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Rendiconti Lincei. Scienze Fisiche e Naturali

, Volume 29, Issue 4, pp 737–753 | Cite as

Rita Levi-Montalcini and her major contribution to neurobiology

  • Piergiorgio Strata
Review

Abstract

In this paper, I shall illustrate the personality of Rita Levi-Montalcini and highlight how she first emerged within a prestigious school, while under the direction of Giuseppe Levi, a most inspiring and stimulating figure. Between 1919 and 1938, he created a rich environment capable of influencing the minds of innumerable students; among them, three future Nobel Prize winners, Renato Dulbecco, Salvador Luria, and Rita Levi-Montalcini, who were companions in the same classroom. In 1938, due to the racial laws for the defence of the race, Luria and Levi-Montalcini were banned from entering the university premises. In a small working space in her bedroom—“a minuscule laboratory not unlike a convent cell”—Levi-Montalcini, supported by Levi, made a most outstanding discovery that opened a new chapter in neurobiology. Further collaborations with Viktor Hamburger and Stanley Cohen led Levi-Montalcini to the discovery of the nerve growth factor (NGF): a remarkable accomplishment which turned out to represent a milestone in the development of modern cell biology.

Graphical abstract

Keywords

Embryonic induction Giuseppe Levi Hans Spemann Nerve growth factor (NGF) Neuroembryology Stanley Cohen 

1 Rita Levi-Montalcini

What I am about to write is based not only on information taken from Levi-Montalcini’s scientific publications, but also from my personal contacts and from the numerous articles she wrote that are rich in autobiographical data (Levi-Montalcini 1975, 1982), and particularly from the book entitled In praise of imperfection (Levi-Montalcini 1988). The story of the family environment, where she grew up as a teenager is very enlightening in revealing the profile of her personality that determined her future success in science and society. For a historical perspective, it is also important to mention the autobiographical report of Rita’s close collaborator Viktor Hamburger (Hamburger 1989, 1993, 1996) and to report the views of qualified scientists deeply involved in the same scientific field (Cowan 1981, 2001; Purves and Sanes 1987; Oppenheim 2001; Chao 2010).

Let me start with the first time I met her. It was in the summer of 1959 when I went to visit her in Turin at the home, where she grew up in Corso Re Umberto 10. I was writing my MD dissertation on avian behaviour and my Professor, Giuseppe Moruzzi, organized the meeting, so that she could enlighten me on the identification of some anatomical lesions of the bird cerebral hemispheres that I had studied in my thesis. When I started to call her “Professor” she stopped me immediately and said: “I am Rita.” This was the way she liked to be called by everyone throughout her life.

This happened at a time when the story that I am going to illustrate on the discovery of a nerve growth factor had its highest rate of success with a series of experiments performed by Rita in collaboration with Stanley Cohen between 1952 and 1959. Rita forgot the purpose of my visit, and with obvious enthusiasm, she described to me the accomplishments she achieved in that period. I was impressed by the possibility of performing a total sympathectomy with simple subcutaneous injections of an antibody against the newly discovered growth factor. The experiments appeared to me very clear and easy to understand. Our talk went on until I had to go and catch my train back to Pisa, where I was a student. My mission had failed, but I went back home full of enthusiasm from the meeting, an experience that contributed to encouraging me to pursue my future activity in research.

2 The young Rita

Rita belonged to a Sephardic Jewish family. Her father, Adamo Levi, had a strict Victorian view of life that at that time was quite widespread in Europe. Accordingly, he saw the role of the woman in society as a wife and as a mother (“two X chromosomes in a Victorian climate,” as Rita herself writes in her autobiography, Levi-Montalcini 1988). Thus, he directed his daughter to a high school, where she was not taught anything that would prepare her to cover important professional roles in life nor any qualification that would allow her to enter a medical faculty.

After completing high school, at age 17, for the next 3 years, Rita felt she was in a dead end and unable to decide her future. She was very attached to her mother, Adele Montalcini, but not to her father with whom she had difficult communications. Such a difficulty was often mentioned by Rita and it is summarized in the dedication to her sister Paola and to her father in her book In praise of imperfection (1988): “To Paola and to the memory of our father whom she adored while he lived and whom I loved and worshiped after his death.” In fact, later, she acknowledged that her father, more than her mother, had a crucial influence on her life through both the inherited genes and his personality that she admired for his tenacity, his energy and his intelligence, not less than for his secular outlook and his Spinozan culture.

Following the illness and death of her governess, Giovanna Bruatto, Rita was attracted by the idea of becoming a medical doctor. She told her mother that she wanted to study medicine and she was encouraged to speak with her father about it. Finally, despite being discouraged by him, she decided that she would not accept the subordinate role of a woman and she convinced herself to give up the possibility of the role of a wife. Therefore, after she had obtained the necessary diploma in a private high school with the help of two tutors, she was allowed to enrol in the Faculty of Medicine and Surgery of the University of Turin (Fig. 1). This was the beginning of a sparkling career as a researcher and in 1936 the Rector of the University, Silvio Pivano, conferred the MD degree summa cum laude to Rita (Fig. 2).
Fig. 1

Photo ID presented by Rita, as requested, together with the application for enrollment in the first year of the Faculty of Medicine and Surgery, dating back to October, 31, 1930.

Courtesy of Historical Archive of the University of Turin

Fig. 2

Fascist University Group, Majoring in Medicine and Surgery, Year 1935–36–XIV. The Faculty (first row): Quarelli, Bizzozzero, Vanzetti, Micheli, Uffredduzzi, Allaria, Cova, Azzi, Negro. Rita is in row 6, number 4 from right.

