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Nanoblades: shuttles for genome surgery

 

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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,[1] 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.

Legend:

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.

[1] Nanoblades have been tested in mice and were patented by Inserm Transfert in 2016.

HIV/tuberculosis co-infection: tunnelling towards better diagnosis

 

Nanotubes linking two macrophages in humans infected with HIV-1 in a TB-associated micro-environment © Shanti Souriant & Renaud Poincloux, IPBS, CNRS/Université Toulouse III-Paul Sabatier

1.2 million people in the world are co-infected by Mycobacterium tuberculosis, the bacteria which causes tuberculosis, and AIDS (HIV-1). This combination is deadly: it makes patient diagnosis and treatment difficult, and increases the pathogenicity of these two infectious agents. An international team led by researchers at the CNRS and Inserm have revealed that in the presence of tuberculosis, HIV-1 moves from one cell to the next via nanotubes which form between macrophages, drastically increasing the percentage of infected cells. These findings appear in the 26 March 2019 edition of Cell Reports.

Researchers at the Institute of Pharmacology and Structural biology (CNRS/Université Toulouse III – Paul Sabatier) and the IM-TB/HIV international laboratory, a consortium between the CNRS and the National Scientific and Technical Research Council (Conicet) (Argentina), together with the Center for Pathophysiology of Toulouse Purpan (CNRS/INSERM/Université Toulouse III – Paul Sabatier), have shown that macrophages – which act as host cells for tuberculosis and HIV-1, join to form nanotubes when exposed to Interleukin-10, a molecule secreted in the presence of tuberculosis. The abundance of these specific M(IL-10) macrophages in the lungs is correlated with the severity of the disease. HIV-1 particles travel through these tunnel-like nanotubes to infect neighbouring cells and multiply. Using different approaches to inhibit their formation, scientists successfully reduced viral transfer between macrophages, leading to a drop in HIV-1 production.

In a case of severe TB, the development of nanotubes between macrophages accelerates, increasing the spread of the AIDS virus and viral production as a result. Because the presence of this specific type of macrophage can be measured, diagnosis and treatment of patients suffering from both illnesses could be made easier. This research paves the way to new therapeutic approaches aimed at limiting viral load increases in tuberculosis patients.

A Gene Therapy Tested in the Treatment of Myotubular Myopathy

 

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 offers major 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.

Découverte d’une réaction immunitaire cruciale lors de la diversification alimentaire pour prévenir l’apparition des maladies inflammatoires

 

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Les microbes colonisent l’ensemble des surfaces de notre corps et participent au bon équilibre de notre système immunitaire. Chez les nouveau-nés, le microbiote intestinal est d’abord formaté par les composants du lait maternel. Lors de la diversification alimentaire, il se développe et de nombreuses bactéries prolifèrent. Des chercheurs de l’Institut Pasteur et de l’Inserm montrent chez la souris qu’une réponse immunitaire importante se produit lors de l’introduction de nourriture solide et du développement du microbiote. Mais surtout, ils ont montré que cette réaction immunitaire était essentielle car elle participe à l’éducation du système immunitaire, et permet, à l’âge adulte, une faible susceptibilité aux maladies inflammatoires (allergies, colites, maladies auto-immunes, cancer). Ces résultats ont été publiés dans la revue Immunity, le 19 mars 2019.

L’introduction d’une hygiène de qualité au milieu du XIXe a drastiquement fait diminuer la mortalité due aux maladies causées par des micro-organismes. Dans nos sociétés industrielles actuelles, l’hypothèse hygiéniste affirme désormais que la réduction de l’exposition en bas âge aux microbes entraînerait une augmentation de la sensibilité aux maladies allergiques ou auto-immunes. De précédentes études ont montré que la perturbation du microbiote, notamment par l’exposition aux antibiotiques, peut se traduire par des réponses allergiques[1].

