New research reveals methods to block the molecular processes responsible for Alzheimer's disease and the memory loss it causes
By Michael S. Wolff, Scientific American
The human brain is an incredibly complex organic computer capable of absorbing a variety of sensory experiences, processing them, storing the information, pulling out pieces of information and connecting them at the right moment. Some liken the destruction wrought by Alzheimer's disease to wiping a hard drive, with the most recent files deleted first. Many times, the first sign of the disease is the inability to recall events that happened in the past few days, such as a phone call with a friend or a repair done at home, while the long-term memories remain intact. However, as the disease progresses, the old memories also begin to disappear, until even the closest people are unrecognizable. The fear of Alzheimer's disease is not a fear of pain or physical suffering but of the persistent loss of memories of a lifetime that actually constitute our identity.
Unfortunately, the comparison to the computer is not complete. You cannot simply reboot the human brain and reload the lost programs and files. The problem is that Alzheimer's disease not only erases the information but also destroys the hardware of the brain, which consists of more than 100 billion nerve cells (neurons), connected by 100 trillion connections. Most of the existing Alzheimer's drugs work based on the fact that many of the neurons affected by the disease release a chemical signal, a neurotransmitter called acetylcholine. The drugs block the enzyme responsible for breaking down acetylcholine, thereby increasing its level which is declining due to the disease. The result is activation of neurons and clearer thinking, but these drugs usually stop working after six months to a year because they cannot stop the destruction of the neurons themselves. Another drug, known as memantine, apparently slows down the rate of cognitive decline in patients with moderate or severe Alzheimer's by inhibiting the excess activity of another neurotransmitter (glutamate), but researchers still do not know if the effect of the drug lasts more than a year.
More than a decade ago, few people were optimistic about the chance of beating Alzheimer's disease. Scientists knew very little about the biology of the disease, and the explanation was that the causes of the disease and the course of its progression were hopelessly complex. However, in recent times the understanding of the molecular events that apparently influence the onset of the disease has greatly expanded, and scientists are now investigating a variety of methods to slow down or stop these destructive processes. It is possible that one of these treatments, or a combination of them, will be able to prevent the degeneration of neurons to the extent that it will be possible to stop Alzheimer's disease. Several drugs are currently in clinical trials and the preliminary results are encouraging. More and more researchers are now sensing hope, a word not usually associated with Alzheimer's disease.
Amyloid hypothesisThe two key signs of the disease, first noted 100 years ago by the German neurologist Alois Alzheimer, are plaques and tangles of proteins in the cerebral cortex and the limbic system, areas responsible for advanced brain functions. Plaques are deposits found outside the neurons and mainly contain a small protein known as amyloid-beta, or A-beta. The tangles are found inside the neurons and inside their branched extensions (axons and dendrites), and they are made of filaments of a protein called tau. When the phenomenon of plaques and tangles was discovered, a debate arose that lasted for most of the 20th century: are the tangles and plaques responsible for the degeneration of neurons in the brain, or do they simply mark the places where neuronal death has already occurred? In the last decade, the accumulating evidence leans towards the amyloid hypothesis, which claims that both A-beta and tau are directly involved in the creation of the disease, but A-beta causes the initial damage.
A-beta is a short fragment of a protein (peptide), which was isolated and characterized for the first time in 1984 by George J. Glenner and Chien W. Wong, who were then at the University of California, San Diego. The peptide originates from a larger protein known as the amyloid-beta precursor protein, or APP. APP molecules are embedded in the cell membrane, one part of the protein is inside the cell and the other part outside the cell. Two protease-type enzymes that cut proteins, called beta-secretase and gamma-secretase, cut APP and create A-beta, a process that occurs normally in all body cells. It is not clear why cells produce A-beta, but according to the available evidence the process is part of a cellular signaling pathway.
