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The NF-κB signaling pathway is crucial during development and inflammatory processes. We have previously shown that NF-κB activation induces dedifferentiation of astrocytes into neural progenitor cells (NPCs). Here, we provide evidence that the NF-κB pathway plays also a fundamental role during the differentiation of NPCs into astrocytes. First, we show that the NF-κB pathway is essential to initiate astrocytic differentiation as its early inhibition induces NPC apoptosis and impedes their differentiation. Second, we demonstrate that persistent NF-κB activation affects NPC-derived astrocyte differentiation. Tumor necrosis factor (TNF)-treated NPCs show NF-κB activation, maintain their multipotential and proliferation properties, display persistent expression of immature markers and inhibit astrocyte markers. Third, we analyze the effect of NF-κB activation on the main known astrocytic differentiation pathways, such as NOTCH and JAK-STAT. Our findings suggest that the NF-κB pathway plays a dual fundamental role during NPC differentiation into astrocytes: it promotes astrocyte specification, but its persistent activation impedes their differentiation.

M1 and M2 are the extremes of the differentiation spectrum of activated macrophages. Since microglia are members of the same cell lineage, we have characterized their transcription profile and their phagocytic activity under different conditions. LPS or IFN-gamma induce a M1-like phenotype, while IL-10 or IL-4 differentiate microglia towards a M2-deactivated or M2-alternatively-activated phenotype respectively. These differentiation processes also affect the Notch pathway. In order to study the polarization induced by Abeta, microglia was stimulated with different forms of the peptide. The oligomeric Abeta is a stronger M1-inductor than the fibrillar form. Moreover, a cytokine-induced anti-inflammatory environment reduces the microglial reactivity towards oligomeric Abeta.

For a long time, the nervous system and the immune system have been studied as isolated entities, but a growing body of evidence shows that there is an extensive crosstalk between both systems. In fact, neurons and immune cells share certain functional features and reside in close proximity within the tissues, enabling them to effectively communicate. T cells are crucial for mounting and controlling almost any kind of immune response. However, when dysregulated, T cells fail to protect the host from invading pathogens or can cause damage to surrounding tissues, leading to autoimmunity-related pathology. In the first part of this cumulative thesis, we aimed at identifying novel genes regulating CD4 T cell responses and identified VIMP, one of the 25 human proteins containing the 21st amino acid selenocysteine, as a gene having anti-inflammatory functions. Furthermore, T cells express various neurotransmitter receptors allowing the integration of neuronal signal for an appropriate response. In the second part, we showed a CD4-T-cell-intrinsic mechanism through which stress hormones mediate their control over the immune system. We identified a previously unrecognized pathway regulating CD4 T cell differentiation that involves the circadian clock gene Per1 and mTORC1 signalling. Finaly, T cells involvement in different neuropathologies has been reported in the past few decades. Emerging evidence indicates the involvement of the immune system and in particular T cells in the pathogenesis of Parkinson’s disease (PD), the 2nd most common neurodegenerative disease. In the 3rd part of the thesis we systematically characterized the immunological status of early-to-mid stage PD patients and matched healthy controls, and identified a distinct peripheral immunological fingerprint in PD patients, especially in the CD8 T-cell compartment. The findings of the studies described in this cumulative thesis advance our understanding of the regulatory nodes of CD4 T cells during a stress response and fill the knowledge gap on the early involvement of CD8 T cells and other immune subsets in neurodegenerative diseases in the case of PD.

