Researchers find mechanism responsible for neuronal death in prion diseases

October 17, 2002

Prion diseases, such as mad cow disease in cattle and Creutzfeldt-Jakob disease (CJD) in humans--have stumped scientists for decades with a complex "whodunnit" complete with many suspects and a missing murder weapon.

The brain of a victim killed by prion diseases is clogged with clumps of prion protein (PrP) in a rare, misfolded state called PrPSc. Controversy has long raged about how these diseases get started and if the clumps of PrPSc actually kill brain cells--in some forms of the disease clumps aren't even there. The culprit responsible for the death of neurons is still a mystery.

Now research from Whitehead Institute Director Susan Lindquist and Jiyan Ma, now at Ohio State University, suggests a unifying theory that can help explain how these devastating diseases get started and how they kill. The results, published in two papers in the October 17, 2002 online issue of the journal Science, show that small amounts of PrP accumulating in the cellular space called the cytosol kill neurons in cultured cells and transgenic mice. Mice suffered from neurodegeneration and loss of muscle control, similar to patients with CJD and other prion diseases.

PrP normally sits on the cell surface. But if it misfolds before it gets to the surface, the cell's quality control mechanism sends it to the cytosol for destruction. The new research shows that if the quality control system becomes overwhelmed, even a small amount of PrP in the cytosol will kill the neuron.

In an accompanying paper, the lab reports also that when misfolded PrP accumulates in the cytosol, it can sometimes convert to the clumpy PrPSc-like form. A key feature of prion hypothesis is the "infectious" nature of its shape change--once PrPSc forms, it can coax other prion proteins into this alternative folding pattern and cause them to clump together. Lindquist and Ma have found a mechanism by which this infectious shape change can get started and demonstrated its self-propagating character.

Together the two papers establish a mechanism for how normal PrP can convert into a highly neurotoxic form that is distinct from the self-propagating PrPSc form of the protein. The low levels of PrP required for cell death would have eluded detection until now.

Mammalian Prion Proteins
Mad cow disease came to the attention of scientists in 1986 when an epidemic erupted in British cattle herds of a new neurological disease. Almost 10 years later, fear accelerated when young people, mostly in Britain, contracted the disease, apparently from eating infected meat. (In people, the disease was previously found only in the elderly.) To date, approximately 150 young people have gotten new-variant Creutzfeldt-Jakob disease. While scientists hope this number will remain small, it is hard to tell since the incubation period can be as long as 20 years. Now, a related disease, known as "wasting disease," is spreading across the United Sates in deer and elk populations.

All mammals, including humans, make the prion protein PrP, but a rare misfolded state called PrPSc is found only in mammals with transmissible prion diseases. Remarkably, this shape change appears to be contagious, converting other normal PrP molecules to the PrPSc conformation and causing them to aggregate. But the mechanism for triggering the initial, rare PrPSc conformation is unknown.

The very idea of prions makes many scientists uneasy because it violates the fundamental principle that the presence of nucleic acids, such as DNA, is essential both to inheritance and infection. Yet prions produce clumps in the brain that are infectious even though no nucleic acid seems to be involved.

PrPSc is widely believed to be the infectious agent in transmissible forms of the disease, responsible for starting the deadly chain of PrP mifolding events. But PrPSc doesn't seem to be directly responsible for killing neurons. PrPSc is not observed in several inherited and experimentally induced forms of disease and PrPSc is not toxic to genetically altered mice that don't make their own PrP protein.

It's All in the Fold
Proteins are the basis of life, providing the building blocks, the moving parts, and the energy that drives the engine. Like simple sheets of paper that fold into origami figures, proteins fold into a vast array of complex and beautiful shapes. Sometimes a protein folds incorrectly by chance, other times a mutation in the protein makes it more likely to misfold. Because misfolded proteins can cause a cell to malfunction, the cell uses a stringent-quality control system to eliminate them.

Misfolded PrP, for instance, doesn't get transported to the surface of the cell, its normal destination. Instead, when the cell detects misfolded PrP, it is spit into the cellular space called the cytosol. There, protein-degrading machines called proteasomes act as garbage disposals to eliminate misfolded proteins. Usually, PrP is degraded so rapidly that it is undetectable. However, when proteasome activity is compromised, as might naturally occur with stress and aging, PrP accumulates in the cytosol.

The Lindquist lab used proteasome inhibitors to block the cell's garbage disposal to see what would happen when misfolded PrP accumulated in the cytosol. Surprisingly, neurons died even before PrPSc clumps formed.

Depending on how fast PrP accumulated in the cytosol, it sometimes converted to a PrPSc like conformation. "When proteins are delivered to the cytosol for destruction, they are unfolded," Lindquist explains. "We think conversion occurs when these wobbly, sticky PrP molecules find others in the same state. If enough of them get together at the same time, they can acquire this new structure."

Once this new conformation appeared, it recruited other PrP molecules to make the same shape change. The shape changes kept occurring even after the inhibitor was removed and the degradation of other proteins resumed.

This work demonstrated the first natural, biological mechanism for producing the PrPSc conformation from scratch. It also shows that the self-perpetuating structural change is an intrinsic--and unusual--property of the PrP protein itself. "That is a very important finding," Lindquist states. "But it doesn't prove that conversion is sufficient for transmission from animal to animal. That has yet to be tested and those experiments can take a long time."

PrPSc, however, did not seem to be killing the neurons, and the fraction of PrP that converts to the PrPSc-like form did not determine cell death. To avoid artifacts from inhibitors, the researchers simply made transgenic mice that would express the normal PrP protein directly in the cytosol. It was deadly--even very small amounts of soluble PrP killed the brain. "This is an entirely new view of the toxic species," said Lindquist. "We think this toxicity wasn't realized before simply because such small amounts of PrP are in the cytosol and other forms of the protein are present at much higher concentrations."

In fact, the toxicity of cytosolic PrP is so great, it has evolved as a way to kill neurons with PrP folding problems, thereby reducing the risk that PrP will accumulate at sufficient levels to produce the infectious PrPSc form.

With a better understanding of how prion diseases initiate and progress, scientists will be better able to design therapeutic approaches for treatment.

Implications for Proteasome Inhibitors
The Lindquist lab used proteasome inhibitors in their initial experiments to clog up the garbage disposal of the cell. These proteasome inhibitors are a different class of drugs, not to be confused with protease inhibitors, which are used in AIDs therapy. This provided the first clues of what happens when PrP accumulates in the cytosol. Several proteasome inhibitors are in clinical trials for their ability to selectively kill cancer cells. The papers by Lindquist and Ma caution that interfering with proteasome degradation, as with proteasome inhibitors, might cause neuronal death and therefore should be handled with care. They also add that because the inhibitors in clinical trials don't cross the blood brain barrier, their potential for harm is greatly reduced.

Whitehead Institute for Biomedical Research

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