Interview with the Father of Collagen VI: Paolo Bonaldo, an excellent scientist and professor of Cell Biology at the University of Padova.
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 at the University of Padova in 1980 and when I started my thesis, I was involved in the workgroup of Prof. Alfonso Colombatti. As an immunologist, Alfonso was interested in the then innovative technique of monoclonal antibodies and he assigned me the task of purifying various collagens from 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 these otherwise disposed organs to the lab. The procedure for isolating extracellular matrix proteins was very laborious, starting from mincing the placentas and then carrying out a number of different purification and extraction steps, which required long working times in a cold room. Among the different collagens, type I is the most abundant, found in bones and skin, and it is the well-known collagen of beauty creams. Type II collagen is found in cartilage. Then there are collagens III, IV, and V… And finally, at that time 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 Cancer 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 larger regions that form globular structures. 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 amounts, or even 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 biosynthesis 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 Padova, 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 technology 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 my interaction with the Telethon Foundation in 1997, thanks to our knockout mice and indeed I obtained my first funding from Telethon.
At that time, patients with collagen VI mutations 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 several different forms of muscular dystrophies, there was Ullrich congenital muscular dystrophy, first described by a German physician in the first half of the past century, and Bethlem myopathy, described by some Dutch neurologists in the ’70s.
In 1996, a team of geneticists in the Netherlands searched for collagen VI mutations and found them in Bethlem patients, publishing their work in Nature Genetics. In 2001, neurologist Dr. Enrico Bertini in Rome showed that Ullrich muscular dystrophy is also due to collagen VI mutations. It thus emerged that different mutations in the three genes coding for collagen VI chain cause different muscular disease, with a very variable pattern in the severity and progression of the pathology. Such heterogeneity is largely due to the fact that in Ullrich there is a very marked functional deficit of collagen VI, while instead in Bethlem collagen VI deficit is partial.
Can you tell me more about myosclerosis?
The identification of muscle alterations in our collagen VI knockout mice led us to start by the end of the ’90s a very intense and fruitful collaboration with Dr. Luciano Merlini, a neurologist clinician at the Rizzoli hospitals in Bologna and a major expert on these diseases. With Dr. Merlini and his collaborator Patrizia Sabatelli, we started to study not only the muscles of the knockout mice but also biopsies from patients with a 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 cases patients display contractures and various alterations of the joints, one of the most distinctive features of these pathologies. The deep 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 remarkable 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. Myosclerosis, 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 collagen VI mutations as causative for the Ullrich congenital muscular dystrophy, there was only a limited number of studies on Bethlem myopathy by few clinicians, including some Italians. Going back to what I was saying earlier, when in 1996 we realized that our knockout mice had muscle weakness and the Dutch group identified mutations of collagen VI genes in patients with Bethlem myopathy, by reading the scientific literature I realized that there were indeed some Italian clinicians who were dealing with this disease, including a neurologist unknown to me and named Luciano Merlini. In 1999 at the scientific congress of Telethon, I met Patrizia Sabatelli and asked her if she could study the muscles of our mice by electron microscopy. I also asked Patrizia if she could introduce me to this Dr. Merlini… She immediately took me to him, who when he saw me sized me up from head to toe, starting to ask me a lot of questions about these mice, very curious and interested. Since then, we became great friends and an intense and very constructive collaboration began, which has been going on for more than two decades.
The results of the studies carried out in collaboration with Patrizia, in which she analyzed the muscles of collagen VI knockout mice under the electron microscope, provided a remarkable and huge surprise, which then led to a number of subsequent developments also for studies on patients. In fact, even though the microscopic images of those muscles did not show any overt alteration of the contractile apparatus of muscle cells (or myofibers), they displayed numerous areas with a sort of “holes” spread inside the cell and that we realized to correspond to altered and dilated mitochondria.
