Dengue virus-elicited tryptase induces endothelial permeability and shock.

Dengue virus (DENV) infection causes a characteristic pathology in humans involving dysregulation of the vascular system. In some patients with dengue hemorrhagic fever (DHF), vascular pathology can become severe, resulting in extensive microvascular permeability and plasma leakage into tissues and organs. Mast cells (MCs), which line blood vessels and regulate vascular function, are able to detect DENV in vivo and promote vascular leakage. Here, we identified that a MC-derived protease, tryptase, is consequential for promoting vascular permeability during DENV infection, through inducing breakdown of endothelial cell tight junctions. Injected tryptase alone was sufficient to induce plasma loss from the circulation and hypovolemic shock in animals. A potent tryptase inhibitor, nafamostat mesylate, blocked DENV-induced vascular leakage in vivo. Importantly, in two independent human dengue cohorts, tryptase levels correlated with the grade of DHF severity. This study defines an immune mechanism by which DENV can induce vascular pathology and shock

Dysregulation of actin dynamics in amyotrophic lateral sclerosis

Empirical thesis.Bibliography: pages 197-237.1. General introduction -- 2. Materials and methods -- 3. C9ORF72 regulates actin dynamics -- 4. Dysregulation of actin dynamics in ALS -- 5. Mechanisms involved in dysregulation of actin dynamics in ALS -- 6. General discussion -- 7. References -- 8. Appendices.Mutations in the C9ORF72 (chromosome 9 open reading frame 72) gene account for 40% of familial cases of Amyotrophic lateral sclerosis (ALS), and pathological forms of TDP-43 in motor neurons are present in almost all cases of ALS. Currently, there is no effective treatment for this disorder. Therefore, given their importance in ALS, understanding the pathological roles of C9ORF72 and TDP-43 is crucial for developing effective therapeutic strategies.The mechanisms underlying neurodegeneration in ALS are still not fully understood. Whilst defects in cytoskeletal organisation and cytoskeletal proteins have been previously associated with ALS, the role of actin filaments and actin binding proteins in ALS has not been previously examined. The overall aim of the studies described in this thesis was to examine the regulation of actin dynamics and actin binding proteins in C9ORF72 and TDP-43 related ALS. Firstly, the normal cellular function of C9ORF72 was examined in Chapter 3. Here it was demonstrated that C9ORF72 is an actin binding protein that regulates actin dynamics. Furthermore, the uDENN domain of C9ORF72 is required for this activity via cofilin-mediated signalling mechanisms. These studies therefore provide novel insights into the normal cellular function of C9ORF72. Secondly, in Chapter 4, it was demonstrated that actin dynamics is disturbed by pathological forms of TDP-43 in cells expressing cytoplasmic TDP-43, in the TDP-43 rNLS mice model and in sporadic ALS (SALS) patient tissues. These studies therefore demonstrate that TDP-43 pathology is associated with increased actin polymerisation, possibly mediated by a direct interaction between actin and TDP-43. Moreover, actin polymerisation causes both mis-localisation of TDP-43 to the cytoplasm and stress granule formation, implying that actin polymerisation induces pathological events relevant to ALS. Finally, the regulation of actin-binding and regulatory proteins in the TDP-43 rNLS mice model and in SALS patients was examined in Chapter 5. Cofilin phosphorylation and profilin-1 expression was enhanced in SALS patients. Furthermore, increased levels of Rac1/cdc42 and Limk1 phosphorylation were also detected, thus providing mechanistic insights into these observations. Similarly, increased levels of profilin-1 and cofilin phosphorylation correlated with disease course pathologically and phenotypically in TDP-43 rNLS mice, consistent with the findings obtained in SALS patients. While actin related proteins; Arp2, Arp3 and ARPC3 were decreased at a later stage in TDP-43 rNLS mice. In conclusion, this thesis provides novel insights into the mechanisms of neurodegeneration in ALS. Importantly, it identifies dysregulation of actin dynamics and actin binding proteins as novel disease mechanisms in both C9ORF72 and TDP-43 associated ALS.Mode of access: World wide web1 online resource (xxvii, 249 pages) colour illustration

Dengue vascular leakage is augmented by mast cell degranulation mediated by immunoglobulin Fcγ receptors

10.7554/eLife.05291.001eLife2015

Th1-Polarized, Dengue Virus-Activated Human Mast Cells Induce Endothelial Transcriptional Activation and Permeability

International audienceDengue virus (DENV), an arbovirus, strongly activates mast cells (MCs), which are key immune cells for pathogen immune surveillance. In animal models, MCs promote clearance of local peripheral DENV infections but, conversely, also promote pathological vascular leakage when widely activated during systemic DENV infection. Since DENV is a human pathogen, we sought to ascertain whether a similar phenomenon could occur in humans by characterizing the products released by human MCs (huMCs) upon direct (antibody-independent) DENV exposure, using the phenotypically mature huMC line, ROSA. DENV did not productively infect huMCs but prompted huMC release of proteases and eicosanoids and induced a Th1-polarized transcriptional profile. In co-culture and trans-well systems, huMC products activated human microvascular endothelial cells, involving transcription of vasoactive mediators and increased monolayer permeability. This permeability was blocked by MC-stabilizing drugs, or limited by drugs targeting certain MC products. Thus, MC stabilizers are a viable strategy to limit MC-promoted vascular leakage during DENV infection in humans

The microglial NLRP3 inflammasome is activated by amyotrophic lateral sclerosis proteins

