Coronavirus disease 2019 (COVID-19) is a global pandemic caused by SARS-CoV-2 infection and is associated with both acute and chronic disorders affecting the nervous system. Acute neurological ...disorders affecting patients with COVID-19 range widely from anosmia, stroke, encephalopathy/encephalitis, and seizures to Guillain-Barré syndrome. Chronic neurological sequelae are less well defined although exercise intolerance, dysautonomia, pain, as well as neurocognitive and psychiatric dysfunctions are commonly reported. Molecular analyses of CSF and neuropathological studies highlight both vascular and immunologic perturbations. Low levels of viral RNA have been detected in the brains of few acutely ill individuals. Potential pathogenic mechanisms in the acute phase include coagulopathies with associated cerebral hypoxic-ischaemic injury, blood-brain barrier abnormalities with endotheliopathy and possibly viral neuroinvasion accompanied by neuro-immune responses. Established diagnostic tools are limited by a lack of clearly defined COVID-19 specific neurological syndromes. Future interventions will require delineation of specific neurological syndromes, diagnostic algorithm development and uncovering the underlying disease mechanisms that will guide effective therapies.
Fiery Cell Death: Pyroptosis in the Central Nervous System McKenzie, Brienne A.; Dixit, Vishva M.; Power, Christopher
Trends in neurosciences (Regular ed.),
January 2020, 2020-Jan, 2020-01-00, 20200101, Volume:
43, Issue:
1
Journal Article
Peer reviewed
Pyroptosis (‘fiery death’) is an inflammatory type of regulated cell death (RCD), which occurs downstream of inflammasome activation. Pyroptosis is mediated directly by the recently identified family ...of pore-forming proteins known as gasdermins, the best characterized of which is gasdermin D (GSDMD). Recent investigations implicate pyroptosis in the pathogenesis of multiple neurological diseases. In this review, we discuss molecular mechanisms that drive pyroptosis, evidence for pyroptosis within the CNS, and emerging therapeutic strategies for its inhibition in the context of neurological disease.
Pyroptosis (‘fiery death’) is a lytic and highly inflammatory regulated cell death program, recently found to be driven by members of the gasdermin family, most notably gasdermin D (GSDMD).GSDMD is a pore-forming executioner protein that is proteolytically cleaved and activated by proinflammatory caspases (caspase-1/4/5/11). These caspases are localized on inflammasomes, large cytosolic protein complexes that serve as platforms upon which activated proinflammatory caspases cleave and activate their protein substrates.Cleaved GSDMD oligomerizes and forms pores in the plasma membrane, leading to disruption of local osmotic potential, formation of pyroptotic bodies, and eventual membrane rupture.During neuroinflammation and neurodegeneration, inflammasome activation and pyroptosis are initiated by acute perturbations in homeostasis (e.g., mitochondrial dysfunction, ion flux) as well as danger signals in the microenvironment (e.g., extracellular ATP, aberrant protein aggregates, double-stranded DNA, microbial molecules) that are present in the injured or diseased CNS.Pyroptosis occurs in multiple CNS cell types under pathological conditions (e.g., multiple sclerosis, Alzheimer’s disease, and stroke), leading to both neuroinflammation and neurodegeneration through multiple mechanisms.Targeting pyroptosis therapeutically shows promise in multiple preclinical models of neurological injury and disease.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Multiple sclerosis (MS) is a progressive inflammatory demyelinating disease of the CNS of unknown cause that remains incurable. Inflammasome-associated caspases mediate the maturation and release of ...the proinflammatory cytokines IL-1β and IL-18 and activate the pore-forming protein gasdermin D (GSDMD). Inflammatory programmed cell death, pyroptosis, was recently shown to be mediated by GSDMD. Here, we report molecular evidence for GSDMD-mediated inflammasome activation and pyroptosis in both myeloid cells (macrophages/microglia) and, unexpectedly, in myelin-forming oligodendrocytes (ODCs) in the CNS of patients with MS and in the MS animal model, experimental autoimmune encephalomyelitis (EAE). We observed inflammasome activation and pyroptosis in human microglia and ODCs in vitro after exposure to inflammatory stimuli and demonstrate caspase-1 inhibition by the small-molecule inhibitor VX-765 in both cell types. GSDMD inhibition by siRNA transduction suppressed pyroptosis in human microglia. VX-765 treatment of EAE animals reduced the expression of inflammasome- and pyroptosis-associated proteins in the CNS, prevented axonal injury, and improved neurobehavioral performance. Thus, GSDMD-mediated pyroptosis in select glia cells is a previously unrecognized mechanism of inflammatory demyelination and represents a unique therapeutic opportunity for mitigating the disease process in MS and other CNS inflammatory diseases.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Zika virus (ZIKV), a member of the
