Défense publique de thèse de doctorat en vue de l’obtention du grade académique de Docteur en Sciences Biomédicales et Pharmaceutiques.

Résumé des objectifs de la thèse :

Mechanical forces are constantly exerted on cells and tissues within the human body and can lead to complex neuroinflammation events in the brain. Among the resident cells, astrocytes and microglia are the first line of defense for preserving the homeostasis of brain tissues and play a significant role in mediating the progression of a mechanical damage. However, both cell types can undergo mechanical deformations during brain injuries, leading to a possible alternative mechanism of activation. While the impact of chemical signaling on astrocytes and microglia function has been studied in much detail, the current understanding of their mechanical signaling is still limited. In the first part of this manuscript, we present an extensive introduction on brain cell mechanics with a specific focus on astrocyte and glial cells. We introduce some scaling concepts about the complex architecture and organization of brain tissues and we describe how physico-chemical changes of the cell matrix can lead to functional modulation of brain cells through mechanosensitive processes.

In the second part of the thesis, we focus our attention on the impact of biochemical compounds released by mechanically activated astrocytes on neuronal networks. We investigate whether cytokines produced by mechanically activated astrocytes can affect growth and synaptic connections of cortical neuronal networks. To this aim, astrocytes were cultivated on thin elastic membranes and subjected to repetitive mechanical stretches, whereas well-defined protein micropatterns were used to form standardized neuronal networks. By staining Glial Fibrillary Acidic Protein (GFAP), we found that astrocytes were mechanically activated after two cycles of stretch and Meso Scale Discovery (MSD) assays indicated that injured astrocytes released four major cytokines. To understand the role of these cytokines, neuronal networks were cultured with the supernatant of healthy or mechanically activated astrocytes and the individual contribution of the proinflammatory cytokine tumor necrosis factor α (TNF-α) was studied. Our findings indicated that the supernatant of two-cycle stretched astrocytes decreased presynaptic terminals and that TNF-α can be considered as a key player of the synaptic loss. Furthermore, our results showed that cytokines released by injured astrocytes significantly modulate the balance between TNFR1 and TNFR2 receptors by enhancing R2 receptors. We demonstrated that TNF-α is not involved in this process, suggesting a predominant role of other secreted cytokines. Altogether, the results presented in this first chapter contribute to a better understanding of the consequences of repetitive astrocyte deformations and highlight the role of inflammatory signaling pathways in synaptic plasticity and modulation of TNFR1 and TNFR2 receptors.

In the third part of this thesis, we used the BV2 cell line and primary microglial cells to study the impact of a mechanical injury on microglial cells. Both cell types were cultivated on thin elastic membranes and mechanically activated by a single uniaxial stretch (< 1 sec) of 20%, that mimics in vitro the mechanical deformations of brain tissues observed during traumatic brain injury (TBI) events. Our findings indicated that a single mechanical stretch exerted on microglial cells induced their activation state through the increase of the ionized calcium binding adapter protein 1 (IBA1) protein level. We observed a significant strengthening of the actin cytoskeleton of mechanically activated microglial cells, leading to their stiffening. Our findings showed that the nuclear volume of stretched glial cells increased significantly and we observed a large amount of DNA double-strand breaks, suggesting that a single mechanical stretch can significantly alter the nuclear compartment. Furthermore, we demonstrated that the phagocytosis activity of mechanically stretched glial cells was enhanced, suggesting a significant impact on their immune activity. To go a step further, we investigated whether a mechanical stretch exerted on microglial cells can change their phagocytosis activity on synapses. To this aim, we used cortical neuronal networks grown into microfluidic chambers that formed synaptic connections. Mechanically activated microglial cells were inserted in the microfluidic device and the quantification of the immunostained synapses indicated a higher phagocytic activity in mechanically activated glial cells. The results were confirmed by the observation of a higher amount of synaptic markers (PSD95 and synaptophysin) inside stretched microglial cells by confocal microscopy.

Altogether, our findings suggest that the mechanical activation of microglial cells could lead to an enhancement of the phagocytosis mechanism and thus to the modulation of neurophagy that contributes to brain development and diseases. It could be very interesting to determine which of the critical steps of phagocytosis (recognition, engulfment and digestion) is enhanced in response to a mechanical insult of microglial cells. Further investigations are needed to understand whether mechanical activation of glial cells can lead to excessive phagocytosis that can contribute to synaptic loss in some brain pathologies, and how synaptic pruning that occurs during development can be affected when glial cells are subjected to mechanical deformations.

Promotrice : Prof. Laurence RIS, Service de Neurosciences

Co-promoteur : Prof. Sylvain GABRIELE

Président du Jury : Prof. Lionel TAFFOREAU

Secrétaire du Jury : Prof. Alexandra TASSIN

Membres du jury :

Dr. Frédéric SAUDOU, CNRS – Grenoble Institut Neurosciences
Dr. Laurent NGUYEN, FNRS – Université de Liège