Sensory, cognitive, and emotional processing are rooted in the cerebral cortex. The cerebral cortex is comprised of six layers defined by the neurons within them that have distinctive connection, both within cortex itself and with other subcortical structures. Although still far from solving the mysteries of the mind, it is clear that networks form by neurons in the cerebral cortex provide the computational substrate for a remarkable range of behaviours. This neuron to neuron activation is mediated through dendritic spines, the postsynaptic target of most excitatory synapses in the cerebral cortex. Dendritic spines are small protrusions found along dendrites of neurons, and their number, as well as structural changes, accompany the development of synapses and establishment of neuronal networks. In fact, dendritic spines undergo rapid structural and functional changes guided by neuronal activity. This marriage between structural and functional plasticity, makes dendritic spines crucial in fine-tuning of networks in the brain; not surprisingly, dendritic spine aberrations are a hallmark of multiple neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. With this in mind, I considered Pannexin 1 (Panx1) an interesting novel candidate for a regulatory role on cortical neuronal network and dendritic spine development, for the following reasons. First, Panx1 transcripts are enriched in the brain and in the cortex are most abundant during the embryonic and early postnatal period. . This timing roughly corresponds to a period of synaptogenesis in the postnatal brain. Second, Panx1 localizes to postsynaptic compartments in neurons and its disruption leads to enhance excitability and potentiation of neuron-to neuron communication. Third, disruption of Panx1 (either knockdown or pharmacological blockade) leads to neurite outgrowth in neuron-like cells. Lastly, genetic variants in PANX1 have been associated with neurodevelopmental disorders. This dissertation explores the role of Panx1 in the development of dendritic spines and neuronal networks, furthering our understanding on cortical development and placing Panx1 as a novel regulator of structural and functional plasticity in the brain. Chapter 1 discusses core concepts on cortical development, with an emphasis on pyramidal neuron, the most abundant and only known projection neurons in the cerebral cortex. Here, I review the embryonic origin of pyramidal neurons, their postnasal development, and how cortical circuits are assembled. I finish this chapter with a brief review on Panx1 and its known expression and involvement in neuronal function. In Chapter 2 I explore the functional properties of neuronal networks and synaptic composition of cortical neurons in the absence of Panx1. Using live cell imaging and analysis of Ca2+ transients in dense primary cortical cultures, revealed that Panx1 knock-out (KO) cultures exhibit more and larger groups of significantly co-activated neurons, known as network ensembles. These network properties were not explained by differences in cell viability or cell-type composition. Examination of protein expression from cortical synaptosome preparations revealed that Panx1 is enriched in synaptic compartments, while also confirming a developmental downregulation. This analysis also revealed increased levels of the postsynaptic scaffolding protein PSD-95, along with the postsynaptic glutamate receptors GluA1 and GluN2A. Lastly, ex vivo tracing of dendritic spines on apical dendrites of Layer 5 pyramidal neurons in global and glutamatergic-specific Panx1 KO brain slices revealed higher spine densities in early and late postnatal development, with no differences in spine length. Analysis of dendritic spine densities in fixed cultured cortical neurons revealed an increase associated with Panx1 KO. Altogether, this work presents for the first time a connection between Panx1 and structural development of dendritic spines and suggest that Panx1 regulates cortical neuronal networks through changes in spine density. Chapter 3 examines the influence of Panx1 on spiny protrusions growth and movement. Spiny protrusion are long, thin, highly dynamic spine precursors. Taking advantage of a fluorescent signal localized to the plasma membrane, I visualized spiny protrusions and quantified their dynamics in wildtype (WT) and Panx1 KO developing cortical neurons, both under fixed and live conditions. I found that transient Panx1 expression is associated with decreased spiny protrusion density both in WT and Panx1 KO neurons. Using live cell imaging, I found that spiny protrusions are more stable and less motile in Panx1 KO neurons, while its transient expression reversed both of these phenotypes. These results suggest that Panx1 regulation of dendritic spines development is rooted partly in the regulation of spiny protrusion dynamics. Overall, this dissertation demonstrates that Panx1 negatively regulates dendritic spines in part by influencing spiny protrusion dynamics. It is reasonable to speculate that Panx1 regulation of dendritic spines underlies its newly discovered role in the formation network ensembles, providing a putative mechanism for previously described roles of Panx1 in synaptic plasticity. Likewise, this body of work furthers our understanding of Panx1 by filling some of the gaps attached to its developmental expression and association with neurodevelopmental disease. / Graduate / 2021-04-16

