Sylvie Syan

Synaptic Pathology in Borna Disease Virus Persistent Infection

ABSTRACT Borna disease virus (BDV) infection of newborn rats leads to a persistent infection of the brain, which is associated with behavioral and neuroanatonomical abnormalities. These disorders occur in the absence of lymphoid cell infiltrates, and BDV-induced cell damage is restricted to defined brain areas. To investigate if damage to synaptic structures anteceded neuronal loss in BDV neonatally infected rats, we analyzed at different times postinfection the expression levels of growth-associated protein 43 and synaptophysin, two molecules involved in neuroplasticity processes. We found that BDV induced a progressive and marked decrease in the expression of these synaptic markers, which was followed by a significant loss of cortical neurons. Our findings suggest that BDV persistent infection interferes with neuroplasticity processes in specific cell populations. This, in turn, could affect the proper supply of growth factors and other molecules required for survival of selective neuronal populations within the cortex and limbic system structures.

Persistent, noncytolytic infection of neurons by Borna disease virus interferes with ERK 1/2 signaling and abrogates BDNF-induced synaptogenesis

Infection of the central nervous system by Borna disease virus (BDV) provides a unique model to study the mechanisms whereby a persistent viral infection can impair neuronal function and cause behavioral diseases reminiscent of mood disorders, schizophrenia, or autism in humans. In the present work, we studied the effect of BDV infection on the response of hippocampal neurons, the main target for this virus, to the neurotrophin BDNF. We showed that persistent infection did not affect neuronal survival or morphology. However, it blocked BDNF‐induced ERK 1/2 phosphorylation, despite normal expression of the TrkB BDNF receptor. In addition, BDNF‐induced expression of synaptic vesicle proteins was abrogated, which resulted in severely impaired synaptogenesis and defects in synaptic organization. Thus, we provide the first evidence that a virus can interfere specifically with neurotrophin‐regulated neuroplasticity, thereby hampering proper neuronal connectivity. These results may help to understand the behavioral disorders associated with BDV infection.

High-quality genome (re)assembly using chromosomal contact data

Closing gaps in draft genome assemblies can be costly and time-consuming, and published genomes are therefore often left ‘unfinished.’ Here we show that genome-wide chromosome conformation capture (3C) data can be used to overcome these limitations, and present a computational approach rooted in polymer physics that determines the most likely genome structure using chromosomal contact data. This algorithm—named GRAAL—generates high-quality assemblies of genomes in which repeated and duplicated regions are accurately represented and offers a direct probabilistic interpretation of the computed structures. We first validated GRAAL on the reference genome of Saccharomyces cerevisiae, as well as other yeast isolates, where GRAAL recovered both known and unknown complex chromosomal structural variations. We then applied GRAAL to the finishing of the assembly of Trichoderma reesei and obtained a number of contigs congruent with the know karyotype of this species. Finally, we showed that GRAAL can accurately reconstruct human chromosomes from either fragments generated in silico or contigs obtained from de novo assembly. In all these applications, GRAAL compared favourably to recently published programmes implementing related approaches.

Borna Disease Virus Persistent Infection Activates Mitogen-activated Protein Kinase and Blocks Neuronal Differentiation of PC12 Cells

Persistence of Borna disease virus (BDV) in the central nervous system causes damage to specific neuronal populations. BDV is noncytopathic, and the mechanisms underlying neuronal pathology are not well understood. One hypothesis is that infection affects the response of neurons to factors that are crucial for their proliferation, differentiation, or survival. To test this hypothesis, we analyzed the response of PC12 cells persistently infected with BDV to the neurotrophin nerve growth factor (NGF). PC12 is a neural crest-derived cell line that exhibits features of neuronal differentiation in response to NGF. We report that persistence of BDV led to a progressive change of phenotype of PC12 cells and blocked neurite outgrowth in response to NGF. Infection down-regulated the expression of synaptophysin and growth-associated protein-43, two molecules involved in neuronal plasticity, as well as the expression of the chromaffin-specific gene tyrosine hydroxylase. We showed that the block in response to NGF was due in part to the down-regulation of NGF receptors. Moreover, although BDV caused constitutive activation of the ERK1/2 pathway, activated ERKs were not translocated to the nucleus efficiently. These observations may account for the absence of neuronal differentiation of persistently infected PC12 cells treated with NGF.

