3D Model Unravels How Beta-amyloid Weakens Brain-Blood Barrier, May Aid in Treatment Discovery

A new 3D model using human cells grown on a chip can mimic what happens in the brain during Alzheimer’s and allow for the screening of possible therapies in a disease where so many fail, including medicines already approved for other conditions.

This tissue model also helps researchers see how the disease’s hallmark protein clumps damage the protective brain–blood barrier (BBB), allowing harmful substances to enter the brain of patients and cause further injury to nerve cells, its developers said.

Their study, “Blood–Brain Barrier Dysfunction in a 3D In Vitro Model of Alzheimer’s Disease,” was published in the journal Advance Science.

The brain–blood barrier (BBB) is a highly selective membrane that shields the central nervous system (brain and spinal cord) from circulating blood, preventing damaging and toxic substances from entering the brain. This protective layer is also responsible for clearing out the toxic beta-amyloid protein that marks this disease.

Evidence suggests that the accumulation of beta-amyloid in Alzheimer’s disrupts several brain functions, kills neurons (nerve cells) and damages blood vessels in the brain, a condition called cerebral amyloid angiopathy (CAA).

Lab (in vitro) models that mimic the interactions of the BBB with other brain cells and with the beta-amyloid protein are key to understanding how Alzheimer’s affects this vital brain barrier, and may help in developing therapies to restore it.

Engineers at Massachusetts Institute of Technology (MIT), together with researchers at Massachusetts General Hospital and Harvard Medical School, developed a 3D model of the human BBB.

This model includes engineered neurons that produce large amounts of beta-amyloid proteins, mimicking what happens in Alzheimer’s disease, grown on a microchip. In a parallel chamber in the same chip, brain endothelial cells that make up the BBB are also grown. Between the two chambers is an empty channel that, after 10 days, is filled with collagen to allow the diffusion of molecules between the two chambers, mimicking what occurs in a person’s brain.

“What we were trying to do from the start was generate a model that we could use to understand the interactions between Alzheimer’s disease neurons and the brain vasculature,” Roger Kamm, a professor of mechanical and biological engineering at MIT and a lead study author, said in a press release.

“Given the fact that there’s been so little success in developing therapeutics that are effective against Alzheimer’s, there has been increased attention paid to CAA over the last couple of years,” he added.

In their model, researchers found that the lab-made BBB was more permeable to substances when grown in the vicinity of Alzheimer’s neurons, much as it is in patients.

They saw that within three to six days, beta-amyloid secreted by nerve cells in one chamber of the microchip started to accumulate in the endothelial cells of the chamber on the other side.

The number of proteins that bind the endothelial cells together, in regions known as tight junctions, were reduced. In particular, the activity of three genes coding for some of these proteins, CLDN1, CLDN5 and CDH5, was significantly lower when endothelial cells were grown close to Alzheimer’s-like neurons.

“We were able to show clearly in this model that the amyloid-beta secreted by Alzheimer’s disease cells can actually impair barrier function, and once that is impaired, factors are secreted into the brain tissue that can have adverse effects on neuron health,” Kamm said.

The endothelial cells that make up the BBB also had higher levels of harmful reactive oxygen species (ROS) in this 3D model. Reactive oxygen species, which are produced as a consequence of oxidative stress, are damaging to cells and are associated with a number of diseases.

Researchers found that the levels of a particular enzyme, called metalloproteinase (MMP) 2 and known to degrade the proteins that ‘glue’ cells together, were also higher than usual in their model. Higher levels of an inflammatory cytokine (small protein) called IFN-gamma were also observed.

Increases in levels of ROS, MMP2 and IFN-gamma are all thought to contribute to the greater permeability of the BBB.

Since a leaky BBB will likely allow neurotoxic molecules to access the brain, researchers then introduced a clotting factor, called thrombin, into their 3D model. Thrombin is normally found in the bloodstream, but can be toxic to neurons and lead to cell death when present in excessive levels. Alzheimer’s patients are known to have elevated levels of thrombin in the brain and cerebral microvasculature.

Within three hours of its introduction, the rate of cell death increased, suggesting that thrombin was able to pass through the disrupted BBB and reach neurons.

“We were able to demonstrate this bidirectional signalling between cell types and really solidify things that had been seen previously in animal experiments, but reproduce them in a model system that we can control with much more detail and better fidelity,” Kamm said.

Using a compound called etodolac, a nonsteroidal anti-inflammatory treatment for pain related to various conditions, researchers were able to restore BBB integrity. After treatment with this FDA-approved compound, the levels of one of the proteins that glues cells together, claudin-5, rose to lessen BBB permeability. Cell death rate was also significantly lower after etodolac’s use.

The researchers suggested their 3D model could be used to test further therapies with a potential to restore the BBB.

“We believe that our AD model will be a useful standardized tool in the study of BBB biology in AD [Alzheimer’s disease] and also provide a platform for moderate-throughput drug screening of drugs to inhibit BBB dysfunction or enhance BBB integrity as an adjunct to other AD therapies,” the researchers wrote.

“We’re starting to use this platform to screen for drugs that have come out of very simple single cell screens that we now need to validate in a more complex system,” Kamm added.  “This approach could offer a new potential form of Alzheimer’s treatment, especially given the fact that so few treatments have been demonstrated to be effective.”