Stress is commonly defined as the response to a non-specific situation presenting a psychological and/or physical challenge. In order to react in an appropriate manner to environmental threats the body will trigger a wide range of defence mechanisms. However cases where challenges are sustained and the individual does not have the ability to cope with the stress are nowadays believed to be a main factor for the onset and exacerbation of a broad range of disorders. Among these are psychiatric disorders such as depression but also pain affections. Pain is described by the International Association for the study of Pain (IASP) as an unpleasant sensory and emotional experience associated with actual or potential tissue damage. As for stress, this system aiming at preserving the body integrity can become defective and enhance pain sensitivity or foster the development of chronic pain. These two health problems categories each represent a considerable issue of public health. Indeed the World Health Organization (WHO) estimates that 27% of the European adult population had experienced at least one episode of mental disorder and the IASP reported that 19% of the pan-European population experienced chronic pain (Macfarlane, Pain 2016). Furthermore, chronic pain and stress-related disorders are greatly comorbid, having deleterious effects on the efficacy of treatments. Despite the raising awareness of clinical and pre-clinical research on their overlapping pathways, common mediators and interactions, the nature of the relationship between chronic pain conditions and stress-related disorders is not yet elucidated. The studies I undertook during my Ph.D. aimed to understand how chronic stress, with an emphasis on early-life stress, is linked to altered nociceptive transmission and to modified chronic pain vulnerability. Early life stress (ELS) was of particular interest as this period of life is subjected to an intense neuronal plasticity of notably stress and pain systems. Furthermore it is increasingly accepted that early life factors are linked to the susceptibility to develop chronic pain conditions in adulthood. As pain is a multidimensional system, I had to restrict my studies to one of the relay stations for the transmission of pain. In the context of chronic stress, most of the work was done on the brain circuits underlying the affective part of pain but little is known about the effect of chronic pain on spinal nociceptive processes. Since chronic stress is a broad phenomenon altering not only processing at the brain level, I focused my studies on spinal dorsal horn noxious transmission. The first stage of my work was to assess the impact of ELS on neuropathic pain, a type of chronic pain arising from nerve lesions. In a second step, I sought to determine if the results obtained in this first study were specific to the type of pain (neuropathic) or also were valid for another type of chronic pain, e.g. chronic inflammatory pain. The third study aimed to determine if ELS would predispose to enhanced vulnerability to stress exposure later in life and to concomitant alterations of chronic pain. To finish, during the last year of my Ph.D. I investigated the possible mechanisms underlying the behavioural results using pharmacological manipulations and initiated a separate project devoted to in vivo electrophysiological characterization of the response behaviour of nociceptive spinal dorsal horn neurons. In the following introduction, I will give an overview of the stress systems, focusing on the hypothalamo-pituitary adrenal (HPA) axis and early life stress. Then I will outline the different part of the pain system and concepts of central sensitization involved in chronic pain.

Neutrophil granulocytes are the first effector cells to be recruited to sites of infection. They rapidly release granule proteins and pro-inflammatory cytokines to efficiently kill intruding pathogens and recruit other immune cells. In exocytotic events, specific interactions of so-called soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins lead to the formation of complexes in order to mediate membrane fusion. Because of the excessive release of neutrophil-derived mediators leading to the development of chronic inflammatory disorders, we aimed in the present scientific work to investigate in more details the regulatory processes of mediator release in neutrophils during inflammation, with emphasis on SNARE proteins. The main objectives were i) to characterize the release of pro-inflammatory mediators, ii) to profile SNARE expression, iii) to determine the functional role of SNAREs and SNARE complexes in cytokine release and degranulation, and iv) to identify the intracellular localization of SNAREs. To characterize the pro-inflammatory response in neutrophils in regard to exocytosis, extensive kinetic studies were performed in a first step on LPS-stimulated primary neutrophils. A novel linear fitting approach was created to correlate the relationship between granule proteins and cytokines secreted to the inflammatory site. In a second step, SNARE expression levels were determined by whole-transcript analysis and the similar profiles in primary neutrophils as well as DMSO-differentiated HL-60 cells (dHL-60 cells), a neutrophil cell model, were underlined. Using an RNAi strategy, the SNARE syntaxin 3 (STX3) was identified as an essential actor in the release of the cytokines IL-1α, IL-1β, IL-12b, and CCL4. It was also involved in MMP-9 exocytosis from gelatinase granules where it could partly be localized. The SNARE SNAP29, which shares common localization with STX3, functionally affects the release of IL-12b, CCL2 and IL-8 as well as MMP-9, and represents a potential candidate to form cognate complexes with STX3. The knockdown of VAMP3, another SNARE candidate, showed deregulated secretion of IL-12b, CCL4, IL-8 as well as MMP-9. However, VAMP3 was located at the plasma membrane and was thus excluded as being part of the STX3-SNAP29 complex. Our findings provide first evidence that SNARE fusion proteins are involved in the release of IL- 12b, IL-1α, IL-1β, CCL4, IL-8, and CCL2 in a neutrophil-like cell model. The impact of SNAREs on gelatinase degranulation led us to hypothesize that cytokines might be packaged in these granules before subsequent exocytosis. !