1) Dynamics of aging and anti-aging compounds:

Aging is a process that remains poorly understood despite significant progress made in the past 3 decades. In addition, given demographic changes in human societies, its deleterious consequences, in particular the increasing incidence of age-related pathologies, are weighing more and more on the socioeconomic level. It is therefore critical to progress in our understanding both of the fundamental mechanisms of aging but also on its implications in terms of associated pathologies, in particular in tissues that are not or not very replicative in humans, such as the nervous system or the heart muscle, which are particularly susceptible to aging.

In recent years, on the basis of pioneering experimental work by Michael Rera carried out in the laboratory, we have developed a new model of theoretical aging based on the existence of 2 discontinuous phases in the life of an individual, with specific characteristics. This model faithfully reproduces the longevity curves observed as well as other characteristics such as the increase in the levels of inflammation genes with age. Interestingly, experimentally, these two phases of aging (characterized by the sudden change in intestinal permeability) are progressively conserved, especially in the vertebrate species analyzed.

Within the framework of this new vision of discontinuous aging, one of our projects consists in researching other earlier stages of aging and in characterizing them molecularly. High throughput sequencing approaches will be used for this purpose.

Simultaneously, in collaboration with the Chinese company Infinitus, we are analyzing the characteristics of different compounds of traditional Chinese medicine (TCM) to counteract aging-related processes (resistance to stress, cardiac dysfunction, reduction in locomotor activity, etc.). Our first results show significant effects for 3 MTC compounds whose molecular mode of action must be characterized.

 

2) Neurodegenerative and cardiac pathologies

Amplifications of triplets in coding or non-coding regions of several genes can lead to degenerative pathologies in different organs. We are studying more particularly 3 of them, focusing on finding new therapeutic strategies. A new research program focused on the LRRK2 protein involved in Parkinson’s disease (PD) has also started with the support of IDEX.

2-a: Polyglutamine-expanding diseases (Huntington’s disease (HD) and Spinocerebellar ataxia type 3 (SCA3):

These two diseases are linked to amplifications of the triplet CAG, coding for glutamine, in the coding regions of the Huntingtin (Htt) and Ataxin 3 (Atxn3) genes respectively. They are both proteinopathies, characterized by the appearance of intranuclear aggregates and the degeneration of specific regions of the central nervous system such as the cerebellum, the pons, the cortex, …

As part of the European TreatPolyQ program, we collaborated with the teams of L. Pereira de Almeida (Coimbra, Portugal) and T. Schmidt (Tübingen, Germany) to study the role of certain microRNAs and nuclear export factors in the SCA3 pathology. Following previous observations that the cytoplasmic ataxin 3 protein, even with pathological CAG expansion, is harmless, we focused on a family of transporters, karyopherins, which transport macromolecules from the cytoplasm to the nucleus, and vice versa. We have thus identified the transporter KPNA3 (also called Importin subunit alpha-4) as being a key element in the nuclear localization of ataxin 3. Thus, thanks to the Drosophila model, we have shown in vivo that the repression of expression of KPNA3 in a pathological context, via RNA interference, leads to the relocation of the mutated ataxin 3 protein (mAtxn3) in the cytoplasm of the cells of the salivary gland of flies. In Drosophila, the specific expression of mAtxn3 in the eye causes large morphological defects of this organ, as well as degeneration of the retina. When we repress KPNA3 in this model of targeted expression of mAtxn3 in the eye, we observed a decrease in the aggregation of the mutant protein and an improvement in the neurodegeneration of the eye of flies (Fig. 1 ). In mice (work of the laboratory of Dr. T. Schmidt), it has also been shown that the decrease in expression of KPNA3 improves the locomotor performance of SCA3 mice, as well as their behavior. These results have highlighted the importance of transport proteins in the progression of this pathology, and more particularly KPNA3 as a potential therapeutic target.

Fig. 1: Under control conditions (SCA3 + control), the mAtx3 is localized in a diffuse manner in the cells of the salivary gland of the Drosophila (A, image on the left). The repression of the expression of KPNA3 (SCA3 + KPNA3 KD) allows the relocation of mAtx3 in the cytoplasm of the cells of the Drosophila salivary gland (A, right image). When mAtx3 is expressed specifically in the Drosophila eye, it causes major morphological defects: depigmentation, necrosis, general disorganization (B, left image). The decrease in KPNA3 induces a clear improvement in these defects (B, right image), the eye is partially repigmented and it is partly reorganized. Looking at the retina of these flies, we can see a clear improvement in the morphology of photoreceptors when the expression of KPNA3 is reduced (C, right pictures). Indeed, under control conditions, there are vacuoles in the retina, as well as a clear disorganization of the photoreceptors. In addition, this rescue of the retina morphology of flies is accompanied by a decrease in the aggregation of mAtx3 (D).

