Scientists at the Gladstone Institute of Neurological Disease (GIND) have identified a new strategy to destroy amyloid-beta (AB) proteins, which are widely believed to cause Alzheimer’s disease (AD). Li Gan, PhD, and her coworkers discovered that the activity of a potent AB-degrading enzyme can be unleashed in mouse models of the disease by reducing its natural inhibitor cystatin C (CysC).

All of us produce AB proteins in the brain. However, in most people, the proteins never build up to dangerous levels because they are cleared away by enzymes that destroy them. Previously Dr. Gan’s laboratory had shown that cathepsin B (CatB) is such an AB-degrading enzyme. In the latest issue of the journal Neuron, the researchers report a highly effective approach to promote CatB-mediated clearance of AB .

“Many groups have developed drugs to block the production of AB, but the efficacy and safety of this approach remains to be demonstrated in clinical trials,” said GIND Director Lennart Mucke, MD “By identifying an effective strategy to enhance the removal of AB, this research provides a very promising alternative or complementary therapeutic avenue.”

High levels of AB in the brain may result from overproduction of AB or from an inability to eliminate it from the brain. While most work has focused on the first option, the latter has been problematic. For example, efforts to develop a vaccine that would trigger the immune system to eliminate AB have shown limited success and resulted in adverse side effects.

“Our strategy to harness the activity of a powerful AB-degrading enzyme takes advantage of the brain’s own defense system to remove the toxic AB build-up,” said Dr. Gan. “In principle, one could boost the activity of CatB by expressing more of it in the brain or by reducing the activity of CysC, its natural inhibitor. We focused on the latter strategy because it has greater long-term therapeutic potential.”

Many enzymes that degrade proteins are kept in check by regulators called protease inhibitors. The activity of CatB is regulated by the protease inhibitor CysC. By reducing CysC activity, the scientists were able to unleash the AB-degrading power of CatB, effectively preventing the build-up of AB in mouse models of AD.

To examine the impact of this manipulation on brain function, Dr. Gan’s team measured brain cell activities that relate closely to learning and memory. Increasing CatB activity by lowering CysC levels prevented AB-induced deficits in those cellular activities. The investigators also tested the modified AD mice for learning and memory in a water maze. Higher levels of CatB activity improved the ability of AD to learn the maze and to retain the new information. Increasing CatB activity also prevented the premature mortality that is typically seen in these Alzheimer models.

“Our results suggest that CysC reduction has major therapeutic potential,” Dr. Gan said. “The next step will be to develop pharmacological approaches to inhibit CysC in the human brain.”

In a paper published today in Nature, the team led by Professor Nigel Robinson have revealed a mechanism that ensures the right metal goes to the right protein. Proteins are essential and involved in just about every process in living cells.

Life, microbe, plant or human, is a painstaking assembly of trillions of atoms. The atoms include metals such as copper and manganese which act as catalysts in proteins. The proteins wrap around the metal atoms.

The research team has shown that to ensure a copper and a manganese protein wrap around the correct metal atoms they do this in different parts of the cell, in zones which contain different metals. Therefore, which protein attaches to which metal is determined by where the folding action takes place in the cell.

Previously, a common view was that the right metals were simply those which were most attracted to the protein, but in this work that is not the case.

Professor Nigel Robinson at Newcastle University who led the research says: “This has taken us one step closer to understanding why metals and proteins assemble in the ways they do.”

“One motive behind the work is pure curiosity, but as so many proteins need metals this type of work has many potential uses - for example, in synthetic biology which is striving to produce green power from bacteria by using energy from sunlight to produce hydrogen gas, a process which needs nickel and iron.

“It may also help in diseases such as Alzheimers where there are unexplained links to proteins binding metals such as copper. There’s also application in controlling infections by Staphylococcus aureus; a bacterium which our bodies defences succeed - or sometimes fail - in killing by removing manganese and zinc from abscesses.”

The researchers have shown that the way the metals attach is identical for a protein that binds manganese to one that binds copper. In both cases the metals bind inside protein barrels with the same type of metal-attractions.

Carrying out the work in a blue-green algae, a cyanobacterium, the team has been able to show that a protein requiring copper transports to the periplasm, the outer area of the cell, where it then folds around the available metal, which is copper.

Conversely, manganese but not copper atoms are found in the cytosol, in the middle of the cell. The team has demonstrated that a protein requiring manganese folds in the cytosol. The manganese protein is then transported to the periplasm having first trapped its manganese.

The cyanobacterium organism was chosen because it has a high demand for these two metals which are required for proteins involved in photosynthesis. These metals were chosen because they lie towards opposite ends of a chemical series called the Irving-Williams series, such that selecting these metals for proteins should be especially demanding.

