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

Summary

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.

Abstract

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer’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.

Introduction

Pathological aggregation of the microtubule-associated protein tau is a defining feature of many neurodegenerative diseases, including AD, frontotemporal dementia (FTD), Pick’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 receptors

Protocol

1. Preparation of Media and Reagents

1. Thaw the basement membrane matrix coating for culture plates at 4 °C (do not allow the basement membrane matrix to warm up or it will solidify). Make 1 mL aliquots and store them at -20 °C or -70 °C.
2. Reconstitute basic fibroblast growth factor (bFGF) in sterile phosphate-buffered saline (PBS) at 10 µg/mL and make 10 µL aliquots. Store them at 4 °C.
3. 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 µL of 10 µg/....

Results

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 β-tubulin III in cul.......

Discussion

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 .......

Materials

List of materials used in this article

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