ARTICLE IN BRIEF
A research team at the University of Pennsylvania has successfully blocked the cell-to-cell transmission of pathologic forms of alpha-synuclein in animal and cellular models of Parkinson's disease.
Alpha-synuclein is to Parkinson's disease (PD) what amyloid-beta is to Alzheimer's — a protein that has a normal function but when overexpressed can damage discrete populations of brain cells. Since its role in PD was described in 1997 by a team of scientists at the University of Pennsylvania, scientists around the world have been working to unravel its role in neurodegenerative disease and develop therapies that can damp down its expression.
Now, a new paper by scientists working in the same laboratory at University of Pennsylvania, led by Virginia M. Y. Lee, PhD, and John Trojanowski, MD, PhD, has successfully blocked the cell-to-cell transmission of pathologic forms of alpha-synuclein in several different experiments reported in the June 26 issue of Cell Reports.
Dr. Lee and Dr. Trojanowski, co-directors at the Center for Neurodegenerative Disease Research, say that the findings offer hope that immunotherapy targeted against abnormal alpha-synuclein could one day alter the neurodegenerative course of the disease in patients.
A NOVEL LABORATORY MODEL
Several years ago, Dr. Lee and her colleagues developed a laboratory model that mimics the hallmark pathology of PD: alpha-synuclein aggregation in Lewy bodies and Lewy neurites. They synthetically created alpha-synuclein preformed fibrils (pff) and showed that the fibrils injected into wild-type mice could act as a template and spread its pathology through their endogenously expressed counterparts. These pathologic forms of alpha-synuclein enter neurons and recruit endogenous alpha-synuclein to misfold and accumulate to form Lewy bodies. This damage leads to synaptic and neuronal loss.
The alpha-synuclein pffs initiated temporal and spatial spread in the wild type mice and this led to loss of dopaminergic neurons in the substania nigra and motor deficits.
In this latest study in Cell Reports, the UPenn team has used the pff model system to test the benefits of immunotherapy targeted against misfolded alpha-synuclein. In a series of experiments, they followed the molecular pathway to show how alpha-synuclein immunotherapy blocks the uptake and propagation of the misfolded protein. They wanted to know whether anti-alpha-synuclein antibodies could reduce the pathology and rescue cell loss and improve motor deficits. The studies were conducted in cell culture and in animal models of sporadic PD triggered by pffs.
ANIMAL STUDIES UNRAVEL PATHWAY
They tested two different antibodies that target pathologic forms of alpha-synuclein — Syn211 and Syn303. Syn 211 is specific for human alpha-synuclein pathological forms and Syn 303 binds a pathologic conformation of human and mouse alpha-synuclein. The antibodies were tested against immunoglobulin, IgG, an antibody that is not specific for alpha-synuclein and has no effect on propagation or cell-to-cell transmission.
To see whether the monoclonal antibodies could prevent the spread of disease, they they administered Syn 303 on the heels of injecting pff into the normal wild type mice. This monoclonal antibody reduced the pathologic spread. The control group had PD pathology that continued to spread.
They also treated another group of PD mice with Syn303 a week after injecting the synthetic fibrils. This was enough time for the animals to have some misfolded alpha-synuclein pathology, a loss of dopaminergic neurons in the substantia nigra that would lead to motor symptoms seen in PD. A month later they measured the amount of alpha-synuclein pathology and saw a significant reduction compared with those animals treated with IgG.
They also tested another group of animals with weekly doses over a six-month period. The monoclonal antibody (Syn303) significantly improved grip strength and motor coordination at all time points tested. There was a 30 percent reduction of alpha-synuclein aggregates in the substantia nigra, as well as a 40 percent reduction in the ipsilateral and contralateral amygdala.
A MODEL FOR CELL-TO-CELL TRANSMISSION
The researchers turned to cell culture studies to figure out how the monoclonal antibodies were working to block cell-to-cell transmission of alpha-synuclein pathology. They developed a microfluid culture device with three independent chambers connected by a series of microgrooves. This allows them to separate three distinct neuronal populations and conduct a variety of tests.
In this latest series of experiments, they plated non-transgenic neurons in all three chambers and initiated alpha-synuclein pathology by adding human wild type alpha-synuclein pffs into the first chamber. They could watch a week later as the propagation occurred to cell bodies and neurites of neurons in the second chamber. Another week later they could observe the propagation in the third chamber.
“Given that the neuronal processes represent the only cellular contact between individual chambers, the delayed presence of alpha-synuclein pathology in chamber 3 supports cell-to-cell transfer of seeding material from C1 [chamber 1] and/or C2 [chamber 2],” the scientists reported in the paper.