family, has recently emerged as an important human pathogen with increasing economic and health impact worldwide. Because of its teratogenic nature and association ...with the serious neurological condition Guillain-Barré syndrome, a tremendous amount of effort has focused on understanding ZIKV pathogenesis. To gain further insights into ZIKV interaction with host cells, we investigated how this pathogen affects stress response pathways. While ZIKV infection induces stress signaling that leads to phosphorylation of eIF2α and cellular translational arrest, stress granule (SG) formation was inhibited. Further analysis revealed that the viral proteins NS3 and NS4A are linked to translational repression, whereas expression of the capsid protein, NS3/NS2B-3, and NS4A interfered with SG formation. Some, but not all, flavivirus capsid proteins also blocked SG assembly, indicating differential interactions between flaviviruses and SG biogenesis pathways. Depletion of the SG components G3BP1, TIAR, and Caprin-1, but not TIA-1, reduced ZIKV replication. Both G3BP1 and Caprin-1 formed complexes with capsid, whereas viral genomic RNA stably interacted with G3BP1 during ZIKV infection. Taken together, these results are consistent with a scenario in which ZIKV uses multiple viral components to hijack key SG proteins to benefit viral replication.
There is a pressing need to understand ZIKV pathogenesis in order to advance the development of vaccines and therapeutics. The cellular stress response constitutes one of the first lines of defense against viral infection; therefore, understanding how ZIKV evades this antiviral system will provide key insights into ZIKV biology and potentially pathogenesis. Here, we show that ZIKV induces the stress response through activation of the UPR (unfolded protein response) and PKR (protein kinase R), leading to host translational arrest, a process likely mediated by the viral proteins NS3 and NS4A. Despite the activation of translational shutoff, formation of SG is strongly inhibited by the virus. Specifically, ZIKV hijacks the core SG proteins G3BP1, TIAR, and Caprin-1 to facilitate viral replication, resulting in impaired SG assembly. This process is potentially facilitated by the interactions of the viral RNA with G3BP1 as well as the viral capsid protein with G3BP1 and Caprin-1. Interestingly, expression of capsid proteins from several other flaviviruses also inhibited SG formation. Taken together, the present study provides novel insights into how ZIKV modulates cellular stress response pathways during replication.
Abstract
Background
Pyroptosis is a type of proinflammatory regulated cell death (RCD) in which caspase-1 proteolytically cleaves gasdermin D (GSDMD) to yield a cytotoxic pore-forming protein. Recent ...studies have suggested that additional cell death pathways may interact with GSDMD under certain circumstances to execute pyroptosis. Microglia/macrophages in the central nervous system (CNS) undergo GSDMD-associated pyroptosis in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) but the contribution of other cell death pathways to this phenomenon is unknown. Herein, we tested the hypothesis that multiple RCD pathways underlie microglial pyroptosis in the context of neuroinflammation.
Methods
A siRNA screen of genes with known RCD functions was performed in primary human microglia to evaluate their role in nigericin-induced pyroptosis using supernatant lactate dehydrogenase activity as a read-out of cell lysis. Activation of apoptotic executioner proteins and their contribution to pyroptosis was assessed using semi-quantitative confocal microscopy, high-sensitivity ELISA, immunoblot, cell lysis assays, and activity-based fluorescent probes. Quantification of pyroptosis-related protein expression was performed in CNS lesions from patients with progressive MS and mice with MOG
35-55
-induced EAE, and in matched controls.
