Tanz Centre findings offer new insight on old prion disease research

Research by a University of Toronto scientist on misfolded proteins in brain cells has prompted a re-examination of decades-old research on prion diseases.

The research, recently published in the journal Acta Neuropathologica, looks at how the characteristic ‘holes’ in brain cells affected by prion disease develop, and has given rise to a new explanation for old images that appear to show virus-like particles previously thought to be responsible for these diseases.

The notion that prion diseases are caused by viruses has long since been dismissed by a majority of prion researchers, but the identity of the “virus-like” particles in brain sections has remained enigmatic until now.

“We found these holes are part of a rescue biology of cells trying to counteract the inhibition of a pump at the cell surface, but then other things go wrong as the disease develops,” says Gerold Schmitt-Ulms, a scientist at the Tanz Centre for Research in Neurodegenerative Diseases.

“We think that what previous researchers believed were virus particles are actually arrangements of inactive ribosomes, the machines responsible for translating the instructions of nucleic acids into proteins. We speculate that these ribosomes fall dormant when the supply lines of nucleic acids to them are interrupted.”

Prion diseases are a group of rare and invariably fatal brain disorders, which include Creutzfeldt-Jakob disease in humans and Bovine Spongiform Encephalopathy in cattle. They are caused by misfolded prion proteins accumulating in the brain.

The misfolded proteins lead to the formation of small holes called vacuoles that cumulatively give rise to a sponge-like disease phenotype known as spongiform degeneration.

Schmitt-Ulms and his team previously found that prion proteins are located in brain cell membranes close to a protein complex that acts as a pump, which moves potassium and sodium ions in and out of the cell. The pump is important for maintaining the brain cells’ electrochemical gradient — the balance of ions and chemicals in the cells, essential for brain cells to function and communicate with one another.

If the pump becomes inhibited, cells will internalize and degrade it, with the prion protein coming along for destruction. Critically, although the pump is rapidly replaced, levels of the prion protein will remain diminished. The Schmitt-Ulms team was interested in exploring this approach as a potential therapeutic strategy aimed at lowering prion protein levels, so they searched the published literature for other studies that looked at pump inhibitors.

“This led us to interesting literature that described how inhibiting the pump in rodent models resulted in brain tissue with similarities to spongiform degeneration in prion diseases,” says Schmitt-Ulms, who is also a professor of laboratory medicine and pathobiology at U of T’s Temerty Faculty of Medicine.

“Initially, people were excited about this, but then they saw that different brain cells were affected: Inhibiting the pump in rodents affected astrocytes, but prion diseases in humans affect neurons. So the idea that the pump might be involved in creating the holes in prion diseases was largely dismissed.”

But Schmitt-Ulms didn’t give up on the idea so easily. His team had recently started work with engineered mice that produce a humanized version of the pump, finding that when they inhibited the pump in these models, it no longer led to vacuoles in astrocytes; rather, the holes shifted to neurons as in humans afflicted with prion diseases.

“We were a bit excited because that could help explain how spongiform degeneration begins and where it starts, but we didn’t want to leave it there,” says Schmitt-Ulms. “We sought to gain a better understanding of the mechanisms behind the formation of the vacuoles, so we launched a project where we really tried to get to the bottom of this.”

The result of that work was the Acta Neuropathologica paper and a follow-up synthetic review currently in pre-print. Their work points toward vacuoles in prion diseases representing dilated cisternae of the endoplasmic reticulum and perinuclear space, a network of membranes inside cells that plays an important role in protein synthesis. Picking up on the 50-year-old data, they documented that a forced overproduction of the pump causes the same structures to dilate.

They also identified ion channels in the endoplasmic reticulum that may be involved in the dilation, and that these channels can be blocked to reverse the dilations. Together, the results provide a clearer picture of the molecular processes that cause the endoplasmic reticulum to dilate, resulting in hole formation and ultimately neurodegeneration.

“The results don’t have a direct impact for therapies at this time, but when we understand how the holes are forming, we can come up with ideas for how these holes could be suppressed, and eventually develop targeted therapies,” says Schmitt-Ulms.

Looking at old microscopy images but with the new perspective that the holes start in the endoplasmic reticulum, Schmitt-Ulms and other researchers — including Tanz Centre scientist Joel Watts — believe that some of the images show excessive production of endoplasmic reticulum, which gives rise to holes or membrane accumulations forming whorls, and others represent inactive ribosomes.

“The cells must be producing a lot of ribosomes in order to replace the pumps and other proteins,” says Schmitt-Ulms. “As the disease progresses, the vacuoles prevent the ribosomes from doing their job. As all attempts of the dying cells to save themselves fail, inactive ribosomes concentrate into arrays that look like packed viruses.”

This hypothesis has not been scientifically proven or peer-review published, but Schmitt-Ulms says it aligns better with the current understanding of prion diseases and how they progress.

“Decades ago, scientists thought that the virus-like structures they observed in electron microscope images were what distinguished brains with prion disease from healthy brains, and they didn’t consider the contributions of the pump and vacuoles,” he says.

“We now have good evidence that dilations of the endoplasmic reticulum and perinuclear space contribute to vacuolation in prion diseases. This finding paved the way for recognizing that the virus-like particles, which have consistently been observed in brain sections of prion-infected brains, represent inactive ribosomes. If further validated, this is a major advance because it resolves a longstanding question regarding the nature of these virus particles, which were held responsible for the infectious nature of these diseases.”