Does the blood we thought to know so well contain elements that had been undetectable until now? The answer is yes, according to a team of researchers from Inserm, Université de Montpellier and the Montpellier Cancer Institute (ICM) working at the Montpellier Cancer Research Institute (IRCM), which has revealed the presence of whole functional mitochondria in the blood circulation. These organelles that are responsible for cellular respiration had hitherto only been found outside cells in very specific cases. The team’s findings, published in The FASEB Journal, will deepen our knowledge of physiology and open up new avenues for treatment.
Mitochondria are organelles that are found in the eukaryotic cells. A place of cellular respiration, they are the cells’ “batteries” and play a major role in energy metabolism and intercellular communication. Their particularity is to possess their own genome, transmitted solely by the mother and separate from the DNA contained in the nucleus. The mitochondria can sometimes be observed outside the cells in the form of fragments encapsulated within microvesicles. Under certain very specific conditions the platelets are also capable of releasing intact mitochondria into the extracellular space.
The work of a team led by Inserm researcher Alain R. Thierry at the Montpellier Cancer Research Institute (Inserm/Université de Montpellier/Montpellier Cancer Institute) has now revolutionized knowledge of this organelle by revealing that whole functioning extracellular mitochondria are in fact found in the bloodstream!
The researchers used previous findings which showed that the plasma of a healthy individual contains up to 50,000 times more mitochondrial DNA than nuclear DNA. They hypothesized that for it to be detectable and quantifiable in the blood in this manner, the mitochondrial DNA had to be protected by a structure of sufficient stability. In order to identify such a structure, plasma samples from around 100 individuals were analyzed.
This analysis revealed the presence in the blood circulation of highly stable structures containing whole mitochondrial genomes. Following examination of their size and density, as well as the integrity of their mitochondrial DNA, these structures observed using electron microscopy (up to 3.7 million per ml of plasma) were revealed to be intact and functional mitochondria.
Throughout the seven-year research period, the scientists used as many technical and methodological approaches as possible to validate this presence of circulating extracellular mitochondria in the blood.
“When we consider the sheer number of extracellular mitochondria found in the blood, we have to ask why such a discovery had not been made before, notes Thierry. Our team has built up expertise in the specific and sensitive detection of DNA in the blood, by working on the fragmentation of extracellular DNA derived from the mitochondria in particular”, he adds.
But what is the role of these extracellular mitochondria? The answer to that could be linked to the structure of the mitochondrial DNA, similar to that of bacterial DNA, which gives it the ability to induce immune and inflammatory responses. Based on this observation, the researchers hypothesize that these circulating mitochondria could be implicated in many physiological and/or pathological processes requiring communication between the cells (such as the mechanisms of inflammation). Indeed, recent studies have demonstrated the ability of certain cells to transfer mitochondria between themselves, such as the stem cells with damaged cells. “The extracellular mitochondria could perform various tasks as messenger for the entire body”, specifies Thierry.
In addition to its importance to our knowledge of physiology, this discovery could lead to improvements in the diagnosis, monitoring and treatment of certain diseases. In fact, the research team is now devoting its attention to evaluating the extracellular mitochondria as biomarkers in non-invasive prenatal diagnosis and cancer.
This research is supported by the Montpellier Integrated Cancer Research Site (SIRIC) (Inserm/CNRS/Université de Montpellier/Montpellier Cancer Institute/Montpellier University Hospital/Université Paul Valéry), funded by Inserm, the National Cancer Institute (INCa) and the Directorate General of Health Care Provision (DGOS).
Reconstruction of a pluristratified epidermis using keratinocytes from human embryonic stem cells, hESC. IStem, Génopole d’Evry.Inserm/Baldeschi, Christine
A CEA, INSERM and the University of Paris team, produced in collaboration with I-Stem, the AFM-Téléthon laboratory, and the University of Évry has just published a paper in which it demonstrates the central role of the transcription factor KLF4 in regulating the proliferation of epidermal stem cells and their ability to regenerate this tissue. This study opens perspectives for regenerative skin medicine. It was published on 21 October in Nature Biomedical Engineering.
Human skin completely renews itself every month thanks to the presence of stem cells in the deepest layer, which generate all the upper layers of this tissue. The deciphering of genes that regulate stemness remains an enigma that is only partially resolved, in particular for human skin.
The discoveries of a French research team from the CEA, INSERM and the University of Paris, produced in collaboration with I-Stem, the AFM-Téléthon laboratory, and the University of Évry, opens perspectives for regenerative cutaneous medicine, in particular for the bio-engineering of skin grafts for tissue reconstruction. Massive ex vivo expansion of epidermal cells (called keratinocytes) is needed for the production of grafts. It is performed using a skin sample from the patient that contains adult keratinocytes and a minority population of keratinocyte stem cells. This expansion phase involves a risk: it may be accompanied by a quantitative loss or degradation of stem cells, leading to a loss of regenerative potential.
The results of the paper published in Nature Biomedical Engineering show that reducing the expression of the KLF4 gene during graft preparation promotes rapid expansion of functional stem cells1, without damaging their genomic stability. Keratinocytes expanded under these conditions have an increased long-term regenerative potential in in vitro epidermal reconstruction models and in vivo grafts2. KLF4 is therefore a new molecular target for preserving the functionality of stem cells and making progress in the bio-engineering of skin grafts. These results constitute a proof of concept, which requires additional developments to envisage clinical applications, including the treatment of severe burns and chronic ulcers, and breast reconstruction.