Les carcinomes épidermoïdes cutanés (CEC) sont des cancers invasifs du kératinocyte qui se développent suite à la subversion du système immunitaire. Une reprogrammation de l’immunité antitumorale par stimulation locale des cellules dendritiques (DC) et des macrophages pourrait être bénéfique. Dans ce contexte, l’objectif de ma thèse a été de comprendre l’hétérogénéité ontogénique, phénotypique et fonctionnelle de ces cellules immunitaires au cours de la carcinogenèse cutanée afin de développer des approches thérapeutiques les stimulant. Nous avons, d’abord, caractérisé les macrophages de la peau de souris et infiltrant les CEC chez l’homme. Nous avons identifié par cytométrie en flux spectrale une population de macrophages matures, autofluorescents et résidents de la peau de souris. Ces macrophages résidents présentent une polarisation fonctionnelle d’homéostasie et de réparation tissulaire. Dans les CEC humains, les macrophages autofluorescents semblent avoir les mêmes caractéristiques que leurs équivalents dans la peau de souris. Dans une seconde étude, nous avons mis en évidence que les DC associées aux tumeurs sont dysfonctionnelles. Une immunothérapie locale composée d’un agoniste de TLR9 et d’un anticorps bloquant la signalisation sous le récepteur à l’IL-10 induit la régression de tumeurs cutanées. Cette approche permet la reprogrammation fonctionnelle des DC et la génération de lymphocytes T CD8+ producteurs d’IFNγ, de TNFα et d’IL-17. Ces résultats mettent en évidence l’hétérogénéité fonctionnelle des cellules myéloïdes dans la peau et les CEC. Leur reprogrammation fonctionnelle permettrait le développement de nouvelles thérapeutiques contre les CEC chez l’homme. / Skin Squamous cell carcinoma (sSCC) are invasive keratinocyte tumor that develop after immune system subversion. The reprogrammation of anti-tumoral immunity using local stimulation of dendritic cells (DC) and macrophages could be useful. In this line, the aim of my thesis was to understand ontogenic, phenotypic and functional heterogeneity of these cell subsets along skin carcinogenesis to develop new immunotherapies. First, we characterized skin macrophage subsets in mouse and those infiltrating sSCC in human. Using spectral flow cytometry, we identified matured autofluorescent tissue-resident macrophages. These macrophages are anti-inflammatory polarized. In human sSCC, autofluorescent macrophages seem to have same properties that their mouse counterparts. In second study, we identified tumor-infiltrating DC with altered functions. We used a local immunotherapy composed by a TLR9 agonist and blocking antibody against α-chain of IL-10 receptor. This combination induced tumor regression through DC reprogrammation and IFNγ+, TNFα+ IL17A+ T CD8+ lymphocyte generation. These results highlight functional myeloid heterogeneity in skin and sSCC. Their reprogrammation could promote the development of immunotherapies against sSCC in human.

No description available.

In the nervous system, billions of neurons undergo a multistep process to establish functional circuits. This entails accurate extension of dendritic and axonal processes and coordinated efforts of pre- and postsynaptic neurons to form synaptic connections. Although many axon guidance molecules and synaptic organizers have been identified, the molecular redundancy and the vast number of synapses in the brain has complicated attempts to define their precise roles. In order to understand the molecular mechanisms that encompass these processes, my studies utilize the genetic strengths and cellular precision available in Caenorhabditis elegans for in vivo investigations of nervous system development. In this work, I unravel cell-specific requirements for the transmembrane receptor integrin in regulating developmental axon guidance of GABAergic motor neurons. Furthermore, I address important questions about mechanisms of synapse formation and maintenance using a novel dendritic spine model in C. elegans. Using high resolution microscopy, I find that the formation of immature presynaptic vesicles and postsynaptic receptors are established prior to the outgrowth of dendritic spines at nascent synapses. During this early period of synapse formation, the kinesin-3 family protein UNC-104/KIF1A transports a transsynaptic adhesion molecule neurexin/NRX-1 to developing active zones, in order to maintain postsynaptic receptors and dendritic spines in the mature circuit. In the absence of nrx-1, spines initially form normally but collapse following their extension. These findings demonstrate that presynaptic NRX-1 is required to maintain postsynaptic structures. Together my work provides new insights into molecular mechanisms that define spatiotemporal characteristics of nervous system development and the maintenance of connectivity.

