Zika virus infection of mature neurons from immunocompetent mice generates a disease-associated microglia and a tauopathy-like phenotype in link with a delayed interferon beta response.

BACKGROUND: Zika virus (ZIKV) infection at postnatal or adult age can lead to neurological disorders associated with cognitive defects. Yet, how mature neurons respond to ZIKV remains substantially unexplored. METHODS: The impact of ZIKV infection on mature neurons and microglia was analyzed at the molecular and cellular levels, in vitro using immunocompetent primary cultured neurons and microglia, and in vivo in the brain of adult immunocompetent mice following intracranial ZIKV inoculation. We have used C57BL/6 and the genetically diverse Collaborative Cross mouse strains, displaying a broad range of susceptibility to ZIKV infection, to question the correlation between the effects induced by ZIKV infection on neurons and microglia and the in vivo susceptibility to ZIKV. RESULTS: As a result of a delayed induction of interferon beta (IFNB) expression and response, infected neurons displayed an inability to stop ZIKV replication, a trait that was further increased in neurons from susceptible mice. Alongside with an enhanced expression of ZIKV RNA, we observed in vivo, in the brain of susceptible mice, an increased level of active Iba1-expressing microglial cells occasionally engulfing neurons and displaying a gene expression profile close to the molecular signature of disease-associated microglia (DAM). In vivo as well as in vitro, only neurons and not microglial cells were identified as infected, raising the question of the mechanisms underlying microglia activation following brain ZIKV infection. Treatment of primary cultured microglia with conditioned media from ZIKV-infected neurons demonstrated that type-I interferons (IFNs-I) secreted by neurons late after infection activate non-infected microglial cells. In addition, ZIKV infection induced pathological phosphorylation of Tau (pTau) protein, a hallmark of neurodegenerative tauopathies, in vitro and in vivo with clusters of neurons displaying pTau surrounded by active microglial cells. CONCLUSIONS: We show that ZIKV-infected mature neurons display an inability to stop viral replication in link with a delayed IFNB expression and response, while signaling microglia for activation through IFNs-I secreted at late times post-infection. In the brain of ZIKV-infected susceptible mice, uninfected microglial cells adopt an active morphology and a DAM expression profile, surrounding and sometimes engulfing neurons while ZIKV-infected neurons accumulate pTau, overall reflecting a tauopathy-like phenotype.

The NSs Protein Encoded by the Virulent Strain of Rift Valley Fever Virus Targets the Expression of Abl2 and the Actin Cytoskeleton of the Host, Affecting Cell Mobility, Cell Shape, and Cell-Cell Adhesion.

Rift Valley fever virus (RVFV) is a highly pathogenic zoonotic arbovirus endemic in many African countries and the Arabian Peninsula. Animal infections cause high rates of mortality and abortion among sheep, goats, and cattle. In humans, an estimated 1 to 2% of RVFV infections result in severe disease (encephalitis, hepatitis, or retinitis) with a high rate of lethality when associated with hemorrhagic fever. The RVFV NSs protein, which is the main virulence factor, counteracts the host innate antiviral response to favor viral replication and spread. However, the mechanisms underlying RVFV-induced cytopathic effects and the role of NSs in these alterations remain for the most part unknown. In this work, we have analyzed the effects of NSs expression on the actin cytoskeleton while conducting infections with the NSs-expressing virulent (ZH548) and attenuated (MP12) strains of RVFV and the non-NSs-expressing avirulent (ZH548ΔNSs) strain, as well as after the ectopic expression of NSs. In macrophages, fibroblasts, and hepatocytes, NSs expression prevented the upregulation of Abl2 (a major regulator of the actin cytoskeleton) expression otherwise induced by avirulent infections and identified here as part of the antiviral response. The presence of NSs was also linked to an increased mobility of ZH548-infected cells compared to ZH548ΔNSs-infected fibroblasts and to strong changes in cell morphology in nonmigrating hepatocytes, with reduction of lamellipodia, cell spreading, and dissolution of adherens junctions reminiscent of the ZH548-induced cytopathic effects observed in vivo Finally, we show evidence of the presence of NSs within long actin-rich structures associated with NSs dissemination from NSs-expressing toward non-NSs-expressing cells.IMPORTANCE Rift Valley fever virus (RVFV) is a dangerous human and animal pathogen that was ranked by the World Health Organization in 2018 as among the eight pathogens of most concern for being likely to cause wide epidemics in the near future and for which there are no, or insufficient, countermeasures. The focus of this work is to address the question of the mechanisms underlying RVFV-induced cytopathic effects that participate in RVFV pathogenicity. We demonstrate here that RVFV targets cell adhesion and the actin cytoskeleton at the transcriptional and cellular level, affecting cell mobility and inducing cell shape collapse, along with distortion of cell-cell adhesion. All these effects may participate in RVFV-induced pathogenicity, facilitate virulent RVFV dissemination, and thus constitute interesting potential targets for future development of antiviral therapeutic strategies that, in the case of RVFV, as with several other emerging arboviruses, are presently lacking.