This work has been extended to other types of cells of interest for cutaneous cell therapy. In the future, keratinocytes produced from pluripotent stem cells could be an alternative to adult stem cells in certain reconstructed tissue bio-engineering applications.
One of the difficulties encountered in this area is that fact that the keratinocytes obtained do not have all the functions of adult stem cells. In particular, they are deficient in terms of their proliferation potential. The study has shown that manipulation of KLF4 expression is also suitable for these cells, as reducing its expression in keratinocytes derived from embryonic stem cells (ESC) improves their proliferation capacity and their ability to reconstruct skin.
Skin cells in culture Skin graft obtained by bio-engineering
1.A single functional stem cell is able to regenerate the skin throughout a person’s life. This is due to its very long-term proliferation capacity, its immature nature and its capacity for three-dimensional organisation.
2. Reconstructed human skin xenograft on an animal model
Today, around 1 in 8 couples seek help because they are struggling to conceive. This is probably linked to the fact that couples are starting families later in life than before, or because they are setting aside the taboos linked to infertility and are more willing to seek help. Infertility has therefore become a public health problem, and the scientific community is rallying in response.
Where are we with research into this area, which lies at the heart of current societal problems? What are the prospects for the transfer of such research into clinical practice? Fertility research covers many different areas. The aim of this press kit is not to tackle them exhaustively, but to highlight the sectors in which research is making progress.
When research makes progress, everyone’s health benefits.
Research into combating infertility
The term infertility is used when a couple are unable to conceive a child naturally after 12 months of trying. This term covers cases of total sterility, where there is no hope of natural conception, and subfertility, the majority of cases, in which couples have a reduced – but not zero – chance of achieving a pregnancy.
Cases of infertility are divided into 4 categories based on their cause:
– 30% are female-related;
– 30% are male-related. In men, azoospermia and oligospermia are the two leading causes of infertility identified to date;
– 30% are combined, meaning that they are caused by reduced fertility in both partners;
– 10% are unexplained.
In women, with the exception of mechanical causes affecting the fallopian tubes – when they are impaired or blocked (usually following an infection) – or uterus, endometriosis and abnormal ovulation are the most common causes of infertility.
Causes of abnormal ovulation include polycystic ovary syndrome (which affects around 10% of women around the world), hyperprolactinemia, and primary ovarian insufficiency (which may also be a side effect of chemotherapy).
Current research seeks to both improve understanding of the causes of infertility, and also to study new therapies or management methods that aim to increase the chances of conception.
Improving understanding of the causes
The genetic approach
Many researchers are studying the genetic causes of ovarian insufficiency. Several fertility problems are caused by certain genes not working, or not working properly. One rapidly growing area of research, due in particular to the improvement in high-throughput screening methods, is the study of genetic variants.
The Inserm laboratory led by Nadine Binart, for example, is working on primary ovarian insufficiency (POI), which is characterized by the inability of ovarian follicles to mature or by diminished ovarian reserve. Based on DNA analysis of women with POI, researchers are working to isolate the genes that are involved or altered in their genetic make-up. This approach is helping to improve understanding of the disease, but does not make it possible to provide specific treatment to these women, as sterility becomes definitive once there are no more eggs left in their ovaries. Preventive management can however be introduced if the genetic abnormality is found before the ovarian reserve is entirely depleted – for example, during family testing. This is the role of clinical research, which makes it possible to lessen the impact of these diseases when mutations are identified in affected families, to inform young patients about the risk of losing their eggs over time, and to introduce fertility preservation methods if appropriate.
The hormonal approach: the example of kisspeptin and prolactin
It is well-established that breastfeeding results in increased secretion of prolactin (PRL) by the pituitary gland, inhibiting a woman’s ability to ovulate. This prevents the onset of a new pregnancy. Some diseases also lead to an increase in PRL, including tumors of the pituitary gland from which this hormone is secreted. These cases of hyperprolactinemia, which result in period problems and infertility, are a leading cause of anovulation. In 2011, the Inserm team led by Jacques Young and Nadine Binart revealed the underlying mechanism that blocks ovarian function. Using a mouse model of the disease, the researchers showed that PRL inhibits secretion of a neurohormone called kisspeptin, which is the starting point for the entire hormone cascade responsible for ovarian cyclicity. In a mouse model, administration of kisspeptin made it possible to restore ovarian cyclicity despite the hyperprolactinemia.
This pathophysiological discovery explains the link between infertility and hyperprolactinemia for the first time, and points the way to developing innovative therapies. The basic concept has recently been validated in women, which will make it possible to offer a therapeutic alternative for patients who do not respond to the drugs currently used.
1.2. Preserving fertility: areas of research and latest findings
Specialist “oncofertility” consultations have developed extensively in recent years and should now be an integral part of the care pathway for all young female patients with cancer. Several “fertility preservation” techniques designed to cryopreserve gametes, or preserve reproductive capacity, are now available, and others are currently in development. In France, since 1994, these methods have been included in various pieces of bioethics legislation. Article L.2141 11, modified by law 2011-814 of July 7, 2011, states that “All persons whose fertility is likely to be impaired by their medical care, or whose fertility risks being prematurely impaired, may have their gametes or reproductive tissue collected and preserved with a view to their later use of assisted reproductive technology, or with a view to preserving and restoring their fertility.” Fertility preservation methods are also included in the 2014-2019 Cancer Plan, which stipulates that “all patients must have access to cancer treatments, and innovative treatments in particular.”
