Biography of Vernon M. Ingram

Inside the brains of Alzheimer's patients, tangles of a misfolded protein crowd against sensitive neurons. These plaques, composed of misfolded β-amyloid protein, are a hallmark of the debilitating neurological disease. Vernon M. Ingram, elected to the National Academy of Sciences in 2002, is using protein chemistry and high-throughput screens to identify novel compounds that obliterate the plaques. His research is likely to have wider implications for other protein-based diseases, such as Huntington's disease and prion diseases. Working with misshapen proteins is not new for Ingram. Early in his career, he discovered that a single amino acid substitution is responsible for the abnormal aggregation pattern of hemoglobin in sickle cell anemia (1).

A Nontraditional Start

Ingram, the John & Dorothy Wilson Professor of Biology at the Massachusetts Institute of Technology (MIT, Cambridge, MA), was born in Breslau, Germany, in 1924. His interest in science was apparent early on. “I knew from the age of 10 that I was interested in science, in how things work,” said Ingram. He was always tinkering with things. Although radio receivers were illegal in Nazi Germany, he constructed them from scratch; he took the risk so he could listen to what was going on outside the country. When he was 14, his family moved to London to escape the environment of Nazi Germany.

Ingram entered London University's Birkbeck College in 1941. Although his degree program focused on chemistry, math, and zoology, it differed from traditional colleges. Birkbeck offered weekend classes, an advantage, Ingram points out, in wartime London. “We were lucky because Birkbeck was the only college not evacuated,” he says. Many of Birkbeck's students were, and still are, professionals with day jobs. During the day, Ingram worked at a chemical factory making drugs, like amphetamines, that supplied the ongoing war effort. Ingram remembers a set of “better than the usual lecturers” brought to the city through their work with the Ministry of Defense. He fondly remembers Graham Jackson, who had such a talent for teaching that he “made the dinosaur walk in front of your eyes.” Alistair Graham's lectures got him interested in animal physiology, which, according to Ingram, “naturally led to biochemistry.” After receiving his B.S. in 1945, Ingram went straight into a doctoral program at Birkbeck, studying physical organic chemistry in the laboratory of Fred Barrow.

Figure 1.

Vernon M. Ingram. Photograph courtesy of Donna Coveney (Massachusetts Institute of Technology).

Upon finishing his Ph.D. in 1949, Ingram completed two postdoctoral research appointments in the United States, the first as a Rockefeller Foundation Fellow at The Rockefeller Institute (New York) with Moses Kunitz to learn protein preparation and the second working on peptide chemistry in Joseph Fruton's laboratory at Yale University (New Haven, CT). By 1952, Ingram was ready to head back to England but was frustrated by conducting a job search 3,000 miles away. After sending out 32 requests for appointment letters, the serendipitous arrival of a new postdoctoral student in the Fruton laboratory spurred Ingram's employment back home. Herbert Gutfreund, fresh from Cambridge, England, encouraged Ingram to apply for a position at the Medical Research Council's Unit for the Study of the Structure of Biological Molecules in the Cavendish Laboratory of Cambridge University.

Back to England

Gutfreund's suggestion proved fruitful, and, by September 1952, Ingram was back in England. This time he was in the laboratory of Max Perutz, who had hired him as a protein biochemist. Perutz needed someone to produce a derivative of hemoglobin with a single heavy atom so that he could determine the protein's crystal structure (2). Ingram applied himself and finished the project quickly and was then free to pursue his own work. The Cambridge neighborhood was ripe with inspiration for protein chemistry.

Francis Crick and Jim Watson occupied the laboratory next door. Frederick Sanger, who had recently sequenced insulin and demonstrated that proteins were made from amino acid chains (3), was just a couple of blocks away in the biochemistry department. Perutz suggested that Ingram build on Sanger's work and Ingram's own interest in characterizing the large fragments of hemoglobin by comparing normal hemoglobin to sickle cell anemia hemoglobin. Using samples of sickle cell hemoglobin left behind by a former visiting scientist, Tony Allison, and samples of normal hemoglobin from his own blood, Ingram set about his project.

Sanger had split the insulin protein into many tiny pieces and identified each piece before putting the jigsaw together. Hemoglobin is 10 times larger than insulin, so Ingram decided to digest the protein into larger, but still manageable, peptides using trypsin. He first looked for the particular segments that might differ between normal and sickle cell hemoglobin. In doing so, he developed the first two-dimensional protein analysis. In the first step, he ran the proteins out in neutral buffer to line the peptides up by charge. After that, he used partition paper chromatography to differentiate the peptides further. The resulting map, albeit crude, showed that only one peptide of 26 differs between normal and sickle cell hemoglobin, a difference caused by a single amino acid change (1). The implications of the work also negated some of the early models of overlapping triplets coding for amino acids (4). If triplet codes actually overlapped, a single base substitution, like that for sickle cell anemia, would affect more than one amino acid.

Encouraged by this breakthrough, Ingram's interest in hemoglobin continued. “The sickle cell work led me to think about the wider implications in molecular genetics,” he says. “For example, what are the effects of mutations?” He went on to study not only the genetics (5) but also the evolution (6) and abnormalities of human hemoglobin (7). He eventually published a book detailing the multitude of abnormalities in human hemoglobin (8). In 1961, after submitting a cohesive body of publications and directing others in research, Ingram received the high degree of D.Sc. from Birkbeck College. He humbly jokes about the degree, “The only benefit is that it entitles you to wear a particularly gaudy gown.” Before long, he was back in the United States, this time for a longer visit.

