For the common form of Alzheimer’s that strikes in old age, the greatest risk factor is, of course, old age. But since 1993, Alzheimer’s researchers have puzzled over another major risk factor, a variant of the apoE gene known as apoE4. Compared with people who have other common apoE variants, those with one copy of apoE4 have three times the Alzheimer’s risk and develop symptoms about five years earlier; while those with two apoE4 copies have roughly 12 times the risk, and develop symptoms about a decade earlier.
How does apoE4 exert such a profound risk-worsening, disease-accelerating effect? It now appears that it does so via multiple biological mechanisms, and this complexity has slowed progress in the field. Moreover, the apoE gene codes for apolipoprotein-E, which carries “lipid” (fat) molecules in the brain, and lipid biochemistry is notoriously tricky. “Many people have been discouraged from pursuing investigations in this area, because it’s technically difficult,” says Thomas Wisniewski, a neurologist and apoE researcher at New York University’s Langone Medical Center.
The good news is that after nearly two decades of research on apoE4’s effects in Alzheimer’s, researchers appear to be zeroing in on the ones that matter, and are getting close to clinical trials of drugs that could reverse those effects.
ApoE4 enhances amyloid beta clumping
In 1992, Wisniewski reported evidence of apoE proteins in a variety of amyloid protein deposits in the brain and the body. He termed apoE a “pathological chaperone protein” because its frequent presence with aggregated proteins suggested that it somehow triggered or enhanced their aggregation.
The year after Wisniewski’s paper appeared, researchers in Allen Roses’s laboratory at Duke University reported that apoE proteins bound tightly to the Alzheimer’s-linked protein amyloid-beta (A-beta), and that Alzheimer’s patients, compared with healthy people of the same age, were much more likely to have the apoE4 variant. In their initial sample, about half of patients were apoE4 carriers, while only about 15 percent of age-matched controls were apoE4 carriers. The apoE4-carrying patients also had heavier A-beta deposits than other patients did, not only in their brain matter but also in cerebral blood vessels.
In lab dish experiments, Wisniewski and others soon determined that apoE somehow made A-beta proteins more likely to stick together in long, plaque-making aggregates called fibrils. “The presence of any apolipoprotein-E tended to enhance fibril formation, and the E4 variant was most likely to do that,” says Wisniewski.
But is that really how apoE4 worsens Alzheimer’s?
ApoE4 impairs microglial clearance of A-beta
As the main transporters of lipids in the brain, apoE proteins perform an important function in bringing these essential building blocks to cell membranes and nerve fibers undergoing development or repair.
“ApoE2 and apoE3 [the other two common variants] form big particles and can accept lots of lipids and are very good transporters of lipids throughout the brain, but apoE4 is not as good at this; it forms smaller particles and it is intrinsically more labile [more likely to be degraded],” says Gary Landreth, an apoE and Alzheimer’s researcher at Case Western Reserve University.
ApoE4’s reduced lipid-carrying efficiency appears to leave the brain broadly more vulnerable to the stresses of aging or acute injuries. Following head traumas and strokes, says Wisniewski, “apoE4 carriers tend to do less well.” This reduced lipid-carrying efficiency also helps to explain why apoE4 is moderately associated with atherosclerosis: In the bloodstream, apoE4 does a relatively poor job of grabbing fat molecules and transporting them for proper disposal.
But it turns out that apoE, when covered with lipids, also transports A-beta, because A-beta as it aggregates develops a strong affinity for lipids. For A-beta, this ride often ends within microglial cells, where the protein and its aggregates are digested by strong proteolytic enzymes. ApoE4’s reduced lipid-carrying capacity thus seems to translate into a reduced A-beta removal capacity. Landreth’s lab reported this in Neuron in 2008, and showed that when they used a drug to enhance apoE production in aged “Alzheimer’s mice,” the mice lost most of their A-beta plaques and memory deficits.
Landreth’s and also David Holtzman’s laboratories at Washington University are now separately doing further tests in animal models, to try to confirm and optimize this A-beta clearing effect using different classes of apoE-boosting drugs. “This is now the primary focus of our lab,” says Landreth. “And I would argue that the more apoE you have in the brain, the less amyloid you have, and that’s now consistent with a very substantial literature.”
Will more apoE4 only make things worse?
There are other hypotheses about apoE4’s principal role in Alzheimer’s, and according to these, boosting apoE4 levels in an Alzheimer’s patient would make things worse, not better. Berislav V. Zlokovic’s group at the University of Rochester, for example, reported in 2008 that all apoE variants serve to slow down another important clearance process, in which A-beta is pushed out of the brain into the bloodstream. Zlokovic’s group found that apoE4 slowed down this process more than apoE3 did.
“Zlokovic’s really good, and there’s no reason to question that work,” says Landreth. “But we still don’t know what fraction of A-beta clearance occurs through its export into the periphery, through this vascular mechanism, and how much is intrinsic due to this proteolytic degradation in the brain.” In other words, more apoE might still be beneficial on balance through its proteolysis-enhancing effect, even if it also reduces A-beta clearance via the bloodstream.
A more radical hypothesis is that apoE4 is actively toxic, so that boosting it in patients would be disastrous. This hypothesis comes from the Gladstone Institute, affiliated to the University of California–San Francisco, where Karl Weisgraber, Robert Mahley and Yadong Huang anchor one of the longest-running apoE research programs.
In 2001, Huang, then a postdoc in Mahley’s lab, reported that apoE proteins could be split by enzymes within neurons to form toxic fragments, and that apoE4 was more likely than other variants to be rendered into such fragments. Transgenic mice that overproduced these fragments developed significant cognitive deficits and even neurofibrillary tangles—amyloid fibrils made of tau protein, which are a marker of severe neuronal stress—like those seen in Alzheimer’s patients.
More recently, Weisgraber’s lab has found evidence, both from lab-dish and mouse studies, that apoE4 can undergo a structural collapse, and in this abnormal “molten globule” form puts stress on the astrocyte cells that make it—effectively shortening their lifespans, and the lifespans of the neurons they service. “Perhaps with age or a second hit such as ischemia [lack of oxygen due to stroke] or a blow to the head, the astrocytes cannot support neurons any longer, and neurons themselves [under stress] start to produce apoE, and that leads to the production of these toxic fragments,” Weisgraber says.
In collaboration with drug-maker Merck, Weisgraber and his colleagues have found compounds that inhibit the apoE-fragment-producing enzyme, as well as compounds that bind to apoE4 and help keep it in a functional structure more like apoE3’s. Merck shelved the project after a recent merger, but Weisgraber says that his group is now trying to get the rights back and restart preclinical development with independent funding. “These are two viable targets that we are going to push forward,” he says.