Courtesy of Piera Levi-Montalcini

In 1938, she obtained the title of “volunteer assistant” in the Nervous and Mental Illness Clinic (Clinica delle Malattie Nervose e Mentali). However, following a Decree of October 18, 1938, signed by the University Rector Azzo Azzi (Fig. 3), as a result of racial laws (Royal Decree Law, September 5, 1938, n. 1390, Provisions for the defence of race in the fascist school) (see also Fig. 4), she had been suspended from service from October 16. However, the same provision, in Article 5, allowed students already enrolled to complete their course of study, provided they were passing their exams successfully and on time, but without the financial help of a scholarship. In this way, in 1939 Rita was able to obtain the Specialist Diploma (Diploma di Specializzazione) in Neuropathology and Psychiatry.
Fig. 3

Veto. THE RECTOR, according to items 3 and 6 of the R.D. (Royal Decree) Law September 5, 1938-XVI, n. 1390; DECREES Dr LEVI RITA, volunteer assistant in the Clinic of Nervous and Mental Diseases of the R. [Royal] University of Turin, is suspended from service, starting on October 16, 1938-XVI. Turin, October 18, 1938—year XVI; THE RECTOR (Prof. Azzo Azzi.

Courtesy of Historical Archive of the University of Turin

Fig. 4

Front page of Corriere della Sera, November 11, 1938, announcing the approval of racial laws. The laws for the protection of the race approved by the Council of the Ministers. Mixed weddings are forbidden—the definition of “Jews”, the discriminations and the annotation of civil status—the exclusion from employment in State, parastatal and public interest agencies—rules concerning elementary and middle schools and teachers

Thus, her scientific activity had begun during the second year of medical school as an internal student at the Anatomy Institute led by Giuseppe Levi. The Institute, as Rita underlined many times, was a pole of attraction for students, not because of the fascination of the discipline, but for the outstanding personality of its Director, renowned for his fame as a scientist, but also for his unconcealed contempt for the fascist regime. This was the start of a fruitful connection that would become very significant in the future of both. It is interesting to note that the first work performed by Rita under the guide of Levi was the object of her MD thesis in July 1936. From the title Research on the in vitro formation of collagen and reticular fibrils from explants of various organs it is clear that she worked on the main topic of her tutor, famous for his in vitro studies. As we shall see, Rita would draw her mentor’s interest to a new field.

3 Giuseppe Levi and his school

Let us now give a brief profile of Giuseppe Levi (Figs. 5 and 6): his personality, his intense and diversified cultural experiences at international level, starting from when he was a university student to his brilliant and multifaceted research activity, to his moral rigor and his charming didactic activity. All this led to the establishment of a true school as a forge capable of forming qualified researchers in a state-of-the-art institute with avant-garde technologies and, above all, of stimulating young people to take up a research career. All this happened at the Human Anatomy Institute (Istituto di Anatomia Umana Normale) of the University of Turin under the direction of this great master in the period 1919–1938. Here, we have a magnificent example of how important it is also to have different and frontier technologies to pursue important achievements.
Fig. 5

Giuseppe Levi’s portrait donated to Rita Levi-Montalcini with a dedication: “Alla sua valente collaboratrice e cara amica. 26 luglio 1943 G. Levi (to his talented collaborator and dear friend. July 26, 1943 G. Levi).” The day before the radio had announced the fall of the fascist government.

Courtesy of Piera Levi-Montalcini

Fig. 6

Giuseppe Levi (left). Tempera on rigid panel by Ottavio Mazzonis, The ages of man, 1994, Institute of Anatomy of Turin.

Courtesy of Giacomo Giacobini

Levi was born in Trieste, but his academic activity started in Florence. In 1889, at the age of 17, Levi entered the Department of Medicine to work under the guidance of Alessandro Lustig, Professor of General Pathology, who had acquired rich international experiences at the University of Vienna, where he had graduated in Medicine, and at the Institute of Physiology at the University of Innsbruck as an Assistant Professor. Levi’s contact with Lustig was the first important step towards his scientific maturation.

During this period, Levi also came into contact with other prominent personalities such as Filippo Bottazzi, Mario Carrara, Gino Galeotti, and Amedeo Herlitzka. After graduating in 1895, he became Assistant to Eugenio Tanzi, and subsequently married his niece. The meeting with Ernesto Lugaro, a pupil of Tanzi, was important, since Lugaro introduced him to the field of neurobiology.

After these early contacts with great personalities, Levi abandoned the clinical medicine to devote himself entirely to experimental biology. Following a brief period (1898–1899) in Berlin with Oscar Hertwig, he moved to Florence (1899–1909) as Assistant to Giulio Chiarugi, Full Professor of Normal Human Anatomy. Then, he moved to the Stazione Zoologica in Neaples and to the University of Sassari. In 1914, he was in Palermo, where he learned how to make use of cell cultures for his research activity. During the First World War, he was a volunteer physician, and in 1919, he finally held the chair of Normal Human Anatomy in Turin until his retirement in 1948.

During the Turin period, he did not have an easy life. In fact, his opposition to the fascist regime saw him among those who signed the Manifesto of anti-fascist intellectuals drawn up by Benedetto Croce in 1925. In addition, in 1926, he sheltered Filippo Turati in his house, and in 1931, he opposed the oath of allegiance to the regime, a position that led him to being imprisoned together with his three sons over the years 1934–1935. Finally, in 1938, he was suspended from service due to the Royal Decree Law that also banned Rita. During this period of ostracism, collaboration with Rita began outside the institutions until 1943 when both were hiding in Florence.

At the end of this troubled period, in 1945, he resumed his activity, and on September 3, he delivered a remarkable opening lecture to his Human Anatomy course students entitled La struttura della sostanza vivente (The structure of the living matter) (Levi 1945). The lecture was dedicated to the memory of Leone Ginzburg, his son-in-law, an anti-fascist political activist, who was arrested at the age of 34, and subjected to severe torture, which led to his death. The lecture was also dedicated to the memory of all the university persons who died defending their freedom. Between 1946 and 1950, he was also Director of the Centre for Studies on Growth and Senescence (Centro di Studi sull’Accrescimento e la Senescenza) of the National Research Council (CNR), a centre that allowed him to obtain research funding, and in 1950 he launched a cell biology laboratory in São Paulo in Brazil.

