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A PRION-LIKE PROTEIN IS ESSENTIAL FOR LONG-TERM MEMORY

Prions may not be all bad, after all. Contrary to the widely held belief that unfolded prion proteins are toxic, scientists have made a startling discovery – a protein that helps store memories undergoes conformational changes like those of the prion proteins implicated in Creutzfeldt-Jakob disease and mad cow disease.

Studies in sea slugs and yeast suggest that the cytoplasmic polyadenylation element binding (CPEB) protein undergoes conformational changes, altering its usual shape and setting off a chain reaction, with one after another molecule unfolding. That is the same behavior exhibited by the destructive prions that cause encephalopathy in animals and people.

There are two forms of the CPEB protein: a less active state and a second, more active prion-like form that is the activator of protein synthesis essential to memory formation. This view is described by Nobel Prize laureate Eric Kandel, MD, at Columbia University and Susan Lindquist, MD, Director of MIT's Whitehead Institute for Biomedical Research, and their associates in back-to-back papers in the December 26th issue of the journal Cell (2003;115:879–891, 893–904).

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Dr. Eric Kandel: “We showed that the normal state of CPEB may be the less active state, and the prion state may be the effective way of utilizing the normal function of the protein.”

“This was a surprising finding,” said co-investigator Kausik Si, PhD, a research fellow in Dr. Kandel's laboratory. “We found a protein that plays a crucial role in stabilizing the information that forms memories, and the protein has some properties that look like the prions so well known for killing cells.”

The research has not been replicated yet in mammals. But the investigators said there is every reason to believe CPEB plays a similar role in humans.

UNRAVELING PRIONS' SECRETS

Integral to the function of any protein is its shape, and most proteins maintain only one shape in the lifetime of an organism. Prions, on the other hand, are proteins that can suddenly change configuration, unraveling from a tight ball into a more linear form; in doing so, they influence other molecules to do the same. Typically, the proteins in these clusters cease to function normally and either die or are deadly to the cell and, ultimately, to the organism.

Until now, Dr. Si said, their studies showed that CPEB, key to maintaining long-term memory, contains certain distinct prion signatures.

The finding of a prion with “good” characteristics was fortuitous, he said; it was discovered during experiments designed to hone in on a molecule that would stabilize memory-related synaptic changes. The research built on previous work in Dr. Kandel's lab of implicit memory in the sea slug Aplysia californica; the evidence implied that cellular changes in the strength of synaptic connections are needed for the protein synthesis that characterizes long-term memory.

“We knew from previous work in Dr. Kandel's lab that protein synthesis in the synapse is critical for stabilizing synaptic plasticity,” Dr. Si said. “The question we asked was, How is protein synthesis activated in the synapse?”

CPEB IN OTHER CELL TYPES

The researchers decided to study CPEB, which had been shown in other cell types, but not neurons, to be an activator of protein synthesis.

In Aplysia, blocking the expression of neuronal CPEB using an antisense oligonucleotide against CPEB mRNA and blocking all cell protein synthesis using anisomycin produced the same phenotype: inhibition of late-phase synaptic facilitation. That is proof, Dr. Si explained, that the neuronal CPEB is the molecule that activates synaptic protein synthesis.

Still, one enigma remained. How could CPEB maintain this activated synthesis over time, as would be required for the formation of memories?

That was when Dr. Si noticed that, compared with CPEB proteins in other organs the particular amino acid composition of the neuronal protein suggested prion-like properties – namely, a high glutamine content and predicted conformational flexibility. “Normally, if you take an amino acid sequence and run a secondary structural prediction program, you'll see a certain structure,” Dr. Si said. “But if you run a prion sequence through this computer-generated program, it does not show any structure. That is very unusual.”

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Yeast cells (top) with active CPEB are blue, and those with inactive CPEB are white. However, inac-tive CPEB can flip into an active, prion-like form, producing blue cells (right), and the reverse con-version can also occur (left).