Dectin-1/CLEC7A is a pattern recognition receptor that recognizes β-1,3 glucans, and its stimulation initiates signaling events characterized by the production of inflammatory cytokines from human dendritic cells (DCs) required for antifungal immunity. β-glucans differ greatly in size, structure, and ability to activate effector immune responses from DC; as such, small particulate β-glucans are thought to be poor activators of innate immunity. We show that β-glucan particle size is a critical factor contributing to the secretion of cytokines from human DC; large β-glucan-stimulated DC generate significantly more IL-1β, IL-6, and IL-23 compared to those stimulated with the smaller β-glucans. In marked contrast, the secretion of TSLP and CCL22 were found to be insensitive to β-glucan particle size. Furthermore, we show that the capacity to induce phagocytosis, and the relative IL-1β production determined by β-glucan size, regulates the composition of the cytokine milieu generated from DC. This suggests that β-glucan particle size is critically important in orchestrating the nature of the immune response to fungi.

The aim of the present study was to develop a dendritic cell (DC) vaccine encoding hepatitis C virus (HCV) multiple cytotoxic T lymphocyte (CTL) epitopes that can stimulate T cell responses in vitro, and can be used for immunization in vivo. DCs were infected with recombinant replication-defective adenoviruses (Ads) expressing 2 HCV sequences fused with green fluorescent protein (GFP) and FLAG tags. One sequence (sequence 1) contained the HCV CTL epitopes, NS4B 1793-1801 and P7 774-782, as well as the HCV Th epitope, NS3 1248-1261. A second sequence (sequence 2) was the positive epitope control which contained HCV core 35-44, core 132-140 and NS3 1248-1261. The efficiency of infection was detected by flow cytometry and the expression of HCV epitopes in the DCs was confirmed by RT-PCR and western blot analysis. Ad infection significantly enhanced DC maturation and interleukin (IL)-12p70 production, resulting in T cell proliferation and increased interferon-γ secretion. The CTLs stimulated by Ad-infected DCs specifically killed Huh7.5 human hepatoma cells. The recombinant Ad-expressing multiple CTL HCV epitopes effectively infected the DCs in vitro and promoted T cell antiviral immune responses, thereby laying the foundation for the development of anti-HCV DC vaccines.

The aim of the present study was to develop a dendritic cell (DC) vaccine encoding hepatitis C virus (HCV) multiple cytotoxic T lymphocyte (CTL) epitopes that can stimulate T cell responses in vitro, and can be used for immunization in vivo. DCs were infected with recombinant replication-defective adenoviruses (Ads) expressing 2 HCV sequences fused with green fluorescent protein (GFP) and FLAG tags. One sequence (sequence 1) contained the HCV CTL epitopes, NS4B 1793-1801 and P7 774-782, as well as the HCV Th epitope, NS3 1248-1261. A second sequence (sequence 2) was the positive epitope control which contained HCV core 35-44, core 132-140 and NS3 1248-1261. The efficiency of infection was detected by flow cytometry and the expression of HCV epitopes in the DCs was confirmed by RT-PCR and western blot analysis. Ad infection significantly enhanced DC maturation and interleukin (IL)-12p70 production, resulting in T cell proliferation and increased interferon-γ secretion. The CTLs stimulated by Ad-infected DCs specifically killed Huh7.5 human hepatoma cells. The recombinant Ad-expressing multiple CTL HCV epitopes effectively infected the DCs in vitro and promoted T cell antiviral immune responses, thereby laying the foundation for the development of anti-HCV DC vaccines.

