ARTICLE IN BRIEF
A new study adds to a growing body of evidence showing that alpha-synuclein (AS) pathology moves through the brain in a prion-like fashion, and is the first to directly link AS accumulation to death of dopaminergic neuronsin vivo.The results also suggest that antibody therapy may be a viable treatment strategy in Parkinson's disease.
A single injection of alpha-synuclein (AS) fibrils into the brain of a normal mouse is enough to cause Parkinson's disease (PD) pathology to spread through the brain, producing Lewy bodies, killing dopaminergic neurons, and causing a movement disorder, according research published Nov. 16, 2012, in the journal Science. The finding is the latest and potentially most significant discovery in a growing body of evidence showing that AS pathology moves through the brain in a prion-like fashion, and is the first to directly link AS accumulation to death of dopaminergic neurons in vivo. The results also suggest that antibody therapy may be a viable treatment strategy in PD.
Unlike in previous studies, no transgenic overexpression of AS was required to set the stage for protein aggregation, suggesting that spread of pathology does not depend on a pre-existing problem with AS overload. Nor were the mice stressed, exposed to environmental toxins, or carrying genes predisposing them to proteinopathy.
“It was actually an astonishing set of experiments that surprised us,” said lead investigator Virginia M. Lee, PhD, professor of pathology and lab medicine at the University of Pennsylvania School of Medicine in Philadelphia.
Dr. Lee had previously shown that purified alpha-synuclein fibrils are endocytosed by healthy neurons in cell culture, and induce the formation of Lewy bodies, ultimately leading to their death. Those findings supported previous autopsy results suggesting a pattern of Lewy body spread over time, from lower brain regions to higher. Autopsies of PD patients who had received fetal grafts also indicated the potential for spread of Lewy bodies into previously unaffected tissue. Other groups showed that pathology would spread from cell to cell in cultured neurons overexpressing human AS. Overexpressing human AS in mice, however, hadn't led to disease, suggesting to Dr. Lee there was a species barrier to the “templated recruitment” process, a phenomenon at work (though perhaps imperfectly) in the prion diseases. [An abnormal (misfolded) alpha-synuclein molecule serves as an initial “seed” and “template,” which catalyzes the conversion of resident normal alpha-synuclein into the pathological isoform.]
All of that led her to try injection of mouse-derived AS fibrils into the striatum of wild-type mice. “The hypothesis we wanted to test was whether we could introduce ‘seeds,’ to see if we could observe recruitment of endogenous normal protein to adopt a misfolded form, and develop the pathology you see in people, and develop disease,” Dr. Lee said.
The protein was created in bacterial culture and allowed to form AS fibrils, which were then purified and sonicated to break the fibrils into multiple smaller forms of the protein, likely including oligomers and misfolded monomers. “We don't know which of them is the most active species,” she said, but that will be a central focus of the lab's future work.
Next, she injected the fibril mix into the dorsal striatum of standard lab mice. Within 30 days, there was evidence of AS deposits at the injection site. At the same time, inclusions reminiscent of Lewy bodies and Lewy neurites were seen in several other brain regions directly connected to the dorsal striatum, including the cortex and olfactory bulb, “confirming the pathological conversion of endogenous mouse alpha-synuclein in wild-type mice,” according to Dr. Lee. These accumulations were seen ipsilateral to the injection site, with the exception of the amygdala, to which the striatum is connected bilaterally, “suggesting that cell-to-cell transmission followed interneuronal connectivity.”
The extent and distribution of pathology spread over the next five months, with a pattern indicating “time-dependent dissemination.” The hippocampus was spared, but was susceptible when directly injected, suggesting that lack of direct connection with the striatum, rather than intrinsic resistance, kept it from developing Lewy bodies.
Substantia nigra pathology was strictly ipsilateral, Dr. Lee said, probably because the nigrostriatal pathway is unilateral. The nigra also showed gradual loss of immunoreactivity for tyrosine hydroxylase, a marker of dopaminergic activity, “suggesting that intraneuronal alpha-synuclein inclusions lead to dopaminergic neuron loss,” she concluded.
Injected mice progressively lost the ability to perform on two motor performance tests, although remained able to walk normally. There were no signs of memory deficits or depressive behavior.
The susceptibility of mouse neurons to AS fibril injection was similar in two different wild-type strains, but was markedly less in animals genetically hemizygous for AS. “There is a dose-dependency in mice,” Dr. Lee said. “If you have only half the amount of alpha-synuclein, the spread is much slower.”
For Dr. Lee, one of the most important conclusions from these experiments is that there is a tight connection between accumulating AS pathology and dopamine cell loss. “We've known about loss of dopamine for 50 years,” and the role of AS in Lewy bodies has been known since the late 1990s, but the field has long debated exactly what role AS plays in the disease. “Our study made that connection: You see the pathology, and then the neuron dies,” Dr. Lee said, indicating it is likely the initiator of the pathologic process within the neuron.
Another key lesson is the importance of the right model. “I am not sure the way we and many labs have tried in the past to generate mouse models of neurodegenerative diseases, by overexpressing the mutant form of the protein, is the right approach,” she said. “Our study shines a light on the fact that perhaps templated recruitment and transmission is really the mechanism of disease.” There are many unanswered questions about the spreading process, including the cellular mechanisms behind it. “Because we have the right model systems now, all of these can be looked at.”
Her study does not address the nature of the initial insult that begins the templated recruitment process: “We don't know how the seeds initiate in people,” she said, and there is no indication that the seeds of the disease can spread between people. But her results do suggest, if the mice are any guide, there need not be any special genetic susceptibility or environmental setting needed to propagate the disease once it starts; you just need alpha-synuclein.
Here, results also point strongly to one way to interrupt the spreading process. “The most obvious approach for therapy is to do passive immunotherapy,” delivering antibodies to the brain to bind to extracellular AS. “It may be realistic to at least initiate those types of therapeutic approaches to halt the spread,” Dr. Lee said.
C. Warren Olanow, MD, professor of neurology and neuroscience at Mount Sinai School of Medicine in New York, commented: “There has been increasing interest in the possibility that alpha-synuclein is a prion and Parkinson's disease a prion disorder,” highlighted most dramatically by the development of Lewy bodies in transplanted fetal tissue in the brains of PD patients.
Dr. Lee's demonstration that wild-type mice develop subcellular, tissue-level, and behavioral effects from the spread of synuclein pathology, he said, “provides additional support for the concept that PD is a prion disorder, and suggests novel targets for putative neuroprotective therapies.”
“This is a very important paper,” agreed Eliezer Masliah, MD, professor of pathology and medicine at the University of California, San Diego School of Medicine, where he specializes in the study of mechanisms of neurodegeneration. “I am pretty excited about it, and most of the community is excited about it. Now we need to understand why this happened, and it may not be a simple explanation,” he said, noting that other groups have tried similar experiments without success.
Whatever the details, however, this model is likely to be useful for understanding propagation of AS pathology. The results point strongly to the conclusion that “it is really a combination of intracellular events and extracellular events that makes Parkinson's disease. This model describes that second part,” and reproduces something intrinsic to the PD disease process.
The study also provides a strong foundation for immunotherapy in PD, Dr. Masliah said, an idea he has been studying in the lab. “Finding therapies that prevent propagation might be of great value, and antibodies are an ideal way to do that.”