Article In Brief
Researchers turned astrocytes into dopamine-producing neurons in an animal model of Parkinson's disease and reversed motor symptoms.
A team of scientists at University of California, San Diego (UCSD) have developed a one-step process that relies on a single RNA-regulating protein to turn astrocytes into dopamine-producing neurons, perhaps bypassing the challenges of other gene and cellular therapy approaches. The technique created a population of mature neurons that helped reverse motor symptoms in an animal model of Parkinson's disease, according to a report published in June in Nature.
Xiang-Dong Fu, PhD, distinguished professor in the department of cellular and molecular medicine at UCSD, is an RNA biology researcher who has been studying an RNA-binding protein called PTB. Several years ago, a post-doc in his lab was trying to grow cell lines and was looking for a short-cut. Once PTB was removed from the cell, it stopped growing. The cells remained in an incubator and he returned two weeks later and was surprised that the majority of the cells in the dishes looked like neurons.
“This was totally unexpected,” said Dr. Fu. Interestingly, PTB is expressed in all cell types except neurons, which is why this protein is so well known in the RNA world but a virtual stranger to neuroscientists. It is thought that it helps determine a cell's function and restricts, or suppresses, cells from becoming neurons.
The UCSD scientists went on to show that PTB targets a transcriptional repressor called REST, which is regulated by a micro-RNA, miR-124. This microRNA targets REST to down-regulate it; REST suppresses the expression of this microRNA. Therefore, more miR-124 will reduce REST, and reducing REST further induces this microRNA.
“This is why we suggest that it works like an engine; once started, it drives itself,” said Dr. Fu. “Sometimes a car battery needs a jump start. PTB is the body's controller used to jump start cells to become neurons. You only need to jump start once for things to begin working normally again,” he explained.
The in vitro studies worked so well that the research team decided to test it in the brains of an animal model of Parkinson's disease. They used a toxin to damage dopamine neurons in the substantia nigral. Astrocytes are abundant in the brain and support a number of functions for the neurons they shepherd, but generally take a quiet role and do not proliferate unless there is damage. Now the scientists were hoping to use their technique to convert some of those proliferating astrocytes into functional dopamine neurons and restore the community of healthy brain cells in the area. It worked.
They used a virus that expresses a short hairpin RNA that binds to PTB in astrocytes and silences its expression. A fine needle was threaded into the substantia nigral and delivered to the region. Over a few weeks, a portion of the mid-brain astrocytes in this region were converted to dopamine neurons and these new cells formed axons that left the substantia nigral and traveled to the striatum, where the cells innervated and repopulated the endogenous neural circuits. Their axons made dopamine and released it. This restored the motor functions that the toxin severely compromised.
“They look and function like endogenous dopamine neurons,” said Dr. Fu.
Dr. Fu said that it is likely that astrocytes from different brain regions convert to different neuronal subtypes, suggesting the strategy might work in different neurological disorders that affect different parts of the brain.
“The technique may work in humans,” he added. “All we need to do is turn down one gene product, PTB. If the general principle works, we should be able to use this to replace neurons lost in other neurodegenerative diseases like Alzheimer's and Huntington's.”
Dr. Fu and colleagues have a lot more work to do before it ever makes it into human trials. The next step is replicating it in larger non-human primates.
Using stem cells to create specific types of neurons has been challenging because those foreign cells are rejected by the host. That these astrocytes are endogenous means that has no risk to create cancer cells or induce inflammation, Dr. Fu said.
“It took someone who is an RNA biologist to stumble on to something that could have significant consequences for people with neurodegenerative disorders,” said William C. Mobley, MD, PhD, a distinguished professor of neurosciences at UCSD and a co-author on the study. “When I saw the data, I was so surprised. It was unexpected. As I came to know the data, I realized that it is a real phenomenon and it is going on in the brain. It is real. And it is really interesting.”