 

In this same program, we characterized in a Drosophila model the cardiac pathology associated, in humans, with HD. We have shown that the inducible expression of a pathological form of huntingtin (mHtt) in the heart is accompanied by cardiac dilation. Building on previous work on Freidreich’s ataxia (see below), we have shown that this dysfunction can be partially corrected by treatment with methylene blue. This new inducible model opens the way to genetic and pharmacological investigations allowing to better understand and to be able to correct the cardiac pathology in HD.

In addition to the cardiac and neuronal models of HD, we have developed an inducible glial model of HD. This model allows us to explore the consequences of the expression of mHtt in the different cell types of the CNS (glia or neurons), in terms of longevity, behavior (locomotor activity, circadian rhythms, ..) and genetic modulators of pathology. We have identified profound differences, at these different levels, between the neural and glial models of HD, which illustrates the complexity of the disease. We are currently characterizing in depth the different signaling pathways involved and, on these bases, are exploring new therapeutic strategies.

We have also developed new Drosophila models of HD, using CRISPR / Cas9 techniques, which are currently under study.

2-b: Freidreich’s ataxia (AF):

Friedreich’s ataxia is a mitochondrial pathology that mainly affects the nervous system and the heart. It is due to an expansion of GAA triplets in the first intron of the FXN gene, which causes the expression of this gene to decrease. FXN encodes frataxine, a small mitochondrial protein involved in the synthesis of iron-sulfur centers and very conserved during evolution. We use Drosophila to better understand the mechanisms involved in this pathology and to seek new therapeutic avenues.

In a project supported by the ANR (FiFA2 project, 2012-2017), we have shown that frataxine is involved in steroidogenesis in Drosophila. In collaboration with Joëlle Cohen-Tannoudji’s team in our unit, we have highlighted a similar function in human ovarian cells, suggesting the existence of hormonal disturbances in Friedreich patients. We have also established a heart model of AF in Drosophila. Heart-specific inactivation of frataxine leads to cardiac dilation with loss of contractility of the cardiomyocytes. We have shown that administration of methylene blue (BM) helps prevent heart dysfunction, possibly by improving the mitochondrial respiratory chain activity of frataxin-deficient hearts. BM being a compound already used in clinical for various applications, it constitutes a promising candidate to prevent cardiac attacks which constitute the first cause of mortality in AF patients.

As part of a project funded by the American agency FARA (2014-2016), we also carried out a medium-scale pharmacological screen (1280 compounds) on this cardiac model. We have therefore optimized our cardiac imaging technique (Fig.2) which allowed us to analyze the cardiac function on several thousand flies (around 20,000 in the context of this study). This screen identified several molecules with a cardioprotective effect.

As part of a project funded by the Rare Disease Alliance (2014-2015), we generated new models of AF in Drosophila by humanizing the Drosophila frataxin gene. To do this, we inserted into the intron of the Drosophila gene of frataxin, intronic sequences of the human gene carrying GAA expansions. These expansions cause Drosophila to decrease the expression of the gene and reproduce symptoms associated with human pathology (locomotor and cardiac deficits, reduced viability).

As part of AFAF-funded projects (2017-2020), we are currently exploiting these new AF models with GAA triplet expansions. In particular, we are evaluating on these models an innovative therapeutic strategy by genome editing based on the CrispR / Cas9 system. We are also developing pharmacological approaches based on national and international collaborations.

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Fig. 2: In vivo cardiac imaging in Drosophila:

Anesthetized Drosophila are placed on a slide under a fluorescent binocular magnifier. They are lit at the excitation wavelength of a fluorescent protein specifically expressed in the heart. The fluorescent light emitted is captured through the dorsal cuticle by a high-speed camera, which allows the cardiac parameters to be extracted.

