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, saidLloyd
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.”
Antonino
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.