Improving gamete preservation
Several techniques for cryopreserving female gametes are currently available. The standard method involves freezing mature eggs or embryos obtained from these eggs. It is not however suitable for prepubescent girls, who need to begin treatment urgently, and can also present problems in patients with hormone-sensitive cancers. Therefore, other techniques, although still considered experimental, may be offered in these situations.
Improving the available methods and developing new strategies is currently a major focus for oncofertility. This is one of the areas of research on which the Inserm team led by Nadine Binard and Charlotte Sonigo is working, in collaboration with Prof. Michael Grynberg.
Using anti-Müllerian hormone
Chemotherapy reduces fertility through a direct toxic effect on the ovaries. Cyclophosphamide, which is commonly used in cancer treatment, causes massive destruction of the germ cells contained in the ovarian follicles. In a mouse model, researchers have recently shown that treatment with anti-Müllerian hormone, which is normally secreted by the ovaries, can limit reduction of follicular reserve during chemotherapy. Use of anti-Müllerian hormone is therefore a promising approach to fertility preservation.
1.3. The role of new technologies: Using artificial intelligence in reproductive research
The store of germ cells contained in the follicles constitutes the ovarian reserve. Assessing the quantity of these germ cells is a common way of providing information on ovarian physiology and of measuring the impact of the environment on the ovaries. The standard method used in mice is time-consuming and tedious. In conjunction with a company specializing in artificial intelligence, Inserm researchers have recently developed an automated artificial intelligence method for follicle counting that uses a deep learning approach. This new tool will be made available to the fertility research scientific community, saving a great deal of time and enabling better reproducibility of data.
Research into combating endometriosis
Endometriosis is a complex disorder characterized by chronic inflammation due to the presence of tissue resembling the uterine lining outside the uterus. This “ectopic uterus” continues to respond to ovarian hormones, which in some women can cause severe pain and sometimes infertility. In response to increased visibility of the disease in the media, notably due to the work of patient organizations, the French health minister has announced an action plan to improve management of endometriosis. In terms of research, there has been a surge in studies of endometriosis over the last 5 years. Around 1,200 articles per year are being produced by researchers around the world, helping to advance understanding of this disorder.
An estimated 1 in 10 women have some form of endometriosis.
The locations of endometriosis lesions vary.
Endometrial cell reflux during periods occurs in 90% of women, but only 10% of them develop disease.
The disease is typically described as having 4 stages, based on the extent and depth of lesions; however, there is no correlation between disease symptoms and severity.
There are 3 forms of endometriosis: superficial peritoneal endometriosis, ovarian endometriosis (or endometrial cyst, or endometrioma), and deep endometriosis.
2.1. Improving understanding of the causes
The epidemiological approach
At present, little is known about the causes of endometriosis, its natural history, and the factors affecting its progression. Epidemiological research plays a crucial role in advancing knowledge in this area. There are only a few large epidemiological cohorts around the world in which these aspects can be explored. The largest cohort for exploring endometriosis risk factors is currently a cohort of 116,430 American female nurses who were between 25 and 42 years old in 1989. The risk factors identified in the literature and confirmed in this cohort include: low birth weight, early menstruation, low body mass index, and short menstrual cycles (under 24 days). However, beyond these factors, little information is available on the causes of the disease, and its natural history is largely unknown. The following table is based on a review of the literature published in August 2018:
*The positive association between smoking and reduced risk of endometriosis may be explained by the antiestrogenic effect of tobacco. This would confirm the therapeutic interest of estrogen blockers, which are available in drug form: far more suitable than cigarettes, whose harmful effects have been widely documented.
In a bid to improve understanding of this disease, several epidemiological studies are being launched in France by the team led by Marina Kvaskoff, Inserm epidemiologist and researcher. These include a recently formed patient cohort dedicated to the study of endometriosis: the ComPaRe-Endometriosis cohort. The study team’s objective is to have enough women in the cohort to obtain robust findings in relation to the many questions that are still unanswered about this disease. In less than 6 months, over 8,000 women have already taken part in the study. The team aims to recruit 15,000 to 20,000 participants, and a broad call for participation has gone out to women with endometriosis or adenomyosis (a form of endometriosis limited to the muscle wall of the uterus) to help speed up research into these disorders simply by completing online questionnaires about their experience of the disease (https://compare.aphp.fr/). The study initially looks to explore the natural history of the disease (change in the symptoms and characteristics of the disease over time), and to identify the factors that determine its progression and result in better response to treatment. This research will also make it possible to describe the circumstances of diagnosis and the patient care pathway, and to assess the impact of the disease on patients’ daily lives.
Endometriosis is also being studied within large French cohorts, such as the CONSTANCES cohort, a prospective study of 200,000 men and women (105,000 women) representative of the French population. Marina Kvaskoff’s team has developed an epidemiological research study to determine the prevalence and incidence of the disease in France, and to explore its risk factors within this cohort. Other studies are currently in development and will be conducted in other cohorts in due course.
The environmental approach
Several epidemiological studies have explored the associations between organochlorine chemicals (solvents, pesticides, insecticides, fungicides, etc.) and endometriosis, but their results have been inconsistent. A French meta-analysis of 17 studies published in February 2019 attempted to draw more robust findings. The risk of developing endometriosis was 1.65 times higher in women exposed to dioxins, 1.70 times higher for those exposed to polychlorinated biphenyls (PCB), and 1.23 times higher for organochlorine pesticides. Although statistically significant, these estimates should be considered with caution due to the significant heterogeneity of the studies and the small estimated effect size. The level of evidence was judged to be “moderate” with a serious risk of bias, supporting the need to conduct further well-designed epidemiological research in order to fill the persistent data gaps.