In the work funded by the BBSRC, the Newcastle University team first developed a new approach to discover metal-binding proteins. This is now being swiftly applied to lots of other types of living cells and other essential metals (zinc, nickel, cobalt, iron). Unexpectedly, x-ray crystal structures showed that the identified proteins, MncA for manganese and CucA for copper, were both cupins (Latin for barrels) with identical sets of atoms for binding to the metals. Consistent with the chemical series, a ten-thousand times excess of manganese over copper was needed to fill the MncA barrel with manganese when folding is done in the laboratory.

Once folded, the manganese site is buried, the metal trapped inside the protein, and so the manganese protein can subsequently co-exist with the copper protein because its’ metal becomes impervious to replacement by metals further up the Irving-Williams series.

The work exemplifies a cell overcoming the metal binding preferences of proteins.

The new discipline of synthetic biology aims to engineer cells to carry out useful tasks, for example to generate valuable compounds. Because metals are the catalysts for so much of biology, knowing how to engineer a supply of the right metals to the right proteins will be important to the success of these ventures.

Amyloid precursor protein (APP), whose cleavage product, amyloid-b (Ab), builds up into fibrous plaques in the brains of Alzheimer’s disease patients, jumps from one specialized membrane microdomain to another to be cleaved, report Sakurai et al.

Although there is no definitive evidence that Ab plaques are the direct cause of Alzheimer’s disease, there is much circumstantial evidence to support this.  And working on this hypothesis, scientists are investigating just how the plaques form and what might be done to stop or reverse their formation.

APP, a protein of unknown function, is membrane associated and concentrates at the neuronal synapse.  Certain factors such as high cellular cholesterol and increased neuronal or synaptic activity are known to drive APP cleavage, and Sakurai and colleagues’ paper pulls these two modes of Ab regulation together.

APP associates with membrane microdomains high in cholesterols (lipid rafts).  These lipid rafts can also contain the enzyme necessary for APP cleavage, BACE.  Synaptic activity is known to involve a very different type of membrane microdomain high in an excytosis-promoting factor called syntaxin.  Sakurai et al.  Now show that although APP preferentially associates with syntaxin microdomains, upon neuronal stimulation APP instead associates with microdomains that contain BACE.

It’s unclear why APP should be associated with syntaxin, though it might suggest a role for APP in vesicle trafficking and exocytosis.  Also unclear is why neuronal activity should cause APP to jump from syntaxin domains to BACE domains.  What is clear, however, is that the process is an active one, requiring a kinase called cdk5.  Furthermore, treating neurons with a cdk5 inhibitor called roscovitine, which is currently in trials for cancer treatment, reduced APP’s association with BACE microdomains and reduced APP cleavage.

Marauding molecules cause the tissue damage that underlies heart attacks, sunburn, Alzheimer’s and hangovers.  But scientists at the Stanford University School of Medicine say they may have found ways to combat the carnage after discovering an important cog in the body’s molecular detoxification machinery.

The culprit molecules are oxygen byproducts called free radicals.  These highly unstable molecules start chain reactions of cellular damage an escalating storm that ravages healthy tissue.

“We’ve found a totally new pathway for reducing the damage caused by free radicals, such as the damage that happens during a heart attack,” said Daria Mochly-Rosen, PhD, professor of chemical and systems biology and the senior author of a study reporting the new findings.  The research will appear in the Sept. 12 issue of Science.

Before the study, scientists knew that heart muscle could be preconditioned to resist heart attack damage for instance, moderate drinkers tend to have smaller, less severe heart attacks than teetotalers.  But scientists didn’t understand how pre-conditioning worked.

To figure out how alcohol protects heart muscle from free-radical damage, Mochly-Rosen’s team tested alcohol pretreatment in a rat heart-attack model.  They compared the enzymes activated during the attacks to those switched on with no alcohol.  Enzymes are the “doers” of the cellular machinery, catalyzing all of the biochemical reactions that form the basis of life.

Surprisingly, the treatment activated aldehyde dehydrogenase 2 (ALDH2), an obscure alcohol-processing enzyme.  Alcohol pretreatment increased the enzyme’s activity during heart attack by 20 percent, leading to a 27 percent drop in the associated damage.

“Although this enzyme was discovered a long time ago, my research group knew nothing about the enzyme except that it helps remove alcohol when people drink,” said Mochly-Rosen, who is also the senior associate dean for research in the School of Medicine and the George D. Smith Professor in Translational Medicine.

ALDH2 wasn’t one of the well-studied antioxidant players that the scientists expected to find fighting free-radical damage.  The enzyme neutralizes an aldehyde molecule, a toxic byproduct of the ethanol in alcoholic beverages.  But aldehydes are also formed in the body when free radicals react with fat molecules.

The body’s cells contain a lot of fat, Mochly-Rosen noted.  “It’s very easy for free radicals to find fat and oxidize it to aldehydes.”