Chez le nouveau-né, la constitution du microbiote se fait lors de l’accouchement par l’acquisition des bactéries de la mère mais aussi, grâce à la composition du lait maternel. Il est alors majoritairement composé de bifidobacteria et de lactobacilles. A l’introduction de nouveaux aliments dans le régime, le microbiote prolifère et le nombre de bactéries augmente de 10 à 100 fois. Des chercheurs (Ziad Al Nabhani et ses collègues) de l’unité Microenvironnement et Immunité (Institut Pasteur/Inserm) dirigée par Gérard Eberl, ont découvert chez la souris que ce phénomène était accompagné d’une réponse immunitaire intense. « Nous avons pu montrer que ce mécanisme se produisait dans une fenêtre de temps très spécifique : entre 2 et 4 semaines chez la souris ce qui correspondrait à 3 et 6 mois chez l’homme » explique Gérard Eberl, principal auteur de l’étude.

« Nous avons ensuite supposé que l’existence d’une fenêtre de temps déterminée indique que la réponse immunitaire est programmée dans le temps et possède de ce fait une fonction unique dans le développement du système immunitaire » poursuit Gérard Eberl. En effet, les chercheurs ont pu démontrer qu’en traitant les souris par antibiotiques sur la fenêtre critique de la réponse immunitaire, elles étaient par la suite plus sujettes à développer certaines maladies inflammatoires : les allergies intestinales, le cancer colorectal et les colites. Ainsi, le microbiote une fois détruit par les antibiotiques, on constate que la réaction immunitaire ne se produit pas. 
« C’est ce que l’on appelle l’empreinte pathogénique » explique Gérard Eberl, « c’est-à-dire que des évènements se produisant dans la prime enfance déterminent une future susceptibilité aux maladies inflammatoires ».

Les chercheurs ont également pu mettre en évidence la présence des cellules spécifiques au moment de cette réaction et qui participent au bon fonctionnement des réponses immunitaires : les cellules T régulatrices (Tregs), des modulateurs clés sans lesquelles les réponses immunitaires sont exacerbées, entraînant par la suite des maladies inflammatoires.

L’ensemble de ces données montre l’importance d’une exposition précoce au microbiote, ciblée dans le temps, pour le développement d’un système immunitaire équilibré. « Nous aimerions maintenant valider ces résultats sur l’influence du microbiote au moment de la diversification alimentaire sur l’apparition d’autres types de pathologies comme les maladies neurodégénératives par exemple » conclut Gérard Eberl.

Ces travaux ont été financés par l’Institut Pasteur et l’Inserm, mais également par l’Association François Aupetit, la Crohn’s Colitis Foundation of America, l’European Crohn’s and Colitis Organisation, la Fondation pour la Recherche Médicale, Janssen, et la Fondation Kenneth Rainin.

Ces travaux ont été menés dans le cadre du programme transversal “Microbiote” mis en place en 2016 dans le cadre du plan stratégique de l’Inserm.

[1] Comment le microbiote bloque les allergies, Science, 2015

Go for a Run or Eat Chocolate: A Choice Dictated by the Cannabinoid Receptors

 

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Physical inactivity is a common factor in lifestyle diseases – and one that is often linked to the excessive consumption of fatty and/or sugary foods. The opposite scenario of excessive physical activity at the expense of caloric intake can also be harmful, as cases of anorexia nervosa illustrate. These data therefore point to the crucial need to research the neurobiological processes that control the respective motivations for exercise and food intake. A study by Inserm and CNRS researchers published on March 7, 2019 in JCI Insight reveals that the cannabinoid type 1 (CB1) receptors play an essential role in the choice between running and eating chocolatey food.

