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), N2 (5 mL), and penicillin-streptomycin (5 mL). Place 50 mL of this neural stem cell (NSC) media in a conical tube and add 10 &#181;L of 10 &#181;g/&#181;L (2 &#181;g/mL final) bFGF. Store the NSC (+)bFGF media at 4 &#176;C.</li>
	<li>To make (-)bFGF media, use the same recipe as described in step 1.3 but do not add bFGF. This media will be used to differentiate NSCs to neurons and to maintain neuronal cultures after differentiation.</li>
</ol>
2. Lentiviral Constructs
NOTE: Before beginning work with lentiviral constructs, ensure that the lab has been approved to use biosafety level-2 (BSL-2) agents. Furthermore, BSL-2 culture hoods, personal protective equipment (PPE), and disposal methods must be used when working with lentiviral vectors.
<ol>
	<li>Obtain tau constructs packaged into lentivirus from a preferred source (see Sanders et al.10 for construct information).</li>
</ol>
3. Culturing Human Neural Stem Cells
NOTE: NSCs are typically seeded at 100,000&#8211;150,000 cells/cm2 and most commercially available NSCs are sold as 1 x 106 cells/vial. This protocol has been optimized for 10 cm cell culture dishes (although other sizes of dishes may be used); therefore, if commercially available NSCs are being used, the NSCs may need to be expanded by first being cultured in six-well dishes in order to result in enough cells to seed 10 cm dishes. This protocol can alternatively be adapted for a variety of cell culture dish sizes (but it does not contain instructions for passaging NSCs as these protocols are available elsewhere11,12).
<ol>
	<li>For the preparation of cell culture plates, remove one aliquot of frozen basement membrane matrix coating for cell culture plates and allow it to thaw at 4 &#176;C (aliquots can be placed at 4 &#176;C overnight the day before the cells are to be seeded). Add 385 &#181;L of basement membrane matrix coating to 5 mL of DMEM/F12 media + penicillin-streptomycin. 
	NOTE: 5 mL of DMEM/F12 media + penicillin-streptomycin is enough to coat a single 10 cm dish (1 mL of this basement membrane matrix coating solution is sufficient to coat one well of a six-well dish). Alternatively, if the cells are to be fixed and immunostained, or stained for thioflavin, culture cells on glass coverslips in 24-well culture dishes.
	<ol>
		<li>Keep the media and the matrix coating cold before and while adding them to culture dishes. Add the basement membrane matrix coating to culture dishes and place the dishes in an incubator (at 37 &#176;C) for 1 h. For optimal coating, do not incubate the basement membrane matrix for more than or less than 1 h. After 1 h, aspirate the basement membrane matrix coating from the cell cultures dishes. Make sure this coincides with the NSCs being ready to be plated. 
		NOTE: If cells are not being thawed from frozen stocks, skip step 3.2 and continue with step 3.3.</li>
	</ol>
	</li>
	<li>If cells are being thawed from frozen NSC stocks, take a vial of frozen cells and warm it in a water bath heated to 37 &#176;C by moving the vial back and forth in the water. Once thawed, spray the vial with 70% ethanol and place it into a cell culture hood. Transfer the cells to ~10 mL of DMEM/F12 + penicillin-streptomycin media and centrifuge the tube at room temperature at 1,000 x g for 5 min. Aspirate the media and resuspend the cells in NSC media.</li>
	<li>Dilute the cells in NSC media to obtain the appropriate seeding density for the cell culture vessel that is being used. 
	NOTE: For example, if NSCs are being plated onto a six-well plate&#8212;one well on a six-well culture dish has a surface area of 9 cm2&#8212;900,000 to 1,350,000 cells are required to seed. So, one vial containing 1,000,000 cells can be resuspended in 2 mL of media and added to a single well of a six-well dish.</li>
	<li>Add enough cells suspended in NSC media to seed the basement membrane matrix-coated culture dishes (100,000 cells/cm2) and change the media every other day (if using a frozen stock, change the media the day after plating and every other day thereafter). Grow the cells until they are 75%&#8211;80% confluent. 
	NOTE: The cells will likely reach 75%&#8211;80% confluence shortly after plating, but this may take longer if frozen stocks are used.</li>
	<li>Once the NSCs are 75%&#8211;80% confluent, begin neuronal differentiation by removing the NSC (+)bFGF media and replacing it with NSC (-)bFGF media. Culture the cells in this media (replacing the media every other day) for at least 4 weeks. 
	NOTE: The cells will continue to divide for a few days following the withdrawal of bFGF; therefore, cells may reach 90&#8211;100% confluence. After culturing for 4 weeks, the cells will have achieved a neuronal fate and will be ready for the treatment of lentivirus.</li>
