Alzheimer’s disease (AD) constitutes the most common form of dementia worldwide and is characterized by the accumulation of extracellular senile plaques mediated by amyloid- (Aβ), intracellular neurofibrillary tangles, synaptic degeneration and neuronal injury. It is considered an urgent social and economic problem due to the lack of effective therapeutic therapies or curative approaches. Until recent times the central nervous system was considered an immune-privileged site, still, the contribution of peripheral immune cells to brain functions highlighted their role in CNS physiology and disease. Neutrophils constitute the most abundant leukocyte population in human circulation and our group has identified a crucial role for these cells in the pathogenesis of AD. Other reports have then further supported this role, emphasizing peripheral neutrophil hyperactivation in AD, although the cause is still unclear. The recent discovery of gastrointestinal dysbiosis in neurodegeneration, including AD, and the subsequent pro-inflammatory response associated with increased circulating microbial products points to a broad systemic dysregulation of the immune system in AD patients. Still, neutrophil dysregulation and its impact on peripheral and brain inflammation in AD are not well understood and were the focus of this doctoral thesis. In the first part, we studied peripheral neutrophil dysregulation and characterized intestinal microbiota changes of 3xTg-AD mice and healthy wild-type (WT) mice. Our results showed a profound dysbiosis characterized by an increase in pro-inflammatory bacteria in 3xTg-AD mice. In addition, we showed that dysbiosis was accompanied by inflammatory changes in the intestinal tract, including a decrease in the number of mucus-producing cells and an increase in permeability of the intestinal tract. Moreover, we identified enteric nervous system (ENS) degeneration and neutrophil infiltration as early events in AD pathogenesis, suggesting a link between neutrophils and intestinal changes in AD mice. To further connect dysbiosis and peripheral inflammation, we performed a phenotype characterization of circulating neutrophils. The analysis of peripheral blood identified a persistent state of neutrophilia and, most importantly, an increase of immature neutrophils in the 3xTg-AD mice, suggesting the activation of “emergency granulopoiesis”. Interestingly, we showed a positive correlation between intestinal permeability and the levels of circulating neutrophils, further showing an interplay between gut dysbiosis and granulopoiesis. To further understand the role of neutrophil pathological changes in AD, in the second part of this project, we studied neutrophil dysfunction from a “central” point of view. Importantly, scRNAseq of brain-infiltrating neutrophils revealed genes associated with neutrophil degranulation, which was later confirmed using flow cytometry. scRNA-seq also identified Ctse as one of the most upregulated genes in infiltrating neutrophils, suggesting its role in the pathogenesis of AD. Interestingly, in vitro stimulation of both human and mouse neutrophils with A led to an increase in the degranulation of neutrophils that was abolished when neutrophils were pretreated with a CTSE inhibitor. To address the potential of CTSE as a therapeutic target, we pre-treated neutrophils with a CTSE inhibitor and cocultured these cells with neurons observing a strong decrease in neuronal death when CTSE was blocked, pointing to CTSE as a key mediator of neutrophil neurotoxicity in AD. Overall, this thesis provides novel insights into neutrophil dysregulation in a mouse model of AD, suggesting that dysbiosis may contribute to peripheral neutrophil responses and neuroinflammation in AD. Furthermore, our data suggest that targeting neutrophil-dependent neurotoxic mechanisms may represent a novel and promising therapeutic strategy for AD.

DISSECTING THE MECHANISMS OF NEUTROPHIL DYSREGULATION AND NEUROTOXICITY IN ALZHEIMER'S DISEASE

dos Santos Lima, Bruno Miguel
2022

Abstract

Alzheimer’s disease (AD) constitutes the most common form of dementia worldwide and is characterized by the accumulation of extracellular senile plaques mediated by amyloid- (Aβ), intracellular neurofibrillary tangles, synaptic degeneration and neuronal injury. It is considered an urgent social and economic problem due to the lack of effective therapeutic therapies or curative approaches. Until recent times the central nervous system was considered an immune-privileged site, still, the contribution of peripheral immune cells to brain functions highlighted their role in CNS physiology and disease. Neutrophils constitute the most abundant leukocyte population in human circulation and our group has identified a crucial role for these cells in the pathogenesis of AD. Other reports have then further supported this role, emphasizing peripheral neutrophil hyperactivation in AD, although the cause is still unclear. The recent discovery of gastrointestinal dysbiosis in neurodegeneration, including AD, and the subsequent pro-inflammatory response associated with increased circulating microbial products points to a broad systemic dysregulation of the immune system in AD patients. Still, neutrophil dysregulation and its impact on peripheral and brain inflammation in AD are not well understood and were the focus of this doctoral thesis. In the first part, we studied peripheral neutrophil dysregulation and characterized intestinal microbiota changes of 3xTg-AD mice and healthy wild-type (WT) mice. Our results showed a profound dysbiosis characterized by an increase in pro-inflammatory bacteria in 3xTg-AD mice. In addition, we showed that dysbiosis was accompanied by inflammatory changes in the intestinal tract, including a decrease in the number of mucus-producing cells and an increase in permeability of the intestinal tract. Moreover, we identified enteric nervous system (ENS) degeneration and neutrophil infiltration as early events in AD pathogenesis, suggesting a link between neutrophils and intestinal changes in AD mice. To further connect dysbiosis and peripheral inflammation, we performed a phenotype characterization of circulating neutrophils. The analysis of peripheral blood identified a persistent state of neutrophilia and, most importantly, an increase of immature neutrophils in the 3xTg-AD mice, suggesting the activation of “emergency granulopoiesis”. Interestingly, we showed a positive correlation between intestinal permeability and the levels of circulating neutrophils, further showing an interplay between gut dysbiosis and granulopoiesis. To further understand the role of neutrophil pathological changes in AD, in the second part of this project, we studied neutrophil dysfunction from a “central” point of view. Importantly, scRNAseq of brain-infiltrating neutrophils revealed genes associated with neutrophil degranulation, which was later confirmed using flow cytometry. scRNA-seq also identified Ctse as one of the most upregulated genes in infiltrating neutrophils, suggesting its role in the pathogenesis of AD. Interestingly, in vitro stimulation of both human and mouse neutrophils with A led to an increase in the degranulation of neutrophils that was abolished when neutrophils were pretreated with a CTSE inhibitor. To address the potential of CTSE as a therapeutic target, we pre-treated neutrophils with a CTSE inhibitor and cocultured these cells with neurons observing a strong decrease in neuronal death when CTSE was blocked, pointing to CTSE as a key mediator of neutrophil neurotoxicity in AD. Overall, this thesis provides novel insights into neutrophil dysregulation in a mouse model of AD, suggesting that dysbiosis may contribute to peripheral neutrophil responses and neuroinflammation in AD. Furthermore, our data suggest that targeting neutrophil-dependent neurotoxic mechanisms may represent a novel and promising therapeutic strategy for AD.
Neutrophils, Alzheimer's disease
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11562/1065264
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