Inside cells, the accumulating aldehydes permanently bind and damage cellular machinery and DNA.  Such damage occurs in many diseases, from heart attack and Parkinson’s to sun-induced aging of the skin.

After learning of ALDH2’s novel role in reducing the damage, the researchers searched for a molecule that could make the enzyme function even better.  They enlisted the Stanford High Throughput Bioscience Center, directed by David Solow-Cordero, PhD, to find a molecule that heightened the enzyme’s activity.

The winner of this contest was a tiny molecule that reduced heart attack damage by 60 percent in the rat model.  The molecule, Alda-1, has a surprising mode of action: it protects ALDH2 itself from aldehyde attack.  The enzyme, it turns out, was being hobbled by the very chemical it removes.

Because Alda-1 is small, it should be easy to adapt for pharmacological use, Mochly-Rosen said.  She expects the new molecule to have many possible drug applications.

“It has a huge potential use,” she said.  So far, Alda-1 has been tested only in the rat model, but Mochly-Rosen’s lab is investigating other possible applications, such as fighting neurodegenerative disease and sun damage on the skin.  The team also hopes to interest drug companies in human trials.

In addition to its lofty medical applications, Alda-1 could also have a much lowlier use: fighting hangovers.  Many nasty hangover symptoms are due to aldehyde buildup.

The tiny molecule may also improve alcohol tolerance and reduce susceptibility to free-radical diseases in people with a common ALDH2 mutation.  The mutation affects 40 percent of people of Asian descent and causes an intolerance for alcohol.

Blocking a common immune system response cleared up plaques associated with Alzheimer’s Disease and enabled treated mice to recover some lost memory, Yale University researchers report Friday in the journal Nature Medicine.Researchers hope the new approach may one day overcome one of the biggest obstacles to development of new dementia medications – the difficulty in finding drugs that can safely cross the blood-brain barrier.

The results of the research surprised the scientists working in the lab of Richard Flavell, senior author of the paper, chairman of the Department of Immunobiology at Yale and investigator with the Howard Hughes Medical Institute. Flavell’s team originally thought that blocking the immune system molecule TGF-β(or transforming growth factor), might actually increase the buildup of amyloid plaques associated with Alzheimer’s Disease

Earlier studies had shown that Alzheimer’s patients tend to have elevated amounts of TGF-β, which plays a key role in activating immune system response to injury. Some had thought the presence of the molecule was simply an attempt to quiet the inflammatory response caused by a buildup of plaque.

Instead, the team found that as much as 90 percent of the plaques were eliminated from the brains of mice genetically engineered to block TGF-β in the peripheral immune cells.

It was like a vacuum cleaner had removed the plaques,” Flavell said.

When the TGF-β pathway was interrupted in mice engineered to have Alzheimer’s, the mice showed an improved ability to perform some tests, including navigating mazes when compared to mice without TGF-β blocked. Scientists also found lower levels of other biological markers associated with the dementia.

When TGF-β was blocked, the immune system seemed to unleash immune cells known as peripheral macrophages. The macrophages passed through the blood-brain barrier and surrounded the neurons and plaques in the brains of mice. “If results from our study in mice engineered to develop Alzheimer’s-like dementia are supported by studies in humans, we may be able to develop a drug that could be introduced into the bloodstream to cause peripheral immune cells to target the amyloid plaques,” said Terrence Town, lead author of the study.

Different types of non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, naproxen, and aspirin, appear to be equally effective in lowering the risk of Alzheimer’s disease, according to the largest study of its kind published in the May 28, 2008, online issue of Neurology®, the medical journal of the American Academy of Neurology. Experts have debated whether a certain group of NSAIDs that includes ibuprofen may be more beneficial than another group that includes naproxen and aspirin.Using information from six different studies, researchers examined data on NSAID use in 13,499 people without dementia. Over the course of these six studies, 820 participants developed Alzheimer’s disease.

Researchers found that people who used NSAIDs had 23 percent lower risk of developing Alzheimer’s disease compared to those who never used NSAIDs. The risk reduction did not appear to depend upon the type of NSAID taken.

“This is an interesting finding because it seems to challenge a current theory that the NSAID group which includes ibuprofen may work better in reducing a person’s risk of Alzheimer’s,” said study author Peter P. Zandi, PhD, with Johns Hopkins Bloomberg School of Public Health in Baltimore, MD. “The NSAID group that includes ibuprofen was thought to target a certain type of plaque in the brain found in Alzheimer’s patients. But our results suggest there may be other reasons why these drugs may reduce the risk of Alzheimer’s.”

The study’s lead author Chris Szekely, PhD, with Cedars Sinai Medical Center in Los Angeles, says the discrepancy between studies such as this one and the negative clinical trials of NSAIDs in treatment or prevention of Alzheimer’s need to be further explored.