The collaboration has led Dr. Mobley and his neuroscience colleagues to write a grant to study the differences in astrocytes from region to region in the brain. When astrocytes convert to neurons, how many are left and why? Are there any problems that are region specific? Can the new neurons innervate their targets in other brain regions? Will this principle prove to be true in older animals, and ultimately in older people?
“The fundamental finding that you can switch an astrocyte into a neuron and establish synaptic connections that are now doing the job of the lost neurons is very exciting,” said Dr. Mobley. “The virus brings this construct into the cell and destroys PTB. Enough dopamine was released into the region and the neurons were communicating with one another well enough that the technique restored motor function in the animals”.
Will it fix everything in Parkinson's? “No, of course not,” Dr. Mobley said. “We are only targeting striatal neurons. Patients have cognitive problems, autonomic issues and other problems but this is not a bad place to start. This is one tangible way to help deal with the motor problems in Parkinson's patients.”
“We have years of work before we have enough evidence to say it will work in humans,” he added. The scientists noted in the Nature paper that the antisense oligonucleotide (ASO)- based experiments illustrate a potentially clinically feasible approach for treatment of patients with Parkinson's disease.
“Eventual application of our approach to humans will need to overcome many obstacles, including age-related limits of reprogramming, understanding potential adverse effects caused by local astrocyte depletion (although we only converted only a small fraction of injury-induced astrocytes), specifically targeting regions that harbor vulnerable neurons, and detecting potential side effects due to mistargeted neurons,” they wrote. “Each of these objectives can now be addressed experimentally to develop this promising therapeutic strategy—one that may be applicable to not only Parkinson's disease, but also other neurodegenerative disorders.”
“A novel feature of this study is that it uses transdifferentiation, in which an adult cell type is directly transformed into a different cell type without a stem cell intermediary, thus potentially reducing the time-frame for creating the new cells and decreasing the risk of tumor formation,” said Melissa J. Nirenberg, MD, PhD, FAAN, professor of neurology at the Icahn School of Medicine at Mount Sinai in New York.
“Another interesting aspect of the study design is that the process occurs in situ in the brain, on autologous cells, and thus may have less immunogenicity.”
“This is an exciting proof-of-principle study, but this is very preliminary data in a rodent model, and there are a number of caveats and many unanswered questions,” Dr. Nirenberg said.
“These include questions about the long-term viability of such cells, potential off-target effects of ASO treatments, potential harm of reducing the number of astrocytes in the substantia nigra, and potential inability of newly-created nigral dopaminergic neurons to reach postsynaptic target neurons in the striatum. While axonal sprouting may be sufficient to achieve this goal in rodents, this would have to occur over a much longer distance in humans. Other dopaminergic cell replacement therapies are implanted directly into the striatum to avoid this issue.”
Dr. Nirenberg said that “like other dopamine cell replacement therapies, such an approach would be symptomatic only, and would not address non-dopaminergic symptoms of Parkinson's disease such as cognitive impairment, postural instability, and speech and swallowing disturbances .”
“The technique is extremely novel,” added Claire Henchcliffe, MD, DPhil, director of the Parkinson's Institute, and vice chair for clinical research in neurology at the NewYork-Presbyterian Hospital/Weill Cornell Medical Center.
“One simple, single intervention can promote a whole cascade of complex changes. It is a very potent tool.”
Dr. Henchcliffe said she was impressed with the extensive characterization of the astrocytes-turned-neurons. In the paper, she said, “about 90 percent of the astrocytes converted and about 30 percent of the new neurons had markers showing that they were dopaminergic. That is a fascinating finding.”
She added: “It is all well to convert astrocytes to neurons but if they don't help the phenotype it wouldn't matter. And they have shown, at least in a mouse, it does. They rescued the phenotype. Of course, it is a very different cellular environment in people with PD versus exposing a wild-type mouse to a toxin that damages dopamine.”
There are so many unanswered questions, she added. “How safe will it be for patients? Can it activate tumorigenic pathways? Are there off-target effects? How do you get it delivered safely into the human brain? No one knows.”