Zika virus propagation and release in human fetal astrocytes can be suppressed by neutral sphingomyelinase-2 inhibitor GW4869.
ABSTRACT: Zika virus (ZIKV) is a neurotrophic flavivirus that is capable of infecting humans, leading to brain abnormalities during fetal development. The ZIKV infectivity in neural target cells remains poorly understood. Here, we found that ZIKV specifically infected glial fibrillary acidic protein- and S100B-positive primary human astrocytes derived from fetal brains. In contrast, neuron-specific Class III ?-tubulin (TuJ1)-positive neurons in the astrocyte cultures and SOX2-positive neural progenitor cells derived from the fetal brains were less susceptible to ZIKV infection compared with astrocytes. The infected astrocytes released competent viral particles and manifested programmed cell death with a progressive cytopathic effect. Interestingly, ZIKV infection in human fetal astrocytes induced a significant increase of extracellular vesicles (EVs). Treatment with GW4869, a specific inhibitor of neutral sphingomyelinase-2, decreased EV levels, suppressed ZIKV propagation, and reduced the release of infectious virions in astrocytes. Therefore, ZIKV infects primary human fetal astrocytes and the infection can be suppressed by neutral sphingomyelinase-2 inhibitor GW4869. Further investigation into sphingomyelin metabolism and EVs may provide insights to the therapeutic treatment of ZIKV infection.
Project description:We present evidence here that exosomes stimulate aggregation of amyloid beta (A?)1-42 in vitro and in vivo and interfere with uptake of A? by primary cultured astrocytes and microglia in vitro. Exosome secretion is prevented by the inhibition of neutral sphingomyelinase 2 (nSMase2), a key regulatory enzyme generating ceramide from sphingomyelin, with GW4869. Using the 5XFAD mouse, we show that intraperitoneal injection of GW4869 reduces the levels of brain and serum exosomes, brain ceramide, and A?1-42 plaque load. Reduction of total A?1-42 as well as number of plaques in brain sections was significantly greater (40% reduction) in male than female mice. Our results suggest that GW4869 reduces amyloid plaque formation in vivo by preventing exosome secretion and identifies nSMase2 as a potential drug target in AD by interfering with exosome secretion.
Project description:Cytoplasmic TDP-43 aggregation is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Here we investigated the role of exosomes in the secretion and propagation of TDP-43 aggregates. TDP-43 was detected in secreted exosomes from Neuro2a cells and primary neurons but not from astrocytes or microglia. Evidence is presented that protein aggregation and autophagy inhibition are factors that promote exosomal secretion of TDP-43. We also report that levels of exosomal TDP-43 full length and C-terminal fragment species are upregulated in human amyotrophic lateral sclerosis brains. Exposure of Neuro2a cells to exosomes from amyotrophic lateral sclerosis brain, but not from control brain, caused cytoplasmic redistribution of TDP-43, suggesting that secreted exosomes might contribute to propagation of TDP-43 proteinopathy. Yet, inhibition of exosome secretion by inactivation of neutral sphingomyelinase 2 with GW4869 or by silencing RAB27A provoked formation of TDP-43 aggregates in Neuro2a cells. Moreover, administration of GW4869 exacerbated the disease phenotypes of transgenic mice expressing human TDP-43A315T mutant. Thus, even though results suggest that exosomes containing pathological TDP-43 may play a key role in the propagation of TDP-43 proteinopathy, a therapeutic strategy for amyotrophic lateral sclerosis based on inhibition of exosome production would seem inappropriate, as in vivo data suggest that exosome secretion plays an overall beneficial role in neuronal clearance of pathological TDP-43.
