One day in 1991, neurologist Warren Strittmatter asked his boss to look at some bewildering data. Strittmatter was studying amyloid-β, the main component of the molecular clumps found in the brains of people with Alzheimer's disease. He was hunting for amyloid-binding proteins in the fluid that buffers the brain and spinal cord, and had fished out one called apolipoprotein E (ApoE), which had no obvious connection with the disease.
Strittmatter's boss, geneticist Allen Roses of Duke University in Durham, North Carolina, immediately realized that his colleague had stumbled across something exciting. Two years earlier, the group had identified a genetic association between Alzheimer's and a region of chromosome 19. Roses knew that the gene encoding ApoE was also on chromosome 19. “It was like a lightning bolt,” he says. “It changed my life.”
In humans, there are three common variants, or alleles, of the APOE gene, numbered 2, 3 and 4. The obvious step, Roses realized, was to find out whether individual APOE alleles influence the risk of developing Alzheimer's disease. The variants can be distinguished from one another using a technique called the polymerase chain reaction (PCR). But Roses had little experience with PCR, so he asked the postdocs in his team to test samples from people with the disease and healthy controls. The postdocs refused: they were busy hunting for genes underlying Alzheimer's, and APOE seemed an unlikely candidate. The feeling in the lab, recalls Roses, was that “the chief was off on one of his crazy ideas”.
Roses then talked to his wife, Ann Saunders, a mouse geneticist who was skilled at PCR. She had just given birth to their daughter and was on maternity leave, so they struck a deal. “She did the experiments while I held the baby,” he says. Within three weeks, they had collected the data that would fuel a series of landmark papers showing that the APOE4 allele is associated with a greatly increased risk of Alzheimer's disease.
Twenty years on, APOE4 remains the leading genetic risk factor for Alzheimer's, the most common form of dementia (see 'Risky inheritance'). Inheriting one copy of APOE4 raises a person's risk of developing the disease fourfold. With two copies, the risk increases 12-fold. Yet Roses' data were largely criticized or ignored. Within a couple of years, interest in ApoE had dwindled as researchers flocked to study amyloid-β. The handful of labs that continued to pursue ApoE did so in the face of indifference from funding agencies and the neuroscience community, and without the resources needed to validate experimental findings with larger studies.
Today, the function of the ApoE protein in the brain remains mostly unknown. This neglect of such a strong lead has puzzled some outside the Alzheimer's field. At a forum on brain diseases in Frankfurt, Germany, Thomas Bourgeron, an autism researcher at the Pasteur Institute in Paris, voiced his confusion. “If I had a risk factor like that, I'd be hot on its trail.”
But interest in the lipoprotein is picking up, in part because attempts to target amyloid-β have repeatedly disappointed in major clinical trials. Pharmaceutical companies are pulling back from amyloid-based approaches and some academics have begun to question the focus on the molecule. For the first time, researchers are developing drugs aimed at the ApoE4 protein and drawing attention from industry.
“The amyloid hypothesis became such a strong scientific orthodoxy that it began to be accepted on the basis of faith rather than evidence,” says Zaven Khachaturian, president of the non-profit campaign Prevent Alzheimer's Disease 2020, and former coordinator of Alzheimer's-related activities at the US National Institutes of Health. Until recently, he says, “no one has stepped back to ask the fundamental question of whether our basic premise about the disease is the correct one”.
Opinions differ as to why Roses' finding was neglected, but many agree that bad timing played a part. In 1991, John Hardy and David Allsop had proposed the 'amyloid cascade hypothesis'. This posits that Alzheimer's disease results from the abnormal build-up of amyloid-β clusters, or plaques, in the brain. Others rallied around the idea and it has won most of the funding available to the field ever since.
But Roses did not subscribe to that theory. “Amyloid is one of many substances that builds up in plaques as a result of dying cells and atrophy in the brain,” he says. “I never did think it was the cause.” In saying so, he may have deterred others from investigating a possible ApoE–amyloid link, and inadvertently set up a competition between the two hypotheses for funding. He never got another grant to work on ApoE.
But there were also technical obstacles to ApoE research. The protein is found throughout the body, making it difficult to target the molecule specifically in the brain. And ApoE is bound to fat, so it tends to stick to other molecules in biochemical assays, says Menelas Pangalos, who leads research on small-molecule discovery at AstraZeneca in Macclesfield, UK, and has long had an interest in ApoE.
Working with such proteins requires an intimate understanding of lipid biochemistry. “If you want to study ApoE biology, you really need to devote a laboratory to understanding the techniques,” says neurologist David Holtzman of Washington University in St. Louis, Missouri. Holtzman did just that, establishing a separate lab dedicated to developing techniques for handling lipoproteins in the central nervous system.
Amyloid was the easier target. Two decades of intensive pursuit have yielded a range of drugs that alter the metabolism of amyloid-β, but these have yet to fulfil expectations. Of the six drugs that were in phase II or III clinical trials in 2012, half have since been dropped because of either safety concerns or lack of effectiveness. This comes against a backdrop of ageing populations, overstretched health-care systems and a dearth of medications for Alzheimer's disease. “The large number of major failed trials in Alzheimer's is quite frightening,” says Lennart Mucke, director of the Gladstone Institute of Neurological Disease at the University of California, San Francisco. “It has really scared off big pharma.”