Genome-wide identification of genic and intergenic neuronal DNA regions bound by Tau protein under physiological and stress conditions.

Tauopathies such as Alzheimer's Disease (AD) are neurodegenerative disorders for which there is presently no cure. They are named after the abnormal oligomerization/aggregation of the neuronal microtubule-associated Tau protein. Besides its role as a microtubule-associated protein, a DNA-binding capacity and a nuclear localization for Tau protein has been described in neurons. While questioning the potential role of Tau-DNA binding in the development of tauopathies, we have carried out a large-scale analysis of the interaction of Tau protein with the neuronal genome under physiological and heat stress conditions using the ChIP-on-chip technique that combines Chromatin ImmunoPrecipitation (ChIP) with DNA microarray (chip). Our findings show that Tau protein specifically interacts with genic and intergenic DNA sequences of primary culture of neurons with a preference for DNA regions positioned beyond the ±5000 bp range from transcription start site. An AG-rich DNA motif was found recurrently present within Tau-interacting regions and 30% of Tau-interacting regions overlapped DNA sequences coding for lncRNAs. Neurological processes affected in AD were enriched among Tau-interacting regions with in vivo gene expression assays being indicative of a transcriptional repressor role for Tau protein, which was exacerbated in neurons displaying nuclear pathological oligomerized forms of Tau protein.

Loss of Tau protein affects the structure, transcription and repair of neuronal pericentromeric heterochromatin.

Pericentromeric heterochromatin (PCH) gives rise to highly dense chromatin sub-structures rich in the epigenetic mark corresponding to the trimethylated form of lysine 9 of histone H3 (H3K9me3) and in heterochromatin protein 1α (HP1α), which regulate genome expression and stability. We demonstrate that Tau, a protein involved in a number of neurodegenerative diseases including Alzheimer's disease (AD), binds to and localizes within or next to neuronal PCH in primary neuronal cultures from wild-type mice. Concomitantly, we show that the clustered distribution of H3K9me3 and HP1α, two hallmarks of PCH, is disrupted in neurons from Tau-deficient mice (KOTau). Such altered distribution of H3K9me3 that could be rescued by overexpressing nuclear Tau protein was also observed in neurons from AD brains. Moreover, the expression of PCH non-coding RNAs, involved in PCH organization, was disrupted in KOTau neurons that displayed an abnormal accumulation of stress-induced PCH DNA breaks. Altogether, our results demonstrate a new physiological function of Tau in directly regulating neuronal PCH integrity that appears disrupted in AD neurons.

ß-Catenin Upregulates the Constitutive and Virus-Induced Transcriptional Capacity of the Interferon Beta Promoter through T-Cell Factor Binding Sites.

Rapid upregulation of interferon beta (IFN-ß) expression following virus infection is essential to set up an efficient innate antiviral response. Biological roles related to the antiviral and immune response have also been associated with the constitutive production of IFN-ß in naive cells. However, the mechanisms capable of modulating constitutive IFN-ß expression in the absence of infection remain largely unknown. In this work, we demonstrate that inhibition of the kinase glycogen synthase kinase 3 (GSK-3) leads to the upregulation of the constitutive level of IFN-ß expression in noninfected cells, provided that GSK-3 inhibition is correlated with the binding of ß-catenin to the IFN-ß promoter. Under these conditions, IFN-ß expression occurred through the T-cell factor (TCF) binding sites present on the IFN-ß promoter independently of interferon regulatory factor 3 (IRF3). Enhancement of the constitutive level of IFN-ß per se was able to confer an efficient antiviral state to naive cells and acted in synergy with virus infection to stimulate virus-induced IFN-ß expression. Further emphasizing the role of ß-catenin in the innate antiviral response, we show here that highly pathogenic Rift Valley fever virus (RVFV) targets the Wnt/ß-catenin pathway and the formation of active TCF/ß-catenin complexes at the transcriptional and protein level in RVFV-infected cells and mice.

Substrate-induced PC12 cell differentiation without filopodial, lamellipodial activity or NGF stimulationa.

