ARTICLE IN BRIEF
Investigators report that the E4 allele of the apolipoprotein gene directly damages blood vessels that feed the brain, compromising the blood-brain barrier and enabling toxic substances to get in the brain and damage brain cells. They propose that blocking the cytokine from degrading the blood-brain barrier may offer a potential therapeutic target for Alzheimer's disease.
Almost 20 years ago, scientists identified the first risk gene for late-onset Alzheimer's disease (AD). One variety of the apolipoprotein E gene (APOE), the E4 allele, was shown to increase the risk for AD three- to four-fold and over ten-fold when someone inherited two copies of the genetic variant. Since then, scientists have been trying to figure out exactly how E4 works to put the brain in a more vulnerable state. Other theories have been put forth but none had taken hold, until now.
A team of scientists led by Berislav V. Zlokovic, MD, PhD, director of the Zilkha Neurogenetic Institute and professor and chair of the department of physiology and biophysics at the Keck School of Medicine at the University of Southern California in Los Angeles, have shown that the E4 allele directly damages blood vessels that feed the brain. The blood-brain barrier becomes leaky and toxic substances get in the brain and damage brain cells. This process does not happen in animals with the other alleles — E2 and E3 — that are not associated with increased risk for AD.
Questions remain about the role of APOE4 and the build-up of amyloid beta (Abeta) in the brain that is one of the hallmark pathological features of the disease. This latest study suggests that APOE4 can damage the neurovascular system in subtle ways — independently of Abeta leading to AD, and that the neurovascular damage makes it harder to clear away Abeta from the brain.
The scientists are now looking into the brains of human subjects with and without the APOE4 allele to see if similar mechanisms are at work. At least in all of the animal models of AD, the variant triggers an inflammatory reaction mediated by the cytokine — cyclophilin A — that weakens the blood-brain barrier, said Dr. Zlokovic.
Cyclophilins are a family of proteins that bind to cyclosporine, an immunosuppressant. The scientists used cyclosporine to block this process and they found that it protected the blood-brain barrier and prevented the pathology associated with AD.
The study, which was funded by the National Institute on Aging and published in the May 16 online edition of Nature, offers the possibility for treatments that block the cytokine from degrading the blood-brain barrier, said Dr. Zlokovic. “If we can block these events that damage the vascular system we may be able to stop or prevent Alzheimer's. It is pretty fascinating.”
Dr. Zlokovic and his colleagues studied several lines of genetically engineered mice — one that lacks the APOE gene altogether and others that produce only human versions of APOE2, APOE3, or APOE4. It was the only the mice with the E4 allele that had the leaky blood-brain barrier. The toxic substances that leach in through the open door through the blood stream end up damaging small blood vessels and synapses. This results in functional changes as well.
Dr. Zlokovic said that they have shown that both APOE2 and APOE3 alleles help control the levels of cyclophilin A, and that APOE4 does not. There was an excess of cyclophilin A in blood vessels, particularly in pericytes — a five-fold increase — in those animals with the APOE4 variant. The cytokine activates an enzyme called MMP-9 in pericytes that breaks down key proteins responsible for the integrity of the blood-brain barrier.
In addition to tests with cyclosporine, the researchers also tried inhibitors of the MMP-9 enzyme and nuclear-factor kB that also protected the blood-brain barrier and the resulting brain damage. The intriguing thing is that the drugs do not have to get into the brain to be effective, Dr. Zlokovic said. “The medicines are taken up by blood vessels.”
This is not a normal inflammatory response. In fact, the inflammation at the scene is minimal, the scientist said. While they did observe functional changes and brain cell death in animals with a leaky blood-brain barrier, this paper did not present information on how the cells die.
They are now developing scanning tests to measure the blood-brain barrier in patients.
“This is an elegant and important paper,” said Rudolph E. Tanzi, PhD, the Joseph P. and Rose F. Kennedy Professor of Neurology at Harvard Medical School and Massachusetts General Hospital. “There have been other ideas about what is going on with the APOE4 allele,” he said, citing one study that shows that APOE2 and APOE3 alleles do a better job at shuttling Abeta out of the brain than APOE4 and another, reporting that truncated APOE4 fragments are toxic and damage the mitochondria that in turn leads to cell death.
“This study offers another possibility: the degradation of the blood-brain barrier in APOE4 carriers... but we have to figure out what is most important to the disease process.”
“No matter which is most relevant, genetics argues that if one could provide a working copy of APOE2 that would help treat the disease,” said Dr. Tanzi. Such a gene expression or gene therapy model could be tested in animals to see if it were possible, he added.
If it is a weakened blood-brain barrier, other types of strategies may work too, added Dr. Tanzi. Cyclosporine would be too strong because of its potential for adverse effects but screening compound libraries could yield a medicine that would work to counter the effects of APOE4.
“These findings point to cyclophilin A as a potential new drug target for Alzheimer's disease,” said Suzana Petanceska, PhD, a program director at the NIH National Institute on Aging. “Many population studies have shown an association between vascular risk factors in mid-life, such as high blood pressure and diabetes, and the risk for Alzheimer's in late-life. We need more research aimed at deepening our understanding of the mechanisms involved and to test whether treatments that reduce vascular risk factors may be helpful against Alzheimer's.”
“The compromise of the blood-brain barrier has important implications for microvascular damage in the brain, which downstream could lead to later neuronal and synaptic degeneration,” said James A. Mortimer, PhD, a professor in the department of epidemiology and biostatistics at the University of South Florida. The study is in mice, so the species difference must be kept in mind. The findings need to be replicated in humans.
“The fact that humans have AD pathology related to APOE4 — and the mice they used for the study lack this pathology — muddies the comparison, but one could certainly compare microvascular damage in E4 and non-E4 carriers adjusting for differences in age at death. We have published results related to this issue using the Nun Study data and see little difference in microvascular damage between E4 vs. non-E4 carriers,” said Dr. Mortimer.
“My main reservation is that this study was performed in mice and it is not clear that its findings apply to humans. Our data on downstream effects (microvascular and other vascular damage) of the proposed blood-brain barrier dysfunction would suggest that E4 carriers differ very little from non-E4 carriers with regard to cerebrovascular pathology; the exception being amyloid angiopathy, which is more severe in E4 carriers. However, the mechanism of this type of vascular damage differs from that described in this paper.”
Norman Relkin, MD, PhD, associate professor of clinical neurology and neuroscience at Weill Cornell Medical College also believes that the limitations are that this study was exclusively carried out in a mouse model and includes no data on human brain tissue.
“It provides no explanation as to why 40–50 percent of AD patients who are non-E4 carriers show pathology that is relatively indistinguishable from that of the E4 carriers,” Dr. Relkin added. “Nevertheless, it's an interesting hypothesis and should inject new vigor in the school that relates the development of AD to vascular insults.”
• Bell RD, Winkler EA, Zlokovic BV,et al. Apolipoprotein E controls cerebrovascular integrity via cyclophilin A. Nature 2012; E-pub 2012 May 16.
• Castellano JM, Kim J, Holtzman DM, et al. Human ApoE isoforms differentially regulate brain amyloid-beta peptide clearance. Sci Transl Med
• Chen HK, Ji ZS, Mahey RW, et al. Apolipoprotein E4 domain interaction mediates detrimental effects on mitochondria and is a potential therapeutic target for Alzheimer disease. J Biol Chem
• Harris FM, Brecht WJ, Huang Y, et al. Carboxyl-terminal-truncated apolipoprotein E4 causes Alzheimer's disease-like neurodegeneration and behavioral deficits in transgenic mice. Proc Natl Acad Sci