Mitochondria are a bit like the power stations of cells, as they produce (in the form of a small molecule called ATP) the energy needed to fuel many cellular processes, and obviously in myofiber mitochondria are fundamental to provide the energy necessary for the contraction of all the different types of muscles. In a lucky coincidence of research, Prof. Paolo Bernardi (who I consider as “the Father of Mitochondria”), had his lab in the floor below mine and in the same building. Driven by the remarkable microscopy findings, I went to my friend Paolo (my namesake and often even confused with me, given the common initials) and asked him if it was possible to study the functionality of mitochondria in muscles of mice lacking collagen VI. Since then, we began a series of very exciting and quite innovative studies that provided some key milestones, identifying a specific mitochondrial defect not only in mice but also in muscle cells of Bethlem and Ullrich patients. The results of those extensive studies 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 were able to build a great collaborative network made of complementary expertise, which in addition to me involved also Merlini, Bernardi and some other researchers, for a number of studies all Italian and carried out entirely in Italy. Under the supervision of Merlini, we carried out 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 displayed by muscle cells of mice and patients. This led us to another completely unexpected surprise, in which we identified a key role of the autophagic process in the onset of muscle cell alterations, wand whose results were published in 2010 in the journal Nature Medicine. The most interesting aspect of these results was that a deficit of autophagy (namely, the dynamic ‘cleaning’ process of cells) is present both in knockout mice and in patients, and that such a deficit is reversible and can be recovered by various strategies aimed at reactivating autophagy, leading to a marked amelioration of muscle structure and strength in mice lacking collagen VI.
This led to a second pilot clinical trial in Ullrich/Bethlem patients, again 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, although we understood some basic aspects. It is like an elaborate puzzle, made up of many small pieces to be added step by step, and on the completion of which we are actively working.
Collagen VI is in the extracellular matrix outside the cells, and the matrix is involved in signaling to the cells a broad number of important function. This happens because the plasma membrane covering each cell contains different types of proteins that function as receptors for various extracellular molecules. This connection is very complex because collagen VI has the capability of regulating a large number of cellular processes, and we still miss several pieces of this puzzle.
In this respect, as a full professor of Cell Biology for many years now, I managed to build a nice research team made up of highly motivated and passionate young scientists. It is thanks to them that so many results could be obtained, as these results can only be achieved thanks to a close-knit and well-organized team, in which each of the young (and less young…) scientist plays an important role.
What is the work completed or goal achieved to date that you feel most satisfied with?
The first one, dating back 26 years ago, in 1998, when we published the study that led us to generate the collagen VI knockout mice. Seen from were we are now, it was a milestone that was only the beginning of a long story that still continues today. That first milestone required almost two years of intense work, weekends included, and served for everything that was following after it.
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 a class of polyamine molecules. It is therefore a natural substance, produced in our bodies and also present in various plants. 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 process within cells. At the dosages at which it is normally taken or used, no negative side effects are reported for spermidine. There are also some studies in humans, some on Parkinson’s for memory loss, one on the elderly to counteract aging and also one on alopecia. Spermidine is being actively studied in various contexts.
Through our studies on collagen VI knockout mice, we found that autophagy could be activated through fasting, an obviously short-term and acute approach completely unthinkable for prolonged treatments. Subsequently, we tested in mice a normocaloric diet with a lower protein content, obtaining several beneficial effects in both muscle structure and function. Such results led to the pilot clinical trial with a controlled low-protein diet 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 our mice, administered either by injection or orally, trying to identify the most effective dosage and treatment times. Through various studies, we observed that spermidine treatment activates autophagy and improves muscle structure in collagen VI knockout mice. In our first studies with spermidine, the overall effect on muscle strength of these treatments was not significant. We therefore carried out further long-term studies in mice, administering spermidine orally at higher concentrations and for longer periods. These subsequent studies provided excellent results, with a marked recovery of the functional ability of knockout mice, and were published in a recent work in 2023.
We are strongly convinced that although gene therapy is the only strategy capable of actually normalizing a diseased gene, the identification of those cellular processes affected by the primary genetic deficit is of great relevance not only for understanding 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 the results strongly support the concept that suggest that combinatory strategies using different approaches and molecules capable of acting on various affected targets and processes, 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 several different diseases, with the aim of increasing the chance of success for combinatorial therapeutic strategies in dystrophies related to collagen VI.
What are the new goals of the laboratory in Padova?
After the Summit in San Sebastian in Spain, organized by the Noelia foundation, I realized that although today there are several neurologists and clinicians interested in these pathologies, few have the extensive basic background on 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 move forward with the basic knowledge on collagen VI and extracellular matrxi. Despite the gratifying results we obtained in three decades of work, we do not stop: we want to understand more, we want to continue to add pieces to the puzzle, with the ultimate goal of closing 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 committee (I don’t know if you knew it, but by accepting this interview you also gave approval for your nomination in the committee of the Col6.it association) would you like to add something? Suggestions, etc.
Thank you very much, needless to say I accept the nomination with much pleasure, and 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 with other patient and family associations at an international level.
Prof. Paolo Bonaldo, bonaldo@bio.unipd.it