1999 : by discovering the gene responsible for the dominant form of the Emery-Dreifuss muscular dystrophy, Gisèle Bonne and colleagues describe the founding member of what will later become a new clinical entity : laminopathies. Only three years later, Nicolas Lévy and his colleagues find that mutations in the very same LMNA gene are responsible for Charcot-Marie-Tooth disease, an inherited neurological disorder characterised by the loss of touch sensation as well as the ability to walk. The team further solve a century-old puzzle by demonstrating in 2003 the implication of the LMNA gene in Progeria, a rare uncurable disorder that results in premature and accelerated aging. Children suffering from this systemic disease present with a wide range of symptoms (lipodystrophy, alopecia, osteoarthritis, osteoporosis, vascular disorders while the cognitive functions remain intact) and die early on (at an average of 13 years), usually from a heart attack or a stroke.
« Progeria was known since the end of the 19th century and yet by the early 21st century, scientist had no clue of the gene that caused this disease», reminds Nicolas Lévy. « Shortly after finding mutations in the LMNA gene among patients suffering from Charcot-Marie-Tooth disease, we noticed that certain patients who carried other mutations in the gene presented with signs of accelerated aging. That’s when we found defects in this gene among Progeria patients ».
Progerin, a toxic variant of lamin A
Located on chromosome 1, the LMNA gene encodes two proteins belonging to the family of intermediate filaments : lamins A and C (every gene can encode several proteins, or isoforms, that are produced by a mechanism called alternative splicing). Once synthesized, lamin A undergoes a series of maturation steps before becoming fully functional. These include the attachement of a farnesyl group to a specific region of the protein (a step called prenylation), after which it enters the nucleus and the farnesyl group is cleaved off. The proteins then combine with one another and with lamins C and B (the latter being encoded by a different gene) to integrate the nuclear matrix and cover the inner surface of the nuclear envelope, thereby forming the nuclear lamina. This structure ensures the dynamic movement of chromosomes and controls the transport of molecules accross the nuclear pores.
In progeria cells, the mutated LMNA gene produces a truncated version of lamin A that bears a deletion of 50 amino acids and is called progerin. The mutant protein remains attached to its farnesyl group, which produces aberrant interactions with other lamins as well as other proteins of the nuclear matrix and lamina. These structures become disorganised causing a series of dysfunctions in the cell. « The progressive accumulation of progerin has several toxic effects, including abnormal shaping of the nucleus and modifications in chromatin structure. As a result, several nuclear functions are disturbed (DNA replication and repair, transcription, RNA splicing, protein transport accross the nuclear pores…) leading to premature aging and cell death » explains Nicolas Lévy. « Given that progerin is ubiquitous, these defects concern almost every differentiated cell of the body, which explains the diversity and severity of clinical signs found in progeria patients ».
The laboratory since works towards further understanding laminopathies as well as other diseases that result from defects in proteins of the nuclear envelope. To date, over 270 mutations in the LMNA gene have been identified, and the number of disorders linked to these mutations is constantly growing. It now includes several porgeroïd syndromes, lipodystrophies, Limb-girdle muscular dystrophy type 1B, dilated cardiomyopathy with cardiac conduction disturbances, in addition to the previously known Emery-Dreifuss muscular dystrophy, Charcot-Marie-Tooth disease and Progeria.
The team’s efforts have also opened the door to potential therapeutic solutions for the treatment of Progeria. Two strategies are currently being developed by the team: the first aims to reduce the toxicity of progerin, while the second aims to limit the protein overflow.
Reducing the toxicity of progerin
The scientists from the AgiPreC department used two existing farnesyl transferase inhibitors to block the prenylation of progerin: Prevastatine (a statin used to lower cholesterol and triglycerides in the blood) and Zoledronate (a drug used to treat osteoporosis as well as pain from bone metastases). They first showed that the combination of the two had beneficial effects in vitro, before demonstrating in 2008 the potential of the bitherapy on the first mouse model of Progeria (a model in which a toxic prenylated prelamin A accumulates in the cells).
« In collaboration with the laboratory of Carlos Lopez−Otin at University of Oviedo, we demonstrated that a treatment based on the association of the two farnesyl transferase inhibitors significantly improved bone density and life expectancy of affected mice (173 days in average) » explains Annachiara De Sandre-Giovannoli.
Based on these pre-clinical results, the team of Nicolas Lévy conducted a clinical trial with the support of the AFM (French Association against Myopathies) and of the Ministry of Health (under the PHRC National Program) between 2008 and 2013. The trial includes 12 european patients from the Timone hospital in Marseille (monocentric, phase II, longitudinal, prospective, open-label, non-randomized clinical trial).