Using the genetic and epigenetic approach for early detection
Detecting endometriosis in the early stages, before patients experience symptoms, would make it possible to improve patient care. Although the heritability of endometriosis has been estimated at 50%, it is highly complex and clearly highly polygenic. Numerous candidate genes have been studied from this perspective in analyses of disease predisposition. Initial results have shown that there is no gene for endometriosis, but that the existence of genetic variants characteristic of the disease could enable it to be diagnosed and to improve patient care. In 2017, efforts by the international community made it possible to identify a total of 14 variants (located on the genes WNT4, GREB1, ETAA1, IL1A, KDR, ID4, CDKN2B-AS1, VEZT, FN1, CCDC170, SYNE1, FSHB, and in the chromosomal regions 7p15.2 and 7p12.3). These 14 genes are involved in proliferation and the cell cycle, adhesion and the extracellular matrix, andinflammation, which makes sense in relation to endometriosis. However, each of the variants identified explain only a small part of the genetic variation in endometriosis. In future, the combination of high-risk alleles in a patient might provide a probability of being affected that could be used to diagnose patients and categorize them based on endometriosis type and severity.
The existence of specific epigenetic markers for endometriosis could also theoretically be used for early detection, with endometrial cells presenting specific epigenetic abnormalities that modify expression of the main transcription factors. However, it is not known how the interactions between the defective epigenomic cells and mutated epithelial cell genes contribute to the pathogenesis of endometriosis.
The microRNA approach
The full complexity of endometriosis cannot however be understood through genetics alone. Genes only influence phenotype through their expression. This expression is regulated by epigenetic molecular mechanisms. As such, most research focuses on studying the microRNA that could be “markers” for the disease. Several have been identified in patients’ plasma thus far, but with very poor reproducibility from one research team to the next. For example, a study published in 2013 identified just four miRNA (miR-199a, miR122, miR145*, and miR-542-3p) as enough to categorize patients, with very few errors. Confirmation of this article’s findings in independent cohorts has however been slow. One possible explanation for this is the fact that extraction of circulating RNA remains very heterogeneous from one study to the next, perhaps due to the technical tools used in extraction. In future, new, more comprehensive approaches could provide more consistent results.
The cellular approach: oxidative stress
Several studies have shown increased oxidative stress in the serum of women with endometriosis. Oxidative stress is a highly general mechanism that induces and is caused by inflammation. It would seem logical to find changes linked to oxidative stress in the context of a painful disease like endometriosis. In mouse models, treatment with antioxidants (N-acetylcysteine) has been seen to reduce endometrial lesions.
Research led by a team from the Institut Cochin has also identified several genes linked to glutathione metabolism within the gene cascades that are deregulated in endometriosis lesions. Glutathione is a peptide that plays a key role in detoxification of hydrogen peroxide, a central molecule in oxidative stress. Down-regulation, particularly of the GCLM and GCLC genes crucial to glutathione synthesis, could explain increased oxidative stress in endometriosis lesions.
The dysfunctional immune system: a possible approach?
The survival of endometrial cells outside the uterus could be linked to poor function of the immune system causing chronic local inflammation. Studies have shown an increase in some immune cells around endometriosis lesions.
2.2. Treatment: areas of research and latest findings
Changing diagnostic methods: phasing out surgery
Before considering treatment, the first stage is to reduce the diagnosis time for endometriosis, which is currently around 7 to 10 years after onset of the initial symptoms. With this in mind, doctors and researchers are working to develop a diagnostic score, based on a dozen questions, from which doctors will be able to provide a diagnosis with 85-90% reliability. This score may be accompanied if necessary by imaging, which can inform endometriosis diagnosis if carried out and interpreted by trained medical personnel.
Doctors and researchers agree that diagnostic surgery is contraindicated for endometriosis.
The 3 pillars of treatment
Drug therapy, surgery, and assisted reproductive technology (ART) are currently the only 3 methods for treating the symptoms of endometriosis and its potential impact on fertility. In the absence of new treatments, the key is to understand the role played by each component in this therapeutic arsenal, so that they can be used effectively.
Drug therapy is based on blocking ovarian function to bring about artificial menopause via continuous administration of contraceptives. These therapies (the combined pill, estrogen pill, or GnRH agonists) must be personalized and adapted to the needs of the patient. These therapies should be prescribed as the first-line treatment for women who are not seeking to become pregnant, in order to reduce the pain caused by the disease.
For patients who want to conceive, ART and surgery may be considered. ART should be used routinely before all surgical procedures in order to maximize the chance of conceiving a child for couples who want to do so. Surgery must not be used in women who do not want to conceive in whom drug therapy is effective. Endometriosis surgery can be highly invasive and debilitating (removing some parts of the colon, with a high risk of ovarian reserve depletion if ovarian cysts are removed, etc.), and does not prevent the disease from recurring, as it does not treat the cause. Doctors and researchers also agree that women who undergo surgery at a young age have a high risk of their endometriosis lesions recurring, and encounter further difficulties if they decide they want to become pregnant.