The internationally renowned School of Giuseppe Levi had among its members a long series of great personalities that were largely anchored in the neurological field in different disciplines: Tullio Terni, Luigi Bucciante, Rodolfo Amprino, Oliviero Mario Olivo, Giovanni Godina, Angelo Bairati, Cornelio Fazio and Edmondo Malan. Three students received the Nobel Prize: Salvador Luria for genetic studies, Renato Dulbecco for research on oncogenic viruses and Rita.

As we shall see, at least two techniques, which were avant-garde at the time, were important to Rita’s success. One was the staining of nerve fibres with silver salts discovered by Camillo Golgi in 1873, later implemented by Santiago Ramón y Cajal and refined by Fernando De Castro. This method was critical for visualizing under the microscope the state of the neuronal cell bodies and of their nerve fibres during development. The second one was the cell culture technology that Levi encountered when he was in Palermo, and that he now introduced to his institute as an important facility focusing his pioneering research on neural cells, mainly in collaboration with Hertha Meyer (Levi and Meyer 1941). Meyer had been trained and had worked in Germany, but when the Nazis seized power, she moved to Italy. Subsequently, when racial persecution became a serious problem in Italy, she moved to Brazil to the most distinguished Biophysics Institute of the University of Rio de Janeiro, led by Carlos Chagas. Many years later Rita visited this same institute and in collaboration with Meyer she performed one of her most significant experiments (Levi-Montalcini et al. 1954).

4 The dual team and the war drama

We left Rita with her academic titles. However, following the above-mentioned legislative measures she was not able to enter the university premises and continue her studies. Therefore, in March 1939 she accepted an invitation from Prof. Lion Laruelle and continued her research activity at the University of Brussels. Giuseppe Levi was already in Belgium invited by Prof. Jean Firket to work in the University of Liège, where he was able to establish a cell culture laboratory in collaboration with Maurice Chèvremont and continue his research activity. At weekends Rita visited Giuseppe Levi and the dual team was again in contact.

However, in September of the same year Poland was occupied by the Germans and Hitler was threatening to invade Belgium. Therefore, Rita returned to Italy for Christmas. On June 10, 1940, Italy entered the Second World War. She was worried by the political situation and unsure of what to do, when Rodolfo Amprino, a brilliant member of the Levi School, encouraged her to set up a small laboratory somewhere to continue her research activity. She did so in a small working space in her bedroom: “a minuscule laboratory not unlike a convent cell” (Levi-Montalcini 1988). Levi, instead, remained in Belgium until the summer of 1941 when he again joined Rita in the new small laboratory. Once again they were in contact.

However, following the heavy bombing of Turin by the Anglo-American air forces in 1941 Rita left the city and took refuge in the countryside near the town of Asti. In 1943 the situation deteriorated considerably. Following the invasion of Italy by the German armed forces and the German plan to send the Jews to concentration camps in Germany, all of Rita’s family escaped to Florence living under a false name until the end of the war, when they could start a new life in Turin and Rita could plan to resume her research activity.

5 Viktor Hamburger

The actual research of Rita and Giuseppe Levi together began in 1941 in her homemade laboratory. Levi had read an article published by Viktor Hamburger (1934) and in 1938 he gave it to Rita to read. She did so in a train during a trip to a small village in the mountain with her friend Guido. Here are her comments: “I was distractedly reading an article Levi had given to me two years before. […] For me, Hamburger’s limpid style and the rigor of his analysisin sharp contrast with those of previous authors who had described the same phenomenon in amphibian larvaecast new light on the problem” (Levi-Montalcini 1988). To better appreciate the scientific achievements made by Rita and Levi, it is worthwhile to outline the scientific profile of Hamburger and his main conclusions that inspired “new light” in Rita.

Hamburger was a world authority in the field of nervous system development. He was born in Germany and after Gymnasium (high school) he spent two summers in the University of Heidelberg. Here he became interested in developmental biology. Soon he moved to the Department of Zoology at the University of Freiburg whose Director, Hans Spemann, assembled an outstanding group of scientists and with whom he obtained his PhD in 1925. The title of his thesis was The relationship between nerve and limb development, thus acquiring interest in neurogenesis (see also Hamburger 1925). This contribution may be considered the starting point for the future investigations that characterized the entire life of Hamburger and led to his collaboration with Rita.

Spemann was awarded the Nobel Prize in 1935 for the discovery of an “inductive factor” in embryology (Spemann and Mangold 1924). Working on amphibian eggs, along with Hilde Mangold, he demonstrated the existence of an area in the embryo which, when transplanted into a still undifferentiated part of another unformed one, induced the formation of a second embryo. In other words, he obtained an embryo within an embryo. Therefore, he postulated the existence of an “organizer”, the name he gave to this region. Later, it has been assumed that several territories are organizers of different regions of the brain. These results inspired many of the embryology experiments conducted in the rest of the century. The role of the amphibian organizer has been identified subsequently in embryos of fish, birds and mammals, including a specific locus in the human embryo (Martinez Arias and Steventon 2018; Martyn et al. 2018; Pourquié 2018).

After short periods in Göttingen and Berlin, in 1927 Spemann offered Hamburger a position as an instructor. This allowed him to return to Freiburg. However, he was dreaming of visiting the United States and spending some time in Chicago with Frank Lillie, a long-term friend and admirer of Spemann and the author of a classic book on chick development (Lillie 1908). Lillie (1903) had performed experiments on chick embryos to ascertain whether a wing primordium might regenerate after extirpation and later (Lillie 1904) he had demonstrated that “the embryo of the chick possesses no greater power of regeneration than the adult” (quoted in Willier 1957).