« The treatment resulted in weight recovery, improved bone metabolism and lower risk of cardiovascular events» discloses Annachiara De Sandre-Giovannoli. For the first time, the scientists were able to slow the progression of the disease and improve quality of life of the patients. « But unfortunately, the effects were only temporary, so we are now in search of more efficient therapeutic approaches » concludes Annachiara De Sandre-Giovannoli.
To refine their strategy, the scientists from AgiPreC and Carlos Lopez-Otin’ s laboratory first decided to build a mouse model that better recapitulated the defects seen in Progeria patients. They introduced a mutation in the mouse Lmna gene that is equivalent to the one they had previously identified in patients, generating only one year later what is now considered to be the best Progeria mouse model available. These animals not only produce progerin by the same mechanism that young patients do, but also present with the same clinical signs, and their life expectancy is of 101 days in average (as compared to two years for a healthy mouse).
Limiting the progerin overflow
The second therapeutic route undertaken by the department consists on lowering the amount of the toxic protein in the cell. To investigate how this goal could be achieved, the scientists turned themselves to neurons. Interestingly, although this systemic disease affects a number of organs in the body, it is not associated with cognitive impairment, suggesting that a mechanism is in place in neurons that protects them from premature aging.
The scientists established a collaboration with the team of Xavier Nissan at the I-Stem Institute, directed by Marc Peschanski, and together, they demonstrated that neurons actually express a micro-RNA that blocks a cryptic splicing site responsible for the production of progerin. This micro-RNA, now called miR-9, is overexpressed in neurons where it limits the synthesis of lamin A, and hence of progerin. Importantly, in vitro expression of miR-9 in cells derived from Progeria patients reduces the amounts of progerin and restores nuclear morphology.
« To mimic the effect of miR-9, we designed antisense vivo-morpholino oligos (short RNA molecules capped with a polymer that ensures their delivery to cells) to either block the synthesis of progerin or promote the production of normal lamin A », says Nicolas Lévy. « Upon treatment with these oligos, progeria mice displayed a significant increase in life expectancy, reaching 190 days for some of the animals ».
Building on these positive results, the researchers at AgiPreC are now testing different vectors and methods of administration to improve efficacy and safety of this procedure. In parallel, they continue their hunt for new molecules that could either limit the production of progerin or increase its degradation.
Beyond premature aging: cancer and male infertility
The department is hence looking for new therapeutic compounds, a search undertaken by combining the use of iPS cells (induced pluripotent stem cells) with high-throughput phenotypic screens (where individual compounds from a molecule collection are tested for their ability to modify one or more cellular traits). In collaboration with Xavier Nissan’s laboratory, the department has recently demonstrated that two compounds that are derived from vitamin A (ATRA and 13-cis RA) can inhibit progerin synthesis and limit aging in iPS cells. They have also found that metformin, which is normally used to treat diabetes, reduces the production of progerin by 50% in differentiated iPS cells (differentiated into osteocytes, keratinocytes and vascular endothelial cells).
Studying pathological forms of nuclear proteins is also important for understanding not only premature aging and the links between atypical progeroïd syndromes and lipodystrophies, but also the mechanisms at work during “normal” aging. In addition, by studying the proteins that physically interact with lamins (signalling proteins, transcription factors, microfilaments, histones…), as well as protein prenylation defects, the researchers of the AgiPreC department hope to decipher mechanisms responsible for other diseases such as cancer and male infertility.
« We are interested in studying the RAS/MAPK signalling pathway, a pathway that is involved in controlling cell proliferation, survival and mobility, and whose defects are often responsible for the development of cancers », describes Sylviane Olschwang. « Several genes mutated in rasopathies, a group of progeroïd syndromes caused by mutations in the RAS/MAPK pathway, actually encode proteins that are prenylated ».
The department is also interested in understanding the dynamics of the nuclear lamina during spermiogenesis, the final stage of spermatogenesis in which spermatids mature into spermatozoa. « Our aim is to understand the spatio-temporal organisation of the nuclear lamina while the cell undergoes the profound structural rearrangements required to form motile spermatozoa », explains Michael Mitchell. « our working hypothesis is that defects in lamins or in proteins they are associated with might cause infertility ».
While therapeutic solutions to treat progeria patients are on the way, researchers and clinicians at the AgiPreC department have made major contributions to the diagnostics of progeroïd syndromes and have enriched patient registries with large amounts of molecular and epidemiological data. In parallel, the laboratory of Anaïs Baudot is developping systems biology approaches to identify new diagnostic or therapeutic markers, by combining data from patient samples, data from omics approaches and bioinformatics.
