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/pubmed?term=((Neurodegenerative+disease+in+2015%3A+Targeting+tauopathies+for+therapeutic+translation%5BTitle%5D)+AND+%22Nature+Reviews+Neurology%22%5BJournal%5D)">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/pubmed?term=((Tau+stabilizes+microtubules+by+binding+at+the+interface+between+tubulin+heterodimers%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((Tau+in+physiology+and+pathology%5BTitle%5D)+AND+%22Nature+Reviews+Neurology%22%5BJournal%5D)">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/pubmed?term=((Characteristics+and+consequences+of+muscarinic+receptor+activation+by+tau+protein%5BTitle%5D)+AND+%22European+Neuropsychopharmacology%22%5BJournal%5D)">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/pubmed?term=((Tau-mediated+NMDA+receptor+impairment+underlies+dysfunction+of+a+selectively+vulnerable+network+in+a+mouse+model+of+frontotemporal+dementia%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Tau+reduction+in+the+presence+of+amyloid-beta+prevents+tau+pathology+and+neuronal+death+in+vivo%5BTitle%5D)+AND+%22Brain%22%5BJournal%5D)">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/pubmed?term=((Tau+reduction+diminishes+spatial+learning+and+memory+deficits+after+mild+repetitive+traumatic+brain+injury+in+mice%5BTitle%5D)+AND+%22PLOS+ONE%22%5BJournal%5D)">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/pubmed?term=((Accelerated+human+mutant+tau+aggregation+by+knocking+out+murine+tau+in+a+transgenic+mouse+model%5BTitle%5D)+AND+%22The+American+Journal+of+Pathology%22%5BJournal%5D)">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/pubmed?term=((Novel+human+neuronal+tau+model+exhibiting+neurofibrillary+tangles+and+transcellular+propagation%5BTitle%5D)+AND+%22Neurobiology+of+Disease%22%5BJournal%5D)">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/pubmed?term=((Trans-cellular+Propagation+of+Tau+Aggregation+by+Fibrillar+Species%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Cell-surface+marker+signatures+for+the+isolation+of+neural+stem+cells%2C+glia+and+neurons+derived+from+human+pluripotent+stem+cells%5BTitle%5D)+AND+%22PLOS+ONE%22%5BJournal%5D)">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/pubmed?term=((Passaging+human+neural+stem+cells%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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/pubmed?term=((Motifs+in+the+tau+protein+that+control+binding+to+microtubules+and+aggregation+determine+pathological+effects%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">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/pubmed?term=((Depletion+of+microglia+and+inhibition+of+exosome+synthesis+halt+tau+propagation%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">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 tau toxicity. This model fills a material need in neurodegenerative research as human tauopathy cell lines are not yet widely available and the use of transgenic tau from mouse-derived neurons requires animal breeding and is limited due to differences in neuronal characteristics between species.
In addition to those listed above, there are a number of critical steps for the success of this procedure. First, ensure that the correct viral titer is used because if the viral titer is too low, cultures will not form aggregates. Second, make sure that NSC cultures are properly maintained and do not exceed ~80% confluence. Overgrowth of NSCs will cause premature differentiation. Last, wait a full 4 weeks for neuronal cultures to differentiate after withdrawing the bFGF media from the NSCs. This duration is necessary to ensure maturation of the neurons.
Although the procedure described above is an efficient method for producing an in vitro tauopathy model, there are some limitations associated with this protocol. First, as described previously, this method produces NFTs in human induced pluripotent stem cell-derived neurons but not in rat embryonic (E18)-derived neurons. Even after using three times more virus to transduce rat neurons than was used for human neurons, rodent cell cultures remained negative for thioflavin staining, which suggests that this method cannot be adapted for rodent cultures. The differences between transduced mouse cells and human neurons may be due to the increased propensity of human tau toward aggregation13. The second limitation of this procedure is that although tau aggregates are formed in cultures, changes in tau phosphorylation between Tau-RDLM-transduced cells and Tau-Wt-YFP-transduced cultures were undetectable using protein PHF-1 (which detects tau phosphorylated at Ser 396/Ser 404) and CP13 antibodies (which detect tau phosphorylated at Ser 202). These findings suggest that tau aggregation in Tau-RDLM-YFP cultures due to P301L and V337M involves a mechanism that is independent of hyperphosphorylation, or the level of endogenous tau phosphorylation is under the detectable level by immunoblot. Because of the lack of differences in PHF-1 and CP13 immunoreactivity between Tau-RDLM-YFP and Tau-Wt-YFP cultures, it is unclear whether or not this model would be useful for studies analyzing the effects of tau kinase/phosphatase activity on pathology. However, both PHF-1 and CP13 antibodies recognize regions outside of the repeat domain harboring the mutations; therefore, additional antibodies raised against various tau phosphorylation sites may be useful for future studies.
In addition to cell culture assays, this model may be a valuable tool for studies beyond the cell culture paradigm. For instance, exosomes isolated from the media of transduced cells contain toxic tau species. Exosomes are small secretory vesicles released from nearly every cell type, and exosomes have been implicated in tau propagation14. Tau-RDLM neuronal exosomes contain human tau which is detectable by western blot9, and these exosomes are sufficient to produce tau inclusions in the na&#239;ve mouse brain. These inclusions are immunoreactive for antibodies specific for human tau (K9JA), but it is unclear whether or not the inclusions include aggregated mouse tau. The finding described here that the procedure does not produce thioflavin-positive staining aggregates in rodent neurons suggests that the inclusions observed in the mouse brain are composed entirely of human tau, although future studies are required to confirm the composition of in vivo deposits.
Given the apparent role of exosome-derived tau in tauopathies, the model described here may be a useful resource for examining the role of the exosome in mediating tau-induced neurodegeneration. In conclusion, this procedure produces an in vitro model of human tauopathy that has significant advantages over transgenic mouse neuronal systems and can be employed for a variety of preclinical analyses.