Hepatitis C virus (HCV) infection is a global health problem characterized by a high rate of chronic infection, which may in part be due to a defect in myeloid dendritic cells (mDCs). This defect appears to be remedied by treatment with interferon-α (IFN-α) -based antiviral therapies; however, the molecular mechanisms underlying mDC dysfunction in HCV infection and restoration by IFN-α treatment are unclear. The ubiquitin-editing protein A20 plays a crucial role in controlling the maturation, cytokine production and immunostimulatory function of mDCs. We propose that the expression of A20 correlates with the function of mDCs during HCV infection and IFN-α therapy. In this study, we observed that A20 expression in mDCs isolated from chronically HCV-infected subjects was significantly higher than healthy subjects or subjects achieving sustained virological responses (SVR) following antiviral treatment. Notably, A20 expression in mDCs from HCV patients during IFN-α treatment was significantly lower than for untreated patients, SVR patients, or healthy subjects. Besides, A20 expression in mDCs stimulated by polyI:C differed between HCV patients and healthy subjects, and this difference could be abrogated by the treatment with IFN-α in vitro. Additionally, A20 expression by polyI:C-activated mDCs, with or without IFN-α treatment, negatively correlated with the expression of HLA-DR, CD86 and CCR7, and the secretion of interleukin-12 (IL-12), but positively associated with the production of IL-10. Importantly, silencing A20 expression using small interfering RNAs increased the production of IL-12 in mDCs of chronically HCV-infected individuals. These findings suggest that A20 plays a crucial role in negative regulation of innate immune responses during chronic viral infection.

OBJECTIVE. To determine if the levels of circulating myeloid-derived suppressor cells increase with progression of prostate cancer (PCa); to determine if such cells could contribute to the relative inefficiency of PCa immunotherapy. MATERIALS AND METHODS. We analyzed peripheral blood mononuclear cells isolated from untreated PCa patients (uPCa; N=18; mean age±SD: 72.1± 6.9 years), tPCa (N = 22; 72.8 ± 9.8 years) and age matched controls (AMC; N = 12; 68.8 ± 7.5 years). We quantified surface marker phenotype, differentiation potential, effects on T cell proliferation and intracellular cytokines. RESULTS. We observed an unexpectedly high percentage of a type of myeloid-derived suppressor cells, CD14+HLA-DR low/- monocytes, in tPCa (30.7±15.0% of CD14+ cells) relative to AMC (4.1+6.5%, P<0.0001) and uPCa (10.6 ± 14.3%, P = 0.0001). The levels of CD14+ HLA-DR low/- cells were significantly correlated with circulating PSA levels and treatment with LHRH-agonist leuprolide in combination with either an antiandrogen or dexamethasone. Monocytes from tPCa inhibited autologous T cell proliferation statistically significantly more effectively than AMC monocytes and were defective in their ability to differentiate into phenotypically mature dendritic cells. Isolated CD14+HLA-DRlow/- cells expressed higher levels of intracellular interleukin-10 and suppressed T cell proliferation more effectively than isolated CD14+HLA-DR+ cells. CONCLUSIONS. This is the first report of CD14+ cells exhibiting reduced expression of HLADRmolecules in PCa patients. These cells suppress immune cell function in vitro and, plausibly, in vivo, a finding that must be factored into the design of immunotherapy protocols for PCa patients.

Schizophrenia postmortem brain is characterized by γ aminobutyric acid downregulation and by decreased dendritic spine density in frontal cortex. Protracted L-methionine treatment exacerbates schizophrenia symptoms, and our earlier work (Tremolizzo et al. and Dong et al.) has shown that L-methionine decreases reelin and GAD67 transcription in mice which is prevented by co-administration of valproate. In this study, we observed a decrease in spine density following L-methionine treatment, which was prevented by co-administration of valproate. Together with our earlier findings conducted under the same experimental conditions, we suggest that downregulation of spine density in L-methionine-treated mice may be because of the decreased expression of reelin and that valproate may prevent spine downregulation by inhibiting the methylation induced decrease in reelin.