Interview with the Father of Collagen VI: Paolo Bonaldo, an excellent professor of Cell Biology at the University of Padua.
Dear Paolo, we have known each other for many years. Fate has fortuitously made us neighbors in the province of Treviso, and thanks to a conference you held 25 years ago that my aunt attended, my family suspected I had something to do with the collagen VI deficiency pathology.
Tell us, firstly, about your academic and educational journey.
As a child, I was fascinated by the books in my home encyclopedia. Attracted by science and nature, I enrolled in biology in 1980 and when I started my thesis, I was involved in the workgroup of Prof. immunologist Alfonso Colombatti. Interested in the innovative technique of monoclonal antibodies, he assigned me the task of purifying various collagens from the placenta, an organ very rich in extracellular matrix.
In 1984 for my thesis, I was tasked with collecting placentas from women in labor at the hospital and bringing them to the lab. The procedure for isolating the proteins of the extracellular matrix was very complex, starting from mincing the placentas and then with many different purification and extraction steps that required long working times in a cold room. Among the different collagens, type I collagen is the most abundant, found in bones and skin, it is the famous collagen of beauty creams. Type II collagen is found in cartilage. Then there are collagens III, IV, and V… And finally, there was a mysterious collagen, which had not yet been well defined and would later become collagen VI.
In 1985, Prof. Colombatti was awarded a position as director at the Oncological Center of Aviano (PN) and I decided to follow him, focusing on studying the various collagens in his Experimental Oncology department. At that time, collagen VI did not yet have a name.
Collagens have a very characteristic structure called a “triple helix” consisting of three chains that wrap around like a rope: they are what form the collagen fibers in the matrix. Unlike all other collagens, type VI collagen has a very short triple helix flanked by much wider regions that form structures folded back on themselves. The purification procedures used at the time for collagens were based on the use of pepsin, an enzyme that degrades other proteins but does not affect the triple helix structure present in the various collagens. For this reason, collagen VI was initially considered a small protein present in low quantities, almost a degradation product.
After graduation, thanks to the use of monoclonal antibodies, we were able to study collagen VI in cells and, for the first time, understand that it was much larger and more abundant than we thought. Until the early ’90s, we studied the different characteristics of collagen VI, discovering that it has a particularly elaborate and complex intracellular synthesis process, based on the assembly of three different chains followed by a series of subsequent steps up to its secretion and deposition in the extracellular matrix.
In 1990, I decided to participate in a researcher competition and thus moved to the University of Padua, but the situation was difficult due to a lack of both funds and adequate facilities. In 1993, I decided to go to Germany to the Max Planck Institute with my whole family, my beautiful wife Orsolina, and my two very young children, Stefano and Cinzia. I chose a department where at the time a pioneering and highly innovative technique for studying the functions of genes in the whole organism was being developed. There I learned the production of knockout mice and understood how to inactivate a gene that produces a certain protein of interest. In 1995 I returned to Italy where I immediately dedicated myself to applying these innovative methods to study the functions of collagen VI in the entire organism. After more than a year of work, in 1996, we managed to reach the final goal, with the generation of knockout mice lacking collagen VI.
The emotion was strong because we did not know what phenotype these mice would have. Even though they seemed completely normal at first glance, we soon realized that these mice had muscle weakness because when lifted by the tail they could not turn around and kept their legs contracted. This led us to deduce that mice lacking collagen VI were affected by a congenital muscle pathology.
At that time there were a lot of orphan dystrophies for which the mutated gene responsible was not known.
A question to interrupt you. But until that time had you never dealt with Telethon?
I started dealing with Telethon thanks to the knockout mice in 1997 and indeed I obtained my first funding from Telethon.
In those years, patients with collagen VI mutation had not yet been identified. The first dystrophy for which the gene was mapped is the well-known Duchenne dystrophy, when towards the end of the ’80s it was discovered that the mutated gene was for a previously unknown protein and that from the disease was named dystrophin. Among the different forms of muscular dystrophies was the congenital muscular dystrophy of Ullrich, described by the German doctor Ullrich in the first half of the 900, and Bethlem myopathy, described by Dutch doctors in the ’70s.
In 1996 in the Netherlands, the team of geneticists, which included Dr. Bethlem now retired, searched for collagen VI mutations and found them in their patients, publishing the work in Nature Genetics. In 2001, neurologist Dr. Enrico Bertini of Rome showed that Ullrich’s muscular dystrophy is also due to a collagen VI mutation. It thus emerged that different mutations in the three genes coding for the chains of collagen VI cause different muscular pathologies, with a very variable and different picture of the severity and progression of the disease. This is due to the fact that in Ullrich there is a very marked deficit of collagen VI, while instead in Bethlem the deficit is partial.