Chemotaxis of Entamoeba histolytica towards the pro‐inflammatory cytokine TNF is based on PI3K signalling, cytoskeleton reorganization and the Galactose/N‐acetylgalactosamine lectin activity

Entamoeba histolytica is the protozoan parasite responsible for human amoebiasis. During invasive amoebiasis, migration is an essential process and it has previously been shown that the pro‐inflammatory compound tumour necrosis factor (TNF) is produced and that it has a migratory effect on E.u2003histolytica. This paper focuses on the analysis of parasite signalling and cytoskeleton changes leading to directional motility. TNF‐induced signalling was PI3K‐dependent and could lead to modifications in the polarization of certain cytoskeleton‐related proteins. To analyse the effect of TNF signalling on gene expression, we used microarray analysis to screen for genes encoding proteins that were potentially important during chemotaxis towards TNF. Interestingly, we found that elements of the galactose/N‐acetylgalactosamine lectin (Gal/GalNAc lectin) were upregulated during chemotaxis as well as genes encoding proteins involved in cytoskeleton dynamics. The α‐actinin protein appeared to be an important candidate to link the Gal/GalNAc lectin to the cytoskeleton during chemotaxis signalling. Dominant negative parasites blocked for Gal/GalNAc lectin signalling were no longer able to chemotax towards TNF. These results have given us an insight on how E.u2003histolytica changes its cytoskeleton dynamics during chemotaxis and revealed the capital role of PI3K and Gal/GalNAc lectin signalling in chemotaxis.

Borna Disease Virus Glycoprotein Is Required for Viral Dissemination in Neurons

ABSTRACT Borna disease virus (BDV) is a nonsegmented negative-strand RNA virus with a tropism for neurons. Infection with BDV causes neurological diseases in a wide variety of animal species. Although it is known that the virus spreads from neuron to neuron, assembled viral particles have never been visualized in the brains of infected animals. This has led to the hypothesis that BDV spreads as nonenveloped ribonucleoproteins (RNP) rather than as enveloped viral particles. We assessed whether the viral envelope glycoprotein (GP) is required for neuronal dissemination of BDV by using primary cultures of rat hippocampal neurons. We show that upon in vitro infection, BDV replicated and spread efficiently in this system. Despite rapid virus dissemination, very few infectious viral particles were detectable in the culture. However, neutralizing antibodies directed against BDV-GP inhibited BDV spread. In addition, interference with BDV-GP processing by inhibiting furin-mediated cleavage of the glycoprotein blocked virus spread. Finally, antisense treatment with peptide nucleic acids directed against BDV-GP mRNA inhibited BDV dissemination, marking BDV-GP as an attractive target for antiviral therapy against BDV. Together, our results demonstrate that the expression and correct processing of BDV-GP are necessary for BDV dissemination in primary cultures of rat hippocampal neurons, arguing against the hypothesis that the virus spreads from neuron to neuron in the form of nonenveloped RNP.

Roles of the H-2D(b) and H-K(b) genes in resistance to persistent Theiler's murine encephalomyelitis virus infection of the central nervous system.

Theilers murine encephalomyelitis virus, a member of the Picornaviridae family, persists in the spinal cord of susceptible strains of mice. Resistant strains of mice, such as the H-2(b) strain, clear the virus infection after an acute encephalomyelitis. The H-2D locus, but not the H-2K locus, has a major effect on this resistance, although both loci code for MHC class I molecules with similar general properties. For the present work, we rendered susceptible H-2(q) FVB/N mice transgenic for either the H-2D(b)gene, the H-2K(b) gene or a chimeric H-2D(b)/K(b) gene in which the exons encoding the peptide-binding groove of the H-2K(b) gene have been replaced by those of the H-2D(b)gene. Mice transgenic for either the H-2D(b)gene or the chimeric H-2D(b)/K(b) gene were significantly more resistant to persistent virus infection than mice transgenic for the H-2K(b) gene, suggesting that the difference in the effects of the H-2D(b)gene and the H-2K(b) gene are due to the nature of the peptides presented by these class I molecules.