All efforts must therefore be made to move away from using surgery as the standard treatment for endometriosis, as has too often been the case in the past.
Some forms of endometriosis – particularly those affecting the ovaries – are now an indication for providing women with access to various fertility preservation techniques.
The role of new technologies: the example of high-intensity focused ultrasound
In Lyon, teams of research clinicians led by Prof. Gil Dubernard (Hospices Civils de Lyon and Inserm unit 1032 LabTAU) have developed an ultrasound-based treatment for bowel endometriosis. When endometriosis infiltrates the rectal wall, it causes debilitating rectal pain that may affect quality of life. After failure of medical treatment, a surgical procedure is often proposed that consists of removing all or part of the rectum and sometimes requires a temporary colostomy (artificial anus).
A phase I clinical trial carried out in 11 patients in 2017 demonstrated that high-intensity focused ultrasound may be a useful alternative to surgery. An ultrasound probe inserted into the rectal passage is able to “desensitize” the lesions within a few minutes. A follow-up trial of 12 patients seeking to confirm these initial results was completed on April 1, 2019. Data analysis is ongoing and will be available shortly.
Meanwhile, in collaboration with the company EDAP TMS (the clinical trial sponsor), the therapeutic ultrasound Inserm laboratory led by Cyril Lafon, LabTAU (Université Claude Bernard Lyon 1/Inserm), is working on optimizing the conditions of ultrasound delivery (insonification) and improving the ergonomics of the probe in order to increase the number of patients eligible for this new treatment.
It is highly likely that this innovative therapy will replace many of the rectal surgeries carried out in this functional disorder that resolves upon menopause.
Millar RP, Sonigo C, Anderson RA, George J, Maione L, Brailly-Tabard S, Chanson P, Binart N, Young J.
Sonigo C, Jankowski S, Yoo O, Trassard O, Bousquet N, Grynberg M, Beau I, Binart N. High-throughput ovarian follicle counting by an innovative deep learning approach. Sci Rep. 2018 Sep 10;8(1):13499. doi: 10.1038/s41598-018-31883-8.
Researchers are now able to edit the genome with precision using the “gene editing scissors” of CRISPR-Cas9, which is a highly promising tool for gene therapy.The technical challenge now is to get this tool into the genome of certain cells.With this in mind, a joint team from Inserm, the CNRS, the Université Claude Bernard Lyon 1, and the École Normale Supérieure de Lyon, working within the International Center for Infectiology Research (CIRI), have developed capsules that allow CRISPR-Cas9 to reach the target DNA: Nanoblades.Described in a recent article in Nature Communications, they open up avenues of research for genome editing in human stem cells.
Since 2012, the scientific community has had access to a revolutionary method for highly precise genome “surgery”: the CRISPR-Cas9 system. These molecular scissors are able to cut DNA at a precise place in a wide variety of cell types. The technique therefore offers significant prospects for research and human health. However, getting these “gene editing scissors” to their target—including the genome of certain stem cells—remains technically challenging.
Tackling this problem has been the focus for research teams from Inserm, the CNRS, the Université Claude Bernard Lyon 1, and the École Normale Supérieure de Lyon, who have developed Nanoblades, particles that enable CRISPR-Cas9 to be delivered into numerous different cells, including human cells.
The scientists had the idea of encapsulating the CRISPR-Cas9 system in structures that strongly resemble viruses as a way to deliver it into target cells, by fusing with the target cell membrane.
In developing Nanoblades, researchers exploited the properties of the retroviral Gag protein, which is able to produce viral particles that have no genome and are therefore non-infectious. The research team fused the Gag protein from a mouse retrovirus with the Cas9 protein—the scissor component of the CRISPR system. This new “fusion” protein is what makes Nanoblades original.
As a result, and unlike classic genome modification techniques, Nanoblades encapsulate a CRISPR/Cas9 complex that is immediately functional rather than delivering a nucleic acid coding for the CRISPR-Cas9 system in the treated cells. “The action of CRISPR-Cas9 on the cells is therefore temporary.It is also more precise and preserves the non-target regions of the genome, which is a particularly important feature in the context of therapeutic applications”, explain the authors.
Représentation schématique d’une particule Nanoblades livrant CRISPR CAS9
Schematic diagram of a Nanoblades particle delivering CRISPR-Cas9
La protéine GAG tapissant l’intérieur des particules rétrovirales
The Gag protein internally lining the retroviral particles
La protéine CAS9, ciseau effecteur du système CRISPR, pouvant cliver l’ADN
The Cas9 protein, the scissor component of the CRISPR system, is able to cleave DNA
L’ARN guide, qui va placer CAS9 sur la région ADN cible. Il a une affinité naturelle pour CAS9
The RNA guides Cas9, then positions it at the target DNA region. It has a natural affinity for Cas9
Les deux enveloppes virales conférant un tropisme large aux particules
The two viral envelopes give the particles a broad tropism
La bicouche lipidique qui entoure la particule
The lipid bilayer surrounding the particle
Finally, researchers used an original combination of two viral envelope proteins on the surface of Nanoblades to enable them to enter a wide range of target cells.
The scientists have demonstrated the efficacy of Nanoblades in vivo, in mouse embryos, for a broad range of applications and in a broad panel of target cells for which other methods have had limited success. “Nanoblades have turned out to be particularly effective for editing the genome of human stem cells. These cells are of major therapeutic interest (particularly in tissue regeneration), but remain difficult to manipulate using standard methods”, explain the study authors.
