Team 07 – D. Dunia / C. Malnou - Institut Toulousain des Maladies Infectieuses et Inflammatoires

Pathogenesis of viral infections of the developing and adult central nervous system

Coordinators: D. Dunia / C. Malnou

D. Dunia

AXIS 1 : Viral interference with neuronal homeostasis

C. Malnou

AXIS 2 : Role of extracellular placental vesicles during congenital viral infections

E. Suberbielle

AXIS 3 : Impact of pathogens on neuronal epigenetics and behavior

G. Ligat

AXIS 4 (emerging axis): Molecular biology of host-human cytomegalovirus interactions in brain tumors and therapeutic innovations

X:

https://twitter.com/ViNeDysLab

Scientific Objectives

To date, the mechanisms whereby pathogens can interfere with brain function are not well understood and the determinants responsible for the associated diseases are poorly defined. Our team has a strong interest in studying the mechanisms and consequences of pathogen infection of the central nervous system (CNS), from the developing to the adult brain.

For our different projects, we use several paradigms of CNS infections: infection by Borna Disease virus (BoDV), congenital infection by human Cytomegalovirus (hCMV) and Zika virus (ZIKV) and persistence of the parasite Toxoplasma gondii in the adult CNS.

In the context of the adult brain, we assess the pathophysiological and behavioral outcome of pathogen persistence in the CNS, either due to the pathogen itself or to the accompanying neuroinflammation. We also aim at identifying processes which could be highjacked by neurotropic pathogens and contribute to neuronal impairment or the resulting neurodegenerative processes.

In the case of congenital infections during pregnancy, we study how viruses can disrupt the normal neurodevelopmental program and lead to severe sequelae in the newborn. In this setting, we examine the modalities of congenital infection and the resulting impact on neonatal brain development. These later aspects are facilitated by the integration in our team of several clinicians from the neonatology unit in Toulouse hospital, who help us to facilitate the clinical translation of our findings.

Our Projects

AXIS 1: Viral interference with neuronal homeostasis

In our laboratory, we study neuronal infection by Borna disease virus (BoDV-1), because we believe that this virus provides an ideal paradigm for studying the behavioral correlates of CNS viral infections. BoDV-1 is the perfect example of a neurotropic virus which achieves long-term persistence, while causing minimal damage to neurons. BoDV-1 infection, however, leads to brain dysfunction and disease, as a result of the selective interference with signaling pathways that are crucial for proper central nervous system (CNS) homeostasis. The ongoing project of our team are:

BoDV-1 as a neuroprotective tool? We recently showed that the BoDV-1 X protein, or a cell-permeable peptide derived from the X protein were able to prevent neurodegeneration in different in vitro and in vivo settings, by acting at the mitochondrial level.

The BoDV-1 X protein is neuroprotective. Top: The X protein protects against rotenone-induced axonal fragmentation. Pictures of the axonal chambers of compartmentalized primary neuronal cultures subjected to rotenone treatment. Bottom: analysis of dopaminergic neurons (staining for tyrosine hydroxylase in the SNpc and the Striatum) in the brains of the MPTP mouse model for Parkinson’s disease.

We are now pursuing different aims : first, to gain further insight on the mechanisms underlying the neuroprotective properties of X, by deciphering its mitochondrial interactome and impact on mitochondrial physiology and dynamics; next, to further document the proof of concept of using X-derived peptides for neuroprotection, by testing its therapeutic potential in various models of chronic neurodegeneration, in the frame in collaborations in Toulouse, Bordeaux or Paris with experts in various neurodegenerative diseases.

Structure-function analysis of the BoDV replication machinery and impact on neuronal epigenetics. Considering the recent identification of BoDV-1 as a potential zoonotic agent, we have established collaborations with structural biologists and virus-host interaction experts. Our goals are to perform a comprehensive analysis of the BoDV replication machinery, a large-scale mapping of virus/host polypeptide interplays and of its consequences on viral fitness and on neuronal physiology. Together with Dr. E. Suberbielle in our team, we notably analyze the resulting consequences on neuronal epigenetics.

Development of a novel nanoelectronics platform to probe neuronal activity. In collaboration with physicists from the LAAS-CNRS laboratory in Toulouse, we have developed nanowire-based devices (Nano-electrode arrays, or NEA) interfaced with living neurons. We are now testing the relevance of such NEA to measure different parameters of neuronal activity (response to stimulation, plasticity, etc..) upon infection, expression of proteins of interest (BoDV-1 P and X proteins, notably), as well as after exposure to other neuronal insults, such as amyloid ß peptide or proinflammatory cytokines. This project is supported by the CNRS MITI (Mission for Transversal and Interdisciplinary Initiatives)

Nano-electrode array (NEA) setup to record neuronal activity. (A) Scanning electron micrograph of one NEA, with an enlarged view (B) of one electrode composed of 7 nanowires. Pictures taken at the LAAS CNRS. (C) NEA mounted on a printed circuit board, before neuronal seeding (D) and setting onto the recording setting (E). Copyright David Villa, Science Image, CNRS. (F) Staining of the neuronal network (using MAP2, shown in red). Copyright Frédérique Sueur, Infinity. (G) Examples of recordings made before and after stimulation of the neurons.