Over the next 4.5 years, the investigators used yeast to determine if this particular signature really acted like a protein, with folding and two conformationally distinct states, each with different physiological functions. They removed CPEB from sea slugs and linked it to the gene for an enzyme that produces a blue color whenever CPEB spurs the translation of mRNA into protein.

When fused to a nonprion reporter protein in yeast, the prion conferred the prototypic epigenetic changes that characterize yeast proteins, Dr. Si said. He explained that first most of the cells turned blue (an identifiable state of the reporter), indicating the presence of active protein. Then the blue form of CPEB started to clump, and when the blue and white yeast cells came together, the blue protein converted the white protein into the blue form.

“If you fuse a prion to another protein, that protein will begin to act just like the prion,” Dr. Si explained, “which is just what happened.”

While full-length CPEB undergoes similar changes, the dominant, self-perpetuating prion-like form works even more vigorously at stimulating translation of CPEB-regulated mRNA, he added.

“What we found is that CPEB altered its form and caused other proteins to follow – functioning exactly like a prion,” Dr. Si said. “But despite its altered form, CPEB carried out its normal function in the synapse and helped generate proteins for memory storage.”

“Neurons come in through these synapses and when new information comes in, the synapse changes itself so that the nature of the synaptic communication is changed and the information would be retained,” Dr. Si said.

SELF-PERPETUATING PROTEINS

“The interesting thing,” he said, “is that prion-like molecules are stabilizing proteins, self-perpetuating. That may explain why once the memory is stored, it stays there forever.”

Dr. Lindquist said in a statement, “This is remarkable not just because the protein executes a positive function in its prion-like state. It also indicates that prions aren't just oddballs of nature but might participate in fundamental processes.”

Added Dr. Kandel in a statement, “We showed that the normal state of CPEB may be the less active state, and the prion state may be the effective way of utilizing the normal function of the protein. We hypothesize that conversion of CPEB to a prion-like state in stimulated synapses helps to maintain the long-term synaptic changes associated with memory storage.”

But, they stressed, it remains to be seen whether CPEB behaves the same way in neurons. “We have shown that CPEB is needed for synaptic protein synthesis and that in yeast, CPEB has the ability to self-perpetuate,” Dr. Si said. “Now we have to connect the two – that is, show that CPEB is indeed self-perpetuating in the synapse.”

The next step, Dr. Si said, is to see if memory in sea slugs is in fact mediated by prion-like molecules, and then try to replicate that work in mice. If it holds up in rodents, the researchers will move to human studies, but that is still years away, according to Dr. Si.

EXPERTS COMMENT

Other prion researchers, who were not involved in the study, applauded the work. Giuseppe Legname, PhD, an Assistant Adjunct Professor of Neurology who works in the lab of prion pioneer Stanley Prusiner, MD, at the University of California-San Francisco, called the research “exciting, clearly a demonstration that prions are not just bad nasty proteins.”

While stressing that the findings still need to be replicated in higher organisms, Dr. Legname said he expects that scientists will soon discover other prions integral to key body functions as well.

“What they have shown is that at least in yeast, different conformations of a prion protein may carry out different functions,” he said. “If true, this opens up a new way of looking at prions.”

Patrick Bosque, MD, Assistant Professor on the Department of Neurology at the University of Colorado Health Sciences Center at Denver, agreed. “Evolution is such that biology usually figures out a good way of using proteins,” he said.

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Dr. Susan Lindquist: “This is remark-able not just because the protein exe-cutes a positive function in its prion-like state. It also indicates that prions arent just oddballs of nature but might participate in fundamental processes.”

“While many diseases are caused by prion-like, cell-propagated misfolding of proteins, this work suggests this property could have normal functions in the cell as well. This is a new way of looking at things and while early, the work is still very exciting.”

ARTICLE IN BRIEF

✓ Dr. Kausik Si and colleagues in the laboratory of Dr. Eric Kandel of Columbia University and Dr. Susan Lindquist of the Whitehead Institute of MIT, have discovered in a sea slug model that a protein that helps store memories undergoes conformational changes like those of the prion proteins implicated in Creutzfeldt-Jakob disease and mad cow disease.