Same Cambridge, Different Continent

Forty-six years have passed since Ingram arrived at MIT for what was originally intended be a one-year sabbatical from the U.K. Medical Research Council. At the time, it was the norm for British scientists to spend a year or two in the U.S. “There were many developments in the States in molecular biology that were different from what was happening in England,” Ingram notes. He was offered a visiting associate professorship at MIT. “MIT was a hotspot at the time,” he says. “I liked it and stayed.”

For 12 years, from 1961 to 1973, Ingram commuted along the east coast between Cambridge and New York. He collaborated with Paul Marks working on hemoglobin and spent a few days each month in Marks's laboratory at Columbia University (New York). Ingram also studied the embryology of hemoglobin, and he was interested in how the structures of fetal and adult hemoglobin differ (9-11).

From Hemoglobin to β -Amyloid

In the 1980s, Ingram began a gradual move toward neuroscience research. By the late part of the decade, he had settled on his current area of research, Alzheimer's disease. The shift was partially inspired from a conversation with his second wife, Beth. She was working as a physician's assistant with the mentally retarded in Boston, including those with Down's syndrome. Ingram explains, “She came home with a story she'd heard that Down's syndrome was a disease of the neurofilaments.” He knew there was a genetic element to the disease and thought it might confer misfolding in the neurofilaments, much like the sickle cell gene codes for a misshapen hemoglobin protein. Ingram hypothesized, “Sickle cell anemia is an inherited mutation. Maybe in Down's syndrome, there is an inherited mutation in different neurofilament proteins.” It turned out not to be the case, but as far as studying a corollary between Alzheimer's disease and Down's syndrome, Ingram confesses, “I was hooked.”

“I am happy to have found candidate compounds for a simply awful disease.”

All Down's syndrome patients develop Alzheimer's disease by the time they reach 40, and there is a clear connection between the two diseases. In each case, β-amyloid is overproduced. In Alzheimer's disease, proteins normally produced by the brain begin to get miscleaved into abnormal peptides before the cell excretes them. One of these peptides, β-amyloid, folds in on itself and aggregates, producing the plaques characteristic of the disease. The result, Ingram explains, can be physiologically devastating: “It very quickly aggregates, and some aggregates—not all—have toxic effects on nerve cells.”

What causes the onset of misregulation of peptide cleavage is unclear. And the exact causes of the pathogenesis are not agreed upon in the scientific community. Ingram believes that the aggregated protein presents a novel surface that interacts with the neuronal cells. His research has suggested that the interaction causes pores to open, resulting in an influx of calcium ions (12, 13). The rapid, uncontrolled influx of this potent signaling molecule hampers the neuron's ability to respond to other stimuli.

Ingram realizes that not all share his view. He points to work done across the Charles River by Ashley Bush at Massachusetts General Hospital (MGH) in Boston that focuses on the creation of toxic reactive oxygen species from the interaction of misfolded peptides with copper and zinc. Referring to Bush's work, Ingram notes, “I'm not saying that it is not so, but I think it is a later effect. The first thing that happens is a calcium influx. Reactive oxygen species have a role to play in pathology, but I don't think they're the first cause.”

Regardless of the nature of the damage, Ingram says, “Most people agree that these fibrils are a toxic agent by some method.” His laboratory is pursuing dual strategies to stop the damage and abrogate a disease that, Ingram notes, is beginning to affect his own generation. “I am happy to have found candidate compounds for a simply awful disease that is beginning to affect people I know,” he says.

One of these strategies has been to develop decoy peptides that bind the aggregated protein and prevent the aftereffects of misfolding (12). The second, detailed in Ingram's Inaugural Article (14) in this issue of PNAS, seeks to obliterate the misfolded proteins. Using a high-throughput screen, Ingram and his colleagues tested more than 3,000 biologically active molecules. They found that the compound 4,5-dianilin-ophthalimide (DAPH) actually destroys the protein aggregates characteristic of Alzheimer's disease, and, by doing so, DAPH reduces the influx of calcium into cells.

With both of these methods offering potentially fruitful treatment options for Alzheimer's disease, Ingram asserts, “As soon as we can, we will go into animal trials.” Currently, a consortium of collaborators at MIT, MGH, and the University of Minnesota (St. Paul) are applying for funding to begin testing the compounds and decoy proteins in vivo.

Still Hard at Work

When asked what he is proud of in his long career, Ingram cites his work on two important diseases and his children, but he also mentions family of a different kind. For 16 years, he and Beth were housemasters in a graduate dorm at MIT. “Some students became friends. Some students became colleagues,” he says.

Although Ingram is happy that his work may generate treatments for Alzheimer's and potentially lend insight into other protein-based diseases like Huntington's disease and Creutzfeldt-Jakob disease, he is still a protein chemist, excited by the questions his success in the laboratory raises: “So we have a compound that destroys fibrils. How does it work? Why does it work?”

This enthusiasm for science is complemented by his interest in art. An admirer of Georgia O'Keefe, Ingram is a passionate photographer, focusing on the inside of flowers. But he does not have as much time to devote to art as he would like. Running a research program takes most of his time, and he still occupies a spot at the bench. “I don't see why everybody else should have all the fun,” he says.