Project description:Malformations of the fetal CNS, known as microcephaly, have been linked to Zika virus (ZIKV) infection. Here, the responses of mammalian and mosquito cell lines, in addition to primary human fetal astrocytes and neurons were studied following infection by ZIKV strains Brazil 2016 (ZIKV-BR), French Polynesia 2013 (ZIKV-FP), and Uganda #976 1947 (ZIKV-UG). Viral production, cell viability, infectivity rate, and mobility of endocytotic ZIKV-laden vesicles were compared. All cell types (SK-N-SH, Vero E6, C6/36, human fetal astrocytes and human fetal neurons) released productive virus. Among primary cells, astrocytes were more susceptible to ZIKV infection than neurons, released more progeny virus and tolerated higher virus loads than neurons. In general, the infection rate of ZIKV-UG strain was the highest. All ZIKV strains elicited differences in trafficking of ZIKV-laden endocytotic vesicles in the majority of cell types, including astrocytes and neurons, except in mosquito cells, where ZIKV infection failed to induce cell death. These results represent a thorough screening of cell viability, infection and production of three ZIKV strains in five different cell types and demonstrate that ZIKV affects vesicle mobility in all but mosquito cells.
Project description:Extracellular vesicles (EVs) are membrane particles secreted from cells into all body fluids. Several EV populations exist differing in size and cellular origin. Using differential centrifugation EVs pelleting at 14,000 g ("microvesicles" (MV)) and 100,000 g ("exosomes") are distinguishable by protein markers. Neutral sphingomyelinase (nSMase) inhibition has been shown to inhibit exosome release from cells and has since been used to study their functional implications. How nSMases (also known as SMPD2 and SMPD3) affect the basal secretion of MVs is unclear. Here we investigated how SMPD2/3 impact both EV populations. SMPD2/3 inhibition by GW4869 or RNAi decreases secretion of exosomes, but also increases secretion of MVs from the plasma membrane. Both populations differ significantly in metabolite composition and Wnt proteins are specifically loaded onto MVs under these conditions. Taken together, our data reveal a novel regulatory function of SMPD2/3 in vesicle budding from the plasma membrane and clearly suggest that - despite the different vesicle biogenesis - the routes of vesicular export are adaptable.
Project description:The Zika virus (ZIKV) epidemic has resulted in congenital abnormalities in fetuses and neonates. Although some cross-reactive dengue virus (DENV)-specific antibodies can enhance ZIKV infection in mice, those recognizing the DENV E-dimer epitope (EDE) can neutralize ZIKV infection in cell culture. We evaluated the therapeutic activity of human monoclonal antibodies to DENV EDE for their ability to control ZIKV infection in the brains, testes, placentas, and fetuses of mice. A single dose of the EDE1-B10 antibody given 3 d after ZIKV infection protected against lethality, reduced ZIKV levels in brains and testes, and preserved sperm counts. In pregnant mice, wild-type or engineered LALA variants of EDE1-B10, which cannot engage Fcg receptors, diminished ZIKV burden in maternal and fetal tissues, and protected against fetal demise. Because neutralizing antibodies to EDE have therapeutic potential against ZIKV, in addition to their established inhibitory effects against DENV, it may be possible to develop therapies that control disease caused by both viruses.
Project description:Outcomes of Zika virus (ZIKV) infection in pregnant women vary from the birth of asymptomatic offspring to abnormal development and severe brain lesions in fetuses and infants. There are concerns that offspring affected in utero and born without apparent symptoms may develop mental illnesses. Therefore, animal models are important to test interventions against in utero infection and health sequelae in symptomatic and likely more widespread asymptomatic offspring. To partially reproduce in utero infection in humans, we directly inoculated selected porcine conceptuses with ZIKV. Inoculation resulted in rapid trans-fetal infections, persistent infection in conceptuses, molecular pathology in fetal brains, fetal antibody and type I interferon responses. Offspring infected in utero showed ZIKV in their fetal membranes collected after birth. Some in utero affected piglets were small, depressed, had undersized brains, and showed seizures. Some piglets showed potentially increased activity. Our data suggest that porcine model of persistent in utero ZIKV infection has a strong potential for translational research and can be used to test therapeutic interventions in vivo.
