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Misfolded Alpha-Synuclein Is Implicated in Multiple System Atrophy, Suggesting the Protein Can Act Like an Infectious Prion

Talan, Jamie

doi: 10.1097/01.NT.0000472960.80306.6e
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ARTICLE IN BRIEF

Transgenic animals injected with postmortem brain tissue from people with multiple system atrophy developed aggregates of alpha-synuclein, triggering neurodegeneration and the spread of the disease. The finding suggests that alpha-synuclein can act like a prion and infect a vulnerable host, the investigators reported.

Transgenic animals that had been injected with postmortem brain tissue from people with multiple system atrophy (MSA) developed aggregates of alpha-synuclein, triggering neurodegeneration and the spread of the disease, investigators at the University of California, San Francisco (UCSF) reported in the Aug. 31 online edition of the Proceedings of the National Academy of Sciences (PNAS).

The finding suggests that alpha-synuclein can act like a prion and infect a vulnerable host, the researchers wrote. Moreover, they said, the data imply that precautions should be put in place for scientists and health care workers who come in contact with MSA tissue.

Many researchers have been looking at neurodegenerative diseases and asking the same question: Could the pathological process of proteins misfolding and aggregating in conditions such as Alzheimer's, Parkinson's, MSA, and other neurodegenerative diseases be the result of prions creating a template of a misshapen protein and seeding other proteins to self-propagate?

If that is the case, it opens the door to the development of new strategies to prevent or slow these diseases, the researchers said. More specifically, the findings could lead to novel ways to treat MSA, since drugs for Parkinson's disease (PD) are not effective.

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STUDY METHODOLOGY

In 2013, a research team led by Stanley B. Prusiner, MD, FAAN, director of the Institute for Neurodegenerative Diseases at UCSF and a 1997 Nobel laureate for his research and discovery of prions, observed that an injection of human MSA tissue from two deceased patients transmitted the disease to a mouse that expressed a mutant form of human alpha-synuclein. When he and his colleagues looked inside the brain of the sick mouse, they found higher levels of the sticky alpha-synuclein protein. The infected mouse brain was then infused into other alpha-synuclein transgenic mice, and these mice got sick as well. They then expanded the study to include tissue from another 12 MSA patients.

For the current study, the investigators collected human tissue from 14 MSA patients and injected it into the brains of transgenic animals expressing human A53T mutated alpha-synuclein. Mice with only one copy of the human A53T mutation do not usually get sick. But when the researchers inoculated these mice with brain homogenates from MSA patients, they became sick and died at four months.

They also introduced the human MSA extracts into cultured cells, and found that the extracts caused aggregation of alpha-synuclein in the lab dish. (A separate paper published on Aug. 18 in PNAS mapped out the work in cultured cells.)

The human tissue was sourced from colleagues at the Massachusetts Alzheimer's Disease Research Center in Boston, the Parkinson's UK Brain Bank at Imperial College London, and the Sydney Brain Bank in Australia. The analysis also included tissue from six patients who had been diagnosed with Parkinson's. None of the samples came from patients with genetic mutations known to trigger MSA or PD.

The UCSF scientists then analyzed the human brain tissue to confirm the diagnosis of MSA and PD, and ran ELISA tests to determine total alpha-synuclein levels from the frozen brain samples. Levels were higher in the non-diseased brains than in the MSA or PD brains.

They then tested the tissue for levels of phosphorylated alpha-synuclein in the insoluble aggregate form, and found that tissue from MSA patients contained higher amounts of phosphorylated alpha-synuclein. Four of the PD brains had similarly elevated amounts of the aggregated phosphorylated alpha-synuclein, and two of the PD patients had small amounts of the aggregated protein.

After transgenic TgM83+/- mice were inoculated with tissue from the MSA samples, they developed central nervous system dysfunction — uncoordinated movement and circling behavior — that ultimately proved fatal. The animals showed signs of neurodegeneration between 100 and 150 days. The brain homogenates from the six patients with PD did not produce signs of neurologic problems in the TgM83+/- mice for more than a year after the inoculation.