Part of the A-beta region in the APP protein is found in the membrane itself, between its outer layer and its inner layer. Since membranes are composed of water-repellent lipid molecules, regions of the protein that cross the membrane are usually composed of water-repellent amino acids. When beta- and gamma-secretase cleave A-beta from APP, it is released into the aqueous environment outside the membrane, and the water-repellent regions of different A-beta molecules stick together to form small, soluble aggregates. In the early 90s, Peter T. Lansbury Jr., now at Harvard Medical School, showed that at high enough concentrations, A-beta molecules can form in vitro a fiber-like structure similar to that found in the plaques of Alzheimer's disease. Both the soluble aggregates and the fibers of A-beta are toxic to neurons in culture, and the fibers can even interfere with the processes essential to learning and memory in mice.
These findings support the amyloid hypothesis, but the strongest support has come from studies of families at particularly high risk for Alzheimer's disease. The members of these families have rare genetic mutations that cause them to get the disease at a relatively young age, usually before the age of 60. In 1991, John A. Hardy, now at the American Institute on Aging, and his research partners discovered the first mutations in the gene containing the code to create APP. The mutations specifically affected the region of A-beta and adjacent regions of the protein. Shortly thereafter, Dennis J. Salkow of Harvard and Steve Younkin of the Mayo Clinic in Jacksonville, Florida, each separately found that these mutations usually increase production of A-beta, or a particular type of A-beta that is particularly prone to forming precipitates. Furthermore, people with Down syndrome, who carry three copies of chromosome 21 instead of the normal two copies, have a much higher incidence of middle-aged Alzheimer's. Because chromosome 21 contains the gene for APP, people with Down syndrome produce higher levels of A-beta from the day they are born, and amyloid deposits can be found in their brains as early as age 12.
Researchers soon discovered other genes associated with Alzheimer's disease and criticized A-beta production. In 1995, Peter St. George-Hyslop and his collaborators at the University of Toronto identified mutations in two similar genes called presenilin 1 and 2. The mutations cause particularly severe Alzheimer's disease that begins at a young age, usually in the 30s or 40s of life. Studies have shown that these mutations increase the relative amount of A-beta that tends to accumulate in clusters. Today we know that the proteins encoded by the presenilin genes are part of the gamma-secretase enzyme.
That is, of the three genes that cause the early onset of Alzheimer's disease, one encodes a precursor protein of A-beta and the other two encode components of the protease enzyme that helps produce the harmful peptide. Furthermore, scientists have discovered that people who carry a certain version of a gene that encodes the protein apolipoprotein E, a protein that helps group the A-beta proteins into clusters and fibers, are at high risk of developing Alzheimer's disease in old age. It is likely that a variety of genetic factors play a role in the onset of the disease, and each of them contributes to a small extent; Studies done on mice show that environmental factors can also affect the risk of getting sick (exercise, for example, reduces the risk).
Scientists still don't understand exactly how the soluble and insoluble fibers of A-beta interfere with neurons and why they kill them. However, the evidence suggests that accumulations of A-beta outside the neurons can activate a pathway of events involving changes in tau proteins inside the neurons. In particular, the A-beta clusters can ultimately change the cellular activity of enzymes called kinases, which add phosphate groups to proteins. The kinases add too many phosphorus groups to the tau proteins, thus affecting the chemical properties of the proteins and causing them to form coiled fibers. Chemically modified tau proteins, in one form or another, kill the neurons, perhaps because they interfere with the system for transporting proteins and other large molecules along axons and dendrites. Mutations in the tau protein itself can also cause the formation of tau fibers and cause other neurodegenerative diseases of the nervous system in addition to Alzheimer's disease. That is, the formation of tau fibers is probably a more general event leading to the death of neurons, while A-beta is a unique cause of Alzheimer's disease.
Disable the molecular scissorsSince A-beta plays a crucial role in the disease process, the proteases that produce it are an obvious target for drugs to inhibit their activity. Protease inhibitors have proven to be highly effective in treating other diseases such as AIDS and hypertension. The first step in creating A-beta is done by beta-secretase, a protease that cleaves the bulk of APP near the outside of the cell membrane. In 1999, this enzyme, which is especially common in brain neurons, was discovered by five separate, unrelated research groups. Although beta-secretase is membrane-anchored, it closely resembles a subset of proteases found in the aqueous environment inside and outside cells. Members of this subgroup, including the protease involved in the replication of HIV, the virus that causes AIDS, use an amino acid known as aspartic acid to carry out the protein-cutting reaction. All proteases use water to cleave their target proteins, and enzymes belonging to the aspartyl protease family use a pair of aspartic acids to activate the water molecule for this purpose.