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>50 mL 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 in cultures. Importantly, fluorescently tagged secondary antibodies should have an excitation/emission spectrum that does not overlap with that of YPF (Cy3, for example). Although yellow fluorescent 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. Additionally, the example in Figure 1 does not include DAPI staining to label the cell nuclei as the excitation/emission of DAPI overlaps with that of thioflavin-S; alternative nuclei stains should be used with thioflavin-S if desired.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59433/59433fig1.jpg" /> 
Figure 1: Thioflavin staining of transduced cultures. Neurons transduced with tau-RDLM-YFP lentivirus were fixed and immunolabeled with the neuron-specific marker &#946;-tubulin III (red). Additionally, tau aggregates (YFP signal in green) were stained for thioflavin (blue). Scale bars = 25 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59433/59433fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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). ...

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5.9K Views.  University of California, San Diego. 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.

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

Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, recent evidence indicated that Tau is present also in other subcellular compartments including the nucleus where it is implicated in DNA protection, in rRNA transcription, in the mobility of retrotransposons and in the structural organization of the nucleolus. We have recently demonstrated that nuclear Tau is involved in the expression of the VGluT1 gene, suggesting a molecular mechanism that could explain the pathological increase of glutamate release in the early stages of Alzheimer&#39;s disease. Until recently, the involvement of nuclear Tau in modulating the expression of target genes has been relatively uncertain and ambiguous due to technical limitations that prevented the exclusion of the contribution of cytoplasmic Tau or the effect of other downstream factors not related to nuclear Tau. To overcome this uncertainty, we developed a method to study the expression of target genes specifically modulated by the nuclear Tau protein. We employed a protocol that couples the use of localization signals and the subcellular fractionation, allowing the exclusion of the interference from the cytoplasmic Tau molecules. Most notably, the protocol is easy and is composed of classic and reliable methods that are broadly applicable to study the nuclear function of Tau in other cell types and cellular conditions.

Tau is a neuronal protein present both in the cytoplasm, where it binds microtubules, and in the nucleus, where it exerts unconventional functions including the modulation of Alzheimer&#39;s disease-related genes. Here, we describe a method to investigate the function of nuclear Tau while excluding any interferences coming from cytoplasmic Tau.

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, ...

Genetics

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-&#946; (A&#946;) peptide.

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are provided.

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

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.

