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SUMMER/FALL 2003
How a Slime Mold Came to the Aid of Alzheimer's Research
by Kathleen Cason

In an ancient Persian fable, three princes from the kingdom of Serendip go out into the world to gain experience. During their adventures, they find they have a gift for making unexpected discoveries.

Like those fabled princes, UGA cell biologist Marcus Fechheimer and his research team have discovered they too have a gift for serendipity.

In the course of trying to understand how proteins “know” where to go in a cell, the researchers unexpectedly stumbled upon an unusual cellular structure normally associated with Alzheimer’s and other neurodegenerative diseases.

“I didn’t think we were working on anything that had to do with Alzheimer’s disease,” said Fechheimer, who uses a slime mold as his lab’s version of a “guinea pig.”

“We didn’t expect to find a structure in a slime mold that is present in increased amounts in people with Alzheimer’s — particularly present in the brain’s major site of learning and memory. That’s where these things accumulate,” he said.

Fechheimer’s team not only chanced upon this unusual structure — called a Hirano body — in a slime mold but also figured out how to force cells to produce them on command in the laboratory. Up till now, these structures could only be studied in brain tissue of people who had died. Because of this discovery, scientists will be able to study Hirano bodies in living cells for the first time and unravel the mysteries of this little-understood protein deposit.

Though Hirano bodies are common in the brains of people diagnosed with dementia, their role in disease, if any, is unclear.

“We don’t know if a Hirano body is good or bad or why it’s there or how it forms,” said Ruth Furukawa, a UGA research scientist who co-directs the study.

Proteins run amuck

What causes neurodegenerative diseases like Alzheimer’s is still largely unknown, but something destroys nerve cells in the brain over a period of time as victims gradually lose their minds.

In the hunt for cause and cure, scientists have focused on various abnormal protein deposits that mar diseased brains. Deposits called plaques and tangles have captured the most attention. Hirano bodies, while not at the forefront of Alzheimer’s research, are another type of protein deposit associated with the disease.

“Hirano bodies are certainly more prevalent in brains from patients who are suffering from dementias, probably any type,” said James Bamburg, one of Fechheimer’s collaborators and a professor of biochemistry and molecular biology at Colorado State University. “Hirano bodies also occur in brains of individuals with normal cognitive function but usually increase in number with age.”

Nearly four decades have passed since Asao Hirano, an eminent neuropathologist at Montefiore Medical Center in New York, first discovered the peculiar deposit in the brain’s memory center. Since then Hirano bodies have been reported in the brains of people with neurodegenerative diseases, as well as diabetes, stroke and alcoholism.

“Making a Hirano body may be a cellular mechanism for dealing with run-amuck proteins,” said Furukawa, who manages the day-to-day operation of the lab. “That’s just a hypothesis.”

Run-amuck proteins certainly seem to play some role in all the diseases where Hirano bodies are found.

“Perhaps Hirano bodies do nothing; perhaps they’re part of cell death; or perhaps they are adaptations to stress that are good for cells,” Fechheimer said. “Because they’re seen in so many diseases, it’s worth finding out.”

After more than 20 years probing basic questions in cell biology, Fechheimer’s team is well positioned to explore the role of Hirano bodies in cells and in human disease.

The path to hirano bodies

But how did a cell biologist studying cell movement and structure — in a lowly slime mold no less — end up investigating something that may have a role in degeneration of the human brain? It seems like a stretch at first.

Basic but complex questions in cell biology have piqued Fechheimer’s curiosity and driven his research program for more than two decades: How do cells move? What gives them shape? How do they eat?

The key to such riddles resides in a fibrous network inside cells, a kind of internal micro-scaffolding called the cytoskeleton. Besides acting like the “bones” of a cell, the cytoskeleton is an intracellular highway system, where specialized proteins scoot along the roadways toting organelles and other materials to the places they need to be.