Since beta-secretase clearly belongs to this family of proteases, researchers have been able to take advantage of the vast knowledge that has accumulated about this group of proteases to understand the function of this enzyme and how it can be inhibited. Indeed, researchers already know the three-dimensional structure of beta-secretase and have used the knowledge to design inhibitory drugs with the help of computer programs. Genetic studies show that inhibiting the activity of the enzyme will not cause harmful side effects, because damage to the gene encoding beta-secretase in mice caused the disappearance of A-beta without causing visible negative effects. But at the moment beta-secretase inhibitors are not yet ripe for clinical trials. The main challenge is to develop effective compounds small enough to penetrate the brain. Unlike blood vessels in other areas of the human body, the capillary walls in the brain are made up of highly dense endothelial cells. Because there are only a few spaces between the cells, the protease inhibitors have to be able to penetrate through the endothelial cell membranes to reach the brain tissue, and most large molecules cannot break through the so-called blood-brain barrier.
The enzyme known as gamma-secretase performs the second step in the formation of A-beta by cutting the APP stump left after the action of beta-secretase. Gamma-secretase performs an unusual reaction that involves using water to cleave the protein in the water "hating" environment of the cell membrane. Two important clues turned out to be essential to understanding this protease. First, Bert de Strupper of the Catholic University of Leuven, Belgium, discovered in 1998 that silencing the presenilin 1 gene in mice significantly reduces APP cleavage by gamma-secretase. This experiment proved that the protein encoded by the gene is essential for the function of the enzyme. Second, in my laboratory, which was then at the University of Tennessee at Memphis, they discovered that compounds similar to the distinct inhibitors of aspartyl proteases could block APP cleavage by gamma-secretase in cells. This result suggests that gamma-secretase, similar to beta-secretase, contains a pair of aspartic acids essential for carrying out protein cleavage reactions.
According to these results, we hypothesize that the presenilin protein is an unusual aspartyl protease found within cell membranes. When I was on sabbatical at Harvard in Salkow's lab and in collaboration with Weiming Xia, we identified two amino acids in the presenilin protein that are supposed to be in the membrane, and showed that both are essential for the generation of A-beta by gamma-secretase. Subsequently, we and others have shown that gamma-secretase inhibitors bind directly to presneylin, and that three other membrane-bound proteins associate with presneylin to carry out the cleavage reaction. Today, gamma-secretase is considered the founding protein of a new group of proteases that apparently use water within cell membranes to carry out their biochemical tasks. Furthermore, gamma-secretase inhibitors are relatively small molecules that can cross membranes, and therefore penetrate through the blood-brain barrier.
Two years ago, I told my youngest son's class, who was in the 10th grade at the time, about the research being conducted in my laboratory, and told about amyloid and the hope of discovering new Alzheimer's drugs that block enzyme activity. One boy interrupted me and said: "But what if the enzyme does something important? You might hurt people!" This problem, identified by a XNUMX-year-old boy, is very real. The potential of gamma-secretase to be used as a therapeutic target is compromised because this enzyme plays a vital role in the maturation of differentiating cells in various areas of the body, such as stem cells in the bone marrow that develop into red blood cells and lymphocytes. Uniquely, gamma-secretase cleaves a protein, at the cell surface, known as the Notch receptor. The part of Notch that is released from the membrane into the cell sends a signal to the nucleus that controls the fate of the cell.
High doses of gamma-secretase inhibitors cause severe toxic effects in mice due to damage to the Notch signaling pathway, and this raises concerns about this method of treatment. Despite this, a drug developed by the pharmaceutical company Eli Lilly passed the safety tests in healthy volunteers. (Phase A in the series of medical tests). The compound can now move to the next phase of testing (phase B) which will be conducted in Alzheimer's patients who are in the early stages of the disease. Furthermore, researchers have identified molecules that regulate gamma-secretase, thereby inhibiting the formation of A-beta without affecting Notch cleavage. These molecules do not bind to the aspartic acids of gamma-secretase but to other areas of the enzyme, and cause a change in its structure.