 Nanoblades have been tested in mice and were patented by Inserm Transfert in 2016.
Inserm and CNRS researchers from the Institute of Genetics and Molecular and Cellular Biology (Inserm/CNRS/Université de Strasbourg) have discovered how myotubularin – a protein deficient in myotubular myopathy – interacts with amphiphysin 2 and suggest targeting the latter in order to treat patients. This research was published on March 20, 2019 in Science Translational Medicine.
Myotubular myopathy is a rare genetic disease affecting around one in 50,000 children. Linked to a mutation in the MTM1 gene located on the X-chromosome, it manifests as reduced muscle-cell adhesion and an alteration of the muscle fibers. This phenomenon causes major muscle weakness – including at the respiratory level – and leads to premature death with two thirds of patients not surviving beyond two years of age. At present, there is no treatment.
When exploring the interactions of myotubularin (coded by the MTM1 gene) with another protein, amphiphysin 2 (coded by the BIN1 gene), which is also expressed in the muscles and involved in similar myopathies, Inserm’s “Pathophysiology of neuromuscular diseases” team, in conjunction with the CNRS at the Institute of Genetics and Molecular and Cellular Biology (CNRS/Inserm/Université de Strasbourg), discovered how these proteins work together and suggests a new therapeutic target. Previous research had shown that myotubularin and amphiphysin 2 can physically interact by binding to each other.
To explore this functional link between the two, the researchers developed a model of MTM1-deficient transgenic mice and crossed these animals with other mice – some of which do not express BIN1 and some of which, on the contrary, overexpress it. They were unable to obtain any animals deficient in both MTM1 and BIN1, proving that at least one of the two proteins is necessary for muscle-fiber development and fetal survival. Conversely – and this came as a pleasant surprise – the overexpression of BIN1 made it possible to correct the myopathy linked to the MTM1 deficiency and obtain life expectancy equivalent to that of wild animals. Upon closer analysis of the muscles, the researchers observed satisfactory muscle-fiber organization and size with good cell adhesion, thereby leading to the hypothesis that MTM1 is an in vivo activator of the bin1 protein and that large quantities of the latter could make it possible to “do without” MTM1.
To verify whether BIN1 is a good therapeutic target, the researchers went on to conduct a gene therapy experiment in MTM1-deficient mice. They administered the human BIN1 gene using an AAV viral vector by systemic (intraperitoneal) injection following the birth of the rodents. A procedure that markedly reduced the symptoms of the condition and increased the survival of the diseased mice to that of healthy mice.
“There we have the proof of concept that the human BIN1 gene offersmajor potential in the treatment of myotubular myopathy linked to myotubularin deficiency, with spectacular results in mice. We would now like to continue this development in the form of preclinical trials and hope in the long-term to be able to propose a treatment for patients currently facing a therapeutic desert”, concludes Jocelyn Laporte, head of the Inserm team having performed this research.
Aggregates of Tau protein in Alzheimer’s disease. Inserm/U837, 2008
The propagation of tau protein aggregates in the brain contributes to the progression of Alzheimer’s disease. Researchers at the Neurodegenerative Diseases Laboratory: mechanisms, therapies, imaging (CNRS/CEA/Université Paris-Sud, MIRCen), working in collaboration with the Ecole Normale Supérieure, Sorbonne University and Inserm, have just identified the targets of these aggregates. Published in the EMBO Journal on 10 January 2019, this work will enable the development of tools capable of blocking these key elements of aggregate propagation and thus combating their pathological effect.
The aggregation of alpha-synuclein proteins in Parkinson’s disease and tau proteins in Alzheimer’s disease is intimately linked to the progression of these neurodegenerative diseases. These aggregates propagate from one neuronal cell to another, attaching themselves to the cells.
They multiply during this propagation. It has already been shown that the propagation and amplification of these protein aggregates are harmful and contribute to the progression of these diseases.
Understanding the formation of these aggregates, their propagation and their multiplication in the cells of the central nervous system offers potential for treatments: it would make it possible to target these processes and to act on their consequences.
The key step in the propagation of the pathogenic aggregates is the attachment of aggregates released from affected neuronal cells to the membranes of unaffected cells. Having already identified the targets of pathogenic aggregates of the alpha-synuclein protein (Shrivastava et al., 2015 EMBO-J), the team at the Neurodegenerative Diseases Laboratory (CNRS/CEA/Université Paris-Sud , MIRCen, Fontenay-aux-Roses), in collaboration with the Ecole normale supérieure, Sorbonne University and Inserm, has just identified the targets of tau protein aggregates. The targets are the sodium / potassium pump and glutamate receptors, two essential proteins for the survival of neurons The experiment was carried out on mouse neurons in culture.
Neuron membrane modification
The researchers also showed that the pathogenic aggregates modify the neuron membranes by redistributing the membrane proteins. The integrity of the membrane—and particularly of the synapses, the essential nodes for communication between neurons—is affected. These changes have a deleterious effect on the neurons because they cause abnormal communication between the neurons, as well as their degeneration.
This work therefore explains the early malfunctioning of the synapses and the degradation of normal communication observed in the neuronal networks as the disease progresses.