In 1932 Hamburger obtained a Rockefeller fellowship to work in Chicago for 1 year (Fig. 7), a period extended to 3 years (1932–1935) when, in April 1933, the Nazis came to power in Germany, and Hamburger received a letter from the Rector of the University of Freiburg, Martin Heidegger, announcing that he no longer had a research position in Germany because of his Jewish ancestry.
Fig. 7

Viktor Hamburger in 1933. Marine Biological Laboratory Copeland-Bloom Photo Album, Marine Biological Laboratory Archives. Licensed as Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported

https://hpsrepository.asu.edu/handle/10776/3264?show=full

Following this difficult period in 1934 he was offered a position at the Washington University in St. Louis. He went briefly back to Germany to bring his wife and daughter to the United States and in 1935 he moved to the new place, where he stayed for the rest of his life. He was Chairman of the Department of Zoology from 1941 to 1966. He became Professor Emeritus in 1969 and continued his laboratory work until the mid-1980s.

6 Peripheral organizers for brain structures

The work published by Hamburger in 1934 is the outcome of the experimental results acquired mainly in Chicago and the interpretation of the results reflects the culture of the Spemann School. Many years before, the young Marian Lydia Shorey, under the suggestion of Lillie, studied the problem of the correlation between the nervous system and peripheral structures during development (Shorey 1909) and this was the real beginning of a new field in embryology. She removed the limb bud on one side of a chick embryo and she found, both in the spinal ganglia and in the anterior and posterior horns of the spinal cord, a reduced number of cells. However, she found that “no degenerating fibres can be observed when the extirpated areas are removed before the nerves reach the region of the operation.” Therefore, she concluded that “the defects which appear in the nervous system are not due to degeneration, but to the failure of certain neuroblasts to develop.” This phenomenon is called hypoplasia, which means deficient development.

In addition, she noticed that a complete destruction of the muscles of a given somite did not lead to the complete suppression of motor cells and motor fibres at this level of the spinal cord, but their number was greatly reduced. However, she observed that the motor fibres were innervating the muscles of the nearby located somite above or below the lesion. Thus, she wrote that “it is safe to assume that all are alike dependent on stimulation from end organs or the products of the activities of the end organs, for differentiation.” In conclusion, her results showed that the periphery is essential for the developmental organization of the nervous system. In a subsequent paper Shorey (1911) suggested that “metabolic products” from the muscles that diffuse into the lymph provide the stimulus that regulates the differentiation of innervating neurons.

Lillie suggested to Hamburger that he should study the mechanisms of such a relationship. First, by careful quantitative assessment at embryonic days 8–9, Hamburger confirmed the previous results illustrated by Shorey on the marked hypoplasia in the spinal cord and in the ganglia and he found a quantitative relation to the degree of muscle destruction. He also noted that when such a deletion was radical it did not result in a complete lack of the anterior horn, where about 40% of cells were still present (Hamburger 1934). Such a survival was not later confirmed by Levi-Montalcini and Levi (1944). The interpretation offered by Hamburger was that these neurons develop independently of the periphery, while the remaining pool is under peripheral control. According to Ross Harrison (1907), embryonic nerve cells send long projections out of the central nervous system and later they take contact with the appropriate peripheral target. Hamburger assigned to these developing neurons the role of “pioneers” to explore the size of the peripheral field and to send back to the neural centres a signal to recruit the appropriate number of central neurons (retrograde recruitment hypothesis) (Fig. 8). The remaining central neuroblasts will degenerate. As to the nature of the retrograde signal, he suggested that specific agents for the sensory and the motor components originate in the peripheral site acting as organizers for brain structures.
Fig. 8

Role of the wing or limb bud on spinal neuron differentiation and maturation. Above: The induction hypothesis by Hamburger. Among a group of neuroblasts, a few of them (pioneers) send an elongation towards the peripheral tissue organizer (a). Here they pick up from the environment a substance that migrate retrogradely (arrows) up to the spinal cord, thus inducing a differentiation and a maturation of the same cell pool (b). Below: Chick embryo submitted to the amputation of the peripheral bud of one side on day 3. The figure on the left shows neurons of the spinal ganglion of the normal side on day 15. The neurons show a high degree of differentiation and maturation (c). The figure on the right side shows the neurons of the ganglion of the amputated side, where numerous cells appear well differentiated, but with clear signs of degeneration (d) (Levi-Montalcini and Levi 1944)

This view was consistent with Spemann’s concept of the “organizer”. In the absence of the periphery the neural centres would go awry and everything would disappear. In summary, it had been proposed that the developing muscles and sensory devices produce two different and specific factors that migrate backwards into their respective nerve centres, namely, the motor columns and spinal ganglia, where they promote the maturation and govern the development of these centres in a precise quantitative manner. The results were good support for the hypothesis elaborated by Spemann that dominated for such a long time.

7 A novel approach

To the eyes of a biologist the explanation proposed on the basis of Spemann hypothesis was widely accepted and possibly had made Lillie, a Spemann admirer, happy. However, to the eyes of Rita, the data represented a way to examine a new paradigm in the study of the brain. Therefore, in collaboration with Levi from the spring of 1941 to the autumn of 1942 she planned to repeat the experiments performed by Hamburger. The results, published in 1942–1944 (see below), signalled the beginning of a new chapter in neurobiology. The aim of the experiments was to observe directly the developing cells and their elongations towards the peripheral region during the developmental phase. This was possible by visualizing the neurons by means of the silver staining, a technological advantage not used by Hamburger. Later, Rita would export the technique to the American lab and Hamburger would acknowledge that she had brought a very important instrument from Turin.