</ol>
4. Transduction and Maintenance of Neuronal Cultures
<ol>
	<li>To transduce neurons with lentivirus, use a titer count of 3.4 x 105 transducible units/cell (a 100% confluent 10 cm dish will contain ~10 million neurons). Dilute the transducible units to the necessary concentration in cell culture media and add them to the cells (use the normal volume of media for the cell culture dish). Two days after adding the lentivirus, wash the cells 1x with fresh (-)bFGF media (without lentivirus) and continue culturing in (-)bFGF media as usual.</li>
	<li>For the post-lentiviral treatment, feed the transduced neurons by changing the cell culture media ([-]bFGF media) every other day. Maintain the cells for ~8 weeks after transduction. Routinely visualize the cells under a light microscope to ensure viability. 
	NOTE: Sparseness or dendritic breakages are signs that the cells are no longer viable.</li>
</ol>
5. Imaging of Cells
<ol>
	<li>Live-cell imaging 
	<ol>
		<li>After transduction (4 days after the addition of lentivirus), observe YFP signals under a fluorescent microscope capable of live-cell imaging. Take the cell culture dishes out of the incubator and make sure the culture wells remain sterile by keeping the lid on top of the culture dishes. Visualize the cells using a 10x objective with an excitation wavelength of ~514 nm and an emission filter of ~527 nm. 
		NOTE: Aggregates are typically visible in transduced cell cultures 6&#8211;8 days after the application of lentivirus.</li>
	</ol>
	</li>
	<li>Fixed-cell staining (&#946;-tubulin III labeling and thioflavin staining)
	<ol>
		<li>To fix the cells in paraformaldehyde (PFA), remove the culture media from transduced neurons grown on glass coverslips. Wash the cells 1x with PBS, remove the wash, and add 300&#8211;500 &#181;L (if using 24-well plates) of 4% PFA (diluted in PBS) to the coverslips for 20 min at room temperature. Remove the PFA and wash the coverslips 2x with PBS.</li>
		<li>Prepare a blocking solution consisting of PBS, 3% bovine serum albumin (BSA), and 0.3% Triton X-100. Add 300&#8211;500 &#181;L of the solution to the wells and incubate the coverslips for 2 h at 4 &#176;C. After 2 h, dilute primary antibodies (at an antibody concentration of 1:500) in blocking solution and add 300&#8211;500 &#181;L of primary antibody solution to each well. Place the plate on a rocking or rotating platform (at low speed) overnight at 4 &#176;C.</li>
		<li>The next day, remove the primary antibody solution and wash the coverslips 3x for 5 min each with PBS. Dilute secondary antibodies in a blocking buffer solution (at a concentration of 1:1,000) and add secondary antibody solution to each well. Select the appropriate secondary antibody by using a fluorescent tag that does not overlap with the YFP signal (CY-3, for example). Incubate the cells at room temperature for 2 h on a rocking or rotating platform. After 2 h, remove the secondary antibody solution and wash the coverslips 3x for 10 min each with PBS.</li>
		<li>Wash the coverslips in double-deionized water (DDW) for 10 min. Remove the DDW and add 300 &#181;L/well of 0.015% thioflavin-S diluted in 50% ethanol for 10 min. Remove the thioflavin solution and wash the coverslips 2x for 4 min each with 50% ethanol, followed by one 4 min wash with 30% ethanol and two washes for 5 min each with 30% ethanol. Wash the coverslips 1x with DDW prior to mounting. 
		NOTE: The thioflavin staining described in step 5.2.4 is optional.</li>
		<li>To mount the coverslips to glass slides, place a single drop of mounting media on the &#8220;+&#8221; side of a glass slide. Remove all liquid from the well containing the glass coverslip and, using fine forceps, carefully remove the glass coverslip, keeping track of which side has the cultured cells.</li>
		<li>Gently press the edge of the coverslip against a Kimwipe to remove any excess moisture. Still gripping the coverslip with fine forceps, touch the edge (with the cell side facing toward the drop of mounting media) of the coverslip to the mounting media, and then, gently place the coverslip on the mounting media (placing the cultured cells into the mounting media and sandwiching them between the coverslip and the glass slide). Allow the mounting media to harden for 30 min prior to imaging. The slides can be stored at -20 &#176;C for future use.</li>
		<li>Image the cells using a fluorescent microscope and the appropriate excitation/emission spectra (YFP = ~514/527 nm, CY-3 = ~555/568 nm, and thioflavin S = ~390/426 nm).</li>
	</ol>
	</li>
</ol>
6. Optional Methods
<ol>
	<li>Every 2 days, collect conditioned media from the cultures and store it at -20 &#176;C for future analyses of tau species released by cultures.</li>
</ol>

