Four years ago, researchers made a splash with the first in vitro model of Alzheimer’s pathology. In that advance, dubbed “Alzheimer’s in a dish,” neurons and astrocytes grown in a three-dimensional gel matrix produced amyloid plaques and hyperphosphorylated tau, proving that amyloid somehow begets tau. Now, Doo Yeon Kim and Rudy Tanzi of Massachusetts General Hospital in Charlestown and Hansang Cho of the University of North Carolina, Charlotte, have taken the model to a new, lethal dimension. Using a sophisticated, multi-chambered microfluidic chip, they mimicked the microglia activation, invasion, and cell death seen in AD brain. Their “AD on a chip” suggests that neuron- and astrocyte-derived chemokines lure microglia to sites of amyloid accumulation, where the microglia then set off a neuroinflammatory pandemonium that leads to severed axons and dead cells. Blocking migration or downstream inflammatory mediators spared neurons, the authors showed. The work appeared June 27 in Nature Neuroscience.
- AD on a chip: Tri-culture of neurons and glia breeds neuroinflammation.
- Chemokines, cytokines spur microglia activation, infiltration, astrogliosis, and neuronal death.
- Microfluidic system can help unravel pathways of neurodegeneration.
“This new and improved three-dimensional culture holds great promise for understanding the contribution of innate immunity to AD evolution and for preclinically evaluating experimental therapeutics that target it,” wrote Terrence Town, University of Southern California, in an email to Alzforum (see full comment below).
The work “lays the foundation for some important future studies,” wrote Christopher Henstridge and Tara Spires-Jones in a commentary accompanying the paper. “Many AD risk factors are in genes highly expressed in microglia, such as ApoE, Trem2 and Clusterin, and the system could be used to discover how these genes affect microglial influence on AD progression,” they wrote.
Clean Cut. In the three-dimensional tri-culture, microglia (red) appear to sever the axon of a neuron (green). [Courtesy of Park et al., Nature Neuroscience 2018.]
In their previous three-dimensional system, Doo and Tanzi demonstrated unequivocally that amyloid deposition came first, and led to formation of tau deposits resembling neurofibrillary tangles (Oct 2014 news). If the scientists blocked amyloid production, they never saw tau pathology emerge. However, the cells happily co-existed with plaques and tangles, giving no hint of neurodegeneration. Clearly, the model lacked a critical piece. Could it be inflammation?
To add that dimension, the researchers used a microfluidic, chip-based system. While a postdoc at MGH, Cho, now an assistant professor in Charlotte, had developed a model for microglia activation and chemotaxis using a circular chip with an inner central chamber connected to a surrounding outer space by narrow channels, like spokes on a wheel. Previously, Cho’s group showed they could load the central chamber with Aβ or other chemoattractants, which would diffuse out through the spokes. That activated microglia waiting in the outer chamber, and induced them to migrate into the central space (Cho et al., 2013).
In the new study, first author and Cho postdoc Joseph Park used the three-dimensional neuron and astrocyte culture as the central lure. He seeded human neuron progenitors overexpressing human APP harboring the Swedish and London familial AD (FAD) mutations into a three-dimensional gel matrix, which he loaded into the middle chamber. There, over three weeks, the cells differentiated into a mix of neurons and astrocytes. After nine weeks, ELISA assays revealed the cells produced excess soluble Aβ40, 42, and phospho-tau. The cells also accumulated aggregated Aβ42. Using PHF-1 and AT8 p-tau antibodies, the investigators found the protein building up in cell bodies and neurites, and the cultures showed neuronal hyperactivity.
Park found that by week nine, the neurons and astrocytes produced immune mediators, including threefold more of the monocyte chemotactic factor CCL2 (aka MCP-1) and eight times more TNFα compared with similar cultures of cells without human APP. The cultures also made IFNγ, a microglia-activating cytokine absent from two-dimensional cultures of the same cells.
To ask if CCL2/MCP-1 made by the three-dimensional cultures could recruit microglia, the investigators added human microglia to the outer chamber, and watched. Within 48 hours, the microglia lost their resting morphology, became elongated and enlarged, upregulated the activation marker CD11b, and began a mass migration though the channels into the central chamber.
A neutralizing antibody to CCL2/MCP-1 partially blocked the migration, indicating that it was one, but not the only, substance responsible for attracting microglia. Other factors, such as Aβ itself and ATP, could also play a role, the authors suggested.
Once the microglia reached their destination, chaos ensued. The cultures ramped up expression of chemotactic factors and inflammatory cytokines, including MCP-1, IL-6, and Il-8. Astrogliosis set in. The microglia attacked neurons outright, damaging axons and causing neurite retraction. At the same time, they boosted levels of neurotoxic TNFα and nitric oxide (NO) by 1.5- and ninefold, respectively. Six days after the microglia invaded, the cultures had lost a fifth of their neurons and astrocytes. This was not seen in otherwise identical tri-cultures in which the neurons did not express APP, or in less-mature cultures where Aβ levels were lower.