Nanoscale gradients in energy of adhesion are physical cues from the extracellular environment that can significantly affect cell functions and enhance the neuronal differentiation of PC12 cells. How such surface effects can trigger differentiation and initiate neurite outgrowth, remains to be elucidated. Here we used surface modification, atomic force microscopy and immunofluorescence to analyze PC12 cells. We studied the kinetics of neurites growth under cytochalasin-B treatment, known as an inhibitor of actin polymerization. We found that neither filopodia nor lamellipodia are involved in detecting the surface effects that induce the differentiation of PC12 cells. This finding suggests that the solution to this problem lies beyond identifying a precise cytoskeleton-associated cell-substrate intermediate. Thus, a more comprehensive model is probably required to identify the mechanism by which cell-substrate interactions are eventually translated into a differentiation signal.

Large-scale chromatin immunoprecipitation with promoter sequence microarray analysis of the interaction of the NSs protein of Rift Valley fever virus with regulatory DNA regions of the host genome.

Rift Valley fever virus (RVFV) is a highly pathogenic Phlebovirus that infects humans and ruminants. Initially confined to Africa, RVFV has spread outside Africa and presently represents a high risk to other geographic regions. It is responsible for high fatality rates in sheep and cattle. In humans, RVFV can induce hepatitis, encephalitis, retinitis, or fatal hemorrhagic fever. The nonstructural NSs protein that is the major virulence factor is found in the nuclei of infected cells where it associates with cellular transcription factors and cofactors. In previous work, we have shown that NSs interacts with the promoter region of the beta interferon gene abnormally maintaining the promoter in a repressed state. In this work, we performed a genome-wide analysis of the interactions between NSs and the host genome using a genome-wide chromatin immunoprecipitation combined with promoter sequence microarray, the ChIP-on-chip technique. Several cellular promoter regions were identified as significantly interacting with NSs, and the establishment of NSs interactions with these regions was often found linked to deregulation of expression of the corresponding genes. Among annotated NSs-interacting genes were present not only genes regulating innate immunity and inflammation but also genes regulating cellular pathways that have not yet been identified as targeted by RVFV. Several of these pathways, such as cell adhesion, axonal guidance, development, and coagulation were closely related to RVFV-induced disorders. In particular, we show in this work that NSs targeted and modified the expression of genes coding for coagulation factors, demonstrating for the first time that this hemorrhagic virus impairs the host coagulation cascade at the transcriptional level.

Interplay between long- and short-range interactions drives neuritogenesis on stiff surfaces.

Substrate factors such as surface energy distribution can affect cell functions, such as neuronal differentiation of PC12 cells. However, the surface effects that trigger such cell responses need to be clarified and analyzed. Here we show that the total surface tension is not a critical parameter. Self-assembled monolayers of alkylsiloxanes on glass were used as culture substrates. By changing the nanoscale structure and ordering of the monolayer, we designed surfaces with a range of dispersive (γ(d) ) and nondispersive (γ(nd) ) potentials, but with a similar value for total free-energy (50 ≤ γ(d) + γ(nd) ≤ 55 mN m⁻¹). When seeded on surfaces displaying γ(d) /γ(nd) ≤ 3.7, PC12 cells underwent low level of neuritogenesis. On surfaces exhibiting γ(d) /γ(nd) ≥ 5.4, neurite outgrowth was greatly enhanced and apparent by only 24 h of culture in absence of nerve growth-factor treatment. These data indicate how the spatial distribution of surface potentials may control neuritogenesis, thus providing a new criterion to address nerve regeneration issues on rigid biocompatible surfaces.

Neuronal adhesion and differentiation driven by nanoscale surface free-energy gradients.

Recent results indicate that, in addition to chemical, spatial and mechanical cues, substrate physical cues such as gradients in surface energy may also impact cell functions, such as neuronal differentiation of PC12 cells. However, it remains to be determined what surface effect is the most critical in triggering PC12 cell differentiation. Here we show that, beyond continuously probing the surface energy landscape of their environment, PC12 cells are highly sensitive to nanoscale chemical heterogeneities. Self-assembled monolayers of alkylsiloxanes on glass were used as a culture substrate. By changing the structure, ordering and chemical nature of the monolayer, the surface energy distribution is altered. While both well-ordered CH(3) terminated substrates and bare glass (OH terminated) substrates did not favor PC12 cell adhesion, PC12 cells seeded on highly disordered CH(3)/OH substrates underwent enhanced adhesion and prompt neuritogenesis by 48 h of culture, without nerve growth factor treatment. These data illustrate that surface free-energy gradients, generated by nanoscale chemical heterogeneities, are critical to biological processes such as nerve regeneration on biomaterials.

Influence of surface energy distribution on neuritogenesis.