“When we look at something that seems very obvious and beautiful like a crawling amoeba, it’s almost beguiling in its simplicity,” Fechheimer said. “But then you think about all the different molecular interactions and changes just in the actin when a cell is crawling forward. There are a lot of different things going on in different places. Figuring out all those dynamics is very complicated and very exciting. And we have great tools today to do it.”

To uncover the secrets of cell structure and movement Fechheimer’s team probed the inner workings of molecules that make up the cytoskeleton. The “roadways” consist of filaments made of three distinct proteins — one of which is actin — along with helper proteins that either anchor or bundle the filaments.

Actin doesn’t work alone. Among its helper molecules is a family of proteins that include one called the 34 kDa actin bundling protein. As its name implies, the 34 kDa protein packages actin filaments into bundles.

Actin bundling proteins play important roles in a variety of processes in the body.

“These actin bundles are ubiquitous,” Fechheimer said. For example, they have a role in hearing.

“The pressure wave that comes into your ear actually deflects actin bundles that are arranged like a staircase in the sensory hair cells and then an electrical signal gets sent to the brain,” he said.

During the past two decades, Fechheimer’s team has systematically answered many questions about the 34 kDa protein. They pinpointed where it is found in cells and what controls its ability to stick to actin. They’ve uncovered the protein’s structure. Along the way, they’ve looked at how it works by chopping the protein into pieces and testing how different snippets bind to actin.

To fine-tune their understanding of what the bundling protein does and how it works, they have altered the gene for the 34 kDa protein in small ways and inserted the altered version back into cells to see what happens.

That’s what Andrew Maselli was doing when he discovered something else.

Serendipity and beyond

Unlikely as it might have seemed, the bundling protein proved to be the key to the team’s discovery of Hirano bodies in the slime mold. Hirano bodies, it turns out, are partially made of actin; a piece of the actin bundling protein triggers cells to make Hirano bodies.

“I was studying the 34 kDa actin bundling protein and I wanted to know how it got where it needed to go in the cell to form the structures for locomotion and eating,” said Maselli, a former UGA doctoral student who is now an assistant professor at Chicago State University.

Other scientists already had demonstrated the bundling protein’s role in making structures that enable cell movement and feeding, Maselli said. But no one had looked at how the protein “knew” to go help form those structures.

Figuring out how a protein like this one works is like figuring out how a clock works: Remove a piece from the clock, tinker with it a bit, put it back in and see what happens.

Maselli took the gene for the bundling protein, removed bits from it, put it back in the slime mold and watched to see what happened. He suspected that calcium helped control the protein’s travels in the cell so he removed the bits that interacted with calcium.

“These structures formed that are very striking,” Maselli said. “It took a while before I was convinced that what I was seeing was interesting biologically and not just some artifact.”

In the slime mold cells with the altered bundling protein, Maselli and colleagues observed large, almond-shaped areas packed with parallel filaments. Nothing like this had ever been seen in a slime mold.

Maselli credits Fechheimer with recognizing that these strange bodies resembled the Hirano bodies previously described in brain tissue. The team set out to confirm Fechheimer’s hunch.

When the researchers examined the mysterious structures with an electron microscope, they discovered a match with Hirano bodies’ characteristics. Filament width and spacing, the pattern that filaments formed and the overall shape of the body all checked out, said doctoral student Rich Davis.

Next, the researchers used antibodies that “recognize” specific proteins and showed that the alleged Hirano bodies were made of actin and a handful of other proteins. Finally, Davis inserted the altered gene into mammalian nerve cells and fibroblasts growing in the lab, and the same structure formed in them.

Fechheimer’s team was convinced that what they were seeing in the slime mold, and now in cultured mammalian cells, were indeed Hirano bodies.

What’s ahead

The scientists now had a way to investigate how Hirano bodies form, what they do and what their fate is: Insert the altered gene in a cell, and “presto” you’ve got Hirano bodies.

“Because it’s novel and a model system, you ask some very basic questions,” Furukawa said. “It’s hard to get to the more subtle questions until you figure out some basic biology.”