Could damping down microglia prevent this destruction? The answer was yes. When the researchers added neutralizing antibodies to IFNγ, or antagonists of toll-like receptor 4 (TLR4) to the tri-culture, they saw a reduction in TNFα and NO, and less cell death. TLR4 knockdown in microglia alone partially protected the tri-cultures. Neither treatment affected microglia infiltration, suggesting that the TNFα and NO released by microglia delivered the deadly blow.
The results begin to unravel the sequence of complex interactions that underlie neuroinflammation and cell death, Tanzi told Alzforum. “This shows us the first release of cytokines and chemokines is starting from neurons and astrocytes. This excites and recruits the microglia, but only when they get to the party does all hell break loose. They start producing tons of cytokines, and only then do you see reactive astrocytes and widespread neuroinflammation and neurodegeneration,” he said.
Importantly, this all starts with amyloid and tau. “If you have wild-type neurons, the microglia hang out and they don’t care about coming in. But if the neurons are making Aβ and tangles, they come swimming in fast,” Tanzi said.
Major Retraction: Microglia (red) swarm a neuron (green), which promptly withdraws its neurite. [Courtesy of Park et al., Nature Neuroscience 2018.]
At the moment, the results raise more questions than answers. “We see now there’s a complicated dance going on, a back-and-forth between neurons, astrocytes, and microglia,” Tanzi said. “Now we can start deconvoluting those relationships, in a system where we can ask questions one at time or several at a time, and genetically or pharmacologically manipulate each of the cells. And we can do those experiments in five or six weeks, rather than waiting a year or more for a mouse.”
Knowing the sequence of events offers new possibilities for intervening early to choke off pathology. For example, Tanzi said his group is exploring small molecules that inhibit MCP-1 release from astrocytes. “That may be a nice early step after neurons have made Aβ and tangles, and prior to microglia infiltration. If you can prevent that chemokine response, you can nip neuroinflammation in the bud,” he said.
The investigators replicated their results using a different source of neurons and astrocytes. They started with neural stem cells newly generated from human IPSCs overexpressing the same APP mutants. When they differentiated the cells in the same culture system, the astrocytes and neurons were able to entice microglia to migrate, and produced cell loss. This result opens the door to using a wider variety of cells, including patient samples, in the system.
An important next step will be to incorporate other types of microglia, Malu Tansey at Emory University in Atlanta wrote to Alzforum (full comment below). In the current configuration, the scientists use a convenience line of immortalized human microglia. Recently, several groups have developed protocols to efficiently generate microglia from human iPSCs (Mar 2018 news; Muffat et al., 2016; Abud et al., 2017; Haenseler et al., 2017). Tanzi said his group is switching over to primary IPSC-derived microglia, which recently became commercially available.
For drug discovery, the new system opens up the possibility of screening for drugs that hit not only plaques and tangles, but also neuroinflammation at the level of microglia and astrocyte activation. The current microfluidic chip harbors 25 individual chambers on a single plate. Cho told Alzforum his group is developing a second-generation version with 120 devices per plate. The technology is generating interest from pharmaceutical companies, he said.—Pat McCaffrey
- Alzheimer’s in a Dish? Aβ Stokes Tau Pathology in Third Dimension
- Derived Human Microglia Manage Without Functional TREM2
- Cho H, Hashimoto T, Wong E, Hori Y, Wood LB, Zhao L, Haigis KM, Hyman BT, Irimia D. Microfluidic chemotaxis platform for differentiating the roles of soluble and bound amyloid-β on microglial accumulation. Sci Rep. 2013;3:1823. PubMed.
- Muffat J, Li Y, Yuan B, Mitalipova M, Omer A, Corcoran S, Bakiasi G, Tsai LH, Aubourg P, Ransohoff RM, Jaenisch R. Efficient derivation of microglia-like cells from human pluripotent stem cells. Nat Med. 2016 Nov;22(11):1358-1367. Epub 2016 Sep 26 PubMed.
- Abud EM, Ramirez RN, Martinez ES, Healy LM, Nguyen CH, Newman SA, Yeromin AV, Scarfone VM, Marsh SE, Fimbres C, Caraway CA, Fote GM, Madany AM, Agrawal A, Kayed R, Gylys KH, Cahalan MD, Cummings BJ, Antel JP, Mortazavi A, Carson MJ, Poon WW, Blurton-Jones M. iPSC-Derived Human Microglia-like Cells to Study Neurological Diseases. Neuron. 2017 Apr 19;94(2):278-293.e9. PubMed.
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No Available Further Reading
- Park J, Wetzel I, Marriott I, Dréau D, D'Avanzo C, Kim DY, Tanzi RE, Cho H. A 3D human triculture system modeling neurodegeneration and neuroinflammation in Alzheimer's disease. Nat Neurosci. 2018 Jul;21(7):941-951. Epub 2018 Jun 27 PubMed.
- Henstridge CM, Spires-Jones TL. Modeling Alzheimer's disease brains in vitro. Nat Neurosci. 2018 Jul;21(7):899-900. PubMed.