Towards new treatments
It also paves the way for the development of new treatment strategies based on protecting the integrity of the synapses, restoring the activity of the tau protein membrane receptors through the use of decoys to prevent harmful interaction between the pathogenic tau protein aggregates and their neuron membrane targets. These therapeutic approaches could be developed using human neurons, since researchers at the laboratory have just developed cultures of this type in collaboration with the I-Stem (Institute for Stem Cell Therapy & Exploration of Monogenic Diseases, AFM-Téléthon/Insem/Génopole/University of Evry-Val-d’Essonne) laboratory and Sorbonne University. This latter study is also published on 10 January 2019, in Stem Cell Reports.
 Propagation of α-Synuclein strains within human reconstructed neuronal network. Simona Gribaudo, Philippe Tixador, Luc Bousset, Alexis Fenyi, Patricia Lino, Ronald Melki, Jean-Michel Peyrin, Anselme Louis Perrier, Stem Cell Reports, 10 January 2019.
 They multiply by recruiting the endogenous alpha-synuclein and tau proteins from the affected cells during this propagation
About the Neurodegenerative Diseases Laboratory: mechanisms, therapies, imaging (LMN), a joint research unit of the CEA, CNRS and Université Paris-Sud.
The laboratory brings together nearly 60 scientists with research interests in neurosciences covering the mechanisms of degeneration, animal models, brain imaging and the study of gene-, cell- and drug-based strategies for treating neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease.
The LMN is located at MIRCen (Molecular Imaging Research Centre), a preclinical research facility developed by the CEA and Inserm. MIRCen is one of the departments of the CEA’s François Jacob Institute of Biology, on the Fontenay-aux-Roses site at CEA Paris-Saclay.
Researchers at CNRS, Université Côte d’Azur and Inserm have demonstrated a new mechanism related to the onset of migraine. In fact, they found how a mutation, causes dysfunction in a protein which inhibits neuronal electrical activity, induces migraines. These results, published in Neuron on December 17, 2018, open a new path for the development of anti-migraine medicines.
Even though 15% of the adult population worldwide suffers from migraines, no long-term, effective, curative treatment has been marketed to date. Migraine episodes are related, among other factors, to electric hyperexcitability in sensory neurons. Their electrical activity is controlled by proteins that generate current called ion channels, specifically by the TRESK channel, which inhibits electrical activity. The researchers have shown that a mutation in the gene encoding for this protein causes a split between two dysfunctional proteins: one is inactive and the other targets other ion channels (K2P2.1) inducing a great stimulation of the neuronal electrical activity causing migraines.
Though researchers had already shown the hereditary nature of migraines, they did not know the mechanism underlying migraine. By demonstrating that the TRESK split induces hyperexcitability in sensory neurons leading to migraine, this work, carried out at the Institut de Biologie Valrose (CNRS/Inserm/Université Côte d’Azur), opens new research path for the development of anti-migraine medicines. A patent application has been filed1: the scope is targeting K2P2.1 channels to reduce the electrical activity of neurons and prevent migraines from being triggered.
What is more, the researchers propose that this new genetical mechanism, causing the formation of two proteins instead of just one, has now to be considered for the study of other genetic diseases and for diagnosing them.
1 Patent PCT/EP2018/067581 “Methods and compositions for treating migraine”
As tumors develop, they evolve genetically. How does the immune system act when faced with tumor cells? How does it exert pressure on the genetic diversity of cancer cells? Scientists from the Institut Pasteur and Inserm used in vivo video techniques and cell-specific staining to visualize the action of immune cells in response to the proliferation of cancer cells. The findings have been published in the journal Science Immunologyon November 23, 2018.
Over time, the uncontrolled proliferation of tumor cells results in the accumulation of new mutations and changes to their genome. This gradual process creates significant genetic diversity among the cancer cells in any given patient. And although the cells in the immune system, especially T cells, are potentially able to eliminate these abnormal cells, tumor diversity can have a harmful effect, complicating the action of the immune system and rendering some therapies ineffective. Understanding this frantic race between tumor development and the immune response is key to the success of future immunotherapy techniques.
Scientists in the Dynamics of Immune Responses Unit (Institut Pasteur/Inserm), directed by Philippe Bousso, in collaboration with Ludovic Deriano, Head of the Genome Integrity, Immunity and Cancer Unit (Institut Pasteur), investigated how spontaneous immune responses to tumors influence this tumor heterogeneity. They demonstrated that the immune system can employ mechanisms to significantly reduce tumor diversity, favoring the emergence of more genetically homogeneous tumor cells.
In their study, the scientists marked each cancer cell subclone with a separate color in a mouse model. By monitoring these different colors they were therefore able to characterize the evolution of tumor heterogeneity in time and space. They were also able to observe the contacts between T cells and cancer cells and determine how some tumor cells are destroyed. Their research highlights the drastic impact the immune system can have on tumors by reducing their heterogeneity.
The same impact on the heterogeneity of tumor cells has also been observed in response to immunotherapies that release the brakes on the immune system, an approach which was awarded the Nobel Prize in Physiology or Medicine this year.
This research shows that taking into account the interaction between immunotherapies and tumor heterogeneity could contribute to the development of optimum therapeutic combinations and sequences.
In addition to the organizations mentioned above, this research was funded by the Fondation de France, the French National Cancer Institute (INCa) and the European Research Council (ERC).
Asymmetry plays a major role in biology at every scale: think of DNA spirals, the fact that the human heart is positioned on the left, our preference to use our left or right hand … A team from the Institute of biology Valrose (CNRS/Inserm/Université Côte d’Azur), in collaboration with colleagues from the University of Pennsylvania, has shown how a single protein induces a spiral motion in another molecule. Through a domino effect, this causes cells, organs, and indeed the entire body to twist, triggering lateralized behaviour. This research is published in the journal Science on November 23, 2018.