Some of the inhibitors even have a unique ability to reduce the formation of the version of A-beta that tends to form aggregates, and encourage the formation of a shorter peptide that does not tend to attach easily. One such drug, Flurizan, developed by a research group led by Eddie Ko from the University of California, San Diego and Todd Gold from the Mayo Clinic in Jacksonville, has shown great promise in Alzheimer's patients in the early stages of the disease and is now entering a more advanced stage of clinical trials (Phase C). which will include more than 1,000 patients across the US.
Removing the cobwebsAnother strategy for fighting Alzheimer's is to cleanse the brain of the toxic accumulations of A-beta after the peptide is formed. One approach is active vaccination, which involves mobilizing the patient's own immune system to attack A-beta. Dale B. Schenck and his partners at the Allen Corporation in South San Francisco broke ground in 1999 with their research on mice genetically engineered to develop plaques. They found that injecting A-beta into these mice triggered an immune response that prevented the formation of plaques in the brains of young mice and cleared the plaques that had already formed in adult mice. The mice produced antibodies that recognized A-beta, and these antibodies apparently encouraged the brain's immune cells, the microglia cells, to attack the peptide clusters (see box on the opposite page). The positive results in mice, which included an improvement in the learning and memory processes, soon led to experiments in humans.
Unfortunately, although the A-beta injection passed the initial safety tests, some patients participating in phase B of the clinical trials developed encephalitis and the study was discontinued at Ibo in 2002. Follow-up studies showed that the treatment may have caused the inflammation because the immune system's T cells were encouraged to over-aggressively attack the A-beta deposits. Nevertheless, the study confirmed that many of the patients developed antibodies against A-beta and these patients showed some signs of improvement in memory and ability to concentrate.
Concerns about an active vaccine have led some researchers to try a tolerized vaccine that aims to clear the peptide by injecting antibodies into patients. These antibodies were created in mouse cells, and a process of genetic engineering prevented their rejection by the human immune system, so it is likely that they will not cause encephalitis because they are not expected to provoke a harmful reaction of T-cells in the brain. A tolerable vaccine treatment developed by the Allen Corporation is already in phase B of the medical tests.
The mechanism by which an active or tolerable vaccine is able to clear A-beta from the brain remains a mystery, because it is not clear to what extent the antibodies are able to penetrate the blood-brain barrier. There is evidence suggesting that there is no need at all for the antibodies to enter the brain. It is possible that clearing A-beta from the body cord leads to the exit of the peptide from the brain, because molecules tend to move from higher to lower concentrations. Although a tolerant vaccine appears to be the most promising at the moment, the active vaccine is not yet out of the race. Preliminary studies led by Cynthia Lemer of Harvard University show that vaccination using selected parts of A-beta, instead of the entire peptide, can stimulate the production of antibodies by B-cells of the immune system without activating the T-cells responsible for encephalitis.
Other researchers are testing non-immune therapies to stop the A-beta molecules from sticking. Several companies have identified compounds that react directly with A-beta, keeping it soluble in the fluid outside the neurons in the brain and preventing the formation of the harmful aggregates. The Quebec company Neurochem is developing Alzhemed, a small molecule that mimics heparin, the natural anticoagulant. In the blood, heparin prevents platelets from clumping together into clots, but when this polysaccharide binds to A-beta, it increases its chances of forming precipitates. Because Alzep binds to the same site on A-beta, it blocks the activity of heparin and therefore reduces the adhesion of the peptide. This compound has very little toxicity even at high doses, and the treatment somewhat improved the cognitive abilities of patients with mild Alzheimer's. This medicine is already in stage C of the medical tests.