The authors of this paper had previously reported that the cannabinoid type-1 (CB1) receptors, present on several types of neurons, play a key role in performance during physical activity in mice. A conclusion based on the performances achieved by animals with free access to an exercise wheel – a model in which it was not possible to distinguish the mechanism involved (motivation, pleasure…). Given that the motivation for a reward can only be estimated by measuring the efforts that the individual – whether human or animal – is prepared to make to get that reward, the researchers devised a model in which each access to the wheel was conditional on a prior effort. This involved the animal repeatedly introducing its snout into a recipient, an essential prerequisite for unlocking the wheel. After a training period during which the level of effort required to unlock the wheel remained the same, the mice were confronted with a test in which the effort required was gradually increased. When exposed to this test, the mice lacking CB1 receptors showed an 80 % deficit in the maximum effort they were prepared to make to unlock the wheel, and without a decrease in performance during their access to it. This finding indicates that the CB1 receptors play a major role in controlling motivation for exercise. The use of other genetically-modified mice also enabled the researchers to demonstrate that these CB1 receptors controlling motivation for exercise are located on GABAergic neurons.

The researchers then examined whether the CB1 receptors in the GABAergic neurons control the motivation for another reward: chocolatey food (like humans, mice love it even when they are otherwise well-fed). While the CB1 receptors also play a role in motivation for food – albeit to a lesser extent than in motivation for exercise – the CB1 receptors located on the GABAergic neurons are not implicated in the motivation for eating chocolatey food.

In our daily life, we are faced with an ongoing choice between various rewards. A fact which has encouraged the researchers to develop a model in which following a learning period the mice had the choice – in return for the efforts described above – between exercise and chocolatey food. The motivation for exercise was greater than that for chocolatey food, with the exception of the mice lacking CB1 – whether generally or just on GABAergic neurons – whose preference was for the food.

In addition to these findings indicating that the cannabinoid receptor is essential for the motivation for exercise, this study opens up avenues for researching the neurobiological mechanisms behind pathological increases in this motivation. One illustration is provided by anorexia nervosa which often combines the decreased motivation to eat with an increased motivation to exercise.

Metastatic Lung Cancer: A Targeted Therapy to Improve Treatment Efficacy

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When faced with the most aggressive forms of lung cancer, how can the efficacy of chemotherapy be increased? Teams from Inserm, Université Paris Descartes and the Paris public hospitals system AP-HP have maybe hit on a solution. They have developed a targeted therapy which aims to improve the response to platinum salts – the standard chemotherapy used in lung cancer – by neutralizing the activity of a receptor that contributes to its aggressiveness. This research, published in Cancer Letters, shows that in mice this therapy restores the response to chemotherapy and reduces the risk of metastasis by one half to two thirds.

In metastatic lung cancer, life expectancy remains very limited, with death occurring within 5 years in 85 % of patients. The new treatments available for some patient populations present genuine efficacy, but this is of limited duration and successive relapses are common. Increasing the efficacy of existing treatments and finding new ones therefore remains a priority.

A priority that is being addressed by the team of Inserm researcher Patricia Forgez who, in collaboration with teams from the Paris public hospitals system AP-HP (Cochin, Lariboisière and Saint-Antoine hospitals) and Université Paris Descartes is developing a targeted therapy to increase the sensitivity of the most aggressive tumors to platinum salts, an essential chemotherapy in lung cancer.

In previous research, Forgez and her co-workers had shown that lung tumors and especially those at a metastatic stage overexpress the neurotensin receptor. Neurotensin is a small molecule produced in the intestines and brain that is also found to be abnormally overexpressed in tumors where, by binding to its receptor it triggers the continuous cascade of signals stimulating tumor cell growth, survival and migration. This renders them much more aggressive and with little or no sensitivity to platinum salts. By correlating the neurotensin receptor overexpression with the poorer prognosis observed in patients, the researchers had demonstrated that this receptor has a role to play in tumor progression.[1]

In this new study, the research team has developed an antibody to specifically neutralize the form of neurotensin produced by the tumors.[2] 

When tested in multiple experimental mouse models, the researchers observed a 40 to 65 % regression in tumor size and a decrease in aggressiveness. The treated mice presented half as many lymph-node and lung metastases as their untreated counterparts. The researchers also showed that concomitant administration of the antibody with a platinum salt restored or improved treatment efficacy by improving the access of the therapeutic molecule to its target.