The primary question was to understand by which mechanism the elimination of the limb causes a reduction of the number of neurons that would normally provide its innervation. The results demonstrated that in a chick embryo, following the removal of the wing or the limb primordium, neurons were able to proliferate to achieve full maturation and to emit elongations toward the peripheral region and reaching the proximity of the missing primordium. They observed a neuron regression during the following days and, starting at about 2 weeks, the complete set of fully differentiated neurons were gradually disappearing. In contrast to what Hamburger postulated originally, the reduced cell number following the bud removal was not a result of hypoplasia. It was the consequence of the death of fully differentiated neurons and not of the failure of cell recruitment from a pool of still uncommitted precursors (Fig. 8). In other words, it was a process of degeneration which had escaped the attention of both Hamburger and Shorey. Rita and Levi concluded that the central neurons develop and differentiate in the absence of the primordium and that the regression of the motor or sensory neurons depends on the impossibility of establishing connections with the peripheral structures, the muscles and the skin. Therefore, for neuron differentiation there was no need of an early interaction with the wing or the limb primordia and this ruled out their role as inductive factors and the retrograde recruitment hypothesis. Instead, it is the lack of a factor released from the primordia that is simply needed for neuron survival. This hypothesis was later confirmed during the collaboration between Rita and Hamburger (Hamburger and Levi-Montalcini 1949).

At that time the results could not be published in Italy, but the authors found space—in short format—in a journal published in Belgium (Levi-Montalcini and Levi 1942, 1943) and—more extensively, with an abstract in Latin—in a Vatican journal (Levi-Montalcini and Levi 1944) (Fig. 9). The results, obtained in the most difficult environmental conditions and in a homemade laboratory, are documented by a rich and magnificent set of handmade illustrations. The conclusions were in contrast to the hypothesis based on the experiments by Spemann and Mangold (1924) and on the interpretation that Hamburger gave for his experiments (Hamburger 1934) on induction in neural development. They should be considered a milestone in neurobiology. In addition, the paper opened a new window for future research in the field. In this fruitful collaboration Rita found in Levi a brilliant mentor and Levi found in Rita an acute and bright mind with outstanding intellectual and technical capacities able to move the main interest of her tutor into a new modern and attractive field. In this outstanding series of revolutionary scientific achievements Rita revealed also a stigma of tenacity and energy that she attributed the genes inherited by the father.
Fig. 9

Front page with Latin abstract of the paper by Levi-Montalcini and Levi (1944)

At the end of the Second World War, Hamburger, Chairman of the Department of Zoology at the Washington University in St. Louis, became aware of the results of his Italian colleagues and he realized the importance of the critical comments they had made about his discoveries. In the spring of 1946 he wrote to Levi to have the young scientist in his laboratory at the Washington University of St. Louis for a period of 6 months to discuss and clarify the different interpretations of their respective experiments. On September 19, 1947, Rita began her 2-week trip on a Polish ship, the Sobieski, to reach the place of the new adventure. Dulbecco was on the same ship on his way to Bloomington, Indiana, to collaborate with Luria. Rita remained at Washington University for 26 years, a period that she recalled as the happiest and most productive of her life. This period was in fact a saga of revolutionary discoveries. The exodus of the three brilliant Turin brains was the start of a heavy and unbalanced flux of Italians which persists until the present day.

8 The new world

At this point of our story the role of Turin becomes of minor importance. However, the subsequent scientific achievements gathered by Rita in the United States remain a wonderful product of the Turin School of Giuseppe Levi. There was a crucial point that divided the line of thought of Rita and Hamburger (Fig. 8). According to Rita, in the absence of the limb or wing buds, the neuronal elements were able to grow and to differentiate, but they were going to degenerate later when their axons arrived close to the missing bud. This view was against the induction hypothesis. According to Hamburger, in fact, the neurons were unable to differentiate due to the lack of an induction mechanism.

Hamburger was interested in examining the effects of limb ablation with Rita. Therefore, as soon as she reached St. Louis the new team repeated the experiments that she and Levi had performed in Turin. They demonstrated that both motor neurons and the neurons of the sensory ganglia were degenerating after removal of the peripheral buds. In addition, they showed that after transplanting an additional primordium in the embryo, the number of spinal neurons increased. Therefore, they concluded that the periphery provided the necessary conditions for both the completion of axonal growth during development and the survival of neurons (Hamburger and Levi-Montalcini 1949). Again, these findings were against Spemann’s induction hypothesis. According to Cowan (2001), these results on development published by Rita and Hamburger in 1949 represent one of the best works in the whole of neuroembryology.

In 1948, at the beginning of their collaboration, Hamburger showed Rita the results of a series of experiments made by one of his former students, Elmer Bueker, who had been working on the topic since 1943. Bueker (1948) had implanted a fragment of a sarcoma on the walls of a 3-day chicken embryo. The student observed that in the following days the sensory ganglia had increased in volume and some fibres of these ganglia elongated to reach the inner region of the tumor. On the contrary, he did not notice any elongation of the fibres of the motor neurons. Bueker interpreted the results as the consequence of an inductive action that the tumor exerted on the nerve centres, the same hypothetical inductive action exerted on the spinal centres by the peripheral buds.

Rita and Hamburger (Levi-Montalcini and Hamburger 1951) repeated the experiment made by Bueker. Using the technique of staining with silver salts that Rita had imported from Turin, they were able to observe the behaviour of the nerve fibres. The results confirmed what Bueker had described. However, thanks to the staining of the nerve fibres, it became clear that growing up inside the tumor were not only the sensory fibres coming from the spinal ganglia, but also fibres that came from the ganglia of the sympathetic nervous system. It was also possible to demonstrate that the new fibres entering the tumor did not make contact with its cells: they were free nerve terminals. These findings demonstrated for the first time the existence of a diffusible agent released by the cells of the tumor into the surrounding tissue.

Rita decided to place the sarcoma on the chorio-allantoic membrane that covers the embryo, thus eliminating the direct contact between the embryo and the tumor. The results were similar and against the retrograde recruitment hypothesis previously postulated by Hamburger. Therefore, the hypothesis of the induction factor and of an organizer was once more not supported by the new experimental evidence. It was evident that the tumor released a soluble factor which, through the embryonic blood circulation, reached the ganglion neurons (Levi-Montalcini 1952; Levi-Montalcini and Hamburger 1953). These results are the birth certificates of the nerve growth-promoting factor, which later became known as NGF.

