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BIOMARKERS FOR AD OFFER NEW CLUES FOR DISEASE DETECTION

A biomarker “bar-code” panel of 23 proteins in the CSF accurately identified Alzheimer disease (AD) patients and differentiated them from healthy subjects or those with non-Alzheimer dementia, according to a new study.

The test demonstrated 94 percent sensitivity and specificity, while a blinded validation analysis of samples from 18 AD patients and 10 non-AD subjects showed 90 percent sensitivity and 80 percent specificity.

The test, which was developed by researchers at Cornell University in Ithaca, NY, and its Weill Medical College in New York City, was reported Dec. 7 in an advance online publication of the Annals of Neurology.

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Investigators predicted that if the results of a biomarker “bar-code” panel of proteins specific for Alzheimer disease could be replicated in a large group of patients, a test could be available for neurologists within the decade.

The study reflected a seven-year collaboration between Kelvin H. Lee, PhD, associate professor and director of Cornell's Institute for Biotechnology and Life Sciences Technologies, and Norman Relkin, MD, PhD, professor of neurology and director of the Memory Disorders Program at the Cornell-Weill Medical College. The team collected CSF samples from patients with autopsy-confirmed AD as well as from age-matched healthy individuals and some with non-Alzheimer dementia.

If the results can be replicated in a larger group of patients, a test could be available for neurologists within the decade, Dr. Lee told Neurology Today in a telephone interview. Currently, the only way to assure the diagnosis is by autopsy.

“Our study is the first to use sophisticated proteomic methods — [which focus on large-scale studies of proteins, particularly their structures and functions] — to hone in on a group of CSF biomarkers that are specific to autopsy-proven Alzheimer disease, and it is the first time proteomic results have been reviewed in a blinded validation using postmortem samples. The pattern of the 23 proteins was more than 90 percent sensitive in identifying people with AD, but more work needs to be done.”

STUDY TECHNIQUES

To separate and quantify the proteins, the scientists used two-dimensional gel electrophoresis, in which proteins are separated according to charge and molecular weight, and then displayed using special imaging software as individual protein “spots” in a two-dimensional image. This provided genetic, sequence, and protein variation information that the team used to identify disease-specific changes in protein expression from complex protein extracts of cells, tissues, and other biological samples.

The researchers discovered in the AD patients that the differences related to the transport of amyloid-beta protein (Abeta), the primary component of AD plaques, as well as inflammatory response and specific neuronal membrane proteins. The identity of 23 proteins unique to AD was determined, and the possible relevance of changes in key pathogenic pathways then analyzed. Although identifying a protein “fingerprint” appears to be a major step forward in developing a diagnostic office test, it could take three or more years before one is available, according to the researchers.

“We're excited, but like any study we need replication and confirmation in more samples,” Dr. Lee said. “The idea is to have a simple and reliable diagnostic test for doctors to use in their offices.”

Even if the test is accurate in further trials, he said it will be only one of several tests that will be used to diagnose AD in the years ahead, noting that many researchers are pursuing other diagnostic approaches.

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Dr. Kelvin H. Lee: “Our study is the first to use sophisticated proteomic methods — [which focus on large-scale studies of proteins, particularly their structures and functions] — to hone in on a group of CSF biomarkers that are specific to autopsy-proven Alzheimer disease, and it is the first time proteomic results have been reviewed in a blinded validation using postmortem samples. The pattern of the 23 proteins was more than 90 percent sensitive in identifying people with AD, but more work needs to be done.”

‘THE SMOKING GUN’?

One such effort is underway at Columbia University's Taub Institute for Research on Alzheimer's Disease and the Aging Brain in New York City. By combining high-resolution spatial imaging with microarray analysis and a novel statistical modeling technique, Scott A. Small, MD, associate professor at the university's Center for Neurobiology and Behavior, reported that expression of the VPS35 molecule in the mouse brain correlates closely with AD development, and blocking its action may make it possible to slow or even halt the shuttling of Abeta to the brain, where it congeals between neurons and causes degenerative plaques.

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Dr. Scott A. Small reported that expression of the VPS35 molecule correlates closely with AD development, and blocking its action may make it possible to slow or even halt the shuttling of amyloid beta to the brain, where it congeals between neurons and causes degenerative plaques.

Dr. Small reported on his research at an early December meeting on neurodegenerative diseases and therapeutics at Cold Spring Harbor Laboratory in New York.