The researchers also inoculated the TgM83+/- mice with a control substance, which did not cause any neurologic deficits. They found that the greater level of infectivity, the shorter the amount of time to onset of disease.

Study author Kurt Giles, PhD, noted that the transgenic animals did not have the same MSA pathology that has been observed in humans. The alpha-synuclein aggregates were found in neuronal cytoplasmic inclusions and in neurites, he said. In contrast, oligodendrocytes are the cell type affected in patients. The mice inoculated with MSA from human patients had phosphorylated deposits in several brain regions, notably the brainstem and reticular formation. There were no aggregates in the cortical regions.

“Our results argue that transmission of MSA to the TgM83+/- mice results in the de novo formation of prions in mouse brain,” the authors concluded in the paper.

After the animals died, the investigators took brain tissue from four of the sick MSA mice and inoculated the material into another group of TgM83+/- mice. They compared these animals to a group of aged TgM83+/- mice. Again, the animals exposed to the brain extracts from the MSA animals got sick, and in a slightly shorter time period compared with those with a primary transmission. They also compared these animals to transgenic animals with two copies of an alpha-synuclein mutation (TgM83+/+), and found that the homozygous animals got sick 40 percent earlier, at around seven to eight months.

Dr. Giles said the findings suggest that there may be several alpha-synuclein prion strains. The evidence of two different strains comes from comparing inoculation of TgM83 (+/-) mice with either MSA or spontaneous TgM83 (+/+) mice. The former get sick in about 120 days, the latter in approximately 200 days.

The investigators then wanted to see whether the alpha-synuclein prions could cause disease in animals without the alpha-synuclein mutation. They repeated the serial transmission studies in wild-type animals and in transgenic mice expressing the normal human alpha-synuclein gene. None of these mice got sick. This suggests that a vulnerable brain is necessary to allow the prions to template onto other prion proteins. “The mutation predisposes the protein to misfold,” Dr. Giles explained.

In another set of experiments, the scientists precipitated alpha-synuclein prions from the human brain samples and put them into a broth of cultured human embryonic kidney (HEK) cells that express full-length alpha-synuclein containing the A53T mutation (fused with yellow fluorescent protein). After four days, they took four images from each of the six wells for each sample. They used automated confocal fluorescence microscopy to determine the percentage of cells that contained aggregated alpha-synuclein.

They reported that 17 of the 18 samples from MSA patient-infected HEK cells had significantly higher levels of protein aggregates than the control wells. In contrast, one in six of the PD samples had higher levels of protein aggregates than the controls. (They went on to manually assess these data and found that the PD sample probably yielded a false positive response and that the MSA sample was a false negative.) The MSA tissue induced significantly more aggregates than the PD cells (p<0.0001).

They also ran tissue from 17 normal brains (people who died between age 56 and 88 with no signs of CNS dysfunction) and found no fluorescent aggregated protein.

“Some people don't want to call these prions,” said Dr. Giles. Still, he and his colleagues wrote in the PNAS paper, the evidence suggests that while “there is no evidence that MSA is a naturally occurring transmissible disease among humans, the unequivocal experimental transmission studies reported here warrant classification of MSA as a novel, bona fide alpha-synuclein prion disorder.” The UCSF team and other groups are working to identify drugs to block prion replication.

Several groups have conducted similar human-to-mouse or human-to-nonhuman primate transmission studies of synucleinopathy using human tissue from diseased brains. But Dr. Giles said that this is the first time that a new prion, alpha-synuclein, was able to trigger a lethal disease when tissue from a patient with MSA was injected into a mouse brain.

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EXPERTS COMMENT

“The idea that alpha-synuclein behaves like a prion has been out there,” said Lary C. Walker, PhD, an associate professor of neurology and a research professor at Emory University School of Medicine's Yerkes National Primate Research Center. “The UCSF group reported a striking difference in the induction of disease and pathology in mice injected with MSA extracts compared with PD extracts. One has to wonder why other groups have been able to induce disease with PD extracts but they did not. They are saying that MSA prions are much more potent and have a different structure and thus may be a different strain.”