And basic questions abound: Do cells grow normally if they have Hirano bodies? What are Hirano bodies made of? What is their purpose?

So far the UGA team has discovered that slime mold cells with Hirano bodies grow normally or a bit more slowly but complete their life cycle.

“Further, mammalian cells with Hirano bodies also grow normally in culture,” Fechheimer said.

Hirano bodies are made of several hundred proteins; only a half dozen or so have been identified. But which ones are required to make the deposits? Graduate and undergraduate students are trying to find out, Furukawa said.

“Even though Hirano bodies look like they are mainly made of actin, there are a lot of other things in there as well,” said doctoral student Paul Griffin. “People speculate there might be growth factors in there, transcription factors, all kinds of things. That’s what I’d like to elucidate.”

But the big question is: What do these things do? Answering that question may finally explain the link to Alzheimer’s and other brain disorders.

“The idea that Hirano bodies are simply a manifestation of cell death is not being born out by our studies,” Fechheimer said.

Davis exposed cells that make Hirano bodies to oxidative stress to see whether they live or die. Early evidence indicates that Hirano bodies actually may have a protective function — bundling up damaged proteins and spitting them out of the cell. Future studies by graduate student Sang Deuk Ha will further probe the protective nature of Hirano bodies in mammalian cells.

“We can make the Hirano bodies in Dicytostelium, in fibroblasts, in epithelial cells, in nerve cells in culture,” Fechheimer said. “So far the answer seems to be that Hirano bodies don’t hurt these cells and perhaps they are protective.”

With one interesting twist.

When Davis introduced the altered bundling protein gene into HeLa cells — cultured human cervical cancer cells — the cells died. In fact, they underwent apoptosis, the scientific name for programmed cell death. Intriguingly, HeLa cells do not appear to form Hirano bodies, unlike the other cell types examined to date.

Fechheimer speculates that the modified HeLa cells undergo apoptosis due to their failure to form Hirano bodies, further evidence to support the idea that Hirano bodies may play a protective role. Now Davis, Ha and undergraduate Dan Del Portal are investigating the relationship between Hirano bodies and this type of cell death.

Still, it could be years before the link between Hirano bodies and neurodegenerative diseases is better understood.

“It’s really about asking the question and solving the problem in such a way that you get a clear answer,” Furukawa said. “When you work in biology, you can ask every question you can think of but that doesn’t mean you can think of a way to get an answer, and some straightforward answer, not some murky answer.”

Future studies in the slime mold and other kinds of cells will uncover what Hirano bodies are made of, how they form and whether they are essential for certain cell processes. Knowing how Hirano bodies behave in cell culture is not enough. For that reason, Fechheimer’s group also plans to study them in entire organisms such as transgenic fish and mice.

“Our findings grew out of research funded by the National Science Foundation,” said Fechheimer, who also received support from the Alzheimer’s Association once the link to neurodegenerative disease was established. “That’s why NSF is very excited by this connection to something that sounded like brain disease. To them it was a wonderful example why you never want to forget about investing in basic research.”

You never know what serendipitous discovery might be around the next bend.

For more information, contact Marcus Fechheimer at fechheim@cellmate.cb.uga.edu oraccess www.uga.edu/cellbio/fechheimer.html

Kathleen Cason is associate director of Research Communications
at the University of Georgia.

What Goes Wrong in Alzheimer’s Disease

By the time British author Iris Murdoch felt that she was “sailing into the darkness,” Alzheimer’s disease had long been at work on her brain.

At one time, Murdoch could compose books — in their entirety — in her mind. But in a handful of years, the disease transformed her brilliant intellect to that of “a very nice 3 year old,” according to her husband, John Bayley.

Her dark descent, captured in the recent movie Iris based on Bayley’s books, illustrates the plight of some 4.5 million Americans, a number expected to more than triple by 2050.

What goes so terribly wrong in the brain of an Alzheimer’s patient?

