In 1906, the German physician Alois Alzheimer noticed changes in the brain of a woman who had died of an unusual mental illness. The abnormal clumps (now called amyloid plaques) and tangled bundles of fibers (now called neurofibrillary tangles) he noted are recognized today as hallmarks of Alzheimer’s disease (AD).

AD, a devastating neurodegenerative disease, is the most common form of dementia among older people. It affects over 4 million Americans, a number that is expected to triple by the year 2050. Its cause remains largely unknown, although mutations are the culprits in a small fraction of cases.

Aside from age, the greatest known risk factor for AD is the gene for one of three apolipoprotein (apo) E isoforms: apoE4. ApoE4 is associated with 40–60% of cases of sporadic and familial AD. In contrast, apoE3 appears to offer some protection against AD, and apoE2 is even more protective.

ApoE has long been studied for its role as a lipid transport protein and its involvement in cardiovascular disease (see side box). Much of the pioneering work on this versatile molecule was done at Gladstone over the past two decades in the laboratories of Robert W. Mahley, Karl H. Weisgraber, Bob E. Pitas, John M. Taylor, Thomas L. Innerarity, Stanley C. Rall, Jr., and Yadong Huang. More recently, Lennart Mucke has joined this cadre of investigators focusing on apoE.

“I have been continually amazed by the diversity of functions of this fascinating protein,” said Dr. Mahley. “The extensive work done on apoE in cardiovascular disease directly translates to understanding its role in neurobiology and particularly in Alzheimer’s disease.”

	AD Risk and ApoE4 Everyone inherits two copies, or alleles, of every gene, one from each parent. As the number of apoE4 alleles increases from 0 to 2, the risk of AD increases from 20 to 90%, and the typical age of onset decreases from 84 years to 68 years. The presence of one apoE4 allele results in an estimated 45% chance of developing AD by 85 years of age. With two apoE4 alleles, the risk increases to 50–90%.		ApoE and Neurobiology In the mid to late 1980s, apoE emerged as a major player in neurological disease, based on observations made at Gladstone. Although synthesized primarily in the liver, apoE is found in abundance in the brain and is the major lipid transport vehicle in the cerebrospinal fluid. In the brain, it is produced by astrocytes and, under various physiological and pathophysiological conditions, by neurons. Interest in apoE’s role in neurobiology got a significant boost in 1993, when researchers at Duke University identified apoE as the major susceptibility factor in AD. Gladstone investigators hypothesized that apoE
is critical in neuronal repair and remodeling. In a series of experiments, they demonstrated that the three isoforms of human apoE (apoE2, apoE3, and apoE4) have different effects in neurobiology and neurodegenerative diseases. For

example, in cultured neuronal cells exposed to a source of lipid, apoE3 stimulates neurite outgrowth, whereas apoE4 inhibits it and causes microtubular instability. ApoE4 also has detrimental effects in transgenic mice expressing apoE4, including behavioral abnormalities, such as deficits in learning and memory, and significant alterations in the hippocampus and cortex—two areas of the brain that are important in cognition and other higher functions and are particularly vulnerable in AD. These findings suggested that apoE4 adversely affects the cytoskeletal system and the ability of neurons to maintain and reestablish connections between cells in the brain.

Production of Amyloid β Peptides

Gladstone scientists also showed that apoE influences amyloid β (Aβ) production in cultured neurons, with apoE4 stimulating production to a greater extent than apoE3 in cells coexpressing both apoE and the amyloid precursor protein (APP). These divergent effects were mediated, it turned out, by differences in the extent to which the two proteins stimulated cell-surface APP recycling. Blocking the low density lipoprotein receptor pathway in the cells abolished these differences, suggesting that this pathway is involved in the more pronounced stimulatory effect of apoE4 on Aβ production.

Structure and Function

In biology, the structure of a protein often determines its function. Gladstone scientists have carefully studied the structures of the apoE isoforms to explain the differences in their functions.

For all their differences in disease processes, apoE3 and apoE4 differ by only a single amino acid. At position 112, apoE3 has cysteine, and apoE4 has arginine. Arg-112 in apoE4 mediates the formation of a salt bridge between Arg-61 in the amino terminus and Glu-255 in the carboxyl terminus, giving apoE4 a more compact structure than apoE3. This so-called domain interaction occurs only in apoE4 and likely contributes to its adverse effects in neurobiology (and in lipid metabolism as well).

In a key study performed at Gladstone, apoE4 was shown to form a reactive molecular intermediate—also known as a molten globule—more readily than apoE3. Molten globules have been associated with several pathological conditions, and an apoE4 molten globule is an intriguing mechanism to explain the pathological functions of apoE4 in various diseases.