Can you tell me more about Miosclerosis?
The identification of muscle alterations in our collagen VI-lacking mice had led us by the end of the ’90s to start a very intense and fruitful collaboration with Dr. Luciano Merlini, a neurologist clinician at the Rizzoli hospitals in Bologna and a great expert on these diseases. With Dr. Merlini and his collaborator Patrizia Sabatelli, we had started to study not only the muscles of the knockout mice but also biopsies from patients with a possible deficit of collagen VI. Despite the heterogeneity of the clinical picture, quite different between Ullrich’s dystrophy and Bethlem’s myopathy, and despite the different severity of muscle weakness, in both patients present contractures and various alterations to the joints, one of the most distinctive characteristics of these pathologies. The long experience of Dr. Merlini with various patients affected by neuromuscular diseases led him to identify some with a different picture from both Ullrich and Bethlem and where the most marked symptom was the presence of very extensive contractures and strong muscle stiffness. It was precisely these that led us to search for mutations in the genes for collagen VI, which were identified thanks to a collaboration with the team of geneticists from the University of Ferrara. Miosclerosis, therefore, as the name suggests, presents a clinical picture characterized by stiffness and contractures, and seems much rarer than either Bethlem or Ullrich.
What was happening in the United States at the same time?
Nothing, no one had dealt with it before the 2000s. Before Dr. Bertini identified the Dystrophy of Ullrich in the mutation of collagen VI, there were only a few studies on Bethlem myopathy by a few clinicians, including some Italians. Going back to what I was saying earlier, when in 1996 we discovered that our mice had muscle weakness and the Dutch group identified the mutations in the collagen VI genes in patients with Bethlem myopathy, searching the scientific literature I realize that there were indeed some Italian clinicians who were dealing with this disease, including a neurologist named Luciano Merlini. In 1999 at the scientific congress of Telethon, I meet Patrizia Sabatelli and ask her if she wants to study the muscles of our mice under the electron microscope. I ask Patrizia if she introduces me to this Dr. Merlini, she immediately takes me to him who when he sees me sizes me up from head to toe, starting to ask me a lot of questions about these mice, very curious and interested. Since then we have become great friends, and an intense and very constructive collaboration has begun that has lasted for more than two decades.
From the studies initiated with Sabatelli, analyzing the collagen VI knockout mice under the electron microscope, emerges an almost incredible huge surprise, which then led to many subsequent developments also for studies on patients. In fact, even though the microscopic images of the muscles of the mice do not show obvious alterations to the contractile apparatus, the muscle cells present numerous areas with a kind of “holes” that we immediately understood to be dilated and completely altered mitochondria.
Mitochondria are a bit like the power stations of cells, because they produce (in the form of a molecule called ATP) the energy necessary for many cellular processes, and obviously in muscle cells, they are fundamental to provide the energy necessary for the contraction of all the different muscles. Fortunately, the Father of Mitochondria, Professor Paolo Bernardi, is on the floor below the building where I work. So I go to my friend Paolo (my namesake and often even confused with me, given the common initials) and ask him if he thinks it is possible to study the functionality of the mitochondria in the muscles of mice lacking collagen VI. From there we begin a series of very exciting and innovative studies that lead us to a series of milestones, identifying a specific mitochondrial defect not only in mice but also in the cells of Bethlem and Ullrich patients, the results of which were published in 2003 in the journal Nature Genetics and in the following years in various other international scientific journals.
Thanks to the support of Telethon, we have therefore built a collaborative network made of complementary skills and areas, which in addition to me involved also Merlini, Bernardi, and other researchers, for a series of studies all Italian and carried out entirely in Italy. Under the supervision of Dr. Merlini, we conducted the first pilot clinical trial in Ullrich/Bethlem patients, based on the use of cyclosporine A to reactivate mitochondrial dysfunction, which was then continued by Bernardi with the study of non-immunosuppressive derivatives of cyclosporine. My team, on the other hand, focused on the connection between the deficit of collagen VI and muscle cell dysfunctions, in order to clarify the molecular mechanisms underlying the alterations that we had identified in the muscle cells of mice and patients. This led us to another completely unexpected surprise, identifying a key role of the autophagy mechanism in the onset of alterations to muscle cells, with work published in 2010 in the journal Nature Medicine. The interesting aspect of these results was that a deficit of autophagy, that is, the ‘cleaning’ process of muscle cells, is present both in knockout mice and in patients, and such a deficit is reversible and can be recovered using various strategies aimed at reactivating autophagy, leading to a recovery of structure and strength in mice lacking collagen VI.