According to Cowan (2001), “There is a hint, however, that Viktor was somewhat uneasy about Rita’s independent report at the meeting of the NY Academy of Sciences, which is referred to in a footnote on the second page of their 1953 paper: ‘A preliminary report of this work has appeared in the Ann. N.Y. Acad. Sci. 55, 1952’.” Later Hamburger, in one of his biographical papers (Hamburger 1989), stated that he participated to the initial phase of the new topic on the soluble factor. He realized that this was the beginning of the story of the nerve growth factor, but he decided to pursue other interests. His collaboration with Rita was close to an end although Hamburger continued to fully support the investigations on this new frontier in his St. Louis Department.

Several years later, however, Hamburger provided further important contributions on the influence of the periphery in the development of the spinal motor column (Hamburger 1958), the timing of cell death (Hamburger 1975), the outcome of a supernumerary limb transplant, where it was shown that it does not lead to an enhancement of cell proliferation, but it reduces cell death (Hollyday and Hamburger 1976). In addition, using a radiolabelled NGF provided by Ralph Bradshaw, he showed that NGF is taken up by sensory nerves and transported in a retrograde direction to their cell body in the spinal ganglia (Brunso-Bechtold and Hamburger 1979). He also provided the first good demonstration that the administration of exogenous NGF to the developing chick is able to reduce the naturally occurring cell death in the spinal ganglia (Hamburger et al. 1981).

9 Rio de Janeiro and the “golden halo”

Rita and Hamburger were aware of the fact that to continue their fascinating story it was necessary to identify the nature of the growth-promoting factors and their mechanisms of action. They tried to see if chemical extracts of the tumor injected into embryos could replicate the effects observed after tumor transplantation. Rita (Levi-Montalcini 1988) reported later that: “The persistent negative results led me to resort to other techniques.” They realized that it would be important to isolate and identify the factor. One possible way was to use the culture technology which Rita had used in Turin for her MD thesis under the supervision of Hertha Meyer. She kept contacts with her and now she asked her old friend to perform the experiments in her lab in Rio de Janeiro. This plan was easier than to set up a new culture facility in St. Louis. Hamburger was able to persuade the Rockefeller Foundation to provide Rita with a travel grant to stay in Rio for 3 months. At the end of the summer of 1952, on her way to Rio, Rita travelled to Italy to visit her family and from there she went to Rio carrying with her, throughout the whole journey, two white mice with transplanted sarcomas S180 and S37 hidden in her purse or her coat.

As soon as she was in Rio, sarcoma cells were grown in a small dish, close, but not in contact, to the sympathetic ganglia neurons. A fascinating picture appeared in front of Rita. After only 24 h of culture, thick bundles of sympathetic fibres emerged radially from the ganglia, like a halo (Fig. 10). There was no doubt that a soluble factor produced by the tumor was driving the fibre growth. Rita kept Hamburger informed of the spectacular results that she carefully recorded by sending handmade outlines of the images. The results of the experiments performed in Rio were soon published (Levi-Montalcini et al. 1954). When back in St. Louis she established a tissue culture facility which was necessary to continue this line of investigation.
Fig. 10

Influence of sarcoma fragments on embryonic sympathetic ganglia of the chicken in vitro. Left (3): control with fragments of chicken heart (C). Right (4): small sarcoma fragments placed at a distance of 1–2 mm from the ganglia. Within 24 h nerve fibres grew radially in all directions forming a dense halo around the ganglion. Reprinted by permission from the American Association for Cancer Research: Levi-Montalcini R, Meyer H, Hamburger V, In vitro experiments on the effects of mouse sarcomas 180 and 37 on the spinal and sympathetic ganglia of the chick embryo. Cancer Res 1954, 14:49-57

10 Stanley Cohen, a new entry

Hamburger and Rita realized that to identify the nature of the newly discovered growth-promoting factor, it would be wise to ask for help from a biochemist. The choice was Stanley Cohen. He was born in Brooklyn in 1922. His parents were both Russian Jewish emigrants who came to America in the early 1900s. In his recollections of his life he writes: “Growing up in the streets of Brooklyn, I remember being interested in how things worked: taking apart an old telephone and the gears of my new 4-speed bicycle was most enjoyable. As a biology major at Brooklyn College in 1945, I was fascinated by embryology. How does an egg turn into a chicken or a frog or a person? My only insight into the problem was the thought that it was necessary to understand the chemical reactions inside the egg and embryo and not simply observe biological structures” (Cohen 2008). He continued his education as a PhD student in the Department of Biochemistry of the University of Michigan under Howard B. Lewis. Then he moved to the Department of Pediatrics at the University of Colorado and in 1952, with a fellowship of the American Cancer Society, he went to the Department of Radiology of the Washington University to work under the guidance of Martin Kamen. As Cohen (2008) recalls: “Upon completion of my fellowship, Dr. Kamen recommended me for a position in the Zoology Department of Washington University in the laboratory of Viktor Hamburger and Rita Levi-Montalcini. This move was critical in determining the direction of my research for the next 40-some odd years: the isolation, structure, and function of the first of the ‘growth factors’, nerve growth factor (NGF) and epidermal growth factor (EGF). […] Neither Dr. Hamburger nor Dr. Levi-Montalcini were biochemists so they went to several of Washington University’s departments looking for a biochemist ‘mad’ enough to study this problem. […] It was a good combination; I knew very little about neuroembryology, and they knew very little about biochemistry so we interacted but never argued.

Hamburger informed Rita, who was still in Rio. Meanwhile, he obtained additional funds from the Rockefeller Foundation to support the new project. Rita answered that Cohen seemed to be the right person. Later she would write (Levi-Montalcini 1988): “I have often asked myself what lucky star caused our paths to cross.