Head: D. Dunia

AXIS 2: Role of extracellular placental vesicles during congenital viral infections

Our research project explores the emerging concept that infection of the placenta with viruses such as human cytomegalovirus (hCMV) or Zika virus (ZIKV) could alter the amount and / or composition of placental extracellular vesicles (EVs) (Figure 1), which could then promote the dissemination of the virus towards the fetus and contribute to the alteration in brain development observed during congenital infections.

Figure 1: Analysis of small extracellular vesicles and their internalization in recipient cell. A) Placental small extracellular vesicles observed by electron microscopy, with gold bead immunostaining directed against CD63 surface molecule (collab. Institut Curie). B) Fluorescence microscopy image of a fibroblast cell after small extracellular vesicles internalization (Blue: DAPI, Red: actin, Green: membrane labeled-vesicles) extracellulaires (Bleu: DAPI, Rouge: actine, Vert: marquage membranaire des vésicules)

Impact of the infection of placental cells on the secretion and composition of EVs and consequences for the development of the fetal central nervous system.

Our results indicate that, upon infection with hCMV, there is a phenotypic modification of exosomes secreted from placental histocultures of early placentas or cultured trophoblast lines. The phenotypic characteristics of EVs isolated from infected cells suggest that they could act in a paracrine manner to facilitate the infection of surrounding or distant cells and that they thus participate in the pathophysiology of the disorders observed during pregnancy and in the fetus. Our work also suggests that EVs could play a role of biomarkers during infection in order to evaluate, via a simple blood test from the mother during pregnancy, the state of the placenta.

Role of the miRNA C19MC cluster carried by placental EVs.

EVs derived from the placenta are capable of conferring antiviral protection on target cells, via the action of the cluster of miRNA C19MC, contained in the EVs. Thanks to an original model of cells deleted for the entire cluster, we are studying the modalities of the antiviral action of these miRNAs. In addition, we are investigating whether placental EVs can also protect human neural stem cells from viral infection. Finally, we are evaluating their expression profile in EVs circulating in pregnant women during infections by hCMV, ZIKV or SARS-CoV2 in order to assess whether these infections modify their expression profile within EVs during pregnancy.

In conclusion, we hope to provide new pathophysiological elements to better understand the etiology of congenital viral infections, in order to improve the diagnosis and also offer new perspectives for therapeutic intervention.

Head: C. Malnou

AXIS 3: Impact of pathogens on neuronal epigenetics and behavior

The hypothesis at the center of our research interests:

Because of their lifelong persistence in the brain or the immune responses raised against them, pathogens in the central nervous system could contribute to neuronal dysfunction, cognitive senescence and neuropsychiatric diseases. From a mechanistic point of view, neuronal epigenetics, which is key to cognitive plasticity, may play a fundamental role in these disorders. In particular, we have shown that DNA double-strand breaks (DSB) represent a critical mode of regulation of neuronal epigenetics. We are interested in deciphering the mechanisms whereby DSB affect neuronal function and in understanding how perturbations in sensing, production and/or repair of DSB may underlie the behavioral impairment that is observed in many inflammatory, infectious, or age-related diseases affecting the CNS.

3 lines of research are currently being pursued:

Role of DSB in the pathophysiology of infection by Borna disease virus (BoDV).

Our recent data indicate that DSB contribute to the regulation of BoDV replication with consequences on the activity of the infected neuronal networks. Understanding how this structurally simple virus disrupts the DSB response will allow us to elucidate the mechanisms of DSB response in neurons.

Infection of the central nervous system by Toxoplasma gondii, a common human parasite.

Studying the impact of various strains of this parasite on behavior and the underlying mechanisms serves as a model for deciphering how a persistent infection in the brain could contribute to cognitive and immune senescence during healthy vs. pathological aging. In collaboration with Team 6 (link).

Epigenetic dysregulations of neuronal function in response to chronic inflammation. Due to aging or a lifelong response to environmental threats, chronic inflammation and in particular, the systemic increases in inflammatory mediators, are thought to have long-lasting impact on behavior. We study the how DSB response and epigenetic changes may be fundamental mechanisms underlying chronic inflammation-induced behavioral deficits.

Finally, the results of our research will allow us to elucidate the key mechanisms of DNA DSB response in neurons under pathophysiological circumstances, to maintain the physiological balance between DNA DSB generation and repair. In the long-term, our research may pave the way for new therapies against chronic neuroinflammation or Alzheimer’s disease.

Head: E. Suberbielle

AXIS 4 (emerging axis): Molecular biology of host-human cytomegalovirus interactions in brain tumors and therapeutic innovations

Our projects focus on the link between human cytomegalovirus (HCMV) infection and glioblastoma, an aggressive form of brain cancer.

Many of these projects have a translational aspect and include the discovery of new biomarkers and therapeutic targets. Through various collaborations* and the support of « Défi Clé Cell Based Biotherapies Occitanie », we are currently working on the vectorization of anti-HCMV therapeutic peptides into extracellular vesicles to improve their efficacy.

We hope to better understand the role of HCMV in the development and progression of brain tumors to identify new prognosis and therapeutic approaches.

*Prof. Sophie Alain, Prof. Sébastien Hantz (NRC Herpesvirus, INSERM UMR 1092, Limoges), Dr Marie Morille (Charles Gerhardt Montpellier Institute – CNRS UMR 5253, Montpellier)

Keywords: HCMV, Glioblastoma, Biotherapies

Head: G. Ligat (gaetan.ligat@inserm.fr)