Tao as a destination
Remember that amyloid is only one component of the Alzheimer's equation. The second component, tau fibers that form the tangles in neurons, is also considered a promising target for preventing the degeneration of nerve cells in the brain. Researchers are now particularly focused on designing inhibitors that will block the kinases that add excess phosphorus groups to tau, which is an essential step in the formation of the fibers. These efforts have not yet yielded potential drugs, but there is reason to hope that such substances will eventually be combined with drugs that target A-beta.
Researchers are even testing whether drugs known as statins, which lower the level of cholesterol in the blood, and are widely used to reduce the risk of heart disease, can also treat Alzheimer's. Epidemiological studies show that people taking statins have a lower risk of developing Alzheimer's. The reason for this is not quite clear. It is possible that by lowering the level of cholesterol, these drugs decrease the production of APP, or it is possible that they directly affect the process of A-beta formation by inhibiting the activity of scaratase. Medical tests in phase C are examining whether statins such as Pfizer's Lipitor can indeed prevent Alzheimer's.
Another exciting development is related to cellular therapy. Mark Toszynski and his collaborators at the University of California, San Diego took skin biopsies from patients with mild Alzheimer's and inserted the gene encoding nerve growth factor (NGF) into these cells. The genetically engineered cells were surgically inserted into the forebrain of the patients from which they were taken. The idea is that the transplanted cells will produce and secrete NGF, thereby preventing the loss of neurons that produce acetylcholine and improving memory. The cellular therapy is a sophisticated method to inject NGF, a large and insoluble protein that cannot enter the brain in other ways. Although this study examined only a handful of patients and did not receive important reviews, a follow-up study showed a significant slowdown in the decline of the patients' cognitive abilities. The encouraging results justified the continuation of the medical tests.
Although some of these potential treatments may not live up to their promise, scientists hope to find at least one drug that will effectively slow or even stop the gradual loss of neurons in the brain. Such a breakthrough could save millions of people from the relentless deterioration of Alzheimer's disease and pave the way for restorative drugs to restore lost mental functions.
Targeting A-beta may be able to prevent the onset of Alzheimer's disease or delay it when it is in the early stages, but it is not clear whether this treatment method can help people in more advanced stages of the disease. Nevertheless, researchers have good reason to be optimistic. The flurry of recent discoveries has convinced many of us that the journey to discover ways to prevent and treat Alzheimer's disease will not be in vain.
Overview/A New Hope for the Elderly
Scientists focused on the hypothesis that a peptide known as amyloid-beta
(A -beta) causes damage and death of brain cells in Alzheimer's patients.
Researchers are currently developing drugs that can inhibit the formation of A-beta and prevent the peptide from damaging neurons.
Several potential drugs are already in clinical trials to determine whether they can slow or stop the brutal mental deterioration caused by Alzheimer's.
The cruelest cut of allAccording to the amyloid hypothesis, Alzheimer's disease begins due to increased accumulation of amyloid-beta
(A-beta) formed from the precursor protein, APP. In the first step, an enzyme called beta-secretase cleaves APP outside the cell membrane using aspartic acids that make the water molecules more active. After that, the protein presenilin, a component of the enzyme gamma-secretase, cuts, inside the membrane, the remaining stump, releasing A -beta. Some promising drugs inhibit the activity of gamma-secretase. Others cause the enzyme to cut APP at a different site, so that a shorter and less harmful version of A-beta is obtained.
About the authorMichael S. Wolfe is an associate professor of neurology at Brigham and Women's Hospital and Harvard Medical School. His work focuses on understanding the molecular basis of Alzheimer's disease and identifying effective therapeutic approaches. He received a PhD in Medicinal Chemistry from the University of Kansas. In January 2006, he founded the Laboratory for Experimental Alzheimer's Drugs at Harvard Medical School, which focuses on the development of potential drugs for Alzheimer's disease.
And more on the subject
Decoding Darkness. Rudolph E. Tanzi and Ann B. Parson. Perseus Books Group, 2000.
Hard to Forget: An Alzheimer's Story. Charles Pierce. Random House, 2000.
Therapeutic Strategies for Alzheimer's Disease. Michael S. Wolfe in Nature Reviews Drug Discovery, Vol. 1, pages 859–866; November 2002
You can find more information on the websites www.alzforum.org and- alz.org
