An Introduction to APOE4
A recent study published in Nature provides new insights into genetic predispositions to Alzheimer’s disease.
The E4 variant of the APOE gene is accepted as the predominant genetic risk factor for Alzheimer’s. Those who contain one copy of the E4 variant are three times more likely to develop Alzheimer’s, while those who contain two copies of the E4 variant are nearly ten times more likely to develop the disease. Unfortunately, while the E4 variant has been associated with Alzheimer’s for decades, it is still unclear exactly how it contributes to the debilitating biological and cognitive symptoms of Alzheimer’s.
Now, a study conducted by Tsai et al. at the Massachusetts Institute of Technology shows that those who contain the E4 variant display abnormalities in cholesterol metabolism. The MIT team suggests that the disruption of cholesterol metabolism could be a fundamental reason why those with the E4 variant are more likely to develop Alzheimer’s disease symptoms.
In my last article, I discussed Tsai et al.’s initial findings using brain tissue samples from Alzheimer’s patients. After characterizing the genetic makeup of tissue samples from thirty-two Alzheimer’s patients, Tsai et al. discovered that the E4 variant of the APOE gene was associated with abnormal cellular activity. These abnormalities particularly affected a subset of brain cells called oligodendrocytes.
Oligodendrocytes are supportive cells in the brain whose main function is to insulate neurons. Neurons send information to each other through electrical signals in the brain. Much like a wire is insulated to allow electricity to travel efficiently, neurons are wrapped with fats called myelin. These fats provide insulation and allow information in the brain to travel more quickly. Oligodendrocytes have the essential function of processing fat molecules to produce myelin for neurons.
Here, we will describe the additional experiments the MIT team conducted to study the relationship between the E4 variant, cholesterol, and oligodendrocytes.
Studying oligodendrocytes with stem cells
Tsai et al. began with a stem cell model. Stem cells are a unique type of cell that can be turned into nearly any other cell type in the body. When stem cells are converted to their embryonic state, they can then be exposed to chemical cues that nudge them into developing into nearly any other cell in the body, including oligodendrocytes.
By using a stem cell model, the team could compare the effects of the E4 variant of the APOE gene in cells from a single patient. This would allow them to confirm that any abnormal activity displayed by oligodendrocytes was due to the E4 variant of APOE. Using gene editing tools like CRISPR, the team could also alter the oligodendrocytes so that they expressed two copies of either the E3 or E4 APOE variants.
To examine the effect of the E4 variant in the stem cell model, the researchers first examined the fats present in the oligodendrocytes. Compared to the E3/E3 cells, the E3/E4 cells exhibited an increase in 88 different fat species. Only one fat species decreased within the E4/E4 cells.
Upon further analysis of the two oligodendrocyte types, researchers found that genes responsible for transporting cholesterol in the E4/E4 oligodendrocytes were less active. This suggested that the E4 variant decreases the ability of oligodendrocytes to distribute cholesterol throughout the cell.
To determine where cholesterol was accumulating, researchers stained the oligodendrocytes to visualize where the cholesterol was localized within the cell. The team found that cholesterol in E4/E4 oligodendrocytes seemed to accumulate more than E3/E3 cells. Surprisingly, researchers found that the cholesterol accumulated within a structure called the endoplasmic reticulum.
Typically, cholesterol is stored within the oligodendrocyte’s cell membrane. These results demonstrate that E4/E4 oligodendrocytes display abnormal activity in cholesterol transport and storage.
The E4 variant, oligodendrocytes, and myelination
Since oligodendrocytes are responsible for myelinating neurons, researchers sought to explore how oligodendrocyte dysfunction may impair its ability to myelinate neurons. Tsai et al. speculated that E4 variant carriers may exhibit decreased myelin production. To explore this, the researchers stained myelin in postmortem tissues from patients that contained a combination of the E3/E4 variants and those who contained two copies of the E3 variant.
The MIT team soon found that samples taken from patients with the E4 variant displayed a significant reduction in myelination. To confirm these results, the researchers then conducted these experiments on mice and found that E4/E4 mice exhibited much fewer myelin proteins than E3/E3 mice. Surprisingly, when inspecting the structure of myelin in E4 mice versus E3 mice, researchers discovered that myelin in E3 mice was much more densely packed than E4 mice, suggesting that there may be structural differences in myelin due to the E4 variant as well as a decrease in myelin production.
This led to a key question: Was the decrease in myelin found in Alzheimer’s patients due to the abnormal activity of oligodendrocytes only? Or were other cells also contributing to the decrease in myelination?
Studying myelination with a 3D stem cell model
Tsai et al. approached this question by using stem cells to form both oligodendrocytes and neurons. The two cell types were co-cultured and grown into 3D tissue that resembled the cellular organization of human brain tissue.
The team created two 3D cultures—one with two copies of the E4 variant and one with two copies of the E3 variant. After observing the model for 6 weeks, Tsai et al. found that in line with their suspicions, the E4/E4 culture produced significantly less myelin than the E3/E3 culture. To determine whether this difference in myelin was due to the oligodendrocytes or due to cellular processes within the neurons themselves, the team then conducted experiments where they co-cultured E4 oligodendrocytes with E3 neurons or E3 oligodendrocytes with E4 neurons.
Surprisingly, samples containing E3 oligodendrocytes engaged in regular myelination even with E4 neurons. In contrast, samples containing E4 oligodendrocytes consistently displayed abnormally low myelination levels. These results confirmed that the decrease in myelination displayed by those with the E4 variant of the APOE gene was, in fact, due to the abnormal activity of oligodendrocyte cells.
So, what do these results mean for potential Alzheimer’s disease treatments? Having confirmed that the E4 variant of the APOE gene promotes abnormal cholesterol accumulation in oligodendrocytes and decreases myelination, Tsai et al. sought to determine how these findings may lead to new treatments for Alzheimer’s.
Cyclodextrin: A potential treatment
Using their stem cell model of oligodendrocytes, the researchers tested specific molecules known to inhibit the production of cholesterol and promote the transport of cholesterol. After two weeks of treatment, researchers found that one molecule called cyclodextrin was particularly effective. In cyclodextrin-treated E4/E4 oligodendrocytes, Tsai et al. found that oligodendrocytes exhibited a decrease in cholesterol production and accumulation.
To examine the effects of cyclodextrin in live organisms, Tsai et al. applied the treatment to mice with the E4 variant. After treating the E4/E4 mice with cyclodextrin for eight weeks, researchers found that the treatment significantly reduced cholesterol production. The treatment also increased the transport of cholesterol and increased healthy myelination.
Interestingly, cyclodextrin not only improved biological symptoms but seemed to support learning and memory. In an additional experiment, researchers conducted several learning and memory tests to see if cyclodextrin could improve cognitive function in E4 mice. To their surprise, E4 mice treated with cyclodextrin exhibited improvements in their learning and memory test results. These results contrasted with a control group of E4 mice who were not treated with cyclodextrin and exhibited decreased cognitive abilities.
Overall, this innovative and exciting study points to a new theory for how the E4 variant of the APOE gene may induce or exacerbate Alzheimer’s disease symptoms by causing abnormal cholesterol metabolism and myelination patterns. Given the success of cyclodextrin in improving the biological and cognitive impacts of the E4 variant in both cells and mice, it will be interesting to see whether cholesterol metabolism and myelin-based treatments can improve the symptoms of Alzheimer’s disease in humans.