Our world is fundamentally asymmetrical: think of the double helix of DNA, the asymmetrical division of stem cells, or the fact that the human heart is positioned on the left … But how do these asymmetries emerge, and are they linked to one another?
At the Institute of biology Valrose, the team led by the CNRS researcher Stéphane Noselli, which also includes Inserm and Université Cote d’Azur researchers, has been studying right–left asymmetry for several years in order to solve these enigmas. The biologists had identified the first gene controlling asymmetry in the common fruit fly (Drosophila), one of the biologists’ favoured model organisms. More recently, the team showed that this gene plays the same role in vertebrates: the protein that it produces, Myosin 1D, controls the coiling or rotation of organs in the same direction.
In this new study, the researchers induced the production of Myosin 1D in the normally symmetrical organs of Drosophila, such as the respiratory trachea. Quite spectacularly, this was enough to induce asymmetry at all levels: deformed cells, trachea coiling around themselves, the twisting of the whole body, and helicoidal locomotive behavior among fly larvae. Remarkably, these new asymmetries always develop in the same direction.
In order to identify the origin of these cascading effects, biochemists from the University of Pennsylvania contributed to the project too: on a glass coverslip, they brought Myosin 1D into contact with a component of cytoskeleton (the cell’s “backbone”), namely actin. They were able to observe that the interaction between the two proteins caused the actin to spiral.
Besides its role in right–left asymmetry among Drosophila and vertebrates, Myosin 1D appears to be a unique protein that is capable of inducing asymmetry in and of itself at all scales, first at the molecular level, then, through a domino effect, at the cell, tissue, and behavioral level.
These results suggest a possible mechanism for the sudden appearance of new morphological characteristics over the course of evolution, such as, for example, the twisting of snails’ bodies. Myosin 1D thus appears to have all the necessary characteristics for the emergence of this innovation, since its expression alone suffices to induce twisting at all scales.
Myosins are a class of proteins that interact with actin (a constituent of cell skeletons or cytoskeletons). The most well-known of them, muscular myosin, makes muscles contract.
Why are some depressed patients more or less totally resistant to the most commonly-prescribed antidepressants? This question was addressed by researchers from Inserm and Sorbonne Université at the Fer à Moulin Institute who were able to reveal the major role of neurons that secrete serotonin – the preferred target for antidepressants – in regulating their own activity. Implicated is a serotonin receptor carried by these neurons whose deficiency could be decisive in the absence of response to the most commonly prescribed antidepressants. This research, published in Neurospychopharmacology, will help elucidate the role of serotonin in psychiatric disorders.
Serotonin is a neurotransmitter – a chemical substance produced by some neurons in order to activate others – which is implicated in a number of psychiatric disorders, such as depression, addiction, impulsiveness and psychosis. It is secreted by specific neurons known as serotonergic neurons.
Releasing serotonin outside the neuronal cell activates neurons which possess receptors specific to this neurotransmitter. When these receptors detect sufficient serotonin in the extracellular environment, they send a message to activate or inhibit the neuron that expresses them. The serotonergic neurons also possess several types of serotonin receptors – called autoreceptors – making it possible to self-regulate their activity.
Researchers from Inserm and Sorbonne Universités/UPMC at the Fer à Moulin Institute (Inserm, UPMC) studied the role of one of the serotonergic neuron autoreceptors – known as 5-HT2B – in the regulation of their activity, in order to elucidate the lack of efficacy of some antidepressant treatments.
Usually, when a serotonergic neuron secretes serotonin in the extracellular environment, it is capable of recapturing some of that serotonin which it will release again later on. This mechanism, ensured by a specific transporter, enables it to regulate the level of serotonin present in the extracellular environment. This transporter is the preferred target of the antidepressant drugs used to treat psychiatric disorders involving serotonin. These drugs are called selective serotonin reuptake inhibitors (SSRIs) because they prevent this recapture by the transporter. In the context of depression in which serotonin secretion is too reduced, SSRIs make it possible to maintain normal levels of serotonin in the extracellular environment.
The research team took as their starting point the observation that, in the mouse, when the serotonergic neuron does not carry any 5-HT2B autoreceptors, there is lower than usual serotonergic neuron activity and that the molecules blocking the activity of the transporter, such as SSRIs, have no effect on extracellular serotonin levels. The researchers therefore showed that in order to have an effect, these molecules needed the presence and normal expression of the 5-HT2B serotonin receptor.
They also discovered that when a neuron secretes serotonin, its 5-HT2B autoreceptor detects the quantity present in the extracellular environment and sends a signal to the neuron for it to secrete more serotonin. To avoid the excessive secretion of serotonin, the serotonergic neuron possesses a negative regulator: the 5-HT1A autoreceptor which also detects the level of extracellular serotonin and sends a signal to the serotonergic neuron to inhibit the secretion. In order to maintain normal neuronal activity, 5-HT2B makes it possible to maintain a certain level of activity by acting as a positive self-regulator.
These findings, which remain to be confirmed in human subjects, reveal a fine serotonergic neuron self-regulation mechanism balanced between the activator autoreceptors and the inhibitor autoreceptors. They constitute a step forward in identifying new drug targets, in elucidating the role of serotonin in certain psychiatric disorders and in understanding the inefficacy of certain antidepressants.