VPS35 is part of the retromer protein complex, which appears to play a pivotal role in shuttling Abeta from cell to cell. The retromer complex, originally described in yeast, mediates intracellular sorting of Vps10p, a receptor that transports vacuolar enzymes. The yeast retromer contains two sub-complexes, one of which includes VPS35. Retromer deficiency leads to an accumulation of Abeta in the hippocampus, a finding with major implications for both diagnosis and potential treatment strategies, Dr. Small told Neurology Today in a telephone interview.

“Declining levels of retromer appear to trigger Abeta accumulation in the brain,” he said, and research indicates that retromer deficiency in the hippocampus affects short-term memory in animal models of AD and in humans. Manipulating VPS35 and the retromer complex may make it possible to reduce local levels of Abeta in the brain. Several studies have shown that a consistent decline in metabolic activity in the prefrontal cortex and the hippocampus is associated with AD; the retromer complex, and VPS35 in particular, may hold the key to preventing this decline, he said.

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Dr. Sam Gandy: “Results of the first phase 3 trials of the anti-amyloids will be coming out soon, and that will be the watershed moment for all of this research — what these findings show about possible vaccines, anti-clumping agents, and secretase inhibitors. They all work well in mice, but we need to have the data in AD patients.”

“The big question has always been what are the primary pathogenic mechanisms involved in late-onset AD, and VPS35 appears to be one of them because we've found that it regulates Abeta — that's the smoking gun.”

The researchers are now taking the molecular findings in several directions simultaneously — developing tests for VPS35 expression, looking for mutations in the VPS35 gene that could predispose some patients to AD, developing transgenic mice with defects in VPS35 activity, and other retromer knockout strains.

“At face value, it looks like retromer might be the one, but we need to resist the temptation until more data are available,” Dr. Small said. “There can always be false positives. We need to confirm the findings, using cell cultures where we can increase or decrease Abeta levels by manipulating this cellular pathway.”

‘WATERSHED’ RESULTS NEAR

Sam Gandy, MD, PhD, professor of neurology at Thomas Jefferson University and director of the university's Farber Institute for Neurosciences in Philadelphia, noted that while the approaches by Drs. Lee and Small are different, these and other strategies have emerged from decades of research in a search for AD diagnostics and possible treatment.

The protein panel data from Drs. Lee and Relkin “look promising,” he told Neurology Today in a telephone interview. “This is the first group to elucidate such biomarkers and their efforts to try to find a fingerprint for AD are encouraging. But the key will be to reproduce the findings in the general population,” said Dr. Gandy, who also serves as vice chair of the national medical and scientific advisory council of the Alzheimer's Association.

“As it is, they have used an enriched patient population and it is important that this is also true in the general population because general practitioners are going to be the ones using the test in patients at clinics, not neurologists.”

Although the sensitivity and specificity of the test are high, in the general population it might be lower, he noted. “What we'd like to see, of course, is 100 percent sensitivity and specificity.”

With regard to Dr. Small's work with the retromer complex, he said the team appears to be “close to what could be a novel strategy” for blocking delivery of Abeta by regulating that specific molecular pathway.

“But again,” he cautioned, “the most important caveat is whether it will work in humans as well as it seems to work in mouse models. This looks promising and fulfills the right criteria in the right way, I think, and if it turns out this does control the movement of Abeta from one cell compartment to the next — especially transport from the endoplasmic reticulum to the Golgi complex — and they can develop animal models to demonstrate this — then they have identified an important pathway and possible target for treatment.”

“Results of the first phase 3 trials of the anti-amyloids will be coming out soon,” Dr. Gandy said, “and that will be the watershed moment for all of this research — what these findings show about possible vaccines, anti-clumping agents, and secretase inhibitors. They all work well in mice, but we need to have the data in AD patients.”

“We're about to find out if amyloid blockers work as well in humans as in mice, and that will tell us if we've been on the right track over the past 20 years or not,” he continued. “There might be other [factors] involved in the disease, but we won't know for sure until we get Abeta out of the way.” Until these results in human subjects are known, it's still a waiting game, he said.

ARTICLE IN BRIEF

  • ✓ Investigators report on two new techniques — one, involving an analysis of proteins and the other, spatial imaging and statistical modeling — that identified possible biomarkers for Alzheimer disease.

REFERENCE

• Finehout EJ, Franck Z, Lee KL, et al. Cerebrospinal fluid proteomic biomarkers for Alzheimer's disease. Ann Neurol 2006; E-pub 7 Dec. 2006.