Microglial NLRP3 inflammasome activation is emerging as a key contributor to neuroinflammation during neurodegeneration. Pathogenic protein aggregates such as β‐amyloid and α‐synuclein trigger microglial NLRP3 activation, leading to caspase‐1 activation and IL‐1β secretion. Both caspase‐1 and IL‐1β contribute to disease progression in the mouse SOD1G93A model of amyotrophic lateral sclerosis (ALS), suggesting a role for microglial NLRP3. Prior studies, however, suggested SOD1G93A mice microglia do not express NLRP3, and SOD1G93A protein generated IL‐1β in microglia independent to NLRP3. Here, we demonstrate using Nlrp3‐GFP gene knock‐in mice that microglia express NLRP3 in SOD1G93A mice. We show that both aggregated and soluble SOD1G93A activates inflammasome in primary mouse microglia leading caspase‐1 and IL‐1β cleavage, ASC speck formation, and the secretion of IL‐1β in a dose‐ and time‐dependent manner. Importantly, SOD1G93A was unable to induce IL‐1β secretion from microglia deficient for Nlrp3, or pretreated with the specific NLRP3 inhibitor MCC950, confirming NLRP3 as the key inflammasome complex mediating SOD1‐induced microglial IL‐1β secretion. Microglial NLRP3 upregulation was also observed in the TDP‐43Q331K ALS mouse model, and TDP‐43 wild‐type and mutant proteins could also activate microglial inflammasomes in a NLRP3‐dependent manner. Mechanistically, we identified the generation of reactive oxygen species and ATP as key events required for SOD1G93A‐mediated NLRP3 activation. Taken together, our data demonstrate that ALS microglia express NLRP3, and that pathological ALS proteins activate the microglial NLRP3 inflammasome. NLRP3 inhibition may therefore be a potential therapeutic approach to arrest microglial neuroinflammation and ALS disease progression

Protein quality control and the amyotrophic lateral sclerosis/frontotemporal dementia continuum

Protein homeostasis, or proteostasis, has an important regulatory role in cellular function. Protein quality control mechanisms, including protein folding and protein degradation processes, have a crucial function in post-mitotic neurons. Cellular protein quality control relies on multiple strategies, including molecular chaperones, autophagy, the ubiquitin proteasome system, endoplasmic reticulum (ER)-associated degradation (ERAD) and the formation of stress granules (SGs), to regulate proteostasis. Neurodegenerative diseases are characterized by the presence of misfolded protein aggregates, implying that protein quality control mechanisms are dysfunctional in these conditions. Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that are now recognized to overlap clinically and pathologically, forming a continuous disease spectrum. In this review article, we detail the evidence for dysregulation of protein quality control mechanisms across the whole ALS-FTD continuum, by discussing the major proteins implicated in ALS and/or FTD. We also discuss possible ways in which protein quality mechanisms could be targeted therapeutically in these disorders and highlight promising protein quality control-based therapeutics for clinical trials

The microglial NLRP3 inflammasome is activated by amyotrophic lateral sclerosis proteins

Microglial NLRP3 inflammasome activation is emerging as a key contributor to neuroinflammation during neurodegeneration. Pathogenic protein aggregates such as β-amyloid and α-synuclein trigger microglial NLRP3 activation, leading to caspase-1 activation and IL-1β secretion. Both caspase-1 and IL-1β contribute to disease progression in the mouse SOD1 model of amyotrophic lateral sclerosis (ALS), suggesting a role for microglial NLRP3. Prior studies, however, suggested SOD1 mice microglia do not express NLRP3, and SOD1 protein generated IL-1β in microglia independent to NLRP3. Here, we demonstrate using Nlrp3-GFP gene knock-in mice that microglia express NLRP3 in SOD1 mice. We show that both aggregated and soluble SOD1 activates inflammasome in primary mouse microglia leading caspase-1 and IL-1β cleavage, ASC speck formation, and the secretion of IL-1β in a dose- and time-dependent manner. Importantly, SOD1 was unable to induce IL-1β secretion from microglia deficient for Nlrp3, or pretreated with the specific NLRP3 inhibitor MCC950, confirming NLRP3 as the key inflammasome complex mediating SOD1-induced microglial IL-1β secretion. Microglial NLRP3 upregulation was also observed in the TDP-43 ALS mouse model, and TDP-43 wild-type and mutant proteins could also activate microglial inflammasomes in a NLRP3-dependent manner. Mechanistically, we identified the generation of reactive oxygen species and ATP as key events required for SOD1 -mediated NLRP3 activation. Taken together, our data demonstrate that ALS microglia express NLRP3, and that pathological ALS proteins activate the microglial NLRP3 inflammasome. NLRP3 inhibition may therefore be a potential therapeutic approach to arrest microglial neuroinflammation and ALS disease progression

ALS/FTD-associated mutation in cyclin F inhibits ER-Golgi trafficking, inducing ER stress, ERAD and Golgi fragmentation

Abstract Amyotrophic lateral sclerosis (ALS) is a severely debilitating neurodegenerative condition that is part of the same disease spectrum as frontotemporal dementia (FTD). Mutations in the CCNF gene, encoding cyclin F, are present in both sporadic and familial ALS and FTD. However, the pathophysiological mechanisms underlying neurodegeneration remain unclear. Proper functioning of the endoplasmic reticulum (ER) and Golgi apparatus compartments is essential for normal physiological activities and to maintain cellular viability. Here, we demonstrate that ALS/FTD-associated variant cyclin FS621G inhibits secretory protein transport from the ER to Golgi apparatus, by a mechanism involving dysregulation of COPII vesicles at ER exit sites. Consistent with this finding, cyclin FS621G also induces fragmentation of the Golgi apparatus and activates ER stress, ER-associated degradation, and apoptosis. Induction of Golgi fragmentation and ER stress were confirmed with a second ALS/FTD variant cyclin FS195R, and in cortical primary neurons. Hence, this study provides novel insights into pathogenic mechanisms associated with ALS/FTD-variant cyclin F, involving perturbations to both secretory protein trafficking and ER-Golgi homeostasis