 

3) Search for efficacy biomarkers for monitoring patients with neurodegenerative diseases: application in case of treatments

Deregulation of the metabolism of monocarbons is associated with an increased incidence of several chronic diseases, such as hyperhomocysteinemia, Alzheimer’s disease and other metabolic diseases such as type 2 diabetes and obesity. Although the molecular mechanisms that trigger Alzheimer’s disease are not fully understood, recent data suggests that dyslipidemia may contribute to its progression. Some patients with Down’s syndrome develop dementia comparable to that of Alzheimer’s disease at the age of 30-40 years. Pathological aging in the subject with trisomy 21 is associated with dementia syndrome which combines, to varying degrees, disorders of cognitive functions and behavior modifying the personality. These patients also have dyslipidemia with deregulations of the metabolism of monocarbons. Among the genes located on chromosome 21, the expression of DYRK1A, a serine threonine kinase, and CBS (cystathionine beta synthase), an enzyme involved in the metabolism of monocarbons, is deregulated in Alzheimer’s disease. We have demonstrated a relationship between DYRK1A and CBS and their involvement in the metabolism of monocarbons, cholesterol and insulin, all metabolisms deregulated in Alzheimer’s disease, hyperhomocysteinemia, trisomy 21 and type 2 diabetes. Targeting these two proteins for pharmacological treatment requires a better understanding of their function and of their interacting partners. The search for interactants, in collaboration with Dr JM Camadro (Jacques Monod Institute, UMR CNRS 7592), has enabled the identification of proteins already shown as potential biomarkers in the serum of patients suffering from Alzheimer’s disease. These different biomarkers are currently being analyzed in plasma and cerebrospinal fluid from patients with Alzheimer’s disease and patients with trisomy 21 with or without dementia and at the prodromal stage (in collaboration with Dr Anne-Sophie Rebillat (Institute Jérôme Lejeune, Paris) and Pr J Fortea (Memory Unit, Department of Neurology, Hospital de la Santa Creu i Sant Pau-Biomedical Research Institute Sant Pau-Universitat Autònoma de Barcelona, Barcelona, Spain).The evaluation of these biomarkers in the patient’s serum combined with imaging parameters and cerebrospinal fluid markers should make it possible to develop a prognostic / diagnostic test with the aim of identifying people at high risk of developing Alzheimer’s disease and who will be able to benefit from treatments.

In addition to validating new blood biomarkers, brain-plasma evaluation in mouse and rat models allows us to study the neurobiological mechanisms underlying deregulation of these biomarkers. To this end, we seek to demonstrate their role in the progression of Alzheimer’s disease by using the first inducible and progressive rat model of the disease, in collaboration with Dr Jérôme Braudeau (AgenT).

These mouse models also allow us to search for treatments targeting DYRK1A and CBS using a computational approach and in vitro and in vivo tests (as part of a consortium, Fig 3).

Fig. 3: Consortium for the research of inhibitors by computational approach and in vitro and in vivo tests

As part of the research for treatments, these biomarkers are also used at the preclinical stage in order to analyze the effects of treatments at an early stage, which we are currently developing in two main directions:

1/a way targeting more specifically the activity of DYRK1A with a specific pharmacological inhibitor (in collaboration with Dr Yann Hérault (Institute of Molecular and Cellular Biology, UMR 7104 of CNRS, U1258 INSERM, Strasbourg) and Dr Laurent Meijer (Perha Pharmaceuticals, Roscoff)).

2/ a way to test a molecule at the embryonic stage (in collaboration with Pr François Vialard (Université Paris-Saclay, UVSQ, INRAE, ENVA, BREED, 78350, Jouy-en-Josas, France) and Pr Anne-Claude Camproux (Université de Paris, BFA, UMR 8251, CNRS, ERL U1133, Inserm)).

As part of a clinical study, a first phase II randomized trial with placebo, which was conducted by different teams including our group, made it possible to demonstrate that EGCG improves cognitive and learning capacities. young adults with Down’s syndrome (Fig. 4). This study is currently being continued in young children with Down’s syndrome, after determining the dose and efficacy using a preclinical model, as part of a phase II study in collaboration with Dr Cécile Cieuta -Walti (Jérôme Lejeune Institute, Paris) and Pr Rafael de la Torre (IMIM-Hospital del Mar Medical Research Institute, Barcelona).

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Fig. 4: Principle of the phase 2 clinical trial with EGCG, a DYRK1A inhibitor, in patients with Down’s syndrome