This much we know: In Alzheimer’s disease, lesions — called tangles and plaques — pepper the brain’s memory center like buckshot. The tangles clog nerve cells with gummed up cytoskeletal proteins. Plaques — accumulations of a toxic snippet of a protein — gunk up the spaces between cells. And brain tissue gradually disappears along with the victim’s memories.

“You’re looking at a disease that has been progressing for 30 years,” said Sangram Sisodia, director of the Center for Molecular Neurobiology at the University of Chicago and a leading Alzheimer’s researcher. “The plaques and tangles are the tombstones of the disease.”

The search for an exact cause has led scientists to suspect that a toxic byproduct of a cell protein damages and kills nerve cells in the process of forming plaques.

“Genetics points me in the direction of a molecule called a-beta and a-beta biology,” Sisodia said. The UGA alumnus earned his doctorate under the direction of Gordhan Patel, who is now UGA’s Vice President for Research.

Plaques form when a normal cell protein called APP — or amyloid precursor protein — gets cut by enzymes in the wrong spot, releasing a toxic fragment called a-beta peptide. The plaques themselves are not thought to kill brain cells; scientists suspect that a wayward form of the a-beta peptide is the culprit.

“The a-beta peptide not only accumulates in plaques but along the way it will form these little fibrils that will be free-floating in the brain,” said Rudolph E. Tanzi, professor of neurology at Harvard Medical School and director of the genetics and aging research unit at Massachusetts General Hospital. “The fibrils can interact with neurons and cause problems, from blocking neural transmission to actually killing nerve cells.”

Sisodia, Tanzi and many other researchers have hunted for genetic evidence by turning to families where Alzheimer’s disease strikes early — in the third or fourth decade of life, sometimes even earlier.

“The pathology in individuals who inherit these genes for early-onset Alzheimer’s disease and the clinical symptoms are almost indistinguishable — with some minor variations — from somebody who gets the disease in the late onset form,” Sisodia said.

So far, the genetic clues support the notion that a-beta peptide production in the brain is the probable cause of Alzheimer’s disease.

“The pathological cascade inside the cell revolves around the life cycle of the a-beta peptide,” said Tanzi, co-author of Decoding Darkness: The Search for the Genetic Causes of Alzheimer’s Disease.

The disease occurs when a person has defective genes that either make too much APP or produce enzymes that cut APP in the wrong place, releasing the “bad” a-beta. But a-beta production alone won’t cause the plaques to form; a-beta has to clump together into fibrils to be toxic to nerve cells. If enzymes chop up a-beta or if a-beta is shuttled into the blood where it can be disposed of, clumping doesn’t occur, Tanzi said.

“If too much a-beta is made at once and the machinery for clearing it or degrading it can’t keep up, it’s going to form fibrils more readily,” Tanzi said. “They’re going to cause neurotoxicity and eventually it’s all going to manifest itself in senile plaques.”

Scientists still have much to learn about this bewildering disease that kills the mind long before the body dies. Several UGA researchers are contributing to understanding Alzheimer’s disease in a variety of studies. They include the following:

• Cell biologist Marcus Fechheimer and his research team developed a system to investigate abnormal cell structures called Hirano bodies and what their role may be in Alzheimer’s and other neurodegenerative diseases.

• Walter Schmidt, assistant professor of biochemistry and molecular biology, studies several enzymes in yeast that bear similarities to enzymes thought to have roles in Alzheimer’s and other diseases. One of those enzymes is similar to insulysin — or IDE — which may remove the a-beta peptide from the brain.

• Alan Przybyla’s biochemistry and molecular biology lab has developed a method to produce a recombinant form of the a-beta peptide. He helped form a start-up company, rPeptide, that provides researchers around the world with molecules involved in Alzheimer’s, Parkinson’s and other diseases.

• William Kisaalita, associate professor of biological engineering, is developing cell-based tests, or biosensors, to screen drugs for Alzheimer’s disease.

• Alvin Terry, associate professor of pharmacy, studies the long-term effects of medication on the brain, particularly medications commonly used to treat people with diseases that affect memory, like Alzheimer’s disease and schizophrenia.