The long-term objective is to develop a targeted therapy to block the neurotensin receptor in order to weaken the tumor cells and improve their sensitivity to platinum salts.

“Almost all patients diagnosed with lung cancer receive platinum salts at some point in their treatment, reiterates Jean Trédaniel, study co-author and head of the Paris Saint-Joseph Hospital Group thoracic oncology unit, whether as first-line treatment or following the failure of a targeted therapy or immunotherapy. However, platinum salts are toxic to the body, making it impossible to increase the doses in the event of resistance. Administering this antibody would render the tumor more sensitive to the treatment. What is more, it has been shown in mice to be very well tolerated over the long-term. “

In collaboration with Inserm Transfert, French technology transfer acceleration company SATT Ile-de-France INNOV, and Fair Journey Biologics, the research team is now working on the development of anti-neurotensin antibodies for use in humans, the objective being to embark on a clinical trial. Encouraging results in lung cancer would enable the extension of this therapy to include the other cancers that express neurotensin and its receptor, such as those of the breast, ovary, endometrium, prostate, pancreas, stomach and colon.

[1] This discovery has been protected by a patent filed by Inserm Transfert, the co-owners of which are Inserm and AP-HP. This patent claims that the neurotensin receptor is a marker of tumor aggressiveness and that neurotensin is a potential therapeutic target.

[2] This antibody has been the subject of several patent applications filed by Inserm Transfert on behalf of Inserm and Université Paris Descartes; one of these patents was issued in the USA at the end of 2018.

Jeudi 14 Février : journée mondiale du rein

Alzheimer’s disease: five new genetic markers identified

Fluorescent marking of the Tau protein in a human cell hNT; the Tau protein, has a role in Alzheimer’s disease, particularly in its familial forms ©Inserm/U837

Knowledge of the genetic component of Alzheimer’s disease continues to deepen. The aim is not to predict the disease but reveal its pathophysiological mechanisms in order to develop new drugs. At Lille’s Institut Pasteur, an Inserm team led by Jean Charles Lambert within Inserm Unit 1167 “Risk factors and molecular determinants of diseases linked to aging” directed by Philippe Amouyel recently hit a new milestone thanks to the Genomics of Alzheimer’s Project (IGAP), in which the genomes of 94,000 people were analyzed. This research revealed five new genetic variants linked to the disease and has been published in Nature Genetics.

Out of the forty genetic variants now linked to Alzheimer’s disease, five have recently been discovered as part of the Genomics of Alzheimer’s Project (IGAP) co-led by the “Identification of molecular determinants of neurodegenerative diseases” team at Inserm unit 1167 at Institut Pasteur in Lille. This major project conducted in partnership with four international consortia*[1] studied 94,000 genomes – 35,000 of which from people with Alzheimer’s disease and 59,000 from healthy controls. The vast majority of cases concerned late-onset Alzheimer’s disease, with the researchers having excluded the early-onset familial forms.

The patients’ genomes were compared with those of the healthy individuals presenting an equivalent average age. The objective was to look for variants, namely distinct DNA sequences between these two groups statistically linked to the development of the disease. The scientists reviewed 11 million of them, revealing five new variants linked to Alzheimer’s disease either in or in proximity to the genes IQCK, ACE, ADAM10, ADAMTS1 and WWOX. One of the five was a rare variant found in less than 1% of the patients. “The discovery of this rare variant validates the strategy of building increasingly large cohorts to obtain a very high statistical power. By increasing their size yet further, we will be able to look for more rare variants which probably represent a large part of the as yet undiscovered genetic component of this disease”, considers Jean-Charles Lambert, Inserm researcher and co-leader of this research.