When Rita was back in St. Louis she started collaborating with Cohen with the aim of identifying the mysterious molecule. In a short time they obtained interesting results that they published immediately. Cell-free homogenates of the tumor developed on embryos reproduced in cultures of sympathetic ganglia the effects observed in the experiments performed in Rio. From this homogenate Cohen could isolate a molecule that appeared to have the characteristics of a nucleoprotein, i.e., made by a protein bound to nucleic acids (RNA, DNA). This still unknown substance was named nerve growth-promoting factor. It marked the beginning of the story of the nerve growth factor or NGF (Cohen et al. 1954).

Cohen, at a daily seminar, where he was presenting his results, asked Arthur Kornberg, a future Nobel Laureate, for advice on how to be sure that the factor isolated from the sarcomas was a nucleoprotein and not a simple protein, and if it were a nucleoprotein, how to see which of the two components was the active one. Kornberg suggested using a snake venom which was known to be rich in an enzyme able to destroy the nucleic acid component. If the fluid was still effective after the treatment, the protein would have been responsible for the action. This proved to be the case. The outcome of the new experiment was successful. After some hours Rita observed a rich halo emerging from the sympathetic ganglia in culture. In addition, now, a remarkable serendipitous discovery had been made. In a control experiment, where only the venom—but not the tumor—was applied to the culture, a similar halo appeared. Thus, the venom contained the same or a similar nerve growth-promoting factor. The most important aspect of this discovery was that: “The biological effects of the venom factor, tested in tissue culture, are very similar to the effects produced by a factor obtained from sarcoma 180. However, our most purified venom preparations have a specific activity (on a protein basis) approximately 1,000 times as high as our purest tumor fraction.” It was Hamburger who presented the results to the National Academy meeting, although he did not appear as an author on the published paper (Cohen and Levi-Montalcini 1956).

Rita recalled that Cohen had an interesting idea. If the protein was present in the snake venom it might be present also in the homologous salivary gland of mice. If so, one would be able to extract a much larger amount of the growth-promoting factor and in a much easier way. The experiment was successful and showed that it is present in large amounts in the submaxillary gland of adult male mice (Cohen 1960). Therefore, the salivary glands of the mouse, especially the male, provided a rich and easily available source of the protein, facilitating subsequent research. Thus, it was possible to isolate a purified form of the protein and to obtain a specific antibody. In addition, Cohen (1960) showed that following a daily subcutaneous injection of the growth factor there was a marked increase in protein, RNA and DNA content of the sympathetic chain ganglia, whereas the injection of an antiserum to the same growth factor resulted in a total destruction of them, not only in the mouse, but also in other mammals. In the same issue of the journal Rita described the impact of the antiserum at cellular level (Levi-Montalcini and Booker 1960a, 1960b). As she commented later, the entire chain of the paravertebral sympathetic ganglia was so small as to appear as a thin string under a magnifying lens and more than 95% of the neurons had disappeared. It was on July 21, 1959, just before Cohen’s departure, when Rita showed him the images under the microscope. In this last collaborative work Rita and Cohen revealed the essential role of NGF in the process of differentiation and in the survival of the neurons and this was another milestone in the NGF history. As commented later by Cattaneo (2013), this was the first phenotypic knockout obtained with antibodies.

On December 1958 Hamburger announced that budget restrictions did not allow him to offer Cohen a position in the Department. Therefore, Cohen moved to the Department of Biochemistry of Vanderbilt University, Tennessee. This marked the end of a short but most productive and happy collaboration. Rita mentions a sentence pronounced by Cohen: “Rita, you and I are good, but together we are wonderful.” In March 1959 Hamburger announced that Rita had been appointed Full Professor in his Department.

In the following years Cohen demonstrated that those mice injected with the factor isolated from the mouse salivary glands had a faster development of eye lid opening and of tooth eruption. This observation was the beginning of a new story developed by Cohen, a story that culminated with the discovery of another growth factor, named epidermal growth factor or EGF (Cohen 1962, 2008).

11 Back to Italy

In 1960, at the time when Cohen left St. Louis, the basic investigations on NGF had been done with great success. Rita realized it was necessary to concentrate her efforts on identifying the structure of the new molecule and the mechanisms of its action both during and after the developmental stage. She was worried that she could not continue the collaboration with Cohen and she felt that collaboration with a biochemist would still be important. She asked a young Italian physician, Pietro Angeletti, an expert biochemist working at the Medical School of Washington University, if he had any possible interest in joining her for the investigation on the NGF and he accepted.

Turin and Giuseppe Levi are now very distant in time, but Rita never meant to stay out of Italy permanently. She was very attached to her family and she was keen to share her life with her sister Paola. In addition, she had in mind to set up a new laboratory in Italy. Therefore, she decided to ask Hamburger for a 3-month leave every year to spend in her native country. Hamburger met her request with a proper teaching schedule, while Pietro Angeletti was in charge of and responsible for the research laboratory.

Satisfied with the agreement, in 1961 Rita went to Washington to ask for financial support for her research activity in Italy and she was successful. She decided not to return to Turin, but she felt that Rome was a more attractive city. In 1962 she had a lab at a Health Institute (Istituto Superiore di Sanità). In 1969 the centre was upgraded to the Laboratory of Cell Biology of the Italian National Research Council. The project was challenging. For 30 years, the investigations on the growth-promoting molecule had been a kind of private hunting reserve of Rita and the three main collaborators: Giuseppe Levi, Viktor Hamburger and Stanley Cohen. Now NGF had an international passport and the number of publications abroad was much more numerous than in Italy. Ralph Bradshaw, for instance, became one of the most deeply involved scientists in the United States addressing the issue of the identification of the molecular composition of NGF (see Shooter 2001). Ruth Hogue-Angeletti and Bradshaw of Washington University identified the aminoacid sequence of NGF, which was made up of two identical subunits consisting of 118 aminoacids (Hogue-Angeletti and Bradshaw 1971; Hogue-Angeletti et al. 1973a, 1973b), and in 1983 the gene coding for NGF was also identified (Scott et al. 1983).