<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>

The authors have nothing to disclose.

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

ヒトニューロンのレンチウイルス媒介性形質導入を用いたタウ凝集を研究するための in Vitro モデル

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). ...

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Neuroscience

5.8K ビュー.  University of California, San Diego. このプロトコルでは、ヒトタウオパチーのインビトロモデルを詳述する。この方法は、薬物スクリーニングおよび疾患メカニズム研究に有用であり得る材料の必要性を満たす。この技術の主な利点は、細胞培養パラダイムが動物研究と比較してタウ毒性および潜在的タウ治療薬のより迅速な評価を可能にすることであるこの手順を開始するには、培養プレートの基基膜?...

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

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

アダルト生まれのニューロンの統合を検討し

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

神経科学

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

嗅神経細胞のレンチウイルスを介した遺伝子操作と可視化 in vivoで</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性のためのアダルト生まれの嗅覚ニューロンの選択的なウイルス形質導入 in vivoで Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

不定冠詞<em&gt;インビトロ</em成体マウスの筋層間神経叢から分離腸ニューロンとグリアの&gt;準備

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell leukodystrophy (GLD) is unclear. This gap in knowledge has been hindered by the lack of an appropriate in vitro model for study. Herein, we describe a primary murine glial culture system in which treatment with psychosine results in multinucleation of microglia resembling the characteristic globoid cells found in GLD. Using this novel system, we defined the conditions and modes of analysis for study of globoid cells. The potential use of this model system was validated in our previous study, which identified a potential role for matrix metalloproteinase (MMP)-3 in GLD. This novel in vitro system may be a useful model in which to study the formation and function, but also the potential therapeutic manipulation, of these unique cells.

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

Globoid cells are a defining pathological feature of Krabbe disease, a leukodystrophy currently lacking an effective long-term therapy. We have developed a cell culture model to study the innate biology and pathogenic potential of activated microglia and their transformation into globoid cells.

アン<em&gt;体外</em球様細胞白質萎縮症における細胞病態の研究のための&gt;モデル

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

アン<em&gt;体外</em筋求心性神経の焼成特性を研究するための&gt;成体マウス筋肉神経の準備

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

根神経節ニューロンと差別化された脂肪由来幹細胞を背：<em&gt;インビトロ</em末梢神経再生を研究するために&gt;共培養モデル

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

微小電極アレイへの3Dエンジニア神経文化のインタフェース：革新的な<em&gt;インビトロ</em&gt;実験モデル

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

蛍光プローブを用いた培養ニューロンにおけるエンドソームおよびリソソームの動力学の定量

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

The tau aggregation assay described in this manuscript mimics the anticipated features of in vivo tau misfolding and aggregation.

体外タウ蛋白と創薬スクリーニングの集計を勉強するための試金

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other ...