Don your IMAX glasses. Researchers have taken a step closer to modeling Alzheimer’s disease in a dish by using a three-dimensional gel matrix. As described in Nature on October 12, researchers led by Doo Yeon Kim and Rudolph Tanzi at Harvard Medical School in Charlestown, Massachusetts, constructed a human neural cell culture system that develops both amyloid plaques and neurofibrillary tangles. Neurons in the three-dimensional culture overexpress disease-associated mutations that ramp up Aβ production, and tau pathology ensues. The system is the first to reveal a direct causal link between Aβ production and tau pathologies, Tanzi told Alzforum, and may serve as a handy tool to screen drugs that target them.
“It’s really beautiful work, and is the first demonstration of a human model of AD,” commented Terrence Town of the University of Southern California in Los Angeles. “The tau pathology appears to be consequent to the amyloid pathology, and that’s key because it supports the amyloid hypothesis.”
The amyloid hypothesis was born 30 years ago, when researchers zeroed in on the identity of the amyloid fibrils that dominated AD brains, and eventually linked familial forms of the disease to APP (see Glenner and Wong, 1984; Yankner et al., 1989; and Levy et al., 1990). The core tenet of the hypothesis is that Aβ drives AD pathology, including the development of neurofibrillary tau tangles (see Hardy and Selkoe, 2002), but modeling this in any one system has been an elusive goal. Mice engineered to ramp up Aβ production and accumulate amyloid develop no tau pathology unless other transgenes are added. Mice expressing mutant forms of tau do develop tauopathy (for example, the Tau P301S and 3xTg lines), but the consensus is that these model frontotemporal dementia rather than AD (see Chin, 2011).
One hint of Aβ being upstream of tau in mice, too, came from a study that detected an age-related tau increase in the CSF of aging mice that express APP mutations (Maia et al., 2013). Town’s lab developed an APP-based rat model that develops tauopathy, likely because rats, unlike mice, express six isoforms of tau—as do humans—but it is not yet widely used (see Cohen et al., 2013).
Cell culture models have fared no better. A range of Aβ oligomers can be produced, but larger insoluble amyloid plaques do not form. “To date, we have not had a model for AD that makes plaques in a dish, let alone a model where plaques actually lead to tangles,” Tanzi told Alzforum.
In an attempt to unite Aβ and tau pathologies into a single model, co-first authors Se Hoon Choi and Young Hye Kim started with a human two-dimensional neuron culture and then added some structure. They selected a commercially available human neural progenitor cell line—monoclonal cells derived from fetal neural stem cells—as the star of their model. They infected the progenitor cells with lentiviral vectors carrying human APP harboring the Swedish and London familial AD (FAD) mutations, with or without the FAD-linked PSEN1ΔE9. After six weeks in culture conditions that promote neuronal differentiation, the neurons overexpressing mutant APP pumped out 19-fold more Aβ42 than control cells, and co-expression of PSEN1Δ9 boosted Aβ production another fivefold.
The researchers hypothesized that if Aβ did not wander away in the culture medium, it might form deposits. To corral Aβ, the researchers grew the neural cultures within a three-dimensional support gel chock-full of brain extracellular matrix proteins. In this structured environment, the progenitor cells differentiated into more mature neurons than those in the two-dimensional culture system, and also expressed higher levels of 4-repeat adult tau isoforms, which are thought to be essential for the development of tauopathy. Six weeks later, confocal microscopy revealed large amyloid deposits lurking in three-dimensional cultures that expressed FAD mutants. The aggregates reacted with several different Aβ-specific antibodies, as well as Congo Red and AmyoGlo, an amyloid-specific dye. Treatment with β- or γ-secretase inhibitors dramatically reduced these deposits.
Tau pathology also developed. By six weeks in culture, western blots revealed an accumulation of soluble and insoluble hyperphosphorylated tau. By immunohistochemistry, the researchers noted some neurons harbored highly elevated levels of phosphorylated tau. They also displayed odd morphologies, such as neurites with the same beaded processes that have been found in the brains of AD patients. To the researchers’ surprise, treatment of the cultures with either β- or γ-secretase inhibitors decreased levels of phospho-tau in extracts as well as the number of cells accumulating these isoforms in culture.
In cultures enriched for cells that expressed the most APP and PSEN1, the researchers found an abundance of phosphorylated tau in neurites and neuronal cell bodies. High molecular weight, hyperphosphorylated forms of tau dominated insoluble fractions (see image below). When the researchers looked at these aggregates with transmission electron microscopy, they observed filaments strikingly similar to those found in AD. Treatment with β- or γ-secretase inhibitors dramatically reduced production of these tau isoforms in the neurons, suggesting a link between Aβ production and tauopathy. By 10 weeks in culture, the neurons contained inclusions detectable by silver staining, a classic measure of tau tangles.
Interestingly, when the researchers treated cultures with an inhibitor of GSK3β, a tau kinase implicated in AD, phosphorylated tau, but not Aβ, fell dramatically. This indicated that the kinase does indeed mediate Aβ-triggered tau pathology and that tau lies downstream of Aβ.
While these cultures may have captured the pathological cascade that can occur among neurons in AD brains, they did not account for the potential role of other cell types, such as microglia and astrocytes. That Aβ induced tau pathology in the absence of immune cells, such as microglia, suggests that inflammation may not be required for Aβ to drive tauopathy, Tanzi said. Such an intermediary step had been proposed by others as part of the amyloid cascade hypothesis. Kim added that the role of microglia should not be overlooked, however. “We cannot discount the impact of microglia on this process, because we may see increases in pathology when we add those cells in,” he said. Kim and Tanzi said they plan to more carefully scrutinize the potential role of neuroinflammation by including microglia in their cultures, among other modifications. “We are just at the beginning,” Tanzi said.
Town agreed that the model was limited in its current form, but could be expanded to include neuroinflammatory or vascular components in the future. “It’s very hard to get one model to study everything,” he said.
The culture model will also allow the researchers to more carefully tease out the links between Aβ production and tauopathy, including how GSK3β serves as an intermediary, which species of Aβ drive tauopathy, and how these factors may influence neurodegeneration, the authors said. Researchers are still not sure which form of Aβ is most toxic. “Given that this is up for debate right now, it is very valuable to have a system where you can actually manipulate plaques and tangles,” commented Asa Abeliovich of Columbia University in New York, who was not involved in the work. Abeliovich was impressed with the level of tau pathology the researchers observed in their system. “There’s a tremendous need to have accurate disease models of neurodegeneration, and it’s tremendously difficult to come up with,” he said. “I like the simplicity of their system.”
Would such a culture model work with AD patient-derived cells, such as iPSCs? Generating amyloid and tau pathology in such models would be difficult without overexpression, Tanzi said. “If you want to recapitulate in two to three months pathology that takes decades to form in the human brain, you most likely have to rely on overexpression,” he said. Other researchers agreed.
Tanzi sees the culture model as a boon to drug discovery, particularly for drugs aimed at knocking down amyloid and/or tau pathologies. “This cell culture model could make drug screening 10 times faster and 10 times cheaper than testing in animal models,” he speculated.
David Holtzman of Washington University in St. Louis, who was not involved in the work, agreed that the culture system would make for a good screen. “This is now a model system to potentially rapidly test new treatments targeting Aβ, tau, or even other targets as new treatments,” he wrote to Alzforum (see full comment below).—Jessica Shugart
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