The CNS/NET department unites a unique set of skills, including clinical expertise at the bedside, molecular genetics, neurophysiology, neuroendocrinology and preclinical research that has been crucial to promote important research breakthroughs and fast-track them into clinical development to treat diseases affecting the central nervous system and/or the neuroendocrine system.
Intellectual deficiency (ID) is defined as a disability characterized by significant limitations both in intellectual functioning and in adaptive behaviour, i.e. a person's social responsibility and independent performance of daily activities. ID touches as much as 3% of the population and represents a major public health problem in all countries. Considerable progress has been made over the past twenty years in the field of early diagnosis, but 50% of ID patients still remain undiagnosed. To improve this situation, the team of Laurent Villard, among others, studies the genetic causes of ID. They have identified several genes responsible for various form of neurodevelopmental disorders, and have made significant contributions in the understanding of the mechanisms that are defective when these genes are mutated.
A unique cohort of 1300 affected patients
In their efforts to improve diagnosis of a group of rare epilepsies associated with severe ID, Developmental and Epileptic Encephalopathies (DEE), the group manages a large cohort of 1300 affected patients. This invaluable resource has already proven instrumental in identifying several new genes associated with DEE. Among them was the unexpected identification of KCNQ2 as the most frequently mutated gene.
“KCNQ2 was traditionally found in patients with benign familial neonatal convulsions (BFNC), a condition for which the neurological prognosis is generally good and development is normal.” explains Mathieu Milh, the neurologist heading the clinical aspects of this research in Laurent Villard’s laboratory. “It was a real surprise to find that KCNQ2 is actually a major EOEE gene, since we identified 68 mutations in the patients in our cohort”.
KCNQ2 encodes a protein that makes potassium channels in neurons and is crucial for proper communication within the brain. In collaboration with INMED (Inserm U1249) and La Timone Neurosciences Institute INT (CNRS), the scientists are currently investigating what might be different at the cellular level in the two populations of patients that are affected by either BFNC or the much more severe condition EOEE. The ultimate goal of this program is to offer EOEE patients new pharmacological therapies to overcome the current lack of effective treatments. These projects were previsouly supported by ANR grants, and are funded again within the 2019 ANR call, for a project called IMprove (2019-2022).
The enterprise is much further along in the field of Rett Syndrome, another intellectual deficiency that has occupied researchers in Laurent Villard’s laboratory for many years.
Rett Syndrome (RTT) was first described in 1966 by the Austrian paediatrician Andreas Rett. It affects mostly girls and is characterized by a normal development up until the age of 6 to 18 months. Brain development then slows down until it eventually stops. As a severe intellectual deficiency settles, head growth stagnates leading to an acquired microcephaly, and previously learned skills such as speech or walk can be lost. Many patients face additional complications including motor defects, epilepsies, seizures and abnormal breathing patterns often leading to hyperventilation or apnoeas.
In 1999, the laboratory of Huda Zoghbi (Houston, Texas, USA) discovered that mutations in a gene called MECP2 are responsible for most cases of RTT. MECP2 is located on the X chromosome, at position Xq28. Importantly, an exhaustive search for mutations in MECP2 among French patients and their families was carried out in the laboratory of Laurent Villard, and revealed that defects in this gene can also causes other types of intellectual disabilities, including severe cases of encephalopathies in boys.
From the molecular mechanisms behind RTT to the development of treatments
To better understand the mechanisms at work during RTT, Laurent Villard and his colleagues have carried a long-lasting characterisation of a mouse model of Rett Syndrome from Adrian Bird’s laboratory (Edinburgh, UK). Their work has lead to major contributions in the field, opening important avenues for treatment of Rett Syndrome patients.
The group has found that Mecp2-deficient mice display an altered breathing pattern, similar to RTT patients. These mice also present low levels of a chemical messenger, norepinephrine (NE), in the area of the brain where the respiratory centres are located: the medulla. At the time, this finding was the first demonstration of a cellular defect caused by the absence of Mecp2 in vivo. Importantly, the experiments carried out by the team suggested that the breathing defects could be corrected with exogenous NE. The team therefore decided to develop a means to artificially increase the levels of NE in the animals by using a chemical compound called desipramine.