Results
Among progressive MS patients, activated caspase-3 was detected in GSDMD immunopositive pyroptotic microglia/macrophages within demyelinating lesions. In the siRNA screen, suppression of caspase-3/7, caspase-1, or GSDMD expression prevented plasma membrane rupture during pyroptosis. Upon exposure to pyroptotic stimuli (ATP or nigericin), human microglia displayed caspase-3/7 activation and cleavage of caspase-3/7-specific substrates (e.g., DFF45, ROCK1, and PARP), with accompanying features of pyroptosis including GSDMD immunopositive pyroptotic bodies, IL-1β release, and membrane rupture. Pyroptosis-associated nuclear condensation and pyroptotic body formation were suppressed by caspase-3/7 inhibition. Pharmacological and siRNA-mediated inhibition of caspase-1 diminished caspase-3/7 activation during pyroptosis. In mice with EAE-associated neurological deficits, activated caspase-3 colocalized with GSDMD immunopositivity in lesion-associated macrophages/microglia.
Conclusions
Activation of executioner caspases-3/7, widely considered key mediators of apoptosis, contributed to GSDMD-associated microglial pyroptosis under neuroinflammatory conditions. Collectively, these observations highlight the convergence of different cell death pathways during neuroinflammation and offer new therapeutic opportunities in neuroinflammatory disease.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Recent studies have revealed that multiple sclerosis (MS) lesions have distinct microRNA (miRNA) expression profiles. miR-181 family members show altered expression in MS tissues although their ...participation in MS pathogenesis remains uncertain. Herein, we investigated the involvement of miR-181a and miR-181b in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis (EAE).
miR-181a and -b levels were measured in the central nervous system (CNS) of patients with MS and mice with EAE as well as relevant leukocyte cultures by real-time RT-PCR. To examine the role of the miRNAs in leukocyte differentiation and function, miR-181a and -b mimic sequences were transfected into cultured primary macrophages and purified CD4
T cells which were then analyzed by RT-PCR and flow cytometry. Luciferase reporter assays were performed to investigate the interaction of miR-181a and -b with the 3'-UTR of potential target transcripts, and the expression of target genes was measured in the CNS of EAE mice, activated lymphocytes, and macrophages.
Expression analyses revealed a significant decrease in miR-181a and -b levels in brain white matter from MS patients as well as in spinal cords of EAE mice during the acute and chronic phases of disease. Suppression of miR-181a was observed following antigen-specific or polyclonal activation of lymphocytes as well as in macrophages following LPS treatment. Overexpression of miR-181a and -b mimic sequences reduced proinflammatory gene expression in macrophages and polarization toward M1 phenotype. miR-181a and -b mimic sequences inhibited Th1 generation in CD4
T cells and miR-181a mimic sequences also promoted Treg differentiation. Luciferase assays revealed Suppressor of mothers against decapentaplegic 7 (Smad7), as a direct target of miR-181a and -b.
Our data highlight the anti-inflammatory actions of miR-181a and -b in the context of autoimmune neuroinflammation. miR-181a and -b influence differentiation of T helper cell and activation of macrophages, providing potential therapeutic options for controlling inflammation in MS.