Rita started to travel back and forth between Rome and St. Louis. On May 12, 1983, Nature (303:109–128) published a supplement Science in Italy compiled by Robert Walgate, Nature’s chief European correspondent, with the title Can order spring from chaos? In her book (Levi-Montalcini 1988) Rita mentions this report to highlight the remarkable differences in the work conditions between the United States and Italy. However, she continued with a series of excellent collaborators in addition to Pietro Angeletti: Pietro Calissano, Luigi Aloe and later Antonino Cattaneo together with a wide team of young investigators. A series of investigations was set up to establish several molecular mechanisms of action of NGF (Angeletti et al. 1968; Levi-Montalcini et al. 1974).

At a Confindustria conference held in Cernobbio in 2001, Rita launched the proposal to create a European Brain Research Institute (EBRI). The Mayor of Turin, Sergio Chiamparino, offered full space in a new infrastructure with all possible support to the initiative, as a kind of repatriation of the lady who honoured Turin in the world. Meanwhile, another offer came from Luigi Amadio, President of the Santa Lucia Foundation, with one of the best Italian hospitals specializing in nervous disorders and Rita chose to stay in Rome. Thus, in 2004 Amadio created in a new premise the European Centre for Brain Research (Centro europeo di ricerche sul cervello, CERC). He moved his neuroscience laboratories, previously hosted in the hospital, and made an agreement to host two National Research Centres, one of them being the one previously directed by Rita. Amadio offered free space to host EBRI and an environment rich in scientists and equipment. However, several problems soon created conflicts that led Rita, as President of EBRI, to ask, and to obtain on February 10, 2010, for a governmental Commissioner to replace the Board of Directors and the Scientific Council which contained three Nobel Laureates. The news aroused amazement and criticism from the national and international scientific community.

12 The Nobel Prize award

In 1986 Rita (Fig. 11) and Cohen received the highest scientific recognition for their outstanding achievements. The Nobel Committee judged the topic of the two growth factors, NGF and EGF, to be the target of the Prize. The importance of the discovery is evident by the fact that it signalled a new era in the study of the brain. NGF was the first of a set of molecules able to modulate the functions of the brain and its cells in health and disease. It took 30 years before other molecules, which are now collectively called neurotrophins, were discovered.
Fig. 11

Nobel Prize award, Stockholm 1986

Among the experts there was a discussion as to why Hamburger was not included in this prestigious award. From what I reported in this long story it is clear that he had a critical role in the different stages leading to the discovery of the growth factors. In fact, Rita said in her Nobel lecture (Levi-Montalcini 1986): “I dedicate this article to Viktor Hamburger, who promoted and took part in this search, and to whom I am forever indebted for invaluable suggestions and generosity. Without him, the Nerve Growth Factor would never have come to our attention.” Rita and Cohen met once more on April 22, 2009, on the occasion of an international meeting held in Rome to celebrate Rita’s 100th birthday (Figs. 12, 13, 14).
Fig. 12

Birthday meeting in Rome, April 22, 2009. Rita and Stanley Cohen.

Courtesy of EBRI

Fig. 13

Birthday meeting in Rome, April 22, 2009. First row from left: Aaron Ciechanover, Torsten Wiesel, Stanley Cohen and Giuliano Amato. Above from left: Moses Chao, Ezio Giacobini, Elena Cattaneo, Anders Björklund and James Fawcett.

Courtesy of EBRI

Fig. 14

Birthday meeting in Rome, April 22, 2009. Rita between Piera Levi-Montalcini and the Mayor of the city Giovanni Alemanno.

Courtesy of EBRI

13 Social and political commitment and final years

In the 1970s Rita supported the campaign for a law in favour of abortion to prevent women having to migrate abroad for such a purpose. Both Rita and Levi were eminent members of the Academy and Rita was very active and influential to promote cultural activities. In addition, she invested a significant fraction of the Nobel Prize in the creation of the Fondazione Levi to be run by the Academy.

In 1992, with her sister Paola, she established the Rita Levi-Montalcini Foundation in memory of their father with the aim of helping young African women to gain a leading role in the scientific and social life of their country through the awarding of scholarships (project called A boarding school for Tuareg girls).

In 2001 Rita was appointed Senator for life and she was regularly present at the Senate sessions. During the Prodi government (2006–2008) she voted always in favour of the Government and in some cases her vote was crucial to avoid the Government’s fall. From 2006 to 2008 she was Honorary President of the National Committee for Bioethics. In 2010 the Minister of Education, University and Research Mariastella Gelmini launched the Montalcini Project for the return of young researchers to Italy.

On December 30, 2012, around lunchtime at her home in Rome that she had shared with her sister Paola until the year 2000, she took a nap in an armchair and silently left us all. She left a huge crowd of Italian and foreign admirers: pupils, collaborators, outstanding personalities and ordinary people. The coffin was exhibited in Rome and visited by academic and political authorities. At her funeral in Turin on January 2, 2013, in addition to local authorities, about 5–7000 people took part. Her ashes are now in the family tomb designed by Gino, her architect brother.

Notes

Acknowledgements

I would like to thank Prof. Germana Pareti and Dr. Robin Harvey for illuminating discussions, and Maria Romanazzo (Fregi e Majuscole, Turin) not only for copy-editing, but especially for the organization of the whole architecture of text and illustrations. A special mention to Piera Levi-Montalcini for precious information and for help in collecting illustrations. This article is dedicated to my father, Gerolamo Strata, MD, who was sentenced to prison in Savona in the late 30s and then confined to Atripalda (Avellino) for criticism of the fascist regime.

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Copyright information

© Accademia Nazionale dei Lincei 2018

Authors and Affiliations

  1. 1.Department of NeuroscienceUniversity of TurinTurinItaly

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