide in media without bFGF.Maintain the cells for about eight weeks after transduction, making sure to change the cell culture media every other day. Use a light microscope to routinely visualize the cells and ensure viability. After transduction, remove the culture dishes from the incubator making sure to keep the lid on them to keep the culture well sterile.Use a fluorescent microscope capable of live cell imaging to visualize the cells under a 10X objective with an excitation of 514 nanometers and an emission filter of 527 nanometers. In this study neurons transduced with tau-RDLM-YFP lentivirus are fluorescently tagged with YFP. RDLM-transduced cultures are seen to display aggregates after transduction.These inclusions stain positive for thioflavin. Neuronal differentiation is confirmed by immunolabeling of the neuron-specific marker beta-tubulin III in cultures. It is important to remember that fluorescently tagged secondary antibodies should have an excitation emission spectrum that does not overlap with that of YFP.Although yellow fluorescence tau aggregates will be visible in the absence of staining, thioflavin should also be used for imaging in order to confirm that the fluorescent signal is aggregated tau and not cellular debris. Following generation of tau overexpressing cells, a number of assessments of neuronal health and tau aggregation can be performed. For example, we have found that these cells display axonal degeneration.In addition to cell culture studies, we have used this method to examine the pathogenicity exosomal-derived tau.

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JoVE Journal

Neuroscience

5.7K vistas.  University of California, San Diego. En este protocolo detallamos un modelo in vitro de tauopatía humana. Este método llena una necesidad de materiales que pueden ser útiles para la detección de fármacos y estudios del mecanismo de la enfermedad. La principal ventaja de esta técnica es que el paradigma del cultivo celular permite una evaluación más rápida de la toxicidad tau y las posibles terapias tau en comparación con los estudios en animales para comenzar este procedimiento descongelar el recubrimiento de la matriz...