Project description:The recent emergence of Zika virus (ZIKV) in Brazil was associated with an increased number of fetal brain infections that resulted in a spectrum of congenital neurological complications known as congenital Zika syndrome (CZS). Herein, we generated de novo from sequence data an early Asian lineage ZIKV isolate (ZIKV-MY; Malaysia, 1966) not associated with microcephaly and compared the in vitro replication kinetics and fetal brain infection in interferon ?/? receptor 1 knockout (IFNAR1-/-) dams of this isolate and of a Brazilian isolate (ZIKV-Natal; Natal, 2015) unequivocally associated with microcephaly. The replication efficiencies of ZIKV-MY and ZIKV-Natal in A549 and Vero cells were similar, while ZIKV-MY replicated more efficiently in wild-type (WT) and IFNAR-/- mouse embryonic fibroblasts. Viremias in IFNAR1-/- dams were similar after infection with ZIKV-MY or ZIKV-Natal, and importantly, infection of fetal brains was also not significantly different. Thus, fetal brain infection does not appear to be a unique feature of Brazilian ZIKV isolates.
Project description:Extracellular vesicles (EVs) can modulate microenvironments by transferring biomolecules, including RNAs and proteins derived from releasing cells, to target cells. To understand the molecular mechanisms maintaining the neural stem cell (NSC) niche through EVs, a new transgenic (Tg) rat strain that can release human CD63-GFP-expressing EVs from the NSCs was established. Human CD63-GFP expression was controlled under the rat Sox2 promoter (Sox2/human CD63-GFP), and it was expressed in undifferentiated fetal brains. GFP signals were specifically observed in in vitro cultured NSCs obtained from embryonic brains of the Tg rats. We also demonstrated that embryonic NSC (eNSC)-derived EVs were labelled by human CD63-GFP. Furthermore, when we examined the transfer of EVs, eNSC-derived EVs were found to be incorporated into astrocytes and eNSCs, thus implying an EV-mediated communication between different cell types around NSCs. This new Sox2/human CD63-GFP Tg rat strain should provide resources to analyse the cell-to-cell communication via EVs in NSC microenvironments.
Project description:Sexual Zika virus (ZIKV) transmission from men to women occurs less frequently than the often-detected high viral loads in semen would suggest, but worries that this transmission route predisposes to fetal damage in pregnant women remain. To better understand sexual ZIKV pathogenesis, we studied the permissiveness of the human female genital tract to infection and the effect of semen on this process. ZIKV replicates in vaginal tissues and primary epithelial cells from the vagina, ectocervix, and endocervix and induces an innate immune response, but also continues to replicate without cytopathic effect. Infection of genital cells and tissues is strongly inhibited by extracellular vesicles (EV) in semen at physiological vesicle-to-virus ratios. Liposomes with the same composition as semen EVs also impair infection, indicating that the EV's lipid fraction, rather than their protein or RNA cargo, is responsible for this anti-viral effect. Thus, EVs in semen potently restrict ZIKV transmission, but the virus propagates well once infection in the recipient mucosa has been established.
Project description:Zika virus (ZIKV) is an emerging pathogen that can cause microcephaly and other neurological defects in developing fetuses. The cellular response to ZIKV in the fetal brain is not well understood. Here, we show that ZIKV infection of human fetal astrocytes (HFAs), the most abundant cell type in the brain, results in elevated expression and secretion of fibroblast growth factor 2 (FGF2). This cytokine was shown to enhance replication and spread of ZIKV in HFAs and human fetal brain explants. The proviral effect of FGF2 is likely mediated in part by suppression of the interferon response, which would represent a novel mechanism by which viruses antagonize host antiviral defenses. We posit that FGF2-enhanced virus replication in the fetal brain contributes to the neurodevelopmental disorders associated with in utero ZIKV infection. As such, targeting FGF2-dependent signaling should be explored further as a strategy to limit replication of ZIKV.