Glucose starvation boosts Entamoeba histolytica virulence.

The unicellular parasite, Entamoeba histolytica, is exposed to numerous adverse conditions, such as nutrient deprivation, during its life cycle stages in the human host. In the present study, we examined whether the parasite virulence could be influenced by glucose starvation (GS). The migratory behaviour of the parasite and its capability to kill mammalian cells and to lyse erythrocytes is strongly enhanced following GS. In order to gain insights into the mechanism underlying the GS boosting effects on virulence, we analyzed differences in protein expression levels in control and glucose-starved trophozoites, by quantitative proteomic analysis. We observed that upstream regulatory element 3-binding protein (URE3-BP), a transcription factor that modulates E.histolytica virulence, and the lysine-rich protein 1 (KRiP1) which is induced during liver abscess development, are upregulated by GS. We also analyzed E. histolytica membrane fractions and noticed that the Gal/GalNAc lectin light subunit LgL1 is up-regulated by GS. Surprisingly, amoebapore A (Ap-A) and cysteine proteinase A5 (CP-A5), two important E. histolytica virulence factors, were strongly down-regulated by GS. While the boosting effect of GS on E. histolytica virulence was conserved in strains silenced for Ap-A and CP-A5, it was lost in LgL1 and in KRiP1 down-regulated strains. These data emphasize the unexpected role of GS in the modulation of E.histolytica virulence and the involvement of KRiP1 and Lgl1 in this phenomenon.

A proteomic and cellular analysis of uropods in the pathogen Entamoeba histolytica.

Exposure of Entamoeba histolytica to specific ligands induces cell polarization via the activation of signalling pathways and cytoskeletal elements. The process leads to formation of a protruding pseudopod at the front of the cell and a retracting uropod at the rear. In the present study, we show that the uropod forms during the exposure of trophozoites to serum isolated from humans suffering of amoebiasis. To investigate uropod assembly, we used LC-MS/MS technology to identify protein components in isolated uropod fractions. The galactose/N-acetylgalactosamine lectin, the immunodominant antigen M17 (which is specifically recognized by serum from amoeba-infected persons) and a few other cells adhesion-related molecules were primarily involved. Actin-rich cytoskeleton components, GTPases from the Rac and Rab families, filamin, α-actinin and a newly identified ezrin-moesin-radixin protein were the main factors found to potentially interact with capped receptors. A set of specific cysteine proteases and a serine protease were enriched in isolated uropod fractions. However, biological assays indicated that cysteine proteases are not involved in uropod formation in E. histolytica, a fact in contrast to the situation in human motile immune cells. The surface proteins identified here are testable biomarkers which may be either recognized by the immune system and/or released into the circulation during amoebiasis.

Astrocyte-to-neuron intercellular prion transfer is mediated by cell-cell contact

Prion diseases are caused by misfolding of the cellular protein PrPC to an infectious conformer, PrPSc. Intercellular PrPSc transfer propagates conversion and allows infectivity to move from the periphery to the brain. However, how prions spread between cells of the central nervous system is unclear. Astrocytes are specialized non-neuronal cells within the brain that have a number of functions indispensable for brain homeostasis. Interestingly, they are one of the earliest sites of prion accumulation in the brain. A fundamental question arising from this observation is whether these cells are involved in intercellular prion transfer and thereby disease propagation. Using co-culture systems between primary infected astrocytes and granule neurons or neuronal cell lines, we provide direct evidence that prion-infected astrocytes can disseminate prion to neurons. Though astrocytes are capable of secreting PrP, this is an inefficient method of transferring prion infectivity. Efficient transfer required co-culturing and direct cell contact. Astrocytes form numerous intercellular connections including tunneling nanotubes, containing PrPSc, often colocalized with endolysosomal vesicles, which may constitute the major mechanism of transfer. Because of their role in intercellular transfer of prions astrocytes may influence progression of the disease.