Down-regulating PTB

SAN DIEGO—Dr. Xiang-Dong Fu has long studied the basic biology of RNA and the proteins that bind it. A single discovery has launched Fu into neuroscience.

Fu and his team at University of California, San Diego (UCSD) School of Medicine have been studying a protein called PTB that is well known for binding RNA and influencing which genes are turned “on” or “off” in a cell. To do this, they used siRNA to manipulate cells to reduce the amount of that protein.

A postdoctoral researcher then convinced Fu to use a different technique to create a stable cell line that permanently lacked PTB. After a few weeks, there were very few fibroblasts left, and the whole dish was filled with neurons. Thus, the team discovered that inhibiting or deleting just a single gene—the gene that encodes PTB—transforms several types of mouse cells directly into neurons.

More recently, Fu and Dr. Hao Qian, another postdoctoral researcher in his lab, took the finding a big step forward, applying it in what could one day be a new therapeutic approach for Parkinson’s disease and other neurodegenerative diseases. Just a single treatment to inhibit PTB in mice converted native astrocytes, star-shaped support cells of the brain, into neurons that produce the neurotransmitter dopamine. As a result, the mice’s Parkinson’s disease symptoms disappeared. The study was published June 24, 2020, in Nature.

“Researchers around the world have tried many ways to generate neurons in the lab, using stem cells and other means, so we can study them better, as well as to use them to replace lost neurons in neurodegenerative diseases,” said Fu, a distinguished professor in the department of cellular and molecular medicine at UCSD’s medical school. “The fact that we could produce so many neurons in such a relatively easy way came as a big surprise.”

Several methods can be used to mimic Parkinson’s disease in mice. The UCSD researchers applied a dopamine look-alike molecule to poison neurons that produce dopamine. As a result, the mice lose dopamine-producing neurons and develop symptoms similar to Parkinson’s disease, such as movement deficiencies. The researchers developed a noninfectious virus that carries an antisense oligonucleotide sequence—an artificial piece of DNA designed to specifically bind the RNA coding for PTB, thus degrading it, preventing it from being translated into a functional protein and stimulating neuron development.

Antisense oligonucleotides, also known as designer DNA drugs, are a proven approach for neurodegenerative and neuromuscular diseases pioneered by study co-author Dr. Don Cleveland. It now forms the basis for a U.S. Food and Drug Administration (FDA)-approved therapy for spinal muscular atrophy and several other therapies currently in clinical trials. Cleveland is chair of the Department of Cellular and Molecular Medicine at UC San Diego School of Medicine and member of the Ludwig Institute for Cancer Research.

“The mechanism is to down-regulate the RNA binding protein PTB, which removes its competition for a specific miRNA (miR-124) to attack the REST complex,” Fu explained. “The REST complex is responsible for suppressing the microRNA as well as a large number of neuronal specific genes. PTB itself is a target of miR-124. Therefore, once PTB is initially down-regulated, miR-124 will be more efficient in dissenting REST, which leads to the induction of miR-124 to further down-regulated REST. During this process, PTB is also further down-regulated by miR-124. This loops keep going until PTB is totally gone and REST largely suppressed, thus a large number of neuronal-specific genes turned on to generate new neurons.”

“Parkinson is characterized by lost dopaminergic neurons in the brain,” he continued. “Dopaminergic neurons originate in a midbrain region called substantia nigra and project their axons to striatum to release dopamine. This circuitry is damaged in Parkinson’s disease patients, which is accompanied with emulsification of surrounding non-neuronal cells called astrocytes. By down-regulating PTB in actrocytes in substantia nigra are converted to dopaminergic neurons to connect to striatum, thereby restoring dopamine to reverse the disease phenotype.”

The next steps include the demonstration of the strategy in large animal models, evaluating the safety issues and further refining the procedure. If all goes well, it will be ready for clinical trials, according to Fu, who adds that, “Because there is no cure for Parkinson’s and many other neurodegenerative diseases, our strategy provides a new and efficient way to generate new neurons in specific brain regions to replace lost ones in specific disease setting and address a wide range of neurological diseases.”

“It’s my dream to see this through to clinical trials, to test this approach as a treatment for Parkinson’s disease, but also many other diseases where neurons are lost, such as Alzheimer’s and Huntington’s diseases and stroke. What if we could target PTB to correct defects in other parts of the brain, to treat things like inherited brain defects? I intend to spend the rest of my career answering these questions,” he concluded.