Cellular senescence – defined by a stable arrest of the cell cycle coupled to stereotyped phenotypic changes – might play a causal role in a variety of lung pathologies, among which chronic obstructive pulmonary disease (COPD), predicted to be the third cause of death worldwide in 2020. COPD is characterized by slowly progressive airflow obstruction and emphysema due to destruction of the lung parenchyma, and is often associated with pulmonary hypertension (PH). No curative treatment is available for this disease. Senescent cells are increased in lungs from COPD patients and express a robust senescence-associated secretory phenotype (SASP), which is pro-inflammatory. Our goal is to critically test the hypothesis that senescent cells drive the lung alterations seen in COPD, to understand the mechanisms, and to develop therapeutic interventions able to slow or delay the cell senescence process in vitro and the lung alterations in vivo. Aim 1- To test the hypothesis that cellular senescence is CAUSAL in lung pathology. Two ways to test this hypothesis: a) Take advantage of a new mouse model that allows induction of senescence in targeted cells and see if this mimicks disease alterations observed in COPD or in PH. We will test the hypothesis that conditional deletion of the specific p53 ubiquitin-ligase Mdm2 (or its homologue Mdm4) in pulmonary endothelial cells (P-EC), alveolar epithelial cells (AEC), or fibroblasts in mice induces cell senescence, lung emphysema, and PH in the absence of lung aggression, or leads to an exaggerated phenotype during exposure to cigarette smoke (CS) or to chronic hypoxia. b) Take advantage of a new mouse model that allows the elimination of senescent cells by combining mouse models of lung pathology with mice expressing a killer gene construct driven by the p16 promoter (p16-INK-ATTAC mice). We will test the hypothesis that treatment of p16-INK-ATTAC mice with AP20187 (which activates caspase 8 in p16 expressing cells), by eliminating p16 positive senescent cells: i) prevents the development of smoke-induced emphysema or reverses it; ii) prevents and reverses PH induced by chronic hypoxia exposure. Parallel studies will be performed with p16-luciferase mice to follow senescent cell appearance. Aim 2- To understand the mechanisms underlying the replicative or premature cell senescence process in COPD and evaluate therapeutic interventions. Two major pathways altered in COPD lungs will be explored: a) Over-activation of the mTOR signaling pathway. We will test the hypothesis that activation of the mTOR complex 1 (mTORC1) is central to the cell senescence process in COPD, and that treatment of cells with mTORC1 inhibitors (low doses of rapamycin and metformin) delays the onset of cell senescence and suppresses the hyper-functional secretory phenotype of lung cells from COPD patients. We will question whether mTOR activation (by deleting TSC1 in targeted cells) in mice increases lung cell senescence and replicates lung alterations seen in patients with COPD and test pharmacological interventions in vivo that inhibit mTOR and suppress the SASP. b) Altered telomere dependent and independent functions of telomerase in COPD and PH. We will question whether mice with deletion of TERT or TERC (protein and RNA components of telomerase, respectively), exhibiting different telomere length, show various emphysema severity in response to CS, whether transgenic mice expressing TERT under the control of the p16 promoter (p16-TERT+ mice) are protected and investigate the modulation of TERT expression in the setting of COPD. We will test the hypothesis that: -TERT overexpression drives pulmonary artery smooth muscle cell growth and PH development through independent telomere elongation function of TERT, - genetic or pharmacological inactivation of TERT protects against hypoxic PH in mice and, - p16-TERT+ mice develop an exaggerated PH phenotype.