Some of these new variants are concentrated in regions implicated in the metabolism of the Tau and amyloid precursor proteins, whose role in Alzheimer’s disease is already known – particularly in its familial forms. “This suggests shared mechanisms in the early or later forms”, explains Lambert. Treatments targeting these mechanisms could therefore be effective against these two forms. But variants are also found in regions implicating immunity, which is very interesting because it could validate a current research avenue. “One hypothesis is that the microglia, made up of immune cells related to the macrophages and playing a role in defending the brain, is implicated in the disease”, clarifies Lambert. For the researchers, it is probable that these variants control shared biological pathways which would lead to multiple impairments and the onset of the disease.

Given the repeated failures of the therapeutic trials in Alzheimer’s disease, better knowledge of the variants linked to the disease and the pathophysiological mechanisms they control is fundamental if new therapeutic strategies are to be developed. A new European project coordinated by Lambert – in which a yet larger number of patients will be enrolled to continue this mission – is already on the drawing board: the European DNA bank for deciphering the missing heritability of Alzheimer’s disease (EADB).

[1] *CHARGE (Heart and Aging Research in Genomic Epidemiology Consortium, USA), EADI (The European Alzheimer’s Disease Initiative), ADGC (Alzheimer Disease Genetics Consortium, USA) and GERAD/PERADES (Cohorts for and Genetic and Environmental Risk in AD/Defining Genetic, Polygenic and Environmental Risk for Alzheimer’s Disease Consortium, France)

Brain Prize 2019 a French team receives international award for his research on CADASIL, a hereditary cerebrovascular disease

Left to right : Pr Elisabeth Tournier-Lasserve, Pr Hugues Chabriat, Pr Marie-Germaine Bousser, Dr Anne Joutel

Awarded by the Danish Lundbeck Foundation, the “Brain Prize” is a major international award for scientific importance of their research in neuroscience. It has a total of one million euros.

He puts this year in honor of the work begun there nearly forty years by four French scientists on CADASIL, a hereditary cerebrovascular disease, which causes migraine attacks, stroke and cognitive decline. Today is the genetic disease of small cerebral vessels most frequently diagnosed.

The four winners of the French “Brain Prize 2019” are:

> Professor Elisabeth Tournier-Lasserve , head of the hospital neurovascular genetics department Lariboisière AP-HP, medical genetics professor at Paris Diderot University and head of the research team “cerebrovascular diseases, genomics , imaging and personalized medicine “within the unit Paris Diderot – Inserm 1141” NeuroDiderot “.

> Professor Hugh Chabriat , head of the neurology department of the hospital Lariboisière AP-HP, coordinator of the reference center for rare vascular diseases of the brain and the eye (CERVCO), professor of neurology at the University Paris Diderot and researcher at the research team “cerebrovascular diseases, genomics, imaging and personalized medicine” in the unit of Paris-Diderot – Inserm 1141 “NeuroDiderot”. Prof. Chabriat also co-coordinates the University Hospital Department (DHU) neurovasc Sorbonne Paris Cité.

> Professor Marie-Germaine Bousser , former head of the neurology department at the hospital Lariboisière AP-HP and emeritus professor of neurology at the University of Paris Diderot.

> Dr. Anne Joutel, Research Director Inserm, director of the team “pathophysiological mechanisms of diseases of the small cerebral vessels” at the Institute of Psychiatry and Neurosciences Paris Descartes – UMR1266 Inserm and professor in the pharmacology department University of Vermont (USA).

The four scientists showed that CADASIL (for “Cerebral Autosomal Dominant arteriopathy with sub-cortical Infarcts and Leukoencephalopathy”) is an inherited cerebrovascular disease caused by mutations in the NOTCH3 gene on chromosome 19. This condition is responsible for migraine attacks and stroke, can lead to severe motor and cognitive disorders. Patients may also suffer from depression, difficulty concentrating, a slowdown and balance disorders.

The discovery of CADASIL has allowed the development of diagnostic tests and the development of mouse models of the disease, essential to understand the mechanisms of brain damage and the development of therapeutic. The identification and clinical and preclinical study of this disease are also a major step to identify and better understand other diseases of cerebral vessels.

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