Nerve Growth Factor

Hamburger sent a copy of his 1934 paper on limb-bud extirpation to an eminent neuroanatomist, Giuseppe Levi, at the University of Turin Medical Faculty. Luckily, Levi passed it on to one of his young post-doctoral fellows, Rita Levi-Montalcini. Like Hamburger, her Jewish descent meant she lost her position in Turin after Mussolini and the Fascists came to power in 1940. By her own account, she got around to reading Hamburger’s paper while sitting on the platform of a cattle car transporting her to exile at a family farm in the country outside of Turin. Although she lacked formal laboratory facilities during the war, using eggs she obtained from local farms, she set up a small work area in her bedroom. Her experiments followed Hamburger’s but her observations differed from Hamburger’s in two important details: 1) She focused on the sensory neurons rather than the motor columns; and 2) She began observing the developing neurons within the two or three days after extirpation of the limb bud, rather than waiting nine days or more as Hamburger had done. She noted that after an initial period of proliferation and growth in the control (non-extirpated) side of the chick spinal cord, a certain percentage of the developing sensory neurons began to degenerate: a process known as neuronal death. This was apparently normal, though somehow Hamburger had missed it. The observation of neuronal death lead Levi-Montalcini to postulate that the limb bud does not have an inductive capacity — that is, the neurons do not need an agent from the developing limb bud to begin their development. What the limb bud does provide is a “maintenance factor” that prevents massive neuronal death.

When Hamburger read Levi-Montalcini’s papers after the war, he was impressed with her work and invited her to St. Louis to work in his lab and try to resolve the discrepancies. With funds provided by the Rockefeller Foundation, Levi-Montalcini came to St. Louis in 1947 for what was intended to be a one-year period. As the work proceeded with exciting results, Hamburger arranged for an extension of the Rockefeller funds and eventually procured for Levi-Montalcini a faculty position at Washington University that she held for the next thirty years (Figure Showing L-M at Wash U).

Repeating Hamburger’s experiments in St. Louis, Levi-Montalcini confirmed that the limb bud must produce a maintenance factor rather than an inducing factor. The obvious next step was to isolate and identify the chemical agent responsible, but that would be difficult because chick limb bud tissue exists in only very small quantities. In what was the first in a series of lucky turns, Hamburger received a paper in 1948 from a former student, Elmer Bueker (then at Georgetown University Medical School), describing an experiment in which he had transplanted a mouse sarcoma (tumor) into a developing chick egg. Shortly afterward, he noted that the tumor had been thickly invaded by sensory neurons, suggesting that the tumor produced some sort of neuron-stimulating factor (Figure of Beuker’s sarcoma 180). With Bueker’s permission, Hamburger and Levi-Montalcini repeated the experiment and observed a clear quantitative relationship between size of sarcoma implant and amount of neuronal growth. This observation became the basis for a bioassay that was used thereafter to quantify the NGF activity of various tissues and/or tissue extracts in vitro.

Hamburger then arranged to hire a young post-doctoral fellow, Stanley Cohen, from the Washington University Medical School Biochemistry Department, to isolate and characterize the active material from the tumor. Cohen quickly managed to isolate a nucleo-protein fraction that stimulated neuronal growth significantly, but it was not clear whether the nucleic acid or protein component was the active agent. Using snake venom as a source of the nucleic-acid digesting enzyme phosphodiesterase, Cohen soon showed that it was the protein fraction that had the nerve-stimulating property. In a second lucky turn, Cohen and Levi-Montalcini found that the snake venom itself had a thousand-fold greater potency for stimulating neuronal growth than the tumor protein. The system for isolating and characterizing the active fraction, which by now they called nerve growth factor, was enhanced further when Cohen realized that snake venom glands are modified salivary glands; he then tried an extract from male mouse salivary glands, which are far cheaper and easier to obtain than snake venom, as a source for NGF. This turned out to be the third lucky turn since neither female mouse salivary glands, nor those of other male rodents, possess NGF activity. Armed with this system, Cohen was eventually able to isolate and characterize the NGF protein. As the NGF work progressed, Hamburger began to step back from the day-to-day activity, leaving the project in the hands of Levi-Montalcini and Cohen. Discovery of this growth factor opened a new way of thinking about embryology, namely, its control and regulation by a variety of other growth factors that either promote growth or prevent cell death in others. Many such growth factors have been identified in the intervening years.