Research on neurodegenerative disease is advancing slowly in part because most models aren’t very good. In the February 15 Journal of Experimental Medicine, researchers led by Todd Golde at the University of Florida, Gainesville, debuted a new system for studying proteinopathies in a dish. They transduced mouse brain slices with genes for mutant human tau, and produced abundant neurofibrillary tangle pathology within one month. Overexpression of human α-synuclein sparked Lewy body formation in the same time frame. In addition, the researchers used different promoters to target expression to specific types of brain cell. The system offers a fast, versatile option for drug screening and mechanistic studies of neurodegenerative disease, the authors suggested.
- Viral transduction of mutant human tau produces robust tauopathy in mouse brain slices.
- Transduction of human α-synuclein gives rise to Lewy bodies.
- Model system may accelerate drug testing and mechanistic studies.
Peter Reinhart at the University of Massachusetts, Amherst, agreed. “This opens the door for new types of studies,” he wrote to Alzforum.
Research on mouse models is not only slow; it’s also expensive. Cell cultures, while offering faster testing, contain neither all the cell types nor the architecture present in the brain, and thus often fail to predict what will happen in vivo. Golde and colleagues wondered if slice cultures could represent a happy medium.
To culture slices, first author Cara Croft dissected the cortices, hippocampi, and connecting regions from eight-day-old wild-type mice, then cut them into 350 μm thick coronal slices. The scientists transduced the slices with recombinant adeno-associated viruses (rAAV). They compared different types of protein coat packaging, or capsids, to identify those that produced the highest gene expression. Distinct promoters restricted transgene expression to neurons, oligodendrocytes, astrocytes, or microglia. Transgene expression lasted from three to nine months.
Could this work to model tauopathy? Using a general promoter that targets all cells, the authors transduced the slices with several different human tau genes: wild-type, the pro-aggregant S320F mutation, a S320F/P301L, and a S320F/P301L/A152T combination. All of these bumped up the abundance of phosphorylated tau after 28 days in culture over vector control, but only S320F/P301L and S320F/P301L/A152T also caused tau inclusions in cell bodies. The deposits stained with Thioflavin S, and electron microscopy confirmed the presence of tau filaments, indicating these were bona fide neurofibrillary tangles. The tangles accumulated throughout the brain slice, with thousands apparent after one month in culture. At this time point, rates of cell death were the same as in control slices; however, by 60 days, about three times as many dead cells had accumulated in the tauopathy slices as in controls.
This slice model could be used to screen drugs and predict their likely in-vivo efficacy, the authors suggested. As a proof of concept, they tested four different GSK3β inhibitors on the tauopathy slice cultures from day 14 to 28. The GSK3β enzyme phosphorylates tau, but only one of the four inhibitor drugs suppressed paired helical filaments and tangles in this milieu. This underlines the idea that only some therapies will work in the brain, the authors noted.
In ongoing work, the authors are examining how tangles change over time and how they might trigger cell death, as well as how different mutations affect seeding. They are collaborating with academic and industry researchers to test potential tau therapeutics in this system. “Our goal is to stress-test this system to see if it is predictive of effects in tau mice,” Croft and Golde wrote to Alzforum.
Could these slice cultures model other protein-aggregation diseases as well? Perhaps. Slices transduced with human wild-type or A53T α-synuclein developed Lewy bodies by 28 days in vitro, and immunostaining and electron microscopy confirmed the presence of α-synuclein fibrils. The authors are collaborating to establish slice models of additional neurodegenerative diseases.
“Using rAAV promoter-capsid combinations, we can flexibly and cost-effectively manipulate expression of genes in all the major central-nervous-system cell types in organotypic brain slice cultures in a manner which was previously not possible. This will no doubt accelerate many research studies in neurodegenerative proteinopathies and likely be leveraged by neuroscientists in many research areas,” they wrote.—Madolyn Bowman Rogers
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- Croft CL, Cruz PE, Ryu DH, Ceballos-Diaz C, Strang KH, Woody BM, Lin WL, Deture M, Rodríguez-Lebrón E, Dickson DW, Chakrabarty P, Levites Y, Giasson BI, Golde TE. rAAV-based brain slice culture models of Alzheimer's and Parkinson's disease inclusion pathologies. J Exp Med. 2019 Mar 4;216(3):539-555. Epub 2019 Feb 15 PubMed.