PC12 cells are a useful model to study neuronal differentiation, as they can undergo terminal differentiation, typically when treated with nerve growth factor (NGF). In this study we investigated the influence of surface energy distribution on PC12 cell differentiation, by atomic force microscopy (AFM) and immunofluorescence. Glass surfaces were modified by chemisorption: an aminosilane, n-[3-(trimethoxysilyl)propyl]ethylendiamine (C(8)H(22)N(2)O(3)Si; EDA), was grafted by polycondensation. AFM analysis of substrate topography showed the presence of aggregates suggesting that the adsorption is heterogeneous, and generates local gradients in energy of adhesion. PC12 cells cultured on these modified glass surfaces developed neurites in absence of NGF treatment. In contrast, PC12 cells did not grow neurites when cultured in the absence of NGF on a relatively smooth surface such as poly-L-lysine substrate, where amine distribution is rather homogeneous. These results suggest that surface energy distribution, through cell-substrate interactions, triggers mechanisms that will drive PC12 cells to differentiate and to initiate neuritogenesis. We were able to create a controlled physical nano-structuration with local variations in surface energy that allowed the study of these parameters on neuritogenesis.

Aggregation of expanded huntingtin in the brains of patients with Huntington disease.

Huntingtin containing an expanded polyglutamine causes neuronal death and Huntington disease. Although expanded huntingtin is found in virtually every cell type, its toxicity is limited to neurons of certain areas of the brain, such as cortex and caudate/putamen. In affected areas of the brain, expanded huntingtin is not found in its intact monomeric form. It is found instead in the form of N-terminal fragments, oligomers and polymers, all of which accumulate in the cortex. Whereas the oligomer is mostly soluble, the polymers and the fragments associate with each other and with other proteins to form the insoluble inclusions characteristic of the disease. It is likely that the aggregates containing expanded huntingtin are toxic to neurons, but it remains to be determined whether the oligomer or the inclusion is the toxic species.

The cytosolic chaperonin CCT associates to cytoplasmic microtubular structures during mammalian spermiogenesis and to heterochromatin in germline and somatic cells.

Spermiogenesis, the haploid phase of spermatogenesis, is characterised by a dramatic cytodifferentiation of spermatids. The two major steps, nuclear shaping and cytoplasmic reorganisation of the organelles, rely on an extensive remodelling of the microtubule cytoskeleton. Folding of alpha- and beta-tubulin is mediated by the cytoplasmic chaperonin containing TCP-1 (CCT), highly expressed in testis. We studied CCT cellular distribution throughout spermatogenesis by immunofluorescence and immunoelectron microscopy. We unveil two main cytoplasmic localisations for CCT: at the centrosome and at the microtubules of the manchette, a structure unique to male germ cells. Both structures are essential for spermatid differentiation and may require CCT function. Although CCT is essentially cytoplasmic, a few reports suggest that a subset may have a nuclear localisation. We demonstrate that in the nucleus of germline and somatic cells, part of CCT associates to heterochromatin. In interphase cells, CCT seems generally confined to constitutive heterochromatin. Nevertheless, in condensing nucleus of future spermatozoon, it is also associated with chromatin undergoing compaction. Finally, in fully-condensed mitotic chromosomes, CCT is located all along the chromosomes. Our finding that CCT is associated with constitutive heterochromatin and to compacting chromatin raises the possibility that it may be implicated in maintenance and remodelling of heterochromatin.

Glutamylated tubulin: diversity of expression and distribution of isoforms.

Glutamylation of alpha and beta tubulin isotypes is a major posttranslational modification giving rise to diversified isoforms occurring mainly in neurotubules, centrioles, and axonemes. Monoglutamylated tubulin isoforms can be differentially recognized by two mAbs, B3 and GT335, which both recognize either polyglutamylated isoforms. In the present study, immunoelectron microscopy and immunofluorescence analyses were performed with these two mAbs to determine the expression and distribution of glutamylated tubulin isoforms in selected biological models whose tubulin isotypes are characterized. In mouse spermatozoa, microtubules of the flagellum contain polyglutamylated isoforms except in the tip where only monoglutamylated isoforms are detected. In spermatids, only a subset of manchette microtubules contain monoglutamylated tubulin isoforms. Cytoplasmic microtubules of Sertoli cells are monoglutamylated. Mitotic and meiotic spindles of germ cells are monoglutamylated whereas the HeLa cell mitotic spindle is polyglutamylated. Three models of axonemes are demonstrated as a function of the degree and extent of tubulin glutamylation. In lung ciliated cells, axonemes are uniformly polyglutamylated. In sea urchin sperm and Chlamydomonas, flagellar microtubules are polyglutamylated in their proximal part and monoglutamylated in their distal part. In Paramecium, cilia are bi- or monoglutamylated only at their base. In all cells, centrioles or basal bodies are polyglutamylated. These new data emphasize the importance of glutamylation in all types of microtubules and strengthen the hypothesis of its role in the regulation of the intracellular traffic and flagellar motility.