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

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Biology

Visualizing Proteins in Primary Hippocampal Neurons by Live&#45;Cell Imaging

Using Live&#45;Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal transport is disrupted in multiple neurodegenerative diseases, and projection neurons are particularly vulnerable because of the need to transport cellular materials over long distances and sustain substantial axonal mass. Pathological modifications of several disease-related proteins negatively affect transport, including tau, amyloid-&#946;, &#945;-synuclein, superoxide dismutase, and huntingtin, providing a potential common mechanism by which pathological proteins exert toxicity in disease. Methods to study these toxic mechanisms are necessary to understand neurodegenerative disorders and identify potential therapeutic interventions.
Here, cultured primary rodent hippocampal neurons are co-transfected with multiple plasmids to study the effects of pathological proteins on fast axonal transport using live-cell confocal imaging of fluorescently-tagged cargo proteins. We begin with the harvest, dissociation, and culturing of primary hippocampal neurons from rodents. Then, we co-transfect the neurons with plasmid DNA constructs to express fluorescent-tagged cargo protein and wild-type or mutant tau (used as an exemplar of pathological proteins). Axons are identified in live cells using an antibody that binds an extracellular&#160;domain of neurofascin, an axon initial segment protein, and an axonal region of interest is imaged to measure fluorescent cargo transport.
Using KymoAnalyzer, a freely available ImageJ macro, we extensively characterize the velocity, pause frequency, and directional cargo density of axonal transport, all of which may be affected by the presence of pathological proteins. Through this method, we identify a phenotype of increased cargo pause frequency associated with the expression of pathological tau protein. Additionally, gene-silencing shRNA constructs can be added to the transfection mix to test the role of other proteins in mediating transport disruption. This protocol is easily adaptable for use with other neurodegenerative disease-related proteins and is a reproducible method to study the mechanisms of how those proteins disrupt axonal transport.

Author Spotlight: Understanding the Impact of Pathological Proteins on Axonal Transport in Neurodegenerative Diseases

Here, we demonstrate how to combine transfection of primary hippocampal rodent neurons with live-cell confocal imaging to analyze pathological protein-induced effects on axonal transport and identify mechanistic pathways mediating these effects.

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal ...

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this principle, the purification of critical proteins required for the functioning of these pathways along with their native interacting partners has not only allowed the mapping of the protein constituents of these pathways, but has also provided a deeper understanding of how these proteins coordinate to regulate these pathways. Within this context, understanding a protein's spatiotemporal localization and its protein-protein interaction network can aid in defining its role within a pathway, as well as how its misregulation may lead to disease pathogenesis. To address this need, several approaches for protein purification such as tandem affinity purification (TAP) and localization and affinity purification (LAP) have been designed and used successfully. Nevertheless, in order to apply these approaches to pathway-scale proteomic analyses, these strategies must be supplemented with modern technological developments in cloning and mammalian stable cell line generation. Here, we describe a method for generating LAP-tagged human inducible stable cell lines for investigating protein subcellular localization and protein-protein interaction networks. This approach has been successfully applied to the dissection of multiple cellular pathways including cell division and is compatible with high-throughput proteomic analyses.

We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks.

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this ...

A Cell-Based Assay to Assess the Tau Protein Uptake by Microglia

Source: De Marco, D. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58576">Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia.</a>&#160;J. Vis. Exp. (2018)
This video showcases a cell-based assay quantifying Tau uptake by murine microglia using pH-sensitive fluorescent-labeled Tau aggregates, analyzed via fluorescence-activated cell sorting for a quantitative assessment.

1. BV-2 Microglia Cells Culture
NOTE: Handle BV-2 cells under Biosafety Level 2 containment. The BV-2 cell line produces an enveloped recombinant ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

Lentiviral-Mediated Transduction of Human Neurons for Tau Protein Aggregation Analysis

Source: Aulston, B., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59433">An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons.</a>&#160;
This video demonstrates the process of transducing human neuronal cells with a lentivirus encoding a mutant human tau protein tagged with yellow fluorescent protein (YFP) to study protein aggregation. It outlines the steps from viral entry to mutant tau expression, culminating in the formation of visible cytoplasmic tau aggregates in the neurons.

Source: Aulston, B., et al. An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons.&#160;
This video ...

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

Summary

Explore More Videos

Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

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

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Using Live-Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

A Cell-Based Assay to Assess the Tau Protein Uptake by Microglia

Lentiviral-Mediated Transduction of Human Neurons for Tau Protein Aggregation Analysis