Then, they added Syn 211 or Syn303 to the second chamber 30 minutes prior to adding the pffs into the first chamber. There was a significant reduction of alpha-synuclein pathology in the first and second chamber observed during day 7 and 14 of the experiment. They found that Syn303 reduced the pathology and propagation in chambers two and three while Syn 211 only blocked pathology in the second chamber.
These cell culture experiments showed that monoclonal antibodies prevented the uptake of misfolded alpha-synuclein fibrils by neurons and sharply reduced the recruitment of endogenous alpha-synuclein into new Lewy body aggregates, Dr. Lee explained. The synthetic fibrils remained outside of the neurons, as did the monoclonal antibody.
This chamber system allowed them to identify the propagation and cell-to-cell transmission because the neuronal processes are the only cellular contact between the chambers. The system affords them the opportunity to test how monoclonal antibodies delivered in one chamber affects neuronal life next door.
Dr. Lee said that the model could be used for other neurodegenerative diseases such as Alzheimer's disease and Huntington's disease that are triggered by the accumulation of other abnormal proteins. Immunotherapy against intracellular proteins involved in these neurodegenerative diseases suggests that targeted antibodies can decrease protein aggregation and neuronal death.
“We believe that the data support the therapeutic potential of alpha-synuclein immunotherapy for PD and other related diseases with Lewy body pathology,” said Dr. Lee. “This is the first demonstration of immunotherapy working in a sporadic PD mouse model.”
Dr. Lee said that timing may be critical to the benefit of these types of treatments. In primary neurons, the monoclonal antibodies were effective when added 30 minutes before and up to a day after the synthetic fibrils were added to the neuron-plated dishes. In the animals, monoclonal antibodies delivered immediately or within a week post pff injection were powerful enough to prevent pathologic spread.
“Immunotherapy against disease proteins may only be efficacious in neurodegenerative diseases early during the course of disease,” they wrote in their Cell Reports paper.
Dr. Lee said that they have also identified several strains of misfolded alpha-synuclein protein that may lead to different clinical symptoms. They are now developing more potent monoclonal antibodies that could be tested in humans.
EXPERTS WEIGH IN
Patrik Brundin, MD, PhD, an associate director of research, professor and director of the Center for Neurodegenerative Science and head of the Laboratory for Translational Parkinson's Disease Research at the Van Andel Institute in Grand Rapids, MI, said that the finding builds on prior work using different model systems. “This is very exciting. The more ways we can model the stronger the case for a clinical trial,” he said.
There are still many unknowns, even though several alpha-synuclein monoclonal antibodies are in development, Dr. Brundin continued. Two antibodies are already in phase 1 clinical trials; one from AffiRis, an active immunotherapy, and the other from Prothena, a passive immunotherapy. In the early days of immunotherapy for Alzheimer's an active antibody immunotherapy triggered inflammation in the brains of some patients. The trials were halted. Now, clinical trials using antibody approaches are underway.
“The use of immunotherapy for PD is extremely promising,” said Anthony E. Lang MD, the Lily Safra chair in movement disorders and the director of the Morton and Gloria Shulman Movement Disorders Clinic and the Edmond J Safra program in Parkinson's disease at Toronto Western Hospital. “But there are many challenges. In this study they delivered the monoclonal antibody very early in the disease process. How early would we have to apply this treatment to be effective? This is not yet clear.”
“This is an important paper in two regards,” added Howard J. Federoff, MD, PhD, executive dean of Georgetown University School of Medicine. “It provides evidence for the locus of alpha-synuclein immunotherapy action in several models. Most significantly, it provides in vivo demonstration that systemic immunotherapy can reduce the spread of pathological aggregates which, in my estimation, is sufficient to compel translation into clinical investigation.”
“Alpha-synuclein is an important protein to go after in Parkinson's disease,” said Kuldip Dave, PhD, director of the Michael J Fox Foundation for Parkinson's Research. And there are many ways to reduce alpha-synuclein expression.
There are drugs being studied that block protein synthesis and others in development that could stop the protein from aggregating, he said, and there are substances that can work on the clearance machinery, and there is the promise of immunotherapy. Dr. Dave said that the attraction of the immunotherapy approaches, as the paper in Cell Reports showed, is that a monoclonal antibody can work outside of the cell. The other treatments will require entry into the cell itself. “You can stop the spread of the disease before it gets into the cell,” he explained.
The Michael J Fox Foundation funds research in all of these different approaches. “We think this is an important way to go after alpha-synuclein,” Dr. Dave said.