This led to a second pilot clinical trial in Ullrich/Bethlem patients, always supervised by Merlini, based on a nutritional approach to reactivate autophagy, which demonstrated the benefits and efficacy of reactivating autophagy, and whose results were published in 2016.
So the connection between mitochondrial dysfunction and collagen VI deficit?
It is not yet entirely clear. It is like a puzzle, made up of many small pieces to identify step by step, and on which we are actively working.
Collagen VI is in the extracellular matrix outside the cells, important for signaling to the cells a series of important effects because the cell covering membrane contains proteins that function as specific receptors for various extracellular molecules. This connection is very complex because collagen VI has the function of regulating more cellular processes, and we still miss several pieces of this puzzle.
In the meantime, as a full professor of Cell Biology for many years now, I managed to build a nice research team, made up of several very motivated and very passionate young people. It is thanks to them that so many results have been obtained, as these results can only be achieved thanks to a close-knit and well-organized group, in which each of the young (and less young…) plays an important role.
What is the work completed or goal achieved to date that you feel most satisfied with?
The first naturally, dating back 26 years ago, in 1998, when we published the study that had led us to obtain the knockout mice. A milestone that was only the beginning of a long story that continues even today. That first milestone had required almost two years of intense work, weekends included, and served for everything that followed after.
Now tell us about spermidine.
Spermidine is not a drug, but a so-called ‘nutraceutical’ (a definition that identifies a natural product, unlike ‘pharmaceutical’) and is part of the polyamines. It is therefore a natural substance, present in various plants and also produced in our bodies. For example, there is a lot of spermidine in some foods, such as soy, wheat germ, and mushrooms, and it is used as a dietary supplement. In recent years, several studies have shown that in invertebrate animals spermidine increases longevity, thanks to its effect in promoting the autophagy mechanism. At the dosages at which it is normally taken or used, no negative side effects are reported. There are few studies on humans, some on Parkinson’s for memory loss, one on the elderly to counteract aging and also one on alopecia. Spermidine is being studied in various contexts.
We, through our studies on mice with a collagen VI deficit, had observed that autophagy could be activated through fasting, an obviously acute approach and unthinkable for prolonged treatment. Subsequently, we tested in mice a normocaloric diet but with a lower protein content, obtaining beneficial effects that led to the pilot clinical trial in patients. With the prospect of developing strategies capable of activating autophagy in a physiological way and without possible side effects, we then focused on nutraceutical substances such as spermidine. We studied the effects of spermidine in knockout mice, administered either by injection or orally, trying to identify the most effective dosage and treatment times. Through various studies, we observed that the treatment activates autophagy and improves muscle structure, with an improvement in the structure of muscle cells. In our first studies with spermidine, the overall effect of these treatments on muscle strength was not significant. We therefore conducted other longer-term studies, administering spermidine to mice by mouth at higher concentrations and for longer periods. These new studies have given excellent results, with a marked recovery of the functional capacity of knockout mice, and were published in a recent work in 2023.
We are strongly convinced that although gene therapy is the only one capable of actually normalizing a diseased gene, the identification of the cellular processes affected by the primary genetic deficit is of great relevance not only for the understanding of the mechanisms underlying the onset and progression of the disease but also for the development of effective therapies. Our studies on the cellular and molecular mechanisms downstream of the primary collagen VI genetic defect have allowed us to identify some targets for therapeutic approaches, and suggest that combinatory strategies with different approaches and molecules capable of acting on various targets and processes involved, such as mitochondria and autophagy, may be effective for their rapid transfer in the treatment of these diseases. In this perspective, we are also conducting other studies aimed at identifying the efficacy of a large number of drugs, already in clinical use for different diseases, with the aim of increasing the chance of success for combinatory therapeutic strategies in dystrophies related to collagen VI.
What are the new goals of the Laboratory of Padua?
After the Summit in San Sebastian, organized by the Spanish foundation Noelia, I realized that although today there are several neurologists and clinicians interested in these pathologies, few have the basic competence background of the extracellular matrix and collagen VI that was instead present in the past. The role of our team, I think, is precisely this: to continue to go forward. Despite the results obtained in these years of work, we do not stop: we want to understand more, we want to continue to add pieces to the puzzle, to close the gap between collagen VI, membrane, and intracellular processes regulated by this protein.
As a biologist professor expert in collagen VI of our medical-scientific commission (I don’t know if you knew but by accepting this interview you consented to your nomination in the CMS of the association col6) would you say something about it? Suggestions, etc.
Thank you very much, I accept the nomination with pleasure. See you at the next meeting of the Collagen VI Italy APS Association.
As for suggestions, I would just add to try to make your voice as an association heard more and more, especially by networking more and more with other patient and family associations at an international level.