Arterial hypertension (HT) is one of the major risk factors for cardiovascular disease and has an important cost for society. Pathogenic mechanisms underlying HT are complex and include genetic and environmental, vascular and hormonal factors. Detection of secondary forms of HT is particularly important since it allows for the targeted management of the underlying disease. Primary aldosteronism (PA) is the most common form of endocrine hypertension. Different adrenal diseases are responsible for PA: i) aldosterone-producing adenoma (APA or Conn's adenoma, ~50% of cases); ii) idiopathic hyperaldosteronism or bilateral adrenal hyperplasia (BAH, 30-40%); iii) unilateral primary adrenal hyperplasia (documented unilateral aldosterone secretion without detectable adenoma, 5-10%) and iv) aldosterone producing adrenal carcinoma in rare cases. Once the diagnosis of PA has been made, it is important to identify its etiology, in order to distinguish between surgically correctable forms (APA and unilateral primary adrenal hyperplasia) and forms to be treated pharmacologically (BAH). Patients with PA exhibit more severe left ventricular hypertrophy and diastolic dysfunction than patients with essential hypertension and a high prevalence of myocardial infarction, stroke and atrial fibrillation. Moreover, among unilateral forms of PA, hypertension is cured after surgery in less than 50% of patients. The understanding of the pathogenic mechanisms underlying PA is then essential to allow for the development of new diagnostic tools and biomarkers and for the identification of new therapeutic targets, concerning up to 10% of the hypertensive population. Increasing evidence shows the relevance of local mechanisms regulating aldosterone production in the adrenal, as opposed to cardiovascular regulation by the renin-angiotensin system. Our research project aims to investigate pivotal aspects of the mechanisms implicated in local control of aldosterone secretion in the adrenal through a strategy that integrates functional genomics, mouse models and clinical studies, with the aim to better understand the physiopathology of PA for improved therapeutic intervention in hypertensive patients. The three French partners and the German partner implicated in the project have a long-lasting and very productive collaboration record in the field of the study of the physiopathology of aldosterone production. In the framework of their previous studies, critical advances have been made in the understanding of the physiopathology of PA, with the characterization of the in vivo role of potassium channels in the regulation of aldosterone secretion by the adrenal cortex and the identification of somatic mutations driving APA phenotype. The first part of our project will focus on the identification of new genes involved in the pathogenesis of APA using the unique model of the Kcnk3 null mice previously characterized by our consortium, which present sex- and sexual hormone-dependent PA, and that allowed to identify a restricted set of genes with a potential role in the regulation of aldosterone secretion in the adrenal. Using a strategy that will integrate genetic, cell and animal studies, we will identify those genes that are relevant for aldosterone secretion in vivo and are associated to PA. In the second part of our project we will tackle the problem of the dissociation of hypersecretion and neoplastic nodule formation in pathological adrenals that overproduce aldosterone. We will produce the first mouse models with an altered function in genes whose homologs are implicated in APA and by genomic analysis we will identify genetic abnormalities in multinodular adrenals whose role in the nodulation process will be validated by producing ad hoc mouse models. We are confident that the results of our project will shed new light on the pathogenetic mechanisms of PA and allow to design new targeted therapies for this disease.

The study of the brain diseases with protein aggregates also called proteinopathies as Prion disorders, Parkinson’s disease or Alzheimer’s disease has opened new questioning. In fact, aggregation of proteins related to these diseases (PrP, alpha-synuclein, amyloid peptide and Tau) is tightly associated to neuronal death. Such protein aggregates would have biochemical and infectious prion-like characteristics allowing them to imprint their intrinsic structure onto the normal forms of their constituting proteins and act by trans-cellular transfer of the pathological property. Such transfer allows for pathology spreading, which is made according to precise cellular pathways. In the project SPREADTAU, we focus our interest on microtubule-associated Tau proteins. These proteins aggregate in numerous neurodegenerative disorders so-called Tauopathies of which the most known is Alzheimer’s disease. In the brain, there are six Tau isoforms having three (3R) or four microtubule-binding domains (4R). Whereas the six Tau isoforms (3R/4R) aggregate in the brain of Alzheimer’ patients, only 4R Tau isoforms form fibrils in other Tauopathies such as progressive supranuclear palsy. Conversely, in Pick’s disease, only 3R Tau isoforms aggregate suggesting the existence of an isoform-specific mechanism. Finally, hierarchical pathways of this neurodegeneration have been well established in Alzheimer’s disease and other sporadic tauopathies such as argyrophilic grain disorder and progressive supranuclear palsy but the molecular and cellular mechanisms supporting this progression are yet not known. Deciphering how and which toxic Tau species are implied in the trans-cellular transfer of Tau pathology will define new perspectives not only in diagnosis of neurodegenerative diseases but also in therapeutical development. To address these fundamental questions, the consortium proposes, first of all, to study the nucleation and fibrillogenesis of the different Tau assemblies either individually or mixed in various environments: test tube, cell culture or rodent brain. Once characterized, Tau assemblies will be used to determine the cellular tropism (either in neurons or glial cells) of Tau isoforms/assemblies and to evaluate their efficiency to be transported in neurons between the soma-dendritic compartment and the axon. Their subsequent secretion in the extra-cellular medium and ability to be transferred from primary cell (donor cells) to secondary cell population (recipient cells) will be done using dedicated microfluidic devices. This will allow to the consortium for deciphering which Tau assemblies and which cell types in the brain are implied in the trans-cellular transfer of Tau. Such findings may help to the understanding of the progressive and hierarchical appearance of Tau pathology in sporadic tauopathies. Finally, we will test the hypothesis of prion-like Tauopathy by validating Tau capacity to pass on its misfolding to normal Tau proteins in keeping the notion of strains likely to be related to 3R and 4R characteristics. All of these questions will be addressed in vitro by using original and dedicated microfluidics systems and validated in vivo in the rodent brain.