“ We administered a daily dose of desipramine to Rett syndrome mice, and noted a remarkable improvement of breathing but also of survival in these mice” indicates Jean-Christophe Roux in charge of the Rett syndrome projects in the Villard lab. “With these promising results, we decided to initiate a clinical trial in collaboration with Prof. Josette Mancini at La Timone Hospital, to study the effects of desipramine on children with Rett Syndrome”. The results of this phase II trial are now published.
More recently, the group also discovered a defect in transport of BDNF along neurons in RTT mice. BDNF is a crucial factor that supports survival and growth of neurons, as well as the establishment of connections between them. Importantly, artificially increasing BDNF secretion using a compound called cysteamine significantly alleviates motor defects of Rett syndrome mice and improves their lifespan. This finding has now been taken to the clinic, where another phase II clinical trial is being initiated to determine the efficacy of a cysteamine treatment in RTT patients.
Efforts to understand the molecular dysfunctions in RTT have proven fruitful. The team is currently exploring new horizons that already appear promising in this same direction, offering new translational perspectives for this disorder.
Neuroendocrine disorders result from a gland secreting either too much or too little of an endocrine hormone. While the vast majority of oversecreting diseases is caused by benign, non-cancerous tumours, called neuroendocrine tumours, undersecreting disorders, or hormone deficiencies, can have a variety of origins. They may affect any gland in the endocrine system, but those that affect the pituitary gland, the master of this system, will have a broader spectrum of consequences on different biological functions. To identify new therapeutic strategies, the laboratory of Thierry Brue has been interested over the past decades in understanding the genetic causes and the molecular mechanisms responsible for these disorders. The scientists have focused on combined pituitary hormone deficiency (CPHD), for which they have identified a number of responsible genes and mutations, partly through the creation of an international collection of DNA samples from over 1200 patients (GENHYPOPIT).
Neuroendocrine disorders: keeping the right balance
“Through a detailed analysis of the DNA samples from the GENHYPOPIT network, we were able to uncover a new syndrome that combines hormone deficiency with a variable immune deficiency, characterized by defective antibody production” declares Thierry Brue. “We have named it DAVID, for Deficit in Anterior pituitary function and Variable Immune Deficiency”. The gene responsible for DAVID syndrome is called NFKB2 and encodes a subunit of the NFB complex, a central activator of genes involved in inflammation and the immune response.
The lab has also widely studied neuroendocrine tumours (NET) to uncover the signals that are responsible for the increase in hormone secretion. Interestingly, the team has identified several factors involved in and with opposite effects on CPHD and NET. Such is the case for the Growth Hormone Receptor (GHR) and for the Ghrelin receptor (GHS-R), as well as for Pit-1, a transcription factor important for pituitary development and hormone expression.
The dual role of Pit-1
Alterations of the PIT-1 gene sequence or expression level have been linked to cases of both dwarfism and gigantism. It encodes a transcription factor that is important for the differentiation of endocrine cells, as well as for regulating the expression of hormones such as Growth Hormone or Prolactin (involved in growth and lactation, respectively). Mutations in the PIT-1 gene had been previously associated with cases of CPHD when, in 2006, Thierry Brue, Anne Barlier and their colleagues demonstrated its importance in NET, where it acts as a growth promoter. Because of the dual role that Pit-1 appeared to have in hormone deficiencies and oversecreting disorders, the team attempted a gene therapy approach where a mutant version of Pit-1 found in CPHD patients, Pit-1-R271W, was used to treat oversecretion and tumoral growth of the opposite disease, pituitary tumours.
“We demonstrated that the Pit1-R271W mutant is able to block hormonal oversecretion of NET cells and to induce cell death both in vitro and in vivo in mice” explains Anne Barlier, who has led this research in Thierry Brue’s laboratory. “This strategy could turn promising for the gene therapy of human pituitary tumours”.
In addition to pursuing the hunt for genetic abnormalities linked to neuroendocrine diseases, the scientists are now investing major efforts in establishing new cellular models to study hormone deficiencies and neuroendocrine tumours, and developing new therapies to treat both of these affections.