Dr. Walker and Mathias Jucker, PhD, a professor in the department of cellular neurology at the Hertie Institute for Clinical Brain Research at the German Center for Neurodegenerative Diseases in Tübingen, wrote about the expanding prion concept in many neurodegenerative diseases in the 2015 Annual Review of Neuroscience.

“One reason that the prion concept has been slow to catch on is that prions are defined as infectious particles,” they wrote. “But these other diseases are not infectious in the usual sense of being easily transmitted from one person to another. The transmission of disease in the MSA experimental paradigm involves the direct introduction of diseased brain matter from one organism into another. There is no evidence that you can catch MSA by interacting with someone who has it.

“The term ‘prion’ is here to stay, but we really need to think about changing the definition of the prion if we are to lessen unnecessary concern that diseases such as MSA and Alzheimer's are infectious,” Drs. Walker and Jucker wrote. “It is important that we understand that all of these neurodegenerative diseases have a common molecular mechanism, but the implication that these disorders are all infectious has been an impediment to fully embracing the explanatory power of the prion paradigm. That mechanism is the corruptive templating of good proteins by bad ones. It is an important concept but we need to do more to get the neurodegenerative disease community on board with it.”

John Trojanowski, MD, PhD, a professor of pathology and laboratory medicine, co-director of the Center for Neurodegenerative Disease Research, and co-director of the Marian S. Ware Alzheimer Drug Discovery Program at the University of Pennsylvania in Philadelphia, has done similar studies taking brain tissue from diseased patients and introducing it into animals, and he reported evidence of pathology. But, he told Neurology Today, “I do not think pathological synuclein proteins are prions, although they share some of the same properties with prions that are better characterized as variants of amyloids.” Dr. Trojanowski said the term “prion” is misused in this context, since prions “were specifically described (by UCSF's Stan Prusiner) as infectious, based on their ability to spread between organisms, not simply from cell to cell.”

Patrik Brundin, MD, PhD, an associate director of research at the Van Adel Research Institute in Michigan and professor and director of the institute's Center for Neurodegenerative Science, is doing similar research using synthetic alpha-synuclein fibrils. He and his colleagues have injected the fibrils into the olfactory bulb in animal models of PD, and have measured the spread from cell to cell. They have also observed defects in olfactory function. (Most PD patients have impaired olfaction about five years before any movement problems are observed.)

“It's very encouraging that the UCSF group has confirmed their earlier findings,” said Dr. Brundin. “This paper is yet another sign that the field is coming of age and that we are learning that alpha-synuclein can occur in a prion-like fashion. However, so far there is no epidemiological evidence to suggest that these neurodegenerative diseases can be transmitted from one person to the next.”

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EXPERTS: ON THE PRION-LIKE SPREAD OF ALPHA-SYNUCLEIN IN MICE

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LINK UP FOR MORE INFORMATION:

•. Prusiner SB, Woerman AL, Mordes DA, et al. Evidence for α-synuclein prions causing multiple system atrophy in humans with parkinsonism http://www.pnas.org/content/112/38/E5308. Proc Natl Acad Sci USA 2015; Epub 2015 Aug. 31.
•. Woerman AL, Stöhr J, Aoyagi A, et al.. Propagation of prions causing synucleinopathies in cultured cells http://www.pnas.org/content/112/35/E4949.abstract. Proc Natl Acad Sci USA 2015 112(35):E4949–4958; Epub 2015 Aug. 18.
•. Walker LC, Jucker M. Neurodegenerative diseases: Expanding the prion concept http://www.annualreviews.org/doi/abs/10.1146/annurev-neuro-071714-033828. Annu Rev Neurosci 2015; 38: 87–103.
•. Boluda S, et al. Differential induction and spread of tau pathology in young PS19 tau transgenic mice following intracerebral injections of pathological tau from Alzheimer's disease or corticobasal degeneration brains http://link.springer.com/article/10.1007%2Fs00401-014-1373-0. Acta Neuropathol. Feb 2015, 129(2): 221–237.
•. Rey N, et al. Transfer of human α-synuclein from the olfactory bulb to interconnected brain regions in mice http://link.springer.com/article/10.1007%2Fs00401-013-1160-3. Acta Neuropathol 2013; 126: 555–573.
© 2015 American Academy of Neurology