HIV-associated neurocognitive disorders (HAND) represent a spectrum neurological syndrome that affects up to 25% of patients with HIV/AIDS. Multiple pathogenic mechanisms contribute to the ...development of HAND symptoms including chronic neuroinflammation and neurodegeneration. Among the factors linked to development of HAND is altered expression of host cell microRNAs (miRNAs) in brain. Here, we examined brain miRNA profiles among HIV/AIDS patients with and without HAND. Our analyses revealed differential expression of 17 miRNAs in brain tissue from HAND patients. A subset of the upregulated miRNAs (miR-500a-5p, miR-34c-3p, miR-93-3p and miR-381-3p), are predicted to target peroxisome biogenesis factors (PEX2, PEX7, PEX11B and PEX13). Expression of these miRNAs in transfected cells significantly decreased levels of peroxisomal proteins and concomitantly decreased peroxisome numbers or affected their morphology. The levels of miR-500a-5p, miR-34c-3p, miR-93-3p and miR-381-3p were not only elevated in the brains of HAND patients, but were also upregulated during HIV infection of primary macrophages. Moreover, concomitant loss of peroxisomal proteins was observed in HIV-infected macrophages as well as in brain tissue from HIV-infected patients. HIV-induced loss of peroxisomes was abrogated by blocking the functions of the upregulated miRNAs. Overall, these findings point to previously unrecognized miRNA expression patterns in the brains of HIV patients. Targeting peroxisomes by up-regulating miRNAs that repress peroxisome biogenesis factors may represent a novel mechanism by which HIV-1 subverts innate immune responses and/or causes neurocognitive dysfunction.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
There is currently no cure for HIV infection although adherence to effective antiretroviral therapy (ART) suppresses replication of the virus in blood, increases CD4
T-cell counts, reverses ...immunodeficiency, and increases life expectancy. Despite these substantial advances, ART is a lifelong treatment for people with HIV (PWH) and upon cessation or interruption, the virus quickly rebounds in plasma and anatomic sites, including the central nervous system (CNS), resulting in disease progression. With recent advances in quantifying viral burden, detection of genetically intact viral genomes, and isolation of replication-competent virus from brain tissues of PWH receiving ART, it has become apparent that the CNS viral reservoir (largely comprised of macrophage type cells) poses a substantial challenge for HIV cure strategies. Other obstacles impacting the curing of HIV include ageing populations, substance use, comorbidities, limited antiretroviral drug efficacy in CNS cells, and ART-associated neurotoxicity. Herein, we review recent findings, including studies of the proviral integration sites, reservoir decay rates, and new treatment/prevention strategies in the context of the CNS, together with highlighting the next steps for investigations of the CNS as a viral reservoir.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Gangliosides are glycosphingolipids highly enriched in the brain, with important roles in cell signaling, cell-to-cell communication, and immunomodulation. Genetic defects in the ganglioside ...biosynthetic pathway result in severe neurodegenerative diseases, while a partial decrease in the levels of specific gangliosides was reported in Parkinson's disease and Huntington's disease. In models of both diseases and other conditions, administration of GM1-one of the most abundant gangliosides in the brain-provides neuroprotection. Most studies have focused on the direct neuroprotective effects of gangliosides on neurons, but their role in other brain cells, in particular microglia, is not known. In this study we investigated the effects of exogenous ganglioside administration and modulation of endogenous ganglioside levels on the response of microglia to inflammatory stimuli, which often contributes to initiation or exacerbation of neurodegeneration.
In vitro studies were performed using BV2 cells, mouse, rat, and human primary microglia cultures. Modulation of microglial ganglioside levels was achieved by administration of exogenous gangliosides, or by treatment with GENZ-123346 and L-t-PDMP, an inhibitor and an activator of glycolipid biosynthesis, respectively. Response of microglia to inflammatory stimuli (LPS, IL-1β, phagocytosis of latex beads) was measured by analysis of gene expression and/or secretion of pro-inflammatory cytokines. The effects of GM1 administration on microglia activation were also assessed in vivo in C57Bl/6 mice, following intraperitoneal injection of LPS.
GM1 decreased inflammatory microglia responses in vitro and in vivo, even when administered after microglia activation. These anti-inflammatory effects depended on the presence of the sialic acid residue in the GM1 glycan headgroup and the presence of a lipid tail. Other gangliosides shared similar anti-inflammatory effects in in vitro models, including GD3, GD1a, GD1b, and GT1b. Conversely, GM3 and GQ1b displayed pro-inflammatory activity. The anti-inflammatory effects of GM1 and other gangliosides were partially reproduced by increasing endogenous ganglioside levels with L-t-PDMP, whereas inhibition of glycolipid biosynthesis exacerbated microglial activation in response to LPS stimulation.
Our data suggest that gangliosides are important modulators of microglia inflammatory responses and reveal that administration of GM1 and other complex gangliosides exerts anti-inflammatory effects on microglia that could be exploited therapeutically.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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