The Discovery and Potential of Nerve Growth Factor (NGF) by Rita Levi-Montalcini

Rita Levi-Montalcini was working with chick embryos in 1938, investigating how neurons find their way to the limbs they are to innervate, when she was barred from the University of Turin, a Jew in Mussolini’s Italy. She continued work in a laboratory she set up in her bedroom in Turin, then in the countryside.

“At the end of this difficult period, there was a seed for what has been one of most fantastic developments in one field of neuroscience”—the identification of nerve growth factor (NGF), said Piergiorgio Strata, president of Italy’s National Institute of Neuroscience, and a member of the European Dana Alliance for the Brain (EDAB).

The occasion was a memorial symposium for Levi-Montalcini, a founding member of EDAB who died in 2012 at the age of 103. Neuroscientists who knew, worked or studied with Levi-Montalcini honored her life by elaborating her legacy—a morning's tour through research that followed the groundbreaking discoveries for which she received The Nobel Prize in Physiology or Medicine in 1986.

Working at Washington University in St. Louis after the war, she and Stanley Cohen (with whom she shared the Nobel) identified a compound, expressed by peripheral cells, that attracted spinal neurons and induced neurite formation, then isolated this substance—NGF—from tumors, snake venom, and mouse salivary glands—all in the face of relentless skepticism from the scientific community.

Speakers at the symposium, presented by the Italian Cultural Institute and Centro Primo Levi in NYC, stressed the characteristics that enabled her to flourish intellectually and prevail in adversity: 

• a powerful, charismatic personality, 
• enormous drive and passion for her work, and 
• an approach that combined intuition with analysis. 

"She often noted that she viewed herself as an artist more than a scientist," said a Neuron obituary.

The importance of this work, speakers said, could hardly be overestimated. "If we look at the history of 20th century neuroscience, Rita ranks with the giants... she was the first major molecular neurobiologist," wrote fellow Nobelist (and member of the Dana Alliance for Brain Initiatives) Eric Kandel, in a tribute read at the meeting. "Her extraordinary discovery of NGF affected all aspects of our field."

As the first identified growth factor, NGF introduced a radically new concept, said Lloyd Greene of Columbia University. "We knew from insulin that organs could communicate via substances that went into the bloodstream. A major implication of Rita's findings was that there was another means of communication between cells, at short range.” Her inquiry into embryonic development illuminated key processes in mature neurons—survival, plasticity, neuroprotection—and beyond, “an explosion of findings within and outside the nervous system."

Ralph Bradshaw of University of California, San Francisco, called NGF a "Rosetta Stone" that helped decode key aspects of nervous system function, proteins, receptors, and cancer biology.

He reviewed some of his involvement in elaborations of the NGF discovery—beginning with the sequencing, in collaboration with Ruth Angeletti (who had been Levi-Montalcini's only PhD student, now at Albert Einstein College of Medicine), of the NGF molecule. The structure, he said, suggested a compound that acted like insulin on target cells. "It turned out we were right, but not for all the right reasons."
“The idea that NGF was an endocrine-like substance led to pursuit of the receptor,” Bradshaw said, summarizing research that eventually characterized not one but two receptors (a fact that “befuddled the field for 15-20 years”) and then to elucidation of the molecular signaling pathways by which NGF and related compounds modulate cellular function.

“The picture started to evolve that these factors were not only involved in growth and development, but also as regulators in growth disorders, namely cancer… that these were very important discoveries,” Bradshaw said.

Greene’s research exemplified this importance. "In science, you start working on one thing and end up with something far different," he said. “NGF led us, in ways we never would have anticipated, to a potential treatment for brain tumors.”

It began with studies in the 1990s to explore how NGF regulates genes. Using serial analysis of gene expression, Greene’s research team identified hundreds of genes that became more or less active after exposure to the compound. The researchers then focused on transcription factors—proteins that determine whether genes are turned on or off. They found that one of these compounds, ATF5, was particularly abundant in neural progenitor cells, but not in mature neurons or astrocytes, and that NGF shut down production of ATF5.

“This led us to the idea that ATF5 is important for proliferation of [stem] cells that eventually give rise to the brain. When they encounter growth factor, they turn into differentiated cells and stop proliferating,” Greene said. Neural progenitor cells that were experimentally deprived of ATF5 differentiated prematurely and failed to migrate. Cells infected with a retrovirus to keep on producing the transcription factor never differentiated and continued to divide—much like a tumor.

“We wondered: is ATF5 present in glioblastomas?” he said.

It was; cells from 29 of these highly virulent, virtually incurable tumors all expressed the transcription factor. When the researchers silenced ATF5 in cultured glioblastoma cells, the cells died.

In subsequent in vivo studies, the researchers gave mice with experimentally induced glioblastomas subcutaneous injections of a molecule that hybridized dominant-negative ATF5 protein, which neutralizes ATF5, with penetratin, a peptide that crosses the blood-brain barrier.

Within days of treatment, tumor cells began to die; 19 days and 6 months later, the tumors had disappeared on MRI. Treated animals all survived for 6 months, while 60% of the others died. There was no apparent kidney, brain, liver, or blood toxicity.

As work proceeds with other animals, “we’re collecting data to go to the FDA for possible clinical trials,” Greene said. The approach “could work for other tumors as well.”

Antonio Cattaneo of the European Brain Research Institute in Rome (which Levi-Montalcini helped establish in 2002), described research linking the NGF system to Alzheimer’s disease pathology, and suggesting a novel treatment strategy.

Using antibodies that target NGF, he showed that neutralizing the growth factor in the brains of adult mice initiated a process of neuroinflammation and neurodegeneration. While the effect on cholinergic neurons—a key population in Alzheimer’s disease (AD)—was first implicated, it became clear that astrocytes and glia were compromised as well.

Further studies characterized this neurodegeneration process as an imbalance between NGF and a precursor protein, proNGF, and showed that the same result could be achieved by modifying mouse brain cells to overexpress proNGF.

Cattaneo has been exploring ways to “strengthen the balance by increasing NGF.” When mice, genetically modified to express AD-like pathology, were given a modified form of NGF intranasally, amyloid plaques regressed, and learning and memory deficits improved.

“This may be a viable candidate for a non-invasive therapeutic approach to AD,” he said. “We’re collaborating with the pharmaceutical industry to get clinical trials.”

Looking toward the future of NGF-related research, Cattaneo cited an "agenda" that Levi-Montalcini proposed in 2009, at the age of 100. In addition to work (like the studies described above) aiming to develop its therapeutic potential, she urged investigations of the NGF system’s role earlier in embryonic development than the nervous system, and in more primitive species.

Her agenda called for studies of NGF in other tissues, particularly the reproductive system. “Rita predicted it would be found to participate in processes like activation of sperm or implantation of ova,” Cattaneo said.

Her scientific intuitions were still reliable, he said. In a paper published three years later, researchers described their work identifying a substance in the semen of diverse mammals that induces ovulation. It was NGF. 

ORIGINAL: DANA Foundation

by Carl Sherman

February 20, 2014